WO2024259624A1 - Protéine f rsv modifiée, nanoparticule, composition et vaccin contre une infection par le virus respiratoire syncytial - Google Patents

Protéine f rsv modifiée, nanoparticule, composition et vaccin contre une infection par le virus respiratoire syncytial Download PDF

Info

Publication number
WO2024259624A1
WO2024259624A1 PCT/CN2023/101642 CN2023101642W WO2024259624A1 WO 2024259624 A1 WO2024259624 A1 WO 2024259624A1 CN 2023101642 W CN2023101642 W CN 2023101642W WO 2024259624 A1 WO2024259624 A1 WO 2024259624A1
Authority
WO
WIPO (PCT)
Prior art keywords
rsv
protein
nanoparticle
cell
plga
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2023/101642
Other languages
English (en)
Inventor
Hongliang LIV
Xiaolin Liu
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Genius Biotech Service Co Ltd
Original Assignee
Shenzhen Genius Biotech Service Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Genius Biotech Service Co Ltd filed Critical Shenzhen Genius Biotech Service Co Ltd
Priority to PCT/CN2023/101642 priority Critical patent/WO2024259624A1/fr
Publication of WO2024259624A1 publication Critical patent/WO2024259624A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0043—Nose
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/006—Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/543—Mucosal route intranasal
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • 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
    • 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
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—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
    • 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
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a modified RSV (Respiratory Syncytial Virus) F protein, a nanoparticle, a composition and a vaccine adapted for mucosal administration, and in particular for intranasal administration.
  • RSV Respiratory Syncytial Virus
  • RSV Human respiratory syncytial virus
  • RSV is an enveloped virus with a non-segmented negative-sense RNA genome of approximately 15,200 nucleotides. The genome is expressed as 10 separate mRNAs encoding 11 proteins.
  • the 3’ to 5’ gene order (identified by encoded proteins) is as follows: nonstructural protein 1 (NS1) , NS2, nucleocapsid protein (N) , phosphoprotein (P) , matrix protein (M) , small hydrophobic protein (SH) , attachment protein (G) , fusion protein (F) , RNA synthesis factors M2-1 and M2-2 (encoded by overlapping open reading frames [ORFs] in the M2 mRNA) , and polymerase protein (L) ; there also are short leader and trailer regions at the 3’ and 5’ genome ends, respectively.
  • NS1 nonstructural protein 1
  • N nucleocapsid protein
  • P phosphoprotein
  • M matrix protein
  • SH small hydrophobic protein
  • F attachment protein
  • F RNA
  • the RSV G protein is the major viral attachment protein.
  • RSV F mediates fusion of the viral envelope with the cellular membrane during viral entry and may also have attachment activity.
  • the RSV F and G proteins are the two RSV neutralization antigens and the major protective antigens. F is generally considered to be a more potent neutralization and protective antigen than G, and its amino acid sequence is much more conserved among RSV strains.
  • RSV F is produced in a prefusion (pre-F) conformation that is metastable and can be readily triggered to undergo a major irreversible conformational rearrangement that drives membrane fusion and leaves F in a highly stable post-fusion (post-F) conformation.
  • Pre-F and post-F share some neutralizing epitopes, but most of the neutralizing activity in convalescent human sera recognizes epitopes specific to pre-F.
  • RSV F can be substantially stabilized in the pre-F conformation by structure-based engineering, such as by the introduction of a disulfide bond called DS and two hydrophobic cavity-filling amino acid substitutions called Cav1.
  • DS-Cav1-stabilized pre-F is substantially more immunogenic in rodents and nonhuman primates than post-F either as a subunit vaccine or expressed by a bacteria vector.
  • RSV candidate vaccines consist of either inactivated whole virus, recombinant subunit vaccine, virus vector attenuated vaccine or mRNA vaccine. Fusion proteins are the antigens to which protective antibody responses are directed, fusion protein being the major protective antigen. Estimates of the efficacy of these parenterally administered vaccines vary greatly. Such vaccines are believed to act primarily by eliciting circulating anti-RSV IgG antibodies that transudate into the lower respiratory tract.
  • Mucosal administration of vaccine would have a number of advantages over traditional parenteral immunization regimes. Paramount amongst these is more effective stimulation of the local mucosal immune system of the respiratory tract and the likelihood that vaccine uptake rates would be increased because the fear and discomfort associated with injections would be avoided. Accordingly, a number of attempts have been made to develop mucosal vaccines. A drawback however is that subunit vaccines are often poorly immunogenic when given mucosally.
  • the present invention provides a modified RSV F protein, a nanoparticle, a composition and a vaccine adapted for mucosal administration, and in particular for intranasal administration.
  • Intranasal administration of the modified RSV F protein, the nanoparticle, the composition and the vaccine elicits mucosal and systemic antiviral immunity, resulting in the reduced virus-associated pathology and reduced viral burdens.
  • the present invention provides a modified RSV F protein, wherein the modified RSV F protein comprises an amino acid sequence having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1.
  • the modified RSV F protein further comprises an inactivated primary fusion cleavage site.
  • the inactivated primary fusion cleavage site is obtained by mutation of arginine residues at positions 133, 135, and 136 of SEQ ID NO: 1 to glutamine.
  • the modified RSV F protein comprises or consists of SEQ ID NO: 5.
  • the modified RSV F protein is a monomeric RSV F protein.
  • the present invention provides a nucleic sequence encoding the modified RSV F protein according to the first aspect.
  • the present invention provides a cell comprising the nucleic sequence according to the second aspect.
  • the cell is E. coli DH5 ⁇ cell.
  • the E. coli DH5 ⁇ cell is named as pCBS220-sF/DH5 ⁇ , classified as E. coli DH5 ⁇ engineering strain of recombinant human respiratory syncytial virus fusion protein F, has been deposited with China General Microbiological Culture Collection Center (CGMCC) (No. 1, West Beichen Rd., Chaoyang District, Beijing 100101, China) under the accession number CGMCC No. 25524 since Aug. 12, 2022.
  • CGMCC General Microbiological Culture Collection Center
  • the present invention provides a nanoparticle comprising a viral protein and at least one polymer (s) , wherein the viral protein consists of RSV antigen or antigens, wherein the RSV antigen contains the modified RSV F protein according to the first aspect.
  • the nanoparticle is immunogenic.
  • the nanoparticle is a RSV antigen or antigens entrapped within the nanoparticle.
  • the polymer is a water soluble, non-adhesive polymer.
  • At least one polymer is selected from the group consisting of poly (lactic-co-glycolic acid) , polyethylene glycol, polyethylene oxide, polyalkylene glycol, polyalkylene oxide and polyethylene glycol-poly (lactic-co-glycolic acid) polymer.
  • the polymer is poly (lactic-co-glycolic acid) (PLA) .
  • the nanoparticle has an average diameter of about 250 nm to about 600 nm as measured by dynamic light scattering.
  • the present invention provides a composition comprising: (i) the nanoparticle according to according to the second aspect; and (ii) an adjuvant.
  • the adjuvant is a Mycobacterium lysate.
  • the adjuvant is a Mycobacterium tuberculosis whole cell lysate.
  • the present invention provides a vaccine comprising the composition according to the fifth aspect and a pharmaceutically acceptable carrier.
  • the present invention provides a method for preventing, ameliorating and/or treating disease caused by infection of the RSV virus comprising administering the effective amount of the composition according to the fifth aspect or the vaccine according to the sixth aspect to a subject in need thereof.
  • the present invention provides a method for eliciting an immune response comprising administering the effective amount of the composition according to the fifth aspect or the vaccine according to the sixth aspect to a subject in need thereof.
  • the composition or vaccine is mucosally administered.
  • the composition or vaccine is administered to mucosal surface.
  • mucosal surface is selected from the group consisting of intratracheal mucosal surface, intranasal mucosal surface, rectal mucosal surface and vaginal mucosal surface.
  • the subject is a human.
  • the subject is a child with ⁇ 5 years of age.
  • Fig. 1 shows PAGE Blue staining results of purified F protein, F monomers, Trimers F.
  • Fig. 2 shows negative stain electron microscopy of RAg.
  • Fig. 3 shows morphology of RAgs entrapped PLGA nanoparticles.
  • Fig. 4 shows r F protein release kinetics from PLGA. Data shown is the cumulative mass fraction of F protein released from PLGA nanoparticles. Data represent mean ⁇ SEM. Results are representative of three independent experiments with duplicate samples.
  • Fig. 5A shows the relative expression of the inflammatory cytokine IL-8 in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 5B shows the relative expression of the inflammatory cytokine IL-12 p40 in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 5C shows the concentration of the inflammatory cytokine INF- ⁇ in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 5D shows the concentration of the inflammatory cytokine IL-1 ⁇ in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 5E shows the concentration of the inflammatory cytokine IL-6 in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 5F shows the concentration of the inflammatory cytokine TNF- ⁇ in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 6A shows the concentration of the inflammatory cytokine INF- ⁇ in the alveolar macrophages stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 6B shows the concentration of the inflammatory cytokine IL-1 ⁇ in the alveolar macrophages stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 6C shows the concentration of the inflammatory cytokine IL-6 in the alveolar macrophages stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Fig. 6D shows the concentration of the inflammatory cytokine TNF- ⁇ in the alveolar macrophages stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.
  • Lung tissue sections were stained with hematoxylin and eosin (H&E) to assess inflammation.
  • A PLGA+WCL (control) ;
  • B RSV-F+WCL (I.N. ) ;
  • C RSV-F-NP (I. N) suspension;
  • D RSV-F-NP+WCL (I.N. ) .
  • Fig. 8 shows the reduced viral Burden in the lungs of vaccinated animals.
  • Fig. 9A shows the increased RSV specific IgA in the nasal fluid.
  • Fig. 9B shows the increased RSV specific IgA in the BAL fluid.
  • Nasal fluid was collected on days 0, 14 and 28 post-vaccination, and on days 3 and 6 post-challenge.
  • BAL fluid was collected during necropsy on day 7 post-challenge.
  • the samples were diluted 1: 2.
  • Fig. 10A shows the enhanced RSV-Specific T cell Response in the Peripheral blood.
  • Fig. 10B shows the concentration of the inflammatory cytokine IFN ⁇ secreted by PBMCs from the BRSV-F nanovaccine-administrated animals in response to whole virus.
  • Fig. 10C shows the concentration of the inflammatory cytokine IL-17A secreted by PBMCs from the BRSV-F nanovaccine-administrated animals in response to whole virus.
  • RAg recombinant F
  • rF recombinant F
  • the recombinant F (rF) comprises one immunogenic RSV proteins and therefore the recombinant F (rF) can be considered a subunit antigen.
  • NP-RAg nanoparticle-recombinant antigen. This represents the nanoparticle encapsulated recombinant rF.
  • treatment or “treating, ” or “alleviating” or “ameliorating” are used interchangeably herein. These terms refers to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that amelioration is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.
  • prevention and “preventing” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a prophylactic benefit.
  • mammals preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans.
  • the combinations of the invention can be administered as described herein to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like) , farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like) .
  • the mammal treated in the methods of the invention is a child with ⁇ 5 years of age.
  • an effective amount of a disclosed composition or the vaccine required for use in therapy varies with the nature of the condition being treated, the length of treatment time desired, the age and the condition of the subejct, and is ultimately determined by the attending physician.
  • doses employed for infants and young children treatment typically are in the range of about one or more doses of composition or the vaccine described herein.
  • the desired dose may be conveniently administered in a single dose consisting of between about 100 ⁇ g and about 150 ⁇ g of composition or the vaccine described herein.
  • modified RSV F protein wherein the modified RSV F protein comprises an amino acid sequence having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1.
  • the modified RSV F protein further comprises an inactivated primary fusion cleavage site.
  • the inactivated primary fusion cleavage site is obtained by mutation of arginine residues at positions 133, 135, and 136 of SEQ ID NO: 1 to glutamine.
  • the modified RSV F protein comprises or consists of SEQ ID NO: 5. Over time, small amount of truncated RSV F peptide may arise due to proteolysis. Advantageously, however, the modified RSV F protein disclosed herein minimizes such degradation and provides extended stability.
  • the modified RSV F protein is a monomeric RSV F protein.
  • Described herein is a nucleic sequence encoding the modified RSV F protein according to the present invention.
  • Described herein is a cell, wherein the cell comprises the nucleic sequence according to the present invention.
  • the cell is E. coli DH5 ⁇ cell.
  • the E. coli DH5 ⁇ cell has been deposited with China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC No. 25524.
  • CGMCC China General Microbiological Culture Collection Center
  • nanoparticle comprises a viral protein and at least one polymer (s) , wherein the viral protein consists of RSV antigen or antigens, wherein the RSV antigen contains the modified RSV F protein according to the present invention.
  • an RSV F nanoparticle comprises one or more the modified RSV F protein monomer encapsulated with PLGA.
  • the RSV F nanoparticle, the nanoparticle has an average diameter of about 200 nm to about 600 nm as measured by dynamic light scattering.
  • each RSV F protein monomer contains an RSV F protein selected from the group consisting of RSV F proteins having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1.
  • each RSV F protein monomer contains an RSV F protein selected from the group consisting of RSV F proteins having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1 and an inactivated primary fusion cleavage site.
  • the modified RSV F protein induces the production of neutralizing antibodies.
  • the neutralizing antibodies recognize the modified RSV F protein in a post-fusion state and/or a pre-fusion state.
  • nanoparticles include, but are not limited to nanoparticles composed of one or more polymers.
  • the one or more polymers is/are a water soluble, non-adhesive polymer.
  • polymer is polyethylene glycol (PEG) or polyethylene oxide (PEO) .
  • the polymer is polyalkylene glycol or polyalkylene oxide.
  • the one or more polymers is/are a biodegradable polymer.
  • the one or more polymers is/are a biocompatible polymer that is a conjugate of a water soluble, non-adhesive polymer and a biodegradable polymer.
  • the biodegradable polymer is poly (lactic-co-glycolic acid) (PLGA) .
  • the nanoparticle is composed of PEG-PLGA polymers.
  • the nanoparticle is formed by self-assembly.
  • Self-assembly refers to the process of the formation of a nanoparticle using components that will orient themselves in a predictable manner forming nanoparticle predictably and reproducably.
  • the nanoparticles are formed using amphiphillic biomaterials which orient themselves with respect to one another to form nanoparticles of predictable dimension, constituents, and placement of constituents.
  • the nanoparticle has a positive zeta potential. In some embodiments, the nanoparticle has a net positive charge at neutral pH. In some embodiments, the nanoparticle comprises one or more amine moieties at its surface. In some embodiments, the amine moiety is a primary, secondary, tertiary, or quaternary amine. In some embodiments, the amine moiety is an aliphatic amine. In some embodiments, the nanoparticle comprises an amine-containing polymer. In some embodiments, the nanoparticle comprises an amine-containing lipid. In some embodiments, the nanoparticle comprises a protein or a peptide that is positively charged at neutral pH. In some embodiments, the nanocarrier is a latex particle. In some embodiments, the nanoparticle with the one or more amine moieties on its surface has a net positive charge at neutral pH.
  • PLGA PLGA-derived mixing about 180 mg of PLGA to about 5 mg of RAg (or about 36 mg PLGA to 1 mg RAg) .
  • the entrapment (encapsulation) efficiency of RAg can vary.
  • the nanoparticle was 50-55%entrapped/encapsulated, calculated based on amount of total RSV protein used in the entrapment.
  • Entrapped recombinant RAg can be administered as mixtures of entrapped/encapsulated and unentrapped/unencapsulated antigen or the entrapped/encapsulated antigens can be further purified.
  • Nanoparticles can aid the delivery of the recombinant RAg and/or can also be immunogenic. Delivery can be to a particular site of interest, e.g. the mucosa.
  • the nanoparticle can create a timed release of the RAg to enhance and/or extend the immune response.
  • the nanoparticle is associated with the RAg such that the composition can elicit an immune response.
  • the association can be, for example, wherein the nanoparticle is coupled or conjugated with the RAg.
  • coupled and conjugated is meant that there is a chemical Linkage between the nanoparticle and the RAg.
  • the recombinant RAg is entrapped or encapsulated within the nanoparticle.
  • the RAg is entrapped within the nanoparticle by a water/oil/water emulsion method.
  • an RSV F nanoparticle comprises one or more RSV F protein monomer encapsulated with PLGA.
  • the RSV F nanoparticle, the nanoparticle has an average diameter of about 250 nm to about 600 nm as measured by dynamic light scattering.
  • a method of manufacturing an RSV F protein nanoparticle comprises preparing an RSV F protein extract from a host cell.
  • the weight ratio of PLGA: RSV F protein is about 180 to about 5.
  • the disclosure provides compositions containing RSV viral protein nanoparticles.
  • the RSV proteins are recombinantly expressed in a suitable host cell.
  • the host cell is an bacteria cell.
  • the bacteria cell is a DH5 ⁇ cell.
  • the composition comprises: (i) the nanoparticle according to the present invention; and (ii) an effective amount of adjuvant.
  • An adjuvant component can increase the strength and/or duration of an immune response to an antigen relative to that elicited by the antigen alone.
  • a desired functional characteristic of an adjuvant component is its ability to enhance an appropriate immune response to a target antigen.
  • the adjuvant can be any composition, pharmacological or immunological agent that modifies the effect of other agents, such as the antigens described herein.
  • adjuvants include, but are not limited to Mycobacterium lysate (including a Mycobacterium tuberculosis whole cell lysate) , a Mycobacterium smegmatis (including Mycobacterium smegmatis whole cell lysate) , cholera toxin B subunit, and E. coli heat labile mutant toxin.
  • adjuvants include evolutionarily conserved molecules, so called PAMPs, which include liposomes, lipopolysaccharide (LPS) , molecular cages for antigen, components of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA (dsRNA) , single-stranded DNA (ssDNA) , and unmethylated CpG dinucleotide-containing DNA.
  • PAMPs evolutionarily conserved molecules
  • LPS lipopolysaccharide
  • dsRNA double-stranded RNA
  • ssDNA single-stranded DNA
  • adjuvants include, but are not limited to aluminum containing adjuvants that include a suspension of minerals (or mineral salts, such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate) onto which antigen is adsorbed.
  • adjuvants include, but are not limited to aluminum- (alum-) free adjuvants, which are formulated in the absence of any such aluminum salts.
  • Alum-free adjuvants include oil and water emulsions, such as water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions) , liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides) , liposomes, Toll-like Receptor agonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists) , and various combinations of such components.
  • the adjuvant is a Mycobacterium tuberculosis whole cell lysate.
  • the disclosure provides immunogenic compositions comprising one or more viral protein species in a nanoparticle structure where the protein is in the form of a monomer and each nanoparticle contains at least one monomer entrapped with PLGA.
  • a nanoparticle consists of an antigen, such as a viral protein, from only one pathogen.
  • a vaccine wherein the vaccine comprises the composition and a pharmaceutically acceptable carrier.
  • the RSV vaccine comprises one RSV F nanoparticle wherein each nanoparticle comprises at least one RSV F protein monomer surrounded by PLGA.
