WO2015019253A2 - Vaccin génétique anti-virus de la dengue à base d'ectodomaines de protéines d'enveloppe - Google Patents

Vaccin génétique anti-virus de la dengue à base d'ectodomaines de protéines d'enveloppe Download PDF

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WO2015019253A2
WO2015019253A2 PCT/IB2014/063588 IB2014063588W WO2015019253A2 WO 2015019253 A2 WO2015019253 A2 WO 2015019253A2 IB 2014063588 W IB2014063588 W IB 2014063588W WO 2015019253 A2 WO2015019253 A2 WO 2015019253A2
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protein
seq
genetic construct
anyone
domain
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Oscar Burrone
Marco Bestagno
Monica POGGIANELLA
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International Centre For Genetic Engineering And Biotechnology - Icgeb
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention refers to the field of genetic engineering and virology.
  • it refers to a genetic vaccine for the dengue virus or for any virus belonging to the Flavivirus genus, more in particular to a vaccine based on the envelope protein E ectodomains, more in particular based on the DIN domain of dengue virus or of other Flavivirus.
  • Dengue virus is a positive-sense RNA virus belonging to the Flavivirus genus of the family Flaviviridae. Dengue virus is widely distributed throughout the tropical and semitropical regions of the world and is transmitted to humans by mosquito vectors. It is estimated to cause in each year more than 50 million cases of dengue fever (DF), 500,000 cases of dengue hemorrhagic fever (DHF) and 25,000 deaths, with 2.5 billion people at risk (Guzman, M.G. et al., Nature Rev. Microbiol., 8, S7-S16, 2010). At the moment there is as yet no dengue vaccine approved for use; a chimeric dengue-yellow fever live vaccine has undergone a phase III clinical trial (Guy, B.
  • DF dengue fever
  • DHF dengue hemorrhagic fever
  • Dengue genome encodes three structural proteins, C (capsid), pr-M (membrane) and E (envelope) and seven non-structural proteins, NS1 , NS2A, NS2B, NS3, NS4A, NS4B and NS5, that include an RNA-directed RNA polymerase, and a protease involved in processing of the long viral polyprotein, produced as a consequence of continuous translation of the RNA viral genome (Chambers, T. J. et al., Ann. Rev. Microbiol. 44, 649- 688, 1990; Bollati, M. et al., Antiviral Res. 87, 125-148, 2010).
  • Protein C encapsidates the RNA genome, while proteins M (obtained after cleavage of the pr-M precursor) and E are associated to the membrane envelope and participate in virus entry into the cell.
  • Protein E is the main component of the viral envelope. It is a membrane-associated glycoprotein containing two trans-membrane domains and an ectodomain composed of three sub-domains (I, II, III) and a stem region. Domain II (E-DII) is responsible for E protein homodimerization; domain III (E-DIII) can fold independently into an immunoglobulin like module and is involved in receptor binding. Most anti-dengue neutralizing antibodies are directed against E-DIII that is therefore considered as a promising target for vaccination (Guzman M.G. et al., Expert Rev. Vaccines, 9, 137-147, 2010).
  • dengue virus There are four serotypes known of dengue virus (“DENV 1" through “DENV 4"), which share approximately 60%-74% amino acid residue identity with one another in the E protein and induce cross- reacting antibodies (Heinz, F.X. & Stiasny, K., Vaccine, 30, 4301-4306, 2012).
  • neutralizing antibodies to the structural proteins of one serotype of dengue provide only limited protection against other serotypes, and in addition appear to predispose to the more severe forms of dengue (DHF) upon re-infection by another serotype (Guzman, M.G. et al., Front, in Dengue Virus Res., Hanley, K.A. & Weaver, S.C.
  • Dengue tetravalent vaccines based on envelope domain III have already been disclosed, for example in the following patent literature.
  • WO2008152652 and WO2007034507 disclose recombinant envelope domain III based proteins wherein the domains DIN of the four serotypes of dengue virus are linked by penta-glycine linkers.
  • the construct can further comprise a secretory peptide from S. cerevisiae.
  • WO2010057159 and US2010291144 disclose DNA vaccines comprising constructs with a DIN consensus sequence, which can further include a leader sequence deriving from IgG or IgE.
  • US6455509 discloses an eukaryotic plasmid expression vector which includes at least part (92%) of the envelope gene and optionally the preM gene of Dengue virus.
  • EP2484376 DIN can be fused to the sequence that codifies the signal peptide of the human tissue plasminogen activator (t-PA).
  • US2009074781 discloses a dengue vaccine active against multiple virus serotypes, comprising a peptide designed on the basis of the DIN domains of 4 serotypes.
  • the construct does not comprise further components.
  • US2013071419 discloses a vaccine comprising a sequence codifying for DIN fused with a sequence codifying for the region p28 of the complement protein c3d.
  • a tetravalent composition may be administered.
