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  • 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
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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2760/16011—Orthomyxoviridae
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16141—Use of virus, viral particle or viral elements as a vector
  • C12N2760/16143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• influenza viruses expressing fluorescent proteins of different colors were generated. Viruses containing the foreign matter gene were passaged. Upon adaptation to mice, stable expression of the fluorescent proteins in infected animals allowed their detection by different types of microscopy and by flow cytometry.
• the use of fluorescent influenza viruses, each of which stably expresses one of four different fluorescent proteins, allows for simultaneous monitoring and live imaging. Using these viruses, several studies were performed to demonstrate the versatility of these viruses. For example, this system was used to analyze the progression of viral spread in mouse lungs, for live imaging of virus-infected ceils, and for differentia! gene expression studies in virus antigen-positive and -negative !ive ceils in the lungs of Color-flu-infected mice.
• Color-flu viruses are powerful tools to analyze virus infections at the cellular level in vivo to better understand influenza pathogenesis.
• different stabilizing mutations in the resulting viruses were identified. These mutations include the T380A in HA protein (numbering is that for HI ) and E712D in PB2 protein of A/PR/8/34 (H 1 N 1 ) virus, and V25A,
• a recombinant virus has one or more stabilizing mutations, e.g., one or more substitutions in one or more influenza virus proteins that enhance the stability or replication (for instance, enhance the titer) of the recombinant virus with the one or more substitutions relative to a corresponding virus without the one or more substitutions (a parental virus) and/or one or more substitutions in one or more influenza virus proteins that enhance the stability or replication of a heterologous gene sequence present on one of the viral segments in the recombinant virus relative to a corresponding virus without the one or more substitutions that has the heterologous gene sequence in the respective viral segment and/or one or more substitutions in one or more influenza virus proteins that enhance the stability or replication of a heteroiogous gene sequence that is present on an additional viral segment in the recombinant virus relative to a corresponding virus without the one or more substitution and that has the additional viral segment with the heteroiogous gene sequence.
• stabilizing mutations e.g., one or more substitutions in one or
• PB1 , PB2, PA, NP, M, and NS encode proteins that are having at least 80%, e.g., 90%, 92%, 95%, 97%, 98%, or 99%, including any Integer between 80 and 99, contiguous amino acid sequence identity to, a polypeptide encoded by one of SEQ ID NOs:1 -6 or 10-15.
• the influenza virus polypeptide has one or more, for instance, 2, 5, 10, 15, 20 or more, conservative amino acids substitutions, e.g , conservative substitutions of up to 10% or 20% of 2, 5, 10,
• the residue at position 443 in PA is K or H.
• the residue at position 180 in PA is R, K or H.
• the residue at position 200 in PA is A, G, I, L or V
• the residue at position 737 in PB1 is H or R.
• the residue at position 149 in PB1 is A, T, G, ! or L.
• the residue at position 684 in FBI is D or N.
• the residue at position 685 in PB1 is E or Q.
• the residue at position 25 in PB2 Is A, L, T, !, or G.
• the residue at position 712 in PB2 Is D.
• a recombinant influenza virus of the disclosure having an extra viral segment with a heterologous gene sequence (a“9 segment” virus) which virus has enhanced stability and/or replication may be prepared by selecting viral segments for inclusion in the recombinant virus having one or more of the stabilizing mutations in an influenza virus protein.
• the extra viral segment may be derived from any of the naturally occurring viral segments.
• the residue at position 443 in PA is K or H. in one embodiment, the residue at position 737 in PB1 is H or R. in one embodiment, the residue at position 25 in PB2 Is A, L, T, I, or G. In one embodiment, the residue at position 180 In PA is R, K or H.
• the residue at position 200 in PA is A, G, I, L or V. in one embodiment, the residue at position 149 in PB1 is A, T, G, I or L. in one embodiment, the residue at position 684 in PB1 is D or N in one embodiment, the residue at position 685 in PB1 is E or Q.
• the heterologous gene in the extra viral segment replaces influenza virus protein coding sequences (e.g., there is a deletion of influenza virus coding sequences without deleting encapsidation (incorporation) sequences in coding sequences that are linked to encapsidation sequences In non-coding sequences at one or both ends of the viral segment).
• the heterologous gene sequence in the extra viral segment is in genomic orientation.
• the heterologous gene sequence in the extra virai segment is fused In frame to N-terminal influenza virus protein coding sequences.
• the heterologous gene sequence in the extra viral segment is fused in frame to C-ierminai influenza virus protein coding sequences.
• a HA viral segment encoding a HA with a residue at position 380 that is not threonine may be selected;
• a PA viral segment encoding a PA with a residue at position 443 that is not arginine may be selected;
• a PB1 viral segment encoding a PB1 with a residue at position 737 that is not lysine may be selected;
• a PB2 viral segment encoding a PB2 with a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid may be selected;
• a NS viral segment enclosing a NS1 with a residue at position 167 that is not proline may be selected; or any combination thereof in one embodiment, the residue at position 443 in PA is K or H.
• the heterologous gene sequence is in the HA viral segment. In one embodiment, the heterologous gene sequence is in the M viral segment in one embodiment, the heterologous gene sequence is in the NS viral segment. In one embodiment, the heterologous gene sequence is in the NR viral segment, e.g., see Liu et a!., 2012; Wang et ai. 2010; Ariior et a!., 2010; Dos Santos Afonso et a!., 2005). In one embodiment, the heterologous gene sequence is in the PA viral segment in one embodiment, the heterologous gene sequence is in the PB1 viral segment in one embodiment, the heterologous gene sequence is in the PB2 viral segment.
