Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/713—Double-stranded nucleic acids or oligonucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • 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
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • 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
  • C07K14/08—RNA viruses
  • C07K14/18—Togaviridae; Flaviviridae
  • C07K14/1816—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus), border disease virus
  • C07K14/1825—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • 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
  • 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
• • 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
  • 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
  • C12N15/861—Adenoviral vectors
  • C12N15/8613—Chimaeric vector systems comprising heterologous sequences for production of another viral vector
• • 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
  • A61K2039/5254—Virus avirulent or attenuated
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/70—Multivalent vaccine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/24011—Flaviviridae
  • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
  • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/24011—Flaviviridae
  • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
  • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • 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
  • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
• • 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
  • C12N2770/24141—Use of virus, viral particle or viral elements as a vector
  • C12N2770/24143—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
  • 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
  • C12N2770/24161—Methods of inactivation or attenuation
• • 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
  • C12N2770/24161—Methods of inactivation or attenuation
  • C12N2770/24162—Methods of inactivation or attenuation by genetic engineering
• • 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

• Embodiments herein report compositions, methods, uses and manufacturing procedures for dengue virus constructs and vaccine compositions thereof.
• compositions that include, but is not limited to, chimeric flavivirus virus constructs that alone or in combination with other constructs can be used in a vaccine composition.
• compositions can include constructs of more than one serotypes of dengue virus, such as dengue- 1 (DEN-1) virus, dengue-2 (DEN-2) virus, dengue-3 (DEN-3) virus and/or dengue-4 (DEN-4) virus.
• dengue- 1 DEN-1
• DEN-2 dengue-2
• DEN-3) virus dengue-3 virus
• DEN-4 virus dengue-4 virus
• Certain embodiments include at least one live, attenuated dengue virus in combination with dengue virus chimeric constructs identified to be both safe and effective in vaccine compositions where the constructs have undergone additional passages in cell cultures.
• dengue fever is caused by infection of a dengue virus.
• Other subtypes may be discovered in the future (e.g. DEN-5).
• Dengue virus serotypes 1-4 can also cause dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). The most severe consequences of infection, DHF and DSS, can be life threatening.
• Dengue viruses cause 50-100 million cases of debilitating dengue fever, 500,000 cases of DHF/DSS, and more than 20,000 deaths each year. To date, there is no effective vaccine to protect against dengue fever and no drug treatment for the disease.
• a composition can include chimeric dengue virus constructs having an attenuated dengue virus backbone with structural genes from at least one other dengue virus serotype.
• Other embodiments concern at least one live, attenuated virus in combination with one or more chimeric dengue viruses.
• Other embodiments can include a composition of chimeric dengue viruses having a modified DEN-2 backbone (e.g.
• PDK-53 as a starting backbone in PI (passage- 1) and passage variability (after passage and growth in vitro on a permissive cell line) as indicated for P2, P3,...P8..P10 etc.) and one or more structural components of DEN- 1, DEN-2, DEN-3 or DEN-4.
• an immunogenic composition is generated where when introduced to a subject, the composition produces an immune response to one or more dengue viruses in the subject. Therefore, constructs contemplated herein can be generated and passaged in vitro, and each of the passages provides an attenuated dengue virus contemplated of use in a pharmaceutically acceptable vaccine composition.
• a live, attenuated virus can be a live, attenuated dengue-2 virus alone or in combination with one or more chimeric dengue viruses.
• chimeric dengue virus constructs of dengue virus serotypes can include passage 7 (P7) live, attenuated viruses or chimeric viruses having nucleic acid sequences identified by SEQ ID NOS: 1, 4, 7 and 10 or polypeptide sequences indicated by SEQ ID NOS: 2, 3, 5, 6, 8, 9, 1 1 and 12. It is contemplated herein that any of the passages for any of the live, attenuated viruses described herein can be used in an immunogenic composition to induce immune responses to the represented dengue viruses (e.g. serotypes 1- 4).
• an immunogenic composition that includes a P-8 isolated live, attenuated virus can be administered to a subject to induce an immunogenic response against one or more dengue virus serotypes depending on the construct selected.
• a live, attenuated virus can be combined with one or more of these chimeric viruses. This is contemplated for each of the live, attenuated viruses isolated/produced in each subsequent cell passages (e.g. African Green Monkey Vero cell production, hereinafter: Vero cells). It is contemplated herein that any cell line (e.g. GMP -produced cell bank, FDA or EMA-approved) capable of producing dengue viruses is of use to passage any of the viral constructs at a manufacturing scale or as appropriate contemplated herein for subsequent use in a vaccine or immunogenic composition against Dengue virus.
• GMP -produced cell bank e.g. GMP -produced cell bank, FDA or EMA-approved
• compositions contemplated herein can be combined with other immunogenic compositions against other Flaviviruses such as West Nile virus, Japanese encephalitis or any other flavivirus chimeric construct and/or live, attenuated virus.
• a single composition can be used against multiple flaviviruses.
• an immunogenic composition of the present invention can include chimeric dengue viruses against one or more of DEN-1, DEN-2, DEN-3 and/or DEN- 4, alone or in combination with a live, attenuated dengue virus composition.
• a construct can include a construct having adaptive mutations in the structural or non-structural regions of the virus that increase growth or production without affecting attenuation or safety of the virus when introduced to a subject.
• any of the contemplated chimeric dengue virus constructs can include a live, attenuated DEN-2 virus having specific mutations used as a backbone where the live attenuated DEN-2 PDK virus further includes structural proteins of one or more of prM (premembrane) and E (envelope) structural proteins of the other dengue virus serotypes.
• a DEN-2 backbone can include additional mutations in order to increase production of or enhance the immune response to a predetermine composition in a subject upon administration (e.g. chimeric Dengue virus 2/1, 2/3 or 2/4).
• structural protein genes can include prM and E genes of DEN- 1, DEN-2, DEN-3 or DEN-4 on a DEN-2 backbone having one or two mutations that are part of a live, attenuated dengue virus.
• a dengue construct in certain embodiments can include those constructs termed DENVax-l-A, DENVax-2-F, DENVax-3- F, and DENVax-4-F (see Example section) where the DEN-2 backbone has one or more mutations (e.g. not found in the PI or other previous passaged virus or PDK-53) from the DEN-2 live, attenuated virus previously demonstrated to be safe and effective to induce an immune response.
• the DEN-2 live, attenuated virus of the instant application is an improved version of the originally used DEN-2 live, attenuated virus.
• a chimeric construct of the instant invention can include a modified attenuated DEN-2 PDK-53 backbone, having one or more structural proteins of the second dengue virus serotype wherein the structural proteins can include additional mutations to increase an immunogenic response to the chimeric construct.
• certain mutations acquired by attenuated DEN-2 PDK-53 can produce a conservative amino acid change or not in a constructs different from the PI construct which can result in desirable traits for production etc.
• a live, attenuated DEN-2 genome can be used to generate constructs of dengue virus serotype 1 (DEN-1) and dengue virus serotype 3 (DEN- 3), dengue virus serotype 4 (DEN-4) where one or more structural protein genes of the DEN- 2 viral genome can be replaced by one or more structural protein genes of DEN- 1 , DEN-3 or DEN-4, respectively.
• a structural protein can be the C, prM or E protein of a second dengue virus.
• structural protein genes include the prM and E genes of DEN-1, DEN-3 or DEN-4. These hybrid viruses express the surface antigens of DEN-1, DEN-3 or DEN-4 while retaining the attenuation phenotypes of the parent attenuated DEN-2.
• Constructs disclosed herein can include chimeric constructs of DEN-4, DEN-2,
• DEN-1, and DEN-3 expressing surface antigens of DEN-1, DEN-3 and DEN-4 using attenuated DEN-2 virus as a backbone.
• compositions of the instant invention can include a composition that comprises a single chimeric dengue virus construct disclosed herein and a pharmaceutically acceptable carrier or excipient.
• compositions of the instant invention can include a composition that comprises two or more, or three or more chimeric dengue virus constructs disclosed herein, and a pharmaceutically acceptable carrier or excipient.
• a one or more dengue virus chimeric constructs contemplated herein can be combined with one or more, live attenuated dengue viruses.
• a live, attenuated virus can be a live, attenuated DEN-2 virus wherein additional mutations in the NCR, NS1 regions or other regions increase the immune response, increase viral growth or other improvement for an improved live, attenuated dengue virus.
• the attenuation loci nucleotide 5'NCR-57-T, NS1-
• Fig. 1 represents an exemplary chart reflecting an exemplary chimeric construct of the instant invention, DEN-2/DEN-4 compared to previously generated constructs and wild type dengue viruses.
• Fig. 2 represents an exemplary histogram plot comparing various responses using a live, attenuated DEN-2 backbone (with additional mutations) and a second dengue virus serotype as structural components substituted for the dengue-2 structural components (e.g. DENVax- 1 MVS).
• This plot illustrates plaque sizes of the DENVax MVS. Wild-type Dengue viruses and previously published research-grade vaccine candidate viruses were included for control and comparison. This plot illustrates improved production of the dengue virus constructs compared to control dengue virus chimeric constructs.
• Fig. 3 represents an exemplary histogram plot that represents temperature sensitivities of DENVax MVS (Master Virus Seed). Wild type dengue viruses and previously published research-grade vaccine candidate viruses were included for comparison with the MVS grade.
• Fig. 4 represents an exemplary histogram plot that represents viral growth of
• DENVax MVS in C6/36 cells compared to controls. Wild-type dengue viruses and research- grade vaccine candidate viruses were included for comparison with the DENVax MVS.
• Figs. 5A-5C represent exemplary plots of neurovirulence in newborn mice.
• Fig. 6 represents an exemplary histogram illustrating plaque size of the
• DENVax MVS, WVS, and BVS Mean plaque diameters ⁇ SD (error bars) of the virus plaques in Vero or LLC-MK2 cells under agarose overlay measured on day 9 pi. Wild type DENVs and previously published research-grade vaccine candidate viruses were included for control and comparison.
• Fig. 7 represents an exemplary histogram plot illustrating growth of DENVax
• Fig. 8 represents an exemplary histogram plotting restricted growth of
• FIGs. 9A-9B represent exemplary graphs of data of neurovirulence of
• Fig. 10 represents an exemplary chart comparing new live, attenuated viruses to previously generated live, attenuated dengue viruses.
• subject or “subjects” may include, but are not limited to, mammals such as humans or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g. hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zoo animals.
• mammals such as humans or mammals, domesticated or wild, for example dogs, cats, other household pets (e.g. hamster, guinea pig, mouse, rat), ferrets, rabbits, pigs, horses, cattle, prairie dogs, wild rodents, or zoo animals.
