MXPA98003840A - Vaccine polivalent anti-den - Google Patents

Vaccine polivalent anti-den

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Publication number
MXPA98003840A
MXPA98003840A MXPA/A/1998/003840A MX9803840A MXPA98003840A MX PA98003840 A MXPA98003840 A MX PA98003840A MX 9803840 A MX9803840 A MX 9803840A MX PA98003840 A MXPA98003840 A MX PA98003840A
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Mexico
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dengue
protein
virus
sequence
polypeptide
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MXPA/A/1998/003840A
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Spanish (es)
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Deubel Vincent
Staropoli Isabelle
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Institut Pasteur
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Publication of MXPA98003840A publication Critical patent/MXPA98003840A/en

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Abstract

Vaccine of polyvalent subunits against the Flaviviridiae especially against the Dengue virus, which comprises the recombinant peptides from each serotype, where the carboxylated extremity is replaced by a peptide containing 2 to 8 histidines, tryptophan or cysteine

Description

ANTI-DENGUE POLIVALENT VACCINE The present invention relates to a vaccine of polyvalent subunits against viruses of the Flaviviridia family and especially against flaviviruses such as Dengue virus, yellow fever, Japanese encephalitis, or other Flaviviridiae, or "Flavi-like" viruses. "like the hepatitis C virus, as well as the mixtures between them (18). Flaviviruses are viruses of the arbovirus family (formerly group B arboviruses). These are the vertebrate viruses usually transmitted by the hematophagous arthropods (mosquitoes, ticks, sandflies), where they multiply before transmission. The maintenance cycle then involves a vertebrate, an arthropod and a receptor. Certain arboviruses may concern man and induce pathologies, the arboviruses that are found mostly in tropical regions. If there is a vaccine against yellow fever since 1930, or more recently against Japanese encephalitis and tick, the other flaviviruses do not currently have any vaccine; This is especially the case of Dengue. Tens of millions of individuals are affected annually by Dengue, particularly in the intertropical regions where the disease rages in the state of uninterrupted pandemic. The virus responsible for Dengue propagates concomitantly to its vector, the Aedes aegypti mosquito. The disease is characterized by a strong fever accompanied by headache, nausea and joint and muscle pain that disappear after a few days. However, the symptoms may intensify to lead to the sometimes fatal hemorrhagic manifestations when the disease is associated with a hypovolemic shock especially in infants. The agent responsible for Dengue is an AR-N flavivirus of the Flaviviridia family, all of them like yellow fever virus or Japanese encephalitis virus. 4 serotypes are known. The hemorrhagic dengue could be a consequence of a poorly known immunological phenomenon, which undoubtedly implies a cascade of cytokines linked to the induction of a cytotoxic cellular immunity, which can result from a sequential infection by many serotypes. The absence of an animal model hinders the conception of a vaccine since it is not possible to explore in detail the factors involved in the serious manifestations of the disease. A first vaccine against Dengue is currently being experimented. It is a vaccine based on 4 living serotypes, attenuated by successive passages of the virus in cell culture. The biggest drawback of this type of vaccine is the possibility of attenuated viruses to fall back towards virulence, by genetic reversion or recombination between genomes. It also presents a potential risk for newborns and for immunosuppressed people. In addition, certain infants vaccinated at half of the tetravalent vaccine appear not to be protected against the 4 serotypes. The structure of the genome and the amino acid sequence of the Dengue virus, especially of serotype 2, has been described by V. Deubel et al. (1). The Dengue virus is a virus with wrapped RNA of approximately 50 nanometers. Like the yellow fever virus, all viral proteins are encoded by a segment of about 10 kilobases whose structure is as follows: 5'C-preM / -E-NSl-NS2A-NS2B-NS3-NS4A-NS4B-NS53 *. The structural proteins are C (capsid), pre-matrix / matrix (preM / M) and envelope protein (E). These proteins are encoded by the 5 'fragment, followed by the non-structural (NS) proteins encoded by the rest of the genome. Proteins are produced by a proteolytic process induced by cellular signaling and other cellular and viral proteinases. The envelope glycoprotein (E) is the majority surface protein. The structure of serotype 1 has been described by Masón and collaborators (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. et al. (3) and serotype type 4 has been described by Zhao et al. (4). Numerous attempts have been made to develop vaccines under units generally expressed as peptides or recombinant proteins by the use of a baculoviral vector expressed in insect cells. Indeed, baculoviruses are very attractive for this type of production thanks to the high level of genetic expression they can reach and 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. Dalenda et al. (10). The sequence homology between the 4 genomes corresponding to the 4 serotypes of the virus is relatively important; by way of example, the E protein of this virus presents a percentage of homology such as that presented in Table 1 below: Although some of the constructions described in the state of the art do not allow to induce an effective immune response against the 4 