  • a tetravalent vaccine for dengue comprising a polypeptide for the DIN domain
  • the fusion protein can further include an adjuvant polypeptide.
  • WO201 11 15583 refers to a platform for retrovirus-like particle (VLP)-display of vaccine antigens.
  • VLP retrovirus-like particle
  • CD16-RigE a chimeric human membrane glycoprotein consisting of the CD 16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE, wherein the CD16 ectodomain can be replaced by the envelope glycoprotein domain III (E:DIII) of serotype I of Dengue virus (DEN1).
  • E:DIII envelope glycoprotein domain III
  • DEN1 Dengue virus
  • the inventors of the present invention have found that when any domain of envelope protein E of all four serotypes of dengue virus, in particular any of the ectodomains Dl, DM and DIN, is fused with at least one carboxyterminal domain of an immunoglobulin constant region, preferably a dimerizing domain, for example the CH3 domain of immunoglobulin G (yCH3) or the CH4 domain of immunoglobulin E (sCH4), the obtained fusion proteins are endowed with a high secretion efficiency.
  • an immunoglobulin constant region preferably a dimerizing domain, for example the CH3 domain of immunoglobulin G (yCH3) or the CH4 domain of immunoglobulin E (sCH4)
  • the new genetic constructs of the present invention encoding the above described fusion proteins can thus be used to efficiently immunise mammals, thanks to the efficient expression of the antigen and subsequent stimulation of the immune response.
  • high titres of neutralising antibodies are generated against all four dengue virus serotypes: a tetravalent vaccine is thus provided with the present invention.
  • the envelope protein domain included in said genetic construct is DI N, it can be the DI N of any virus belonging to the Flavivirus genus.
  • a genetic construct comprising a nucleotide sequence encoding a secretion leader peptide, a nucleotide sequence encoding any domain of envelope protein E of dengue virus and a nucleotide sequence encoding one or more carboxyterminal domains from the constant regions of an immunoglobulin.
  • said domain of envelope protein E is anyone of the ectodomains Dl , DM and DI N . In a more preferred embodiment it is DI N.
  • the envelope protein domain included in said genetic construct is DI N , it can be the DIN of any virus belonging to the Flavivirus genus.
  • the genetic construct comprises a nucleotide sequence encoding a secretion leader peptide, a nucleotide sequence encoding the domain DI N of envelope protein E of a virus belonging to the Flavivirus genus, provided that the envelope protein of said virus is subdivided in three subdomains, and a nucleotide sequence encoding one or more carboxyterminal domains from the constant regions of an immunoglobulin.
  • Said immunoglobulin can be of human, murine or other animal origin.
  • said carboxyterminal domain is selected from the group consisting of yCH3, aCH3, sCH4, ⁇ 4, CK and C domains from any immunoglobulin subclass.
  • the construct of the invention can further comprise a nucleotide sequence encoding a tag, said sequence being preferably included between the sequence encoding the domain of protein E and the sequence encoding one or more carboxyterminal domains.
  • a genetic construct comprising a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3 and 4. aagcttgtcgaccATGGGCTGGAGCCTGATCCTCCTGTTCCTCGTCGCTGTGGCTACAGGTGTGCA
  • Sec is the secretory domain
  • E-DIII is the E-DIII domain
  • SV5 is the exemplary SV5 tag
  • hCH3 is the hCH3 IgG domain.
  • said construct does not contain any kind of tag.
  • the protein codified by any genetic construct according to the invention is also within the scope of the present invention.
  • Said protein is also herein referred to as "fusion protein” or “chimeric protein”.
  • a protein comprising an amminoacidic sequence selected from the group consisting of SEQ ID NO: 5, 6, 7 and 8, respectively coded by gene sequences SEQ ID NO. 1 , 2, 3 and 4, is a preferred object of the present invention.
  • Sec is the secretory domain
  • E-DIII is the E-DIII domain
  • SV5 is the exemplary SV5 tag
  • hCH3 is the hCH3 IgG domain.
  • said construct does not contain any kind of tag.
  • Antibodies raised against any protein codified by a genetic construct according to the invention are further objects of the present invention.
  • the genetic constructs of the invention, the proteins codified by said genetic constructs and the antibodies induced by said constructs can be used as medicaments.
  • the genetic constructs of the invention and the proteins codified by said genetic constructs can be used as a medicament for the prevention of dengue infections, in particular of all the four serotypes.
  • they can be used as a medicament for the prevention of infections of any virus belonging to the Flavivirus genus, provided that the envelope protein of said virus is subdivided in three subdomains and that the genetic construct comprises a sequence encoding the DIN domain of said virus.
  • a vaccine comprising at least one genetic construct of the invention or at least one protein codified by said genetic construct, provided that said genetic construct or protein induces neutralizing antibodies, and pharmaceutically acceptable carriers, excipients and optionally adjuvants is also an object of the present invention.