• virus chimera can mean a construct comprising a portion of the nucleotide sequence of a dengue-2 virus and further nucleotide sequence that is not from dengue-2 virus or is from a different flavivirus.
• a "dengue chimera” comprises at least two different dengue virus serotypes but not a different flavivirus.
• examples of other dengue viruses or flaviviruses include, but are not limited to, sequences from dengue- 1 virus, dengue-3 virus, dengue-4 virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, tick-borne encephalitis virus, yellow fever virus and any combination thereof.
• nucleic acid chimera can mean a construct of the invention comprising nucleic acid comprising a portion of the nucleotide sequence of a dengue-2 virus and further nucleotide sequence that is not of the same origin as the nucleotide sequence of the dengue-2 virus.
• any chimeric flavivirus or flavivirus chimera disclosed herein can be recognized as an example of a nucleic acid chimera.
• a live, attenuated virus can mean a wild-type virus, mutated or selected for traits of use in vaccine or other immunogenic compositions wherein some traits can include reduced virulence, safety, efficacy or improved growth etc.
• Embodiments herein concern compositions, methods and uses for inducing immune responses against one or more dengue virus serotypes in a subject, individually or simultaneously.
• attenuated dengue viruses and nucleic acid chimeras are generated and used in vaccine compositions disclosed herein.
• Some embodiments concern modified or mutated dengue constructs or chimeras. Other embodiments concern introducing mutations to modify the amino acid sequences of structural proteins of dengue viruses wherein the mutation increase immunogenicity to the virus.
• live, attenuated dengue-2 PDK-53 vaccine virus contained a mixture of viruses, with the population containing varying nucleotide differences. After genetic characterization of the attenuating mutations, certain attenuating characteristics were outlined and engineered into a cDNA infectious clone. RNA was transcribed from this infectious clone and introduced into Vero cells as a passage 1 of the newly characterized and derived PDK-53-Vero-DEN-2-P 1 virus (see for example, Table 1).
• DEN-2 PDK-53 has the lowest infectious dose (50% minimal infectious dose of 5 plaque forming units or PFU) in humans, is strongly immunogenic, and produces no apparent safety concerns.
• the DEN-1 PDK-13, DEN-3 PGMK-30/FRhL-3 and DEN-4 PDK-48 vaccine virus candidates have higher 50% minimal infectious doses of 10,000, 3500, and 150 PFU, respectively, in humans.
• DEN-2 PDK-53 virus vaccine candidate also abbreviated PDK-53
• PDK-53 has several measurable biological markers associated with attenuation, including temperature sensitivity, small plaque size, decreased replication in mosquito C6136 cell culture, decreased replication in intact mosquitoes, loss of neurovirulence for suckling mice and decreased incidence of viremia in monkeys.
• Clinical trials of the candidate PDK-53 vaccine have demonstrated its safety and immunogenicity in humans.
• the PDK-53 vaccine induces dengue virus-specific T-cell memory responses in human vaccine recipients.
• Immunogenic flavivirus chimeras having a dengue-2 virus backbone and at least one structural protein of another dengue virus serotype can be used for preparing the dengue virus chimeras and methods for producing the dengue virus chimeras are described.
• the immunogenic dengue virus chimeras are provided, alone or in combination, in a pharmaceutically acceptable carrier as immunogenic compositions to minimize, inhibit, or immunize individuals against infection by one or more serotypes, such as dengue virus serotypes DEN-1, DEN-2, DEN-3 and DEN-4, alone or in combination.
• the immunogenic dengue virus chimeras may be used as multivalent vaccines (e.g.
• the dengue virus chimeras are combined in an immunogenic composition useful as a bivalent, trivalent or tetravalent vaccine against the known dengue virus serotypes or confer immunity to other pathogenic flaviviruses by including nucleic acids encoding one or more proteins from a different flavivirus.
• avirulent, immunogenic dengue virus chimeras provided herein contain the nonstructural protein genes of the attenuated dengue-2 virus (e.g. PDK-53), or the equivalent thereof, and one or more of the structural protein genes or immunogenic portions thereof of the flavivirus against which immunogenicity is to be induced in a subject.
• the nonstructural protein genes of the attenuated dengue-2 virus e.g. PDK-53
• structural protein genes or immunogenic portions thereof of the flavivirus against which immunogenicity is to be induced in a subject e.g. PDK-53
• some embodiments concern a chimera having attenuated dengue-2 virus PDK-53 genome as the viral backbone, and one or more structural protein genes encoding capsid, premembrane/membrane, or envelope of the PDK-53 genome, or combinations thereof, replaced with one or more corresponding structural protein genes from DEN-1, DEN-3 or DEN-4 or other flavivirus to be protected against, such as a different flavivirus or a different dengue virus serotype.
• a nucleic acid chimera disclosed herein can have functional properties of the attenuated dengue-2 virus and is avirulent, but expresses antigenic epitopes of the structural gene products of DEN-1, DEN-3 or DEN-4 in addition to other flaviviruses and is immunogenic (e.g. induces an immune response to the gene products in a subject). Then, these DNA constructs are used to transcribe RNA from an infectious clone, this RNA is introduced into Vero cells again producing a new progeny virus at P I. These new progeny viruses are distinguishable from PDK-53. (See e.g. P 1-P10).
• a nucleic acid chimera can be a nucleic acid chimera having, but not limited to, a first nucleotide sequence encoding nonstructural proteins from an attenuated dengue-2 virus, and a second nucleotide sequence encoding a structural protein from dengue-4 virus alone or in combination with another flavivirus.
• the attenuated dengue-2 virus can be vaccine strain PDK-53 having one or more mutated amino acids (see Examples). These additional mutations confer desirable traits of use as live, attenuated dengue-2 or as chimeric constructs described herein.
• Some embodiments include structural proteins of one or more of C, prM or E protein of a second dengue virus.
• chimeric viruses can include nucleotide and amino acid substitutions, deletions or insertions for example, in the control PDK-53 dengue-2 genome to reduce interference with immunogenicity responses to a targeted dengue virus serotype.
• modifications can be made in structural and nonstructural proteins alone or in combination with the example modifications disclosed herein and can be generated by passaging the attenuated virus and obtaining an improved composition for inducing an immune response against one or more dengue virus serotypes.
• a dengue-2 backbone used herein can include one or more mutations presented in Table 3.
• a dengue-dengue chimera of the instant application can include one or more mutations as presented in Table 3.
• a dengue-dengue chimera can include all of the mutations for each chimera as represented in Table 3 for Den-2/Den-l, Den-2/Den-3 or Den-2/Den-4.
• compositions that include a live, attenuated virus represented by the constructs of Table 3 are contemplated.
• a live, attenuated virus represented by the constructs of Table 3 are contemplated.
• mono-, di-, tri- or tetravalent compositions are contemplated of use herein using chimeras and live, attenuated dengue-2 viruses as presented in Table 3.
• a live, attenuated DEN-2 variant contemplated herein can be formulated into a pharmaceutical composition wherein the pharmaceutical composition can be administered alone or in combination with dengue-dengue chimeras or dengue-flavivirus chimeras.
• a bi-, tri or tetravalent compositions can be administered in a single application or in multiple applications to a subject.
• Dengue virus types 1-4 are mosquito-borne flavivirus pathogens.
• the flavivirus genome contains a 5'-noncoding region (5'-NC), followed by a capsid protein (C) encoding region, followed by a premembrane/membrane protein (prM) encoding region, followed by an envelope protein (E) encoding region, followed by the region encoding the nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) and finally a 3' noncoding region (3TSTC).
• the viral structural proteins are C, prM and E, and the nonstructural proteins are NS1-NS5.
• the structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.
• Flavivirus chimeras can be constructs formed by fusing non-structural protein genes from one type, or serotype, of dengue virus or virus species of the flaviviridae, with protein genes, for example, structural protein genes, from a different type, or serotype, of dengue virus or virus species of the flaviviridae.
• a flavivirus chimera of the invention is a construct formed by fusing non-structural protein genes from one type, or serotype, of dengue virus or virus species of the flaviviridae, with further nucleotide sequences that direct the synthesis of polypeptides or proteins selected from other dengue virus serotypes or other viruses of the flaviviridae.
• avirulent, immunogenic flavivirus chimeras provided herein contain the nonstructural protein genes of the attenuated dengue-2 virus, or the equivalent thereof, and one or more of the structural protein genes, or antigenic portions thereof, of the flavivirus against which immunogenicity is to be conferred.
• Suitable flaviviruses include, but are not limited to those listed in Table 1.
• Suitable dengue viruses for use in constructing the chimeras can be wild-type, virulent DEN-1 16007, DEN-2 16681, DEN-3 16562 and DEN-4 1036 and attenuated, vaccine-strain DEN-1 PDK-13, DEN-2 PDK-53, DEN-3 PMK-30/FRhL-3 and DEN-4 PDK-48. Genetic differences between the DEN-1, DEN-2, DEN-3 and DEN-4 wild type/attenuated virus pairs are contemplated along with changes in the amino acid sequences encoded by the viral genomes.
• Sequence listings for DEN-2 PDK-53 correspond to the DEN-2 PDK-53-V variant, wherein genome nucleotide position 5270 is mutated from an A to a T and amino acid position 1725 of the polyprotein or amino acid position 250 of the NS3 protein contains a valine residue.
• the DEN-2 PDK-53 variant without this nucleotide mutation, DEN-2 PDK-53-E differs from PDK-53-V only in this one position.
• DEN-2 PDK-53-E has an A at nucleotide position 5270 and a glutamate at polyprotein amino acid position 1725, NS3 protein amino acid position 250. It is understood that embodiments herein include modified PDK 53 that include one or more passages in a separate host cell (e.g. Vero cells, see Table 1) where desirable traits of use in vaccine compositions contemplated herein are generated.
• designations of the chimeras can be based on the
• DEN-2 virus-specific infectious clone modified backbones and structural genes insert of other dengue viruses or other flaviviruses.
• DEN-2 for the dengue-2 backbone followed by the strain from which the structural genes are inserted.
• One DEN-2 backbone variant is reflected in the next letter after the number designation.
• DEN-2 backbone variant from which the chimera was constructed is indicated by the following letter placed after a hyphen, parent 16681 (P), PDK-53 -E (E), or PDK-53 -V (V); the last letter indicates the C-prM-E structural genes from the parental (P) strain or its vaccine derivative (V) or the prM-E structural genes from the parental (P) or its vaccine derivative (VI).
• DEN-2/1-VP denotes the chimera comprising the attenuated DEN-2 PDK-53V backbone comprising a valine at NS3-250 and the C-prM-E genes from wild-type DEN-1 16007
• DEN-2/1-VV denotes the DEN-2 PDK-53V backbone with the vaccine strain of dengue- 1, DEN-1 PDK-13
• DEN-2/1-VP 1 denotes the DEN-2 PDK-53V backbone and the prM-E genes from wild-type DEN-1 16007
• DEN-2/3-VP1 denotes the DEN-2 PDK-53 V backbone and the prM-E genes from wild-type DEN-3 16562
• DEN- 2/4VP 1 denotes the DEN-2 PDK-53V backbone and the prM-E genes from wild-type DEN- 4 1036.
• Other chimeras disclosed herein are indicated by the same manner.
• chimeras disclosed herein contain attenuated dengue-2 virus PDK-53 genome as the viral backbone, in which the structural protein genes encoding C, prM and E proteins of the PDK-53 genome, or combinations thereof, can be replaced with the corresponding structural protein genes from dengue- 1, dengue-3 or dengue-4 virus and optionally, another flavivirus to be protected against, such as a different flavivirus or a different dengue virus strain.
• a Gly-to-Asp wild type-to-PDK-53 mutation was discovered at nonstructural protein NS l-53 (genome nucleotide position 2579); a Leu-to-Phe (wild type-to-PDK-53) mutation was discovered at nonstructural protein NS2A-181 (genome nucleotide position 4018); a Glu-to-Val (wild type-to-PDK-53) mutation was discovered at nonstructural protein NS3-250 (genome nucleotide position 5270); and a Gly-to-Ala mutation (wild type-to-PDK-53) was discovered at nonstructural protein NS4A-75 (genome nucleotide position 6599).
• the live, attenuated DEN-2 virus of the instant invention further includes mutations as presented in any chimera or live, attenuated dengue-2 virus of Table 3.
• PDK-53 virus strain has a mixed genotype at genome nucleotide 5270. A significant portion (approximately 29%) of the virus population encodes the non-mutated NS3-250-Glu that is present in the wild type DEN-2 16681 virus rather than the NS3-250- Val mutation. As both genetic variants are avirulent, this mutation may not be necessary in an avirulent chimera.
• avirulence of the attenuated PDK-53 virus strain can be attributed to mutations in the nucleotide sequence encoding nonstructural proteins and in the 5' noncoding region. For example, a single mutation at NSl-53, a double mutation at NSl-53 and at 5TSTC-57, a double mutation at NSl-53 and at NS3-250 and a triple mutation at NSl-53, at 5TSTC-57 and at NS3-250, result in attenuation of the DEN-2 virus.
• the genome of any dengue-2 virus containing such non-conservative amino acid substitutions or nucleotide substitutions at these loci can be used as a base sequence for deriving the modified PDK-53 viruses disclosed herein.
• Another mutation in the stem of the stem/loop structure in the 5' noncoding region will provide additional avirulent phenotype stability, if desired. Mutations to this region disrupt potential secondary structures important for viral replication.
• a single mutation in this short (only 6 nucleotide residues in length) stem structure in both DEN and Venezuelan equine encephalitis viruses disrupts the formation of the hairpin structure. Further mutations in this stem structure decrease the possibility of reversion at this locus, while maintaining virus viability.
• Mutations disclosed herein can be achieved by any method known in the art including, but not limited to, naturally-occurring or selected clones having additional features once passaged in a cell line of interest (e.g. Vero cells). It is understood by those skilled in the art that the virulence screening assays, as described herein and as are well known in the art, can be used to distinguish between virulent and avirulent backbone structures.
• Flavivirus chimeras described herein can be produced by splicing one or more of the structural protein genes of the flavivirus against which immunity is desired into a PDK-53 dengue virus genome backbone, or other methods known in the art, using recombinant engineering to remove the corresponding PDK-53 gene and replace it with a dengue- 1, dengue-3 or dengue-4 virus gene or other gene known in the art.
• the nucleic acid molecules encoding the flavivirus proteins may be synthesized using known nucleic acid synthesis techniques and inserted into an appropriate vector. Avirulent, immunogenic virus is therefore produced using recombinant engineering techniques known to those skilled in the art.
• a target gene can be inserted into the backbone that encodes a flavivirus structural protein of interest for DEN-1, DEN-3, DEN-4 or other flavivirus.
• a flavivirus gene to be inserted can be a gene encoding a C protein, a PrM protein and/or an E protein.
• the sequence inserted into the dengue-2 backbone can encode both PrM and E structural proteins.
• the sequence inserted into the dengue-2 backbone can encode all or one of C, prM and E structural proteins.
• Suitable chimeric viruses or nucleic acid chimeras containing nucleotide sequences encoding structural proteins of other flaviviruses or dengue virus serotypes can be evaluated for usefulness as vaccines by screening them for the foregoing phenotypic markers of attenuation that indicate avirulence and by screening them for immunogenicity.
• Antigenicity and immunogenicity can be evaluated using in vitro or in vivo reactivity with flavivirus antibodies or immunoreactive serum using routine screening procedures known to those skilled in the art.
• chimeric viruses and nucleic acid chimeras can provide live, attenuated viruses useful as immunogens or vaccines. Some embodiments include chimeras that exhibit high immunogenicity to dengue-4 virus while producing no dangerous pathogenic or lethal effects.
• a tetravalent vaccine is needed to provide simultaneous immunity for all four serotypes of the virus.
• a tetravalent vaccine is produced by combining a live, attenuated dengue-2 virus of the instant application with dengue-2/1, dengue-2/3, and dengue-2/4 chimeras described above in a suitable pharmaceutical carrier for administration as a multivalent vaccine.
• the chimeric viruses or nucleic acid chimeras of this invention can include structural genes of either wild-type or live, attenuated virus in a virulent or an attenuated DEN-2 virus backbone.
• the chimera may express the structural protein genes of wild-type DEN-4 1036 virus, its candidate vaccine derivative in either DEN-2
• Viruses used in the chimeras described herein can be grown using techniques known in the art. Virus plaque titrations are then performed and plaques counted in order to assess the viability and phenotypic characteristics of the growing cultures. Wild type viruses can be passaged through cultured cell lines to derive attenuated candidate starting materials.
• Chimeric infectious clones can be constructed from the various dengue serotype clones available.
• the cloning of virus-specific cDNA fragments can also be accomplished, if desired.
• the cDNA fragments containing the structural protein or nonstructural protein genes are amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from dengue virus RNA with various primers. Amplified fragments are cloned into the cleavage sites of other intermediate clones. Intermediate, chimeric dengue virus clones are then sequenced to verify the accuracy of the inserted dengue virus-specific cDNA.
• RT-PCR reverse transcriptase-polymerase chain reaction
• DEN-2 PDK-53 vaccine virus is significant for the attenuated phenotype of this virus, because the NSl-53-Gly of the DEN-2 16681 virus is conserved in nearly all flaviviruses, including the tick-borne viruses, sequenced to date.
• DEN-4 vaccine virus can also contain an amino acid mutation in the NS 1 protein at position 253. This locus, which is a Gln-to-His mutation in DEN-4 PDK-48 vaccine virus, is Gin in all four wild serotypes of dengue virus. This Gin residue is unique to the dengue viruses within the flavivirus genus.
• the NS1 protein is a glycoprotein that is secreted from flavivirus -infected cells. It is present on the surface of the infected cell and NS1 -specific antibodies are present in the serum of virus-infected individuals. Protection of animals immunized with NS 1 protein or passively with NS1 -specific antibody has been reported. The NS 1 protein appears to participate in early viral RNA replication.
• the mutations that occurred in the NS2A, NS2B, NS4A, and NS4B proteins of the DEN-1, -2, -3 and -4 attenuated strains are conservative in nature.
• the flaviviral NS3 protein possesses at least two recognized functions: the viral proteinase and RNA helicase/NTPase.
• the 698-aa long (DEN-2 virus) NS3 protein contains an amino-terminal serine protease domain (NS3-51-His, -75-Asp, -135-Ser catalytic triad) that is followed by sequence motifs for RNA helicase/NTPase functions (NS3-196-GAGKT (SEQ ID NO: 147), -284-DEAH, -459-GRIGR). None of the mutations in the NS3 proteins of DEN-1, DEN-2, or DEN-3 virus occurred within a recognized motif.
• the NS3-510 Tyr-to-Phe mutation in DEN-1 PDK-13 virus was conservative. Since the wild-type DEN-2, -3 and -4 viruses contain Phe at this position, it is unlikely that the Tyr- to-Phe mutation plays a role in the attenuation of DEN-1 virus.
• the NS3-182 Glu-to-Lys mutation in DEN-1 PDK-13 virus occurred at a position that is conserved as Asp or Glu in most mosquito-borne flaviviruses and it may play some role in attenuation. This mutation was located 15 amino acid residues upstream of the GAGKT helicase motif. As noted in previous reports, the NS3-250-Glu in DEN-2 16681 virus is conserved in all mosquito- borne flaviviruses except for yellow fever virus.
• Nucleic acid probes selectively hybridize with nucleic acid molecules encoding the DEN-1, DEN-3 and DEN-4 viruses or complementary sequences thereof.
• selective or “selectively” is meant a sequence which does not hybridize with other nucleic acids to prevent adequate detection of the dengue virus. Therefore, in the design of hybridizing nucleic acids, selectivity will depend upon the other components present in a sample.
• the hybridizing nucleic acid should have at least 70% complementarity with the segment of the nucleic acid to which it hybridizes.
• the term “selectively hybridizes” excludes the occasional randomly hybridizing nucleic acids, and thus, has the same meaning as “specifically hybridizing.”
• the selectively hybridizing nucleic acid of this invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, and 99% complementarity with the segment of the sequence to which it hybridizes, preferably 85% or more.
• probes which selectively hybridize to the encoding nucleic acid or the complementary, or opposite, strand of the nucleic acid are contemplated. Specific hybridization with nucleic acid can occur with minor modifications or substitutions in the nucleic acid, so long as functional species-specific hybridization capability is maintained.
• probe nucleic acid sequences that can be used as probes or primers for selective hybridization with complementary nucleic acid sequences for their detection or amplification, which probes can vary in length from about 5 to 100 nucleotides, or preferably from about 10 to 50 nucleotides, or most preferably about 18-24 nucleotides.
• the composition preferably includes at least two nucleic acid molecules which hybridize to different regions of the target molecule so as to amplify a desired region.
• the target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions.
• the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from other organisms.
• Nucleic acid sequences encoding the DEN-4, DEN-3 or DEN-1 virus can be inserted into a vector, such as a plasmid, and recombinantly expressed in a living organism (e.g. into a dengue-2 backbone) to produce recombinant dengue virus peptides and/or polypeptides and/or viruses.
• a rapid genetic test that is diagnostic for each of the vaccine viruses described herein is provided by the current invention.
• This embodiment of the invention enhances analyses of viruses isolated from the serum of vaccinated humans who developed a viremia, as well as enhancing characterization of viremia in nonhuman primates immunized with the candidate vaccine viruses.
• sequences include a diagnostic TaqMan probe that serves to report the detection of the cDNA amplicon amplified from the viral genomic RNA template by using a reverse-transciptase/polymerase chain reaction (RT/PCR), as well as the forward and reverse amplimers that are designed to amplify the cDNA amplicon, as described below.
• RT/PCR reverse-transciptase/polymerase chain reaction
• the forward and reverse amplimers that are designed to amplify the cDNA amplicon, as described below.
• one of the amplimers has been designed to contain a vaccine virus- specific mutation at the 3 '-terminal end of the amplimer, which effectively makes the test even more specific for the vaccine strain because extension of the primer at the target site, and consequently amplification, will occur only if the viral RNA template contains that specific mutation.
• the TaqMan assay is a highly specific and sensitive assay that permits automated, real time visualization and quantitation of PCR-generated amplicons from a sample nucleic acid template. TaqMan can determine the presence or absence of a specific sequence.
• a forward and a reverse primer are designed to anneal upstream and downstream of the target mutation site, respectively.
• a specific detector probe which is designed to have a melting temperature of about 10. degree. C. higher than either of the amplimers and containing the vaccine virus-specific nucleotide mutation or its complement (depending on the strand of RT/PCR amplicon that is being detected), constitutes the third primer component of this assay.
• a probe designed to specifically detect a mutated locus in one of the vaccine viral genomes will contain the vaccine-specific nucleotide in the middle of the probe. This probe will result in detectable fluorescence in the TaqMan assay if the viral RNA template is vaccine virus-specific.
• genomic RNA templates from wild-type DEN viruses will have decreased efficiency of probe hybridization because of the single nucleotide mismatch (in the case of the parental viruses DEN viruses) or possibly more than one mismatch (as may occur in other wild-type DEN viruses) and will not result in significant fluorescence.
• the DNA polymerase is more likely to displace a mismatched probe from the RT/PCR amplicon template than to cleave the mismatched probe to release the reporter dye (TaqMan Allelic Discrimination assay, Applied Biosystems).
• the molecular beacon strategy also utilizes primers for RT/PCR amplification of amplicons, and detection of a specific sequence within the amplicon by a probe containing reporter and quencher dyes at the probe termini. In this assay, the probe forms a stem-loop structure.
• the molecular beacons assay employs quencher and reporter dyes that differ from those used in the TaqMan assay.
• Embodiments herein provide for administration of compositions to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo.
• biologically compatible form suitable for administration in vivo is meant a form of the active agent (e.g. pharmaceutical chemical, protein, gene, of the embodiments) to be administered in which any toxic effects are outweighed by the therapeutic effects of the active agent.
• Administration of a therapeutically active amount of the therapeutic compositions is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
• a therapeutically active amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage periods may be adjusted to provide the optimum therapeutic response.
• the compound e.g. pharmaceutical chemical, protein, peptide etc. of the embodiments
• the compound may be administered in a convenient manner, for example, subcutaneous, intravenous, by oral administration, inhalation, intradermal, transdermal application, intravaginal application, topical application, intranasal or rectal administration.
• the active compound may be contained in a protective buffer (e.g. FTA, F127/trehalose/albumin).
• a composition may be orally administered.
• the composition may be administered intravenously.
• the composition may be administered intranasally, such as inhalation.
• the composition may be administered
• a composition may be administered to a subject in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
• pharmaceutically acceptable carrier as used herein is intended to include diluents such as saline and aqueous buffer solutions. It may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
• the active agent may also be administered parenterally, or intraperitoneally.
• Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms or other stabilizing formulation (e.g. FTA).
• compositions suitable for injectable use may be administered by means known in the art.
• sterile aqueous solutions where water soluble
• dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion may be used.
• the composition can be sterile and can be fluid to the extent that easy syringability exists. It might be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the pharmaceutically acceptable carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• Prevention of microorganisms can be achieved by heating, exposing the agent to detergent, irradiation or adding various antibacterial or antifungal agents.
• Sterile injectable solutions can be prepared by incorporating active compound (e.g. a compound that induces an immune response to one or more dengue virus serotypes) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• active compound e.g. a compound that induces an immune response to one or more dengue virus serotypes
• compositions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
• the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. It is contemplated that compositions are especially suitable for intramuscular, subcutaneous, intradermal, intranasal and intraperitoneal administration. A particular ratio may be sought such as a 1 : 1, 1 :2 or other ratio (e.g. PFUs of a given dengue virus serotype)
• the active therapeutic agents may be formulated within a mixture
• Single dose or multiple doses can also be administered on an appropriate schedule for a given situation (e.g. prior to travel, outbreak of dengue fever).
• nasal solutions or sprays, aerosols or inhalants may be used to deliver the compound of interest.
• Additional formulations that are suitable for other modes of administration include suppositories and pessaries.
• compositions can include excipients, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like.
• a pharmaceutical composition may be prepared with carriers that protect active ingredients against rapid elimination from the body, such as time-release
• Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others are known.
• compositions are administered in an amount, and with a frequency, that is effective to inhibit or alleviate side effects of a transplant and/or to reduce or prevent rejection.
• the precise dosage and duration of treatment may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Dosages may also vary with the severity of the condition.
• a pharmaceutical composition is generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. In general, dose ranges from about 10 2 to 10 6 PFU can be administered initially and optionally, followed by a second administration within 30 days or up to 180 days later, as needed.
• a subject can receive dual administration of a mono, bi-, tri or tetravalent composition disclosed herein wherein the composition is a single composition mixture or has predetermined compositions of different dengue virus serotypes.
• a DEN2/4 chimera can be present in higher concentrations than other dengue virus serotypes such as a live, attenuated dengue- 1.
• composition disclosed herein can be administered to a subject subcutaneously or intradermally.
• compositions containing live, attenuated dengue viruses may be administered to individuals, particularly humans, for example by subcutaneously, intramuscularly, intranasally, orally, topically, transdermally, parenterally, gastrointestinally, transbronchially and transalveolarly.
• Topical administration is accomplished via a topically applied cream, gel, rinse, etc. containing therapeutically effective amounts of inhibitors of serine proteases.
• Transdermal administration is accomplished by application of a cream, rinse, gel, etc. capable of allowing the inhibitors of serine proteases to penetrate the skin and enter the blood stream.
• osmotic pumps may be used for administration. The necessary dosage will vary with the particular condition being treated, method of administration and rate of clearance of the molecule from the body.
• the subject may be a mammal such as a human or a veterinary and/or a domesticated animal or livestock or wild animal.
• methods provide for inducing an immune response to dengue virus serotype(s) using a mono, bi-, tri or tetravalent formulation of live, attenuated and/or chimeric viral constructs contemplated herein.
• compositions used to generate as referred to herein as "master virus seeds (MVS)" are disclosed. These compositions may be derived from one or more live, attenuated dengue viruses, such as DEN-1, DEN-2, DEN-3, and DEN-4. In certain methods, compositions may be derived from one or more live attenuated Dengue viruses that include but are not limited to, specific constructs disclosed herein referred to as DENVax- 1, DENVax-2, DENVax-3, and DENVax-4. In other exemplary methods, strategies used to generate and characterize these compositions are provided. In yet other embodiments, tetravalent dengue virus formulations and genetic and phenotypic characterization of these formulations are provided.
• DENVax viruses were re-derived by transfection of viral RNA transcribed from the full- length recombinant DENVax cDNA into production-certified cells (e.g. Vero cells), resulting in PI (passage 1) virus seed.
• production-certified cells e.g. Vero cells
• PI passage 1 virus seed.
• the four PI viruses from each of dengue- 1 to dengue-4 were then amplified and plaque purified to obtain the candidate pre-master vaccine P7 seeds (see Table 1).
• Certain tests were performed to analyze passages of dengue viruses.
• P7 DENVax seeds Some tests were further performed to characterize P7 DENVax seeds, such as analysis of genome sequences and plaque phenotypes of the P7 seeds, and comparison with P2 seeds (Table 2). Plaque phenotypes of the P7 viruses were generally similar to those of the P2 seeds. In some exemplary experiments, virus titers were monitored. Virus titers reached over 6.0 log pfu/ml for most of the P7 seeds, except for 5 viruses.
• Genome sequencing of more than 60 candidate vaccine virus seeds after 10 or more serial passages in Vero cells identified no reversion event at NS1-53 and NS3-250 of the three major attenuation determinants of the DENV-2 PDK-53 genetic vector, suggesting that these 2 loci are quite stable in candidate vaccine virus seeds. All sequence chromatograms of the 24 candidate strains generated from both forward and reverse sequencing for these two sites were homogenous without any minor nucleotide populations evident at the NSl -53 and NS3-250 genetic loci.
• TaqMAMA mismatch amplification assay
• the sensitivity limit of the TaqMAMA ranged between 0.01% and 0.07% reversion, which is much more sensitive than the 10-30% reversion sensitivity limit detectable by consensus genome sequence analysis.
• the resulting data illustrates that 15 of the 24 P7 viruses had minimal or undetectable reversion ( ⁇ 0.07%), one virus (DENVax-3-D) had almost 100% reversion, and 8 viruses (1 DENVax-1, 1 DENVax-2, 2 DENVax-3, and 4 DENVax-4) had partial reversion ranging from 0.08% to 12.85% (Table 2).
• DENVax- 1 -A, DENVax-2-F, and DENVax-4-F had two non-synonymous mutations, and the DENVax-3-F had one.
• the evidence suggests these additional mutations observed in these 4 pre-master seeds do not cause safety concerns or immunogenicity alterations for the viruses.
• These pre-master seeds were further amplified to generate the MVS (master seed, designated as P7, Table 1).
• RNA from cloned cDNA plasmid as the pure source to transfect vaccine-certified Vero cells to generate vaccine virus.
• Serial plaque purifications and full-genome sequence analyses were incorporated into the manufacturing procedures to ensure manufactured vaccine seeds with optimal purity and genetic stability.
• Six cloned viruses were prepared as potential pre- master seeds for each serotype of DENVax.
• pre-master seeds were chosen to advance to master virus seeds production for each serotype (serotypes 1-4).
• the selected pre-master seeds had undetectable reversions ( ⁇ 0.01% or ⁇ 0.07%) at the 5'NCR-57 locus, with 1 or 2 amino acid substitutions in their genomes, and retained the small plaque phenotypes previously observed.
• Table 2 Characterizations of pre-master (P7) seeds
• P2 similar to P2 virus
• L larger than P2 virus
• D similar size, but appear somewhat different in clearness of the plaques
• S smaller than P2.
• Example 2 [000100] In some exemplary methods, compositions of master virus seeds, working virus seeds and bulk virus seeds as well as their genetic and phenotypic characterization are described. These compositions are provided for manufacture of clinical materials and ultimately commercial vaccine supplies. Serial plaque purifications and full-genome sequence analyses were incorporated into the manufacturing process to ensure compositions of vaccine seeds with optimal safety and genetic stability for manufacture of clinical trial materials.
• MVS of the 4 DENVax were produced by amplifying the pre-master P7 seed in certified Vero cells. In other studies, MVS were used to make large amount of WVS in cell factories. Further, the BVS stocks of DENVax were amplified from the WVS and were formulated into tetravalent drug product mixtures to be used used for human clinic trials. Quality controls for product release were performed in some exemplary methods, including, but not limited to, testing all of the MVS, WVS, and BVS for identity, infectious titer, sterility, mycoplasma, and in vitro and in vivo adventitious agents.
• the DENVax-2 MVS was identical to its pre-master seed, and the WVS and BVS had 2 additional mutations at NS4A-36 and NS4B-11 1. Both mutations were partial in WVS and were the major genotype in the BVS.
• the MVS of DENVax-3 was again identical to the pre-master seed, but the WVS and BVS contained an additional aa substitution at NS4A-23.
• the DENVax-4 MVS acquired an additional amino acid mutation, at locus NS2A-99 (from Lys to Lys/Arg mixed genotype) during production of the MVS (Table 3).
• NS2A-66 mutation of the DENVax-4 is in the nonstructural backbone part of the DENV-2 PDK-53. Although NS2A-66 locus is usually Asp among various strains of DENV-2, it is usually Gly for DENV-4. It is possible that the Asp to Gly change in the DENVax-4 is relevant for fitness of the DENVax-4 in Vero cells.
• the DENVax-2 prM-52 mutation resides in the C-terminal portion of the prM that is cleaved out from the mature virus particles.
• phenotypic characterization was performed to confirm that none of the mutations in the MVS seeds significantly altered the attenuation phenotypes of the vaccine.
• the DENVax viruses demonstrated high genetic stability during the manufacturing process.
• the three defined DENV-2 PDK-53 attenuation loci located in 5'NCR, NS1-53, and NS3-250 remained stable in the consensus genome sequence upon serial passage of the DENVax from pre-Master strains to bulk vaccine preparations.
• the highly sensitive TaqMAMA of the 5'NCR-57 locus demonstrated minimal or undetectable reversion in the MVS, WVS (P9/Working), and BVS (Bulk Virus Seed for vaccines) of dengue virus serotypes.
• the 5'NCR-57 reversion rates of the DENVax BVS preparations were significantly lower than the 5'NCR-57 reversion rates that evolved in research-grade vaccine candidates after 10-serial passages in Vero cells (4-74% reversion).
• the strategy for large-scale manufacturing of the DENVax seeds provided herein resulted in a genetically stable vaccine seed which retained the attenuation markers in the candidate vaccine viruses.
• plaque phenotypes of the DENVax MVS were compared with wild type Dengue viruses and their homologous research-grade chimeric viruses in Vero cells (Fig. 2). All of the MVS of DENVax- 1, -2, and -3 produced plaques that were significantly smaller than their wild type homologs and very similar (within 0.4- mm differences) to their homologous research-grade viruses in Vero cells.
• DENVax-4 MVS was also significantly smaller than the wild type DENV-4, but was slightly larger (0.9 mm difference) than the original lab derived D2/4-V chimera.
• Fig. 2 represents an exemplary histogram illustrating plaque sizes of the DENVax MVS in contrast with control wild type viruses and research-grade vaccine candidate viruses.
• temperature sensitivity was tested in Vero cells for the DENVax MVS and compared with their homologous wild type and the original research-grade chimeric vaccine virus.
• the wild type (wt) DENV-3 16562 was not temperature sensitive.
• the wt dengue virus serotype 1 and dengue virus serotype-4 were moderately temperature sensitive at 39°C (titers were approximately 1.0 logiopfu/ml lower at 39°C than at 37°C, Fig. 3).
• Wt Dengue virus serotype-2 16681 was the most temperature sensitive of the wt Dengue viruses tested, and resulted in a 100-fold titer drop at 39°C.
• DENVax- 1, -2, and -3 were as temperature sensitive as their original homologous research- grade chimeric vaccine viruses (Fig. 2). Titers at 39°C dropped between 2.0 and 3.0 log 10 pfu/ml for these DENVax strains. DENVax-4 also was temperature sensitive,
• DENVax-4 MVS contained F 127 (and other agents known to stabilize these formulations (FTA)), which was shown to enhance thermal stability of the Dengue viruses.
• FTA agents known to stabilize these formulations
• the presence of the F127 in DENVax-4 MVS likely contributed to the less pronounced temperature sensitivity of the virus in the Vero culture assay.
• temperature sensitivity of an MSV-derived DENVax-4 strain in the absence of F 127 was further evaluated. To remove the F127 from the strain, viral RNA was isolated from a DENVax-4 bulk virus preparation and was transfected into Vero cells. This DENVax-4 virus appeared to be as temperature sensitive as the D2/4 V research strain (titer reduced 1.5 log 10 pfu/ml) on day 3 pi in the absence of F 127 (Fig. 3).
• Fig. 3 illustrates an exemplary histogram illustrating temperature sensitivities of DENVax MVS.
• the wild type Dengue viruses and previously published research-grade vaccine candidate viruses were included for comparison.
• the DENVax-4 MVS contains additional F-127 that can mask the temperature sensitivity results of the virus in this assay.
• a separate experiment analyzing a surrogate DENVax-4 in the absence of F 127 was also included.
• the DENVax MVS were grown in C6/36 cells to verify their retention of the in vitro attenuation phenotype, with the knowledge that the research-grade chimeric vaccine viruses retained the attenuation phenotype of the backbone DENV-2 PDK53 virus in these mosquito cells.
• DENVax- 1, DENVax-2 and DENVax-4 MVS showed significant growth reduction (at least 3 logio pfu/ml reduction) in C6/36 cells on day 6 pi (Fig. 4).
• the DENVax-3 MSV also exhibited reduced growth compared to the wt DENV-3 16562, but the reduction was not as marked (1-2 log 10 pfu/ml reduction).
• the C6/36 titers of the DENVax-3 seed lots were similar (within 1 logiopfu/ml difference) to the C6/36 titer of the original research- grade chimeric D2/3-V vaccine virus.
• Fig. 4 illustrates an exemplary histogram plotting restricted growth of DENVax MVS (grey bars) in C6/36 cells in comparison with wt Dengue viruses (black bars) and research-grade vaccine viruses (white bars). Mean titers ⁇ SD (error bars) of the viruses replicated in C6/36 cells 6 days pi.
• the infection and dissemination rates of the DENVax were compared with their parental wt Dengue viruses.
• oral infection experiments were conducted i Ae. aegypti mosquitoes.
• DENVax-4 did infect some mosquitoes through oral feeding (2 out of 55), the infection rate was significantly lower (p ⁇ 0.05) than its parental wt virus, DENV-4 1036 (8 out of 50).
• DENVax-3 did not infect any mosquitoes in two experiments with blood meal viral titers of 5.2 ⁇ 0.02 logio pfu/ml (Table 4), and in a separate experiment with blood meal viral titer of 6.0 logio pfu/ml, only 1 out of 30 mosquitoes became infected (data not shown).
• wt Dengue virus-3 16562 also had a very low infection rate (8%) at 5.2 logio pfu/ml, and the rate did not increase in a separate experiment with a higher blood meal viral titer at 6.2 logio pfu/ml (3%, 1 positive out of 30 mosquitoes, data not shown).
• the wild type (wt) Dengue virus-3 and Dengue virus-4 had significantly lower infection rates than the wt Dengue virus- 1 and Dengue virus-2, the mean virus titers in the infected mosquitoes were similar (3.1 to 3.9 logio pfu/mosquito).
• DENVax-4 titers from the two infected mosquitoes were both minimal (0.7 logio pfu/mosquito), which was 1,000-fold lower than the titer from the mosquitoes infected by wt Dengue virus serotype-4 1036 (3.9 ⁇ 1.5 pfu/mosquito).
• Table 4 Virus infection, dissemination, and transmission rates in whole mosquitoes DENV-1 44.0% 3.6 ⁇ 36.3% 100% 4.7 ⁇ 43%
• Vector competence is an important safety component for live-attenuated flavivirus vaccine viruses.
• the research-grade DENV-2 PDK-53-VV45R virus and wt derivatives were tested in Ae. aegypti, and found that the NS 1-53 -Asp attenuating mutation was the dominant determinant for impaired mosquito replication.
• the other two major attenuation loci of the DENV-2 PDK-53 vaccine, nucleotide 5'NCR-57-T and NS3- 250-Val also exhibited some inhibiting effect on replication in mosquitoes, thus providing additional, redundant restrictions for mosquito vector competence.
• Some exemplary methods described herein were used to test the mosquito oral and IT infection and replication for all four DENVax strains. DENVax- 1, -2, and -3 did not infect any Ae.
• the infection rates for the DENVax- 1, -2, and -4 were all significantly less than their wt counterparts, but the difference was not significant between DENVax-3 and wt DENV-3 16562 due to the very low infection rates for both viruses.
• the parental wt Dengue virus strains used for engineering DENVax appeared to have lower infectious and dissemination rates by oral infection.
• the wt DENV-1 PU0359, DENV-2 PU0218, DENV-3 PaH881/88, and DENV-4 1288 used for engineering the Yellow Fever (YF) 17D vaccine-based ChimeriVax-DEN vaccines had infection rates ranging 47-77%. In contrast, the YF 17D vaccine cannot infect Ae. aegypti. Although the ChimeriVax strains contained the prM-E from these highly infectious wt DENV, the ChimeriVax retain the mosquito attenuation phenotype of their YF 17D replicative backbone. Results provided herein also indicated that the mosquito attenuation of DENV-2 PDK-53 backbone was maintained in the DENVax strains. In addition, using the wt Dengue virus strains with lower mosquito-infectivity in constructs included in compositions described herein provides an additional safety feature.
• the oral infection results illustrate that the DENVax had minimum mosquito infectivity and dissemination capacity.
• the more sensitive and stringent IT infection experiments were performed to further analyze the potential of DENVax to be transmitted by Ae. aegypti.
• the IT results demonstrated that all four DENVax viruses had non-detectable or minimal mosquito transmission potential compared to their wt counterparts.
• DENVax transmission could only theoretically occur if (1) vector feeds on a vaccinee with a sufficient viremia titer to infect mosquito midgut, (2) the virus is capable of replicating in the midgut epithelium and able to subsequently disseminate out of the midgut, and (3) the disseminated virus can replicate in salivary gland and expectorate sufficient virus in saliva for transmission.
• the threshold of human viremia required to infect mosquitoes has not been established adequately, but human viremia can be 10 6 -10 8 mosquito infectious doseso (MID5o)/ml after natural wt DENV infection. This MID50 was based on direct IT inoculation of mosquitoes with diluted human plasma.
• any of the passages of any of the serotypes could be used in a composition to generate a safe and effective vaccine against one, two, three or all four dengue virus serotypes.
• 5A summarizes the neurovirulence of wt Dengue virus serotype-2 16681 in CDC-ICR colony and Taconic-ICR newborn mice challenged ic with 10 4 pfu of the virus.
• the Taconic-ICR mice (100% mortality in 32 mice, average survival time of 8.3 ⁇ 0.5 days) were more susceptible to ic Dengue virus serotype-2 16681 challenge than the previous CDC-ICR mice (91% fatalities in 72 mice, average survival time of 14.6 ⁇ 2.3 days).
• D2/3-V research grade virus, as well as DENVax-1, and DENVax-3 MVS exhibited fully attenuated neurovirulence phenotypes (no illness or mortality).
• wt Dengue virus serotype-2 was found to be "fatal", with average mouse survival time (AST) of 8.3 ⁇ 0.8 days.
• AST average mouse survival time
• the D2 PDK-53-VV45R research grade virus resulted in 81.3% mortality.
• the DENVax-2 MVS and DENVax-4 MVS were uniformly fatal in the Taconic-ICR, showing AST values of 9.8 ⁇ 1.7, 10.2 ⁇ 1.4, and 1 1.3 ⁇ 0.4 days, respectively.
• the neurovirulence of wt Dengue virus serotype-2 16681 virus was compared with that of D2 PDK-53 VV45R, DENVax-2 MVS and DENVax-4 MVS, as well as D2/4-V research grade virus, at a 10-fold lower dose (10 3 pfu, Fig. 5C).
• the wt Dengue virus serotype-2 retained a uniformly fatal neurovirulent phenotype, with AST of 9.0 ⁇ 1.4 days, at this lower challenge dose.
• the other 4 viruses exhibited intermediate neurovirulence phenotypes, and the degree of neurovirulence was serotype-specific.
• the D2 PDK-53-VV45R virus and its DENVax-2 MVS cognate showed significant attenuation (32.3% survival with AST of 13.1 ⁇ 3.8 days and 31.2% survival with AST of 10.5 ⁇ 3.4 days, respectively).
• Both the DENVax-4 MVS and the research grade D2/4-V virus were highly attenuated for neurovirulence (81.3% survival with AST of 18.8 ⁇ 5.8 days and 100% survival, respectively).
• DENVax-4 MVS was also significantly smaller than the wt DENV-4, but was slightly (0.9 mm) larger than the original lab derived D2/4-V chimera.
• DENVax-2 all of the WVS and BVS of the DENVax- 1, -3, -4 retained significantly smaller plaque sizes than those produced from their wt homologs.
• the DENVax-2 WVS and BVS produced plaques that were similar to the plaques of wt DENV- 2 virus in Vero cells, but when tested in LLC-MK 2 cells all of the DENVax-2 manufactured seeds produced plaques that were somewhat smaller than those of the wt DENV-2 (1.4 ⁇ 0.4) and similar to the lab derived D2 PDK-53-VV45R (1.0 ⁇ 0.3) (Fig. 6).
• Fig. 6 represents an exemplary histogram illustrating plaque size of the DENVax MVS, WVS, and BVS. Mean plaque diameters ⁇ SD (error bars) of the virus plaques in Vero or LLC-MK 2 cells under agarose overlay measured on day 9 pi. The wt DENVs and previously published research-grade vaccine candidate viruses were included for control and comparison.
• the DENVax-3 seeds also exhibited reduced growth compared to the wt DENV-3 16562, but the reduction was not as marked (1-2 log 10 PFU/ml reduction). However, the titers of the DENVax-3 seed lots were similar (within 1 log 10 PFU/ml difference) to the original research-grade chimeric D2/3-V vaccine virus.
• Fig. 8 represents an exemplary histogram plotting restricted growth of DENVax MVS, WVS, and BVS in C6/36 cells. Mean titers ⁇ SD (error bars) of the viruses replicated in C6/36 cells 7 days pi. The wt Dengue viruses and previously published research-grade vaccine candidate viruses were included for comparison.
• Figs. 9A-9B represent exemplary graphs of data of neurovirulence of DENVax MVS in newborn ICR mice.
• the highly sensitive TaqMAMA of the 5'-NCR-57 locus showed minimal or undetectable reversion in the MVS, WVS, and BVS of DENVax.
• the highest reversion rate of 0.21% was identified in the DENVax-2 BVS.
• the reversion rates of the P10-equivalent BVS ( ⁇ 0.07% to 0.21%) were significantly lower than the reversion rates that evolved in other vaccine candidates after serial passages in Vero cells (4-74% reversion by P 10). This suggests that this strategy for large scale manufacturing of the DENVax seeds is successful, regarding maintaining genetic stability and retention of attenuation markers in the candidate vaccine viruses.
• MVS stocks disclosed herein will be used for future manufacturing of WVS and BVS lots, full panels of virus attenuation phenotype evaluations, including small plaque phenotype, temperature sensitivity, reduced replication in mosquito cells, reduced infection/dissemination/transmission in whole mosquitoes, and reduced neuro virulence in newborn ICR mice, were conducted for all MVS or their equivalent surrogate stocks.
• virus attenuation phenotype evaluations including small plaque phenotype, temperature sensitivity, reduced replication in mosquito cells, reduced infection/dissemination/transmission in whole mosquitoes, and reduced neuro virulence in newborn ICR mice.
• For the WVS and BVS stocks plaque size, infectivity in mosquito cells, were also performed to confirm their attenuations.
• Exemplary methods are provided herein to demonstrate manufacture of DENVax MVS, WVS, and BVS stocks under cGMP.
• the BVS stocks were used to formulate the tetravalent DENVax currently in human clinical trial evaluations.
• a unique manufacture strategy to optimize the genetic stability and safety of the manufactured MVS was provided in some exemplary methods. Since the main attenuation loci of the DENVax have been well characterized previously and a highly sensitive and quantifiable SNP assay, TaqMAMA was developed to integrate genome sequence and the TaqMAMA to identify optimal pre-master seeds for making the MVS. The genetic and phenotypic
• Fig. 10 represents an exemplary table comparing new live, attenuated viruses to previously generated live, attenuated dengue viruses. Mutations are indicated where different from a control virus (e.g. 16681), or other live, attenuated dengue-2 viruses. Materials and methods
• DENV-1 16007, DENV-2 16681, DENV-3 16562, and DENV-4 1034 served as wild-type (wt) DENV controls, and they were the parental genotype viruses for the four recombinant DE Vax vaccine candidates.
• DENVax progenitor research-grade viruses designated as D2/1-V, D2 PDK-53-VV45R, D2/3-V, and D2/4-V, were prepared and characterized previously.
• Vero African green monkey kidney cells used for making the master and working cell banks for vaccine production were originated from the American Type Culture Collection (ATCC) CCL81 cell line that has been characterized by the World Health Organization (WHO) for vaccine manufacture (WCB-Vero cells).
• ATCC American Type Culture Collection
• WHO World Health Organization
• RNA A-cap analog m7G(5')ppp(5')A (New England BioLabs) was incorporated during the 2-hr transcription reaction to add the 5 '-terminal A-cap to the RNA transcript.
• the samples were then treated with DNase I to digest the template cDNA, followed by low pH phenol/chloroform extraction and ethanol precipitation to remove residual DNA and proteins.
• the purified RNA transcripts, suspended in RNase-free water, were distributed in 20- ⁇ 1 aliquots and stored at -80°C until ready for transfection of cells. The integrity and concentration of the RNA transcripts were analyzed by agarose gel electrophoresis. Each 20- ⁇ 1 aliquot was estimated to contain sufficient genome-length viral RNA to permit transfection of 0.4-1 x 10 7 production-certified Vero cells by electroporation.
• RNA transcripts were thawed, mixed with 400 ⁇ of the Vero cell suspension (1 x 10 7 cells/ml), and transferred to a pre-chilled sterile electroporation cuvette (4-mm gap) for electroporation by a Gene Pulser Xcell total system (BioRad Laboratories).
• Each sample was pulsed once at 250V/ ⁇ Ohms/500 ⁇ , incubated for 10-15 min at room temperature, transferred to a 75-cm 2 flask containing 30 ml of cell growth medium (MEM with 10% FBS), and incubated at 36°C ⁇ 1°C, 5% C0 2 for 6 to 11 days.
• the culture medium was harvested, clarified by centrifugation, stabilized, and stored in small aliquots below -60°C.
• the viral titers of candidate vaccine stocks (termed PI for passage level 1) resulting from transfection were determined by plaque titration assay in Vero cells and used for further propagation of the DENVax seeds.
• PI virus seeds were used to propagate DENVax pre-master, master, working, and bulk virus seed lots through a strategy designed to ensure the optimal genetic stability and safety of the manufactured lots.
• This strategy included three serial plaque purifications, as well as genetic analyses of viruses at various passage levels to select the optimal clonal virus population for continued seed production (Table 1). Briefly, the PI seeds harvested from transfected cells were amplified once by infection of Vero cells at a MOI of 0.001 to generate the P2 seeds. Aliquots of the P2 seed stocks were evaluated by plaque morphology and complete viral genomic sequencing. The genetically confirmed P2 stocks were plated on Vero cell monolayers with overlay medium as described in the plaque titration section below to generate well-isolated plaques.
• plaque clones A to F plaque clones A to F
• passage P3 0.5 ml of culture medium
• Each of the six plaque suspensions was subjected to two additional rounds of plaque purification, resulting in twice- and thrice- plaque purified virus seeds at passages P4 and P5, respectively.
• the P5 viruses were amplified through two sequential Vero passages to produce P7 seed stocks.
• Each selected pre-master seed was expanded to master virus seed (MVS or P8) by a one-time passage of the virus at MOI of 0.001 in multiple 175 cm 2 flasks of Vero cells. Except for the DENVax-4 MVS, the master virus seeds were harvested at 8-10 days post infection (pi). The MVS stocks were harvested at 6-10 days post infection (pi), clarified by centrifugation, stabilized by the addition of sucrose/phosphate/glutamate solution (final concentration 7.5 % sucrose, 3.4 mM potassium dihydrogen phosphate, 7.2 mM dipotassium hydrogen phosphate, 5.4 mM monosodium glutamate, respectively) and 0.95 to 1.90% FBS (final concentration).
• sucrose/phosphate/glutamate solution final concentration 7.5 % sucrose, 3.4 mM potassium dihydrogen phosphate, 7.2 mM dipotassium hydrogen phosphate, 5.4 mM monosodium glutamate, respectively
• DENVax-4 MVS was prepared differently to optimize its yield. Briefly, multiple flasks of cells were infected with DENVax-4 pre-master seed at a MOI of 0.001 in the presence of 0.1% F-127TM, poloxamer 407, (other EO-PO block copolymers have been assessed and may substitute here, see issued patent ) that have been demonstrated to enhance DENV virus thermal stability. Infectious media was harvested days 6-10 pi, and stabilized with 17% FBS (final concentration), pooled, and frozen. All four DENVax MVS stocks were stored as 1-ml aliquots below -60°C.
• the DENVax working virus seeds were prepared by one-time passage in Vero cell culture of the MVS at a MOI of 0.001. The procedures were similar to the production of MVS, except they were cultured in multiple-layer cell factories (6360 cm 2 ). The WVS stocks were filtered through 10 ⁇ and 0.45 ⁇ filters, stabilized with the same stabilizers used for the MVS, aliquoted into 30ml PETG bottles or 2.0 ml cryovials, and stored below-60°C.
• BVS bulk virus seeds
• Viruses were harvested between day 5 and day 10 pi, and immediately clarified by filtration through a 0.45 um pore size filter, and 1L of each clarified virus pool was stabilized by addition of 500 ml of 3x FTA buffer (final concentrations of 15% trehalose, 1.0% Pluronic ® F-127TM poloxamer 407, 0.1% human albumin USP in PBS, pH 7.4).
• the stabilized virus was distributed into 1-L PETG bottles and stored frozen below -60°C for subsequent pooling and quality control testing. All stabilized virus harvests with a virus titer above 10 5 PFU/ml and an acceptable level of residual DNA were rapidly thawed in a water bath at 32°C, then aseptically pooled and mixed. Each pooled monovalent BVS was distributed into labeled PETG containers and stored at below -60°C until further use.
• the MVS, WVS, and BVS seeds were tested for identity, sterility, and detectable adventitious agents.
• the identity of each vaccine stock was confirmed by RT- PCR with DENVax serotype-specific primers.
• the amplified cDNA fragments contained the E/NS1 chimeric junction site to permit identification of each of the four DENVax serotypes.
• Each seed was tested in all 4 serotype-specific RT-PCR reactions to confirm viral identity and freedom from cross contamination with heterologous DENVax serotypes.
• Sterility testing was performed in accordance with USP 71 (United States Pharmacopeia, section 71). Mycoplasma testing was performed.
• the culture supernatant was removed and replaced with 10 mL of a guinea pig red blood cell (RBC) solution (3 mL of 0.5% guinea pig RBC in phosphate buffered saline, made up to 10 mL with cell growth medium).
• RBC red blood cell
• the flasks were then incubated at 5 ⁇ 3°C for 30 minutes followed by incubation at room temperature for 30 minutes.
• the monolayers were washed with PBS and observed under 10 X magnification for the presence of any star-shaped clumps of RBCs for hemadsorption.
• Post-weaning mice were observed for a total of 28 days, and guinea pigs were observed for a total of 42 days following inoculation.
• the test articles met the acceptance criterion if >80% of the inoculated animals remained healthy throughout the observation period.
• the in vivo testing for contaminants was also performed in embryonated chicken eggs and was conducted. For every sample, 10 embryonated hen eggs (9 days old) were each inoculated with 0.5 mL of the DENV antiserum-neutralized sample into the allantoic fluid and incubated at 35°C for 3 days. The allantoic fluids from these 10 eggs were harvested, pooled and passaged into the allantoic fluid of 10 fresh embryonated eggs (10-11 days old; 0.5mL/egg) and incubated at 35°C for a further 3 days.
• Virus titers were measured by plaque assay or immunofocus assay using Vero cells. Plaque assays were performed in double agarose overlays in six-well plates of confluent Vero cells as previously described, and they were also used to evaluate the plaque phenotypes of the DENVax seeds. For accurate comparison, plaque sizes of all viruses were measured and compared in the same experiment. After visualization with neutral red on day 9 pi, up to 10 well isolated plaques for each virus were measured for mean plaque size calculation. Fewer plaques were measured for wt DENV-1, -3, and -4, whose larger plaque sizes often did not permit measurement of 10 well-separated plaques.
• DENVax MVS were compared with the plaque assays to ensure virus titration results were comparable between the two assays.
• the immunofocus assay was conducted in 6-well plates of confluent Vero cells infected with serially diluted viruses. Cells were overlayed with a balanced salt medium (BSS/YE-LAH medium) containing 0.7% high viscosity carboxymethyl cellulose (Sigma) and incubated for 7 days at 37°C with 5% CO 2 .
• BSS/YE-LAH medium balanced salt medium containing 0.7% high viscosity carboxymethyl cellulose (Sigma)
• cell sheets were washed 3 times with PBS, fixed with cold 80% acetone for 30min at -20°C, washed once with PBS, and blocked with a blocking buffer containing 2.5% (w/v) nonfat dry milk, 0.5% Triton X-100, 0.05% Tween-20 in PBS at 37°C for 30 min.
• Blocked cells were incubated with diluted DENV serotype-specific MAbs, 1F1 (DENV-1), 3H5 (DENV-2), 8A-1 (DENV-3), or 1H10 (DENV-4) in blocking buffer at 37°C for 1 hour or 4°C overnight, washed 3 times with washing buffer (0.05% Tween-20 in PBS), and incubated with alkaline phosphatase- or horse radish peroxidase (HRP)- conjugated affinity-pure goat anti-mouse IgG (Jackson Immuno Research Laboratories) at 37°C for 45-60 min.
• HRP horse radish peroxidase
• Taqman-based mismatch amplification mutation assay (TaqMAMA)
• TaqMAMA is a sensitive, quantitative single nucleotide polymorphism assay developed to permit finer assessment of the level of reversion at the 5'NC-57 locus of attenuation, and was further optimized for this study.
• Extracted viral RNA from MVS and WVS were analyzed by the TaqMAMA with both sets of primers/Taqman probe that are specific to wt or the vaccine 5TSTC-57 region.
• the forward primers used to detect DENV-2 wt and vaccine sequences were D2-41-GC and D2-40-TT, respectively.
• each forward primer matched the specific 5'NCR-57 nucleotide for each virus, while the nucleotide adjacent to the 3 '-terminal nucleotide in each primer differed from the DENV-2 viral genomic sequence to enhance the mismatch effect.
• the reverse primer, CD- 207, and the Taqman probe, CD-169F, for both wt and vaccine sets were identical.
• RT-PCR was performed with the iQ5 or CFX-95 system (BioRad), using a BioRad iScript RT-PCR (for probes) kit, in a 25- ⁇ 1 reaction containing 5 ⁇ of viral RNA template, 0.4 uM of each primer, and 0.2 uM of the probe. Triplicate reactions for each wt- and vaccine-specific assay were conducted for each sample. Genome copy numbers were determined relative to a standard curve prepared for each viral genotype, where the RNA standards were transcripts derived from plasmids containing nt 1-2670 of each genotype- specific cDNA.
• the specificity of the assay was confirmed by testing each RNA standard with the heterologous genotype primer/probe sets to ensure minimum cross- reactivity in every experiment. The results were reported as the percentage of viral genomes showing reversion. Previously, due to higher cross-reactive backgrounds that limited the input RNA levels for this assay, the original detection sensitivity was about 0.1% reversion (discrimination power). Since then, the assay has been further optimized using improved real-time PCR equipment and reaction kits, and the cross-reactive background was decreased considerably at much high levels (7-8 logio copies) of RNA template input. This optimization resulted in significant improvement of the detection sensitivity, down to 0.01-
• C6/36 mosquito cells (Aedes albopictus).
• C6/36 cells grown in 6-well plates were infected in duplicate with each virus at a MOI of 0.001 and incubated with 4 ml/well of DMEM medium containing 2% FBS in a 5% CO 2 incubator at 28°C. Small aliquots of the culture supernatant were collected for each virus on day 6 pi, mixed with an equal volume of medium containing 40% FBS, and stored at -80°C until ready for virus plaque titration.
• Temperature sensitivity was conducted by comparing viral growth at 39°C versus growth at 37°C at five days pi of Vero cells in 6-well plates. Cells were infected in quadruplicate with each virus at a MOI of 0.001 at 37°C. Following adsorption of virus, the infected cultures were incubated with 4 ml/well of DMEM medium containing 2% FBS in 2 separate 5% CO 2 incubators, one set (duplicate plates) at 37°C and the other at 39°C.
• Aedes aegypti mosquitoes used for the study were from a colony established in 2002 from a village near Mae Sot (16' N, 33' E), Thailand. After emerging from larvae, adult mosquitoes were maintained at 28°C at a 16:8 (ligh dark) photoperiod with 10% sucrose solution provided ad libitum. Five-to-seven day old female mosquitoes were used for infectious blood meal feeding or intrathoracic (IT) inoculations. Aliquots of freshly cultured DENVax and wt DENV were used immediately upon harvest (without any freeze- thaw cycle) to make virus blood meals as indicated below for oral infection.
• Remaining virus supernatants were supplemented with FBS to a final concentration of 20%, and aliquots were stored at -80°C for future virus plaque titration and IT inoculation experiments.
• the freshly prepared DENVax seeds for these experiments were amplified from the pre- master seeds in Vero cells, and were considered DENVax MVS equivalents.
• Infectious blood meals were prepared by mixing fresh virus at a ratio of 1 : 1 with defribrinated chicken blood (Colorado Serum Company) on the day of oral infection. Mosquitoes were sugar-starved overnight and then offered the virus :blood mixture for 1 hour using a Hemotek membrane feeding system (Discovery Workshops). A 50- ⁇ 1 aliquot of the blood meal was retained at -80°C for back-titration of virus doses. Fully-engorged females were sorted under cold anesthesia and placed into cartons with 10% sucrose solution provided ad libitum. Cartons were placed at 28°C with a photoperiod of 16:8 h
• body and leg samples were homogenized with copper coated BBs (Crossman Corporation, NY) at 24 cycles/second for 4 min using a mixer mill, and then clarified by centrifuging at 3,000 x g for 3 min.
• Saliva samples were centrifuged at 3,000 x g for 3 minutes to expel fluid from capillary tubes.
• Ten-fold dilutions of the body and leg homogenates and saliva samples were tested for presence of infectious virus by plaque assay. Results from bodies, legs, and saliva were used for determining the infection, dissemination, and transmission rates, respectively.
• Nucleotide sequence of the chimeric viral genome and deduced amino acid sequence of the translated protein are provided herein.
• Most of the prM-E gene (nt 457 to - 2379, underlined) is wild-type (wt) DEN-1 16007 virus specific; the remaining genome is DEN-2 PDK-53 virus specific. All engineered substitutions differ from wt virus (Dl 16007 or D2 16681), as well as extra mutations (changes from engineered cDNA clone) detected in the MVS are marked.
• Ngo IV (nt 2380-2385): engineered mutations, nt-2381/2382 TG-to-CC (resulted in E-482 Val-to-Ala change)
• nt-8571 C-to-T silent mutation of PDK-53 is not engineered in the vaccine virus
• Engineered silent mutation remove the native DEN-1 virus-specific xbal site
• Nucleotide sequence of the recombinant viral genome and deduced amino acid sequence of the translated protein are provided herein.
• the engineered virus is based on D2 PDK-53 virus. All engineered substitutions that are different from wild-type DEN-216681 virus (also the parental virus for PDK-53), as well as extra mutations (changes from engineered cDNA clone) detected in the MVS are marked.
• NCR 5'-noncoding region
• nt-57 C-to-T major attenuation locus
• prM-29 Asp-to-Val nt-524 A-to-T
• NS5-412 Ile-to-Val (nt-8803 A-to-G), in bold 130 140 150 160 170 180 190 200 210 220 230 240
• Nucleotide sequence of the chimeric viral genome and deduced amino acid sequence of the translated protein are provided herein.
• Most of the prM-E gene (nt- 457 to -2373, underlined) is wild-type (wt) DEN-3 16562 virus-specific; the remaining nucleotide sequence is DEN-2 PDK-53 virus-specific.
• the E protein of DEN-3 virus has two fewer amino acids than the E protein of DEN-2. Therefore, nt position starting from NgoMIV is 6 nt less than the original DEN-2 PDK-53 nt position. All engineered substitutions differ from wt virus (DEN-3 16562 or DEN-2 16681), as well as extra mutations (changes from engineered cDNA clone) are marked.
• NgoM IV (nt 2374-2379): engineered mutations, nt-2375/2376 TG-to-CC
• D2 PDK-53 virus backbone (change from wt D2 16681): in bold
• NS3-250 Glu-to-Val (nt-5264 A-to-T): major attenuation locus (in red) e. nt-5541 (NS3 gene) T-to-C silent mutation
• nucleotide sequence of the chimeric viral genome and deduced amino acid sequence of the translated protein is wild-type (wt) DEN -4 1036 virus-specific; the remaining nucleotide sequence is DEN-2 PDK-53 virus-specific. All engineered substitutions differ from wt virus (DEN-3 16562 or DEN-2 16681), as well as extra mutations (changes from engineered cDNA clone) are marked.
• D2 PDK-53 virus backbone (change from wt D2 16681)
• NS3-250 Glu-to-Val (nt-5270 A-to-T): major attenuation locus (in red) e. nt-5547 (NS3 gene) T-to-C silent mutation (in bold)
• Engineered C-100 Arg-to-Ser may improve viral replication in culture

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Organic Chemistry (AREA)
• Virology (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Biochemistry (AREA)
• Engineering & Computer Science (AREA)
• Epidemiology (AREA)
• Immunology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Zoology (AREA)
• Microbiology (AREA)
• Gastroenterology & Hepatology (AREA)
• Biotechnology (AREA)
• General Engineering & Computer Science (AREA)
• Wood Science & Technology (AREA)
• Biomedical Technology (AREA)
• Communicable Diseases (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Mycology (AREA)
• Oncology (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Tropical Medicine & Parasitology (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (31)

Applications Claiming Priority (2)

Publications (2)

Family

ID=50678274

Family Applications (1)

Country Status (32)

Cited By (14)

Families Citing this family (3)

Family Cites Families (53)

• 2014
  • 2014-03-12 KR KR1020157029897A patent/KR102389908B1/ko active Active
  • 2014-03-12 PE PE2015001953A patent/PE20160027A1/es unknown
  • 2014-03-12 EP EP19168475.2A patent/EP3539565B1/en active Active
  • 2014-03-12 SI SI201432055T patent/SI4129330T1/sl unknown
  • 2014-03-12 LT LTEP22182269.5T patent/LT4129330T/lt unknown
  • 2014-03-12 KR KR1020257006481A patent/KR20250033325A/ko active Pending
  • 2014-03-12 PE PE2021000217A patent/PE20211814A1/es unknown
  • 2014-03-12 CA CA3166063A patent/CA3166063A1/en active Pending
  • 2014-03-12 CN CN202010617988.1A patent/CN111778263B/zh active Active
  • 2014-03-12 NZ NZ630869A patent/NZ630869A/en unknown
  • 2014-03-12 SG SG10201913435TA patent/SG10201913435TA/en unknown
  • 2014-03-12 HR HRP20231581TT patent/HRP20231581T1/hr unknown
  • 2014-03-12 EP EP14722014.9A patent/EP2968516B1/en active Active
  • 2014-03-12 CN CN202110718694.2A patent/CN113637693B/zh active Active
  • 2014-03-12 SG SG11201507460PA patent/SG11201507460PA/en unknown
  • 2014-03-12 PH PH1/2015/502120A patent/PH12015502120B1/en unknown
  • 2014-03-12 MY MYPI2015002317A patent/MY187796A/en unknown
  • 2014-03-12 CA CA3177572A patent/CA3177572A1/en active Pending
  • 2014-03-12 MX MX2015012893A patent/MX374519B/es active IP Right Grant
  • 2014-03-12 ES ES22182269T patent/ES2965652T3/es active Active
  • 2014-03-12 EP EP22204593.2A patent/EP4183411A1/en active Pending
  • 2014-03-12 SG SG10201913387XA patent/SG10201913387XA/en unknown
  • 2014-03-12 CA CA2903231A patent/CA2903231A1/en active Pending
  • 2014-03-12 CN CN202110723336.0A patent/CN113637086B/zh active Active
  • 2014-03-12 PT PT191684752T patent/PT3539565T/pt unknown
  • 2014-03-12 AU AU2014235476A patent/AU2014235476B2/en active Active
  • 2014-03-12 FI FIEP19168475.2T patent/FI3539565T3/fi active
  • 2014-03-12 CN CN202110718703.8A patent/CN113637694B/zh active Active
  • 2014-03-12 HU HUE19168475A patent/HUE061507T2/hu unknown
  • 2014-03-12 CN CN201480028001.5A patent/CN105451763B/zh active Active
  • 2014-03-12 PL PL19168475.2T patent/PL3539565T3/pl unknown
  • 2014-03-12 EP EP20154012.7A patent/EP3689374B1/en active Active
  • 2014-03-12 KR KR1020247001384A patent/KR102775714B1/ko active Active
  • 2014-03-12 WO PCT/US2014/024603 patent/WO2014150939A2/en not_active Ceased
  • 2014-03-12 DK DK22182269.5T patent/DK4129330T5/da active
  • 2014-03-12 EP EP22182269.5A patent/EP4129330B1/en active Active
  • 2014-03-12 ES ES20154012T patent/ES2933970T3/es active Active
  • 2014-03-12 KR KR1020227013084A patent/KR102626270B1/ko active Active
  • 2014-03-12 CA CA3177574A patent/CA3177574A1/en active Pending
  • 2014-03-12 JP JP2016501585A patent/JP6818548B2/ja active Active
  • 2014-03-13 US US14/209,808 patent/US9783579B2/en active Active
  • 2014-03-14 TW TW108114540A patent/TWI726312B/zh active
  • 2014-03-14 TW TW103109624A patent/TWI733646B/zh active
  • 2014-03-14 TW TW110111747A patent/TWI894248B/zh active
  • 2014-03-17 AR ARP140101223A patent/AR095598A1/es unknown
  • 2014-03-17 UY UY35489A patent/UY35489A/es active IP Right Grant
• 2015
  • 2015-09-11 DO DO2015000232A patent/DOP2015000232A/es unknown
  • 2015-09-14 MX MX2023000349A patent/MX2023000349A/es unknown
  • 2015-09-14 MX MX2019003602A patent/MX2019003602A/es unknown
  • 2015-09-14 MX MX2023000353A patent/MX2023000353A/es unknown
  • 2015-09-14 MX MX2023000354A patent/MX2023000354A/es unknown
  • 2015-10-15 CR CR20150569A patent/CR20150569A/es unknown
• 2017
  • 2017-04-20 US US15/492,981 patent/US10449231B2/en active Active
• 2019
  • 2019-06-05 JP JP2019105307A patent/JP7050031B2/ja active Active
  • 2019-08-16 AU AU2019216724A patent/AU2019216724C1/en active Active
  • 2019-09-05 US US16/561,755 patent/US20200061151A1/en not_active Abandoned
• 2021
  • 2021-09-17 US US17/478,537 patent/US12186363B2/en active Active
  • 2021-10-06 AR ARP210102772A patent/AR123702A2/es unknown
• 2022
  • 2022-12-12 US US18/064,775 patent/US11931399B2/en active Active
  • 2022-12-16 FR FR22C1064C patent/FR22C1064I2/fr active Active
• 2023
  • 2023-02-24 EC ECSENADI202313715A patent/ECSP23013715A/es unknown
  • 2023-03-08 NL NL301223C patent/NL301223I2/nl unknown
  • 2023-06-20 HU HUS2300022C patent/HUS2300022I1/hu unknown
  • 2023-06-27 FI FIC20230021C patent/FIC20230021I1/fi unknown
  • 2023-07-04 NO NO2023026C patent/NO2023026I1/no unknown
• 2024
  • 2024-02-28 AR ARP240100498A patent/AR132009A2/es unknown
  • 2024-03-05 LT LTPA2024503C patent/LTPA2024503I1/lt unknown
  • 2024-07-10 US US18/768,774 patent/US20250018006A1/en active Pending
  • 2024-07-16 AR ARP240101841A patent/AR133264A2/es unknown

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events