WO2011138586A1 - Dengue virus vaccine - Google Patents

Dengue virus vaccine Download PDF

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Publication number
WO2011138586A1
WO2011138586A1 PCT/GB2011/000688 GB2011000688W WO2011138586A1 WO 2011138586 A1 WO2011138586 A1 WO 2011138586A1 GB 2011000688 W GB2011000688 W GB 2011000688W WO 2011138586 A1 WO2011138586 A1 WO 2011138586A1
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WO
WIPO (PCT)
Prior art keywords
denv
virus
prm
nucleic acid
protein
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Application number
PCT/GB2011/000688
Other languages
French (fr)
Inventor
Gavin Screaton
Prida Malasit
Wanwisa Dejnirattisai
Juthathip Mongkolsapaya
Original Assignee
Imperial Innovations Limited
National Center For Genetic Engineering And Biotechnology (Biotec)
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Application filed by Imperial Innovations Limited, National Center For Genetic Engineering And Biotechnology (Biotec) filed Critical Imperial Innovations Limited
Publication of WO2011138586A1 publication Critical patent/WO2011138586A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • 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
    • C12N2770/24123—Virus like particles [VLP]
    • 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 invention relates to a composition
  • a composition comprising a chimeric flavivirus comprising a nucleic acid sequence encoding a non-DENV flavivirus prM protein, a nucleic acid sequence encoding at least one epitope of a DENV virus envelope protein and a nucleic acid sequence encoding the non-structural flavivirus proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 and uses thereof.
  • Dengue virus is a single-stranded positive-polarity RNA virus of the
  • DENV is a mosquito borne virus infection found in tropical and subtropical areas of the world, with an estimated 50-100 million infections per annum. Sequence variation of 30-35% allows DENV to be divided into four serotypes (DEN-1 , DEN-2, DEN-3 and DEN-4) and infection with one serotype does not provide protection to infection with the other serotypes meaning secondary infections are common.
  • DENV envelope contains 180 copies of the E glycoprotein, which can be found in either dimeric or trimeric (pre-fusion) conformation.
  • Precursor-membrane (prM) protein is a 166 amino acid protein intimately associated in a 1 :1 fashion with domain II of E, and is believed to act as a chaperone for the folding of E and to prevent the premature fusion of virus to membranes inside the producing cell.
  • prM contains a furin cleavage site, and is cleaved into a C-terminal M portion containing a transmembrane domain that remains associated with the virus particle, and an N-terminal 91 amino acid fragment that dissociates upon release of the virus from the infected cell.
  • ADE antibody dependent enhancement
  • monocytes/macrophages a major site of DENV replication in vivo, leading to increased vims replication.
  • a flavivirus vaccine that does not contain the wild-type DENV prM protein but does comprise at least an epitope from the DENV envelope reduces the incidence of ADE in a vaccinated subject.
  • the first aspect of the invention relates to a composition
  • a composition comprising a chimeric flavivirus comprising a first nucleic acid sequence encoding a non-DENV flavivirus prM protein, a second nucleic acid sequence encoding at least one epitope of a DENV virus envelope protein (preferably a complete DENV virus envelope protein) and a third sequence encoding the non-structural flavivirus proteins NSl, NS2A, NS2B, NS3, NS4A, NS4B and/or NS5.
  • the three sequences are preferably part of a single sequence.
  • the three sequences are preferably contiguous.
  • the three sequences are contiguous with the most 5' end being the prM protein and the most 3' end being the specified non-structural flavivirus proteins.
  • the chimeric flavivirus of the invention further comprises a sequence encoding a flavivirus capsid (C) protein or a fragment thereof which encodes a functioning capsid protein.
  • the sequence encoding the capsid is a fourth nucleic acid sequence.
  • it forms part of the single nucleic acid sequence which the other three nucleic acids form. Most preferably it is present at the furthest 5' end (immediately preceding prM).
  • the composition is a live attenuated virus vaccine and is capable of eliciting an immune response to DENV virus.
  • the immune response can be characterised by reduced generation of antibodies to DENV prM.
  • the prM protein can be from yellow fever virus, West Nile virus, Tick-borne
  • Encephalitis Japanese Encephalitis, Murray Valley Encephalitis, St. Louis Encephalitis or Hepatitis C virus, Langat virus, Kunjin virus, Powassan virus or any other flavivirus that is not DENV virus.
  • the first sequence comprises a nucleic acid sequence encoding a prM protein comprising the following sequence:
  • the invention extends to nucleic acid sequences encoding a prM protein that is at least 80% homologous with or identical to the sequences above.
  • sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequences above.
  • Homologous nucleic acid sequences of this aspect of the invention encode non-DENV prM proteins of the invention that do not substantially cross react with DE V antibodies and act as functioning prM proteins when expressed to enable correct assembly and production of infectious viruses.
  • the prM protein can include all or a fragment of the prM signal protein.
  • the prM signal is 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids in length.
  • the chimeric flavivirus of the invention can comprise a prM signal from the same flavivirus as the non-structural genes.
  • the composition of the invention comprises a sequence encoding one or more yellow fever non- structural genes
  • the composition can also comprise a sequence encoding a yellow fever prM signal sequence and a prM protein from a different flavivirus.
  • the at least one epitope of a DENV virus envelope protein (preferably the complete protein) can be from the envelope protein of any one of the four serotypes DEN-1, DEN-2, DEN-3 and DEN-4.
  • the second nucleic acid sequence encodes the DENV virus envelope protein.
  • the invention extends to nucleic acid sequences encoding an envelope protein from DENV serotype 1 (DEN-1) that comprises the following amino acid sequence:
  • the invention extends to nucleic acid sequences that encode a DEN-1 envelope which is at least 80% homologous with or identical to the sequence directly above.
  • sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequence directly above.
  • the invention extends to nucleic acid sequences that encode a DEN-2 envelope which is at least 80% homologous with or identical to the sequence directly above.
  • sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequence directly above.
  • the invention extends to nucleic acid sequences encoding an envelope protein from DENV serotype 3 (DEN-3) that comprises the following amino acid sequence:
  • the invention extends to nucleic acid sequences encoding an envelope protein from DENV serotype 4 (DEN-4) that comprises the following amino acid sequence:
  • the invention extends to nucleic acid sequences that encode a DEN-4 envelope which is at least 80% homologous with or identical to the sequence directly above.
  • sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequence directly above.
  • DENV envelope proteins of the invention are immunogenic.
  • DENV envelope proteins of the invention raise antibodies that neutralise DENV infection.
  • the composition comprises a sequence encoding the domain III, the fusion loop regions and/or other domains or sequences of the DENV envelope protein that carry protective epitopes or epitopes that allow generation of neutralising antibodies.
  • the sequence encodes an epitope from more than one DENV serotype.
  • the serotype can encode a DENV virus envelope epitope from 2, 3 or all 4 serotypes in which case the composition is a bivalent, trivalent or tetravalent vaccine, respectively.
  • the composition can be used as a monovalent live attenuated virus vaccine when the second nucleic acid sequence encodes one or more epitopes from one DENV serotype envelope.
  • the composition of the invention can comprise a first nucleic acid sequence encoding a yellow fever prM protein, a second nucleic acid sequence encoding at least one epitope from a DENV virus serotype 1, 2, 3 or 4 envelope protein (preferably the complete protein) and a third sequence encoding at least one non-structural yellow fever protein (preferably all nonstructural yellow fever proteins).
  • the three sequences are preferably part of a single sequence.
  • the three sequences are preferably contiguous. Most preferably the three sequences are contiguous with the most 5' end being the prM protein and the most 3' end being the non-structural yellow fever protein(s).
  • the composition can be used as a bivalent live attenuated virus vaccine when the second nucleic acid encodes for one or more epitopes from one DENV serotype envelope protein and one or more epitopes from a second DENV serotype envelope protein.
  • the second nucleic acid encodes for one or more epitopes from a DENV serotype 2 envelope protein and one or more epitopes from a DENV serotype 3 envelope protein.
  • composition can also be used as a trivalent live attenuated virus vaccine when the second nucleic acid encodes one or more epitopes from a first DENV serotype envelope protein, one or more epitopes from a second DENV serotype envelope protein and one or more epitopes from a third serotype envelope protein.
  • a composition will comprise a nucleic acid sequence encoding an envelope epitope from at least three different DENV serotypes.
  • the composition can also be used as a tetravalent live attenuated virus vaccine when the second nucleic acid encodes one or more epitopes from a first DENV serotype envelope protein, one or more epitopes from a second DENV serotype envelope protein, one or more epitopes from a third DENV serotype envelope protein and one or more epitopes from a fourth DENV serotype envelope protein.
  • the chimeric flavivirus can further comprise a sequence that encodes for a signalase cleavage site at the envelope/non-structural protein junction, wherein upstream of the envelope/non-structural protein junction comprises a sequence of a cleavage site from DENV or a sequence at least 80% identical thereof and downstream of the
  • envelope/non-structural protein junction comprises a sequence of a cleavage site from the flavivirus backbone or a sequence 80% identical thereof.
  • nucleic acid sequence encoding the non-structural proteins can be termed
  • Encephalitis Murray Valley Encephalitis, St. Louis Encephalitis, Hepatitis C virus, Langat virus, Kunjin virus, Powassan virus or a combination thereof.
  • the YF17D strain can comprise a number of substrains, for example 17D-204, 17D- 213 and 17DD.
  • the third nucleic acid sequence can encode Yellow Fever non-structural proteins NS 1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 with the following amino acid sequences:
  • the third nucleic acid sequence can encode Japanese
  • Encephalitis non-structural proteins NS 1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 with the following amino acid sequences: Japanese Encephalitis virus nakayama strain GenBank: ABQ52691.1
  • the third nucleic acid sequence can encode DENV nonstructural proteins NS1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 with the following amino acid sequences:
  • Dengue serotype 4 prototype strain GenBank: AAX48017.1
  • this aspect of the invention extends to nucleic acid sequences which are at least 80% homologous with or identical to the sequences above relating to non-structural proteins.
  • sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequences above relating to non-structural proteins.
  • Homologous nucleic acid sequences of this aspect of the invention encode functioning non-structural proteins.
  • the non-structural proteins are from the same flavivirus as the prM protein. In a further embodiment, the non-structural proteins are from different a flavivirus to the prM protein.
  • the chimeric flavivirus of the invention further comprises a sequence encoding a flavivirus capsid (C) protein or a fragment thereof which encodes a functioning capsid protein.
  • the sequence encoding the capsid is a fourth nucleic acid sequence.
  • it forms part of the single nucleic acid sequence which the other three nucleic acids form. Most preferably it is present at the furthest 5' end
  • a nucleic acid sequence encoding the flavivirus capsid (C) protein can be selected from any encoding the following protein sequences (or any having 90% identity): Dengue serotype 1 strain DENV-1/KH/BID-V2003/2006 GenBank: ACN42698.1 mmqrkkta sfhmlkramrvstvsqlakrfskgllsgqgpmklvm
  • the capsid can be from any flavivirus, preferably the same virus this from which the prM is from.
  • the chimeric flavivirus can further comprise a sequence that encodes for a protease recognition site at the C/prM protein junction that is the NS2B-NS3 protease recognition site.
  • the chimeric flavivirus can comprise a sequence that encodes for a signalase cleavage site at the C/prM junction, wherein upstream of the C/prM junction comprises a sequence of a cleavage site from the flavivirus backbone or a sequence at least 80% identical thereof and downstream of the C/prM junction comprises a sequence of a cleavage site from the same virus as the prM sequence or a sequence 80% identical thereof.
  • the chimeric flaviviruses defined above can be produced using techniques known in the art.
  • nucleic acid sequences of the present invention may be recombinant or provided as an isolate, in isolated and/or purified form.
  • Nucleic acids of the present invention can be readily prepared by the skilled person, for example using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, "Molecular Cloning", A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992), given the nucleic acid sequences and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences.
  • PCR polymerase chain reaction
  • DNA encoding the polypeptide may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preferences in the host cells used to express the nucleic acid.
  • the nucleic acid sequence of the invention can additionally comprise a promoter or other regulatory sequence which controls expression of the nucleic acid.
  • Promoters and other regulatory sequences which control expression of a nucleic acid have been identified and are known in the art. It may not be necessary to utilise the whole promoter or other regulatory sequence. Only the minimum essential regulatory element may be required and, in fact, such elements can be used to construct chimeric sequences or other promoters. The essential requirement is, of course, to retain the tissue and/or temporal specificity.
  • the promoter may be any suitable known promoter, for example, the human cytomegalovirus (CMV) promoter, the CMV immediate early promoter, the HSV thymidinekinase, the early and late SV40 promoters or the promoters of retroviral LTRs, such as those of the Rous Sarcoma virus (RSV) and metallothionine promoters such as the mouse metallothionine-I promoter.
  • the promoter may comprise the minimum comprised for promoter activity (such as a TATA elements without enhancer elements) for example, the minimum sequence of the CMV promoter.
  • Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the nucleic acid sequence.
  • a promoter nucleotide sequence is operably linked to a nucleic acid sequence if the promoter nucleotide sequence controls the transcription of the nucleic sequence.
  • the polymerase chain reaction (PCR) procedure may be employed to isolate and amplify a nucleic acid sequence encoding a desired protein sequence.
  • Oligonucleotides that define the desired termini of the nucleic acid sequence are employed as 5' and 3' primers.
  • the oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified nucleic acid sequence into an expression vector.
  • PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).
  • homology at the amino acid level is generally in terms of amino acid similarity or identity.
  • Percent homology of sequences may be determined by visual inspection and
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST or ALIGN.
  • protein searches can be performed with the XBLAST program to obtain amino acid sequences homologous to protein molecules of the invention.
  • Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • homologous nucleic acid sequences which hybridises to a sequence in accordance with the first aspect of the invention under stringent conditions, a complement of said sequence or a nucleic acid sequence which is homologous to or would hybridise under stringent conditions to such a sequence but for the degeneracy of the genetic code, or an oligonucleotide sequence specific for any such sequence.