  • the carrier may be any suitable carrier known to a person skilled in the art, for example a protein or an antigen presenting cell, such as a dendritic cell (DC) .
  • Carrier proteins include keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier protein may be tetanus toxoid or diphtheria toxoid.
  • the carrier may be a dextran such as sepharose. The carrier must be physiologically acceptable to humans and safe.
  • the immunogenic composition and vaccine disclosed herein are suitable for preventing, ameliorating and/or treating disease caused by infection of the RSV virus.
  • Disclosed herein is a method for preventing, ameliorating and/or treating disease caused by infection of the RSV virus, wherein the method comprises administering the effective amount of the composition or the vaccine to a subject in need thereof.
  • Disclosed herein is a method for eliciting an immune response, wherein the method comprises administering the effective amount of the composition or the vaccine to a subject in need thereof.
  • a method of preventing, ameliorating and/or treating disease caused by infection of the RSV virus or for eliciting an immune response comprises administering one or more doses of the vaccine composition.
  • each dose consists of between about 100 ⁇ g and about 150 ⁇ g of the protein antigen.
  • the composition or vaccine is mucosally administered.
  • the composition or vaccine is administered to mucosal surface.
  • mucosal surface is selected from the group consisting of intratracheal mucosal surface, intranasal mucosal surface, rectal mucosal surface and vaginal mucosal surface.
  • the subject is a human.
  • the subject is a child with ⁇ 5 years of age.
  • nanoparticles for inducing immune responses, methods for producing and administering mucosally them and composition or vaccine containing them.
  • the nanoparticle provides antigen surrounded with PLGA that result in a structure that provides enhanced the stimulation of a protective IgA mucosal immune response and an IgG systemic immune response.
  • the nanoparticles offer especially good antigen presentation to immune systems.
  • a plasmid with the DNA encoding the vaccine candidate was prepared, amplified and purified from E. coli cells and were transformed.
  • the protein was expressed and purified from cell fermentation.
  • the amino acid sequence of the RSV F protein was shown as SEQ ID NO: 1:
  • amino acid sequence of the signal peptide was shown as SEQ ID NO: 2
  • the amino acid sequence of the TEV cleavage site was shown as SEQ ID NO: 3
  • amino acid sequence of the solubility enhancer 6 ⁇ his Tag was shown as SEQ ID NO: 4:
  • amino acid sequence of the modified RSV F protein was shown as SEQ ID NO: 5:
  • the additional amino acid residues flanking the N-and C-terminal ends of the F were added for restriction sites (Hind III/Age I/Kpn I and Xma I/Nsi I, respectively) to facilitate transfer of the gene into different expression vectors. Following cleavage of the signal peptide, the C-terminal E residue is expected to remain on the final antigen. Additional non-coding sequences were used at the ends of the gene to ensure optimal Kozak sequence and compatibility with the plasmid pCBS220. The final construct was sequenced to confirm intactness of the F gene.
  • the plasmid pCBS220-F was transformed into E. coli DH5 ⁇ (Bio-RAD) according to the manufacturer’s instructions.
  • the designated pCBS220-sF/DH5 ⁇ has been deposited with China General Microbiological Culture Collection Center (CGMCC) on August 12, 2022 under the accession number CGMCC No. 25524.
  • the resulting strains were tested first at the shake flask scale. Induced bands of the expected size for processed and unprocessed (56 kDa and 180 kDa, respectively) were detected by SDS-PAGE. About half of the protein was processed (indicating localization to the periplasm) , and of the processed about half was in the soluble fraction and half in the insoluble fraction. Expression studies were scaled up to 20-L bioreactors. Densitometry of the Coomassie-stained SDS-PAGE gels showed that 18%of the total RSV F produced was processed and soluble. The strain produced 3.2 g/L of all forms of RSV F processed and soluble was 0.6 g/L. Recombinant F was isolated from the cell extract of a shake flask experiment using the Qiagen Ni-NTA protocol. This F was found to be active against RSV in an ELISA assay.
  • Lysis buffer comprised the following components: Tris HCl, 50 mM, pH 7.5 final; NaCl, 200 mM; glycerol, 5%v/v; EDTA, 20 mM; Triton X-100, 5%v/v; and, added last, DTT, 1 mM.
  • Screw-cap microfuge tubes (2 mL) were filled about 3/4 full with 0.1 mm glass beads and topped off with cell suspension.
  • the tubes were given a quick shake to mix the beads and removed air bubbles, and further filled to the top with cell suspension.
  • the tubes were capped, sealed, and inserted into a BioSpec mini bead-beater for 60 seconds at 5000 rpm.
  • the samples were kept on ice between beatings and beat 3 to 5 times until about 90%of the cells were lysed.
  • Cell lysis was observed by microscopic observation.
  • a volume of 0.025 mL of the lysed cell preparation was pipetted from each tube, minus beads, into new microfuge tubes and centrifuged for 5 minutes. The supernatant fraction was carefully transferred to another tube with 0.075 mL LSB, and 0.100 mL LSB was added to the pellet fraction.
  • SARS-COV F The solubility of SARS-COV F in cells was tested bacteria and indicated that most, if not all, of the SARS-COVF remained in soluble form. To do these solubility-tests, viable un-amended bacteria cells were broken in a French Press (or mini-bead beater) , and centrifuged to separate cell debris and any inclusion bodies from soluble proteins. SDS gels of these two fractions indicated that SARS-COV F was retained in the soluble portion.
  • bacteria is a good biofactory, capable of producing up to 40%or more of total cell protein as recombinant protein, such as SARS-COV F and the cells produced active protein.
  • Cell culture medium was harvested, loaded into the Ni-NTA column and washed with washing buffer (50 mM Na 2 HPO 4 , 300 nM NaCl, 20 mM imidazole, pH 8.0) , and eluted with elution buffer washing buffer containing 250 nM imidazole) , and purified from the soluble fraction by denaturing Ni-NTA chromatography.
  • washing buffer 50 mM Na 2 HPO 4 , 300 nM NaCl, 20 mM imidazole, pH 8.0
  • elution buffer washing buffer containing 250 nM imidazole purified from the soluble fraction by denaturing Ni-NTA chromatography.
  • the purified fusion protein was incubated with (Factor Xa +TEV 1: 1) protease (Novagen) at room temperature in a buffer containing 20 mM Tris, pH 8.0, 0.2 M NaCl and 5 mM CaCl 2 .
  • One unit of Factor Xa was used for 75–100 ⁇ g of target protein.
  • the reaction was allowed to proceed for 36–48 h for maximal cleavage (90–95%) of the fusion protein.
  • the fractions were concentrated using Amicon Ultra concentrator (Millipore) with a 10 kDa cut-off filter. Protein concentration was determined by measuring absorbance at 280 nm using multi-mode microplate reader (BioTek Synergy 2) .
  • Protein purity and integrity were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by visualization of the protein using PAGE Blue stain (Invitrogen) .
  • PAGE Blue staining results of purified F protein, F monomers, Trimers F were shown in Fig. 1.
  • Negative stain electron microscopy of Rag was shown in Fig. 2, indicating that the RAg was highly homogenous.
  • PLGA mol wt 40-75 kDa
  • polyvinyl alcohol mol wt. 30-70 kDa
  • Dicholoro methane Acros Organics
  • BCA bisinchoninic acid protein assay kit
  • Mycobacterium species can be grown in liquid medium as recommended by ATCC (American Type Culture Collection) .
  • BCG Bacillus Calmette-Guerin
  • WCL can be prepared.
  • Live bacteria can be harvested and washed twice using PBS (pH 7.4) and suspended (2 g/ml) in PBS containing 8 mM EDTA, proteinase inhibitors, DNase and RNase.
  • Cells can be disrupted by using the Bead Beater until approximately 90%breakage was obtained (monitored by acid fast staining) , centrifuged at 3,000 ⁇ g to pellet unbroken cells and insoluble cell wall components, and the supernatant (WCL) can be harvested.
  • the protein content and endotoxin levels in the WCL can be quantified using the kits, and the aliquots will be stored at -70°C.
  • the mycobacterial WCL preparation contains water-soluble proteins, lipids, and carbohydrates.
  • Nanoparticles were prepared by a standard double emulsion solvent evaporation method. Briefly, 15%of PLGA (1500 mg) was dissolved in 5 ml of dichloromethane and 10 mg F protein was added. The mixture was homogenized for 90 seconds using a homogenizer at 6000 rpm. The homogenized mixture was added to 120 ml of aqueous solution of polyvinyl alcohol (10%PVA) , and homogenized for 5 min. Finally, the preparation was stirred overnight at room temperature (RT) to allow solvent evaporation. The nanoparticles were washed in distilled water for three times and the wet nanoparticles were freeze-dried and stored at 4 °C.
  • RSV F antigens as described previously used in the Example 1 PLGA NPs entrapped with RAg (NP-RAg) or M. tb (Mycobacterium tuberculosis) WCL (NP-M tb WCL) were prepared by double emulsion method (w/o/w) .
  • nanoparticle size and morphology of nanoparticle was detected using scanning electron microscopy. Freeze-dried nanoparticles were mounted on an adhesive stub coated with gold platinum under vacuum using an ion coater. The coated specimen was examined under the microscope at 10 KV. The amount of entrapped r F in the nanoparticles was determined.
  • Entrapped protein in NPs was estimated. Morphology of the Nano-RAg was visualized using the Philips XL30-FEG scanning electron microscope (SEM) at 20 kV at 30,000 ⁇ magnification. Size distribution of the sham or RAg entrapped NPs was measured using NICOMP 370 particle sizer (Particle Sizing Systems, CA) . The zeta potential of the NPs was determined by ZetaPALS (Brookhaven Instruments Corp., NY) .
  • the assay was performed, 100 mg NP-RAg was suspended in two ml PBS and the supernatant was collected immediately to estimate the burst release.
  • the pellet of NP-RAg was resuspended repeatedly with two ml PBS and the supernatants were collected at 1, 5, 10, 15, 20, 25, and 30 days and stored at -20 °C. On day 30, undegraded NPs were lysed to recover the protein, and all the samples were estimated for protein concentration by BCA method.
  • ELISA plates were coated with 5 ⁇ g/mL (2.5 ⁇ g/mL each) of the recombinant F proteins (BRSV-F) , or with 100 ⁇ L/well of RSV virus stock ( ⁇ 10 4 TCID 50 ) grown in Hep4 cells.
  • Recombinant RSV F proteins were encapsulated in PLGA particles and released as described. The released proteins were also coated onto ELISA plates at ⁇ 5 ⁇ g/mL (BRSV-F NP) .
  • Sera from RSV-immune rats were diluted 1: 1000 and added to the plates. The binding of rats IgG to the virus or recombinant proteins was measured by absorbance.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • PSV F proteins 5 ⁇ g/mL recombinant RSV F proteins that were encapsulated and released from the PLGA particles; or 0.01 MOI of RSV strain Long.
  • Pokeweed Mitogen was used at a concentration of 1 ⁇ g/mL as a positive control.
  • Mock stimulated samples (negative control wells) were cultured with cRPMI and were used to correct for background proliferation.
  • Nano-RAg vaccine containing 4 ⁇ g of RSV RAg were suspended in one ml of RPMI and incubated with PAM (ATCC) for 0, 5, 20, 30 min, 3, 12, and 24 hr.
  • RAg (4 ⁇ g) , sham NPs, and HEp-2 cells infected for 24 hr with RSV strain Long (ATCC) were included as controls.
  • Cells were washed and fixed with acetone and incubated with RSV F mAb followed by anti-mouse IgG (H+L) Alexa-488 (Life Technologies, Grand Island, N. Y. ) , and observed under the inverted fluorescent microscope.
  • Monocyte-derived dendritic cells were prepared from adult rats. The moDC were generated. Recombinant rats IL-4 and recombinant rats GM-CSF were purchased from (JDK, Beijing) . Rat moDC were seeded at 5 ⁇ 10 5 cells per well in a 24-well plate and stimulated with 10 ⁇ g/mL ‘empty’ or RSV-F-loaded PLGA particles. Mock cultures were treated with media only. After 18 hours, RNA was isolated from the cells and analyzed by qPCR for expression of IL-8 and IL-12p40. For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples.
  • NP-RAg vaccine was rapidly endocytosed by cotton rat alveolar macrophages (MAMs) and sustained release of entrapped NP-RAg over several weeks. Potency of NP mediated delivery of drugs or vaccines depend on their loading capacity and size.
  • the entrapment efficiency of RAg (rRSV F) and M. tb WCL in NPs was about 50-60%. Sham or entrapped NP particles were circular with smooth surface.
  • Dynamic light scattering (DLS) of NPs measured their diameter based distribution, and the mean diameter ⁇ SD of sham, NP-RAg, and 151 NP-M. tb WCL were 490 ⁇ 45, 510 ⁇ 44, and 670 ⁇ 76 nm, respectively.
  • NPs 85%of sham NPs, 92%of NP-M. tb WCL, and 78%of NP-RAg were in the size range of 400-700 nm.
  • surface electrostatic potential -26 mV
  • Fig. 3 Scanning electronic photomicrograph of PLGA nanoparticles prepared by a standard multiple emulsion method. The size of the nanoparticles appears to be variable ranging 400–700 nm. The encapsulation efficiency of F protein in then nanoparticles was 20%.
  • the surface associated protein in nanoparticles was equivalent to the amount of protein released at time zero (i.e., immediately after reconstitution in PBS) , called as burst release and it was 9.5%in NP-RAg vaccine.
  • the release during first 24 hr after reconstitution was 30.5%, and after 30 days 61%of the entrapped protein was released (as shown in Fig. 4) .
  • the remaining 39%of viral Ag was recovered in the un-lysed NPs.
  • NP-RAg Even one year old NP-RAg stored at -20 °C, NP-RAg had 13.6%burst release, and 76%of released antigens by 30 days, indicating that PLGA NPs retain the entrapped vaccine beyond one year and allow its sustained release over a period of several weeks under normal physiological conditions. Thus, the results indicated that as expected PLGA nanoparticles could efficiently retain and permit sustained release of entrapped RSV Ag over a long period of time.
  • Immunogenicity of recombinant RSV-F was preserved following release from PLGA nanoparticles.
  • the recombinant RSV F was immunogenic and stable following encapsulation and release from the PLGA nanoparticles.
  • CD4 T cells from RSV-immune cows underwent clonal expansion in recall response to RSV, recombinant RSV-F and the released RSV-F/G proteins. We observed no significant differences in the response to the encapsulated and released proteins compared to the unencapsulated recombinant proteins.
  • RSV is host-specific, infects cotton rats and the virus infects pulmonary alveolar macrophages (PAM) .
  • PAM pulmonary alveolar macrophages
  • alveolar macrophages were stimulated with 10 ⁇ g/mL PLGA nanoparticles that were ‘empty’ .
  • Inflammatory cytokine production was measured by multiplex immunoassay.
  • Increased inflammatory cytokine expression by nanovaccine-stimulated alveolar macrophages compared to untreated controls were observed and observed no difference in the response to ‘empty’ or RSV-F loaded nanoparticles.
  • the PLGA nanoparticles were able to activate rat APC and demonstrated that this response was largely independent of the antigen payload, as ‘empty’ particles and particles loaded with recombinant F proteins elicit similar inflammatory responses.
  • HEp-2 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, ) with 10%fetal rat serum (FBS, GIBCO-BRL) , 2mM glutamine, penicillin and streptomycin (GIBCO-BRL) at 37°C with 5%CO 2 .
  • DMEM Dulbecco’s modified Eagle’s medium
  • FBS GIBCO-BRL
  • FBS fetal rat serum
  • GIBCO-BRL penicillin and streptomycin
  • RSV infected Hep-2 cells were cultured for 3 days, harvested and centrifuged for 10min at 2000 rpm in a table-top centrifuge at 4 °C. RSV within supernatants wascollected.
  • Post-fusion (F) stabilized F and pre-fusion stabilized F proteins and pre-fusion specific 5C4 mAb were generously provided by Dr XuWenbo (National Institute of Viral Diseases Prevention and Control, Beijing) .
  • Palivizumab mAb was kindly provided by Dr XuWenbo (National Institute of Viral DiseasesPrevention and Control, Beijing) .
  • D25 mAb was purchased from Creative Biolabs (Shirley, NY, USA) .
  • Rats were euthanized on day 7 post-infection (p.i. ) by barbiturate overdose.
  • Pathological evaluation was performed.
  • Bronchoalveolar lavage fluid (BAL) was collected by introducing 500 mL of sterile, ice-cold PBS through the trachea.
  • Samples of affected and unaffected lungs were collected from multiple sites for histopathological evaluation.
  • Histopathology Lung tissues collected from cotton rats at 5 days after RSV challenge were fixed with 10%neutral buffered-formalin. Lung tissue histology was performed by staining with hematoxylin and eosin (H&E) , periodic acid–Schiff (PAS) , and Congo red (C&R) and analyzed under light microscopy. The tissue slides were examined for lymphocytes and eosinophils in peribronchiolar, perivascular, interstitial, and alveolar spaces. At least 10 sections per lung tissue from individual rats were obtained and blind scoring was performed
  • Inflammation and focal aggregates of infiltrating cells in the airways of the lung were blindly examined and measured using a severity score system defined as 0 (normal) , 1 (mild inflammation, 60%lung affected with tissue necrosis or damage) .
  • the mucin expression of goblet cell hyperplasia was identified in 50 randomly selected lung airways in the PAS stained slides. Eosinophils were counted per viewing PAS-positive areas within the airway epithelium (400 ⁇ magnification) and annotated using the magnetic lasso tool of Adobe Photoshop CS5.1 software.
  • PFU plaque forming units
  • cDNA synthesis and PCR was performed in a single tube using the SuperScript III Platinum One-Step Quantitative RT-PCR kit (Invitrogen) with 5 ⁇ L of eluted RNA, 10 ⁇ M of each primer, and 50 ⁇ M of the probe (primers and probes from Integrated DNA Technologies) .
  • Forward primer TTGGATCTGCAATCGCCA (SEQ ID NO: 10) .
  • Reverse primer CTTTTGATCTTGTTCACTTCTCCTTCT (SEQ ID NO: 11) .
  • Probe 5'-carboxyfluorescein (FAM) -TGGCACTGCTGTATCTAAGGTCCTGCACT-tetramethylcarboxyrhodamine (TAMRA) -3' (SEQ ID NO: 12) .
  • Amplification and detection was performed with an ABI Prism 7900HT or 7500 (Applied Biosystems) .
  • a threshold cycle value (Ct) is defined for each sample as the cycle number at which the fluorescent signal first becomes detectable above a set threshold. PFU equivalents for each sample is then determined based on a standard curve of Ct verses the logarithm of defined copy number of viral RNA.
  • ELISAs were performed according to kit manufacturer’s instructions. Indirect ELISAs were used to quantify IgA in the nasal and BAL fluid. Indirect ELISAs were also used to determine the immunogenicity of the RSV F proteins prior to encapsulation and following release from the PLGA nanoparticles. For the IgA quantification, ELISA plates were coated overnight at 4 °C with 3 ⁇ g/mL F protein, or with 100 ⁇ l/well of RSV stock ( ⁇ 10 5 TCID50) .
  • the ELISA plates were coated overnight at 4 °C with 5 ⁇ g/mL total of the F protein (2.5 ⁇ g/mL of each) , with ⁇ 5 ⁇ g/mL of the RSV F proteins that had been encapsulated and released from the PLGA nanoparticles, or with 100 ⁇ l/well of RSV stock ( ⁇ 10 5 TCID50) .
  • Negative control wells were coated with 100 ⁇ l/well cell culture media prepared from uninfected HEp-2 cells. Nasal fluid samples were diluted 1: 2 and treated with 10mM dithiothreitol (DTT) for 1 hour at 37 °C prior to performing the ELISAs.
  • DTT dithiothreitol
  • BAL samples were diluted 1: 2 but were not treated with DTT. Serum samples were diluted 1: 1000. All samples were plated in duplicates, incubated for 2 hours at room temperature and then washed. Sheep anti-rat IgA-HRP (Bethyl Laboratories) was used at 0.5 ⁇ g/mL. Sheep anti-rat IgG-HRP (Bethyl Laboratories) was used at 0.5 ⁇ g/mL for the immunogenicity experiments. Plates were developed using Pierce 1-Step Ultra TMP Substrate (Termo Scientifc Pierce) . The reaction was stopped with the addition of 0.2M H 2 SO 4 and plates were read at an optical density of 450nm and 540nm using an automated plate reader.
  • a multiplex immunoassay (Invitrogen) was used to quantify cytokine secretion in supernatants from rats alveolar macrophages.
  • IL-17A, IFN ⁇ , IL-6, IL-1 ⁇ and TNF ⁇ Set ELISA Development kits were purchased from King fisher Biotech, Inc.
  • the bovine IL-4 ELISA kit was purchased from Termo Fisher Scientific ELISAs were performed according to kit manufacturer’s instructions.
  • Indirect ELISAs were used to quantify IgA in the nasal and BAL fluid.
  • Indirect ELISAs were also used to determine the immunogenicity of the BRSV F and G proteins prior to encapsulation, and following release from the CPTEG: CPH nanoparticles.
  • ELISA plates were coated overnight at 4 °C with 3 ⁇ g/mL F or G protein, or with 100 ⁇ l/well of BRSV stock ( ⁇ 10 4 TCID50) .
  • the ELISA plates were coated overnight at 4 °C with 5 ⁇ g/mL total of the F and G protein (2.5 ⁇ g/mL of each) , with ⁇ 5 ⁇ g/mL of the BRSV F and G proteins that had been encapsulated and released from the CPTEG: CPH nanoparticles, or with 100 ⁇ l/well of BRSV stock ( ⁇ 10 4 TCID50) .
  • Negative control wells were coated with 100 ⁇ l/well cell culture media prepared from uninfected BT. Nasal fluid samples were diluted 1: 2 and treated with 10mM dithiothreitol (DTT) for 1 hour at 37 °C prior to performing the ELISAs. BAL samples were diluted 1: 2 but were not treated with DTT. Serum samples were diluted 1: 1000. All samples were plated in duplicates, incubated for 2 hours at room temperature and then washed. Sheep anti-bovine IgA-HRP (Bethyl Laboratories) was used at 0.5 ⁇ g/mL.
  • DTT dithiothreitol
  • Sheep anti-bovine IgG-HRP (Bethyl Laboratories) was used at 0.5 ⁇ g/mL for the immunogenicity experiments. Plates were developed using Pierce 1-Step Ultra TMP Substrate (TermoScientifc Pierce) . The reaction was stopped with the addition of 0.2M H 2 SO 4 and plates were read at an optical density of 450nm and 540nm using an automated plate reader
  • ELISA enzyme-linked immunosorbent assay
  • HRP horse radish peroxidase
  • OD optical density
  • GraphPad Prism nonlinear regression
  • Serum samples were tested for the presence of neutralizing antibodies by a plaque reduction neutralization test (PRNT) .
  • PRNT plaque reduction neutralization test
  • Two-fold serial dilutions of HI-serum (in PBS with 5%HI-FBS) were added to an equal volume of RSV Long previously titered to give approximately 115 PFU/25 ⁇ l.
  • Serum/virus mixtures were incubated for 2 hours at 37 °C and 5%CO 2 , to allow virus neutralization to occur, and then 25 ⁇ l of this mixture (containing approximately 115 PFU) was inoculated on duplicate wells of HEp-2 cells in 96 well plates.
  • the cells were over-laysed with 0.75%Methyl Cellulose/EMEM 5%HI-FBS and incubated for 42 hours.
  • the number of infectious virus particles was determined by detection of syncytia formation by immunostaining followed by automated counting.
  • the neutralization titer is defined as the reciprocal of the serum dilution producing at least a 60%reduction in number of synctia per well, relative to controls (no serum) .
  • mice were vaccinated IN with 10 ⁇ g RSV-F plus WCL, 10 ⁇ g ‘empty’ PLGA nanoparticles plus WCL, 10 ⁇ g RSV-F -loaded nanoparticles plus WCL, and 10 ⁇ g ‘empty’ PLGA nanoparticles plus WCL, respectively.
  • rats were challenged via intranasal inoculation with ⁇ 10 5 RSV strain Long. Rats were monitored daily for clinical signs and rectal temperatures.
  • FIG. 7 Representative micrographs from an uninfected control rats (i) , an unvaccinated positive control rats (ii) , an ‘empty’ nanoparticle-administered rats (iii) and a RSV-F/vaccinated rats (iv) are shown in Fig. 7. Cumulative histopathology scores were depicted in Table 5. Pulmonary lesions were most pronounced in the unvaccinated control animals and included thickened alveolar septa with infiltrates of macrophages, lymphocytes and occasional neutrophils; and bronchioles filled with neutrophils, sloughed epithelial cells and necrotic cell debris.
  • RSV-F nanoparticle-administered rats Nasal swabs and lung tissues were assessed for virus isolation.
  • RSV was isolated from 5/6 PLGA+WCL vaccinated cotton rats on day 3 p. i and 6/6 animals on day 6 p.i.
  • Virus was isolated from the lungs of all 6 PLGA+WCL vaccinated controls at necropsy.
  • RSV was isolated from 0/6 PLGA-RAg+WCL vaccinated cotton rats on day 3, and 1/6 on day 6 p.i.
  • Virus was isolated from the lungs of this same animal on day 7 p.i.
  • RSV was isolated from the nasal swabs of 4/6 cotton rats in the ‘empty’ PLGA+WCL administered group on days 3 and 6 after infection, and from the lung tissues of 3/6 cotton rats on day 7 p.i. Virus from the nasal swabs collected from any of the animals on day 0 (prior to RSV challenge) was not isolated , nor RSV from the nasal swabs or lung tissues of the uninfected control cotton rats.
  • RSV-F nanoparticle vaccine i.n. administered cotton rats demonstrated significantly reduced quantities of viral RNA compared to their PLGA+WCL cohorts (Fig. 8) .
  • Fig. 8 showed the reduced viral burden in the lungs of BRSV-F nano vaccine-administered rats.
  • Samples were collected from 2–3 representative lesion and non-lesion sites of the lungs on day 7 post-challenged and preserved in RNA later.
  • the RNA was extracted using Trizol reagent.
  • the RNA from multiple sites was then pooled and was analyzed by qPCR for the RSV NS2 gene.
  • the graph depicts mean ⁇ SEM of each group. *p ⁇ 0.05 compared to PLGA+WCL control rats.
  • RSV-specific cellular responses in the lungs and peripheral blood of RSV-F nanovaccine administered rats Prior to challenge, we detected significant RSV-F protein specific CD4 or CD8 T cell proliferation in PBMCs from any groups. By day 6 p.i., we measured significant antigen-specific proliferation by CD4 T cells from RSV-F nanovaccine-administered rats in response to live virus a (Fig. 10A) . PBMCs from the BRSV-F nanovaccine-administrated animals also secreted IFN ⁇ and IL-17A in response to whole virus (Fig. 10B-10C) . Levels of IL-4 were below the limit of detection for all groups (data not shown) .
  • Our PLGA nanovaccine platform which is based upon PLGA copolymers, offers a number of advantages over other polymeric nanoparticle systems, including: inherent adjuvant properties the tendency of the particles to surface erode, thus stabilizing the encapsulated proteins and maintaining the structural and biological features of the antigens for longer periods of time; the ability to provide sustained and tunable antigen release kinetics; and the ability to degrade at a neutral pH, into non-toxic and non-mutagenic carboxylic acids.
  • the in vitro antigenicity of the RSV F proteins was preserved following encapsulation and release from the PLGA particles (Fig. 4) .
  • IgA responses can wane and the IgG response may be more important in long-term protection.
  • RSV infection causes inflammation in the upper respiratory tract, thus the increase in neutralizing antibody titers could also potentially be attributed to increased levels of serum antibodies leaking into the nasal secretions of the ‘empty’ nanovaccine administered rats.
  • the RSV F protein is a type I viral fusion protein that is synthesized as a precursor that is proteolytically cleaved by furin into disulfde-linked fragments. It is highly conserved between virus strains, as well as between HRSV and BRSV, demonstrating approximately 80%homology.
  • the F protein exists in two forms on the virion surface: a metastable pre-fusion form and a stable post-fusion trimer.
  • the post-fusion form of the F protein contains two major neutralizing epitopes, antigenic sites II and IV7. High-affinity, site-II directed neutralizing antibodies are protective in cotton rats.
  • protein stability is of paramount concern, and the post-fusion F is advantageous due to its highly stable nature.
  • the pre-fusion F protein contains antigenic site and recent evidence suggests that this epitope is the primary target for neutralizing antibodies in humans.
  • Vaccine formulations incorporating the post-fusion form of the F protein are highly efficacious in rodent models and a highly efficacious post-fusion F vaccine was also recently reported for RSV, suggesting that the antigenic site is conserved and of immunologic relevance to the cotton rat model as well.
  • Stable expression of the post-fusion F protein is not trivial and hurdles exist with respect to affordable, consistent production of sufficient quantities of stable, post-fusion F for use in subunit vaccines.
  • the availability of the crystal structure and improved strategies to express and stabilize the post-fusion F significantly enhance the feasibility of a subunit based pre-fusion F vaccine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Nutrition Science (AREA)
  • Mycology (AREA)
  • Inorganic Chemistry (AREA)
  • Otolaryngology (AREA)
  • Physiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

La présente invention concerne une protéine F du VRS modifiée, une nanoparticule, une composition et un vaccin conçus pour une administration par voie muqueuse, et en particulier pour une administration intranasale. L'administration intranasale de la protéine F RSV modifiée, de la nanoparticule, de la composition et du vaccin peut provoquer une immunité antivirale muqueuse et systémique, conduisant à une réduction de la pathologie associée au virus et à une réduction des charges virales, ce qui permet de prévenir, d'améliorer et/ou de traiter une maladie provoquée par une infection par le virus RSV.
PCT/CN2023/101642 2023-06-21 2023-06-21 Protéine f rsv modifiée, nanoparticule, composition et vaccin contre une infection par le virus respiratoire syncytial Pending WO2024259624A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/101642 WO2024259624A1 (fr) 2023-06-21 2023-06-21 Protéine f rsv modifiée, nanoparticule, composition et vaccin contre une infection par le virus respiratoire syncytial

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2023/101642 WO2024259624A1 (fr) 2023-06-21 2023-06-21 Protéine f rsv modifiée, nanoparticule, composition et vaccin contre une infection par le virus respiratoire syncytial

Publications (1)

Publication Number Publication Date
WO2024259624A1 true WO2024259624A1 (fr) 2024-12-26

Family

ID=93934653

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/101642 Pending WO2024259624A1 (fr) 2023-06-21 2023-06-21 Protéine f rsv modifiée, nanoparticule, composition et vaccin contre une infection par le virus respiratoire syncytial

Country Status (1)

Country Link
WO (1) WO2024259624A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008110627A1 (fr) * 2007-03-14 2008-09-18 Pierre Fabre Medicament Nouvelle composition de vaccin destinée au traitement de maladies infectieuses respiratoires
WO2013049342A1 (fr) * 2011-09-30 2013-04-04 Novavax, Inc. Vaccin f de rsv à nanoparticule recombinante pour le virus respiratoire syncytial
WO2017041100A2 (fr) * 2015-09-03 2017-03-09 Novavax, Inc. Compositions vaccinales ayant une stabilité et une immunogénicité améliorées
WO2017070622A1 (fr) * 2015-10-22 2017-04-27 Modernatx, Inc. Vaccin contre le virus respiratoire syncytial
WO2017172890A1 (fr) * 2016-03-29 2017-10-05 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Protéines f du vrs modifiées par substitutions et sous conformation de pré-fusion, et leur utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008110627A1 (fr) * 2007-03-14 2008-09-18 Pierre Fabre Medicament Nouvelle composition de vaccin destinée au traitement de maladies infectieuses respiratoires
WO2013049342A1 (fr) * 2011-09-30 2013-04-04 Novavax, Inc. Vaccin f de rsv à nanoparticule recombinante pour le virus respiratoire syncytial
WO2017041100A2 (fr) * 2015-09-03 2017-03-09 Novavax, Inc. Compositions vaccinales ayant une stabilité et une immunogénicité améliorées
WO2017070622A1 (fr) * 2015-10-22 2017-04-27 Modernatx, Inc. Vaccin contre le virus respiratoire syncytial
WO2017172890A1 (fr) * 2016-03-29 2017-10-05 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Protéines f du vrs modifiées par substitutions et sous conformation de pré-fusion, et leur utilisation

Similar Documents

Publication Publication Date Title
EP2601970B1 (fr) Formulations de vaccin contre un norovirus
Garlapati et al. Enhanced immune responses and protection by vaccination with respiratory syncytial virus fusion protein formulated with CpG oligodeoxynucleotide and innate defense regulator peptide in polyphosphazene microparticles
CN1268393C (zh) 经改良修饰的brsv或疫苗
ES2932952T3 (es) Composiciones y métodos para tratar y prevenir el síndrome reproductivo y respiratorio porcino
Sokolova et al. The potential of nanoparticles for the immunization against viral infections
JP5551774B2 (ja) インフルエンザワクチン、組成物、および使用方法
CN1391483A (zh) 作为佐剂的聚氧乙烯脱水山梨醇酯和辛苯昔醇组合及其在疫苗中的应用
JP6122083B2 (ja) ナノエマルションワクチン
WO2022110099A1 (fr) Vaccins à coronavirus et leurs utilisations
KR20160055075A (ko) 수족구 백신 및 그의 제조 방법 및 용도
Zheng et al. Comparison of immune responses in guinea pigs by intranasal delivery with different nanoparticles-loaded FMDV DNA vaccine
Boley et al. Enhanced mucosal immune responses and reduced viral load in the respiratory tract of ferrets to intranasal lipid nanoparticle-based SARS-CoV-2 proteins and mRNA vaccines
JP2009523722A (ja) ポリイノシン酸−ポリシチジル酸を基礎としたアジュバントを含む粘膜免疫原性物質
WO2024259624A1 (fr) Protéine f rsv modifiée, nanoparticule, composition et vaccin contre une infection par le virus respiratoire syncytial
US20240299528A1 (en) A dna plasmid sars-corona virus-2/covid-19 vaccine
TW202120123A (zh) 類屈公病毒顆粒疫苗及使用其之方法
WO2025189658A1 (fr) Vaccin protéique recombinant pour la prévention et le traitement des variants jn.1, ba.2.86 et xbb du sars-cov-2, médicament pour usage combiné et utilisation
WO2008037033A1 (fr) Vaccin contre la grippe constitué d'un mélange de mannane et d'antigène de la grippe
Li et al. Plug-and-display nanoparticle immunization of the core epitope domain induces potent neutralizing antibody and cellular immune responses against PEDV
JP5995329B2 (ja) インフルエンザワクチン、組成物、および使用方法
KR102743672B1 (ko) 점막에서 항원 특이적 면역 반응을 증진시키는 신규한 펩타이드 및 상기 펩타이드를 유효성분으로 포함하는 점막 백신 보조제
KR20210068429A (ko) 점막 애주번트
Kumar et al. Nasal Vaccine for the Control of Emerging Variants of SARS-CoV-2
WO2023159082A2 (fr) Vaccin intranasal à base de nanotechnologie contre la covid-19 comprenant du chitosane
Raina et al. Immune responses to polyethylenimine delivered plasmid DNA encoding a Fasciola gigantica fatty acid binding protein in mice and rabbits

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23941928

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE