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NZ712752B2 - Influenza virus-like particle production in plants - Google Patents
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NZ712752B2 - Influenza virus-like particle production in plants - Google Patents

Influenza virus-like particle production in plants Download PDF

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Publication number
NZ712752B2
NZ712752B2 NZ712752A NZ71275214A NZ712752B2 NZ 712752 B2 NZ712752 B2 NZ 712752B2 NZ 712752 A NZ712752 A NZ 712752A NZ 71275214 A NZ71275214 A NZ 71275214A NZ 712752 B2 NZ712752 B2 NZ 712752B2
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New Zealand
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seq
sequence
modified
cpmv
plant
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NZ712752A
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NZ712752A (en
Inventor
Manon Couture
Aoust Marc Andre D
Louis Philippe Vezina
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Medicago Inc
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Priority claimed from PCT/CA2014/050326 external-priority patent/WO2014153674A1/en
Publication of NZ712752A publication Critical patent/NZ712752A/en
Publication of NZ712752B2 publication Critical patent/NZ712752B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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Abstract

method of producing a virus like particle (VLP) in a plant comprising modified hemagglutinin is provided. The method comprises introducing a nucleic acid comprising a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) protein into the plant, or portion of the plant, the modified HA protein comprises a modified proteolytic loop. Followed by incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acids, thereby producing the VLP. The modified proteolytic loop may comprise one or more protease cleavage sites exhibiting reduced or abolished cleavage by a protease. The nucleotide sequence encoding the HA may be selected from the group consisting of B HA, C, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. Also described is a virus like particle (VLP) produced by the method, and plants expressing the VLP. The virus like particle (VLP) may comprise plant-specific N-glycans, or modified N- glycans. HA) protein into the plant, or portion of the plant, the modified HA protein comprises a modified proteolytic loop. Followed by incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acids, thereby producing the VLP. The modified proteolytic loop may comprise one or more protease cleavage sites exhibiting reduced or abolished cleavage by a protease. The nucleotide sequence encoding the HA may be selected from the group consisting of B HA, C, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16. Also described is a virus like particle (VLP) produced by the method, and plants expressing the VLP. The virus like particle (VLP) may comprise plant-specific N-glycans, or modified N- glycans.

Description

Influenza Virus-Like Particle Production in Plants FIELD OF INVENTION id="p-1" id="p-1" id="p-1" id="p-1" id="p-1"
[0001] This invention relates to producing virus like particles in plants.
BACKGROUND OF THE INVENTION id="p-2" id="p-2" id="p-2" id="p-2" id="p-2"
[0002] Influenza is caused by an RNA virus of the orthomyxoviridae family. There are three types of these s and they cause three ent types of influenza: type A, B and C. Influenza virus type A s infect mammals (humans, pigs, ferrets, horses) and birds. This is very important to mankind, as this is the type of virus that has caused worldwide pandemics. za virus type B (also known simply as 10 influenza B) infects only humans. It occasionally causes local outbreaks of flu.
Influenza C viruses also infect only humans. They infect most people when they are young and rarely causes serious illness. id="p-3" id="p-3" id="p-3" id="p-3" id="p-3"
[0003] Vaccination provides protection against disease caused by a like agent by inducing a subject to mount a defense prior to infection. Conventionally, this has been 15 accomplished through the use of live ated or whole vated forms of the infectious agents as gens. To avoid the danger of using the whole virus (such as killed or attenuated viruses) as a vaccine, recombinant viral proteins, for example subunits, have been pursued as vaccines. Both peptide and subunit vaccines are subject to a number of potential limitations. Subunit vaccines may exhibit poor 20 immunogenicity, owing to incorrect folding or poor antigen presentation. A major m is the difficulty of ensuring that the conformation of the ered proteins mimics that of the ns in their natural environment. Suitable nts and, in the case of peptides, carrier proteins, must be used to boost the immune response. In addition these vaccines elicit primarily humoral responses, and thus may fail to evoke 25 effective immunity. Subunit vaccines are often ineffective for diseases in which whole inactivated virus can be demonstrated to provide protection. id="p-4" id="p-4" id="p-4" id="p-4" id="p-4"
[0004] Virus—like particles (VLPs) are potential ates for inclusion in immunogenic compositions. VLPs closely resemble mature virions, but they do not contain viral genomic material. Therefore, VLPs are nonreplicative in nature, which 30 make them safe for administration as a vaccine. In addition, VLPs can be engineered WO 2014/153674 PCT/CA2014/050326 to express viral glycoproteins on the surface of the VLP, which is their most native physiological uration. Moreover, since VLPs resemble intact s and are multivalent particulate structures, VLPs may be more effective in inducing neutralizing antibodies to the glycoprotein than e pe protein antigens. id="p-5" id="p-5" id="p-5" id="p-5" id="p-5"
[0005] VLPs have been produced in plants (W02009/009876; WO 2009/076778; WO 2010/003225; WO 2010/003235; WO 2011/03522; WO 2010/148511; which are incorporated herein by reference), and in insect and mammalian systems (Noad, R. and Roy, R, 2003, Trends Microbiol 11: 438—44; Neumann et al., 2000, J. Virol., 74, 547—551). Latham and Galarza (2001, J. Virol., 75, 6154—6165) reported the 10 formation of za VLPs in insect cells infected with recombinant baculovirus co— sing hemagglutinin (HA), neuramindase (NA), M1, and M2 genes. This study demonstrated that influenza virion proteins self-assemble upon co—expression in eukaryotic cells and that the M1 matrix protein was required for VLP production.
Gomez—Puertas et al., (1999, J. Gen. Virol, 80, 1635—1645) showed that 15 overexpression of M2 completely blocked CAT RNA transmission to MDCK cultures. id="p-6" id="p-6" id="p-6" id="p-6" id="p-6"
[0006] The spike glycoprotein hemagglutinin (HA) of influenza viruses is of great ance for the uptake of virus particles by the host cell. It is sible for their ment to sialic acid-containing cellular receptors, and it is involved in virus 20 penetration through fusion of the virus envelope with cellular membranes. Fusion activity and consequently virus infectivity depend on cleavage of the HA precursor molecule, HAO, into the disulfide-linked polypeptide chains, HA1 and HA2.
Cleavage a uent endent conformational change result in the exposure and relocation of a highly conserved hydrophobic peptide at the amino terminus of the 25 transmembrane polypeptide HA2, which mediates ne fusion. id="p-7" id="p-7" id="p-7" id="p-7" id="p-7"
[0007] HA is synthesised as a precursor protein HAO, which undergoes proteolytic processing into two subunits (HA1 and HA2) linked er by a disulfide bridge.
Two structural features are thought to be involved in HA cleavability: in HAs of restricted cleavability, the linker usually consists of a single arginine, whereas HAs 30 cleavable in a broad range of different cell types have an insertion of a series of multiple basic residues in this position with the main enzyme recognition motif Arg- WO 53674 PCT/CA2014/050326 Arg-Arg, whereby X is a nonbasic amino acid. HAs with a multiple basic cleavage site are cleaved on the ic transport route before they reach the g site on the cell surface, in contrast to HAs with a sic linker that are activated on virus particles either in the extracellular space or, as shown for the WSN strain, at the stage of virus entry. A second determinant of HA cleavage appears to be a carbohydrate side chain that is present in close vicinity of the cleavage site and eres with protease accessibility. Loss of this carbohydrate resulted in enhanced HA cleavability and viral pathogenicity. id="p-8" id="p-8" id="p-8" id="p-8" id="p-8"
[0008] Mammalian and apathogenic avian influenza virus strains cause anatomically 10 zed ions as a result of the restricted range of cells secreting a protease that can cleave the HAO precursor extracellularly (Chen J, et. al. 1998, Cell. Vol 95 :409— 417). The ses responsible for cleavage of HAO in influenza ions of humans, are secreted by cells of the respiratory tract, or by coinfecting bacteria or mycoplasma, or they may be produced in inflammatory responses to infections. A 15 major protease candidate is the tryptase Clara, which is produced by Clara cells of the iolar epithelium, and has limited tissue distribution (upper respiratory tract).
The protease is specific for the monobasic sequence Q/E—X—R found at the cleavage site of the H1, H2, H3, and H6. HA from H9 and B strains show a slightly different monobasic cleavage site with SSR and KER sequence respectively. No protease has 20 been identified for the majority of influenza viruses that cause enteric and atory infection seen in aquatic birds. Most cell lines do not support multi-cycle replication unless exogenous protease (usually trypsin) is added. id="p-9" id="p-9" id="p-9" id="p-9" id="p-9"
[0009] In highly pathogenic avian strains, however, HAO are cleaved by a family of more widespread intracellular proteases, ing in systemic flu infections. This 25 difference in pathogenicity correlates with structural differences at the HAO cleavage site. Pathogenic strains have inserts of polybasic amino acids within, or next to, the monobasic site. Cleavage in this case occurs intracellularly and the proteases involved have been identified as furin, and other subtilisin-like enzymes, found in the Golgi and involved in the post-translational processing of hormone and growth factor 30 precursors. The furin recognition sequence R—X-lUK—R is a frequent ion amino acid at the HAO cleavage sites of H5 and H7. The wide tissue distribution of the WO 2014/153674 PCT/CA2014/050326 enzyme, and the efficiency of intracellular cleavage, contribute to the wide—spread and virulent systemic infection caused by these viruses. id="p-10" id="p-10" id="p-10" id="p-10" id="p-10"
[0010] The HA cleavage site is a target for virus ation, since activation cleavage of the HAO precursor into the HA1 and HA2 fragments by host proteases is a step in the replication cycle of all influenza A and B virus strains. Only the cleaved HA can undergo a conformational change in the acidic milieu of the endosome after or—mediated endocytosis to expose the hydrophobic N terminus of the HA2 fragment for mediating fusion between endosomal and virion membranes. id="p-11" id="p-11" id="p-11" id="p-11" id="p-11"
[0011] Horimoto T, et.al. (2006, Vaccine, Vol 24 : 3669—3676) describes the abolition 10 of the polybasic cleavage site of H5 (RERRRKKRiG) in H5. Selected mutants were submitted to immunogenicity study in mice, ing a mutant with a deletion of the 4 first charged amino acids (RERR) and a modification to inactivate the sic cleavage site (RKKR with TETR). Abolition of the cleavage site did not affect the genic properties of the mutant H5. ion the polybasic site 15 (GERRRKKRiG replaced by RETR) to produce mutant NIBSC 05/240 NIBSC influenza reference virus NIBG-23, has also been reported. Hoffman et. al. (2002, 2002, Vaccine, Vol 20 3170) replaced the sic cleavage site of a H5 HA with the monobasic site of H6 in order to boost the expression in eggs. The f1rst4 residues were deleted and replaced the four last amino acids of the polybasic site by 20 IETR (replacement of RERRRKKRiG with IETRiG). This mutant H5 showed a high sion level, potential proteolysis and conformational change at low pH required for viral replication and production in the host cell, immunogenicity data were not reported. These s show that modification of the cleavage site can be employed to diminishes the virulence of the viral particle (in cases where the true viruses is 25 replicated), allowing the virus to replicate without killing the host egg. Without such mutations, viruses kill the egg before reaching high titers. id="p-12" id="p-12" id="p-12" id="p-12" id="p-12"
[0012] W02013043067 by Sirko et al. describe a DNA vaccine for chicken which contains the cDNA encoding a modified H5 haemagglutinin (HA) protein n the proteolytic ge site between HA subunits is deleted. Sirko et al. state that this 30 provides for greater safety of the vaccines and the expression of a "super antigen" in the form of a long, non-processed polypeptide. Sirko et al. further state that the encoding region of the HA is modified in such a way that protein production in bird cells achieves maximal yield. The main modification is codon optimisation for chicken and deletion of the proteolysis sensitive region of HA. id="p-13" id="p-13" id="p-13" id="p-13" id="p-13"
[0013] WO 2013/044390 describes a method of producing a virus like particle (VLP) in a plant 5 with modified hemagglutinin (HA) n the ed HA protein comprises a ed proteolytic loop. The modified HA is expressed in the presence of the regulatory region Cowpea mosaic virus (CPMV) HT and the geminivirus amplification element from Bean Yellow Dwarf Virus (BeYDV). id="p-14" id="p-14" id="p-14" id="p-14" id="p-14"
[0014] US 2008/0069821 by Yang et al. discloses ptides and cleotides variants of 10 influenza HA for use in the production of influenza viruses as vaccines. Reassortant influenza viruses are obtained by introducing a subset of vectors corresponding to genomic segments of a master influenza virus, in combination with complementary ts derived from the variants of influenza HA. Typically, the master strains are selected on the basis of desirable properties relevant to vaccine administration. For example, for vaccine production, e.g., for production of a live 15 ated vaccine, the master donor virus strain may be selected for an attenuated ype, cold adaptation and/or temperature sensitivity.
SUMMARY OF THE INVENTION id="p-15" id="p-15" id="p-15" id="p-15" id="p-15"
[0015] This invention s to producing virus like particles and modified HA ns in plants. 20 [0016] It is an advantage that in one or more embodiments the invention may provide improved production of virus like particles and HA proteins in plants. id="p-17" id="p-17" id="p-17" id="p-17" id="p-17"
[0017] Described herein is a nucleic acid comprising an sion enhancer active in a plant and operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) comprising a modified proteolytic loop. 25 [0017a] In particular, in an aspect of the invention there is provided a nucleic acid comprising a regulatory region active in a plant and an expression enhancer active in a plant, in the absence of a Bean Yellow Dwarf Virus ) amplification element, the regulatory region and the expression enhancer operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) selected from nza type B HA and influenza type A subtype H3 and H7 30 HA, the modified HA comprising a fully deleted proteolytic loop between subunits HA1 and HA2, -5a- the fully deleted proteolytic loop comprising a monobasic or a multi-basic cleavage site, wherein the expression enhancer is not CPMV-HT. [0017b] In another aspect of the invention there is provided a method of producing nza virus like particle (VLP) in a plant comprising, 5 a) introducing a nucleic acid into the plant or portion of the plant, or providing a plant, or a portion of a plant, comprising the nucleic acid, wherein the nucleic acid comprises a regulatory region active in the plant and an expression enhancer active in the plant, the regulatory region and the expression enhancer ively linked to a nucleotide sequence ng a modified influenza type A subtype H3 or H7 hemagglutinin (HA), the modified HA comprising a fully deleted 10 proteolytic loop between subunits HA1 and HA2, the fully d lytic loop comprising a monobasic or a multi-basic cleavage site, and n the nucleic acid does not comprise a Bean Yellow Dwarf Virus (BeYDV) ication element, and wherein the expression enhancer is not CPMV-HT; and b) incubating the plant or portion of the plant under ions that permit the expression 15 of the nucleic acids, thereby producing the VLP. [0017c] In another aspect of the invention there is provided a method of producing a modified HA protein comprising a fully deleted proteolytic loop between subunits HA1 and HA2, the proteolytic loop comprising one or more than one monobasic or multi-basic cleavage sites exhibiting reduced or abolished cleavage in a plant comprising, 20 a) ucing a nucleic acid into the plant, the nucleic acid comprising a regulatory region active in the plant and an expression enhancer active in the plant, the regulatory region and the expression enhancer ively linked to a nucleotide sequence ng a modified influenza type A subtype H3 or H7 HA, the modified HA comprising the fully d proteolytic loop between subunits HA1 and HA2, the fully deleted proteolytic loop comprising a monobasic or a 25 multi-basic cleavage site, wherein the nucleic acid does not comprise a Bean Yellow Dwarf Virus ) amplification element, and wherein the expression enhancer is not CPMV-HT; b) incubating the plant or portion of the plant under conditions that permit the expression of the HA n, thereby producing the modified HA protein, and c) harvesting the plant and purifying the modified HA protein. -5b- [0017d] In a related embodiment there is ed a modified influenza hemagglutinin (HA) comprising a fully deleted proteolytic loop between subunits HA1 and HA2, the fully deleted proteolytic loop comprising a monobasic cleavage site, wherein the modified influenza HA is derived from an unmodified H3 HA comprising 90% to 100% sequence identity to a wild type H3 5 sequence defined by SEQ ID NO: 19. [0017e] In another aspect of the invention there is provided a modified nza hemagglutinin (HA) sing a fully d proteolytic loop between subunits HA1 and HA2, the fully deleted proteolytic loop comprising a multi-basic cleavage site, wherein the modified influenza HA is derived from an unmodified H7 HA comprising 90% to 100% sequence identity to a wild type H7 10 sequence defined by amino acids 25 to 566 of SEQ ID NO: 150. id="p-18" id="p-18" id="p-18" id="p-18" id="p-18"
[0018] Furthermore bed herein is a method (A) of producing a virus like particle (VLP) in a plant comprising, a) ucing a nucleic acid comprising an expression enhancer active in a plant and operatively linked to a *Followed by page 6 WO 2014/153674 PCT/CA2014/050326 hemagglutinin (HA) comprising a modified proteolytic loop into the plant, or portion of the plant; b) incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLP. id="p-19" id="p-19" id="p-19" id="p-19" id="p-19"
[0019] In addition, described herein is a method (B) for producing influenza virus like particles (VLPs) in a plant comprising: a) providing a plant, or a portion of a plant, comprising a nucleic acid comprising an expression enhancer active in a plant and operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) comprising a 10 modified proteolytic loop, and b) incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLPs id="p-20" id="p-20" id="p-20" id="p-20" id="p-20"
[0020] Furthermore described herein is a method (C) of ing a modified HA protein comprising a modified proteolytic loop comprising one or more protease 15 cleavage sites exhibiting reduced or abolished cleavage in a plant comprising, a) introducing a nucleic acid comprising an expression enhancer active in a plant and operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) sing a ed lytic loop into the plant, or portion of the plant; 20 b) incubating the plant or portion of the plant under conditions that permit the expression of the HA protein, thereby ing the modified HA n, c) harvesting the plant and ing the modified HA protein. id="p-21" id="p-21" id="p-21" id="p-21" id="p-21"
[0021] The methods (A), (B) or (C) as described above may further comprising the steps of 25 c) harvesting the plant, and d) ing the VLPs, wherein the VLPs range in size from 80—300 nm.
WO 2014/153674 PCT/CA2014/050326 id="p-22" id="p-22" id="p-22" id="p-22" id="p-22"
[0022] Furthermore, described herein is a method (D) of increasing the t yield of a HA protein in a plant, comprising , a) introducing a c acid comprising an expression enhancer active in a plant and operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) comprising a modified proteolytic loop into the plant, or portion of the plant; b) incubating the plant or portion of the plant under ions that permit the expression of the HA protein, thereby producing the modified HA protein, c) harvesting the plant and purifying the HA n. 10 [0023] The expression enhancer may be CPMVX, CPMVX+ or CPMV—HT+.
Furthermore, the nucleotide acid may not comprise a geminivirus amplification element. The nucleotide acid therefore may not se a Bean Yellow Dwarf Virus long intergenic region (BeYDV LIR), and a BeYDV short enic region (BeYDV SIR). The modified proteolytic loop may comprises one or more protease cleavage 15 sites exhibiting reduced or abolished cleavage by a protease. The protease may be Clara-like or Furin—like. Furthermore the modified proteolytic loop may ses a linker sequence and the linker sequence may have the amino acid sequence GG, TETQ or TETR. The modified HA may comprise a native or a non-native signal peptide. Furthermore the the nucleotide sequence encoding the modified HA may 20 comprise a chimeric nucleotide sequence encoding, in series, a ed HA ectodomain comprising a modified proteolytic loop, an influenza transmembrane domain, and a cytoplasmic tail, wherein the ed HA ectodomain is from a first influenza strain and the transmembrane domain and the cytoplasmic tail are from a second influenza strain. 25 [0024] The modified proteolytic loop may comprise one or more protease ge sites exhibiting reduced or abolished cleavage by a protease. Furthermore, the nucleotide ce ng the HA is selected from the group consisting of B HA, C, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, and H16.
Also described herein is a virus like particle (VLP) produced by the method (A). The virus like particle (VLP) may comprise plant-specific N-glycans, or modified N-glycans. id="p-25" id="p-25" id="p-25" id="p-25" id="p-25"
[0025] The present disclosure also provides a composition comprising an effective dose of the VLP as bed herein for inducing an immune response, and a ceutically acceptable carrier. 5 [0026] Also described herein is a nucleic acid comprising a tide sequence encoding an influenza hemagglutinin (HA), the nucleotide sequence operatively linked with a tory region that is active in a plant, wherein the HA comprises a modified proteolytic loop sequence. The nucleic acid may encode an HA comprising a modified proteolytic loop, where in the protein has hemagglutinin (HA) activity. A plant comprising the nucleic acid is also provided. Also included is 10 a virus like particle (VLP) produced in a plant, the VLP sing an influenza virus hemagglutinin (HA) encoded by the nucleic acid and one or more than one lipid derived from a plant. [0026a] In particular, in another aspect there is ed a plant comprising a nucleic acid embodied by the invention. 15 [0026b] In another aspect there is provided an influenza virus like particle (VLP) comprising a modified nza HA embodied by the invention. [0026c] In another aspect there is provided a vaccine comprising an effective dose of a VLP embodied by the invention for ng an immune response. [0026d] In another aspect there is ed the use of a VLP embodied by the invention in the 20 preparation of a ment for inducing immunity to an influenza virus infection in a subject. [0026e] In still another aspect there is provided a method of increasing the product quality of an HA protein expressed in a plant, comprising: a) introducing a nucleic acid embodied by the invention into the plant; b) incubating the plant or portion of the plant under conditions that permit the expression 25 of the HA protein, thereby producing a modified HA protein, c) harvesting the plant and purifying the modified HA protein, n the modified HA protein has increased product quality, when compared to a native HA. [0026f] -8a- or step, or group of elements integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. [0026g] Any discussion of documents, acts, als, devices, articles or the like which has been included in this specification is solely for the purpose of providing a context for the present 5 invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in New Zealand or elsewhere before the priority date of this ation. id="p-27" id="p-27" id="p-27" id="p-27" id="p-27"
[0027] This summary of the invention does not arily describe all es of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS 10 [0028] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings n: id="p-29" id="p-29" id="p-29" id="p-29" id="p-29"
[0029] Figure 1 shows components used to prepare A-2X35S/CPMV-HT/H5 Indonesia/NOS (Construct number 489). Figure 1A shows primer IF-H5A-I-05.s1+3c (SEQ ID NO: 2). Figure 1B shows primer IF-H5dTm.r (SEQ ID NO: 3). Figure 1C shows a schematic entation of 15 construct 1191. Figure 1D shows Construct 1191 (SEQ ID NO 4). Figure 1E shows expression cassette number 489 (SEQ ID NO 5). Figure 1F shows amino acid sequence of H5 from influenza A/Indonesia/5/2005 (H5N1) (SEQ ID NO: 6). Figure 1G shows a nucleotide sequence encoding H5 from nza A/Indonesia/5/2005 (H5N1) (SEQ ID NO: 42). id="p-30" id="p-30" id="p-30" id="p-30" id="p-30"
[0030] Figure 2 shows components used to prepare B-2X35S/CPMV HT/M2 New 20 Caledonia/NOS (Construct number 1261). Figure 2A shows primer IF-S1 - *Followed by page 9 WO 2014/153674 PCT/CA2014/050326 M1+M2ANC.c (SEQ ID N017). Figure 2B shows primer IF-S1M2ANC.r (SEQ ID NO: 8). Figure 2C shows the nucleotide sequence for the synthesized M2 gene (corresponding to nt 1—26 joined to 715—982 from k accession number DQ508860) (SEQ ID NO: 9). Figure 2D shows the sion cassette number 1261 from 2X35S promoter to NOS terminator. M2 from influenza A/New Caledonia/20/1999 (H1N1) is ined. (SEQ ID NO: 10). Figure 2E shows the amino acid sequence of M2 from influenza A/New Caledonia/20/1999 (H1N1) (SEQ ID NO: 11). id="p-31" id="p-31" id="p-31" id="p-31" id="p-31"
[0031] Figure 3 shows components used to prepare C—2X35S/CPMV—HT/M2 Puerto 10 Rico/NOS (Construct number 859). Figure 3A shows the nucleotide sequence of the sized M2 gene (corresponding to nt 26-51 joined to nt 740-1007 from Genebank accession number EF467824) (SEQ ID NO: 12). Figure 3B shows the expression cassette number 859 from 2X35S promoter to NOS terminator. M2 from Influenza A/Puerto Rico/8/1934 (H1N1) is underlined. (SEQ ID NO: 13). Figure 3C 15 shows the amino acid sequence of M2 from influenza A/Puerto Rico/8/1934 (H1N1) (SEQ ID NO:14). id="p-32" id="p-32" id="p-32" id="p-32" id="p-32"
[0032] Figure 4 shows components used to prepare G—2X35S/CPMV—HT/PDISP/HA B Brisbane/NOS into BeYDV+Replicase amplification system (Construct number 1008). Figure 4A shows a schematic representation of uct 1194. SacII and StuI 20 restriction enzyme sites used for plasmid linearization are annotated on the representation. Figure 4B shows construct 1194 from left to right t-DNA borders (underlined). 2X35S/CPMV—HT/PDISP/NOS into BeYDV+Replicase amplification system with cyanine-Pl9-Plastocyanine silencing inhibitor expression cassette (SEQ ID NO: 31). Figure 4C shows expression cassette number 1008 from BeYDV 25 left LIR to BeYDV right LIR. PDISP/HA from influenza B/Brisbane/60/2008 is underlined. (SEQ ID NO: 32). id="p-33" id="p-33" id="p-33" id="p-33" id="p-33"
[0033] Figure 5 shows ents used to prepare I—2X35S/CPMV—HT/PDISP/HA B ne with deleted proteolytic loop/NOS into BeYDV+Replicase amplification system ruct number 1059). Figure 5A shows primer 1039+1059.r (SEQ ID 30 NO: 38). Figure 5B shows primer 1039+1059.c (SEQ ID NO: 39). Figure 5C shows expression cassette number 1059 from BeYDV left LIR to BeYDV right LIR.
WO 2014/153674 PCT/CA2014/050326 -10, HA from influenza B/Brisbane/60/2008 with deleted proteolytic loop is underlined (SEQ ID NO: 40). Figure 5D shows amino acid sequence of PDISP/HA from influenza B/Brisbane/60/2008 with deleted proteolytic loop (SEQ ID NO: 41).
