WO2017044890A1 - Bird flu vaccine combination comprising virus-like particles and novel adjuvants - Google Patents

Bird flu vaccine combination comprising virus-like particles and novel adjuvants Download PDF

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Publication number
WO2017044890A1
WO2017044890A1 PCT/US2016/051160 US2016051160W WO2017044890A1 WO 2017044890 A1 WO2017044890 A1 WO 2017044890A1 US 2016051160 W US2016051160 W US 2016051160W WO 2017044890 A1 WO2017044890 A1 WO 2017044890A1
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influenza
vlp
virus
vaccine composition
proteins
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PCT/US2016/051160
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French (fr)
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Shu-Mei Liang
Pei-Wen Hsiao
Ming-Chu Cheng
Yu-Chih Yang
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Academia Sinica
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Priority to EP16845223.3A priority Critical patent/EP3347044B1/en
Priority to CN201680052472.9A priority patent/CN108367066B/en
Priority to US15/756,774 priority patent/US10391163B2/en
Publication of WO2017044890A1 publication Critical patent/WO2017044890A1/en

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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
    • A61K39/145—Orthomyxoviridae, e.g. influenza 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
    • 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
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • 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
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • 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
    • 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
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16123—Virus like particles [VLP]
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
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    • C12N2770/00011—Details
    • C12N2770/00034—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
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    • 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
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—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
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32123—Virus like particles [VLP]

Definitions

  • the present invention relates generally to vaccines, more specifically to bird flu vaccines.
  • Avian flu is a major disease in poultry.
  • the disease is caused by avian influenza virus (AIV).
  • Highly pathogenic avian influenza such as H5N1 and H5N2 strain resulted in the deaths of hundreds of millions of birds, including poultry.
  • Current commercial vaccine against the highly pathogenic avian influenza virus (HP AIV) is made from inactivated viruses. This vaccine may not be as effective against a newly mutated highly pathogenic strain.
  • birds vaccinated with inactivated virus vaccine cannot be distinguished from infected birds, which can cause great difficulty to monitor the epidemics. Therefore, development of new and effective vaccine against AIV to control the spread of HP AIV is needed.
  • subunit vaccine is a preferred design over the whole viral vaccine.
  • Virus-like particle (VLP) which represents the structure and antigen compositions of whole virus is a potent vaccine antigen and lacks the risks of growing virus.
  • the invention relates to a vaccine composition
  • a vaccine composition comprising:
  • VLP influenza virus-like particle
  • influenza Ml influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins
  • influenza Ml influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins
  • the invention relates to a vaccine composition
  • a vaccine composition comprising:
  • VLP influenza virus-like particle
  • influenza Ml influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins;
  • FMDV Foot-and-mouth disease virus
  • influenza HA and NA proteins are avian influenza HA and NA proteins.
  • the avian influenza HA and NA proteins are selected from the group consisting of H5N2, H5N1 and H5N8.
  • the invention relates to a method of formulating a vaccine composition of the invention, in which the method comprises adding the therapeutically effective amount of the influenza VLP to the VP3, rVP3, VP3 peptide, or SUMO VP3 and alum.
  • the invention relates to use of a vaccine composition of the invention in the manufacture of a medicament for inducing an immunogenic response in a subject in need thereof.
  • the invention also relates to a vaccine composition of the invention for use in inducing an immunogenic response in a subject in need thereof.
  • the invention relates to a method for inducing an immunogenic response in a subject in need thereof, comprising administering to the subject in need thereof a vaccine composition of the invention to induce an immunogenic response.
  • the subject may be at least one selected from the group consisting of mammalian and avian animals.
  • influenza virus-like particle comprising influenza M 1 , influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins.
  • influenza virus-like particle comprising:
  • influenza HA and NA proteins expressed on the surface of the VLP (b) influenza HA and NA proteins expressed on the surface of the VLP;
  • influenza VLP is non-replicative and noninfectious and does not contain intact viral nucleic acids.