  • DIN is the DIN domain of the envelope protein E of dengue virus or of any virus belonging to the Flavivirus genus whose envelope protein is subdivided in three subdomains.
  • DIN ⁇ -DW
  • domain III are used in the present invention as synonyms.
  • a “fusion protein” is a protein created through the joining of two or more genes which originally coded for separate proteins. It is also herein referred to as “chimeric protein”.
  • a “genetic construct” is a functional unit necessary for the expression of a gene. It is characterized by a nucleotide sequence and it can be a DNA or RNA sequence.
  • a "secretion leader peptide” is a short peptide sequence, usually present at the N- terminus of a protein, necessary for the secretion of the linked protein or polypeptide.
  • Figure 1 Schematic representation of the SV5-tagged recombinant E-DIII domain alone or combined with the human yCH3 domain; sec: secretion signal, b: Western blotting analysis of cell culture supernatants (S) or cellular extracts (E) from HEK-293 cells transfected with vectors expressing E-DIII of the indicated serotypes, fused or non-fused with the CH3 domain, c: Western blotting analysis of cell culture supernatants from cells transfected with vectors encoding E-DIII-yCH3 chimeras of the indicated serotypes, where the DIN sequence is either the original viral sequence (V) or the codon-optimized one (O). E-DIII containing proteins were detected with anti-SV5 antibodies.
  • Figure 2. Scheme of the ELISA test to evaluate the immunologic response of mice immunized with E-DIII-yCH3. Streptavidin-coated plates were used to capture biotinylated chimeras E-DIII-sCH4-BAP and mouse sera were assayed at different dilutions to evaluate their anti-Dill titres; sec: secretion signal; BAP: biotin acceptor peptide.
  • mice immunized with E-DIII-yCH3 of the indicated serotypes at 1 :2000 dilutions, against the corresponding E-DIII-sCH4, compared with the activity of a preimmune serum (PI), at the same dilution against the same antigens.
  • PI preimmune serum
  • Figure 3 a: Scheme of the E-DIII-sCH4-GPI chimeras expressed on cell surface that are used in cytofluorimetry experiments; sec: secretion signal; GPI: glycosyl-phosphatidyl- inositol anchor sequence, b: FACS analysis of Sp2/0 cells expressing on their surface E- DIII-sCH4-GPI proteins of the indicated serotypes. Each cell line was assayed with sera from mice immunized against the different serotypes, as well as with preimmune serum (PI). It is evident that sera from mice immunized against each serotype cross-react efficiently with Dills of the other serotypes.
  • PI preimmune serum
  • Figure 4. a: Scheme of the complete Env protein ectodomain fused to the transmembrane domain of human MHC-la allele A2 (TM); sec: secretion signal, b: FACS analysis of transiently transfected HEK293 cells expressing on their surface serotype 3 E- DIII-sCH4-GPI (sDIII-GPI), or serotype 3 Env ectodomain (m3E), or mock transfected (mock); cells were assayed with anti-serotype 3 DIN sera (anti-3DI 11) or preimmune sera (PI).
  • TM human MHC-la allele A2
  • b FACS analysis of transiently transfected HEK293 cells expressing on their surface serotype 3 E- DIII-sCH4-GPI (sDIII-GPI), or serotype 3 Env ectodomain (m3E), or mock transfected (mock); cells were assayed with anti-serotype 3 DIN
  • FIG. 1 Plaque Reduction Neutralization Test (PRNT). Vero cells were infected with dengue virus serotype 2 (DENV-2) and treated with sera from mice immunized with E-DIII from the corresponding serotype. The result of a typical assay for serotype 2 is represented in a, where plaque reduction on cells treated with anti-E-DIII serum (right) is visible by comparison with cells treated with preimmune serum (center); on the left are non infected cells. In b is indicated the level of neutralization in comparison with the preimmune serum of eight individual sera from anti-serotype 2 E-DIII mice.
  • It is an object of the present invention a genetic construct comprising a nucleotide sequence encoding a secretion leader peptide, a nucleotide sequence encoding any domain of envelope protein E of dengue virus and a nucleotide sequence encoding one or more carboxyterminal domains from the constant regions of an immunoglobulin.
  • carboxyterminal domain from the constant regions of an immunoglobulin is also herein referred to as "carboxyterminal domain”.
  • the secretion leader peptide comprised in said construct can be one of the secretion leader peptides known in the art.
  • a suitable peptide has the following amminoacidic sequence: MGWSLI LLFLVAVATGVHS (SEQ ID NO: 9), encoded by the following nucleotide sequence: atgggctggagcctgatcctcctgttcctcgtcgtggctacaggtgtgcactct (SEQ ID NO. 10).
  • the domain of protein E is DIN, it is preferably the DIN of one of the following dengue virus strains: DENV1 strain Nauru Island, DENV2 strain NGC, DENV3 strain 3H87 and DENV4 strain Dominica.
