AU718740B2

AU718740B2 - Polyvalent anti-dengue vaccine

Info

Publication number: AU718740B2
Application number: AU76286/96A
Authority: AU
Inventors: Vincent Deubel; Isabelle Staropoli
Original assignee: Institut Pasteur de Lille
Current assignee: Institut Pasteur de Lille
Priority date: 1995-11-14
Filing date: 1996-11-14
Publication date: 2000-04-20
Anticipated expiration: 2016-11-14
Also published as: CA2235334A1; EP0861324A1; WO1997018311A1; KR19990067581A; FR2741077A1; JP2000500969A; CN1202201A; AU7628696A; BR9611477A; FR2741077B1

Description

1 POLYVALENT VACCINE AGAINST DENGUE The present invention relates to a polyvalent subunit vaccine against viruses from the Flaviviridiae family, in particular against flaviviruses such as dengue, yellow fever, Japanese encephalitis, or other Flaviviridiae or "Flavi-like" viruses such as hepatitis C virus, also mixtures thereof (18).

Flaviviruses are viruses from the arbovirus family (old arbovirus group B).

They are vertebrate viruses generally transmitted by haematophagous arthropods (mosquitos, ticks, Phlebotomi) in which they multiply before transmission. The maintenance cycle thus involves a vertebrate and an arthropod reservoir. Certain arboviruses, particularly those found in tropical regions, may involve man and cause diseases. While a vaccine against yellow fever has existed since 1930, and, more recently, one against Japanese encephalitis and tick encephalitis, other flaviviruses currently have no vaccine; this is particularly the case for dengue.

Tens of millions of individuals are affected annually by dengue, in particular in intertropical regions where the disease is rife and has assumed the proportions of an uninterrupted pandemic disease. The virus responsible for dengue is propagated concomitantly with its vector, the Aedes aegypti mosquito.

The disease is characterized by a high fever accompanied by headaches, nausea and muscular and pains in the joints which disappear after several days. However, the symptoms can intensify to lead to haemorrhaging which may be fatal when the disease is associated with hypovolemic shock, in particular in children. The agent responsible for dengue is an RNA flavivirus from the Flaviviridiae family, like the yellow fever virus or that of Japanese encephalitis. 4 serotypes are known.

Haemorrhaging dengue could be a consequence of a little known immunological phenomenon, doubtless involving a cytokine cascade linked to induction of cytotoxic cellular immunity which can result from sequential infection by several serotypes. The absence of an animal model has slowed down the design of a vaccine as it is not possible to explore the factors involved in severe occurrences of the disease in detail.

A first vaccine against dengue is currently being investigated. The vaccine is based on 4 live serotypes attenuated by successive cell culture of the virus. The major disadvantage of this type of vaccine is the possibility that attenuated viruses can revert to virulence by genetic reversion or recombination between genomes.

There is also a potential risk to new-borns and immunodepressed individuals.

Further, some children vaccinated using a 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, in particular that of serotype 2, has been described by V. Deubel et al. The dengue virus is an enveloped RNA virus of about 50 nanometres. As in the yellow fever virus, all of the viral proteins are coded by a segment of about 10.5 kilobases with the following structure: 5' C-preM/M-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5 3' The structural proteins are C (capsids), prematrix/matrix (preM/M) and the envelope protein These proteins are coded by the 5' fragment, followed by non structural (NS) proteins coded by the remainder of the genome. The proteins are produced by a proteolytic process induced by a cellular signalase and other cellular and viral proteins.

The envelope glycoprotein is the major surface protein. The structure of serotype 1 has been described by Mason et al. Serotype 2 has been described in and is the best known of the 4 serotypes; serotype 3 has been described by Osatomi K. et al. and serotype type 4 has been described by Zhao et al. A number of attempts have been made to develop subunit vaccine generally expressed as recombinant proteins or peptides using a baculoviral vector expressed in insect cells.

Baculoviruses are very attractive for this type of production due to the high level of genetic expression which they can achieve and for their absence of pathogenicity in man. Baculovirus has already been used to express flavivirus proteins: dengue serotype 1 dengue serotype 4 (DEN-4) and serotype 2 (DEN-2) have been described, and serotypes 2 and 3 have been described by C. Delenda et al. The sequence homology between the 4 genomes corresponding to the 4 virus serotypes is relatively high; as an example, the E protein of these viruses has a homology percentage as shown in Table 1 below: Dengue 2 Dengue 3 Dengue 4 Dengue 1 78% 82% 67% Dengue 2 78% 73% Dengue 3 68% Despite that, none of the constructions described in the prior art has been able to induce an effective immunological response against the 4 serotypes: generally, the titre obtained is low, and the resistance to infection induced by the virulent virus challenge cannot be considered to be sufficient.

