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, NI22HMFPXXXXG005, NI20HMFPXXXXG023, NI21HMFPXXXXG031, NI20HFPXXXXXG055, and NI21HFPXXXXXG004 awarded by the National Institute of Food and Agriculture. The government has certain rights in the invention. CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C.119(e) to U.S. Application No.63/471,614 filed June 7, 2023. TECHNICAL FIELD This disclosure generally relates to methods and compositions for vaccine development and delivery. BACKGROUND 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. Due to the cost and the time demand of the vaccine licensing process, veterinary vaccines are not updated in time to cope with the continual evolution of the viruses. Therefore, methods and compositions that can be used to rapidly develop and deliver new vaccines would be very useful. SUMMARY This disclosure describes a non-replicating, non-viable vaccine platform for the rapid development and delivery of vaccines (e.g., veterinary vaccines).
F&R Ref No.: 24742-0144WO1 In one aspect, vaccine compositions are provided that 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. In some embodiments, 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. In some embodiments, 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). 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). In some embodiments, 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). In some embodiments, the nucleic acid vector is a DNA plasmid. In some embodiments, 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. In some embodiments, 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. In another aspect, 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. In some embodiments, the administration comprises an intra-muscular injection. In some embodiments, 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. In some embodiments, 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. In some embodiments, the animal is selected from swine, avian, cow, horse, goat, sheep, dog, and cat. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which 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. DESCRIPTION OF DRAWINGS 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 log10 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. Data are expressed as the IFN-gamma secreting cells (IFN-gamma- SC) per 106 cells. Data were analyzed by one-way ANOVA, followed by Tukey’s multiple comparisons test. 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 µL of the sample. FIG.3B is data showing viral RNA copies in bronchoalveolar lavage fluid (BALF) collected on day 5 post-challenge infection. The horizontal dotted lines at y = 0.8 indicate the limit of detection of the RT-qPCR assay. For graphical and statistical purposes, samples with undetectable levels of viral RNA were assigned a value of 0.8 log10 which is equivalent to the assay detection limit. 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. 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.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 µL of the sample. FIG.8B is data showing viral RNA copies in bronchoalveolar lavage fluid (BALF) collected on day 5 post-challenge infection. The horizontal dotted lines at y = 0.8 indicate the limit of detection of the RT-qPCR assay. For graphical and statistical purposes, samples with undetectable levels of viral RNA were assigned a value of 0.8 log10 which is equivalent to the assay detection limit. 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®). To enhance antigenic coverage, commercial WIV vaccines often contain multiple strains (e.g., multiple Influenza A strains) representing different subtypes (e.g., different Influenza A subtypes) that are co-circulating in the host population. Even so, commercial influenza vaccines often fail to confer optimal levels of heterologous protection, because the virus is constantly evolving and new virus variants frequently emerge. In this study, a nucleic acid encoding the 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. These findings suggest that 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. See, for example, Sun et al. (2019, Vet. Microbiol., 239:108451) and Vander Veen et al. (2012, Vaccine, 30(11):1944- 50). In addition to HA, 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. It would be appreciated that 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. A DNA plasmid expressing a protein (e.g., an antigenic protein (an “antigen”)) is non-infectious, non-viable, and safe to use in animals. However, 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. Recently, lipid nanoparticles (LNPs) have emerged as a promising carrier for nucleic acid-based vaccines. It would be appreciated that 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. Trapped in endosomes, in which the pH is lower than in the extracellular environment, 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 LαC to HIIC 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. It would be appreciated that 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. It would be appreciated that 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. It would be appreciated that 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. 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (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. The 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). Specifically, for example, 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).
F&R Ref No.: 24742-0144WO1 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. One example of 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. As used herein, 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. Many methods for introducing nucleic acids into host cells, both in vivo and in vitro, 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. In accordance with the present disclosure, there may be employed 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.
F&R Ref No.: 24742-0144WO1 EXAMPLES 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 μg/mL streptomycin. 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. AEK70342.1), H1N1 (GenBank Accession No. AFM46639.1) and H1N2 (GenBank Accession No. KR715130.1) were codon optimized for optimal expression in swine cells (Sus scrofa). 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.
