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US20180243397A1 - Norovirus vaccine - Google Patents

Norovirus vaccine Download PDF

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US20180243397A1
US20180243397A1 US15/754,807 US201615754807A US2018243397A1 US 20180243397 A1 US20180243397 A1 US 20180243397A1 US 201615754807 A US201615754807 A US 201615754807A US 2018243397 A1 US2018243397 A1 US 2018243397A1
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norovirus
vaccine
gii
antigen
vlp
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Ron Cobb
Michael Springer
Yawei Ni
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Resilience Government Services Inc
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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
    • 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
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1682—Processes
    • A61K9/1694—Processes resulting in granules or microspheres of the matrix type containing more than 5% of excipient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • 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
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16023—Virus like particles [VLP]
    • 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
    • C12N2770/16011—Caliciviridae
    • C12N2770/16034—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
    • C12N2770/16011—Caliciviridae
    • C12N2770/16071—Demonstrated in vivo effect

Definitions

  • the present invention is generally related to vaccines for prevention of infectious diseases and more specifically a vaccine for prevention and/or alleviation of norovirus infections and norovirus-related diseases and symptoms.
  • Norovirus a single-stranded RNA virus in the Caliciviridae family, is the primary cause of nonbacterial gastroenteritis worldwide, accounting for 96% of all cases of viral gastroenteritis [1]. It is estimated, on average, that norovirus is responsible for 19 to 21 million infections per year [2] and up to 200,000 deaths in children under 5 years of age in developing countries [3, 4]. Norovirus is transmitted primarily through the fecal-to-oral route [5] making norovirus particularly threatening to individuals who occupy a high density, communal environments such as schools, nursing homes, cruise ships, and in the military [6]. Norovirus is also stable ex vivo which makes decontamination after an outbreak laborious and time consuming.
  • norovirus The robustness of norovirus, along with a low infectious dose ( ⁇ 10 virions per individual) [7], makes norovirus a highly infectious virus with dramatic socio-economic impacts. This disease burden strongly indicates the need for an effective vaccine; however, currently there is no FDA-approved norovirus vaccine available.
  • Norovirus is distributed among at least five different genogroups GI, GII, GIII, GIV, and GV. Only genogroups I, II, and IV are infectious to humans, with GI and GII being most prevalent [8, 9]. Recently, genogroup II has become the most prevalent, accounting for 81.4% of norovirus outbreaks worldwide [10]. Each genogroup is subdivided further into genoclusters. Full-length genomic sequencing of various norovirus strains indicate that norovirus can vary by 3% to 31% within genogroups and 49% to 54% between genogroups [11]. Due to this wide variation, development of a broadly effective vaccine remains a challenge as the antibodies from humans immunized against one genogroup do not cross react with noroviruses from other genogroups [12].
  • virus-like particles As vaccine antigens has been demonstrated by the licensure of hepatitis B virus VLP and human papilloma-virus VLP vaccines.
  • Extensive research has focused on the development of norovirus VLPs as vaccine antigens that can be delivered parenterally, orally, or mucosally [13, 14].
  • Clinical evidence has demonstrated that norovirus VLPs administered orally or intranasally were well tolerated and modestly immunogenic [15, 16].
  • norovirus VLPs As a potential vaccine, there is still a great need to produce a norovirus vaccine that is multivalent, targeting the wide variation of norovirus strains. Moreover, the vaccine should be suitable for multiple routes of administrations in order to minimize the number of invasive injections. In addition, a vaccine in a form a dry power is preferred over a traditional liquid form as a dry powder can be stably stored at a room temperature for a long period.
  • the present invention relates to formulations of a dry powder norovirus vaccine comprising one, two or more antigens from different genogroups of noroviruses.
  • the norovirus vaccine formulation is monovalent, comprising genogroup GII VLP antigens.
  • the norovirus vaccine formulation is multivalent, containing multiple norovirus VLP antigens derived from multiple genogroups of norovirus.
  • the vaccine formulation is a multivalent norovirus vaccine comprising two norovirus VLP antigens from GI and GII noroviruses, respectively.
  • the vaccine formulation is a multivalent norovirus vaccine comprising three norovirus VLP antigens from GI, GII and GIV noroviruses respectively.
  • norovirus VLP antigens are recombinant VLPs.
  • Recombinant norovirus virus-like particles are obtained by expressing the virus-like particles in an expression system selected from a group consisting of viruses, baculovirus expression systems, tobacco mosaic virus vector systems, prokaryotic cells, E. coli systems, yeast ( S. cerevisiae ), eukaryotic expression systems, Sf9 insect cells, mammalian cells, HEK 293 and CHO cells.
  • Formulations of a dry powder norovirus vaccine may further comprise anionic polysaccharide.
  • anionic polysaccharide is sodium polygalacturonate.
  • Sodium polygalacturonate is an Aloe vera L.-derived polysaccharide polymer with mucoadhesive properties.
  • the dry powder formulation of this compound improves mucosal residence time of administered vaccines.
