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WO2024254461A2 - Procédés et compositions pour le développement et la distribution de vaccins - Google Patents

Procédés et compositions pour le développement et la distribution de vaccins Download PDF

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WO2024254461A2
WO2024254461A2 PCT/US2024/033024 US2024033024W WO2024254461A2 WO 2024254461 A2 WO2024254461 A2 WO 2024254461A2 US 2024033024 W US2024033024 W US 2024033024W WO 2024254461 A2 WO2024254461 A2 WO 2024254461A2
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virus
vaccine composition
lipids
porcine
vaccine
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WO2024254461A3 (fr
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Hiep Vu
The Nhu NGUYEN
Sarah SILLMAN
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NuTech Ventures Inc
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NuTech Ventures 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • 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
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • F&R Ref No.: 24742-0144WO1 METHODS AND COMPOSITIONS FOR VACCINE DEVELOPMENT AND DELIVERY FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
  • This invention was made with government support under NI19HMFPXXXXG032, NI22HMFPXXXG005, NI20HMFPXXXXG023, NI21HMFPXXXG031, NI20HFPXXXXXG055, and NI21HFPXXXXXG004 awarded by the National Institute of Food and Agriculture. The government has certain rights in the invention.
  • RNA viruses with an RNA genome evolve rapidly due to the lack of proof-reading activity of their RNA-dependent RNA polymerase.
  • the highly variable nature of RNA viruses poses a great challenge to the development of vaccines with a broad spectrum of protection.
  • One way to improve vaccine efficacy against RNA viruses is to frequently update the vaccine immunogens to match with viral strains circulating in the field.
  • vaccine compositions include a nucleic acid vector expressing an antigen encapsulated in a lipid nanoparticle (LNP), wherein the LNP comprises a plurality of lipids at a suitable ratio.
  • LNP lipid nanoparticle
  • the one or more of the plurality of lipids is a cationic lipid. In some embodiments, the one or more of the plurality of lipids is a permanently charged cationic lipid. In some embodiments, the one or more of the plurality of lipids is conditionally ionized.
  • the plurality of lipids includes at least one of O-(Z,Z,Z,Z- heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (D-Lin-MC3-DMA or MC3), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000).
  • D-Lin-MC3-DMA or MC3 1,2-dioleoyl-3-trimethylammonium-propane
  • DSPC distearoylphosphatidylcholine
  • cholesterol 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000
  • a representative composition includes a plurality of lipids including O- (Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (D-Lin-MC3-DMA or MC3), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000).
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-propane
  • DSPC distearoylphosphatidylcholine
  • cholesterol 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000).
  • the ratio of the lipids is 30 to 50 : 2.5 to 15 : 5 to 15 : 30 to 55 : 0.5 to 5 (MC3 : DOTAP : DSPC : cholesterol : DMG-PEG2000).
  • One representative ratio of the lipids is about 35:5:10:48:2 (MC3 : DOTAP : DSPC : cholesterol : DMG-PEG2000).
  • Another representative ratio of the lipids is about 42:10:8:38:2 (MC3 : DOTAP : DSPC : cholesterol : DMG-PEG2000).
  • the nucleic acid vector is a DNA plasmid.
  • the antigen is hemagglutinin (HA), neuraminidase of influenza virus, spike protein of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, capsid protein of porcine circovirus, envelope protein of classical swine fever virus, atypical porcine pestivirus, VP1, VP2 VP3 and PV4 of foot-and-mouth disease virus, and senecavirus.
  • HA hemagglutinin
  • influenza virus spike protein of porcine epidemic diarrhea virus
  • transmissible gastroenteritis virus capsid protein of porcine circovirus
  • envelope protein of classical swine fever virus atypical porcine pestivirus
  • VP1, VP2 VP3 and PV4 of foot-and-mouth disease virus
  • senecavirus senecavirus
  • the antigen is from a pathogen selected from the group consisting of swine influenza virus, avian influenza virus, rotavirus, porcine circovirus, porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), classical swine fever virus (CSFV), atypical porcine pestivirus (APPV), porcine F&R Ref No.: 24742-0144WO1 epidemic diarrhea virus (PEDV), porcine circovirus, foot-and-mouth disease virus, and bovine viral diarrhea virus.
  • methods of vaccinating an animal are provided. Such methods include administering a vaccine composition as described herein to the animal. In some embodiments, the methods further include identifying an animal in need of a vaccine.
  • the administration comprises an intra-muscular injection.
  • the vaccine is against swine influenza virus, avian influenza virus, rotavirus, porcine circovirus, porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), classical swine fever virus (CSFV), atypical porcine pestivirus (APPV), porcine epidemic diarrhea virus (PEDV), porcine circovirus, foot-and-mouth disease virus, and bovine viral diarrhea virus.
  • PRRSV African swine fever virus
  • CSFV classical swine fever virus
  • APPV atypical porcine pestivirus
  • PEDV porcine epidemic diarrhea virus
  • porcine circovirus porcine circovirus
  • foot-and-mouth disease virus and bovine viral diarrhea virus.
  • the method is used for viral pathogens that cannot be grown in cell culture. In some embodiments, the method is used for newly emerging viruses.
