WO1997018311A1

WO1997018311A1 - Polyvalent anti-dengue vaccine

Info

Publication number: WO1997018311A1
Application number: PCT/FR1996/001801
Authority: WO
Inventors: Vincent Deubel; Isabelle Staropoli
Original assignee: Institut Pasteur
Priority date: 1995-11-14
Filing date: 1996-11-14
Publication date: 1997-05-22
Also published as: BR9611477A; AU718740B2; CA2235334A1; FR2741077A1; KR19990067581A; FR2741077B1; JP2000500969A; AU7628696A; CN1202201A; EP0861324A1

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

VERSATILE ANTI-DENGUE VACCINE

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

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.

The 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).

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'.

The structural proteins are C (capsids), pre-matrix / matrix (preM / M) and the envelope protein (E).

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.

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).

Numerous 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.

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).

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:

Despite 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

Finally, 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

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

In 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

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

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 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

These 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.

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

Advantageously, 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

A 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 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

So 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

The 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 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

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 1-1497, 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,

- 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,

- 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,

- 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

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 "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

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 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

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) I Construction of recombinant baculoviruses

1 ° Preparation of the gene fragments coding for the envelope protein E of the Dengue viruses

The Dengue virus strains that were used to clone the E genes are listed in Table 2 below. table 2

2 ° Preparation of the recombinant baculoviruses expressing the truncated proteins of the Dengue virus:

The 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

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;

5'CGGGATCCATGGGGATCATTTTCATπTGCTGATG-3 '

Dengue 2:

5'-GGCCTTGATTTTCATCTTAC-3 '

Dengue 3:

5'-CGGGATCCATGGTGGTTA I I M I ATACTATTAATG-3 '

Dengue 4: δ'-CGGGATCCATGACTGTCTTCTTTGTCCTAATG-S '

3 'end (negative direction)

Dengue 1:

5'-CGGAGCTCAATGATGATGATGATGATGTCCTTTCTTGAACCAGCTTAG-3 '

Dengue 2:

S'-GGAGCTCAATGATGATGATGATGATGTCCTTTCTTAAACCAGTTG-S '

Dengue 3:

5'-CGGAGCTCAATGATGATGATGATGATGTCCCTTCTTGTACCAGTTAAT-3 '

Dengue 4:

5'-CGGAGCTCAATGATAATGATAATGATACCCTTTCCTGAACCAATGGAG-3 '

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 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

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) or 50 (Dengue 2) nucleodides.

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:

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

II. Expression and purification of DEΔ100Hιs6 proteins

1) 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) 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) 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

Immunogens: 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

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 ml of viral suspension at different dilutions The lethal dose killing 50% of the mice (LD50) is calculated by the method of

Reed and Muench

Immunization 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

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 souπs are observed for <_. weeks and signs of paralysis or death are noted

Results: The results obtained are presented in Table 3 below

This 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

The mice were immunized with the tetravalent mixture in the 3 cases of figures

table 3

These results indicate that the formulation containing the 4 recombinant proteins induces neutralizing antibodies directed against the 4 virus serotypes.

This mixture can therefore constitute the basis of a composition or of an active principle of a vaccine in polyvalent units against Dengue

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 ° 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.

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). The title corresponds to the reverse of the dilution neutralizing more than 50% of the outbreaks.

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:

NEUTRAL READING TITLE

Immunogen Dengue 1 Dengue 2 Dengue 3 Dengue 4

D1EΔ100His6 20 <20 <20 <20

D2EΔ100His6 <20> 100 20 <20

D3EΔ100His6 <20 100 100 <20

D4EΔ100His6 <20 <20 <20 100

4xDEΔ100His6 100> 100 100> 100

PBS <20 <20 <20 <20

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.

TABLE 5: Immune response of cynomolgus monkeys vaccinated against dengue 2 and infected with a wild strain of virus -1

00 Ul

Monkeys J28 J56 J70 J98

ELISA PRNT ELISA PRNT ELISA PRNT ELISA PRNT

A1 <100 <20 <100 ND <100 <20 400 160

A2 <100 <20 <100 ND <100 20 630 160

B1 <100 <20 <100 ND <100 <20 250 80

B2 <100 <20 <100 ND <100 <20 630> 640

C1 100 <20 800 20 1000 160 12000 2560

C2 <100 <20 300 20 800 160 400 320

D1 <100 <20 <100 ND <100 <20 100 80

D2 <100 <20 <100 ND <100 <20 300 80

E1 500 80 600 20 400 40 5000> 1280

E2 160 20 200 20 300 20 <100 <40

TJ π

H

Groups of 2 monkeys received different immunogenic preparations:

Japanese encephalitis vaccine (A); nonapeptide KKGH6 (B); Recombinant envelope protein D2EΔ100His6 (C); PBS

(D); dengue 2 vaccine attenuated (E). PRNT: Test neutralization reduction plate (ref. 10). o oo

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.

Obtaining immunogens ^'

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).

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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Claims

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 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 or glycopeptide according to claims 1 or

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 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 according to claims 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 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 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 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 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 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 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 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. 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 serotype r ° 2 in which the COOH end has been deleted from 100 amino acids and substituted by 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. Expression vector according to claims 15 or 16 characterized in that it is a recombinant baculovirus deposited at the

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. 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. 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. 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. 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. 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. 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.