  • the nucleotide sequence encoding any domain of envelope protein E of dengue virus is a nucleotide sequence encoding for domain III.
  • said nucleotide sequence is selected from the group consisting of
  • the carboxyterminal domain is from the constant region of an immunoglobulin selected from the group consisting of human, murine or other animal origin.
  • said carboxyterminal domain is selected from the group consisting of yCH3, aCH3, sCH4, ⁇ 4, CK and C domains from any immunoglobulin subclass, said immunoglobulin being of human, murine or other animal origin, preferably of human origin.
  • the human yCH3 domain has the following amminoacidic sequence: QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ I D NO. 15).
  • a tag can be inserted between the sequence of the domain from protein E and the sequence of the one or more carboxyterminal domains, for example between the DI N and the CH3 domain, to facilitate the detection of the obtained recombinant proteins.
  • Suitable tags are, for example, SV5, FLAG, c-myc, HA, His6, HSV, T7, which are all tags known in the art.
  • a preferred tag is the SV5, which has the amminoacidic sequence GKPI PNPLLGLD (SEQ I D NO: 16).
  • the genetic construct comprises a nucleotide sequence encoding a secretion leader peptide, a nucleotide sequence encoding the domain I I I of envelope protein E of dengue virus and a nucleotide sequence encoding one or more carboxyterminal domains from the constant regions of an immunoglobulin.
  • the genetic construct comprises a nucleotide sequence encoding a secretion leader peptide, a nucleotide sequence encoding the domain III of envelope protein E of dengue virus and a nucleotide sequence encoding the yCH3 domain from immunoglobulin G (construct E-DIII-yCH3).
  • the genetic construct comprises a nucleotide sequence encoding a secretion leader peptide, a nucleotide sequence encoding the domain III of envelope protein E of dengue virus and a nucleotide sequence encoding the sCH4 domain from immunoglobulin E (construct E-DIII-sCH4).
  • the genetic construct comprises a nucleotide sequence encoding a secretion leader peptide, a nucleotide sequence encoding the domain III of envelope protein E of any virus belonging to the Flavivirus genus, provided that the envelope protein of said virus is subdivided in three subdomains, and a nucleotide sequence encoding one or more carboxyterminal domains from the constant regions of an immunoglobulin.
  • the DNA fragments encoding the viral DIN of the invention are preferably codon-optimized in order to improve their expression in mammalian cells. Codon-optimization is achieved by the methods known in the art for chemical synthesis of defined DNA sequences (Jestin, J.L. & Kempf, A., J. Mol. Evol.. 69, 452-457, 2009, Wang, P.G. et al., PLoSOne 4(12): e8325, 2009).
  • the genetic construct comprises a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 1 , 2, 3 and 4.
  • Expression vectors comprising a construct according to the present invention are within the scope of the present invention. Suitable vectors are commercially available. More specifically, the genetic constructs of the present invention can be inserted in any expression vector able to sustain expression in mammalian cells.
  • Exemplary vectors are pcDNA 3 (Life Technologies) and pVAX (Life Technologies).
  • the skilled person knows how to insert the construct of the invention in the desired expression vector using suitable restrictions sites; for example the genetic construct can be included in the Hindlll/EcoRI restriction sites of an expression vector.
  • the genetic construct of the invention is introduced into a host cell.
  • the host cell can be a prokaryotic or a eukaryotic cell. Preferably, it is a mammalian cell.
  • a suitable prokaryotic expression vector should be used.
  • the construct can be introduced into a host cell by transfection with any procedure suitable for entry into a cell, for example biological or physical methods, so that the molecules of the present invention are expressed by the host cell.
  • a host cell transfected with an expression vector comprising the genetic construct of the invention is also within the scope of the present invention.
  • the corresponding fusion protein When transfecting a host cell with the construct of the invention, the corresponding fusion protein is expressed. A properly folded antigen is thus obtained and secreted.
  • Induction of antibody responses following genetic immunization requires that the antigen is well expressed and secreted from the cells of the host, in order to produce significant availability of antigen for effective stimulation of B cells.
  • the construct of the invention solves the problem of inefficiency of secretion, providing a fusion protein comprising any domain of envelope protein E that is efficiently secreted from mammalian cells, mainly thanks to the presence of one or more carboxyterminal domains from the constant regions of an immunoglobulin.
  • a particularly efficient secretion has been obtained by a fusion protein comprising DIN fused with the IgG CH3 domain.
  • the construct of the present invention thus encodes for an engineered recombinant protein consisting (from N to C termini) of three essential elements:
  • proteins codified by the genetic constructs of the invention are within the scope of the present invention.
  • said protein comprises the amino acid sequence of domain III of envelope protein E of dengue virus.
  • said amino acid sequence is selected from the group consisting of
  • the protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 5, 6, 7 and 8.
  • Said protein is codified, for example, by the polynucleotides of SEQ ID NO. 1 , 2, 3 and 4.