F&R Ref No.: 24742-0144WO1 SEQ ID NO: 1 atgaagactatcatcgcactgtcatatatcctctgcttagtttttgcccagaaattaccgggaaatga taatagcacagccacattatgtctgggccaccatgcagtgcccaacggtaccctggtcaaaacaataa caaacgatcagatcgaggtgactaacgccaccgagcttgtacagagttcaagtacgggccgtatctgt gactctccccacaggatcctcgatggcaagaactgtacactcattgatgctctgcttggggaccctca ctgtgatggttttcaaaataaagaatgggacctcttcatcgaacgctccaaggcctactcaaattgtt acccttatgacgtgccagactatagcagtttgagatcacttgtagcctccagtgggacgctcgaattc accaatgaagatttcaactggaccggagtcgcacaggacggtggatcctatagctgcaagcgggggag tgttaaatctttcttcagcaggcttaactggctgcacaagctcgagtacaaatatccggcactgaatg tgaccatgccaaacaatgataaatttgataagctgtacatctggggcgtccatcatccttccactgat tcagagcagaccagcttgtacgtacaggccattggccgggttacagtcagcaccaagagttctcaaca gacagtgattccaaacattggttccaggccctgggtgcgtggcatctcgtctcgaatatctatctatt ggacgattgtgaagccaggagacattttgctcattagctccacggggaacctgattgcccctcgcggc tacttcaaaatcagaaatgggaaatcaagcatcatgcgatcggatgcgcccatcgacaactgctactc cgagtgcatcaccccaaatggatctatacctaatgacaagcccttccagaatgttaacagaattacat atggtgcgtgccccaagtatgtcaagcaaaagactttgaaactggccactggaatgaggaatgtccca gagaaacagacacggggtatctttggggctatcgctggctttattgagaatgggtgggagggcatggt ggacggctggtacggcttccggcaccagaactcagaagggactggccaggctgctgacctgaaatcga ctcaagcagcaattgaccaggtgaacgggaagctcaaccgactgatagaaaagaccaatgagaaattc catcagatcgaaaaggagttcagcgaagttgagggaaggatccaggatcttgagaagtacgtggaaga caccaaaattgatctgtggagttataatgcagaactgctggtggccctggagaaccagcacaccatcg acttaacggacagtgagatgaacaagctgtttgaaaaaactaggaagcagctgagagaaaatgctgag gacatgggcaacggctgcttcaagatttaccacaaatgtgacaatgcctgcattggaagcatacgcaa cggcacctatgaccatgatgtgtacagagatgaggccctgaacaaccgcttccaaataaaaggggtcg agctgaagtccgggtacaaggactggattctctggatctcctttgctatctcctgctttttgctttgt gtggttctattgggatttataatgtgggcgtgccagaagggaaacatccggtgcaatatttgtataGA CTACAAAGACGATGACGACAAGTAATAGTGA SEQ ID NO: 2 atggaagcaatccttgtagttctcctatacacttttgcagccgccaatgccgacacactgtgcatcgg ctaccatgccaacaactcaacagatactgttgacaccgtgcttgagaagaacgtgacagtgacccact ctgtgaacctactggaggacaagcacaacggaaagctctgcaagctcagaggtgtggctccactgcac ctgggcaagtgcaatatagctggatggatcctcggaaatcctgagtgtgaatccttgtcaacggctag ttcctggtcctacatagtggaaacaagctcatcagataatgggacctgctacccgggggacttcatag actatgaagagcttagagaacagctgagcagtgtatctagctttgagcgctttgaaatttttcctaag acatcctcctggcccaaccacgacagcaacaagggtgtcacagcagcttgtcctcatgcaggagcaaa gagcttctacaagaacttaatatggctggttaaaaagggcaacagctatcctaaactttccaaaagtt atatcaatgacaaaggcaaggaagtgctggttctgtggggcatccaccaccccagcacgtccgcggac cagcagggcttgtaccaaaatgctgatgcttatgtctttgtcggaagttcaaggtattccaagaagtt caaaccagaaatcgccattcggccaaaggtgcgagatcaagaagggcggatgaactactactggacgc tggtggagcctggtgataaaataactttcgaggccactgggaacttagtggtgccccgatacgccttt gccatggaaaggaatgcaggctctgggatcatcatttctgatgcccctgtgcatgactgtaataccac ctgtcagaccccaaatggggcgattaacacgtctctacccttccacaacatccatcccatcaccattg ggaagtgtccgaagtatgtcaaatctacaaagctgcgcttggccacaggtctcagaaatgtaccatct atccagagccgtggactcttcggggctatagcaggctttattgaaggaggctggaccggcatggtgga cggctggtacggataccaccaccagaatgagcagggaagcggctatgctgcggacctgaaatcaaccc agaacgccattgatgaggtcaccaacaaagtgaactccgtaattgaaaagatgaatacacagttcacc gcggtggggaaggagttcaaccatctagagacccgcatcgagaacctgaataaaaaagttgatgatgg gtttctggacatctggacttacaatgctgagctgcttgttttgttggaaaatgaacggactctggatt