Figure 5E shows nucleotide sequence of PDISP/HA from influenza B/Brisbane/60/2008 with deleted proteolytic loop (SEQ ID NO: 43). id="p-34" id="p-34" id="p-34" id="p-34" id="p-34"
[0034] Figure 6 shows components used to prepare B—2X35S/CPMV—HT/HA B Wisconsin/NOS into BeYDV(m)+Replicase amplification system (Construct number 1462). Figure 6A shows primer 110.S1+3c (SEQ ID NO: 49). Figure 6B shows primer IF—HAB110.s1—4r (SEQ ID NO: 50). Figure 6C shows the nucleotide 10 sequence of synthesized HA B sin nk accession number IN993010) (SEQ ID NO: 51). Figure 6D shows a schematic entation of uct 193.
Figure 6E shows construct 193 from left to right t-DNA borders (underlined). 2X35 S/CPMV—HT/NOS into BeYDV(m)+Replicase amplification system with Plastocyanine-Pl9-Plastocyanine silencing inhibitor expression cassette (SEQ ID NO: 15 52). Figure 6F shows the nucleotide sequence of expression cassette number 1462 from 2X35S er to NOS terminator. HA from influenza B/Wisconsin/1/2010 is underlined (SEQ ID NO: 53). Figure 6G shows the amino acid sequence of HA from influenza B/Wisconsin/1/2010 (SEQ ID NO: 54). Figure 6H shows a schematic representation of construct 1462. 20 [0035] Figure 7 shows components used to prepare C—2X35S/CPMV—HT/HA B Wisconsin with deleted proteolytic loop/NOS into BeYDV(m)+Replicase amplification system (Construct number 1467). Figure 7A shows primer HAB110(PrL-).r (SEQ ID NO: 55). Figure 7B shows primer HAB110(PrL-).c (SEQ ID NO: 56). Figure 7C shows the nucleotide sequence of expression cassette number 25 1467 from 2X35S promoter to NOS terminator. HA from influenza B/Wisconsin/1/2010 with deleted proteolytic loop is underlined (SEQ ID NO: 57).
Figure 7D shows the amino acid ce of influenza B/Wisconsin/1/2010 with deleted proteolytic loop (SEQ ID NO: 58). Figure 7E shows a schematic representation of construct 1467. 30 [0036] Figure 8 shows components used to prepare A—2X35S/CPMV—HT/PDISP/HA B Brisbane with deleted proteolytic loop/NOS (Construct number 103 9). Figure 8A WO 2014/153674 PCT/CA2014/050326 -11, shows the nucleotide sequence of expression cassette number 1039 from 2X35S promoter to NOS terminator. HA from influenza B/Brisbane/60/2008 with deleted proteolytic loop is underlined (SEQ ID NO: 15). Figure 8B shows a schematic representation of construct 1039. id="p-37" id="p-37" id="p-37" id="p-37" id="p-37"
[0037] Figure 9 shows the plasmid map of construct number 1008. Construct number 1008 directs the expression of wild—type HA from za strain B/Brisbane/60/2008. This construct ses derived elements for DNA amplification. id="p-38" id="p-38" id="p-38" id="p-38" id="p-38"
[0038] Figure 10 shows the plasmid map of construct number 1059. Construct 10 number 1059 directs the expression of a mutant HA from influenza strain B/Brisbane/60/2008 with deleted proteolytic loop. This construct comprises BeYDV— derived elements for DNA amplification. id="p-39" id="p-39" id="p-39" id="p-39" id="p-39"
[0039] Figure 11 shows the plasmid map of construct number 1261. Construct number 1261 directs the expression of wild-type M2 from influenza strain A/New 15 Caledonia/20/99 (HlNl). id="p-40" id="p-40" id="p-40" id="p-40" id="p-40"
[0040] Figure 12 shows the plasmid map of construct number 859. Construct number 859 directs the expression of wild—type M2 from influenza strain A/Puerto Rico/8/34 (HlNl). id="p-41" id="p-41" id="p-41" id="p-41" id="p-41"
[0041] Figure 13A shows a Western blot analysis of HA protein expression in 20 agroinfiltrated ana benthamiana leaves. "1008": sion of wild—type HA from B/Brisbane/60/2008 in the presence of amplification elements (BeYDV); "1008+126l": co-expression of wild-type HA from B/Brisbane/60/2008 in the ce of amplification elements (BeYDV) with M2 from A/New Caledonia/20/99; "1059": expression of mutant HA from B/Brisbane/60/2008 in the presence of 25 ication elements (BeYDV); "1059+126l": co-expression of mutant HA from B/Brisbane/60/2008 in the ce of amplification elements (BeYDV) with M2 from A/New Caledonia/20/99. Plants from three separate infiltrations were analyzed (A, B and C). Ratios indicate the proportion of cterium cultures used in co— expression experiments. Figure 13B shows a comparison of hemagglutination 30 capacity of crude protein ts from HA-producing plants.
W0 53674 PCT/CA2014/050326 -12, id="p-42" id="p-42" id="p-42" id="p-42" id="p-42"
[0042] Figure 14 shows a Western blot analysis of HA protein expression in agroinfiltrated Nicotiana benthamiana leaves. "1059": expression of mutant HA from bane/60/2008 in the presence of amplification elements ); "1059+1261": co—expression of mutant HA from B/Brisbane/60/2008 in the ce of amplification elements (BeYDV) with M2 from A/New Caledonia/20/99. "1059+859": co—expression of mutant HA from B/Brisbane/60/2008 in the presence of amplification elements (BeYDV) with M2 from A/Puerto /34. Plants from three separate infiltrations were analyzed (A, B and C). Ratios indicate the proportion ofAgrobacterium cultures used in co—expression experiments. 10 [0043] Figure 15 shows the amino acid sequence alignment of the region surrounding the linker of HAs from several s of influenza: H1 New Cal (SEQ ID NO:22); H1 Brisbane (SEQ ID NO:23); H1 Sol Islands (SEQ ID NO:24); H2A Singapore (SEQ ID NO:25); H3A Brisbane (SEQ ID NO:26); H3A WCN (SEQ ID NO:27); H5 Anhui (SEQ ID ; H5 Indo (SEQ ID NO:29); H5 Vietnam (SEQ ID N030); H6 Teal 15 HK (SEQ ID N033); H7 Eq Prague (SEQ ID NO34); H9A HK (SEQ ID N035); B Florida (SEQ ID N036); B Malaysia (SEQ ID N037). The cleavage site of the precursor HAO is indicated by an arrow. id="p-44" id="p-44" id="p-44" id="p-44" id="p-44"
[0044] Figure 16A shows a Western blot analysis of HA n expression in agroinfiltrated Nicotiana benthamiana leaves. HA from B/Wisconsin/1/2010 is co— 20 expressed with M2 from A/New Caledonia/20/99. Ten micrograms of protein extract were loaded per lane. "C+": positive control, semi—purified B/Wisconsin/1/2010 virus from the National Institute for Biological Standards and Control, United Kingdom; "1462": expression of wild—type HA from B/Wisconsin/1/2010 in the presence of ication elements (BeYDV); "1467": expression of the mutant HA from 25 onsin/1/2010 in the presence of amplification elements (BeYDV); "1462+1261": co—expression of wild—type HA from onsin/1/2010 in the presence of amplification elements (BeYDV) with M2; "1467+1261": co-expression of the mutant HA from B/Wisconsin/1/2010 in the presence of amplification ts (BeYDV) with M2. Ratios indicate the optical density for each Agrobacterium culture 30 used in expression and co—expression experiments. Figure 16B shows a comparison of hemagglutination capacity of crude protein extracts from plants transformed with AGLl/l462, 467, AGLl/l462+AGLl/l26l and AGLl/l467+AGLl/l26l.
WO 2014/153674 PCT/CA2014/050326 -13, id="p-45" id="p-45" id="p-45" id="p-45" id="p-45"
[0045] Figure 17A and 17B shows a Western blot is of HA protein expression in agroinfiltrated Nicotiana benthamiana . "1008": expression of wild—type HA from B/Brisbane/60/2008 in the presence of amplification elements (BeYDV); "1008+1261": co-expression of ype HA from B/Brisbane/60/2008 in the presence of amplification elements (BeYDV) with M2 from A/New Caledonia/20/99;"l039":expression of mutant HA from bane/60/2008 in the absence of amplification elements (BeYDV). "1039+1261": co—expression of mutant HA from B/Brisbane/60/2008 in the absence of ication elements (BeYDV) with M2 from A/New Caledonia/20/99.HA from A/Brisbane/59/2007 (HlNl). "1059": 10 sion of mutant HA from B/Brisbane/60/2008 in the presence of amplification elements (BeYDV); "1059+126l": co—expression of mutant HA from B/Brisbane/60/2008 in the presence of amplification elements (BeYDV) with M2 from A/New Caledonia/20/99.
Figure 18A shows the schematic entation of the cleavage of the HAO by Clara- 15 like and/or Furin like protease into HA1 and HA2. Figure 18B shows the sequence alignment of HAs from several strains of influenza: H1 New Cal (SEQ ID N0122); H1 Brisbane (SEQ ID N0123); H1 Sol Islands (SEQ ID N0:24); H2A ore (SEQ ID ; H3A Brisbane (SEQ ID N0:26); H3A WCN (SEQ ID N0:27); H5 Anhui (SEQ ID N0:28); H5 Indo (SEQ ID N0:29); H5 Vietnam (SEQ ID N030); 20 H6 Teal HK (SEQ ID N033); H7 Eq Prague (SEQ ID N034); H9A HK (SEQ ID N035); B Florida (SEQ ID N036); B Malaysia (SEQ ID N037). Figure 18C shows deletion of part of the cleavage site in H5 strains, A/Anhui/1/2005 (H5N1) SEQ ID NO: 69, A/Indonesia/5/2005 (H5N1) SEQ ID NO: 70, A/Vietnam/1194/2004 (H5N1) SEQ ID NO: 71, and type B strains, B/Florida/4/2006 SEQ ID NO: 72, and 25 B/Malaysia/2506/2004 SEQ ID N0:73. id="p-46" id="p-46" id="p-46" id="p-46" id="p-46"
[0046] Figure 19 shows the on of the cleavage site in H5/Indo. Native sequence (SEQ ID NO: 44); o modified cleavage site comprising TETR (SEQ ID N0:45); H5/Indo modified cleavage site comprising TETQ (SEQ ID N0:46). id="p-47" id="p-47" id="p-47" id="p-47" id="p-47"
[0047] Figure 20 shows the titers found in initial biomasses after enzyme-extraction 30 of different modified H5/Indo HA’s comprising mutations within the protelolytic loop. H5/Indo control (construct 489); H5/Indo proteolytic loop replaced with GG WO 2014/153674 PCT/CA2014/050326 -14, linker (construct 928); H5/Indo proteolytic loop replaced with TETR linker (construct 676); H5/Indo proteolytic loop ed with TETQ linker (construct 766). id="p-48" id="p-48" id="p-48" id="p-48" id="p-48"
[0048] Figure 21 shows various approaches for modifying the proteolytic loop of type B HA. Figure 21A shows the amino acid sequence of native B/Brisbane/60/2008 (SEQ ID NO: 16). The underlined portion is the proteolytic loop and the HA2 domain. Figure 21B shows the amino acid sequence of B/Brisbane/60/2008 with the proteolytic loop modified (SEQ ID NO: 17), wherein 19 amino acid residues comprising the sequence AKLLKERGFFGALAGFLEG have been replaced with a GG linker (italics). Figure 21 C shows the amino acid sequence 10 of B/Brisbane/60/2008 (SEQ ID NO: 18), wherein 9 amino acid comprising the ce KER has been replaced with a —GSSSGSSSG— linker (italics).
Figure 21 D shows the amino acid sequence of native H3 A/Perth/l6/2009 (SEQ ID NO: 19). Figure 21 E shows the amino acid ce of H3 A/Perth/16/2009 (SEQ ID NO: 20) with 12 amino acid residues comprising the sequence QTRGIF 15 replaced by a GS linker (italics). Figure 21 F shows the amino acid sequence of H3 A/Perth/l6/2009 (SEQ ID NO: 21) with 9 amino acid residues comprising the sequence QTR replaced by a GSSGSSGSS— linker (italics). id="p-49" id="p-49" id="p-49" id="p-49" id="p-49"
[0049] Figure 22 shows a n blot is of HA protein expression in filtrated Nicotiana benthamiana leaves. HA from o. Upper panel listing 20 of components loaded in each lane ; lower panel Western blot. C: Recombinant H5 Indonesia/5/05 S—STD—0002; primary antibody: Anti—HA A/Indonesia/05/2005 CBER # S—BIO—0003 1/50 000; blot. Lanes 1 and 2: H5/Indo control ruct 489); lanes 3 and 4 H5/Indo proteolytic loop replaced with GG linker (construct 928); lanes 5 and 6 H5/Indo proteolytic loop replaced with TETR linker (construct 676); lanes 7 25 and 8 H5/Indo proteolytic loop replaced with TETQ linker (construct 766); lane 9 MW marker; lane 10 H5 control. id="p-50" id="p-50" id="p-50" id="p-50" id="p-50"
[0050] Figure 23 shows components used to prepare B—2X35S/CPMV—HT/H5 from A/Indonesia/5/2005 with TETR cleavage site mutation (Construct number 676).
Figure 23A shows primer sequence MutCleavage—H5(Indo).r (SEQ ID NO:74). 30 Figure 23B shows primer sequence MutCleavage—H5(Indo).c (SEQ ID NO:75).
Figure 23C shows the nucleotide sequence (SEQ ID NO: 76) for expression cassette WO 2014/153674 PCT/CA2014/050326 -15, 676 from the 2X35S promoter to NOS terminator. H5 from influenza A/Indonesia/5/2005 (H5N1) TETR cleavage site mutant is underlined. Figure 23D shows the amino acid sequence (SEQ ID NO:77) of a TETR ge site mutant of H5 from influenza A/Indonesia/5/2005 (H5N1). Figure 23E shows a schematic representation of uct number 676. id="p-51" id="p-51" id="p-51" id="p-51" id="p-51"
[0051] Figure 24 shows components used to prepare B—2X35S/CPMV—HT/H5 from A/Indonesia/5/2005 with TETQ cleavage site mutation (Construct number 766).
Figure 24A shows primer sequence H51505_TETQ.r (SEQ ID NO:78). Figure 24B shows primer sequence H51505_TETQ.c (SEQ ID NO:79). Figure 24C shows the 10 nucleotide sequence (SEQ ID NO: 80) for expression cassette 766 from the 2X35S er to NOS terminator. H5 from influenza A/Indonesia/5/2005 (H5N1) TETQ cleavage site mutant is underlined. Figure 24D shows the amino acid sequence (SEQ ID NO:81) of a TETQ cleavage site mutant of H5 from za A/Indonesia/5/2005 (H5N1). Figure 24E shows a schematic representation of construct number 766. 15 [0052] Figure 25 shows components used to prepare S/CPMV—HT/H5 from A/Indonesia/5/2005 with a deleted proteolytic loop (Construct number 928). Figure 25A shows primer sequence H51505(PrL—).r (SEQ ID NO: 82). Figure 25B shows primer sequence H51505(PrL—).c (SEQ ID NO: 83). Figure 25C shows the nucleotide sequence (SEQ ID NO:84) for expression cassette 928 from the 2X35S 20 er to NOS terminator. H5 from influenza A/Indonesia/5/2005 (H5Nl) the deleted proteolytic loop is underlined. Figure 25D shows the amino acid sequence (SEQ ID NO:85) of a mutant of H5 from influenza A/Indonesia/5/2005 (H5Nl) comprising a deleted proteolytic loop. Figure 25E shows a schematic representation of construct number 928. 25 [0053] Figure 26A shows a general schematic of an e of several enhancer sequences, CPMVX, and CPMVX+ (comprising CPMVX, and a stuffer fragment, which in this non-limiting example, comprises a multiple cloning site and plant kozak sequence), as bed herein. CPMCX and CPMVX+ are each shown as operatively linked to plant regulatory region at their , and at their 3’ ends, in 30 series, a nucleotide sequence of interest (including an ATG initiation site and STOP site), a 3’UTR, and a terminator sequence. An example of construct CPMVX as WO 2014/153674 PCT/CA2014/050326 -16, bed herein, is CPMV160. An example of construct CPMVX+ as described herein, is CPMV160+. Figure 26B shows the relative hemagglutination titres (HMG) in crude protein extracts of modified HA ns produced in plants comprising T expression constructs, and CPMV160+ based expression constructs. Data for the expression of HA B Brisbane/60/08 with deleted proteolytic loop and with a PDI signal peptide (construct number 1039, 5’UTR: CMPV HT; and construct number 1937, 5’UTR: CMPV160+; see Example 5.7), B Brisbane/60/08+H1Tm, with deleted proteolytic loop, with transmembrane domain and cytoplasmic tail replaced by those of H1, B Massachusetts/2/2012 2012 with deleted proteolytic loop and with 10 a PDI signal peptide (construct number 2072, 5’UTR: CMPV HT; and construct number 2050, 5’UTR: CMPV160+; see Example 5.14), B Massachusetts/2/2012+H1Tm with deleted proteolytic loop, with transmembrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009 and with a PDI signal peptide (construct number 2074, 5’UTR: CMPV HT; and construct 15 number 2060, 5’UTR: CMPV160+; see Example 5.15), B Wisconsin/1/2010 with deleted lytic loop and with the native signal peptide (construct number 1445, 5’UTR: CMPV HT; and construct number 1975, 5’UTR: CMPV160+; see Example 5.16), and B Wisconsin/1/2010+H1Tm with deleted proteolytic loop, with transmembrane domain and cytoplasmic tail ed by those of H1 20 A/California/07/2009 and with the native signal peptide (construct number 1454, 5’UTR: CMPV HT; and uct number 1893, 5’UTR: CMPV160+; see Example 5.18), are shown. id="p-54" id="p-54" id="p-54" id="p-54" id="p-54"
[0054] FIGURE 27A shows a general schematic of the enhancer sequence of the CPMV HT and CPMV HT+ fused to a nucleotide ce of interest. Not all of the 25 elements shown in this figure may be required within the enhancer sequence.
Additional elements may be included at the 3’end of the nucleotide sequence of interest (not shown) including a sequence encoding a comovirus 3’ untranslated region (UTR), a plastocyanin 3’ UTR, or a combination of the comovirus 3’ UTR and the plastocyanin 3’ UTR. FIGURE 27B shows the relative hemagglutination 30 titre (HMG) in crude n extracts of ns produced in plants comprising CPMV—HT sion constructs, and CPMV HT+ based sion constructs, operatively linked with a nucleotide sequence of interest. Data for the expression of WO 2014/153674 PCT/CA2014/050326 -17, HA B Brisbane/60/08 with deleted lytic loop and with a PDI signal peptide (construct number 1039: CPMV HT; see e 5.7 and construct number 1829: CPMV HT+; see example 5.12), B Brisbane/60/08 + HlTM with deleted proteolytic loop, with transmembrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009, and with a PDI signal peptide (construct number 1067: CPMV HT; see Example 5.14 and construct number 1875: CPMV HT+; see example 5.19), B Massachusetts/2/2012 with deleted proteolytic loop, and with a PDI signal peptide (construct number 2072: CMPV HT; see Example 5.15 and construct number 2052: CMPV HT+; see Example 5.20), B Massachusetts/2/2012+H1Tm with deleted 10 proteolytic loop, with transmembrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009 and with a PDI signal peptide (construct number 2074: CMPV HT; see Example 5.16 and construct number 2062: CMPV HT+; see Example 5.21), B Wisconsin/1/2010 with deleted proteolytic loop and with the native signal peptide (construct number 1445: CMPV HT; see Example 5.17 and construct number 15 1839: CMPV HT+; see Example 5.22), and B sin/1/2010+H1Tm with deleted proteolytic loop, with transmembrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009 and with the native signal e (construct number 1454: CMPV HT; see Example 5.18 and uct number 1860: CMPV HT+; see Example 5.23), are shown. 20 [0055] FIGURE 28A shows a Western blot analysis of H3 Perth n sion in agroinfiltrated Nicotiana benthamiana leaves. Lane 1: (2019+1261) co—expression of native (wildtype) HA from H3 Perth-l6-09 in the presence of expression enhancer (CPMV-HT+) with M2 from A/New Caledonia/20/99; Lane 2: (2139+1261) co— expression of native (wildtype) HA from H3 Perth-l6-09 in the presence of 25 expression enhancer (CPMV 160+) with M2 from A/New Caledonia/20/99; Lane 3 (2039+1261) co—expression of mutant (modified) HA from H3 Perth-l6-09 in the presence of expression enhancer (CPMV HT+) with M2 from A/New Caledonia/20/99; Lane 4: (2159+1261) co—expression of mutant (modified) HA from H3 Perth-l6-09 in the presence of expression enhancer (CPMV 160+) with M2 from 30 A/New Caledonia/20/99. id="p-56" id="p-56" id="p-56" id="p-56" id="p-56"
[0056] FIGURE 28B shows a Western blot is of B Malaysia protein sion in agroinfiltrated ana benthamiana leaves. Lane 2: (2013+1261) co— WO 2014/153674 PCT/CA2014/050326 -18, expression of native ype) HA from B Malaysia 2506-04 in the presence of expression enhancer (CPMV—160+) with M2 from A/New Caledonia/20/99; Lane 2: (2014+1261) co-expression of mutant ed) HA from B Malaysia 2506-04 in the presence of expression enhancer (CPMV 160+) with M2 from A/New Caledonia/20/99. id="p-57" id="p-57" id="p-57" id="p-57" id="p-57"
[0057] FIGURE 28C shows a Western blot analysis of H9 Hong Kong protein expression in agroinflltrated Nicotiana miana leaves. Lane 1: (1610+1261) co— sion of native (wildtype) HA from H9 Hong Kong —1037—99 in the presence of expression enhancer HT) with M2 from A/New Caledonia/20/99; Lane 2: 10 (1630+1261) co—expression of native (wildtype) HA from H9 Hong Kong 99 in the presence of expression enhancer HT+) and amplification element BEYDV with M2 from A/NeW Caledonia/20/99; Lane 3: (2244+1261) co—expression of native (wildtype) HA from H9 Hong Kong -1037—99 in the presence of expression enhancer (CPMV—HT+) with M2 from A/New nia/20/99; Lane 4: 15 (2226+1261): co—expression of native (wildtype) HA from H9 Hong Kong —1037—99 in the presence of expression enhancer (CPMV 160+) with M2 from A/New Caledonia/20/99. Lane 6: (2246+1261) ression of native (wildtype) HA from H9 Hong Kong —1037—99 in the presence of expression enhancer (CPMV—160+) and amplification element BeYDV with M2 from A/New Caledonia/20/99; Lane 7: 20 (2225+1261) co—expression of mutant (modified) HA from H9 Hong Kong —1037—99 in the presence of expression enhancer (CPMV-HT+) with M2 from A/NeW Caledonia/20/99; Lane 8: (2245+1261) co—expression of mutant (modified) HA from H9 Hong Kong —1037—99 in the presence of expression er (CPMV HT+) and amplification element BeYDV with M2 from A/New Caledonia/20/99. Lane 9: 25 (2227+1261) co—expression of mutant (modified) HA from H9 Hong Kong —1037—99 in the presence of expression enhancer (CPMV 160+) with M2 from A/New nia/20/99. Lane 10: (2247+1261) co—expression of mutant ied) HA from H9 Hong Kong —1037—99 in the presence of expression enhancer (CPMV 160+) and amplification element BeYDV with M2 from A/NeW Caledonia/20/99. 30 [0058] FIGURE 28D shows a n blot analysis of B Massachusetts protein expression in agroinflltrated Nicotiana benthamiana leaves. Lane 1: (2070+1261) co— expression of native (wildtype) HA from B Massachusetts 12 in the presence of WO 2014/153674 PCT/CA2014/050326 -19, expression er (CPMV-HT) with M2 from A/New Caledonia/20/99; Lane 2: (2080+126l) co—expression of native (wildtype) HA from B Massachusetts —2—12 in the ce of expression enhancer (CPMV—HT+) with M2 from A/New Caledonia/20/99; Lane 3: (2090+1261) co—expression of native (wildtype) HA from B Massachusetts 12 in the ce of expression er (CPMV-160+) with M2 from A/New Caledonia/20/99; Lane 4: (2072+1261) co—expression of mutant (modified) HA from B Massachusetts 12 in the presence of expression enhancer (CPMV HT) with M2 from A/Nevv Caledonia/20/99; Lane 5: (2052+126l) co— expression of mutant (modified) HA from B Massachusetts 12 in the presence of 10 expression er (CPMV HT+) with M2 from A/New Caledonia/20/99; Lane 6: (2050+1261) co—expression of mutant (modified) HA from B Massachusetts 12 in the ce of expression enhancer (CPMV 160+) with M2 from A/New Caledonia/20/99. id="p-59" id="p-59" id="p-59" id="p-59" id="p-59"
[0059] FIGURE 28E shows a Western blot analysis of H2 Sin protein expression in 15 agroinfiltrated Nicotiana benthamiana leaves. Lane 1: 126l) co—expression of native (wildtype) HA from H2 Singapore —1—57 in the presence of expression enhancer (CPMV—HT+) with M2 from A/New Caledonia/20/99; Lane 2: (2222+1261) co-expression of native (wildtype) HA from H2 Singapore -l-57 in the presence of expression enhancer (CPMV 160+) with M2 from A/Nevv 20 Caledonia/20/99. Lane 3: (2221+1261) co-expression of mutant ed) HA from H2 Singapore -l-57 in the presence of expression enhancer HT+) with M2 from A/NeW Caledonia/20/99; Lane 4: (2223+1261) co-expression of mutant (modified) HA from H2 Singapore -l-57 in the presence of expression enhancer (CPMV 160+) with M2 from A/Nevv Caledonia/20/99. 25 [0060] FIGURE 28F shows a Western blot is of B/Forida protein expression in agroinfiltrated Nicotiana miana leaves. Lane 1: (1004+126l) co—expression of native (wildtype) HA from B/Florida in the presence of expression enhancer (CPMV— HT) with M2 from A/Nevv Caledonia/20/99; Lane 2: (1003+1261) co—expression of native (wildtype) HA from B/Florida in the presence of expression enhancer (CPMV 30 HT) and ication element BeYDV with M2 from A/Nevv Caledonia/20/99. Lane 3: (2102+1261) co-expression of mutant (modified) HA from B/Florida in the presence of expression enhancer (CPMV—HT+) with M2 from A/New WO 2014/153674 PCT/CA2014/050326 -20, Caledonia/20/99; Lane 4: (2104+1261) co-expression of mutant (modified) HA from B/Florida in the presence of expression enhancer (CPMV HT+) and amplification element BeYDV with M2 from A/New Caledonia/20/99. Lane 5: (2106+1261) co— expression of mutant (modified) HA from B/Florida+H1 California TMCT in the presence of expression enhancer (CPMV HT+) with M2 from A/New Caledonia/20/99.Lane 6: (2108+1261) co—expression of mutant (modified) HA from B/Florida+H1 California TMCT in the presence of expression enhancer (CPMV HT+) and amplification element BeYDV with M2 from A/New Caledonia/20/99. id="p-61" id="p-61" id="p-61" id="p-61" id="p-61"
[0061] Figure 29A shows the relative HA titer of modified HA from various 10 influenza strains that were expressed in the presence of er element CPMV HT, CPMV HT+ or CPMV 160+. Activity is ed to the native HA protein expressed with the same er element. H3 A Perth/16/09 (H3 Perl 609), H3 Victoria/36l/ll (H3 Vic26l 11), B Brisbane 60/2008 (HB Bris60008), B Malaysia 2506/04 (HB Mal 250604) and B Massachusetts 2/12 (Ma212) were co-expressed with influenza M2 15 n. id="p-62" id="p-62" id="p-62" id="p-62" id="p-62"
[0062] Figure 30A shows primer IF—S2+S4—B Bris.c (SEQ ID NO: 86). Figure 30B shows primer IF—S1a4—B Bris.r (SEQ ID NO: 87). Figure 30C shows the nucleotide sequence of synthesized HA B ne gene (corresponding to nt 34—1791 from Genbank accession number FJ766840) (SEQ ID NO: 88). Figure 30D shows the 20 nucleotide sequence of expression cassette number 1029 from 2X35S promoter to NOS terminator. HA from influenza B/Brisbane/60/2008 is ined. (SEQ ID NO: 89). Figure 30E shows the amino acid sequence of PDISP/HA from influenza B/Brisbane/60/2008 (SEQ ID NO: 90). Figure 30F shows a schematic entation of uct 1029. SacII and StuI restriction enzyme sites used for 25 plasmid linearization are annotated on the representation. id="p-63" id="p-63" id="p-63" id="p-63" id="p-63"
[0063] FIGURE 31 shows sequence components used to prepare construct numbers 1039 and 1829 (2X35S/CPMV HT HA B Brisbane (PrL-) NOS and 2X35S/CPMV HT+ PDISP/HA B Brisbane (PrL—) NOS, respectively; see Example 5.12). Construct number 1039 incorporates a prior art T sequence (CMPV 30 S’UTR with mutated start codon at position 161 fused to a sequence encoding an incomplete M protein) and does not comprise a heterologous kozak sequence between WO 2014/153674 PCT/CA2014/050326 -21, the 5’UTR and the nucleotide sequence of interest (PDISP/HA B Brisbane (PrL-)).