serotypes: generally the titer obtained is low, and the resistance induced by an incitement against an infection by the virulent virus can not be considered as enough. Some constructions have still allowed a good induction of immunogenicity, and especially in the case described in (11) where an envelope glycoprotein deleted in its carboxy-terminal part of 100 amino acids, allows to obtain an increased immunogenicity. But this is not effective against the other Dengue serotypes. Finally, Dalenda et al (9, 10) have effectively shown that a truncation in the carboxy-terminal part of the protein is capable of increasing the immune response. The purification of recombinant proteins obtained by expression in a heterologous system always poses a problem of compatibility between the performance achieved and the conservation of the structure of said protein and especially of its glycosylated motifs. One test technique is the affinity purification of peptides or proteins covalently coupled to a short sequence of amino acids that play the role of tag (label). Examples of techniques that exploit this form of approximation are: glutathione-S-transferase (GST) purified on sepharose-glutathione beads (12), the association of Mal E protein to maltose and its derivatives (13), a fusion of protein A with the immunoglobulin columns (14), or the mark for histidine that has an affinity for the chelated metal columns (15). Schmidt et al. (16) describes in a recent article the advantages and limitations of this technique. But it should be noted that the person skilled in the art systematically seeks to eliminate histidine residues prior to the purification step especially using carboxypeptidase A. The present invention results from the discovery that a polypeptide or a glycolypeptide consisting of a polypeptide or glycoprotein emitted of a Flaviviridiae protein, namely, the Dengue virus in which a deletion of its carboxylated limb has been replaced by a residue of amino acids selected from histidine, tryptophan or cysteine have an immunogenic character much more important than the same polypeptide or glycopeptide having only amino acid residues attached, that residue having the effect of improving the antigenic presentation and consequently inducing a cellular and humoral response much more important than in absence of the residue. In all that precedes and follows, the term "polypeptide" should be understood in the broad sense, that is, including the sequences of at least 15 amino acids comprising or not glycosylated motifs or glycopeptides. and whatever its structure, primary, secondary or tertiary. The present invention is more generally directed to the polypeptides, with increased immunogenic power, derived from Flaviviridiae, characterized by the existence of an amino acid residue at its carboxy-terminal extremity, an amino acid residue which contains from 2 to 8 amino acids selected from the histidine, the tiptophane and cysteine. In 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 tryptophan or 2 to 8 cysteines. It can also be constituted by a mixture of 2 or 3 of these amino acids; can finally comprise any amino acid other than histidine, tryptophan or cysteine from the moment when their number does not exceed percent of the residue. These constructions make it possible to solve the problem of the lack of immunogenicity of some peptides derived from this virus, such as, for example, certain serotypes of Dengue virus against which current techniques do not allow making subunit-type vaccines. The present invention is directed to such immunogenic polypeptides, consisting of a polypeptide derived from the E protein, from the preM protein, from the NS1 or NS3 protein of Dengue virus and in which the carboxylated E protein has been deleted from 70 to 105 amino acids to be substituted by a residue of 2 to 8 amino acids selected from histidine, cysteine or tryptophan. It can also be the same for the deleted preM protein of 30 to 50 amino acids at its carboxy-terminal extremity to be replaced by the same type of residue. For the other proteins this suppression is not necessary; and the polypeptide of the invention derived from NS1 or Ns3 comprises a residue of amino acids selected from histidine, cysteine or tryptophan. Conveniently, the polypeptide leaves the E protein and the deletion is about 100 amino acids; otherwise, the amino acid residue contains from 2 to 8 histidines, this confers on the peptide the property of being fixed on the divalent or trivalent cations. The polypeptides or the peptides of the invention can be emitted of any of the 4 serotypes of Dengue, knowing that the percentage of homology between the 4 serotypes is high and is between 67 percent and 80 percent for protein E as indicated in table 1 above. The present invention is also directed to the compositions consisting of mixtures of polypeptides of the invention. These mixtures can constitute an active principle of polyvalent vaccine, either against different serotypes of the same virus, such as Dengue, or against different viruses of the same family of Flaviviridiae or Flavi-like "and susceptible to affect the same populations. present invention is also related to a composition suitable for stimulating the immunity of individuals susceptible 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 one contai at least one polypeptide of the invention. The invention comes from a Dengue protein from one of the 4 serotypes, the E protein is a good candidate: it has an essential role in viral infection and is the intermediary between the virus and cellular receptors, protein E seems likewise be an essential target of the protective immune response within the guests infected to the extent that it induces neutralizing antibodies and an immunocellular response.