  • a formulation of a dry powder norovirus vaccine is produced as one powder formulation with a mixture of two or more norovirus VLP antigens.
  • the vaccine is formulated as a combination of two or more monovalent vaccine powders with each containing one norovirus VLP antigen.
  • the present invention further provides methods of producing a dry powder norovirus vaccine that is multivalent.
  • the methods may include a lyophilization-milling method.
  • the methods comprise a spray-drying method.
  • the sodium polygalacturonate comprises at least 0.1% (w/w).
  • the norovirus virus-like particle comprises about 1 ⁇ g to 100 ⁇ g of the vaccine formulation.
  • the present invention further provides methods of immunization against norovirus infections which comprises at least one immunization via a parenteral and/or a mucosal route.
  • one or more immunizations via a mucosal route is followed by one or more immunizations via a parenteral route or vice versa, to maximize both mucosal and systemic immune responses and protection against norovirus infection.
  • a dry powder vaccine is used for both parenteral and mucosal immunizations, i.e., the mucosal immunization is performed directly with the dry powder vaccine by intranasal delivery, whereas the parenteral immunization with the reconstituted dry powder vaccine by intramuscular (IM) injection.
  • IM intramuscular
  • FIG. 1 Transmission electron microscopy of norovirus VLPs.
  • GI (A) and GII.4 (B) VLPs were dissolved in water and imaged at 150,000 ⁇ magnification (scale bar 100 ⁇ m). VLP particles were spherical in appearance at the expected size of 23 nm to 38 nm.
  • FIG. 2 Thermal stability evaluation of norovirus VLPs using SYPRO Orange. VLPs were diluted in 4 ⁇ SYPRO orange solution and the melt curve was analyzed using a fluorescent thermocycler. Data is plotted as the change in Fluorescence per unit Temperature.
  • FIG. 3 Thermal stability evaluation of norovirus VLPs using capture ELISA.
  • VLP samples (0.2 ⁇ g/mL) were treated at varying temperatures for 5 minutes. Each sample was then analyzed by capture ELISA.
  • FIG. 4 SDS-PAGE and western blot analysis of GelVacTM GI (A) and GII.4 (B) vaccine powders.
  • Vaccine powders were reconstituted and analyzed by SDS-PAGE and western blot to confirm the presence of VLPs. Order from left to right: 100 ⁇ g, 50 ⁇ g, 15 ⁇ g, 5 ⁇ g, 1 ⁇ g, 0.1 ⁇ g, 0, reference standard. Both GI and GII had observable bands at ⁇ 55 kDa, consistent with the size of VP1 capsid protein.
  • FIG. 5 Serum norovirus-specific IgG production following intranasal immunization with GelVacTM vaccine powder.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of powder formulation containing various amounts of VLP on days 0 and 21. Serum samples were collected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B) Norovirus-specific IgG antibodies. *P ⁇ 0.05 as compared to the placebo control group.
  • FIG. 6 Serum norovirus-specific IgG1 and IgG2 production following intranasal immunization with GelVacTM vaccine powder.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of powder formulation containing various amounts of VLP on days 0 and 21. Serum samples were collected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B) Norovirus-specific IgG1 and IgG2 antibodies.
  • FIG. 7 Serum norovirus specific IgA production following intranasal immunization with GelVacTM vaccine powder.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of powder formulation containing various amounts of VLP on days 0 and 21. Serum samples were collected on day 56 and analyzed for GI (A) and GII.4 (B) Norovirus-specific IgA antibodies.
  • FIG. 8 Neutralizing antibody production following intranasal immunization with GelVacTM vaccine powder.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of powder formulation containing various amounts of VLP on days 0 and 21. Serum samples were collected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B) neutralizing antibodies. *P ⁇ 0.05 as compared to the placebo control group.
  • FIG. 9 Vaginal Norovirus-specific IgG production following intranasal immunization with GelVacTM vaccine powder.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of powder formulation containing various amounts of VLP on days 0 and 21.
  • Vaginal lavages samples were collected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B) Norovirus-specific IgG antibodies. *P ⁇ 0.05 as compared to the placebo control group.
  • FIG. 10 Serum and vaginal norovirus specific IgG production after 2 intranasal immunizations on day 0 and 21 followed by a parenteral immunization on day 42 of GelVacTM vaccine powder.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of powder formulation containing 100 ⁇ g of either GI or GII.4 VLP on days 0 and 21.
  • animals were immunized via an intramuscular (IM) injection of 20 mg of powder formulation containing 100 ⁇ g of GI or GII.4 VLP following reconstitution with water.
  • IM intramuscular
  • Vaginal lavages samples were collected on days 0, 14, 21, 42, and 56 and analyzed for Norovirus VLP specific IgG antibodies in serum (A) and Norovirus VLP specific IgG antibodies from vaginal swabs derived (B). *P ⁇ 0.05 as compared to the placebo control group.
  • FIG. 11 Serum norovirus-specific IgG and IgA production following intranasal immunization with GelVacTM monovalent and bivalent vaccine powders.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of a bivalent vaccine powder formulation containing various amounts of GI and GII.4 VLPs on days 0 and 21.