Construct number 1829 includes a CPMV 5’UTR comprising 160 nucleotides, a r fragment comprising an lete M protein ,a multiple cloning site, and a plant kozak sequence and is an example of a CPMV HT+ based construct. PDISP: protein ide isomerase signal peptide; NOS: nopaline se terminator; PrL-: deleted proteolytic loop. FIGURE 31A shows the nucleotide sequence of PDISP/HA B ne (PrL-) (SEQ ID NO: 91). FIGURE 31B shows the amino acid sequence of PDISP/HA B Brisbane (PrL-); SEQ ID NO: 92). FIGURE 31C shows a schematic representation of construct number 1829 (2X35S/CPMV HT+). 10 [0064] FIGURE 32 shows sequence components used to e construct numbers 1039 and 1937 (2X35S/CPMV HT PDISP/HA B ne (PrL-) NOS and 2X35S/CPMV160+ PDISP/HA B Brisbane (PrL-) NOS, respectively; see Example 5.7). Construct number 1039 incorporates a prior art CPMV—HT sequence (CMPV S’UTR with mutated start codon at position 161 fused to a sequence encoding an 15 incomplete M protein) and does not comprise a heterologous kozak sequence between the 5’UTR and the nucleotide sequence of interest (PDISP/HA B Brisbane (PrL-)).
Construct number 1937 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer fragment (multiple cloning site), and a plant kozak sequence (this construct does not comprise a ce encoding an incomplete M protein) and is an example 20 of a CPMV160+ (CPMVX+, where X=160) based construct. PDISP: protein disulfide isomerase signal peptide; NOS: nopaline se terminator; PrL-: deleted proteolytic loop. FIGURE 32A shows a schematic representation of construct number 1937 (2X35 S/CPMV160+; a CPMVX+ based construct, where X=160). id="p-65" id="p-65" id="p-65" id="p-65" id="p-65"
[0065] FIGURE 33 shows sequence components used to prepare construct numbers 25 1067 and 1977 (2X35S/CPMV HT HA B ne (Prl-)+H1 California TMCT NOS and 2X35S/CPMV160+ PDISP/HA B ne (PrL—)+H1 California TMCT NOS, respectively; see Example 5.14). Construct number 1067 orates a prior art CPMV-HT sequence (CMPV 5’UTR with mutated start codon at position 161 fused to a sequence encoding an incomplete M protein) and does not comprise a 30 heterologous kozak sequence between the 5’UTR and the tide sequence of interest (PDISP/HA B Brisbane (PrL-)+H1 California TMCT). Construct number 1977 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer fragment WO 2014/153674 PCT/CA2014/050326 -22, (multiple cloning site), and a plant kozak sequence (this construct does not se a sequence ng an incomplete M protein) and is an example of a CPMV160+ (CPMVX+, where X=160) based construct. PDISP: protein disulfide isomerase signal peptide; NOS: nopaline synthase terminator; PrL-: d lytic loop; TMCT: transmembrane domain cytoplasmic tail. FIGURE 33A shows the nucleotide sequence of PDISP/HA B Brisbane (PrL—)+H1 California TMCT (SEQ ID NO: 95). FIGURE 33B shows the amino acid sequence of PDISP/HA B Brisbane (PrL—)+H1 rnia TMCT (SEQ ID NO: 96). FIGURE 33C shows a schematic representation of construct number 1067 (2X35S/CPMV HT; reference construct). 10 FIGURE 33D shows a tic representation of construct number 1977 (2X35S/CPMV160+; a CPMVX+ based uct, where X=160). id="p-66" id="p-66" id="p-66" id="p-66" id="p-66"
[0066] FIGURE 34 shows sequence components used to prepare construct numbers 2072 and 2050 (2X35S/CPMV HT PDISP/HA B Massachusetts (PrL-) NOS and 2X35S/CPMV160+ PDISP/HA B Massachusetts (PrL—) NOS, respectively; see 15 Example 5.15). Construct number 2072 incorporates a prior art CPMV—HT sequence (CMPV S’UTR with mutated start codon at position 161 fused to a sequence encoding an incomplete M n) and does not comprise a heterologous kozak sequence between the 5’UTR and the nucleotide sequence of interest (PDISP/HA B Massachusetts (PrL—)). Construct number 2050 includes a CPMV 5’UTR comprising 20 160 nucleotides, a stuffer fragment (multiple cloning site), and a plant kozak sequence (this construct does not comprise a sequence encoding an incomplete M protein) and is an example of a CPMV160+ (CPMVX+, where X=160) based construct. PDISP: protein disulfide isomerase signal peptide; NOS: nopaline synthase terminator; PrL-: d proteolytic loop. FIGURE 34A shows the tide sequence of 25 HA B Massachusetts (PrL-) (SEQ ID NO: 97). FIGURE 34B shows the amino acid sequence of PDISP/HA B Massachusetts (PrL—) (SEQ ID NO: 98).
FIGURE 34C shows a schematic representation of construct number 2072 (2X35S/CPMV HT; reference construct). FIGURE 34D shows a schematic representation of construct number 2050 (2X35S/CPMV160+; a CPMVX+ based 30 construct, where X=160). id="p-67" id="p-67" id="p-67" id="p-67" id="p-67"
[0067] FIGURE 35 shows sequence components used to e construct numbers 2074 and 2060 (2X35S/CPMV HT PDISP/HA B Massachusetts (PrL—)+H1 California WO 2014/153674 PCT/CA2014/050326 -23, TMCT NOS and 2X35S/CPMV160+ PDISP/HA B husetts (PrL-)+H1 California TMCT NOS, respectively; see Example 5.16). Construct number 2074 incorporates a prior art CPMV-HT sequence (CMPV 5’UTR with mutated start codon at position 161 fused to a sequence encoding an lete M protein) and does not comprise a logous kozak sequence between the 5’UTR and the nucleotide sequence of interest (PDISP/HA B Massachusetts (PrL—)+H1 California TMCT).
Construct number 2060 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer fragment (multiple cloning site), and a plant kozak sequence (this construct does not comprise a sequence encoding an incomplete M protein) and is an example 10 of a CPMV160+ (CPMVX+, where X=160) based construct. PDISP: n disulfide isomerase signal peptide; NOS: nopaline synthase terminator; PrL-: deleted proteolytic loop; TMCT: transmembrane domain cytoplasmic tail. FIGURE 35A shows the nucleotide sequence of PDISP/HA B Massachusetts +H1 California TMCT (SEQ ID NO: 99). FIGURE 35B shows the amino acid ce of 15 PDISP/HA B Massachusetts (PrL—)+H1 California TMCT (SEQ ID NO: 100).
FIGURE 35C shows a tic representation of construct number 2074 (2X35S/CPMV HT; reference uct). FIGURE 35D shows a schematic representation of construct number 2060 (2X35S/CPMV160+; a CPMVX+ based construct, where X=160). 20 [0068] FIGURE 36 shows sequence components used to e construct numbers 1445, 1820 and 1975 (2X35S/CPMV HT HA B Wisconsin (PrL—) NOS, 2X35S/CPMV160+ HA B Wisconsin (PrL-) NOS and CPMV160 HA B Wisconsin (PrL—) NOS, respectively; see Example 15.17). uct number 1445 incorporates a prior art CPMV-HT sequence (CMPV 5’UTR with mutated start codon 25 at position 161 fused to a sequence encoding an incomplete M protein) and does not comprise a heterologous kozak sequence between the 5’UTR and the nucleotide sequence of interest (HA B Wisconsin (PrL—)). Construct number 1820 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer fragment (multiple cloning site), and a plant kozak sequence (this construct does not comprise a sequence encoding an 30 incomplete M protein) and is an example of a 0+ (CPMVX+, where X=160) based construct. Construct number 1975 includes a CPMV 5’UTR comprising 160 nucleotides, and does not include a stuffer fragment (multiple cloning site), or a plant WO 2014/153674 PCT/CA2014/050326 -24, kozak sequence (this construct also does not comprise a sequence encoding an incomplete M protein) and is an example of a "CPMV160" (CPMVX) based construct. PrL—: deleted proteolytic loop; NOS: nopaline synthase terminator.
FIGURE 36A shows the nucleotide sequence ofHA B Wisconsin (PrL—) (SEQ ID NO: 101). FIGURE 36B shows the amino acid ce ofHA B Wisconsin (PrL—) (SEQ ID NO: 102). FIGURE 36C shows a schematic representation of construct number 1445 (2X35S/CPMV HT; reference construct). FIGURE 36D shows a tic representation of construct number 1820 (2X35S/CPMV160+; a CPMVX+ based construct). FIGURE 36E shows a tic representation of construct 10 number 1975 (2X35 S/CPMV160; a CPMVX based construct, where X=160). id="p-69" id="p-69" id="p-69" id="p-69" id="p-69"
[0069] FIGURE 37 shows sequence components used to prepare construct numbers 1454 and 1893 (2X35S/CPMV HT HA B Wisconsin (PrL-)+H1 rnia TMCT NOS and 2X35S/CPMV160+ HA B Wisconsin (PrL-)+H1 California TMCT NOS, respectively; see Example 5.18). Construct number 1454 incorporates a prior art 15 CPMV—HT sequence (CMPV 5’UTR with mutated start codon at position 161 fused to a sequence encoding an lete M protein) and does not comprise a heterologous kozak sequence between the 5’UTR and the nucleotide sequence of interest (HA B Wisconsin (PrL—)+H1 rnia TMCT). Construct number 1893 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer fragment (multiple 20 cloning site), and a plant kozak sequence (this uct does not comprise a sequence encoding an incomplete M n) and is an example of a CPMV160+ (CPMVX+, where X=160) based construct. NOS: nopaline synthase terminator; PrL-: deleted proteolytic loop; TMCT: transmembrane domain cytoplasmic tail. FIGURE 37A shows the nucleotide sequence of HA B Wisconsin (PrL—)+H1 California TMCT 25 (SEQ ID NO: 103). FIGURE 37B shows the amino acid sequence of PDISP/HA B Wisconsin (PrL—)+H1 California TMCT (SEQ ID NO: 104). FIGURE 37C shows a schematic representation of construct number 1454 /CPMV HT; nce uct). FIGURE 37D shows a schematic representation of construct number 1893 /CPMV160+; a CPMVX+ based construct, where X=160). 30 [0070] FIGURE 38 shows sequence components used to prepare construct numbers 1067 and 1875 (2X35S/CPMV HT PDISP/HA B Brisbane (Prl-)+H1 California TMCT NOS and 2X35S/CPMV HT+ PDISP/HA B ne (PrL—)+H1 California WO 2014/153674 PCT/CA2014/050326 -25, TMCT NOS, respectively; see Example 5.19). uct number 1067 incorporates a prior art CPMV-HT sequence (CMPV 5’UTR with mutated start codon at position 161 fused to a sequence encoding an incomplete M protein) and does not comprise a heterologous kozak sequence between the 5’UTR and the nucleotide sequence of interest /HA B Brisbane +Hl rnia TMCT). uct number 1875 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer fragment comprising an incomplete M protein, a multiple cloning site, and a plant kozak sequence and is an example of a CPMV HT+ based construct. PDISP: protein disulfide ase signal peptide; NOS: nopaline synthase ator; PrL-: deleted 10 proteolytic loop; TMCT: transmembrane domain asmic tail. FIGURE 38A shows the nucleotide sequence of PDISP/HA B Brisbane (PrL—)+H1 California TMCT (SEQ ID NO: 105). FIGURE 38B shows the amino acid sequence of PDISP/HA B Brisbane (PrL-)+Hl California TMCT (SEQ ID NO: 106). FIGURE 38C shows a schematic representation of uct number 1875 15 (2X35 l 60+). id="p-71" id="p-71" id="p-71" id="p-71" id="p-71"
[0071] FIGURE 39 shows sequence components used to prepare construct numbers 2072 and 2052 (2X35S/CPMV HT PDISP/HA B Massachusetts (PrL-) NOS and 2X35S/CPMV HT+ PDISP/HA B Massachusetts (PrL—) NOS, respectively; see Example 5.20). Construct number 2072 incorporates a prior art CPMV—HT sequence 20 (CMPV S’UTR with d start codon at position 161 fused to a sequence encoding an incomplete M protein) and does not comprise a heterologous kozak sequence between the 5’UTR and the nucleotide sequence of interest (PDISP/HA B Massachusetts (PrL—)). Construct number 2052 includes a CPMV S’UTR comprising 160 nucleotides, a r fragment comprising an incomplete M protein, a multiple 25 cloning site, and a plant kozak sequence and is an example of a CPMV HT+ based construct. PDISP: protein disulfide isomerase signal peptide; NOS: nopaline synthase terminator; PrL—: deleted proteolytic loop. FIGURE 39A shows the nucleotide sequence of PDISP/HA B Massachusetts (PrL—) (SEQ ID NO: 107).
FIGURE 39B shows the amino acid sequence of PDISP/HA B Massachusetts (PrL-) 30 (SEQ ID NO: 108). FIGURE 39C shows a schematic representation of construct number 2052 (2X35S/CPMV HT+).
WO 53674 2014/050326 -26, id="p-72" id="p-72" id="p-72" id="p-72" id="p-72"
[0072] FIGURE 40 shows sequence components used to prepare construct numbers 2074 and 2062 (2X35S/CPMV HT PDISP/HA B Massachusetts (PrL—)+H1 rnia TMCT NOS and 2X35S/CPMV HT+ PDISP/HA B Massachusetts (PrL—)+H1 California TMCT NOS, respectively; see Example 5.21). Construct number 2074 orates a prior art CPMV-HT ce (CMPV 5’UTR with mutated start codon at on 161 fused to a sequence encoding an incomplete M protein) and does not comprise a heterologous kozak sequence between the 5’UTR and the nucleotide sequence of interest (PDISP/HA B Massachusetts (PrL—)+H1 California TMCT).
Construct number 2062 includes a CPMV 5’UTR comprising 160 nucleotides, a 10 stuffer fragment comprising an incomplete M protein, a multiple cloning site, and a plant kozak sequence and is an example of a CPMV HT+ based construct. PDISP: protein disulfide isomerase signal peptide; NOS: ne synthase terminator; PrL-: d lytic loop; TMCT: transmembrane domain cytoplasmic tail. FIGURE 40A shows the nucleotide sequence of PDISP/HA B Massachusetts (PrL—)+H1 15 rnia TMCT (SEQ ID NO: 109). FIGURE 40B shows the amino acid sequence of PDISP/HA B Massachusetts (PrL—)+H1 California TMCT (SEQ ID NO: 110).
FIGURE 40C shows a schematic representation of construct number 2062 (2x35S/CPMV HT+). id="p-73" id="p-73" id="p-73" id="p-73" id="p-73"
[0073] FIGURE 41 shows sequence ents used to prepare construct numbers 20 1445 and 1839 (2X35S/CPMV HT HA B Wisconsin (PrL—) NOS, and 2X35S/CPMV HT+ HA B Wisconsin (PrL—) NOS, respectively; see Example 5.22). Construct number 1445 incorporates a prior art CPMV-HT sequence (CMPV 5’UTR with mutated start codon at position 161 fused to a sequence encoding an incomplete M protein) and does not comprise a heterologous kozak sequence between the 5’UTR 25 and the nucleotide sequence of interest (HA B Wisconsin (PrL—)). Construct number 1839 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer fragment comprising an incomplete M protein, a multiple cloning site, and a plant kozak sequence and is an e of a CPMV HT+ based construct. PrL—: deleted proteolytic loop; NOS: nopaline synthase terminator. FIGURE 41A shows the 30 nucleotide sequence of HA B sin (PrL—) (SEQ ID NO: 111). FIGURE 41B shows the amino acid sequence of HA B Wisconsin (PrL—) (SEQ ID NO: 112).
WO 53674 PCT/CA2014/050326 -27, FIGURE 41C shows a schematic representation of construct number 1839 (2x35S/CPMV HT+). id="p-74" id="p-74" id="p-74" id="p-74" id="p-74"
[0074] FIGURE 42 shows sequence components used to e construct numbers 1454 and 1860 (2X35S/CPMV HT HA B Wisconsin (PrL-)+H1 California TMCT NOS and 2X35S/CPMV HT+ HA B sin (PrL-)+H1 California TMCT NOS, tively; see Example 5.23). Construct number 1454 incorporates a prior art CPMV—HT sequence (CMPV 5’UTR with mutated start codon at position 161 fused to a sequence encoding an lete M protein) and does not se a heterologous kozak sequence between the 5’UTR and the nucleotide sequence of 10 interest (HA B Wisconsin +H1 California TMCT). Construct number 1860 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer fragment comrpsing n incomplete M protein, a multiple cloning site, and a plant kozak sequence and is an example of a CPMV HT+ based construct. NOS: nopaline synthase terminator; PrL- : deleted proteolytic loop; TMCT: transmembrane domain cytoplasmic tail. 15 FIGURE 42A shows the nucleotide sequence ofHA B Wisconsin (PrL—)+H1 California TMCT (SEQ ID NO: 113). FIGURE 42B shows the amino acid sequence of PDISP/HA B Wisconsin (PrL-)+H1 California TMCT (SEQ ID NO: 114).
FIGURE 42C shows a schematic representation of construct number 1893 (2x35S/CPMV HT+). 20 [0075] FIGURE 43 shows sequence ents used to prepare construct numbers 489 /CPMV HT H5 Indonesia NOS see Example 5.24). Construct number 489 incorporates a CPMV—HT sequence (CMPV S’UTR with mutated start codon at position 161 fused to a sequence encoding an incomplete M protein) and does not comprise a heterologous kozak sequence between the 5’UTR and the nucleotide 25 ce of interest (PDISP/H1 California). FIGURE 43A shows the nucleotide sequence of native H5 Indonesia (SEQ ID NO: 115). FIGURE 43B shows the amino acid ce of native H5 Indonesia (SEQ ID NO: 116). FIGURE 43C shows a schematic representation of construct number 489 (2X35S/CPMV HT; reference construct). 30 [0076] FIGURE 44 shows the sequence components used to prepare construct number 1800 (A-2X35S CPMV160+ PDISP H3Victoria NOS; see example 5.25).
WO 2014/153674 PCT/CA2014/050326 -28, Construct number 1800 includes a CPMV 5’UTR comprising 160 nucleotides, a stuffer nt (multiple cloning site), and a plant kozak sequence (this construct does not comprise a sequence encoding an incomplete M protein) and is an example of a CPMV160+ (CPMVX+, where X=160) based construct. PDISP: protein disulfide isomerase signal peptide. NOS: nopaline synthase terminator. FIGURE 44A shows primer sequence IF**(SacII)-PDI.s1+4c (SEQ ID NO:117). FIGURE 44B shows primer sequence IF-H3V36111.s1-4r (SEQ ID NO: 118). FIGURE 44C shows the sequence of PDISP/H3 Victoria (SEQ ID NO:119). FIGURE 44D shows a schematic representation of construct 2171 (SacII and StuI restriction enzyme sites 10 used for plasmid linearization are indicated). FIGURE 44E shows construct 2171 from left to right t-DNA s (underlined), 2X35 S/CPMV160+/NOS with Plastocyanine-Pl9-Plastocyanine silencing inhibitor expression cassette, an H1 California transmembrane cytoplasmic tail, and the CPMV3’UTR (SEQ ID NO: 120).
FIGURE 44F shows expression cassette number 1800 from 2X35S promoter to NOS 15 terminator. H3 Victoria nucleotide ce is underlined; 5’UTR is shown in bold; plant kozak sequence double underline; a r fragment (multiple cloning site) of 16 base pairs is oned between the S’UTR and plant kozak sequence (SEQ ID NO:121). FIGURE 44G shows the amino acid ce of PDISP/H3 Victoria (SEQ ID NO: 122). FIGURE 44H shows a tic representation of 20 construct number 1800 (a CPMVX+ based construct, where X=160). id="p-77" id="p-77" id="p-77" id="p-77" id="p-77"
[0077] FIGURE 45 shows the sequence components used to prepare construct number 1819 (2X35S CPMV-HT+ PDISP H3Victoria NOS). uct number 1819 incorporates a CPMV—HT+ sequence (CMPV S’UTR with mutated start codon at position 161 fused to a r fragment encoding an incomplete M protein, a multiple 25 g site, and comprises a plant kozak sequence between the multiple cloning site and the tide sequence of interest (PDISP/H3 Victoria)). PDISP: protein disulfide isomerase signal peptide. NOS: nopaline synthase terminator. FIGURE 45A shows primer sequence IF(SacII)-Kozac_PDI.c (SEQ ID NO: 123). FIGURE 45B shows primer sequence IF-H3V36111.s1-4r (SEQ ID NO: 124). FIGURE 45C 30 shows a schematic representation of construct 2181. FIGURE 45D shows the sequence for construct 2181 (from left to right t-DNA borders, underlined; 2X35 -HT+/NOS with Plastocyanine-P 1 9-Plastocyanine silencing inhibitor WO 2014/153674 PCT/CA2014/050326 -29, expression cassette; SEQ ID ). FIGURE 45E shows expression cassette number 1819 from 2X35S promoter to NOS terminator. The PDISP/H3 Victoria nucleotide sequence is underlined (SEQ ID NO:127). FIGURE 45F shows a schematic representation of construct 1819. id="p-78" id="p-78" id="p-78" id="p-78" id="p-78"
[0078] Figure 46A shows the ve hemagglutination activity of native H7 Hangzhou HA and modified H7 Hangzhou HA, with the proteolytic loop deleted when co—expressed with M2. Native and modified H7 ou HA (constructs #2142 and 2152, see es 5.33 and 5.34) were expressed in the presence of M2 (construct #1261 see Example 5.1) and purified from plants. Figure 46B shows the 10 protein yield of native H7 Hangzhou HA (construct #2142) and modified H7 Hangzhou HA, with the proteolytic loop deleted (construct #2152). Figure 46C shows an SDS-PAGE analysis, with lane 2 showing purified modified H7 Hangzhou HA with a removed proteolytic loop (construct #2152) and lane 3 showing the purified native H7 Hangzhou HA (construct #2142). For each lane, 2ug of total 15 protein were loaded on the gel. The purity of the proteins profiles are r for both constructs. id="p-79" id="p-79" id="p-79" id="p-79" id="p-79"
[0079] Figure 47A shows the Trypsin resistance between native HA protein and modified HA with the proteolytic loop replaced with a GG liker (prl-), modified HA with the proteolytic loop replaced with a TETQ liker (TETQ) and ed HA with 20 the proteolytic loop replaced with a TETR liker (TETR). Native (# 489), PRL- (# 928), TETQ (#766) and TETR (#676) H5 Indonesia HA VLP constructs were purified. For each lot, two samples of HA VLPs were resuspended in buffer (100 mM Na/KP04, 150 mM NaCl, 0.01% TWEEN 80) at pH 7.4 at a target concentration of 150ug/mL. n was added in a 1:100 protein ratio to one resuspended sample. 25 Samples were incubated for 30, 60 and 120 minutes at room ature. 30uL of the non—digested extract (control) and 30uL of the digested extracts were loaded on SDS— PAGE gel, which was stained with sie blue. Figure 47B shows immunogenicity (HI titer) of native H5 VLP and its mutant counterparts (prl-, TETQ and TETR) in mice after two doses. Bars ent relative (%) HI titers comparison 30 of each H5 mutants VLP with the native H5 VLP.