• the method includes serially passaging an isolate of an influenza virus in an individual host organism, and identifying individual viruses with the altered property and optionally moiecu!ariy characterizing the individual viruses.
• Sequential images In lower panel (1 -4) show an enlarged view of the box in the upper right panel.
• J Differentially expressed (DE) transcripts were identified by comparing gene expression levels in naive macrophages with those in Venus(+) macrophages from infected mice. Likewise, gene expression levels were compared for naive macrophages and Venus(-) macrophages obtained from infected mice.
• FIGS 4A-4E Characterization of MA-Venus-HPAI virus
• A Four B6 mice per group were intranasally inoculated with MA-Venus-HPAI virus. Mouse body weight and survival were monitored for 14 days
• B Lungs, spleens, kidneys, and brains were harvested from B6 mice at day 3 p.i. with 10 5 PFU of MA-Venus-HPAI virus.
• C, D Lung tissues were harvested from B6 mice at day 1 and day 2 p.i.
• FIG. 1 Polymerase activity of different RNP combinations derived from the WT-Venus-H5N1 and MA-Venus-H5N1 viruses. 293 ceils were transfected in triplicate with a !uciferase reporter plasmid and an Interna! control plasmid, together with plasmids expressing PB1 , PB2, PA, and NP from either WT- Venus-H5N1 or MA-Venus-H5N1 virus. Segments derived from WT-Venus-H5N1 virus are shown in white, whereas those derived from MA-Venus-H5N1 virus are in green.
• Ceils were incubated at 37°C for 24 hours, and ceil lysates were analyzed to measure firefly and Reni!la !uciferase activities.
• the values shown are means ⁇ SD of the three independent experiments and are standardized to the activity of WT- Venus-H5N1 (100%).
• * P ⁇ 0.05 compared with that of WT-Venus-H5N1 virus.
• ** P ⁇ 0.01 compared with that of WT-Venus-H5N1 virus.
• Lane 1 WT+MA-NS; lane 2, WT+MA-M; lane 3, WT+MA-NA; lane 4, WT+MA-PA; lane 5, WT+MA-PB1 ; lane 6, WT+MA-PB2; lane 7, WT+MA-(PB2+PA); lane 8, WT-Venus-H5N1 ; lane 9, RG-MA; lane 10, PR8; and lane 1 1 , 1 -kb DNA marker.
• FIG. 17 The stability of Venus expression by NS1 -Venus PR8 MA virus in vitro and in vivo.
• the positive rate of Venus expression was examined in MDCK cells and in mouse lung.
• Left panel MDCK cells were infected with NS1 -Venus PR8 MA virus at an MOI of 0.001 , and supernatants were collected every 24 hours. The positive rate of Venus expression was estimated by dividing the number of plaques that expressed Venus by the total number of plaques.
• Middle panel NS1 -Venus PR8 MA virus was serially passaged in MDCK cells five times and the positive rates of Venus expression were estimated.
• Right panel Nine mice were infected with 10 3 PFU of NS1 -Venus PR8 MA virus. Three mice were euthanized at each time point and plaque assays were performed using lung homogenates. The positive rates of Venus expression were estimated as described above.
• MDGK cells were infected with each virus at an MO! of 1. Twelve hours after infection, ceils were fixed, and Venus expression was observed. Representative resuits of two independent experiments are shown indicated viruses were used to infect MDGK cells (MOI of 1 ) and confocai microscopy was performed 12 hours later (G) HEK293 cells were Infected with viral protein expression plasmids for NR, PA, PB1 and PB2 or PB-2-E712D, together with a plasmid expressing a vRNA encoding firefly !uciferase.
• FIGS. 20A-20L Time-course observation of Venus-expressing cells in transparent lungs.
• FIGS 21A-21 B Analysis of Venus expression in CC10 4 DC cells in lungs.
• Lung sections from mice infected with NS1 -Venus PR8 MA virus were stained with several antibodies specific for the epithelial ce!!s in the lung. Mice were infected with 10 4 PFU of NS1 -Venus PR8 MA virus and lungs were collected at 3 and 5 days post-infection.
• Figures 24A-24D Genes differentially expressed between Venus-positive and -negative F4/80 + cells. Mice were infected with 10 5 PFU of NS1 -Venus PR.8 MA virus and lungs were collected at 3 days post-infection. Single cell suspensions were stained in the same manner as described in Fig. 10. Venus- positive and -negative cells were separately harvested by using FACSAria !l and subjected to microarray analysis. F4/80 + cells isolated from the lungs of mice inoculated with PBS were used as a control.
• Figure 26 Schematic of fusion protein comprising a heterologous protein.
• Figures 29A-29B Effect of PB2-E712D on the mutation rate.
• A Each virus was passaged five times in MDCK cells, and the mutations introduced into each segment during the passages were counted.
• B The mutation number per nucleotide In each segment was calculated, and the mean values for all eight segments are shown.
• FIGS 32A-32D Additional mutations that stabilizes the Venus gene inserted into the NS segment.
• A identified amino acid mutations were mapped onto the influenza polymerase complex (PDB ID 4WSB).
• B Polymerase internal tunnels (shown as yellow tubes). The vRNA promotewr binds to the polymerase, and the template vRNA enters the polymerase complex. The template vRNA passes through the active site, where RNA synthesis occurs, and then leaves via the template exit. The RNA products synthesized at the active site, leave via the product exit.
• C Each mutant Venus-PR8 virus was passaged four times in MDCK cells, and the proportion of Venus-expressing plaques after passaging was determined in MDCK ceils by using fluorescence microscopy.
• D Percentages of influenza A virus strains containing mutations that stabilize the Venus gene in Venus-PRS (i.e., the number of strains containing the indicated amino acid/tota! number of strains available in the influenza Research Database).