  • Said proteins can be obtained by a genetic construct according to the invention by methods known in the art.
  • Antibodies raised against the E protein domain encoded by any genetic construct according to the invention are a further object of the present invention. Said antibodies can be obtained following immunization starting from the constructs of the invention or from the codified fusion proteins according to methods known in the art.
  • Said antibodies can be used, for example, for Passive Humoral Immunotherapy for preventing or treating dengue virus or other Flavivirus infections.
  • the delivery to a subject of a genetic construct comprising the DIN sequence from dengue virus induces for all virus serotypes high anti-Dill antibody titres.
  • Sera of the subjects vaccinated with said genetic construct have high titres of virus infectivity neutralising activity.
  • constructs according to the invention are efficient in inducing neutralizing responses against any of the four dengue virus serotypes.
  • vaccination of a subject with four constructs of SEQ ID NO: 1 , 2, 3 and 4 induces neutralising antibodies to all four serotypes with comparable activity.
  • the genetic construct according to the invention can be used as a medicament.
  • the protein codified by said construct can be used as a medicament.
  • said construct or protein can be used as a medicament for the prevention of dengue infections of all the four serotypes.
  • it can be used as a medicament for the prevention of infections of any virus belonging to the Flavivirus genus, provided that the envelope protein of said virus is subdivided in three subdomains and that said genetic construct comprises a nucleotide sequence encoding the domain DIN of said virus.
  • the construct can be administered alone or in combination with other therapeutic agents to enhance antigenicity, for example adjuvants.
  • a vaccine against any of the four dengue virus serotypes comprising at least one genetic construct according to the invention or at least one protein codified by said genetic construct, provided that said genetic construct or protein induces neutralizing antibodies, and pharmaceutically acceptable carriers, excipients and optionally adjuvants is also within the scope of the present invention.
  • a vaccine against any of the four dengue virus serotypes comprising at least one genetic construct according to the invention or at least one protein codified by said genetic construct, provided that said genetic construct or protein induces cross-neutralizing antibodies, and pharmaceutically acceptable carriers, excipients and optionally adjuvants is also within the scope of the present invention.
  • said vaccine comprises at least four constructs, each one comprising a nucleotide sequence encoding domain III of a different dengue serotype.
  • a vaccine against any virus belonging to the Flavivirus genus comprising at least one genetic construct according to the invention or at least one protein codified by said genetic construct, wherein the nucleotide sequence encoding any domain of envelope protein E encodes the DIN domain, provided that said genetic construct or protein induces cross- neutralizing antibodies, and pharmaceutically acceptable carriers, excipients and optionally adjuvants, is a further object of the present invention.
  • Said vaccine can be used for preventing infections from any virus belonging to the Flavivirus genus, provided that the envelope protein of said virus is subdivided in three subdomains.
  • Examples of said virus are Dengue hemorrhagic fever virus, West Nile virus, Tick-borne encephalitis virus, Japanese encephalitis virus, yellow fever virus, Kyasanur Forest Disease virus, Omsk hemorrhagic fever virus, St. Louis encephalitis virus, Powassan virus, Murray Valley encephalitis virus.
  • said genetic vaccine comprises a construct comprising a nucleotide sequence encoding domain DIN and a nucleotide sequence encoding the CH3 domain of IgG.
  • the vaccine comprises at least one construct comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO. 1 , 2, 3 and 4.
  • the vaccine comprises four constructs, one comprising the polynucleotide sequence of SEQ ID NO. 1 , one comprising the polynucleotide sequence of SEQ ID NO. 2, one comprising the polynucleotide sequence of SEQ ID NO. 3 and one comprising the polynucleotide sequence of SEQ ID NO. 4.
  • This embodiment of the vaccine is also herein referred to as tetravalent formulation.
  • neutralizing antibodies means fully neutralizing antibodies to all four dengue virus serotypes.
  • the genetic construct in the vaccine can be in the form of naked DNA or RNA.
  • viral vectors for example, adeno-associated virus (AAV)
  • AAV adeno-associated virus
  • DNA vaccines are within the framework of general knowledge. Reference can be made to Saltzman W. M., Shen H., Brandsma J.L. eds. (2006) DNA Vaccines. Methods and Protocols. 2 nd Edition. Totowa, NJ, Humana Press.
  • the vaccine can be in the form of a preparation for intradermal, intramuscular, intraperitoneal, parenteral, intravenous, subcutaneous, intranasal or oral administration but other forms are equally suitable.
  • Administration is generally achieved by injection.
  • the vaccine doses can be determined properly by a physician considering the type of dosage form, method of administration, the age of the patient, weight, etc. This activity falls within the normal activities of a person skilled in the art.
  • a vaccine according to the invention can be used for immunizing an animal subject. Therefore, the vaccine of the invention for veterinary use is also an object of the present invention.
  • virus infections that can be treated with the present invention are: Japanese encephalitis virus, Kyasanur Forest disease virus, Louping ill virus, Murray Valley encephalitis virus, Omsk hemorrhagic fever virus, St. Louis encephalitis virus, Tick-borne encephalitis virus, Wesselsbron virus, West Nile virus, Yellow fever virus.
  • a method for immunizing a subject against dengue virus infection of any of the four serotypes comprising administration of the genetic construct of the invention to a subject is also provided.
  • a method for immunizing a subject against Flavivirus infection comprising administration of the genetic construct of the invention, wherein said genetic construct comprises a nucleotide sequence encoding DIN, to a subject is also provided.
  • Said subject is an animal, preferably a mammalian, more preferably it is a human.
  • DENV1 strain Nauru Island GenBank accession number U88535.1
  • DENV2 strain New Guinea C NCT, GenBank accession number AF038403
  • DENV3 strain 3H87 GenBank accession number M93130
  • DENV4 strain Dominica GenBank accession number AF326573.
  • E-DIII sequences of dengue virus serotype 1 , 2, 3 and 4 were produced as synthetic genes by GenScript (Piscataway, NJ, USA). E-DIII sequences of DENV serotype 2, 3 and 4 were further codon optimized to improve expression in mammalian cells.
  • E-DIII coding fragments from each serotype were inserted in pcDNA 3 vectors (Life Technologies) already containing sequences encoding a secretion signal from mouse immunoglobulins (sec) and the SV5 tag, or sec, SV5 and the CH3 domain of human IgG (yCH3), to yield the pCDNA 3 -sec-E-DIII-SV5 and pCDNA 3 -sec-E-DIII-SV5-yCH3, respectively, that were used for mice vaccination.
  • the E-DIII domain of each serotype was engineered by adding at the C-terminus the CH4 domain from human IgE (sCH4) fused either with GPI anchor signal (to produce a membrane E- DIII-sCH4-GPI protein) or with BAP (Biotin Acceptor Peptide) sequence (to obtain a secreted biotinylated E-DIII-sCH 4 molecule).
  • sCH4 human IgE
  • Hindlll-Bsu36l fragments from the four plasmids pCDNA 3 -sec-E-DIII-sCH4-GPI were excised and inserted in the corresponding sites of the previously described bigenic vector pCDNA 3 -sec-SV5-CyssCH3CH4 BAP-BirA (Predonzani A.
  • the ectodomain was engineered with the exogenous trans-membrane domain of human MHC-la allele A2.
  • the complete envelope ectodomains (composed of domains Dl, DM and DIN) of the four serotypes were synthetized and inserted into pcDNA 3 vectors (pcDNA 3 -Env).
  • the sequence coding for the MHC-la transmembrane domain (TMD) was amplified from the template pcDNA-BAP-MHC-la (Petris G.
  • HEK293 and HEK293T/17 cells from American Type Culture Collection (Rockville, MD, numbers CRL-1573 and CRL-1 1268, respectively) were cultured in Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Paisley, UK) supplemented with 10% heat inactivated foetal calf serum (FCS) (Life Technologies), 50 ⁇ g/ml gentamycin (Life Technologies) and 2 mM L-glutamine (Sigma).
  • FCS heat inactivated foetal calf serum
  • FCS heat inactivated foetal calf serum
  • 50 ⁇ g/ml gentamycin Life Technologies
  • 2 mM L-glutamine Sigma
  • BHK-21 cells were grown in DMEM medium supplemented with 10% heat inactivated FCS, 50 ⁇ g/ml gentamycin and 2 mM L-glutamine.
  • Vero cells were maintained in DMEM medium supplemented with 10% heat inactivated FCS, 50 ⁇ g/ml gentamycin, 2 mM L-glutamine and 4.5 g/l glucose.
  • Sp2/0 cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with 10% heat inactivated FCS, 50 ⁇ g/ml gentamycin, 2 mM L-glutamine, 1 mM sodium pyruvate; Sp2/0 stable transfected clones were grown in selective medium containing 0.4 mg/ml Geneticin.
  • DENV1 strain Hawaii A, DENV2 strain NGC, DENV3 strain TC3 and DENV4 strain TC25 were used for plaque neutralization reduction test.
  • DENV strains were propagated in BHK21 cells, cultivated in DMEM supplemented with 2% heat inactivated FCS, 50 ⁇ g/ml gentamycin, 2 mM L-glutamine and maintained at 36°C and 5% C0 2 .
  • Transient transfections of HEK293T/17 cells and stable transfections of HEK293 cells were performed essentially as described by Sambrook et al., using circular or linearized plasmids respectively.
  • HEK 293T/17 cells were seeded in 6-well plates at 5x10 5 cells/well density and transfected using standard calcium phosphate method with between 2.5 and 5 ⁇ g of plasmids. 24 h after transfection the culture medium was replaced with a serum-free medium or with a serum free medium supplemented with 100 ⁇ biotin (Sigma), to produce the biotinylated protein in vivo, according to the method described (Predonzani A. et al., BMC Biotechnology 8:41 , 2008), and 48 h post-transfection cellular extracts and supernatants from transiently transfected cells were collected.
