WO2012027473A2 - Compositions immunogènes comprenant des antigènes de la protéine e du virus de la dengue à vecteurs d'alphavirus - Google Patents

Compositions immunogènes comprenant des antigènes de la protéine e du virus de la dengue à vecteurs d'alphavirus Download PDF

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WO2012027473A2
WO2012027473A2 PCT/US2011/048971 US2011048971W WO2012027473A2 WO 2012027473 A2 WO2012027473 A2 WO 2012027473A2 US 2011048971 W US2011048971 W US 2011048971W WO 2012027473 A2 WO2012027473 A2 WO 2012027473A2
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virus
alphavirus
dengue virus
vector
dengue
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PCT/US2011/048971
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WO2012027473A3 (fr
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Laura J. White
Robert Edward Johnston
Wahala M.P.B. Wahala
Aravinda De Silva
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The University Of North Carolina At Chapel Hill
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Publication of WO2012027473A3 publication Critical patent/WO2012027473A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • 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
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36141—Use of virus, viral particle or viral elements as a vector
    • C12N2770/36143—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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention was made, in part, with the support of grant numbers
  • the present invention relates to compositions and methods directed to producing an immune response against dengue virus.
  • Dengue fever is a major emerging human disease in the tropics with -50 to 100 million people infected annually.
  • the disease is caused by one of 4 dengue virus serotypes (DENl, DEN2, DEN3 and DEN4).
  • Infection by any one serotype confers life-long protective immunity against the same serotype, but only short-lived cross-protective immunity to the other serotypes.
  • Epidemiological studies suggest that during a secondary infection, preexisting cross-reactive antibodies can enliance the risk of severe and potentially lethal forms of the disease, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS),
  • the present invention provides an innovation in the field of dengue vaccines.
  • the inventors have developed a platform based on an alphavirus vector expressing a dengue virus E protein antigen.
  • the inventors have shown that a tetravalent formulation of a an alphavirus vector expressing a dengue virus E protein antigen induces in mice and macaques a balanced immune response against all four serotypes of dengue virus. No evidence of interference was found among the components of the tetravalent formulation (DEN1 , DEN2, DEN3 and DEN4), and anti-vector immunity against the first dose did not seem to reduce the effectiveness of a second dose.
  • the balanced response to the tetravalent formulation is advantageous because it minimizes the risk of the subject becoming sensitized to severe disease with subsequent infection.
  • the present invention can be practiced to produce an immune response against one or more dengue virus strains, genotypes and/or serotypes in young animals that have maternal antibodies present against the dengue virus(es).
  • An alphavirus vector comprising a heterologous nucleotide sequence encoding a dengue virus E protein antigen.
  • the alphavirus is Venezuelan Equine Encephalitis (VEE) virus.
  • VEE Venezuelan Equine Encephalitis
  • an alphavirus particle comprises VEE structural proteins.
  • the invention provides a nucleic acid (e.g. , DNA) comprising or encoding an alphavirus vector of the invention.
  • a nucleic acid e.g. , DNA
  • the nucleic acid is a viral DNA
  • compositions comprising two or more different alphavirus vectors according to the invention, wherein the composition comprises: (a) a first alphavirus vector comprising a heterologous nucleotide sequence encoding a first dengue virus E protein antigen from a first dengue virus serotype, strain and/or genotype; and (b) a second alphavirus vector comprising a heterologous nucleotide sequence encoding a second dengue virus E protein antigen from a second dengue virus serotype, strain and/or genotype.
  • the composition comprises two or more alphavirus vectors comprising a heterologous nucleotide sequence encoding a dengue virus E protein antigen from DEN 1 , DEN2, DEN3 and DEN4.
  • compositions comprising an alphavirus vector, nucleic acid, or composition of the invention in a pharmaceutically acceptable carrier,
  • the invention provides a method of inducing an immune response to dengue virus in a subject, the method comprising administering to the subject an effective amount of an alpliavirus vector, nucleic acid, composition or pharmaceutical formulation of the invention.
  • the invention provides a method of treating dengue virus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of an alphavirus vector, nucleic acid, composition or pharmaceutical formulation of the invention,
  • the invention also encompasses a method of preventing infection with dengue virus in a subject, the method comprising administering to the subject an effective amount of an alphavirus vector, nucleic acid, composition or pharmaceutical formulation of the invention.
  • Also contemplated by the invention is a method of protecting a subject from the effects of dengue virus infection, the method comprising administering to the subject an effective amount of an alphavirus vector, nucleic acid, composition or pharmaceutical formulation of the invention.
  • Figure 1 is a schematic representation of dengue virus immunogen configurations.
  • Figure 2 shows neutralizing antibody responses in monovalent versus tetravalent DEN prM/E-VRP vaccines in mice.
  • Panels A to D mice immunized with 10 6 IU of monovalent DEN1 , DNE2, DEN3 or DEN4 prM/E-VRP, respectively. Each serum was tested for its ability to neutralize virus from the 4 DEN serotypes.
  • Panel E mice immunized with a tetravalent cocktail, containing 1 x 10 6 IU of each serotype prM/E-VRP.
  • Figure 3 shows the kinetics of neutralizing antibody responses to all 4 DEN serotypes in BALB/c mice immunized with tetravalent prM/E-VRP cocktail at two doses.
  • Panel (A) Low Dose: 7.2 x 10 5 IU (1.8 x 10 s IU of each serotype); panel (b), high dose: 7.2 x 10 6 IU (1.8 x 10 6 IU of each serotype). Mice were immunized at week 0 and boosted at week 8.
  • Figure 4 shows the kinetics of neutralizing antibody responses to DEN3 in rhesus macaques immunized with DEN3 prM/E-VRP or DEN3 E85-VRP at the dose of 1 x 10 8 IU. Macaques were immunized at week 0 and boosted at week 7 and 25.
  • Figure 5 shows serotype-specific neutralizing antibody titers in macaques immunized with DEN3 E85-VRP. Each dot represents one monkey sample, the bar represents the geometric mean neutralization titer, Limit of detection is 1 : 10. Neutralization titers against DENl , DEN2 and DEN4 are below the level of detection of the assay.
  • Figure 6 shows typical neutralization curves of undepleted, MBP depleted or
  • DEN3EDIII-MBP depleted macaque serum.
  • Panel (A) neutralization curves for 2 macaque sera 4 weeks post-challenged with DEN3 virus.
  • Panel (b) neutralization curves for 2 macaque sera 3 weeks after immunization with DEN3 E85 VRP3.
  • Figure 7 shows the effect of anti-vector immunity on the ability of the second dose to boost.
  • Figures 8A and 8B show the amino acid sequences of prM/E immunogens.
  • Figure 9 shows the amino acid sequences of E85 and ED III immunogens.
  • Figures 10A, 10B, 10C and 10D shows the nucleotide sequences encoding the prM/E and E85 immunogens.
  • Figures 11A and 11B show sequences of DEN2 strain New Guinea C immunogens: prM/E (nucleotide and amino acid), E85, E81 and EDIII (amino acid). The full length nucleotide and amino acids sequences are found at GenBank Accession No. AF038403. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention provides an innovation in the field of dengue vaccines.
  • the inventors have developed a platform based on an alphavirus vector expressing a dengue virus E protein antigen.
  • the invention provides a balanced response against multiple serotypes of dengue virus (e.g. , DENl, DEN2, DEN3 and DEN4), which reduces the risk of sensitizing the subject to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) in response to secondary infection.
  • the present invention can be practiced to produce an immune response against one or more dengue virus strains, genotypes and/or serotypes in young animals that have maternal antibodies present against the dengue virus(es).
  • a can mean one or more than one.
  • a cell can mean a single cell or a multiplicity of cells.
  • the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 211 1.03, Thus, the term “consisting essentially of when used in. a claim of this invention is not intended to be interpreted to be equivalent to "comprising.”
  • nucleic acid encompasses both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g. , chemically synthesized) DNA and chimeras of RNA and DNA.
  • the nucleic acid may be double-stranded or single-stranded.
  • the nucleic acid may be synthesized using nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such nucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • polypeptide encompasses both peptides and proteins
  • fusion protein is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different polypeptides not found fused together in nature are fused together in the correct translational reading frame.
  • an “isolated” polynucleotide e.g. , an “isolated nucleic acid” or an “isolated nucleotide sequence” means a polynucleotide at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • the "isolated" e.g. , an "isolated nucleic acid” or an “isolated nucleotide sequence
  • polynucleotide is present at a greater concentration (i.e. , is enriched) as compared with the starting material (e.g. , at least about a two-fold, three-fold, four-fold, ten- fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousand-fold or greater concentration).
  • the isolated polynucleotide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
  • an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.
  • the "isolated" polypeptide is present at a greater concentration (i.e. , is enriched) as compared with the starting material (e.g. , at least about a two-fold, three-fold, four-fold, ten-fold, twenty-fold, fifty-fold, one-hundred-fold, five-hundred-fold, one thousand-fold, ten thousandfold or greater concentration).
  • the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more pure.
  • an "isolated" cell is a cell that has been partially or completely separated from other components with which it is normally associated in nature.
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier.
  • immunogen and "antigen” are used interchangeably herein and mean any compound (including polypeptides) to which a cellular and/or humoral immune response can be directed,
  • an immunogen or antigen can induce a protective immune response against the effects of dengue virus infection.
  • Effective amount refers to an amount of a vector, nucleic acid, composition or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect.
  • the effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art, As appropriate, an “effective amount” in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.
  • immunogenic amount or "effective immunizing dose,” as used herein, unless otherwise indicated, means an amount or dose of a nucleic acid, alphavirus vector, population of alphavirus vectors and/or composition sufficient to induce an immune response (which can optionally be a protective response) in the treated subject that is greater than the inherent immunity of non-immunized subjects.
  • An immunogenic amount or effective immunizing dose in any particular context can be routinely determined using methods known in the art.
  • vaccine means vacuna
  • vaccination means a process or composition that increases a subject's immune reaction to an immunogen (e.g. , by providing an active immune response), and therefore its ability to resist, overcome and/or recover from infection (i.e. , a protective immune response).
  • treat By the term “treat,” “treating” or “treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • the term “treat,”, “treating” or “treatment of (and grammatical variations thereof) refer to a reduction in the severity of viremia and/or a delay in the progression of viremia, with or without other signs of clinical disease.
  • a “treatment effective” amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • prevent refers to prevention and/or delay of the onset and/or progression of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset and/or progression of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention.
  • the term “prevent,”, “preventing” or “prevention of (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of viremia in the subject, with or without other signs of clinical disease. The prevention can be complete, e.g.
  • the prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset and/or the progression is less than what would occur in the absence of the present invention.
  • prevention effective amount is an amount that is sufficient to prevent (as defined herein) the disease, disorder and/or clinical symptom in the subject. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.
  • the efficacy of treating and/or preventing dengue virus infection by the methods of the present invention can be determined by detecting a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters (e.g. , viremia), as would be well known to one of skill in the art.
  • a clinical improvement as indicated by a change in the subject's symptoms and/or clinical parameters (e.g. , viremia), as would be well known to one of skill in the art.
  • “protective” encompass both methods of preventing and treating dengue virus infection in a subject, whether against one or multiple strains, genotypes or serotypes of dengue virus.
  • protective immune response or “protective” immunity indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence and/or severity and/or duration of disease or any other manifestation of infection.
  • a protective immune response or protective immunity results in reduced viremia, whether or not accompanied by clinical disease.
  • a protective immune response or protective immunity may be useful in the therapeutic treatment of existing disease.
  • an “active immune response” or “active immunity” is characterized by “participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both.” Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody
  • an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination. Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host.” Id.
  • a "subject" of the invention includes any animal susceptible to dengue virus infection.
  • a subject is generally a mammalian subject ⁇ e.g. , a laboratory animal such as a rat, mouse, guinea pig, rabbit, primates, etc.), a farm or commercial animal (e.g. , a cow, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g. , cat, dog, ferret, etc.).
  • the subject is a primate subject, a non-human primate subject (e.g. , a chimpanzee, baboon, monkey, gorilla, etc.) or a human.
  • Subjects of the invention can be a subject known or believed to be at risk of infection by dengue virus.
  • a subject according to the invention can also include a subject not previously known or suspected to be infected by dengue virus or in need of treatment for dengue virus infection.
  • Subjects may be treated for any purpose, such as for eliciting a protective immune response or for eliciting the production of antibodies in that subject, which antibodies can be collected and used for other purposes such as research or diagnostic purposes or for administering to other subjects to produce passive immunity therein, etc.
  • Subjects include males and/or females of any age, including neonates, juvenile, mature and geriatric subjects. With respect to human subjects, in representative
  • the subject can be an infant (e.g. , less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, or younger), a toddler (e.g. , at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months), or a child (e.g. , at least about 1 , 2, 3, 4 or 5 years of age and/or less than about 14, 12, 10, 8, 7, 6, 5, or 4 years of age).
  • an infant e.g. , less than about 12 months, 10 months, 9 months, 8 months, 7 months, 6 months, or younger
  • a toddler e.g. , at least about 12, 18 or 24 months and/or less than about 36, 30 or 24 months
  • a child e.g. , at least about 1 , 2, 3, 4 or 5 years of age and/or less than about 14, 12, 10, 8, 7, 6, 5, or 4 years of age.
  • the subject is a human subject that is from about 0 to 3, 4, 5, 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 3 to 6, 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 6 to 9, 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 9 to 12, 15, 18, 24, 30, 36, 48 or 60 months of age, from about 12 to 18, 24, 36, 48 or 60 months of age, from about 18 to 24, 30, 36, 48 or 60 months of age, or from about 24 to 30, 36, 48 or 60 months of age.
  • the subject has maternal antibodies to dengue virus.
  • a "subject in need" of the methods of the invention can be a subject known to be, or suspected of being, infected with, or at risk of being infected with, dengue virus.
  • the present invention provides alphavirus vectors expressing one or more dengue virus antigens.
  • the dengue virus antigen is a dengue virus envelope (E) protein antigen, including without limitation prM/E, the full-length E protein, or immunogenically active fragments thereof.
  • E dengue virus envelope
  • immunogenically active fragment is a truncated E protein, including soluble (e.g. , lacking the transmembrane region(s)), truncated E proteins such as E85, E81 and EDIII (E domain HI).
  • a "soluble" E protein antigen does not span the cell membrane, e.g. , because it lacks functional forms of one or both transmembrane anchoring domains (see Figure 1).
  • the soluble E protein antigen is secreted.
  • the soluble E protein antigen lacks one or both transmembrane regions, e.g. , due to a C-terminal truncation of the dengue virus E protein.
  • the inventors have unexpectedly found that the alphavirus vectors, nucleic acids, compositions and pharmaceutical formulations of the invention induce a strong antibody response to EDIII.
  • EDIII is believed to contain the major neutralizing epitope(s) on the E protein; however, existing vaccines and even dengue virus infection fail to elicit a strong antibody response to this epitope(s).
  • the superior properties of the alphavirus vectors, nucleic acids, compositions and pharmaceutical formulations of the invention may be due, at least in part, to the strong anti- EDIII neutralizing antibody response that is elicited in the subject.
  • the present invention provides compositions and methods to increase the immunogenicity of a dengue viral E protein by partially or completely truncating the transmembrane domain of the protein such that the protein is no longer anchored in the membrane.
  • the dengue virus antigens of the invention can be derived from any dengue virus, including all serotypes, strains and genotypes, now known or later identified.
  • the dengue virus E protein antigen is derived from UNC1017 strain (DEN1), West Pacific 74 strain (DEN1), S 16803 strain (DEN2), UNC2005 strain (DEN2), UNC3001 strain (DEN3), UNC3043 (DEN3 strain 059.AP-2 from
  • the dengue isolate from which the antigen is derived can be selected so as to enhance the breadth of protection across different strains or genotypes within each serotype (e.g. , by using alignment methods). Naturally occurring antigens can also be modified to enhance cross-protection.
  • a dengue virus "E protein antigen” refers to a molecule (e.g. , polypeptide) that comprises, consists essentially of or consists of all or an immunologically active fragment of a dengue virus E protein.
  • An "immunogenically active fragment" of a dengue virus E protein is a fragment that induces an immune response in subject, optionally, a protective immune response.
  • an "immunogenically active fragment" of a dengue virus antigen comprises, consists essentially of or consists of at least about 6, 8, 10, 12, 15, 20, 30, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450 or more amino acids, optionally contiguous amino acids, and/or less than about 495, 475, 450, 425, 400, 350, 300, 250, 200, 150, 100, 75 or 50 amino acids, optionally contiguous amino acids, including any
  • the immunogenically active fragment induces an immune response in a host, optionally a protective immune response, that is at least about 50%, 75%, 80%, 85%, 90%, or 95% or more of the immune response induced by the full-length antigen or epitope, or induces an immune response that is the same as or essentially the same as the full-length antigen, or induces an immune response that is even greater than the immune response induced by the full-length antigen.
  • the immunogenically active fragment can stimulate humoral and/or cellular immune responses.
  • the immunogenically active fragment can stimulate humoral and/or cellular immune responses.
  • immunogenically active fragment comprises one or more B-epitope and/or T epitope.
  • Portions of a given polypeptide that include a B-cell epitope can be identified using any number of epitope mapping techniques that are known in the art. (See, e.g. , Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed., 1996, Humana Press, Totowa, N.J.).
  • linear epitopes can be determined by, e.g. , . concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports.
  • Such techniques are known in the art and described in, e.g. , U.S.
  • This computer program employs the Hopp/Woods method (Hopp et al,, Proc. Natl. Acad. Sci USA (1981 ) 78:3824-3828) for determining antigenicity profiles and the Kyte-Doolittle technique (Kyte et al., J. Mol. Biol (1982) 157: 105- 132) for hydropathy plots.
  • T-cell epitopes that are involved in stimulating the cellular arm of a subject's immune system are short peptides of about 8-25 amino acids.
  • a common way to identify T-cell epitopes is to use overlapping synthetic peptides and analyze pools of these peptides, or the individual ones, that are recognized by T cells from animals that are immune to the antigen of interest, using, for example, an enzyme-linked immunospot assay
  • ELISPOT overlapping peptides
  • MHC histocompatibility
  • the E protein antigen comprises, consists essentially of, or consists of the full-length E protein.
  • the E protein antigen comprises, consists essentially of, or consists of a soluble (e.g. , truncated at the C-terminus) E protein antigen. It is known in the art that the dengue virus E protein comprises two transmembrane domains ( Figure 1). In embodiments of the invention, the soluble E protein antigen lacks one or both
  • the soluble E protein antigen comprises one or both helical coil domains (HI and H2), optionally with the conserved sequence (CS) between the two ( Figure 1). In embodiments of the invention, the soluble E protein antigen comprises the HI domain, and optionally all or a portion of the conserved CS domain.
  • the soluble E protein antigen comprises, consists essentially of or consists of a dengue virus E85 immunogen and/or E81 immunogen ( Figure 1).
  • the E protein antigen comprises, consists essentially of, or consists of a dengue virus EDIII immunogen ( Figure 1) or an immunologically active fragment thereof.
  • the immunologically active fragment of EDM comprises, consists essentially of, or consists of at least about 6, 8, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids, optionally contiguous amino acids and/or less than about 103, 102, 101 , 100, 90, 80, 70, 60, 50, 40, 30, 20, 15 or 12 amino acids, optionally contiguous amino acids, including any combination of the foregoing as long as the lower limit is less than the upper limit and induces an immune response (e.g.
  • the immunologically active fragment of EDM comprises, consists essentially of, or consists of the IgG like domain found in EDM.
  • the dengue virus E protein antigen further comprises the EDI and/or EDII domains.
  • E protein antigen can optionally be fused directly or indirectly (e.g. , with intervening sequences) to all or a portion of the dengue virus membrane (M) protein or M protein precursor (prM) or a portion thereof (e.g. , at least about 6, 8, 10, 12, 15, 20, 30, 40, 50 or more amino acids, optionally contiguous amino acids).
  • M dengue virus membrane
  • prM M protein precursor
  • the E protein antigen is a C-terminally truncated form of the dengue virus E protein corresponding to about 81% (E81) or 85% (E85) of the N terminus of the protein.
  • the E protein antigen comprises, consists essentially of, or consists of the ectodomain (e.g. , residues 1 to 394 of DEN1 , DEN2 or DEN4; 1-392 of DEN3), the first helical coil domain HI (e.g. , residues 401 to 420 of DEN1 , DEN2 or DEN4 or residues 399 to 418 of DEN3) and part of the conserved sequence (CS) region (e.g.
  • CS conserved sequence
  • residues 420-424 of DEN1 , DEN2 or DEN4 or residues 418 to 422 of DEN3 but lacks the C terminal amino acids corresponding to the H2 domain and two transmembrane anchor domains (TM) (residues 425-495 in DEN1 , DEN2 or DEN4 or residues 423 to 493 of DEN3).
  • TM transmembrane anchor domains
  • the dengue virus E protein antigen comprises, consists essentially of, or consists of the ectodomain, and optionally, the predicted alpha- helix domain HI and/or all or a portion of the conserved sequence region at residues.
  • the dengue E protein antigen lacks all or part of one or both of the two transmembrane anchor domains such that the antigen is not membrane-anchored,
  • the E protein antigens lacks all or part of the H2 helical coil region.
  • the antigen can further comprise, consist essentially of, or consist of a modified form of any of the foregoing.
  • a "dengue virus antigen,” “dengue virus E protein antigen” or similar terms include, without limitation, naturally occurring dengue virus antigens and modified forms thereof that induce an immune response in a subject, optionally a protective immune response, against one or more dengue virus strains, genotypes and/or serotypes.
  • a native polypeptide antigen can be modified to increase safety and/or immunogenicity and/or as a result of cloning procedures or other laboratory manipulations.
  • the amino acid sequence of the modified form of the dengue virus antigen can comprise one, two, three or fewer, four or fewer, five or fewer, six or fewer, seven or fewer, eight or fewer, nine or fewer, ten or fewer, 12 or fewer, 15 or fewer, 20 or fewer, 25 or fewer, 30 or fewer, 35 or fewer or 40 or fewer modifications as compared with the amino acid sequence of the naturally occurring antigen and induce an immune response (optionally a protective immune response) against dengue virus in the host.
  • Suitable modifications encompass deletions (including truncations), insertions (including N- and/or C-terminal extensions) and amino acid substitutions, and any combination thereof.
  • the dengue virus antigen is a polypeptide antigen that is substantially similar or substantially identical at the amino acid level to the amino acid sequence of a naturally occurring dengue virus antigen and induces an immune response (optionally a protective immune response) against dengue virus in a host.
  • a "modified" dengue virus antigen induces an immune response in a host (e.g. , IgG and/or IgA that react with the native antigen), optionally a protective immune response, that is at least about 50%, 75%, 80%, 85%, 90%, or 95% or more of the immune response induced by the native antigen, or induces an immune response that is the same as or essentially the same as the native antigen, or induces an immune response that is even greater than the immune response induced by the native antigen.
  • a host e.g. , IgG and/or IgA that react with the native antigen
  • a protective immune response that is at least about 50%, 75%, 80%, 85%, 90%, or 95% or more of the immune response induced by the native antigen, or induces an immune response that is the same as or essentially the same as the native antigen, or induces an immune response that is even greater than the immune response induced by the native antigen.
  • amino acid sequence that is “substantially identical” or
  • substantially similar to a reference amino acid sequence is at least about 75%, 80%, 85%, 90%, 95%), 97%, 98% or 99% identical or similar, respectively, to the reference amino acid sequence.