• Virus produced by the host cell may be highly purified prior to vaccine or gene therapy formulation. Generally, the purification procedures result in extensive removal of cellular DNA and other cellular components, and adventitious agents. Procedures that extensively degrade or denature DNA may also be used. Influenza Vaccines
• a vaccine of the disclosure includes an Isolated recombinant influenza virus of the disclosure, and optionally one or more other isolated viruses including other Isolated influenza viruses, one or more immunogenic proteins or glycoproteins of one or more isolated influenza viruses or one or more other pathogens, e.g., an immunogenic protein from one or more bacteria, non-influenza viruses, yeast or fungi, or isolated nucleic acid encoding one or more viral proteins (e.g., DNA vaccines) including one or more immunogenic proteins of the isolated inf!uenza virus of the disclosure.
• the influenza viruses of the disclosure may be vaccine vectors for influenza virus or other pathogens.
• Viruses are available that are capable of reproducibly attenuating influenza viruses, e.g., a cold adapted (ca) donor virus can be used for attenuated vaccine production. See, for example, !sakova-Siva!! et a!., 2014. Live, attenuated reassortant virus vaccines can be generated by mating the ca donor virus with a virulent replicated virus. Reassortant progeny are then selected at 25 C, C (restrictive for replication of virulent virus), in the presence of an appropriate antiserum, which inhibits replication of the viruses bearing the surface antigens of the attenuated ca donor virus.
• C restrictive for replication of virulent virus
• Attenuating mutations can be introduced into influenza virus genes by site-directed mutagenesis to rescue infectious viruses bearing these mutant genes. Attenuating mutations can be introduced into non-coding regions of the genome, as well as into coding regions. Such attenuating mutations can also be introduced into genes other than the HA or NA, e.g , the PB2 polymerase gene. Thus, new donor viruses can also be generated bearing attenuating mutations introduced by site-directed mutagenesis, and such new donor viruses can be used in the production of live attenuated reassortants vaccine candidates in a manner analogous to that described above for the ca donor virus. Similarly, other known and suitable attenuated donor strains can be reasserted with influenza virus to obtain attenuated vaccines suitable for use in the vaccination of mammals.
• Such known methods include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of the donor virus; chemical selection (e.g., amantadine or rimantidine); HA and NA activity and inhibition; and nucleic acid screening (such as probe hybridization or PGR) to confirm that donor genes encoding the antigenic determinants (e.g., HA or NA genes) are not present in the attenuated viruses.
• the disclosure provides an isolated recombinant influenza virus having PA, PB1 , PB2, NP, NS,
• the viral segments is a PB2 viral segment encoding PB2 with residue at position 540 that is not asparagine, a PA virai segment encoding PA with a residue at position 180 that Is not glutamine or a residue at position 200 that is not threonine, or a PB1 viral segment encoding FBI with a residue at position 149 that is not valine, a residue at position 684 that is not glutamic acid or a residue at position 685 that is not aspartic acid, or any combination thereof, wherein the recombinant influenza virus has enhanced genetic stability or enhanced replication relative to a corresponding recombinant influenza virus with a residue at position 540 in PB2 that is asparagine, a residue at position 180 in PA that is glutamine, a residue at position 200 in PA that is threonine, a residue at position 149 in PB1 that is valine, a residue at position 684 in PB1 that is glut
• the residue at position 685 in PB1 is E, N, R, H, K, S, T, Y, C, or Q.
• the residue at position 540 of PB2 is K, R, H, D, S, H, T, Y, or C
• the residue at position 712 of PB2 is D, K, H, R, Q, or N
• the residue at position 180 in PA is R, K, D, N, S, H, T, Y, or H
• the residue at position 200 in PA is A, i, L, G, S, M, or V
• the residue at position 149 in PB1 is A, T, i, L, S, M, or G
• the residue at position 684 is D, Q, H, L, R or N
• the residue at position 685 in PB1 is E, N, R, H, K or Q.
• the residue at position 540 of PB2 is K, R or H
• the residue at position 712 of PB2 is D or N
• the residue at position 180 in PA is R, K or H
• the residue at position 200 in PA is A, !, L, G or V
• the residue at position 149 in FBI is A, T, I, L or G
• the residue at position 884 is D or N
• the residue at position 685 in PB1 is E or Q.
• the heteroiogous sequence is 5' or 3' to the PA coding sequence in the PA viral segment, 5' or 3' to the PB1 coding sequence in the PB1 viral segment. In one embodiment, the heteroiogous sequence is 5' or 3' to the PB2 coding sequence in the PB2 viral segment. In one embodiment, the heterologous sequence is 3 or 3' to the NS1 coding sequence in the NS viral segment.
• the residue at position 200 in PA is A, !, L, G, S, M, or V
• the residue at position 149 in PB1 is A, T, I, L, S, M, or G
• the residue at position 684 is D, Q, H, L, R or N, or the residue at position 685 in PB1 Is E, N, R, H, K or Q
• the residue at position 540 of PB2 is K, R or H
• the residue at position 712 of PB2 is D or N
• the residue at position 180 in PA is R, K or H
• the residue at position 200 in PA is A, !, L, G or V
• the residue at position 684 is D or N
• the residue at position 685 In PB1 is E or Q.