WO 2014/153674 2014/050326 -30, id="p-80" id="p-80" id="p-80" id="p-80" id="p-80"
[0080] FIGURE 48 shows sequence components used to prepare construct numbers 2220 /CPMV HT+/ PDISP/H2 Singapore/ NOS see e 5.27). FIGURE 48A shows the tide sequence of primer IF**—H2S157.s1—6r (SEQ ID NO: 127).
FIGURE 48B shows the nucleotide sequence of PDISP/H2 Singapore (SEQ ID NO: 128) FIGURE 48C shows the nucleotide sequence of expression cassette number 2220 from 2X35S promoter to NOS terminator. PDISP/H2 Singapore nucleotide sequence is underlined. FIGURE 48D shows the Amino acid sequence of PDISP/H2 Singapore. FIGURE 48E shows a schematic representation of construct number 2220. 10 [0081] FIGURE 49 shows sequence components used to prepare construct numbers 2221 (2X35S/CPMV HT+/ PDISP/H2 Singapore with d proteolytic loop/ NOS see e 5.28). FIGURE 49A shows the nucleotide sequence of primer H2S157(Prl—).r (SEQ ID NO: 131). FIGURE 49B shows the nucleotide sequence of primer H2S157(Prl—).c (SEQ ID NO: 132) FIGURE 49C shows the nucleotide 15 sequence of expression cassette number 2221 from 2X35S promoter to NOS terminator. PDISP/H2 Singapore nucleotide sequence is underlined (SEQ ID NO: 133). FIGURE 49D shows the Amino acid sequence of PDISP/H2 Singapore with deleted lytic loop (SEQ ID ) . FIGURE 49E shows a schematic entation of construct number 2221. 20 [0082] FIGURE 50 shows sequence components used to prepare construct s 2222 (2X35S/CPMV 160+/ PDISP/H2 ore) and 2223 (2X35S/CPMV 160+/ PDISP/H2 Singapore with deleted proteolytic loop/ NOS) see Example 5.29).
FIGURE 50A shows the nucleotide sequence of expression cassette number 2222 from 2X35S promoter to NOS terminator. PDISP/H2 Singapore nucleotide sequence 25 is underlined (SEQ ID NO:l35). FIGURE 50B shows the nucleotide sequence of expression cassette number 2223 from 2X35S promoter to NOS terminator.
PDISP/H2 Singapore with deleted proteolytic loop nucleotide sequence is underlined (SEQ ID NO: 136). FIGURE 50C a schematic representation of construct number 2222 FIGURE 50D a schematic representation of construct number 2223. 30 [0083] FIGURE 51 shows sequence components used to prepare construct numbers 2219 (2X35S/CPMV HT+ H3 Perth) and 2139 (2X35S/CPMV 160+/ WO 53674 PCT/CA2014/050326 -31, PDISP/H3 Perth) see Example 5.30). FIGURE 51A shows the nucleotide sequence of PDISP/H3 Perth (SEQ ID NO: 137). FIGURE 51B shows the nucleotide sequence of primer IF**-H3P1609.S1-6r (SEQ ID NO: 138). FIGURE 51Cshows the Amino acid sequence of PDISP/H3 Perth (SEQ ID NO: 139). Figure 51D shows a schematic entation of construct number 2219. FIGURE 51E shows a schematic representation of construct number 2139. id="p-84" id="p-84" id="p-84" id="p-84" id="p-84"
[0084] FIGURE 52 shows sequence ents used to prepare construct numbers 2039 (2X35S/CPMV HT+ H3 Perth with deleted proteolytic loop) and 2159 (2X35S/CPMV 160+ PDISP/H3 Perth with d proteolytic loop) see Example 10 5.31). FIGURE 52A shows the nucleotide sequence of H3 Perth with deleted proteolytic loop (SEQ ID NO: 140). FIGURE 52B shows the nucleotide sequence of primer H3P1609(Prl—)#2.r (SEQ ID NO: 141). FIGURE 52C shows the nucleotide sequence of primer 9(Prl—)#2.c (SEQ ID NO: 142). FIGURE 52Dshows the amino acid sequence of PDISP/H3 Perth with deleted proteolytic loop (SEQ ID NO: 15 143). Figure 52E shows a schematic entation of construct number 2039.
FIGURE 52F shows a schematic representation of construct number 2159. id="p-85" id="p-85" id="p-85" id="p-85" id="p-85"
[0085] FIGURE 53 shows sequence components used to prepare construct numbers 2230 (2X35S/CPMV HT+ PDISP/H3 Victoria with deleted proteolytic loop) and 2250 (2X35S/CPMV 160+ PDISP/H3 Victoria with deleted proteolytic loop) see 20 Example 5.32). FIGURE 53A shows the tide sequence of nucleotide sequence of PDISP/H3 Victoria with deleted proteolytic loop (SEQ ID NO: 144). FIGURE 53B shows the nucleotide sequence of primer H3V36111(Prl—).r (SEQ ID NO: 145).
FIGURE 53C shows the nucleotide sequence of primer H3V36111(Prl—).c (SEQ ID NO: 146). FIGURE 53Dshows the amino acid sequence of PDISP/H3 Victoria with 25 d proteolytic loop (SEQ ID NO: 147). FIGURE 53E shows a schematic representation of construct number 2230. FIGURE 53F shows a schematic representation of construct number 2250. id="p-86" id="p-86" id="p-86" id="p-86" id="p-86"
[0086] FIGURE 54 shows sequence components used to e construct number 2142 (2X35S/CPMV HT+/PDISP/H7 Hangzhou/NOS) see Example 5.33). FIGURE 30 54A shows the nucleotide sequence of PDISP/H7 Hangzhou (SEQ ID NO: 148).
FIGURE 54B shows the nucleotide sequence of primer IF*-H7H1 13.s1—6r (SEQ ID WO 2014/153674 PCT/CA2014/050326 -32, NO: 149). FIGURE 54C shows the amino acid sequence of PDISP/H7 Hangzhou (SEQ ID NO: 150). FIGURE 53D shows a schematic representation of construct number 2142. id="p-87" id="p-87" id="p-87" id="p-87" id="p-87"
[0087] FIGURE 55 shows sequence ents used to prepare construct number 2152 (2X35S/CPMV HT+/PDISP/H7 Hangzhou with deleted proteolytic loop/NOS) see Example 5.34). FIGURE 55A shows the nucleotide sequence of H7 Hangzhou with deleted proteolytic loop (SEQ ID NO: 151). FIGURE 55B shows the nucleotide sequence of primer H7H113(PrL—).r (SEQ ID NO: 152). FIGURE 55C shows the nucleotide sequence of primer (PrL—).c (SEQ ID NO: 153). 10 FIGURE 55D shows the amino acid sequence of PDISP/H7 Hangzhou with deleted proteolytic loop (SEQ ID NO: 154). FIGURE 53E shows a schematic representation of construct number 2152. id="p-88" id="p-88" id="p-88" id="p-88" id="p-88"
[0088] FIGURE 56 shows sequence components used to prepare construct numbers 2224 (2X35S/CPMV HT+ PDISP/H9 Hong Kong) and 2226 (2X35S/CPMV 160+ 15 PDISP/H9 Hong Kong) see Example 5.35). FIGURE 56A shows the nucleotide ce of PDISP/H9 Hong Kong (SEQ ID NO: 155). FIGURE 56B shows the nucleotide ce of primer IF**—H9HK107399.S1—6r (SEQ ID NO: 156).
FIGURE 56C shows the amino acid sequence of PDISP/H9 Hong Kong (SEQ ID NO: 157). FIGURE 56D shows a schematic representation of construct number 2224. 20 FIGURE 56E shows a schematic entation of construct number 2226. id="p-89" id="p-89" id="p-89" id="p-89" id="p-89"
[0089] FIGURE 57 shows sequence components used to prepare construct numbers 2225 (2X35S/CPMV HT+ PDISP/H9 Hong Kong with d proteolytic loop) and 2227 /CPMV 160+ PDISP/H9 Hong Kong with deleted proteolytic loop) see Example 5.36. FIGURE 57A shows the nucleotide sequence of PDISP/H9 Hong 25 Kong with deleted proteolytic loop (SEQ ID NO: 158). FIGURE 57B shows the nucleotide sequence of primer H9HK107399(Prl—).r (SEQ ID NO: 159). FIGURE 57C shows the nucleotide sequence of primer H9HK107399(Prl—).c (SEQ ID NO: 160). FIGURE 57D shows the amino acid sequence of PDISP/H9 Hong Kong with deleted lytic loop (SEQ ID NO: 161). FIGURE 57E shows a schematic 30 representation of construct number 2225. FIGURE 57F shows a schematic representation of construct number 2227.
WO 2014/153674 PCT/CA2014/050326 -33, id="p-90" id="p-90" id="p-90" id="p-90" id="p-90"
[0090] FIGURE 58 shows sequence components used to prepare uct number 2013 (2X35S/CPMV 160+/PDISP/HA B ia/NOS) see Example 5.37.
FIGURE 58A shows the nucleotide sequence of PDISP/HA B Malaysia (SEQ ID NO: 162). FIGURE 58B shows the nucleotide sequence of primer IF**— 604.S1—6r (SEQ ID NO: 163). FIGURE 58C shows the amino acid sequence of PDISP/HA B Malaysia (SEQ ID NO: 164). FIGURE 58D shows a schematic representation of construct number 2013. id="p-91" id="p-91" id="p-91" id="p-91" id="p-91"
[0091] FIGURE 59 shows ce components used to prepare construct number 2014 (2X35S/CPMV 160+/PDISP/HA B Malaysia with deleted proteolytic 10 OS) see Example 5.38. FIGURE 59A shows the nucleotide sequence of PDISP/HA B ia with deleted proteolytic loop (SEQ ID NO: 165). FIGURE 59B shows the nucleotide sequence of primer HBM250604(PrL-).r (SEQ ID NO: 166). FIGURE 59C shows the nucleotide ce of primer HBM250604(PrL—).c (SEQ ID NO: 167). FIGURE 59D shows the amino acid sequence of PDISP/HA B 15 Malaysia with deleted proteolytic loop (SEQ ID NO: 168). FIGURE 59E shows a schematic representation of construct number 2014. id="p-92" id="p-92" id="p-92" id="p-92" id="p-92"
[0092] FIGURE 60 shows sequence components used to prepare construct numbers 2070 (2X35S/CPMV HT PDISP/HA B Massachusetts), 2080 (2X35S/CPMV HT+ HA B Massachusetts) and 2090 /CPMV 160+ PDISP/HA B 20 Massachusetts) see Example 5.39. FIGURE 60A shows the nucleotide sequence of PDISP/HA B husetts (SEQ ID NO: 169). FIGURE 60B shows amino acid sequence of PDISP/HA B Massachusetts (SEQ ID NO: 170). FIGURE 60C shows a schematic representation of construct number 2070. FIGURE 60D shows a schematic representation of construct number 2080. FIGURE 60E shows a schematic 25 representation of construct number 2090. id="p-93" id="p-93" id="p-93" id="p-93" id="p-93"
[0093] FIGURE 61 shows sequence components used to prepare construct numbers 2102 (2X35S/CPMV HT PDISP/HA B Florida with proteolytic loop deleted) and 2104 (2X35S/CPMV HT+ /BeYDV/PDISP/HA B Florida with proteolytic loop deleted) see Example 5.40. FIGURE 61A shows the nucleotide sequence of primer 30 HBF406(PrL—).r (SEQ ID NO: 190). FIGURE 61B shows the nucleotide sequence of primer HBF406(PrL-).c (SEQ ID NO: 191). FIGURE 61C shows the nucleotide WO 2014/153674 2014/050326 -34, sequence of primer IF*—HBF406.s1—6r (SEQ ID NO: 192). FIGURE 61D shows the nucleotide sequence of PDISP/HA B Florida with deleted proteolytic loop. FIGURE 61E shows the amino acid sequence of PDISP/HA B a with deleted proteolytic loop. FIGURE 61F shows the nucleotide sequence of expression cassette number 2102. FIGURE 61G shows the tic representation of construct number 2102.
FIGURE 61H shows the nucleotide sequence of expression cassette number 2104.
FIGURE 611 shows the schematic representation of construct number 2104. id="p-94" id="p-94" id="p-94" id="p-94" id="p-94"
[0094] FIGURE 62 shows sequence components used to prepare construct numbers 2106 (2X35S/CPMV HT+/ PDISP/B Florida +Hl California TMCT with proteolytic 10 loop deleted / NOS) and 2108 (2X35S/CPMV HT+/ BeYDV/PDISP/B Florida +H1 rnia TMCT with lytic loop deleted/ NOS) see Example 5.41. FIGURE 62A shows the nucleotide sequence of primer IF—chTMCT.S1—4r (SEQ ID NO: 197). FIGURE 62B shows the nucleotide sequence of PDISP/HA B a+H1Cal TMCT with d proteolytic loop (SEQ ID NO: 198). FIGURE 62C shows the 15 amino acid sequence of PDISP/HA B a+HlCal TMCT with deleted proteolytic loop. FIGURE 62D shows the nucleotide sequence of expression cassette number 2106. FIGURE 62E shows the schematic representation of construct number 2106.
FIGURE 62F shows the tide sequence of expression cassette number 2108.
FIGURE 62G shows the schematic representation of construct number 2108. 20 DETAILED DESCRIPTION id="p-95" id="p-95" id="p-95" id="p-95" id="p-95"
[0095] The following description is of a preferred embodiment. id="p-96" id="p-96" id="p-96" id="p-96" id="p-96"
[0096] The present invention relates to virus-like particles (VLPs) and methods of producing and increasing VLP yield, accumulation and production in plants. id="p-97" id="p-97" id="p-97" id="p-97" id="p-97"
[0097] The present invention provides, in part, a method of ing a virus like 25 particle (VLP) in a plant, or portion of the plant. The method involves introducing a nucleic acid into the plant or portion of the plant. The c acid comprises comprising a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding an influenza hemagglutinin (HA). The HA comprises a modified proteolytic loop or cleavage site. The plant or portion of the plant is 30 incubated under conditions that permit the expression of the nucleic acid, thereby WO 2014/153674 2014/050326 -35, producing the VLP. If desired, the plant or portion of the plant may be harvested and the VLP purified. id="p-98" id="p-98" id="p-98" id="p-98" id="p-98"
[0098] The present invention also provides a VLP produced by this method. The VLP may comprise one or more than one lipid derived from a plant. id="p-99" id="p-99" id="p-99" id="p-99" id="p-99"
[0099] The VLP may be used to prepare a ition comprising an effective dose of the VLP for ng an immune response, and a pharmaceutically acceptable carrier. id="p-100" id="p-100" id="p-100" id="p-100" id="p-100"
[00100] Also provided herein is a modified lutinin, wherein the proteolytic loop or cleavage site has been ed. 10 [00101] The present invention also provides plant matter comprising the VLP produced by sing the nucleic acids described above. The plant matter may be used in inducing immunity to an influenza virus infection in a subject. The plant matter may also be admixed as a food supplement. id="p-102" id="p-102" id="p-102" id="p-102" id="p-102"
[00102] The VLP of the present ion may also be produced by providing a 15 plant or portion of the plant comprising a nucleic acid as defined above, and incubating the plant or portion of the plant under ions that permit the expression of the nucleic acid, thereby producing the VLP. The VLP may comprise one or more than one lipid derived from a plant. The VLP may be used to prepare a composition comprising an effective dose of the VLP for inducing an immune response, and a 20 pharmaceutically acceptable carrier. The present invention also provides plant matter comprising the VLP ed by expressing the first and second nucleic acids. The plant matter may be used in inducing immunity to an influenza virus infection in a subject. The plant matter may also be admixed as a food supplement. id="p-103" id="p-103" id="p-103" id="p-103" id="p-103"
[00103] The VLP of the present invention comprises one or more modified 25 influenza hemagglutinin (HA). The modified HA may be d from any HA, for example an H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA as described in WO 2009/009876; WO 2009/076778; WO 2010/003225; WO 2010/003235; WO 2011/03522; which are incorporated herein by reference).
WO 2014/153674 PCT/CA2014/050326 -36, id="p-104" id="p-104" id="p-104" id="p-104" id="p-104"
[00104] The current invention includes VLPs sing HA sequences of influenza strains, where the HA sequences comprise modified polybasic cleavage sites including for example, the modifications as described herein.
HA Protein id="p-105" id="p-105" id="p-105" id="p-105" id="p-105"
[00105] The term "hemagglutinin" or "HA" as used herein refers to a glycoprotein found on the outside of influenza viral particles. HA is a homotrimeric membrane type I glycoprotein, generally comprising a signal peptide, an HA1 , and an HA2 domain sing a membrane-spanning anchor site at the C- terminus and a small cytoplasmic tail. Nucleotide sequences encoding HA are well 10 known and are available--see, for example, the BioDefence Public Health base (Influenza Virus; see URL: biohealthbase.org) or National Center for Biotechnology Information (see URL: ncbi.nlm.nih.gov), both of which are orated herein by reference. HA may include any HA, for example an H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA as described in WO 15 2009/009876; WO 2009/076778; WO 2010/003225; WO 2010/003235; WO 2011/03522; which are incorporated herein by reference). Furthermore, the HA may be based on the sequence of a hemagglutinin that is isolated from one or more ng or newly-identified influenza viruses. The present invention also includes VLPs that comprise modified HAs obtained from one or more than one influenza 20 subtype. id="p-106" id="p-106" id="p-106" id="p-106" id="p-106"
[00106] The HA monomer may be subdivided in three functional domains - a stem domain, or stem domain cluster (SDC), a globular head domain, or head domain cluster (HDC) and a transmembrane domain r (TDC). The SDC and HDC may be collectively referred to as the 'ectodomain'. A ation by Ha et al. 2002 25 (EMBO J. 21 1865—875; which is incorporated herein by reference) illustrates the relative ation of the s subdomains of the SDC and HDC in several influenza subtypes, based on Xray crystallographic structures. id="p-107" id="p-107" id="p-107" id="p-107" id="p-107"
[00107] HA protein is synthesized as a sor protein (HAO) of about 75 kDa, which assembles at the surface into an elongated ic protein. The precursor 30 n is cleaved at a conserved activation cleavage site into 2 polypeptide chains, HA1 and HA2 (comprising the transmembrane region), linked by a disulfide bond.
WO 2014/153674 PCT/CA2014/050326 -37, Figure 15 provides non-limiting examples of amino acid ces of the linker region for several HAs. id="p-108" id="p-108" id="p-108" id="p-108" id="p-108"
[00108] The term "homotrimer" or "homotrimeric" indicates that an oligomer is formed by three HA protein les. Without wishing to be bound by theory, HA protein is synthesized as ric precursor protein (HAO) of about 75 kDa, which assembles at the surface into an elongated trimeric protein. Before trimerization occurs, the precursor protein is cleaved at a conserved activation cleavage site (also referred to as fusion peptide) into 2 polypeptide chains, HA1 and HA2 (comprising the transmembrane region), linked by a disulfide bond. The HA1 t may be 328 10 amino acids in length, and the HA2 segment may be 221 amino acids in length.
Although this cleavage may be important for virus infectivity, it may not be ial for the ization of the protein or for immunogenicity. ion of HA within the asmic reticulum (ER) membrane of the host cell, signal peptide cleavage and protein glycosylation are co-translational events. Correct refolding of HA requires 15 glycosylation of the protein and formation of 5-6 chain disulfide bonds. The HA trimer les within the cis- and trans-Golgi complex, the transmembrane domain playing a role in the trimerization process. The crystal structures of bromelain—treated HA proteins, which lack the transmembrane domain, have shown a highly ved structure amongst influenza strains. It has also been established that HA undergoes 20 major conformational changes during the infection process, which requires the precursor HAO to be cleaved into the 2 polypeptide chains HA1 and HA2. The HA protein may be processed (i.e., se HA1 and HA2 s), or may be unprocessed (i.e. comprise the HAO domain). The unprocessed precursor protein of HA is synthesized as a precursor protein (HAO) of about 75 kDa, which assembles at 25 the surface into an elongated trimeric protein. The precursor protein is cleaved at a conserved cleavage site (also known as a proteolytic loop) into 2 polypeptide chains, HA1 and HA2 (comprising the transmembrane region), linked by a disulfide bond. id="p-109" id="p-109" id="p-109" id="p-109" id="p-109"
[00109] The HA protein as described herein may further be a modified HA (also referred to as "mutant HA") protein, for example a modified precursor protein 30 (HAO), in which the proteolytic loop or cleavage site is ed.
Modified HA/cleavage Site WO 2014/153674 PCT/CA2014/050326 -38, id="p-110" id="p-110" id="p-110" id="p-110" id="p-110"
[00110] ing cleavage of HAO, HA becomes sensitive to pH, undergoing irreversible conformational change at the pH of endosome ( id="p-111" id="p-111" id="p-111" id="p-111" id="p-111"
[00111] In order to optimize the production of vaccine in eggs and maintain an active but attenuated virus, modification of the polybasic cleavage site of H5 (RERRRKKRiG) has been d (Horimoto T, et. al, 2006, Vaccine, Vol 24 : 3669— 15 3676). Mutants of st contained a deletion of the 4 first charged amino acids (RERR) and a replacement of amino acids RKKR with TETR that inactivate the polybasic cleavage site but maintained the ility to process HAO to HA1-HA2 through the Arginin residue of the TETR motif (see Figure 19). A similar strategy to produce attenuated virus is employed by NIBSC to abolish the polybasic site ng 20 producing at high yields the A/Turkey/Turkey/1/2005 H5N1 strain without killing the eggs. The polybasic site sequence (GERRRKKRiG) is replaced by RETR in their mutant (NIBSC 05/240 NIBSC influenza reference virus NIBG—23). The polybasic cleavage site of a H5 HA has also been replaced by the monobasic site of H6 for expression in eggs. In this example, the first 4 residues and the four last amino acids 25 of the polybasic site are replaced by IETR (replacement of RERRRKKRiG with IETRiG; Hoffman E, et. al., 2002, Vaccine, Vol 20 :3165—3170). In each of the examples provided above, the modification was performed to ate the virus while ining production of the HA within eggs. That is, the cleavage of HAO precursor was not totally inactivated in order to allow the HAO to be processed to 30 HA1-HA2 and undergo pH conformational change, thereby permitting virus replication in the host cell.
WO 2014/153674 PCT/CA2014/050326 -39, id="p-112" id="p-112" id="p-112" id="p-112" id="p-112"
[00112] As used herein, the term "modified hemagglutinin" or "modified HA", "mutated hemagglutinin" or "mutated HA" refers to an HA in which the HA has a modification or mutation, for example a substitution, insertion, deletion, or a combination thereof, that s in an altered amino acid sequence in the proteolytic loop or cleavage site of the HA n. id="p-113" id="p-113" id="p-113" id="p-113" id="p-113"
[00113] The crystal structure of HAO from A/Hong Kong/68 has been determined (Chen, J., 1998. Cell 95:409—417; incorporated herein by reference).
Residues that are d to solvent are generally thought of being part of the cleavage site which forms an extended, highly exposed surface loop. A consensus 10 sequence may be determined in this chosen region for e, but not limited to: A/H3/HAO Consensus: NVPEKQTIUGIFGAIAGFIE (SEQ ID NO: 66) AO Consensus: SIUGLFGAIAGFIE (SEQ ID NO: 67) Avian H5 Consensus QRESRRKKIUGLFGAIAGFIEG (SEQ ID NO: 1) B/HAO Consensus: PAKLLKEIUGFFGALAGFLE (SEQ ID NO: 68) 15 Where the cleavage between HA1 and HA2 is indicated by "/" (see Bianchi et al., 2005, Journal of Virology, 79:73 80—73 88; incorporated herein by reference), and also Figures 15 and 18A. id="p-114" id="p-114" id="p-114" id="p-114" id="p-114"
[00114] The HA protein may be an influenza type B hemagglutinin or Influenza type A hemagglutinin protein with a modification in the proteolytic loop 20 region, for example a deletion, insertion, tution or a combination thereof of the proteolytic loop (cleavage site). Without wishing to be bound by theory, modification of the proteolytic loop may ensures that the HA molecule is maintained as an HAO precursor. y producing a more homogenous and consistent VLP comprising HAO ns. 25 [00115] By "proteolytic loop" or "cleavage site" is meant the consensus sequence of the proteolytic site that is involved in precursor HAO cleavage.
"Consensus" or "consensus sequence" as used herein means a sequence (either amino acid or nucleotide sequence) that ses the sequence variability of related WO 2014/153674 PCT/CA2014/050326 -40, sequences based on analysis of alignment of multiple ces, for example, subtypes of a particular influenza HAO sequence. Consensus sequence of the influenza HAO cleavage site may include influenza A consensus hemagglutinin amino acid sequences, including for example consensus Hl, consensus H3, consensus H5, or influenza B consensus hemagglutinin amino acid ces, for example but not limited to B Florida and B Malaysia. Non limiting examples of sequences of the proteoloytic loop region are shown in Figure 15 and 18B (and see i et al., 2005, Journal of Virology, 79:73 80—73 88; incorporated herein by reference).