  • the animal is selected from swine, avian, cow, horse, goat, sheep, dog, and cat.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
  • FIG.1A-1E are experimental results showing the physical characterization of the LNP-DNA vaccines based on the H3 antigen of the influenza virus and in vitro transfection efficiency.
  • FIG.1A is a graph showing the particle diameter determination by the nanoparticle tracking analysis (NTA).
  • FIG.1B is a graph showing the polydispersity index.
  • FIG.1C is a graph showing the zeta potential determination by electrophoretic light F&R Ref No.: 24742-0144WO1 scattering analysis.
  • FIG.1D is a graph showing the encapsulation efficiency (EE%).
  • FIG.1E are photographs showing the transfection efficiency of the indicated construct (LNP1-H3, LNP2-H3, PEI-H3, Naked) in HEK-293T cells.
  • FIG.2A-2D are experimental results showing immune responses following vaccination.
  • FIG.2A is a graph showing the anti-H3 IgG titers in blood samples collected on various days post-vaccination. Data are expressed as the log 10 of the reciprocal of the highest plasma dilution at which anti-H3 antibodies were observed. The samples were initially diluted at 1:100 and samples with undetectable antibodies at this dilution were considered negative and assigned a titer of 1:10.
  • FIG.2B is a graph showing hemagglutinin inhibition (HI) antibody titers measured against the H3N2 virus. Data are expressed as the reciprocal of the highest plasma dilution at which hemagglutination inhibition was observed. The samples were initially diluted at 1:10 and samples with undetectable HI activity at this dilution were considered negative and assigned a titer of 1:5.
  • FIG.2C is a graph showing the anti-NP antibody levels measured using a commercial ELISA kit. Data are presented as the sample to negative (S/N) ratio.
  • FIG.2D is a graph showing IFN-gamma-secreting cell responses following vaccination.
  • FIG.3A-3B are experimental results showing viral shedding after challenge infection with the homologous H3N2 influenza virus.
  • FIG.3A is a graph showing viral RNA in nasal swabs as determined by RT-qPCR. Data are presented as log10 viral RNA copies per 100 ⁇ L of the sample.
  • BALF bronchoalveolar lavage fluid
  • FIG.4A-4D are experimental results showing lung pathology and the presence of virus-infected cells in tissue samples.
  • FIG.4A are representative photos of lungs taken during necropsy. Black arrows indicate areas of the lungs with typical consolidation caused by IAV-S. The graph indicates the percentage of lung consolidation calculated based on the weighted proportions of each lobe to the total lung volume.
  • FIG.4B shows representative F&R Ref No.: 24742-0144WO1 images of lung sections stained with H&E and the graph indicates the composite microscopic lesion scores based on the parameters described herein.
  • FIG.4C shows representative images of lung sections stained with in situ hybridization (ISH) for the detection of viral NP-based mRNA transcript and the graph indicates the composite ISH scores.
  • FIG.4D shows representative images of tracheal sections stained with ISH for the detection of viral NP- based mRNA transcript and the graph indicates the composite ISH scores.
  • FIG.5A-5F are experimental results showing the physical characterization of the LNP-DNA vaccine based on the H1 antigen of the influenza virus and in vitro transfection efficiency.
  • FIG.5A is a graph showing the zeta potential determination by electrophoretic light scattering analysis.
  • FIG.5A is a graph showing the particle diameter determination by the nanoparticle tracking analysis (NTA).
  • FIG.5B is a graph showing the polydispersity index.
  • FIG.5C is a graph showing the zeta potential determination by electrophoretic light scattering analysis.
  • FIG.5D is a graph showing the encapsulation efficiency (EE%).
  • FIG.5E are photographs showing the transfection efficiency of the indicated construct (LNP3- H1pdm09 or mock transfected) in HEK-293T cells.
  • FIG.6 illustrates the transfection efficiency of the LNP3-H1pdm09 vaccine in HEK- 293T cells when stored at room temperature or 4°C for different time intervals.
  • FIG.7A-7B are experimental results showing the immune responses of pigs following vaccination with the LNP3-H1pdm09 vaccine.
  • FIG.7A is a graph showing the anti-H1 IgG titers in blood samples collected on various days post-vaccination. Data are expressed as the log10 of the reciprocal of the highest plasma dilution at which anti-H1 antibodies were observed. The samples were initially diluted at 1:100 and samples with undetectable antibodies at this dilution were considered negative and assigned a titer of 1:10.
  • FIG.7B is a graph showing hemagglutinin inhibition (HI) antibody titers measured against the H1N1 virus. Data are expressed as the reciprocal of the highest plasma dilution at which hemagglutination inhibition was observed.
  • HI hemagglutinin inhibition
  • FIG.8A-8B are experimental results showing viral shedding of pigs vaccinated with the LNP3-H1pdm09 vaccine followed by challenge infection with the homologous H1N1 influenza virus.
  • FIG.8A is a graph showing viral RNA in nasal swabs as determined by RT- F&R Ref No.: 24742-0144WO1 qPCR. Data are presented as log10 viral RNA copies per 100 ⁇ L of the sample.
  • FIG.8B is data showing viral RNA copies in bronchoalveolar lavage fluid (BALF) collected on day 5 post-challenge infection.
