EP2858668A1 - Zusammensetzungen und verfahren zur verabreichung von impfstoffen gegen den dengue-virus - Google Patents

Zusammensetzungen und verfahren zur verabreichung von impfstoffen gegen den dengue-virus

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Publication number
EP2858668A1
EP2858668A1 EP13729909.5A EP13729909A EP2858668A1 EP 2858668 A1 EP2858668 A1 EP 2858668A1 EP 13729909 A EP13729909 A EP 13729909A EP 2858668 A1 EP2858668 A1 EP 2858668A1
Authority
EP
European Patent Office
Prior art keywords
dengue
vaccine
dengue virus
subject
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13729909.5A
Other languages
English (en)
French (fr)
Inventor
Dan T. Stinchcomb
Jorge E. Osorio
Charalambos D. Partidos
Joseph N. Brewoo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Takeda Vaccines Inc
Original Assignee
Takeda Vaccines Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US13/492,884 external-priority patent/US8968996B2/en
Application filed by Takeda Vaccines Inc filed Critical Takeda Vaccines Inc
Publication of EP2858668A1 publication Critical patent/EP2858668A1/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N2770/24143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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 of the present invention report compositions and methods for administering a vaccine to a subject against all dengue virus strains.
  • vaccine compositions may be administered by subcutaneous, intradermal, intramuscular or other injection or introduction methods.
  • injection in a subject of a vaccine against all dengue virus types includes multiple anatomical sites at day 0.
  • Other embodiments include follow-on injections from within days of the first treatment to up to 12 months after initial injection(s). In other embodiments, no additional injections are needed other than the day 0 treatment.
  • Vaccines for protection against viral infections have been effectively used to reduce the incidence of human disease.
  • One of the most successful technologies for viral vaccines is to immunize animals or humans with a weakened or attenuated strain of the virus (a "live, attenuated virus"). Due to limited replication after immunization, the attenuated strain does not cause disease. However, the limited viral replication is sufficient to express the full repertoire of viral antigens and can generate potent and long-lasting immune responses to the virus. Thus, upon subsequent exposure to a pathogenic strain of the virus, the immunized individual is protected from disease.
  • live, attenuated viral vaccines are among the most successful vaccines used in public health.
  • Embodiments of the present invention generally relate to methods and compositions for inducing protection in a subject against multiple dengue viruses by, for example, administering a multivalent dengue vaccine to a subject.
  • Some embodiments can include introducing a vaccine composition to a subject via intradermal (ID) injection.
  • the vaccine composition can be introduced to a subject intradermally to, for example, to induce neutralizing antibodies against three or more dengue virus serotypes.
  • a vaccine composition can include, but is not limited to, a single dose of one formulation of a multivalent dengue serotype vaccine having a predetermined ratio administered to a subject.
  • a vaccine composition may include, but is not limited to; an initial dose of one formulation of dengue vaccine (e.g. tetravalent formulations such as DENVaxTM) and then one or more boosts of the same, or a different formulation can be administered to a subject.
  • dengue vaccine e.g. tetravalent formulations such as DENVaxTM
  • compositions disclosed can be administered intradermally to a subject for modulating neutralizing antibody production in the subject against three or more dengue virus serotypes.
  • predetermined composition ratios e.g.
  • any ratio of three or more serotypes is contemplated) of the various serotypes of dengue virus or fragments thereof or attenuated compositions thereof in a single vaccine composition in order to increase cross protection and levels of neutralizing antibodies in a subject against at least three dengue virus serotypes when the subject is administered the single vaccine composition.
  • some advantages of using intradermal introduction of a vaccine against dengue virus can include, but are not limited to, multiple protection (cross protection) against some or all dengue virus serotypes in a subject, reduced cost by using reduced volumes of vaccine doses compared to subcutaneous injection, modulation of antibodies produced against some or all dengue virus serotypes in a subject and reduced pain at a site of administration in a subject administered a composition of vaccine against dengue virus.
  • a single dose vaccine against dengue virus can include one or more dengue virus serotype(s).
  • certain embodiments concern treating a subject with at least one additional injection(s) of a vaccine containing multiple dengue viruses administered at a separate site from the first injection, for example, in close proximity to the initial injection or in a distant anatomical site on the subject.
  • at least one additional intradermal injection(s) may be performed less than 30 days after the first administration to the subject while others are performed 30 days and up to 12 months after the first administration of the vaccine.
  • inventions disclosed herein relate to methods and compositions for inducing protection in a subject against all dengue virus serotypes by, for example, administering a vaccine to a subject against all dengue virus serotypes in two or more doses on one or more than one anatomical location consecutively within a short interval of time.
  • Some embodiments can include introducing a vaccine composition to a subject via intradermal (ID), subcutaneous (SC), or intramuscular (IM) injection in one location and consecutively in another anatomical location by ID, SC, IM or by other introduction method at a second different anatomical location.
  • ID intradermal
  • SC subcutaneous
  • IM intramuscular
  • inventions include using any combination of modes of administration for introducing a dengue virus vaccine of all dengue virus serotypes to a subject where administration of the vaccine occurs at two or more anatomical sites or by two or more different routes consecutively on the same day to the subject.
  • Some embodiments include treating a subject in need of dengue virus tetravalent vaccinations consecutively at two or more anatomical locations.
  • a subject may need two consecutive administrations in a single day to induce adequate levels of neutralizing antibodies which will protect against dengue infection.
  • a subject may be administered dengue virus multivalent vaccinations consecutively at two or more anatomical locations, then the subject can be administered at least a third vaccine within 30 days such as about 7, about 14, about 21 or about 28 days later with a composition comprising dengue virus serotypes which may or may not have all serotypes.
  • a subject may be administered dengue virus tetravalent vaccinations consecutively at two or more anatomical locations on day 0, then the subject can be administered at least a third vaccine within 30 days such as about 7, about 14, about 21 or about 28 days later with a composition comprising dengue virus serotypes which may or may not have all serotypes.
  • Vaccine compositions of these and other embodiments disclosed herein may include two or more dengue virus serotypes at a predetermined ratio for the subsequent administrations beyond the initial dual vaccination. These subsequent vaccinations may depend on personalized titers of antibodies post dual injection or other criteria such as results of test populations.
  • a subsequent vaccination may only include a single dengue serotype (e.g. DEN-4).
  • the composition introduced to the subject comprises vaccines against all dengue virus serotypes, for example tetravalent DENVaxTM or another similar formulation.
  • DENVaxTM comprises a tetravalent dengue vaccine of predetermined ratio where the vaccine is made up of constructs on an attenuated DEN-2 backbone (see for example, PCT Application Number PCT/USOl/05142 filed on February 16, 2001 incorporated herein by reference in its entirety for all purposes).
  • all dengue vaccine virus serotypes are in equal proportions in the composition.
  • each dengue vaccine virus serotype may be in a particular ratio to one another such that introduction of the composition induces sufficient levels of neutralizing antibodies which would provide the subject with sufficient protection against infection with three or more dengue viruses (e.g. DEN-1 , DEN-2, DEN-3 and/or DEN-4).
  • DEN-1 , DEN-2, DEN-3 and/or DEN-4 dengue viruses
  • a booster for that subject can contain a multiple (more than two) vaccine components or a single vaccine component to improve immune responses to all four dengue viruses in the subject.
  • samples from a subject may be analyzed for resistance to dengue infection using standard means known in the art.
  • the vaccine composition can be introduced to a subject by any route in multiple anatomical locations to, for example, protect against three or more dengue serotypes after consecutive administrations.
  • a vaccine composition can include, but is not limited to, a single dose of a formulation containing all serotypes of dengue virus (e.g. DENVaxTM) administered to a subject capable of providing protection against at least three dengue virus serotypes.
  • a vaccine composition can include attenuated dengue virus serotypes in combination with other anti- pathogenic compositions (e.g. Japanese encephalitis, yellow fever, West Nile, influenza, Chikungunya or other).
  • compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingested, and any other method.
  • Introduction in two or more anatomical sites can include any combination administration including by the same mode in two or more anatomical sites or by two or more different modes that include two or more separate anatomical sites.
  • two or more anatomical sites can include different limbs.
  • vaccinations can be delivered to a subject using any device known in the art including, but not limited to, a needle and syringe, jet injection, microneedle injection, patch delivery (e.g. skin), intradermal delivery devices, inhalation device, intranasal device, slow release microparticles, and any other acceptable vaccine-delivery device.
  • a vaccine composition for dual administration of dengue virus vaccines can include a composition comprising more than one chimeric dengue viruses in a single composition.
  • the chimeric constructs used in such a composition are made up of dengue-dengue serotypes such as a dengue- 1, dengue-3, and/or dengue-4 on a dengue-2 backbone.
  • a single vaccine composition can include live, attenuated dengue viruses where an immune response is induced in a subject receiving such a compositions to at least three and up to all four dengue virus serotypes.
  • Constructs contemplated herein include live, attenuated dengue viruses comprising one or more live, attenuated dengue viruses and one or more dengue-dengue chimeric viruses further comprising capsid and non-structural proteins of the attenuated dengue virus and pre-membrane and envelope proteins of at least a second dengue virus in a single construct.
  • the capsid and non-structural proteins are from an attenuated dengue- 1, dengue-2, dengue-3 or dengue-4 virus.
  • pre- membrane and envelope proteins of at least a second dengue virus are dengue-2, dengue-3 or dengue-4 when the attenuated dengue virus is dengue- 1; or dengue- 1, dengue-3 or dengue-4 when the attenuated dengue virus is dengue-2; or dengue- 1, dengue-2 or dengue-4 when the attenuated dengue virus is dengue-3; or dengue- 1, dengue-2 or dengue-3 when the attenuated dengue virus is dengue-4.
  • dengue-dengue chimeric viruses can include the capsid and non- structural proteins of an attenuated dengue-2 virus and the pre-membrane and envelope proteins are dengue- 1, dengue-3 or dengue-4.
  • dengue-2 can include any dengue-2 strain.
  • dengue-2 comprises PDK-53 strain.
  • a chimera is a nucleic acid chimera including a first nucleotide sequence encoding nonstructural proteins from an attenuated dengue-2 virus, and a second nucleotide sequence encoding a structural protein from a second flavivirus.
  • the structural protein can be the C, prM or E protein of a flavivirus.
  • flaviviruses from which the structural protein may be selected include, but are not limited to, dengue- 1 virus, dengue-2 virus, dengue-3 virus, dengue-4 virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, yellow fever virus and tick-borne encephalitis virus.
  • the structural protein may be selected from non-flavivirus species that are closely related to the flaviviruses, such as hepatitis C virus.
  • amino acid substitution mutations in the nonstructural proteins and a nucleotide substitution mutation in the 5' noncoding region can be present.
  • This nucleotide substitution mutation occurs in the stem of a stem-loop structure that is conserved in all four dengue serotypes.
  • 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 can provide the attenuated DEN-2 virus disclosed herein
  • the genome of any dengue-2 virus containing non- conservative amino acid substitutions at these loci can be used as the backbone in the avirulent chimeras described herein.
  • other flavivirus genomes containing analogous mutations at the same loci, after amino acid sequence or nucleotide sequence alignment and stem structure analysis can also be used as the backbone structure and are defined herein as being equivalent to attenuating mutations of the dengue-2 PDK-53 genome.
  • the backbone that region of the chimera that includes 5' and 3' noncoding regions and the region encoding the nonstructural proteins, can also contain further mutations to maintain stability of the avirulent phenotype and to reduce the possibility that the avirulent virus or chimera might revert back to the virulent wild-type virus.
  • a second mutation in the stem of the stem/loop structure in the 5' non-coding region can provide additional stability, if desired.
  • chimeric viruses can include nucleotide and amino acid substitutions, deletions or insertions in their structural and nonstructural proteins in addition to those specifically described herein.
  • Structural and nonstructural proteins disclosed herein are to be understood to include any protein including or any gene encoding the sequence of the complete protein, an epitope of the protein, or any fragment comprising, for example, two or more amino acid residues thereof.
  • Embodiments disclosed herein provide a method for making chimeric viruses of embodiments described herein using recombinant techniques, by inserting the required substitutions into the appropriate backbone genome.
  • compositions can include a pharmaceutically acceptable carrier and attenuated chimeric viruses which contain amino acid sequences derived from other dengue virus serotypes, other flavivirus species or other closely related species, such as hepatitis C virus, proteins or polypeptides comprising the amino acid sequences derived from other dengue virus serotypes, other flavivirus species or other closely-related species, can act as immunogens and, thus, be used to induce an immunogenic response against other dengue virus serotypes, other flavivirus species or other closely related species.
  • nucleic acid chimeras including nucleotide sequence from an attenuated dengue-2 virus and nucleotide sequence from a second dengue virus (or other flavivirus), wherein the nucleotide sequence from the second flavivirus directs the synthesis of flavivirus antigens are contemplated of use for dual administration at day 0.
  • compositions for vaccines comprising three or more dengue virus serotypes is contemplated.
  • Another object of the invention is to provide compositions and methods for imparting immunity against three or more dengue virus serotypes simultaneously using dual administration in different anatomical areas to induce other lymph nodes of a subject receiving such a regimen.
  • Another object of the invention is to provide nucleic acid probes and primers for use in any of a number of rapid genetic tests that are diagnostic for each of the vaccine viruses of the current invention.
  • This object of the invention may be embodied in polymerase chain reaction assays, hybridization assays or other nucleic acid sequence detection techniques known to the art.
  • One embodiment includes using an automated PCR-based nucleic acid detection system.
  • a composition can include chimeric dengue viruses capable of eliciting an immune response to all four dengue virus serotypes wherein a single composition is introduced in two anatomical locations of a subject. Certain embodiments concern targeting populations of people visiting dengue endemic countries for short periods of time such as tourists. Brief Description of the Drawings
  • Fig. 1 represents an example of an intradermal injection device currently available.
  • Fig. 2 represents examples of injection sites in a non-human primate subject having intradermal introduction of a vaccine against dengue virus.
  • Fig. 3 represents a bar graph comparison of neutralizing antibody titer produced against different ratios of dengue virus serotypes after a one (primary) administration via the subcutaneous (SC) versus intradermal (ID) route of injection of a vaccine against dengue virus.
  • Fig. 4 represents a bar graph comparison of neutralizing antibody titer produced against different dengue virus serotypes after a second, boosting administration via the subcutaneous (SC) versus intradermal (ID) injection of a vaccine against dengue virus.
  • Fig. 5 represents a histogram plot of neutralizing antibody titers after subcutaneous and intradermal immunizations with a vaccine against a dengue virus serotype-4 in mice.
  • FIGs. 6 A and 6B represent graphic depictions of mouse survival after vaccination with a dengue vaccine followed by a challenge with wild-type dengue virus. Mice were vaccinated by SC or ID route of infection with a dengue vaccine (e.g. DENVax- 4) or a buffer/placebo (e.g. TFA).
  • a dengue vaccine e.g. DENVax- 4
  • a buffer/placebo e.g. TFA
  • Fig. 7 represents neutralizing antibody titers for DEN-1, DEN-2, DEN-3 and
  • DEN-4 at day 28 and day 56 after two day-0; or 1 day-0 and 1 day-42 injections (e.g. DENVaxTM; 4:3:4:5 ratio).
  • Fig. 8 represents neutralizing antibody titers for DEN-1, DEN-2, DEN-3 and
  • DEN-4 at day 28 and day 56 after two day-0; or 1 day-0 and 1 day-42 injections (e.g. DENVaxTM; 3:3:3:3, approximately equivalent amounts used).
  • Figs. 9A-9D represent graphs comparing neutralizing antibody titers achieved in non-human primates after SC immunization with tetravalent dengue virus vaccines. Two groups were vaccinated with the needle-free device via the subcutaneous route either twice on the same day (0,0) or once on day 0 and again on day 60 (0,60).
  • Figs. 10A-10B represent data obtained from a human clinical trial.
  • Seronegative humans (humans demonstrating little to no antibodies to dengue virus serotypes at the onset of the trial) were given two doses of a tetravalent serotype formulation of dengue vaccine either subcutaneously or intradermally (day 0 and day 90).
  • Antibody levels against each of the dengue serotypes were analyzed on days 0, 30, 60, 90 and 120.
  • Figs. 11A-11D represent a graph comparing neutralizing antibody titers achieved in non-human primates after subcutaneous immunization with a tetravalent serotype dengue vaccine. Two groups were vaccinated either twice on the same day (0,0) or once on day 0 and again on day 60 (0,60). Serum was analyzed for presence of antibodies on days 0, 28, 58, 73 and 90, and the detection of antibodies against all four dengue serotypes were analyzed (DEN-1 , DEN-2, DEN-3, DEN-4).
  • Fig. 12 represents gene expression levels in samples obtained from a subject having single administration or dual (double) administration at separate anatomical sites of dengue virus vaccines. This data represents gene cluster variability where changes in levels of various gene transcripts in a subject are analyzed after exposure to a composition using certain regimens disclosed herein.
  • Figs. 13A-13F represent levels of certain genes after single or dual (double) administration.
  • the genes represented in cluster 2 Fig. 12 and A-F are associated with innate immunity.
  • vessel can include, but is not limited to, test tube, mini- or micro-fuge tube, channel, vial, microtiter plate or container.
  • subject or “subjects” may include but are not limited 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, 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, or zoo animals.
  • Attenuated virus can mean a virus that demonstrates reduced or no clinical signs of disease when administered to a subject such as a mammal (e.g., human or an animal).
  • a subject e.g., human or an animal.
  • "consecutively” can mean in close temporal proximity, usually within a single patient visit and within 24 hours.
  • administration can mean delivery of a vaccine or therapy to an individual animal or human by any one of many methods such as intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, oral, buccal, by inhalation, intranasally, or any others known in the art.
  • Certain aspects of the present invention include, but are not limited to, administration of vaccine compositions against dengue virus.
  • Embodiments of the present invention generally relate to methods and compositions for inducing protective neutralizing antibodies in a subject against three or more dengue virus serotypes.
  • Other embodiments can include introducing a vaccine composition to a subject via any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, orally, intranasally, vaginal, intravenous, ingested, and any other method wherein the vaccine composition so introduced induces neutralizing antibodies against three or more dengue virus serotypes.
  • the vaccine composition comprises a dose of a vaccine against three or more dengue virus serotypes administered to a subject.
  • the vaccine composition comprises an initial dose against all four dengue serotypes then, one or more other vaccine compositions administered to a subject.
  • Vaccines against dengue virus may include a composition comprising predetermined ratios of all four live, attenuated dengue vaccine viruses, recombinant dengue vaccine viruses, chimeric viruses or mutants thereof .
  • the ratios of various dengue serotypes may be equivalent or nearly equal in representation or certain serotypes may be represented at higher concentrations than others depending on need or ability to induce a balanced neutralizing antibody response in the subject.
  • ratios of different dengue vaccines may differ by 2 to 100,000 fold (e.g. plaque forming units) between any two serotypes.
  • any dengue vaccine virus serotype formulation may be used to generate a vaccine (e.g. attenuated virus etc.) of use in consecutive administration to a subject in need thereof where the composition includes, but is not limited to, three or more dengue virus serotypes.
  • compositions of dengue virus vaccine formulations may be introduced to a subject prior to, during or after exposure to dengue virus by the subject.
  • a subject may receive more than one administration consecutively or more than one administration comprising a dengue virus formulation, optionally, followed by one or more additional administrations at a later time.
  • Intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, oral, and any other method of applications of formulations described herein may be combined with any other anti-viral treatment.
  • intradermal, subcutaneous, intramuscular introduction of a formulation contemplated herein may be administered to any appropriate region of a subject's body (e.g. arm, shoulder, hip, intranasally etc).
  • parenteral administration of vaccine formulations may be combined with other modes of administration such as intranasal, pulmonary, oral, buccal, or vaginal in consecutive administrations.
  • primary or booster administrations may occur consecutively on the same day, consecutive days, weekly, monthly, bi-monthly or other appropriate treatment regimen.
  • Dengue is endemic in Asia, Central and South America including Colombia, the Caribbean, the Pacific Islands, and parts of Africa and Australia. It is estimated that 3.6 billion people (55% of the world's population) live in areas at risk of dengue virus transmission (DVI). Infection with a dengue virus can result in a range of symptoms, from subclinical disease to debilitating but transient dengue fever to life-threatening dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). Currently, there is no therapeutic treatment or prophylactic vaccine for dengue fever. Given the impact of dengue on populations in endemic countries and on travelers to those regions, a vaccine to prevent dengue is needed.
  • Dengue is a mosquito borne viral disease, transmitted from human to human primarily by the mosquito, Aedes aegypti.
  • Dengue viruses contain a single-stranded, positive-sense RNA genome of approximately 11 kb. The genome consists of three structural proteins, capsid (C), premembrane (prM), and envelope (E), and seven nonstructural proteins, NSl, NS2A, NS2B, NS3, NS4A, NS4B, and NS5.
  • C capsid
  • prM premembrane
  • E envelope
  • a multivalent (e.g. tetravalent) vaccine that stimulates immunity against more than one and up to all four serotypes of DENV is needed.
  • Several DENV vaccine candidates attenuated by classical serial passage in cell culture have proven unsafe or poorly immunogenic.
  • Chimeric live-attenuated, recombinant DENV vaccines candidates, including viruses based on the attenuated genetic background of yellow fever 17D (YF-17D)vaccine virus, DENV-2 PDK- 53 vaccine virus, or DENV -4 containing a 30-nucleotide 3' non-coding region (NCR) deletion are known in the art.
  • a challenging issue in the development of an effective live-attenuated dengue virus (DENV) vaccine is the interference between the four dengue vaccine viruses when administered as a tetravalent formulation. Interference is manifest when one or more components of a multivalent mixture will induce lower immune responses than those elicited by each individual monovalent vaccine. Interference has been observed with vaccines for diseases with multiple pathogenic serotypes, such as polio, dengue or others. Due in part to this interference, it was previously discovered that three dose regimen of oral polio vaccine is required to induce adequate immune responses to the three key serotypes.
  • intradermal route for dengue virus vaccine delivery will favor the induction of more potent and balanced immune responses to all four dengue virus serotypes.
  • the presence of an increased number of natural host cells in the skin for virus replication may reduce interference and permit replication of the less dominant viruses in tetravalent formulations.
  • intradermal immunization of multivalent, live, attenuated dengue vaccines can be used to induce more balanced immune responses to dengue virus exposure in a subject.
  • DENVaxTM is a dengue vaccine that consists of a mixture of four recombinant dengue virus strains designed to generate immune responses to the four dengue serotypes (DEN-1, DEN-2, DEN-3 and DEN-4).
  • DENVax the dengue serotype 2 vaccine component (DENVax-2) corresponds to an attenuated DEN-2 PDK-53 strain. This construct has already been investigated in many clinical studies.
  • the other dengue vaccine strains are chimeras consisting of the DEN-1, DEN-3 or DEN-4 structural pre-membrane (prM) and envelope (E) protein genes cloned into a DEN-2 PDK-53 non-structural gene backbone. These recombinant viruses express the surface antigens of DEN-1, DEN-3 or DEN-4 and retain the genetic alterations responsible for the attenuation of the DEN-2 PDK-53 strain.
  • DENVaxTM can be used as an example of a multivalent live, attenuated dengue vaccine having all four dengue virus serotypes represented in one vaccine composition at various ratios. Other embodiments relate to optimizing tetravalent vaccine administrations. Yet other embodiments relate to DENVaxTM immunization methods.
  • immunization regimens with multivalent, live attenuated vaccines at shorter intervals in more than one anatomical site were not considered a viable option for treating a subject in need of such a treatment. It is contemplated herein that multiple site administration, by accessing larger numbers of antigen presenting cells and/or more than one draining lymph node, permits immune responses to less dominant components of a multivalent, live attenuated vaccine and effectively reduces vaccine interference.
  • the composition introduced to the subject comprises vaccines against all dengue virus serotypes (DEN-1 , DEN-2, DEN-3, DEN-4).
  • a composition contemplated herein can include DENVaxTM or other similar formulation.
  • vaccine viruses against all dengue serotypes are in equal proportions in the composition.
  • each dengue vaccine virus serotype may be in a particular ratio to one another such that introduction of the composition provides the subject with sufficient levels of neutralizing antibodies against all dengue viruses (e.g. DEN-1, DEN-2, DEN-3, DEN-4).
  • a vaccine composition for dual administration of dengue virus vaccines can include a composition comprising more than one chimeric dengue viruses in a single composition.
  • the chimeric constructs used in such a composition are made up of dengue-dengue serotypes such as a dengue- 1 , dengue-3, and/or dengue-4 on a dengue-2 backbone.
  • a single vaccine composition can include live, attenuated dengue viruses where an immune response is induced in a subject receiving such a compositions to at least three and up to all four dengue virus serotypes.
  • Constructs contemplated herein include live, attenuated dengue viruses comprising one or more live, attenuated dengue viruses and one or more dengue-dengue chimeric viruses further comprising capsid and non-structural proteins of the attenuated dengue virus and pre-membrane and envelope proteins of at least a second dengue virus in a single construct.
  • the capsid and non-structural proteins are from an attenuated dengue- 1, dengue-2, dengue-3 or dengue-4 virus.
  • pre- membrane and envelope proteins of at least a second dengue virus are dengue-2, dengue-3 or dengue-4 when the attenuated dengue virus is dengue- 1 ; or dengue- 1 , dengue-3 or dengue-4 when the attenuated dengue virus is dengue-2; or dengue- 1 , dengue-2 or dengue-4 when the attenuated dengue virus is dengue-3; or dengue- 1 , dengue-2 or dengue-3 when the attenuated dengue virus is dengue-4.
  • dengue-dengue chimeric viruses can include the capsid and non- structural proteins of an attenuated dengue-2 virus and the pre-membrane and envelope proteins are dengue- 1, dengue-3 or dengue-4.
  • dengue-2 can include any dengue-2 strain.
  • dengue-2 comprises PDK-53 strain.
  • a chimera is a nucleic acid chimera including a first nucleotide sequence encoding nonstructural proteins from an attenuated dengue-2 virus, and a second nucleotide sequence encoding a structural protein from a second flavivirus.
  • the structural protein can be the C, prM or E protein of a flavivirus.
  • flaviviruses from which the structural protein may be selected include, but are not limited to, dengue- 1 virus, dengue-2 virus, dengue-3 virus, dengue-4 virus, West Nile virus, Japanese encephalitis virus, St. Louis encephalitis virus, yellow fever virus and tick-borne encephalitis virus.
  • the structural protein may be selected from non- flavivirus species that are closely related to the flaviviruses, such as hepatitis C virus.
  • amino acid substitution mutations in the nonstructural proteins and a nucleotide substitution mutation in the 5' noncoding region can be present.
  • This nucleotide substitution mutation occurs in the stem of a stem-loop structure that is conserved in all four dengue serotypes.
  • 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 can provide the attenuated DEN-2 virus disclosed herein
  • the genome of any dengue-2 virus containing non- conservative amino acid substitutions at these loci can be used as the backbone in the avirulent chimeras described herein.
  • other flavivirus genomes containing analogous mutations at the same loci, after amino acid sequence or nucleotide sequence alignment and stem structure analysis can also be used as the backbone structure and are defined herein as being equivalent to attenuating mutations of the dengue-2 PDK-53 genome.
  • the backbone that region of the chimera that includes 5' and 3' noncoding regions and the region encoding the nonstructural proteins, can also contain further mutations to maintain stability of the avirulent phenotype and to reduce the possibility that the avirulent virus or chimera might revert back to the virulent wild-type virus.
  • a second mutation in the stem of the stem/loop structure in the 5' non-coding region can provide additional stability, if desired.
  • chimeric viruses can include nucleotide and amino acid substitutions, deletions or insertions in their structural and nonstructural proteins in addition to those specifically described herein.
  • Structural and nonstructural proteins disclosed herein are to be understood to include any protein including or any gene encoding the sequence of the complete protein, an epitope of the protein, or any fragment comprising, for example, two or more amino acid residues thereof.
  • Embodiments disclosed herein provide a method for making chimeric viruses of embodiments described herein using recombinant techniques, by inserting the required substitutions into the appropriate backbone genome.
  • compositions can include a pharmaceutically acceptable carrier and attenuated chimeric viruses which contain amino acid sequences derived from other dengue virus serotypes, other flavivirus species or other closely related species, such as hepatitis C virus, proteins or polypeptides comprising the amino acid sequences derived from other dengue virus serotypes, other flavivirus species or other closely-related species, can act as immunogens and, thus, be used to induce an immunogenic response against other dengue virus serotypes, other flavivirus species or other closely related species.
  • nucleic acid chimeras including nucleotide sequence from an attenuated dengue-2 virus and nucleotide sequence from a second dengue virus (or other flavivirus), wherein the nucleotide sequence from the second flavivirus directs the synthesis of flavivirus antigens are contemplated of use for dual administration at day 0.
  • compositions for vaccines comprising three or more dengue virus serotypes is contemplated.
  • Another object of the invention is to provide compositions and methods for imparting immunity against three or more dengue virus serotypes simultaneously using dual administration in different anatomical areas to induce other lymph nodes of a subject receiving such a regimen.
  • Another object of the invention is to provide nucleic acid probes and primers for use in any of a number of rapid genetic tests that are diagnostic for each of the vaccine viruses of the current invention.
  • This object of the invention may be embodied in polymerase chain reaction assays, hybridization assays or other nucleic acid sequence detection techniques known to the art.
  • One embodiment includes using an automated PCR-based nucleic acid detection system.
  • a composition can include chimeric dengue viruses capable of eliciting an immune response to all four dengue virus serotypes wherein a single composition is introduced in two anatomical locations of a subject. Certain embodiments concern targeting populations of people visiting dengue endemic countries for short periods of time such as tourists.
  • Certain embodiments disclosed herein relate to methods and compositions for a rapid induction of protection in a subject against all dengue virus serotypes by, for example, administering a vaccine to a subject against all dengue virus serotypes in more than one anatomical location consecutively on the same day.
  • Some embodiments can include introducing a vaccine composition to a subject via intradermal (ID) or subcutaneous (SC) injection or other administration mode in one anatomical location then introducing at least a second vaccine composition at another anatomical location by ID, SC or other administration mode.
  • ID intradermal
  • SC subcutaneous
  • Some embodiments include using any combination of modes of administration for introducing a dengue virus vaccine of all dengue virus serotypes to a subject where administration of the vaccine occurs at two or more anatomical sites or by two or more different routes on day 0 to the subject. Some embodiments include using the same mode of administration but at different anatomical locations.
  • Some dengue virus vaccine compositions described herein range in dosage from from 10 2 to 5 x 10 6 PFU for each serotype in a composition.
  • Other compositions e.g. follow-on vaccinations contemplated herein include compositions that have dosages less than or more than this range based on immune response in the subject after primary immunization.
  • ratios can vary for the various Dengue vaccine virus serotypes depending on need and immune response in a subject.
  • compositions introduced on the first vaccination or in any follow-on vaccination contemplated herein may include one tetravalent dengue virus composition.
  • the composition can include DENVaxTM or other similar tetravalent formulation of equal or equivalent ratios or at predetermined serotype ratios.
  • Other embodiments can include using different formulations (e.g. serotype ratios) for each of the vaccine compositions administered at the primary vaccination or any follow-on vaccinations (e.g. less than 30 days later).
  • Some embodiments herein include treating a subject in need of such a vaccine, on day 0 at two or more anatomical locations then administering at least a second vaccine within 30 days such as about 7, about 14, about 21 or about 28 days later with a composition comprising dengue virus serotypes which may or may not have all serotypes.
  • each vaccination has all dengue virus serotypes represented in the vaccine formulation.
  • Vaccine compositions of follow-on administration disclosed herein may include two or more dengue virus serotypes at a predetermined ratio for the subsequent administration(s) .
  • the composition introduced to the subject comprises all dengue virus serotypes.
  • vaccine compositions comprise various formulations of DENVaxTM or other similar formulation.
  • the ratio of DEN-1 :DEN-2:DEN-3 :DEN-4 can be 3 :3 :3 :3, 4:3 :4:5, 5 :4:5 :5, 5 :4:5 :5, 5 :5 :5; 10, 10: 1 : 10: 100 or other ratio where the ratio between 2 serotypes can be about 2 to about 100,000 fold difference (e.g. DENVax 4:3:4:5TM etc.) in a single composition.
  • a dengue serotype ratio can be DEN-1 at 2 xlO 4 : DEN-2 at 5 xlO 4 : DEN-3 at 1 xlO 5 : DEN-4 at 3 xlO 5 PFUs or DEN-1 at 8 xlO 3 : DEN-2 at 5 xlO 3 : DEN-3 at 1 xlO 4 : DEN-4 at 2 xlO 5 PFUs.
  • all dengue vaccine virus serotypes are in equal proportions in the composition.
  • each dengue vaccine virus serotype may be in a particular ratio to another serotype such that introduction of the composition provides the subject with adequate or more than adequate levels of neutralizing antibodies which confer protection against all dengue viruses (e.g. Dengue 1, 2, 3 and 4).
  • a booster for that subject can contain an increased concentration of the one or more dengue vaccine virus serotype (that demonstrated lower neutralizing antibodies) to provide better protection against all dengue virus types.
  • samples from a subject may be analyzed for an immune response to dengue serotype infection (e.g. Dengue- 1 , -2, -3, -4) using standard means known in the art.
  • the vaccine composition can be simultaneously or consecutively introduced to a subject intradermally in multiple anatomical locations to, for example, protect against all dengue serotypes (e.g. cross protection).
  • a vaccine composition can include, but is not limited to, a single formulation of all dengue vaccine virus serotypes (e.g. DENVaxTM) administered to a subject capable of providing full protection against infection by all dengue virus serotypes.
  • a vaccine composition can include attenuated dengue virus serotypes in combination with other anti- pathogenic compositions (e.g. Japanese encephalitis, West Nile, influenza etc.).
  • compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingested, and any other method.
  • Introduction in two or more anatomical sites can include any combination administration including by the same mode in two or more anatomical sites or by two different modes that include two separate anatomical sites.
  • two or more anatomical sites can include different limbs.
  • a booster vaccination for that subject can contain an increased concentration of the one or more dengue vaccine virus serotype (that demonstrated lower levels of neutralizing antibodies) to provide complete protection against infection by all dengue virus types.
  • samples from a subject may be analyzed for resistance to dengue infection using standard means known in the art.
  • doses of the vaccine composition can be consecutively introduced to a subject in multiple anatomical locations to, for example, to protect against all dengue serotypes (e.g. cross protection) at day 0.
  • a vaccine composition can include, but is not limited to, a single composition of three or four dengue virus serotypes (e.g. DENVaxTM) administered to a subject capable of inducing neutralizing antibodies to levels which would provide full protection against infection by all dengue virus serotypes.
  • DENVaxTM dengue virus serotypes
  • a particular subject may need to visit a clinic only one time to receive enough protection to visit or remain in a region having dengue virus for a predetermined period of time (e.g. 30 days).
  • a vaccine composition can include attenuated dengue virus serotypes in combination with vaccine compositions against other pathogens (e.g. flaviviruses such as Japanese encephalitis, West Nile, or other viruses such as influenza etc.).
  • Compositions contemplated herein can be administered by any method known in the art including, but not limited to, intradermal, subcutaneous, intramuscular, intranasal, inhalation, vaginal, intravenous, ingested, and any other method.
  • Introduction in two or more anatomical sites can include any combination administration including by the same mode in two or more anatomical sites or by two or more different modes that include two or more separate anatomical sites.
  • two or more anatomical sites can include different limbs, different tissues, intranasally, as drops (e.g. for the eye), intramuscular in two or more locations.
  • vaccine compositions disclosed herein can be chimeric constructs that can include a mixture of constructs that make up at least 3 dengue serotypes in a vaccine composition for administration to a subject.
  • dengue virus vaccines can include constructs having an attenuated flavivirus backbone with various dengue serotype substitutions representing each of the four serotypes where the constructs can be mixed in a composition for administration as a vaccine.
  • Chimeras contemplated and 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 a dengue virus genome backbone (e.g. PDK-53), or the equivalent thereof as described above, using recombinant engineering techniques well known to those skilled in the art to remove the corresponding structural genes and replace it with the desired structural gene.
  • a dengue virus genome backbone e.g. PDK-53
  • 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.
  • the gene to be inserted into the backbone encodes a flavivirus (e.g. other dengue virus serotype) structural protein.
  • the flavivirus gene to be inserted is 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 the PrM and E structural proteins.
  • the sequence inserted into the dengue-2 backbone can encode the C, prM and E structural proteins.
  • the dengue virus backbone is the PDK-53 dengue-2 virus genome and includes either the spliced genes that encode the C, PrM and/or E structural proteins of dengue-1 (DEN-2/1), the spliced genes that encode the PrM and/or E structural proteins of dengue-3 (DEN-2/3), or the spliced genes encode the PrM and/or E structural proteins of dengue-4 (DEN-2/4).
  • the spliced gene that encodes the structural protein of dengue-3 virus directs the synthesis of an E protein that contains a leucine at amino acid position 345.
  • a chimera of encodes the C structural protein of dengue-2 virus and directs the synthesis of a C protein that contains a serine at amino acid position 100 and comprises a spliced gene encoding the structural proteins of dengue-4 which directs the synthesis of an E protein that contains a leucine at amino acid position 447.
  • a chimera can encode the C structural protein of dengue-2 virus and directs the synthesis of a C protein that contains a serine at amino acid position 100 and comprises a spliced gene encoding the structural proteins of dengue-4 which directs the synthesis of an E protein that contains a leucine at amino acid position 447 and a valine at amino acid position 364.
  • the structural proteins described herein can be present as the only flavivirus structural protein or in any combination of flavivirus structural proteins in a viral chimera of this invention.
  • Chimeras can be engineered by recombination of full genome-length cDNA clones derived from both DEN-2 16681 wild type virus and either of the PDK-53 dengue-2 virus variants.
  • Uncloned PDK-53 vaccine contains a mixture of two genotypic variants, designated herein as PDK53-E and PDK53-V.
  • the PDK53-V variant contains all nine PDK- 53 vaccine-specific nucleotide mutations, including the Glu-to-Val mutation at amino acid position NS3-250.
  • the PDK53-E variant contains eight of the nine mutations of the PDK-53 vaccine and the NS3-250-Glu of the parental 16681 virus.
  • Infectious cDNA clones are constructed for both variants, and viruses derived from both clones are attenuated in mice.
  • the phenotypic markers of attenuation of DEN-2 PDK-53 virus include small plaque size, temperature sensitivity (particularly in LLC-MK.sub.2 cells), limited replication (particularly in C6/36 cells), attenuation for newborn mice (specifically loss of neurovirulence for suckling mice) and decreased incidence of viremia in monkeys.
  • the chimeras that are useful as vaccine candidates are constructed in the genetic backgrounds of the two DEN-2 PDK-53 variants which all contain mutations in nonstructural regions of the genome, including 5TSTC- 57 C-to-T (16681-to-PDK-53) in the 5' noncoding region, as well as mutations in the amino acid sequence of the nonstructural proteins, such as, for example, NS1-53 Gly-to-Asp and NS3-250 Glu-to-Val.
  • 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 provide live, attenuated viruses useful as immunogens or vaccines. These chimeras exhibit high immunogenicity while at the same time producing no dangerous pathogenic or lethal effects.
  • the chimeric viruses or nucleic acid chimeras can include structural genes of either wild-type or attenuated virus in a virulent or an attenuated DEN-2 virus backbone.
  • the chimera may express the structural protein genes of wild-type DEN-1 16007 virus or its candidate PDK-13 vaccine derivative in either of the DEN-2 PDK-53 backgrounds.
  • Tetravalent formulations e.g. DENVaxTM
  • the remaining volume of the tetravalent DENVaxTM vaccine can be composed of diluent containing Trehalose (15%) F127 (1%) and human serum albumin (0.1%) in a saline buffer to stabilize the live, attenuated vaccine formulation.
  • a predetermined ratio of at least three dengue virus serotypes can be represented in a single composition.
  • dengue- 1 thru dengue-4 constructs may be represented in a single composition where more of one serotype of a live, attenuated virus can be present compared to the other constructs.
  • dengue-4 can be several fold pfu higher than other dengue viruses because it can demonstrate a reduced response.
  • Nucleic acids may be used in any formulation or used to generate any formulation contemplated herein.
  • Nucleic acid sequences used as a template for amplification can be isolated viruses ⁇ e.g. dengue viruses), according to standard methodologies.
  • a nucleic acid sequence may be genomic DNA or fractionated or whole cell RNA. Where RNA is used, it may be desired to convert the RNA to a complementary cDNA. In some embodiments, the RNA is whole cell RNA and is used directly as the template for amplification. Any method known in the art for amplifying nucleic acid molecules is contemplated ⁇ e.g., PCR, LCR, Qbeta Replicase, etc).
  • Genes can be expressed in any number of different recombinant DNA expression systems to generate large amounts of the polypeptide product, which can then be purified and used in methods and compositions reported herein. Any method known in the art for generating and using constructs is contemplated. In certain embodiments, genes or gene fragments encoding one or more polypeptide may be inserted into an expression vector by standard cloning or subcloning techniques known in the art.
  • Proteins, peptides and/or antibodies or fragments thereof may be detected or analyzed by any means known in the art.
  • methods for separating and analyzing molecules may be used such as gel electrophoresis or column chromatography methods.
  • Electrophoresis may be used to separate molecules ⁇ e.g., large molecules such as proteins or nucleic acids) based on their size and electrical charge. There are many variations of electrophoresis known in the art. A solution through which the molecules move may be free, usually in capillary tubes or it may be embedded in a matrix or other material known in the art. Common matrices can include, but are not limited to, polyacrylamide gels, agarose gels, mass spec, blotting and filter paper.
  • a gene or gene fragment encoding a polypeptide may be inserted into an expression vector by standard subcloning techniques.
  • An expression vector may be used which produces the recombinant polypeptide as a fusion protein, allowing rapid affinity purification of a peptide or protein.
  • fusion protein expression systems are the glutathione S-transferase system (Pharmacia, Piscataway, NJ), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (IBI, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA).
  • a formulation can contain one or more dengue virus serotype in various ratios in a single vaccine. It is contemplated that formulations can contain other agents of use in vaccination of a subject including, but not limited to other active or inactive ingredients or compositions known to one skilled in the art.
  • vaccinal viruses herein can be administered in the form of vaccinal compositions which can be prepared by any method known to one skilled in the art.
  • the virus compositions are lyophilized and are mixed with a pharmaceutically acceptable excipient (e.g. water, phosphate buffered saline (PBS), wetting agents etc.)
  • a pharmaceutically acceptable excipient e.g. water, phosphate buffered saline (PBS), wetting agents etc.
  • vaccine compositions can include stabilizers that are known to reduce degradation of the formulation and prolong shelf-life of the compositions.
  • an adjuvant may be added to the composition to induce, increase, stimulate or strengthen a cellular or humoral immune response to administration of a vaccination described herein. Any adjuvant known in the art that is compatible with compositions disclosed herein is contemplated.
  • Some embodiments herein concern amounts or doses or volumes of administration of a tetravalent dengue virus composition and the amount or dose can depend on route of administration and other specifications such as the subject getting the vaccine (e.g. age, health condition, weight etc.).
  • compositions described can be administered to a subject living in an area having dengue virus, a subject traveling to an area having dengue virus or other subject such as any human or animal capable of getting dengue fever or other dengue virus condition.
  • a subject traveling to an area having dengue virus is administered one or more vaccine compositions ⁇ e.g. two or more on Day 0) about 1 to about 3 months prior to dengue virus exposure.
  • Vaccines herein can be administered as a prophylactic treatment to prevent infection in adults and children.
  • a subject can be na ' ive or non-na ' ive subject with respect to exposure to dengue virus and vaccine regimens disclosed herein. Kits
  • kits of use with the methods ⁇ e.g. methods of application or administration of a vaccine) and compositions described herein.
  • Some embodiments concern kits having vaccine compositions of use to prevent or treat subjects having been exposed or suspected of being exposed to one or more dengue viruses.
  • a kit may contain one or more than one formulation of dengue virus serotype(s) ⁇ e.g. attenuated vaccines, trivalent or tetravalent formulations, DENVaxTM) at predetermined ratios.
  • Kits can be portable, for example, able to be transported and used in remote areas such as military installations or remote villages in dengue endemic areas.
  • Other kits may be of use in a health facility to treat a subject having been exposed to one or more dengue viruses or suspected of being at risk of exposure to dengue virus.
  • Kits can also include a suitable container, for example, a vessel, vials, tubes, mini- or micro fuge tubes, test tube, flask, bottle, syringe or other container. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. Kits herein will also typically include a means for containing the agent (e.g. a vessel), composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • one or more additional agents such as immunogenic agents or other anti-viral agents, anti-fungal or anti-bacterial agents may be needed for compositions described, for example, for compositions of use as a vaccine against one or more additional microorganisms.
  • kits can include devices for administering one or more vaccination to a subject such as an ID, SQ, IM, an inhaler, intranasal applicator or other device for administering a vaccine composition disclosed herein.
  • a single vaccine composition of at least three serotypes of live, attenuated dengue virus, or fragments thereof for use in rapidly inducing an immune response in a subject against at least three dengue virus serotypes wherein at least two doses of the single vaccine composition are to be administered in two or more anatomical locations on the same day of a subject in need thereof, inducing neutralizing antibodies in the subject against at least three dengue virus serotypes.
  • the single vaccine composition can contain at least one additional booster administration of a formulation of a live, attenuated dengue vaccine is to be administered 1 to 180 days after the simultaneous administrations.
  • the single vaccine composition can include a predetermined ratio of monovalent vaccines for the three or more dengue virus serotypes in the single vaccine composition.
  • the single vaccine composition comprises equivalent ratios of monovalent vaccines for three or more dengue virus serotypes in the single vaccine composition.
  • a single vaccine composition can include the formulation of the live, attenuated dengue vaccine for the at least one additional booster administration can be the same or a different formulation as the first formulation. If different than the first formulation, a vaccine composition can include a pre-determined concentration of one or more monovalent vaccines for the dengue virus serotypes. Further, concentration of dengue virus serotypes can include a higher concentration of one or more dengue virus serotype than the formulation used for the same day administrations, wherein the higher concentration is 2 to 100,000 fold greater concentrations than that used in the single formulation first administered.
  • two or more anatomical locations comprise different anatomical locations using the same mode of administration. Two or more anatomical sites can include different anatomical locations using different modes of administration.
  • a composition can include a tetravalent single vaccine composition that represents all four dengue virus serotypes.
  • a single vaccine composition can include all four dengue virus serotype(s) at a predetermined ratio.
  • the live, attenuated dengue viruses can include one or more dengue-dengue chimeric viruses further comprising capsid and non- structural proteins of the attenuated dengue virus and pre- membrane and envelope proteins of at least a second dengue virus.
  • the capsid and nonstructural proteins are from an attenuated dengue-1, dengue-2, dengue-3 or dengue -4 virus.
  • kits of one or more of the above referenced compositions and one or more device for administration by any mode contemplated herein.
  • Fig. 1 represents an intradermal inject (e.g., PharmaJet® or other intradermal device) device used for intradermal inoculations.
  • ID intradermal
  • One exemplary method of intradermal administration was performed on four Cynomologous macaques administered a DENVaxTM ((DENVax-1 : l xl0 5 PFU, DENVax-2; lxl0 5 PFU, DENVax3: lxl0 5 PFU, DENVax4: lxl0 5 PFU) Dengue virus vaccine) by intradermal administration.
  • DENVaxTM Cynomologous macaques administered a DENVaxTM ((DENVax-1 : l xl0 5 PFU, DENVax-2; lxl0 5 PFU, DENVax3: lxl0 5 PFU, DENVax4: lxl0 5 PFU) Dengue virus vaccine
  • 0.15 ml of vaccine was deposited ID
  • Fig. 2 illustrates inoculation sites on Cynomolgus macaques post vaccination with PharmaJet device. Animals were boosted 60 days later with the same formulation by the same route. Serum samples were collected at predetermined intervals, days 15, 30, 58, 74, and 91 and were tested for the presence of neutralizing antibodies directed against the four Dengue serotypes. PRNT (plaque reduction neutralization test, known in the art for quantifying levels of anti-DEN neutralizing antibodies) were performed on the sera samples.
  • PRNT plaque reduction neutralization test, known in the art for quantifying levels of anti-DEN neutralizing antibodies
  • the neutralizing antibody titers to all four dengue viruses were higher after intradermal versus subcutaneous administration.
  • the number of animals that demonstrated neutralizing antibody responses (“seroconversion” defined as PRNT > 10) was greater after the first dose of vaccine (see Table 1, the percentage of animals that seroconverted to each of the four Dengue serotypes is shown after primary and secondary immunization).
  • Viremia is given as the number of days that live DEN-1 virus could be isolated from blood samples (“Duration”) and the loglO of the peak titer isolated from each animal.
  • Viral RNA is given as the number of days viral RNA could be detected in the serum samples (“Duration”) and peak viral RNA levels in each monkey, expressed as the log 10 of the number of viral RNA genomes detected. Table 2: Responses after challenge with DEN-1
  • an optimized DENVaxTM formulation delivered in different locations and with different timings will be tested in non-human primates.
  • Groups of eight Cynomolgus macaques will be immunized with a DENVaxTM formulation containing 1 x 10 5 plaque forming units (pfu), 1 x 10 4 pfu, 1 x 10 5 pfu and 1 x 10 5 pfu of DENVaxTM- 1, DENVaxTM-2, DENVaxTM-3 and DENVaxTM-4, respectively (abbreviated 5:4:5:5).
  • Two doses will be administered in 0.1 ml ID.
  • Groups will be immunized with either one dose in each arm at Day 0, one dose in one arm at Day 0 and one dose in the other arm at Day 7, or one dose in one arm at Day 0 and one dose in the other arm at Day 60. These groups will be compared to a group that receives the same dose (5:4:5:5) in three sites in the same are on Day 0 and three sites in the other arm on Day 60 as well as a group that receives the same dose in a single 0.5 ml SC immunization in one arm at Day 0 and in the other arm at Day 60.
  • a control group will be immunized with vaccine excipients only (no vaccine viruses).
  • PBMCs collected on days 30, 60, 90 will be also monitored for IFN- ⁇ secretion by an ELISPOT assay.
  • ELISPOT assay On day 90, two animals from each group will be challenged with wild type of DEN-1, DEN-2, DEN-3, or DEN-4 viruses. Challenged animals will be monitored for clinical signs and temperature (twice daily), changes in food consumption (once daily) and body weight (weekly). In addition, all animals will be bled daily for 11 days post-challenge to monitor viremia and hematological parameters.
  • DENVaxTM formulations administered in 0.1 ml either by ID or SC injection.
  • Groups of 12 individuals will be immunized with for example, a low dose DENVaxTM formulation (8 x 10 3 pfu, 5 x 10 3 pfu, 1 x 10 4 pfu and 2 xlO 5 pfu of DENVaxTM-l , -2, -3 and -4, respectively) or a high dose (2 x 10 4 pfu, 5 x 10 4 pfu, 1 x 10 5 pfu and 3 xlO 5 pfu of DENVaxTM- 1 , -2, -3 and -4, respectively) of DENVaxTM ID or SC on Days 0 and 90.
  • Two control groups will be injected SC or ID with phosphate-buffered saline. Patients will be monitored for any adverse events, and for any significant changes in hematological or blood chemistry parameters. Serum samples will be collected to measure vaccine virus replication and neutralizing antibody responses at periodic intervals.
  • mice from each treatment group were boosted via the corresponding ID or SC route with 10 5 PFU of DENVaxTM-4 or TFA. Mice were bled on Day 31 and 58 and collected sera were pooled to measure neutralizing antibody responses.
  • Fig. 5A represents survivals of DENVaxTM-4 immune AG129 mice following challenge with DEN-1 (a) or DEN-2 (b) viruses. Challenged animals were monitored for clinical signs of disease and survival rates were recorded over a period of 5 weeks.
  • mice have an "intact" immune system; deficient for the interferon ( ⁇ )- ⁇ / ⁇ and - ⁇ receptors. Dengue infection has been described for this model. Other studies: pathogenesis, cell tropism, and ADE have also been examined. This model permits challenge with DEN-1 and DEN-2.
  • Nonhuman primates Cynomolgus, rhesus macaques carry virus (viremia), but no disease manifests.
  • the high dose formulation of the tetravalent formulation (e.g. DENVaxTM) was used for immunization in this study.
  • This vaccine lot is the same material used for two Phase 1 studies being conducted.
  • the high dose tetravalent formulation vaccine consists of 2xl0 4 pfu of DEN-1, 5xl0 4 pfu of DEN-2, lxlO 5 pfu DEN-3 and 3xl0 5 pfu DEN-4.
  • the study design for the nonhuman primate study is shown in Table 5.
  • Serum samples were collected after each vaccination and wild type dengue virus challenge on Days 0, 3, 5, 7, 10, 12, 14, 53, 64, 67, 88, 91, 93, 95, 97, 99, 101, 102 and 104 to analyze the samples for dengue viremia. Serum samples were also collected on Days 0, 30, 53, 75, 88 and 104 to determine the levels of neutralizing antibodies induced by the tetravalent formulation administered by needle/syringe or the ID injector.
  • Serum samples were collected at specified intervals during the course of the study. Sera collected on Days 0, Day 30 and Day 88 (pre-boost) have been assayed for neutralizing antibodies to Dengue-1, Dengue-2, Dengue-3 and Dengue-4.
  • the GMT antibody titers are shown below in Table 6.
  • Neutralizing antibody titers of ⁇ 10 are reported as "5" . Serum dilutions started at 1: 10 Seroconversion (values in parenthesis) is defined as titer >10 over Day 0 ⁇ 10 baseline titer or a >4-fold rise in titer if baseline titer on Day 0 was >10.
  • Neutralizing antibody titers of ⁇ 10 are reported as a value of "5".
  • Serum dilutions for analysis started at 1 :10.
  • PJ exemplary PharmaJet needle-free injector
  • N/S needle/syringe
  • vaccine virus replication after immunization is an important measure of vaccine uptake and vaccine safety.
  • Vaccine virus replication in the nonhuman primates was evaluated after the first and second immunization with a live attenuated tetravalent formulation vaccine (DENVaxTM). Serum samples collected on Days 0, 3, 5, 7, 10, 12, 14 after the first immunization were tested for the presence of viral RNA from the vaccine strains using a qRT-PCR assay (see Table 7). [000128] Table 7. DENVax-2 RNA detected in the serum after primary immunization with DENVax.
  • Results are averages from duplicate or triplicate data.
  • N/S needle/syringe
  • PJ PharmaJet needle-free injector
  • Viral RNA of the wild-type challenge viruses was detected only in Group 7 that had received PBS.
  • For Dengue -2 viral RNA was detected in 3 of 3 animals on Days 93 to 97.
  • For Dengue-4 viral RNA was detected in only 1 of 3 animals on Day 95.
  • One important observation of the groups that were immunized with the tetravalent formulation is that no viral RNA for either the Dengue-2 or the Dengue-4 challenge viruses was observed.
  • This protocol was performed in part to activate immune cells and antigen presenting cells in two different lymph nodes on Day 0 to induce higher levels and more robust dengue-specific immune responses compared to administering two doses intradermally 7, 14 or 42 days apart.
  • two routes of administration were compared, SC and ID routes using a conventional 42-day interval between vaccinations.
  • the mice were immunized with a low dose formulation of a tetravalent formulation (DENVaxTM; 3 :3 :3 :3 ratio of each of the serotypes) which consisted of 10 3 PFU of each Dengue-1 , -2, -3, and -4 (e.g.
  • the in live portion of this study was conducted prior to initiation of this contract.
  • the study design is shown in Table 9 below.
  • the neutralizing antibody titers to Dengue 1-4 present in the collected mouse sera were determined by a microneutralization assay. Sera were collected at specified time points throughout the study and the longevity of the immune responses was studied by maintaining the study groups until Day 160 (longer than 5 months after study start). The results obtained from sera collected on Days 28 and 56 post-immunization are illustrated in Table 10. Table 10. Neutralizing antibody titers (GMTs) to DEN-1, -2, -3 and -4
  • mice were dosed with either a low dose formulation of DENVaxTM (3 :3 :3 :3) which consisted of 10 3 PFU of each of DENVaxTM- 1 , -2 , -3, and 4 in a 0.05 mL volume given via the intradermal route (in the foot pad) or a medium dose formulation of DENVaxTM (4:3 :4:5) which contained 10 4 PFU of DENVaxTM- 1 , 10 3 PFU of DENVaxTM-2, 10 4 PFU of DENVaxTM-3, and 10 5 PFU of DENVaxTM-4 in a 0.05 mL volume.
  • mice On Day 0 all mice were immunized and Groups 2 and 4 were boosted on Day 42.
  • Sera for antibody analysis were collected on Days 14, 41 and 56 post-primary vaccination and analyzed using a plaque reduction microneutralization assay to determine the neutralizing antibody levels to all four dengue serotypes. Immunogenicity results obtained from pooled mouse serum samples are shown in Table 12.
  • immunization with either the low or medium dose tetravalent vaccine e.g. DENVaxTM
  • tetravalent vaccine e.g. DENVaxTM
  • the medium dose DENVaxTM formulation induced slightly higher neutralizing antibody titers by Day 28 for Groups 1 and 3 particularly for DEN-1 and DEN-3, that received two doses on Day 0 compared to groups that received only a single dose on Day 0 (Groups 2 and 4).
  • ELISPOT dengue virus neutralizing titers calculated using 50% NMS cutoff at a starting dilution of 1 :20. Serum from individual animals within a group were pooled and tested in triplicate.
  • Figs. 9A-9D represent graphs comparing neutralizing antibody titers achieved in non-human primates after immunization with tetravalent DENVax containing DENVax-1 (lxl0 5 pfu); DENVax-2 (lxl0 4 pfu); DENVax-3 (lxl0 5 pfu); DENVax-4 (lxl0 6 pfu).
  • Two groups were vaccinated with the needle-free PharmaJet device via the subcutaneous route either twice on the same day (0,0) or once on day 0 and again on day 60 (0,60). Serum was analyzed for presence of antibodies on days 0, 30, 53, 75 and 88, and the detection of antibodies against four dengue serotypes were analyzed (DEN-1, DEN-2, DEN- 3, DEN-4).
  • seronegative human subjects were immunized with two doses of a tetravalent formulation of DENVax containing DENVax-1 (lxl0 4 pfu); DENVax- 22 (lxl0 3 pfu); DENVax3 (lxl0 4 pfu); DENVax-4 (lxl0 5 pfu).
  • the route of immunization was subcutaneous or intradermal, and the vaccinations were given 90 days apart.
  • Antibody levels against each of the dengue serotypes were analyzed on days 0, 30, 60, 90 and 120.
  • the vaccine induced neutralizing antibodies to all four serotypes.
  • the levels of seroconversion were different when comparing the routes of immunization.
  • the intradermal route of immunization produced appeared to be more "balanced" immune responses in this study, with the levels of antibodies being more equivalent as compared to the subcutaneous route.
  • Fig. 10 represents the data obtained from a human clinical trial in Colombia. Seronegative humans were given two doses of a tetravalent formulation of DENVax containing DENVax-1 (lxl0 4 pfu); DENVax-2 (lxl0 3 pfu); DENVax-3 (lxl0 4 pfu); DENVax-4 (lxl0 5 pfu) subcutaneously or intradermally. Antibody levels against each of the dengue serotypes were analyzed on days 0, 30, 60, 90 and 120.
  • non-human primates were immunized with two doses of a tetravalent vaccine (e.g. DENVaxTM DENVax-1 : 2xl0 4 pfu, DENVax-2: 5xl0 4 pfu, DENVax-3: lxl0 5 pfu, DENVax-4: 3xl0 6 pfu) either simultaneously on Day 0, or two separate doses on days 0 and 60.
  • the vaccine induced neutralizing antibodies to all four Dengue serotypes.
  • the neutralizing antibody titers of the two groups were relatively equal (Fig. 11).
  • the kinetics of the immune response was more rapid in the group which received two immunizations on day 0.
  • the results obtained in this study further support the application of the novel dosing schedule of administering two doses on Day 0 at two immunologically distinct sites.
  • Fig. 11 represents a graph comparing neutralizing antibody titers achieved in non-human primates after subcutaneous immunization with tetravalent DENVax containing DENVax-1 (lxl0 5 pfu); DENVax-2 (lxl0 4 pfu); DENVax-3 (lxl0 5 pfu); DENVax-4 (lxl0 6 pfu).
  • DENVax-1 lxl0 5 pfu
  • DENVax-2 lxl0 4 pfu
  • DENVax-3 lxl0 5 pfu
  • DENVax-4 lxl0 6 pfu
  • Fig. 12 represents analysis of single dose administration versus dual administration in separate anatomical locations of dengue virus vaccines. This data represents changes in levels of various gene transcripts in a subject after 0,0 or single injection where the magnitude of change in gene expression is greater for dual administration (double dose).
  • the genes included in the cluster analysis, represented in Cluster 2 include several genes that change in relation to induction of innate immunity. Thus, induction of innate immunity and related genes can be greater in a subject having dual administration at separate anatomical locations of compositions disclosed herein. It is likely that the composition induces a response in multiple anatomical regions (e.g. lymph nodes etc.) reducing interference and affecting multiple genes that participate in innate immune responses.
  • RIS rapid immunization strategy
  • compositions disclosed herein include chimeric dengue virus compositions where a backbone of one dengue virus can accommodate one or more of the other dengue virus components. Mixtures of these chimeric compositions can be used to generate trivalent or tetravalent formulations of use in methods disclosed herein.
  • Figs. 13A-13F represent expression levels over time of various genes associated with innate immunity in response to single or dual administration of dengue virus vaccines. This data illustrates that expression of certain genes are increased in response to a dual administration (in separate anatomical locations such as each arm) versus single administration of a vaccine composition disclosed herein (e.g. tetravalent dengue virus composition).
  • Various genes illustrated include A. interferon- induced 17 kDa protein (ISG15), a 15-kDa protein of unique primary amino acid sequence; B. IF144 (interferon- induced protein 44); C. XAF1, XAF1 antagonizes the anticaspase activity of XAP1 ; D.
  • OASL The human 2',5'-oligoadenylate synthetase-like gene (OASL) encoding the interferon-induced 56-kDa protein
  • E. MX1 is interferon-induced GTP-binding protein
  • Mxl is a protein that in humans is encoded by the MX1 gene
  • EIF2AK2 Eukaryotic translation initiation factor 2-alpha kinase 2 1.59

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