  • Stringent conditions of hybridisation may be characterised by low salt concentrations or high temperature conditions.
  • highly stringent conditions can be defined as being hybridisation to DNA bound to a solid support in 0.5M NaHP0 4 , 7% sodium dodecyl sulfate (SDS), ImM EDTA at 65°C, and washing in O. lxSSC/ 0.1%SDS at 68°C (Ausubel et al eds. "Current Protocols in Molecular Biology" 1, page 2.10.3, published by Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, (1989)). In some circumstances less stringent conditions may be required.
  • moderately stringent conditions can be defined as comprising washing in 0.2xSSC/0.1%SDS at 42°C (Ausubel et al (1989) supra).
  • Hybridisation can also be made more stringent by the addition of increasing amounts of formamide to destabilise the hybrid nucleic acid duplex.
  • convenient hybridisation temperatures in the presence of 50% formamide are 42°C for a probe which is 95 to 100% homologous to the target DNA, 37°C for 90 to 95% homology, and 32°C for 70 to 90% homology.
  • compositions according to the invention may comprise, in addition to an active ingredient (i.e. the chimeric flavivirus of the invention), a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration.
  • the composition is an injectable composition.
  • the formulation may be a liquid, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze dried powder.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • Suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, Lactated Ringer's injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • composition may also be in the form of microspheres, liposomes, other microparticulate delivery systems or sustained release formulations for administration to certain tissues including blood.
  • sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g.
  • Implantable or microcapsular sustained release matrices include polylactides (US Patent No. 3, 773, 919; EP-A-0058481) copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22(1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981 , and Langer, Chem. Tech. 12:98-105, 1982).
  • composition/polypeptide is preferably administered to an individual in a
  • the composition can be formulated as a sterile aqueous solution containing between 10 2 and 10 8 infectious units per serotype in a dose volume of 0.1 to l.OmL to be administered intravenously.
  • the invention includes a composition comprising a chimeric flavivirus comprising a first nucleic acid sequence encoding a DENV flavivirus prM protein, a second nucleic acid sequence encoding at least one epitope of a DENV virus envelope protein and a third sequence encoding the non-structural flavivirus proteins NS1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5, wherein the DENV prM protein comprises a mutation, addition or deletion.
  • the mutation, addition or deletion is such that the prM protein does not contribute significantly to the immunogenic response elicited by the composition upon vaccination of a subject.
  • the second aspect of the invention relates to a polypeptide encoded by the chimeric flavivirus defined above.
  • the third aspect of the invention relates to the composition of the invention for use in inducing an immunological response to DENV virus.
  • the composition is for use as a vaccine against DENV virus.
  • the fourth aspect of the invention relates to the use of the composition of the invention in manufacturing a vaccine for the treatment of DENV virus.
  • the fifth aspect of the invention relates to a method of treating a subject comprising administering a composition of the first aspect of the invention to a subject.
  • composition comprises a live attenuated virus as defined above.
  • composition of the invention can be administered to a subject that has not been infected with DENV virus as a preventative measure against an initial DENV infection.
  • composition can also be administered after a subject has been infected with DENV virus to protect the subject against secondary infections.
  • treatment' is used herein to refer to any regimen that can benefit a human or non-human animal.
  • the human or non-human animal is in need of such treatment.
  • treatment includes “therapeutic” and “prophylactic” and these types of treatment are to be considered in their broadest context. Accordingly, therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • the term “prophylactic” may be considered as reducing the severity of or preventing the onset of a particular condition. “Prophylactic” also includes preventing reoccurrence of a particular condition in a patient previously diagnosed with the condition. “Prophylactic” does not necessarily mean that the subject will not eventually contract a disease condition. “Therapeutic” may also reduce the severity of an existing condition and does not necessarily imply that a subject is treated until total recovery.
  • the composition of the invention may be administered to a patient in need of treatment or that might benefit from such treatment via any suitable route.
  • Route of administration may include; parenterally (including subcutaneous, intramuscular, intravenous, by means of, for example a drip patch), some further suitable routes of administration include (but are not limited to) oral (including buccal and sublingual), nasal, topical, infusion, intradermal, intraperitoneally, intracranially, intrathecal and epidural administration.
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated and the precise nature of the form of the vaccine to be administered. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
  • the sixth aspect of the invention relates to a virus-like particle (VLP) comprising a non-DENV flavivirus prM protein and at least one epitope of a DENV virus envelope protein (preferably the complete protein) and sequences encoding such particles.
  • VLP may optionally contain a capsid protein, but this is not essential.
  • VLPs refer to structures that resemble a virus in terms of proteins but that are not infectious. VLPs in accordance with the invention do not carry genetic information encoding for the proteins of VLPs. In general, VLPs lack a viral genome and are, therefore, non-infectious.
  • FIGURE 1 illustrates neutralisation and ADE of anti-E and anti-prM antibodies.
  • FIGURE 3 illustrates Anti-prM responses during primary DENV infection.
  • FIGURE 4 illustrates reactivity of human anti-prM monoclonal antibodies.
  • FIGURE 5 illustrates partial neutralisation but potent enhancement by human anti-prM monoclonal antibodies.
  • Neutralisation assays (A) and enhancement assays (B) were performed with the six human anti-prM mAbs (clones 3-147, 58/5, 2F5, 2G4, 5F9 and 135.3), murine anti-E mAb (4G2) and purified Ig from pooled DENV convalescent serum (PCS) and pooled non DENV immune serum (PND) were used as controls; meaniSE from three independent experiments;
  • FIGURE 6 illustrates that anti-prM can rescue infectivity in virus containing high densities of prM.
  • DENV produced in the presence of NH4C1.
  • the density of uncleaved prM and E were measured by ELISA and expressed as the prM:E ratio (A).
  • Infectivity was determined in Vero cells, expressed as FFU, and the amount of total virus- eauivalent particles was calculated based upon the concentration of E protein measured by a sensitive sandwich ELISA. Data is presented as virus-equivalent particles/FFU ratio (B).
  • Enhancement assays of U937 cells read out by FACS based intracellular staining for DENV antigens (4G2) using either a constant amount of infectious virus (D) or constant number of virus particles (E); mean ⁇ SE from three independent experiments;
  • FIGURE 7 illustrates infectivity of virus produced in furin deficient LoVo cells.
  • LoVo produced virus has a high prM:E ratio measured by ELISA (A).
  • Silver stained gel (non-reduced) of DENV immunoprecipitated with 4G2 showing absence of M in LoVo cells and reduction in M in virus produced in the presence of ammonium chloride (B).
  • Infection of U937 cells was detected by FACS with mAb 4G2 at 3 days post infection; and
  • FIGURE 8 illustrates the roles of anti-prM on neutralisation and enhancement of DENV infection of PBMC.
  • PBMC were infected with DENV2 in the presence of human anti-prM mAbs, at 24hrs DENV Ag was stained intracellularly (4G2) and detected by flow cytometry in gated monocytes (A).
  • PCS, PND and irrelevant human mAb were used as control.
  • the density of prM on DENV from C6/36 cells and DC were detected by ELISA and presented as prM:E ratio (B).
  • C Neutralisation
  • D antibody-dependent enhancement of infection
  • C6/36 a cell line derived from the mosquito Aedes albopictus was cultured in Leibovitz L-15 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS),
  • TPB tryptose phosphate broth
  • Vero a cell line derived from the kidney of African green monkeys and U937, a human monocytic cell line, were grown in MEM and RPMI1640, respectively. The media were supplemented with 10% FBS, 100 units/ml penicillin, 100 ⁇ g/ml streptomycin and 2 mM L-Glutamine in a 37°C humidified 5% C02 incubator.
  • Monocyte-derived dendritic cells (DC) were prepared as previously described.
  • LoVo cells were cultured in Nutrient mixture (Ham) F12 medium containing 20% FBS. Conjugated antibodies against human or mouse Ig (DAK.0) were used. Pooled convalescent DENV hyperimmune human serum (PCS) (hemagglutination titre > 1/25600), pooled non-DENV immune serum (PND) (hemagglutination inhibition titre and anti-DENV Ab ELISA negative) and mouse anti-DENV envelope, 4G2, were kindly provided by AFRIMS, Thailand. NS1-F3, 2G6 and 1H10 are anti-NSl and anti- prM mAb, respectively.
  • PCS convalescent DENV hyperimmune human serum
  • PND pooled non-DENV immune serum
  • mouse anti-DENV envelope 4G2
  • NS1-F3, 2G6 and 1H10 are anti-NSl and anti- prM mAb, respectively.
  • DENV serotype 1 (Hawaii), serotype 2 (16681), serotype 3 (H87) and serotype 4 (H241) were propagated in C6/36 cells and virus supernatant was collected and stored at -80°C.
  • the DENV stock propagated from C6/36 and MDDC's were free from endotoxin and mycoplasma detected by Limulus amebocyte lysate assay (Whittaker M.A.) and PCR using the mycoplasma detection set (TAKARA BIO INC),
  • C6/36 cells were infected with DENV2.
  • culture medium was replaced by fresh L-15 containing 1.5% FBS and 0.26% TPB with 10 or 20 mM NH4C1 for 2 hrs and the medium was then replaced again.
  • virus particles were harvested and precipitated 2 by 10% PEG 8000.
  • Fully immature virus was produced on LoVo cells as previously described. Briefly, virus was produced by infecting LoVo cells with DENV2 strain 16681 at MOI of 10 and virus was harvested at 2 days.
  • virus was determined by a focus forming assay on Vero cells and expressed as focus-forming units (FFU) per ml. Briefly, virus was serially diluted and incubated with Vero cells for 2 hrs at 37°C. The monolayers were then overlaid with 1.5% carboxymethylcellulose and incubated at 37°C for 3 days. Virus foci were stained with anti-E antibody (4G2) followed by peroxidase-conjugated anti-mouse Ig and visualized by the addition of DAB substrate.
  • FFU focus-forming units
  • IgG+ memory B cells were positively selected from PBMC through magnetic sorting using MACS CD22 microbeads (Miltenyibiotec) followed by depletion of IgA, IgD and IgM expressing cells by FACS-sorting. Isolated IgG+ memory B cells were then transformed with EBV and cultured in RPMI containing 10% FCS, 2.5 ug ml CpG, 10 ng/ml, IL-2, 30 ug/ml holo-Transferrin and irradiated allogeneic PBMC.
  • DENV serotypes were captured onto a MAXISORP immunoplate (NUNC) coated with mouse anti-E antibody (4G2) or anti-NSl antibody (NS1-F3).
  • DENV captured wells were then incubated with B cell line (BCL) culture supematants followed by alkaline phosphatase (AP)-conjugated anti-human IgG Abs.
  • BCL B cell line
  • AP alkaline phosphatase
  • lysates from DENV infected C6/36 cells treated with 1% triton X-100 in PBS were run on 12% SDS polyacryramide gels without heating under nonreducing conditions and electroblotted onto nitrocellulose membranes (Amersham).
  • dot enzyme immunoassay culture supematants from mock, DENV or JEV infected C6/36 cells were dotted onto nitrocellulose membranes. The membranes were then blocked with PBS containing 5% skimmed milk and probed with BCL supematants or plasma samples diluted 1 :100 followed by peroxidase-conjugated anti-human IgG Abs. Finally, membranes were developed with enhancement chemiluminescence substrate (Amersham).
  • Serially diluted antibody was incubated with an equal volume of virus for 1 hr at 37°C then transferred to U937 cells and incubated at 37°C for 4 days. Supematants were then harvested and viral titre assessed by a focus forming assay as described above.
  • PBMC DENV infected PBMC and FACS analysis PBMC were infected with endotoxin-free DENV in the presence of different concentrations of antibodies for 24 hrs. Cells were then fixed, permeabilised, and stained for DENV antigen by intracellular staining as described above.
  • the supernatants from DENV2 infected culture were pre-cleared with protein A- agarose beads for 1 hr at 4 °C. After centrifugation, supernatant was incubated with 10 ⁇ g of 4G2 for 2 hrs at 4 °C. Protein A-agarose beads were then added and incubated for 1 hr at 4 °C. After washing, proteins were eluted with non-reducing loading buffer and run on a 15% 4 SDS-polyacrylamide gel followed by silver staining according to the manufacture protocol (SilverQuest staining kit, Invitrogen).
  • B cells from seven DENV infected individuals were used to produce human mAb.
  • Culture supernatants were screened against structural antigens using whole vims and against non-structural protein 1 (NS l) by ELISA.
  • 301 of 3020 cell lines screened positive; 73% reacted to the whole virus ELISA for structural antigens and 27% to NSl .
  • Positive supernatants were tested for reactivity to specific DENV antigens by non-reducing Western blot (Fig. 2A).
  • JEV Japanese encephalitis virus
  • ELISA assays were performed to measure E and prM in the virus preparation; the E assay was calibrated by plotting a standard curve using recombinant E protein produced in Sf9 cells.
  • a measure of the number of potential virus particles (virus equivalent particles) was derived assuming each particle contained 180 copies of E and the relative density of uncleaved prM, calculated by the ratio of prM:E.
  • the prM:E ratio was increased by roughly 40% and 80% when virus was cultured in 10 mM and 20 raM ammonium chloride respectively (Fig. 6A).
  • the infectivity of virus produced in the presence of NH4C1 was markedly reduced from 46 to 555 virus-equivalent particles/FFU (Fig. 6B). Although infectivity was reduced, infectious virus produced under each condition remained partially susceptible to neutralisation as before, and the titration curves for virus produced in 0, 10 and 20 mM NH4C1 were similar (Fig. 6C).
  • Virus was generated in the insect cell line C6/36, which is known to cleave prM inefficiently, and the results we have obtained are therefore analagous to the first encounter with DENV i.e., insect-produced virus following a bite from an infected mosquito. Finally, we set out to determine whether virus produced in primary mammalian cells contained non-cleaved prM and whether anti-prM had any enhancing capacity on such virus. Virus was produced in immature dendritic cells (DC) where cleavage of prM was more efficient than in the insect cell culture, but still not complete (Fig. 8B). As with insect-produced virus, the anti-prM antibodies were unable to fully neutralise DC-produced virus with a clear plateau in efficacy (Fig. 8C), but were still able to enhance infection, although to a lesser degree (Fig. 8D).
  • DC dendritic cells

Abstract

The invention relates to a composition comprising a chimeric flavivirus comprising a nucleic acid sequence encoding a non-DENV flavivirus prM protein, a nucleic acid sequence encoding at least one epitope of a DENV virus envelope protein and a nucleic acid sequence encoding the non-structural flavivirus proteins NSl, NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 and uses thereof.

Description

Dengue Virus Vaccine
The invention relates to a composition comprising a chimeric flavivirus comprising a nucleic acid sequence encoding a non-DENV flavivirus prM protein, a nucleic acid sequence encoding at least one epitope of a DENV virus envelope protein and a nucleic acid sequence encoding the non-structural flavivirus proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 and uses thereof.
Dengue virus (DENV) is a single-stranded positive-polarity RNA virus of the
Flaviviridae family. DENV is a mosquito borne virus infection found in tropical and subtropical areas of the world, with an estimated 50-100 million infections per annum. Sequence variation of 30-35% allows DENV to be divided into four serotypes (DEN-1 , DEN-2, DEN-3 and DEN-4) and infection with one serotype does not provide protection to infection with the other serotypes meaning secondary infections are common.
DENV envelope contains 180 copies of the E glycoprotein, which can be found in either dimeric or trimeric (pre-fusion) conformation. Precursor-membrane (prM) protein is a 166 amino acid protein intimately associated in a 1 :1 fashion with domain II of E, and is believed to act as a chaperone for the folding of E and to prevent the premature fusion of virus to membranes inside the producing cell. prM contains a furin cleavage site, and is cleaved into a C-terminal M portion containing a transmembrane domain that remains associated with the virus particle, and an N-terminal 91 amino acid fragment that dissociates upon release of the virus from the infected cell.
In 1977 Halstead (Halstead and O'Rourke, J Exp Med, 146, 201) suggested antibody dependent enhancement (ADE) to explain severe DENV infections. ADE occurs when a patient already infected by one DENV serotype is infected by a second DENV serotype. Serious complications of DENV haemorrhagic fever (DHF) are more likely during secondary versus primary infections. ADE has been widely studied and results from the high sequence divergence between DENV such that antibody to the first infection may not be of sufficient avidity to neutralise a secondary infection. The partial cross reactivity may cause a degree of opsonisation that promotes virus uptake into Fc bearing cells such as
monocytes/macrophages, a major site of DENV replication in vivo, leading to increased vims replication.
There are currently no approved vaccines for DENV infection. The inventors have surprisingly discovered that a flavivirus vaccine that does not contain the wild-type DENV prM protein but does comprise at least an epitope from the DENV envelope reduces the incidence of ADE in a vaccinated subject.
The first aspect of the invention relates to a composition comprising a chimeric flavivirus comprising a first nucleic acid sequence encoding a non-DENV flavivirus prM protein, a second nucleic acid sequence encoding at least one epitope of a DENV virus envelope protein (preferably a complete DENV virus envelope protein) and a third sequence encoding the non-structural flavivirus proteins NSl, NS2A, NS2B, NS3, NS4A, NS4B and/or NS5. The three sequences are preferably part of a single sequence. The three sequences are preferably contiguous. Most preferably, the three sequences are contiguous with the most 5' end being the prM protein and the most 3' end being the specified non-structural flavivirus proteins. The chimeric flavivirus of the invention further comprises a sequence encoding a flavivirus capsid (C) protein or a fragment thereof which encodes a functioning capsid protein. Preferably, the sequence encoding the capsid is a fourth nucleic acid sequence. Preferably it forms part of the single nucleic acid sequence which the other three nucleic acids form. Most preferably it is present at the furthest 5' end (immediately preceding prM).
The composition is a live attenuated virus vaccine and is capable of eliciting an immune response to DENV virus. The immune response can be characterised by reduced generation of antibodies to DENV prM. The prM protein can be from yellow fever virus, West Nile virus, Tick-borne
Encephalitis, Japanese Encephalitis, Murray Valley Encephalitis, St. Louis Encephalitis or Hepatitis C virus, Langat virus, Kunjin virus, Powassan virus or any other flavivirus that is not DENV virus.
In particular, the first sequence comprises a nucleic acid sequence encoding a prM protein comprising the following sequence:
Japanese Encephalitis virus strain Nakayama GenBank: ABQ52691.1
mklsnfqgkllmtvnntdiadviviptskgerircwvraidvgymcedtityecpkltrngndpedvdcwcdnqevyv qygrctrtrhslcrsnrsvsvqthgesslvnkkeawldstkatrylmkte^
lllvapays
Murray Valley Encephalitis virus strain MVE-1-51 Swiss-Prot: P05769.2
lmligfaaalklstfqgkimmtvnatdiadviaiptpkgpnqcwim
qavyvnygrctrarhskj'siTsitA'qthgestlvnld dawldstkatryltktenwiimpgyalvawlgwrnlgsntgqk viftvllllvapays
Yellow Fever Virus strain 17D/Tiantan GenBank: ACN41908.1
vtlvrkrimlllnvtsedlgktfsvgtgncttnileakywcpdsmeyncpnlspreepddidcwcygvenvrvaygkcd sagrsirsrraidlpthem glktrqekwmtgrmgerqlqkierwfVmpffavtaltiaylvgsnm
ays
West Nile virus clone 33/G8; 34/F6 GenBank: AAA48498.2
vtlsnfqgkvmmtvnatdvtdvitiptaagknlcivramdvgylcedtityecpvlaagndpedidcwctkssvyvrygr ctktrhsrrsiTsltvqthgestlankkgawldstkatrylvkteswilmpgyalvaavigwmlgsntmqrvvfaillllvap ays
Langat virus GenBank: AAA02740.1
atvrrerdgsmviraegrdaatqvrvengtcvilatdmgswcddslayecvtidqgeepvdvdcfcrgvekvtleygrcg rregsrsrrsvlipshaqrdltgrghqwlegeavkahltrvegwvwlmklftlslvmvawlmvdgllprilivvvalalvp aya
Kunjin virus strain MRM61C GenBank: BAA00176.1
vtlsnfqgkvmmtvnatdvtdiitippaagknlcivramdvghmcddtityecpvlsagndpedidcwctklavyvry grctktrhsiTsrrsltvqthgestlsnkkgawm
vapays Tick-borne Encephalitis virus strain Sofjin GenBank: CAA30581.1
atvrkerdgttviraegkdaatqvrvengicvilatdmgswcddsltyecvtidqgeepvdvdcscrnvdgvyleygrcg kqegsrtrrsvlipshaqgdltgrglikwlegdslrthltrvegwvwkrikvltlaviavvw
ya
Powassan virus strain LB GenBank: AAA02739.1
ttihrdregym\m rasgrdaasqvrvqngtcvilatdmge
grqagsrgkrswipthaqkdmvgrghawlkgdnirdhvtrvegwmwkjiklltaaivalawlmvdswm alslgpvya In a further embodiment of this aspect the invention extends to nucleic acid sequences encoding a prM protein that is at least 80% homologous with or identical to the sequences above. In further embodiments such sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequences above.
Homologous nucleic acid sequences of this aspect of the invention encode non-DENV prM proteins of the invention that do not substantially cross react with DE V antibodies and act as functioning prM proteins when expressed to enable correct assembly and production of infectious viruses.
The prM protein can include all or a fragment of the prM signal protein. In one embodiment, the prM signal is 20, 19, 18, 17, 16, 15, 14, 13 or 12 amino acids in length. If the prM protein is from a different flavivirus to the one or more non-structural genes, the chimeric flavivirus of the invention can comprise a prM signal from the same flavivirus as the non-structural genes. Thus, for example, when the composition of the invention comprises a sequence encoding one or more yellow fever non- structural genes, the composition can also comprise a sequence encoding a yellow fever prM signal sequence and a prM protein from a different flavivirus. The at least one epitope of a DENV virus envelope protein (preferably the complete protein) can be from the envelope protein of any one of the four serotypes DEN-1, DEN-2, DEN-3 and DEN-4. In a further embodiment, the second nucleic acid sequence encodes the DENV virus envelope protein.
In a further embodiment of this aspect the invention extends to nucleic acid sequences encoding an envelope protein from DENV serotype 1 (DEN-1) that comprises the following amino acid sequence:
Envelope of Dengue serotype 1 prototype strain (Hawaii) GenBank: AAN32773.1 1 mrcvgignrd fveglsgatw vdwlehgsc vttmakdkpt ldiellktev tnpavlrklc
61 ieakisnttt dsrcptqgea tlveeqdanf vcrrtfvdrg wgngcglfgk gslitcakfk
121 cvtklegkiv qyenlkysvi vtvhtgdqhq vgnettehgt iatitpqapt seiqltdyga
181 ltldcsprtg ldfnemvllt mkekswlvhk qwfldlplpw tsgastpqet wnredllvtf
241 ktahakkqev avlgsqegam htaltgatei qtsgttkifa ghlkcrlkmd kltlkgmsyv
301 mctgsfklek evaetqhgtv lvqvkyegtd apckipfstq dekgvtqngr litanpivtd
361 kekpvnieae ppfgesyiw gagekalkls wfkkgssigk mleatargar rmailgdtaw
421 dfgsiggvft svgklvhqif gtaygvlfsg vswtmkigig illtwlglns rsaslsmtci
481 avgmvtlylg vmvqa
In a further embodiment of this aspect the invention extends to nucleic acid sequences that encode a DEN-1 envelope which is at least 80% homologous with or identical to the sequence directly above. In further embodiments such sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequence directly above.
In a further embodiment of this aspect the invention extends to nucleic acid sequences encoding an envelope protein from DENV serotype 2 (DEN-2) that comprises the following amino acid sequence: Envelope of Dengue serotype 2 prototype strain (New Guinea C) GenBank:
AAC59275.1
1 mrcigisnrd fvegvsggsw vdivlehgsc vttmaknkpt ldfeliktea kqpatlrkyc
61 ieakltnttt dsrcptqgep slneeqdkrf vckhsmvdrg wgngcglfgk ggivtcamft
121 ckknmkgkw qpenleytiv itphsgeeha vgndtgkhgk eikitpqssi teaeltgygt
181 vtmecsprtg ldfhemvllq menkawlvhr qwfldlplpw lpgadtqgsn wiqketlvtf
241 knphakkqdv wlgsqegam htaltgatei qmssgnllft ghlkcrlrmd klqlkgmsys
301 mctgkfkwk eiaetqhgti virvqyegdg spckipfeim dlekrhvlgr litvnpivte
361 kdspvnieae ppfgdsyiii gvepgqlkln wfkkgssigq miettmrgak rmailgdtaw
421 dfgslggvft sigkalhqvf gaiygaafsg vswtmkilig viitwigmns rstslsvslv
481 lvgwtlylg vmvqa
In a further embodiment of this aspect the invention extends to nucleic acid sequences that encode a DEN-2 envelope which is at least 80% homologous with or identical to the sequence directly above. In further embodiments such sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequence directly above.
In a further embodiment of this aspect the invention extends to nucleic acid sequences encoding an envelope protein from DENV serotype 3 (DEN-3) that comprises the following amino acid sequence:
Envelope of Dengue serotype 3 prototype strain (H87) GenBank: AAA99437.1
1 mrcvgvgnrd fveglsgatw vdwlehggc vttmaknkpt ldielqktea tqlatlrklc
61 iegkitnitt dsrcptqgea ilpeeqdqny vckhtyvdrg wgngcglfgk gslvtcakfq
121 clesiegkw qhenlkytvi itvhtgdqhq vgnetqgvta eitsqastae ailpeygtlg
181 lecsprtgld fnemilltmk nkawmvhrqw ffdlplpwts gattktptwn rkellvtfkn
241 ahakkqewv lgsqegamht altgateiqt sggtsifagh lkcrlkmdkl klkgmsyamc
301 lntfvlkkev setqhgtili kveykgedap ckipfstedg qgkahngrli tanpwtkke
361 epvnieaepp fgesnivigi gdkalkinwy rkgssigkmf eatargarrm ailgdtawdf
421 gsvggvlnsl gkmvhqifgs aytalfsgvs wimkigigvl ltwiglnskn tsmsfsciai
481 giitlylgvv vqa In a further embodiment of this aspect the invention extends to nucleic acid sequences that encode a DEN-3 envelope which is at least 80% homologous with or identical to the sequence directly above. In further embodiments such sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequence directly above.
In a further embodiment of this aspect the invention extends to nucleic acid sequences encoding an envelope protein from DENV serotype 4 (DEN-4) that comprises the following amino acid sequence:
Envelope of Dengue serotype 4 prototype strain (H241) GenBank: AAX48017.1 1 mrcvgvgnrd fvegvsggaw vdlvlehggc vttmaqgkpt ldfeliktta kevallrtyc
61 ieasisnitt atrcptqgep ylkeeqdqqy icrrdwdrg wgngcglfgk ggwtcakfs
121 csgkitgnlv qienleytvv vtvhngdtha vgndipnhgv tatitprsps vevklpdyge
181 ltldceprsg idfnemilmk mkkktwlvhk qwfldlplpw aagadtsevh wnykermvtf
241 kvphakrqdv tvlgsqegam hsaltgatev dsgdgnhmfa ghlkckvrme klrikgmsyt
301 mcsgkfsidk emaetqhgtt wkvkyegag apckvpieir dvnkekvvgr iisstpfaey
361 tnsvtniele ppfgdsyivi gvgdsaltlh wfrkgssigk mlestyrgak rmailgetaw
421 dfgsvggllt slgkavhqvf gsvyttmfgg vswmvrilig flvlwigtns rntsmamtci
481 avggitlflg ftvha
In a further embodiment of this aspect the invention extends to nucleic acid sequences that encode a DEN-4 envelope which is at least 80% homologous with or identical to the sequence directly above. In further embodiments such sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequence directly above.
DENV envelope proteins of the invention are immunogenic. In particular, DENV envelope proteins of the invention raise antibodies that neutralise DENV infection. In one embodiment of the invention, the composition comprises a sequence encoding the domain III, the fusion loop regions and/or other domains or sequences of the DENV envelope protein that carry protective epitopes or epitopes that allow generation of neutralising antibodies.
In one embodiment, the sequence encodes an epitope from more than one DENV serotype. The serotype can encode a DENV virus envelope epitope from 2, 3 or all 4 serotypes in which case the composition is a bivalent, trivalent or tetravalent vaccine, respectively.
In particular, the composition can be used as a monovalent live attenuated virus vaccine when the second nucleic acid sequence encodes one or more epitopes from one DENV serotype envelope. For example, in this embodiment, the composition of the invention can comprise a first nucleic acid sequence encoding a yellow fever prM protein, a second nucleic acid sequence encoding at least one epitope from a DENV virus serotype 1, 2, 3 or 4 envelope protein (preferably the complete protein) and a third sequence encoding at least one non-structural yellow fever protein (preferably all nonstructural yellow fever proteins). The three sequences are preferably part of a single sequence. The three sequences are preferably contiguous. Most preferably the three sequences are contiguous with the most 5' end being the prM protein and the most 3' end being the non-structural yellow fever protein(s).
The composition can be used as a bivalent live attenuated virus vaccine when the second nucleic acid encodes for one or more epitopes from one DENV serotype envelope protein and one or more epitopes from a second DENV serotype envelope protein. For example, where the second nucleic acid encodes for one or more epitopes from a DENV serotype 2 envelope protein and one or more epitopes from a DENV serotype 3 envelope protein.
The composition can also be used as a trivalent live attenuated virus vaccine when the second nucleic acid encodes one or more epitopes from a first DENV serotype envelope protein, one or more epitopes from a second DENV serotype envelope protein and one or more epitopes from a third serotype envelope protein. Thus, such a composition will comprise a nucleic acid sequence encoding an envelope epitope from at least three different DENV serotypes.
In a further embodiment, the composition can also be used as a tetravalent live attenuated virus vaccine when the second nucleic acid encodes one or more epitopes from a first DENV serotype envelope protein, one or more epitopes from a second DENV serotype envelope protein, one or more epitopes from a third DENV serotype envelope protein and one or more epitopes from a fourth DENV serotype envelope protein.
Multivalent live attenuated virus vaccines may also be obtained by combining individual monovalent, bivalent or trivalent vaccines. For example, four compositions, each comprising a nucleic acid sequence encoding for at least one epitope from a different DENV virus serotype, can be administered to an individual at the same time, at substantially the same time or sequentially. Similarly, two bivalent vaccines can be administered as a tetravalent vaccine at the same time, substantially the same time or sequentially.
The chimeric flavivirus can further comprise a sequence that encodes for a signalase cleavage site at the envelope/non-structural protein junction, wherein upstream of the envelope/non-structural protein junction comprises a sequence of a cleavage site from DENV or a sequence at least 80% identical thereof and downstream of the
envelope/non-structural protein junction comprises a sequence of a cleavage site from the flavivirus backbone or a sequence 80% identical thereof.
The nucleic acid sequence encoding the non-structural proteins can be termed
"backbone". Thus, the composition of the invention can be defined as comprising a flavivirus backbone in which the sequence encoding at least one epitope of the envelope protein is replaced with a sequence encoding at least one epitope from a DENV sequence (any one of serotypes 1-4). In one embodiment, the non-structural proteins are from one or more of the following types of viruses or live attenuated versions of these viruses: Yellow Fever, for example, from the Yellow Fever 17D (YF17D) virus strain, Japanese Encephalitis, DENV (any one of serotypes 1 -4 or a combination thereof), West Nile virus, Tick-borne
Encephalitis, Murray Valley Encephalitis, St. Louis Encephalitis, Hepatitis C virus, Langat virus, Kunjin virus, Powassan virus or a combination thereof.
The YF17D strain can comprise a number of substrains, for example 17D-204, 17D- 213 and 17DD.
In a particular embodiment, the third nucleic acid sequence can encode Yellow Fever non-structural proteins NS 1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 with the following amino acid sequences:
Yellow fever virus strain 17D/TiantanGenBank: ACN41908.1
NS1
dqgcainfgkrelkcgdgififrdsddwlnkysyypedpvklasivkasfeegkcglnsvdslehemwrsradeinaife enevdisvvvqdpbivyqrgthpfsrirdglqygwktwgknlvfspgrlmgsfiidgksrkecpfsnrvwnsfqieefg tgvfttrvymdavfeytidcdgsilgaavngkJ sahgsptfwmgshevngtwmihtlealdykecewplthtigtsvee semfiriprsiggpvssrinhipgykvqtngpwmqvplevkreacpgtsviidgncdgrgkstrsttdsgkvipewccrsc tmpp vsfhgsdgcwypmeirprkaheshl vrs wvta
NS2A
geihavpfglvsmmiamewlrkrqgpkqmlvggwllgamlvgqvtlldllkftvavglhfhemnnggdamymal iaafsirpglligfglrtlwsprerlvltlgaamveialggvmgglwkylnavslciltinavasrkasntilplmalltpvtma evrlaamffcawiigvlhqnfkdtsmqktiplvaltltsylgltcipflglcaflatrifgrr
NS2B
sipvnealaaaglvgvlaglafqemenflgpiavggllmmlvsvagrvdglelkklgevsweeeaeisgssarydvalse qgefkllseekvpwdqwmtslalvgaalhpfalllvlagwlfhvrrarr
NS3
sgdvlwdiptpkiieecehledgiygifqstflgasqrgvgvaqggvmtmwhvtrgaflvmgkklipswasvkedlva yggswklegrwdgeeevqliaavpgknwnvqtkpslfkvrnggeigavaldypsgtsgspivnrngeviglygngilv gdnsfvsaisqtevkeegkeelqeiptmlkkgmttvldmpgagktrr^
dvkmtqafsahgsgrevidamchatltynrileptrvvnweviimdeahfldpasiaargwaahraranesatilmtatp pgtsde hsngeiedvqtdi sepwntghdwiladk tawfl siraanvmaaslrkagksvwl rktfereyptik qkkpdfilatdiaemganlcve ldcrtafkpvlvdegrkvaikgplrisassaaqiTgrigrnpnrdgdsyyyseptsen nahhvcwleasmlldnmevrggmvaplygvegtktpvspgemrlrddqrkvfrelvrncdlpvwlswqvakaglkt ndrkwcfegpeeheilndsgetvkcrapggakkplrprwcdervssdqsalsefikfaegrr
NS4A
gaaevlwlselpdflakkggeamdtisvflhseegsraymalsmmpeamtiv^
smamgtmagcgylmflggvkpthisyimliff mv
NS4B
nelgmlektkedlfgkknlipssasp swpdldlkpgaawtvyvgivtmlspmlh wikveygnlslsgiaqsas mdkgipfhikmnisvimllvsg vnsitvmpllcgigcamlhwslilpgikaqqsklaqn^
ieeapempalyekklalylllalslasvamcrtpfslaegivlasaalgpliegntsllwngpmavsmtgvmrgnhyafvg vmynlwkmktgrr
NS5
gsangktlgevwkxelnlldkiqfelykrtdivevdrdtarrhlaegkvdtgvavsrgiaklmfhergyvklegrvidlgc grggwcyyaaaqkevsgvkgftlgrdghekpmnvqslgwniitfkdktdihrlepvkcdtllcdigesssssvtegertvr vldtvek lacgvdnfcvkvlapympdvlekleUqrrfggtvimplsmsthemyyvsgarsnvtftvnqtsrllmrrm
^tgkvtleadvilpigtrsvetdkgpldkeaieerverikseymtswfydndnpyrtwhycgsyvtktsgsaasmvng vikiltypwdrieevtimamtdttpfgqqrvfkekvdtra^
shaaigayleeqeqwktaneavqdpkfvvelvdeerklhqqgrcrtcvynmmgkreldilsefgkakgsraiwymwlg arylefealgflnedhwasrensgggvegiglqylgyvirdlaamdgggfyaddtagwdtriteadlddeqeilnymsp hkJ laqavmemtyki kwkvl^apggkaymdvisiTdqrgsgqvvtyalntitnlkvqlim
dcdesvltrleawltehgcnΓlkrmavsgddcw iddrfglalshlnamskvrkdisewqpskgwndwenvpfcsh hfhelqlkdgrrivvpcreqdeligrgrvspgngwmiketaclskayanmwslmyfhkrdmrllsla
qgrttwsihgkgewmttedmlev nrvwitrmphmqdktmvkkwrdvpyltlo-qdklcgsligm
vihrirtligqekytdyltvmdrysvdadlqlgeli
In a further embodiment, the third nucleic acid sequence can encode Japanese
Encephalitis non-structural proteins NS 1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 with the following amino acid sequences: Japanese Encephalitis virus nakayama strain GenBank: ABQ52691.1
NS1
dtgcaidvtrkemrcgsgifvhndveawvdrykylpetprslakivhkahkegvcgvrsvtrlehqmweavrd kenavdlsx^nkpvgrvrsaplalsmtqekfemgwkawgksilfapelanstiVvdgpetkecpdehrawnsmq edfgfgitstrvwlkireestdecdgaiigtavkghvavhsdlsywiesryndtwkleravfgevksctwpethtlwgd eeseliiphtiagpkskhnrregyktqnqgpwdengivldfdycpgtk^
pplrfrtengcwygmeirpvrhdettlvrsqvda
NS2A
fngemvdpfqlgllvmflatqevlrkrwtarltipavlgallvlmlggitytdlary lvaaafaeansggdvlhlaliavfk icipaflvmnmlstrwtnqenwlvlgaaffqlasvdlqigv^
ldtyriillvigicsllqerkJctmakkkgavllglaltstgwfspttiaaglmvcnpnkkr
NS2B
g pateflsavglmfaivgglaeldiesmsipfmlaglmavsyvvsgkatdmwleraadiswemdaaitgssrr^ dddgdfhliddpgvpwkvwvlrmsciglaaltpwaivpaafgywltlkttkr
NS3
ggvfwdtpspkpcskgdtttgvyrimargilgtyqagvgvmyenvfhtlwhttrgaaimsgegkltpywgsvkedria yggpwrfdrkwngtddvqvivvepgkaavniqtkpgvfrtpfgevgavsldyprgtsgspildsngdiiglygngvelg dgsyvsaivqgdrqeepvpeaytpnmlrkrqmtvldlhpgsgktrkilpqiikdaiqqrlrtavlaptrvvaaemaealrg lpvryqtsavqrehqgneivdvmchatlthrlmspnrvpnynlfvmdeahfldpasiaargyiatkvelgeaaaife ppgttdp^dsnapihdlqdeipdrawssgyewiteyagktvwfvasvlangneiam
kcloigdwdfvittdisemganfgasrvidcrksvkptileegegivilgnpspitsasaaqn-givgrnpnqvgdeyhyg gatseddsnlah teakimldnihmpnglvaqlygperekaltmdgeyrlrgeeklmflellrtadlpvwlaykvasngi qytdrkwcfdgprtnailednteveivtrmgerkilkpr ldarvyadhqalkwfkdfaagkr
NS4A
savsfievlgrmpehfiiigktrealdtmylvataekggkahrmaleelpdaletitlivaitvmtggffllmmqrk glgalvltlatfflwaaevpgtkiagtllialllmwlipepekqrsqtdnqlavflicvltwgmvaa
NS4B
neygmlektkadlksmfggktqasgltglpsmald^atawalyggstvvltpllkhlitseyvttslasinsqagslfvlpr gvpftdldltvglvflgcwgqitlttvltamvlatlhygymlpgwqaealraaqirtaagimknawdgmvatdvpelert tplmqkkvgqvlligvsvaaflvnpnvttvreagvlvtaatltlwdngasavwnsttatglchvmrgsylaggsiawtlik nadkpslkr NS5
^ggrtlgeqwkeklnamsreeffcyrreaiievdrte^
grggwsyyaatlkkvqevrgytkggagheepmlmqsygwnlvslksgvdvfykpsepsdtlfcdigesspspeveeq rtlrvlemtsdwlhrgprefcikvlcpympkviekmevlqrrfggglvrlplsmsnhemy^
qvllgrmdrtvwrgpkyeedvnlgsgtravgkgevhsnqekikl riqklkeefattwhkdpehpyrtwtyhgsyevka tgsasslvngvvklmskpwdaianvttmamtdttpfgqqrvfkekvdtkapeppagakevlnettnwlwahlsrekrp rlctkeefikkxnsnaalgavfaeqnqwstareavddprfwemvdeerenhlrgecM
kgsraiwfinwlgarylefealgflnedhwlsrensgggvegsgvqklgyilrdiagkqggkmyaddtagwdtritrtdle neakvlelldgehrmlaraiieltyrhkvvkvmrpaaegktvmdvisredqrgsgqvvtyalntftniavqlvrta vigpqhleqlprknkiavrtwlfengee trmaisgddcvvkplddrfatalhflnamskvrkdiqewkpshgwh qqvpfcsnhfqeivmkdgrsivvpcrgqdeligrarispgagwnvkdtaclakayaqmwlllyfhrrdlrlmanaicsa vpvdwvptgrtswsihskgewmttedmlq nrvwieenewmmdktpitswtdv^