• the PA further comprises a residue at position 443 that is not arginine
• the PB1 further comprises a residue at position 737 that is not iysine
• the PB2 further comprises a residue at position 25 that is not valine or a residue at position 712 that is not glutamic acid
• the NS virai segment encodes a NS1 with a residue at position 167 that is not pro!ine
• the HA virai segment encodes a HA with a residue at position 380 that is not threonine, or any combination thereof in one embodiment
• the residue at position 443 of PA is K or H
• the residue at position 737 of PB1 is H or R
• the residue at position 25 of PB2 is A, L, T, i, or G
• the residue at position 712 of PB2 is D
• the residue at position 167 of NS1 is S, C, M, A, L, i, G or T, or any combination thereof in one embodiment
• the residue at position 540 of PB2 is K, R or H
• the residue at position 712 of PB2 is D or N
• the residue at position 180 in PA is R, K or H
• the residue at position 149 in PBl is A, T, i, L or G
• the residue at position 684 is D or N
• the residue ai position 685 in FBI is E or Q.
• At least one of the viral segments includes a heterologous gene sequence encoding a gene product in one embodiment, the heterologous sequence Is in the NS viral segment, M viral segment, NP viral segment, PA viral segment, FBI viral segment, or the PB2 viral segment. In one embodiment, the heterologous sequence is 5' or 3' to the PA coding sequence in the PA viral segment, 5' or 3' to the PB1 coding sequence in the PB1 viral segment.
• the disclosure also provides a vaccine having the Isolated recombinant virus.
• the disclosure provides a piuraiity of influenza virus vectors for preparing a reassortant, comprising
• a vector for vRNA production comprising a promoter operabiy linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operab!y linked to an influenza virus PB1 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an Influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an Influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operabiy linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising a promoter
• the residue at position 540 of PB2 is K, R or H
• the residue at position 180 in PA is R, K or H
• the residue at position 200 in PA is A, I, L, G or V
• the residue at position 149 in FBI is A, T, i, L or G
• the residue at position 684 is D or N
• the residue at position 685 in PB1 is E or Q.
• at least one of the viral segments includes a heterologous gene sequence encoding a gene product.
• the vectors comprise a further vector having a viral segment comprising a heterologous gene sequence encoding a gene product.
• a method to prepare influenza virus comprising: contacting a ceil with a vector for vRNA production comprising a promoter operably linked to an influenza virus PA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus PBi DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an Influenza virus PB2 DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus HA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NP DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus NA DNA linked to a transcription termination sequence, a vector for vRNA production comprising a promoter operably linked to an influenza virus M DNA linked to a transcription termination sequence, and a vector for vRNA production comprising comprising
• the heterologous sequence e.g., for a therapeutic or prophylactic gene of interest, which may be in an additional Influenza segment, e.g., in one of the segments of a 9 segment influenza A or B virus, in one of 8 viral segments, or In one of the segments in a 7 segment virus, may be an Immunogen for a cancer associated antigen or for a pathogen such as a bacteria, a noninfluenza virus, fungus in one embodiment, the influenza viruses of the disciosure may be vaccine vectors for influenza virus and for at least one other pathogen, such as a viral or bacteria!
• pathogen or for a pathogen other than influenza virus, pathogens including but not limited to, ientiviruses such as HIV, hepatitis B virus, hepatitis C virus, herpes viruses such as GMV or HSV, Foot and Mouth Disease Virus, Measles virus, Rubella virus,
• ientiviruses such as HIV, hepatitis B virus, hepatitis C virus, herpes viruses such as GMV or HSV, Foot and Mouth Disease Virus, Measles virus, Rubella virus,
• Mumps virus human Rhinovirus
• Parainfluenza viruses such as respiratory syncytial virus and human parainfluenza virus type 1 , Coronavirus, Nipah virus, Hantavirus, Japanese encephalitis virus, Rotavirus, Dengue virus, West Nile virus, Streptococcus pneumoniae, Mycobacterium tuberculosis, Bordeteiia pertussis, or Haemophilus influenza.
• the biologically contained influenza virus of the disciosure may include sequences for H protein of Measles virus, viral envelope protein E1 of Rubella virus, HN protein of Mumps virus, RV capsid protein VP1 of human Rhinovirus, G protein of Respiratory syncytia!
• the gene therapy vector may include a heterologous sequence useful to inhibit or treat, e.g., cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamy!ase deficiency and centra! nervous system tumors, or pathogens, or may encode an antibody or fragment thereof, e.g., scFv or a single chain antibody.
• a heterologous sequence useful to inhibit or treat e.g., cancer, AIDS, adenosine deaminase, muscular dystrophy, ornithine transcarbamy!ase deficiency and centra! nervous system tumors, or pathogens
• an antibody or fragment thereof e.g., scFv or a single chain antibody.
• Conventional vaccines generally contain about 0.1 to 200 pg, e.g., 30 to 100 pg, 0.1 to 2 pg, 0.5 to 5 pg, 1 to 10 pg, 10 pg to 20 pg , 15 pg to 30 pg, or 10 to 30 pg, of HA from each of the strains entering into their composition.
• the vaccine forming the main constituent of the vaccine composition of the disclosure may comprise a single Influenza virus, or a combination of influenza viruses, for example, at least two or three influenza viruses, including one or more reassortant(s).
• Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary agents or excipients known in the art.
• compositions of the present disclosure when used for administration to an individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition.
• adjuvants substances which can augment a specific immune response, can be used. Normally, the adjuvant and the composition are mixed prior to presentation to the immune system, or presented separately, but Into the same site of the organism being immunized.
• a pharmaceutical composition according to the present disclosure may further or additionally comprise at least one chemotherapeutic compound, for example, for gene therapy, immunosuppressants, anti-inflammatory agents or immune enhancers, and for vaccines, chemotherapeutics including, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazoie, interferon-a, interferon-b, interferon-y, tumor necrosis factor-alpha, thiosemicarbarzones, methisazone, rifampin, ribavirin, a pyrimidine analog, a purine analog, foscarnet, phosphonoacetic acid, acyclovir, dideoxynucieosides, a protease inhibitor, or ganciclovir.
• chemotherapeutics including, but not limited to, gamma globulin, amantadine, guanidine, hydroxybenzimidazoie, interferon-
• a composition is said to be“pharmacologically acceptable” if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered In a“therapeutically effective amount” if the amount administered is physiologically significant.
• a composition of the present disclosure is
• physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, e.g., enhances at least one primary or secondary humoral or cellular immune response against at least one strain of an infectious influenza virus.
• a composition of the present disclosure may confer resistance to one or more pathogens, e.g., one or more influenza virus strains, by either passive immunization or active immunization.
• active immunization an attenuated live vaccine composition is administered prophylactica!!y to a host (e.g., a mammal), and the host’s immune response to the administration protects against infection and/or disease.
• passive immunization the elicited antisera can be recovered and administered to a recipient suspected of having an infection caused by at least one influenza virus strain.
• a gene therapy composition of the present disclosure may yield prophylactic or therapeutic levels of the desired gene product by active immunization.
• a composition having at least one influenza virus of the present disclosure including one which is attenuated and one or more other isolated viruses, one or more isolated viral proteins thereof, one or more isolated nucleic acid molecules encoding one or more viral proteins thereof, or a combination thereof, may be administered by any means that achieve the intended purposes.
• administration of such a composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transderma! routes.
• Parenteral administration can be accomplished by bolus injection or by gradual perfusion over time.
• a typical regimen for preventing, suppressing, or treating an influenza virus related pathology comprises administration of an effective amount of a vaccine composition as described herein, administered as a single treatment, or repeated as enhancing or booster dosages, over a period up to and including between one week and about 24 months, or any range or value therein.
• an“effective amount” of a composition is one that is sufficient to achieve a desired effect it is understood that the effective dosage may be dependent upon the species, age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect wanted.
• the ranges of effective doses provided below are not intended to limit the invention and represent dose ranges.
• the dosage of a live, attenuated or killed virus vaccine for an animal such as a mammalian adult organism may be from about 10 2 -1 0 20 , e.g., 10 3 -10 12 , 10 2 -1 0 1 °, 10 5 -10 11 , 10 6 -1 G 15 , 10 2 -1 G i0 , or 1 Q i5 -10 20 plaque forming units (PFUV'kg, or any range or value therein.
• the dose of one viral isolate vaccine may range from about 0 1 to 1000, e.g., 0.1 to 10 pg, 1 to 20 pg, 30 to 100 pg, 10 to 50 pg, 50 to 200 pg, or 150 to 300 pg, of HA protein.
• the dosage should be a safe and effective amount as determined by conventional methods, using existing vaccines as a starting point.
• the dosage of immunoreactive HA in each dose of replicated virus vaccine may be standardized to contain a suitable amount, e.g., 0.1 pg to 1 pg, 0.5 pg to 5 pg, 1 pg to 10 pg, 1 0 pg to 20 pg, 15 pg to 30 pg, or 30 pg to 100 pg or any range or value therein, or the amount recommended by government agencies or recognized professional organizations.
• the quantity of NA can also be standardized, however, this glycoprotein may be labile during purification and storage.
• Each 0.5-mi dose of vaccine may contain approximately 0.1 to 0.5 billion viral particles, 0.5 to 2 billion viral particles, 1 to 50 billion virus particles, 1 to 10 billion viral particles, 20 to 40 billion viral particles, 1 to 5 billion viral particles, or 40 to 80 billion viral particles.
• Sections and cells were visualized by using a confocai microscope (Nikon Al , Nikon, Tokyo, Japan), controlled by NIS-E!ements software.
• a confocai microscope Nikon Al , Nikon, Tokyo, Japan
• NIS-E!ements software For quantitative multi-color imaging analysis, the slides were visualized by use of an inverted fluorescence microscope (Nikon Eclipse TS100) with a Nuance FX muitispectrai imaging system with InForm software (PerkinE!mer, Waltham, MA).
• a total of 1 Q 5 PFU of MA-eGFP-PR8 was intranasa!!y inoculated into B6 mice.
• 50 m ⁇ of PE-CD1 1 b (M1/70, BioLegend, San Diego, CA) was injected intravenously to the mice at day 3 p.i.
• the lungs of the mice were harvested.
• the kinetics of eGFP- and PE-positive cells in the lungs were imaged with a multi -photon microscope (LSM 710 NLG, Carl Zeiss, Oberkochen, Germany). During the analysis, the lungs were maintained in complete medium (RPM!
• anti-CD45 (30-F11 : eBioscience, San Diego, CA), anti-CDU b (Ml/70: BioLegend), anti-F4/80 (BM8: eBioscience), and anti-CDU c (HL3: BD Biosciences).
• Aii samples were also incubated with 7-aminoactinomycin D (Via-Probe, BD Biosciences) for dead cell exclusion. Data from labeled ceils were acquired on a FACSAria P (BD Biosciences) and analyzed with FiowJo software version 9.3.1 (Tree Star, San Carlos, CA). To Isolate Venus-positive and -negative macrophages from lungs, stained cells were sorted using a FACSAria ii (BD Biosciences).
• Arrays were scanned with a DNA Microarray Scanner with Su reScan High-Resolution Technology, (G2565CA; Agilent Technologies), and data were acquired using Agilent Feature Extraction software ver. 10.7.3.1. (Agiient Technologies). Probe annotations were provided by Agilent Technologies (AMAD!D 028005) Probe intensities were background corrected and normalized using the normai- exponentiai and quantile methods, respectively. The log2 0f the intensities were then fit to a linear model that compared the groups of interest 34 . Ail reported p values were adjusted for multiple hypothesis comparisons using the Benjammati-Hocbberg method.
