WO2012178196A2 - Protection contre le virus de la dengue et prévention des formes graves de la dengue - Google Patents

Protection contre le virus de la dengue et prévention des formes graves de la dengue Download PDF

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Publication number
WO2012178196A2
WO2012178196A2 PCT/US2012/044071 US2012044071W WO2012178196A2 WO 2012178196 A2 WO2012178196 A2 WO 2012178196A2 US 2012044071 W US2012044071 W US 2012044071W WO 2012178196 A2 WO2012178196 A2 WO 2012178196A2
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WIPO (PCT)
Prior art keywords
dengue virus
protein
subject
infection
denv2
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PCT/US2012/044071
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English (en)
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WO2012178196A3 (fr
Inventor
Sujan Shresta
Lauren Yauch
Alessandro Sette
Daniela Weiskopf
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La Jolla Institute For Allergy And Immunology
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Priority claimed from PCT/US2011/041889 external-priority patent/WO2011163628A2/fr
Application filed by La Jolla Institute For Allergy And Immunology filed Critical La Jolla Institute For Allergy And Immunology
Priority to EP12801934.6A priority Critical patent/EP2723372A4/fr
Priority to US14/128,268 priority patent/US20150150960A1/en
Publication of WO2012178196A2 publication Critical patent/WO2012178196A2/fr
Publication of WO2012178196A3 publication Critical patent/WO2012178196A3/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
    • 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
    • A61K2039/5252—Virus inactivated (killed)
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to Dengue virus proteins, subsequences and portions thereof, including DENV epitopes and modifcations of DENV proteins, subsequences and portions thereof, and uses and methods for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection.
  • Dengue virus (DENV, or DV) is a mosquito-borne RNA virus in the Flaviviridae family, which also includes West Nile Virus (WNV), Yellow Fever Virus (YFV), and Japanese Encephalitis Virus (JEV).
  • WNV West Nile Virus
  • YFV Yellow Fever Virus
  • JEV Japanese Encephalitis Virus
  • the four serotypes of DENV (DENV 1 -4) share approximately 65-75% homology at the amino acid level (Fu, et al. Virology 188:953 (1992)). Infections with DENV can be
  • DF dengue fever
  • DHF dengue hemorrhagic fever
  • DSS dengue shock syndrome
  • DF and DHF DSS are a significant cause of morbidity and mortality worldwide, and therefore a DENV vaccine is a global public health priority.
  • vaccine development has been challenging, as a vaccine should protect against all four DENV serotypes (Whitehead, et al. Nat Rev Microbiol 5:518 (2007)).
  • DHF/DSS Severe dengue disease
  • serotype cross-reactive memory T cells may respond sub-optimally during secondary infection and contribute to the pathogenesis (Mathew, et al. Immunol Rev 225 :300 (2008)). Accordingly, studies have shown serotype cross-reactive T cells can exhibit an altered phenotype in terms of cytokine production and degranulation (Mangada, et al . J Immunol 175 :2676 (2005); Mongkolsapaya, et al. Nat Med 9:921 (2003); Mongkolsapaya, et al. J Immunol 1 76:3821 (2006)). However, another study found the breadth and magnitude of the T cel l response during secondary DENV infection was not significantly associated with disease severity (Si mmons, et al. J Virol 79:5665 (2005)).
  • CD4 + T cells can contribute to the host response to pathogens in a variety of ways. They produce cytokines and can mediate cytotoxicity. They also help B cell responses by inducing immunoglobulin class switch recombination (CSR), and help prime the CD8 + T cell response. CD4 + T cells can help the CD8 + T cell response indirectly by activating APCs, for example via
  • CD40L/CD40 (Bevan, Nat Rev Immunol 4:595 (2004)). CD40L on CD4 + T cells is important in activating B cells as well (Elgueta, et al. Immunol Rev 229: 152 (2009)). CD4 + T cells can also induce chemokine production that attracts CD8 + T cells to sites of infection (Nakanishi, et al. Nature 462:510 (2009)). However, the requirement for CD4 + T cell help for antibody and CD8 + T cell responses is not absolute, and may be specific to the pathogen and/or experimental system. For instance, it has been shown that CSR can occur in the absence of CD4 + T cells (Stavnezer, et al.
  • HLA human leukocyte antigens
  • mice transgenic for human leukocyte antigens are widely used to study T cell responses restricted by human MHC molecules and studies in other viral systems have shown the valuable impact of HLA transgenic mice in epitope identification (Kotturi, et al. Immunome Res 6:4 (201 0); Kotturi, et al. Immunome Res 5 :3 (2009); Pasquetto, et al. J Immunol 1 75 :5504 (2005)).
  • a mouse-passaged DENV2 strain, S221 which does not replicate to detectable levels in wild-type C57BL/6 mice, was reported to replicate in IFN-a/ R' mice (Yauch, et al. J Immunol 1 82 :4865 (2009)).
  • ADE has been demonstrated to enhance viremia and severity of dengue disease in non-human primate (Goncalvez, et al. Proc Natl Acad Sci U S A 104:9422-9427 (2007); Halstead J Infect Dis 140:527-533 ( 1979); Halstead, et al. J Infect Dis 128: 15-22 (1973)) and mouse (Balsitis, et al . PLoS Pathog 6:e l 000790 (2010); Zellweger, et al. Cell Host Microbe 7: 128- 139 (2010)) models, respectively.
  • the invention is based, in part, on the discovery that DENV vaccine-induced antibody response can mediate ADE and enhance (worsen) DENV disease severity.
  • the invention is also based, in part, on the discovery that CD8+ T cell responses dictate the extent of dengue vaccine- mediated protection.
  • the invention is further based, in part, on the discovery that CD8+ T cell responses can provide protection against DENV infection, including protection against heterologous DENV serotypes, even in the presence of enhancing antibodies.
  • a use or method includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to elicit an anti-Dengue virus T cell response in the subject.
  • a use or method elicits, stimulates, induces, promotes, increases, or enhances an anti-Dengue virus T cell response in a subject without sensitizing the subject to severe dengue disease (e.g., ADE) upon a secondary or subsequent Dengue virus exposure or infection.
  • severe dengue disease e.g., ADE
  • a use or a method of vaccinating a subject against or providing a subject with protection against a Dengue virus infection includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to vaccinate the subject against or protect the subject against the Dengue virus infection.
  • the use or method does not sensitize the subject to severe dengue disease upon a secondary or subsequent Dengue virus exposure or infection.
  • a use or method of treating a subject for a Dengue virus infection includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to treat the subject for the Dengue virus infection.
  • the use or method does not sensitize the subject to severe dengue disease upon a secondary or subsequent Dengue virus exposure or infection.
  • compositions including an amount of a Dengue virus protein or subsequence or portion or modification thereof.
  • these compositions are for use in: eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject, optionally without elicting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection or exposure; in providing a subject with protection against a Dengue virus infection or pathology, or one or more physiological disorders, illness, diseases or symptoms caused by or associated with Dengue virus infection or pathology, optional ly without elicting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection; in vaccinating a subject against a Dengue virus infection without el icting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection or exposure; and in treating a subject for a Dengue virus infection, optionally without elicting or sensitizing the
  • the uses, methods and compositions are useful for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus CD8 + T cell response, optionally without elicting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection or exposure.
  • anti-Dengue virus CD8+ T cell response is directed and/or protective against a plurality of different Dengue virus serotypes.
  • the anti-Dengue virus CD8+ T cell response is directed and/or protective against at least two Dengue virus serotypes selected from DEN V I , DENV2, DENV3 and DEN V4.
  • the protein comprises or consists of a Dengue virus serotype 1 , 2, 3 or 4 protein.
  • a Dengue virus protein is a non-structural protein such as, for example, NS 1 , NS2A, NS2B, NS3, NS4A, NS4B or NS5.
  • a Dengue virus protein is a structural protein such as, for example, Dengue virus envelope (E) protein, membrane (M) protein or core protein.
  • methods and compositions of the invention include those that do not substantially sensitize a subject to severe dengue disease (e.g., via ADE), or elicit, induce, stimulate or promote severe dengue disease, upon a secondary or subsequent Dengue virus infection or exposure.
  • severe dengue disease is mediated by antibody dependent enhancement (ADE).
  • ADE antibody dependent enhancement
  • the severe Dengue virus disease comprises antibody-dependent enhancement of infection.
  • the protein administered consists of a single Dengue virus serotype. In other embodiments of the uses, methods and compositions, protein administered comprises a plurality of single Dengue virus serotype proteins administered. In still further embodiments of the uses, methods and compositions, protein administered comprises or consists of one or more Dengue virus serotype 1 , 2, 3 or 4 proteins.
  • protein administered comprises or consists of one or more Dengue virus serotype 1 proteins, and not a Dengue virus serotype 2, 3 or 4 protein; protein administered comprises or consists of one or more Dengue virus serotype 2 proteins, and not a Dengue virus serotype 1 , 3 or 4 protein; protein administered comprises or consists of one or more Dengue virus serotype 3 proteins, and not a Dengue virus serotype 1 , 2 or 4 protein; or protein administered comprises or consists of one or more Dengue virus serotype 4 proteins, and not a Dengue virus serotype 1 , 2 or 3 protein.
  • administration of a protein of a first Dengue virus serotype is effective to vaccinate or provide the subject with protection against one or more Dengue virus serotypes distinct from the first Dengue virus serotype.
  • administration of a Dengue virus serotype 1 protein is effective to vaccinate or provide the subject with protection against one or more of Dengue virus serotypes 2, 3 or 4; administration of a Dengue virus serotype 2 protein is effective to vaccinate or provide the subject with protection against one or more of Dengue virus serotypes 1 , 3 or 4; administration of a Dengue virus serotype 3 protein is effective to vaccinate or provide the subject with protection against one or more of Dengue virus serotypes 1 , 2 or 4; or administration of a Dengue virus serotype 4 protein is effective to vaccinate or provide the subject with protection against one or more of Dengue virus serotypes 1 , 2 or 3.
  • administration of a protein of a first Dengue virus serotype is effective to treat the subject for infection with one or more Dengue virus serotypes distinct from the first Dengue virus serotype.
  • administration of a Dengue virus serotype 1 protein is effective to treat the subject for infection with one or more of Dengue virus serotypes 2, 3 or 4;
  • administration of a Dengue virus serotype 2 protein is effective to treat the subject for infection with one or more of Dengue virus serotypes 1 , 3 or 4;
  • uses, methods and compositions reduce Dengue virus titer, increasing or stimulating Dengue virus clearance, reduce or inhibit Dengue virus proliferation, reduce or inhibit increases in Dengue virus titer or Dengue virus proliferation, reduce the amount of a Dengue virus protein or the amount of a Dengue virus nucleic acids, or reduce or inhibit synthesis of a Dengue virus protein or a Dengue virus nucleic acid.
  • uses, methods and compositions prevent, reduce, improve or inhibit one or more adverse physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated with Dengue virus infection or pathology.
  • uses, methods and compositions reduce or inhibit susceptibility to Dengue virus infection or pathology or protect a subject from adverse physiological conditions, disorders, illnesses, diseases, symptoms or complications caused by or associated with an antibody response to a Dengue virus infection.
  • invention uses, methods and compositions may be performed or administered prior to exposure to or infection of the subject with the Dengue virus, or substantially contemporaneously with exposure to or infection of the subject with the Dengue virus, or following exposure to or infection of the subject with the Dengue virus.
  • exposure or infection includes secondary or subsequent DENV infections (e.g., reinfection).
  • invention uses and methods include administering a Dengue virus protein or subsequence or portion or modification thereof in combination with a T-cell stimulatory molecule.
  • a composition includes a combination of a Dengue virus protein or portion or modification thereof and a T-cell stimulatory molecule.
  • a T-cell stimulatory molecule is OX40 or CD27.
  • the subject is a mammal, for example, a human.
  • a subject has not previously been infected with Dengue virus.
  • a subject prior to administration of the Dengue virus protein, produces antibodies against one or more Dengue virus serotypes.
  • a subject has previously been infected with Dengue virus.
  • NS 1 , NS2A, NS2B, NS3, NS4A, NS4B, NS5 proteins were identified.
  • Numerous CD4 + T cell and CD8+ T cell epitopes from the structural and non-structural (NS) proteins are also disclosed herein (e.g., Tables 1 -4).
  • T cell epitopes such as CD8 + or CD4 + T cell epitopes
  • CD8 + or CD4 + T cell epitopes While CD4 + T cells do not appear to be required for controlling primary DENV infection, immunization contributes to viral clearance.
  • DENV proteins include or consist of a subsequence, portion, or an amino acid modification of Dengue virus (DV) structural or non-structural (NS) polypeptide sequence from any of DENV serotypes 1 , 2, 3 or 4, and the protein elicits, stimulates, induces, promotes, increases, or enhances an anti-DV CD8 + T cell response or an anti-DV CD4 + T cell response.
  • DV Dengue virus
  • NS non-structural
  • a protein includes or consists of a subsequence, portion, or an amino acid modification of Dengue virus (DV) structural core (C), membrane (M) or envelope (E) polypeptide sequence, for example, based upon or derived from a DENV l , DENV2, DENV3 or DENV4 serotype.
  • a protein includes or consists of a subsequence, portion, or an amino acid modification of Dengue virus (DV) NS 1 , NS2A, NS2B, NS3, NS4A, NS4B or NS5 polypeptide sequence, for example, based upon or derived from a DENVl , DENV2, DENV3 or DENV4 serotype.
  • a protein includes or consists of a structural or non-structural (NS) polypeptide sequence from a DENV serotype 1 , 2, 3 or 4.
  • a protein includes or consists of a sequence set forth in Tables 1 -4, or a subsequence thereof or a modification thereof. Exemplary modifications include 1 , 2, 3, 4, 5 or 6, 7, 8, 9, 10 or more conservative, non- conservative, or conservative and non-conservative amino acid substitutions.
  • an anti-DV response includes a CD8 T cell response and/or a CD4 T cell response.
  • Such responses can be ascertained, for example, by increased IFN- gamma, TNF-alpha, IL- 1 alpha, 1L-6 or 1L-8 production by CD8 + T cells in the presence of the protein; and/or increased CD4 + T cell production of IFN-gamma, TNF, IL-2, or CD40L in the presence of the protein, or killing of peptide-pulsed target cells.
  • compositions including the proteins, subsequences, portions, or modifications thereof (e.g., T cell epitopes), such as pharmaceutical compositions.
  • Compositions can include one or more proteins, subsequences, portions, or modifications thereof, such as peptides selected from Tables 1 -4, or a subsequence or portion thereof, or a modification thereof, as well as optionally adjuvants.
  • Proteins, subsequences, portions, and modifications thereof can be used for stimulating, inducing, promoting, increasing, or enhancing an immune response against Dengue virus (DV) in a subject.
  • a method includes administering to a subject an amount of a DENV protein, subsequence, portion, or a modification thereof sufficient to stimulate, induce, promote, increase, or enhance an immune response against Dengue virus (DV) in the subject, and/or provide the subject with protection against a Dengue virus (DV) infection or pathology, or one or more physiological conditions, disorders, illness, diseases or symptoms caused by or associated with DV infection or pathology.
  • DENV proteins, subsequences, portions, and modifications thereof can also be used for treating a subject for a Dengue virus (DV) infection.
  • a method includes administering to a subject an amount of a DENV protein, subsequence, portion, or a modification thereof sufficient to treat the subject for the Dengue virus (DV) infection.
  • Exemplary responses, in vitro, ex vivo or in vivo, elicited by proteins, subsequences, portions, or modifications thereof, such as T cell epitopes include, stimulating, inducing, promoting, increasing, or enhancing an anti-DV CD8 + T cell response or an anti-DV CD4 + T cell response.
  • CD8 + T cells produce IFN-gamma, TNF-alpha, IL- 1 alpha, IL-6 or IL-8 in response to T cell epitope
  • CD4 + T cells produce IFN-gamma, TNF, IL-2 or CD40L, or kill peptide-pulsed target cells in response to a T cell epitope.
  • proteins, subsequences, portions, and modifications thereof can also be used for inducing, increasing, promoting or stimulating anti-Dengue virus (DV) activity of CD8 + T cells or CD4+ T cells in a subject.
  • DV anti-Dengue virus
  • a Dengue virus (DV) protein such as a T cell epitope, includes or consists of one or more sequences set forth in Tables 1-4, or a
  • Figure 1 shows a schematic of an immunization protocol.
  • FIGS 2A-2B show levels of viral RNA in the liver of AG 129 mice that were immunized with UV-inactivated DENV2 in alum and then challenged with DENV2.
  • the control groups represent non-immunized AG 129 mice that were treated i.p.
  • FIGS 3A-3B show levels of viral RNA in the liver (A) and survival (B) of AG 129 mice that were immunized with VRP-DENV2E and then challenged with DENV2.
  • AG129 mice were immunized with VRP-DENV2E ( 10 6 GE) via i.f. (IF vaccinated, black circles) or i.p. (IP vaccinated, black triangles) route on days - 14 and -5, followed by challenge with 5xl 0 8 GE of S221 i.v. on day 0.
  • the control groups represent non-immunized mice that were treated i.p.
  • FIG. 4 shows levels of viral RNA in the liver of AG 129 mice that were immunized with VRP-GFP or VRP-DENV2E and then challenged with DENV2.
  • AG 129 mice were immunized i.f. with 10 6 GE of VRP-GFP (white triangles) or VRP-DENV2E (black triangles) on days - 14 and - 5, followed by challenge with 5x10 8 GE of S221 i.v. on day 0.
  • the control groups represent non- immunized AG 129 mice that were treated i.p. with 15 ⁇ g of 2H2 (ADE, black squares) or C I .18 (baseline, white squares) 1 hour before viral challenge.
  • DENV RNA levels in the liver were measured 72 hours after infection by qRT-PCR. Each symbol represents a mouse.
  • FIG. 5 shows data indicating that DENV2E provides protection against ADE-DENV challenge.
  • AG 129 mice were immunized i.p. with 10 6 GE of VRP-DENV2 (VRP2) on days -14 and -5, followed by challenge with 5xl 0 8 GE of S221 i.v. on day 0 in the presence of isotype control mAb C I .1 8 (baseline, white circles) or anti-DENV mAb 2H2 (ADE, black circles).
  • Control groups represent non-immunzed AG 129 mice that were treated i.p. with 15 ⁇ g of 2H2 (ADE, black squares) or C I .1 8 (baseline, white squares) 1 hour before viral challenge.
  • DENV RNA levels in the liver were measured 72 hours after infection by qRT-PCR. Each symbol represents a mouse.
  • FIGS 6A-6B show a comparison of antibody (Ab) responses induced by UV- inactivated DENV2 plus alum versus VRP-DENV2E.
  • AG129 mice were immunized i.p. with 10" GE of UV-inactivated S221 in alum (diamonds) or DENV2E (triangles) on days -14 and -5, followed by harvest of serum on day - 1 , as per our standard immunization protocol.
  • A) DENV2- reactive IgG in the sera harvested from the immunized mice was measured by ELISA on plates coated with sucrose gradient purified S221.
  • B) Neutralization activity of the sera used in A was examined by measuring their ability to reduce infection of C6/36 cells by S221.
  • Figure 7 shows a schematic of T cell depletion from immunized mice.