A particularly convenient composition of the invention could be a mixture of 3 or 4 polypeptides or glycopeptides described hereinabove, where one part leaves the E protein of any of the 4 suppressed serotypes of a hundred amino acids, to which it is grafted. covalently a residue contai from 2 to 8 histidines, and preferably from each of the known serotypes of the virus. In order that the immunogenic composition of the invention be effective against each other with the 3 or 4 serotypes in question or more if new serotypes are about to appear, each peptide or glycopeptide is present in a proportion of 10 to 50 weight percent. with respect to the assembly of the peptides of the composition, and preferably a substantially equal percentage for each of the peptides. The immunogenic compositions of the invention can also be constituted of a mixture of polypeptides derived from protein E, preM, NS1, or NS3 and directing at least 3 antigens of 3 subtypes of Dengue virus, each one being provided with amino acid residues in the carboxy-terminal extremity. More generally, the invention is directed to immunogenic compositions consisting of a mixture of polypeptides or glycopeptides derived from the E protein, preM, NS1 or NS3 of the Dengue virus and carrying at least 3 antigens of 3 serotypes of that virus and more generally an antigen corresponding to each of the known serotypes, not necessarily comprising the amino acid residue at its carboxy-terminal extremity, but characterized because each of the peptides, polypeptides or glycopeptides presents the ability to induce an immune reaction in the host. The composition according to the invention may otherwise contain the polypeptides, glycopeptides or proteins from other flaviviruses such as yellow fever, Japanese encephalitis, tick encephalitis or hepatitis C or more generally from other Flaviviridiae of the orrágicas fevers present in the tropical zones of Africa, Asia or America, as well as the case of what is emitted by other viral, bacterial or parasitic antigens, from the moment they are provided with amino acid residue such as the one defined hereinabove in the carboxy-terminal extremity. The invention is also directed to the recombinant nucleic acid sequences encoding the immunogenic peptides of the Flaviviridiae and especially the Dengue virus, deleted in 3 'from 210 to 315 nucleotides, to which a sequence coding has been linked in 3'. from 2 to 8 amino acids selected from histidine, tryptophan or cysteine in the sense defined above. The nucleic acid sequences according to the invention that encode more particularly the glycopeptides derived from the Dengue E protein of serotype 1, 2, 3 or 4 and the 3 'linked sequence preferentially encodes a sequence containing 6 histidines. The invention also addresses the appropriate nucleic acid vectors for use in the expression of exogenous genes in a eukaryote; these vectors carry another nucleic acid sequence such as that described hereinabove a homologous or heterologous signal sequence of the encoded protein, the assembly being dependent on a promoter capable of being active in said eukaryotic cell. A preferred type of eukaryotic cell is that of insect cells and especially the cells of Spodoptera frugiperda SF 9. The vector is a pVLd shuttle vector capable of homologous recombination with a baculovirus, and carrier, upstream of the sequence of the signal, from a promoter of genes endogenous to the baculovirus, for example, that of polyhedrin. Recombinant baculoviruses can be obtained by different techniques known to those skilled in the art but a particularly convenient technique is that of homologous recombination using the shuttle vectors between E. coli and the baculovirus. Expression vectors such as those obtained by this technique, and described more in detail in the part experimental of the present, later, have been deposited in the CNCM; it is: a recombinant baculovirus deposited in the CNCM under the number 1-1497, the lo. December 1994 and carrier of a sequence encoding a peptide constituted of the E gene of the Dengue virus, serotype no. 2 at whose COOH end 100 amino acids were deleted and replaced with 6 histidines; of a recombinant baculovirus deposited in the CNCM under the number 1-1624, on October 11, 1995 and carrying a sequence that encodes a peptide constituted by the E gene of the Dengue virus, serotype no. 4 at whose COOH end 100 amino acids were deleted and replaced with 6 histidines; of a recombinant baculovirus deposited in the CNCM under the number 1-1625, on October 11, 1995 and carrying a sequence encoding a peptide constituted by the E gene of the Dengue virus, serotype no. 3 at whose COOH end 100 amino acids were deleted and replaced with 6 histidines; - of a recombinant baculovirus deposited in the CNCM under the number 1-1626, on October 11, 1995 and carrying a sequence that encodes a peptide constituted by the E gene of the Dengue virus, serotype no. 1 at whose COOH end 100 amino acids were deleted and replaced with 6 histidines; The invention is also directed on the compositions capable of inducing an immune, cellular or humoral response in a host susceptible to being infected by the Dengue virus and comprising a mixture of nucleic acids that respectively encode the polypeptides of the invention, namely, those derived from the Dengue protein , such as protein E, preM, NS1 or NS3, and therefore at its carboxy-terminal extremity a residue of 2 to 8 amino acids