  • Serum samples were collected on day 0, 14, 21, 42, and 56 and analyzed for specific IgG antibodies against GI (A) and GII.4 (B).
  • Serum samples were also analyzed for specific IgA antibodies against GI (C) and GII.4 (D). Error bars are provided as geometric standard error. *p ⁇ 0.05 as compared to the placebo control group.
  • FIG. 12 Serum norovirus-specific IgG1 and IgG2 production following intranasal administration with GelVacTM dry powder monovalent and bivalent vaccine. Serum samples were analyzed for norovirus-specific IgG1 antibodies against GI (A) and GII.4 (B), and norovirus-specific IgG2 antibodies against GI (C) and GII.4 (D).
  • FIG. 13 Neutralizing antibody production following intranasal immunization with GelVacTM dry powder monovalent and bivalent vaccine.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of a bivalent vaccine powder formulation containing various amounts of GI and GII.4 VLPs on days 0 and 21. Serum samples were collected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B) neutralizing antibodies. Error bars are provided as geometric standard error. *p ⁇ 0.05 as compared to the placebo control group.
  • FIG. 14 Mucosal norovirus-specific antibody production following intranasal immunization with GelVacTM dry monovalent and powder bivalent vaccine.
  • Female Hartley guinea pigs were immunized intranasally with 20 mg of a bivalent vaccine powder formulation containing various amounts of GI and GII.4 VLPs on days 0 and 21.
  • Vaginal lavage samples were collected on days 0, 14, 21, 42, and 56 and analyzed for GI (A) and GII.4 (B) norovirus-specific antibodies.
  • animals were euthanized and intestinal lavage samples were analyzed for GI (C) and GII.4 (D) norovirus-specific antibodies. Error bars are provided as geometric standard error. *p ⁇ 0.05 as compared to the placebo control group.
  • the present invention includes formulations of a norovirus vaccine in a form of a dry powder, methods of producing such vaccine, and methods of performing immunization by administering such vaccine.
  • the present invention provides a formulation comprising at least two norovirus virus-like particle antigens.
  • “Norovirus” herein refers to members of the genus Norovirus of the family Caliciviridae.
  • norovirus includes a group of viruses that cause acute gastroenteritis in human and can be infectious to mammals including, but not limited to, human.
  • Norovirus may include at least five genogroups (GI-GV) defined by nucleic acid and amino acid sequences known in the art [40].
  • norovirus refers to a subset of genogroups.
  • norovirus refers to GI and GII. genogroups.
  • norovirus refers to GI, GII and GIV genogroups.
  • norovirus A number of examples of norovirus is known in the art. The examples include, but not limited to, Norwalk virus, Southampton virus, Desert Shield virus, and Hawaii virus. New strains of norovirus are routinely discovered [41]. Use of a combination of norovirus genogroups such as GI and GII or synthetic constructs representing combinations or portions thereof are considered in some embodiments.
  • Norovirus may refer to recombinant norovirus virus-like particles (VLPs).
  • VLPs are structurally similar and immunogenic as native norovirus, but lack the viral RNA genome of norovirus that is required for infection.
  • Virus-like particles” or “VLPs” herein refer to virus-like particles or fragments thereof, produced using methods known in the art [18, 19]. In some embodiments, VLPs are produced using baculovirus or tobacco mosaic virus [18, 19, 23].
  • Nevirus antigen or “antigen” herein refers to any form of proteins or peptides of norovirus VLPs and fragments thereof, that elicit immune response in vivo.
  • Norovirus VLPs may contain norovirus capsid proteins or fragments thereof such as, but not limited to, VP1 and VP2.
  • norovirus antigen comprises norovirus VLPs.
  • norovirus VLPs may be monovalent or multivalent. As used herein, “monovalent” refers to antigens derived from a single genogroup of norovirus. “Multivalent” refers to antigens derived from two or more genogroups of norovirus.
  • the formulation used herein comprises antigens derived from different genogroups of norovirus.
  • norovirus VLPs may have capsid proteins or derivatives such as VP1 and VP2 from different genogroups of norovirus.
  • a combination of monovalent or multivalent norovirus VLPs may be used in a formulation of a norovirus vaccine.
  • the resulting vaccine is referred as multivalent, comprising norovirus VLPs derived from different genogroups of norovirus.
  • a multivalent vaccine is bivalent, when it comprises two norovirus VLPs from two different genogroups; trivalent, when it comprises three norovirus VLP from three different genogroups.
  • antigens may be isolated and purified from organisms as naturally occurred. Antigens may be produced by recombinant techniques.
  • norovirus VLPs can be produced from cells such as prokaryotic or eukaryotic cells. Those cells include, but not limited to, E. coli, S. cerevisiae , insect cells such as Sf9, and mammalian cells such as HEK293 cells and CHO cells.
  • an antigen is a recombinant norovirus VLP derived from GI and/or GII genogroups. The recombinant norovirus VLPs may be expressed using baculovirus or tobacco mosaic virus [18, 19].