  • FIGS 8A-8C show the role of T cells in DENV2E vaccine-mediated protection.
  • Each symbol represents a mouse.
  • DENV RNA levels were measured 72 hours after infection by qRT-PCR.
  • FIG. 9 shows RNA levels in the liver of AG 129 mice adoptively transferred with homologous or heterologous T cells and then challenged with DENV.
  • A129 mice were infected with 10 10 GE of S221 or DENV4 strain H421 (Philippino clinical isolate). 6 weeks later, total T cells from spleens of the DENV-immune mice were isolated by negative selection (Miltenyi MACS system) and transferred i.v. into AG 129 mice 1 day before challenge with 5xl 0 8 GE of S221 i.v. Liver DENV2 RNA levels on day 3 after infection were measured by qRT-PCR.
  • FIG. 10 shows viral RNA levels in the liver of CD8+ T cell-sufficient or -depleted AG 129 mice with heterologous secondary DENV infection.
  • AG129 mice were infected with 5xl 0 10 GE of DENV3 strain UNC3001 (Sri Lankan clinical isolate). 21 days later, DENV3-immune mice were depleted (or not) of T cells by injecting i.p. with 250 ⁇ g of SFR3 (isotype control) or 2.43 (anti-CD8) in PBS 3 days and 1 day before infection with 5xl 0 8 GE of S221 i.v. Liver DENV2 RNA levels on day 3 after infection were measured by qRT-PCR.
  • Figu re 11 shows a schematic of the basic immunization protocol using the AB6 mouse model of DENV2 infection.
  • FIG. 12 shows a schematic for varying the immunization protocol.
  • Figure 13 shows that adoptively transferred wild-type T cells protect against DENV in
  • Figures 14A-14D show that DENV2 infection results in CD4+ T cell activation and expansion in IFN-a/pR-/- mice.
  • C) Blood lymphocytes were obtained from IFN-a/pR -/- mice on days 3, 5, 7, 10, and 14 after infection with 10 10 GE of DENV2. The percentage of CD44 hl CD62L l0 cells (gated on CD4+ T cells) ⁇ SEM (n 6) is shown.
  • FIGS 15A-15B show the identification of DENV2-derived epitopes recognized by CD4 + T cells.
  • A) Splenocytes were obtained from IFN-a/pR " ' " mice 7 days after infection with 10 10 GE of DENV2 and re-stimulated in vitro with DENV2-derived 15-mer peptides predicted to bind I- A b . Cells were then stained for surface CD4 and intracellular IFN- ⁇ and analyzed by flow cytometry. The 4 positive peptides identified are shown. In the dot plots, the percentage of CD4 + T cells producing IFN- ⁇ is indicated. The responses of individual mice as well as the mean and SEM are also shown (n 7- 1 1 ).
  • FIG. 16 shows that DENV2-specific CD4 + T cells are polyfunctional.
  • Splenocytes were obtained from IFN-a/pR " ' " mice 7 days after infection with l O 10 GE of DENV2 and stimulated in vitro with individual peptides. Cells were then stained for surface CD4, and intracellular IFN- ⁇ , TNF, IL-2, and CD40L, and analyzed by flow cytometry. The response of unstimulated cells was subtracted from the response to each DENV2 peptide, and the net percentages of the CD4 + T cells that are expressing at least one molecule are indicated. The mean and SEM of 3 mice is shown.
  • Figure 17 shows that depletion of CD4 + T cells prior to DENV2 infection does not affect viral RNA levels.
  • IFN-ct/pR "A mice were depleted of CD4 + or CD8 + cells, or both, by administration of GK 1.5 or 2.43 Ab, respectively, (or given an isotype control Ab) 2 days before and 1 day after infection with l O 10 GE of DENV2. Mice were sacrificed 5 days later, and DENV2 RNA levels in the serum, spleen, small intestine, brain, and kidney were quantified by real-time RT-PCR. Data are expressed as DENV2 copies per ml of sera, or DENV2 units normalized to 18S rRNA levels for the organs.
  • Each symbol represents one mouse, the bar represents the geometric mean, and the dashed line is the limit of detection.
  • FIGS 18A-18C show that CD4 + T cells are not required for the anti-DENV2 antibody response.
  • IFN-a/pR " ' " mice control or CD4-depleted mice were infected with 10 I0 GE of DENV2.
  • A) IgM and IgG titers in the sera at day 7 were measured by ELISA (n 5 control and 6 CD4-depleted mice). Data are combined from two independent studies.
  • FIGs 19A-19C show that CD4 + T cells are not required for the primary DENV2- specific CD8 + T cell response.
  • Figure 20 shows cytotoxicity mediated by DENV2-specific CD4 + T cells.
  • IFN-a/pR "A mice control, CD4-depleted, or CD8-depleted infected 7 days previously with 10 10 GE of DENV2 were injected i.v.
  • mice Separate groups of peptide-immunized mice were depleted of CD4 + or CD8 + T cells prior to infection.
  • DENV2 RNA levels in the tissues were quantified by real-time RT-PCR and are expressed as DENV2 units normalized to 18S rRNA. Each symbol represents one mouse and the bar represents the geometric mean. * p ⁇ 0.05, * * p ⁇ 0 .01.
  • Figure 22A-22D show identification of DENV-derived epitopes recognized by CD8 + T cells.
  • DENV specific epitope identification was performed in four different HLA transgenic mouse strains (A) A*0201 ; (B) A * 1 101 ; (C) A*0101 ; and (D) B*0702.
  • IFNy ELISPOT was performed using splenic T cells isolated from HLA transgenic IFN-a/pR ⁇ mice (black bars) and H LA transgenic IFN-a/pR t + mice (white bars). Mice were infected i.v. retro- orbitally with i x l 0 l o GE of DENV2 (S221 ) in 100 ⁇ PBS.
  • CD8 + T cells were puritled and tested against a panel of S221 predicted peptides.
  • the data are expressed as mean number of SFC/1 0 6 CD8 + T cells of two independent studies. Error bars represent SEM. Responses against peptides were considered positive if the stimulation index (SI) exceeded double the mean negative control wells (effector cells plus APCs without peptide) and net spots were above the threshold of 20 SFCs/10 6 CD8 + T cells in two independent studies. Asterisks indicate peptides, which were able to elicit a significant IFNy response in each individual study, according to the criteria described above.
  • FIG 23 shows identification of DENV-derived epitopes recognized by CD4 + T cells.
  • IFNy ELISPOT was performed using CD4 + T cells isolated from DRB 1 *0101 transgenic IFN-a/pR ' " (black bars) and IFN-a/pR + + (white bars) mice. Mice were infected i.v. retro-orbitally with l xlO 10 GE of DENV2 (S221 ) in 100 ⁇ PBS. Seven days postinfection, CD4 + T cells were purified and tested against a panel of S221 predicted peptides. The data are expressed as mean number of SFC/10 6 CD4 + T cells of two independent studies. Error bars represent SEM.
  • Figures 24A-24B show the determination of optimal epitope studies.
  • HLA-transgenic IFN-a/pR 7" mice were infected with 1 x 10'° GE of DENV2 (S221 ) and spleens harvested 7 days post infection.
  • CD8 + T cells were purified and incubated for 24 hours with ascending concentrations of nested peptides.
  • A) shows pairs of peptides where the 9-mer and the l Omer were able to elicit a significant T cell response;
  • B) shows the 3 B*0702 restricted peptides which did show an IC 50 > 1000nM in the respective binding assay.
  • Peptides were retested in parallel with their corresponding 8-, 10- and 1 1 -mers. The peptides, which were able to elicit stronger IFNy responses at various concentrations, were then considered the dominant epitope.
  • FIGS 25A-25B show MHC-restriction of identified epitopes.
  • Purified CD8 + T cells from DENV2 (S221 ) infected HLA A*A0201 and HLA A* l 10 IFN-a/pR ⁇ mice were incubated with increasing concentrations of peptides and tested for IFNy production in an ELISPOT assay.
  • Figures 26A-26F show antigenicity of identified epitopes in human donors.
  • Epitopes ⁇ g/ml individual peptide for 7 days
  • IFN-a/pR "7* mice were validated by their capacity to stimulate PBMC (2x10 6 PBMC/ml) from human donors and then tested in an IFNy ELISPOT assay.
  • A-E) show IFNy responses/10 6 PBMC after stimulation with A*0101 , A*0201 , A* l 101 , B*0702 and DRB 1 *0101 restricted peptides, respectively.
  • Donors seropositive for DENV, were grouped in HLA matched and non-HLA matched cohorts, as shown in panels 1 and 2 of each figure. All epitopes identified were further tested in DENV seronegative individuals. The average IFNy responses elicited by PBMC from DENV seropositive non-HLA matched and DENV seronegative donors plus 3 times the standard deviation (SD) was set as a threshold for positivity, as indicated by the dashed line. F) shows the mean IFNy response /10 6 T cells from HLA transgenic mice (black bars) and HLA matched donors (white bars) grouped by HLA restriction of the epitopes tested.
  • Figure 27 shows subproteinlocation of identified epitopes from Table 2. All identified epitopes were grouped according to the DENV subprotein they are derived from. Black bars show the total IFNy response all epitopes of a certain protein could elicit. Numbers in parenthesis indicate the number of epitopes that have been detected for this protein.
  • T cells contribute towards protection against primary Dengue virus (DENV) infection in cl inically relevant mouse models of Dengue virus (Yauch, et al. J Immunol 1 85:5405-5416 (2010); Yauch, et al. J Immunol 182:4865-4873 (2009)).
  • the studies disclosed herein demonstrate that CD8+ T cells play a critical role in vaccine-mediated protection against DENV infection.
  • the findings disclosed herein reveal that CD8+ T cell immunity is required for vaccine-mediated protection against DENV, which is contrary to the general consensus in the field that antibodies are essential for immunization or vaccination against Dengue virus.
  • the studies disclosed herein demonstrate that the responsive CD8+ T cells after administration of a particular DENV serotype can provide the animal with protection against other distinct (heterologous) DENV serotypes.
  • the studies disclosed herein reveal that a protein or subsequence of a given DENV serotype can be used to provide protection against other distinct DENV serotypes in vaccination and immunization methods and uses.
  • a DENV3 serotype protein or subsequence or portion can be administered to provide a subject with protection against a DENV 1 , DENV2 and/or DENV4 serotype infection.
  • CD8+ T cells that provide protection against distinct DENV serotypes can also provide protection against other distinct DEN V serotypes, even in the presence of enhancing antibodies.
  • the studies disclosed herein also reveal that a protein or subsequence of a given DENV serotype can be used to provide (broad spectrum) protection in subjects who already have developed antibodies against DENV, as a consequence of a prior DENV infection or exposure to DENV (e.g., vaccination or immunization), for example.
  • ADE mediated DHF or DSS subjects that are at risk of severe dengue disease
  • DHF or DSS subjects having Dengue virus antibodies
  • transfer e.g., maternal transfer or passive immunization or vaccination with against Dengue virus
  • a use or method for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to elicit, stimulate, induce, promote, increase or enhance an anti-Dengue virus T cell response in the subject.
  • a use or method for vaccinating or providing a subject with protection against a Dengue virus infection without eliciting or sensitizing the subject to severe dengue disease upon a secondary or subsequent Dengue virus infection includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to vaccinate or provide the subject with protection against the Dengue virus infection.
  • a use or method for treating a subject for a Dengue virus i nfection without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection includes administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to treat the subject for the Dengue virus infection.
  • severe dengue disease e.g., ADE mediated DHF or DSS
  • sensitize refers to causing a subject to acquire or develop a condition, disease or disorder or the symptoms or complications caused by or associated with the condition, disease or disorder, or to be susceptible to acquiring or developing a condition, disease or disorder or the symptoms or complications cause by or associated with the condition, disease or disorder.
  • stressitize or “sensitizing” may refer to increasing the susceptibility of a subject to acquiring or developing a condition, disease or disorder or the symptoms or complications cause by or associated with the condition, disease or disorder.
  • sensitizing a subject to severe dengue disease upon a secondary or subsequent Dengue virus infection may refer to causing the subject to acquire or develop severe dengue disease or the symptoms or complications caused by or associated with severe dengue disease upon subsequent Dengue virus infection.
  • Sensitizing a subject to severe dengue disease may also refer to causing the subject to be susceptible to acquiring or developing severe dengue disease or one or more other symptoms or complications caused by or associated with severe dengue disease upon a secondary or subsequent Dengue virus infection.
  • sensitizing a subject to severe dengue disease may also refer to increasing the susceptibil ity of the subject to acquiring or developing severe dengue disease, one or more other symptoms or complications of severe dengue disease, or more severe symptoms or complications of severe dengue disease, caused by or associated with severe dengue.
  • a "severe dengue disease” refers to conditions, disease and disorders caused by or associated with Dengue virus infection, including but not limited to dengue hemorrhagic fever (DHF), dengue shock syndrome (DSS) and any symptoms or complications cause by or associated with DHF and DSS including but not limited to increased vascular permeability, thrombocytopenia, hemorrhagic manifestions and death.
  • DHF dengue hemorrhagic fever
  • DSS dengue shock syndrome
  • any symptoms or complications cause by or associated with DHF and DSS including but not limited to increased vascular permeability, thrombocytopenia, hemorrhagic manifestions and death.
  • the development of severe dengue disease may be mediated by antibody dependant enhancement (ADE).
  • ADE antibody dependant enhancement
  • the term antibody (Ab) dependent enhancement of infection refers to a phenomenon in which a subject who has antibodies against Dengue virus, due to a previous Dengue virus infection or exposure to Dengue virus or antigen (e.g, vaccination, immunization, receipt of maternal anti-Dengue virus antibodies, etc.), suffers from enhanced or a more severe illness after a secondary or subsequent infection with a Dengue virus, or after a Dengue virus vaccination or immunization.
  • the more severe symptoms include one or more of hemorrhagic fever/Dengue shock syndrome, increased viral load, increased vascular permeability, increased hemorrhagic manifestations, thrombocytopenia, and shock, compared to the acute self- limited illness typically caused by Dengue virus in subjects who have not been vaccinated, immunized or previously infected with Dengue virus.
  • ADE is believed to be a consequence of the presence of serotype cross-reactive antibodies enhancing viral infection of FcyR + cells resulting in higher Dengue viral loads and a more severe ill ness upon subsequent exposure or infection of the subject to a Dengue virus or antigen.
  • Methods and uses of the invention therefore include methods and uses that do not substantially or detectably cause, elicit or stimulate one or more symptoms characteristic of ADE, or more broadly ADE, in a subject.
  • ADE there may be other adverse symptoms that result from, or be enhanced or more severe, when a subject who has antibodies against Dengue virus (e.g., due to a prior infection, exposure, vaccination, immunization, maternal antibodies etc.) becomes infected with Dengue virus, or receives a Dengue virus vaccination or immunization, as compared to a subject that has not been vaccinated, immunized or previously infected with a Dengue virus.
  • adverse symptoms that may result from, or may be enhanced or more severe include, for example, fever, headache, rash, liver damage, diarrhea, nausea, vomiting or abdominal pain.
  • the methods and uses of the invention therefore also include methods and uses that do not substantially elicit, enhance or worsen one or more such other adverse symptoms that may be elicted, enhanced or be more severe in a subject who has antibodies against a Dengue virus, as compared to a subject that does not have antibodies against a Dengue virus.
  • a Dengue virus protein of the uses, methods and compositions may be a non-structural or structural Dengue virus protein, subsequence or portion or modification thereof.
  • the Dengue virus protein is a non-structural Dengue virus protein, for example, NS l , NS2A, NS2B, NS3, NS4A, NS4B or NS5.
  • the Dengue virus protein is a NS3, NS4B or NS5 protein, subsequence or portion or modification thereof.
  • the Dengue virus protein is a structural Dengue virus protein, for example, Dengue virus envelope protein, membrane protein or core protein, subsequence or portion or modification thereof.
  • a DENV protein, subsequence, portion or modification thereof elicits a cellular or humoral immune response.
  • a DENV protein, subsequence, portion or modification thereof elicits, stimulates, promotes or induces a CD8+ T cell and/or CD4+ T cell response.
  • Such responses can provide protection against (e.g., prophylaxis) an initial DENV infection, or a secondary or subsequent DENV infection.
  • T cell responses can also be effective in treatment (e.g., therapeutic) of an initial DENV infection, or a secondary or subsequent DENV infection.
  • T cell responses can occur without detectably or substantial ly eliciting, inducing or promoting severe dengue disease (e.g., ADE mediated DHF or DSS) in a subject having anti-DENV antibodies, or detectably or substantially sensitizing a subject to developing severe dengue disease (e.g., ADE mediated DHF or DSS) upon a subsequent DENV infection.
  • severe dengue disease e.g., ADE mediated DHF or DSS
  • DSS severe dengue disease
  • a DENV protein, subsequence, or portion thereof may be derived from or based upon any sequence from any DENV strain or serotype, such as wild-type.
  • Exemplary serotypes are DEN V 1 , DENV2, DENV3 and DEN V4.
  • a DENV protein, subsequence, portion or modification thereof is derived from or based upon a DENV 1 , DENV2, DENV3 or DENV4 sequence.
  • a protein, subsequence, portion or modification thereof van be derived from or is based upon West Pacific 74 strain (DENV 1 ), UNC 1017 strain (DENV l ), UNC 2005 strain (DENV2), S 16803 strain (DENV2), UNC 3001 strain (DENV3), UNC 3043 (DE V3 , strain 059.AP-2, Philippines), UNC 3009 strain (DENV3, D2863, Sri Lanka), UNC3066 (DENV3, strain 1342 from Puerto Rico 1977), CH 53489 strain (DENV3), TVP-360 (DENV4), or UNC 4019 strain (DENV4).
  • a DENV protein, subsequence, or portion thereof may also be a modified or variant form (hereinafter referred to as a "modification").
  • modified forms such as amino acid deletions, additions and substitutions, can also be used in the invention uses, methods and compositions for eliciting, inducing, promoting, increasing or enhancing a T cell response, protecting, vaccinating or immunizing a subject, or treatment of a subject, as set forth herein.
  • a subsequence of a Dengue virus protein includes or consists of one or more amino acids less than the full length Dengue virus protein.
  • the term "subsequence" means a fragment or part of the full length molecule.
  • a subsequence of a Dengue virus protein has one or more amino acids less than the full length Dengue virus protein (e.g. one or more internal or terminal amino acid deletions from either amino or carboxy-termini). Subsequences therefore can be any length up to the full length native molecule, provided said length is at least one amino acid less than full length native molecule.
  • Subsequences can vary in size, for example, from a polypeptide as small as an epitope capable of binding an antibody (i. e. , about five amino acids) up to a polypeptide that is one amino acid less than the entire length of a reference polypeptide such as a Dengue virus protein
  • a dengue virus protein subsequence is characterized as including or consisting of a NS 1 sequence with less than 380 amino acids in length identical to NS 1 , a NS2A sequence with less than 159 amino acids in length identical to NS2A, a S2B sequence with less than 130 amino acids in length identical to NS2B, a NS3 sequence with less than 618 amino acids in length identical to NS3, a NS4A sequence with less than 127 amino acids in length identical to NS4A, a NS4B sequence with less than 248 amino acids in length identical to NS4B, a NS5 sequence with less than 900 amino acids in length identical to NS5, a dengue virus envelope protein sequence with less than 495 amino acids in length identical to dengue virus envelope protein, a dengue virus membrane protein sequence with less than 166 amino acids in length identical to dengue virus membrane protein, a dengue virus core protein sequence with less than 96 amino acids in length identical to dengue virus core protein
  • Non-limiting exemplary subsequences less than full length NS 1 sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, or 300 to 380 amino acids in length.
  • Non-limiting exemplary subsequences less than full length NS2Asequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 1 59 amino acids in length.
  • Non-limiting exemplary subsequences less than full length NS2Bsequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 130 amino acids in length.
  • subsequences less than full length NS3sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 618 amino acids in length.
  • Non-limiting exemplary subsequences less than full length NS4Asequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 127 amino acids in length.
  • Non-limiting exemplary subsequences less than full length NS4Bsequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200 to 248 amino acids in length.
  • Non-limiting exemplary subsequences less than full length NS5sequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900 amino acids in length.
  • Non-limiting exemplary subsequences less than full length dengue virus envelope proteinsequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 1 50, 150 to 200, 200 to 300, 300 to 400, 400 to 495 amino acids in length.
  • Non-limiting exemplary subsequences less than full length dengue virus membrane proteinsequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 100, 100 to 1 50, 1 50 to 166 amino acids in length.
  • Non-l imiting exemplary subsequences less than full length dengue virus core proteinsequence include, for example, a subsequence from about 5 to 10, 10 to 20, 20 to 30, 30 to 50, 50 to 96 acids in length.
  • subsequences may also include or consist of one or more amino acid additions or deletions, wherein the subsequence does not comprise the full length native/wild type Dengue virus protein sequence. Accordingly, total subsequence lengths can be greater than the length of the full length native/wild type Dengue virus protein, for example, where a Dengue virus protein subsequence is fused or forms a chimera with another polypeptide.
  • the uses, methods and compositions may comprise an Dengue virus protein or peptide comprising or consisting of a subsequence, or an amino acid modification of Dengue virus structural or non-structural protein sequence, wherein the protein or peptide elicits, stimulates, induces, promotes, increases or enhances and anti-Dengue virus CD8 + T cell response or an anti-Dengue virus CD4 + T cell response, as described herein.
  • a non-limiting example of a protein, subsequence or portion of a Dengue virus (DV) polypeptide sequence includes or consists of a subsequence or portion of Dengue virus (DV) structural Core, Membrane or Envelope polypeptide sequence.
  • a non-limiting example of a protein, subsequence or portion of a Dengue virus (DV) polypeptide sequence includes or consists of a protein, subsequence or portion of Dengue virus (DV) non-structural (NS) NS 1 , NS2A, NS2B, NS3, N S4A, NS4B or NS5 polypeptide sequence.
  • a non-limiting Core sequence of or from which a protein, subsequence, portion or modi fication can be based upon is a sequence set forth as:
  • a non-limiting Membrane (M) sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • a non-limiting Envelope (E) sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • a non-limiting non-structural NS 1 sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • a non-limiting non-structural NS2A sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • a non-limiting non-structural NS2B sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • a non-limiting non-structural NS3 sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • a non-limiting non-structural NS4A sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • a non-limiting non-structural NS4B sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • a non-limiting non-structural NS5 sequence of or from which a protein, subsequence, portion or modification can be based upon is a sequence set forth as:
  • Structural proteins E and prM are major targets of anti-DENV antibody response.
  • NS proteins in particular NS3, NS4B and NS5 are more conserved across the four DENV serotypes than E, and NS proteins are not expressed in DENV virions (unlike E and PrM proteins).
  • NS3, NS4B, or NS5 will be better at inducing cross-protective (heterologous) CD8+ T cell responses and at avoiding ADE.
  • DENV vaccines expressing NS3, NS4B, or NS5 will likely provide superior CD8+ T cell immunity against DENV infection, or secondary or subsequent infection (reinfection) than Envelope and Membrane proteins.
  • Dengue virus (DV) proteins, subsequences, portions and modifications thereof of the invention include those having all or at least partial sequence identity to one or more exemplary Dengue virus (DV) proteins, subsequences, portions or modifications thereof (e.g., sequences set forth in Tables 1 -4).
  • the percent identity of such sequences can be as little as 60%, or can be greater (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, etc.).
  • the percent identity can extend over the entire sequence length or a portion of the sequence.
  • the length of the sequence sharing the percent identity is 2, 3, 4, 5 or more contiguous amino acids, e.g., 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 1 8, 19, 20, etc. contiguous amino acids.
  • the length of the sequence sharing the percent identity is 20 or more contiguous amino acids, e.g., 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, etc. contiguous amino acids.
  • the length of the sequence sharing the percent identity is 35 or more contiguous amino acids, e.g., 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 45, 47, 48, 49, 50, etc., contiguous amino acids.
  • the length of the sequence sharing the percent identity is 50 or more contiguous amino acids, e.g., 50-55, 55-60, 60- 65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95- 100, 100- 1 10, etc. contiguous amino acids.
  • identity and grammatical variations thereof, mean that two or more referenced entities are the same.
  • DV proteins Dengue virus (DV) proteins, subsequences, portions and modi fications thereof are identical, they have the same amino acid sequence.
  • the identity can be over a defined area (region or domain) of the sequence. "Areas, regions or domains" of homology or identity mean that a portion of two or more referenced entities share homology or are the same.
  • BLAST e.g. , BLAST 2.0
  • search algorithm see, e.g. , Aitschui et al., J Mol. Biol. 215 :403 ( 1990), publicly available through NCBl
  • exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2.
  • a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM 100, PAM 250, BLOSUM 62 or BLOSUM 50.
  • FASTA e.g., FASTA2 and FASTA3
  • SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85 :2444 (1988); Pearson, Methods Mol Biol. 1 32: 185 (2000); and Smith et al., J. Mol. Biol. 147: 195 (1981 )).
  • Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).
  • modified and variant forms of Dengue virus (DV) proteins, subsequences and portions there are provided.
  • Such forms referred to as “modifications” or “variants” and grammatical variations thereof, are a Dengue virus (DV) protein, subsequence or portion thereof that deviates from a reference sequence.
  • certain sequences set forth in Tables 1 -4 are considered a modification or variant of Dengue virus (DV) protein, subsequence or portion thereof.
  • Such modifications may have greater or less activity or function than a reference Dengue virus (DV) protein, subsequence or portion thereof, such as ability to elicit, stimulate, induce, promote, increase, enhance or activate a CD4+ or a CD8+ T cell response.
  • Dengue virus (DV) proteins, subsequences and portions thereof include sequences having substantially the same, greater or less relative activity or function as a T cell epitope than a reference T cell epitope (e.g., any of the sequences in Tables 1 -4), for example, an ability to elicit, stimulate, induce, promote, increase, enhance or activate an anti-DV CD4 + T cell or anti-DV CD8 + T cell response in vitro or in vivo.
  • Non-limiting examples of modifications include one or more amino acid substitutions (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50- 100, or more residues), additions and insertions (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 , 12, 1 3, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50-100, or more residues) and deletions (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 , 12, 1 3, 14, 1 5, 16, 17, 18, 19, 20, 20-25, 25-30, 30-50, 50- 100) of a reference Dengue virus (DV) protein, subsequence or portion thereof.
  • DV Dengue virus
  • a modified or variant sequence retains at least part of a function or an activity of unmodified sequence, and can have less than, approximately the same, or greater, but at least a part of, a function or activity of a reference sequence, for example, the ability to elicit, stimulate, induce, promote, increase, enhance or activate an anti-DV CD4 V T cell or anti-DV CD8 + T cell response in vitro or in vivo.
  • Such CD4 T cell and CDS ' T cell responses elicited include, for example, among others, induced, increased, enhanced, stimulate or activate expression or production of a cytokine (e.g., IFN-gamma, TNF, 1L-2 or CD40L), release of a cytotoxin (perforin or granulysin), or apoptosis of a target (e.g., DV infected) cell.
  • a cytokine e.g., IFN-gamma, TNF, 1L-2 or CD40L
  • a cytotoxin perforin or granulysin
  • apoptosis of a target e.g., DV infected
  • substitutions include conservative and non- conservative amino acid substitutions.
  • a "conservative substitution” is the replacement of one amino acid by a biologically, chemically or structurally similar residue.
  • Biologically similar means that the substitution does not destroy a biological activity.
  • Structurally similar means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size.
  • Chemical similarity means that the residues have the same charge, or are both hydrophilic or hydrophobic.
  • Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, serine for threonine, and the like.
  • An addition can be the covalent or non-covalent attachment of any type of molecule to the sequence.
  • Specific examples of additions include glycosylation, acetylation, phosphorylation, amidation, formylation, ubiquitination, and derivatization by protecting/blocking groups and any of numerous chemical modifications. Additional specific non-limiting examples of an addition are one or more additional amino acid residues.
  • DV sequences including DENV proteins, T cell epitopes, subsequences, portions, and modifications thereof can be a part of or contained within a larger molecule, such as another protein or peptide sequence, such as a fusion or chimera with a different DV sequence, or a non-DV protein or subsequence or portion or modification thereof.
  • an addition is a fusion (chimeric) sequence, an amino acid sequence having one or more molecules not normally present in a reference native (wild type) sequence covalently attached to the sequence.
  • chimera when used in reference to a sequence, means that the sequence contains one or more portions that are derived from, obtained or isolated from, or based upon other physical or chemical entities.
  • a chimera of two or more different proteins may have one part a Dengue virus (DV) peptide, subsequence, portion or modi fication, and a second part of the chimera may be from a different Dengue virus (DV) protein sequence, or a non-Dengue virus (DV) sequence.
  • DV Dengue virus
  • DV Dengue virus
  • heterologous functional domain is attached (covalent or non-covalent binding) that confers a distinct or complementary function.
  • heterologous functional domains are not restricted to amino acid residues.
  • a heterologous functional domain can consist of any of a variety of different types of small or large functional moieties. Such moieties include nucleic acid, peptide, carbohydrate, lipid or small organic compounds, such as a drug (e.g., an antiviral), a metal (gold, silver), and radioisotope.
  • the invention provides Dengue virus (DV) proteins, subsequences, portions and modi fications thereof and a heterologous domain, wherein the heterologous functional domain confers a distinct function, on the Dengue virus (DV) proteins, subsequences, portions and modifications thereof.
  • DV Dengue virus
  • Such constructs containing Dengue virus (DV) proteins, subsequences, portions and modifications thereof and a heterologous domain are also referred to as chimeras.
  • Linkers such as amino acid or peptidomimetic sequences may be inserted between the sequence and the addition (e.g., heterologous functional domain) so that the two entities maintain, at least in part, a distinct function or activity.
  • Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character, which could promote or interact with either domain.
  • Amino acids typically found in flexible protein regions include Gly, Asn and Ser. Other near neutral amino acids, such as Thr and Ala, may also be used in the l inker sequence.
  • the length of the linker sequence may vary without significantly affecting a function or activity of the fusion protein (see, e.g., U.S. Patent No.
  • Linkers further include chemical moieties and conjugating agents, such as sulfo- succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS),
  • DSG disuccinimidyl glutarate
  • DST disuccinimidyl tartrate
  • the invention provides Dengue virus (DV) proteins, subsequences and portions thereof that are detectably labeled.
  • detectable labels include fluorophores, chromophores, radioactive isotopes (e.g., S 35 , P 32 , I 125 ), electron-dense reagents, enzymes, ligands and receptors. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert a substrate such as 3,3-',5,5-'-tetramethylbenzidine (TMB) to a blue pigment, which can be quantified.
  • TMB 3,3-',5,5-'-tetramethylbenzidine
  • an addition is an insertion of an amino acid within any Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., any DV sequence set forth herein, such as in Tables 1 -4).
  • an insertion is of one or more amino acid residues inserted into a Dengue virus (DV) protein, subsequence portion or modification thereof, such as any sequence set forth herein, such as in Tables 1 -4.
  • Modified and variant Dengue virus (DV) proteins, subsequences and portions thereof also include one or more D-amino acids substituted for L-amino acids (and mixtures thereof), structural and functional analogues, for example, peptidomimetics having synthetic or non-natural amino acids or amino acid analogues and derivatized forms. Modifications include cyclic structures such as an end-to-end amide bond between the amino and carboxy-terminus of the molecule or intra- or inter-molecular disulfide bond.
  • Dengue virus (DV) proteins, subsequences and portions thereof may be modified in vitro or in vivo, e.g., post-translationally modified to include, for example, sugar residues, phosphate groups, ubiquitin, fatty acids, lipids, etc.
  • Dengue virus proteinsubsequences or portions include an amino acid sequence comprising at least one amino acid deletion from full length Dengue virus (DV) protein sequence.
  • a protein subsequence or portion is from about 5 to 300 amino acids in length, provided that said subsequence or portion is at least one amino acid less in length than the full-length Dengue virus (DV) structural sequence or the non-structural (NS) sequence.
  • a protein subsequence or portion is from about 2 to 5, 5 to 10, 10 to 15, 1 5 to 20, 20 to 25, 25 to 50, 50 to 100, 100 to 150, 150 to 200, or 200 to 300 amino acids in length, provided that said subsequence or portion is at least one amino acid less in length than the full-length Dengue virus (DV) structural protein sequence or non-structural (NS) protein sequence.
  • DV Dengue virus
  • NS non-structural
  • Dengue virus (DV) proteins, subsequences and portions thereof including modified forms can be produced by any of a variety of standard protein purification or recombinant expression techniques.
  • a Dengue virus (DV) protein, subsequence, portion or modification thereof can be produced by standard peptide synthesis techniques, such as solid-phase synthesis.
  • a portion of the protein may contain an amino acid sequence such as a T7 tag or polyhistidine sequence to facilitate purification of expressed or synthesized protein.
  • the protein may be expressed in a cell and purified.
  • the protein may be expressed as a part of a larger protein (e.g., a fusion or chimera) by recombinant methods.
  • Dengue virus (DV) proteins, subsequences and portions thereof including modified forms can be made using recombinant DNA technology via cell expression or in vitro translation.
  • Polypeptide sequences including modified forms can also be produced by chemical synthesis using methods known in the art, for example, an automated peptide synthesis apparatus (see, e.g. , Applied Biosystems, Foster City, CA).
  • the invention provides isolated and/or purified Dengue virus (DV) proteins, including or consisting of a protein, subsequence, portion or modification of a structural core (C), membrane (M) or envelope (E) polypeptide sequence, or a non-structural (NS) NS 1 , NS2A, NS2B, NS3, NS4A, NS4B or NS5 polypeptide sequence.
  • an isolated and/or purified protein, subsequence, portion or modification of the Dengue virus (DV) polypeptide sequence includes a T cel l epitope, e.g., as set forth in Tables 1 -4.
  • isolated when used as a modifier of a composition (e.g., Dengue virus (DV) proteins, subsequences, portions and modifications thereof, nucleic acids encoding same, etc.), means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, cell membrane.
  • DV Dengue virus
  • isolated does not exclude alternative physical forms of the composition, such as fusions/chimeras, multimers/oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.
  • An "isolated" composition e.g., Dengue virus (DV) protein, subsequence, portion or modi fication thereof
  • DV Dengue virus
  • an isolated Dengue virus (DV) protein, subsequence, portion or modification thereof, that also is substantially pure or purified does not include polypeptides or polynucleotides present among millions of other sequences, such as peptides of an peptide library or nucleic acids in a genomic or cDNA library, for example.
  • a “substantially pure” or “purified” composition can be combined with one or more other molecules.
  • “substantially pure” or “purified” does not exclude combinations of compositions, such as combinations of Dengue virus (DV) proteins, subsequences, portions and modifications thereof (e.g., multiple, T cell epitopes), and other antigens, agents, drugs or therapies.
  • DV Dengue virus
  • the invention also provides nucleic acids encoding Dengue virus (DV) proteins, subsequences, portions and modifications thereof.
  • Such nucleic acid sequences encode a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, etc.) identical to a Dengue virus (DV) protein, subsequence or portion thereof.
  • a nucleic acid encodes a sequence having a modification, such as one or more amino acid additions (insertions), deletions or substitutions of a Dengue virus (DV) protein, subsequence or portion thereof, such as any sequence set forth in Tables 1 -4.
  • nucleic acid refers to at least two or more ribo- or deoxy-ri bonucleic acid base pairs (nucleotides/nucleosides) that are linked through a phosphoester bond or equivalent.
  • Nucleic acids include polynucleotides and
  • Nucleic acids include single, double or triplex, circular or linear, molecules.
  • nucleic acids include but are not limited to: RNA, DNA, cDNA, genomic nucleic acid, naturally occurring and non naturally occurring nucleic acid, e.g., synthetic nucleic acid.
  • Nucleic acids can be of various lengths. Nucleic acid lengths typically range from about 20 bases to 20 i lobases (Kb), or any numerical value or range within or encompassing such lengths, 10 bases to 10Kb, 1 to 5 Kb or less, 1000 to about 500 bases or less in length. Nucleic acids can also be shorter, for example, 100 to about 500 bases, or from about 12 to 25, 25 to 50, 50 to 100, 1 00 to 250, or about 250 to 500 bases in length, or any numerical value or range or val ue within or encompassing such lengths.
  • Kb i lobases
  • a nucleic acid sequence has a length from about 10-20, 20-30, 30-50, 50- 1 00, 100- 1 50, 150-200, 200-250, 250-300, 300-400, 400-500, 500- 1000, 1000-2000 bases, or any numerical value or range within or encompassing such lengths.
  • Shorter nucleic acids are commonly referred to as "ol igonucleotides” or “probes” of single- or double- stranded DNA. However, there is no upper limit to the length of such oligonucleotides.
  • Nucleic acid sequences further include nucleotide and nucleoside substitutions, additions and deletions, as well as derivatized forms and fusion/chimeric sequences (e.g., encoding recombinant polypeptide).
  • nucleic acids include sequences and subsequences degenerate with respect to nucleic acids that encode Dengue virus (DV) proteins, subsequences and portions thereof, as well as variants and modifications thereof (e.g., substitutions, additions, insertions and deletions).
  • DV Dengue virus
  • Nucleic acids can be produced using various standard cloning and chemical synthesis techniques. Techniques include, but are not limited to nucleic acid amplification, e.g., polymerase chain reaction (PCR), with genomic DNA or cDNA targets using primers (e.g., a degenerate primer mixture) capable of annealing to the encoding sequence. Nucleic acids can also be produced by chemical synthesis (e.g., solid phase phosphoramidite synthesis) or transcription from a gene.
  • PCR polymerase chain reaction
  • primers e.g., a degenerate primer mixture
  • Nucleic acids can also be produced by chemical synthesis (e.g., solid phase phosphoramidite synthesis) or transcription from a gene.
  • sequences produced can then be translated in vitro, or cloned into a plasmid and propagated and then expressed in a cell (e.g., a host cell such as eukaryote or mammalian cell, yeast or bacteria, in an animal or in a plant).
  • a cell e.g., a host cell such as eukaryote or mammalian cell, yeast or bacteria, in an animal or in a plant.
  • Nucleic acid may be inserted into a nucleic acid construct in which expression of the nucleic acid is influenced or regulated by an "expression control element.”
  • An “expression control element” refers to a nucleic acid sequence element that regulates or influences expression of a nucleic acid sequence to which it is operatively linked. Expression control elements include, as appropriate, promoters, enhancers, transcription terminators, gene silencers, a start codon
  • An expression control element operatively linked to a nucleic acid sequence controls transcription and, as appropriate, translation of the nucleic acid sequence.
  • Expression control elements include elements that activate transcription constitutively, that are inducible (i.e., require an external signal for activation), or derepressible (i. e. , require a signal to turn transcription off; when the signal is no longer present, transcription is activated or "derepressed"), or specific for cell-types or tissues (i.e., tissue-specific control elements).
  • Nucleic acid can also be inserted into a plasmid for propagation into a host cell and for subsequent genetic manipulation.
  • a plasmid is a nucleic acid that can be propagated in a host cell, plasmids may optional ly contain expression control elements in order to drive expression of the nucleic acid encoding Dengue virus (DV) proteins, subsequences, portions and modifications thereof in the host cel l.
  • a vector is used herein synonymously with a plasmid and may also include an expression control element for expression in a host cell (e.g., expression vector). Plasmids and vectors generally contain at least an origin of replication for propagation in a cell and a promoter.
  • Plasmids and vectors are therefore useful for genetic manipulation and expression of Dengue virus (DV) proteins, subsequences and portions thereof. Accordingly, vectors that include nucleic acids encoding or complementary to Dengue virus (DV) proteins, subsequences, portions and
  • particles e.g., viral particles
  • transformed host cells that express and/or are transformed with a nucleic acid that encodes and/or express Dengue virus (DV) proteins, subsequences, portions and modifications thereof.
  • Particles and transformed host cells include but are not limited to virions, and prokaryotic and eukaryotic cells such as bacteria, fungi (yeast), plant, insect, and animal ⁇ e.g., mammalian, including primate and human, CHO cells and hybridomas) cells.
  • bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors for example, bacteria transformed with recombinant bacteriophage nucleic acid, plasmid nucleic acid or cosmid nucleic acid expression vectors; yeast transformed with recombinant yeast expression vectors; plant cell systems infected with recombinant virus expression vectors (e.g.
  • the cells may be a primary cell isolate, cel l culture (e.g., passaged, established or immortalized cell line), or part of a plurality of cells, or a tissue or organ ex vivo or in a subject (in vivo).
  • transformed or "transfected” when used in reference to a cell (e.g. , a host cel l) or organism, means a genetic change in a cell following incorporation of an exogenous molecule, for example, a protein or nucleic acid (e.g. , a transgene) into the cell.
  • transfected or transformed cell is a cell into which, or a progeny thereof in which an exogenous molecule has been introduced by the hand of man, for example, by recombinant DNA techniques.
  • the nucleic acid or protein can be stably or transiently transfected or transformed (expressed) in the host cel l and progeny thereof.
  • the cell(s) can be propagated and the introduced protein expressed, or nucleic acid transcribed.
  • a progeny of a transfected or transformed cell may not be identical to the parent cel l, since there may be mutations that occur during replication.
  • DV Dengue virus
  • nucleic acid in particles or introduction into target cells e.g., host cells
  • Non-limiting examples include osmotic shock (e.g., calcium phosphate), electroporation, microinjection, cell fusion, etc.
  • osmotic shock e.g., calcium phosphate
  • electroporation e.g., electroporation
  • microinjection e.g., cell fusion
  • Introduction of nucleic acid and polypeptide in vitro, ex vivo and in vivo can also be accomplished using other techniques.
  • a polymeric substance such as polyesters, polyamine acids, hydrogel, polyvinyl pyrrolidone, ethylene-vinylacetate, methylcellulose, carboxymethylcellulose, protamine sulfate, or lactide/glycolide copolymers, polylactide/glycolide copolymers, or ethylenevinylacetate copolymers.
  • a nucleic acid can be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin- microcapsules, or poly (methylmethacrolate) microcapsules, respectively, or in a colloid system.
  • Colloidal dispersion systems include macromolecule complexes, nano-capsules, microspheres, beads, and l ipid-based systems, including oi l-in-water emulsions, micelles, mixed micelles, and liposomes.
  • Liposomes for introducing various compositions into cells are known in the art and include, for example, phosphatidylcholine, phosphatidylserine, lipofectin and DOTAP (e.g., U.S. Patent Nos. 4,844,904, 5,000,959, 4,863,740, and 4,975,282; and GIBCO-BRL, Gaithersburg, MD).
  • Piperazine based amphilic cationic lipids useful for gene therapy also are known (see, e.g., U.S. Patent No. 5,861 ,397).
  • Cationic lipid systems also are known (see, e.g., U.S. Patent No. 5,459, 127).
  • Polymeric substances, microcapsules and colloidal dispersion systems such as liposomes are collectively referred to herein as "vesicles.” Accordingly, viral and non-viral vector means delivery into cells are included.
  • Dengue virus proteins, subsequences, portions and modifications thereof can be employed in various methods and uses. Such methods and uses include, for example, use, contact or administration of one or more DENV proteins, subsequences or moficiations thereof, such as the proteins and subsequences set forth herein (e.g., Tables 1 -4), in vitro and in vivo.
  • methods for eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection comprising administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to elicit an anti-Dengue virus T cell response in the subject.
  • a method for providing a subject with protection against a Dengue virus infection or pathology, or one or more physiological disorders, illness, diseases or symptoms caused by or associated with Dengue virus infection or pathology without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection comprising administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to protect the subject against Dengue virus infection.
  • a method of vaccinating a subject against a Dengue virus infection without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection comprising administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to vaccinate the subject against the Dengue virus infection.
  • a method of treating a subject for a Dengue virus infection without sensitizing the subject to severe dengue disease upon subsequent Dengue virus infection comprising administering to the subject an amount of a Dengue virus protein or subsequence thereof sufficient to treat the subject for the Dengue virus infection.
  • the terms "protect” and grammatical variations thereof when used in reference to a Dengue virus infection or pathology, means preventing a DENV infection, or reducing or decreasing susceptibility to a DENV infection, or preventing or reducing one or more symptoms or pathologies caused by or associated with DENV infection or pathology, such as ADE.
  • a subject may be protected from one or more DENV serotypes, e.g. any or all of DENV 1 , 2, 3 or 4, or any variant serotype.
  • a protected subject may also have been previously exposed to or infected with a DENV, and have developed antibodies against DENV. Protection in this context would therefore include, but not be limited to, protection from a secondary or subsequent DENV infection.
  • a method includes administering to a subject an amount of a Dengue virus (DV) protein, subsequence or portion or modification thereof, such as a T cell epitope, sufficient to stimulate, induce, promote, increase, or enhance an immune response against Dengue virus (DV) in the subject.
  • DV Dengue virus
  • Such immune response methods can in turn be used to provide a subject with protection against a Dengue virus (DV) infection or pathology, or one or more physiological conditions, disorders, illness, diseases or symptoms caused by or associated with DV infection or pathology.
  • treatment uses and methods include therapeutic (following Dengue virus (DV) infection) and prophylactic (prior to Dengue virus (DV) exposure, infection or pathology) uses and methods.
  • therapeutic and prophylactic uses and methods of treating a subject for a Dengue virus (DV) infection include but are not limited to treatment of a subject having or at risk of having a Dengue virus (DV) infection or pathology, treating a subject with a Dengue virus (DV) infection, and methods of protecting a subject from a Dengue virus (DV) infection (e.g., provide the subject with protection against Dengue virus (DV) infection), to decrease or reduce the probability of a Dengue virus (DV) infection in a subject, to decrease or reduce susceptibility of a subject to a Dengue virus (DV) infection, to inhibit or prevent a Dengue virus (DV) infection in a subject, and to decrease, reduce, inhibit or suppress transmission of the Dengue virus (DV) from a host (e.g., a host), e.g., a host, a
  • Such methods include, for example, administering Dengue virus (DV) protein, subsequence, portion or modification thereof to therapeutically or prophylactically treat (vaccinate or immunize) a subject having or at risk of having a Dengue virus (DV) infection or pathology.
  • DV Dengue virus
  • uses and methods can treat a Dengue virus (DV) infection or pathology, or provide a subject with protection from infection (e.g., prophylactic protection).
  • DV Dengue virus
  • a method includes administering to a subject an amount of Dengue virus (DV) protein, subsequence, portion or modification thereof sufficient to treat the subject for the Dengue virus (DV) infection or pathology.
  • a method includes administering to a subject an amount of a Dengue virus (DV) protein, subsequence, portion or modification sufficient to provide the subject with protection against the Dengue virus (DV) infection or pathology, or one or more physiological conditions, disorders, illness, diseases or symptoms caused by or associated with the virus infection or pathology.
  • a method includes administering a subject an amount of a Dengue virus (DV) protein, subsequence, portion or modification sufficient to treat the subject for the Dengue virus (DV) infection.
  • Dengue virus (DV) proteins, subsequences, portions and modifications thereof include T cell epitopes.
  • a method includes administering an amount of Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., a T cell epitope) to a subject in need thereof, sufficient to provide the subject with protection against Dengue virus (DV) infection or pathology.
  • a method includes administering an amount of a Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., a T cell epitope) to a subject in need thereof sufficient to treat, vaccinate or immunize the subject against the Dengue virus (DV) infection or pathology.
  • a method includes administering to a subject an amount of a Dengue virus (DV) protein, subsequence or portion, or modification thereof, such as a T cell epitope, sufficient to induce, increase, promote or stimulate anti-Dengue virus (DV) activity of CD8 + T cells or CD4 + T cells in the subject.
  • DV Dengue virus
  • any appropriate Dengue virus (DV) protein, subsequence, portion or modification thereof can be used or administered.
  • Non-limiting examples include Dengue virus (DV) protein, subsequence, portion or modification thereof of a DENV 1 , DENV2, DENV3 or DENV4 serotype protein, subsequence or portion or modification thereof, such as a T cell epitope.
  • Additional non-limiting examples include a Dengue virus structural protein (e.g., C, M or E) or nonstructural (NS) protein (e.g., NS 1 , NS2A, NS2B, NS3, NS4A, NS4B or NS5), or a subsequence or portion or modification thereof, such as a T cell epitope, in or of such structural and non-structural (NS) proteins.
  • NS structural and non-structural
  • Particular non-limiting examples include a DENV protein, or a protein subsequence, such as a sequence set forth in Tables 1 -4, or a subsequence or a modification thereof.
  • one or more disorders, diseases, physiological conditions, pathologies and symptoms associated with or caused by a Dengue virus (DV) i nfection or pathology will respond to treatment.
  • treatment uses and methods reduce, decrease, suppress, limit, control or inhibit Dengue virus (DV) numbers or titer; reduce, decrease, suppress, limit, control or inhibit pathogen proliferation or replication; reduce, decrease, suppress, limit, control or inhibit the amount of a pathogen protein; or reduce, decrease, suppress, limit, control or inhibit the amount of a Dengue virus (DV) nucleic acid.
  • treatment uses and methods include an amount of a Dengue virus (DV) protein, subsequence or portion or modification thereof sufficient to increase, induce, enhance, augment, promote or stimulate an immune response against a Dengue virus (DV); increase, induce, enhance, augment, promote or stimulate Dengue virus (DV) clearance or removal; or decrease, reduce, inhibit, suppress, prevent, control, or limit transmission of Dengue virus (DV) to a subject (e.g., transmission from a host, such as a mosquito, to a subject).
  • DV Dengue virus
  • treatment uses and methods include an amount of Dengue virus (DV) protein, subsequence or portion or modification thereof sufficient to protect a subject from a Dengue virus (DV) infection or pathology, or reduce, decrease, limit, control or inhibit susceptibility to Dengue virus (DV) infection or pathology.
  • DV Dengue virus
  • Uses and methods of the invention include treatment uses and methods, which result in any therapeutic or beneficial effect.
  • Dengue virus (DV) infection, proli feration or pathogenesis is reduced, decreased, inhibited, limited, delayed or prevented, or a use or method decreases, reduces, inhibits, suppresses, prevents, controls or limits one or more adverse (e.g., physical) symptoms, disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology (e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • adverse e.g., physical
  • symptoms e.g., disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection
  • proliferation or replication e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite.
  • treatment uses and methods include reducing, decreasing, inhibiting, delaying or preventing onset, progression, frequency, duration, severity, probability or susceptibility of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology (e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • DV Dengue virus
  • treatment uses and methods include improving, accelerating, facilitating, enhancing, augmenting, or hastening recovery of a subject from a Dengue virus (DV) infection or pathogenesis, or one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology (e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • DV Dengue virus
  • pathology e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite.
  • treatment uses and methods include stabi lizing infection, proliferation, replication, pathogenesis, or an adverse symptom, disorder, illness, disease or complication caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology, or decreasing, reducing, inhibiting, suppressing, limiting or controll ing transmission of Dengue virus (DV) from a host (e.g., mosquito) to an uninfected subject.
  • a host e.g., mosquito
  • a therapeutic or beneficial effect of treatment is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject.
  • a therapeutic or beneficial effect can but need not be complete ablation of all or any particular adverse symptom, disorder, illness, disease or complication caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology (e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite).
  • DV Dengue virus
  • a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in an adverse symptom, disorder, illness, disease or complication caused by or associated with Dengue virus (DV) infection, proliferation or replication, or pathology, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illnesses, diseases or complications caused by or associated with Dengue virus (DV) infection, Dengue virus (DV) numbers, titers, proliferation or replication, Dengue virus (DV) protein or nucleic acid, or Dengue virus (DV) pathology, over a short or long duration (hours, days, weeks, months, etc.).
  • a therapeutic or beneficial effect also includes reducing or eliminating the need, dosage frequency or amount of a second active such as another drug or other agent (e.g., anti-viral) used for treating a subject having or at risk of having a Dengue virus (DV) infection or pathology.
  • a second active such as another drug or other agent (e.g., anti-viral) used for treating a subject having or at risk of having a Dengue virus (DV) infection or pathology.
  • reducing an amount of an adjunct therapy for example, a reduction or decrease of a treatment for a Dengue virus (DV) infection or pathology, or a vaccination or immunization protocol is considered a beneficial effect.
  • reducing or decreasing an amount of a Dengue virus (DV) antigen used for vaccination or immunization of a subject to provide protection to the subject is considered a beneficial effect.
  • Adverse symptoms and complications associated with Dengue virus (DV) infection and pathology include, for example, e.g., fever, rash, headache, pain behind the eyes, muscle or joint pain, nausea, vomiting, loss of appetite, etc. Thus, the aforementioned symptoms and complications are treatable in accordance with the invention.
  • Other symptoms of Dengue virus (DV) infection and pathology include ADE, which occurs upon a secondary or subsequent DENV infection of a subject, which had been previously infected with or exposed to DENV.
  • ADE as set forth herein or known to one of skill in the art, can be minimized or avoided (i.e., a subject would not be sensitized to ADE), or ADE would not be substantially elicited, induced, stimulated or promoted in a subject, in accordance with the invention uses and methods. Additional symptoms of Dengue virus (DV) infection or pathogenesis are known to one of skill in the art and treatment thereof in accordance with the invention is provided.
  • DV Dengue virus
  • Uses, methods and compositions of the invention also include increasing, stimulating, promoting, enhancing, inducing or augmenting an anti-DENV CD4 + and/or CD8 + T cell responses in a subject, such as a subject with or at risk of a Dengue virus infection or pathology.
  • a use or method includes administering to a subject an amount of Dengue virus (DV) protein, subsequence, portion or modification thereof sufficient to increase, stimulate, promote, enhance, augment or induce anti-DENV CD4 + or CD8 + T cell response in the subject.
  • DV Dengue virus
  • a method in another embodiment, includes administering to a subject an amount of Dengue virus (DV) protein, subsequence, portion or modification thereof, and administering a Dengue virus (DV) antigen, live or attenuated Dengue virus (DV), or a nucleic acid encoding all or a portion (e.g., a T cell epitope) of any protein or proteinaceous Dengue virus (DV) antigen sufficient to increase, stimulate, promote, enhance, augment or induce anti-Dengue virus (DV) CD4 + T cell or CD8 + T cell response in the subject.
  • DV Dengue virus
  • Uses and methods of the invention additionally include, among other things, increasing production of a Th l cytokine (e.g., IFN-gamma, TNF-alpha, IL- 1 alpha, IL-2, IL-6, IL-8, etc.) or other signaling molecule (e.g., CD40L) in vitro or in vivo.
  • a Th l cytokine e.g., IFN-gamma, TNF-alpha, IL- 1 alpha, IL-2, IL-6, IL-8, etc.
  • CD40L signaling molecule
  • a method includes administering to a subject in need thereof an amount of Dengue virus (DV) protein, subsequence or portion or modification thereof sufficient to increase production of a Thl cytokine in the subject (e.g., IFN-gamma, TNF-alpha, IL- 1 alpha, IL-2, IL-6, IL-8, etc.) or other signaling molecule (e.g., CD40L).
  • DV Dengue virus
  • Uses, methods and compositions of the invention include administration of Dengue virus (DV) protein, subsequence, portion or modification thereof to a subject prior to contact, exposure or infection by a Dengue virus, administration prior to, substantially contemporaneously with or after a subject has been contacted by, exposed to or infected with a Dengue virus (DV), and administration prior to, substantially contemporaneously with or after Dengue virus (DV) pathology or
  • Methods, compositions and uses of the invention also include administration of Dengue virus (DV) protein, subsequence, portion or modification thereof to a subject prior to, substantially contemporaneously with or following an adverse symptom, disorder, illness or disease caused by or associated with a Dengue virus (DV) infection, or pathology.
  • a subject infected with a Dengue virus (DV) may have an infection over a period of 1 -5, 5- 10, 10-20, 20-30, 30-50, 50-100 hours, days, months, or years.
  • invention compositions e.g., Dengue virus (DV) protein, subsequence or portion or modification thereof, including T cell epitopes
  • uses and methods can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect.
  • Exemplary combination compositions and treatments include multiple DENV proteins, subsequences, portions or modifications thereof, such as T cell epitopes as set for the herein, second actives, such as anti- Dengue virus (DV) compounds, agents and drugs, as well as agents that assist, promote, stimulate or enhance efficacy.
  • second actives such as anti- Dengue virus (DV) compounds, agents and drugs, as well as agents that assist, promote, stimulate or enhance efficacy.
  • Such anti-Dengue virus (DV) drugs, agents, treatments and therapies can be administered or performed prior to, substantially contemporaneously with or following any other use or method of the invention, for example, a therapeutic use or method of treating a subject for a Dengue virus (DV) infection or pathology, or a use or method of prophylactic treatment of a subject for a Dengue virus (DV) infection.
  • DV Dengue virus
  • Dengue virus (DV) proteins, subsequences, portions and modifications thereof can be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) administering a second active, to a subject.
  • the invention therefore provides combinations in which a use or method of the invention is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, such as an anti-viral (e.g., Dengue virus (DV)) or immune stimulating, enhancing or augmenting protocol, or pathogen vaccination or immunization (e.g., prophylaxis) set forth herein or known in the art.
  • an anti-viral e.g., Dengue virus (DV)
  • immune stimulating e.g., enhancing or augmenting protocol
  • pathogen vaccination or immunization e.g., prophylaxis
  • the compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of one or more Dengue virus (DV) proteins, subsequences, portions or modifications thereof, or a nucleic acid encoding all or a portion (e.g., a T cell epitope) of a Dengue virus (DV) protein, subsequence, portion or modification thereof, to a subject.
  • DV Dengue virus
  • Specific non-limiting examples of combination embodiments therefore include the foregoing or other compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition.
  • An exemplary combination is a Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g.. a CD4 + or CD8 + T cell epitope) and a different Dengue virus (DV) protein, subsequence, portion or modification thereof (e.g., a different T cell epitope) such as a DENV protein or T cell epitope, antigen (e.g., Dengue virus (DV) extract), or live or attenuated Dengue virus (DV) (e.g., inactivated Dengue virus (DV)).
  • Another exemplary combination is a Dengue virus (DV) protein, subsequence, portion or modification thereof and a T-cell stimulatory molecule, including for example an OX40 or CD27 agonist.
  • Such Dengue virus (DV) proteins, antigens and T cell epitopes set forth herein or known to one skilled in the art include Dengue virus (DV) proteins and antigens that increase, stimulate, enhance, promote, augment or induce a proinflammatory or adaptive immune response, numbers or activation of an immune cell (e.g., T cell, natural killer T (NKT) cell, dendritic cell (DC), B cell, macrophage, neutrophil, eosinophil, mast cell, CD4 + or a CD8 + cell, B220 + cell, CD14 + , CD 1 1 b + or CD1 lc + cells), an anti-Dengue virus (DV) CD4 + or CD8 + T cell response, production of a Th l cytokine, a T cell mediated immune response, such as activation of CD8+ T cells, or induction of CD8+ memory T cells, etc.
  • an immune cell e.g., T cell, natural killer T (NKT) cell, dendritic cell (DC), B cell
  • Combination methods and use embodiments include, for example, second actives such as anti-pathogen drugs, such as protease inhibitors, reverse transcriptase inhibitors, virus fusion inhi bitors and virus entry inhibitors, antibodies to pathogen proteins, live or attenuated pathogen, or a nucleic acid encoding all or a portion (e.g., an epitope) of any protein or proteinaceous pathogen antigen, immune stimulating agents, etc., and include contact with, administration in vitro or in vivo, with another compound, agent, treatment or therapeutic regimen appropriate for pathogen infection, vaccination or immunization
  • second actives such as anti-pathogen drugs, such as protease inhibitors, reverse transcriptase inhibitors, virus fusion inhi bitors and virus entry inhibitors, antibodies to pathogen proteins, live or attenuated pathogen, or a nucleic acid encoding all or a portion (e.g., an epitope) of any protein or proteinaceous pathogen antigen, immune stimulating agents,
  • Uses and methods of the invention also include, among other things, uses and methods that result in a reduced need or use of another compound, agent, drug, therapeutic regimen, treatment protocol, process, or remedy.
  • a use or method of the invention has a therapeutic benefit if in a given subject a less frequent or reduced dose or elimination of an anti-Dengue virus (DV) treatment results.
  • uses and methods of reducing need or use of a treatment or therapy for a Dengue virus (DV) infection or pathology, or vaccination or immunization are provided.
  • invention uses and methods in which there is a desired outcome, such as a therapeutic or prophylactic method that provides a benefit from treatment, vaccination or immunization, a Dengue virus (DV) protein, subsequence, portion or modification thereof can be administered in a sufficient or effective amount.
  • a desired outcome such as a therapeutic or prophylactic method that provides a benefit from treatment, vaccination or immunization, a Dengue virus (DV) protein, subsequence, portion or modification thereof can be administered in a sufficient or effective amount.
  • DV Dengue virus
  • a "sufficient amount” or “effective amount” or an “amount sufficient” or an “amount effective” refers to an amount that provides, in single (e.g., primary) or multiple (e.g., booster) doses, alone or in combination with one or more other compounds, treatments, therapeutic regimens or agents (e.g., a drug), a long term or a short term detectable or measurable improvement in a given subject or any objective or subjective benefit to a given subject of any degree or for any time period or duration (e.g., for minutes, hours, days, months, years, or cured).
  • An amount sufficient or an amount effective can but need not be provided in a single administration and can but need not be achieved by Dengue virus (DV) protein, subsequence, portion or modification thereof alone, optionally in a combination composition or method that includes a second active.
  • an amount sufficient or an amount effective need not be sufficient or effective if given in single or multiple doses without a second or additional
  • additional doses, amounts or duration above and beyond such doses, or additional antigens, compounds, drugs, agents, treatment or therapeutic regimens may be included in order to provide a given subject with a detectable or measurable improvement or benefit to the subject.
  • additional "'boosters" of one or more Dengue virus (DV) proteins, subsequences, portions or modifications thereof can be administered to increase, enhance, improve or optimize immunization and/or vaccination.
  • Such subsequent "booster" administrations can be of the same or a different formulation, dose or concentration, route, etc.
  • prophylactically effective in each and every subject treated, nor a majority of subjects treated in a given group or population An amount sufficient or an amount effective means sufficiency or effectiveness in a particular subject, not a group of subjects or the general population. As is typical for such methods, different subjects will exhibit varied responses to a use or method of the invention, such as immunization, vaccination and therapeutic treatments.
  • subject refers to a subject at risk of DEN V exposure or infection as well as a subject that has been exposed or already infected with DENV.
  • Such subjects include mammalian animals (mammals), such as a non human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), experimental animal (mouse, rat, rabbit, guinea pig) and humans.
  • Subjects include animal disease models, for example, mouse and other animal models of pathogen (e.g., DENV) infection known in the art.
  • subjects appropriate for treatment include those having or at risk of exposure to Dengue virus infection or pathology, also referred to as subjects in need of treatment.
  • Subjects in need of treatment therefore include subjects that have been exposed to or contacted with Dengue virus (DV), or that have an ongoing infection or have developed one or more adverse symptoms caused by or associated with Dengue virus (DV) infection or pathology, regardless of the type, timing or degree of onset, progression, severity, frequency, duration of the symptoms.
  • DV Dengue virus
  • Target subjects and subjects in need of treatment also include those at risk of Dengue virus (DV) exposure, contact, infection or pathology or at risk of having or developing a Dengue virus (DV) infection or pathology.
  • the invention uses, methods and compositions are therefore applicable to treating a subject who is at risk of Dengue virus (DV) exposure, contact, infection or pathology, but has not yet been exposed to or contacted with Dengue virus (DV).
  • Prophylactic uses and methods are therefore included.
  • Target subjects for prophylaxis can be at increased risk
  • Subjects for prophylaxis need not be at increased risk but may be from the general population in which it is desired to vaccinate or immunize a subject against a Dengue virus (DV) infection, for example.
  • a subject that is desired to be vaccinated or immunized against a Dengue virus (DV) can be administered Dengue virus (DV) protein, subsequence, portion or modi fication thereof.
  • a subject that is not specifically at risk of exposure to or contact with a Dengue virus (DV), but nevertheless desires protect against infection or pathology can be administered a Dengue virus (DV) protein, subsequence, portion or modification thereof.
  • Such subjects are also considered in need of treatment.
  • At risk subjects appropriate for treatment also include subjects exposed to environments in which subjects are at risk of a Dengue virus (DV) infection due to mosquitos.
  • Subjects appropriate for treatment therefore include human subjects exposed to mosquitos, or travelling to geographical regions or countries in which Dengue virus (DV) is known to infect subjects, for example, an individual who risks exposure due to the presence of DENV in a particular geographical region or country or population, or transmission from mosquitos present in the region or country.
  • At risk subjects appropriate for treatment also include subjects where the risk of Dengue virus (DV) infection or pathology is increased due to changes in infectivity or the type of region of Dengue virus (DV) carrying mosquitos. Such subjects are also considered in need of treatment due to such a risk.
  • Prophylaxis and grammatical variations thereof mean a use or a method in which contact, administration or in vivo delivery to a subject is prior to contact with or exposure to DENV or DENV infection.
  • DV Dengue virus
  • administration or in vivo delivery to a subject can be performed prior to infection or manifestation of pathology (or an associated adverse symptom,, condition, complication, etc. caused by or associated with a Dengue vims (DV)).
  • a subject can be immunized or vaccinated with a Dengue virus (DV) protein, subsequence, portion or modification thereof.
  • a use or method can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a Dengue virus (DV) infection or pathology, or an adverse symptom, condition or complication associated with or caused by or associated with a Dengue virus (DV) infection or pathology.
  • DV Dengue virus
  • Prophylaxis can also refer to a use or a method in which contact, administration or in vivo delivery to a subject is prior to a secondary or subsequent exposure or infection.
  • a subject may have had a prior DENV infection, or have been contacted with or exposed to Dengue virus (DV).
  • DV Dengue virus
  • an acute DENV infection may but not need be resolved.
  • Such a subject typically has developed anti-DENV antibodies due to the prior exposure or infection.
  • Immunization or vaccination, by administration or in vivo delivery to such a subject can be performed prior to a secondary or subsequent DENV infection or exposure.
  • Such a use or method can eliminate, prevent, inhibit, suppress, limit, decrease or reduce the probability of or susceptibility towards a secondary or subsequent Dengue virus (DV) infection or pathology, or an adverse symptom, condition or complication associated with or caused by or associated with a Dengue virus (DV) infection or pathology, or an adverse symptom or pathology associated with the development of anti-DENV antibodies, such as ADE.
  • DV Dengue virus
  • Treatment of an infection can be at any time during the infection.
  • Dengue virus (DV) protein, subsequence or portion or modification thereof can be administered as a combination (e.g., with a second active), or separately concurrently or in sequence (sequentially) in accordance with the uses and methods as a single or multiple dose e.g., one or more times hourly, daily, weekly, monthly or annually or between about 1 to 10 weeks, or for as long as appropriate, for example, to achieve a reduction in the onset, progression, severity, frequency, duration of one or more symptoms or complications associated with or caused by Dengue virus (DV) infection, pathology, or an adverse symptom, condition or complication associated with or caused by a Dengue virus (DV).
  • DV Dengue virus
  • a method can be practiced one or more times (e.g., 1 - 10, 1 -5 or 1 -3 times) an hour, day, week, month, or year.
  • a non-limiting dosage schedule is 1 -7 times per week, for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more weeks, and any numerical value or range or value within such ranges.
  • Uses and methods of the invention may be practiced by any mode of administration or delivery, or by any route, systemic, regional and local administration or delivery.
  • Exemplary administration and delivery routes include intravenous (i.v.), intraperitoneal (i.p.), intrarterial, intramuscular, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, intra-cranial, intra-spinal, rectal, oral (alimentary), mucosal, inhalation, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, or intralymphatic.
  • Doses can be based upon current existing protocols, empirically determined, using animal disease models or optionally in human clinical trials. Initial study doses can be based upon animal studies set forth herein, for a mouse, which weighs about 30 grams, and the amount of Dengue virus (DV) protein, subsequence, portion or modification thereof administered that is determined to be effective. Exemplary non-limiting amounts (doses) are in a range of about 0.1 mg kg to about 100 mg/kg, and any numerical value or range or value within such ranges. Greater or lesser amounts (doses) can be administered, for example, 0.01 -500 mg/kg, and any numerical value or range or value within such ranges.
  • DV Dengue virus
  • the dose can be adjusted according to the mass of a subject, and will generally be in a range from about 1 -10 ug/kg, 10-25 ug/kg, 25-50 ug/kg, 50- 100 ug/kg, 100-500 ug/kg, 500-1 ,000 ug/kg, 1 -5 mg/kg, 5- 10 mg/kg, 10-20 mg/kg, 20-50 mg/kg, 50- 100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, or more, two, three, four, or more times per hour, day, week, month or annually.
  • a typical range will be from about 0.3 mg/kg to about 50 mg/kg, 0-25 mg/kg, or 1.0- 10 mg/kg, or any numerical value or range or value within such ranges.
  • Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, whether a subject has been previously exposed to, infected with our suffered from Dengue virus (DV), the onset, progression, severity, frequency, duration probability of or susceptibility of the symptom, condition, pathology or complication, or vaccination or immunization to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.
  • DV Dengue virus
  • Dengue virus (DV) protein, subsequence, portion or modification thereof wi ll be administered as soon as practical, typically within 1 -2, 2-4, 4-12, 12-24 or 24-72 hours after a subject is exposed to or contacted with a Dengue virus (DV), or within 1 -2, 2- 4, 4- 12, 12-24 or 24-48 hours after onset or development of one or more adverse symptoms, conditions, pathologies, complications, etc., associated with or caused by a Dengue virus (DV) infection or pathology.
  • Dengue virus (DV) protein, subsequence, portion or modification thereof can be administered for a duration of 0-4 weeks, e.g., 2-3 weeks, prior to exposure to, contact or infection with Dengue virus (DV), or at least within 1 -2, 2-4, 4-12, 12-24, 24-48 or 48-72 hours prior to exposure to, contact or infection with Dengue virus (DV).
  • Dengue virus (DV) protein, subsequence, portion or modification thereof is administered at any appropriate time.
  • the dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by the status of the subject. For example, whether the subject has a pathogen infection, whether the subject has been exposed to, contacted or infected with pathogen or is merely at risk of pathogen contact, exposure or infection, whether the subject is a candidate for or will be vaccinated or immunized.
  • the dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy.
  • the route, dose, number and frequency of administrations, treatments, immunizations or vaccinations, and timing/intervals between treatment, immunization and vaccination, and viral challenge can be modified.
  • rapid induction of immune responses is desired for developing protective emergency vaccines against DENV
  • a desirable DENV vaccine will elicit robust, long-lasting immunity.
  • invention uses, methods and compositions provide long-lasting immunity to DENV. Immunization strategies provided can provide long-lived protection against DENV challenge, depending on the level of vaccine-induced CD8+ T cell response.
  • the invention also provides an amount of a Dengue virus protein, subsequence or portion, or modification thereof for use in: eliciting, stimulating, inducing, promoting, increasing, or enhancing an anti-Dengue virus T cell response in a subject without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection; providing a subject with protection against a Dengue virus infection or pathology, or one or more physiological disorders, illness, diseases or symptoms caused by or associated with Dengue virus infection or pathology without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection; vaccinating a subject against a Dengue virus infection without eliciting or sensitizing the subject to severe dengue disease (e.g., ADE mediated DHF or DSS) upon a secondary or subsequent Dengue virus infection; and treating a subject
  • the term "pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact.
  • Such formulations include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil- in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical
  • Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents.
  • Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals.
  • Supplementary active compounds e.g., preservatives, antibacterial, antiviral and antifungal agents
  • compositions can be formulated to be compatible with a particular route of administration.
  • pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, opthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.
  • Formulations suitable for parenteral administration comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.
  • Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
  • Dengue virus (DV) proteins, subsequences, portions and modifications thereof can be coupled to another protein such as ovalbumin or keyhole limpet hemocyanin ( LH), thyroglobulin or a toxin such as tetanus or cholera toxin.
  • Dengue virus (DV) proteins, subsequences, portions and modifications thereof can also be mixed with adjuvants.
  • Adj uvants include, for example: Oil (mineral or organic) emulsion adjuvants such as Freund's complete (CFA) and incomplete adjuvant (IFA) (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/1 1241 ; and U .S. Patent No.
  • CFA Freund's complete
  • IFA incomplete adjuvant
  • metal and metallic salts such as aluminum and aluminum salts, such as aluminum phosphate or aluminum hydroxide, alum (hydrated potassium aluminum sulfate); bacterially derived compounds, such as Monophosphoryl lipid A and derivatives thereof (e.g., 3 De-O-acylated monophosphoryl lipid A, aka 3D-MPL or d3-MPL, to indicate that position 3 of the reducing end glucosamine is de-O-acylated, 3D-MPL consisting of the tri and tetra acyl congeners), and enterobacterial lipopolysaccharides (LPS); plant derived saponins and derivatives thereof, for example Quil A (isolated from the Quilaja Saponaria Molina tree, see, e.g., "Saponin adj uvants", Archiv.
  • Quil A isolated from the Quilaja Saponaria Molina tree, see, e.g., "Saponin adj uvants", Archiv.
  • QS7 and QS21 also known as QA7 and QA21
  • surfactants such as, soya lecithin and oleic acid
  • sorbitan esters such as sorbitan trioleate
  • polyvinylpyrrolidone oligonucleotides such as CpG (WO 96/02555, and WO 98/16247), polyriboA and polyriboU; block copolymers
  • immunostimulatory cytokines such as GM-CSF and IL- l , and Muramyl tripeptide (MTP).
  • Cosolvents may be added to a Dengue virus (DV) protein, subsequence, portion or modification composition or formulation.
  • Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol;
  • compositions may therefore include
  • preservatives antioxidants and antimicrobial agents.
  • Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation.
  • Suitable preservatives include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate.
  • Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.
  • An antimicrobial agent or compound directly or indirectly inhibits, reduces, delays, halts, eliminates, arrests, suppresses or prevents contamination by or growth, infectivity, replication, proli eration, reproduction, of a pathogenic or non- pathogenic microbial organism.
  • Classes of antimicrobials include antibacterial, antiviral, antifungal and antiparasitics.
  • Antimicrobials include agents and compounds that kill or destroy (-cidal) or inhibit (-static) contamination by or growth, infectivity, replication, proliferation, reproduction of the microbial organism.
  • antibacterials include penicillins (e.g., penicillin G, ampicillin, methicillin, oxacillin, and amoxicillin), cephalosporins (e.g., cefadroxil, ceforanid, cefotaxime, and ceftriaxone), tetracyclines (e.g., doxycycline, chlortetracycline, minocycline, and tetracycline), aminoglycosides (e.g., amikacin, gentamycin, kanamycin, neomycin, streptomycin, netilmicin, paromomycin and tobramycin), macrolides (e.g., azithromycin, clarithromycin, and erythromycin), fluoroquinolones (e.g., ciprofloxacin, lomefloxacin, and norfloxacin), and other antibiotics including chloramphenicol, clindamycin,
  • anti-virals include reverse transcriptase inhibitors; protease inhibitors; thymidine kinase inhibitors; sugar or glycoprotein synthesis inhibitors; structural protein synthesis inhibitors; nucleoside analogues; and viral maturation inhibitors.
  • anti-virals include nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, zidovudine (AZT), stavudine (d4T), larnivudine (3TC), didanosine (DDI), zalcitabine (ddC), abacavir, acyclovir, penciclovir, ribavirin, valacyclovir, ganciclovir, 1 ,-D- ribofuranosyl-l ,2,4-triazole-3 carboxamide, 9->2-hydroxy-ethoxy methylguanine, adamantanamine, 5-iodo-2'-deoxyuridine, trifluorothymidine, interferon and adenine arabinoside.
  • compositions and methods of the invention are known in the art (see, e.g. , Remington: The Science and Practice of Pharmacy (2003) 20 th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences ( 1990) 18 th ed., Mack Publishing Co., Easton, PA; The Merck Index ( 1996) 12 th ed., Merck
  • Dengue virus (DV) proteins, subsequences, portions, and modifications thereof, along with any adjunct agent, compound drug, composition, whether active or inactive, etc., can be packaged in unit dosage form (capsules, tablets, troches, cachets, lozenges) for ease of
  • a "unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active ingredient optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect).
  • Unit dosage forms also include, for example, ampules and vials, which may include a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo.
  • Unit dosage forms additionally include, for example, ampules and vials with liquid compositions disposed therein. Individual unit dosage forms can be included in multi-dose kits or containers. Pharmaceutical formulations can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.
  • reference to a range of 90- 100% includes 91 -99%, 92-98%, 93-95%, 91 -98%, 91 - 97%, 91 -96%, 91 -95%, 91 -94%, 91 -93%, and so forth.
  • Reference to a range of 90- 100% includes 91 %, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1 %, 91.2%, 91.3%, 91 .4%, 91 .5%, etc., 92.1 %, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.
  • Reference to a range of 1 -5 fold therefore includes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, fold, etc., as well as 1 .1 , 1.2, 1.3, 1.4, 1.5, fold, etc., 2.1 , 2.2, 2.3, 2.4, 2.5, fold, etc., and so forth.
  • reference to a series of ranges of 2-72 hours, 2-48 hours, 4-24 hours, 4- 18 hours and 6- 12 hours includes ranges of 2-6 hours, 2, 12 hours, 2- 18 hours, 2-24 hours, etc., and 4-27 hours, 4-48 hours, 4-6 hours, etc.
  • a series of range formats are used throughout this document.
  • the use of a series of ranges includes combinations of the upper and lower ranges to provide a range. Accordingly, a series of ranges include ranges which combine the values of the boundaries of different ranges within the series. This construction applies regardless of the breadth of the range and in all contexts throughout this patent document.
  • reference to a series of ranges such as 5-10, 10-20, 20-30, 30-40, 40-50, 50-75, 75-100, 100- 150, and 1 50- 171 includes ranges such as 5-20, 5-30, 5-40, 5-50, 5-75, 5- 100, 5- 150, 5-171 , and 10-30, 10-40, 10-50, 10-75, 10- 100, 10-150, 10- 171 , and 20-40, 20-50, 20-75, 20- 100, 20-150, 20-171 , and so forth.
  • the invention is generally disclosed herein using affirmative language to describe the numerous embodiments and aspects.
  • the invention also specifically includes embodiments in which particular subject matter is excluded, in full or in part, such as substances or materials, method steps and conditions, protocols, procedures, assays or analysis.
  • substances or materials, method steps and conditions, protocols, procedures, assays or analysis such as antibodies or other materials and method steps are excluded.
  • a Dengue virus (DV) protein, subsequence, portion, or modification thereof is excluded.
  • DV Dengue virus
  • Example 1 This example includes a description of an ADE mouse model that reflects ADE in humans.
  • Antibody (Ab)-induced dengue disease is a severe condition that affects humans having existing Dengue virus antibodies.
  • a clinically relevant model of antibody (Ab)-induced dengue disease (ADE) in mice is disclosed. The model demonstrates, for the first time, ADE in vivo (Zel lweger, et al . Cell Host Microbe 7: 128-1 39 (2010)).
  • mice were passively administered 15 ⁇ ig of mouse mAb of subclass lgG2a (clone 2H2; DENVl -4 cross-reactive) before infection with 5x10 s genomic equivalents (GE) ( ⁇ 10 4 PFU) of the DENV2 strain S221.
  • mice treated with 2H2 succumbed early to S221 infection day 4-6 and featured the hallmarks of severe dengue disease in humans (high viral load, elevated hematocrit, cytokine storm, low platelet count, increased vascular permeability, hemorrhagic manifestations, and shock-induced death).
  • mice treated with isotype control Ab developed paralysis at later times after infection (day 10-30).
  • This example includes data demonstrating that vaccination with inactivated Dengue virus mediates ADE.
  • mice were injected with 10" GE ( «2 x 10 6 PFU) of UV-inactivated DENV2 strain S221 via a subcutaneous (s.c.) or intraperitoneal (i.p.) route 14 and 5 days before a sublethal intravenous (i.v.) infection with S221 ( 5 xlO 8 GE or - 104 PFU) (schematized in Figure 1).
  • Control groups included a baseline/isotype group (i.p. injected with 15 ⁇ g of an irrelevant isotype control Ab prior to viral challenge) and an ADE group (i.p. injected with 15 ⁇ g of DENV prM/M-specific IgG2a mAb clone 2H2 prior to viral challenge).
  • DENV RNA levels in the liver at day 3 after viral challenge were measured by qRT-PCR analysis.
  • control mice with enhancing Ab i.e. the ADE group
  • both the s.c. and i.p. groups of vaccinated animals contained high viral RNA levels ( Figure 2A), and most of the vaccinated animals died between days 4-5 post-infection, thereby demonstrating ADE effect upon immunization with UV-inactivated DENV2 in alum.
  • This example includes data demonstrating that Dengue Virus protein can provide protective immunity, without substantially inducing ADE, and even in the presence of enhancing antibodies.
  • VEE Venezuelan Equine Encephalitis
  • VRP virus replicon particles
  • UV-inactivated DENV2 plus alum regimen is shown in Figure 1.
  • AG 129 mice were immunized i.p. or intra-foot pad (i.f.) with 10 6 GE of VRP-DENV2E (White, et al. Journal of Virol. 81 : 10329- 10339 (2007)) on 14 and 5 days prior to challenge with the sub-lethal dose of S221.
  • mice were immunized with VRP-DENV2E on 14 and 5 days before viral challenge (i.e. the same immunization protocol as all studies described thus far), but the immunized mice were administered anti-DENV mAb ( 15 ⁇ g of clone 2H2) just prior to i.v. inoculation with S22 1. It was found that DENV2E- vaccination reduced viral RNA levels in the liver on day 3 after challenge with virus alone or with virus plus anti-DENV Ab, indicating that DENV2 immunization strategy offers protection even in the presence of enhancing Abs (Figure 5).
  • This example includes data demonstrating that cell-mediated immunity contributes to the DENV2E- mediated protection against DENV.
  • This example includes data demonstrating that CD8+ T cells provide early protective capacity against Dengue virus.
  • mice were immunized with VRP-DENV2E as described above, followed by depletion of CD4+ and/or CD8+ T cells before challenge with S221 (Figure 7).
  • Figure 7 On day 3 post-challenge, viral RNA levels in l iver and cytokine levels in the serum were measured ( Figure 8).
  • Figure 8A Depletion of both CD4+ and CD8+ T cells from immunized animals abolished protection
  • Figure 8B depletion of CD4+ T cel ls alone had little to no effect on DENV viral load, compared to immunized but non-depleted mice
  • This Example includes studies demonstrating that adoptively transferred wild-type T cells protect against DENV in AG 129 mice.
  • the data indicate a rapidly protective, CD8+ T cel l-dependent DENV immunization strategy using DENV2E in a clinically relevant model of DENV infection.
  • a desirable dengue vaccine should elicit robust, long-lasting immunity.
  • length of protection and uses and methods to augment magnitude and duration of CD8+ T cell immunity, if such augmentation is desired, can be obtained by adjusting one or more of the following parameters.
  • activated CD8+ T cells are CD44 hl CD62L low i-67+ Bcl-21ow; effector CD8+ T cells are CD107a + granzyme B + perforin + ; short-lived effector cells (SLECs) are
  • LRG 1 + CD127 " memory precursor effector cells (MPECs) are KLRG 1 " CD127 + ; central memory T (TCM) are CD62L + CD 127 + ; and effector memory T (TEM) are CD62L " CD127 + .
  • DENV2E dose translating to a greater antigenic load over time
  • TCM central memory T
  • TEM effector memory T
  • the greater CD8+ T cell response should respond faster upon viral challenge and correlate with better protection (i.e. the immunized mice should have increased survival and decreased levels of viral RNA in the liver and cytokines in the serum upon viral challenge).
  • Days between immunization can be optimized, for example, if 30 days between immunizations is too short due to delayed T cell contraction upon repeated immunizations, longer intervals between immunizations, such as 45, 60, or 90 days can be employed.
  • CD4+ T cells are not necessary for CD8+ T cel l-dependent protection provided by DENV2E immunization (Figure 9). Based on these observations and without being limited to or bound by any particular theory, it appears that CD4+ T cel ls may not be required for recall immunity mediated by DENV2E-elicited CD8+ T cells.
  • the level of CD8+ T cell response should correlate with protection against DENV.
  • C57BL/6 (H-2 b ) mice were obtained from The Jackson Laboratory and subsequently bred.
  • IFN-a/pR ⁇ ' ⁇ mice on the C57BL/6 background were obtained from Dr. Wayne Yokoyama (Washington U niversity, St. Louis, MO) via Dr. Carl Ware.
  • HLA-A*0201/ b, A* 1 101/ b, A*0101 , B*0702 and DRB 1 *0101 transgenic mice were bred at LIAI as previously described (Kotturi et al., Immunome Res 6:4 (2010); Pasquetto et al., J Immunol 175:5504 (2005); Alexander et al., J
  • LIAI.B6.SJL mice were purchased from Taconic. Mice were used between 5 and 10 weeks of age.
  • mice were infected intravenously (i.v.) in the lateral tail vein or retro-orbital ly (r.o.) with 200 ⁇ of the DENV2 strain, S221 , in 5% FBS/PBS. Blood was obtained from anesthetized mice by r.o. puncture.
  • mice were infected i.v.r.o. with 10 10 genomic equi valents (GE) of S221 in l OOuL PBS.
  • GE genomic equi valents
  • mice were sacrificed and splenic CD8+ or CD4+ T cells, respectively, were used in mouse lFNy ELISPOT assays. All mouse studies were approved by the Animal Care Committee.
  • hybridoma clones SFR3, GK 1.5, and 2.43 which produce rat anti-human HLA- DR5, anti-mouse CD4, and anti-mouse CD8 IgG2b Ab, respectively, were from the American Type Culture Collection, and were grown in Protein-Free Hybridoma Medium supplemented with penicillin, streptomycin, HEPES, GlutaMAX, and 2-ME (all from Invitrogen) at 37°C, 5% C0 2 . C6/36, an A.
  • albopictus mosquito cell line was cultured in Leibovitz's L- 15 Medium (Invitrogen) supplemented with 10% FBS (Gemini Bio-Products), penicillin, streptomycin, and HEPES at 28°C in the absence of C0 2 .
  • S221 a plaque-purified DENV2 strain, was derived from the clinical isolate, PL046 (Lin et al., J Virol 72:9729 ( 1998)), as described previously (Yauch et al., J Immunol 182:4865 (2009)). Viral stocks were amplified in C6/36 cells and purified over a sucrose gradient as previously described (Prestwood et al., J Virol 82:841 1 (2008)). Infectious doses were determined based on GE, which were quantified by real-time RT-PCR. There are approximately 5 x 10 4 GE/PFU for S221 , based on plaque assay on baby hamster kidney cells.
  • Candidate epitopes were identified using a consensus approach (Wang et al ., PLoS Comput Biol 4:el 000048 (2008)). Briefly, all 15-mer peptides that are encoded in the DENV2 PL046 polyprotein were predicted for binding to H-2 I-A b . Two independent algorithms (Zhang et al., Nucleic Acids Res 36:W513 (2008)) were used to rank the peptides by predicted binding affinity. The median of the two ranks was used to select the top 73 out of 3383 peptides, corresponding to the top 2% of all peptides.
  • binding predictions For human M HC class I binding predictions all 9 and l Omer peptides were predicted for their binding affinity to their respective alleles. Binding predictions were performed using the command-line version of the consensus prediction tool available on the 1EDB web site (Zhang et al ., Nucleic Acids Res 36:W5 1 3 (2008)). Peptides were selected if they are in the top 1 % of binders in a given strain. For human MHC class II binding predictions all 1 5mer peptides were predicted for their binding affinity to the DRB 1 *0101 allele. As with class I, binding predictions were performed using the command-l ine version of the consensus prediction tool available on the IEDB web site. The top 2% of predicted binders were then selected for synthesis.
  • Peptides utilized in initial screening studies were synthesized as crude material by A and A Labs. A total of 73 1 5-mer peptides were ordered and synthesized twice in different (alphabetical vs. predicted IC 5 r>) order. Positive peptides were re-synthesized by A and A Labs and purified to >90% homogeneity by reverse-phase HPLC. Purity of these peptides was determined using mass spectrometry. The HPLC-purified peptides were used for all subsequent studies.
  • MHC class I All peptides using human MHC class I or 11 sequences were synthesized by Mimotopes (Victoria, Australia). MHC class I predictions led to the synthesis of a total of 431 9-mer and 10- mer peptides. Peptides were made as crude material and combined into pools of 10 individual peptides, according to their predicted HLA restriction. MHC class II predictions resulted in the synthesis of 12 15-mers, which were tested individual ly.
  • splenocytes were stained with anti-B220- Alexa Fluor 647 (Biolegend), anti-CD4-PerCP (BD Biosciences), GL7-FITC (BD Biosciences), anti-IgD-eFluor 450 (eBioscience), and anti-Fas-PE (BD Biosciences).
  • ICS intracellular cytokine staining
  • 2 x 10 6 splenocytes were plated in 96-well U-bottom plates and stimulated with individual DENV2 peptides (3 ⁇ ) for 2 h (hours).
  • Brefeldin A (GolgiPlug, BD Biosciences) was then added and cells were incubated for another 5 h (hours). Cells were washed, incubated with supernatant from 2.4G2-producing hybridoma cells, and labeled with anti-CD4- eFluor 450 (eBioscience) and anti-CD8a-PerCP-eFluor 710 (eBioscience) or PE-Cy7 (BD B iosciences).
  • the cells were then fixed and permeabilized using the BD Cytofix/Cytoperm Kit, and stai ned with various combinations of anti-IFN-y-APC (eBioscience), anti-TNF-PE-Cy7 (BD Biosciences), anti-IL-2-Alexa Fluor 488 (BD Biosciences) or -PE (Biolegend), and anti-CD40L-PE (eBioscience). Foxp3 staining was done using the mouse regulatory T cell staining kit from eBioscience.
  • CD4 + T cell epitope identification The criteria for positivity in CD4 + T cell epitope identification were: 2x the percentage of IFN- ⁇ produced by stimulated cells compared with unstimulated cells, positive in two independent crude peptide orders, and positive when ordered as HPLC-purified (>90% pure).
  • splenocytes (2 x 10 6 ) were stimulated in 96-well U-bottom plates for 5 h (hours) in the presence of 1 ig/m ⁇ H-2 b -restricted epitopes identified previously: M 6 o-67, NS2A g _ i 5 , and NS4B 9 9_ i o7 (Yauch et al ., J Immunol 1 82:4865 (2009)).
  • Anti-CD 107a-FITC (BD Biosciences) was added to the wells during the stimulation. Cells were then stained as described for CD4 + T cell ICS. Samples were read on an LSR II (BD Biosciences) and analyzed using FloJo software (Tree Star).
  • Tissues were embedded in O.C.T. compound (Sakura). Sections (6 ⁇ ) were cut and stored at -80° C. Frozen sections were thawed and fixed for 10 minutes in acetone at 25° C, followed by 8 minutes in 1 % paraformaldehyde (EMS) in 100 mM dibasic sodium phosphate containing 60 niM lysine and 7 mM sodium periodate pH 7.4 at 4° C. Sections were blocked first using the Avidin/Biotin Blocking Kit (Vector Labs) followed by 5% normal goat serum (Invitrogen) and 1 % BSA (Sigma) in PBS. Sections were stained overnight with anti-F4/80-biotin (clone BM8,
  • Hybridoma supernatants were clarified by centrifugation, dialyzed against PBS, sterile- filtered, and quantified by BCA Protein Assay Reagent (Thermo Scientific).
  • IFN- / R "/" mice were injected i.p. with 250 ⁇ g of SFR3, or GK1.5, or 2.43 in PBS (250 ⁇ total volume) 3 days and 1 day before or 1 day before and 1 day after infection, which resulted in depletion of >90% of CD8 + cells and >97% of CD4 + cells.
  • PBS 250 ⁇ total volume
  • Serum was harvested from control and CD4-depleted IFN-a/pR " ' " mice 7 days after infection with 10 10 GE of DENV2, or naive mice.
  • EIA/RIA 96-well plates (Costar) were coated with DENV2 (10 9 GE per well) in 50 ⁇ 0. 1 M NaHCO 3 . The virus was UV-inactivated and plates left overnight at 4°C. The plates were then washed to remove unbound virus using 0.05% (v/v) Tween 20 (Sigma) in PBS.
  • Blocker Casein Blocking Buffer (Thermo Scientific) for 1 h at room temperature, 1 :3 serial dilutions of serum in a total volume of 100 ⁇ were added to the wells. After 1 .5 h, wells were washed and bound antibody was detected using HRP-conjugated goat anti-mouse IgG Fc portion or HRP-conjugated donkey anti-mouse ⁇ chain (Jackson
  • Serum was heat-inactivated at 56°C for 30 min. Three-fold serial dilutions of serum were then incubated with 5x l 0 8 GE of DENV2 for 1 h at room temperature in a total vol ume of 100 ⁇ PBS. Next, approximately 6 x 10 5 C6/36 cells per well of a 24-well plate were infected with 100 ⁇ of the virus-antibody mix for one hour at 28°C. Cells were washed twice with 500 ⁇ of PBS, and then incubated at 28°C in 500 ⁇ L-1 5 Medium containing 5% FBS, penicillin, and streptomycin for 24 h.
  • the percentage of infected cells was determined by flow cytometry as previously described (Lambeth et al., J Clin Microbiol 43:3267 (2005)) using 2H2-biotin (IgG2a anti-prM/M, DENV l -4 reactive) and streptavidin-APC (Biolegend). The percentage of infected cells was normalized to 100% (infection without serum).
  • mice were infected with 10 10 GE of DENV2. Some mice were depleted of CD4 ' T cells before infection. Splenocytes (targets) were harvested from donor B6.SJL congenic mice (CD45. 1 ) 7 days later. RBC were lysed, and the target cells were pulsed with varying concentrations of a pool of 4 H-2 b -restricted DENV2 peptides (M6o-67, NS2A 8 -i5, S4B 9 _i 0 7, NS5 237 - 245) or DMSO for 1 h at 37°C.
  • M6o-67, NS2A 8 -i5, S4B 9 _i 0 7, NS5 237 - 245) or DMSO for 1 h at 37°C.
  • CFSE Invitrogen
  • PBS/0.1 % BSA PBS/0.1 % BSA for 10 min at 37°C.
  • Cells were labeled with 1 ⁇ CFSE (CFSE high )or 100 nM CFSE (CFSE low ) or left unlabeled.
  • the cell populations were mixed and 5 x 10 6 cells from each population were injected i.v.into naive or infected recipient mice. After 4 h, the mice were sacrificed and splenocytes stained with anti-CD45.1 -APC (eBioscience) and analyzed by flow cytometry, gating on CD45.1 + cells.
  • the percentage killing was calculated as follows: 100 - ((percentage DENV peptide-pulsed in infected mice/percentage DMSO-pulsed in infected mice) / (percentage DENV peptide-pulsed in na ' ive mice/percentage DMSO-pulsed in naive mice) x 100).
  • IFN-a/pR " ' " mice were infected with 10 10 GE of DENV2. Some mice were depleted of CD4 + or CD8 + cells before infection. Splenocytes (targets) were harvested from donor B6.SJL congenic mice (CD45.1 ) 7 days later. RBC were lysed and the target cells were pulsed with 1 .7 ⁇ g (approximately 1 ⁇ ) each of NS2B
  • the cells were then washed and labeled with CFSE in PBS/0.1% BSA for 10 min at 37°C.
  • DENV2 peptide-pulsed cells were labeled with 1 ⁇ CFSE (CFSE high ) and DMSO-pulsed cells with 100 nM CFSE (CFSE low ).
  • the two cell populations were mixed and 5 x 10 6 cells from each population were injected i. v. into na ' ive or infected recipient mice. After 16 h, the mice were sacrificed and splenocytes stained and the percentage killing calculated as described for the CDS in vivo cytotoxicity assay.
  • the DENV2 standard curve was generated with serial dilutions of a known concentration of DENV2 genomic RNA which was in vitro transcribed (MAXIscriptKit, Ambion) from a plasmid containing the cDNA template of S221 3' UTR. After transcription, DNA was digested with DNase I, and RNA was purified using the RNeasy Mini Kit and quantified by spectrophotometry. To control for RNA quality and quantity when measuring DENV in tissues, the level of 18S rRNA was measured using 18S primers described previously (Lacher, et al., Cancer Res 66: 1648 (2006)) in parallel real-time RT-PCR reactions. A relative 18S standard curve was made from total splenic RNA.
  • mice were immunized s.c.with 50 ⁇ g each of NS2Biog-i 22 , NS3 i 98 . 2 i 2 , and NS3 2 37-25 i emulsified in CFA (Difco). After 1 1 days, mice were boosted with 50 ⁇ g peptide emulsified in IFA (Difco). Mock-immunized mice received PBS/DMSO emulsified in CFA or IFA. Mice were infected 13 days after the boost with 10 " GE of DENV2 (some mice were depleted of CD4 + or CD8 + T cells 3 days and 1 day before infection).
  • mice Four days later, the mice were sacrificed and tissues harvested, RNA isolated, and DENV2 RNA levels measured as described above.
  • mice were immunized instead with 50 ⁇ g each of C51.59, NS2A 8 - i5, NS4B 99 .107, and NS5 237 -2 5 as described in Yauch et al., J Immunol 1 82:4865 (2009).
  • binding of the radiolabeled peptide to the corresponding MHC molecule was determined by capturing MHC/peptide complexes on Greiner Lumitrac 600 microplates (Greiner Bio-One, Monroe, NC) coated with either the W6/32 (HLA class 1 specific) or L243 (HLA DR specific) monoclonal antibodies. Bound cpmwere then measured using the Topcountmicroscintillation counter (Packard Instrument, Meriden, CT). The concentration of peptide yielding 50% inhibition of the binding of the radiolabeled probe peptide (IC 5 o) was then calculated.
  • the tumor cell line 721.221 (Shimizu et al, J Immunol 142:3320 ( 1989), which lacks expression of HLA -A, -B and C class I genes, was transfected with the HLA-A*0201 /Kb or HL- A* 1 101 chi meric genes, and was used as APC in the restriction assays.
  • the non-transfected cell line was used as a negative control.
  • Peripheral blood samples were obtained from healthy adult blood donors from the National Blood Center in Colombo, Sri Lanka.
  • PBMC peripheral blood samples were purified by density gradient centrifugation (Ficoll-Hypaque, Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions.
  • Cell were suspended in fetal bovine serum (Gemini Bio-products, Sacramento, CA) containing 10% dimethyl sulfoxide, and cryo-preserved in liquid nitrogen.
  • DE V seropositivity was determined by ELISA.
  • a flow cytometry-based neutralization assays was performed for further characterization of seropositve donors, as previously described (Kraus et al, J Clin Microbiol 45 :3777 (2007)).
  • Genomic DNA isolated from PBMC of the study subjects by standard techniques was use for HLA typing.
  • High resolution Luminex-based typing for HLA Class I and Class II was utilized according the manufacturer's protocol (Sequence-Specific Oligonucleotides (SSO) typing; One Lambda, Canoga Park, CA). Where needed, PCR based methods were used to provide high resolution sub-typing. (Sequence-Specific Primer (SSP) typing; One Lambda, Canoga Park, CA).
  • splenic CD4 + or CD8 + T cells were isolated by magnetic bead positive selection (MiltenyiBiotec, BergischGladbach, Germany) 7 days after infection.
  • 2 x l O 5 T cel ls were stimulated with 1 ⁇ 10 5 uninfected splenocytes as APCs and 10 ⁇ / ⁇ 1 of individual DENV peptides in 96-well flat-bottom plates (Immobilon-P; Millipore, Bedford, MA) coated with anti-IFNy mAb (clone AN 18; Mabtech, Sweden). Each peptide was evaluated in triplicate.
  • This example includes data demonstrating CD4 + T cell activation and expansion following DENV2 infection.
  • the cells were activated, as measured by CD44 upregulation and CD62L downregulation on splenic CD4 + T cells (Figure 14B) and on circulating blood CD4 + T cells, with the peak on day 7 after infection ( Figure 14C).
  • CD4 + T cell response in the spleen in more detail, immunohistochemistry on spleen sections obtained from na ' ive mice and mice 3, 5, and 7 days after DENV2 infection was performed. Sections were stained for CD4, CD8, B220 to highlight B cell follicles, and F4/80 to show red pulp macrophages.
  • CD4 + and CD8 + T cells were dispersed throughout the spleen, but preferentially in T cell areas, also known as the periarteriolar lymphoid sheath (PALS).
  • PALS periarteriolar lymphoid sheath
  • CD8 + T cells differed from the CD4 + T cells mainly in that at day 5 after infection, many of the CD8 + T cells had left the T cell area and were found distributed throughout the red pulp and marginal zone (MZ). By day 7, the CD8 + T cells were observed in the PALS, MZ, and also the red pulp.
  • Tregs Regulatory T cells
  • Regulatory T cells are a subset of CD4 + T cells that are characterized by the expression of the transcription factor, Foxp3 (Josefowicz, et al. Immunity 30:616 (2009)), and have been found to facilitate the early host response to HSV-2 (Lund, et al. Science 320: 1220 (2008)) and help control WNV infection (Lanteri, et si. J Clin Invest 1 19:3266 (2009)).
  • the number of CD4 + Foxp3 + cells in the spleen 7 days after infection was determined. There was a decrease in the percentage of Tregs among total CD4 + cells, and no change in the number of Tregs, demonstrating that DENV2 infection does not lead to an expansion of Tregs in the spleen ( Figure 14D).
  • This example includes data for the identification of DENV2 CD4 + T cell epitopes and phenotype of DENV2-specific CD4 + T cells.
  • NS3 2 oo-2 i4 has been identified as a human HLA-DR15-restricted CD4 + T cell epitope (Simmons, et al. J Virol 79:5665 (2005); Zeng, et al. J Virol 70:3108 (1996)). It was also of interest that NS4B 96 .
  • U0 contains a CD8 + T cell epitope ( S4B 99 .107) that was identified as the immunodominant epitope in both wild-type and IFN-a/pR " C57BL/6 mice infected with DENV2 (Yauch, et al. J Immunol 182:4865 (2009)).
  • Multicolor flow cytometry was performed to study the phenotype of DENV2-specific CD4 + T cells. These cells produced lFN- ⁇ , TNF, and 1L-2 ( Figure 16). No intracellular IL-4, IL-5, IL- 17, or IL- 10 were detected. The DENV2-specific CD4 + T cells also expressed CD40L, suggesting they are capable of activating CD40-expressing cells, which include DCs and B cells. The four DENV2-derived CD4 + T cell epitopes induced responses that differed in magnitude, but were similar in terms of phenotype. The most polyfunctional cells (those expressing IFN- ⁇ , TNF, 1L-2, and CD40L) also expressed the highest levels of the cytokines and CD40L. These results demonstrate that DENV2 infection elicits a virus-specific Thl CD4 + T cell response in IFN-a/pR 7" mice.
  • This example includes a description of studies of the effects of CD4 + and/or CD8 + T cell depletions on DENV2 viral RNA levels, and data showing that CD4 + T cells are not required for the anti- DENV2 antibody response, and are also not necessary for the primary DE V2-specific CD8 + T cell response.
  • CD4 + T cells CD4 + T cells, CD8 + T cells, or both were depleted from IFN-a/pR " ' " mice and DENV2 RNA levels 5 days after infection with 10'° GE of DENV2 was measured. No difference in viral RNA levels between control undepleted mice and CD4-depleted mice in the serum, kidney, small intestine, spleen, or brain was observed ( Figure 17). CD8-depleted mice had significantly higher viral loads than control mice. Depletion of both CD4 + and CD8 + T cells resulted in viral RNA levels that were significantly higher than those in control mice in all tissues examined, and equivalent to the viral RNA levels in CD8-depleted mice. These data show that CD4 + T cells are not required to control primary DENV2 infection in IFN-a/pR "7" mice, and confirm an important role for CD8 + T cells in viral clearance.
  • CD4 + T cells were not required for controlling DENV2 infection, the contribution to the anti-DENV immune response, for example by helping the B cell and/or CD8 + T cell responses, was investigated.
  • CSR the process by which the immunoglobulin heavy chain constant region is switched so the B cell expresses a new isotype of Ab, can be induced when CD40L-expressing CD4 + T cells engage CD40 on B cells (Stavnezer, et al. Annu Rev Immunol 26:261 (2008)).
  • CSR can also occur in the absence of CD4 + T cell help.
  • DENV2-specific IgM and IgG titers in the sera of control and CD4-depleted mice was measured 7 days after infection with 10 10 GE of DENV2. As expected, there was no difference in IgM titers at day 7 between control and CD4- depleted mice ( Figure 18A). There was also no difference in IgG titers between control and CD4- depleted mice.
  • a flow cytometry- based neutralization assay was performed, in which C6/36 mosquito cells were infected with DE V2 in the presence of heat-inactivated sera obtained from control and CD4-depleted mice 7 days after infection.
  • CD4 + T cells were assessed by examining the DENV2-specific CD8 + T cell response in control and CD4-depleted DENV2-infected mice.
  • the numbers of splenic CD8 + T cells were equivalent in control and CD4-depleted mice.
  • IFN- ⁇ ICS was performed using DENV2-derived H-2 b -restricted immunodominant peptides identified (M 60 -67, NS2Ag_i 5 , and NS4B 99 -io7) (Yauch, et al. J Immunol 182:4865 (2009)).
  • This example is a description of studies of in vivo killing of I-A b -restricted peptide-pulsed target cells in DENV2-infected mice, and data showing that vaccination with DENV2 CD4 + T cell epitopes controls viral load.
  • CD4 + T cells could still be contributing to the anti-DENV2 host response by killing infected cells.
  • In vivo cytotoxicity assay was performed using splenocytes pulsed with the three peptides that contain only CD4 + T cell epitopes (NS2B 1 0 8-i22, NS3 i 98 -2i 2 , and NS3 2 37- 25 ) ) and not NS4B 96 _uo to measure only CD4 + , not CD8 + T cell-mediated killing. Approximately 30% killing of target cells was observed (Figure 20).
  • CD4 + T cell peptide immunization requires both CD4 + and CD8 + T cells.
  • CD4 + T cells elicited by immunization protect by helping the CD8 + T cell response.
  • CD4 + T cells are not required for the primary CD8 + T cell or antibody response, and the absence of CD4 + T cells had no effect on viral RNA levels, vaccination with CD4 + T cell epitopes can reduce viral loads.
  • This example includes a discussion of the data and a summary of the implications.
  • CD8 + T cells play an important protective role in the response to primary DENV2 infection, whereas CD4 + T cells do not.
  • CD4 + T cells expanded, were activated, and were located near CD8 + T cells and B cells in the spleen after DENV2 infection, yet they did not seem to affect the induction of the DENV2-specific CD8 + T cell or antibody responses. In fact, CD4 + T cell depletion had no effect on viral clearance.
  • the data demonstrate that vaccination with CD4 + T cell epitopes prior to DENV infection can provide significant protection, demonstrating that T cell peptide vaccination is a strategy for DENV immunization without the risk of ADE.
  • CD4 + T cell epitopes recognized epitopes from the NS2B, NS3, and NS4B proteins, and displayed a Th l phenotype.
  • CD4 + T cell epitopes have been identified in mice infected with other flaviviruses, including YFV, for which an l-A b -restricted peptide from the E protein was identified (van der Most, et al. Virology 296: 1 17 (2002)), and WNV, for which six epitopes from the E and NS3 proteins were identified (Brien, et al. J Immunol 1 81 :8568 (2008)).
  • DENV-derived epitopes recognized by human CD4 + T cells have been identified, primarily from NS proteins, including the highly conserved NS3 (Mathew, et al. Immunol Rev 225:300 (2008)).
  • one of the NS3-derived epitopes identified herein is also a human CD4 + T cell epitope, which may bind human HLAs promiscuously, making it a good vaccine candidate.
  • Another finding was that one of the CD4 + T cell epitopes identified in this study contained the most immunodominant of the CD8 + T cell epitopes identified previously. Overlapping epitopes have also been found in LCMV (Homann, et al. Virology 363 : 1 1 3 (2007); Mothe, et al. J Immunol 179: 1058- 1067 (2007); Dow, et al. J Virol 82: 1 1734 (2008)).
  • overlapping epitopes The significance of overlapping epitopes is unknown, but is likely related to homology between MHC class I and MHC class II, and may be associated with proteasomal processing. Overlapping epitopes may turn out to be common once the complete CD4 + and CD8 + T cell responses to other pathogens are mapped.
  • CD4 + T cells are classically defined as helper cells, as they help B cell and CD8 + T cell responses.
  • inflammatory stimuli can override the need for CD4 + T cell help, and therefore, the responses to many acute infections are CD4-independent (Bevan, Nat Rev Immunol 4:595 (2004)).
  • DENV2 replicates to high levels in IFN-a/pR " ' " mice, the mice appear hunched and ruffled at the time of peak viremia, and they have intestinal inflammation, suggesting that there is a significant inflammatory response to DENV2.
  • CD4 + T cells did not play a critical role in the immune response to primary DENV2 infection. The contribution of CD4 + T cells has been examined during infections with other flaviviruses.
  • Antibody responses can be T cell-dependent or T cell-independent.
  • the formation of GCs is thought to be CD4 + T cell-dependent, and is where high-affinity plasma cells and memory B cells are generated and where CSR can occur (Stavnezer, et al. Annu Rev Immunol 26:261 (2008); Allen, et al. Immunity 27: 190 (2007); Fagarasan et al. Science 290:89 (2000)).
  • T- independent antibody responses to viruses have been demonstrated for vesicular stomatitis virus (Freer, et al. J Virol 68:3650 ( 1994)), rotavirus (Franco, et al.
  • EBV via LM P1
  • CD40-independent CSR He, et al. J Immunol 171 :521 5 (2003)
  • mice deficient for CD40 or CD4 + T cells are able to mount an influenza-specific IgG response that is protective (Lee, et al. J Immunol 175 :5827 (2005)).
  • CD4-independent CD8 + T cell responses have been demonstrated for Listeria monocytogenes (Sun, et al. Science 300:339 (2003); Shedlock, et al. J Immunol 170:2053 (2003)), LCMV (Ahmed, et al. J Virol 62:2102 ( 1988)), and influenza (Belz, et al. J Virol 76: 12388 (2002)). Recently a mechanism for how DCs can activate CD8 + T cells in the absence of CD4 + T cell help has been described (Johnson, et al. Immunity 30:218 (2009)).
  • the primary CD8 + T cell response to DENV2 did not depend on CD4 + T cells.
  • an enhanced DENV2-specific CD8 + T cell response in CD4-deficient mice compared with control mice at day 7 was observed, which has also been reported for influenza- (Belz, et al. J Virol 76: 12388 (2002)) and WNV- (Sitati, et al. J Virol. 80: 12060 (2006)) specific CD8 + T cell responses. This could be due to the depletion of Tregs, or an increased availability of cytokines (e.g. IL-2) in mice lacking CD4 + T cells.
  • This enhanced CD8 + T cell response may explain why CD4-depleted mice have no differences in viral titers despite the fact that DENV2-specific CD4 + T cells demonstrate in vivo cytotoxicity.
  • CD4 + T cells did not play an important role in helping antibody or CD8 + T cell responses, DENV2-specific CD4 + T cells could kill peptide-pulsed target cells in vivo.
  • CD4 + T cells specific for other pathogens including HIV (Norris, et al. / Virol 78:8844 (2004)) and influenza (Taylor, et al. Immunol Lett 46:67 (1995)) demonstrate in vitro cytotoxicity. In vivo cytotoxicity assays have been used to show CD4 + T cell-mediated killing following infection with LCMV (Jellison, et al. J Immunol 174:614 (2005)) and WNV (Brien, et al.
  • Type I IFNs are known to help T cell and B cell responses through their actions on DCs, and can act directly on T cells (Iwasaki, et al. Nat Immunol 5 :987 (2004)).
  • Type I IFNs were found to contribute to the expansion of CD4 + T cells following infection with LCM V, but not Listeria monocytogenes (Havenar-Daughton, et al. J Immunol 176:33 1 5 (2006)).
  • Type 1 IFNs can induce the development of Th l IFN- ⁇ responses in human CD4 + T cells, but cannot substitute for 1L-12 in promoting Th l responses in mouse CD4 + T cells (Rogge, et al.
  • An alternative approach would be a peptide vaccine that induces cell-mediated immunity, including both CD4 + and CD8 + T cell responses, which, although not able to prevent infection, would reduce viral loads and disease severity, and would eliminate the risk of ADE.
  • a vaccine should target highly conserved regions of the proteome, for example NS3, NS4B, and/or NS5, and ideally include epitopes conserved across all four serotypes.
  • a vaccine containing only peptides from these particular NS proteins would also preclude the induction of any antibody against epitopes on the virion, which could enhance infection, or antibody against NS 1 , which could potentially contribute to pathogenesis (Lin, et al. Viral Immunol 19: 127 (2006)).
  • Peptide vaccination was given along with CFA, which is commonly used in mice to induce Thl responses (Billiau, et al. J Leukoc Biol 70:849
  • CFA is not a vaccine adjuvant approved for human use, and thus, any peptide vaccine developed against DENV wi ll be formulated with an adjuvant that is approved for human use.
  • CD4 + T cells do not make a significant contribution to controlling primary DENV2 infection
  • the characterization of the primary CD4 + T cell response and epitope identification allows the determination of the role of CD4 + T cells during secondary homologous and heterologous infections.
  • CD4 + T cells are often dispensable for the primary CD8 + T cell response to infection, but have been shown to be required for the maintenance of memory CD8 T cell responses after acute infection (Sun, et al. Nat Immunol 5 :927 (2004)).
  • the data herein support a DENV vaccine strategy that induces CD4 + T cell, in addition to CD8 + T cell, responses.
  • This example includes a description of additional studies showing that vaccination with DENV CD8 + T cell epitopes controls viral load.
  • mice were immunized with four dominant DENV epitopes (C51.59, NS2A 8 -i 5, NS4B 9 9- io7, and NS5 23 7-24s) (Yauch et al, J Immunol 182:4865 (2009)) in an attempt to induce a multispecific T cell response, which is desirable to prevent possible viral escape through mutation (Welsh et al., Nat Rev Microbiol 5 :555 (2007)).
  • viremia in the serum was measured by real-time RT-PCR, as described above.
  • the peptide-immunization resulted in enhanced control of DENV infection, with 350-fold lower serum DENV RNA levels in peptide-immunized mice than mock-immunized mice (Yauch et al ., J Immunol 182:4865 (2009)).
  • CD8 + T cells were depleted from a group of peptide- immunized mice prior to infection, and it was found that this abrogated the protective effect (Yauch et al., J Immunol 182:4865 (2009)).
  • the data demonstrate that a preexisting DENV-specific CD8 + T cell response induced by peptide vaccination enhances viral clearance.
  • Results from the Examples described herein reveal a critical role for CD8 + T cells in the immune response to an important human pathogen, and provide a rationale for the inclusion of CD8 + T cell epitopes in DENV vaccines. Furthermore, identification of the CD8 + T cell epitopes recognized during DENV infection in combination with the disclosed mouse model can provide the foundation for elucidating the protective versus pathogenic role of CD8 + T cells during secondary infections.
  • This example is a description of a novel system to identify DENV specific HLA*0201 epitopes.
  • Mouse-passaged DENV is able to replicate to significant levels in IFN-a/pR 7" mice.
  • HLA*0201 transgenic and IFN-a/pR ⁇ mice strains were backcrossed to study DENV-specific HLA restricted T cell responses. These mice were then infected with mouse adapted DENV2 strain S221 , and purified splenic T cells were used to study the anti-DENV CD8 + T cell responses.
  • a panel of 1 16 predicted A*0201 binding peptides were generated using bioinformatics (Moutaftsi, et al. Nat Biotechnol 24:817 (2006)). Predicted HLA A*0201 binding peptides were combined into pools of 10 individual peptides and tested in an IFNy ELISPOT assay using CD8 + T cells from HLA transgenic IFN-a/pR " ' " and IFN-a/pR +/+ , S221 infected mice, respectively. Positive pools were deconvoluted and the individual peptides were tested in two independent studies.
  • This example describes population coverage by additional HLA transgenic mice IFN-a/pR-/- strains.
  • IFN-a/pR mice were backcrossed with HLA A*0101 , A* 1 101 , and B*0702 transgenic mice. These alleles were chosen as representatives of three additional HLA class I supertypes (A l , A3 and B7, respectively).
  • the responses observed in the HLA B*0702 transgenic IFN-a/pR "A mice were not only broader but also more than ten-fold higher in magnitude.
  • the one epitope recognized in the IFN-a/pR +/+ strain elicited an IFNy response of 50 SFC/10 6 CD8 + T cells compared to an average of 857 SFC/10 6 CD8 + T cells in the IFN-a/pR "A mice.
  • This example describes Dengue virus specific T cell responses in an MHC class II transgenic mouse model.
  • HLA DRB 1 *0101 DENV predicted binding peptides in HLA DRB 1 *0101 , IFN-a/pR " ' " and IFN-a/pR +/+ mice, respectively, was determined.
  • HLA DRB 1 *0101 , IFN-a/pR "A and IFN-a/pR +/+ mice were infected with DENV2 (S221), and CD4 + T cells were isolated 7 days post infection.
  • This example is a description of mapping optimal epitopes with respect to peptide length, and further characterization of the identified epitopes. J ,
  • This example includes a description of validation studies of the identified epitopes in human DENV seropositive donors.
  • Figures 26A-26D show the capacity of the identified epitopes to stimulate PBMC from the various donor categories.
  • A*0101 and A*0201 epitopes was detected at least once in an FILA matched donor, although the magnitude as well as the frequency of responses was higher for the A*0201 restricted epitopes ( Figures 26A-26B and Table 2).
  • the B *0702 restricted epitopes 10 out of the 12 have been detected in one or more HLA matched donors as shown in Figure 26D and Table 1 .
  • an IEDB query was performed with the epitopes identified in the mouse model.
  • 13 of the 42 epitopes previously described to elicit an IFNy in DENV seropositive individuals were identified, as indicated in Table 2.
  • the 30% overlap with known epitopes contributes to the validation of our mouse model and shows on the other hand that 70% of the epitopes identified are novel, contributing to an extended knowledge of T cell mediated responses to DENV.
  • This example includes studies showing dominance of B7 responses.
  • mice B*0702 restricted epitopes were able to el icit the strongest IFNy responses, reaching an average of 688 SFC/ 10 6 CDS ' T cells, followed by an average of 530, 423 and 1 1 9 SFC/10 6 CD8 T cells for HLA* 1 1 01 , A*0202 and A*0101 restricted epitopes, respectively.
  • the fact that the mouse model described herein reflects response patterns observed in humans makes it an especially suitable model to identify and study epitopes of human relevance to DENV infection.
  • This example includes a description of studies showing the subprotein location of identified epitopes, and the conservancy of identified epitopes within the DENV2 serotype.
  • the identified epitopes are derived from 9 of the 10 DENV proteins, with the membrane protein being the only protein where no epitope could be detected (Figure 27).
  • the majority of epitopes are derived from the seven nonstructural proteins. 39 out of 42 of the identified epitopes (93%) originate from the nonstructural proteins, accounting for 97% of the total IFNy response observed.
  • NS3 and NS5 alone account for 67% of the total response, representing a total number of 23 epitopes detected from these two proteins.
  • NS5 is furthermore the only subprotein where at least one derived epitope has been identified in all five HLA transgenic mouse strains.
  • the DENV2 epitopes identified in Table 2 were analyzed for their respective homologues in DEN V 1 , DENV3 and DENV4. 162 DENV1 , 171 DENV2, 169 DENV3 and 53 DENV4 sequences from the NCBI Protein database were analyzed for conservancy. Table 3 shows the sequences of the epitopes identified after infection with DENV2 (bold letters). "Counts" indicate the number of strains in which the epitope is conserved within the respective serotype. Listed for each epitope are variants of the epitope in the DENV1 , 3 and 4 serotypes and their respective counts. Epitopes are sorted according to their appearance in Table 2. These sequences help determine the cross-reactivity patterns of the identified epitopes.
  • LGDGLAIGI DENV1 1 ILPSIVREA DENV4 53 LGDGFAMGI DENV1 1 1
  • LGDGLAMGI DENV1 160 NS32013-2022 DLMRRGDLPV DENV2 157 LTDAIALGI DENV2 13 DLLRRGDLPV DENV1 1 LTDAWALGM DENV2 1
  • ELMRRGDLPV DENV2 3 MANGVALGL DENV3 2 DLMRRGDLPV DENV2 157 MANGIALGL DENV3 167
  • ELMRRGHLPV DENV3 2 LISGISLGL DENV4 1
  • AIDLDPVVY DENV1 156 ALSELAETL DENV2 1
  • IMAVGWSI DENV1 2 TQVGVGIQK DENV3 3 VMAVGIVSI DENV1 1 TQVGVGVHK DENV3 2 1MA1G1VS1 DENV1 64 TQVGVGVQK DENV3 164 IMAVGIVSI DENV1 95 TQVGVGIHI DENV4 4
  • VMAVGMVSI DENV2 14 TQVGVGIHT DENV4 1 IMAVGMVSI DENV2 157 TQVGVGIHM DENV4 47
  • GMLITCYVI DENV1 1 GTSGSPHDK DENV2 49 GMLIACYVI DENV1 161 GTSGSPIADK DENV2 1 GPLTVCYVL DENV2 1 GTSGSPIVDR DENV2 75 GLLTVCYVL DENV2 170 GTSGSPIVDK DENV2 46 GMLIACYVI DENV3 2 GTSGSPIINK DENV3 1 GLLIACYVI DENV3 167 GTSGSPII NR DENV3 168 GLLLAAYMM DENV4 1 GSSGSPI INR DENV4 1 GLLLAAYVM DENV4 52 GTSGSPIVNR DENV4 1
  • RVYADPMALQ DENV4 1 KTFNTEYQK DENV3 1
  • VPNYNMIIV DENV1 1 GVTYLALLATFKVRP DENV2 1
  • VPNYNMIVM DENV1 1 GVTYLAL L AAFRVRP DENV2 12
  • VPNYNLIIM DENV2 171 GVTYLALLAAFKVRP DENV2 156
  • VPNYNLVIM DENV3 6 GVTYLALIATFKVQP DENV3 1
  • AIIQDEERDI DENV1 1 GQIHLAIMTMFKMSP DENV4 1
  • APIMDDEREI DENV2 1 MAIGIVSILLSSLLK DENV1 64
  • APIQDEEKDI DENV3 2 MAIGLVSILASSLLR DENV3 3
  • This example includes a discussion of the foregoing data and conclusions based upon the data.
  • Wild-type mice are resistant to DENV-induced disease, and therefore, development of mouse models for DENV infection to date has been challenging and has had to rely on infection of immunocompromised mice, non-physiologic routes of infection, and mouse-human chimeras (Yauch, et al . Antiviral Res 80:87 (2008)). Due to the importance of the 1FN system in the host antiviral response, mice lacking the lFNR- ⁇ / ⁇ support a productive infection. A mouse-passaged DENV2 strain, S221 , is highly immunogenic and also repl icates to high levels in IFNR- ⁇ / ⁇ - ⁇ - mice, thus al lowing the study of CD4 + and CD8 + T cell responses in DENV infection.
  • HLA transgenic mice are fairly accurate models of human immune responses, especially when peptide immunizations are utilized. Numerous studies to date show that these mice develop T cell responses that mirror the HLA restricted responses observed in humans in context of various pathogens (Gianfrani, et al . Hum Immunol 61 :438 (2000); Wentworth, et al. Eur J Immunol 26:97 ( 1 996); Shirai, et al . J Immunol 1 54:2733 (1995); Ressing, et al . J Immunol 154:5934 ( 1995);
  • HLA transgenic IFNRa/ ⁇ ' ' ' mice are a valuable model to identify DENV epitopes recognized in humans. Not only were a number of HLA- restricted T cell responses identified, but the genome wide screen provided further insight into the subproteins targeted by T cells during DENV infection. The majority of DENV responses (97%) were derived from the nonstructural proteins; more than half of the epitopes identified originate from the NS3 and NS5 protein. The data show the immunodominant role of the highly conserved NS3 protein (Rothman ⁇ v Virus Res 60:397 (2003); Duangchinda, et al.
  • RNA viruses Another unique challenge in vaccine development is the high degree of sequence variation in a pathogen, characteristically associated with RNA viruses. This is of particular relevance in the case of DEN V infections, where it is well documented that prior exposure to a different serotype may lead to more severe disease and immunopathology (Sangkawibha, et al. Am J Epidemiol 120:653 (1984)). The fact that there is also significant genetic variation within each serotype adds to the complexity of successful vaccinations (Twiddy, et al. Virology 298:63 (2002); Holmes, et al. Trends Microbiol 8:74 (2000)).
  • peptide variants derived from the original antigen in the primary infection can induce a response that is qualitatively different from the response induced by the original antigen (for example inducing a different pattern of lymphokine production; Partial agonism), or even actively suppressing the response (TCR antagonism).
  • variants associated with this phenotype are often collectively referred to as Altered Peptide Ligands (APLs) (Yachi, et al.
  • T cell response directed at the APL may lead to altered or aberrant patterns of lymphokine production, and TCR antagonist mediated inhibition of T cell responses ( ast, et al. J Immunol 152:3904 (1994)). Therefore, immunity to all four serotypes would provide an optimal DENV vaccine. It is generally recognized that conserved protein sequences represent important functional domains (Valdar Proteins 48:227 (2002)), thus mutations at these important protein sites could be detrimental to the survival of the virus. T cell epitopes that target highly conserved regions of a protein are therefore likely to target the majority of genetic variants of a pathogen (Khan, et al. Cell Immunol 244: 141 (2006)).
  • HLA polymorphism adds to the complexity of studying T cell responses to DENV.
  • MHC molecules are extremely polymorphic, with several hundred different variants known in humans (Klein, Natural History of the Major Histocompatibility Complex ( 1986); Hughes, et al. Nature 355:402 (1992)). Therefore, selecting multiple peptides matching different MHC binding specificities will increase coverage of the patient population for diagnostic and vaccine applications alike.
  • different MHC types are expressed at dramatically different frequencies in different ethnicities.
  • IFNR- ⁇ / ⁇ - ⁇ mice were backcrossed with mice transgenic for HLA A*0101 , A*0201, A* l 101 , B*0702 and
  • HLA supertypes are not limited to class 1 molecules.
  • Several studies have demonstrated the existence of H LA class II supertypes (Doolan, et al. J Immunol 165: 1 123 (2000); Wilson, et al. J Virol 75 :41 95 (2001 ); Southwood, et al.
  • HLA B responses have been shown in context of several other viruses, such as HIV, EBV, CMV, and Influenza ( iepiela, et al. Nature 432:769 (2004); Bihl, et al. J Immunol 176:4094 (2006); Boon, et al. J Immunol 172:4435 (2004); Lacey, et al. Hum Immunol 64:440 (2003)), suggesting that this observation is not limited to RNA viruses, and in fact, it has even been described for an intracellular bacterial pathogen, Mycobacterium Tuberculosis
  • HLA B restricted T cell responses have been described to be of higher magnitude (Bihl, et al. J Immunol 176:4094 (2006)) and to influence infectious disease course and outcome.
  • DENV one particular B07 epitope was reported to elicit higher responses in patients with DHF compared to patients suffering from DF only and could therefore be associated with disease outcome (Zivna, et al. J Immunol 168:5959 (2002)).
  • HLA B44, B62, B76 and B77 alleles suggest a role for HLA B44, B62, B76 and B77 alleles in protection against developing clinical disease after secondary DENV infection, whereas other alleles were associated with contribution to pathology (Stephens, et al. Tissue Antigens 60:309 (2002); Appanna, et al. PLoS One 5 (2010). Accordingly, HLA alleles appear to be associated with clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals. The fact that the stronger B*07 response occurs in our human samples as well as in our mouse model of DENV infection validates the relevance of this mouse model, since it even mimics patterns of immuno-dominance observed in humans.
  • This example includes a description of the identification of T cell responses against additional DENV-derived peptides in human donors.
  • Peripheral blood samples were obtained from healthy adult blood donors from the National Blood Center in Colombo, Sri Lanka. DENV-seropositivity was determined by ELISA. Those samples that are positive for DENV-specific IgM or IgG are further examined by the FACS based neutral ization assay to determine whether the donor may have been exposed to single or multiple DENV serotypes. For MHC class I binding predictions all 9- and 10-mer peptides were predicted for their binding affinity to their respective alleles. Binding predictions were performed usi ng the command-line version of the consensus prediction tool available on the 1EDB web site. Peptides were selected if they were in the top 1 % of binders.

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Abstract

La présente invention concerne des utilisations, des procédés et des compositions permettant de provoquer, de stimuler, d'induire, de favoriser, d'accroître ou d'améliorer une réponse des lymphocytes T contre le virus de la dengue chez un sujet.
PCT/US2012/044071 2010-06-24 2012-06-25 Protection contre le virus de la dengue et prévention des formes graves de la dengue WO2012178196A2 (fr)

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US20150126386A1 (en) * 2012-01-24 2015-05-07 Biomerieux Method for the in vitro prediction of the probability of a patient developing severe dengue, based on a blood sample
EP2959915A1 (fr) * 2014-06-23 2015-12-30 Institut Pasteur Polyépitope chimérique du virus de la dengue composé de fragments de protéines non structurelles et son utilisation dans une composition immunogène contre l'infection par le virus de la dengue
CN115804362A (zh) * 2023-02-08 2023-03-17 中国医学科学院医学生物学研究所 一种IFN-α/βR-/-小鼠抗体依赖性增强感染的注射液及其制备方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003102166A2 (fr) * 2002-02-26 2003-12-11 Maxygen, Inc. Nouveaux antigenes de flavivirus
CU23578A1 (es) * 2005-09-16 2010-09-30 Ct Ingenieria Genetica Biotech Proteína de la cápsida del virus dengue inductora de respuesta protectora y composición vacunal
GB0620894D0 (en) * 2006-10-20 2006-11-29 Univ Southampton Human immune therapies using a CD27 agonist alone or in combination with other immune modulators
US20110150914A1 (en) * 2008-06-09 2011-06-23 La Jolla Institute For Allergy And Immunology Compositions and methods for dengue virus (dv) treatment and vaccination
WO2011163628A2 (fr) * 2010-06-24 2011-12-29 La Jolla Institute For Allergy And Immunology Séquences polypeptidiques du virus de la dengue (dv), épitopes de lymphocytes t et leurs procédés et leurs utilisations

Non-Patent Citations (1)

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Title
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US10823732B2 (en) 2012-01-24 2020-11-03 Biomerieux Method for the in vitro prediction of the probability of a patient developing severe dengue, based on a blood sample
US9863948B2 (en) * 2012-01-24 2018-01-09 Biomerieux Method for the in vitro prediction of the probability of a patient developing severe dengue, based on a blood sample
US20150126386A1 (en) * 2012-01-24 2015-05-07 Biomerieux Method for the in vitro prediction of the probability of a patient developing severe dengue, based on a blood sample
JP2017520252A (ja) * 2014-06-23 2017-07-27 アンスティテュ・パストゥール 非構造タンパク質断片から構成されるデングウイルスのキメラポリエピトープ及びデングウイルス感染症に対する免疫原性組成物におけるその使用
US10316066B2 (en) 2014-06-23 2019-06-11 Institut Pasteur Dengue virus chimeric polyepitope composed of fragments of non-structural proteins and its use in an immunogenic composition against dengue virus infection
CN107074913A (zh) * 2014-06-23 2017-08-18 巴斯德研究院 包含非结构蛋白的片段的登革热病毒嵌合多表位及其在抗登革热病毒感染的免疫原性组合物中的用途
EP2959915A1 (fr) * 2014-06-23 2015-12-30 Institut Pasteur Polyépitope chimérique du virus de la dengue composé de fragments de protéines non structurelles et son utilisation dans une composition immunogène contre l'infection par le virus de la dengue
KR20170020513A (ko) * 2014-06-23 2017-02-22 앵스티띠 파스퇴르 비-구조 단백질의 단편으로 구성된 뎅기 바이러스 키메라 폴리에피토프 및 이의 뎅기 바이러스 감염에 대한 면역원성 조성물에서의 용도
US11034730B2 (en) 2014-06-23 2021-06-15 Institut Pasteur Dengue virus chimeric polyepitope composed of fragments of non-structural proteins and its use in an immunogenic composition against dengue virus infection
WO2015197565A1 (fr) * 2014-06-23 2015-12-30 Institut Pasteur Polyépitope chimérique du virus de la dengue composé de fragments de protéines non structurales et son utilisation dans une composition immunogène contre une infection par le virus de la dengue
AU2015279375B2 (en) * 2014-06-23 2019-06-13 Centre National De La Recherche Scientifique A dengue virus chimeric polyepitope composed of fragments of non-structural proteins and its use in an immunogenic composition against dengue virus infection
CN107074913B (zh) * 2014-06-23 2022-02-25 巴斯德研究院 包含非结构蛋白的片段的登革热病毒嵌合多表位及其用途
KR102557390B1 (ko) * 2014-06-23 2023-07-19 앵스티띠 파스퇴르 비-구조 단백질의 단편으로 구성된 뎅기 바이러스 키메라 폴리에피토프 및 이의 뎅기 바이러스 감염에 대한 면역원성 조성물에서의 용도
CN115804362A (zh) * 2023-02-08 2023-03-17 中国医学科学院医学生物学研究所 一种IFN-α/βR-/-小鼠抗体依赖性增强感染的注射液及其制备方法

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