selected from histidine, cysteine or tryptophan, or more generally encoding polypeptides exiting from other Flaviviridiae such as yellow fever, Japanese encephalitis, tick-borne encephalitis or other "Flavi-like" diseases such as hepatitis C virus. Indeed, it has been shown that injecting a plasmid containing the necessary sequences to the animal the expression of proteins or polypeptides triggers a cellular and humoral immune response in the host allowing in certain animal models is to protect the latter against a lethal viral load; such results have been described by J.A. Olff et al. (1990), Science 247: 1465-1468 or by E.F. Fynan et al. (1993), DNA cell biol. 12: 785-789. The immunogenic compositions comprising such nucleic acid sequences and the vaccines constituted by these compositions, if an auxiliary is presented, form part of the invention as well. The invention is also related to the vaccines of polyvalent subunits against the Dengue virus especially against the 4 serotypes of said virus and comprises an immunogenic composition as described hereinabove, with the case if given, of a vaccination assistant that can be especially an auxiliary carrier of divalent or trivalent ions such as aluminum hydroxide or calcium phosphate or also an auxiliary of the type of Freund's adjuvant, a muramylpeptide derivative of an "iscom" (Immunostimulatory Complex), (19). The immunogenic effect of the compositions of the invention can be the consequence of an uptake of the polypeptides or glycoproteins of the invention by divalent or trivalent ions and the eventual formation of multimers that have an increased immunogenic power in relation to the polypeptides exiting the Dengue and already described in the literature (references 3, 7, 8, 9). The subunit vaccines of the invention may also be constituted by compositions capable of inducing an immunogenic reaction against other Flaviviridiae, which may be intended for the prophylaxis or therapy of the infection by one or many viral species simultaneously.A polyvalent subunit vaccine according to the invention, and containing from 1 to 4 glycopeptides or immunogenic polypeptides of the invention, in case they are associated with immunogenic glycopeptides from other flaviviruses or virus of hemorrhagic fevers, present at the same time the advantage of the polyvalence linked to the immunogenic effect of active principle against the different serotypes and that of the total innocuousness. In fact, there are currently vaccines against Dengue based on 4 live serotypes attenuated by successive passages of the virus in culture, but as we have seen previously, this vaccine sometimes presents a risk for immunosuppressed people or for newborns, and It is not always effective against different serotypes and especially about the 4 Dengue serotypes. It can also be envisaged in the vaccines of the invention to add to the vaccines currently in clinical experimentation 1, 2, 3 or 4 polypeptides according to the invention. The detailed description hereinafter of a particular embodiment using the E protein of serotypes 1 to 4 (DEN1 to DEN4) illustrates, without being limiting, the structural and functional properties of the polypeptides, polypeptide compositions and vaccines of the invention. Figure 1 represents the electrophoretic profile of the recombined E polypeptides DEN1 to DEN4 secreted in the supernatant of the infected Sf9 cells. The radioimmunoprecipitated proteins have been treated by endoglycosidase F (F) or H (H) or untreated (O). 1. Construction of recombinant baculoviruses: the. Preparation of the fragments of the gene encoding the envelope protein E of Dengue virus The strains of the Dengue virus that served for the cloning of the E genes are inscribed in table 2 hereinafter: Table 2 2o. Preparation of the recombinant baculoviruses expressing the truncated proteins of the Dengue virus: The preparation of the genes of E has been carried out by reverse transcription / chain reaction by polymerase (RT / PCR) from the RNA extracted from Aedes mosquito cells AP61 pseudoscutellaris infected by the different viruses. The size in amino acids reached by protein E, suppressed 100 amino acids in its C-terminal extremity (E? 100) is indicated in the right column of table 2. The oligonucleotide bait located 5 'of the gene to be amplified possesses a restriction site (except for Dengue 2), the ATG start codon followed by the nucleotides encoding the first 6 amino acids among the 15 that correspond to the signal sequence of each protein. The oligonucleotide bait located 3 'of the gene to be amplified possesses the sequence corresponding to 6 amino acids of the E protein of the virus followed by 6 codons for histidine, of a stop codon and of a uri restriction site. The sequence of the oligonucleotides is as indicated hereinafter: 5 'extremity (positive sense) Dengue 1: 5' CGGGATCCATGGGGATCATTTTCATTTTGCTGATG-3 'Dengue 2: 5' -GGCCTTGATTTTCATCTTAC-3 'Dengue 3: 5' -CGGGATCCATGGTGGTTATTTTTATACTATTAATG-3 ' Dengue 4: 5 '-CGGGATCCATGACTGTCTTCTTTGTCCTAATG-3 • Tip 3"(negative sense) Dengue 1: 5 '-CGGAGCTCAATGATGATGATGATGATGATGTCCTTTCTTGAACCAGCTTAG-3' Dengue 2: 5 '-GGAGCTCAATGATGATGATGATGATGTCCTTTCTTAAACCAGTTG-3' Dengue 3: 5 '-CGGAGCTCAATGATGATGATGATGATGTCCCTTCTTGTACCAGTTAAT-3' dengue 4: 5 '-CGGAGCTCAATGATAATGATAATGATACCCTTTCCTGAACCAATGGAG-3' The amplified fragments from viral RNA extracted from Aedes pseudoscutellaris AP61 cells were cloned in the shuttle vector pVL-poly (17).