However, certain constructions have induced good immunogenicity, in particular in the case described in (11) where an envelope glycoprotein truncated at its carboxyl terminal by 100 amino acids resulted in increased immunogenicity.

However, this was not effective against the other dengue serotypes.

Finally, Delenda et al., 10) have effectively shown that a truncation in the carboxyl-terminal portion of the protein could increase the immune response.

The purification of recombinant proteins obtained by expression in a heterologous system always poses problems of compatibility between the desired yield and preserving the structure of the protein, particularly the glycosylated moieties. A powerful technique is affinity purification of peptides or proteins covalently coupled to a short amino acid sequence which acts as a tag. Examples of techniques which exploit this approach are: glutathione-S-transferase (GST) purified on sepharose-glutathion beads the association of the Mal E protein with maltose and its derivatives a fusion of protein A with immunoglobulin columns or tagging with histidine which has an affinity for metal chelate columns In a recent article, Schmidt et al. (16) have described the advantages and limitations of this technique. However, it should be noted that the skilled person is constantly seeking to eliminate histidine residues after the purification step, in particular by using carboxypeptidase A.

The present invention results from the discovery that a polypeptide or a S glycopeptide constituted by a polypeptide or a glycoprotein from a Flaviviridiae 4 protein, namely the dengue virus in which a deletion in its carboxyl end has been substituted by an amino acid residue selected from histidine, tryptophan or cysteine, has an immunogenic character which is much higher than the same polypeptide or glycopeptide without this added amino acid residue, the residue improving the antigenic presentation and as a result inducing a far greater cellular and humoral response than in the absence of this residue.

Throughout this text, the term "polypeptide" must be construed in its broad sense, as including sequences of at least 15 amino acids which may or may not comprise glycosylated moieties or glycolipids, whether the structure is primary, secondary or tertiary.

More generally, the present invention pertains to polypeptides with increased immunogenic power, from Flaviviridiae, characterized by the existence of an amino acid residue at its carboxyl-terminal extremity, the amino acid residue containing 2 to 8 amino acids selected from histidine, tryptophan and cysteine.

In the test of the present application, the amino acid residue can be homogeneous in the sense that it is constituted by 2 to 8 histidines, 2 to 8 tryptophans or 2 to 8 cysteines. It can also be constituted by a mixture of 2 or 3 .of these amino acids; finally, it can comprise several amino acids other than histidine, tryptophan or cysteine as long as they do not exceed 25% of the residue.

These constructions can solve the problem of a lack of immunogenicity in certain peptides from these viruses, such as certain serotypes of the dengue virus against which current techniques cannot produce subunit type vaccines.

The present invention covers such immunogenic polypeptides, constituted by a polypeptide from the E protein, the preM protein, the NS1 or NS3 protein of the dengue virus. Preferably, for the E protein, 70 to 105 amino acids have been deleted from the carboxyl end and substituted with a residue of 2 to 8 amino acids selected from histidine, cysteine or tryptophan. Preferably for the preM protein, 30 to 50 amino acids are deleted from its carboxy-terminal extremity to be substituted by the same type of residue as for the E protein. For other proteins this deletion is not preferred; and the polypeptide of the invention from NS1 or NS3 comprises a residue of 2 to 8 amino acids selected from histidine, cysteine and tryptophan.

The polypeptide is advantageously from the E protein and about 100 amino acids are deleted; if, further, the amino acid residue contains 2 to 8 histidines, this confers on the peptide the property of fixing to divalent or trivalent cations. The polypeptides or peptides of the invention can issue from each of the 4 dengue serotypes, knowing that the percentage homology between the 4 serotypes is high at between 67% and 80% for the E protein as shown in Table 1 above.

The present invention also relates to compositions constituted by 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 Flaviviridiae or Flavi-like family which may affect the same populations.

The present invention also relates to a composition which can stimulate immunity in individuals susceptible to being affected by the dengue virus, characterized in that it contains at least 3 polypeptides or glycopeptides of the invention. A preferred composition contains at least one polypeptide of the invention from a dengue protein originating from each of the 4 serotypes; the E protein is a good candidate: it has an essential function in viral infectivity and is intermediate between the virus and the cell receptors; the E protein also appears to be an essential target in the protective immune response in infected hosts in that it induces neutralising antibodies and an immunocellular response.

A particularly advantageous composition of the invention is a mixture of 3 or 4 polypeptides or glycopeptides described above, of which a portion is from the E protein of each of the 4 serotypes, with about one hundred amino acids deleted, to which a residue containing 2 to 8 histidines is covalently grafted, preferably to each of the known virus serotypes.