] Residues in the proteolytic loop or ge site might be either 10 d, for example but not limited to point mutation, substitution, insertion, or deletion. The term "amino acid mutation" or "amino acid modification" as used herein is meant to ass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, ion, and modification can be made to arrive at the final construct, provided that the final construct possesses 15 the desired characteristics, e. g., reduced or abolished cleavage of the proteolytic loop or cleavage site by a protease. id="p-117" id="p-117" id="p-117" id="p-117" id="p-117"
[00117] By "modified proteolytic loop" it is meant that the proteolytic loop may e one or more point mutations, be partially deleted, fully deleted, lly replaced with a linker sequence, fully replaced by a linker sequence, comprise a 20 partial or complete ement of amino acids within the cleavage site with one or more otein amino acids, or a combination thereof. Similarly, by "modified cleavage site", it is meant that the cleavage site within the proteolytic loop may include one or more point mutations, be partially deleted, fully deleted, partially replaced with a linker sequence, fully ed by a linker sequence, comprising a 25 partial or complete replacement of amino acids within the cleavage site with one or more non-protein amino acids, or a ation thereof. Modifications to the proteolytic loop or both, may also involve the deletion, replacement, , cleavage site, or substitution of one or more amino acids that are located outside of, or adjacent to, the proteolytic loop or cleavage site sequence. By "linker" it is meant an amino acid 30 sequence comprising one or more amino acids that may be introduced within a proteolytic loop or a cleavage site, or that may replace some or all of the amino acids with the proteolytic loop or cleavage site. A linker may be designed to ensure that WO 53674 PCT/CA2014/050326 -41, any amino acids deletions within the proteolytic loop or cleavage site do not disrupt the expression or subsequent activity of the modified HA. id="p-118" id="p-118" id="p-118" id="p-118" id="p-118"
[00118] By stabilizing the HA protein by modifying or deleting the proteolytic loop increased product or protein yields may be achieved, when expressing the ed HA in a plant, when ed to a native HA sed in a plant under the same ions. Furthermore, by modifying or deleting the proteolytic loop the variability of expression of the expressed modified HA is reduced and the consistency of the produced modified HA is increased, when compared to a native HA expressed in a plant under the same conditions. 10 [00119] ore, the present invention also includes a method of increasing the product yield of a HA protein in a plant. Without wishing to be bound by theory, it is believed that by modifying or deleting the proteolytic loop in an HA protein, improved stability against proteolytic degradation in the plant, stabilization during passage of the HA in the golgi apparatus secretion process, and during the purification 15 process is provided.
] Furthermore, the present invention also includes a method of increasing the product quality of an HA protein expressed in a plant. By product quality, it is meant for example an increased product yield of an HA expressed in a plant, stability of the t for example increased stability of the HA sed in a 20 plant, consistency of the product for example the production of a homogenous product for example HAO or a combination f. id="p-121" id="p-121" id="p-121" id="p-121" id="p-121"
[00121] By an increase in product or protein yield, it is meant an increase in relative protein yield by about 20% to about 100%, or any amount therebetween as determined using rd techniques in the art, for e, from about 40% to 25 about 70% or any value therebetween for example about 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 55, 56, 58, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or any amount therebetween, when compared to the product or protein yield of the same HA protein that does not have its proteolytic loop removed. id="p-122" id="p-122" id="p-122" id="p-122" id="p-122"
[00122] As shown in Figures 13A and 14, HA from B/Brisbane/60/2008 is 30 poorly expressed in agroinfiltrated Nicotiana benthamiana leaves (see lane 1008).
WO 2014/153674 2014/050326 -42, However, expression of HA type B that has been modified to delete the proteolytic loop (see lane 1059, Figure 13A, Figure 14) resulted in increased expression.
Furthermore, co-expression of HA-type B with M2 from A/New Caledonia/20/99, results in an increase in HA expression (see lanes "1008+1261"; and 1059+1261"). ression of HA type B comprising a deletion in the proteolytic loop, with a M2 from A/Puerto Rico/8B4 also resulted in increased sion (1059+859; Figure 14). id="p-123" id="p-123" id="p-123" id="p-123" id="p-123"
[00123] As further shown in Figure 46B, protein yield of HA n from H7 A/Hangzhou/l/l3 expressed in agroinfiltrated ana benthamiana is increased in HA that has been ed to delete or modify the proteolytic loop. Co—expression of 10 native (wildtype) HA H7 A/Hangzhou/l/ 13 with M2 from A/New Caledonia/20/99 lead to a 100% relative n yield, whereas co—expression of HA H7 A/Hangzhou/l/l3 comprising a deletion in the proteolytic loop, with M2 from A/New nia/20/99, lead to a 182% relative protein yield (Figure 46B). The increase of relative protein yield of HA comprising a deletion in the proteolytic loop is, however, 15 not dependent on M2. As for example shown in Figure 29A, H7 zhou/l/l3 comprising a deletion in the proteolytic loop showed increased expression (as measure by the relative HA titer) when compared to native H7 A/Hangzhou/l/l3. id="p-124" id="p-124" id="p-124" id="p-124" id="p-124"
[00124] Several strategies were evaluated in order to inactivate the cleavage of HAO for both A and B strains. The consensus sequence that is recognized by 20 proteases is enclosed on a extended loop, exposed to the solvent, and closed to the membrane distal part to the protein. In the B strain, this loop ns 2 sequence motifs recognized by proteases and the first inal amino acids of the HA2 domain. A point mutation approach (for examples see Table 2, below) to inactivate the cleavage of HAO precursor resulted in HAO production, without an increase 25 accumulation of B strain VLP. Deletion of the sequence motifs comprising the 2 protease cleavage motifs (7 amino acids) abolished the lation of the B HA.
Remove the entire 18-amino acid loop from the HA n of the B strain and inserting a linker to conserve structural features (beta strands) of the protein structure was effective (see below; Figures 13A, 14, 16A, 17B). Removal or replacement of 30 the proteolytic loop in HA protein ofA strains was also effective (see Figures 20, 22).
WO 2014/153674 PCT/CA2014/050326 -43, id="p-125" id="p-125" id="p-125" id="p-125" id="p-125"
[00125] Amino acid sequence deletions and insertions include amino acid deletions and insertions of amino acids. A non-limiting example of a deletion in influenza B is the deletion of 17 amino acids (AKLLKERGFFGAIAGFLE) from position 340 to 357 of mature HA protein for example as shown in Figure 18C for influenza B Florida and B Malaysia. This on may be replaced by an riate linker to link the polypeptide chains for proper sion, for example but not limited to, using the sequence "GG", as shown in Figure 21B (SEQ ID NO:17; modified bane/60/2008; ing AKLLKERGFFGALAGFLEG with GG; e. g.
Construct 1059, Figures 5D, 10; Construct 1039, Figure 8B, or Construct 1467; 10 Figure 7D, 7E). An alternate replacement make comprise replacing "PPAKLLKER" with "GSSSGSSSG", as shown in Figure 21C (SEQ ID NO: 18). Furthermore, the sequence "RESRRKKR" may be replaced with "TETR" or "TETQ", as shown in Figure 19 for influenza H5/Indonesia. id="p-126" id="p-126" id="p-126" id="p-126" id="p-126"
[00126] Alternate amino acid mutations for HA from the A strain include 15 amino acid substitutions, insertions and deletions, for example but not limited to a deletion in the proteolytic loop region of H5 Anhui of the amino acid sequence "RERRRKRGLFGALAGFIE", a deletion of the amino acid ce of the proteolytic loop region of H5 Indo comprising "RESRRKKRGLFGAIAGFIE" or a deletion of the amino acid sequence of the proteolytic loop region of H5 Vietnam 20 "RERRRKKRGLFGAIAGFIE". For H3, the sequence "RNVPEKQTRGIF" may be deleted and replaced by an appropriate linker sequence, for example but not limited to "GS" as shown in Figure 21E (SEQ ID NO:20). Alternatively, the sequence "RNVPEKQTR" in H3 may be replaced by "GSSGSSGSS" as shown in Figure 21F (SEQ ID NO: 21; modified H3 h/16/2009). 25 [00127] Furthermore, modifying or altering the proteolytic loop or cleavage site of a HA to reduce or h cleavage of the proteolytic loop or cleavage site by a protease, may also se non—conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and/or chemical ties. Non—protein amino acids may also be used for substitution. For example, 30 amino acid substitutions may include ing a hydrophobic by a hydrophilic amino acid. Amino acid substitutions may include replacement by non-naturally occurring WO 53674 PCT/CA2014/050326 -44, amino acids or by naturally occurring amino acid derivatives of the protein amino acids. id="p-128" id="p-128" id="p-128" id="p-128" id="p-128"
[00128] Amino acid mutations for HA from the B strain and/or A strains may include amino acid deletions. For example in order to reduce or abolish cleavage of the proteolytic loop or cleavage site by a protease, one or more amino acid are deletion or removal within the proteolytic loop or cleavage site sequence. Non— limiting examples of deletions include removal of amino acids 323 to 341 of native HA H5 protein, for example H5 Anhui KRGLFGAIAGFIE), H5 Indo (RESRRKKRGLFGAIAGFIE), or H5 Vietnam (RERRRKKRGLFGAIAGFIE), as 10 shown in Figure 18C. For H3, the sequence "RNVPEKQTRGIF" may be replaced by "GS" (Figure 21E; SEQ ID , or the H3 sequence "RNVPEKQTR" may be ed by "GSSGSSGSS" (Figure 21F; SEQ ID NO: 21). For B strains, the sequence "AKLLKERGFFGAIAGFLE" may be deleted and/or replaced by the ce "GG", as shown in Figure 21B (SEQ ID NO:17), the sequence 15 "AKLLKERGFFGAIAGFLEG" may be replaced with "GG"), or the sequence "PPAKLLKER" replaced with "GSSSGSSSG"(Figure 21C; SEQ ID NO: 18). id="p-129" id="p-129" id="p-129" id="p-129" id="p-129"
[00129] Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may e site—directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of 20 altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be . id="p-130" id="p-130" id="p-130" id="p-130" id="p-130"
[00130] Therefore the hemagglutinin (HA) sequences of the ion may comprise ed proteolytic loop sequences or cleavage sites, thereby having reduced or abolished cleavage of the proteolytic loop or cleavage site by a protease. 25 The hemagglutinin polypeptide sequences may comprise modified proteolytic loop or modified cleavage site sequences as, for example, set forth in Figures 5D, 7D, 8A, 18C, 19, 21B, 21C, 21E, 21F, 24D, 25D, and 26D. The cleavage sites of any hemagglutinin polypeptide sequence of any influenza strain can be determined or predicted using any number of methods known in the art, including ce 30 ent (see for example Figure 15).
WO 2014/153674 PCT/CA2014/050326 -45, id="p-131" id="p-131" id="p-131" id="p-131" id="p-131"
[00131] Analysis of sequence from H1, H3 and B HAs s that H1 s one monobasic proteolytic site (Clara type monobasic: Q/EXR) that directly precedes the fusion peptide, whereas H3 and B HAs have 2 lytic sites, one that is recognized by like proteases (as found in H1), and another site recognized by trypsine and chymotrypsine—like proteases (P—E/A—K). The consensus sequence for cleavage of these HA is presented in Table 1.
Table 1 : Consensus sequence of the proteolytic site for precursor HA0 cleavage. The sequences recognized by Clara tryptases or trypsine/chimotrypsine are italicized, and bolded respectively. Several HA strains comprise polybasic Furin type cleavage sites 10 (RKKR; plain text, underlined).
H1 N PS QSRiGLF SEQ ID NO: 47 NVPEKQTRiGIIF SEQ ID NO: 48 id="p-132" id="p-132" id="p-132" id="p-132" id="p-132"
[00132] In order to avoid a potential proteolytic cleavage of HAO precursor of the HA, only one proteolytic site may need to be modified from the sequence of H1, 15 whereas, in the case of H3 and B, two monobasic sites may need to be ed. id="p-133" id="p-133" id="p-133" id="p-133" id="p-133"
[00133] For example, a first cleavage site of HA0 of B/Florida and bane may for example be eliminated by replacing the Lys 341 (mature protein numbering) with an Ile (see Table 2). The second monobasic site may be hed by replacing three amino acids prior to the fusion peptide, KER 46), with NIQ. Sequences of 20 several modified proteolytic loops of HA are provided in Table 2.
Table 2: ration of examples of mutations to destroy the cleavage of the precursor HA0. The monobasic site are italicized (Clara-like recognition) and in bold (no underlining; trypsine/chymotrypsine-like). The mutation are shown as bolded and underlined. The arrow represents the site for cleavage for conversion of HA0 into 25 HA1 —HA2.
WO 2014/153674 2014/050326 -46, Strain Natural sequence Abolition of precursor cleavage site H5/Indo TGLRNSPQRESRRKKRiGLF TGLRNSPQEGLF sao D NO:6O SfiQ D NO:6l TGLRNSPQLTQGLF «2Q D NO:62 Hl/Brisbane QSRiGLF H3/Brisbane NVPEKQTRiGIZF B/Florida, PAKLLKERiGFF PAELLNIQGFF B/Brisbane SrlQ D NO:59 SrlQ D NO:65 id="p-134" id="p-134" id="p-134" id="p-134" id="p-134"
[00134] In further examples, the sequences comprising the proteolytic loop in HAO may be ed or deleted. For example, an H3 variant ning a deletion of the sequence RNVPEKQT at the C-terminus of HA1 in addition of deletion of the N- us amino acids GIFGLA of HA2 is provided in Figure 21 E. The shortened HA1-HA2 may be linked together by a GS linker.
] In another example, the loop contain the proteolytic cleavage site in, for example H3, may have been ed by a flexible linker, and the HA2 part may be left intact. A (GSS)3 linker may be designed in order to accommodate the 10 shortened HA1 to HA2. (see Figure 21F). id="p-136" id="p-136" id="p-136" id="p-136" id="p-136"
[00136] In another example, HA from influenza B may n a deletion of sequence ALKLLKER at the C-terminus of HA1 in addition of deletion of the N- terminus amino acids GFFGAIAGFLEG of HA2. The shortened HA1-HA2 may be linked together by a GG linker (see for example Figure 21B; Construct 1008). The 15 expression of this construct is shown in Figures 13A and B. id="p-137" id="p-137" id="p-137" id="p-137" id="p-137"
[00137] In another example, HA from za B the loop containing the proteolytic site may have been replaced by a flexible linker, and the HA2 part was left WO 2014/153674 PCT/CA2014/050326 -47, intact. A longer GSSS linker may be designed in order to accommodate the shortened HA1 to HA2. (see for example Figure 21 C). id="p-138" id="p-138" id="p-138" id="p-138" id="p-138"
[00138] As shown in Figures 13A and 14, HA from B/Brisbane/60/2008 is poorly expressed in agroinfiltrated Nicotiana miana leaves (see lane 1008).
However, expression of HA type B that has been modified to delete the proteolytic loop (see lane 1059, Figure 13A, Figure 14) resulted in increased expression.
Furthermore, co—expression of HA—type B with M2 from A/New Caledonia/20/99, results in an increase in HA expression (see lanes "1008+1261"; and 1059+1261").
Co—expression of HA type B comprising a on in the proteolytic loop, with a M2 10 from A/Puerto Rico/8/34 also resulted in increased expression (1059+859; Figure 14). id="p-139" id="p-139" id="p-139" id="p-139" id="p-139"
[00139] In a similar manner, deletion of the proteolytic loop in H5/Indo, and replacement with either a "GG" (Construct 928; see Figure 46D), "TETR" (Construct 676; also see Figures 19, 24D) or "TETQ" (Construct 766; also see Figures 19, 25D) sequence resulted in expression levels that matched or increased over the level of 15 expression observed with native H5/Indo ruct 489; see Figures 20 and 23). id="p-140" id="p-140" id="p-140" id="p-140" id="p-140"
[00140] As show in Figure 13B, by deleting the proteolytic loop of HAO (sequence shown in Figure 21B), the resultant HAO protein exhibits an increased activity as shown by a greater hemagglutination capacity, when compared to a HA protein that does not have its proteolytic loop removed. 20 [00141] By an increase in ty, it is meant an se in hemagglutination capacity by about 2% to about 100%, or any amount therebetween as determined using rd techniques in the art, for e, from about 10% to about 50% or any value etween for example about 2, 5, 8, 10, 12, 15, 18, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 55, 56, 58, 60, 65, 70, 75, 25 80, 85, 90, 95, or 100%, or any amount etween, when compared to the activity of the same HA protein that does not have its proteolytic loop removed. id="p-142" id="p-142" id="p-142" id="p-142" id="p-142"
[00142] The present invention also includes nucleotide sequences encoding modified HA from for example modified Hl,H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA, or any nucleotide sequences that 30 hybridize to Hl,H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, WO 2014/153674 PCT/CA2014/050326 -48, H16 or type B HA under stringent conditions, or a nucleotide sequence that hybridizes under stringent hybridisation conditions to a compliment of H1,H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA, wherein the nucleotide sequence encodes a hemagglutinin protein that when expressed forms a VLP, and that the VLP s the production of an antibody. For example, expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP may be used to produce an antibody that is capable of g HA, including mature HA from B or H3. The VLP, when administered to a t, induces an immune response. Preferably, the VLP s the production of an dy and the 10 VLP, when administered to a subject, induces an immune response. id="p-143" id="p-143" id="p-143" id="p-143" id="p-143"
[00143] For example, expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP may be used to e an antibody that is capable of binding a virus protein such for example HA, including but not d to HAO, HAO protein with its proteolytic loop deleted or modified, HA1 or HA2 of one or more 15 influenza types or subtypes, such for example but not d to subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, type B HA. The VLP, when administered to a subject, induces an immune se. id="p-144" id="p-144" id="p-144" id="p-144" id="p-144"
[00144] Hybridization under stringent hybridization conditions is known in the art (see for example Current ols in lar Biology, Ausubel et al., eds. 1995 20 and supplements; Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982; Sambrook and Russell, in Molecular Cloning: A Laboratory Manual, 3rd edition 2001; each of which is incorporated herein by reference). An example of one such stringent hybridization conditions may be about 16-20 hours hybridization in 4 X SSC at 65°C, followed by washing in 0.1 X SSC at 25 65°C for an hour, or 2 washes in 0.1 X SSC at 65°C each for 20 or 30 minutes.
Alternatively, an exemplary stringent ization condition could be overnight (16— 20 hours) in 50% formamide, 4 X SSC at 42°C, followed by washing in 0.1 X SSC at 65°C for an hour, or 2 washes in 0.1 X SSC at 65°C each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church aqueous phosphate buffer (7% 30 SDS; 0.5M NaPO4 buffer pH 7.2; 10 mM EDTA) at 65°C, with 2 washes either at 50°C in 0.1 X SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65°C in 2 X SSC, 0.1% SDS for 20 or 30 minutes each.
WO 2014/153674 PCT/CA2014/050326 -49, id="p-145" id="p-145" id="p-145" id="p-145" id="p-145"
[00145] Additionally, the present invention includes nucleotide sequences that are characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100% or any amount therebetween, sequence identity, or sequence similarity, with the nucleotide sequence encoding H1,H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA, wherein the nucleotide sequence encodes a hemagglutinin protein (modified HA) with a modified proteolytic loop ces or cleavage sites which has reduced or abolished cleavage of the proteolytic loop or cleavage site by a se. When nucleotide sequence ng the modified HA is sed it forms a VLP, and the VLP induces the production of an antibody. For 10 example, expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP may be used to produce an antibody that is capable of binding HA, including unprocessed HA (HAO) or unprocessed wherein the proteolytic loop has been deleted.
The VLP, when administered to a subject, s an immune response. id="p-146" id="p-146" id="p-146" id="p-146" id="p-146"
[00146] Additionally, the present invention includes nucleotide ces that 15 are characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100% or any amount therebetween, ce identity, or sequence similarity, with the nucleotide sequence of SEQ ID NO: 43, 91, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 137, 140, 144, 151, 158, 165, wherein the nucleotide sequence encodes a ed HA protein that when expressed forms a VLP, and that the VLP induces the 20 production of an antibody that is capable of binding HA, including unprocessed HA (HAO) or unprocessed wherein the proteolytic loop has been d or modified. The VLP, when administered to a subject, induces an immune response. id="p-147" id="p-147" id="p-147" id="p-147" id="p-147"
[00147] Furthermore, the t invention includes amino acid sequences that are terized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93 94, 95, 96, 97, 98, 25 99, 100% or any amount therebetween, sequence identity, or sequence similarity, with the amino acid sequences of SEQ ID NO: 17, 18, 20, 21, 41, 58, 77, 81, 85, 92, 96, 98,100,102,104,106,108,110,112,114,134,143,147,154,161,168,194 and 199. wherein the amino acid sequence encodes a ed HA protein that when expressed forms a VLP, and that the VLP induces the production of an dy that is capable 30 of binding HA, including unprocessed HA (HAO) or unprocessed wherein the proteolytic loop has been deleted or modified. The VLP, when administered to a subject, induces an immune response.
WO 2014/153674 PCT/CA2014/050326 -50, id="p-148" id="p-148" id="p-148" id="p-148" id="p-148"
[00148] Sequence identity or sequence similarity may be determined using a nucleotide sequence comparison program, such as that provided within DNASIS (for example, using, but not limited to, the following parameters: GAP penalty 5, #of top als 5, fixed GAP y 10, k-tuple 2, floating gap 10, and window size 5).
However, other methods of alignment of sequences for comparison are well-known in the art for example the algorithms of Smith & Waterman (1981, Adv. Appl. Math. , man & Wunsch (J. Mol. Biol. 48:443, 1970), Pearson & Lipman (1988, Proc. Natl. Acad. Sci. USA 4), and by computerized implementations of these algorithms (e.g. GAP, BESTFIT, FASTA, and BLAST)., or by manual alignment and 10 visual inspection. An e of sequence alignment of HAs from different strains of influenza can be found in Figure 24.
For example, but is not limited to, nucleotide sequences encoding: - a type B HA with a modified proteolytic loop as defined by SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID 15 NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114 and SEQ ID NO: 168, or nucleotide sequences encoding type B HAs comprising modified lytic loop regions as defined in SEQ ID NO: 65, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO:95, SEQ ID NO: 20 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113 and SE ID NO: 165. - an H1 with a modified proteolytic loop include sequences comprising a ed cleavage site as defined by SEQ ID NO: 63. 25 - an H2 with a modified proteolytic loop include sequences comprising a modified cleavage site as defined by SEQ ID NO: 134. - an H3 with a modified proteolytic loop include sequences defined by SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO 143, SEQ ID NO: 147 or comprising a modified ge site as defined by SEQ ID NO: 64.
WO 53674 PCT/CA2014/050326 -5], an H5 with a deleted proteolytic loop include sequences comprising a modified cleavage site as d by SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71. - an H7 with a modified proteolytic loop include sequences comprising a modified cleavage site as defined by SEQ ID NO: 154 or nucleotide sequences ng type H7 HAs comprising modified proteolytic loop s as d in SEQ ID NO: 151. - an H9 with a modified proteolytic loop include sequences comprising a modified cleavage site as defined by SEQ ID NO: 161 or nucleotide sequences 10 encoding type H9 HAs comprising modified proteolytic loop regions as defined in SEQ ID NO: 158.
] The present invention pertains to the use of an HA protein comprising the transmembrane domain and includes HA1 and HA2 domains, for example the HA protein may be HAO, or processed HA comprising HA1 and HA2. The HA n 15 may be used in the production or formation of VLPs using a plant, or plant cell, sion system.
Amplification Elements and Enhancer Elements/Regulatory Elements id="p-150" id="p-150" id="p-150" id="p-150" id="p-150"
[00150] In another example the modified HA protein may be expressed in an expression system that comprises amplification elements and/or regulatory elements 20 or regions (also referred to herein as enhancer elements). For example an amplification element from a geminivirus such as for example, an ication element from the bean yellow dwarf virus (BeYDV) may be used to express the modified HA. BeYDV belongs to the viruses genus adapted to dicotyledonous plants. BeYDV is monopartite having a single-strand circular DNA genome and can 25 ate to very high copy numbers by a rolling circle mechanism. BeYDV—derived DNA replicon vector systems have been used for rapid high—yield protein tion in plants. id="p-151" id="p-151" id="p-151" id="p-151" id="p-151"
[00151] As used herein, the phrase "amplification elements" refers to a nucleic acid segment comprising at least a portion of one or more long intergenic regions 30 (LIR) of a geminivirus genome. As used herein, "long intergenic region" refers to a WO 2014/153674 PCT/CA2014/050326 -52, region of a long intergenic region that contains a rep binding site capable of mediating excision and replication by a geminivirus Rep protein. In some aspects, the c acid segment comprising one or more LIRs, may further comprises a short intergenic region (SIR) of a geminivirus genome. As used herein, "short intergenic region" refers to the complementary strand (the short IR (SIR) of a Mastreviruses). Any suitable geminivirus—derived amplification element may be used herein. See, for e, W02000/20557; W02010/025285; Zhang X. et al. (2005, Biotechnology and Bioengineering, Vol. 93, 271—279), Huang Z. et al. (2009, Biotechnology and Bioengineering, Vol. 103, 706—714), Huang Z. et al.(2009, Biotechnology and 10 Bioengineering, Vol. 106, 9—17); which are herein incorporated by reference). If more than one LIR is used in the construct, for example two LIRs, then the er, CMPV—HT regions and the nucleic acid sequence of st and the terminator are bracketed by each of the two LIRs. id="p-152" id="p-152" id="p-152" id="p-152" id="p-152"
[00152] As described herein, co—delivery of bean yellow dwarf virus (BeYDV)— 15 derived vector and a Rep/RepA—supplying vector, by agroinflltration of Nicotiana benthamiana leaves s in efficient replicon amplification and robust protein production. n blot analysis of protein extracts from plants transformed with gene constructs driving the sion of modified influenza B HA (from B/Brisbane/60/2008) with or without the proteolytic loop removed (see Figure 17A 20 for constructs) and in the presence or absence of the amplification element BeYDV (construct no. 1059 and 1039) showed that in the e of BeYDV no accumulation of influenza B HA could be detected (Figure 17B), when the regulatory element was T. id="p-153" id="p-153" id="p-153" id="p-153" id="p-153"
[00153] As shown in Figures 17B, expression of HA from B/Brisbane/60/2008 25 with the proteolytic loop removed in the absence of BeYDV does not lead to detectable expression by Western Blot analysis (see lane 1039 in Figure 17B).
However, expression of HA type B with the proteolytic loop removed in the presence of amplification element BeYDV, results in sed expression (see lane 1059 ). Similarly, in the absence of BeYDV, co-expression of mutant HA-type B 30 comprising a deletion in the lytic loop, with M2 from A/New Caledonia/20/99, does not result in detectable HA expression (see lanes "1039+1261" in Figure 17B).
Co-expression of mutant HA type B comprising a deletion in the proteolytic loop in WO 2014/153674 PCT/CA2014/050326 -53, the presence of BeYDV, with a M2 from A/New Caledonia/20/99 on the other hand resulted in sed expression (see lane "1 059+1261"; Figure 17B).
] However, the presence of BeyDV is not required when an enhancer element is present in the expression system and when the enhance element is not CPMV-HT. As for example shown in Figures 29A, sion of various B HA strains under the control of an enhancer element, such for example CPMV 160, CPMV160+ or CPMV HT+, leads to the production ofHA proteins that show increased hemagglutination titre (HMG) in the absence of BeYDV. id="p-155" id="p-155" id="p-155" id="p-155" id="p-155"
[00155] Therefore, the mutant (modified) HA protein may be expressed in the 10 absence of an amplification t, such as a geminivirus-based amplification t for example BeYDV, but in the presence of an er element, such for example CPMV 160, CPMV160+ or CPMV HT+. id="p-156" id="p-156" id="p-156" id="p-156" id="p-156"
[00156] The mutant (modified HA) may be expressed in the presence of an enhancer element, such for example CPMV 160, CPMV160+ or CPMV HT+, but in 15 the e or presence of an amplification element, such for example BeYDV. As shown in Figures 28B, 28C and 28F mutant (modified) HA may be sed in the presence of an enhancer element, with or without the presence of an amplification element. Therefore the present invention is also directed to the expression of a mutant (modified) HA in the presence of an enhancer element and optionally an amplification 20 element. id="p-157" id="p-157" id="p-157" id="p-157" id="p-157"
[00157] HA constructs comprising an er element (either CMPV HT+ or CMPV 160+) and a proteolytic loop replaced with a GG linker (deleted proteolytic loop) exhibit increased expression when compared to wild type or HA constructs comprising CPMV HT (Figure 28A, H3 Per; Figure 28B, B Malaysia; Figure 28C, H9 25 HK; Figure 29D, B Mass; Figure 28E, H2 Sin). id="p-158" id="p-158" id="p-158" id="p-158" id="p-158"
[00158] Figure 29A present summary data for hemagglutination titre of modified HA ns produced in plants comprising CPMV HT, CPMV HT+ , CPMV 160 or CPMV160+, based enhancer ts operatively linked with a nucleotide sequence encoding either modified HA with a d proteolytic loop (GG 30 linker) or a native HA. In most cases, the expression (determined as WO 2014/153674 PCT/CA2014/050326 -54, lutination titer) were higher for the CPMV HT+, CPMV 160 or CPMV160+ based uct demonstrates significant expression . id="p-159" id="p-159" id="p-159" id="p-159" id="p-159"
[00159] Enhancer elements may be used to achieve high level of transient expression of mutant (modified) HA proteins with modified proteolytic loops.
Enhancer elements may be based on RNA plant viruses, including comoviruses, such as Cowpea mosaic virus (CPMV; see, for example, W02007/135480; W02009/087391; US 2010/0287670, Sainsbury F. et al., 2008, Plant Physiology; 148: 121-1218; Sainsbury F. et al., 2008, Plant Biotechnology Journal; 6: 82—92; Sainsbury F. et al., 2009, Plant Biotechnology l; 7: 682—693; Sainsbury F. et al. 10 2009, Methods in Molecular Biology, Recombinant ns From Plants, vol. 483: 25-39).
CPMV160 (CPMVX) and CPMV 160+ (CPMVX+) ] In one embodiment the er Elements are "CPMVX" (also referred as "CPMV 160") and/ or "CPMVX+" (also referred to as "CPMV 160+") as 15 described in US 61/925,852, which is incorporated herein by reference. id="p-161" id="p-161" id="p-161" id="p-161" id="p-161"
[00161] Expression enhancer "CPMVX" comprises a comovirus cowpea mosaic virus (CPMV) 5’ untranslated region (UTR). The 5’UTR from nucleotides 1- 160 of the CPMV RNA —2 sequence (SEQ ID NO: 93), starts at the transcription start site to the first in frame initiation start codon (at position 161), which serve as the 20 initiation site for the production of the longer of two y coterminal proteins encoded by a wild—type comovirus genome segment. Furthermore a 'third' tion site at (or corresponding to) position 115 in the CPMV RNA—2 genomic sequence may also be mutated, deleted or otherwise altered. It has been shown that removal of AUG 115 in addition to the removal ofAUG 161 es expression when 25 combined with an incomplete M protein (Sainsbury and Lomonossoff, 2008, Plant Physiology; 148: 1212—1218; WO 2009/087391; which are incorporated herein by reference). id="p-162" id="p-162" id="p-162" id="p-162" id="p-162"
[00162] CPMVX comprises X nucleotides of SEQ ID NO:93, where X=160, 155, 150, or 114 of SEQ ID NO:93, or a sequence that comprises between 80% to 30 100% ce similarity with CPMVX, where X=160, 155, 150, or 114 of SEQ ID NO:93. This expression enhancer is generally referred to as CPMVX (see Figure 26A).
] The expression enhancer CPMVX, where X=160, consists of nucleotides 1-160 of SEQ ID NO: 93: 1 aatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 5 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc 121 tcttcaac gttgtcagat cgtgcttcgg caccagtaca (SEQ ID NO:93) id="p-164" id="p-164" id="p-164" id="p-164" id="p-164"
[00164] The CPMVX enhancer sequence may further be fused to a stuffer sequence, wherein the CMPVX comprises X nucleotides of SEQ ID NO:93, where X=160, 155, 150, or 114 of SEQ ID NO:93, or a sequence that comprises n 80 to 100 % sequence similarity with CPMVX, 10 where X=160, 155, 150, or 114 of SEQ ID NO:93, and the stuffer sequence comprises from 1-100 comprise from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides, or any number of nucleotides therebetween. 15 [00165] If the CMPVX sequence comprises a stuffer fragment, then this expression er may be referred to as CPMVX+ (see Figure 26A), where X=160, 155, 150, 114 of SEQ ID NO:93, it may also be referred to as CMPVX comprising a stuffer sequence, or it may be referred to as CPMV160+; CPMV155+; CPMV150+; CPMV114+, when X-160, 155, 150, or 114, respectively. ucts comprising CPMVX that do not comprise a stuffer sequence may be termed CPMVX+, 20 where X=160, 155, 150, 114 of SEQ ID NO:93, and where the stuffer sequence is of 0 nucleotides in length.
] The stuffer sequence may be modified by truncation, on, or replacement of the may be removed, replaced, truncated or shortened when compared to the initial or unmodified (i.e. 25 G.P., 2008, Plant Physiol. 148: pp. 1212-1218). The stuffer sequence may comprise a one or more restriction sites (polylinker, le cloning site, one or more cloning sites), one or WO 2014/153674 PCT/CA2014/050326 -56, more plant kozak sequences, one or more linker ces, one or more recombination sites, or a combination f. For example, which is not to be considered limiting, a stuffer sequence may comprise in series, a multiple cloning site of a d length fused to a plant kozak sequence. The stuffer sequence does not comprise a nucleotide sequence from the native 5’UTR sequence that is oned 3’ to nucleotide 160 of the native CPMV 5’UTR, for example nucleotides 161 to 512 as shown in Figure 1 of ury F., and Lomonossoff GR (2008, Plant Physiol. 148: pp. 1212—1218; which is incorporated herein by reference), or nucleotides 161—509 of SEQ ID N014. That is, the incomplete M protein present in the prior art CPMV HT 10 sequence (Figure 1; of Sainsbury F., and Lomonossoff GR, 2008) is removed from the 5’UTR in the present invention. id="p-167" id="p-167" id="p-167" id="p-167" id="p-167"
[00167] Plant Kozak sus sequences are known in the art (see for example Rangan et al. Mol. Biotechnol., 2008, July 39(3), pp. 207-213). Both naturally occurring and synthetic Kozak sequences may be used in the expression enhancer or 15 may be fused to the nucleotide sequence of interest as described herein. id="p-168" id="p-168" id="p-168" id="p-168" id="p-168"
[00168] The plant kozak sequence may be any known plant kozak sequences (see for example L. Rangan et. al. Mol. hnol.2008), including, but not limited to the following plant consensus sequences: caA(A/C)a (SEQ ID NO:174; plant kingdom) 20 aaA(A/C)a (SEQ ID NO:175; dicots) )(A/C)a (SEQ ID NO:176; arabidopsis) The plant kozak sequence may also be selected from the group of: AGAAA (SEQ ID NO: 177) AGACA (SEQ ID NO: 178) 25 AGGAA (SEQ ID NO: 179) AAAAA (SEQ ID NO: 180) AAACA (SEQ ID NO: 181) AAGCA (SEQ ID NO: 182) AAGAA (SEQ ID NO: 183) 30 AAAGAA (SEQ ID NO: 184) AAAGAA (SEQ ID NO: 185) (A/—)A(A/G)(A/G)(A/C)A.(SEQ ID NO: 186; Consensus sequence) 35 WO 2014/153674 PCT/CA2014/050326 -57, id="p-169" id="p-169" id="p-169" id="p-169" id="p-169"
[00169] The expression enhancer CPMVX, or , may be operatively linked at the 5’end of the enhancer sequence with a regulatory region that is active in a plant, and ively linked to a nucleotide sequence of interest at the 3’end of the sion enhancer (Figure 26A), in order to drive expression of the nucleotide sequence of st within a plant host.
CPMVHT+ ,CPMVHT+[WTII5], CPMVHT+ [511] id="p-170" id="p-170" id="p-170" id="p-170" id="p-170"
[00170] In r embodiment the Enhancer Elements is "CPMV HT+" as described in US 61/971,274, which is incorporated herein by reference. Expression enhancer "CPMV HT+" (see Figure 27A) comprises a comovirus 5’ untranslated 10 region (UTR) and a modified, lengthened, or truncated r sequence. id="p-171" id="p-171" id="p-171" id="p-171" id="p-171"
[00171] A plant expression system comprising a first nucleic acid sequence comprising a regulatory region, operatively linked with one or more than one expression enhancer as described herein (e.g. CPMV HT+, CPMV HT+[WT115], CPMV HT+ [511]), and a nucleotide sequence encoding a ed HA is also 15 provided. Furthermore, a nucleic acid comprising a er (regulatory region) sequence, an expression er (e.g. CPMV HT+ or CPMV HT+[WT115]) comprising a comovirus 5’UTR and a stuffer sequence with a plant kozak sequence fused to one or more nucleic acid sequences encoding a modified HA are described.
The nucleic acid may further comprise a sequence comprising a comovirus 3’ 20 untranslated region (UTR), for example, a plastocyanin 3’ UTR, or other 3’UTR active in a plant, and a terminator sequence, for example a NOS terminator, operatively linked to the 3’end of the nucleotide sequence encoding a modified HA (referred to as nucleotide of interest in Figure 27A), so that the nucleotide sequence encoding the modified HA is inserted upstream from the comovirus 3’ slated 25 region (UTR), plastocyanin 3’ UTR, or other 3’UTR sequence. id="p-172" id="p-172" id="p-172" id="p-172" id="p-172"
[00172] SEQ ID NO:173 comprises a "CPMV HT" expression enhancer as known in the prior art (e. g. Figure 1 of Sainsbury and Lomonossoff 2008, Plant Physiol. 148: pp. 1212-1218; which is incorporated herein by reference). CPMV HT includes the 5’UTR sequence from tides 1—160 of SEQ ID NO: 173 with 30 ed tides at position 115 (cgt), and an incomplete M protein with a VV()2014/153674 PCT/CA2014/050326 -58, modified nucleotide at position 162 (acg), and lacks a plant kozak sequence (5’UTR: nucleotides 1—160; lete M protein underlined, nucleotides 161 — 509). SEQ ID NO: 173 also includes a multiple cloning site (italics, nucleotides 510—528) which is not present in the prior art CPMV HT sequence: 1 tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc gagc 121 gatcttcaac gttgtcagat cgtgcttcgg caccagtaca acggtttctt tcactgaagc 181 gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241 ctattcttgt ngtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 10 301 atacattact gatt ctgctgactt ggtg caatatctct acttctgctt 361 gacgaggtat tgttgcctgt acttctttct tcttcttctt tggt tctataagaa 421 atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481 taagcttctg ctgc ccaaatttgt cgggccc SEQ ID NO: 173 ] CPMV HT+ with a plant kozak consensus ce is provided in 15 SEQ ID NO:187 (nucleotide 1—160, 5’UTR, including ed ATG at positions 115 (QTG) lower case bold and italics; stuffer fragment comprising: an lete M protein underlined, nucleotides 161 — 509, with ed nucleotide at 162 (AQG); a multiple cloning site, italics, nucleotides 510-528; and a consensus plant kozak sequence, caps and bold, nucleotides 529—534). 20 1 tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc 121 caac agat cgtgcttcgg caccagtaca acggtttctt tcactgaagc 181 gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241 ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct cccc 25 301 atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361 gacgaggtat tgttgcctgt acttctttct tcttcttctt tggt tctataagaa 421 atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481 taagcttctg tatattctgc ccaaatttgt tcgggcccaa taccgcgg(A/—)A(A/G) WO 2014/153674 2014/050326 -59, (A/G) (A/C)A (SEQ ID ) id="p-174" id="p-174" id="p-174" id="p-174" id="p-174"
[00174] SEQ ID NO:l88 ("CPMV HT+ 511") comprises a segment of the native sequence of the CPMV RNA 2 genome from nucleotides 1-154. The S’UTR sequence from nucleotides 1—511 of SEQ ID NO: 1 88 comprises modified "atg" sequences at positions 115 ("g" in place of "a"; italics bold) and 162 ("c" in place of "t"; italics bold), and an incomplete M protein (underlined) from tides 161 — 511. CPMV HT+ 511 comprises a native M protein kozak consensus sequence (nucleotides 508—51 1; bold): 1 tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 10 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc 121 caac gttgtcagat cgtgcttcgg taca acggtttctt tcactgaagc 181 gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241 ctattcttgt cggtgtggtc ttgggaaaag tgct ggaggctgct gttcagcccc 301 atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 15 361 gacgaggtat ctgt acttctttct tcttcttctt gctgattggt tctataagaa 421 atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga ttgt 481 taagcttctg tatattctgc ccaaatttga gm SEQ ID NO: 188 ] Another non-limiting example of a CPMV HT+ enhancer sequence is provided by the sequence of SEQ ID NO: 1 89 (CPMV HT+[WT115]). Expression 20 cassettes or vectors comprising CPMV HT+ and including a plant regulatory region in operative association with the sion enhancer sequence of SEQ ID NO: 189, and the transcriptional start site (ATG) at the 3’ end fused to a nucleotide sequence encoding modified HA are also part 0 the present invention. id="p-176" id="p-176" id="p-176" id="p-176" id="p-176"
[00176] SEQ ID NO: 189 (CPMV HT+[WT115]) nucleotide 1—160, 5’UTR, 25 with an ATG at position 115-117, lower case bold; stuffer fragment sing: an incomplete M protein underlined, nucleotides 161 - 509; with a modified ATG at position 161—153 lower case bold, and underlined, a multiple cloning site, italics, nucleotides 510—528; and a plant kozak sequence, caps and bold, nucleotides 529— 534).
WO 2014/153674 PCT/CA2014/050326 -60, tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 61 actc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcatgagc 121 caac gttgtcagat cgtgcttcgg caccagtaca acggtttctt tcactgaagc 181 gaaatcaaag atctctttgt gtag tgcggcgcca ttaaataacg tgtacttgtc 241 ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301 atacattact gatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361 gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421 tatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481 taagcttctg tatattctgc ccaaatttgt tcgggcccaa taccgcggAG AAAA 10 (SEQ ID NO:189) id="p-177" id="p-177" id="p-177" id="p-177" id="p-177"
[00177] The plant kozak sequence of SEQ ID N01189 may be any plant kozak sequence, including but not limited, to one of the sequences of SEQ ID NO’s: 174— 186. ric protein " 15 [00178] The modified HA might further be a chimeric protein. By ric Virus n" or "chimeric Virus polypeptide", also referred to as "chimeric protein" or "chimeric polypeptide", or "chimeric HA" it is meant a protein or polypeptide that comprises amino acid sequences from two or more than two sources, for example but not limited to, two or more influenza types or subtypes, or influenza's of a different 20 origin, that are fused as a single polypeptide. The chimeric protein or polypeptide may include a signal peptide that is the same as, or heterologous with, the remainder of the ptide or protein. The chimeric protein or chimeric polypeptide may be produced as a transcript from a chimeric tide sequence, and following synthesis, and as required, may associate to form a multimeric protein. Therefore, a 25 chimeric protein or a chimeric polpypeptide also includes a protein or polypeptide comprising subunits that are associated Via hide s (i.e. a multimeric protein). For example, a chimeric polypeptide comprising amino acid sequences from two or more than two sources may be processed into subunits, and the subunits associated Via disulphide bridges to e a chimeric protein or chimeric WO 53674 PCT/CA2014/050326 -61, polypeptide. A chimeric HA protein may also comprises an antigenic protein or a fragment thereof of a first influenza virus, and a transmembrane domain x (TDC) from an second virus influenza HA, including a transmembrane domain and lic tail domains ). The polypeptide may be a modified HA, and each of the two or more than two amino acid sequences that make up the polypeptide may be ed from different HA's to produce a chimeric HA, chimeric influenza HA, chimeric modified HA or chimeric modified za HA. A chimeric HA may also include an amino acid ce comprising heterologous signal peptide (a chimeric HA preprotein) that is cleaved after or during protein synthesis. Preferably, the 10 chimeric polypeptide, or chimeric influenza HA is not naturally occurring. A nucleic acid encoding a chimeric polypeptide may be described as a "chimeric nucleic acid", or a "chimeric nucleotide sequence". For e a chimeric nucleic acid may comprise a nucleotide ce encoding the modified HA comprises a chimeric nucleotide sequence encoding, in series, a modified HA ectodomain comprising a 15 modified proteolytic loop, an influenza transmembrane domain, and a asmic tail, wherein the modified HA ectodomain is from a first influenza strain and the transmembrane domain and the cytoplasmic tail are from a second influenza strain. es of chimeric nucleotide acids, wherein the ed HA ectodomain is from a first influenza strain and the transmembrane domain and the cytoplasmic tail are 20 from a second influenza strain are given in Examples 5.14, 5.16, 5.18, 5.19, 5. 21 and 5.23. A virus—like particle comprised of chimeric HA may be described as a ric VLP". id="p-179" id="p-179" id="p-179" id="p-179" id="p-179"
[00179] As described above, the chimeric protein, chimeric polypeptide, or chimeric HA may include a signal peptide that is the same as, or heterologous with, 25 the remainder of the polypeptide or protein. The term "signal peptide" is well known in the art and refers generally to a short (about 5—30 amino acids) sequence of amino acids, found generally at the N—terminus of a polypeptide that may direct translocation of the newly-translated polypeptide to a particular organelle, or aid in positioning of specific domains of the polypeptide chain relative to others. As a non-limiting 30 example, the signal peptide may target the translocation of the protein into the endoplasmic reticulum and/or aid in positioning of the N—terminus proximal domain relative to a membrane-anchor domain of the nascent ptide to aid in cleavage WO 2014/153674 PCT/CA2014/050326 -62, and folding of the mature protein, for example a modified HA or chimeric modified HA. id="p-180" id="p-180" id="p-180" id="p-180" id="p-180"
[00180] The HA may also be a chimeric HA or chimeric modified HA, wherein a native transmembrane domain of the HA or modified HA is replaced with a logous transmembrane . The transmembrane domain of HA proteins is highly conserved (see for e Figure 1C of WO 2010/14851 1; which is incorporated herein by reference). The heterologous embrane domain may be obtained from any HA transmembrane domain, for example but not limited to the transmembrane domain from H1 California, B/Florida/4/2006 (GenBank Accession 10 No. ACA3 3 4 9 3 . l), B/Malaysia/25 06/2004 (GenBank Accession No.
Ai3U9 9 l 9 4 . l), H1/Bri (GenBank Accession No. ADEZ 8 7 5 O . 1), H1 A/Solomon Islands/3/2006 (GenBank Accession No. AZ3U9 9 l O 9 . l) , H1/NC (GenBank Accession No. AAP3 4 32 4 . 1), H2 A/Singapore/1/1957 (GenBank Accession No.
AAA6 4 3 6 6 . l ) H3 bane/10/2007 (GenBank Accession No. AC 22 2 63 l 8 , . l) , 15 H3 A/Wisconsin/67/2005 (GenBank Accession No. ABO37599.1), H5 A/Anhui/1/2005 (GenBank Accession No. ABD28180.1), H5 nam/1194/2004 (GenBank Accession No. ACR48874.1), H5—Indo (GenBank Accession No.
ABW06108.1),. The transmembrane domain may also be defined by the following consensus amino acid sequence: 20 iLXiszthiSlelXXmlangmecs (SEQ ID NO:94) id="p-181" id="p-181" id="p-181" id="p-181" id="p-181"
[00181] Examples of constructs comprising a chimeric HA with a heterologous trans-membrane domain include: construct number 1875 (CPMV—HT+ B Brisbane/60/08 with deleted proteolytic loop + H1TM, with membrane domain 25 and cytoplasmic tail replaced by H1 fornia/07/2009; see example 5.19), construct number 1977 (CPMV—160+ B Brisbane/60/08 with deleted proteolytic loop + H1TM, with trans-membrane domain and cytoplasmic tail replaced by H1 A/California/07/2009; see example 5.14), construct number 1067 (CPMV—HT B Brisbane/60/08 with deleted proteolytic loop + H1TM, with trans-membrane domain 30 and asmic tail replaced by H1 A/California/07/2009; see example 5.14), construct number 2074 (CPMV HT B Massachusetts/2/2012+H1Tm, with trans- WO 53674 PCT/CA2014/050326 -63, membrane domain and cytoplasmic tail ed by those of H1 A/California/07/2009; see Example 5.16), construct number 2060 (CPMV HT160+ Massachusetts/2/2012+H1Tm, with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009; see Example 5.16), construct number 2062 (CPMV 160+ B Massachusetts/2/2012+H1Tm, with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/Califomia/07/2009; see Example 5.21), construct number 1860 (CPMV HT+ B sin/1/2010+H1Tm with trans— membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009; see Example 5.23), construct number 1454 (CPMV HT B 10 Wisconsin/1/2010+H1Tm with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009, see Example 5.18) and construct number 1893 (CPMV 160+ B sin/1/2010+H1Tm with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009) see e 5.18. . ty of these chimeric modified HA’s is shown in Figures 26B and 27B. 15 Signal Peptide ] A signal e (SP) may be native to the modified HA or chimeric modified HA, or a signal peptide may be heterologous with respect to the primary sequence of the modified HA being expressed. The modified HA may comprise a signal peptide from a first influenza type, subtype or strain with the balance of the HA 20 from one or more than one different influenza type, subtype or strain. For example the native signal peptide of HA subtypes H1, H2, H3, H5, H6, H7, H9 or influenza type B may be used to s the modified HA in a plant system. In some embodiments of the invention, the SP may be of an influenza type B, H1, H3 or H5; or of the subtype Hl/Bri, Hl/NC, H5/Indo, H3/Bri or B/Flo. 25 [00183] Furthermore, the modified HA or chimeric modified HA may comprise a native, or a non-native signal peptide; the non-native signal peptide may be of plant origin or obtained from an animal or bacterial polypeptide. The native signal peptide may correspond to that of the HA or modified HA being expressed, additionally, the signal peptide may be from a structural n or hemagglutinin of a virus other than 30 influenza. Non-limiting examples of a signal peptide that may be used is that of alfalfa protein disulfide isomerase (PDI SP; nucleotides 32—103 of Accession No.
WO 2014/153674 PCT/CA2014/050326 -64, 211499 also see WO 2009/076778; WO 2010/148511, or WO 2010/003235), or the patatin signal peptide (PatA SP; d nucleotides 1738 — 1806 of k Accession number A08215). The nucleotide sequence of PatA SP for this ion number is: ATGGCAACTACTAAAACTTTTTTAATTTTATTTTTTATGATATTAGCAACTACTAGTTCAACA TGTGCT (SEQ ID NO: 171) the amino acid sequence of patatin A signal peptide is : MATTKTb'T. mm TATTSSTCA (SEQ ID NO: 172) id="p-184" id="p-184" id="p-184" id="p-184" id="p-184"
[00184] . The present invention therefore provides for a modified HA or 10 chimeric ed HA comprising a native, or a non-native signal peptide, and nucleic acids encoding such chimeric modified HA ns.
Co-expression with channel protein id="p-185" id="p-185" id="p-185" id="p-185" id="p-185"
[00185] The mutant (modified) HA may be produced in a plant by co- expressing a first nucleic acid encoding the modified HA With a second nucleic acid 15 encoding a channel protein, for example but not limited to a proton channel protein.
The first and second nucleic acids may be introduced to the plant in the same step, or they may be introduced to the plant sequentially. The first and second nucleic acids may be introduced in the plant in a transient manner, or in a stably manner. rmore, a plant that expresses a first nucleic acid ng the modified HA may 20 transformed with a channel protein, for example but not limited to a proton channel protein, (second nucleic acid) so that both the first and the second c acids are co-expressed in the plant. Alternatively, a plant that expresses a l protein, for example but not limited to a proton channel protein, (second nucleic acid) may transformed with a first nucleic acid encoding the modified HA so that both the first 25 and the second nucleic acids are co—expressed in the plant. onally, a first plant expressing the first nucleic acid encoding modified HA, may be crossed With a second plant expressing the second c acid encoding the channel proteinfor example but not limited to a proton channel protein, to produce a progeny plant that co- expresses the first and second nucleic acids encoding the modified HA and the 30 channel protein, for example but not limited to a proton channel protein, respectively.