D (1,2,3 or 4) E? L00His6 corresponds to protein E of serotype 1, 2, 3, 4 deletion of 100 amino acids at its carboxyl extremity and to which 6 residues have been bound histidine. The genes encoding the D (1, 2,3 or 4) E? L00 His6 proteins and their signal sequence are thus placed under the control of the polyhedrin promoter. The number of nucleotides comprised between the end of the promoter and the initiation codon is 42 (Dengue 1, Dengue 3, Dengue 4) or 50 (Dengue 2) nucleotides. The recombinant baculoviruses Ac.D (1, 2, 3) E? L00His6 have been obtained by homologous recombination in Spodoptera frugiperda Sf9 cells between shuttle vectors pVL.D (1, 2,3 or 4) E? L00His6 and baculovirus AcRP231acZ linearized in a single site Bsu361. Recombinant baculoviruses have been selected by 3 successive cloning using the beach method. 3rd Structural characteristics of the obtained recombinant proteins: Figure 1 shows the electrophoretic profile of the 4 recombinant proteins corresponding to the Dengue 1, 2, 3 and 4 viruses. The profile of the proteins treated by endoglycosidase F or H indicates that all they are glycosylated, no doubt by only one of two glycosylation sites since the molecular weight difference does not exceed 4000 daltons. II. Expression and purification of proteins 1) Infection of Sf9 cells: ten thousands of Sf9 cells in suspension are infected by the recombinant baculovirus AcD (1, 2,3 or 4) E? 100His6s: in 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 TClOO medium (GIBCO) without fetal calf serum. 2) Preparation of supernatants from infected cells: 2 liters of cell culture supernatant are recovered 3 days after infection, centrifuged at 2500 rpm at 4 ° C for 30 minutes. To the supernatant is added 400 grams / liter of ammonium acetate under agitation between 3 and 14 hours at 4 ° C. The precipitate is centrifuged at 10000 rpm for 20 minutes at 4 ° C and redissolved in 1/20 of the initial volume (100 milliliters) of chromatography plug (0.5M NaCl; 20mM Tris-HCl pH 7.9) containing aprotinin at a final concentration of 20 μg / milliliters and phenyl methyl sulfonyl fluoride at 1 M final. The concentrated supernatant is then dialyzed twice against 100 volumes of chromatography plug. 3) Chromatography: 5 mM final imidazole is added to the dialyzed medium, then incubated for 30 minutes (under gentle agitation) at laboratory temperature in the presence of 5 milliliters of TALON resin (Clontech). The resin is washed three times with 30 milliliters of chromatography plug containing 10 mM imidazole. The resin is then placed in a column, lava with 10 volumes of chromatography plug, and the protein is eluted with 10 milliliters of chromatography plug containing imidazole at 0 mM. The fractions of 1 milliliter are recovered with a fraction collector. A second elution is carried out for the aid of the chromatography cap containing imidazole at 100 mM. The fractions containing the recombinant protein E are determined by spot staining with the aid of mouse anti-E antibodies and anti-mouse conjugate with alkaline phosphatase or with the aid of the Ni-NTA conjugate coupled to alkaline phosphatase (Qiagen). 4) Purification control: An aliquot of each fraction is deposited on polyacrylamide gel. The presence of the protein of interest is verified by Western staining and after staining of silver nitrate. Immunogens: Viral proteins in concentration of 5 μg of pure protein are mixed in 100 μg of aluminum hydroxide (Alugel-S Serva) and inoculated to SWISS mice intraperitoneally. The viral strains of Dengue having been used for immunization are titrated on AP61 cells (detection of foci for immunocytochemistry) and inoculated at a rate of 103 particles forming beach (PFU) per mouse. The control antigen injected into the mice corresponds to a peptide containing the last three amino acids of the E protein (KKG) followed by six histidines (KKG His6). The inoculum of each injection contains 100 μg of mixed peptide in the Alu-gel. Calculation 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 milliliters of viral suspension in different dilutions. The fatal lethal dose for 50% of the mice (DLd50 is calculated by the method of Reed and Muench.) Immunization of the mice: The three-week-old mice were vaccinated by an intraperitoneal injection of a mixture of products corresponding to the 4 serotypes. of Dengue (DE? 100His6 or virus) at 3 weeks, 4 weeks and 6 weeks, then individually bled one week after the last injection.The negative control vaccination peptide is identical for all 4 serotypes. These sera were titrated with respect to each homologous virus.Serums also serve as a protection test after passive transfer to the mouse Neutralization test: Antibodies from vaccinated mice were titrated by neutralization in the 24-well plates in the presence of 50 PFU of homologous virus on Vero cells The neutralizing antibody titer corresponds to the neutral dilution We found 80 percent of the virus foci detected by immunocytochemistry with the help of viral antiprotein antibodies and anti-IgG conjugate from mice marked with peroxidase.