twaeniyaainqvravigkenyvdymtslrryedvliqedrvi In a particular embodiment, the third nucleic acid sequence can encode DENV nonstructural proteins NS1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 with the following amino acid sequences:
Dengue serotype 1 strain DENV-1/KH/BID-V2003/2006 GenBank: ACN42698.1 NS1
dsgcvinwkgrelkcgsgifvtnevhtwteqykfqadspkrlsaaigkaweegvcgirsatrlenimwkqisnelnhill endmkftvwgdvagilaqgkkmirpqpmeykyswrswgkakiigadvqnttfiidgpntpecpddqrawniwev edygfgifttniwlklrdsytqvcdhrlmsaaikdskavhadmgywieseknetwklarasfievktciwpkshtlwsn gvlesemiipkiyggpisqhny gyftqtagpwhlgkleldfdlcegίtv dehcgnrgpslrtttvtgkiihewccrs tlpplrfrgedgcwygmeirpvkekeenlvksmvsa
NS2A
gsgevdsfslgllcisimieevmrsmsrkmlmtgtlavflllimgqltwndlirlcimvganasdrmgmgttylalmatf lαnφmfavgllίrrltsrevllltiglslvasvelpnsleelgdglamgimilklltdfqshqlwatlls]tflkttfslhyawkt mamvlsivslfplclsttsqkttwlpvllgslgckpltmfliaenkiwgrr
NS2B
swplnegimavgivsillssllkndvplagpliaggmliacyvisgssadlslekaaevsweeeaehsgashnilvevqdd gtmkikdeerddtltillkatllavsgvyplsipatlf yfwqkkkqr NS3
sgvlwdtpsppeveravlddgiyrimqrgllgrsqvgvgvfqenvfhtmwhvtrgavlmyqgkrlepswasv ygggwrlqgswntgeevqviavepgknpknvqtapgtfktpegevgaialdfkpgtsgspivnregkivglygngvvtt sgtyvsaiaqakasqegplpeiedevfrkmltimdlhpgsgktrrylpaivreaikrklrtlilaptrwase
iryqttavksehtgkeivdlmchatftmrllspvrvpnymniimdeahftdpssiaargyistrvgmgeaaa sveafpqsnaviqdeerdiperswnsgyewitdfygkt fVpsiksgndi^^
dwdyvvttdisemganfradrvidprrclkpvilkdgpervilagpmpvtvasaaqrrgrigrnhnkegd
nndedhahwteakmlldnintpegiipalfepereksaaidgeyrlrgearktfvelmiTgdlpvwlsykvasegfqysd iTwcfdgerrinqvleenmdveiwtkegerkklrprwldartysdplalrefkefaagrr
NS4A
svsgdlileigklpqhltqraqnaldnlvmlhnseqggrayrhameelpdtietlmllaliavltggvtlfflsgrglgktsi cvmassvllwmasvephwiaasiilefflmvllipepdrqrtpqdnqlayvvigllfiTiiltvaa
NS5
nemgllettkkdlgighvavgnhlihaamldvdlhpasawtlyavattiitpmmrhtienttanisl^
gwpislandigvpllalgcysqvnpltltaavlmlvahyaiigpglqakatreaqkrtaagimknptvdgivaidldpvvy dakfekqlgqimllilctsqillmrttwalcesitlatgplttlwegspgkfwnttiavsmanifrgsylagaglafslm ggrrgtgaqgetlgekwkrqlnqlsksefntykjsgimevdrseakeglkrgeitldiavsrgtaklnvfVernlvkpegkv idlgcgrggwsyycaglkkvtevkgytkggpgheepipmatygwnlvklhsgkdvffvppekcdtllcdigesspnpt ieegrtlrvlkmvepwlrgnqfcikilnpympswetleqmqrk^
mtsrmlliirftmahrkptyerdvdlgagtrhvavepevanldiigqrienik ehkstwhydednpyktwa kpsgsassmvng klltkpwdvipm\ qiamtdttpfgqqrvfkekvdtrtpkakrgtaqimevtakwlwgflsrnk kprictreeftrkvrsnaaigavfvdenqwnsakeavederfwdlvhrerelhkqgkcatcvynmmgkrekklgefgk akgsraiwym lgarflefealgimnedhwfsrenslsgvegeglhklgyilrdiskipggnmyaddtagwdtri lqnearitdimepehallaksifkltyqnk rvqrpakngtvmdvisrrdqrgsgqvgtyglntftrmeaqlirqmeseg ifspseletpnlaervldwlekygverlkrmaisgddcvvkpiddrfataltalndmgkvrkdipqwepskgwndwqq vpfcshhfhqlimkdgreiwpcmqdelvgrarvsqgag^slretaclgksyaqmwqlmyfhirdl^
vdwiptsrttwsihahhqwmttedmlsvwnrvwieenpwmedkthisswgdvpylgkredqwcgsliglta^ tniqvainqvrrlignenyldymtsmkrfknesdpegalw Dengue serotype 2 prototype strain (New Guinea C)GenBank: AAC59275.1 NS1
dsgcwswkrikelkcgsgifitdnvhtwteqykfqpespsklasaiqkaheegicgirsvtrlenlmwkqitpelnhilse nevkltimtgdikgimqagkrslqpqptelkyswktwgkakmlsteshnqtflidgpetaecpntnrawnslevedygf gvfttniwlklrekqdvfcdsklmsaaikdnravhadmgywiesalndtwkiekasfievkschwpkshtlwsngvle semiiplaifagpvsqhnyrpgyhtqtagpwhlgklemdfdfcegttvvvtedcgnrgpslm
plryrgedgcwygmeirplkekeenlvnslvta
NS2A
ghgqidnfslgvlgmalfleemlrtrvgtkJiaillvavsfVtlrt
kv tfaaglllrkltskelmmttigivllsqstipetileltda
wkvsctilawsvsplfltssqqkadwiplaltikglnptaiflttlsrtnkkr
NS2B
swplneaimavgmvsilassllkndipmtgplvagglltvcyvltgrsadleleraadvkwedqaeisgsspilsitisedg smsikneeeeqtltilirtgllvisglQ vsipitaaawylwevkkqr
NS3
agvlwdvpspppvgkaeledgayrikqkgilgysqigagvykegtfhtmwh\ rgavlm
sygSgwklegewkegeevqvlalepgknpravqtkpglfktnagtigavsldfspgtsgspiidkkgkvvglygngvvt rsgayvsaiaqteksiednpeieddifrkrkltimdlhpgagktkrylpaivreaikrglrtlilaptrvvaaemeealrglp yqtpairaehtgreivdlmchatftmrnspvr pnynliimdeaM
p^qsnapimdeereiperswssghewvtdfkgktvwfVpsikagndiaaclrkngkJcviqlsrktfdseyvktrt dfVvttdisemganfkae dprrcmkpviltdgeervik^
dedcahwkeakmlldnintpegiipsmfeperekvdaidgeyrlrgearktfvdlmrrgdlp layrvaaeginyadr rwcfdgiknnqileenveveiwtkegerkklkprwldariysdpltlkefkefaagrk
NS4A
sltlnlitemgrlptfmtqkardaldnlavlhtaeag
iitasillwyaqiqphwiaasiilefflivllipepekqrtpqdnqltyvviailtvvaatma
NS4B
nemgflektldcdlglgsittqqpesnildidlrpasawtlyavattfVtpmlrhsienssvnvsltaianqatvlmglgkgw plskmdigvpllaigcysqvnpitltaalfllvahyaiigpglqakatreaqkraaagimknptvdgitvidldpipydpkfe kqlgqvmllvlc^qvlmmrttwalcealtlatgpistlwegnpgrfwnttiavsmanifrgsylagagllfsim NS5
gtgnigetlgekwksrlnalgksefqiykksgiqevdrtlakegikrgetdhhavs^
gcgrggwsyycgglknvrevkgltkggpgheepipmstygwnlvrlqsgvdvfftppekcdtllcdigesspnptvea grtln'lnlvenwlnimtqfcikvlnpympsviekmealqrkyggalvmplsmsthemywvs
mlinrftmrhkkatyepdvdlgsgtmigieseipnldiigkriekikqehetswhydqdhpyktwayhgsyetk^ ssmvng vrlltkpwdwpmvtqmamtdttpfgqqrvfkekvdtrt^
ctreeftrkvrsnaalgaiftdenkwksareavedsrf elvdkemlhleg
iwymwlgarflefealgflnedhwfsrenslsgvegeglhJ lgyilrdvskkeggamyaddtagwdtritledllmeem vtnhmegehkklaeaifkltyqnkwrvq tprgtvmdiisrrdqrgsgqvgtyglntftnmea
qhltvteeiavqnwlarvgrerlsrrnaisgddcvvkplddrfasaltalndmgkvrkdiqqwepsrgwndwtqvpfcs hhfhelimkdgrvlvvpcmqdeligrarisqgagwslretaclgksy
srttwsihak ewmttedmltvwnrvwiqenpwmedktpvesweeipylgkredqwcgsligltsratwalm^ nqvrsligneeytdympsmkrfrreeeeagvlw Dengue serotype 3 prototype strain(H87) GenBank: AAA99437.1
NS1
dmgcvinwkgkelkcgsgifvtnevhtwteqykfqadspkrvataiagawengvcgirsttirnenllwkqianelnyil wendikltv gditgvleqgkrtltpqpmelkyswktwglakivtaetqnssfiidgpstpecpsasrawnvwevedy gfgvfttniwlklrevytqlcdhrlmsaavkderavhadmgywiesqkrigswklekaslievktctwpkshtlwsngvl esdmiipkslagpisqhnhrpgyhtqtagpwhlgkleldfayce
lrymgedgcwygmeirpinekeenmvkslasa
NS2A
gsgkvdnftmgvlclailfeevmrgkfgkl hmiagvlftfvlllsgqitwrgmahtn
fkiqpflalgfflrkltsrenlllgvglamaatlrlpedieqmangialglmalklitqfetyqlwtalvsltcsntiftltvawrta tlilagisllpvcqsssmrktdwlpmtvaamgvpplplfifslkdtlkrr
NS2B
swplnegvmavglvsilassllmdvpmagplvagglliacyvitgtsadltvekaad\ weeeaeqtgvshnlmitvdd dgtmrikddeteniltvllktallivsgifpysipatmlvwhtwqkqtqr
NS3
sgvlwdvpsppetqkaeleegvyrikqqgifgktqvgvgvqkegvfhtmwhvtrgavlthngkrlepnwasvldidli ygggwrlsaqwqkgeevqviavepgknpknfqtmpgifqtttgeigaialdfkpgtsgspiinregkvvglygngwtk npgyvsgiaqtnaepdgptpeleeemfkkmltimdlhpgsgktrkylpaivreaikxrlrtlilapti va lpiryqttatksehtgreivdlmchatftmrllspvrvpnynliimdeahftdpasiaargyistrvgmgeaaai tada^qsnapiqdeerdiperswnsgnewitdfvgktvwfvpsikagnviancl^
dwdfv tdisemganfiadrvidpn-clkpviltdgpervilagpm
nkdedhahwteal mlldnintpegiipalfepereksaaidge^
rkwcfdgernnqileenmdveiwtkegekkklrprwldartysdplalkefkdfaagrk
NS4A
sialdlvteigrvpshlahrtmaldnlvmlhtsehggrayrha
cviassgmlwmadvplqwiasaivleffmmvllipepekqrtpqdnqlayvvigiltlaaivaa
NS4B
nemgllettl dlgmskepg^sptsyldvdlhpasawtlyavattvitpmlrhtienstanvslaaianqavvlmgldkg wpiskmdlgvpllalgcysqvnpltliaavlllvthyaiigpglqakatreaqkxtaagimknptvdgimtidldpviyd^ fekqlgqvmllvlcavqlllmrtswalcevltlatgpittlwegspgkfwnttiavsmanifrgsylagaglalsimksvgtg kr
NS5
gtgsqgetlgekwklddnqlsrkefdlykksgitevdrteakeglkrgeitM
cgrggwsyycaglkkvtevrgytkggpgheepvpmstygwnivklmsgkdvfylppekcdtllcdigesspsptvee srtirvlkmvepwlknnqfcikvlnpymptviehlerlqrkhggmlvmplsmsthemywisng^
llnrftmth^tiekdvdlgagtrhvnaepetpnmdvigerikrikeehsstwhyddenpyktwayhgsyevkatgsas sming klltkpwd pmvtqmamtdttpfgqqrvfkekvdtrtp mpgtrkvme^taewlwrtlgrrlkrp^ eeftkkvrtnaamgavfteenqwdsaraavedeefwklvdrerelhklgkcgscvynmmgkrekklgefgkakgsra iwymwlgarylefealgflnedhwfsrensysgvegeglhklgyilrdiskipggamyaddtagwdtriteddlhneeki tqqmdpehrqlanaifkltyqnkvvkvqrptpkgtvmdiisrkdqrgsgqvgtyglntftnmeaqlirqniegegvlska dlenphplekJiitqwletkgverlkirnaisgddcwkpiddrfanallalndmgkvrkdipqwqpskgwhdwqqvp fcslΛftelimkdgrklwpc qdeligrarisqgagwslretaclgkayaq
vptsrttwsihahhqwmttedmltvwnr iednpwmedktpvttwedvpylgkredqwcgsligltsratwa^ taiqqvrsligneefldympsmkrfrkeeesegaiw
Dengue serotype 4 prototype strain (H241) GenBank: AAX48017.1
NS1
dtgcavswsgkelkcgsgifvidnvhtwteqykfqpesparlasailnahedgvcgirsttrlenimwkqitnelnyvlw egghdltwagdvkgvlskgkralappvndlkyswktwgkakiftpeaknstflidgpdtsecpnerrawnflevedyg fpmfttniwmkfregssevcdhrlmsaaikdqkavhadmgywiessknqtwqiekaslievktclwpkth lesqmlipkayagpfsqhnyrqgyatqtvgpwhlgkleidfgecpgttvtiqedcdhrgpslrtttasgklvtqwccrsct mpplrflgedgcwygmei lsekeenmvksqvsa
NS2A
gqgtsetfsmgllcltliVeeclrrrvtrkhmilvvvttlcaiilggltwmdllralimlgdu^
pgyvlgiflrkltsretalmvigmamttvlsiphdlmefidgislglilllonvthfdntqvgtlalsltfi
mavlfVvtliplcrtsclqkqshwveitalilgaqalpvylmtlmkgaskr
NS2B
swplnegimavglvsllgsallkndvplagpmvagglllaayvmsgssadlslekaanvqwdemaditgsspiievkq dedgsfsirdieetnmitllvklalitvsglyplaipvtmtlwymwqvktqr
NS3
sgalwdvpspaaaqkatltegvyrimqrglfgktqvgvgihmegvfhtmwhvtrgsvichetgrlepswadvmdmi sygggwrlgdkwdkeedvqvlaiepglmpkhvqtkpglfktltgeigavtldfkpgtsgspiinrkgkviglygngvvt ksgdyvsaitqaertgepdyevdediirkkrltimdlhpgagktkrilpsivrealkirlrtli
yqtpavksehtgreivdlmchatfttrllsstrvpnynlivmdeahftdpssvaargyistrvemgeaaaifmtatppgatd p qsnspiediereiperswntgfdwitdyqgktvwfvpsikagndianck^
wttdisemganfragrvidprrclkpvistdgpervilagpipvtpasa
ahwtealanlldniytpegiiptlfgpereknqaidgefrlrgeqrktfVelmiTgdlp lsykvasagisy ernnqileenmeveiwtregekkklrpkwldarvyadpmalkdfl efasgrk
NS4A
sitldilteiaslptylssraklaldni\milhtterggkayqhalnelpesletlmlvallgamtagiflffh qgkgi aiavasgllwvaeiqpqwiaasiilefflmvllipepekqrtpqdnqliyviltiltiigliaa
NS4B
nemgliektktdfgfyqvktettildvd^asawtlyavattiltpmlrhtientsanlslaaianqaavlmglgkgwplhr mdlgvpllamgcysqvnpttliaslvmllvhyaiigpglqakatreaqkrtaagjmknptvdgitvidlepisydpk lgqvmllvlcagqlllmrttwafcevltlatgpvltlwegnpgxf nttiavstanifrgsylagaglafsliknaqtprr NS5
gtgttgetlgekwkrqlnsldrkefeeykrsgilevdrteaksalkdgskildiavsrgsskirwivergmvkpkgkvvdlg cgrggwsyymatllmvtevkgytkggpgheepipmatygwnlvklhsgvdvfykpteqvdtllcdigesssnptieeg rtlrvlkmvepwlsskpefcikvlnpymptvieeleklqrkliggslvrcplsmsthemywvsgvsgni lnrfttrhrkptyekdvdlgagtrsvstetekpdmtiigrrlqrlqeehketwhydqenpyrtwayhgsyeapstgsassm vng klltkpwdvipmvtqlamtdttpfgqqrvfkekvdtrtpqpkpgtimvmtttanwlwallgkkknprlctreef iskvrsnaaigavfqeeqgwtsaseavndsrfwelvdkeralhqegkcescvynmmgkrekklgefgrakgsraiwy mwlgarflefealgflnedhwfgrenswsgvegeglhrlgy^
aplihkilakaifkltyqnkwkvlrptpkgavmdiisrkd
pkglkervekwlkecgvdrlloinaisgddcwkplderfstsllflndmgkvrkdipqwepskgwknwqevpfcshh mkifmkdgrslwpcmqdeligrarisqgagwslretaclgkayaqmwslmvfhrrdl^
twsihahhqwmttedmlkvwnrvwiednpiimtdktpvh^
ml ligkeeyvdympvmkrysapfesegvl
In a further embodiment, this aspect of the invention extends to nucleic acid sequences which are at least 80% homologous with or identical to the sequences above relating to non-structural proteins. In further embodiments such sequences may be at least 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, 99.4% or 99.5% homologous with/identical to the sequences above relating to non-structural proteins.
Homologous nucleic acid sequences of this aspect of the invention encode functioning non-structural proteins.
In one embodiment, the non-structural proteins are from the same flavivirus as the prM protein. In a further embodiment, the non-structural proteins are from different a flavivirus to the prM protein.
The chimeric flavivirus of the invention further comprises a sequence encoding a flavivirus capsid (C) protein or a fragment thereof which encodes a functioning capsid protein. Preferably, the sequence encoding the capsid is a fourth nucleic acid sequence. Preferably it forms part of the single nucleic acid sequence which the other three nucleic acids form. Most preferably it is present at the furthest 5' end
(immediately preceding prM).
In a particular embodiment, a nucleic acid sequence encoding the flavivirus capsid (C) protein can be selected from any encoding the following protein sequences (or any having 90% identity): Dengue serotype 1 strain DENV-1/KH/BID-V2003/2006 GenBank: ACN42698.1 mmqrkkta sfhmlkramrvstvsqlakrfskgllsgqgpmklvm
gfkkeisnmlnimnrrkrsvtmllmlmptala
Dengue serotype 2 strain New Guinea C (prtotype strain) GenBank: AAC59275.1 mmqrkkamtpfnmlkrernrvstvqqltkrfslgmlqgrgplklfmalvaflrfltipptagi
frkeigrmlnilnrnTtagmiimliptvma
Dengue serotype 3 strain H87 (prtotype strain) GenBank: AAA99437.1
mnnqrkktgkpsirunlkivnirvstgsqlakrf^
kgfkkeisnmlsiinkrkktslclmmmlpatla
Dengue serotype 4 strain H241 (prtotype strain) GenBank: AAX48017.1
mnqrld vAappfhmlla mrvstpqglvkrfe^
keigrmlnilngrkrstmtllcliptama
Japanese Encephalitis virus strain Nakayama GenBank: ABQ52691.1
mtkkpggpgkrιrairιmlkrglprv lvgvkr mslldgrgpvrfvlalitffkft
ltsfkrelgtlidavnkrgrkqnkrggnegsimwlaslaviiacaga
Murray Valley Encephalitis virus strain MVE-1-51 Swiss-Prot: P05769.2
mskkpggpgkpr nmlkrgiprv^lvgvkrwmnlldgrgpirfvlallaffrftalaptk
hltsfkkelgtlidvvnkrgkkqkkrggsetsvlmli
Yellow Fever Virus strain 17D/Tiantan GenBank: ACN41908.1
msgrkaqgktlgvrimvrrgvrslsnkikqktkqign
kvkrwaslmrglssrkrrshdvltvqflilgmllmtgg
West Nile virus clone 33/G8; 34/F6 GenBank: AAA48498.2
mskJcpggpgknravmnlkrgmprglsliglla'amlslidgkgpirfvlallaffrftai
lsfkkelgtltsainrrstkqkkrggtagftillgliacaga
Langat virus GenBank: AAA02740.1
magkavlkgkgggpprraskvapl ktrqlrvqmpnglvlmrmlgvlwhaltgtarspvlkafwkvvplkqatlalrki krtvstlmvglhrrgsrrttidwmtpllitvmlgmclt
Kunjin virus strain MRM61C GenBank: BAA00176.1
msldcpggpgksravnmlkrgmprvlsltglkramlslidgrgptrfvlallaffrftaiaptravld
sfkkelgtltsainrrsskqkkrggktgiafmigliagvga Tick-borne Encephalitis virus strain Sofjin GenBank:
CAA30581 magkailkgkgggpprrvsketakktrqsrvqmpnglvlmrrnmgilwhavagtarspvlksi\v svplkqataalrkikkavstlmvglqrrgkrrsavdwtgwllvwllgvtla
Powassan virus strain LB GenBank: AAA02739.1
mmttskgkgggpprrklkvtanks atspmpkgfvlsrmlgilwha tgta p
vignlmqslhmrgrrrsgvdwtwifltmalrntmarna
The capsid can be from any flavivirus, preferably the same virus this from which the prM is from.
The chimeric flavivirus can further comprise a sequence that encodes for a protease recognition site at the C/prM protein junction that is the NS2B-NS3 protease recognition site. In addition, the chimeric flavivirus can comprise a sequence that encodes for a signalase cleavage site at the C/prM junction, wherein upstream of the C/prM junction comprises a sequence of a cleavage site from the flavivirus backbone or a sequence at least 80% identical thereof and downstream of the C/prM junction comprises a sequence of a cleavage site from the same virus as the prM sequence or a sequence 80% identical thereof. The chimeric flaviviruses defined above can be produced using techniques known in the art. For example, those described in Bray and Lai, 1991 (Proc Natl Acad Sci, 88: 10342-10346), Chen et al, 1995 (J Virol, 69: 5186-5190), Huang et al, 2000 (J Virol, 74: 3020-3028) and Pletnev and Men, 1998 (Proc Natl Acad Sci, 95: 1746-1751). The nucleic acid sequences of the present invention may be recombinant or provided as an isolate, in isolated and/or purified form.
Nucleic acids of the present invention can be readily prepared by the skilled person, for example using the information and references contained herein and techniques known in the art (for example, see Sambrook, Fritsch and Maniatis, "Molecular Cloning", A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, and Ausubel et al, Short Protocols in Molecular Biology, John Wiley and Sons, 1992), given the nucleic acid sequences and clones available. These techniques include (i) the use of the polymerase chain reaction (PCR) to amplify samples of such nucleic acid, e.g. from genomic sources, (ii) chemical synthesis, or (iii) preparing cDNA sequences. DNA encoding the polypeptide may be generated and used in any suitable way known to those of skill in the art, including by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system. Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers. Modifications to the sequences can be made, e.g. using site directed mutagenesis, to lead to the expression of modified peptide or to take account of codon preferences in the host cells used to express the nucleic acid.
The nucleic acid sequence of the invention can additionally comprise a promoter or other regulatory sequence which controls expression of the nucleic acid. Promoters and other regulatory sequences which control expression of a nucleic acid have been identified and are known in the art. It may not be necessary to utilise the whole promoter or other regulatory sequence. Only the minimum essential regulatory element may be required and, in fact, such elements can be used to construct chimeric sequences or other promoters. The essential requirement is, of course, to retain the tissue and/or temporal specificity. The promoter may be any suitable known promoter, for example, the human cytomegalovirus (CMV) promoter, the CMV immediate early promoter, the HSV thymidinekinase, the early and late SV40 promoters or the promoters of retroviral LTRs, such as those of the Rous Sarcoma virus (RSV) and metallothionine promoters such as the mouse metallothionine-I promoter. The promoter may comprise the minimum comprised for promoter activity (such as a TATA elements without enhancer elements) for example, the minimum sequence of the CMV promoter.
Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the nucleic acid sequence. Thus, a promoter nucleotide sequence is operably linked to a nucleic acid sequence if the promoter nucleotide sequence controls the transcription of the nucleic sequence. The polymerase chain reaction (PCR) procedure may be employed to isolate and amplify a nucleic acid sequence encoding a desired protein sequence. Oligonucleotides that define the desired termini of the nucleic acid sequence are employed as 5' and 3' primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases, to facilitate insertion of the amplified nucleic acid sequence into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).
As is well understood, homology at the amino acid level is generally in terms of amino acid similarity or identity.
Percent homology of sequences may be determined by visual inspection and
mathematical calculation. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways using publicly available computer software such as BLAST or ALIGN. For example, protein searches can be performed with the XBLAST program to obtain amino acid sequences homologous to protein molecules of the invention. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Also included within the scope of the present invention are homologous nucleic acid sequences which hybridises to a sequence in accordance with the first aspect of the invention under stringent conditions, a complement of said sequence or a nucleic acid sequence which is homologous to or would hybridise under stringent conditions to such a sequence but for the degeneracy of the genetic code, or an oligonucleotide sequence specific for any such sequence.
Stringent conditions of hybridisation may be characterised by low salt concentrations or high temperature conditions. For example, highly stringent conditions can be defined as being hybridisation to DNA bound to a solid support in 0.5M NaHP04, 7% sodium dodecyl sulfate (SDS), ImM EDTA at 65°C, and washing in O. lxSSC/ 0.1%SDS at 68°C (Ausubel et al eds. "Current Protocols in Molecular Biology" 1, page 2.10.3, published by Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, (1989)). In some circumstances less stringent conditions may be required. As used in the present application, moderately stringent conditions can be defined as comprising washing in 0.2xSSC/0.1%SDS at 42°C (Ausubel et al (1989) supra). Hybridisation can also be made more stringent by the addition of increasing amounts of formamide to destabilise the hybrid nucleic acid duplex. Thus particular hybridisation conditions can readily be manipulated, and will generally be selected according to the desired results. In general, convenient hybridisation temperatures in the presence of 50% formamide are 42°C for a probe which is 95 to 100% homologous to the target DNA, 37°C for 90 to 95% homology, and 32°C for 70 to 90% homology.
Compositions according to the invention may comprise, in addition to an active ingredient (i.e. the chimeric flavivirus of the invention), a pharmaceutically acceptable excipient, carrier, buffer stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration. In a preferred embodiment, the composition is an injectable composition.
The formulation may be a liquid, for example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-7.6, or a lyophilised or freeze dried powder.
For intravenous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, Lactated Ringer's injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
The composition may also be in the form of microspheres, liposomes, other microparticulate delivery systems or sustained release formulations for administration to certain tissues including blood. Suitable examples of sustained release carriers include semipermeable polymer matrices in the form of shared articles, e.g.
suppositories or microcapsules. Implantable or microcapsular sustained release matrices include polylactides (US Patent No. 3, 773, 919; EP-A-0058481) copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, Biopolymers 22(1): 547-556, 1985), poly (2-hydroxyethyl-methacrylate) or ethylene vinyl acetate (Langer et al, J. Biomed. Mater. Res. 15: 167-277, 1981 , and Langer, Chem. Tech. 12:98-105, 1982).
The composition/polypeptide is preferably administered to an individual in a
"therapeutically effective amount", this being sufficient to show benefit to the individual.
The composition can be formulated as a sterile aqueous solution containing between 102 and 108 infectious units per serotype in a dose volume of 0.1 to l.OmL to be administered intravenously. In a further embodiment, the invention includes a composition comprising a chimeric flavivirus comprising a first nucleic acid sequence encoding a DENV flavivirus prM protein, a second nucleic acid sequence encoding at least one epitope of a DENV virus envelope protein and a third sequence encoding the non-structural flavivirus proteins NS1 , NS2A, NS2B, NS3, NS4A, NS4B and/or NS5, wherein the DENV prM protein comprises a mutation, addition or deletion. The mutation, addition or deletion is such that the prM protein does not contribute significantly to the immunogenic response elicited by the composition upon vaccination of a subject. The second aspect of the invention relates to a polypeptide encoded by the chimeric flavivirus defined above.
The third aspect of the invention relates to the composition of the invention for use in inducing an immunological response to DENV virus. Thus, the composition is for use as a vaccine against DENV virus.
The fourth aspect of the invention relates to the use of the composition of the invention in manufacturing a vaccine for the treatment of DENV virus. The fifth aspect of the invention relates to a method of treating a subject comprising administering a composition of the first aspect of the invention to a subject.
The composition comprises a live attenuated virus as defined above. In a preferred embodiment of the invention, the composition of the invention can be administered to a subject that has not been infected with DENV virus as a preventative measure against an initial DENV infection.
The composition can also be administered after a subject has been infected with DENV virus to protect the subject against secondary infections.
The term 'treatment' is used herein to refer to any regimen that can benefit a human or non-human animal. In one embodiment, the human or non-human animal is in need of such treatment.
More specifically, treatment includes "therapeutic" and "prophylactic" and these types of treatment are to be considered in their broadest context. Accordingly, therapeutic and prophylactic treatment includes amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylactic" may be considered as reducing the severity of or preventing the onset of a particular condition. "Prophylactic" also includes preventing reoccurrence of a particular condition in a patient previously diagnosed with the condition. "Prophylactic" does not necessarily mean that the subject will not eventually contract a disease condition. "Therapeutic" may also reduce the severity of an existing condition and does not necessarily imply that a subject is treated until total recovery. The composition of the invention may be administered to a patient in need of treatment or that might benefit from such treatment via any suitable route.
Route of administration may include; parenterally (including subcutaneous, intramuscular, intravenous, by means of, for example a drip patch), some further suitable routes of administration include (but are not limited to) oral (including buccal and sublingual), nasal, topical, infusion, intradermal, intraperitoneally, intracranially, intrathecal and epidural administration.
The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated and the precise nature of the form of the vaccine to be administered. Prescription of treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and at the discretion of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners.
Single or multiple vaccinations of the compositions can be carried out with dose levels and patterns being selected by the treating physician. The sixth aspect of the invention relates to a virus-like particle (VLP) comprising a non-DENV flavivirus prM protein and at least one epitope of a DENV virus envelope protein (preferably the complete protein) and sequences encoding such particles. The VLP may optionally contain a capsid protein, but this is not essential.
VLPs refer to structures that resemble a virus in terms of proteins but that are not infectious. VLPs in accordance with the invention do not carry genetic information encoding for the proteins of VLPs. In general, VLPs lack a viral genome and are, therefore, non-infectious.
Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by a person who is skilled in the art in the field of the present invention.
Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.
The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is made to a number of Figures in which:
FIGURE 1 illustrates neutralisation and ADE of anti-E and anti-prM antibodies.
Culture supernatants from 20 anti-E and 20 anti-prM cell lines, all of which were specific to DENV2 and cross-reactive with other DENV serotypes, were assayed in neutralization and ADE assays to DENV 2 strain 16681. Neutralisation was performed by focus forming assay on Vero cells using a 1 :2 dilution of supernatant while ADE was performed using a 1 : 100 dilution on U937 cells and infection read by FACS using 4G2 (A and B). Control experiment showing roughly equal levels of infection of monocytes in the presence of the 6 anti-prM monoclonal antibodies when assayed by intracellular staining for either DENV envelope (4G2) or DENV non-structural protein- 1 (2G6). Two irrelevant human antibodies are also shown (C). Histograms showing higher amplitude staining with 4G2 (anti-E) compared to 2G6 (anti-NSl), 4G2 was selected for the intracellular staining assays (D); FIGURE 2 illustrates specificity of human antibodies (n=30I).
Western Blot of infected cell lysates (non-reduced) showing reactivity of antibodies with DENV NS 1 , E and prM proteins (A). Cross-reactivity of human mAb within the DENV serotypes (B-D) or between the DENV group and JEV (E-G);
FIGURE 3 illustrates Anti-prM responses during primary DENV infection.
Western blots of C6/36 cell lysates infected with DENVl -DENV4 and probed with primary immune DENV serum (A). Plasma diluted 1 in 100 from five cases each of DENVl , DENV2 and DENV3 were analysed by Western blot for anti-prM against the four DENV serotypes and reactivity was then scored (B);
FIGURE 4 illustrates reactivity of human anti-prM monoclonal antibodies.
Reactivity of the 6 anti-prM antibodies to reduced (R) and non-reduced (N) DENV infected cell lysates showing loss of reactivity on reduced Western blots (A). To show reactivity to the pr peptide, western blotting of culture supernatant (non- heat/nonreduced) from DENV (B) and mock (C) infected cells was preformed. Non reduced gels were run in loading buffer lacking 2-mercaptoethanol which were also non-heat treated.
FIGURE 5 illustrates partial neutralisation but potent enhancement by human anti-prM monoclonal antibodies. Neutralisation assays (A) and enhancement assays (B) were performed with the six human anti-prM mAbs (clones 3-147, 58/5, 2F5, 2G4, 5F9 and 135.3), murine anti-E mAb (4G2) and purified Ig from pooled DENV convalescent serum (PCS) and pooled non DENV immune serum (PND) were used as controls; meaniSE from three independent experiments;
FIGURE 6 illustrates that anti-prM can rescue infectivity in virus containing high densities of prM. DENV produced in the presence of NH4C1. The density of uncleaved prM and E were measured by ELISA and expressed as the prM:E ratio (A). Infectivity was determined in Vero cells, expressed as FFU, and the amount of total virus- eauivalent particles was calculated based upon the concentration of E protein measured by a sensitive sandwich ELISA. Data is presented as virus-equivalent particles/FFU ratio (B). Neutralisation assays with purified human anti-prM mAb (3-147) (C).
Enhancement assays of U937 cells read out by FACS based intracellular staining for DENV antigens (4G2) using either a constant amount of infectious virus (D) or constant number of virus particles (E); mean±SE from three independent experiments;
FIGURE 7 illustrates infectivity of virus produced in furin deficient LoVo cells.
LoVo produced virus has a high prM:E ratio measured by ELISA (A). Silver stained gel (non-reduced) of DENV immunoprecipitated with 4G2 showing absence of M in LoVo cells and reduction in M in virus produced in the presence of ammonium chloride (B). Enhancement of infection of LoVo produced virus by anti-prM antibody (C). Infection of U937 cells was detected by FACS with mAb 4G2 at 3 days post infection; and
FIGURE 8 illustrates the roles of anti-prM on neutralisation and enhancement of DENV infection of PBMC. PBMC were infected with DENV2 in the presence of human anti-prM mAbs, at 24hrs DENV Ag was stained intracellularly (4G2) and detected by flow cytometry in gated monocytes (A). PCS, PND and irrelevant human mAb were used as control. The density of prM on DENV from C6/36 cells and DC were detected by ELISA and presented as prM:E ratio (B). Neutralisation (C) and antibody-dependent enhancement of infection (D), performed on Vero and U937 cells respectively, of DENV generated from either C6/36 cells or DC in the presence of PCS or anti-prM mAb (3-147); mean±SE from three independent experiments.
EXAMPLES
Material and Methods
Samples
Blood samples were taken from patients after consent and approvals from the ethical committee of Siriraj, Khon Kaen and Songkhla hospitals, Thailand. The study was also approved by the Riverside Ethics Committee in the UK. Acute DENV infection was identified by RT-PCR-based DENV gene identification or DENV-specific IgG and IgM capture ELISA. Disease severity was classified according to the World Health Organization criteria. Of the patients enrolled in the study, 1 patient was classified as DF, 6 as DHF. Blood samples were collected in heparin (BD). PBMC were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation and cryopreserved until tested. Characteristics of DENV-infected cases enrolled in the study are summarised in table S 1.
Table SI. Summary of DENV-infected patients enrolled in the study
Serotype No. of positive
Day after
Patient ID Severity of BCL
defervescence
infection (n= 301)
1 DF DENV2 19 19
2 DHF1 DENV2 23 54
3 DHF1 DENV2 17 53
4 DHF1 Unknown 15 76
5 DHF1 DENV1 17 69
6 DHF2 DENV4 24 20
7 DHF3 DE V2 20 10
Cells and Antibodies
C6/36, a cell line derived from the mosquito Aedes albopictus was cultured in Leibovitz L-15 medium supplemented with 10% heat-inactivated foetal bovine serum (FBS),
0.26% tryptose phosphate broth (TPB), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-Glutamine at 28°C. For endotoxin-free conditions, cells were grown in the absence of TPB. Vero, a cell line derived from the kidney of African green monkeys and U937, a human monocytic cell line, were grown in MEM and RPMI1640, respectively. The media were supplemented with 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-Glutamine in a 37°C humidified 5% C02 incubator. Monocyte-derived dendritic cells (DC) were prepared as previously described. LoVo cells were cultured in Nutrient mixture (Ham) F12 medium containing 20% FBS. Conjugated antibodies against human or mouse Ig (DAK.0) were used. Pooled convalescent DENV hyperimmune human serum (PCS) (hemagglutination titre > 1/25600), pooled non-DENV immune serum (PND) (hemagglutination inhibition titre and anti-DENV Ab ELISA negative) and mouse anti-DENV envelope, 4G2, were kindly provided by AFRIMS, Thailand. NS1-F3, 2G6 and 1H10 are anti-NSl and anti- prM mAb, respectively.
Virus stock
DENV serotype 1 (Hawaii), serotype 2 (16681), serotype 3 (H87) and serotype 4 (H241) were propagated in C6/36 cells and virus supernatant was collected and stored at -80°C. The DENV stock propagated from C6/36 and MDDC's were free from endotoxin and mycoplasma detected by Limulus amebocyte lysate assay (Whittaker M.A.) and PCR using the mycoplasma detection set (TAKARA BIO INC),
respectively. For poorly infectious DENV, C6/36 cells were infected with DENV2. Four days after infection, culture medium was replaced by fresh L-15 containing 1.5% FBS and 0.26% TPB with 10 or 20 mM NH4C1 for 2 hrs and the medium was then replaced again. At 24 hrs after the medium containing NH4C1 was added, virus particles were harvested and precipitated 2 by 10% PEG 8000. Fully immature virus was produced on LoVo cells as previously described. Briefly, virus was produced by infecting LoVo cells with DENV2 strain 16681 at MOI of 10 and virus was harvested at 2 days.
Focus forming assay
The titres of virus were determined by a focus forming assay on Vero cells and expressed as focus-forming units (FFU) per ml. Briefly, virus was serially diluted and incubated with Vero cells for 2 hrs at 37°C. The monolayers were then overlaid with 1.5% carboxymethylcellulose and incubated at 37°C for 3 days. Virus foci were stained with anti-E antibody (4G2) followed by peroxidase-conjugated anti-mouse Ig and visualized by the addition of DAB substrate.
Generation of DENV-specific human monoclonal Abs
DENV-specific human mAb's were generated as previously described (E. Traggiai et al, Nat Med 10, 871 (2004)). Briefly, IgG+ memory B cells were positively selected from PBMC through magnetic sorting using MACS CD22 microbeads (Miltenyibiotec) followed by depletion of IgA, IgD and IgM expressing cells by FACS-sorting. Isolated IgG+ memory B cells were then transformed with EBV and cultured in RPMI containing 10% FCS, 2.5 ug ml CpG, 10 ng/ml, IL-2, 30 ug/ml holo-Transferrin and irradiated allogeneic PBMC. After 2 weeks, culture supematants were screened for anti- DENV specific antibodies. Human EBV-transformed B cells producing anti-DENV antibodies were then cloned by limiting dilution. All human monoclonal antibodies used in this study are summarised in table S4.
Table S4. Characteristics of the human antibodies
Monoclonal Isotype Specificity
Immunoblot
antibody (ELISA) DENV1 DE V2 DENV3 DENV4 JEV
IgGl
3-147 prM + + + + - (Kappa)
IgGl
58/5 prM + + + + - (Lamda)
IgGl
2F5 prM + + + + - (Lamda)
IgGl
2G4 prM + + + + - (Kappa)
IgGl
5F9 prM + + + + - (Lamda)
IgGl
135.3 prM + + + + - (Kappa)
Detection of DENV-specific human Abs by ELISA
Mixtures of all four DENV serotypes were captured onto a MAXISORP immunoplate (NUNC) coated with mouse anti-E antibody (4G2) or anti-NSl antibody (NS1-F3). DENV captured wells were then incubated with B cell line (BCL) culture supematants followed by alkaline phosphatase (AP)-conjugated anti-human IgG Abs. The reaction was visualized by the addition of PNPP substrate. The reaction was stopped with NaOH.
ELISA for prM:E ratio and the number of E molecules of poorly infectious DENV To determine the prM:E ratio and to estimate the number of virus-equivalent particles, DENV was captured onto plates coated with anti-E antibody (4G2) and then detected " :th human anti-prM or E mAb. Recombinant E protein (a gift from Dr. Jonathan Grimes, University of Oxford, UK), produced in Sf9 cells was used to plot a standard curve to calculate the concentration of E. The relative concentration of prM was expressed as the OD ratio of prM:E measured by ELISA and the number of virus equivalent particles estimated assuming each particle would contain 180 copies of the E protein.
Western blot and Dot blot analysis
For western blot analysis, lysates from DENV infected C6/36 cells treated with 1% triton X-100 in PBS were run on 12% SDS polyacryramide gels without heating under nonreducing conditions and electroblotted onto nitrocellulose membranes (Amersham). For dot enzyme immunoassay, culture supematants from mock, DENV or JEV infected C6/36 cells were dotted onto nitrocellulose membranes. The membranes were then blocked with PBS containing 5% skimmed milk and probed with BCL supematants or plasma samples diluted 1 :100 followed by peroxidase-conjugated anti-human IgG Abs. Finally, membranes were developed with enhancement chemiluminescence substrate (Amersham).
Focus reduction neutralisation test (FRNT)
Neutralizing activity of DENV-specific Abs was determined by the focus reduction neutralisation test (FRNT). Briefly, serially diluted antibody was mixed with an equal volume of virus and incubated for 1 hr at 37°C. The mixtures were then transferred to Vero cell monolayers followed by the focus forming assay described above. Antibody-dependent infection enhancement assay
Serially diluted antibody was incubated with an equal volume of virus for 1 hr at 37°C then transferred to U937 cells and incubated at 37°C for 4 days. Supematants were then harvested and viral titre assessed by a focus forming assay as described above.
Alternatively, after 24 hrs, U937 was fixed and permeabilised in 4% paraformaldehyde and 0.5% saponin. DENV antigens were then stained intracellularly with anti-DENV
(4G2) followed by phycoerythrin-conjugated anti-mouse IgG Abs and analysed by flow cvtometry. In preliminary experiments, to check that intracellular staining with anti-E antibody- 4G2 represented DENV infection we compared 4G2 staining with 2G6 an antibody to NSl (Fig. 1C). Staining of infected monocytes was similar, but as 4G2 showed a larger shift in staining (Fig. 1 D) this was chosen for use in further assays.
DENV infected PBMC and FACS analysis PBMC were infected with endotoxin-free DENV in the presence of different concentrations of antibodies for 24 hrs. Cells were then fixed, permeabilised, and stained for DENV antigen by intracellular staining as described above.
DENV immunoprecipitation
The supernatants from DENV2 infected culture were pre-cleared with protein A- agarose beads for 1 hr at 4 °C. After centrifugation, supernatant was incubated with 10 μg of 4G2 for 2 hrs at 4 °C. Protein A-agarose beads were then added and incubated for 1 hr at 4 °C. After washing, proteins were eluted with non-reducing loading buffer and run on a 15% 4 SDS-polyacrylamide gel followed by silver staining according to the manufacture protocol (SilverQuest staining kit, Invitrogen).
Results
As set out above, B cells from seven DENV infected individuals (Table SI) were used to produce human mAb. Culture supernatants were screened against structural antigens using whole vims and against non-structural protein 1 (NS l) by ELISA. 301 of 3020 cell lines screened positive; 73% reacted to the whole virus ELISA for structural antigens and 27% to NSl . Positive supernatants were tested for reactivity to specific DENV antigens by non-reducing Western blot (Fig. 2A). When the
supernatants reacting to whole DENV were tested, 78% gave a positive signal by Western blot, all of these reacted to either E or prM with no reactivity to capsid.
Interestingly, the anti-prM response was dominant (60% CI 67.3-52.2%) compared with the response to E (40%); subgroup analysis of each of the individual cases is shown in table S2. Table S2a . Antibody responses against prM and E
Patient ID Serotype anti-structural (n) anti-prM(n) anti-E(n) %prM
1 2 16 1 1 5 68.8
2 2 30 12 18 40.0
3 2 33 23 10 69.7
4 unknown 43 26 17 60.5
5 1 44 29 15 65.9
6 4 3 0 3 0.0
7 2 3 2 1 66.7 total 172 103 69 59.9
Table S2b. Antibody cross reactivity among DENV serotypes
prM NSl
Patient
Serotype full* partial** specific*** full partial specific fall partial specific ID
1 2 9 2 0 5 0 0 2 1 0
2 2 1 1 0 1 18 0 0 4 4 8
3 2 23 0 0 10 0 0 12 3 1
4 unknown 24 2 0 12 4 1 6 10 2
5 1 28 0 1 13 2 0 7 4 1
6 4 0 0 0 2 1 0 6 8 1
7 2 2 0 0 1 0 0 1 0 0
♦cross reacts with all 4 serotypes
** cross reacts with 2-3 serotypes
*** reacts only one serotype
Table S2c Antibody cross reactivity between DENV and JEV
prM NSl
Patient
Serotype DENV+JEV* DENV** DENV+JEV DENV DENV+JEV DENV ID
1 2 1 10 2 3 0 3
2 2 0 12 17 1 2 14
3 2 1 22 8 2 1 15
4 unknown 0 26 9 8 0 18
5 1 1 28 6 9 0 12
6 4 0 0 2 1 3 12
7 2 0 2 0 1 0 1
♦cross reacts between DENV and JEV
** no cross-reaction to JEV
We next assessed the serotype specificity of the human antibodies by dot blot against the E. Traggiai et al, Nat Med 10, 871 (2004) four viral serotypes, which showed a divergence in the cross reactivity between the anti- NSl and structural (anti-E and anti- prM) groups of antibodies. Half of the anti-NSl showed limited cross reactivity among DENV, while most of the structural antibodies showed full cross reactivity against all virus serotypes (Fig. 2B-D). As these antibodies were made from secondary cases of DENV we investigated primary anti-prM responses. Western blotting of DEN V infected cell lysates demonstrate that cross-reactive anti- prM responses are made during the primary infection (Fig. 3) although as has been reported before, the anti-prM response is amplified following secondary infection. Finally, we tested cross reactivity to the related flavivirus Japanese encephalitis virus (JEV) which co circulates with DENV in some parts of SE Asia (Fig. 2E-G).
Interestingly, only 3% of the anti-prM antibodies cross reacted with JEV in contrast to the antibodies recognizing envelope which showed 64% cross reactivity. The relative specificity of anti-prM to the DENV may reflect the lower sequence conservation between prM sequences (35% DENV vs. JEV) compared with E (50%); a comparison of sequence conservation among other members of the family flaviviridae can be found in Table S3.
Table S3. Amino acid sequence homology among members of Flaviviridae family
DENVl
prM E NSl
DENV l 100 100 100
DENV 2 73 69 73
DENV 3 80 77 78
DENV 4 65 63 69
JEV 35 50 51
KUN 33 50 50
SLE 35 49 53
TBE 22 38 37
WN 34 51 51
YF 33 42 42
Six monoclonal anti-prM mAbs were produced, Western blotting showed that at least 5/6 react with the cleaved pr peptide and reactivity was lost to reduced antigen implying that they recognize conformational epitopes (Fig. 4). In general, the anti-prM antibodies were unable to completely neutralise infection (Fig. 5A). Instead, neutralisation plateaued between 10 and 60% and the partial neutralisation was largely cross-reactive among the four virus serotypes, the only exceptions being mAb
5F9 and 135.3 which showed almost 100% neutralisation of DENV4 at high antibody concentrations. This partial neutralisation was in contrast to results seen with pooled convalescent DENV serum (PCS) or anti-envelope monoclonal antibodies, where neutralisation approached or reached 100%. Next, we performed ADE assays using U937 cells as targets where virus was preincubated with an increasing titre of antibody before addition to the Fc-receptor- bearing cells. Enhancement of infection was seen with all six human anti-prM monoclonal antibodies with a peak of nearly 105 fold (Fig. 5B) which is consistent with a report of ADE with murine anti-prM mAb.
To ascertain whether the results with these six anti-prM mAbs were representative we tested the enhancing and neutralising capacity of a further 20 anti-prM and 20 anti-E cell lines as well as two irrelevant human antibodies (Fig. 1A&B). None of the anti- prM antibodies showed a high level of neutralisation (19/19 <80%), whereas 12/20 anti-E showed >90% and 6/20 showed 100% neutralisation. All the anti-prM antibodies showed ADE of 10-800 fold, while the anti-E antibodies showed more variable ADE.
The failure of anti-prM antibodies to fully neutralise DENV viruses with a clear plateau in the response was puzzling and suggested the virus may exist in two populations, one susceptible to neutralisation and another not. Cleavage of prM during viral maturation is believed to be a prerequisite for viral replication, which is exemplified by the very low infectivity of DENV and tick borne encephalitis viral particles with wholly uncleaved prM. In many virus preparations prM cleavage is incomplete and cyro- electron microscopy yields particles that contain both full length prM and processed M protein, suggesting that a distribution of virus maturation may be present in virus cultures. The demonstration here that the human anti-prM antibodies can show partial neutralisation implies that some prM containing particles remain infectious. The propensity towards incomplete cleavage of prM in DENV leads to two interesting predictions, first that the density of prM at the surface of the virus may not be high enough to allow full neutralisation with most anti-prM antibodies. Instead, viruses with low levels of prM may be susceptible to ADE. Second, viruses which are inherently noninfectious by virtue of displaying a high density of prM may be rendered infectious by ADE. To investigate the effect of prM cleavage on neutralisation and enhancement, virus was produced in cells cultured in the presence of ammonium chloride to raise intracellular pH and reduce the efficiency of furin cleavage. ELISA assays were performed to measure E and prM in the virus preparation; the E assay was calibrated by plotting a standard curve using recombinant E protein produced in Sf9 cells. A measure of the number of potential virus particles (virus equivalent particles) was derived assuming each particle contained 180 copies of E and the relative density of uncleaved prM, calculated by the ratio of prM:E. The prM:E ratio was increased by roughly 40% and 80% when virus was cultured in 10 mM and 20 raM ammonium chloride respectively (Fig. 6A). As expected, the infectivity of virus produced in the presence of NH4C1 was markedly reduced from 46 to 555 virus-equivalent particles/FFU (Fig. 6B). Although infectivity was reduced, infectious virus produced under each condition remained partially susceptible to neutralisation as before, and the titration curves for virus produced in 0, 10 and 20 mM NH4C1 were similar (Fig. 6C).
We next tested enhancement of these viruses using either a constant amount of infectious virus i.e. fixed focus forming units (Fig. 6D) or a constant amount of virus equivalent particles (Fig 6E). These results show that the relatively poorly infectious virus cultured in the presence of NH4C1 can be rendered much more infectious in the presence of enhancing anti-prM antibodies and indeed can be restored nearly to the level of control virus (Fig. 6E). These results were further exemplified using virus produced in LoVo cells that lack functional furin and therefore produce virus with very low levels of cleaved prM (Fig. 7). Virus produced in LoVo cells as expected had a high prM:E ratio and very low infectivity (< 10x10-5 ffu/virus equivalent particle) that could be enhanced in the presence of anti-prM antibody.
Three populations of DENV virus appear to be produced: first, a population containing relatively high levels of prM that are inherently non-infectious but that can be made infectious in the presence of enhancing anti-prM antibodies; second, a population with an intermediate density of prM at the surface that can infect, but are susceptible to neutralisation at high antibody titre; third, a population with low or absent prM at the surface that would not normally be susceptible to neutralisation. To test the relative roles of the anti-prM antibodies in neutralisation and enhancement of primary cells we looked at human monocytes, which are thought to be a major site of virus replication in vivo. Monocytes can be infected in the absence of antibody and as they express Fc receptors infection can be increased by ADE. To our surprise, human anti-prM mAbs failed to show any neutralisation activity on primary monocytes and instead, even at concentrations of antibody as high as 30 μg/ml, enhanced infection from 20% to 70% (Fig. 8A) over a large range of antibody concentration.
Virus was generated in the insect cell line C6/36, which is known to cleave prM inefficiently, and the results we have obtained are therefore analagous to the first encounter with DENV i.e., insect-produced virus following a bite from an infected mosquito. Finally, we set out to determine whether virus produced in primary mammalian cells contained non-cleaved prM and whether anti-prM had any enhancing capacity on such virus. Virus was produced in immature dendritic cells (DC) where cleavage of prM was more efficient than in the insect cell culture, but still not complete (Fig. 8B). As with insect-produced virus, the anti-prM antibodies were unable to fully neutralise DC-produced virus with a clear plateau in efficacy (Fig. 8C), but were still able to enhance infection, although to a lesser degree (Fig. 8D).

Claims

Claims
1. A composition comprising a first nucleic acid sequence encoding a non-DENV flavivirus prM protein, a second nucleic acid sequence encoding at least one epitope of a DENV virus envelope protein and a third nucleic acid sequence encoding the nonstructural flavivirus proteins NSl, NS2A, NS2B, NS3, NS4A, NS4B and/or NS5 and optionally a fourth nucleic acid sequence encoding a flavivirus capsid protein.
2. The composition of claim 1, wherein the third nucleic acid encodes non- structural proteins from Yellow Fever virus, Japanese Encephalitis virus and/or DENV virus or from attenuated versions of these viruses.
3. The composition of claim 1, wherein the second nucleic acid encodes an epitope from DENV virus serotypes 1, 2, 3 and 4.
4. A chimeric polypeptide encoded by the sequences defined in claim 1.
5. The composition of any one of claims 1 to 3 and 8 for use in inducing an immunological response to DENV virus.
6. Use of the composition of any one of claims 1 to 3 and 8 in the manufacture of a vaccine for treating DENV virus.
7. A method of treating a subject comprising administering the composition of any one of claims 1 to 3 and 8 to a subject.
8. A virus-like particle comprising a non-DENV flavivirus prM protein and at least one epitope of a DENV virus envelope protein
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WO2014016362A1 (en) 2012-07-24 2014-01-30 Sanofi Pasteur Vaccine compositions for prevention against dengue virus infection
WO2014016360A1 (en) 2012-07-24 2014-01-30 Sanofi Pasteur Vaccine compositions
EP3932422A1 (en) 2012-07-24 2022-01-05 Sanofi Pasteur Vaccine compositions for the prevention of dengue virus infection
WO2014083194A1 (en) 2012-11-30 2014-06-05 Sanofi Pasteur Methods for inducing antibodies
WO2019069130A1 (en) 2017-10-05 2019-04-11 Sanofi Pasteur Compositions for booster vaccination against dengu

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