WO 2014/153674 PCT/CA2014/050326 -65, id="p-186" id="p-186" id="p-186" id="p-186" id="p-186"
[00186] t wishing to be bound by theory, the pH of a cellular compartment comprising modified HA, including the Golgi apparatus, may be important for the folding, stability and /or lysis of HA. Proton channel proteins, such as for example za M2 and BM2 protein may regulate the pH in cellular compartments. For example, M2 regulates the potentiation of membrane fusion by buffering intracellular compartments both in late and early stages of influenza viral replication. id="p-187" id="p-187" id="p-187" id="p-187" id="p-187"
[00187] By ressing a channel protein, for example but not limited to a proton l protein, along with a modified HA, the pH within the Golgi apparatus 10 may increase, and result in an se in stability, reduction of degradation, or a combination thereof, and increase expression levels and yield of modified HA and/or VLPs. id="p-188" id="p-188" id="p-188" id="p-188" id="p-188"
[00188] By co—expressing a modified HA along with a channel n, for example but not limited to a proton channel protein, in a plant, increased yield of HA 15 and/or VLPs are observed, when ed to a plant that sed the modified without co-expression of the l protein, for example but not limited to a proton channel protein (see Figures 13A and 14). As shown for example in Figure 13A, the co-expression of M2 with the modified influenza B HA increased HA accumulation level (Figure 13A, 1059 vs 1059+1261) . 20 [00189] Furthermore, the efficacy of M2 from influenza A/Puerto Rico/8/1934 to increase accumulation of the modified influenza B HA and H3 was compared to that of M2 from influenza A/New nia/20/1999. For the modified influenza B HA, the comparison was undertaken by western blot analysis of protein extracts from plants transformed with constructs 1059, 1059+1261 and 1059+859. The results 25 obtained demonstrated that the co-expression of M2 from influenza A/Puerto /1934 (encoded by construct no. 859) was as ent as the co—expression of M2 from influenza A/New Caledonia/20/1999 (encoded by construct no. 1261) for increasing accumulation of the modified influenza B HA (Figure 14). id="p-190" id="p-190" id="p-190" id="p-190" id="p-190"
[00190] As used herein, the terms "M2," "M2 protein," "M2 sequence" and 30 "M2 domain" refer to all or a portion of an M2 protein sequence isolated from, based upon or present in any naturally occurring or cially produced influenza virus WO 2014/153674 PCT/CA2014/050326 -66, strain or isolate. Thus, the term M2 and the like include naturally occurring M2 sequence variants produced by mutation during the virus ycle or produced in response to a selective pressure (e. g., drug therapy, expansion of host cell tropism or infectivity, etc.), as well as recombinantly or synthetically produced M2 sequences.
Non-limiting e of sequences that may be used with the present invention include M2 from A/Puerto /1934 and M2 from A/New Caledonia/20/1999.
Immune Response id="p-191" id="p-191" id="p-191" id="p-191" id="p-191"
[00191] An "immune response" generally refers to a response of the adaptive immune system. The adaptive immune system generally comprises a humoral 10 se, and a cell—mediated response. The humoral response is the aspect of immunity that is mediated by secreted antibodies, produced in the cells of the B lymphocyte lineage (B cell). Secreted antibodies bind to antigens on the surfaces of invading es (such as s or bacteria), which flags them for destruction.
Humoral immunity is used generally to refer to antibody production and the processes 15 that accompany it, as well as the effector functions of antibodies, including Th2 cell activation and cytokine production, memory cell generation, opsonin promotion of phagocytosis, pathogen elimination and the like. The terms "modulate" or "modulation" or the like refer to an increase or decrease in a particular response or parameter, as determined by any of several assays generally known or used, some of 20 which are exemplified . id="p-192" id="p-192" id="p-192" id="p-192" id="p-192"
[00192] A ediated response is an immune response that does not involve antibodies but rather involves the activation of macrophages, natural killer cells (NK), antigen—specific cytotoxic T—lymphocytes, and the release of various cytokines in response to an antigen. Cell-mediated ty is used generally to refer to some Th 25 cell activation, Tc cell activation and T—cell mediated responses. Cell mediated immunity is of particular ance in responding to viral infections.
] For example, the induction of n ic CD8 positive T lymphocytes may be measured using an T assay; stimulation of CD4 positive T-lymphocytes may be measured using a proliferation assay. Anti-influenza antibody 30 titres may be quantified using an ELISA assay; isotypes of antigen—specific or cross reactive antibodies may also be measured using anti-isotype antibodies (e.g. anti -IgG, WO 2014/153674 PCT/CA2014/050326 -67, 1gA, 1gE or 1gM). Methods and techniques for ming such assays are well— known in the art.
] Cross—reactivity HA1 titres may also be used to demonstrate the efficacy of an immune response to other strains of virus related to the vaccine subtype. For example, serum from a subject immunized with a vaccine composition of a first strain (e.g. VLPs of A/1ndonesia 5/05) may be used in an HA1 assay with a second strain of whole virus or virus particles (e. g. A/Vietnam/1194/2004), and the HA1 titer determined. id="p-195" id="p-195" id="p-195" id="p-195" id="p-195"
[00195] Cytokine presence or levels may also be quantified. For example a T— 10 helper cell response (Th1/Th2) will be characterized by the measurement of 1FN—y and 1L-4 ing cells using by ELISA (e.g. BD Biosciences OptE1A kits).
Peripheral blood mononuclear cells (PBMC) or splenocytes obtained from a subject may be cultured, and the supernatant analyzed. T lymphocytes may also be fied by fluorescence—activated cell sorting (FACS), using marker specific fluorescent 15 labels and methods as are known in the art. id="p-196" id="p-196" id="p-196" id="p-196" id="p-196"
[00196] A microneutralization assay may also be conducted to characterize an immune response in a t, see for example the methods of Rowe et al., 1973.
Virus neutralization titers may be obtained several ways, including: 1) enumeration of lysis s (plaque assay) following crystal violet fixation/coloration of cells; 2) 20 microscopic observation of cell lysis in culture; 3) ELISA and spectrophotometric detection ofNP virus protein (correlate with virus infection of host cells). id="p-197" id="p-197" id="p-197" id="p-197" id="p-197"
[00197] The term "virus like particle" (VLP), or "virus-like les" or "VLPs" refers to structures that self—assemble and comprise virus proteins for example an influenza HA protein or ed HA protein such for example an HAO 25 protein, wherein the proteolytic loop has been modified. VLPs are generally morphologically and antigenically similar to virions produced in an infection, but lack genetic information ient to replicate and thus are non-infectious. In some es, VLPs may comprise a single protein species, or more than one protein species. For VLPs comprising more than one protein species, the protein species 30 may be from the same s of virus, or may comprise a protein from a different species, genus, subfamily or family of virus (as designated by the 1CTV WO 2014/153674 2014/050326 -68, nomenclature). In other examples, one or more of the protein species comprising a VLP may be modified from the naturally occurring sequence, such for e a modified HA as described herein. VLPs may be produced in suitable host cells including plant and insect host cells. Following extraction from the host cell and upon ion and further purification under suitable conditions, VLPs may be purified as intact structures. id="p-198" id="p-198" id="p-198" id="p-198" id="p-198"
[00198] Furthermore, VLPs may be produced that comprise a combination of HA subtypes. For example, VLPs may comprise one or more than one HA or one or more than one modified HA from the subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, 10 H10, H11, H12, H13, H14, H15, H16, subtype B HA or a combination f. ion of the combination of HAs or modified HAs may be determined by the intended use of the vaccine prepared from the VLP. For example a vaccine for use in inoculating birds may comprise any combination of HA subtypes or modified HA subtypes, while VLPs useful for inoculating humans may comprise subtypes one or 15 more than one of subtypes or ed subtype of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, subtype B HA. However, other HA subtype or ed HA subtype combinations may be prepared depending upon the use of the VLP. In order to e VLPs comprising combinations of HA subtypes or modified subtype HAs, the desired HA subtype or modified HA subtype may be 20 co—expressed within the same cell, for example a plant cell. id="p-199" id="p-199" id="p-199" id="p-199" id="p-199"
[00199] The VLPs produced from influenza derived proteins, in accordance with the present invention do not comprise M1 protein. The M1 protein is known to bind RNA ield and Brownlee, 1989) which is a contaminant of the VLP preparation. The presence of RNA is undesired when obtaining regulatory approval 25 for the VLP t, therefore a VLP preparation lacking RNA may be advantageous. id="p-200" id="p-200" id="p-200" id="p-200" id="p-200"
[00200] The VLPs produced as described herein do not lly comprise neuramindase (NA). However, NA may be co—expressed with HA should VLPs comprising HA and NA be desired. id="p-201" id="p-201" id="p-201" id="p-201" id="p-201"
[00201] The ion also includes, but is not limited to, virus derived VLPs 30 that obtain a lipid envelope from the plasma membrane of the cell in which the VLP WO 53674 PCT/CA2014/050326 -69, proteins are expressed. For example, if the VLP is expressed in a plant-based system, the VLP may obtain a lipid envelope from the plasma membrane of the cell. id="p-202" id="p-202" id="p-202" id="p-202" id="p-202"
[00202] Generally, the term "lipid" refers to a fat-soluble (lipophilic), naturally—occurring molecules. The term is also used more specifically to refer to fatty-acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids), as well as other fat-soluble sterol-containing metabolites or sterols.
Phospholipids are a major component of all biological membranes, along with glycolipids, sterols and proteins. Examples of phospholipids include phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, 10 phosphatidylserine, and the like. Examples of sterols include zoosterols (e. g., cholesterol) and phytosterols. Over 200 phytosterols have been fied in various plant species, the most common being campesterol, stigmasterol, ergosterol, brassicasterol, deltastigmasterol, deltaavenasterol, terol, sitosterol, 24- methylcholesterol, cholesterol or itosterol. As one of skill in the art would 15 understand, the lipid composition of the plasma membrane of a cell may vary with the culture or growth conditions of the cell or organism from which the cell is obtained. id="p-203" id="p-203" id="p-203" id="p-203" id="p-203"
[00203] Cell membranes generally se lipid bilayers, as well as proteins for various functions. Localized concentrations of particular lipids may be found in the lipid bilayer, referred to as ‘lipid rafts". Without wishing to be bound by theory, 20 lipid rafts may have significant roles in endo and exocytosis, entry or egress of viruses or other infectious agents, inter-cell signal transduction, interaction with other ural ents of the cell or organism, such as intracellular and extracellular matrices. id="p-204" id="p-204" id="p-204" id="p-204" id="p-204"
[00204] In plants, influenza VLPs bud from the plasma membrane therefore the 25 lipid composition of the VLPs reflects their origin. The VLPs ed according to the present invention comprise HA of one or more than one type or subtype of influenza, complexed with plant derived lipids. Plant lipids can stimulate specific immune cells and enhance the immune response induced. Plant membranes are made of , atidylcholine (PC) and phosphatidylethanolamine (PE), and also 30 contain glycosphingolipids, saponins, and terols. Additionally, lipid rafts are also found in plant plasma membranes - these omains are enriched in WO 2014/153674 PCT/CA2014/050326 -70, olipids and s. In plants, a variety of phytosterols are known to occur, including stigmasterol, erol, 24-methylcholesterol and cholesterol (Mongrand et al., 2004). id="p-205" id="p-205" id="p-205" id="p-205" id="p-205"
[00205] PC and PE, as well as phingolipids can bind to CD1 molecules expressed by mammalian immune cells such as antigen-presenting cells (APCs) like dendritic cells and macrophages and other cells including B and T lymphocytes in the thymus and liver (Tsuji M,. 2006). CD1 molecules are structurally similar to major histocompatibility complex (MHC) molecules of class I and their role is to present glycolipid antigens to NKT cells (Natural Killer T cells). Upon activation, NKT cells 10 activate innate immune cells such as NK cells and tic cells and also activate ve immune cells like the antibody-producing B cells and T—cells. id="p-206" id="p-206" id="p-206" id="p-206" id="p-206"
[00206] A variety of phytosterols may be found in a plasma membrane — the specific complement may vary depending on the species, growth conditions, nutrient resources or pathogen state, to name a few factors. Generally, beta-sitosterol is the 15 most abundant phytosterol. id="p-207" id="p-207" id="p-207" id="p-207" id="p-207"
[00207] The phytosterols present in an influenza VLP complexed with a lipid bilayer, such as an plasma-membrane derived envelope may provide for an advantageous vaccine composition. Without wishing to be bound by theory, plant— made VLPs complexed with a lipid bilayer, such as a -membrane derived 20 envelope, may induce a stronger immune reaction than VLPs made in other expression systems, and may be similar to the immune reaction induced by live or attenuated whole virus vaccines. id="p-208" id="p-208" id="p-208" id="p-208" id="p-208"
[00208] The VLP as described herein may be xed with a plant-derived lipid bilayer. In some embodiments the plant-derived lipid bilayer may comprise the 25 envelope of the VLP. The plant derived lipids may comprise lipid components of the plasma membrane of the plant where the VLP is produced, including, but not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phingolipids, terols or a combination thereof. A plant-derived lipid may alternately be referred to as a ‘plant lipid’. Examples of phytosterols are known in the art, and 30 include, for example, stigmasterol, sitosterol, 24-methylcholesterol and cholesterol — see, for example, Mongrand et al., 2004.
WO 53674 PCT/CA2014/050326 -71, id="p-209" id="p-209" id="p-209" id="p-209" id="p-209"
[00209] VLPs may be assessed for structure and size by, for example, hemagglutination assay, electron microscopy, or by size exclusion chromatography. id="p-210" id="p-210" id="p-210" id="p-210" id="p-210"
[00210] For size exclusion chromatography, total soluble proteins may be extracted from plant tissue by homogenizing (Polytron) sample of frozen—crushed plant material in extraction buffer, and insoluble material removed by centrifugation.
Precipitation with PEG may be used. The soluble protein is quantified, and the extract passed through a size exclusion matrix, for example but not limited to SephacrleM. Following chromatography, fractions may be further analyzed by immunoblot to determine the protein complement of the fraction. 10 [00211] Without wishing to be bound by theory, the capacity of HA to bind to RBC from different animals is driven by the affinity of HA for sialic acids a2,3 or a2,3 and the presence of these sialic acids on the surface of RBC. Equine and avian HA from influenza s agglutinate erythrocytes from all several species, ing turkeys, chickens, ducks, guinea pigs, humans, sheep, horses and cows; whereas 15 human HAs will bind to erythrocytes of turkey, chickens, ducks, guina pigs, humans and sheep (see also Ito T. et al, 1997, Virology, vol 227, 99; and Medeiros R et al, 2001, Virology, vol 289 p.74—85). id="p-212" id="p-212" id="p-212" id="p-212" id="p-212"
[00212] Correct folding of the expressed virus protein may be important for stability of the protein, formation of multimers, ion of VLPs, on of the 20 virus n and recognition of the virus protein by an antibody, among other characteristics. Folding and accumulation of a protein may be influenced by one or more s, including, but not limited to, the sequence of the protein, the relative abundance of the protein, the degree of intracellular crowding, the pH in a cell compartment, the availability of cofactors that may bind or be transiently associated 25 with the folded, partially folded or unfolded protein, the presence of one or more one proteins, or the like. id="p-213" id="p-213" id="p-213" id="p-213" id="p-213"
[00213] Heat shock proteins (Hsp) or stress proteins are es of chaperone proteins, which may ipate in various cellular processes including protein synthesis, intracellular trafficking, prevention of misfolding, prevention of protein 30 aggregation, assembly and embly of protein complexes, protein folding, and protein disaggregation. es of such chaperone proteins include, but are not WO 2014/153674 PCT/CA2014/050326 -72, limited to, Hsp60, Hsp65, Hsp 70, Hsp90, Hsp100, 30, Hsp10, Hsp100—200, Hsp100, Hsp90, Lon, TF55, FKBPs, cyclophilins, ClpP, GrpE, ubiquitin, calnexin, and protein disulfide isomerases (see, for example, Macario, A.J.L., Cold Spring Harbor tory Res. 25:59—70. 1995; Parsell, D.A. & Lindquist, S. Ann. Rev.
Genet. 27:437—496 (1993); U.S. Patent No. 5,232,833). As described herein, chaperone ns, for example but not limited to Hsp40 and Hsp70 may be used to ensure folding of a virus protein. id="p-214" id="p-214" id="p-214" id="p-214" id="p-214"
[00214] Examples of Hsp70 e Hsp72 and Hsc73 from mammalian cells, DnaK from bacteria, particularly mycobacteria such as Mycobacterium leprae, 10 Mycobacterium tuberculosis, and Mycobacterium bovis (such as Bacille—Calmette Guerin: referred to herein as Hsp7l). DnaK from Escherichia coli, yeast and other prokaryotes, and BiP and Grp78 from eukaryotes, such as A. thaliana (Lin et al. 2001 (Cell Stress and Chaperones 6:201—208). A particular e of an Hsp70 is A. thaliana Hsp70 (encoded by Genbank ref: AY120747.1). Hsp70 is capable of 15 ically g ATP as well as unfolded polypeptides and peptides, thereby participating in n g and unfolding as well as in the ly and disassembly of protein complexes. id="p-215" id="p-215" id="p-215" id="p-215" id="p-215"
[00215] Examples of Hsp40 include DnaJ from prokaryotes such as E. coli and mycobacteria and HS] 1, HDJl and Hsp40 from eukaryotes, such as alfalfa (Frugis et 20 al., 1999. Plant Molecular Biology -408). A particular example of an Hsp40 is M. sativa Mle (Genbank ref: AJ000995.1). Hsp40 plays a role as a molecular chaperone in protein folding, thermotolerance and DNA replication, among other cellular activities. id="p-216" id="p-216" id="p-216" id="p-216" id="p-216"
[00216] Among Hsps, Hsp70 and its co—chaperone, Hsp40, are involved in the 25 stabilization of translating and newly synthesized polypeptides before the synthesis is complete. Without wishing to be bound by theory, Hsp40 binds to the hydrophobic patches of unfolded (nascent or newly transferred) polypeptides, thus facilitating the interaction of Hsp70-ATP complex with the polypeptide. ATP hydrolysis leads to the formation of a stable complex between the polypeptide, Hsp70 and ADP, and release 30 of Hsp40. The association of Hsp70-ADP complex with the hydrophobic s of the polypeptide prevents their interaction with other hydrophobic s, preventing WO 2014/153674 PCT/CA2014/050326 -73, the incorrect folding and the formation of aggregates with other proteins (reviewed in Hartl, FU. 1996. Nature 381:571—579). id="p-217" id="p-217" id="p-217" id="p-217" id="p-217"
[00217] Native chaperone proteins may be able to facilitate correct folding of low levels of recombinant protein, but as the sion levels increase, the abundance of native chaperones may become a limiting factor. High levels of expression of virus protein in the agroinfiltrated leaves may lead to the accumulation of virus protein in the cytosol, and co—expression of one or more than one chaperone proteins such as Hsp70, Hsp40 or both Hsp70 and Hsp40 may reduce the level of misfolded or aggregated proteins, and se the number of ns exhibiting 10 tertiary and quaternary structural characteristics that allow for ion of virus-like particles. id="p-218" id="p-218" id="p-218" id="p-218" id="p-218"
[00218] Therefore, the present invention also provides for a method of ing virus protein VLPs in a plant, wherein a first nucleic acid encoding a virus protein is co—expressed with a second nucleic acid encoding a channel protein, for 15 example but not limited to a proton channel n, and a third nucleic acid encoding a one. The first, second and third nucleic acids may be introduced to the plant in the same step, or may be uced to the plant sequentially.
] The VLP produced within a plant may induce an virus protein comprising plant-specific N-glycans. Therefore, this invention also es for a 20 VLP comprising virus protein having plant ic N—glycans. id="p-220" id="p-220" id="p-220" id="p-220" id="p-220"
[00220] Furthermore, modification ycan in plants is known (see for example WO 2008/151440; WO 2010/006452; or U.S. 60/944,344; which are incorporated herein by reference) and virus protein having modified N—glycans may be produced. Virus protein comprising a modified ylation pattern, for example 25 with reduced fucosylated, xylosylated, or both, fucosylated and xylosylated, N— glycans may be obtained, or virus protein having a modified glycosylation pattern may be obtained, wherein the protein lacks fucosylation, xylosylation, or both, and comprises increased galatosylation. Furthermore, modulation of post-translational modifications, for example, the addition of terminal galactose may result in a 30 reduction of fucosylation and xylosylation of the expressed virus protein when ed to a wild-type plant expressing virus protein.
WO 2014/153674 PCT/CA2014/050326 -74, id="p-221" id="p-221" id="p-221" id="p-221" id="p-221"
[00221] For e, which is not to be considered limiting, the synthesis of virus protein having a modified glycosylation pattern may be achieved by co— expressing the protein of interest along with a nucleotide sequence encoding beta— 1.4galactosyltransferase (GalT), for example, but not limited to mammalian GalT, or human GalT however GalT from r sources may also be used. The catalytic domain of GalT may also be fused to a CTS domain (i.e. the cytoplasmic tail, transmembrane domain, stem region) ofN—acetylglucosaminyl transferase (GNTl), to produce a GNTl—GalT hybrid enzyme, and the hybrid enzyme may be co—expressed with virus protein. The virus protein may also be co-expressed along with a 10 nucleotide sequence encoding N—acetylglucosaminyltrasnferase III (GnT-HI), for example but not limited to mammalian GnT-HI or human GnT-HI, GnT-HI from other sources may also be used. Additionally, a GNTl—GnT—III hybrid enzyme, comprising the CTS of GNTl fused to GnT—III may also be used. id="p-222" id="p-222" id="p-222" id="p-222" id="p-222"
[00222] Therefore the present invention also es VLP’s comprising one or 15 more virus protein having modified N—glycans.
] Non-limiting e of sequences that may be used with the t invention to produce modified HA’s also include those described in W0 2009/009876; W0 2009/076778; W0 03225; W0 2010/148511; W0 2010/003235; W0 2010/006452 (which are herein incorporated by reference), for 20 example, but not limited to: H1 protein encoded by the c acid molecule for example from A/Brisbane/59/2007 (HlNl), A/New Caledonia/20/99 (HlNl) mon s , 3/2006 (HlNl), /PuertoRico/8/34 (HlNl), A/Brisbane/59/2007 (HlNl), strain; H2 protein encoded by the nucleic acid molecule may be from the 25 A/Singapore/1/57 (H2N2) ; H3 protein encoded by the nucleic acid molecule may be from the A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2) strain, A/Victoria/361/2011 (H3N2) or A/Perth/16/2009 (H3N2); H5 protein encoded by the nucleic acid molecule may be from the 30 A/Anhui/l/ZOOS (H5N1), A/Indonesia/5/2005 (H5N1), A/Vietnam/l 194/2004 (H5N1) WO 2014/153674 PCT/CA2014/050326 , _ 75 H6 protein encoded by the nucleic acid molecule may be from the A/Teal/HongKong/W3 12/97 (H6N 1) ; H7 protein encoded by the c acid molecule may also be from the A/ Hangzhou/1/13 (H7N9), A/Equine/Prague/56 (H7N7) strain; H9 protein encoded by the nucleic acid le may be from the A/HongKong/1073/99 (H9N2) strain; HA protein from B subtype encoded by the c acid may be from the B/Florida/4/2006, B/Massachusetts/2/l2, B/Malaysia/2506/2004, B/Wisconsin/1/2010, or B/Brisbane/60/2008 strain. 10 [00224] Table 3: Examples of constructs that have been prepared as described herein: Constr. Expression Ampl. Description Example Enhancer Element _A/New Caledonia/20/1999 HlNl 5.1 _A/Puerto Rico/8/1934 HlNl 5.2 CPMV-HT BeYDV B/Brisbane/60/2008 with deleted Example 5.4 nroteol tic 100 n CPVIV HT BeYDV B/Wisconsin/1/2010 5.5 CPi V-HT BeYDV B/Wisconsm/1/2010 With deleted. Example 5 .6 nroteol tic 100 n C MV-HTP B/Brisbane/60/2008 with deleted Example 5.7 proteolytic loop CPMV-HT B/Brisbane/60/2008 Examnle 5.11 1829 CPMV- HT+ nroteol tic loo nroteol tic loo B/Brisbane/60/2008 with deleted Example 5.14 l i0100n--H1 California TMCT B/Brisbane/60/2008 with deleted Example 5.14 proteolytic loop --H1 rnia TMCT bane/60/2008 with d Example 5.19 rnoteol tic 100n --H1 California TMCT cleavae site cleavae site H5 A/lndonesia/5/2005 with deleted Example 5.10 nroteol tic 100 n 489 CPMV-HT H5 A/lndones1a/5/2005 e) Example 5.24 2220 CPMV- H2 A/Sinanore/1/57 native Examnle 5.27 WO 2014/153674 PCT/CA2014/050326 , _ 76 HT-- 2221 CPMV- H2 apore/1/57 With deleted Example 5.28 roteol tic loo .
H2 A/Singapore/1/57 (native) Example5.29 H2 A]Singapore/1/57 With deleted Example5.29 roteol tic loo . 2019 CPMV- H3 A/Perth/16/09 (native) e 5.30 HT-- 2039 CPMV- H3 A/Perth/16/09 With deleted Example 5.31 HT-- roteol tic loo . 2139 CPMV- H3 A/Perth/16/09 (native) Example 5.30 160- 2159 CPMV- H3 A/Perth/16/09 With deleted Example 5.31 160-- roteol tic loo . 1 819 CPMV- H3 A/Victoria /361/11 (native) Example 5.26 HT 2230 CPMV- H3 oria /361/11 With deleted Example 5 .32 HT-- roteol tic loo . 1800 CPMV- H3 A/Victoria /361/11 (native) Example 5.25 160- 2250 C MV-P H3 A/Victoria 1 With d Example 5.32 160-- proteolytic loop 2142 CPMV- H7 A/Hangzhou/1/13 (native) Example 5.33 HT-_ 2152 CPMV- H7 A/Hangzhou/1/13 With deleted Example 5.34 HT-- proteolytic loop 2224 CPMV- H9 A/Hong 073/99 (native) Example 5.35 HT-- 2225 CPMV- H9 A/Hong Kong/1073/99 With d Example 5.36 HT-- roteol tic loo . 2226 CPMV- H9 A/Hong Kong/1073/99 (native) Example 5 .35 160-- 2227 H9 A/Hong Kong/1073/99 With deleted Example 5.36 160-- roteol tic loo .