  • mice immune responses elicited by DNA vaccines using an ELISA assay recombinant biotinylated E-DIII molecules, fused to human sCH4, or recombinant biotinylated envelope ectodomain were expressed in stable transfected HEK293 cells. 10 7 HEK 293 cells were transfected with 15 ⁇ g of DNA using calcium phosphate technique. Stable clones were selected by culture in medium containing 0.4 mg/ml G418 and secretion of biotinylated proteins was confirmed by western blot. Supernatants from stable transfected cells were collected after 72 h of culture in serum-free medium supplemented with biotin and dialyzed against PBS.
  • mice Female Balb/c mice, 5-6 weeks old, were purchased from Harlan (Milan, Italy). They were housed at the Animal House of ICGEB (Trieste, Italy). All animal procedures were performed in accordance with institutional guidelines and in compliance with national and international laws and policies.
  • mice Four groups of 10 female Balb/c mice were vaccinated with each monovalent dengue DNA vaccine using Gene Gun (Bio-Rad, Hercules, CA, USA) delivery as previously described (Cesco-Gaspere M. et al., Scand. J. Immunol., 68, 58-66, 2008). A group of non-immunized mice was also used as negative control and a group of mice was vaccinated with all four serotypes. Animals were immunized three times at fifteen days intervals with 1 ⁇ g DNA for monovalent vaccines or 2 ⁇ g DNA (0.5 ⁇ g each) for tetravalent vaccines and blood was collected at 45 th day and 60 th day. Animals were bled by submandibular puncture. Serum samples were collected and stored at -20°C until use.
  • Gene Gun Bio-Rad, Hercules, CA, USA
  • a group of non-immunized mice was also used as negative control and a group of mice was
  • mice sera were analyzed for the presence of anti-E-DIII antibodies by ELISA as well as by flow-cytometry.
  • E-DIII immunized mouse sera for each dengue virus serotype was confirmed using in vivo biotinylated E-DIII as coating antigen.
  • MaxiSorp Nunc Immuno-Plates (Nunc, Roskilde, Denmark) were coated with 100 ⁇ /well of avidin diluted at 5 ⁇ g/ml in 50mM Na 2 C0 3 /NaHC0 3 buffer pH 9.5 and incubated overnight at 4°C.
  • the anti-dengue IgG titres were determined as the reciprocal of the highest serum dilution at which OD 450 was ten times higher than that of the non-immune serum.
  • PRNT was carried out on Vero cells in 24 multi-well plates, following procedures described in "Guidelines for plaque reduction neutralization testing of human antibodies to dengue virus” (World Health Organization, Geneva, Switzerland, 2007).
  • mice sera samples (30 min at 56°C) were serially diluted (from 1 : 12.5 to 1 :400) in DMEM serum free medium followed by addition of an equal volume of dengue virus containing 50 PFU/reaction.
  • the neutralization reaction was performed in a final volume of 60 ⁇ and it was incubated 1 h 30 min at 36°C.
  • Vero cells were infected in duplicate with 25 ⁇ of neutralization mixture for 1 h at 36°C and after this time, the viral inoculum was discarded and cells were overlaid with 1 ml of DMEM supplemented with 2% FCS and 3% carboxymethylcellulose (Sigma). Plates were incubated at 36°C for 8-10 days, depending on the serotype. After this period, cells were washed twice with PBS solution, fixed for 1 h with paraformaldehyde (Sigma) 3.7% and stained with 1 % crystal violet for 20 min.
  • Plaques were counted and percentage of plaque reduction was calculated as [(average number of plaques in pre-immune control wells - average number of plaques in test well)/ average number of plaque in pre-immune control well] x 100.
  • Neutralizing antibody titres were expressed as maximum serum dilution yielding a 50% plaque number reduction (PRNTso)
  • Induction of antibody responses following genetic immunization requires that the antigen is well expressed and secreted from the mammalian cells of the host, for effective stimulation of B cells.
  • E-DIII sequences from the four dengue serotypes (from aa 298 to 416, or 296 to 414 for serotype 3, of the viral envelope protein).
  • Each E-DIII was also provided, at its N-terminus, with a sequence encoding a secretion leader peptide to allow translocation into the ER and the secretory pathway and at the C-terminus with the dimerising yCH3 domain from the IgG Heavy-chain ( Figure 1a).
  • Figure 1a the 1 1 amino acids long SV5-tag was also included.
  • Serotype 1 >1 :200 >1 :4000
  • Serotype 2 >1 :100 >1 :5000
  • Serotype 3 >1 :200 >1 :4000
  • Serotype 4 >1 :50 >1 :3000
  • the induced antibodies were able to recognise the different Dills also when expressed as part of the full-length E-ectodomain (containing also E domains Dl and DM, Figure 4a).