In order for the immunogenic composition of the invention to be effective against the 3 or 4 serotypes in question or if novel serotypes were to appear, each peptide or glycopeptide is present in a proportion of 10% to 50% by weight with respect to the ensemble of peptides in the composition, preferably in a substantially equal percentage for each peptide.

The immunogenic compositions of the invention can also be constituted by a mixture of polypeptides from the E, preM, NS1 or NS3 protein comprising at least 3 antigens of 3 sub-types of the dengue virus, each being provided with an amino acid residue at the carboxyl-terminal extremity.

More generally, the invention provides immunogenic compositions constituted by a mixture of polypeptides or glycopeptides from the E, preM, NSI or NS3 protein 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 extremity, but characterized in that each of the peptides, polypeptides or glycopeptides have the capacity to induce an immune reaction in the host.

The composition of the invention can also contain polypeptides, glycopeptides or proteins originating from other flavivirus such as those of yellow fever, Japanese encephalitis, tick encephalitis or hepatitis C or more generally, other Flaviviridiae of haemorrhaging fevers present in the tropical zones of Africa, Asia or America, and if necessary originating from other viral, bacterial or parasitic antigens, from the moment when they are provided with an amino acid residue as defined above at their carboxy-terminal extremity.

The invention also provides sequences of recombinant nucleic acids coding for peptide immunogens of Flaviviridiae, in particular the dengue virus, with 210 to 315 nucleotides deleted from the 3' end, to which a sequence coding for 2 to 8 amino acids selected from histidine, tryptophan and cysteine as defined above has been added to the 3' end. More particularly, the nucleic acid sequences of the invention code for glycopeptides from the E protein of serotype 1, 2, 3 or 4 dengue and the sequence added to the 3' end preferably codes for a sequence containing 6 histidines.

The invention also provides nucleic acid vectors suitable for use in expressing exogenic genes in a eukaryote; in addition to a sequence of nucleic acids as described above, these vectors carry a signal sequence which is homologous or heterologous to that of the coded protein, the ensemble being dependent on a promoter which can be activated in the eukaryotic cell. A preferred eukaryotic cell type is that of insect cells, in particular cells of Spodopterafrugiperda SF9. The vector is a pVLd shuttle vector which can carry out homologous recombination with a baculovirus and which carries, upstream of the signal sequence, a promoter of endogenic baculovirus genes for example that ofpolyhedrin.

Recombinant baculoviruses can be obtained using different techniques which are known to the skilled person, but one particularly advantageous technique is homologous recombination using shuttle vectors between E. Coli and the baculovirus.

Expression vectors obtained by this technique and described in more detail in the experimental section below have been deposited at the CNCM [Collection Nationale de cultures de Microorganismes, National Collection of Microorganism cultures], namely: A recombinant baculovirus deposited at the CNCM with registration number 1-1497 on 1 s December 1994, carrying a sequence coding for a peptide constituted by the E gene of the serotype 2 dengue virus from which 100 amino acids have been deleted from the COOH extremity and substituted with 6 histidines; A recombinant baculovirus deposited at the CNCM with registration number 1-1624 on 11 th October 1995, carrying a sequence coding for a peptide constituted by the E gene of the serotype 4 dengue virus from which 100 amino acids have been deleted from the COOH extremity and substituted with 6 histidines; A recombinant baculovirus deposited at the CNCM with registration number 1-1625 on 11" October 1995, carrying a sequence coding for a peptide constituted by the E gene of the serotype 3 dengue virus from which 100 amino acids have been deleted from the COOH extremity and v 6 histidines; A recombinant baculovirus deposited at the CNCM with registration number 1-1626 on 11 t October 1995, carrying a sequence coding for a peptide constituted by the E gene of the serotype 4 dengue virus from which 100 amino acids have been deleted from the COOH extremity and substituted with 6 histidines.

The invention also provides compositions which can induce a cellular or humoral immune response in a host which is susceptible of being infected by the dengue virus and comprising a mixture of nucleic acids respectively coding for the polypeptides of the invention, namely those from the dengue protein, such as the E, preM, NS1 or NS3 protein, and carrying a residue of 2 to 8 amino acids at its carboxy-terminal extremity selected from histidine, cysteine and tryptophan, or more generally coding for polypeptides from other Flaviviridiae such as yellow fever, Japanese encephalitis, tick encephalitis or other "flavi-like" viruses such as hepatitis C. It has been shown that injection of a plasmid containing the sequences necessary for protein or polypeptide expression in an animal sets off a cellular and humoral immune response in the host which, in certain animal models, protects the latter against a lethal viral charge; such results have been described by J. A. Wolff et al., (1990), Science 247: 1465-1468 or by E. F. Fynan et al., (1993), DNA cell biol. 12: 785-789. Immunogenic compositions comprising such nucleic acid sequences and vaccines constituted by these compositions, with an adjuvant if necessary, also form part of the invention.