• the effect of the PB2, PA, or M genes derived from WT-Venus-H5N1 on the virulence of MA-Venus- H5N1 was also examined by generating three sing!e-gene recombinant viruses, each containing the PB2, PA, or M gene from WT-Venus-H5N 1 virus and the remaining segments from MA-Venus-H5N1 virus (designated MA+WT-PB2, MA+WT-PA, or MA+WT-M).
• H5N1 virus H5N1 virus, RG-MA virus, or WT+MA-(PB2+PA) virus.
• Lungs were collected on day 4 p.L, before the mice died, and were homogenized in PBS. The supernatants were inoculated into MDCK ceils, and at 48 hpi Venus-negative plaques were picked up and amplified in MDCK cells. It should be noted that sometimes the Venus signal of the plaque correlates with the condition of the cultured ceils and the detection time. Therefore the Venus expression of amplified Venus-negative plaques was rechecked in MDCK ceils to exclude false negatives.
• V25A of PB2 also significantly increased Venus expression and viral replication in MDGK ceils and in mice, and that R443K of PA further enhanced these effects.
• the stability of different reassortants was examined in vitro, the reassortants with MA-PB2, MA-PA, or MA-NS were found to be more stable. These results suggest that the PB2 and PA proteins play roles in the pathogenicity and Venus stability of Venus-expressing H5N1 viruses in mammalian hosts.
• Residues 1 -37 at the N-terminus of the PB2 protein play a vital role in binding to the PB1 protein and affect the RNA polymerase activity, and these residues are highly conserved among ail subtypes of influenza virus (Sugiyama et al., 2009).
• the amino acid at position 25 of PB2 Is located within the third a- helix (amino acids 25 to 32) of its PB1 - binding domain (Sugiyama et ai., 2009).
• the amino acid at position 25 in PB2 was found to be changeable, and V25A in PB2 was found to increase viral replication in mammalian cells and in mice, resulting in higher pathogenicity of the H5N1 virus in mice.
• MDGK Madin-Darby canine kidney
• MCS minimum essential medium
• HEK293T Human embryonic kidney 293T
• FGS fetal calf serum
• A/Puerto Rico/8/34 (H1 1 ; PR8) (Horimoto et a!., 2007) and each NS1 -Venus PR8 virus were generated by using reverse genetics and were propagated in MOCK ceils at 37°G for 48 hours in MEM containing L-(tosyiamidc-2-phenyi) ethyl ch!oro ethyl ketone (TPCK) -treated trypsin (0.8 jjg/mL) and 0.3% bovine serum albumin (BSA) (Sigma Aldrich).
• Sequence analysis Sequence analysis of viral RMA was performed as described previously (Sakabe et ai., 201 1 ). Briefly, viral RNAs were extracted by using a QIAamp Viral RNA mini kit (QIAGEN) and Superscript IIITM reverse transcriptase (Invitrogen) and an oligonucleotide complementary to the 12-nucleotide sequence at the 3’ end of the viral RNA (Katz et a!., 1990) were used for reverse transcription of viral RNAs. Each segment was amplified by using PCR with Phusion High Fidelity DNA polymerase (Finnzymes) and primers specific for each segment of the PR8 virus. The PCR products were purified and their sequences determined by using ABI 3130xi (Applied Biosystems).
• Poiykaryon formation assay was performed as described previously (! ai et a! , 2012) with modifications.
• HEK293 cells propagated in 24-weil plates were infected with wiid-type PR8 or PR8 possessing the hemagglutinin (HA) mutation found in NS1 -Venus PR8 MA virus in DMEM containing 10% FCS at a multiplicity of infection (MOI) of 10.
• HA hemagglutinin
• the sections were incubated 'with species-specific fluorescence dye-conjugated secondary antibodies at room temperature for 30 minutes. Nuclei were stained with Hoechst33342 (invitrogen). A Nikon A1 coniocal microscope (Nikon) was used to observe the sections.
• Flow cytometry To prepare single-cell suspensions, lungs were minced with scissors and digested with 20 mg of coiiagenase D (Roche) and 200 units of DNase (Worthington) for 30 minutes at 37°C. Samples were then passed through 100-mth ceil strainers and red blood cells were lysed by red blood cell lysis buffer (Sigma Aldrich). Single-cell suspensions were stained with a combination of the following antibodies:
• Cy3-!abe!ed complementary RNA probe synthesis was initiated with 100 ng of total RNA by using the Agilent Low Input Quick Amp Labeling kit, one color (Agilent Technologies) according to the manufacturer’s instructions.
• the Agilent SurePrint G3 Gene Mouse GE 8 c 60 K microarray was also used. Slides were scanned with an Agilent’s High-Resolution Microarray Scanner, and image data were processed by using Agilent Feature Extraction software ver. 10.7.3.1 . All data were subsequent!/ uploaded into Gene-Spring GX ver 12.5 for data analysis.
• each gene expression array data set was normalized to the in siiico pool for samples from mice inoculated with PRS.
• NS 1 -Venus PRS MA virus The pathogenicity of NS1 -Venus PRS MA virus was higher than that of NS1 -Venus PRS WT virus (MLDsoi 2.1 c 1 0 4 PFU).
• MDCK ceils were infected with these viruses at an MOi of 0.001 and viral titers in supernatants were determined every 12 hours by means of a p!aque assay ( Figure 14)
• NS1 -Venus PR8 WT virus grew to 10 6 5 PFU/mL
• NS1 -Venus PR8 MA virus grew to more than 10 8 PFU/mL, comparable to wild-type PR8 virus.
