WO2021262095A1 - Vaccin viral chimerique - Google Patents

Vaccin viral chimerique Download PDF

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Publication number
WO2021262095A1
WO2021262095A1 PCT/SG2021/050353 SG2021050353W WO2021262095A1 WO 2021262095 A1 WO2021262095 A1 WO 2021262095A1 SG 2021050353 W SG2021050353 W SG 2021050353W WO 2021262095 A1 WO2021262095 A1 WO 2021262095A1
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polynucleotide
nucleic acid
vacdz
virus
sequence
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PCT/SG2021/050353
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English (en)
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Jang Hann Justin CHU
Wei-Xin CHIN
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National University Of Singapore
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • 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
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24241—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24243—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24241—Use of virus, viral particle or viral elements as a vector
    • C12N2770/24244—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24261—Methods of inactivation or attenuation
    • C12N2770/24262—Methods of inactivation or attenuation by genetic engineering
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates generally to the field of immunology.
  • the specification teaches, in particular, an isolated polynucleotide comprising a) a tetracycline -responsive promoter; and b) a nucleic acid sequence encoding a live attenuated chimeric virus.
  • Dengue is a prevalent mosquito-borne flavivirus causing significant human disease ranging from dengue fever to life-threatening dengue hemorrhagic fever/dengue shock syndrome. It is estimated that 2.5 billion people are at risk of dengue virus infection with 50-100 million cases of dengue fever annually causing approximately 25,000 deaths (predominantly among children). Japanese encephalitis virus accounts for up to 50,000 cases of encephalitis in humans annually (with case fatality rates of about 25%). Yellow fever virus causes a wide spectrum of disease ranging from mild symptoms to kidney/liver failure and hemorrhaging of the gastrointestinal tract.
  • Zika virus Zika virus
  • Zika virus is an emerging mosquito-borne pathogen from the genus of Flavivirus. It is typically spread by the Aedes aegypti and Aedes albopictus mosquitoes, although past outbreaks have also been linked to the Aedes hensilli mosquito. In the past two decades, ZIKV first emerged during smaller outbreaks in several Pacific islands, before it emerged in the Americas to cause a larger outbreak. Between January 2015 and May 2017, ZIKV is estimated to have caused an estimated 8.5 million symptomatic infections in Brazil alone.
  • ZIKV infection is reported to be asymptomatic in a majority of patients, with the reported asymptomatic rate varying from 29% to 82%, and with 80% being the commonly cited figure.
  • Symptomatic ZIKV infection typically presents as an acute and self-limiting febrile disease, with symptoms such as maculopapular rash, joint pain, conjunctivitis.
  • viable ZIKV can be recovered from patient semen and serum weeks after the acute phase of the infection, indicating that ZIKV can also cause a persistent infection. During this persistent phase ZIKV remains transmissible through sexual contact. ZIKV infection is also associated with other severe complications.
  • Flaviviruses share a common genome organisation, consisting of a positive sense single-stranded RNA genome that encodes for a single ORF that is flanked by 5' and 3' untranslated regions (5' UTR and 3'UTR).
  • the ORF encodes for a polyprotein that is processed by host and viral proteases into the capsid, premembrane and envelope structural proteins (cap, prM and Env), as well as 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).
  • the flavivirus structural proteins are functionally interchangeable to a certain extent, and it is possible to construct viable chimeric viruses which express the heterologous structural proteins of another flavivirus or other viruses.
  • an isolated polynucleotide comprising: a) a tetracycline-responsive promoter; and b) a nucleic acid sequence encoding a live attenuated chimeric virus, wherein the tetracycline-responsive promoter is positioned immediately adjacent to the N terminus of the 5’- noncoding region of the nucleic acid sequence encoding the live attenuated chimeric virus.
  • an expression construct comprising a polynucleotide as defined herein.
  • a vector comprising a polynucleotide as defined herein.
  • a chimeric virus encoded by a polynucleotide as defined herein, an expression construct as defined herein or a vector as defined herein.
  • an immunogenic composition comprising a polynucleotide as defined herein, an expression construct as defined herein or a vector as defined herein.
  • a method of modulating an immune response in a subject comprising administering a therapeutically effective amount of a vector as defined herein or an immunogenic composition as defined herein to the subject.
  • Disclosed herein is a method of preventing or treating a viral infection in a subject, the method comprising administering a therapeutically effective amount of a vector as defined herein or an immunogenic composition as defined herein to the subject.
  • FIG. 1 Genomic and plasmid map of chimeric Zika/dengue virus VacDZ.
  • A Genomic map of VacDZ. 5' & 3'UTR: untranslated regions. Protein coding regions: C, capsid protein; prM, premembrane protein; Env, envelope protein; NS1 to NS5, non-structural proteins 1 to 5. ZIKV derived sequences are shown in colour.
  • B Premembrane protein signal sequence at the capsid- premembrane junction. The C-terminus of the capsid protein contains the premembrane signal sequence, and in VacDZ this signal sequence is ZIKV derived.
  • C Plasmid map of pVacDZ (not drawn to scale). The plasmid carries the cDNA sequence of VacDZ, flanked by promoter sequences necessary for DNA-launch.
  • TRE-minCMV tetracycline regulatory element fused to minimal CMV promoter.
  • HDVr hepatitis D virus self-cleaving ribozyme.
  • SV40-polyA SV40 polyadenylation sequence.