  • influenza VLP may further comprise influenza M2 protein expressed on the surface of the influenza VLP.
  • the invention relates to a host cell comprising an influenza VLP of the invention.
  • the host cell is a Vero cell or 293T cell.
  • FIG. 1 shows characterization of H5N2-VLP produced by Vero E6 cell.
  • A The titer of hemagglutination.
  • B Quantification of HA proteins by western blotting.
  • C Morphology of H5N2- VLP observed by transmission electron microscope.
  • FIG. 2 shows that rVP3 protein significantly stimulates the proliferation of chicken B cells (A, left panel) and CD8+ T cells (A, right panel) and induces chicken immune cells to produce IFN- ⁇ (B).
  • FIG. 3 shows that after being immunized with VLP (with rVP3 and alum as adjuvants), chicken elicit a higher amount of IFN- ⁇ (B), survive fully from a virus challenge (A) and shed less virus than those immunized with inactivated virus (C).
  • VP3 is one of the capsid proteins of FMDV.
  • Four structural proteins , VP1, VP2, VP3 and VP4, comprise the viral capsid of the FMDV.
  • rVP3 is a recombinant DNA derived VP3-like protein expressed and purified from E.coli. A T7 tag and a His tag were attached to the N-terminus and the C-terminus of the VP3 protein, respectively.
  • VP3 peptide is a polypeptide consisting of FMDV VP3 residues 91 -150.
  • SUMO VP3 is a VP3 fusion protein in which VP3 is fused to a hexahistidine-tagged yeast SUMO (small ubiquitin-related modifier) protein.
  • VLP Virus-like particle
  • H5N2-VLP adjuvant with rVP3 and alum protected 100% of chicken from H5N2 virus challenge resulting in less virus shedding than chicken immunized with inactivated virus adjuvant with rVP3 and alum.
  • Our data suggest that VLP adjuvant with rVP3 and alum as novel vaccines against flu.
  • FMDV VP3 protein was expressed and purified from E.coli. Briefly, the gene encoding FMDV VP3 was cloned into a pET-24a (Novagen, Madison, WI, USA) expression vector and transformed into BL21-CodonPlus (DE3)-RIL strain (Stratagene, CA, USA). The E.coli expressing recombinant VP3 was broken up with a microfluidizer (M-l 10Y Cell Disruption) in TEN buffer (20 mM Tris- HC1, pH 8.0, 1 mM EDTA, 100 mM NaCl).
  • M-l 10Y Cell Disruption M-l 10Y Cell Disruption
  • the bacterial lysate was centrifuged, and the pellet was sequentially washed with 0.05% deoxycholate in TEN buffer, 25% sucrose in TEN buffer and TEN buffer alone.
  • the pellet was finally solubilized by binding buffer (20 mM Tris-HCl, 100 mM NaCl, 8 M urea) and the VP3 was purified by cobalt affinity column with a step gradient of imidazole.
  • the fractions containing VP3 proteins were pooled together and then diluted in a drop-wise fashion with 10X volume of refolding buffer (20 mM Tris, 5 ⁇ ⁇ -Mercaptoethanol) and incubated at 4°C for 1 h.
  • rVP3 protein sequence (SEQ ID NO: 1), 241 amino acids:
  • H5N2-VLP producer cell line clones Based on an established platform disclosed in U.S. Patent No. 8980281, we successfully created H5N2-VLP producer cell line clones by sequential stable transfection of pCI6/TO-Ml-M2 and pCI4/TO-HA-NA plasmids into 293T cells.
  • Vero E6 cells We also used Vero E6 cells to generate influenza H5N2- VLPs in Vero cells.
  • Vero E6 cells to generate influenza H5N2- VLPs in Vero cells.
  • Vero E6 cells to generate influenza H5N2- VLPs in Vero cells.
  • a plasmid harboring two expression cassettes to allow tetracycline-inducible expression of H5 and N2 of avian influenza virus.