F&R Ref No.: 24742-0144WO1 atcatgattccaatgtgaagaatctctacgagaaagtacgttctcaactcaaaaacaatgccaaagag attggtaacggatgtttcgaattttatcacaagtgtgacaacacttgcatggagtctgtcaagaatgg aacttatgactatcccaaatacagcgaggaggccaagctgaaccgggaggagatagatggcgtcaaat tagaatccaccaagatctaccagattttggctatctatagtactgtggccagcagcctggtcctggtc gtctcgcttggggcaattagtttctggatgtgctcgaacggcagcctccagtgcaggatctgcattgg tagtgcccaccatcaccatcatcactga SEQ ID NO: 3 atgaaggtcaagttactcatactgctgtgtaccttcactgccgcctacgctgatactatctgcatcgg ctatcatgcgaacaactccacagacaccgtggacacggtcctggagaagaacgtcacagtgacccact cggtgaacctgcttgaggattcacacaacggtaagctgtgcctcctcaagggcatggctcccctgcaa ctcgggaactgttcggtggccgggtggatcctgggtaacccagaatgtgaaagtctcatatccaagga gtcctggagctacatcgtggaggcacccaattcagacaacggagcctgctaccctggccagtttgcag attatgaagaactgcgagagcagctctctagtgtctcgtcctttgagcgctttgaaatcttcccgcgg gaatcttcttggcccaaccacaccgttacgggggtgtctgcgtcttgctctcataatggcaaacgctc tttctatcggaacttgatttggctcacagtgaaaaatggcctgtatccaaatctaagcaagtcctacg aaaatgacaagggcaaagaggtgctcatattatggggcgtgcatcacccttccaacattggggaccag agaactttgtaccacacagagaatgcgtatgtgtcagtcatgtcctctcactatagtcgtcgtttcac accagagattaccaagcggccaaaagttagagaccaagagggccgaattaattactactggacgctgc tagaacctggagatactattatttttgagaccaatggaaacctgatcgcgccttggtacgcctttgct cttagtaggggtcttggtagtgggatcatcacctcaaaggcccccatggacaaatgtgatgccaagtg ccagacccccaaaggagcaatcaacagcaatctccccttccagaacgttcatcctgtgactattggag agtgtccaaaatacgtccgcagtacaaagctgaggatggttacagggcttaggaatattccgtcgatc cagagccgggggctctttggagctatcgcaggatttattgagggggggtggacaggaatggtggacgg ctggtatggctaccaccaccagaatgaacagggcagcggttacgcagctgatcaggagagtacgcaaa atgcaataaacgggatcactaataaagtcaactctgttatagaaaaaatgaatactcagttcaccgca gtgggcaaggaattcaacaaactggagaggagaatggagaacctgaacaagaaggttgatgatggctt cttggatatctggacctacaatgctgagctgctggtattgcttgaaaacgaacgcaccctggactttc atgacagtaatgtgaaatcactgtatgagaaggtaaagtcccaactgaagaataatgctaaagaaata ggtaatggctgcttcgaattttatcacaagtgtaacaatgagtgcatggaaagcgtaagaaatggaac atatgactacccgaagtactccgaggagagcaaattgaacagagagaaaattgatggagtgaaactgg actccatgggagtgtatcgaattctggccatctacagcaccgtagccagctcattagtcttattggtt tctttgggggccatcagcttctggatgtgcagcaacggctcactgcagtgcaggatctgtattggcag tgccgactacaaagacgatgacgacaagtaatagtga Example 3—Preparation of LNPs Three different LNP-DNA formulations were prepared following a method described previously with some modifications (Roces et al., 2020, Pharmaceutics, 12). In formula 1 (LNP1-H3), specific volumes of MC3, DOTAP, DSPC, cholesterol, and DMG-PEG2000 were mixed at a molar ratio of 35:5:10:48:2 to form a final mixture with a total lipid concentration of 28 mM, and the DNA plasmid encoding the H3 antigen was diluted in 50 mM citrate buffer pH 4.0. In formula 2 (LNP2-H3), MC3, DOTAP, DSPC, cholesterol, and DMG-PEG2000 were mixed at a molar ratio of 42:10:8:38:2 to form a final mixture with a total lipid concentration of 25 mM, and the DNA plasmid encoding the H3 antigens was
F&R Ref No.: 24742-0144WO1 diluted in 25 mM sodium acetate buffer pH 4.0 for LNP2. In formula 3 (LNP3-H1pdm09), 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 NanoGenerator™ 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-LyzerTM G2 dialysis cassettes with the molecular weight cut-off of 10 kDa (Thermo Scientific). After dialysis, LNPs were passed through 0.45 µm PES filters and the encapsulated DNA plasmid concentration was adjusted to 100 µg/mL. 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. At 48 hr post-transfection, 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. After three washes in PBS, the cells were incubated with Alexa FluorTM 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. The fluorescent images were acquired using a Nikon Eclips Ts2R-FL operated by Nikon NIS Elements (ver 5.02). The images were captured separately using either a green or a blue filter and then merged to create the final composite images.
F&R Ref No.: 24742-0144WO1 Example 5—Animal Experiment No.1 Eighteen weaned pigs, approximately 4 weeks old and seronegative for porcine reproductive and respiratory syndrome virus (PRRSV) and IAV-S, were obtained from Midwest Swine Research and housed in the animal biosafety level 2 (ABSL2) research facility at the University of Nebraska-Lincoln (UNL). The pigs were randomly assigned into three groups of six pigs. Pigs in Groups 1 and 2 were intramuscularly injected with the LNP1-H3 or LNP2-H3 vaccines, respectively. Each dose of the LNP vaccine contained 500 µg of encapsulated DNA. Group 3 was injected with PBS to serve as a non-vaccination control. Whole-blood samples were collected from all pigs before immunization and weekly after immunization using vacuum tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant. The blood tubes were then centrifuged at 1200 x g for 15 minutes, and the plasma was collected and stored at -20°C for evaluation of humoral immune responses. The cells were re-suspended in PBS and transferred to SepMate-50 tubes (Stemcell Technologies) prefilled with 10 mL of density gradient medium. After centrifugation at 1200 x g for 15 min at room temperature, the top layer containing peripheral blood mononuclear cells (PBMCs) was collected and washed twice with PBS containing 2% FBS. 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. On day 35 post-vaccination, all pigs were challenged with a combination of intratracheal and intranasal inoculation of 2 × 105 TCID50 of the H3N2 TX98 virus diluted in 4 mL serum-free DMEM. To administer the challenge inoculation, the pigs were first sedated by intramuscular injection with telazol (Zoetis), ketamine (Zoetis), and xylazine (Vet One). Two mL of the virus inoculum then were administered via an endotracheal tube inserted into the tracheal tract, and 1 mL of the inoculum was administered into each nostril. Nasal swabs were taken from all pigs daily post-challenge to measure viral shedding. On day 5 post-challenge, the pigs were humanely euthanized with an overdose of sodium pentobarbital. During the necropsy, the lungs were removed, and bronchioalveolar lavage fluid (BALF) samples were collected using 50 mL of cold PBS to measure viral load in the lungs. Gross lung lesion was visually estimated and samples of the trachea (approximately 1
F&R Ref No.: 24742-0144WO1 inch above the carina), left apical, middle, and caudal lung lobes were collected and fixed in 10% buffered formalin for histopathologic examination. Example 6—Animal Experiment No.2 Ten weaned pigs, approximately 4 weeks old and seronegative for porcine reproductive and respiratory syndrome virus (PRRSV) and IAV-S, were obtained from Midwest Swine Research and housed in the Animal Biosafety Level 2 (ABSL2) research facility at the University of Nebraska-Lincoln (UNL). The pigs were randomly assigned into two groups 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 μg 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. On day 49 post-vaccination, the pigs were challenged with a combination of intratracheal and intranasal inoculation of 2 × 105 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. On day 5 post-challenge, the pigs were humanely euthanized and necropsy was performed as described in Example 5. Example 7—Animal Experiment No.3 Twenty-four weaned pigs, approximately 4 weeks old and seronegative for porcine reproductive and respiratory syndrome virus (PRRSV) and IAV-S, were obtained from Midwest Swine Research and housed in the Animal Biosafety Level 2 (ABSL2) research facility at the University of Nebraska-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. with the HA protein mixed with an oil-in-water adjuvant (referred to as protein-H1pdm09). Groups 3 and 4 received an i.m. immunization with 500 ^g of the LNP3-H1pdm09 and LNP3-H1d1 vaccines, respectively. It should be noted that 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. 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). For the evaluation of microscopic lung lesions, sections of the apical, middle, and caudal lung lobes were stained with hematoxylin and eosin (H&E) using routine procedures. Each section was scored for six parameters, including peribronchiolar lymphocytic cuffing, interstitial pneumonia, bronchial and bronchiolar epithelial cell changes, bronchiolitis, edema, and epithelial exocytosis, using the scoring scale described previously (Gauger et al., 2014, Virology, 471-3:93-104). The average composite scores of the three lobes are reported. 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). The frequency of virus-infected cells in airway epithelium and pulmonary parenchyma was estimated for each lung component using a 5-point scale: 0 - no signals, 1 - minimal occasional signals, 2 - mild scattered signals, 3 - moderate scattered signals, and 4 - abundant signals.
F&R Ref No.: 24742-0144WO1 Example 9—Immunological Assays Antibody responses against IAV-S nucleoprotein (NP) were measured by using a commercial blocking ELISA (IDEXX, Montpellier, France), following the manufacturer’s recommendation. Results are expressed as a sample to negative control ratio (S/N ratio). Samples with an S/N ratio below 0.6 were considered positive. IgG antibodies specific to the H3 protein was measured using indirect ELISAs. Briefly, flat-bottom 96-well plates (Immulon™ 2HB) were coated with 100 µL of the H3 or H1pdm09 protein diluted in PBS pH 7.4. After washing with the phosphate buffer saline containing 0.1% tween 20 (PBS-T20), the plates were blocked with PBS-T20 containing 10% skim milk for 2 hr at room temperature. Plasma samples were serially 10-fold diluted in a sample dilution buffer (PBS-T20 containing 5% skim milk), and 100 µL of each diluted sample was added to each well of the plates in duplicate, followed by 1 hr incubation at room temperature and five washes with PBS-T20. One hundred µL of HRP-labeled goat anti-pig IgG (Sera Care, Milford, MA) diluted 1:2000 in a dilution buffer was added to each well. The plates were incubated at room temperature for 30 min, and washed five times with PBS- T20, followed by incubation with 100 µL/well of the ABTS substrate (Sera Care, Milford, MA, USA). The reaction was stopped at 10 min by adding 100 µL of 1% sodium dodecyl sulfate diluted in distilled water. Optical density (OD) was measured at 405 nm wavelength using an ELISA plate reader (Bio-Tek ELx808, Winooski, VT, US). An arbitrary cutoff value equivalent to the mean plus five standard deviations of the OD values of samples from the non-immunized control animals was calculated. Antibody titers were determined at the highest dilutions with OD values above the cutoff value. Hemagglutination inhibition (HI) assay and interferon-gamma (IFN-gamma) ELISpot were performed as previously described (Sun et al., 2019, supra). Example 10—Quantification of Viral Load 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. The number of viral genomic copies was quantified using a commercial real-time reverse transcription PCR (RT-qPCR) (VetMax-Gold SIV Detection Kit, Life Technologies, Austin, TX). A standard curve was established using a chemically synthesized 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 μL 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. Example 12—Generation 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). Both 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). Overall, LNP1 and LNP2 had similar physical characteristics. To evaluate the transfection efficiency, 0.5 µg 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. At 48 hrs post-transfection, the cells were fixed and stained with an anti-flag tag antibody to detect HA-expressing cells. As expected, 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. We first measured H3-specific IgG using an indirect ELISA. As expected, 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). On the other hand, 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 1:106 on day 14 post-vaccination, and maintained at the similar titer until the end of the 35-day observation period (FIG.2A). There were no significant differences in kinetics and magnitude of anti-H3 IgG antibody responses between the LNP1-H3 and LNP2-H3 groups. Next, we measured hemagglutination inhibition (HI) antibody titers which are an important component of the protective immunity against IAV-S. 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. We also measured antibodies against the viral nucleoprotein (NP) using a commercially available ELISA kit that is commonly used for IAV-S serodiagnosis. All samples collected on days 0 and 35 post-vaccination were negative for the NP-based antibodies (FIG.2C). The results demonstrated that the pigs were not exposed to IAV-S and that the anti-H3 IgG or HI antibodies detected in the LNP1-H3 and LNP2-H3 groups were due to vaccination. The frequencies of IFN-gamma secreting cells in PBMCs were evaluated using the IFN-gamma ELISPOT assay. No IFN-gamma spots were detected in PBMCs collected before vaccination or even in PBMCs collected from the PBS group on day 35 post- vaccination (FIG.2D). 