  • D The DNA sequence of the junction between the TRE-minCMV promoter and the cDNA sequence of VacDZ. The small letters represent the 3' end of the promoter sequence. The capital letters represent the 5' end of the VacDZ cDNA sequence, with the underlined A representing the putative transcription start site.
  • FIG. 1 Immunofluorescence assay for ZIKV-Env expression.
  • BHK-21 cells were infected with ZIKV, DENV2- 16681, or VacDZ. The cells were fixed and immunofluorescence assay was used to analyse expression of Flavivirus NS1 protein (red), or ZIKV envelope protein (green). Host nuclei were stained with DAPI (blue).
  • Figure 3 Plaque formation and temperature sensitivity assay. VacDZ was investigated for markers of attenuation in cell culture.
  • A Plaque formation assay in BHK-21 cells for VacDZ and parental DENV2- 16681 to investigate small plaque phenotype. Incubation time for both viruses was six days.
  • FIG. 4 Survival curve for neurovirulence and pathogenicity studies in mice.
  • B Pathogenicity studies in adult AG129 mice.
  • ZIKV neutralising antibody titres in vaccinated AG129 mice 5-8 weeks old AG129 mice were inoculated intraperitoneally with the indicated vaccine or control and then boosted four weeks later with the same dose of their respective vaccine or control. Four weeks after boosting mouse serum was harvested. ZIKV neutralising antibody titres were determined using PRNT, and is defined as the highest serum dilution that reduced the ZIKV plaque count by at least 50% in three technical replicates. Each point represents the neutralising antibody titre for one mouse.
  • FIG. 6 ELIspot assay of T helper cell response. 5-8 weeks old AG129 mice were inoculated intraperitoneally with the indicated vaccine or control and then boosted four weeks later with the same dose of their respective vaccine or control. Four weeks after boosting the mouse splenocytes were harvested. IFNy-ELIspot was used to analyse the Thl response, and IL-4- ELIspot was used to analyse the Th2 response. Each point represents one mouse and is the mean spot forming unit (SFU) count of three technical replicates. Magenta circles: activated with ZIKV, black squares: activated with RPMI medium control.
  • SFU mean spot forming unit
  • FIG. 7 Survival curve for protective immunity studies in AG129 mice. 5-8 weeks old AG129 mice were inoculated intraperitoneally with the indicated vaccine or control. Four weeks after vaccination, they were challenged with a lethal dose of 10 s PFU of ZIKV per mouse. The mice were kept for four weeks and observed daily for clinical symptoms and euthanised when they reached a humane endpoint.
  • FIG. 8 ZIKV neutralising antibody titres in vaccinated AG129 mice. 5-8 weeks old AG129 mice were inoculated intraperitoneally with the indicated vaccines (or their respective control). Four weeks after vaccination, they were challenged with a lethal dose of 10 s PFU of ZIKV per mouse. The mice were kept for four weeks and observed daily for clinical symptoms and euthanised when they reached a humane endpoint. Four weeks after lethal challenge, serum was harvested from the surviving mice. ZIKV neutralising antibody titres were determined using PRNT, and is defined as the highest serum dilution that reduced the ZIKV plaque count by at least 50% in three technical replicates. Each point represents the neutralising antibody titre for one mouse.
  • N numbers are the number of mice surviving four weeks after lethal challenge.
  • Figure 9 Construction of chimeric dengue/Zika virus VacDZ infectious clone.
  • the infectious clone of VacDZ called pVacDZ, was constructed using PCR, fusion PCR and conventional molecular cloning techniques.
  • A, B, C, D, E and F indicate PCR amplicons that were amplified using primers, fusion PCR primers or site-mutagenesis primers.
  • the PCR amplicons were fused by fusion PCR to ultimately form amplicon ABCDEF, which was cloned into a DENV2 infectious clone using the Notl and Xmall restriction digestion sites.
  • the present specification teaches an isolated polynucleotide comprising a) a tetracycline- responsive promoter; and b) a nucleic acid sequence encoding a live attenuated chimeric virus.
  • the tetracycline -responsive promoter may be positioned immediately adjacent to the N terminus (or upstream) of the 5 ’-noncoding region of the nucleic acid sequence encoding the live attenuated chimeric virus.
  • an isolated polynucleotide comprising a) a tetracycline -responsive promoter; and b) a nucleic acid sequence encoding a live attenuated chimeric virus, wherein the tetracycline-responsive promoter is positioned immediately adjacent to the N terminus of the 5’- noncoding region of the nucleic acid sequence encoding the live attenuated chimeric virus.
  • the positioning of the tetracycline -responsive promoter immediately adjacent (i.e. without any spacer sequence in between) to the N terminus of the 5’-non-coding region of the nucleic acid sequence encoding the live attenuated chimeric virus promotes efficient virus production and virus replication in a mammalian cell. Unlike typical expression plasmids, the exact sequence at this region is defined without any additional or missing nucleotides between the promoter and the 5’ non-coding region.
  • VacDZ is a DNA-launched live attenuated chimeric vaccine candidate against Zika virus and other viruses.
  • the inventors created the chimeric dengue/Zika virus, VacDZ, as a live attenuated vaccine candidate against Zika virus, using a clinically validated dengue virus vaccine strain as the backbone.
  • VacDZ may be administered as a DNA-launched vaccine (or as a traditional live virus vaccine) that produces live VacDZ after in vivo delivery.
  • the DNA-launched vaccine is easier to produce, has minimal reversion risks, and utilizes a novel delivery formulation that does not require in vivo electroporation.
  • the exact sequence and positioning of the promoter in relation to the cDNA sequence of a flavivirus is selected to ensure sufficiently high virus production and virus replication efficiency.
  • the inventors were able to determine a specific position for a TRE- minCMV promoter that allows efficient virus production from the DNA infectious clone during the DNA-launch process. It also allowed production of a virus that replicates efficiently. The virus production is efficient enough that it allows the infectious clone to be used as a DNA- launched vaccine.
  • polynucleotide is used herein interchangeably with “nucleic acid” to indicate a polymer of nucleosides.
  • a polynucleotide of this invention is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) joined by phosphodiester bonds.
  • nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules may be preferred for certain applications.
  • this application refers to a polynucleotide it is understood that both DNA, RNA, and in each case both single and double-stranded forms (and complements of each single-stranded molecule) are provided.
  • Polynucleotide sequence as used herein can refer to the polynucleotide material itself and/or to the sequence information (e.g., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated.
  • the polynucleotide is a DNA polynucleotide.
  • Polypeptide,” “peptide,” “protein” and “proteinaceous molecule” are used interchangeably herein to refer to molecules comprising or consisting of a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally- occurring amino acid polymers.
  • recombinant polynucleotide refers to a polynucleotide formed in vitro by the manipulation of nucleic acid into a form not normally found in nature.
  • the recombinant polynucleotide may be in the form of an expression vector.
  • expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleotide sequence.
  • recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant polynucleotide.
  • the term “recombinant virus” will be understood to be a reference to a “parent virus” comprising at least one heterologous nucleic acid sequence.
  • isolated refers to a biological material, such as a virus, a nucleic acid or a protein, which is substantially free from components that normally accompany or interact with it in its naturally occurring environment.
  • the isolated material optionally comprises material not found with the material in its natural environment, e.g., a cell. For example, if the material is in its natural environment, such as a cell, the material has been placed at a location in the cell (e.g., genome or genetic element) not native to a material found in that environment.
  • a naturally occurring nucleic acid e.g., a coding sequence, a promoter, an enhancer, etc.
  • a locus of the genome e.g., a vector, such as a plasmid or virus vector, or amplicon
  • Such nucleic acids are also referred to as “heterologous” nucleic acids.
  • An isolated virus for example, is in an environment (e.g., a cell culture system, or purified from cell culture) other than the native environment of wild-type virus (e.g., the nasopharynx of an infected individual).
  • chimeric when referring to a virus, indicates that the virus includes genetic and/or polypeptide components derived from more than one parental viral strain or source.
  • chimeric when referring to a viral protein, indicates that the protein includes polypeptide components (i.e., amino acid subsequences) derived from more than one parental viral strain or source.
  • encode refers to the capacity of a nucleic acid to provide for another nucleic acid or a polypeptide.
  • a nucleic acid sequence is said to “encode” a polypeptide if it can be transcribed and/or translated to produce the polypeptide or if it can be processed into a form that can be transcribed and/or translated to produce the polypeptide.
  • Such a nucleic acid sequence may include a coding sequence or both a coding sequence and a non-coding sequence.
  • the terms “encode”, “encoding” and the li e include a RNA product resulting from transcription of a DNA molecule, a protein resulting from translation of a RNA molecule, a protein resulting from transcription of a DNA molecule to form a RNA product and the subsequent translation of the RNA product, or a protein resulting from transcription of a DNA molecule to provide a RNA product, processing of the RNA product to provide a processed RNA product (e.g., mRNA) and the subsequent translation of the processed RNA product.
  • a processed RNA product e.g., mRNA
  • coding sequence is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene.
  • non-coding sequence refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.
  • RNA transcript e.g., mRNA
  • expression of a coding sequence results from transcription and translation of the coding sequence.
  • promoter refers to a DNA sequence that determines the site of transcription initiation for an RNA polymerase.
  • Promoter sequences comprise motifs which are recognized and bound by polypeptides, i.e. transcription factors.
  • the said transcription factors shall upon binding recruit RNA polymerases II, preferably, RNA polymerase I, II or III, more preferably, RNA polymerase II or III, and most preferably, RNA polymerase II. Thereby will be initiated the expression of a nucleic acid operatively linked to the transcription control sequence.
  • expression as meant herein may comprise transcription of DNA sequences into RNA polynucleotides (as suitable for, e.g., anti-sense approaches, RNAi approaches or ribozyme approaches) or may comprise transcription of DNA sequences into RNA polynucleotides followed by translation of the said RNA polynucleotides into polypeptides (as suitable for, e.g., gene expression and recombinant polypeptide production approaches).
  • the transcription control sequence is located immediately adjacent to the nucleic acid to be expressed, i.e. physically linked to the said nucleic acid at its 5' end.
  • sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C,
  • sequence identity will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window”, “sequence identity,” “percentage of sequence identity” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length.
  • two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • 5' untranslated region or “5' UTR” refers to a sequence located 3' to promoter region and 5' of the downstream coding region. Thus, such a sequence, while transcribed, is upstream (i.e., 5') of the translation initiation codon and therefore is generally not translated into a portion of the polypeptide product.
  • 3' untranslated region refers to a nucleotide sequences downstream (i.e., 3') of a coding sequence. It extends from the first nucleotide after the stop codon of a coding sequence to just before the poly(A) tail of the corresponding transcribed mRNA.
  • the 3' UTR may contain sequences that regulate
  • the live attenuated chimeric virus is a live attenuated chimeric Dengue virus.
  • the nucleic acid sequence encoding the live attenuated chimeric Dengue virus may comprise nucleic acid sequences derived from the dengue virus genome as well as one or more nucleic acid sequences derived from a non-dengue virus genome.
  • the one or more nucleic acid sequences derived from the non-dengue virus genome encodes a premembrane protein and/or an envelope protein from the non-dengue virus genome.
  • the 5 ’-noncoding region of the nucleic acid sequence encoding the live attenuated chimeric virus may be one that is derived from the 5 ’-noncoding region (or 5’ UTR) of the dengue virus genome.
  • the 5 ’-noncoding region may comprise or consist of a nucleic acid sequence having at least 70% (80%, 90%, 95% or 99%) sequence identity to SEQ ID NO: 3.
  • an isolated polynucleotide comprising a) a TRE-minCMV promoter; and b) a nucleic acid sequence encoding a live attenuated chimeric virus.
  • the nucleic acid sequence may comprise a 5’ non-coding region.
  • the TRE-minCMV promoter is positioned immediately adjacent to the N terminus of the 5’-noncoding region (i.e. without any spacer sequence in between). Unlike typical expression plasmids, the exact sequence at this region is defined without any additional or missing nucleotides between the TRE- minCMV promoter and the 5’ non-coding region.
  • the TRE-minCMV promoter comprises a nucleic acid sequence having at least 70% (including 80%, 90%, 95%, or 99%) identity to SEQ ID NO: 2.
  • an intron sequence is inserted to stabilize the infectious clone during propagation in bacteria.
  • the specific positioning of the intron had to be determined as insertion of an intron in the wrong position will not contribute to stability.
  • the inventors were able to determine a specific position for the insertion of an intron sequence that allowed the construction of a stable DNA infectious clone, allowing it to be used as a DNA-launched vaccine.
  • the polynucleotide comprises an intron sequence (e.g. SEQ ID NO: 12) inserted into the non-structural sequence of NS1.
  • the intron sequence may be inserted between codon 130 and 131 of NS1.
  • the nucleic acid sequence encoding the live attenuated chimeric virus may comprises nucleic acid sequences from the dengue (e.g. DenV-2) genome.
  • the nucleic acid sequences from the dengue genome may be a 5 ’-noncoding region, a nucleic acid encoding a capsid, a nucleic acid encoding a replication machinery and/or a 3’ noncoding region.
  • the nucleic acid sequence encoding the live attenuated chimeric virus comprises a) a 5 ’-noncoding region, a nucleic acid encoding a capsid, a nucleic acid encoding a replication machinery and a 3’ noncoding region, each from the Denv-2 genome; and b) a nucleic acid sequence encoding a premembrane protein and an envelope protein from a non-dengue viral genome.
  • the nucleic acid sequence encoding the live attenuated chimeric virus comprises nucleic acid sequence from the dengue (e.g. Denv-2) genome; and b) a nucleic acid sequence from a non-dengue viral genome encoding, for example, a structural protein (e.g. a spike protein) from a non-dengue viral genome.
  • a structural protein e.g. a spike protein
  • the non-dengue viral genome as referred to herein may be a viral genome from Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae
  • the nucleic acid sequence encoding the live attenuated chimeric virus comprises: a) a 5 ’-noncoding region, a nucleic acid encoding a capsid, a nucleic acid encoding a replication machinery and a 3 ’-noncoding region, each from the Denv-2 genome; and b) a nucleic acid sequence encoding the premembrane protein and envelope protein derived from the Zika genome.
  • the nucleic acid sequence encoding the live attenuated chimeric virus may further comprise a nucleic acid sequence encoding a signal sequence peptide from the Zika virus.
  • the capsid comprises a portion of a premembrane signal sequence from the Zika genome.
  • the replication machinery comprises non-structural sequences of NS1, NS2A, NS2B, NS3, NS4A NS4B and NS5.
  • the Denv-2 genome comprises a C to T mutation at position 57 of the 5’ non-coding region. In one embodiment, the Denv-2 genome comprises a nucleotide mutation that results in the presence of an aspartate residue at position 53 of the NS1 protein. In one embodiment, the Denv-2 genome comprises a nucleotide mutation that results in the presence of a valine at amino acid position 250 of the NS3 protein.
  • the polynucleotide comprises a sequence having at least 70% (including 80%, 90%, 95%, or 99%) sequence identity to SEQ ID NO: 28.
  • the polynucleotide comprises a sequence having at least 70% (including 80%, 90%, 95%, or 99%) sequence identity to SEQ ID NO: 1.
  • the nucleic acid sequence encoding the live attenuated chimeric virus comprises: a) a 5 ’-noncoding region, a nucleic acid encoding a replication machinery and a 3’- noncoding region, each from the Denv-2 genome; and b) a nucleic acid sequence encoding the an amino acid derived from the Spike protein of a Coronavirus (such as SARS-CoV-2).
  • the amino acid derived from the Spike protein of a Coronavirus comprises or consists of the receptor binding domain (RBD) of the Spike protein.
  • the nucleic acid sequence encoding the live attenuated chimeric virus may further comprise a nucleic acid sequence encoding a signal sequence peptide from the Coronavirus.
  • an expression construct comprising a polynucleotide as defined herein.
  • vector comprising a polynucleotide as defined herein.