Protection test of mice: 100 LD50 of virus are inoculated to groups of mice of 5 days having received the day before an intraperitoneal injection of 0.05 milliliters of serum from immunized mice (passive transfer). Each group of passive transfer mice was subjected to a test dose for one of the 4 virus serotypes. Mice were observed for 3 weeks and signs of paralysis or death were recorded. Results: The results obtained are presented in table 3 below in the present. This table concentrates the results obtained in terms of the neutralizing antibody titer on the one hand, and on the percentage of surviving mice after a stimulus by the virulent viruses according to the conditions described hereinbefore. For some of the serotypes, the results obtained are compared with the peptides of the invention comprising the histidine residue, with the live viruses, and with the KKG His6 peptides comprising the histidine residue, and at the respective doses of antigens of 5μg 103 Pfu and 100 μg. The neutralization titer was measured on the previously discounted sera of 7 weeks after the 3 antigen injections. The neutralization titre corresponds to the inverse of the serum dilution that leads to an 80 percent reduction of the units that form foci (UFF) of the homologous virus strain on the VERO cells. The homologous viruses have been used for the neutralization and stimulation test. The mice were immunized by the tetravalent mixture in the 3 model cases. Table 3: These results indicate that the formulation containing the 4 recombinant proteins induce neutralizing antibodies directed against the 4 virus serotypes. This mixture can then form the basis of a composition or an active principle of a vaccine of polyvalent subunits against Dengue.
The elevated titer of neutralizing antibodies produced after 3 injection of 5μg of protein is remarkable. None of the recombinant proteins produced previously without histidine had led to the induction of such high titers of neutralizing antibodies. Two hypotheses are advanced to explain these results: either the imperfect purification of the preceding proteins that contain the immunosuppressive constituents, either the sequence of 6 histidines contained in the present proteins in the presence of particles formed of divalent or trivalent ions that confer a multimeric spatial presentation and consequently of the particular immunogenic properties. III. Induction of neutralizing antibodies by the recombinant envelope proteins of each of the four serotypes: lo. The envelope proteins of each viral serotype suppressed (?) 100 amino acids at its C-terminal extremity have been expressed by the baculovirus system and purified by chromatography on a cobalt column thanks to the segment of six histidines (His6) that make up its extremity C -terminal. The vaccinating antigen has been injected to the mice in the monotypic form and as a comparison the results obtained after the injection of the tetravalent form such as those presented in table 3 previously in the present they are called back to the level of the line 4 x DE? lOOisd. The PBS line represents a witness represented by the plug of the same name. Immunization of the mice: Groups of 10 BALB / c mice of three weeks were immunized on days Jl, J7 and J21 with the help of 5 μg of recombinant antigen coupled to 100 μg of aluminum hydroxide taken as adjuvant. The mice were bled on day J28 and examined in serum neutralization with respect to 200 Focusing Formers of each of the reference strains (Dengue 1: Hawaii 1944, Dengue 2: New Guinea C, 1944, Dengue 3 HB7 Philippines 1956 Dengue 4 H251, Philippines 1951). The title corresponds to the inverse of the neutralizing dilution plus 50 percent of the foci. The results indicated in Table 4 hereinafter indicate that both the immunogen D2E? L00His6 and D3E? L00His6 induce the formation of neutralizing antibodies against the Dengue 2 serotype while the Dengue 3 and Dengue 4 serotypes are not neutralized respectively. that by the recombinant protein D3E? l00His6 and D4E? l00His6. Finally the DlE? 100is6 has no neutralizing effect. table 4 The recombinant proteins derived from serotypes D2, D3 and D4 then seem particularly interesting and the titre of neutralizing antibodies 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 in the present.
TABLE 5 Immune response of cynomolgus monkeys vaccinated against Dengue 2 and infected by a native strain of virus Groups of 2 monkeys received different immunogenic preparations: Japanese encephalitis vaccine (A); nonapeptide KKGH6 (b); Recombinant envelope protein D2? L00His6 (C); PBS D; dengue 2 attenuated vaccine (E). PRNT: Neutralization reduction plate test (ref. 10). The preparations were injected on day JO (A and E) or days JO, J28, J56 after preparation of plasma units. The monkeys were subjected to a test dose on J83 day by intradermal injection of 2 x 10 5 infective particles of dengue 2 virus, strain Jamaica 1983. The plasmas were collected after, on day J98.
Obtaining immune cells: Monkeys receive three inoculations of 100 μg of adjuvant peptides monthly with 1 milligram of aluminum adjuvant (group B). Three inoculations of 100 μg of recombinant envelope proteins of Dengue 2 D2? L00 His6 mixed with 1 milligram of aluminum adjuvant (group C); three inoculations of PBS plug with the adjuvant (group D) or one inoculation of 0.5 milliliters of live attenuated Dengue 2 type THAI vaccines prepared by PASTEUR MEREUX SERUMS & VACCINS (group E). The sera are harvested four weeks after the last inoculation and tested for their neutralizing antibodies against 200 plaque-forming units (PFU) of the Dengue 2 Jamaica homologous strain. Titers corresponding to dilutions of serum that neutralize 50 percent of the plates. Table 5 indicates the specificity of induction of neutralizing antibodies of the recombinant protein released from Dengue serotype 2 in cynomolgus monkeys, with a response at least four times more important than that obtained with the attenuated virus.
REFERENCES (1) V.Deubel, R.-M. Kinney, D.W. Trent (1986). Nucleotide secuence and deduced amino acid sequence of the structural proteins of Dengue type 2 virus, Jamaica genotype. Virology, 155: 365-377. (2) Mason P.W., Mc Ada P.C., Mason T.L., Fournier M.J. (1987). Virology 161: 262-267. (3) OSATOMI K. and collaborators. (1990) . Virology 176: 643-647. (4) Zhao B. et al. (1986) Cloning full cength Dengue type 4 virol DNA sequences: analysis of genes coding for structural proteins Virology: 155 .: 77-88. (5) Putnak R. et al. (1991). Dengue 1 virus envelop glycoprotein gene expressed in recombinant baculovirus elicits 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. Mc Carty, D.R. D bois, 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 resistance to Dengue virus encephalitis. J. Virol. 62: 3027-3031. (7) Deubel V. and collaborators. (1991). Processing secretion and immunoreactivity of carboxiterminally truncated Dengue 2 enveloppe protheins 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. Tro. 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. (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 immunogenic properties in mice. J. Gen. Virol., 75: 1569-1578. (11) R. Men, M. Bray, C.J. 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) Nikolo D.B. and collaborators (1992). Nature 360: 4046. (13) Maina E. et al (1988). A vector to express and purify foreign proteins in E. Coli by fusion to, and separation from, maltose binging protein. Gene 74: 365-373. (14) Moks T. and collaborators (1987). Biotechnology 5: 379-382. (15) Hochuli E. et al. (1987). J. chromatography 411: 177-184. (16) J. Schmitt, H. Hess, H.G. Stunnenberg.Affinity purification of histidine-tagged proteins. Mol. Biol. Reports, 18: 223-230 (17) Fla and M. et al. (1992) Expression and secretion of Japanese encephalitis virus nonstructural protein NSl by insect cells using a recombinant baculovirus virology 191: 826-836. (18) Brevet européen no. 0 318 216. (19) Morein B. (1987). Potentiation of immune response by immunization with antigens in defined multimeric physical forms. Vet. Immunol. Immunopathol. 17: 153-159.

Claims (30)

1. A polypeptide or immunogenic glycopeptide characterized in that it consists of a polypeptide or glycoprotein derived from a Fliviviridiae, especially of the E protein, or preM, or NSl or NS3 of the Dengue virus, said polypeptide carries in its carboxylated extremity a residue of 2 to 8 selected amino acids between histidine, tryptophan or cysteine.
2. Polypeptide or glycopeptide according to claim 1, characterized in that the glycopeptide is constituted by the envelope protein E deleted from 70 to 105 amino acids at its carboxylated extremity.
3. Polypeptide or glycopeptide according to claim 1 or 2, characterized in that the sequence in the carboxylated extremity is constituted by a residue of 6 histidines, thus conferring on it the property of being fixed on the divalent or trivalent cations.
4. Composition suitable to stimulate the immunity of individuals susceptible to be infected by the virus of the Dengue, 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 leaves the glycoprotein E of the Dengue virus.
Composition according to claim 4 or 5, characterized in that the polypeptides or glycopeptides are a mixture of at least 3 polypeptides according to one of claims 1 to 3 issued from at least 3 serotypes of the Dengue virus, and preferably from each of the known serotypes.
7. Composition according to claims 4 to 6, characterized in that it contains the D3E? L00His6 polypeptide and / or the D2E? L00His6 polypeptide.
8. Composition according to claims 4 to 7 constituted by polypeptides or glycopeptides derived from protein E or preM or NSl or NS3 of Dengue virus, and preferably protein E.
9. Composition according to claims 6 or 7, characterized in that each peptide is present in a proportion of 10 to 50 weight percent relative to the peptide assembly of said composition.
10. Composition according to one of claims 4 to 9, characterized in that it contains, on the other hand, polypeptides, glycopeptides or proteins from other flaviviruses such as yellow fever or Japanese encephalitis or hepatitis C.