  • recombinant norovirus VLPs are expressed and produced from plants such as Nicotiana benthamiana , as described previously [23]. Briefly, clarified leaf extracts containing norovirus VLPs are filtered and concentrated. The extracts further run through a sepharose column, allowing the recovery of the VLPs. Endotoxins and other impurities may be further removed by a fractionation. In some embodiments, the purity of norovirus VLPs is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 100%. Norovirus VLPs as used herein preferably do not interfere with the efficacy of each VLP to elicit immunogenicity in vivo when used in combination.
  • vaccine or “vaccine formulation” refers to a formulation containing norovirus antigens that can be administered to mammals including human and elicit immune response in vivo.
  • the vaccine formulation of this invention may prevent and/or ameliorate an infection of norovirus.
  • the vaccine formulation may reduce at least one symptom related to norovirus infection.
  • the vaccine formulation may further enhance the efficacy of another dose of norovirus antigen.
  • immunogenicity refers to humoral and/or cell-mediated immune response. Humoral response leads to production of antibodies from B lymphocytes.
  • Cell-mediated immune response refers to response mediated by T lymphocytes or other cells such as macrophages.
  • norovirus vaccine is formulated as a form of a dry powder, capable of being administered by a mucosal route.
  • the dry powder vaccine is delivered via an intranasal way using an intranasal delivery device.
  • the dry powder vaccine formulation may be administered as a dry powder form or optionally be reconstituted in an aqueous solution prior to administration to mammals.
  • the vaccine formulation may be soluble in an aqueous solution.
  • the aqueous solution includes, but not limited to, water and saline buffer.
  • Other routes to administer the vaccine formulation are considered in some embodiments, and include, but not limited to, dermal and parenteral methods.
  • the vaccine is delivered by intramuscular injection. Detailed routes and options to administer the formulation of the present invention will be discussed in immunization methods section.
  • the present invention includes a formulation of norovirus vaccine that is monovalent or multivalent.
  • a multivalent norovirus vaccine may be formulated as one powder formulation containing one multivalent norovirus VLP antigen, or at least two monovalent or multivalent norovirus VLP antigens.
  • the multivalent vaccine may be formulated as a mixture of at least two dry powder formulations, each containing one monovalent or multivalent norovirus VLP antigen.
  • norovirus vaccine as used herein may comprise one or more norovirus VLP antigens derived from different genogroups of norovirus.
  • norovirus vaccine comprises two norovirus VLP antigens derived from different genogroups of norovirus.
  • norovirus VLPs may be derived from GI and GII. Norovirus VLPs may be present from 0.01 ⁇ g to 1,000 ⁇ g per 20 mg of dry powder vaccine formulation, depending on the desired dose. In some embodiments, norovirus VLPs are 10 ⁇ g to 50 ⁇ g per 20 mg of dry powder vaccine formulation.
  • a formulation of norovirus vaccine as used herein may further comprise at least one or more excipients.
  • Excipients used in the formulation preferably do not interfere with norovirus VLPs.
  • the excipient further enhances the therapeutic efficacy of the vaccine formulation by increasing mucosal residence time of administered vaccine formulation.
  • the formulation of the present invention may comprises at least one or more excipients categorized in the type of including, but not limited to, preservatives, viscosity adjusting agents, tonicity adjusting agents, and buffering agents.
  • the formulation in a form of a dry powder may also contain one or more excipients.
  • the vaccine formulation comprises a polymer with mucoadhesive properties.
  • the vaccine formulation comprises anionic polysaccharides.
  • Anionic polysaccharides include, but not limited to, dextran, guar gum, ben gum, methyl cellulose, and sodium polygalacturonate.
  • the vaccine formulation comprises sodium polygalacturonate and/or GelSite®.
  • GelSite® is a chemically and functionally distinct high molecular weight anionic polysaccharide (sodium polygalacturonate) extracted from an Aloe vera L. An exemplary method to extract a polymer from Aloe vera L.
  • the dry powder vaccine formulation may contain GelSite® in amounts of at least 0.01%, 0.1%, 0.25%, 0.5%, or 1% (w/w). In certain embodiments, the dry powder vaccine formulation contains 0.25% (w/w) of GelSite®.
  • GelSite® refers to a dry powder vaccine formulation comprising GelSite′ and at least one norovirus VLP antigen.
  • the formulation of the present invention may comprise one or more excipients with mucoadhesive properties, the formulation may not require immune adjuvants such as alum adjuvants.
  • the vaccine formulation may further comprise excipients such as, but not limited to, povidone and lactose.
  • Povidone polyvinylpyrrolidone
  • Lactose is also a commonly used excipient in the pharmaceutical industry.
  • Norovirus vaccine formulation of the present invention may be produced as a form of a dry powder and stored anhydrous until it is ready to be used.
  • Various methods to dry a formulation are known in the art [42]. The methods include, but not limited to, precipitation, crystallization, jet milling, spray-drying and lyophilizing (freeze-drying). Downstream operations may be further required, such as drying, milling and sieving.
  • the formulation can be freeze-dried, producing powders with desirable characteristics.
  • Cryo-milling may be further required in order to produce a homogenous mixture.
  • An exemplary lyophilized-milling method is incorporated herein as a reference (U.S. Pat. No. 8,074,906).
  • the formulation may be produced as a powder by a spray-drying method. Once it is in a form of a dry powder, norovirus vaccine may have an average diameter particle size from 1 ⁇ m to 100 ⁇ m.
  • At least two norovirus VLP antigens derived from different genogroups of norovirus are added in the formulation in order to produce a multivalent dry powder norovirus vaccine.
  • at least three norovirus VLP antigens from different norovirus genogroups are added in the formulation in order to produce a multivalent dry powder vaccine.
  • a multivalent dry powder norovirus vaccine is generated by mixing at least two dry powders, each containing one norovirus VLP antigen. Drying constitutes desiccating, dehydrating, or substantially dehydrating the formulation, such that a dry powder formulation is prepared.
  • each dried formulation may be milled using a mortar and pestle under a controlled, low-humidity ( ⁇ 10% RH) environment, and the formulation is optionally passed through a 70 ⁇ m filter to sterilize the formulation.
  • the amount of antigen in each antigenic or vaccine formulation dose is selected as an amount which induces a robust immune response without significant, adverse side effects.
  • the dose administered to a subject in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the subject over time, or to induce the production of antigen-specific antibodies.
  • the vaccine formulation is administered to a patient in an amount sufficient to elicit an immune response to the specific antigens and/or to alleviate, reduce, or cure symptoms and/or complications from the disease or infection. An amount adequate to accomplish this is defined as a “therapeutically effective dose.”
  • the vaccine formulation of the present invention may be administered through such as, but not limited to, mucosal, dermal, and parenteral routes.
  • Exemplary detailed routes further include, but not limited to, oral, topical, subcutaneous, intranasal, intravenous, intramuscular, intranasal, sublingual, transcutaneous, subdermal, intradermal, and suppository routes.
  • the vaccine formulation may be administered as a form of a dry power or reconstituted in an aqueous solution prior to administration.
  • at least two immunizations are given to a subject at once or separated by a few hours, days, months, or years.
  • one or more immunizations are administered by a mucosal route or a parenteral route.
  • one or more immunizations via a mucosal route is followed by one or more immunizations via a parenteral route or vice versa, to maximize both mucosal and systemic immune responses and protection against norovirus infection.
  • a dry powder vaccine is used for both parenteral and mucosal immunizations, i.e., the mucosal immunization is performed directly with the dry powder vaccine by intranasal delivery, whereas the parenteral immunization with the reconstituted dry powder vaccine by intramuscular injection.
  • the vaccine formulation of the invention may be administered to a subject to reduce the risk of norovirus infection prior to any future exposure to norovirus, ameliorate and/or treat symptoms of norovirus infection.
  • Symptoms of norovirus infection are well known in the art and include, but not limited to, nausea, vomiting, diarrhea, stomach cramping, a low-grade fever, headache, chills, muscle aches, and fatigue.
  • the invention encompasses a method of inducing an immune response in a subject not exposed to norovirus at the time of administration of the vaccine formulation of this invention.
  • the formulation may be administered to a subject currently experiencing a norovirus infection such that at least one symptom associated with norovirus infection is alleviated and/or reduced after administration.
  • a therapeutically effective dose for immunizing a subject with the vaccine is one that results in the generation of specific antibodies against the vaccinated antigen. Additionally, a therapeutically effective dose for immunizing a subject with the vaccine is one that results in increase in norovirus neutralizing antibodies in the subject. Increase in specific antibodies against norovirus antigens in the serum and mucosa of the subject confers active immunity. Similarly, generation of norovirus neutralizing antibody in the subject confers active immunity against a norovirus infection. Increase in the titer of specific antibody is usually proportional to the degree of protective immunity conferred by the vaccine.
  • a reduction in a symptom may be determined subjectively or objectively, e.g., self-assessment by a subject, by a clinician's assessment or by conducting an appropriate assay or measurement including, but not limited to, body temperature, a level of norovirus infection, antibody titer, and T cell counts.
  • Recombinant norovirus GI and GII VLPs expressed in Nicotiana benthamiana were obtained from Kentucky Bioprocessing (Owensboro, Ky.) as previously described [23] and used for powder formulations.
  • recombinant Norovirus GI and GII VLPs expressed and purified from Sf9 insect cells using the baculovirus expression system were also used [18, 19].
  • the GelVacTM vaccine powders were made with a lyophilization-milling method. Liquid formulations were first prepared using a formulation that is comprised of the recombinant VLP in a solution with GelSite® polymer, povidone and lactose. They were then lyophilized. Following lyophilization, dried formulation contain 0.25% (w/w) GelSite®, 99% lactose and 0.05% povidone and 0 ⁇ g to 100 ⁇ g (based on ELISA data) of VLP per 20 mg of formulation, depending on the desired dose. The GI and GII VLPs were added together to the formulation to produce the multivalent powder or individually to produce the monovalent powders.
  • the multivalent powder can also be produced by mixing together two or more monovalent powders.
  • Each dried formulation could be milled using a mortar and pestle under a controlled, low-humidity ( ⁇ 10% RH) environment and passed through a 70 ⁇ m filter.
  • the powder formulations can be made using a spray-drying apparatus as well. Powders were stored in sealed containers under desiccation at room temperature until use.
  • GI and GII VLP stocks were analyzed for the presence of intact VLPs by transmission electron microscopy prior to powder manufacturing. The results confirmed the presence of intact VLPs of the expected sizes (38 nm) for both GI and GII VLP stocks ( FIG. 1 ).
  • VLP stability was established by determining the melt temperature of norovirus VLPs with SYPRO Orange. Briefly, SYPRO Orange (Sigma-Aldrich, St. Louis, Mo.) was diluted in PBS to make a final 4 ⁇ concentration of SYPRO Orange. Each VLP was diluted in 4 ⁇ SYPRO Orange to a final concentration of 1 mg/mL. Each sample was then placed in a fluorescent thermocycler and was run through a 25′C-95′C gradient while reading the fluorescent signal. The derivative of the signal was determined by taking the difference between successive points in the fluorescent signal. The noise was reduced by using a 4-point moving average filter. Melt curve plots are shown for GI ( FIG. 2A ) and GII ( FIG. 2B ).
  • GI VLPs had two melt peaks, one minor peak at 43° C. and a major peak at 65° C.
  • GII VLPs showed a major peak at 65′C.
  • the major peak observed in the GII VLP melt curve is consistent with the major peak for GI VLPs.
  • VLP stability was also evaluated based on antigenicity.
  • Norovirus VLPs were incubated at various temperatures and tested in a capture ELISA.
  • Mouse monoclonal IgG2 anti-norovirus antibodies (Maine Biotech, MAB228 (GI); MAB227 (GII)) diluted 1:2000 in PBS were coated on Nunc MaxiSorp 96-well plates (Fisher Scientific, Pittsburgh, Pa.) overnight at 4° C. The wells were washed 5 times with wash buffer, and then blocked for 1 hr at room temperature in blocking buffer.
  • Norovirus VLPs were diluted in blocking buffer, and allowed to incubate on the plate at room temperature for 1 hr.
  • the wells were washed 3 times with wash buffer, followed by incubation with corresponding mouse monoclonal IgG1 anti-norovirus antibodies (Millipore, MAB80143 (GI); Maine Biotech MAB226 (GII)) diluted 1:2000 in blocking buffer for 1 hr at room temperature.
  • the wells were washed 3 times with wash buffer, followed by incubation with a polyclonal anti-mouse IgG1:HRP (Abcam, Cambridge, Mass.) diluted 1:2000 in blocking buffer for 1 hr at room temperature.
  • the wells were washed 3 times with wash buffer and were developed using 1-step Ultra TMB according to manufacturer's protocol (Thermo Scientific, Waltham, Mass.). The OD at 450 nm was measured and plotted against known VLP concentrations.
  • the GelVacTM vaccine powder was manufactured through a manual milling process under nitrogen gas.
  • Laser diffraction particle size distribution confirmed the volumetric mean particle size to be 24 ⁇ m to 37 ⁇ m for all powders, which was determined using a laser diffraction particle size analyzer with a liquid module (Beckman Coulter L513 320, Pasadena, Calif.). Furthermore, the d10 for the powders was approximately 5 ⁇ m for all powders, thus minimizing the amount of powders ( ⁇ 5 ⁇ m) which can reach deep lung.
  • a representative particle distribution result for each antigen can be found in Table 1.
  • the mean particle diameter for the GI VLP formulation was 29.73 ⁇ m and for GII VLP formulation was 25.2 ⁇ m.
  • Capture ELISAs were used to quantify the VLP dose content of each vaccine powder.
  • GI and GII capture ELISAs were performed with a 15 ⁇ g dose formulated vaccine. 10 mg of each 15 ⁇ g VLP dose powders were dissolved in 1 mL of water. Each powder was tested in the capture ELISA and compared to the VLP reference standard. Using a 4-PL fit, the antigen concentration of each powder was then quantified based on the weight of vaccine powder (Table 2). The capture ELISA was established using the GI- and GII-specific monoclonal antibodies. No cross-reactivity was observed between these two genogroups. In addition, no interference was observed with the powder formulation excipients.
  • the vaccine powders were administered intranasally using Aptar Unit Dose Spray (UDS) Devices (Aptar Pharma, Congers, N.Y.), one per nare with half of the total antigen dose per nare (10 mg total powder per nare).
  • the control group was administered the same amount of a placebo powder formulation.
  • Serum and vaginal lavage samples were collected from the animals on days 0 (preimmunization), 21, 42 and 56.
  • Serum samples were analyzed for norovirus VLP specific IgG by ELISA.
  • Norovirus GI or GII VLPs (2 ⁇ g/mL) in PBS were incubated on Nunc MaxiSorp 96-well plates (Fisher Scientific) for 4 hrs at room temperature. The plates were blocked overnight at 4° C. in blocking buffer. All samples were diluted in blocking buffer and serially diluted 2-fold down the plate. Samples were allowed to incubate at room temperature for 1 hr. The wells were washed 5 times with wash buffer, followed by incubation with anti-guinea pig IgG-HRP secondary antibodies (Southern Biotech, Birmingham, Ala.) at 1:1000 for 1 hr at room temperature.
  • the wells were washed 5 times with wash buffer.
  • the wells were developed using 1-step Ultra TMB according to manufacturer's protocol. End-point titers were reported as the reciprocal of the highest dilution that produced an OD of 0.1 above background.
  • a positive control serum generated in guinea pigs against GI or GII VLP was included in each test run to confirm reproducibility.
  • the total antigen specific IgG antibodies present in the serum exhibited a dose-dependent increase with both GI and GII vaccine powders ( FIG. 5 ).
  • serum IgG titers increased on day 21 and peaked by day 42 at all doses greater than 1 ⁇ g.
  • GI IgG titers increased by >600-fold for all dose groups of ⁇ 15 ⁇ g and GII IgG titers increased by >300-fold for all dose groups of ⁇ 5 ⁇ g.
  • a dose of the vaccine which corresponds to >100-fold increase in antigen-specific IgG titers in the subject confers active immunity against a norovirus infection.
  • both GI and GII vaccine powders induced a dose dependent antibody response.
  • Serum antigen specific IgG antibody production was correlated with amounts of both GI and GII VLP antigens present in the powders and reached a maximal level at 15 ⁇ g to 50 ⁇ g of VLP antigen.
  • Administration of higher doses of VLPs did not result in significantly higher levels of antigen specific IgGs. It is important to note that the boosting effect on systemic and mucosal IgGs was observed for each VLP antigen after the second dose on day 21.
  • IgG1 and IgG2 subclasses were also analyzed using the serum samples from each group ( FIG. 6 ). Both IgG1 and IgG2 exhibited similar response profiles as the total IgG described above. The IgG2 titers were apparently higher than IgG1 titers with both GI and GII VLP powders, especially at low dose levels (1 and 5 ⁇ g) with a difference of up to 100 fold ( FIG. 6 ).
  • Antigen-specific IgA serum levels were also investigated. At day 56, anti-GI and anti-GII VLP IgA antibodies were observed at all doses that were administered when compared to mock dose controls, except for the 1.0 ⁇ g dose group with GII ( FIG. 7 ). They also showed an overall trend of higher levels at higher antigen doses. These results showed that the VLP formulations with GelVacTM nasal powder were highly immunogenic and significant antigen specific antibody can be induced with GelVacTM nasal powders with GI at 15 ⁇ g and GII VLP at 5 ⁇ g.
  • HBGA histo-blood group antigen
  • GI neutralizing antibody titers were elevated in 15 ⁇ g dose group by day 21, in 15 ⁇ g, 50 ⁇ g, and 100 ⁇ g dose groups by day 42, and in all dose groups greater than 1 ⁇ g by day 56.
  • GII neutralizing antibody titers were elevated for the 100 ⁇ g dose group by day 21, elevated by day 42 in the 5 ⁇ g, 50 ⁇ g, and 100 ⁇ g dose groups, and elevated in all dose groups greater than 1 ⁇ g by day 56.
  • GI neutralizing antibody titers increased by >5-fold for all dose groups of >5 ⁇ g and GII neutralizing antibody titers increased by >10-fold for all dose groups >1 ⁇ g, consistent with the findings with serum IgG titers.
  • the lowest dose that produced a detectable neutralization titer at day 56 was 5 ⁇ g for both GI and GII.
  • the highest neutralizing antibody titers at day 56 occurred in the 15 ⁇ g dose group for GI and 100 ⁇ g dose group for GII.
  • mucosal antibody titers were evaluated in the reproductive tracts with vaginal lavage ( FIG. 9 ).
  • GI vaginal antibody titers were elevated in 50 ⁇ g dose group by day 21 and in all dose groups greater than 1 ⁇ g by day 56.
  • GII vaginal antibody titers were elevated in the 5 ⁇ g, 50 ⁇ g, and 100 ⁇ g dose groups by day 42.
  • the lowest dose that elicited a mucosal IgG response was 5 ⁇ g for both GI and GII.
  • the highest vaginal antibody titers occurred at 15 ⁇ g for GI and 100 ⁇ g for GII.
  • Antigen specific IgG antibody production in the vaginal tract showed similar trends to what was observed for both antigen specific IgG antibodies and neutralizing antibodies detected in serum. Presence of mucosal IgG antibodies is most likely conferred through transudation of serum IgG antibodies [39]. These results demonstrate that the GelVacTM vaccine powder is capable to inducing a mucosal response along with a neutralizing antibody response.
  • Serum VLP specific titers further increased by an additional 10-fold after the IM immunization for both GI and GII ( FIG. 10 ).
  • VLP specific IgG titers were also further increased in the vaginal lavage samples.
  • 2- to 4-fold increases in antigen specific IgG titers were observed in the vaginal lavage for both GI and GII VLPs.
  • VLP specific titers in vaginal lavage increased by 10-fold for both GI and GII VLPs.
  • a relatively larger increase in the mucosal antibodies was obtained after the IM immunization.
  • the present disclosure further shows that immune responses induced by a norovirus vaccine can be further enhanced by immunization via both parenteral and mucosal routes.
  • animals were first immunized with the norovirus VLP powder vaccine intranasally twice followed by an additional immunization via IM injection with the reconstituted powder vaccine, and a significant increase in both systemic and mucosal immune responses was obtained after the additional immunization by IM injection.
  • a norovirus vaccine can be administered with one or more doses via a mucosal route followed by one or more doses via a parenteral route or vice versa to further enhance the immune responses. There may be an interval of 2 to 4 weeks between the two routes of immunization.
  • the first dose is administered by the mucosal route and the second dose by the parenteral route.
  • the dry powder vaccine is particularly advantageous for immunization by the combinatorial routes, i.e., the mucosal immunization can be performed directly with the dry powder vaccine by intranasal delivery, whereas the parenteral immunization by IM injection is performed with the same dry powder vaccine after reconstitution, which may simply be carried out with sterile water.
  • the total antigen specific IgG antibodies present in the serum exhibited a dose-dependent increase with both GI and GII vaccine powders ( FIG. 11 ).
  • serum IgG titers increased on day 14 and a further increase was observed on days 21 and 42 at all doses >5 ⁇ g.
  • GI IgG titers increased by at >4-fold compared to controls. Further increases in GI IgG titers were observed at day 21 compared to day 14. Similar results were observed for GII IgG titers at day 14 and 21.
  • GI IgG titers increased by >600-fold compared to day 21 for all dose groups of ⁇ 5 ⁇ g and GII IgG titers increased by >300-fold compared to day 21 for all dose groups of ⁇ 5 ⁇ g.
  • the lowest dose that elicited an antigen specific IgG response was 5 ⁇ g for both GI and GII which corresponded to a titer of 30800 and 245840 on day 56, respectively.
  • Antigen specific IgA serum levels were also investigated.
  • anti-GI and anti-GII VLP IgA antibodies were observed at all doses that were administered when compared to the mock dose controls ( FIG. 11 ). They also exhibited an overall trend of higher levels at higher antigen doses.
  • the IgG1 and IgG2 subclasses were also analyzed using the pooled serum samples from each group ( FIG. 12 ). As shown, GI and GII IgG2 specific antibody titers were observed at day 14 but not IgG1 specific titers. GI and GII IgG2 specific titers were also shown to be higher than GI or GII IgG1 specific titers at day 21. IgG1 and IgG2 Boosting effects were also observed for both GI and GII at day 42. Overall, the IgG2 titers were higher than IgG1 titers for both GI and GII VLPs ( FIG. 12 ). These results show that the bivalent GI/GII VLP vaccine formulations were highly immunogenic and capable of producing a wide range of antibody responses.
  • Antigen specific antibodies were investigated for their ability to inhibit the binding of the norovirus VLPs to porcine gastric mucin.
  • the neutralizing antibodies present in the serum exhibited a dose-dependent response similar to that observed for antigen specific IgG antibody titers ( FIG. 13 ).
  • GI neutralizing antibody titers were elevated in the 15 ⁇ g, 50 ⁇ g, and 100 ⁇ g dose groups by day 42 with similar titers observed at day 56.
  • GII neutralizing antibody titers were elevated for all groups by day 42 with similar titers observed at day 56.
  • GI neutralizing antibody titers increased by >50-fold for all dose groups ⁇ 5 ⁇ g and GII neutralizing antibody titers increased by >190-fold for all dose groups ⁇ 5 ⁇ g, consistent with the findings with serum IgG titers.
  • the lowest dose that produced a detectable neutralization titer at day 56 was 15 ⁇ g for GI and 5 ⁇ g for GII.
  • mucosal antibody titers were evaluated in the reproductive tracts and intestines ( FIG. 14 ).
  • GI vaginal antibody titers were elevated in 50 ⁇ g and 100 ⁇ g dose group by day 21 and in all dose groups greater than 5 ⁇ g by day 56.
  • GII vaginal antibody titers were elevated in the 5 ⁇ g, 15 ⁇ g, 50 ⁇ g, and 100 ⁇ g dose groups by day 42.
  • the lowest dose that elicited a mucosal IgG response was 5 ⁇ g for both GI and GII.
  • the highest vaginal antibody titers occurred at 50 ⁇ g for both GI and GII.
  • vaginal IgG antibody titers exhibited a dose-dependent response.
  • GI and GII specific IgG titers were also observed in the intestines at day 56 ( FIGS. 14 C and D). As shown, antibody titers were observed in all treatment groups for both GI and GII specific antibodies.

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