  • BALF bronchoalveolar lavage fluid
  • FIG.9A-9C are experimental results showing gross lung lesions of pigs vaccinated with the LNP3-H1pdm09 vaccine followed by challenge infection with the homologous H1N1 influenza virus.
  • FIG.9A and 9B are representative photos of the lungs of pigs in the LNP3-H1pdm09 group or PBS control group, respectively. Black arrows indicate areas of the lungs with typical consolidation caused by IAV-S.
  • FIG 9C is the graph that indicates the percentage of lung consolidation calculated based on the weighted proportions of each lobe to the total lung volume.
  • FIG.10A-10C are experimental results showing microscopic lung lesions of pigs vaccinated with the LNP3-H1pdm09 vaccine followed by challenge infection with the homologous H1N1 influenza virus.
  • FIG.10A and 10B are representative H&E images of the lungs of pigs in the LNP3-H1pdm09 group or PBS control group, respectively.
  • FIG.10C is the graph that indicates the composite microscopic lesion scores based on the parameters described herein.
  • FIG.11A-11B is a graph showing hemagglutinin inhibition (HI) antibody titers measured against the H1N1 and H1N2 viruses, respectively. Data are expressed as the reciprocal of the highest sera dilution at which hemagglutination inhibition was observed. The samples were initially diluted at 1:10 and samples with undetectable HI activity at this dilution were considered negative and assigned a titer of 1:5.
  • FIG.12A-12D are experimental results showing protection results after challenge infection with the the H1N2 influenza strain.
  • FIG.12A is a graph showing viral RNA copies in nasal swabs after challenge infection with the H1N2 virus.
  • FIG.12B is a graph indicating viral RNA copies in BALF collected at necropsy.
  • FIG.12C is a graph indicating the percentage of lung surface exhibiting consolidation based on the weighted proportions of each lobe to the total lung volume.
  • FIG.12D are representative lung photos of pigs taken F&R Ref No.: 24742-0144WO1 during necropsy. The oval dashed lines indicate areas with characteristic consolidation caused by IAV-S. DETAILED DESCRIPTION Currently, whole-inactivated virus (WIV) vaccines are commonly used for the control of Influenza A virus of swine (IAV-S) as well as other types of influenza viruses in swine and in other animals (e.g., FLUSURE XP®).
  • WIV whole-inactivated virus
  • hemagglutinin (HA) antigen of the IAV-S H3N2 strain and H1N1 were used as model vaccine immunogens to assess the effectiveness of three different LNP formulations, each with a unique combination of permanently and conditionally ionized cationic lipids.
  • a single-dose intramuscular administration of the LNP- DNA vaccine induced high levels of antibodies against the H3, H1pdm09 or H1d1 antigen within 7-14 days post-vaccination. Furthermore, pigs vaccinated with the LNP-DNA vaccine based on H3, H1pdm09 or H1d1 antigen were completely protected against challenge infection with the respective homologous strains. Moreover, the group received LNP-DNA vaccine based on H1pdm09 antigen conferred partial protection against the heterologous H1N2 influenza strain and did not induce Vaccine-Associated Enhanced Respiratory Disease as compared to the protein-H1pdm09 group.
  • LNP-DNA can serve as an effective platform for the development of vaccines against any number of different pathogens (e.g., viruses, bacteria, etc.).
  • IAV-S is a very well-suited model antigen for studying vaccine efficacy in pigs, at least because the influenza A virus is an important respiratory pathogen that causes significant economic losses to swine producers, the viral hemagglutinin (HA) antigen has been the primary target for vaccine development and immunization of pigs, the HA protein alone is sufficient to induce complete protection, hemagglutinin inhibition antibody titers are a reliable immune correlate to predict vaccine-induced protection, and a swine model has F&R Ref No.: 24742-0144WO1 been developed to evaluate the protective efficacy of vaccine.
  • HA hemagglutinin
  • any number of antigens can be used in the methods and compositions described herein including, for example, neuraminidase of influenza virus, spike protein of porcine epidemic diarrhea virus, transmissible gastroenteritis virus, envelope protein of classical swine fever virus, atypical porcine pestivirus, or capsid protein of porcine circovirus.
  • the method described herein can utilize an antigen from any number of viruses, including, without limitation, swine influenza virus, avian influenza virus, rotavirus, porcine circovirus, porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), foot-and-mouth disease virus, or bovine viral diarrhea virus, thereby developing a vaccine against such a virus.
  • the methods described herein also can be used with viral pathogens that cannot readily be grown in cell culture (e.g., porcine circovirus type 3, atypical porcine pestivirus (APPV)) as well as newly emerging viruses.
  • porcine circovirus type 3, atypical porcine pestivirus (APPV) as well as newly emerging viruses.
  • a DNA plasmid expressing a protein is non-infectious, non-viable, and safe to use in animals.
  • a protein e.g., an antigenic protein (an “antigen”)
  • an antigenic protein an “antigen”
  • vaccination of animals with a naked DNA plasmid often results in poor immune responses due to ineffective cellular uptake.
  • Various methods have been developed to improve DNA plasmid delivery, including gene guns or in vivo electroporation, but the currently used methods of developing and delivering DNA vaccines do not induce adequate immune responses.
  • lipid nanoparticles LNPs
  • LNPs lipid nanoparticles
  • the lipids can be cationic lipids (i.e., DOTAP, DDAB, DOTMA, a head group having permanent positive charges) and/or can be conditionally ionized lipids.
  • Ionizable lipids generally are protonated at lower than its pKa, which makes them positively charged, but they remain in the neutral range at physiological pH.
  • the pH-sensitivity of ionizable lipids is beneficial for in vivo mRNA delivery because neutral particles have less interactions with the anionic membranes of blood cells, thus, improving the biocompatibility of lipid nanoparticles.
  • ionizable lipids can be protonated and, F&R Ref No.: 24742-0144WO1 therefore, become positively charged, it would be able to make the particles undergoing L ⁇ C to HII C transition, which can promote membrane destabilization and facilitate endosomal escape of the nanoparticles.
  • O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) also is known as D-Lin-MC3-DMA or simply MC3 (CAS 1224606-06-7).
  • MC3 is a highly potent cationic lipid.
  • KC2, SM102, or DODAP could be used in the compositions described herein in place of or in conjunction with MC3.
  • 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP or 18:1TAP) (CAS 113669-21- 9) is a lipid with cationic helper lipid properties.
  • ethyl phosphatidylcholine could be used in place of or in conjunction with DOTAP.
  • Distearoylphosphatidylcholine (DSPC) (CAS 4539-70-2) is a phophatidylcholine that is naturally found in cell membranes. It serves as a neutral helper lipid.
  • DOPE or DOPC could be used in place of or in conjunction with DSPC.
  • Cholesterol (CAS 57-88-5) is well known and is an essential structural component of animal cell membranes. Cholesterol, as an uncharged amphiphile, does not directly interact with nucleic acids, however, cholesterol plays a crucial role in facilitating the interaction between cationic lipids and nucleic acids, and contributes to the process of endosome escape. It would be appreciated that beta-sitosterol could be used in place of or in conjunction with cholesterol.
  • DMG-PEG2000 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (CAS 160743-62-4) is a synthetic lipid that is formed by the PEGylation of myristoyl diglyceride. It would be appreciated that DSPE-PEG2000 could be used in place of or in conjunction with DMG-PEG2000. In general, due to its hydrophilic property, PEGylation can offer a mechanism to reduce from nonspecific uptake and can further contribute to enhance particles stability by decreasing particles aggregation.
  • lipids described herein can be used in a nanoparticle in ratios of about 30 to 50 : 2.5 to 15 : 5 to 15 : 30 to 55 : 0.5 to 5 (MC3 : DOTAP : DSPC : cholesterol : DMG- PEG2000).
  • a lipid nanoparticle can include a ratio of 35 : 5 : 10 : 48 : 2 (MC3 : DOTAP : DSPC : cholesterol : DMG-PEG2000) or 42 : 10 : 8 :38 : 2 (MC3 : DOTAP : DSPC : cholesterol : DMG-PEG2000).
  • a vector containing a nucleic acid (e.g., a nucleic acid that encodes an antigen) also is provided.
  • Vectors, including expression vectors are commercially available or can be produced by recombinant DNA techniques routine in the art.
  • a vector containing a nucleic acid can have expression elements operably linked to such a nucleic acid, and further can include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene).
  • a vector containing a nucleic acid can encode a chimeric or fusion polypeptide (i.e., a polypeptide operatively linked to a heterologous polypeptide, which can be at either the N- terminus or C-terminus of the polypeptide).
  • Representative heterologous polypeptides are those that can be used in purification or detection of the encoded polypeptide (e.g., 6xHis tag, glutathione S-transferase (GST)).
  • Expression elements include nucleic acid sequences that direct and regulate expression of nucleic acid coding sequences.
  • an expression element is a promoter sequence.
  • Expression elements also can include introns, enhancer sequences, response elements, or inducible elements that modulate expression of a nucleic acid.
  • Expression elements can be of bacterial, yeast, insect, mammalian, or viral origin, and vectors can contain a combination of elements from different origins.
  • operably linked means that a promoter or other expression element(s) are positioned in a vector relative to a nucleic acid in such a way as to direct or regulate expression of the nucleic acid.
  • nucleic acids are well known to those skilled in the art and include, without limitation, electroporation, calcium phosphate precipitation, polyethylene glycol (PEG) transformation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer.
  • electroporation calcium phosphate precipitation
  • PEG polyethylene glycol
  • heat shock heat shock
  • lipofection lipofection
  • microinjection and viral-mediated nucleic acid transfer.
  • molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. Certain methods and compositions will be described in the following examples, which do not limit the scope of the methods and compositions described in the claims.
  • Example 1 Cells, Viruses, Lipids, and Other Reagents
  • HEK-293T (ATCC CRL-3216) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
  • Madin-Darby Canine Kidney (MDCK) cells (ATCC CCL-34) were cultured in DMEM supplemented with 10% FBS, 0.2% bovine serum albumin (BSA) fraction V, 25 mM HEPES, 100 U/mL penicillin, and 100 ⁇ g/mL streptomycin.
  • DMEM Modified Eagle Medium
  • FBS fetal bovine serum
  • MDCK Madin-Darby Canine Kidney
  • BSA bovine serum albumin
  • H3N2 The IAV-S A/swine/Texas/4199-2/1998 (herein referred to as H3N2), A/swine/Iowa/A01202099/2011 (herein referred to as H1N1) and A/swine/Minnesota/A01392045/2013 (H1N2) (herein referred to as H1N2) were obtained from the National Veterinary Services Laboratories (NVSL, Ames, IA). The influenza strains were propagated and titrated in MDCK cells as previously described (WHO, 2011).
  • the lipids used in this study included DLin-MC3-DMA (MC3) (Nanosoft Polymer, Winston-Salem, NC), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (Cayman Chemical, Ann Arbor, MI), cholesterol (Sigma Aldrich), distearoylphosphatidylcholine (DSPC), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2000) (Avanti Polar Lipids, Birmingham, AL). The lipids were separately dissolved in absolute ethanol.
  • Example 2 DNA Plasmid Construction The HA sequences of the influenza virus H3N2 (GenBank Accession No.
  • H1N1 GenBank Accession No. AFM46639.1
  • H1N2 GenBank Accession No. KR715130.1
  • a flag-epitope sequence (DYKDDDDK (SEQ ID NO:4)) was fused in-frame to the 3’ end of both the H3 gene (SEQ ID NO:1) and H1d1 gene (SEQ ID NO:3), while a 6x histidine tag was fused in-frame to the 3’ end of the H1pdm09 gene (SEQ ID NO:2) to facilitate protein detection.
  • the gene fragment were chemically synthesized using a commercial DNA synthesis service (GenScript, Piscataway, NJ, USA) and subsequently cloned into the pCI plasmid (Promega). Large-scale DNA plasmid were amplified in Escherichia coli DH5a and purified by using a plasmid giga prep kit (Zymo Research). DNA sequencing was performed to confirm the plasmid sequence’s authenticity.
  • MC3, DOTAP, DSPC, cholesterol, and DMG-PEG2000 were mixed at a molar ratio of 42:10:8:38:2 like formula 2, but the total lipid molar concentration was adjusted to 15 mM instead of 25 mM.
  • the DNA plasmid encoding the H1 antigen was diluted in 25 mM sodium acetate buffer pH 4.0.
  • the lipids (organic phase) and DNA solution (aqueous phase) were mixed by using a mixer-4 chip (Precigenome, San Jose, CA) and the NanoGeneratorTM Flex-M Nanoparticle Synthesis System (PreciGenome, San Jose).
  • the DNA and lipid flow rates were set at 3:1 (v/v) and the total flow rate was set at 4 mL per minute.
  • the nitrogen-to-phosphate (N/P) ratio (mol/mol) was fixed at 4.5.
  • the resulting products were immediately dialyzed against 100 mM Tris-Cl buffer at pH 7.4 (LNP1-H3) or 100 mM Tris-Cl buffer containing 10% sucrose at pH 7.4 (LNP2-H3, LNP3-H1pdm09, LNP3-H1d1) using the Slide-A-Lyzer TM G2 dialysis cassettes with the molecular weight cut-off of 10 kDa (Thermo Scientific).
  • Example 4 Evaluation of Transfection Efficiency In Vitro To assess the transfection efficiency of the LNP vaccines, 500 ng of encapsulated DNA was directly added to one well of the 24-well plate containing HEK-293T cells. An equal amount of naked-DNA plasmid was added to another well to serve as a negative control while DNA plasmid mixed with PEI transfectant (PEI-DNA) was used as a positive control.
  • PEI-DNA PEI transfectant
  • the cells were washed with phosphate-buffered saline (PBS) and fixed with a mixture of methanol and acetone (1:1 v/v). The cells were then rehydrated in PBS and incubated with an anti-flag tag antibody to detect H3N2 and H1N2 antigen or with a mouse anti-H1 polyclonal antibody to detect H1 antigen for 1 hr at room temperature.
  • PBS phosphate-buffered saline
  • the cells were incubated with Alexa Fluor TM 488 labeled goat anti-mouse IgG (H+L) (Thermo Scientific) for 1 hr at room temperature, washed thrice in PBS, and treated with DAPI (4′,6′-diamidino-2-phenylindole) to view the cell nuclei.
  • Alexa Fluor TM 488 labeled goat anti-mouse IgG H+L
  • DAPI 4′,6′-diamidino-2-phenylindole
  • PBMCs peripheral blood mononuclear cells
  • the cells were then suspended in a cell freezing medium containing 50% RPMI, 40% FBS, and 10% DMSO, and cryopreserved for evaluation of T cell responses.
  • a cell freezing medium containing 50% RPMI, 40% FBS, and 10% DMSO, and cryopreserved for evaluation of T cell responses.
  • all pigs were challenged with a combination of intratracheal and intranasal inoculation of 2 ⁇ 10 5 TCID 50 of the H3N2 TX98 virus diluted in 4 mL serum-free DMEM.
  • the pigs were first sedated by intramuscular injection with telazol (Zoetis), ketamine (Zoetis), and xylazine (Vet One).
  • Group 1 received an intramuscular injection of PBS to serve as a control, while Group 2 received an intramuscular injection of 500 ⁇ g of the LNP3-H1pdm09 vaccine.
  • Blood samples were collected from all pigs before immunization and weekly after immunization following the same method as described in Example 5.
  • the pigs were challenged with a combination of intratracheal and intranasal inoculation of 2 ⁇ 10 5 TCID50 of the H1N1 pdm09 virus diluted in 4 mL serum-free DMEM following the same method as described in Example 5.
  • Nasal swabs were taken post-challenge from all pigs daily to measure viral shedding.
  • Example 7 Animal Biosafety Level 2 (ABSL2) research facility at the University of Kansas-Lincoln (UNL). The pigs were randomly assigned into four treatment groups of six pigs. Group 1 received an intramuscular injection of PBS to serve as a control. Group 2 was injected i.m.
  • PRRSV porcine reproductive and respiratory syndrome virus
  • IAV-S Animal Biosafety Level 2
  • protein-H1pdm09 an oil-in-water adjuvant
  • Groups 3 and 4 received an i.m. immunization with 500 ⁇ g of the LNP3-H1pdm09 and LNP3-H1d1 vaccines, respectively.
  • the HA sequence of the protein-H1pdm09 vaccine was derived from the 2009 pdm09 H1N1 virus, which is identical to that of the LNP3-H1pdm09 DNA vaccine.
  • the F&R Ref No.: 24742-0144WO1 LNP-DNA vaccines were administered once, while the protein-H1pdm09 vaccine was given twice, three weeks apart.
  • Example 8 Blood samples were collected from all pigs before immunization and weekly after immunization following the same method as described in Example 5. On day 42 p.v., all pigs were challenged with the H1N2 influenza strain. The virus dose and route of challenge were identical to the ones described in Example 5. Nasal swabs were taken daily post-challenge from all pigs to measure viral shedding. On day 5 post- challenge, the pigs were humanely euthanized and necropsy was performed as described in Example 5.
  • Example 8 Pathological Analysis Evaluation of pathology parameters was performed by a veterinary pathologist blinded to the treatment groups. For the evaluation of gross lung lesions, the percentage of purple-red consolidation typical of IAV-S infection was visually estimated for each lung lobe.
  • the total percentage of lung surface affected was then calculated based on the weighted proportions of each lobe to the total lung volume (Halbur et al., 1995, Vet. Pathol., 32:648- 60).
  • H&E hematoxylin and eosin
  • Virus-infected cells were detected in lung and trachea sections using the RNA in situ hybridization (ISH) assay as previously described (Sun et al., 2019, Vet. Microbiol., 239:108451).
  • ISH RNA in situ hybridization
  • RNA was extracted from nasal swabs and BALF samples using the Quick RNA viral Kit (Zymo Research, Costa Mesa, CA) following the manufacturer’s protocol.
  • RNA fragment with known F&R Ref No.: 24742-0144WO1 copy numbers, based on which the absolute copy numbers of viral RNA in each sample were estimated (Sun et al., 2019, supra).
  • the viral loads were reported as log10 copies per 100 ⁇ L of samples. Samples with a cycle threshold value above 38 were considered negative and assigned a value of 0.8 log10, equivalent to the assay limit of detection.
  • Example 11 Statistical Analysis The statistical analyses were performed using GraphPad Prism 9.0.
  • the HI antibody titers were log2 transformed and analyzed using the mixed-effects model. Univariate data including lung consolidation score, lung microscopic lesion score, virus titers in BALF samples, and IFN-gamma ELISpot data, were analyzed by ordinary one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test.
  • ANOVA ordinary one-way analysis of variance
  • Tukey Tukey’s multiple comparison test.
  • Example 12 Geneation and In Vitro Characterization of the Lipid Nanoparticle-DNA Vaccines Based on the H3 Antigen
  • the average diameter of LNP1-H3 was 81 nm, which was slightly larger than LNP2- H3 (76 nm) (FIG.1A).
  • LNP1 and LNP2 formulations had a polydispersity index below 0.1, indicating a monodispersity distribution (FIG.1B).
  • the respective mean zeta potentials of LNP1 and LNP2 were -1.25 mV and +2.5 mV in 100 mM Tris-Cl pH 7.4, which were in the neutral range (FIG.1C).
  • the mean encapsulation efficiencies of LNP1 and LNP2 were 62% and 82%, respectively (FIG.1D).
  • LNP1 and LNP2 had similar physical characteristics.
  • 0.5 ⁇ g encapsulated DNA in LNP1-H3 or LNP2-H3 was added directly into the medium of HEK-293T cells in a 24-well plate.
  • Naked DNA plasmid was used as a negative control, while DNA plasmid complexed with PEI (PEI- H3) was used as a positive control.
  • PEI- H3 DNA plasmid complexed with PEI
  • the cells were fixed and stained with an anti-flag tag antibody to detect HA-expressing cells.
  • no fluorescent positive cells were detected from cells transfected with naked DNA plasmid, while approximately 90% positive cells were detected from cells treated with PEI-H3 (FIG. 1E).
  • the number of positive cells in LNP2-H3-treated wells was slightly lower than in PEI- H3 transfected wells but was significantly greater than in LNP1-H3-treated wells. Thus, LNP2-H3 had greater in vitro transfection efficiency than LNP1-H3.
  • F&R Ref No.: 24742-0144WO1 Example 13—Pigs Vaccinated with LNP-DNA Vaccines Based on the H3 Antigen Mounted Robust Immune Responses
  • a vaccination / challenge experiment was conducted in 4-week-old pigs to assess the immunogenicity and protective efficacy of LNP1-H3 and LNP2-H3 vaccines.
  • anti-H3 IgG antibodies were not detected in the PBS control group at any sampling dates at 1:100 plasma dilution and were assigned 1 log10 using for drawing graph and statistical purposes (FIG.2A).
  • anti-H3 IgG antibodies were detected in both LNP1-H3 and LNP2-H3 vaccinated pigs on day 7 post-vaccination and the antibody titers sharply increased, reaching the highest titers of 1:10 6 on day 14 post-vaccination, and maintained at the similar titer until the end of the 35-day observation period (FIG.2A).
  • FOG.2A 35-day observation period
  • HI-antibodies were detected on day 7 post-vaccination and gradually increased over time. By day 35 post-vaccination, HI titers exceeded 1:640 (FIG.2B). No significant difference in HI antibody titers was observed between the LNP1-H3 and LNP2-H3 groups. The PBS control group showed low HI titers of 1:10, which might be attributed to non-specific inhibition.
  • NP viral nucleoprotein
  • Example 14 The number of IFN-gamma spots in PBMCs collected from LNP1-H3 F&R Ref No.: 24742-0144WO1 or LNP2-H3 groups on day 35 post-vaccination ranged between 50 and 350 spots per 10 6 PBMCs, with no significant difference observed between these two groups (FIG.2D).
  • Example 14 Pigs Vaccinated with LNP-DNA Vaccines Based on the H3 Antigen were Protected against Challenge Infection with the Homologous H3N2 Influenza Strain
  • all pigs were challenged with an intranasal/intratracheal inoculation with the homologous H3N2 strain. Daily nasal swabs were collected to measure viral shedding.
  • PBS group viral RNA was detected from all samples starting from day 1 post-challenge, reached a maximal titer of 10 8 copies/100 ⁇ L nasal swab at day 3 post-challenge, and declined to 10 6 copies/100 ⁇ L at day 5 post-challenge (FIG.3A).
  • LNP1-H3 group only one pig had a low copy number of viral RNA in samples collected at days 3 and 4 post-challenge (FIG.3A). None of the pigs in the LNP2-H3 group had detectable levels of viral RNA at any sampling dates post-challenge (FIG.3A).
  • Sections of three lung lobes (apical, middle, and caudal) from each pig were stained with H&E to evaluate microscopic changes.
  • Lung sections from pigs in the PBS group exhibited variable, but typically moderate peribronchiolar lymphocytic cuffing with F&R Ref No.: 24742-0144WO1 interstitial pneumonia, necrosis, and attenuation of epithelial cells in bronchioles, and areas of suppurative bronchiolitis.
  • the multifocal interstitial pneumonia and consolidation were characterized by the infiltration of macrophages, lymphocytes, and neutrophils in the alveolar septae, sometimes spilling out into the alveolar lumens (FIG.4B).
  • ISH in situ hybridization
  • the lipid mixture (organic phase) and DNA solution (aqueous phase) were mixed by using a mixer-4 chip (Precigenome, San Jose, CA) and the NanoGeneratorTM Flex-M Nanoparticle Synthesis F&R Ref No.: 24742-0144WO1 System (PreciGenome, San Jose) as described in Example 3.
  • the resulting LNP-DNA vaccine was designated LNP3-H1pdm09.
  • the LNP3-H1pdm09 exhibited an average size of 65 nm, a polydispersity index of 0.1, a zeta potential of +1 mV, and an encapsulation efficiency of 90% (FIG.5A-5D). These physical characteristics of LNP3-H1pdm09 were found to be consistent with the results obtained in the previous study involving the H3 antigens (FIG.1A-1D). Next, we conducted a stability assessment of the LNP3-H1pdm09 under two storage conditions: room temperature and 4°C. No statistically significant changes were observed in the particle size, polydispersity index, and encapsulation efficiency throughout the 56-day observation period (FIG.5A-5B and 5D).
  • Example 16 Pigs Vaccinated with the LNP-DNA Vaccines Based on the H1pdm09 Antigen Mounted Robust Antibody Responses
  • a vaccination / challenge experiment was conducted in seronegative, 4-week-old pigs to evaluate the immunogenicity and protective efficacy of the LNP3-H1pdm09 vaccine. The study included two groups, each consisting of five pigs.
  • Group 1 received an intramuscular injection of PBS to serve as a control, while Group 2 received an intramuscular injection of 500 ⁇ g of the freshly prepared LNP3-H1pdm09 vaccine.
  • F&R Ref No.: 24742-0144WO1 To assess the humoral immune response, we initially measured H1-specific IgG antibodies in sera samples using an indirect ELISA. As expected, the PBS control group did not show any detectable levels of anti-H1pdm09 IgG antibodies at any of the sampling time points at 1:100 plasma dilution and were assigned 1 log10 using for drawing graph and statistical purposes (FIG.7A).
  • pigs vaccinated with the LNP3-H1pdm09 displayed detectable high levels of anti-H1pdm09 IgG antibodies on day 14 post-vaccination.
  • FIG.7A The antibody titers gradually increased, reached the titer of 1:10 5 on day 35 post- vaccination, and slightly decreased to the titer of 1:10 4 at the end of the 48-day observation period (FIG.7A).
  • FIG.7B we measured the HI antibody titers against the homologous H1N1 influenza strain. In the LNP3-H1pdm09 group, the sera HI antibodies were first detected on day 14 post-vaccination and showed a progressive increase (FIG.7B).
  • Example 17 Pigs Vaccinated with the LNP3-DNA Vaccines Based on the H1pdm09 Antigen were Protected Against Challenge Infection with the Homologous H1N1 Influenza Strain
  • all pigs were challenged by intranasal / intratracheal inoculation with the homologous virus H1N1 influenza strain.
  • Daily nasal swabs were collected to monitor virus shedding.
  • Viral RNA was not detected in samples collected before the challenge infection (FIG.8A). High numbers of viral RNA genome were detected from all pigs of the PBS group, while only low copy numbers of viral RNA genome were detected from pigs of the LNP3-H1pdm09 group (FIG.8A).
  • the PBS group exhibited severe microscopic lesions, including moderate to intense peribronchiolar and perivascular lymphocytic cuffing, moderate necrotic and attenuated epithelial lining, and moderate to intense suppurative bronchiolitis.
  • the multifocal interstitial pneumonia and prominent consolidation with the infiltration of macrophages, lymphocytes, and granulocytes was observed in several affected areas (FIG.10B).
  • the lung sections of pigs from the LNP3-H1pdm09 had minor histopathological changes, consisting of mild interstitial pneumonia and mild vascular edema (FIG.10A).
  • the mean composite microscopic score of the LNP3-H1pdm09 group was significantly lower than that of the PBS group (FIG.10C).
  • the results demonstrate that pigs vaccinated with LNP3-H1 were fully protected against challenge infection with the homologous H1N1 virus.
  • Example 18 Pigs Vaccinated with the LNP-DNA Vaccines Based on the H1pdm09 Antigen Conferred Partial Heterologous Protection against the H1N2 Strain and Did Not Induce Vaccine-Associated Enhanced Respiratory Disease It has been extensively documented that pigs vaccinated with either a WIV vaccine or an HA protein-based subunit vaccine may display more pronounced lung pathology than non-vaccinated pigs when exposed to an antigenically mismatched IAV-S strain. This phenomenon is commonly referred to as vaccine-associated enhanced respiratory disease (VAERD). The presence of non-neutralizing antibodies that cross-react with the heterologous IAV-S strain is the primary factor driving the development of VAERD.
  • VAERD vaccine-associated enhanced respiratory disease
  • the HA antigen is synthesized inside the animal cells; thus, it is an intracellular antigen and will undergo a different process of antigen- processing and presentation to the antigen-pressing cells than the HA antigen in the protein- based vaccine, which is an exogenous antigen.
  • the LNP-DNA vaccine will not induce VAERD.
  • this HA sequence of the H1N2 virus belongs to the delta-1 lineage.
  • the resulting LNP-DNA vaccine was designated LNP3-H1d1.
  • pigs vaccinated with the LNP3-H1pdm09 exhibited high HI titers against the homologous H1N1 strain (FIG.11A).
  • pigs vaccinated with the protein-H1pdm09 vaccine did not develop HI antibodies against the H1N1 virus.
  • Pigs vaccinated with the LNP3-H1d1 vaccine had high HI titer against the homologous H1N2 strain (FIG.11B).
  • the LNP3-H1pdm09 group also had similar viral RNA copies in their nasal swabs for the first four days p.c., but the viral RNA copies were reduced on day 5 p.c. (FIG 12A). All pigs in the PBS group had high viral RNA copies in BALF collected at necropsy (FIG.12B). Only two of six pigs in the LNP3-H1d1 group had low viral RNA copies in BALF. The LNP3-H1pdm09 group exhibited a 10-fold lower viral RNA copy in BALF than the PBS group (FIG.12B). The H1N2 strain used in this study was virulent and induced severe lung consolidation in the PBS group (FIG.12C and 12D).
  • the LNP3-H1d1 vaccine F&R Ref No.: 24742-0144WO1 protected pigs from the homologous challenge.
  • pigs in the LNP3-H1pdm09 group exhibited a significantly lower percentage of lung consolidation than those in the PBS group (FIG.12C).
  • pigs in the protein-H1pdm09 group had significantly more severe lung consolidation than those in the PBS group, indicating the development of VAERD (FIG.12C and 12D).
  • the results of this third animal study demonstrated that the LNP3-H1d1 vaccine conferred solid protection against the homologous H1N2 virus.
  • the HA protein- based vaccine did not confer heterologous protection against the H1N2 strain; instead, it induced VAERD, consistent with a previous report.
  • the LNP3-H1pdm09 vaccine provided partial protection against the H1N2 virus, as evidenced by lower viral RNA in BALF and lower lung lesions compared to the PBS group.
  • compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions.
  • These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

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Abstract

Ce document décrit des procédés et des compositions qui peuvent être utilisés pour développer et administrer rapidement de nouveaux vaccins.
PCT/US2024/033024 2023-06-07 2024-06-07 Procédés et compositions pour le développement et la distribution de vaccins Pending WO2024254461A2 (fr)

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