  • stable transfection of the expression plasmid in the founder cell line followed by clone isolation we screened and identified a producer cell line for H5N2-VLP.
  • VLP hemagglutinin
  • FIG. 1C the particle morphologies (FIG. 1C), average sizes, and hemagglutination abilities of secreted VLPs were characterized, and the VLP constituents were quantified by total protein amount.
  • Vero cells in this manner were better VLP producers than 293T cells. They contained higher abundance of hemagglutinin (HA) protein and as much as 9% of total VLP protein (FIG. 1 A and B).
  • VLP produced by Vero E6 cell was 4 folds higher than that produced by 293T cell. Therefore, H5N2-VLP produced in Vero E6 cells were used as antigen for bird flu vaccine.
  • H5N1-VLP producer cell line clones by sequential stable transfection of pCI6/TO-Ml-M2 and pCI4/TO-HA-NA plasmids into 293 T cells.
  • Vero E6 cells to generate influenza H5Nl-VLPs in Vero cells.
  • Vero E6 cells to generate influenza H5Nl-VLPs in Vero cells.
  • a plasmid harboring two expression cassettes to allow tetracycline-inducible expression of H5 and Nl of avian influenza virus.
  • stable transfection of the expression plasmid in the founder cell line followed by clone isolation we screened and identified a producer cell line for H5N1-VLP.
  • VLP VLP from the producer cells was performed in microcarrier suspension culture system with ensuing doxycycline induction. After centrifugation purification of conditioned media, the particle morphologies, average sizes, and hemagglutination abilities of secreted VLPs are characterized, and the VLP constituents are quantified by total protein amount. H5N1-VLP produced in Vero E6 cells are used as antigen for bird flu vaccine.
  • H5N8-VLP producer cell line clones by sequential stable transfection of pCI6/TO-M 1 -M2 and pCI4/TO-HA-N A plasmids into 293 T cells.
  • Vero E6 cells to generate influenza H5N8-VLPs in Vero cells.
  • Vero E6 cells to generate influenza H5N8-VLPs in Vero cells.
  • stable transfection of the expression plasmid in the founder cell line followed by clone isolation we screened and identified a producer cell line for H5N8-VLP.
  • VLP VLP from the producer cells was performed in microcarrier suspension culture system with ensuing doxycycline induction. After centrifugation purification of conditioned media, the particle morphologies, average sizes, and hemagglutination abilities of secreted VLPs are characterized, and the VLP constituents are quantified by total protein amount. H5N8- VLP produced in Vero E6 cells are used as antigen for bird flu vaccine.
  • This new avian flu vaccine consists of avian flu VLPs as antigen and rVP3 and alum as adjuvants. This vaccine can induce high titers of neutralizing antibodies and cell mediated immunity in immunized chicken. It protects chicken against high pathogenic avian virus challenge with less virus shedding. This vaccine may be used as a vaccine against avian flu virus to protect poultry industry, poultry egg industry and human from the outbreak of avian flu virus epidemic.

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Abstract

A vaccine composition is disclosed. The vaccine composition comprises: (a) a therapeutically effective amount of an influenza virus-like particle (VLP) comprising: (i) influenza M1, influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins; (b) Foot-and-mouth disease virus (FMDV) capsid protein VP3, recombinant FMDV VP3 (rVP3), VP3 peptide, or SUMO VP3; and (c) alum. Also disclosed is use of a vaccine composition according to the invention in the manufacture of a medicament for inducing an immunogenic response in a subject in need thereof.

Description

BIRD FLU VACCINE COMBINATION COMPRISING VIRUS-LIKE PARTICLES
AND NOVEL ADJUVANTS
FIELD OF THE INVENTION
The present invention relates generally to vaccines, more specifically to bird flu vaccines.
BACKGROUND OF THE INVENTION
Avian flu is a major disease in poultry. The disease is caused by avian influenza virus (AIV). Highly pathogenic avian influenza such as H5N1 and H5N2 strain resulted in the deaths of hundreds of millions of birds, including poultry. Current commercial vaccine against the highly pathogenic avian influenza virus (HP AIV) is made from inactivated viruses. This vaccine may not be as effective against a newly mutated highly pathogenic strain. Furthermore, birds vaccinated with inactivated virus vaccine cannot be distinguished from infected birds, which can cause great difficulty to monitor the epidemics. Therefore, development of new and effective vaccine against AIV to control the spread of HP AIV is needed. For these concerns, subunit vaccine is a preferred design over the whole viral vaccine. Virus-like particle (VLP) which represents the structure and antigen compositions of whole virus is a potent vaccine antigen and lacks the risks of growing virus.
SUMMARY OF THE INVENTION
In one aspect, the invention relates to a vaccine composition comprising:
(a) a therapeutically effective amount of an influenza virus-like particle (VLP) comprising:
(i) influenza Ml, influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins;
(b) a recombinant capsid VP3 protein (rVP3) of foot-and-mouth disease virus; and
(c) alum.
In another aspect, the invention relates to a vaccine composition comprising:
(a) a therapeutically effective amount of an influenza virus-like particle (VLP) comprising:
(i) influenza Ml , influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins;
(b) Foot-and-mouth disease virus (FMDV) capsid protein VP3, recombinant FMDV VP3 (rVP3), VP3 peptide, or SUMO VP3; and
(c) alum.
In one embodiment of the invention, the influenza HA and NA proteins are avian influenza HA and NA proteins. In another embodiment, the avian influenza HA and NA proteins are selected from the group consisting of H5N2, H5N1 and H5N8.
In another aspect, the invention relates to a method of formulating a vaccine composition of the invention, in which the method comprises adding the therapeutically effective amount of the influenza VLP to the VP3, rVP3, VP3 peptide, or SUMO VP3 and alum.
Further in another aspect, the invention relates to use of a vaccine composition of the invention in the manufacture of a medicament for inducing an immunogenic response in a subject in need thereof.
The invention also relates to a vaccine composition of the invention for use in inducing an immunogenic response in a subject in need thereof. Alternatively, the invention relates to a method for inducing an immunogenic response in a subject in need thereof, comprising administering to the subject in need thereof a vaccine composition of the invention to induce an immunogenic response.
The subject may be at least one selected from the group consisting of mammalian and avian animals.
Further in another aspect, the invention relates to an influenza virus-like particle (VLP) comprising influenza M 1 , influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins.
Alternatively, the invention relates to an influenza virus-like particle (VLP) comprising:
(a) an influenza virus M1 core protein that can self assemble into a VLP core; and
(b) influenza HA and NA proteins expressed on the surface of the VLP;
wherein the VLP is non-replicative and noninfectious and does not contain intact viral nucleic acids. The influenza VLP may further comprise influenza M2 protein expressed on the surface of the influenza VLP.
Further in another aspect, the invention relates to a host cell comprising an influenza VLP of the invention.
In one embodiment, the host cell is a Vero cell or 293T cell.
These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected without departing from the spirit and scope of the novel concepts of the disclosure.
The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows characterization of H5N2-VLP produced by Vero E6 cell. (A) The titer of hemagglutination. (B) Quantification of HA proteins by western blotting. (C) Morphology of H5N2- VLP observed by transmission electron microscope.
FIG. 2 shows that rVP3 protein significantly stimulates the proliferation of chicken B cells (A, left panel) and CD8+ T cells (A, right panel) and induces chicken immune cells to produce IFN-γ (B).
FIG. 3 shows that after being immunized with VLP (with rVP3 and alum as adjuvants), chicken elicit a higher amount of IFN-γ (B), survive fully from a virus challenge (A) and shed less virus than those immunized with inactivated virus (C).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. Referring to the drawings, like numbers indicate like components throughout the views. As used in the description herein and throughout the claims that follow, the meaning of "a", "an", and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Moreover, titles or subtitles may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present invention.
Additionally, some terms used in this specification are more specifically defined below.
DEFINITIONS
The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a term is the same, in the same context, whether or not it is highlighted. It will be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control.
As used herein, "around", "about" or "approximately" shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range.
Numerical quantities given herein are approximate, meaning that the term "around", "about" or "approximately" can be inferred if not expressly stated.
VP3 is one of the capsid proteins of FMDV. Four structural proteins , VP1, VP2, VP3 and VP4, comprise the viral capsid of the FMDV.
rVP3 is a recombinant DNA derived VP3-like protein expressed and purified from E.coli. A T7 tag and a His tag were attached to the N-terminus and the C-terminus of the VP3 protein, respectively.
VP3 peptide is a polypeptide consisting of FMDV VP3 residues 91 -150.
SUMO VP3 is a VP3 fusion protein in which VP3 is fused to a hexahistidine-tagged yeast SUMO (small ubiquitin-related modifier) protein.
Development of new and effective vaccines to protect humans and animals from influenza virus infection and to control the spread of influenza viruses is an important quest worldwide. Both antigen preparation and adjuvant are important components of vaccine. Virus-like particle (VLP) which represents the structure and antigen compositions of whole virus is a potent vaccine antigen and lacks the risks of growing virus. We found the combination of VLP and rVP3 and alum as vaccines against bird flu yielded the best protection. Chicken immunized with H5N2-VLP adjuvant with rVP3 and alum protected 100% of chicken from H5N2 virus challenge resulting in less virus shedding than chicken immunized with inactivated virus adjuvant with rVP3 and alum. Our data suggest that VLP adjuvant with rVP3 and alum as novel vaccines against flu.
EXAMPLES
Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the examples for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action. VP3 and VLP were prepared according to the U.S. Patent Nos. 8,980,281 and 8,795,678, which are enclosed herein by reference in their entireties.
FMDV VP3 protein was expressed and purified from E.coli. Briefly, the gene encoding FMDV VP3 was cloned into a pET-24a (Novagen, Madison, WI, USA) expression vector and transformed into BL21-CodonPlus (DE3)-RIL strain (Stratagene, CA, USA). The E.coli expressing recombinant VP3 was broken up with a microfluidizer (M-l 10Y Cell Disruption) in TEN buffer (20 mM Tris- HC1, pH 8.0, 1 mM EDTA, 100 mM NaCl). The bacterial lysate was centrifuged, and the pellet was sequentially washed with 0.05% deoxycholate in TEN buffer, 25% sucrose in TEN buffer and TEN buffer alone. The pellet was finally solubilized by binding buffer (20 mM Tris-HCl, 100 mM NaCl, 8 M urea) and the VP3 was purified by cobalt affinity column with a step gradient of imidazole. The fractions containing VP3 proteins were pooled together and then diluted in a drop-wise fashion with 10X volume of refolding buffer (20 mM Tris, 5 μΜ β-Mercaptoethanol) and incubated at 4°C for 1 h. The refolded VP3 was dialyzed against 10X volume of dialysis buffer (20 mM Tris, 100 mM NaCl, 2μΜ β-Mercaptoethanol) at 4°C overnight. rVP3 protein sequence (SEQ ID NO: 1), 241 amino acids:
Figure imgf000006_0001
Figure imgf000007_0001
EXAMPLE 1
Expression and Purification of Influenza VLPs from Vero Cells. Establishment of Vero Cell System Producing Influenza VLPs is according to U.S. Patent No. 8980281.
Based on an established platform disclosed in U.S. Patent No. 8980281, we successfully created H5N2-VLP producer cell line clones by sequential stable transfection of pCI6/TO-Ml-M2 and pCI4/TO-HA-NA plasmids into 293T cells. We also used Vero E6 cells to generate influenza H5N2- VLPs in Vero cells. In brief, we constructed a plasmid harboring two expression cassettes to allow tetracycline-inducible expression of H5 and N2 of avian influenza virus. By stable transfection of the expression plasmid in the founder cell line followed by clone isolation, we screened and identified a producer cell line for H5N2-VLP. Expression and secretion of the VLP from the producer cells was performed in microcarrier suspension culture system with ensuing doxycycline induction. After centrifugation purification of conditioned media, the particle morphologies (FIG. 1C), average sizes, and hemagglutination abilities of secreted VLPs were characterized, and the VLP constituents were quantified by total protein amount. We found that the Vero cells in this manner were better VLP producers than 293T cells. They contained higher abundance of hemagglutinin (HA) protein and as much as 9% of total VLP protein (FIG. 1 A and B). Moreover, the titer of hemagglutination in VLP produced by Vero E6 cell was 4 folds higher than that produced by 293T cell. Therefore, H5N2-VLP produced in Vero E6 cells were used as antigen for bird flu vaccine.
Both antigen preparation and adjuvant are important components of vaccine. To search for better adjuvants, we cloned VP3 gene of capsid protein of foot and mouth virus. The plasmid containing VP3 gene was over-expressed in E.coli, purified and refolded to have adjuvant activity. We then investigated whether rVP3 could activate immune responses in chicken immune cells. We found that rVP3 stimulated the proliferation of chicken B cells and CD8+ T cells (FIG. 2A). rVP3 also induced these chicken immune cells to produce IFN^y (FIG. 2B). These results indicated that rVP3 induces cellular immune responses of chicken immune cells in vitro.
To examine the vaccine efficacy of H5N2-VLP and rVP3, we first immunized chicken with H5N2-VLP alone and found that it did not elicit sufficient antibody titers suggesting that H5N2-VLP required assistance of adjuvants) to induce full immune responses. Chicken immunized with low dose of H5N2-VLP adjuvanted with alum were sicker 10 days after challenge than chicken immunized with same dose of H5N2-VLP adjuvanted with rVP3 and alum or same dose of inactivated virus with alum. Chicken primed and boosted with higher dose of H5N2-VLP adjuvant with rVP3 and alum elicited higher hemagglutination inhibition (HI) and serum neutralization (SN) titers similar to that of inactivated virus adjuvant with rVP3 and alum. In addition, chicken immunized with H5N2-VLP adjuvant with rVP3 and alum fully survived from H5N2 virus challenge (FIG. 3A), yielded less virus shedding that were even better than inactivated virus vaccine adjuvant with rVP3 and alum (FIG. 3B). In consistent with our chicken immune cell in vitro study, chicken immunized with H5N2-VLP adjuvant with rVP3 and alum induced IFN-γ and IL18 production higher than that induced by inactivated virus adjuvant with rVP3 and alum (FIG. 3C).
EXAMPLE 2
Using the similar method as described above, we generate H5N1-VLP producer cell line clones by sequential stable transfection of pCI6/TO-Ml-M2 and pCI4/TO-HA-NA plasmids into 293 T cells. We also used Vero E6 cells to generate influenza H5Nl-VLPs in Vero cells. In brief, we constructed a plasmid harboring two expression cassettes to allow tetracycline-inducible expression of H5 and Nl of avian influenza virus. By stable transfection of the expression plasmid in the founder cell line followed by clone isolation, we screened and identified a producer cell line for H5N1-VLP. Expression and secretion of the VLP from the producer cells was performed in microcarrier suspension culture system with ensuing doxycycline induction. After centrifugation purification of conditioned media, the particle morphologies, average sizes, and hemagglutination abilities of secreted VLPs are characterized, and the VLP constituents are quantified by total protein amount. H5N1-VLP produced in Vero E6 cells are used as antigen for bird flu vaccine.
EXAMPLE 3
Using the similar method as described above, we generate H5N8-VLP producer cell line clones by sequential stable transfection of pCI6/TO-M 1 -M2 and pCI4/TO-HA-N A plasmids into 293 T cells. We also used Vero E6 cells to generate influenza H5N8-VLPs in Vero cells. In brief, we constructed a plasmid harboring two expression cassettes to allow tetracycline-inducible expression of H5 and N8 of avian influenza virus. By stable transfection of the expression plasmid in the founder cell line followed by clone isolation, we screened and identified a producer cell line for H5N8-VLP. Expression and secretion of the VLP from the producer cells was performed in microcarrier suspension culture system with ensuing doxycycline induction. After centrifugation purification of conditioned media, the particle morphologies, average sizes, and hemagglutination abilities of secreted VLPs are characterized, and the VLP constituents are quantified by total protein amount. H5N8- VLP produced in Vero E6 cells are used as antigen for bird flu vaccine.
In conclusion, we have discovered a new vaccine against high pathogenic avian influenza virus.
This new avian flu vaccine consists of avian flu VLPs as antigen and rVP3 and alum as adjuvants. This vaccine can induce high titers of neutralizing antibodies and cell mediated immunity in immunized chicken. It protects chicken against high pathogenic avian virus challenge with less virus shedding. This vaccine may be used as a vaccine against avian flu virus to protect poultry industry, poultry egg industry and human from the outbreak of avian flu virus epidemic.
The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.
The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.
Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is "prior art" to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.

Claims

CLAIMS What is claimed is:
1. A vaccine composition comprising:
(a) a therapeutically effective amount of an influenza virus-like particle (VLP) comprising:
(i) influenza Ml, influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins;
(b) a recombinant capsid VP3 protein (rVP3) of foot-and-mouth disease virus; and
(c) alum.
2. A vaccine composition comprising:
(a) a therapeutically effective amount of an influenza virus-like particle (VLP) comprising:
(i) influenza Ml, influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins;
(b) Foot-and-mouth disease virus (FMDV) capsid protein VP3, recombinant FMDV VP3 (rVP3), VP3 peptide, or SUMO VP3; and
(c) alum.
3. The vaccine composition of claim 1 or 2, wherein the influenza HA and NA proteins are avian influenza HA and NA proteins.
4. The vaccine composition of claim 3, wherein the avian influenza HA and NA proteins are selected from the group consisting of H5N2, H5N1 and H5N8.
5. A method of formulating a vaccine composition as claimed in claim 1 or 2, comprising: adding the therapeutically effective amount of the influenza VLP to the VP3, rVP3, VP3 peptide, or SUMO VP3 and alum.
6. Use of a vaccine composition as claimed in claim 1 or 2 in the manufacture of a medicament for inducing an immunogenic response in a subject in need thereof.
7. The use of claim 6, wherein the subject is at least one selected from the group consisting of mammalian and avian animals.
8. Use of a vaccine composition as claimed in claim 3 in the manufacture of a medicament for inducing an immunogenic response in a subject in need thereof.
9. The use of clam 8, wherein the subject is at least one selected from the group consisting of mammalian and avian animals.
10. Use of a vaccine composition as claimed in claim 4 in the manufacture of a medicament for inducing an immunogenic response in a subject in need thereof.
11. An influenza virus-like particle (VLP) comprising:
influenza Ml, influenza M2, influenza hemagglutinin (HA), and influenza neuraminidase (NA) proteins.
12. An influenza virus-like particle (VLP), comprising:
(a) an influenza virus Ml core protein that can self-assemble into a VLP core; and
(b) influenza HA and NA proteins expressed on the surface of the VLP;
wherein the VLP is non-replicative and noninfectious and does not contain intact viral nucleic acids.
13. An influenza VLP as claimed in claim 12, further comprising influenza M2 protein expressed on the surface of the influenza VLP.
14. A host cell comprising an influenza VLP as claimed in any one of claims 11, 12, and 13.
15. The host cell of claim 14, wherein the host cell is a Vero cell or a 293T cell.
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