  • vector is meant a polynucleotide molecule, suitably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned.
  • a vector may contain one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome.
  • the vector can contain any means for assuring self-replication.
  • the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • the vector is a DNA vector.
  • the vector as defined herein is delivered with a cationic polymer.
  • the cationic polymer may be polyethylenimine.
  • the vector is deliver using an in vivo-jetPEI transfection reagent.
  • the DNA-launched VacDZ requires co-transfection of two plasmids.
  • the first plasmid is the infectious clone of the chimeric dengue/Zika virus itself.
  • the second plasmid is an accessory plasmid that is required to activate the infectious clone plasmid.
  • the default delivery method utilizes a tet-off approach, whereby the vaccine is activated by default after co transfection.
  • the alternative approach is a tet-on approach, where the vaccine requires the further administration of a small dose of doxycycline or tetracycline to activate the vaccine after co transfection.
  • the plasmid comprising the polynucleotide as referred to herein is a pDL (a heavily modified pSmart plasmid; 1.9 kbp).
  • the ratio of the chimeric virus DNA plasmid and the accessory plasmid was optimized.
  • the inventors were able to perform the optimization without the need for mice by using a combination of cell culture, high throughput imaging equipment, and machine learning. They were able to arrive at an optimal ratio of about 1:4 (1 mg of accessory plasmid, 4 mg of chimeric virus DNA plasmid), and this was the ratio was used in our final formulation.
  • a method of producing or expressing a chimeric virus in a cell comprises transfecting a polynucleotide or vector as defined herein into a cell.
  • the cell may be a host cell for production of the chimeric virus or a cell in an animal body.
  • an immunogenic composition comprising a polynucleotide, an expression construct, a vector or a chimeric virus as defined herein.
  • An immunogenic composition as defined herein may comprise a pharmaceutically acceptable carrier or excipient.
  • a pharmaceutically acceptable carrier or excipient according to the present invention means any solvent or dispersing medium etc., commonly used in the formulation of pharmaceuticals and immunogenic compositions to enhance stability, sterility and deliverability of the active agent and which does not produce any secondary reaction, for example an allergic reaction, in humans.
  • the excipient is selected on the basis of the pharmaceutical form chosen, the method and the route of administration. Appropriate excipients, and requirements in relation to pharmaceutical formulation, are described in “Remington's Pharmaceutical Sciences” (19th Edition, A. R. Gennaro, Ed., Mack Publishing Co., Easton, Pa. (1995)).
  • An immunogenic composition of the present invention may optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, human serum albumin, essential amino acids, nonessential amino acids, L-arginine hydrochlorate, saccharose, D-trehalose dehydrate, sorbitol, tris (hydroxymethyl) aminomethane and/or urea.
  • the immunogenic composition may optionally comprise pharmaceutically acceptable additives including, for example, diluents, binders, stabilizers, and preservatives. Preferred stabilizers are described in WO 2010/003670.
  • An immunogenic composition of the present invention may comprise one or more adjuvants to enhance the immunogenicity of the live attenuated viruses.
  • adjuvants may be used in a vaccine composition of the invention comprising a live attenuated virus, as long as said adjuvant does not impact replication.
  • Suitable adjuvants include an aluminum salt such as aluminum hydroxide gel, aluminum phosphate or alum, but may also be a salt of calcium, magnesium, iron or zinc. Further suitable adjuvants include an insoluble suspension of acylated tyrosine or acylated sugars, cationically or anionically derivatized saccharides, or polyphosphazenes. Alternatively, the adjuvant may be an oil-in-water emulsion adjuvant (EP 0 399 843B), as well as combinations of oil-in-water emulsions and other active agents (WO 95/17210; WO 98/56414; WO 99/12565 and WO 99/11241).
  • oil emulsion adjuvants have been described, such as water-in-oil emulsions (U.S. Pat. No. 5,422,109; EP 0480982 B2) and water-in-oil-in- water emulsions (U.S. Pat. No. 5,424,067; EP 0 480 981 B).
  • water-in-oil emulsions U.S. Pat. No. 5,422,109; EP 0480982 B2
  • water-in-oil-in- water emulsions U.S. Pat. No. 5,424,067; EP 0 480 981 B.
  • examples of such adjuvants include MF59, AF03 (WO 2007/006939), AF04 (WO 2007/080308), AF05, AF06 and derivatives thereof.
  • the adjuvant may also be a saponin, lipid A or a derivative thereof, an immunostimulatory oligonucleotide, an alkyl glucosamide phosphate, an oil in water emulsion or combinations thereof.
  • saponins include Quil A and purified fragments thereof such as QS7 and QS21.
  • a immunogenic composition of the present invention is suitably formulated to be compatible with the intended route of administration.
  • suitable routes of administration include for instance intramuscular, transcutaneous, subcutaneous, intranasal, oral or intradermal.
  • the route of administration is subcutaneous.
  • the immunogenic compositions of the present invention may be administered using conventional hypodermic syringes or safety syringes such as those commercially available from Becton Dickinson Corporation (Franklin Lakes, N.J., USA) or jet injectors.
  • conventional hypodermic syringes may be employed using the Mantoux technique or specialized intradermal delivery devices such as the BD SoluviaTM microinjection system (Becton Dickinson Corporation, Franklin Lakes, N.J., USA), may be used.
  • the volume of an immunogenic composition of the present invention administered will depend on the method of administration. In the case of subcutaneous injections, the volume is generally between 0.1 and 1.0 ml, preferably approximately 0.5 ml.