The invention also provides polyvalent subunit vaccines against the dengue virus in particular against the 4 serotypes of that virus and comprising an immunogenic composition as described above, if necessary with a vaccination adjuvant which can in particular be an adjuvant carrying divalent or trivalent ions such as aluminium hydroxide or calcium phosphate or a Freund type adjuvant, a muramylpeptide derivative or an iscom (immunostimulating complex), The immunogenic effect of the compositions of the invention can be the consequence of capture of the polypeptides or glycopeptides of the invention by divalent or trivalent ions and the possible formation of multimers which have an increased immunogenic power with respect to polypeptides from dengue and already described in the literature (references 3, 7, 8, 9).

The subunit vaccines of the invention can also be constituted by compositions which can induce an immunogenic reaction against other Flaviviridiae. They may be prophylactic or for infection therapy using one or more viral species simultaneously.

A polyvalent subunit vaccine of the invention containing 1 to 4 immunogenic glycopeptides or polypeptides of the invention, if necessary associated with immunogenic glycopeptides originating from other flaviviruses or viruses of haemorrhaging fevers, has both the advantage of polyvalence linked to the immunogenic effect of the active principle against the different serotypes of the virus and that of total innocuousness. Vaccines against dengue based on 4 live serotypes attenuated by successive culture of the virus currently exist; but as we have seen above, this vaccine is sometimes a risk for immunodepressed individuals or new-borns, and is not always effective against the different serotypes in particular the 4 dengue serotypes. The vaccines of the invention can also encompass vaccines under current clinical investigation with the addition of 1, 2, 3 or 4 polypeptides in accordance with the invention.

The detailed description below of a particular embodiment using the E protein of serotypes 1 to 4 (DEN1 to DEN4) is a non limiting illustration of the structural and functional properties of the polypeptides, polypeptide compositions and vaccines of the invention.

Figure 1 shows the electrophoretic profile of recombined DEN1 to DEN4 E polypeptides secreted in the supernatant fluid of infected SF9 cells. The radioimmunoprecipitated proteins were treated with endoglycosidase F or H or were untreated I. CONSTRUCTION OF RECOMBINANT BACULOVIRUSES Preparation of gene fragments coding for the envelope protein E of dengue virus Strains of dengue virus used to clone E genes are described in Table 2 below: TABLE 2 VIRUS ORIGIN YEAR SIZE E (aa) Native Recombined Dengue 1: French 1989 495 395 FGA/89 Guyana Dengue 2: Jamaica 1983 495 395 JAM/83 Dengue 3: Thailand 1988 493 393 PaH881 Dengue 4: Burma 1976 494 394 63 632 Preparation of recombinant baculoviruses expressing truncated proteins of dengue virus: The E genes were prepared by reverse transcription/polymerase chain reaction (RT/PCR) of RNA extracted from cells of the Aedes pseudoscutellaris AP61 mosquito infected with the different viruses. The expected number of amino acids in E proteins with 100 amino acids deleted from their C-terminal extremity (EA100) is shown in the right hand column of Table 2.

The oligonucleotidic primer located in the 5' end of the gene to be amplified had a restriction site (apart from dengue the start codon ATG followed by nucleotides coding for the first 6 amino acids of the 15 corresponding to the signal sequence of each protein. The oligonucleotide primer located at the 3' end of the gene to be amplified had a sequence corresponding to 6 amino acids of the E protein of the virus followed by 6 histidine codons, a stop codon and a restriction site. The oligonucleotide sequence is as shown below: end (positive direction) dengue 1: 5'CGGGATCCATGGGGATCATTTTCATTTTGCTGATG-3' dengue 2: 5'-GGCCTTGATTTTCATCTTAC-3' dengue 3: 5'-CGGGATCCATGGTGGTTATTTTTATACTATTAATG-3' dengue 4: 5'-CGGGATCCATGACTGTCTTCTTTGTCCTAATG-3' 3' end (negative direction) dengue 1: 5 '-CGGAGCTCAATGATGATGATGATGATGTCCT1TCTTGAACCAGCTTAG-3' dengue 2: 5'-GGAGCTCAATGATGATGATGATGATGTCCTTTCTTAAACCAGTTG-3' dengue 3: 5'-CGGAGCTCAATGATGATGATGATGATGTCCCTECTTGTACCAGTTAAT-3' dengue 4: 5'-CGGAGCTCAATGATAATGATAATGATACCCTITCCTGAACCAATGGAG-3' The fragments amplified from the viral RNA extracted from Aedes pseudoscutellaris AP61 cells were then cloned in the shuttle vector pVL-poly (17).