• the viral titers of NS1 -Venus PR8 PB2 virus and NS1 - Venus PR8 HA virus reached approximately 1 G 7 5 PFU/mL, these were lower than that of NS1 -Venus PR8 MA virus. Therefore, the growth capability of NS1 -Venus PR8 MA virus was remarkably Improved in MDCK ceils, and the mutations in the PB2 and HA genes acted in an additive manner.
• mice infected with 10 4 PFU of NSl -Venus PR8 HA and NS1 -Venus PR8 WT virus decreased slightly, all of the mice survived in the case of infection with 1 Q 3 PFU, while the body weights of the mice infected with NS1 -Venus PR8 PB2 and NS1 -Venus PR8 MA decreased slightly, ail of these mice also survived.
• Mice infected with 10 3 PFU of NSl -Venus PR8 WT and NS1 -Venus PR8 HA showed little body weight loss, and ail of the mice survived.
• the viral titers of these viruses were determined in mouse !ung ( Figure 16).
• mice were infected with 10 3 PFU of the viruses and lungs were collected on days 3, 5, and 7 after infection.
• the maximum virus lung titer from mice infected with NS1 -Venus PR8 PB2 virus was > 1 Q 6 PFU/g, which was similar to that from mice infected with NS1 -Venus PR8 MA virus in contrast, virus titers in iungs from mice infected with NSl -Venus PR8 WT and NS1 -Venus PR8 HA virus were significantly lower than those in iungs from mice infected with NSl -Venus PR8 PB2 and NS1 -Venus PR8 MA virus at all time points.
• the PB2-E712D substitution is responsible for high Venus expression.
• the Venus expression level of NS1 -Venus PR8 MA virus was substantially higher than that of NS1 -Venus PR8 WT virus. Since PB2 is one of the subunit of the influenza virus polymerase, it was hypothesized that the PB2-E712D substitution was important for the augmentation of Venus expression.
• western blots of the viral protein and Venus in infected cells were performed ( Figure 18A).
• HEK293 cells were transfected with viral protein expression plasmids for NP, PA, PB1 , and PB2 or PB2- E712D, together with a plasmid expressing a vRNA encoding the firefly luciferase gene; the pRL-null luciferase protein expression plasmid (Promega) served as a transfection control. Luciferase activities were measured by using a Dua!-G!o luciferase assay system (Promega) at 48 hours post-transfection (Ozawa et al., 2007). Unexpectedly, the polymerase activity of PB2-E712D was lower than that of the parental PB2 ( Figure 18D).
• the HA-T380A substitution raises the threshold for membrane fusion.
• the HA vRNA of MA-Venus- PR8 did not significantly Increase the virulence of WT-Venus-PR8 In mice; however, HA-Venus-PR8 virus grew more efficiently In MDCK cells than WT-Venus-PR8 ( Figure 14), suggesting a contribution of the HA- T380A mutation to, at least, virus replication In cultured ceils. Because the HA-T380A substitution is located on an a-he!ix in the HA2 subunit (Gamblin et a!., 2004), its effect on HA membrane-fusion activity was evaluated by using a po!ykaryon formation assay (Imai et a!., 2012).
• HEK293 cells were infected with WT-PR8 or a mutant PR8 virus encoding HA-T380A at an MOI of 10. Eighteen hours iater, ceils were treated with TPGK-treated trypsin (1 pg/mL) for 15 minutes at 37°C, exposed to !ow-rH buffer (145 mM NaC!, 20 mM sodium citrate (pH 6.0-5.4)) for 2 minutes, incubated for 2 hours In maintenance medium at 37°G, fixed with methanol, and stained with Giemsa's solution.
• NS 1 -Venus PR8 MA virus allows the observation of virus-infected cells without immunostaining because the Venus expression by this virus is sufficiently high to permit the visualization of infected cells with a microscope.
• SCALEVIEW A2 a reagent that make samples optically transparent without decreasing fluorescence intensity were used ( Figure 20) Mice were intranasally infected with 10 5 PFU of PR8, NS1 -Venus PR8 WT, and NS1 -Venus PR8 MA virus, and lungs were collected on days 1 , 3, and 5 after infection. After treatment with SCALEVIEW A2, the samples were observed using a stereo fluorescence microscope.
• NS1 -Venus PR8 MA virus identification of the target cells of NS1 -Venus PR8 MA virus in mouse lung.
• Transparent lungs infected with NS1 -Venus PR8 MA virus revealed that influenza virus first infected the bronchia! epithelium and subsequent!/ invaded the alveoli over time.
• an immunofluorescence assay of frozen sections was performed using several antibodies specific for lung cells ( Figure 21 ).
• the epithelial cells of the bronchi and bronchioles include G!ara cells, ciliated cells, goblet ceils, and a small number of neuroendocrine cells, whereas alveoli comprise type I and type P alveolar epithelial cells. Of these cel!
• Flow cytometry was performed to determine whether alveolar macrophages and monocytes were infected with NS1 -Venus PR8 MA virus, because these immune ceils are present In lung and function as the first line of defense against inhaled microbes and particulates.
• Alveolar macrophages were distinguished monocytes on the basis of the CD1 1 b expression level in the F4/80 + population ( Figure 22A). Mice were infected with 10 5 PFU of PR8 or MSI -Venus PR8 MA virus and the total number of these cells were compared.
• Live mononuclear cells were gated as CD45 + and via-probe cells. As shown in Figure 22A, the ceils were confirmed as alveolar macrophages and monocytes on the basis of CD1 1 b expression levels In the F4/80 + population. Venus-positive and - negative F4/8Q ceils were sorted from a fraction of the live mononuclear cells by FACSAria II. Since CD1 1 c high alveolar macrophages possess high autofluorescence, the possibility existed for overlap with the Venus signal. Therefore, CD1 1 c high alveolar macrophages with intermediate expression of Venus were excluded from the Venus-positive fraction ( Figure 23A). From confoca!