  • booster administrations of an immunogenic composition according to the present invention may be used, for example between six months and ten years, for example six months, one year, three years, five years or ten years after initial immunization (i.e. after administration of the last dose scheduled in the initial immunization regimen).
  • a method of modulating an immune response in a subject comprising administering a therapeutically effective amount of a vector, a chimeric virus or an immunogenic composition as defined herein to the subject.
  • modulating an immune response may comprise inducing an immune response in a subject. This includes stimulating an immune response and/or enhancing a previously existing immune response.
  • a method of preventing or treating a viral infection in a subject comprising administering a therapeutically effective amount of a vector, a chimeric virus or an immunogenic composition as defined herein to the subject.
  • the viral infection is a Zika virus infection.
  • subject refers to an animal, in particular a mammal and more particularly a primate including a lower primate and even more particularly, a human who can benefit from the present disclosure.
  • a subject regardless of whether a human or non-human animal or embryo may be referred to as an individual, subject, animal, patient, host or recipient.
  • an "animal” specifically includes livestock animals such as cattle, horses, sheep, pigs, camelids, goats and donkeys, as well as domestic animals, such as dogs and cats.
  • laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates.
  • the subject is human.
  • treating may refer to (1) preventing or delaying the appearance of one or more symptoms of the disorder; (2) inhibiting the development of the disorder or one or more symptoms of the disorder; (3) relieving the disorder, i.e., causing regression of the disorder or at least one or more symptoms of the disorder; and/or (4) causing a decrease in the severity of one or more symptoms of the disorder.
  • the methods as disclosed herein may comprises the administration of a “therapeutically effective amount” of an agent (e.g. a vector, chimeric virus or an immunogenic composition as defined herein) to a subject.
  • an agent e.g. a vector, chimeric virus or an immunogenic composition as defined herein
  • therapeutically effective amount includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation.
  • kits comprising an immunogenic composition as defined herein and instructions for the use of said immunogenic composition in a method of modulating an immune response in a subject.
  • the kit can comprise at least one dose (typically in a syringe) of any immunogenic composition contemplated herein.
  • the kit may comprises a multi-dose formulation (typically in a vial) of any immunogenic composition as described herein.
  • the kit further comprises a leaflet mentioning the use of the said immunogenic composition for modulating an immune response in a subject.
  • an agent includes a plurality of agents, including mixtures thereof.
  • the infectious clone of VacDZ ( Figure 1), the chimeric dengue/Zika virus, was constructed by modifying an existing DENV2-16681 infectious clone.
  • the infectious clone of VacDZ (pVacDZ) was constructed using conventional molecular cloning techniques.
  • the 16681 strain of DENV2 is the parent strain of PDK-53. While PDK-53 differs from the parental wildtype DENV2- 16681 by several mutations, only three mutations are necessary and sufficient for the attenuation of PDK-53: 5'UTR-c57t, NS1-G53D and NS3-E250V. Therefore, these three attenuating mutations were introduced into pVacDZ ( Figure 1A). The region encoding for the prM signal sequence, prM protein, and Env protein were also replaced with their counterparts from ZIKV strain PRVABC59 ( Figure 1A and IB).
  • the prM signal sequence at the capsid-prM junction was replaced because prior studies with dengue/Zika virus and dengue West Nile virus chimeras showed that the prM protein requires a homologous signal sequence upstream for correct processing.
  • Flaviviral infectious clone plasmids can be highly unstable, and this instability can prevent the construction of infectious clones for chimeric vaccines. Therefore, in order to further stabilise the pVacDZ infectious clone, an intron sequence was cloned between codon 130 and 131 of the NS1 coding region (Figure 1C).
  • the infectious clone includes promoter sequences that allow virus rescue by DNA launch. This includes the TRE-minCMV promoter to drive RNA transcription in mammalian cells ( Figure 1C and ID).
  • a AGDD mutant of VacDZ was also constructed as a control for DNA vaccination studies.
  • VacDZ-AGDD has a lethal deletion of the GDD catalytic triad of the NS5 RNA-dependent RNA polymerase (RDRP). While VacDZ-AGDD can express a small quantity of viral proteins, it cannot enter into the exponential RNA-replication and protein expression stage of the virus replication cycle.
  • RDRP RNA-dependent RNA polymerase
  • Virus rescue for the infectious clones takes place by DNA-launch, whereby the infectious clone plasmid is co-transfected with a pTet-Off Advanced accessory plasmid. After transfection, host nuclear machinery transcribes the viral RNA, which is eventually processed into a functional viral genome that can establish a normal virus replication cycle.
  • VacDZ was rescued by DNA- launch in BHK-21 cells to produce a passage 1 stock.
  • VacDZ viral stock was then amplified by passaging the virus in BHK-21 cells to produce a working stock, which was then titrated by plaque assay.
  • VacDZ expresses ZIKV envelope protein.
  • VacDZ was expressing ZIKV Env protein.
  • BHK-21 cells were infected with VacDZ or with parental DENV2-16681 or ZIKV. Mock infected cells were used as a control. The cells were then fixed and viral protein expression was analysed by using immunofluorescence assay. Cells that were infected with VacDZ, DENV2-16681 or ZIKV were found to express Flaviviral NS1 protein, whereas mock infected cells did not ( Figure 2). Cells that were infected with VacDZ or ZIKV were also found to express ZIKV Env protein, while mock infected cells and DENV2- 16681 infected cells did not ( Figure 2). This demonstrates that VacDZ expresses ZIKV Env protein as intended.