Dengue 1 Dengue 2 Dengue 3 Dengue 4 Cleavage sites BamH1/Sacl -I BamHI/Sacl BamHI/of amplified Sadcl fragment Cleavage sites BamHI/Sacl Smal/Sacl BamHI/Sacl BamHI/Smal of pVL-poly vector D (1,2,3 or 4)EA100His6 corresponded to the E protein of serotype 1, 2, 3, 4 with 100 amino acids deleted from its carboxyl extremity and to which 6 histidine residues had been added.

The genes coding for proteins D (1,2,3 or 4)EA100His6 and their signal sequence were then placed under the control of the polyhedrin promoter. The number of nucleotides between the end of the promoter and the start codon was 42 (dengue 1, dengue 3, dengue 4) or 50 (dengue 2) nucleotides.

Recombinant baculoviruses AcD(1,2,3 or 4)EA00 His6 were obtained by homologous recombination in Spodoptera frugiperda SI9 cells between shuttle vectors pVL.D(1,2,3 or 4) EA100His6 and linearised AcRP23LacZ baculovirus at the unique Bsu361 site. The recombinant baculoviruses were selected by 3 successive clonings using a plaque method.

Structural characteristics of recombinant proteins obtained Figure 1 shows an electrophoretic profile of 4 recombinant proteins corresponding to dengue 1, 2, 3 or 4 virus. The profile for proteins treated with F or H endoglycosidase indicated that they were all glycosylated, clearly at only one of the two glycosylation sites as the molecular weight difference did not exceed 4000 daltons.

H EXPRESSION AND PURIFICATION OF DEA100His6 PROTEINS 1) Infection of Sf9 cells: ten billion Sf9 cells in suspension were infected with the recombinant baculovirus acD(1,2,3 or 4)EA100 His6 at an infection multiplication of 2 to 5. The infection duration was 1 hour at 28 0 C. The inoculum was then withdrawn and the cells were incubated for 3 days at 28 0 C in 2 litres of TC100 (GIBCO) medium without foetal calf serum.

2) Preparation of supernatants of infected cells: 2 litres of cell culture supernatant was recovered 3 days after infection, and centrifuged at 2500 rpm at 4 0 C for 30 minutes. 400 g/litre of ammonium acetate was added to the supernatant with stirring for 3 and 14 hours at 4 0 C. The precipitate was centrifuged at 10000 rpm for 20 minutes at 4 0 C and re-dissolved in 1/20 of the initial volume (100 ml) of chromatography buffer (0.5 M NaCI; 20 mM tris-HC1, pH 7.9) containing aprotinin at a final concentration of 20 tg/ml and phenylmethylsulphonyl fluoride at 1 mM final. The concentrated supernatant fluid was then dialysed twice against 100 volumes of chromatography buffer.

3) Chromatography: 5 mM final of imidazole was added to the dialysed medium and incubated for 30 min (with gentle stirring) at laboratory temperature in the presence of 5 ml of TALON resin (Clontech). The resin was washed three times with 30 ml of chromatography buffer containing 10 mM of imidazole. The resin was then placed in a column, washed with 10 volumes of chromatography buffer, and the protein was eluted with 10 ml of chromatography buffer containing 50 mM of imidazole. 1 ml fractions were recovered with a fraction collector. A second elution was carried out using a chromatography buffer containing 100 mM imidazole. The fractions containing the recombinant E protein were determined by dot-blot using anti-E mouse antibody and anti-mouse conjugate coupled to alkaline phosphatase, or using a Ni-NTA conjugate coupled to alkaline phosphatase (Qiagen).

4) Purification monitoring: An aliquot of each fraction was deposited on polyacrylamide gel. The presence of the protein of interest was verified by Western blot after staining with silver nitrate.

Immunogens: Viral proteins in a concentration of 5 pg of pure protein were mixed with 100 pg of aluminium hydroxide (Alugel-S, Serva) and intraperitoneally inoculated into SWISS mice. The viral dengue strains which were used for immunisation were titrated onto AP61 cells (detection of colonies by immunocytochemistry) and inoculated in a proportion of 103 plate forming particles (PFU) per mouse. The control antigen injected into the mice corresponded to a peptide containing the last three amino acids of the E protein (KKG) followed by six histidines (KKGHis6). The inoculum for each injection contained 100 gg of peptide mixed with Alugel.

Calculation of test dose: The lethal activity of the dengue viruses for a day mouse was analysed by intracerebral inoculation of 0.02 ml of viral suspension at different dilutions. The lethal dose killing 50% of the mice was calculated using the Reed and Muench method.

Immunisation of mice: Three week old mice were vaccinated by intraperitoneal injection of a mixture of products corresponding to the 4 dengue serotypes (DEA100His6 or virus) at 3 weeks, 4 weeks and 6 weeks, then individually bled one week after the last injection. The vaccinating peptide of the negative control was identical for the 4 serotypes. The neutralising antibodies of these serums were titrated against each homologous virus. The serums also acted as a protection test after passive transfer to the young mouse.

Neutralisation test: The antibodies of vaccinated mice were titrated by seroneutralisation in 24 cell plaques in the presence of 50 PFU of homologous virus on Vero cells. The titre of neutralising antibodies corresponded to the dilution neutralising 805 of the virus colonies detected by immunocytochemistry using viral anti-protein antibodies and mouse anti-IgG conjugate tagged with peroxidase.

Young mouse protection test: 100 LD50 of virus was inoculated into groups of 5 day old mice which had received an intraperitoneal injection of 0.05 ml of immunised mouse serum the day before (passive transfer).

Each group of young mice for passive transfer was subjected to a test dose with one of the 4 virus serotypes. The mice were observed for 3 weeks and death or signs of paralysis were noted.

Results: The results obtained are shown in Table 3 below.

This table shows the results obtained regarding the neutralising titre of antibodies and the percentage of young mice surviving after virulent virus challenge under the conditions described above.

For each serotype, the results obtained with the peptides of the invention comprising a histidine residue are compared with live viruses, and with KKGHis6 peptides comprising the histidine residue, in respective antigen doses of 5 lg 103 PFU and 100 gg.

The neutralisation titre was measured on serums removed at 7 weeks after 3 injections of antigen. The neutralisation titre corresponded to the inverse of the serum dilution leading to a reduction of 80% of colony forming units (FFU) in the homologous viruses on VERO cells.

The homologous viruses were used for the neutralisation test and for the challenge.

The mice were immunised with the tetravalent mixture in the 3 cases under consideration.

TABLE 3 Mixture of 4 DEA100His6 Virus Peptide serotypes: KKG H6 Quantity of (5 pg) (10 PFU) (100 Rg) antigens: Serotype: D1 D2 D3 D4 Dl D2 D3 D4 D1 D2 D3 D4 Neutralising 200 800 100 400 800 800 200 400 10 510 <10 510 antibodies in vaccinated mouse young mice 80 90 80 90 90 100 90 100 0 20 0 surviving post challenge These results indicate that the formulation containing the 4 recombinant proteins induced neutralising antibodies directed against the 4 serotypes of the virus.

This mixture can thus constitute the base of a composition or an active principle of a polyvalent subunit vaccine against dengue.

The high titre of neutralising antibodies produced after 3 injections of 5 Ig of proteins is remarkable. None of the recombinant proteins produced previously without histidine has resulted in the induction of such high titres of neutralising antibodies. Two hypotheses have been advanced to explain these results: either imperfect purification of the previous proteins contained immunosuppressing constituents, or the sequence of 6 histidines contained in the current proteins in the presence of particles formed from divalent or trivalent ions confers on it a multimeric spatial presentation and as a result, particular immunogenic properties.

III. INDUCTION OF NEUTRALISING ANTIBODIES BY RECOMBINANT ENVELOPE PROTEINS FOR EACH OF THE FOUR

SEROTYPES

1° Envelope proteins for each viral serotype with 100 amino acids deleted from their C-terminal extremity were expressed by the baculovirus system and purified by column chromatography on a cobalt column using the six histidine segment (His6) which composed their C-terminal extremity. The vaccinating antigen was injected into mice in monotype form, and by way of comparison the results obtained after injection of the tetravalent form as shown in table III above is shown in the line 4 x DEA100His6. The line PBS represents a control represented by the buffer with the same name.

Immunisation of mice: Groups of 10 three week old BALB/c mice were immunised on days Dl, D7 and D21 using 5 ig of recombinant antigen coupled with 100 gig of aluminium hydroxide acting as an adjuvant. The mice were bled on day D28 and seroneutralisation tested against 200 colony forming units of each of the reference strains (dengue 1: Hawaii 1944; dengue 2: New Guinea C, 1944; dengue 3: H87 Philippines 1956; dengue 4: H251, Philippines 1951). The titre corresponded to the inverse of the dilution neutralising more than 50% of the foyers.

The results shown in table 4 below indicate that both immunogen D2EA100His6 and D3EA100His6 induced the formation of neutralising antibodies against the dengue 2 serotype while the dengue 3 and dengue 4 serotypes were only neutralised by the recombinant protein D3EA100His6 and D4EA100His 6 respectively. Finally, DIEA100His6 had no neutralising effect.

TABLE 4 NEUTRALISING TITRE Immunogen Dengue 1 Dengue 2 Dengue 3 Dengue 4 DIEA100His6 20 <20 <20 D2EA100His6 <20 2100 20 D3EA100His6 <20 100 100 D4EA100His6 <20 <20 <20 100 4xDEA100His6 100 >100 100 >100 PBS <20 <20 <20 The recombinant proteins from serotypes D2, D3 and D4 thus appear to be of particular interest and the neutralising antibody titre in monkey serum vaccinated with a purified recombinant envelope protein was compared with that obtained with an attenuated virus; the results are shown in Table 5 below.

TABLE Immune response of cynomolgus monkeys against dengue 2 and infected with a wild virus strain Monkeys D28 D56 D70 D98 ELISA PRNT ELISA PRNT ELISA PRNT ELISA PRNT Al <100 <20 <100 ND <100 <20 400 160 A2 <100 <20 <100 ND <100 20 630 160 BI <100 <20 <100 ND <100 <20 250 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 Dl <100 <20 <100 ND <100 <20 100 D2 <100 <20 <100 ND <100 <20 300 El 500 80 600 20 400 40 5000 >1280 E2 160 20 200 20 300 20 <100 Groups of 2 monkeys received different immunogenic preparations: Japanese encephalitis vaccine nonapeptide KKGH6 recombinant envelope protein D2EA100His6 PBS attenuated dengue vaccine PRNT: Plaque reduction neutralisation test (ref. The preparations were injected on day DO (A and E) or on days DO, D28, D56 after sampling plasma. The monkeys were then subjected to a test dose on day D83 by intradermal injection of 2 x 105 infectious particles of dengue 2 virus, Jamaica 1983 strain.

Plasma was then collected on D98.

Obtaining immunogens: The monkeys received monthly: three inoculations of 100 lg of peptides with 1 mg of aluminium adjuvant (group Three inoculations of 100 lg of recombinant dengue 2 envelope proteins D2EA100His6 mixed with 1 mg of alum adjuvant (group three inoculations ofPBS buffer with the adjuvant (group D) or an inoculation of 0.5 ml of live attenuated THAI type dengue 2 vaccines prepared by PASTEUR MERIEUX SERUMS VACCINS (group E).

The sera were collected four weeks after the last inoculation and tested for their neutralising antibodies against 200 plaque forming units (PFU) of the homologous Janaque dengue 2 strain. The titres corresponded to serum dilutions which neutralised 50% of the plates. Table 5 indicates the specificity of induction of neutralising antibodies of the recombinant protein from dengue serotype 2 in cynomolgus monkeys, with a response which was at least four times larger than that obtained with the attenuated virus.

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Claims

1. A polypeptide or glycopeptide immunogen, characterized in that it is constituted by a polypeptide or glycoprotein from Flaviviridiae, in particular from the E or preM or NS1 or NS3 protein of the dengue virus, said polypeptide carrying a residue of 2 to 8 amino acids selected from histidine, tryptophan or cysteine at its carboxyl extremity.

2. A polypeptide or glycopeptide according to claim 1, characterized in that the glycopeptide is constituted by the envelope protein with 70 to 105 amino acids deleted from its carboxyl extremity.

3. A polypeptide or glycopeptide according to claim 1 or claim 2, characteried in that the sequence at the carboxyl extremity is constituted by a residue of 6 histidines which thus confer on it the property of fixing to divalent or trivalent cations.

4. A composition for stimulating the immunity of individuals susceptible of being infected with the dengue virus, characterized in that it contains at least one polypeptide or glycopeptide according to any one of claims 1 to 3. A composition according to claim 4, characterized in that the glycopeptide is from the E glycoprotein of the dengue virus.

6. A composition according to claim 4 or claim 5, characterized in that the 20 polypeptides or glycopeptides are a mixture of at least 3 polypeptides according to any one of claims 1 to 3 from at least 3 dengue virus serotypes, preferably from each of the known serotypes.

7. A composition according to claims 4 to 6, characterized in that it contains the polypeptide D3EA100His6 and/or the polypeptide D2EA100His6.

8. A composition according to claims 4 to 7 constituted by polypeptides or glycopeptides from the E or preM or NS1 or NS3 protein of the dengue virus, preferably the E protein. A composition according to claim 6 or claim 7, characterized in that each peptide is present in a proportion of 10% to 50% by weight with respect to the ensemble of the peptides of said composition. A composition according to any one of claims 4 to 9, characterized in that it also contains polypeptides, glycopeptides or proteins originating from other flavivirus such as yellow fever or Japanese encephalitis or hepatitis C.

11. A composition according to any one of claims 4 to 9, characterized in that it also contains viral, bacterial or parasitic antigens carrying a residue of 2 to 8 amino acids selected from histidine, tryptophan and cystein at their carboxy-terminal extremity.

12. A nucleic acid sequence constituted by a sequence coding for a protein of the dengue virus, with 210 to 315 nucleotides deleted from the 3' end, to which a sequence coding for 2 to 8 amino acids selected from histidine, tryptophan or cystein has been added to the 3' end.

13. A nucleic acid sequence according to claim 12, characterized in that the sequence codes for an E protein of serotype 1, 2, 3 or 4 dengue.

14. A nucleic acid sequence according to claim 12 or claim 13, characterized in that the 3' end sequence codes for 6 histidines. A nucleic acid vector suitable for use in expressing exogenic genes in a eukaryote, characterized in that it contains a nucleic acid sequence according to any one of claims 12 to 14, provided at its 5' end with a signal sequence homologous and/or heterologous to that of the protein coded by said sequence, the ensemble being dependent on a promoter which can be activated in said eukaryotic cell.

16. An 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 which can effect homologous recombination with a baculovirus and carries, upstream of the signal sequence, a promoter of a gene which is endogenic to baculovirus in particular that of polyhedrin.

17. An expression vector according to claim 15 or claim 16, characterized in that it is a recombinant baculovirus deposited at the CNCM with registration number 1-1497 on 1 t December 1994, carrying a sequence coding for a peptide constituted by the E gene of the serotype 2 dengue 23 virus from which 100 amino acids have been deleted from the COOH extremity and substituted with 6 histidines.

18. An expression vector according to claim 15 or claim 16, characterized in that it is a recombinant baculovirus deposited at the CNCM with registration number 1-1624 on 11 October 1995, carrying a sequence coding for a peptide constituted by the E gene of the serotype 4 dengue virus from which 100 amino acids have been deleted from the COOH extremity and substituted with 6 histidines.

19. An expression vector according to claim 15 or claim 16, characterized in that it is a recombinant baculovirus deposited at the CNCM with registration number 1-1625 on 11 i October 1995, carrying a sequence coding for a peptide constituted by the E gene of the serotype 3 dengue virus from which 100 amino acids have been deleted from the COOH extremity and substituted with 6 histidines.

20. An expression vector according to claim 15 or claim 16, characterized in that it is a recombinant baculovirus deposited at the CNCM with registration number 1-1626 on 11 October 1995, carrying a sequence coding for a peptide constituted by the E gene of the serotype 1 dengue virus from which 100 amino acids have been deleted from the COOH extremity and substituted with 6 histidines.

21. A composition for inducing a cellular or humoral immune response in a host which is susceptible of being infected by the dengue virus, characterized in that it contains a nucleic acid coding for a polypeptide according to any one of claims 1 to 3.

22. A process for preparing a polypeptide or glycopeptide according to any one of claims 1 to 4, characterized in that it comprises an affinity chromatography step carried out on a support with an affinity for the sequence of 2 to 8 amino acids.

23. A process according to claim 22, characterized in that the support is a column containing divalent or trivalent ions in particular nickel, copper, cobalt or zinc, with an affinity for electron-rich histidine,- tryptophan or cystein.

24. A polyvalent vaccine against the dengue virus, characterized in that it comprises an imunogenic composition according to any one of claims 4 to 11 and 21, if necessary with a vaccination adjuvant which can in particular be an adjuvant carrying divalent or trivalent ions such as aluminium hydroxide or calcium phosphates, or a Freund type adjuvant, or a muramylpeptide derivative or an iscom. A polyvalent vaccine according to claim 24, characterized in that it also contains immunogenic polypeptides or glycopeptides originating from other flavivirus such as yellow fever or Japanese encephalitis, or tick encephalitis or hepatitis C.

26. A polyvalent vaccine according to 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 divalent or trivalent ions.

27. A 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 .attenuated viruses. *28. A polyvalent vaccine according to claim 24, characterized in that in addition to polypeptides according to any one of claims 1 to 3, it contains a 20 plurality of viral, bacterial or parasitic antigens.

29. A polyvalent vaccine, characterized in that it comprises a mixture of nucleic acids carrying sequences coding for a polypeptide according to any one of claims 1 to 3. 00. 30. An antigenic composition comprising polypeptides or glycopeptides according to any one of claims 1 to 3, characterized in that it comprises at least i 3 antigens of 3 different serotypes of that virus, and preferably an antigen for each of the 4 serotypes. S.. 0.o 0 0: