WO2018076001A1 - Polypeptide synthétique, composition vaccinale le comprenant, et leurs utilisations - Google Patents

Polypeptide synthétique, composition vaccinale le comprenant, et leurs utilisations Download PDF

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Publication number
WO2018076001A1
WO2018076001A1 PCT/US2017/057796 US2017057796W WO2018076001A1 WO 2018076001 A1 WO2018076001 A1 WO 2018076001A1 US 2017057796 W US2017057796 W US 2017057796W WO 2018076001 A1 WO2018076001 A1 WO 2018076001A1
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polypeptide
subject
synthetic polypeptide
denv
vaccine composition
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PCT/US2017/057796
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English (en)
Inventor
Trai-Ming Yeh
Yen-chung LAI
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National Cheng Kung University
Dcb-Usa Llc
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Publication of WO2018076001A1 publication Critical patent/WO2018076001A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/18—Togaviridae; Flaviviridae
    • C07K14/1816—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus), border disease virus
    • C07K14/1825—Flaviviruses or Group B arboviruses, e.g. yellow fever virus, japanese encephalitis, tick-borne encephalitis, dengue
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24151—Methods of production or purification of viral material
    • C12N2770/24152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure in general relates to the field of preventing viral infection. More particularly, the present disclosure relates to a synthetic polypeptide and its applications in the prophylaxis and/or treatment of dengue virus (DENV) infection.
  • DEV dengue virus
  • Dengue virus is a mosquito-borne single positive-stranded RNA virus of the family Flaviviridae and genus Flavivirus. Dengue is an enveloped virus, 40-60 nm in size, having an isometric nucleocapsid of 25-30 nm and an about 10.7 kb, linear, positive-sense RNA genome. Dengue virus exists as five serotypes (Dengue 1-5, the fifth is reported in 2013) and is genetically related to other flaviviruses such as yellow fever and tick-borne encephalitis viruses. DENV infection may cause life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS).
  • DHF dengue hemorrhagic fever
  • DFS dengue shock syndrome
  • ADE antibody-dependent effect
  • NS1 DENV nonstructural protein 1
  • the synthetic polypeptide comprises a first polypeptide, which has the amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 1.
  • the first polypeptide has the amino acid sequence of SEQ ID NO: 1.
  • the N-terminus of the synthetic polypeptide is acetylated and/or the C-terminus of the synthetic polypeptide is amidated.
  • the synthetic polypeptide further comprises a second polypeptide that is disposed at the N- or C- terminus of the first polypeptide, wherein the second polypeptide is selected from the group consisting of, ovalbumin (OVA), bovine serum albumin (BSA), keyhole limpet haemocyanin (KLH), ⁇ -galactosidase, thyroglobulin (TGB), heat shock protein (HSP) and a combination thereof.
  • OVA ovalbumin
  • BSA bovine serum albumin
  • KLH keyhole limpet haemocyanin
  • TGB thyroglobulin
  • HSP heat shock protein
  • the second aspect of the present disclosure pertains to a vaccine composition for preventing a DENV infection in a subject.
  • the vaccine composition comprises the present synthetic polypeptide and a pharmaceutically acceptable adjuvant.
  • the pharmaceutically acceptable adjuvant is selected from the group consisting of, Emulsigen-D, aluminum hydroxide, incomplete Fruend's adjuvant (IFA), complete Fruend's adjuvant (CFA), endotoxin based adjuvant, mineral oil, mineral oil and surfactant, Ribi adjuvant, Titer-max, syntax adjuvant formulation, aluminium salt adjuvant, nitrocellulose adsorbed antigen, immune stimulating complexe, Gebru adjuvant, super carrier, elvax 40w, L-tyrosine, montanide, Adju prime, Squalene, sodium phthalyl lipopolysaccharide (SPLPS), calcium phosphate, saponin, and muramyl dipeptide (MDP).
  • SPLPS sodium phthalyl lipopoly
  • the synthetic polypeptide of the present composition comprises a first polypeptide having the amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 1, and optionally, a second polypeptide disposed at the N- or C- terminus of the first polypeptide.
  • the second polypeptide may be any of OVA, BSA, KLH, ⁇ -galactosidase, TGB, HSP or a combination thereof.
  • the third aspect of the present disclosure is directed to a method of preventing a subject from being infected by a DENV.
  • the method comprises administering to the subject an effective amount of the present vaccine composition so as to vaccinate the subject against the DENY
  • the vaccine composition may give rise to about 0.8 ⁇ g to 80 mg of the polypeptide per kilogram (Kg) of body weight per dose (0.8 ⁇ g to 80 mg/Kg/dose); preferably, 8 to 8 mg/Kg/dose; more preferably, 80 to 800 ⁇ g/Kg/dose.
  • the vaccine composition is administered to the subject at least 2 times in the course of vaccination.
  • the vaccine composition is administered to the subject 3 to 5 times in the course of vaccination.
  • the present vaccine composition can be administered by a route selected from the group consisting of transmucosal, subcutaneous, intradermal, intramuscular, intravenous, and intraperitoneal injection.
  • the present method is useful in protection the subject against the infection caused by DENV serotype 1, 2, 3 or 4.
  • the subject is a mammal.
  • the subject is a human.
  • Figure 1A is the data depicting the binding of the modified NS1 wing domain (NSl-WD) immune sera of mice to the surface of different serotypes of DENV-infected HuH-7 cells according to one embodiment of the present disclosure.
  • DENV 1-4 or mock-infected HuH-7 cells were respectively stained the specified immune sera of mice (1 : 100 dilution) and analyzed by flow cytometry: KLH-immune sera (the sera isolated from the mice immunized with KLH control), NS1 -immune sera (the sera isolated from the mice immunized with full length NS1 protein), modified NSl-WD-immune sera (the sera isolated from the mice immunized with the modified NSl-WD polypeptide (SEQ ID NO: 1)), and 2° Ab (stained with secondary antibody only, serving as the negative control in the experiment).
  • KLH-immune sera the sera isolated from the mice immunized with KLH control
  • NS1 -immune sera the sera
  • Figure IB is the line chart depicting the binding affinity of purified pAbs from modified NSl-WD polypeptide-immunized mouse sera to NS1 polypeptide, modified NSl-WD polypeptide-conjugated BSA or BSA according to one embodiment of the present disclosure.
  • Figure 2A is the histogram depicting the binding affinity of different doses of purified pAbs from KLH control, wildtype NSl-WD polypeptide (SEQ ID NO: 2), modified NSl-WD polypeptide immune sera to HUVECs according to another embodiment of the present disclosure.
  • the shift ratio (%) represents the percentage of binding compared with the control group (Anti-mouse IgG Alexa 488 only). All data are presented as the mean ⁇ S.D. from at least three independent experiments. *P ⁇ 0.05, **P ⁇ 0.01.
  • Figure 2B is the histogram depicting the binding affinity of different doses of purified pAbs from KLH control, Full length NS1, modified NSl-WD polypeptide immune sera to human platelets according to another embodiment of the present disclosure.
  • FIGS. 3 are histograms respectively depicting antibodies against modified NSl-WD polypeptide caused cytotoxicity only in DENV-infected cells by complement-dependent cytolysis manner according to one embodiment of the present disclosure.
  • Figures 4A and 4B are histograms respectively depicting antibodies against modified NSl-WD polypeptide inhibit viral propagation by complement-dependent cytolysis of DENV-infected cells and directly reduce viral replication by complement-independent manner respectively according to one embodiment of the present disclosure.
  • Figures 5A-5D are dot plots respectively depicting the active immunization of modified NSl-WD polypeptide protects mice against DENV 1, DENV 2, DENV 3 or DENV 4-elicited prolonged bleeding time according to another embodiment of the present disclosure. *P ⁇ 0.05, ***P ⁇ 0.001.
  • Figures 6A-6B are photographs and histograms respectively depicting the active immunization of modified NSl-WD polypeptide protects mice against specified DENV-elicited hemorrhage according to another embodiment of the present disclosure.
  • Figure 6A representative images of the skins of mice respectively treated with specified treatments.
  • Figure 6B the quantitative results of hemorrhage of mice challenged with DENV 1 (panel A), DENV 2 (panel B), DENV 3 (panel C) or DENV 4 (panel D). The clinical score of hemorrhage was quantified and determined as digital hemorrhage severity by ImageJ. Arrows indicate hemorrhage in the skin lesions.
  • Figure 7 is the data depicting the active immunization of modified NSl-WD polypeptide protects mice against DENV 2-elicited hemorrhage according to another embodiment of the present disclosure.
  • Hematoxylin and eosin (H&E) staining was performed to analyze local skin hemorrhage in the skin lesions.
  • the arrows indicate the intact blood vessels.
  • the scale bar indicated 40 ⁇ .
  • Figures 8A and 8B are photographs and histograms respectively depicting active immunization of modified NSl-WD polypeptide reduces NS3 expression in mice according to still another embodiment of the present disclosure.
  • DENV NS3 expression was detected by IHC staining. Arrows indicate local DENV NS3 expression. The scale bar indicates 40 ⁇ . ***P ⁇ 0.001, ns indicates no significance.
  • Figures 9A-9C are dot plots and line chart respectively depicting the protective effect protection of the antibody produced by the modified NSl-WD polypeptide on DENV infection in STATl _/ ⁇ mice according to one embodiment of the present disclosure.
  • Figure 9A viremia in STATl _/ ⁇ mice 2 days post-infection.
  • Figure 9B NS1 expression in the sera of STATl _/ ⁇ mice 2 days post-treatment.
  • Figure 9C survival rate of mice administered with specified treatment. **P ⁇ 0.01, ***P ⁇ 0.001.
  • polypeptide refers to a polymer of amino acids without regard to the length of the polymer; thus, “peptides,” “oligopeptides”, and “proteins” are included within the definition of polypeptide and used interchangeably herein. This term also does not specify or exclude chemical or post-expression modifications of the polypeptides of the invention, although chemical or post-expression modifications of these polypeptides may be included or excluded as specific embodiments. Therefore, for example, modifications to polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
  • polypeptides with these modifications may be specified as individual species to be included or excluded from the present invention.
  • positions of any specified amino acid residues within a polypeptide are numbered starting from the N terminus of the polypeptide.
  • amino acids are not designated as either D-or L-amino acids, the amino acid is either an L-amino acid or could be either a D- or L- amino acid, unless the context requires a particular isomer.
  • the notation used herein for the polypeptide amino acid residues are those abbreviations commonly used in the art.
  • synthetic polypeptide refers to a polypeptide which does not comprise an entire naturally occurring protein molecule.
  • the polypeptide is “synthetic” in that it may be produced by human intervention using such techniques as chemical synthesis, recombinant genetic techniques, or fragmentation of whole antigen or the like.
  • vaccine refers to a composition which when inoculated into an animal has the effect of stimulating an immune response (e.g., antigen-specific antibody) in the animal, which serves to fully or partially protect the animal against a disease (e.g., DENV infection) or its symptoms.
  • an immune response e.g., antigen-specific antibody
  • the term vaccine encompasses prophylactic as well as therapeutic vaccines.
  • a combination vaccine is one which combines two or more vaccines.
  • adjuvant refers to any substance or mixture of substances that enhances, increases, upwardly modulates, diversifies or otherwise facilitates the immune response (e.g., humoral or cellular immune response) to an antigen.
  • antigen refers to any agent that, when introduced into an immunocompetent human or animal, stimulates a humoral and/or cellular immune response.