  • VacDZ retains the small plaque phenotype of PDK-53.
  • VacDZ In order to validate the potential safety of VacDZ, it was investigated if VacDZ retains key markers of attenuation of the DENV2-PDK-53 vaccine strain, namely small plaque phenotype, temperature sensitivity, attenuation of neurovirulence in suckling mice, and attenuation of pathogenicity in AG129 mice.
  • VacDZ retains the temperature sensitivity phenotype, which is an attenuation marker that is defined as a reduction of the viral titre of 1-log or more at 39°C relative to 37°C.
  • the growth kinetics of VacDZ, DENV2-16681, and ZIKV were compared in BHK-21 baby hamster kidney cells.
  • the cells were infected with VacDZ, DENV2-16681, or ZIKV. After infection, the supernatant was harvested daily, and the extracellular virus titre was determined by using plaque assay.
  • ZIKV had the highest peak titre, followed by VacDZ and then DENV2-16681 ( Figure 3C, 3D and 3E). Both VacDZ and ZIKV were temperature sensitive, whereas DENV2- 16681 was not ( Figure 3C, 3D and 3E).
  • VacDZ is genetically stable.
  • VacDZ The potential safety of VacDZ is dependent on its three attenuating mutations: 5'UTR-c57t, NS1- G53D and NS3-E250V. These attenuating mutations are known to be stable for the component viruses of the TAK-003 live dengue vaccine. Therefore, it was investigated if these attenuating mutations were also stable in VacDZ.
  • VacDZ was serially passaged in BHK-21 cells. At passage 10 the viral RNA was extracted and the reversion rates of the attenuating mutations was analyzed by using next-generation sequencing. All three attenuating muations had reversion rates of less than 1% (Table 1). This demonstrates that VacDZ is genetically stable, with a low risk of reversion.
  • VacDZ retains attenuation of neuro virulence in suckling mice
  • mice that were challenged with ZIKV or DENV2- 16681 had weight loss, wobbling gait, limb weakness or dragging, paralysis, hunching, or inactivity.
  • Mice that were challenged with 1, 10, or 100 PFU of ZIKV per mouse had a mortality rate of 80%, 100% or 100% respectively.
  • Mice that were challenged with 10 or 100 PFU of DENV2-16681 per mouse had a mortality rate of 66.7% or 75% respectively.
  • mice that were challenged with 1, 10, or 100 PFU of VacDZ per mouse or with the PBS vehicle control had a mortality rate of 0%. This demonstrates that VacDZ has highly attenuated neurovirulence in suckling mice compared to the parental wildtype DENV2- 16681 and ZIKV.
  • AG129 mice are commonly used for flavivirus vaccine studies because they are highly susceptible to lethal flavivirus challenge, but retain the ability to develop an adapative immune response.
  • ZIKV strain PRVABC59 (ZIKV-PRVABC59) is known to be lethal for AG129 mice, whereas DENV2-16681 is not.
  • the pathogenicity of ZIKV was investigated in AG129 in order to determine a suitable dose for subsequent lethal challenge studies. Because VacDZ encodes the prM and Env proteins of ZIKV-PRVABC59, it was also investigated if expression these ZIKV proteins resulted in any gain of pathogenicity.
  • AG129 mice were challenged by intraperitoneal inoculation with different doses of VacDZ, ZIKV, or PBS vehicle control. The mice were then observed daily for clinical symptoms for a period of four weeks (Figure 4B). Mice that were challenged with 10 3 or 10 4 PFU of ZIKV per mouse had a mortality rate of 100%. In contrast, mice that were challenged with 10 6 PFU of VacDZ per mouse or with the PBS vehicle control had a mortality rate of 0%. This demonstrates that VacDZ has attenuated pathogenicity in AG129 mice as well. VacDZ is immunogenic in AG129 mice
  • VacDZ was tested as both a live virus vaccine (live VacDZ) and also as a DNA-launched vaccine (DNA-launched VacDZ).
  • the DNA-launched vaccine is essentially an alternative delivery method, whereby the pVacDZ infectious clone is transfected in vivo, after which the production of live VacDZ occurs in vivo. This live, DNA-launched VacDZ can then proceed to establish an attenuated viral infection in the host as per normal.
  • the dose for vaccination or boosting was 10 4 PFU of VacDZ per mouse, and PBS was used as a vehicle control.
  • the dose for vaccination or boosting was 80 pg of pVacDZ and 20 pg of pTet-Off Advanced per mouse, while a dose of 80 pg of pVacDZ-AGDD mutant and 20 pg of pTet-Off Advanced per mouse was used for the RDRP-defective control, and a 5% glucose solution was used as a vehicle control.
  • mice were first vaccinated with live VacDZ, DNA-launched VacDZ, or their respective controls via intraperitoneal inoculation. Four weeks after primary vaccination, the mice were boosted with the same dose of their respective vaccine or control. Four weeks after boosting, the mice were euthanised and their blood and organs were harvested.
  • mice that were vaccinated with live VacDZ developed a significant Thl and Th2 response ( Figure 6A and 6B). However, the Thl response was stronger compared to the Th2 response. For the mice vaccinated with live VacDZ the Thl response was 10.2-fold higher compared to the mice vaccinated with the PBS control, and the Th2 response was 2-fold higher.