  • E-ectodomain containing also E domains Dl and DM, Figure 4a.
  • the reactivity against the E-ectodomain was comparable to the one against DIN alone, as exemplified for serotype 3 in Figure 4b.
  • these antibodies proved to be directed mostly against conformational epitopes, since sera positive in ELISA and FACS on native DIN or E-ectodomain recombinant proteins were not reactive in western blots on denatured proteins.
  • Example 3 - Anti-Dill antibodies have virus neutralising activity
  • the neutralising activity of the serotype specific serum was tested implementing the plaque reduction neutralisation test (PRNT) for each virus serotype on Vero cells.
  • Figure 5a shows a representative set of plates with plaques for dengue virus serotype 2 (DENV- 2), treated with a mouse pre-immune serum (centre) or a mouse anti-2DIII serum (right). The neutralisation activities of each group of animal sera against the homologous dengue serotype were determined.
  • Plaque Reduction Neutralization Test titres of sera from mice immunized against the E-DIII-yCH3 of each serotype, assayed on the corresponding dengue virus serotype. Titre is calculated as the dilution producing a 50% reduction in the number of plaques in comparison with the preimmune serum at the same dilution.
  • Example 4 Neutralising antibodies of a tetravalent formulation
  • a tetravalent formulation containing the four genetic serotype-specific constructs was used to vaccinate a group of animals.
  • the total amount of DNA per cartridge was increased to 2 micrograms.
  • sera from the vaccinated animals were then tested by ELISA and PRNT.
  • the tetravalent vaccine was able to induce total Ab titres (measured by ELISA, Table 3) and neutralising activity (PRNT, Table 4) against all four serotypes with titres comparable to the individual ones and, more importantly, the four serotypes.
  • Titres of sera from mice immunized with the tetravalent vaccine, tested in ELISA on E-DII I-£CH4 from each serotype Titre is calculated as the dilution giving an OD 450 10 times higher than the preimmune serum at the same dilution.
  • Plaque Reduction Neutralization Test titres of sera from mice immunized with the tetravalent vaccine, assayed on each dengue virus serotype. Titre is calculated as the dilution producing a 50% reduction in the number of plaques in comparison with the preimmune serum at the same dilution.
  • the present invention provides industrial application for the manufacturing of a vaccine against Flaviviridae viruses, in particular dengue virus.
  • the vaccine can neutralise all 4 serotypes with comparable activity.

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Abstract

La présente invention concerne un vaccin génétique pour le virus de la dengue de tous les sérotypes ou pour tout virus appartenant au genre Flavivirus. Ledit vaccin est basé sur les ectodomaines de protéine E d'enveloppe, plus particulièrement sur le domaine DIII du virus de la dengue ou d'autres Flavivirus. La présente invention concerne une construction génétique codant pour une protéine de fusion dans laquelle ledit domaine de la protéine E d'enveloppe est fusionné à un domaine carboxy-terminal provenant de la région constante d'une immunoglobuline. L'utilisation de ladite construction permet une sécrétion efficace de la protéine de fusion à partir des cellules et par conséquent pour une réponse élevée en anticorps.
PCT/IB2014/063588 2013-08-05 2014-07-31 Vaccin génétique anti-virus de la dengue à base d'ectodomaines de protéines d'enveloppe WO2015019253A2 (fr)

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WO2017079625A1 (fr) * 2015-11-06 2017-05-11 University Of South Alabama Nouvelle plateforme de vaccins à adn
WO2018071405A1 (fr) * 2016-10-11 2018-04-19 University Of Miami Vecteurs et cellules de vaccin pour immunité contre le virus zika
CN108503696A (zh) * 2017-02-27 2018-09-07 中国科学院上海巴斯德研究所 一种酵母细胞表达的寨卡病毒亚单位疫苗
WO2021026372A1 (fr) * 2019-08-06 2021-02-11 The University Of North Carolina At Chapel Hill Méthodes et compositions pour dimères de protéine e de flavivirus recombinée stabilisée

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017079625A1 (fr) * 2015-11-06 2017-05-11 University Of South Alabama Nouvelle plateforme de vaccins à adn
WO2018071405A1 (fr) * 2016-10-11 2018-04-19 University Of Miami Vecteurs et cellules de vaccin pour immunité contre le virus zika
US11666649B2 (en) 2016-10-11 2023-06-06 University Of Miami Vectors and vaccine cells for immunity against Zika virus
CN108503696A (zh) * 2017-02-27 2018-09-07 中国科学院上海巴斯德研究所 一种酵母细胞表达的寨卡病毒亚单位疫苗
WO2021026372A1 (fr) * 2019-08-06 2021-02-11 The University Of North Carolina At Chapel Hill Méthodes et compositions pour dimères de protéine e de flavivirus recombinée stabilisée

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