  • Sequence similarity or identity may be determined using standard techniques laiown in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad, Sci.
  • BLAST algorithm Another suitable algorithm is the BLAST algorithm, described in Altschul et al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad, Sci. USA 90, 5873-5787 (1993).
  • a particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996);
  • WU-BLAST-2 uses several search parameters, which are optionally set to the default values.
  • the parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • alphavirus has its conventional meaning in the art, and includes the various species of alphaviruses including, but not limited to, Eastern Equine Encephalitis virus (EEE), Venezuelan Equine Encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, Western Equine Encephalitis virus (WEE), Sindbis virus, South African Arbovirus No.
  • EEE Eastern Equine Encephalitis virus
  • VEE Venezuelan Equine Encephalitis virus
  • Everglades virus Venezuelan Equine Encephalitis virus
  • Mucambo virus Pixuna virus
  • WEE Western Equine Encephalitis virus
  • Sindbis virus South African Arbovirus No.
  • alphavirus vectors which may optionally be a propagation-incompetent alphavirus vector, for example an alphavirus replicon vector (as described below).
  • alphavirus replicon vectors are described in U.S. Patent No.
  • the alphavirus vector comprises one or more heterologous nucleic acids.
  • at least one of the heterologous nucleic acids encodes an antigen.
  • Alphavirus vectors can be transcribed in vitro from cDNA molecules, for example, from a bacterial or viral promoter. Alternatively, they can be produced in vivo from a nucleic acid (e.g. , DNA), for example, from a viral or eukaryotic promoter (see, e.g. , U.S. Patent Nos. 5,814,482 and 6,015,686). Thus, the alphavirus vectors of the invention can be administered as a nucleic acid (e.g. , DNA) that expresses the alphavirus vector in vivo.
  • a nucleic acid e.g. , DNA
  • the alphavirus vector has a VEE virion shell.
  • the alphavirus may be a chimeric alphavirus and have a genomic RNA from another alphavirus.
  • the alphavirus virion comprises a VEE El glycoprotein and may comprise structural proteins (e.g. , capsid and/or E2 glycoprotein) from other alphaviruses.
  • the alphavirus is a VEE virus with both a VEE genomic RNA and virion coat.
  • Alphavirus vectors elicit a strong host response to the antigen(s) encoded by the heterologous sequence(s) in the vector. While not wishing to be held to any particular theory of the invention, it appears that alphavirus vectors induce a more balanced and comprehensive immune response (i.e. , cellular and humoral immunity) than do conventional vaccination methods. Moreover, it appears that alphavirus vectors induce a strong immune response, in part, because they directly infect and replicate within dendritic cells, The resulting presentation of antigen to the immune system induces a strong immune response.
  • the alphavirus 26S subgenomic promoter also appears to give high level of expression of a heterologous nucleic acid encoding an immunogen.
  • the alphavirus vector preparation may be partially or highly purified, or may be a relatively crude cell lysate or supernate from a cell culture, as known in the art.
  • the alphavirus vector can comprise, consist essentially of, or consist of a recombinant alphavirus genomic nucleic acid.
  • the alphavirus vector further comprises alphavirus structural proteins (e.g. , capsid and the El and E2 glycoproteins), e.g., the alphavirus vector is an alphavirus particle (e.g. , virion).
  • the invention also provides nucleic acids (e.g. , DNA), such as viral nucleic acid that comprise or encode an alphavirus vector of the invention encoding a dengue virus antigen(s).
  • nucleic acids e.g. , DNA
  • the invention further provides cells that comprise the nucleic acids and alphavirus vectors of the invention. Any suitable cell can be used including mammalian, avian, insect, yeast, bacterial and/or plant cells.
  • the alphavirus genomic RNA is a double promoter vector that is both replication and propagation competent. Double promoter vectors are described in United States Patent Nos. 5,185,440, 5,505,947 and 5,639,650, the disclosures of which are incorporated in their entireties by reference.
  • the alphavirus genomic RNA used to construct the double promoter vector is a VEE, Semliki Forest Virus, S.A.AR86, Girdwood S.A., TR339, Sindbis or Ockelbo genomic RNA.
  • the double promoter vector contains one or more attenuating mutations in the genomic RNA. Attenuating mutations are described in more detail hereinbelow.
  • the double promoter vector is constructed so as to contain a second subgenomic promoter ( . e. , 26S promoter) inserted 3' to the virus RNA encoding the structural proteins.
  • the heterologous RNA is inserted between the second subgenomic promoter, so as to be operatively associated therewith, and the 3' UTR of the virus genome.
  • Heterologous RNA sequences of less than about 3 kilobases, preferably those less than about 2 kilobases, and more preferably those less than about 1 kilobase, can be inserted into the double promoter vector.
  • the double promoter vector is derived from a VEE genomic RNA
  • the second subgenomic promoter is a VEE subgenomic promoter
  • the double promoter vector is derived from a Sindbis (e.g. , TR339) genomic RNA
  • the second subgenomic promoter is a Sindbis (e.g., TR339) subgenomic promoter.
  • Replicon vectors which are infectious, propagation-defective, virus vectors can also be used to carry out the present invention.
  • Replicon vectors are described in more detail in WO 96/37616 to Johnston et al., U.S. Patent No. 5,505,947 to Johnston et al., and U.S. Patent No. 5,792,462 to Johnston et al; the disclosures of which are incorporated by reference herein in their entireties.
  • Alphaviruses for constructing the replicon vectors according to the present invention include, but are not limited to, VEE, Semliki Forest Virus, S.A.AR86, Girdwood S.A., Sindbis (e.g. , TR339), and Ockelbo.
  • one or more foreign gene(s) to be expressed is/are inserted in place of at least a portion of one or more of the viral structural protein genes in a transcription vector containing the viral sequences necessary for viral replication (e.g., the nspl- 4 genes).
  • RNA transcribed from this vector contains sufficient viral sequences (e.g. , the viral nonstructural genes) to be competent for RNA replication and transcription.
  • This RNA can be transcribed in vitro or in vivo. In the case of in vitro transcribed RNA, the RNA is first transfected into susceptible cells by any method known in the art, wherein it is replicated and translated to give the replication proteins.
  • RNA messenger RNA
  • transgene(s) is/are operatively associated with the alphavirus 26S subgenomic promoter, which will produce high level of the transcript and, in the case of a translated RNA, the protein of interest.
  • the autonomously replicating RNA (/ ' . e. , replicon) can only be packaged into virus particles if the deleted alphavirus structural protein genes are provided.
  • the deleted alphavirus structural protein genes may be provided by any suitable means, e.g. , by a stably transformed packaging cell line (see, e.g. , U.S. Patent No. 5,789,245), or by one or more helper nucleic acid molecules (RNA or DNA), which are provided to the cell along with the replicon vector, and are then expressed in the cell so that new replicon particles are produced in the cell.
  • the helper nucleic acids do not contain the viral nonstructural genes for replication, but these functions are provided in trans by the replicon molecule.
  • the non-structural proteins translated from the replicon molecule transcribe the structural protein genes on the helper nucleic acid molecule, resulting in the synthesis of viral structural proteins and packaging of the replicon into virus-like particles.
  • the alphavirus packaging or encapsidation signals are located within the nonstructural genes, the absence of these sequences in the helper nucleic acids precludes their incorporation into virus particles.
  • the replicon molecule is "propagation defective," as described hereinabove inasmuch as the replicon RNA in these particles does not include all of the alphavirus structural proteins required for encapsidation, at least a portion of at least one of the required structural proteins being deleted therefrom.
  • the replicon RNA therefore only initiates an abortive infection; no new viral particles are produced, and there is no spread of the infection to other cells.
  • the replicon molecule comprises an alphavirus packaging signal.
  • the replicon molecule is self-replicating. Accordingly, the replicon molecule comprises sufficient coding sequences for the alphavirus nonstructural polyprotein so as to support self- replication. In embodiments of the invention, the replicon encodes the alphavirus nsPl, nsP2, nsP3 and nsP4 proteins.
  • the replicon molecules of the invention "do not encode” one or more of the alphavirus structural proteins.
  • do(es) not encode one or more structural proteins, it is intended that the replicon molecule does not encode a functional form of one or more structural proteins and, thus, a complementing sequence is provided by a helper or packaging cell to produce new virus particles.
  • the replicon molecule does not encode any of the alphavirus structural proteins.
  • the replicon may not encode the structural protein(s) because the coding sequence is partially or entirely deleted from the replicon molecule. Alternatively, the coding sequence is otherwise mutated so that the replicon does not express the functional protein. In embodiments of the invention, the replicon lacks all or substantially all of the coding sequence of the structural protein(s) that is not encoded by the replicon, e.g., so as to minimize recombination events with the helper sequences.
  • the replicon molecule may encode at least one, but not all, of the alphavirus structural proteins.
  • the alphavirus capsid protein may be encoded by the replicon molecule.
  • one or both of the alphavirus glycoproteins may be encoded by the replicon molecule.
  • the replicon may encode the capsid protein and either the El or E2 glycoprotein.
  • none of the alphavirus structural proteins are encoded by the replicon molecule.
  • all or essentially all of the sequences encoding the alphavirus capsid protein and glycoproteins may be deleted from the replicon molecule.
  • the invention provides a composition comprising a population of replicon particles containing no detectable replication-competent alphavirus particles.
  • Replication-competent virus may be detected by any method known in the art, e.g. , by neurovirulence following intracerebral injection into suckling mice, or by passage twice on alphavirus-permissive cells (e.g. , BHK cells) and evaluation for virus induced cytopathic effects.
  • the present invention also provides alphavirus virion coats (e.g. , VEE virion coats) including attenuating mutations (as defined above) and genomic RNA and DNA constructs encoding the same.
  • alphavirus virion coats e.g. , VEE virion coats
  • attenuating mutations as defined above
  • genomic RNA and DNA constructs encoding the same.
  • the alphaviruses of the invention may further comprise attenuating mutations in the nonstructural protein coding region or other regions of the alphavirus genome.
  • the attenuating mutation(s) reduces ⁇ e.g. , by at least 25%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the neurovirulence of the alphavirus vector ⁇ e.g. , as determined by intracerebral injection in weanling or adult mice). It is not necessary that the attenuating mutations of the invention eliminate all pathology or adverse effects associated with virus administration, as long as there is some improvement or benefit ⁇ e.g., increased safety and/or reduced morbidity and/or reduced mortality) as a result of the attenuating mutation.
  • Exemplary attenuating mutations include, but are not limited to, those described in United States Patent No. 5,505,947 to Johnston et al., U.S. Patent No. 5,185,440 to Johnston et al span U.S. Patent No. 5,643,576 to Davis et al., U.S. Patent No. 5,792,462 to Johnston et al, and U.S. Patent No. 5,639,650 to Johnston et al., the disclosures of which are incorporated herein in their entirety by reference.
  • Attenuating mutations of particular interest include attenuating mutations in the El glycoprotein of the alphavirus virion shell ⁇ e.g. , VEE virion shell). While not wishing to be bound by any theory of the invention, the E2 glycoprotein is believed to bind to cellular virus receptors and, thus, El mutants may advantageously achieve attenuation without disrupting cellular targeting. Accordingly, in embodiments of the invention, the attenuating mutation is a mutation in the E1 glycoprotein ⁇ e.g. , the VEE E1 glycoprotein) that does not unduly interfere ⁇ e.g. , reduce by more than 25%, 35% or 50%o) with cellular targeting, receptor binding and/or infectivity, for example, to or in dendritic cells.
  • the attenuating mutation is in the putative fusogenic peptide region in the alphavirus El glycoprotein ⁇ e.g., the fusogenic peptide region of the VEE El glycoprotein).
  • This region is from about amino acid 80 to about amino acid 93 of the El glycoprotein and contains a stretch of uncharged and hydrophobic amino acids ⁇ see, e.g. , Davis et al., (1994) Arch Virol [Suppl] 9:99).
  • the glycoproteins rearrange and this hydrophobic domain is exposed and is believed to facilitate entry of the virus across the cellular membrane.
  • the alphavirus virion shell has an attenuating mutation at El glycoprotein amino acid position 81.
  • the attenuating mutation may be a phenylalanine to leucine or isoleucine mutation in Sindbis virus ⁇ e.g. , strain TR339) or a mutation from tyrosine to leucine or isoleucine in Semliki Forest Virus or Ross River Virus. Similar mutations in the El fusogenic region may be made in any alphavirus (as defined above).
  • the alphavirus comprises a VEE virion shell comprising an attenuating mutation at El glycoprotein amino acid position 81 and/or 253.
  • the VEE virion shell may additionally contain other attenuating mutations.
  • Attenuating mutations may be selected as described below.
  • the attenuating mutation at amino acid position 81 is a mutation from phenylalanine to leucine or isoleucine.
  • the attenuating mutation at amino acid position 253 is a mutation from phenylalanine to serine or threonine.
  • Another particular attenuating mutation is an attenuating mutation in the VEE virion shell at El amino acid position 83.
  • this attenuating mutation is used together with a second site suppressor mutation to avoid lethality.
  • the present invention advantageously provides immunogenic compositions comprising attenuated alphavirus particles with improved efficacy (e.g. , provides protection at a lower dosage) as compared with other attenuated alphaviruses.
  • Methods of assessing the effectiveness of immunogenic compositions are well known in the art and include but are not limited to methods of evaluating protection against a challenge pathogen and indirect methods such as determination of antibody titers.
  • the present invention provides alphaviruses having attenuating mutations that achieve attenuation without significantly reducing (e.g. , by more than 25%, 35% or 50%) immunogenicity, thereby resulting in a need for a corresponding increase in dosage.
  • the present invention provides an attenuated alphavirus having a VEE shell, where the alphavirus is substantially as immunogenic as, or is even substantially more immunogenic than, a comparable alphavirus having a wild-type VEE virion shell (for example, the VEE 3000 described herein), / ' . e. , a substantially similar number of infectious virus particles or even substantially less virus is required to provide an
  • the attenuated alphavirus is as immunogenic as an alphavirus having a wild-type VEE coat (e.g. , VEE 3000) at a dosage that is about 50% to 200% of the dosage of the vims having the wild- type VEE coat, i.e., one-half to two times as much attenuated virus is needed to elicit the same immune response as an alphavirus having a wild-type coat.
  • the alphavirus is "substantially more immunogenic" than a comparable alphavirus comprising a wild-type VEE coat, i.e. , a substantially lower dosage (e.g. , less than about 50%) of the attenuated virus provides the same immune response as the alphavirus comprising the wild-type VEE coat.
  • the attenuated alphavirus is as immunogenic as an alphavirus having a wild-type VEE coat (e.g. , VEE 3000) at a dosage that is about 250% or more of the dosage of a comparable alphavirus having a wild-type VEE coat, i. e. , 2.5 -times or more attenuated virus is needed to elicit the same immune response as an alphavirus having a wild-type coat.
  • a wild-type VEE coat e.g. , VEE 3000
  • alphaviruses having attenuated VEE coats that are less immunogenically effective than a comparable alphavirus having a wild-type VEE virion shell can nonetheless be advantageous and are encompassed by the present invention, e.g. , attenuated viruses that require a dosage that is less than about 5ive-fold, less than about 7.5-fold, less than about 10-fold, less than about 15- fold, less than about 25-fold, less than about 50-fold higher, or even less than about 100-fold higher than the dosage of a comparable virus having a wild-type VEE virion shell to elicit a similar immune response.
  • the attenuated vims is more immunogenic than a comparable attenuated virus comprising the 3014 VEE coat described below, i. e. , a lower dosage of the attenuated virus of the invention produces an immunogenically effective response as compared with the dosage of an alphavirus comprising the 3014 coat.
  • the immunogenically effective dosage of the attenuated virus of the invention is less than about 25%, about 50%, or about 75% of the dosage of a comparable virus having a 3014 VEE virion shell.
  • the immunogenically effective dosage of the attenuated virus is reduced by about one order of magnitude, two orders of magnitude, or even three orders of magnitude or more as compared with the dosage of a comparable vims having a 3014 VEE coat.
  • the relative immunogenicity of the attenuated alphavirus as compared with a suitable non-attenuated control virus may vary depending upon the particular dosage, route of administration, species and age of the subject, and the like.
  • VEE virion shell only interacts poorly with heparin, whereas some attenuated VEE mutants (e.g., the 3014 mutant having an Ala ⁇ Thr mutation at E1 position 272, a Glu ⁇ Lys mutation at E2 position 209, and a Ile ⁇ Asn mutation at E2 position 239) bind relatively strongly to heparin.
  • Methods of detecting viral interaction with heparin are known to those skilled in the art, for example, binding to immobilized heparin (e.g. , a heparin column or beads) or inhibition of cell infectivity or binding by heparin (e.g. , to BHK cells or dendritic cells), which are described in Bernard et al., (2000) Virology 276:93).
  • the attenuated viruses of the invention do not exhibit detectable binding to, or only weakly bind to, heparin or heparan sulfate.
  • the attenuated viruses of the invention are more similar to the wild-type virus than the 3014 mutant described above with respect to heparin binding. While not wishing to be bound by any particular theory, it appears that binding to heparin and/or heparan sulfate may increase viral clearance rates and reduce infectivity, with a resulting loss of immunogenicity.
  • the attenuated virus e.g.
  • an attenuated alphavirus with a VEE virion shell does not exhibit detectable binding to glycosaminoglycans (e.g. , heparin, heparan sulfate, chondroitin, chondroitin sulfate and/or dextran sulfate) or only exhibits weak binding thereto.
  • glycosaminoglycans e.g. , heparin, heparan sulfate, chondroitin, chondroitin sulfate and/or dextran sulfate
  • the alphavirus comprises a VEE virion shell comprising an attenuating mutation in the El glycoprotein, where the alphavirus exhibits no detectable binding or only weak binding to heparin.
  • the alphavirus comprises a VEE virion shell comprising an attenuating mutation in the fusogenic peptide region of the El glycoprotein (as described above), wherein the alphavirus exhibits no detectable binding or only weak binding to heparin.
  • the virion shell can further comprise additional attenuating mutations in the E2 and/or E3 glycoproteins (exemplary mutations in the E2 and E3 glycoproteins are discussed below).
  • the alphavirus comprises a VEE virion shell comprising an attenuating mutation at El amino acid position 81 and/or El 253 (each as described above), and exhibits no detectable binding or only weak binding to heparin.
  • the 3042 mutation has a Phe ⁇ Ile mutation at El position 81.
  • the alphavirus comprises a VEE coat comprising an attenuating mutation that results in the deletion of the furin cleavage site in the E3 glycoprotein (e.g. , deletion of E3 amino acids 56-59), and exhibits no detectable binding or only weak binding to heparin.
  • This type of attenuating mutation may be present in conjunction with a second site mutation to maintain viability (e.g.
  • the attenuated mutant comprises a mutation (e.g. , Phe ⁇ Ser) at El position 253 and a deletion of the furin cleavage site (e.g. , deletion of E3 amino acids 56-59), and exhibits no detectable binding or only weak binding to heparin.
  • a mutation e.g. , Phe ⁇ Ser
  • a deletion of the furin cleavage site e.g. , deletion of E3 amino acids 56-59
  • the attenuated alphavirus comprises a VEE virion shell comprising an attenuating mutation at El amino acid 272 (e.g. , an Ala - Thr mutation).
  • the attenuated alphavirus comprises a VEE virion shell comprising attenuating mutations at E2 amino acids 76 and 166 (e.g. , Glu ⁇ Lys mutation at E2 position 76 and a Lys ⁇ Glu mutation at E2 position 116).
  • virus interaction with heparin may be assessed by inhibition of virus infectivity.
  • a vims that "exhibits (only) weak binding" to heparin does not demonstrate a substantial reduction (/. e. , more than about 50%) in infectivity (e.g. , in BHK cells or dendritic cells) in the presence of relatively low concentrations of heparin (e.g. , concentrations of about 50, 100, 150 or 200 ⁇ ig/ml or less).
  • heparin binds to the attenuated vims comprising the VEE virion shell (e.g.
  • interfering with infectivity of the virus with an affinity that is similar to or less than the affinity of heparin for the wild-type virus or, alternatively, is less than about two-fold, three-fold, four-fold, or five-fold greater than the affinity of the wild-type virion shell for heparin.
  • the affinity of heparin binding to the alphavirus comprising the attenuated VEE virion shell is less than about 25%, 20%, 15%, 10%, 5% or less than the affinity of the 3014 coat for heparin, e.g., interference of virus infectivity by heparin is less than about 25%, 20%, 15%, 10%), 5% or less than the interference of infectivity by a virus comprising the 3014 coat.
  • Attenuating mutations other than those specifically disclosed herein using methods known to those skilled in the art (see, e.g., Olmsted et al, (1984) Science 225:424 and Johnston and Smith (1988) Virology 162:437).
  • Olmsted et al. describes a method of identifying attenuating mutations in Sindbis virus by selecting for rapid growth in cell culture.
  • the Johnston and Smith publication describes the identification of attenuating mutations in VEE by applying direct selective pressure for accelerated penetration of BHK cells.
  • Attenuating mutations having the desired characteristics for example, improved immunogenicity as compared with laiown attenuating alphaviruses
  • tecliniques for assessing immunogenicity known in the art e.g. , antibody titers may be measured by ELISA assay, hemagglutinin inhibition, virus neutralization and plaque reduction neutralization assays
  • ELISA assay hemagglutinin inhibition, virus neutralization and plaque reduction neutralization assays
  • the present invention also includes methods for identification of attenuating mutations that lack the ability to bind heparin and have increased immunogenicity.
  • One such method involves the selection of virus particles with the ability to infect cell monolayers in vitro in the presence of heparin or heparan sulfate.
  • other glycosaminoglycans can be used for this selection, including, but not limited to dextran sulfate, chondroitin sulfate A, chondroitin sulfate B as described in Klimstra el al. (1998) J Virol 72:7357-7366.
  • a spectrum of mutations are first engineered into the El and/or E2 glycoproteins of the alphavirus by methods well known in the art, such as random, site-directed or saturation mutagenesis.
  • This heterogeneous population of mutated viral particles is then incubated with a permissive (i.e. a cell line that can be infected by the alphavirus) cell line in vitro in the presence of glycosaminoglycans at a sufficient concentration as to be inhibitory to the infection of the cell line by viral particles known to bind heparin, e.g. , between 20 and 300 microgram/per ml.
  • a permissive i.e. a cell line that can be infected by the alphavirus
  • the viral population can be incubated with the
  • glycosaminoglycan prior to exposure of the cell line to the mutant particles.
  • This screening method selectively prohibits the entry of viral particles with significant affinity for the particular glycosaminoglycan and imposes selective pressure, allowing identification of low or non-binding glycosaminoglycan mutants that are able to enter the cell and establish a productive infection.
  • These mutants are then passed for multiple passages through the cell line, under the same or increased stringencies of selection for non-glycosaminoglycan binding alphaviral shells.
  • the selected mutant populations are isolated by plaque assay, plaque purified by methods known in the art to produce clonal populations of viral particles that are sequenced to identify individual and/or combinations of non-glycosaminoglycan binding mutations.
  • These mutations either separately or in combination, are introduced into the wild-type virus and further selected for their attenuation and potential increased immunogenicity by methods known in the art, e.g. Davis et al. 1991 ; U.
  • Another method for selecting attenuating mutations encompassed by this invention is to take the mutagenized viral population described above, which consists of a mixed population of alphaviral shell-mutated viruses, and select within this population using affinity-based chromatographic techniques, for example glycosaminoglycan matrix chromatographic columns (specifically heparin or any other glycosaminoglycan as described above). Low or non-glycosaminoglycan-binding mutant virus particles will pass through or elute from the column in the early fractions. Individual clonal viral populations are then isolated from these fractions by plaque purification.
  • affinity-based chromatographic techniques for example glycosaminoglycan matrix chromatographic columns (specifically heparin or any other glycosaminoglycan as described above).
  • Purified viral clones are sequenced by standard methods to identify the specific mutations that can be introduced into the wild-type virus shell, and virus or replicon particles made with such mutated shells are assayed for both attenuation and immunogenicity.
  • the overall stringency of this column selection method can be increased or decreased by methods known in the art such as altering column conditions, e.g. buffer pH, salt concentration, column length, and chromatographic matrix choice, to optimize the retention of glycosaminoglycan binding mutants and to expand the range of mutations that might be usefully employed in this invention.
  • the present invention encompasses other attenuating mutations that do not substantially reduce immunogenicity (i. e. , the attenuated virus is essentially as immunogenic as, or more immunogenic than, a comparable alphavirus having a wild-type coat).
  • alphavirus structural proteins are from VEE
  • other suitable attenuating mutations may be selected from the group consisting of codons at E2 amino acid position 76 which specify an attenuating amino acid, preferably lysine, arginine, or histidine as E2 amino acid 76; codons at E2 amino acid position 120 which specify an attenuating amino acid, preferably lysine as E2 amino acid 120; codons at E2 amino acid position 209 which specify an attenuating amino acid, preferably lysine, arginine or histidine as E2 amino acid 209; codons at El amino acid 272 which specify an attenuating amino acid, preferably threonine or serine as El amino acid 272, as provided above.
  • Attenuated alphavirus vectors comprise an attenuating mutation in the capsid protease that reduces, preferably ablates, the autoprotease activity of the capsid and results, therefore, in non-viable virus.
  • Capsid mutations that reduce or ablate the autoprotease activity of the alphavirus capsid are known in the art, see e.g. , WO 96/37616 to Johnston et al., the disclosure of which is incorporated herein in its entirety.
  • the alphavirus vector comprises a VEE capsid protein in which the capsid protease is ablated, e.g. , by introducing an amino acid substitution at VEE capsid position 152, 174, or 226.
  • one or more of the homologous positions in other alphaviruses may be altered to reduce capsid protease activity.
  • the attenuating mutation may be a mutation at capsid amino acid position 215 (e.g., a Ser- ⁇ Ala) that reduces capsid autoprotease activity (see, Hahn et al., (1990) J. Virology 64:3069).
  • the alphavims structural proteins are from S.A.AR86.
  • Exemplary attenuating mutations in the S.A.A 86 structural proteins are known in the art (see, e.g. , International Application No. PCT/US03/09121 ; incorporated by reference herein in its entirety).
  • amino acid substitutions may be based on any characteristic loiown in the art, including the relative similarity or differences of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Amino acid substitutions other than those disclosed herein may be achieved by changing the codons of the genomic RNA sequence (or a DNA sequence), according to the following codon table:
  • the hydropathic index of the amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art ⁇ see, yte and Doolittle, (1982) J Mol. Biol. 157: 105; incorporated herein by reference in its entirety). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.
  • tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the hydropathic index of the amino acid may be considered when identifying additional attenuating mutations according to the present invention
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine ( ⁇ 3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0);
  • threonine (-0.4); proline (-0.5 ⁇ I); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • hydrophilicity of the amino acid may be considered when identifying additional attenuating mutations according to the present invention.
  • the attenuating mutations may be located in any of the structural proteins.
  • the alphavirus vectors may contain two or more attenuating mutations within one structural protein or may contain two or more attenuating mutations distributed among the structural proteins. Further, additional attenuating mutations may be located on the replicon RNA in either the non- structural or structural coding regions as well as in non-coding regions,
  • Mutations may be introduced into the alphavirus vector by any method known in the art.
  • mutations may be introduced into the alphavirus RNA by performing site-directed mutagenesis on the cDNA which encodes the RNA, in accordance with known procedures (see, Kunkel, Proc. Natl. Acad Sci. USA 82, 488 (1985), the disclosure of which is incorporated herein by reference in its entirety).
  • mutations may be introduced into the RNA by replacement of homologous restriction fragments in the cDNA which encodes for the RNA, in accordance with known procedures.
  • helper cells for producing alphavirus particles in vitro.
  • Methods and helper cells for producing alphavirus stocks, ' including double-promoter alphaviruses and alphavirus replicon particles are known in the art. See, e.g. , Patent No. 5,185,440 to Davis et al., U.S. Patent No. 5,505,947 to Johnston et al.; U.S. Patent No. 5,792,462 to Johnston et al., and Pushko et al. (1997) Virol. 239:389-401 ; the disclosures of which are incorporated herein by reference in their entireties.
  • the methods and helper cells are used to produce propagation-incompetent alphavirus particles, for example, propagation-incompetent alphavirus replicon particles.
  • the helper cells of the invention contain one or more helper nucleic acid sequences (e.g. , as DNA and/or RNA molecules) encoding the alphavirus structural proteins (e.g. , VEE structural proteins).
  • the combined expression of the replicon molecule and the one or more helper molecules in the helper cell results in the production of an assembled alphavirus particle comprising a replicon RNA packaged within a virion comprising alphavirus structural proteins, which is able to infect a cell, but is unable to produce a productive infection (i. e. , produce new virus particles).
  • the population of alphavirus particles produced according to the invention contains no detectable propagation-competent alphavirus particles.
  • Propagation-competent virus may be detected by any method known in the art, e.g. , by neurovirulence following intracerebral injection into suckling mice, or by passage twice on alphavirus-permissive cells (e.g. , BHK cells) and evaluation for virus induced cytopathic effects.
  • the helper cells are typically alphavirus-permissive cells.
  • Alphavirus-permissive cells employed in the methods of the present invention are cells that, upon transfection with the viral RNA transcript, are capable of producing viral particles.
  • Alphaviruses have a broad host range. Examples of suitable host cells include, but are not limited to fibroblasts, Vero cells, baby hamster kidney (BHK) cells, 293 cells, 293T cells, and chicken embryo fibroblast cells (e.g., DF-1 cells).
  • helper cells of the invention may comprise sequences encoding the alphavirus structural proteins sufficient to produce an alphavirus particle, as described herein.
  • the helper cell may comprise a replicon RNA comprising one or more heterologous sequences, also as described herein,
  • sequences encoding the alphavirus structural proteins are distributed among one or more helper molecules
  • one or more structural proteins may be encoded by the replicon RNA, provided that the replicon RNA does not encode at least one structural protein such that the resulting alphavirus particle is propagation-incompetent in the absence of the helper sequence(s).
  • At least one of the alphavirus structural and/or non- structural proteins encoded by the replicon and helper molecules contain one or more attenuating mutations, as described herein.
  • the replicon molecule encodes at least one, but not all, of the alphavirus structural proteins (e.g. , the El and/or E2 glycoproteins and/or the capsid protein).
  • the replicon encodes the capsid protein, and the El and E2 glycoproteins are encoded by one or more separate helper molecules. It may be advantageous to provide the glycoproteins by two separate helper molecules, so as to minimize the possibility of recombination to produce replication-competent vims.
  • the replicon does not encode any of the El glycoprotein, the E2 glycoprotein, or the capsid protein.
  • the capsid protein and alphavirus glycoproteins are encoded by one or more helper molecules, preferably two or more helper molecules. By distributing the coding sequences for the structural proteins among two, three or even more helper molecules, the likelihood that recombination will result in replication- competent virus is reduced.
  • the replicon does not encode any of the alphavirus structural proteins, and may lack the sequences encoding the alphavirus structural proteins.
  • the replicon may not encode the structural protein(s) because of a partial or complete deletion of the coding sequence(s) or otherwise contains a mutation that prevents the expression of a functional protein(s).
  • all or substantially all of the coding sequences for the structural protein(s) that is not encoded by the replicon are deleted from the replicon molecule.
  • the El and E2 glycoproteins are encoded by one helper molecule, and the capsid protein is encoded by another helper molecule.
  • the El glycoprotein, E2 glycoprotein, and capsid protein are each encoded by separate helper molecules.
  • the capsid protein and one of the glycoproteins are encoded by one helper molecule, and the other glycoprotein is encoded by a second helper molecule.
  • helper and replicon sequences are RNA molecules that are introduced into the cell, e.g. , by lipofection or electroporation.
  • Uptake of helper RNA and replicon RNA molecules into packaging cells in vitro can be carried out according to any suitable means known to those skilled in the art. Uptake of RNA into the cells can be achieved, for example, by treating the cells with DEAE-dextran, treating the RNA with LIPOFECTINTM before addition to the cells, or by electroporation, with electroporation being the currently preferred means.
  • electroporation being the currently preferred means.
  • helper and/or replicon molecules are DNA molecules, which are either stably integrated into the genome of the helper cell or expressed from an episome ⁇ e.g. , an EBV derived episome).
  • the DNA molecule may be any vector known in the art, including but not limited to a non-integrating DNA vector, such as a plasmid, or a viral vector.
  • the invention provides recombinant alphavirus vectors comprising one or more nucleic acids encoding one or more dengue virus antigens as described herein.
  • the alphavirus vector can comprise one or more (e.g. , two, three, four, five, etc.) heterologous nucleic acid sequences, optionally each encoding a dengue virus antigen according to the present invention.
  • the alphavirus vector can also encode other polypeptides, such as immunomodulatory polypeptides and/or an antigen from any other organism of interest.
  • each heterologous nucleic acid sequence will typically be operably associated with a promoter.
  • an internal ribosome entry site (IRES) sequence(s) can be placed downstream of the first heterologous nucleic acid sequence and upstream of a second or additional heterologous nucleic acid sequence(s).
  • IRS internal ribosome entry site
  • heterologous nucleic acid sequence(s) can be associated with a constitutive or inducible promoter.
  • An exemplary promoter is an alphavirus 26S subgenomic promoter (e.g. , VEE 26S subgenomic promoter),
  • the S.A.AR86 26S subgenomic promoter can be used with S.A.AR86 replication proteins
  • the VEE 26S subgenomic promoter can be used with VEE replication proteins, and the like.
  • an alphavirus vector comprises nucleotide sequences encoding two or more E protein antigens from DENl, DEN2, DEN3 and/or DEN4.
  • the alphavirus vector comprises a nucleotide sequence encoding an E protein antigen from DENl, DEN2, DEN3 and DEN4.
  • the dengue virus E protein antigen can be any antigen as described herein.
  • the E protein antigen is a soluble E protein antigen and comprises, consists essentially of, or consists of E85, E81 or EDIII or an immunogenically active fragment thereof. Alternatively, any other E protein antigen as described herein can be employed in this embodiment.
  • the nucleotide sequences encoding the multiple E protein antigens can be fused into a polyprotein or can each be operably associated with a different promoter (e.g. , alphavirus 26S subgenomic promoter) or, alternatively, an IRES sequence can be used as described above and as understood in the art.
  • a different promoter e.g. , alphavirus 26S subgenomic promoter
  • an IRES sequence can be used as described above and as understood in the art.
  • a nucleic acid encoding the recombinant alphavirus vector is administered to the subject and the alphavirus vector(s) expressed in vivo.
  • the alphavirus vector comprises the alphavirus structural proteins, e.g. , is an alphavirus particle (e.g. , virion).
  • alphavirus structural proteins e.g. , is an alphavirus particle (e.g. , virion).
  • the alphavirus vector can further comprise one or more heterologous nucleotide sequences encoding a dengue virus antigen from two or more different dengue serotypes, strains and/or genotypes.
  • an alphavirus vector may comprise nucleotide sequences encoding antigens from two different dengue strains of the same serotype.
  • the present invention can advantageously be practiced to induce an immune response against one, two, three or all four of DENl, DEN2, DEN3 and DEN4.
  • the immune response may be predominantly directed against only some of the target serotypes. Multiple vaccinations are then required to try to achieve a response against all serotypes; however, in the case of dengue virus, this approach can be dangerous because repeated administrations to a subject with pre-existing antibodies can lead to dengue hemorrhagic fever.
  • the inventors have unexpectedly found that in embodiments of the present invention a more balanced immune response against all targeted serotypes is achieved with the additional advantage that for each serotype the antibody response is strongly directed against EDIII.
  • the invention provides an alphavirus vector or nucleic acid encoding the same that comprises one or more heterologous nucleic acids encoding a dengue virus E protein antigen of the invention from two, three or all four dengue serotypes (in any combination).
  • the alphavirus vector e.g. , VEE
  • the alphavirus vector can encode an antigen comprising, consisting essentially of, or consisting of prM/E, E, E85, E81 and/or EDIII from two or more of DEN1 , DEN2, DEN3 and DEN4.
  • the invention also provides a composition comprising a population of alphavirus vectors, where the population as a whole expresses a dengue antigen of the invention from two, three or all four dengue serotypes (in any combination). For example, there can be four different alphavirus vectors in the population, each encoding a dengue virus antigen from a different serotype. However, one or more of the alphavirus vectors in the population may encode antigens from two or more dengue virus serotypes, strains and/or genotypes. The population as a whole can further express a dengue virus antigen of the invention from two or more different dengue serotypes, strains and/or genotypes.
  • the invention provides a composition comprising two or more different alphavirus vectors comprising a dengue virus E protein antigen of the invention, wherein the composition comprises: (a) a first alphavirus vector comprising a heterologous nucleotide sequence encoding a first dengue virus E protein antigen from a first dengue virus serotype, strain and/or genotype; and a second alphavirus vector comprising a heterologous nucleotide sequence encoding a second dengue virus E protein antigen from a second dengue virus serotype, strain and/or genotype.
  • the two or more different alphavirus vectors comprise nucleotide sequences encoding E protein antigens from two or more of DEN1 , DEN2, DEN3 and DEN4.
  • the two or more different alphavirus vectors comprise nucleotide sequences encoding E protein antigens from DEN1 , DEN2, DEN3 and DEN4.
  • the composition can comprise one or more nucleic acids encoding the alphavirus vectors.
  • the composition comprises: (a) a first alphavirus vector comprising a heterologous nucleotide sequence encoding a dengue virus E protein antigen from DEN1; (b) a second alphavirus vector comprising a heterologous nucleotide sequence encoding a dengue virus E protein antigen from DEN2; (c) a third alphavirus vector comprising a heterologous nucleotide sequence encoding a dengue virus E protein antigen from DEN3; and (d) a fourth alphavirus vector comprising a heterologous nucleotide sequence encoding a dengue virus E protein antigen from DEN4.
  • the composition can comprise one or more nucleic acids encoding the alphavirus vectors, for example, four nucleic acids encoding each of the alphavirus vectors delivering the DEN1 , DEN2, DEN3 and DEN4 E protein antigens.
  • the composition is a tetravalent composition comprising two bivalent alphavirus vectors, each encoding two E protein antigens from DEN1 , DEN2, DEN3 and/or DEN4, such that nucleotide sequences encoding E protein antigens from all four serotypes are present in the tetravalent composition.
  • the dengue virus E protein antigen can be any antigen as described herein.
  • the E protein antigen is a soluble E protein antigen and comprises, consists essentially of, or consists of E85, E81 or EDIII or an immunogenically active fragment thereof. Alternatively, any other E protein antigen as described herein can be employed in this embodiment.
  • the multiple E protein antigens can be fused into a polyprotein or can each be operably associated with a different promoter (e.g. , alphavirus 26S subgenomic promoter) or, alternatively, an IRES sequence can be used as described above and as understood in the art.
  • a different promoter e.g. , alphavirus 26S subgenomic promoter
  • an IRES sequence can be used as described above and as understood in the art.
  • a tetravalent composition can comprise one, two, three, four or more alphavirus vectors such that nucleotide sequences encoding E protein antigens from DEN1, DEN2, DEN3 and DEN4 are present in the one, two, three, four or more alphavirus vectors.
  • a nucleic acid encoding the alphavirus vector(s) is administered to the subject and the alphavirus vector(s) expressed in vivo.
  • the present invention can be practiced for prophylactic and/or therapeutic purposes, in accordance with known techniques.
  • the invention can be practiced to produce antibodies for any purpose, such as diagnostic or research purposes, or for passive immunization by transfer to another subject.
  • a further aspect of the present invention is a method of eliciting an immune response in a subject, comprising administering to the subject an effective amount of a nucleic acid(s), alphavirus vector(s) or composition of the invention.
  • a still further aspect of the invention is a method of treating a dengue virus infection, comprising administering to the subject an effective amount of a nucleic acid(s), alphavirus vector(s) or composition of the invention.
  • a still further aspect of the invention is a method of preventing a dengue virus infection, comprising administering to the subject an effective amount of a nucleic acid(s), alphavirus vector(s) or composition of the invention.
  • a still further aspect of the invention is a method of protecting a subject from the effects of dengue virus infection, comprising administering to the subject an effective amount of a nucleic acid(s), alphavirus vector(s) or composition of the invention.
  • kits comprising one or more compositions, alphavirus vectors, nucleic acids and/or pharmaceutical formulations of this invention.
  • the kit of this invention can comprise one or more containers and/or receptacles to hold the reagents (e.g., antibodies, antigens, nucleic acids) of the kit, along with appropriate buffers and/or diluents and/or other solutions and directions for using the kit, as would be well known in the art.
  • reagents e.g., antibodies, antigens, nucleic acids
  • kits can further comprise adjuvants and/or other immunostimulatory or
  • immunomodulating agents as are well known in the art.
  • compositions and kits of the present invention can also include other medicinal agents, pharmaceutical agents, carriers, diluents, immunostimulatory cytokines, etc. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art.
  • Administration of a nucleic acid, alphavirus vector, composition or pharmaceutical formulation of the invention can be by any route known in the art.
  • the route of administration can be by inhalation (e.g. , oral and/or nasal inhalation), oral, buccal (e.g. , sublingual), rectal, vaginal, topical (including administration to the airways), intraocular, transdermal, by parenteral (e.g. , intramuscular [e.g. , administration to skeletal muscle], intravenous, intra- arterial, intraperitoneal and the like), subcutaneous (including administration into the footpad), intradermal, intrapleural, intracerebral, and/or intrathecal routes.
  • parenteral e.g. , intramuscular [e.g. , administration to skeletal muscle], intravenous, intra- arterial, intraperitoneal and the like
  • subcutaneous including administration into the footpad
  • intradermal, intrapleural, intracerebral, and/or intrathecal routes e.g.
  • the alphavirus vectors of the invention can be delivered per se or by delivering a nucleic acid (e.g. , DNA) that encodes the alphavirus vector.
  • Immunomodulatory compounds such as immunomodulatory chemokines and cytokines (preferably, CTL inductive cytokines) can be administered concurrently to a subject.
  • Cytokines may be administered by any method known in the art. Exogenous cytokines may be administered to the subject, or alternatively, a nucleic acid encoding a cytokine may be delivered to the subject using a suitable vector, and the cytokine produced in vivo. In particular embodiments, a viral adjuvant expresses the cytokine.
  • the methods of administering the alphavirus vectors, nucleic acids, compositions and/or pharmaceutical formulations of the invention do not result in detectable pathogenicity (e.g. , Dengue Shock Syndrome/Dengue Hemorrhagic Fever).
  • multiple dosages e.g. , two, three or more
  • the alphavirus vectors, nucleic acids, compositions and/or pharmaceutical formulations of the invention can be administered without detectable pathogenicity (e.g. , Dengue Shock
  • the multivalent vaccines of the invention do not result in immune interference, e.g. , a balanced immune response is induced against all antigens presented.
  • the balanced response results in protective immunity against DEN1, DEN2, DEN3 and DEN4.
  • the multivalent vaccine can be administered to a subject that has anti-dengue maternal antibodies present.
  • compositions comprising the nucleic acids, compositions and alphavirus vectors of the invention and a pharmaceutically acceptable carrier are also provided.
  • the nucleic acids, compositions and alphavirus vectors of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (latest edition).
  • the composition of the invention is typically admixed with, inter alia, a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is meant a carrier that is compatible with other ingredients in the pharmaceutical composition and that is not harmful or deleterious to the subject.
  • the carrier may be a solid or a liquid, or both, and is preferably formulated with the composition of the invention as a unit-dose formulation, for example, a tablet, which may contain from about 0.01 or 0.5% to about 95% or 99% by weight of the composition.
  • the pharmaceutical compositions are prepared by any of the well-known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients.
  • the pharmaceutically acceptable carrier is sterile and would be deemed suitable for administration into human subjects according to regulatory guidelines for pharmaceutical compositions comprising the carrier.
  • a "pharmaceutically acceptable” component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the composition.
  • Non-limiting examples of pharmaceutically acceptable components include any of the standard
  • phosphate buffered saline solutions water
  • emulsions such as oil/water emulsion, microemulsions and various types of wetting agents.
  • the compositions of the invention can further comprise one or more than one adjuvant.
  • the adjuvants of the present invention can be in the form of an amino acid sequence, and/or in the form or a nucleic acid encoding an adjuvant.
  • the adjuvant can be a component of a nucleic acid encoding the polypeptide(s) or fragment(s) or epitope(s) and/or a separate component of the composition comprising the nucleic acid encoding the polypeptide(s) or fragment(s) or epitope(s) of the invention.
  • the adjuvant can also be an amino acid sequence that is a peptide, a protein fragment or a whole protein that functions as an adjuvant, and/or the adjuvant can be a nucleic acid encoding a peptide, protein fragment or whole protein that functions as an adjuvant.
  • adjuvant describes a substance, which can be any immunomodulating substance capable of being combined with a composition of the invention to enhance, improve or otherwise modulate an immune response in a subject.
  • the adjuvant can be, but is not limited to, an
  • immuno stimulatory cytokine including, but not limited to, GM/CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, inteiieukin-1 , hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules
  • SYNTEX adjuvant formulation 1 SAF-1) composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.
  • Suitable adjuvants also include an aluminum salt such as aluminum hydroxide gel (alum), aluminum phosphate, or algannmulin, but may also be a salt of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized polysaccharides, or polyphosphazenes.
  • aluminum salt such as aluminum hydroxide gel (alum), aluminum phosphate, or algannmulin
  • alum aluminum hydroxide gel
  • aluminum phosphate aluminum phosphate
  • algannmulin algannmulin
  • adjuvants are well known in the art and include without limitation MF 59, LT- K63, LT-R72 (Pal et al, Vaccine 24(6):766-75 (2005)), QS-21, Freund's adjuvant (complete and incomplete), aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor- MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dipalmitoyl-sn - glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, tre
  • Additional adjuvants can include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl. lipid A (3D-MPL) together with an aluminum salt.
  • An enhanced adjuvant system involves the combination of a monophosphoryl lipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in PCT publication number WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in PCT publication number WO
  • nucleic acid compositions of the invention can include an adjuvant by comprising a nucleotide sequence encoding the antigen and a nucleotide sequence that provides an adjuvant function, such as CpG sequences.
  • CpG sequences, or motifs are well known in the art.
  • an adjuvant for use with the present invention such as, for example, an adjuvant for use with the present invention, such as, for example, an adjuvant for use with the present invention
  • immunostimulatory cytokine can be administered before, concurrent with, and/or within a few hours, several hours, and/or 1 , 2, 3, 4, 5, 6, 7, 8, 9, and/or 10 days before and/or after the administration of a composition of the invention to a subject.
  • any combination of adjuvants such as immunostimulatory cytokines
  • immunostimulatory cytokines can be co-administered to the subject before, after and/or concurrent with the administration of an immunogenic composition of the invention.
  • combinations of immunostimulatory cytokines can consist of two or more immunostimulatory cytokines, such as GM/CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1 , hematopoietic factor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2 co-stimulatory molecules.
  • an adjuvant or combination of adjuvants can be determined by measuring the immune response produced in response to administration of a composition of this invention to a subject with and without the adjuvant or combination of adjuvants, using standard procedures, as described herein and as known in the art.
  • the adjuvant comprises an alphavirus adjuvant as described, for example in U.S. 7,862,829.
  • the dosage of each alphavirus vector in the pharmaceutical formulations of the invention is greater than or equal to about 10, 10 4 , 10 5 or 10 6 International Units (IU) and/or less than or equal to about 10 6 , 10 7 , 10 8 , 10 9 , 10 10 IU or more (encompassing any combination as long as the lower limit is less than the upper limit).
  • IU International Units
  • Boosting dosages can further be administered over a time course of days, weeks, months or years. In chronic infection, initial high doses followed by boosting doses may be advantageous.
  • the pharmaceutical formulations of the invention can optionally comprise other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, diluents, salts, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the carrier will typically be a liquid.
  • the carrier may be either solid or liquid.
  • the carrier will be respirable, and is typically in a solid or liquid particulate form.
  • compositions of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (9 th Ed. 1995).
  • the VLPs are typically admixed with, inter alia, an acceptable canier.
  • the carrier can be a solid or a liquid, or both, and is optionally formulated with the compound as a unit-dose formulation, for example, a tablet.
  • aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.), and the like.
  • aqueous carriers e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.), and the like.
  • These compositions can be sterilized by conventional techniques.
  • the formulations of the invention can be prepared by any of the well-known techniques of pharmacy.
  • the pharmaceutical formulations can be packaged for use as is, or lyophilized, the lyophilized preparation generally being combined with a sterile aqueous solution prior to administration.
  • the compositions can further be packaged in unit/dose or multi-dose containers, for example, in sealed ampoules and vials.
  • compositions can be formulated for administration by any method Icnown in the art according to conventional techniques of pharmacy.
  • the compositions can be formulated to be administered intranasally, by inhalation (e.g. , oral inhalation), orally, buccally (e.g. , sublingually), rectally, vaginally, topically, intrathecally, intraocularly, transdermally, by parenteral administration (e.g. , intramuscular [e.g. , skeletal muscle], intravenous, subcutaneous, intradermal, intrapleural, intracerebral and intra-arterial, intrathecal), or topically (e.g. , to both skin and mucosal surfaces, including airway surfaces).
  • parenteral administration e.g. , intramuscular [e.g. , skeletal muscle], intravenous, subcutaneous, intradermal, intrapleural, intracerebral and intra-arterial, intrathecal
  • topically e.g. , to both skin
  • the pharmaceutical formulation can be formulated as an aerosol (this term including both liquid and dry powder aerosols).
  • the pharmaceutical formulation can be provided in a finely divided form along with a surfactant and propellant. Typical percentages of the composition are 0.01 -20% by weight, preferably 1-10%.
  • the surfactant is generally nontoxic and soluble in the propellant.
  • esters or partial esters of fatty acids containing from 6 to 22 carbon atoms such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters, such as mixed or natural glycerides may be employed.
  • the surfactant may constitute 0.1-20%) by weight of the composition, preferably 0.25-5%o.
  • the balance of the composition is ordinarily propellant.
  • a carrier can also be included, if desired, as with lecithin for intranasal delivery.
  • Aerosols of liquid particles can be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as is Icnown to those of skill in the art. See, e.g. , U.S. Patent No. 4,501,729. Aerosols of solid particles can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. Intranasal administration can also be by droplet administration to a nasal surface.
  • Injectable formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one can administer the pharmaceutical formulations in a local rather than systemic manner, for example, in a depot or sustained-release formulation.
  • Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.
  • an injectable, stable, sterile formulation of the invention in a unit dosage form in a sealed container can be provided.
  • the formulation can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject.
  • the unit dosage form can be from about 1 ⁇ g to about 10 grams of the formulation.
  • a sufficient amount of emulsifying agent which is pharmaceutically acceptable, can be included in sufficient quantity to emulsify the formulation in an aqueous carrier.
  • emulsifying agent is phosphatidyl choline.
  • compositions suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tables, as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in- water or water- in-oil emulsion.
  • Oral delivery can be performed by complexing a compound(s) of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art.
  • Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the protein(s) and a suitable carrier (which may contain one or more accessory ingredients as noted above).
  • the pharmaceutical formulations are prepared by uniformly and intimately admixing the compound(s) with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture.
  • a tablet can be prepared by compressing or molding a powder or granules, optionally with one or more accessory ingredients.
  • Compressed tablets are prepared by compressing, in a suitable machine, the formulation in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface
  • Molded tablets are made by molding, in a suitable machine, the powdered protein moistened with an inert liquid binder.
  • compositions suitable for buccal (sub-lingual) administration include lozenges comprising the compound(s) in a flavored base, usually sucrose and acacia or tragacanth; and pastilles in an inert base such as gelatin and glycerin or sucrose and acacia.
  • compositions suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended recipient.
  • Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents.
  • nonD aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water,
  • alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • compositions suitable for rectal administration are optionally presented as unit dose suppositories. These can be prepared by admixing the active agent with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.
  • compositions suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil.
  • Carriers that can be used include, but are not limited to, petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • topical delivery can be performed by mixing a pharmaceutical formulation of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.
  • a lipophilic reagent e.g., DMSO
  • compositions suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time.
  • Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3:318 (1986)) and typically take the form of a buffered aqueous solution of the compound(s).
  • Suitable formulations can comprise citrate or bis ⁇ tris buffer (pH 6) or ethanol/water and can contain from 0.1 to 0.2M active ingredient.
  • composition can be formulated as a liposomal formulation.
  • the lipid layer employed can be of any conventional composition and can either contain cholesterol or can be cholesterol-free.
  • the liposomes that are produced can be reduced in size, for example, through the use of standard sonication and homogenization techniques.
  • the liposomal formulations can be lyophilized to produce a lyophilizate which can be reconstituted with a pharmaceutically acceptable carrier, such as water, to regenerate a liposomal suspension.
  • the immunogenic formulations of the invention can optionally be sterile, and can further be provided in a closed pathogen-impermeable container.
  • BHK-21 and Vero-81 cells were obtained from the American Type Culture Collection (ATCC). BHK-21 cells were maintained in alpha minimal essential medium containing 10% donor calf serum, 10% tryptose phosphate broth, and 0.29 mg of glutamine per ml. Vero-81 cells were maintained in D-MEM/F12 medium supplemented with 10% fetal calf serum, 0.29 mg of glutamine per ml. Insect C6/36 cells were obtained from ATCC and maintained in alpha minimal essential medium containing 10% fetal calf serum.
  • HMAF Hyperimmune mouse ascetic fluid
  • mice-adapted, neurovirulent New Guinea C (NGC) strain of DEN2 virus used in the monovalent pilot study in mice was provided by the late Robert Shope, UTMB,
  • VRP VEE replicon particle
  • DENl WP The strains of DEN used to challenge the macaques in the efficacy studies were provided by Dr. Steve Whitehead from NIAID: DENl WP, DEN2 NGC, DEN3 Sleman/78, and DEN4 814669. These strains were amplified once in Vero cells and used as described in Blaney et al., (2005) J Virol. 79:5516.
  • the viruses used for neutralization assays were the WHO reference strains for each DEN serotype, and were obtained from Aravinda de Silva's laboratory, and propagated two or three times in C6/36 cells, titrated on Vero cells and stored at -80°C.
  • the virus preparations used as antigen to coat ELISA plates were obtained by further purification and concentration of the C6/36 grown virus stock. Briefly, DEN infected C6/36 culture supernatants were subjected to centrifugation at 72,000 x g for 5 h through a 5 -ml cushion of 20% (w/v) sucrose.
  • Sedimented virus was further purified by density gradient centrifugation in a 10-40% iodixanol gradient at 163,700 x g for 120 minutes. Virus-containing fractions were pooled and purified virus was concentrated by centrifugation at 72,000 g for 5 h. Virus was resuspended in PBS 1% fetal bovine serum and stored at -80°C.
  • VEE Venezuelan Equine Encephalitis
  • Viral RNA was extracted from virus growth culture media and used as a template to generate cDNA by reverse transcription-PCR.
  • the forward PCR primer was design to insert an EcoRV restriction site at the 5' end, where the last 3'G constitutes the first nucleotide of the first methionine, followed by the second and third nucleotides of the MET start codon (AT).
  • the Reverse primer was designed to add a stop codon followed by an Ascl site at the 3 ' end.
  • the amplified regions were initially cloned into PCR cloning plasmids and their sequences confirmed.
  • the dengue sequences were cloned into the multicloning site of the VEE replicon vector pVK21 EcoRV using EcoRV and Ascl sites, upstream and downstream of the 26S subgenomic RNA transcription start site, respectively.
  • the clones were linearized at a unique Notl site downstream of the VEE 3 'untranslated region and poly(A) tract, and full-length T7 transcripts were generated in vitro using an mMessagemMachine kit (Ambion).
  • helper RNAs are deleted for the replicase genes and the cis-acting packaging signal. Transcripts were co-transfected into BHK or Vero cells by electroporation. The culture medium was harvested at 22-24 hours post electroporation.
  • VRP-containing culture medium was clarified by centrifugation at 12,000 x g for 30 min, and the VRP were partially purified and concentrated by sedimentation at 72,000 x g for 3 h through a 5-ml cushion of 20% (w/v) sucrose dissolved in PBS.
  • Pelleted VRP were resuspended overnight in endotoxin-free PBS with 1% human serum albumin at 4°C followed by storage at -80°C.
  • Each VRP preparation was safety tested to ensure the absence of replication competent virus that could have arisen by non-homologous recombination.
  • CPE cytopathic effect
  • Vaccines were formulated in PBS with 1% human serum albumin. The 4 components of the tetravalent formulations were stored separately at -80°C and only thawed and mixed immediately before administering to the animals. Immunogenicity studies in mice.
  • BALB/c mice Specific pathogen-free adult (6-week-old) BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA). Animal housing and care at UNC were in accordance with all UNC-CH Institutional Animal Care and Use Committee guidelines.
  • mice were immunized at weeks 0 and 8 with a tetravalent cocktail containing 1.8 x 10 6 IU of each serotype prM/E- VRP.
  • Vaccines were administered by subcutaneous (s.c.) inoculation in both of the rear footpads in a volume of 10 ⁇ per footpad. Serum was collected at 4 and 8 wks post prime, and periodically between wks 3 and 71 post boost.
  • mice in groups of 6 were immunized at 0 and 4 wks with 2 x 10 6 IU of either DEN E85-VRP representing each serotype, or a cocktail containing 2 x 10 6 IU of each of the four E85-VRP monovalent vaccines.
  • Vaccines were administered by s.c. inoculation in the both rear footpads in a volume of 10 ⁇ l per footpad.
  • Neutralizing antibody titers were determined at 4 wks post prime and 3 wks post boost.
  • Recombinant DEN3 EDIII protein (AA 295-398) from a WHO reference strain was fused to the N-terminus of maltose binding protein (MBP) by cloning the PCR amplified EDIII to the pMAL c2X vector (NEB) to generate MBP-EDIII.
  • MBP-EDIII was expressed in E. coli DH5a (Invitrogen) and purified using amylose resin affinity chromatography (MEB). Purified recombinant DEN3 EDIII-MBP and MBP control were used to deplete EDIII- reactive antibodies in macaque sera.
  • EDIII-MBP and MBP were incubated with amylose resin in column buffer containing 3% of normal monkey serum, and incubated overnight at 4 C. The resin was washed 3 times with column buffer and 3 times with PBS. After blocking the resin with 5% normal macaque serum, the resin was incubated with 1.5 ml of immunized or DEN challenged macaque serum diluted 1 : 10 in PBS for 4 h at 37 C. The amylose resin was pelleted and the EDIII depleted serum was subjected to another round of depletion, for a total of 6 to 8 depletion cycles. Depletion of EDIII-reactive antibodies was confirmed by ELISA. The MBP and EDIII-MBP depleted serum were analyzed for neutralizing antibodies.
  • Serum viremia post-challenge Serum viremia post-challenge.
  • Macaque serum samples collected during 10 consecutive days after the challenge were tested for the presence of infectious DEN on Vero cells.
  • Virus foci titration was performed under 1% carboxmethylcellulose overlay in a DEN focus assay on Vero cells.
  • Vero cells seeded in 24 well plates were infected with macaque serum diluted 1 :5, 1 : 10 and 1 :20 in triplicates. After 1 hr at 37 C, 1 ml of 1% carboxymethylcellulose in optimum containing 2% FBS, was added to each well. After 5 days at 37C, the overlay was removed and the cell monolayers fixed with 80% methanol.
  • DEN serotype specific neutralizing antibodies in immunized mouse and macaque serum were quantified using a flow cytometry-based neutralization assay (F-NEUT) on Vero cells.
  • F-NEUT flow cytometry-based neutralization assay
  • This neutralization assay was adapted from a flow cytometry-based DEN titration assay.
  • the ability of immune serum to neutralize the infectivity of DEN on Vero cells is measured at 24 hpi by enumerating cells that are positive for intracellular staining of DEN prM or E protein by flow cytometry.
  • the DEN used to test the neutralizing capacity of the sera were reference strains representing each serotype provided by WHO to Aravinda de Silva.
  • heat inactivated control or immune sera were diluted four-fold (six dilutions) with DMEM-F12 containing 1 % bovine serum albumin, and each dilution mixed with an equivalent volume of DEN2 NGC in a total volume of 1 10 ⁇ .
  • the dilution of DEN chosen ensured an infection rate within the linear range of the dose response curve, i.e. 7 to 15 % at 24 h, and allowed antibody to be in excess.
  • the mixture was incubated for 1 h at 37°C in 5% CO 2 before transferring it to Vero cell monolayers seeded the day before in 24-well plates at 10 5 cells/well. Each serum dilution was tested in duplicate wells.
  • VEE specific neutralization titers in serum from VRP-immunized mice were determined using a flow cytometry-based neutralization assay. Briefly, BHK cell monolayers s were seeded in 24 well plates at a density of 10 5 cells per well. Control or immune sera were diluted two-fold with MEM containing 1 % bovine serum albumin, and each dilution mixed with an equivalent volume of a VRP expressing green fluorescent protein GFP-VRP (36) to give a m.o.i. of 0.1 in a total volume of 1 10 ⁇ . The mixture was incubated for 1 h at 37°C in 5% C0 2 before transferring it to BHK cell monolayers in 24-well plates. Each serum dilution was tested in duplicate wells.
  • Antibody titers were evaluated for statistically significant differences by the Mann- Whitney test (INSTAT; GraphPad, San Diego). P ⁇ 0.05 was considered significant.
  • the nucleotide sequences encoding the prM/E and E85 immunogens are shown in Figures 10A, 10B, IOC and 10D.
  • Example 2 Results -Increased immunogenicity of an alphavirus replicon particle- based dengue vaccine expressing soluble forms of dengue envelope protein.
  • VRP Venezuelan equine encephalitis replicon particles
  • the dengue virus (DEN) E protein is the major viral antigen inducing protective neutralizing antibodies.
  • the in vitro expression and immunogenicity in mice of different configurations of DEN E immunogen using the VRP vector were characterized in this example ( Figure 1).
  • DEN2 New Guinea C (NGC) strain was used to make the constructs.
  • DEN2 full-length E in the presence of prM (prM/E) previously shown to induce protective neutralizing antibodies in mice, was compared to three truncated forms of the protein that lack the transmembrane domains: E85 (residues 1 -424), E81 (1 -397) and E domain III (296- 400).
  • the E81 construct lacks residues 401 to 420 that are predicted to form an alpha-helical domain (HI), and which is present in the E85 construct.
  • HI alpha-helical domain
  • the increased immunogenicity of E85 and E81 compared to the full-length E in the presence of prM may allow the use of VRP vaccine vectors at doses up to 10-fold lower.
  • a DEN2 NGC EDIII construct delivered with a VRP vaccine vector was also immunogenic in mice with geometric mean titers comparable to those of prM/E (data not shown).
  • Example 3 Results - Rapid, balanced and long-lasting neutralizing antibodies in mice immunized with tetravalent DEN-VRP.
  • mice were immunized by subcutaneous inoculation with 7.2 x 10 6 IU of each monovalent formulation and with a tetravalent cocktail containing 7.2 x 10 6 or 7.2 x 10 5 IU of each.
  • Booster immunizations were given at 4 or 8 weeks post-prime, and serum was collected periodically between 3 weeks post prime and 71 weeks post boost (Figure 3).
  • Neutralizing antibodies against each of the four DEN serotypes were measured using a flow cytometry- based neutralization assay. All mice vaccinated with the tetravalent cocktail, including those that received the lower dose, had neutralizing antibodies to all four serotypes after a single immunization. The neutralizing activity to all 4 serotypes persisted in the serum for up to 71 weeks post boost.
  • Example 4 Results - E85 is more immunogenic than prM/E in mice.
  • glycoprotein corresponding to 85% of the N terminus of the protein was cloned into the VRP vector for DEN1 , DEN2, DEN3 and DEN4 using strains from Aravinda deSilva's laboratory ⁇ see Example 1).
  • This truncated E protein contains sequences encoding E signal peptide, domains I, II and III of the ectodomain, but lacks the C terminal amino acids 425 to 495 (for DEN1 , DEN2 and DEN4; 423 to 493 for DEN3) corresponding to the H2 and two transmembrane anchor domains (TM).
  • the resulting truncated E protein has at its C terminus two regions: a) residues 401 to 420 (for DEN1, DEN2 and DEN4; 399 to 418 for DEN3), predicted to form an alpha-helical domain (HI), and b) residues 420 to 424 (for DEN1, DEN2 and DEN4; 418 to 422 for DEN3) which are part of a highly conserved sequence among flaviviruses (CS).
  • the truncation at this position (at residue 424 for DEN1 , DEN2 and DEN4; 422 for DEN3) renders the protein membrane-anchor-free and subject to traffic through the secretory pathway, while able to associate with the plasma membrane through interactions with HI domain.
  • a viral vector such as VRP
  • the immunogenicity of VRP expressing E85 was compared to prM/E for each serotype in B ALB/c mice (6 per group) immunized with 10 6 IU of each construct.
  • E85 was a better immunogen than prM/E for serotypes 1 , 2 and 3.
  • the response induced by DEN4 E85 was lower than that of the other E85 serotypes, and not better than that of prM/E DEN4.
  • the in vitro expression of E in DEN4 E85-VRP infected Vero cells we found that the lower immunogenicity correlated with lower in vitro expression and secretion.
  • Example 5 Results -Monovalent VRP vaccines in macaques.
  • E85-VRP was also better than prM/E-VRP in monkeys
  • Dengue sequences from serotype 2 strain New Guinea C were used in experiment #1 (see Figures 11A and 11B), and sequences from serotype 3 strain UNC3001 were used in experiment #2 (see Figures 8B, 9 and 10C). Animals (2 per group) were immunized with 3 doses each containing 10 7 IU of dengue serotype 2 prM/E-VRP or E85-VRP in experiment #1 .
  • animals (6 per group) were immunized with 3 doses each containing 10 8 IU of dengue serotype 3 prM/E-VRP or E85-VRP in experiment #2.
  • Blood was collected periodically after each immunization and right before a challenge with live dengue virus serotype 2 (experiment # 1) or serotype 3 (experiment #2), done 16-20 weeks after the last immunization.
  • Control groups of unvaccinated macaques were also challenged. Blood was collected daily for 10 days after the challenge to determine the presence of dengue virus in the serum.
  • E85-VRP showed increased immunogenicity and protective efficacy than prM/E in both experiments.
  • E85-VRP induced serotype-specific neutralizing antibodies in all the animals after the first dose while prM/E required more than one dose.
  • peak titers and pre-challenge titers were significantly higher in E85-VRP vaccinated macaques ( Figure 4).
  • Protective efficacy was determined as the ability of the vaccine to reduce or eliminate the presence of virus in the blood (viremia) when monkeys received a virus challenge (Table 1). Unvaccinated control macaques had dengue virus in the serum between days 2 and 8 post challenge.
  • the contribution of EDIII reactive antibodies to the protective response was determined by depleting DEN2 EDIII and DEN3 EDIII binding antibodies from the sera of DEN2 E85 and DEN3 E85-VRP vaccinated macaques, and comparing the ability of the undepleted versus depleted sera to neutralize infection in vitro. The results were compared to those of unvaccinated macaques after DEN2 and DEN3 challenges, as representatives of responses after natural infection with the virus.
  • MBP, and MBP alone were expressed in E. coli, purified and bound to amylose resin beads.
  • Serum from immunized macaques was incubated with EDIII-MBP and to MBP alone, resulting in binding and removal of EDIII binding antibodies from the serum. After 6 cycles of depletion, there were no residual EDIII binding antibodies in the serum, as determined by ELISA using rEDIII-coated plates.
  • Typical neutralization curves are shown in Figure 6.
  • the neutralization titers are summarized in Table 2. The results show that EDIII-reactive antibodies play only a minor role in neutralization in DEN3 virus infected macaques, but represent the majority of the neutralizing capacity of the sera from DEN3 E85-VRP vaccinated animals.
  • DEN2 E85-VRP and DEN3 E85-VRP when administered as monovalent vaccines protected rhesus macaques from challenge, and induced strong serotype specific neutralizing antibodies directed predominantly to EDIII.
  • rhesus macaques infected with live dengue virus developed neutralizing antibodies that recognize mostly epitopes outside EDIII.
  • neutralizing antibodies Although in human convalescent sera, only a small proportion of neutralizing antibodies are directed to EDIII, it may be desirable in a dengue vaccine to target strongly neutralizing type-specific epitopes, masked during natural infection.
  • a tetravalent formulation consisting of a mix of 1 x 10 6 IU of eacli DENlE85-VRP, DEN2E85-VRP, DEN3E85-VRP and DEN4E85-VRP was tested in mice to address kinetics of induction, duration and balanced neutralizing antibodies.
  • the E85-VRP vaccine induced neutralizing antibodies to all 4 serotypes after one dose, and robust long term neutralizing antibody responses after 2 doses (data not shown).
  • study # 3 To determine whether the tetravalent cocktail of E85-VRP induce a balanced and protective immune response against all 4 serotypes in macaques.
  • tetravalent live attenuated vaccine candidates have shown to be less immunogenic when administered in the mix, than when they are given separately as monovalent vaccine.
  • the tetravalent cocktail elicited high neutralizing antibody titers against DEN4, which in mice had shown to be less immunogenic.

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Abstract

Cette invention concerne un vecteur d'alphavirus comprenant un antigène de la protéine E du virus de la dengue. Cette invention concerne également des acides nucléiques codant ou comprenant ce vecteur. L'invention concerne également des compositions multivalentes et des préparations pharmaceutiques comprenant au moins deux vecteurs d'alphavirus ou acides nucléiques selon l'invention. L'invention concerne par ailleurs des méthodes d'administration de ces vecteurs d'alphavirus, de ces acides nucléiques, de ces compositions et/ou de ces préparations pharmaceutiques chez un sujet, notamment pour induire une réponse immunitaire dirigée contre le virus de la dengue, pour traiter l'infection par le virus de la dengue, pour prévenir l'infection par le virus de la dengue et/ou pour protéger un sujet des effets d'une infection par le virus de la dengue.
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CN113736750B (zh) * 2021-09-22 2023-03-31 中牧实业股份有限公司 一株盖他病毒毒株及其应用

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