  • mice that were vaccinated with DNA-launched VacDZ developed a significant Thl response, but no significant Th2 response (Figure 6C and 6D).
  • mice that were vaccinated with the AGDD mutant control did not develop any significant Thl or Th2 response compared to the 5% glucose solution vehicle control ( Figure 6C and 6D).
  • the Thl response was 12.2-fold higher compared to the mice vaccinated with the 5% glucose solution control.
  • VacDZ confers protective immunity against lethal ZIKV challenge
  • mice were vaccinated with live VacDZ, DNA-launched VacDZ, or their respective controls.
  • ZIKV 10 s PFU per mouse
  • the mice were then observed daily for clinical symptoms for a period of four weeks.
  • the mice were euthanised and their blood and organs were harvested.
  • Mice that were vaccinated with the PBS or 5% glucose solution vehicle controls had a mortality rate of 100% ( Figure 7).
  • Mice that were vaccinated with the replication defective pVacDZ- AGDD mutant control had a mortality rate of 80% ( Figure 7B).
  • Live attenuated vaccines are the gold standard for the control and prevention of flavivirus infection. Therefore, it is not surprising that several studies have reported the development of live attenuated ZIKV vaccine candidates. However, there are risks involved with the development of such live attenuated vaccines. Finally, while live viral vaccines have superior immunogenicity, they face several disadvantages in terms of genetic stability, storage, and scalability of production during outbreaks.
  • VacDZ chimeric dengue/Zika virus
  • VacDZ utilises the clinical validated DENV2-PDK-53 vaccine strain as the backbone of the chimeric virus. This was to ensure that the vaccine would be sufficiently attenuated.
  • the attenuating mutations of DENV2-PDK- 53 vaccine are known, and they are not affected by chimerisation because they are located outside of the structural proteins.
  • VacDZ retained the key attenuation markers of the DENV2-PDK-53 vaccine strain: small plaque phenotype, temperature sensitivity, attenuation of neurovirulence in suckling mice, and attenuation of pathogenicity in AG129 mice. This indicates that VacDZ is sufficiently attenuated.
  • VacDZ is designed to induce an immune response against ZIKV. It was found that both live VacDZ and DNA-Iaunched VacDZ could induce a protective immune response against ZIKV. This immune response included neutralising antibodies against ZIKV, a strong Thl response against ZIKV, and a weaker or negligible Th2 T-cell response.
  • Thl response and negligible Th2 response is similar to the anti-DENV response that is induced by the Sanofi- Pasteur CYD-TDV live dengue vaccine.
  • the robust IHNg response in VacDZ vaccinated mice is also similar to the anti-DENV response in DENV2-PDK-53 vaccinated A129 mice, and also in dengue patients with milder disease outcomes.
  • this Thl biased response is favourable because it is associated with milder disease outcomes during flavivirus infections, and the robust IHNg response is also favourable because it is associated with a protective CD8+ T-cell response.
  • Live VacDZ had better efficacy compared to DNA-launched VacDZ, with live VacDZ offering better protective immunity. Furthermore, in vivo DNA transfection were performed using a PEI based reagent instead of an in vivo electroporator. Electroporation usually offers better DNA transfection efficiency compared to PEI or liposome based transfection reagents, and therefore prior studies on DNA-launched flaviviral vaccines have utilised electroporation for in vivo transfection.
  • DNA-launched VacDZ offers many advantages over live viral vaccines.
  • DNA plasmids are more stable and easier to store, with simplified cold chain requirements.
  • the yellow fever vaccine only has a shelf life of three years, which limits the size of stockpiles. Each batch of the live yellow fever vaccine also takes up to 6 months to produce.
  • DNA-launch is an alternative method of getting a live attenuated virus into the host, which it accomplishes by getting the host itself to produce the live virus in vivo.
  • DNA-launched live vaccines are far more immunogenic than traditional DNA vaccines which only express non-replicating antigens. Therefore, unlike traditional DNA vaccines, DNA-launched VacDZ does not require any adjuvant. Even though DNA-launched VacDZ has a lower efficacy compared to live VacDZ, it is still immunogenic enough to give 100% seroconversion rate with a prime + boost strategy.
  • VacDZ is a safe and effective vaccine candidate against ZIKV.
  • VacDZ can be delivered as a live virus formulation or as a DNA-Iaunched formulation. This convenience and flexibility would greatly benefit the future development and deployment of the vaccine vulnerable populations. All this makes VacDZ a promising vaccine candidate against ZIKV and other viruses.

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Abstract

La présente invention concerne un polynucléotide isolé comprenant a) un promoteur sensible à la tétracycline ; et b) une séquence d'acide nucléique codant pour un virus chimère atténué vivant. Le virus chimère atténué vivant peut comprendre ou se composer de : i) une région de non-codage 5', un acide nucléique codant pour une capside, un acide nucléique codant pour une machine de réplication et une région de non-codage 3', chacune à partir du génome de Denv-2 ; et ii) une séquence d'acide nucléique codant pour une protéine de prémembrane et une protéine d'enveloppe dérivée du génome de Zika, le promoteur sensible à la tétracycline étant un promoteur TRE-minCMV, en outre, l'invention concerne une composition immunogène comprenant ledit polynucléotide et une méthode de traitement ou de prévention d'une infection virale par Zika.
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WO2018009604A1 (fr) * 2016-07-08 2018-01-11 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vaccins de virus zika à virus de la dengue/zika chimère vivants-atténués

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