11. Composition, according to one of claims 4 to 9 characterized in that it contains, on the other hand, the viral, bacterial or parasitic antigens that carry in their carboxy-terminal extremity a residue of 2 to 8 amino acids selected from histidine, tryptophan or cysteine.
12. Nucleic acid sequence consisting of a sequence encoding a Dengue virus protein, deleted in 3 'from 210 to 315 nucleotides, to which a sequence encoding 2 to 8 amino acids selected from histidine has been added in 3'. , tryptophan and cysteine.
13. Nucleic acid sequence according to claim 12, characterized in that the sequence encodes a Dengue E protein of serotype 1, 2, 3 or 4.
14. Nucleic acid sequence according to claims 12 or 13 characterized in that the sequence in 3 '. encodes 6 histidines.
15. Vector of nucleic acids 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 5 'of a homologous signal sequence and / or heterologous of the protein encoded by said sequence, the assembly being dependent on a promoter capable of being active in said eukaryotic cell.
16. Expression vector according to claim 15, characterized in that the eukaryotic cell is an insect cell, especially of Spodoptera frugiperda SF9, the vector is a shuttle vector capable of carrying out a homologous recombination with a baculovirus, and carrier upstream of the signal sequence, of a promoter of a gene endogenous to the baculovirus, especially that of polyhedrin.
17. Expression vector according to the claims 15 or 16 characterized in that it is a recombinant baculovirus deposited in the CNCM under the number 1-497, lo. December 1994 and carrier of a sequence encoding a peptide constituted by an E gene of Dengue virus, serotype no. 2 at whose end COOH 100 amino acids have been deleted and replaced by 6 histidines.
18. Expression vector according to claims 15 or 16 characterized in that it is a recombinant baculovirus deposited in the CNCM under the number 1-624, on October 11, 1995 and carrying a sequence encoding a peptide constituted by an E gene. of the Dengue virus, serotype no. 4 at whose COOH end 100 amino acids have been deleted and replaced by 6 histidines.
Expression vector according to claims 15 or 16 characterized in that it is a recombinant baculovirus deposited in the CNCM under the number 1-625, on October 11, 1995 and carrying a sequence that encodes a peptide constituted by an E gene of the Dengue virus, serotype no. 3 at whose COOH end 100 amino acids have been deleted and replaced by 6 histidines.
20. Expression vector according to claims 15 or 16 characterized in that it is a recombinant baculovirus deposited in the CNCM under the number 1-626, on October 11, 1995 and carrying a sequence encoding a peptide constituted by an E gene of the virus of Dengue, serotype no. 1 at whose COOH end 100 amino acids have been deleted and replaced by 6 histidines.
21. A composition suitable for inducing a cellular or humoral immune response in a host susceptible to being infected with Dengue virus, characterized in that it contains a nucleic acid encoding a polypeptide according to one of claims 1 to 3.
22. Process for the preparation of a polypeptide or a glycopeptide according to one of claims 1 to 4, characterized in that it comprises an affinity chromatography step on a support having an affinity for the sequence of 2 to 8 amino acids.
Method according to claim 22, characterized in that the support is a column containing the divalent or trivalent ions, in particular nickel, copper, cobalt or zinc, which has an affinity for electron-rich histidine, tryptophan or cysteine.
24. A polyvalent vaccine against Dengue virus, characterized in that it comprises an immunogenic composition according to one of claims 4 to 11 and 21, with, if necessary, a vaccination adjuvant which may especially be an adjuvant carrying divalent or trivalent ions such as aluminum hydroxide or calcium phosphate, or an adjuvant of the Freund's adjuvant type, or a muramyl peptide derivative or an iscom.
25. A polyvalent vaccine according to claim 24, characterized in that it contains, on the other hand, immunogenic polypeptides or glycopeptides from other flaviviruses such as yellow fever or Japanese encephalitis or tick encephalitis or hepatitis C.
26. Polyvalent vaccine according to the claim 24, characterized in that it contains 4 peptides or glycopeptides corresponding to each of the serotypes of the Dengue virus with an adjuvant containing the divalent or trivalent ions.
27. Polyvalent vaccine according to claim 24, characterized in that it contains 1, 2, 3 or 4 peptides or polypeptides, according to claims 1 to 3 and the attenuated viruses.
28. Polyvalent vaccine according to claim 24, characterized in that it contains, on the other hand, the polypeptides according to one of claims 1 to 3, one or more viral, bacterial or parasitic antigens.
29. Polyvalent vaccine characterized in that it comprises a mixture of nucleic acids carrying sequences encoding a polypeptide according to one of claims 1 to 3.
30. Antigenic composition consisting of polypeptides or glycopeptides derived from the E protein, preM, NSl 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.
MXPA/A/1998/003840A 1995-11-14 1998-05-14 Vaccine polivalent anti-den MXPA98003840A (en)

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FR95/13489 1995-11-14
FR9513489 1995-11-14

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