• Madin-Darby canine kidney (MDCK) cells were cultured in minimal essential medium (Gibco) with 5% newborn calf serum at 37°C in 5% CQg.
• Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum.
• WT- Venus-PR8 and Venus-PR8 mutants with MS segments encoding the Venus fluorescent protein (Fukuyama et ai., 2015) were generated by using reverse genetics (Neumann et ai., 1999) and propagated in MDCK ceils at 37°C.
• Deep sequencing analysis WT-PR8, PR8-PB2-E712D, PR8-PB1 -V43I (Cheung et ai., 2014; Naito et a!., 2015), and PR8-PB1 -T123A (Pauly et ai., 2017) were generated by reverse genetics (Neumann et ai.,
• DNA amp!icons were purified by Q.45x of Agencourt AMpure XP magnetic beads (Beckman Coulter), and 1 ng was used for barcoded library preparation with a Nextera XT DNA kit (li!umina). After bead-based normalization (!!iumina), libraries were sequenced on the MISeq platform in a paired-end run using the MiSeq v2, 300 cycle reagent kit (liiurnina).
• the raw sequence reads were analyzed by using the ViVan pipeline (Isakov et ai., 2015). Here, a cutoff of 1 % as the minimum frequency was used. Moreover, we defined an empirical cutoff for the minimum read coverage: for a variant with 1 % frequency, at least 1 ,000 reads should cover that region. Likewise, for a variant with 0.1 % frequency, 10,000 reads should cover that region. Namely, if the coverage was
• RNA samples were infected with WT-Venus-PR8 or Venus-PR8-PB2- E712D at an MQ! of 1 or mock infected with medium.
• the total RNA was extracted from ceils at 9 h postinfection by using an RNeasy minikit (Qiagen). Quantification of RNA was performed as described previously (Kawakami et a!., 201 1 ).
• the primers for IFN-b, NS vRNA, NP vRNA, and b-actin were described previously (Kawakami et ai., 201 1 ; Kupke et a! , 2018; Park et ai., 2015).
• Proteins on SDS-PAGE gels were transferred to a polyvinyiidene fluoride membrane (Miiilpore) and detected by using the indicated primary antibodies (rabbit anti-NS1 [GeneTex], mouse anti-Aichi NP [2S 347/4], mouse anti-p-actin [Sigma-Aidrich]), followed by secondary antibodies (sheep horseradish peroxidase [HRP]-conjugated anti-mouse IgG [GE Healthcare] or donkey HRP-conjugated anti-rabbit IgG [GE Healthcare]). Signals of specific proteins were detected by using ECL Prime Western blotting detection reagent (GE Healthcare).
• the PB2-E712D mutation does not cause an appreciable change in polymerase fidelity.
• PR8-PB1 -V43I which has been reported to be a high-fideiity mutant virus (Cheung et a!., 2014; Naito et ai., 2015), and PR8-PB1 -T123A, which has been reported to be a low-fidelity mutant virus (Pauly et a!., 2017), were also generated by reverse genetics and used as controls. To estimate the mutation rates, these viruses were passaged in MDCK cells at an MOI of 0.001 and deep sequencing of the entire genome performed; the sequencing data for the five-times passaged viruses were compared to those of viruses before passaging. The number of nucleotide changes in the five-times passaged viruses that were not present before passaging were counted.
• MDCK cells were infected with WT- Venus-PRS or Venus-PR8-PB2-E712D at an MOI of 1 or mock infected with medium only, and the relative expression levels of IFN-b in Infected ceils were quantified at 9 h postinfection.
• WT-Venus-PRS induced a higher level of IFN-b expression than did Venus-PR8-PB2-E712D (Fig 3QA). This result suggests that WT- Venus-PRS does not efficient!/ inhibit IFN-b expression.
• NS1 plays a key role in suppressing IFN expression and !FM-mediated antiviral responses in the host (Garcia-Sastre et ai., 1998; Opitz et ai., 2007), the NS vRNA in infected cells was quantified by using influenza virus strand-specific real-time PGR
• PA-180 and PA-200 are located on the surface of the polymerase complex, as is PB2-712, whereas PB2-540, PB1 -149, and PB 1 -684 are located inside the complex.
• PA-180 and PA-200 are located in the endonuclease domain, PB1 -149 and PB1 -684 are located near the exit of the RNA template, and PB2-540 is located near the exit of newly synthesized RNA products (Fig. 32B), while the function of the region around PB2-712 has remained unclear ( Reich et a!.,
• Venus-PR8-PB2-E712D restores the transcription/replication efficiency of the NS segment, leading to efficient virus replication. Therefore, viruses expressing Venus are not purged by selective pressure in the presence of the PB2-E712D mutation, which enables Venus-PR8- PB2-E712D to stably maintain the inserted Venus gene.
• Additional mutations in the influenza virus polymerase complex were identified that stabilize the inserted Venus gene, which may help us to further understand the stabilization mechanisms based on the positions of these mutations in the viral polymerase complex.
• Some of the identified amino acids are located near the polymerase internal tunnels, which are near the RNA template or newly synthesized RNA product during the transcription/replication reactions (Reich et al., 2014; Pf!ug et al., 2017; Gerlach et al., 2015). These amino acids might directly affect the binding affinity between the polymerase complex, template, and product.
• amino acids may not necessarily cause the stabilization of a foreign gene in all influenza virus strains, since PB2-V25A, which stabilizes the Venus gene in Venus-H5N1 , had a negative effect on virus replication in Venus-PR8 and did not cause Venus stabilization (our unpublished data).

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