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 106 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 On day 35 post-vaccination, all pigs were challenged with an intranasal/intratracheal inoculation with the homologous H3N2 strain. Daily nasal swabs were collected to measure viral shedding. Viral RNA was not detected in any samples collected before the challenge infection (FIG.3A). In the PBS group, viral RNA was detected from all samples starting from day 1 post-challenge, reached a maximal titer of 108 copies/100 µL nasal swab at day 3 post-challenge, and declined to 106 copies/100 µL at day 5 post-challenge (FIG.3A). In 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). On day 5 post-challenge, the pigs were humanely euthanized and necropsied and BALF samples were collected to evaluate viral loads within the lungs. High copy numbers of viral RNA, ranging from 106 to 108 copies/100 µL of BALF, were detected in all pigs from the PBS group (FIG.3B). In contrast, viral RNA was not detected in any pigs from the LNP1-H3 or LNP2-H3 groups (FIG.3B). The gross lung lesions were evaluated by a board-certified pathologist who was blinded to the experimental design at the time of necropsy. Lungs from the PBS group presented with purple-red consolidation typical of IAV-S infection, with the total lung surface consolidation ranging from 0.4 to 4.6% (FIG.4A). The consolidation was more noticeable in the apical and middle lobes (FIG.4A). In contrast, only one pig in the LNP2- H3 group exhibited lung consolidation (2.5%), while none of the pigs in the LNP1-H3 group displayed visible lung consolidation (FIG.4A). The percentage of lung consolidation was not statistically different between the LNP1-H3 and LNP2-H3 groups, and both groups had significantly lower lung consolidation than the PBS group (FIG.4A). 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). In contrast, the lung tissues of pigs from the LNP1-H3 and LNP2-H3 groups showed only mild interstitial pneumonia and peribronchial lymphocytic infiltration (FIG.4B). The composite microscopic scores of pigs from the LNP1-H3 and LNP2-H3 groups were statistically similar and significantly lower than those of the PBS group (FIG.4B). The in situ hybridization (ISH) assay was used to detect viral infected cells in the trachea and the middle lung lobe. Numerous viral infected cells were detected in the tracheal, bronchial, and bronchiolar epithelium of pigs from the PBS group (FIG.4C and 4D). On the other hand, viral-infected cells were rarely detected in either tracheal or lung sections of pigs from the LNP1-H3 and LNP2-H3 groups (FIG.4C and 4D). Collectively, the data clearly indicate that vaccination with either the LNP1-H3 or LNP2-H3 vaccines fully protected pigs against challenge infection with the homologous H3N2 influenza strain. Example 15—Generation and In Vitro Characterization of a Lipid Nanoparticle-DNA Vaccine Based on the 2009 Pandemic H1N1 Virus In the previous studies, we generated two different formulations of LNP-DNA vaccines based on the HA gene of the H3N2 influenza virus (designated LNP1-H3 and LNP2-H3). We successfully demonstrated that both formulations of LNP-DNA vaccines were highly effective in inducing protective immune responses in pigs. To further assess the efficacy of LNP as a nanocarrier for the delivery of nucleic acid-based vaccines, in this study, we encapsulated the DNA plasmid encoding the HA antigens of the 2009 pandemic H1N1 (designated H1pdm09). The 5 lipids: 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 H1pdm09 antigen diluted in 25 mM sodium acetate buffer pH 4.0. The lipid mixture (organic phase) and DNA solution (aqueous phase) were mixed by using a mixer-4 chip (Precigenome, San Jose, CA) and the NanoGenerator™ 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). However, there was a gradual decrease in the zeta potential over time, with the zeta potential of LNP3-H1pdm09 stored at room temperature exhibiting a greater reduction compared to that stored at 4°C (FIG.5C). Nevertheless, by the end of the 56-day observation period, the zeta potentials of LNP3-H1pdm09 under both storage conditions remained within the neutral range. We assessed the transfection efficacy of the LNP3-H1pdm09 in HEK-293T cells. The freshly prepared LNP3-H1pdm09 demonstrated excellent transfection efficiency, as evidenced by the detection of a significant number of cells expressing the H1 protein at 48 hrs post-transfection (FIG.5E and 5F). Visual examination revealed that the frequencies of H1-expressing cells were remarkably similar in HEK-293T cells transfected with LNP3- H1pdm09 stored at either room temperature or 4°C for various durations, up to 56 days (FIG. 6). These findings indicate that the storage conditions did not appear to have any noticeable impact on the transfection efficiency. 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 μg 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). In contrast, 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 1:105 on day 35 post- vaccination, and slightly decreased to the titer of 1:104 at the end of the 48-day observation period (FIG.7A). Next, 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). By day 35 post-vaccination, the mean HI titer reached 1:640, followed by a slight reduction to 1:320 on day 48 post- vaccination (FIG.7B). In the PBS control group, all pigs exhibited HI antibody titers below 1:10 and were assigned a titer of 1:5 for graphical and statistical purposes. These findings collectively demonstrate that pigs vaccinated with the LNP3-H1pdm09 mounted a robust antibody response. 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 On day 49 post-vaccination, 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). On day 5 post-challenge, the pigs were euthanized in a humane manner and necropsies were performed. Bronchoalveolar lavage fluid (BALF) samples were collected to assess viral loads within the lungs. In the PBS group, all pigs exhibited high levels of viral
F&R Ref No.: 24742-0144WO1 RNA, ranging from 104.8 to 106.5 copies/100 μL of BALF, while none of the pigs of the LNP3-H1pdm09 group exhibited detectable levels of viral RNA in their BALF (FIG.8B). During necropsy, gross lung lesion was scored by a board-certified pathologist who was blinded to the experimental design. The lungs of pigs of the PBS group presented with purple-red consolidation typical of IAV-S infection, with the percentage of total lung surface consolidation ranging from 4.0% to 17.6% (FIG.9A-9C). In contrast, only one pig of the LNP3-H1pdm09 group had lung consolidation (0.3%) (FIG.9C). The percentage of lung consolidation of the LNP3-H1pdm09 group was statistically lower than that of the PBS group (FIG.9C). Histopathological lesions were evaluated using lung tissues from the right apical, middle, and caudal lobes of each pig. 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). Conversely, 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). In summary, 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.
F&R Ref No.: 24742-0144WO1 In the case of the LNP-DNA vaccine, 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. We hypothesized that the LNP-DNA vaccine will not induce VAERD. To test this hypothesis, we developed another LNP-DNA vaccine containing the HA gene of an H1N2 strain. The LNP formulation used to encapsulate this HA gene is the same as the one used to encapsulate the H1pdm09 antigen. Based on phylogenic analysis, this HA sequence of the H1N2 virus belongs to the delta-1 lineage. Thus, the resulting LNP-DNA vaccine was designated LNP3-H1d1. Consistent with the previous experiment, pigs vaccinated with the LNP3-H1pdm09 exhibited high HI titers against the homologous H1N1 strain (FIG.11A). In contrast, 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). It is important to note that the H1N1 and H1N2 strains used in this share only 79% similarity in their HA sequences. Consequently, sera from pigs vaccinated with the LNP3-H1pdm09 vaccine did not exhibit any cross-HI titers against the H1N2 virus and vice versa (FIG.11). Following the challenge infection with the H1N2 influenza strain, the PBS group showed high viral RNA copies in nasal swabs, while the LNP3-H1d1 group had minimal viral RNA copies in three out of six pigs (FIG.12A). The protein-H1pdm09 group exhibited similar viral RNA copies in their nasal swabs as the PBS group. 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). In contrast, none of the pigs in the LNP3-H1d1 group displayed visible lung consolidation. Thus, the LNP3-H1d1 vaccine
F&R Ref No.: 24742-0144WO1 protected pigs from the homologous challenge. Interestingly, pigs in the LNP3-H1pdm09 group exhibited a significantly lower percentage of lung consolidation than those in the PBS group (FIG.12C). On the other hand, 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). In conclusion, 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. It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims. Disclosed are methods and 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.