  • the antigen may be a pure substance, a mixture of substances, or particulate material (including cells, cell fragments, or cell derived fragments) or a live, usually attenuated, organism or virus.
  • suitable antigens include, but are not limited to, a protein, glycoprotein, lipoprotein, polypeptide, peptide, carbohydrate/polysaccharide, lipopolysaccharide, toxin, virus, bacterium, fungus, and parasite.
  • antibody refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • Antibodies are typically tetramers of immunoglobulin molecules.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies (pAb), monoclonal antibodies (mAb), Fv, Fab and F(ab) 2 , as well as single chain antibodies and humanized antibodies.
  • the term “treat”, “treating” and “treatment” are interchangeable, and encompasses partially or completely ameliorating, mitigating and/or managing a symptom, a secondary disorder or a condition associated with DENV infection, in which inducing a DNEV-specific immune response (e.g., DENV-specific antibody) provides a benefit to the subject having or suspected of having such symptom, disorder or condition.
  • a DNEV-specific immune response e.g., DENV-specific antibody
  • treating refers to application or administration of the present polypeptide or vaccine composition to a subject, who has a symptom, a secondary disorder or a condition associated with DENV infection, with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms, secondary disorders or features associated with DENV infection.
  • Symptoms, secondary disorders, and/or conditions associated with DENV infection include, but are not limited to, fever, severe headache, pain behind the eyes, muscle and joint pains, nausea, vomiting, swollen glands, rash, severe abdominal pain, rapid breathing, bleeding gums, fatigue, restlessness and blood in vomit.
  • Treatment may be administered to a subject who exhibits only early signs of such symptoms, disorder, and/or condition for the purpose of decreasing the risk of developing the symptoms, secondary disorders, and/or conditions associated with DENV infection.
  • Treatment is generally "effective” if one or more symptoms or clinical markers are reduced as that term is defined herein.
  • a treatment is "effective” if the progression of a symptom, disorder or condition is reduced or halted.
  • prevent refers to the prophylactic treatment of a subject who is at risk of developing a symptom, a secondary disorder or a condition associated with DENV infection, so as to decrease the probability that the subject will develop the symptom, secondary disorder or condition.
  • the term “prevent”, “preventing” or “prophylaxis” refers to inhibit the occurrence of a symptom, a secondary disorder or a condition associated with DENV infection, that is, to reduce the inc
  • idence or the frequency of occurrence of the symptom, secondary disorder or condition is idence or the frequency of occurrence of the symptom, secondary disorder or condition.
  • the term “prevent”, “preventing” or “prophylaxis” as used herein referring to a polypeptide, a composition comprising the same and/or a method does not mean or imply that use of the polypeptide, the composition comprising the same and/or the method will provide a guarantee that the symptom, secondary disorder or condition will never occur, but rather that the polypeptide, the composition comprising the same and/or the method will inhibit the occurrence of the symptom, secondary disorder or condition, and that the incidence and/or frequency of the symptom, secondary disorder or condition will be reduced.
  • the term "effective amount" as referred to herein designate the quantity of a component which is sufficient to yield a desired response.
  • the effective amount is also one in which any toxic or detrimental effects of the component are outweighed by the therapeutically beneficial effects.
  • the specific effective or sufficient amount will vary with such factors as the particular condition being treated, the physical condition of the patient (e.g., the patient's body mass, age, or gender), the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. Effective amount may be expressed, for example, in grams, milligrams or micrograms or as milligrams per kilogram of body weight (mg/Kg).
  • the effective amount can be expressed in the concentration of the active component (e.g., the synthetic polypeptide of the present disclosure), such as molar concentration, mass concentration, volume concentration, molality, mole fraction, mass fraction and mixing ratio.
  • the term "therapeutically effective amount" used in connection with the synthetic polypeptide described herein refers to the quantity of the synthetic polypeptide, which is sufficient to alleviate or ameliorate the symptoms associated with the cancer in the subject.
  • Persons having ordinary skills could calculate the human equivalent dose (HED) for the medicament (such as the present synthetic polypeptide) based on the doses determined from animal models. For example, one may follow the guidance for industry published by US Food and Drug Administration (FDA) entitled "Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers" in estimating a maximum safe dosage for use in human subjects.
  • FDA US Food and Drug Administration
  • subject refers to a mammal including the human species that is treatable with methods of the present invention.
  • subject is intended to refer to both the male and female gender unless one gender is specifically indicated.
  • the first aspect of the present disclosure is directed to a polypeptide for preventing or treating DENV infection in a subject, in which the polypeptide comprises an amino acid sequence at least 85% identical to the sequence of "YKDWSEWGKAC" (SEQ ID NO: 1); that is, the amino acid sequence comprised in the present polypeptide may be 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1.
  • the present polypeptide has the amino acid sequence at least 85% identical to SEQ ID NO: 1.
  • the present polypeptide has the amino acid sequence of SEQ ID NO: 1.
  • the N-terminus of the present polypeptide is acetylated. Additionally or alternatively, the C-terminus of the present polypeptide is amidated.
  • the N- or C-terminus of the present polypeptide is conjugated with a carrier molecule so as to increase the immunogenicity of the polypeptide.
  • exemplary carrier molecules include, but are not limited to, OVA, BSA, KLH, ⁇ -galactosidase, TGB, HSP or a combination thereof.
  • the carrier molecule is KLH.
  • the synthetic polypeptides of the invention can be synthesized by commonly used methods such as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the polypeptide.
  • Polypeptides of the invention can also be synthesized by the well-known solid phase peptide synthesis methods.
  • the synthetic polypeptides of the invention can be produced by host cells (e.g., FIEK293 cells), which is transfected with a nucleic acid encoding the polypeptide.
  • the present polypeptide possesses an immunogenic function to stimulate an immune response (i.e., the production of antibody) against four serotypes (serotype 1-4) of DENV (hereinafter respectively designated as DENV 1, DENV 2, DENV 3, and DENV 4).
  • DENV 1, DENV 2, DENV 3, and DENV 4 four serotypes of DENV (hereinafter respectively designated as DENV 1, DENV 2, DENV 3, and DENV 4).
  • the second aspect of the present disclosure pertains to a vaccine composition that comprises the polypeptide according to any of the above-mentioned aspect and embodiments of the present disclosure, and a pharmaceutically acceptable adjuvant.
  • the adjuvant is a substance that enhances the immune response against an antigen (e.g., the present polypeptide).
  • Suitable examples of adjuvant for enhancing the present polypeptide include, but are not limited to, Emulsigen-D, aluminum hydroxide, incomplete Fruend's adjuvant (IFA), complete Fruend's adjuvant (CFA), endotoxin based adjuvant, mineral oil, mineral oil and surfactant, Ribi adjuvant, Titer-max, syntax adjuvant formulation, aluminium salt adjuvant, nitrocellulose adsorbed antigen, immune stimulating complexe, Gebru adjuvant, super carrier, elvax 40w, L-tyrosine, montanide, Adju prime, Squalene, sodium phthalyl lipopolysaccharide (SPLPS), calcium phosphate, saponin, and muramyl dipeptide (MDP).
  • the adjuvant is CFA.
  • an antibody e.g., a mAb or a pAb
  • a host animal such as a mouse, a rat, or a rabbit
  • the immunization may be performed in accordance with commonly adopted procedures.
  • the immunization interval is not particularly limited. Immunization may be carried out at intervals of several days to several weeks, preferably one week, for 2-10 times, until a desired antibody titer is reached.
  • the host animals are vaccinated by subcutaneously or intradermally injecting with the present vaccine composition on weekly basis for 3-5 consecutive weeks.
  • Blood samples are taken regularly after immunization and subject to centrifugation to separate sera.
  • the resultant sera are then subject to measurement of antibody titers by any suitable method, which includes, but is not limited to, ELISA, enzyme immunoassay (EIA), or radio immunoassay (RIA).
  • EIA enzyme immunoassay
  • RIA radio immunoassay
  • the blood is collected from the immunized animals, and then separated and purified by an ordinary method, including centrifugation, precipitation using ammonium sulfate or polyethylene glycol, chromatography (such as gel filtration chromatography, ion exchange chromatography, or affinity chromatography) etc., so as to obtain a pAb recognizing the present polypeptide in the form of a polyclonal antiserum.
  • an ordinary method including centrifugation, precipitation using ammonium sulfate or polyethylene glycol, chromatography (such as gel filtration chromatography, ion exchange chromatography, or affinity chromatography) etc.
  • the present polypeptide or vaccine composition induces an immune response (e.g., the production of pAb) against DENY
  • an immune response e.g., the production of pAb
  • the thus produced antibody exhibits binding affinity and specificity to the present polypeptide.
  • the thus produced antibody is capable of binding and/or neutralizing the DENVs (including DENV 1, DENV 2, DENV 3 and DENV 4).
  • the third aspect of the present disclosure is directed to a method for protecting a subject from being infected by a DENV.
  • the present method comprises administering to the subject an effective amount of the present vaccine composition so as to vaccinate the subject against the DENV (i.e., stimulate an immune response against DENV in the subject).
  • the effective amount of the active component (i.e., the present polypeptide) comprised in the vaccine composition may vary with various factors, such as the physical condition of the patient (e.g., the patient's body mass, age, or gender), the vaccinated subject, and the immunogenicity of the active component.
  • the subject is a mouse.
  • the present polypeptide is administered to the subject in the amount of about 0.01 to 1,000 mg/Kg body weight per dose; for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,
  • 0.1 to 100 mg/Kg body weight per dose More preferably, 1 to 20 mg/ Kg body weight per dose.
  • 2-2.5 mg/Kg of the present polypeptide per dose is sufficient to induce an immune response (e.g., the production of antibody) in the subject.
  • the effective HED of the present polypeptide is about 0.8 ⁇ g/Kg to 80 mg/Kg body weight per dose for human; in other words, the effective HED of the present polypeptide may be any of, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 or 950 ⁇ g/Kg, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,
  • the effective HED of the present polypeptide is about 160 to 200 ⁇ g/Kg per dose.
  • the present polypeptide or vaccine composition is administered to the subject at least 2 times, such as, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times, in the course of vaccination.
  • the vaccine composition may be administered to the subject for 2-10 times with an interval from several days to several years.
  • the subject is a mouse, and the present vaccine composition is administered to the subject 4-5 times within one month so as to induce the polypeptide-specific immune response in the subject.
  • the subject is primed with the vaccine composition comprising an effective amount of the present polypeptide and CFA adjuvant, and then boosted 3-4 times with the vaccine composition comprising an effective amount of the present polypeptide and IFA adjuvant.
  • the present vaccine composition can be administered to the subject by an appropriate route, such as transmucosal, subcutaneous, intradermal, intramuscular, intravenous, and intraperitoneal injection. According to one specific example, the present vaccine composition is administered to the subject via intraperitoneal injection.
  • the present method can be applied to the subject, alone or in combination with additional therapies that have some beneficial effects on the treatment of DENV infection. Depending on the intended/therapeutic purpose, the present method can be applied to the subject before, during, or after the administration of the additional therapies.
  • the present method is useful in protecting the subject against DENV (including DENV 1, DENV 2, DENV 3 and DENV 4) infection.
  • DENV DENV-associated illness/disorder
  • the antibody/polyclonal antiserum produced by the present method is capable of inducing a complement dependent cytolysis (CDC) response to DENV, and accordingly, decreasing the viral titer in the vaccinated subject.
  • CDC complement dependent cytolysis
  • the antibody/polyclonal antiserum produced by the present method dose not cross react to the endothelial cell and the platelet of the subject.
  • the present method is useful in prolonging the survival of DENV-infected subject.
  • the subject treatable by the present method is a mammal, for example, a human, a mouse, a rat, a hamster, a guinea pig, a rabbit, a dog, a cat, a cow, a goat, a sheep, a monkey, and a horse.
  • the subject is a human.
  • Human hepatoma cell line Huh-7, Baby hamster kidney cell line BHK-21 and Aedes albopictus cell line C6/36 purchased from the Japanese Collection of Research Bioresources (Japan) and the American Type Culture Collection (ATCC, Manassas, Virginia), were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone, Logan, UT).
  • DMEM Dulbecco's Modified Eagle's Medium
  • HJVECs Human umbilical vein endothelial cells
  • BCRC Bioresource Collection and Research Center
  • EMB-2 endothelial Basal Medium-2
  • FBS FBS
  • SingleQuotsTM Kit SingleQuotsTM Kit
  • the fluorescent focus assay was used to determine the virus titer.
  • supernatants containing infectious virus were collected and stored below -70°C until use.
  • Supernatant was serially diluted and incubated with BHK-21 cells for 2 hours at 37°C.
  • the monolayers were then overlaid with DMEM containing 2% FBS and 1% methylcellulose and incubated at 37°C for 2-3 days.
  • Virus foci were stained with anti-NSl antibody followed by Alexa 488-conjugated goat anti-mouse IgG (Invitrogen, Carlsbad, CA) and visualized with a fluorescence microscope (Leica Geosystems AG, St. Gallen, Switzerland).
  • HRP horseradish peroxidase
  • TMB tetramethylbenzidine
  • C3H/HeN wildtype mice were used in a DENV-induced disease murine model.
  • the KLH-conjugated modified NS1-WD polypeptide SEQ ID NO: 1; 50 ⁇ g; ChinaPeptides, Shanghai, China
  • the control protein KLH
  • CFA, IF A Complete or Incomplete Freund's Adjuvant
  • mice 6-week-old mice were intraperitoneally immunized with one dose of the CFA-emulsion; two weeks later, the immunized mice were boosted with 3 doses of IFA-emulsion with an interval of one week between each dose (each dose contained 50 ⁇ g of KLH-conjugated modified NS1-WD polypeptide or KLH control protein). Three days following the last immunization, the mice were challenged intradermally with concentrated DENV (2* 10 8 PFU/mouse) or concentrated C6/36 medium as a control at four sites on the upper back.
  • concentrated DENV 2* 10 8 PFU/mouse
  • concentrated C6/36 medium as a control at four sites on the upper back.
  • mice were used in a lethal infection murine model.
  • a DENV 2 lethal strain (454009 A) (4 ⁇ 10 7 PFU/mouse) or C6/36 control medium were inoculated intravenously (i.v.) into STAT] ⁇ mice.
  • Sera isolated from the immunized mice were injected intraperitoneally one day before the DENV challenge. Two days later, the sera were collected from the DENV-challenged mice to determine the viremia and NS1 secretion by FFA and quantitative NS1 ELISA. The body weight and survival rates of mice were monitored for 14 days.
  • H&E stain Hematoxylin and Eosin staining
  • IHC Immunohistochemistry
  • the degree of murine skin hemorrhage was image-processed by Photoshop ® and digitally quantified by ImageJ software.
  • the samples of mice skin were collected and adjusted to the same image sizes.
  • the hemorrhagic areas were isolated and created as new images in Photoshop 6.0 (Adobe, San Jose, CA).
  • the processed images were loaded into ImageJ software, and converted into 16-bit images. After image type was set to black and white, the total hemorrhage volume was calculated and analyzed by Prism software.
  • Example 1 Protective effect of modified NS1-WD polypeptide against DENV infection in vitro
  • the sera isolated from mice respectively immunized with specified antigens including full length NSl protein, the KLH-conjugated modified NSl -WD polypeptide, and the KLH control protein
  • specified antigens including full length NSl protein, the KLH-conjugated modified NSl -WD polypeptide, and the KLH control protein
  • modified NSl-WD pAb modified NSl-WD
  • the cell-protective effect of the modified NSl-WD pAb was determined by LDH release assay.
  • the data of Fig. 3 indicated that the modified NSl-WD pAb activated complement that induced significant LDH release in the DENV-infected HUVECs, but not the uninfected HUVECs, in which the anti-NSl 2E8 monoclonal antibody (mAb) and the KLH polyclonal antibody (hereinafter designated as "KLH pAb”) respectively served as the positive and negative control antibodies.
  • mAb monoclonal antibody
  • KLH pAb KLH polyclonal antibody
  • Example 2 Protective effect of modified NSl-WD pAb on DENV infection by active immunization
  • mice Since immunodeficient mice are more susceptible to DENV infection, a lethal infection mouse model in STAT 1 -deficient mice (STAT V'- mice) was established so as to test the prophylactic effects of the modified NSl-WD pAb against lethal DENV 2 infection.
  • the non-mouse adapted DENV 2 strain 454009 A actively replicated in STAT] ⁇ mice, and the viremia in the sera of these mice could reach 10 3 — 10 4 PFU/ml at 2-3 days post infection (data not shown). In addition, mice died within two weeks after the DENV challenge (data not shown).
  • Administration of anti-NSl-WD pAb significantly reduced the DENV-induced viremia, NS1 secretion and lethality in STATl ⁇ mice. (Figs. 9A-9C).
  • the present invention demonstrated that a modified NSl-WD polypeptide can stimulate an immune response against DENV infection, including DENV 1, DENV 2, DENV 3 and DENV 4. Accordingly, the present vaccine composition comprising the modified NSl-WD polypeptide and an adjuvant may provide a potential means to efficiently prevent a subject from being infected by DENV.

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Abstract

L'invention concerne un polypeptide comprenant la séquence d'acides aminés au moins identique à 85% à la séquence de SEQ ID NO: 1. Elle concerne également une composition vaccinale comprenant ledit polypeptide et un adjuvant. La composition vaccinale est utile pour la prévention d'une infection par DENV et/ou l'atténuation de la maladie associée à DENV chez le sujet.
PCT/US2017/057796 2016-10-21 2017-10-23 Polypeptide synthétique, composition vaccinale le comprenant, et leurs utilisations WO2018076001A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013003579A1 (fr) * 2011-06-29 2013-01-03 Immunotope, Inc. Immunogènes inducteurs de lymphocytes t cytotoxiques pour la prévention, le traitement et le diagnostic d'une infection par le virus de la dengue
WO2014064943A1 (fr) * 2012-10-25 2014-05-01 Osaka University Peptide antigène dérivé du virus de la dengue
WO2014144600A2 (fr) * 2013-03-15 2014-09-18 Viktor Roschke Complexes multispécifiques multivalents et monovalents et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013003579A1 (fr) * 2011-06-29 2013-01-03 Immunotope, Inc. Immunogènes inducteurs de lymphocytes t cytotoxiques pour la prévention, le traitement et le diagnostic d'une infection par le virus de la dengue
WO2014064943A1 (fr) * 2012-10-25 2014-05-01 Osaka University Peptide antigène dérivé du virus de la dengue
WO2014144600A2 (fr) * 2013-03-15 2014-09-18 Viktor Roschke Complexes multispécifiques multivalents et monovalents et leurs utilisations

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank 29 August 2014 (2014-08-29), SPANU, P. ET AL.: "rRNA Biogenesis Protein [Blumeria Graminis f. sp. Hordei DH14", XP055478269, Database accession no. CCU76378.1. *

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