B/Malaysia 04 (native) Example 5.37 160-- B Malaysia /2506/04 With deleted/ Example 5.38 160-- roteol tic loo . 2070 B/Massachusetts/2/12 native Exam - le 5.39 2072 C VIV HTP1 _ B/Massachusetts/Z/12 With deleted Example 5.15 roteol tic loo . 2080 CPMV- B/Massachusetts/2/12 (native) Example 5.39 HT 2052 CPMV- B Massachusetts/Z/12 With deleted/ Example 5.20 HT-- roteol tic loo . 2090 CPMV— B/Massachusetts/2/12 (native) Example 5.39 160-- B/Massachusetts/Z/12 With deleted Example 5.15 160-- proteolytic loop HA B/Massachusetts/2/12 (PrL-)--H1 Example 5.16 California TMCT 2060 CPMV- HA B/Massachusetts/2/12 PrL- --H1 Examle 5.16 WO 2014/153674 PCT/CA2014/050326 , _ 77 _—_CahformaTMCT — 2062 CPMV- HA B/Massachusetts/Z/12 (PrL-)+H1 Example 5 .21 HT+ rnia TMCT 1445 CPMV HT B/Wisconsin/1/2010 with deleted Example 5.17 proteolytic loop (PrL-) 1839 CPMV- B/Wisconsin/1/2010 with deleted Example 5 .22 HT+ rnoteol tic 100 n 1 820 CPMV160+ B/Wisconsin/1/2010 with d Example 5.17 nroteol tic loo n PrL- 1975 CPMV160 B/Wisconsin/1/2010 with deleted Example 5.17 nroteol tic loo n PrL- 1454 CPMV-HT loo PrL- --H1 California TMCT 1893 CPMV- HA B Wisconsin with deleted proteolytic 160 loo PrL- --H1 California TMCT CPMV- HA B Wisconsin with deleted proteolytic HT-- loo PrL- --H1 California TMCT HT-- PrL- HT-- PrL- 2016 CPMV- B Florida --H1 California TMCT with Example 5.41 HT-- deleted proteolytic loop (PrL-) 2108 CPMV- BeYDV B a --H1 California TMCT with Example 5.41 HT-- deleted lytic loop (PrL-) Table: 4: Description of sequences Description Description Avian H5 Amino acid ce of HA proteolytic loop B Wisconsin (PrL-). consensus sequence IF-H5A-I-05.si+3c A Nucleotide sequence of HA B Wisconsin (PrL-)+H1 California TMCT IF-H5dTm.r 1B Amino acid sequence of HA B Wisconsin (PrL-)+H1 California TMC.
Construct 1191 Nucleotide sequence of PDISP/HA B Brisbane (PrL- )+H1 rnia TMCT. 5 Cassette 489 106 Amino acid sequence of 38B PDISP/HA B Brisbane PrL- WO 2014/153674 PCT/CA2014/050326 , _ 78 ption Description )+H1 California TMCT.
Amino acid _ '11 107 Nucleotide sequence of 39A sequence H5 PDISP/HA B A/Indonesia/5/2005 hussetts (PrL-).
(H5N1) IF-Sl- N',> Amino acid sequence of M1+M2ANC.c PDISP/HA B Massachussetts (PrL-).
IF-S 1 NC.r U0 Nucleotide sequence of PDISP/HA B Massachussetts (PrL-)+H1 California TMCT.
Synthetic M2 (nt 1- Amino acid sequence of 26 joined to 715- PDISP/HA B 982 from Massachussetts +H1 DQ508860) California TMCT.
Cassette 1261 2D 111 tide sequence ofHA 41A B Wisconsin (PrL-).
M2 from influenza [\J Amino acid sequence of HA 41B A/New B Wisconsin (PrL-).
Caledonia/20/1999 (H1N1) Synthetic M2 (nt L» ',> Nucleotide sequence of HA 26-51 joined to nt B Wisconsin (PrL-)+H1 740-1007 from California TMCT EF467824) Cassette 859 Amino acid sequence ofHA 42B B Wisconsin (PrL-)+H1 California TMC.
Amino acid Nucleotide sequence of ce M2 native H5 Indonesia. influenza A/Puerto Rico/8/1934 (H1N1) 15 Cassette 1039 8A 116 Amino acid sequence of 43B native H5 Indonesia 16 Amino acid 21A 117 IF**(SacH)-PDI.s1+4c 4A se uence WO 2014/153674 PCT/CA2014/050326 2 _ 79 ption Description B/Brisbane/60/2008 Amino acid 21B IF-H3V36111.s1-4r sequence delta- proteolytic loop of type B HA (with linker GG) Amino acid Nucleotide sequence of sequence replacing PDISP/H3 Victoria. cleavage site of B HA with linker Amino acid Construct 2171 sequence H3 A/Perth/16/2009 Amino acid Construct 1800 sequence delta- proteolytic loop H3 (with linker GS) Amino acid Amino acid sequence of sequence Replacing PDISP/H3 Victoria cleavage site of H3 with linker H1 New Cal linker IF(SacH)-Kozac7PDI.c region H1 Brisbane linker 36111.s1-4r region H1 Sol Islands Construct 2181 linker region H2A Singapore 15 Construct 1819 linker region H3A Brisbane 15 IF** -H2S157.s1 6—r linker region H3A WCN linker ._i U} Nucleotide sequence of region PDISP/H2 Singapore.
H5 Anhui linker sion cassette number region 2220 H5 Indo linker Amino acid sequence of WO 2014/153674 PCT/CA2014/050326 7 _ 80 Description Figure NO: PDISP/H2 Smgapore 3 0 H5 Vietnam linker 15 131 H2s1 57(Prl-).r 49A region 3 1 Construct 1 194 4B 1 32 H2Sl57(Prl-).c 49B 32 Cassette 1008 4C 133 Expression cassette number 49C 2221 H6 Teal HK linker Amino acid ce of 49D region PDISP/H2 Singapore with deleted proteolytic loop H7 Eq Prague linker ._1 U} Expression cassette number region 2222 35 H9A HK linker 15 136 Expression cassette number 50B region 2223 36 B Florida linker 15 137 Nucleotide sequence of 51A region PDISP/H3 Perth 37 B Malaysia ._1 U} 138 IF**-H3P1609.Sl-6r 51B 3 8 103 9-- 1 059.r 5A 139 Amino acid ce of 51C PDISP/H3 Perth 39 103 9.c 5U0 1 40 Nucleotide sequence of 52A PDISP/H3 Perth with deleted proteolytic loop 40 Cassette 1059 5C 141 H3P1609(Prl-)#2.r 5213 41 Amino acid U1U H3P1609(Prl-)#2.c sequence PDISP/HA influenza B/Brisbane/60/2008 (deleted proteolytic loop) Nucleotide 5 Amino acid sequence of sequence H5 PDISP/H3 Perth with A/Indonesia/5/2005 deleted proteolytic loop (H5N1) nucleotide sequence 5E 144 Nucleotide sequence of 53A PDISP/HA H3 Victoria with influenza WO 2014/153674 PCT/CA2014/050326 7 _ 81 Description Description B/Brisbane/60/2008 deleted proteolytic loop (deleted proteolytic loop) H5/Indo cleavage ._1 \0 H3V36111(Prl-).r site natural sequence H5/Indo modified ._1 \O H3V36111(Prl-).c cleavage site (TETR) H5/Indo modified _ \O Amino acid sequence of ge site PDISP/H3 Victoria with (TETQ) deleted proteolytic loop H1 cleavage site Table 1 148 Nucleotide ce of 54A PDISP/H7 Hangzhou H3 cleavage site Table 1 IF*-H7H113.s1-6r IF-HAB110.Sl+3c 6A Amino acid sequence of PDISP/H7 Hangzhou IF-HAB 1 4r Nucleotide sequence of PDISP/H7 Hangzhou with deleted proteolytic loop Synthetic HA B O\0 H7H113(PrL-).r Wisconsin uct 193 153 H7H113(PrL-).c 55C te 1462 O\ '11 Amino acid sequence of PDISP/H7 Hangzhou with d proteolytic loop Amino acid 0\ Nucleotide sequence of sequence HA PDISP/H9 Hong Kong za B/Wisconsin/1/201 HAB110(PrL)r IF**-H9HK107399Sl-6r HAB110(PrL-)c Amino acid sequence of PDISP/H9 Hong Kong Cassette 1467 7C Nucleotide sequence of PDISP/H9 Hon; Kon with WO 2014/153674 PCT/CA2014/050326 7 _ 82 Description Description deleted lytic loop Amino acid H9HK107399(Prl)1 sequence HA influenza B/Wisconsin/1/201 0 (deleted PL) B ge site Table 1 160 H9HK107399(Prl-).c 57C HS/Indo natural Table 2 Amino acid sequence of cleavage site PDISP/H9 Hong Kong with d proteolytic loop 61 HS/Indo modified Table 2 Nucleotide sequence of cleavage site PDISP/HA B Malaysia HS/Indo modified Table 2 IF**-HBM250604$1 -61 cleavage site H1/Brisbane Table 2 Amino acid ce of modified cleavage PDISP/HA B Malaysia site H3/Brisbane Nucleotide sequence of modified cleavage PDISP/HA B Malaysia with site deleted proteolytic loop B/Florida, HBM250604(PrL-).r B/B1isbane d ge site A/H3/HAO 167 HBM250604(PrL-).c 59C Consensus A/Hl/HAO Amino acid sequence of Consensus PDISP/HA B Malaysia with deleted proteolytic loop B/HAO Consensus 169 Nucleotide sequence of 60A HA B Massachusetts H5 Anhui Amino acid sequence of proteolytic loop PDISP/HA B Massachusetts deletion H5 Indo proteolytic 81C 171 nucleotide sequence of PatA loop deletion SP WO 53674 PCT/CA2014/050326 7 _ 83 Description Description H5 Vietnam amino acid sequence of proteolytic loop n A signal peptide deletion B a CPMV HT sequence proteolytic loop deletion B Malaysia Plant consensus kozak proteolytic loop sequence - plant kingdom deletion MutCleavage- 23A Plant consensus kozak H5(Indo)1 sequence- dicots MutCleavage- 23B Plant consensus kozak H5(Indo)c sequence -arabidopsis Cassette 676 23C Plant consensus kozak sequence Amino acid Plant consensus kozak sequence influenza sequence A/Indonesia/5/2005 (H5N1) TETR cleavage site mutant.
H51505TETQr Plant consensus kozak sequence H51505TETQc Plant consensus kozak sequence Cassette 766 25C Plant consensus kozak sequence Amino acid 25D Plant sus kozak sequence influenza sequence A/Indonesia/5/2005 (H5N1) TETQ cleavage site mutant.
H51505(PrL)r Plant consensus kozak sequence H51505(PrL)c Plant sus kozak sequence WO 2014/153674 2014/050326 -84, Description Description Figure Cassette 928 Plant consensus kozak sequence Amino acid Kozak consensus sequence sequence influenza nesia/5/2005 (H5N1) with deleted proteolytic loop.
IF-S2+S4-B Bris.c tide sequence of CPMV HT-- IF-Sla4-B Bris.r Nucleotide sequence of CPMV HT-- 511 Synthesized HA B 30C 189 Nucleotide sequence Brisbane gene ofCPMV HT+[WT115] Construct 1029 190 HBF406(PrL-).r Amino acid 191 HBF406(PrL-).c sequence of PDISP/HA from influenza B/Brisbane/60/2008 Nucleotide IF*-HBF406.s1-6r sequence of PDISP/HA B Brisbane (PrL-).
Amino acid Nucleotide sequence of sequence of PDISP/HA B Florida with HA B deleted proteolytic loop Brisbane (PrL-) Nucleotide Amino acid sequence of sequence of PDISP/HA B Florida with CPMVX/CPMVX+ deleted proteolytic loop consensus amino Expression cassette number acid sequence of 2102 transmembrane domain Nucleotide Expression cassette number ce of 2104 PDISP/HA B Brisbane PrL- +H1 WO 2014/153674 PCT/CA2014/050326 -85, Description California TMCT.
Amino acid sequence of PDISP/HA B ne (PrL-)+H1 California TMCT.
Nucleotide Nucleotide sequence of sequence of PDISP/HA B Florida+H1Cal PDISP/HA B TMCT with deleted hussetts proteolytic loop (PrL-) Amino acid Amino acid sequence of sequence of PDISP/HA B Florida+H1Cal PDISP/HA B TMCT with deleted Massachussetts proteolytic loop (PrL-) Nucleotide Expression cassette number sequence of 2106 PDISP/HA B Massachussetts (PrL-)+H1 California TMCT.
Amino acid Expression cassette number ce of 2108 PDISP/HA B Massachussetts (PrL-)+H1 rnia TMCT.
Nucleotide sequence ofHA B Wisconsin (PrL-).
Examples Example 1 Agrobacterium transfection WO 2014/153674 PCT/CA2014/050326 -86, id="p-225" id="p-225" id="p-225" id="p-225" id="p-225"
[00225] Agrobacterium strain AGLl was transfected by electroporation with the DNA constructs using the methods described by D’Aoust et al 2008 (Plant Biotechnology Journal 6:93 0—940). Transfected Agrobacterium were grown in YEB medium supplemented with 10 mM 2-(N—morpholino)ethanesulfonic acid (MES), 20 uM acetosyringone, 50 ug/ml kanamycin and 25 ug/ml of carbenicillin pH5.6 to an OD600 between 0.6 and 1.6. cterium suspensions were centrifuged before use and resuspended in infiltration medium (10 mM MgClz and 10 mM MES pH 5.6). ation of plant biomass, inoculum and agroinfiltration id="p-226" id="p-226" id="p-226" id="p-226" id="p-226"
[00226] The terms "biomass" and "plant matter" as used herein are meant to 10 reflect any material derived from a plant. Biomass or plant matter may comprise an entire plant, tissue, cells, or any fraction thereof. Further, biomass or plant matter may comprise intracellular plant components, extracellular plant ents, liquid or solid ts of plants, or a combination thereof Further, s or plant matter may comprise plants, plant cells, tissue, a liquid extract, or a ation thereof, 15 from plant leaves, stems, fruit, roots or a combination thereof. A portion of a plant may comprise plant matter or biomass. id="p-227" id="p-227" id="p-227" id="p-227" id="p-227"
[00227] Nicotiana benthamiana plants were grown from seeds in flats filled with a commercial peat moss substrate. The plants were allowed to grow in the greenhouse under a 16/8 photoperiod and a temperature regime of 25°C day/20°C 20 night. Three weeks after seeding, individual plantlets were picked out, transplanted in pots and left to grow in the greenhouse for three additional weeks under the same environmental conditions. id="p-228" id="p-228" id="p-228" id="p-228" id="p-228"
[00228] Agrobacteria transfected with each construct were grown in a YEB medium supplemented with 10 mM 2-(N—morpholino)ethanesulfonic acid (MES), 20 25 uM acetosyringone, 50 ug/ml cin and 25 ug/ml of icillin pH5.6 until they reached an OD600 n 0.6 and 1.6. Agrobacterium suspensions were fuged before use and resuspended in infiltration medium (10 mM MgClz and 10 mM MES pH 5.6) and stored overnight at 4°C. On the day of infiltration, culture batches were diluted in 2.5 e volumes and allowed to warm before use. Whole 30 plants of N. benthamiana were placed upside down in the bacterial suspension in an WO 2014/153674 PCT/CA2014/050326 -87, air-tight stainless steel tank under a vacuum of 20-40 Torr for 2-min. Plants were returned to the greenhouse for a 2—6 day incubation period until harvest.
Leaf harvest and total protein extraction id="p-229" id="p-229" id="p-229" id="p-229" id="p-229"
[00229] Following incubation, the aerial part of plants was harvested, frozen at —80°C and crushed into pieces. Total soluble proteins were extracted by nizing (Polytron) each sample of frozen-crushed plant al in 3 volumes of cold 50 mM Tris pH 8.0, 0.5 M NaCl, 0.1% Triton X-100 and 1 mM phenylmethanesulfonyl fluoride. After homogenization, the slurries were centrifuged at 10,000 g for 10 min at 4°C and these clarified crude extracts (supernatant) kept for analyses. 10 n analysis and immunoblotting id="p-230" id="p-230" id="p-230" id="p-230" id="p-230"
[00230] The total protein content of clarif1ed crude extracts was determined by the Bradford assay (Bio—Rad, Hercules, CA) using bovine serum albumin as the reference standard. Proteins were separated by SDS-PAGE and electrotransferred onto polyvinylene difluoride (PVDF) membranes (Roche Diagnostics Corporation, 15 apolis, IN) for immunodetection. Prior to immunoblotting, the membranes were d with 5% skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS-T) for 16—1 8h at 4°C. id="p-231" id="p-231" id="p-231" id="p-231" id="p-231"
[00231] Immunoblotting was med with a first incubation with a y antibody (Table 4 presents the antibodies and conditions used for the detection of 20 each HA), in 2 ug/ml in 2% skim milk in TBS—Tween 20 0.1%. Secondary dies used for chemiluminescence detection were as indicated in Table 4, diluted as indicated in 2% skim milk in TBS—Tween 20 0.1%. Immunoreactive complexes were detected by chemiluminescence using luminol as the substrate (Roche Diagnostics Corporation). Horseradish peroxidase—enzyme ation of human IgG antibody 25 was carried out by using the EZ-Link Plus® Activated Peroxidase conjugation kit (Pierce, Rockford, IL).
Table 4: Electrophoresis ions, antibodies, and dilutions for immunoblotting of expressed proteins.
WO 2014/153674 PCT/CA2014/050326 2 _ 88 Electro- Primary Secondary Influenza strain phoresis . Dilution antlbody antlbody. on lon. .
Rabbit anti- . Non- TGA, B/Brisbane/60/2008 1.20000. sheep (JIR ng AS397 313_035_ Rabbit anti- . . Non- NIBSC sheep (JIR B/Wisconsm/l/2010 reducing 07/356 313—035— Rabbit anti- . Non- NIBSC sheep (JIR B/Malaysra/2506/2004 reducing 07/184 313_035_ Rabbit anti- A/Perth/16/2009 Non- TGA, sheep (JIR 1.20000 (H3N2) reducing AS400 ' 313—035— Rabbit anti- AN"0113/361/201 1. . Non- TGA, sheep (JIR reducing AS400 313—035— Goat anti- A/Califomia/07/2009 mouse Reducing (HlNl) (JIR 1 1 5 - Rabbit anti- A/Indonesia/05/2005 sheep (JIR Reducing (H5N1) 313—035- JIR: n ImmunoResearch, West Grove, PA, USA; CBER: Center for Biologics Evaluation and Research, Rockville, MD, USA.
Sino: Sino Biological inc., Beijing, China.
TGA: eutic Goods Administration, Australia.
NIBSC: National Institute for ical Standards and Control, United Kingdom Hemagglutination assay id="p-232" id="p-232" id="p-232" id="p-232" id="p-232"
[00232] Hemagglutination assay was based on a method described by Nayak and Reichl (2004). Briefly, serial double ons of the test samples (100 uL) were made in V—bottomed 96-well iter plates containing 100 uL PBS, leaving 100 uL of diluted sample per well. One hundred microliters of a 0.25% turkey red blood cells suspension (Bio Link Inc., Syracuse, NY) were added to each well, and plates were incubated for 2h at room temperature. The reciprocal of the highest dilution showing complete hemagglutination was recorded as HA activity. In parallel, a recombinant HA standard (A/Vietnam/1203/2004 H5N1) (Protein Science Corporation, Meriden, CT) was diluted in PBS and run as a control on each plate.
WO 2014/153674 PCT/CA2014/050326 -89, VLP extraction by cell wall digestion id="p-233" id="p-233" id="p-233" id="p-233" id="p-233"
[00233] Leaf tissue was collected from the Nicotiana benthamiana plants and cut into ~1 cm2 pieces. The leaf pieces were soaked in 500 mM mannitol for 30 s at room temperature (RT). The mannitol on was then removed and changed with the enzyme mix (mixture of ases from derma viride (Onozuka R-10; 3% v/v) and a mixture of pectinases from Rhizopus sp.
(MACEROZYMETM; 0.75% v/v; both from Yakult Pharmaceuticals) in protoplasting solution (500 mM mannitol, 10mM CaClz and 5 mM MES/KOH (pH 5.6)). The ratio used was 20 g of leaf pieces per 100 mL solution. This preparation was spread evenly 10 into a shallow vessel (~11x18 cm) and incubated for 16 hours on a rotary shaker at 40 rpm and 26°C. id="p-234" id="p-234" id="p-234" id="p-234" id="p-234"
[00234] Alternately, VLP extraction may be med as follows: plants were agroinfiltrated with AGL1/#489, 928, 676 and 766. Leaf tissue was collected from the N. benthamiana plants at day 7 post-infiltration and cut into ~l cm2 pieces. 15 Pectinase 162L (Biocatalysts), Multifect CX CG and Multifect CX B (Genencor) were added to a 200 mM Mannitol, 75 mM Citrate, 0.04% sodium bisulfite pH 6.0 buffer; digestion buffer. The biomasses were digested in duplicate overnight at room temperature in an orbital shaker. . id="p-235" id="p-235" id="p-235" id="p-235" id="p-235"
[00235] Following enzyme—assisted tion, leaf debris was removed by 20 filtration (nylon filter of 250 or 400 um mesh). The coarse filtered extract was centrifuged at 5000 xg for 5 minutes. Supernatant was submitted to detection of HA expression (hemagglutination activity (see Figure 20) and Western blotting (see Figure 22).
Example 2: 25 Effect of modified lytic loop on lation of HA id="p-236" id="p-236" id="p-236" id="p-236" id="p-236"
[00236] As shown in Figure 13A, expression of native B/Brisbane (Construct No:1008) was lower than the expression of B/Brisbane comprising a modified proteolytic loop (Construct No: 1059). Increased hemagglutination activity was also observed with B/Brisbane comprising a ed lytic loop (Construct No: WO 2014/153674 PCT/CA2014/050326 -90, 1059) when ed to the native B/Brisbane HA (Construct No: 1008; Figure 13B). id="p-237" id="p-237" id="p-237" id="p-237" id="p-237"
[00237] Similar results were observed in the accumulation level of B/ sin comprising a modified proteolytic loop (Construct No: 1467), which is greater than that observed for the native B/Wisconsin HA (Construct No: 1462; Figure 16A). Increased hemagglutination activity was also observed with B/Wisconsin sing a modified proteolytic loop (Construct No: 1467) when compared to the native B/Wisconsin HA (Construct No: 1462; Figure 16B) indicating a greater accumulation for the mutant protein. 10 [00238] Expression of H5/Indo comprising a modified proteolytic loop was also observed with modifications including a proteolytic loop comprising a GG linker (Construct No: 928; SEQ ID NO:85), a TETR linker (Construct No: 676; SEQ ID NO:77), or a TETQ linker (Construct No: 766; SEQ ID NO: 8; Figure 22).
Effect of influenza M2 co-expression on the accumulation level of HA 15 [00239] The co—expression of M2 was ted for its impact on the accumulation level of a modified influenza B HA. Construct no. 1059 encodes an influenza B HA in which the proteolytic loop is replaced by a 2 amino acid linker (GG in place of aa 341—359). The results fiom western blot analysis presented in figure 13A show that the removal of the proteolytic loop resulted in increased 20 influenza B HA accumulation level (compare 1008 with 105 9) and that the co— expression of M2 with the modified za B HA also sed HA accumulation level e 13A, 1059 vs 1059+126l). An analysis of hemagglutination activity on crude protein extracts from plants transformed with influenza B HA with or without cation and with or t co-expression of M2 confirmed the positive effect 25 of M2 co—expression on the lation level of the native influenza B HA (Figure 13B, 1008 vs 1008+126l) and the modified za B HA (Figure 13B, 1059 vs 1059+1 261). id="p-240" id="p-240" id="p-240" id="p-240" id="p-240"
[00240] Co—expression of M2 with type A HA comprising a modified proteolytic loop also resulted in HA expression. For example, co—expression of WO 53674 PCT/CA2014/050326 -91, modified H3, with the proteolytic loop replaced with a GS linker or a (GSS)3 linker (see Figure 21E, 21F), along with M2 may also result in HA accumulation in a plant. id="p-241" id="p-241" id="p-241" id="p-241" id="p-241"
[00241] The efficacy of M2 from influenza A/Puerto Rico/8/1934 to increase accumulation of the modified influenza B HA and H3 was compared to that of M2 from influenza A/New Caledonia/20/1999. For the modified influenza B HA, the comparison was undertaken by western blot analysis of protein extracts from plants transformed with constructs 1059, 1059+1261 and 1059+859. The results ed demonstrated that the co-expression of M2 from influenza A/Puerto Rico/8/1934 (encoded by construct no. 859) was as efficient as the co—expression of M2 from 10 influenza A/New nia/20/1999 (encoded by construct no. 1261) for increasing accumulation of the modified influenza B HA (Figure 14).
Effect of influenza M2 co-expression on the accumulation level of different strains of B HA ] Western blot analysis of protein extracts from plants transformed with 15 gene constructs driving the expression of influenza B HA (from B/Wisconsin/1/2010) (constructs no. 1462) in the ce or absence of M2—expression construct (construct no. 1261) showed that M2 ression results in increased accumulation of influenza B HA (Figure 16A). id="p-243" id="p-243" id="p-243" id="p-243" id="p-243"
[00243] The co—expression of M2 was also evaluated for its impact on the 20 accumulation level of a modified influenza B HA. Construct no. 1467 encodes an influenza B HA in which the proteolytic loop is replaced by a 2 amino acid linker (GG in place of aa 341—359). The s from western blot analysis presented in figures 16A show that the removal of the proteolytic loop resulted in increased influenza B HA accumulation level (compare 1462 with 1467) and that the co- 25 expression of M2 with the modified influenza B HA also increased HA accumulation level (Figure 16A, 1467 vs 1467+1261). An analysis of hemagglutination activity on crude protein extracts from plants ormed with influenza B HA with or without modification and with or without co-expression of M2 confirmed the positive effect of M2 ression on the lation level of the native influenza B HA (Figure WO 2014/153674 PCT/CA2014/050326 -92, 16B, 1462 vs 26l) and the modified influenza B HA (Figure 16B, 1467 vs l467+l26l).
Effect of amplification element BeYDV and modified proteolytic loop on accumulation of HA id="p-244" id="p-244" id="p-244" id="p-244" id="p-244"
[00244] n blot analysis of protein extracts from plants transformed with gene constructs driving the expression of modified influenza B HA (from B/Brisbane/60/2008) with or without the proteolytic loop removed (see Figure 17A for constructs) and in the ce or absence of the amplification element BeYDV (construct no. 1059 and 1039) showed that in the absence of BeYDV no accumulation 10 of za B HA could be detected (Figure 17B), when the regulatory element was CPMV—HT.
Effect of modified proteolytic loop on relative HA titer and hemagglutination id="p-245" id="p-245" id="p-245" id="p-245" id="p-245"
[00245] With nce to Figure 29A, there is shown a comparison of the activity of d HA proteins produced in plants comprising CPMV HT, CPMV 15 HT+ CPMV 160 or CPMV160+, based enhancer elements operatively linked with a , nucleotide sequence encoding either modif1ed HA with the proteolytic loop deleted (replaced with a GG linker) or a native HA. In most cases, the expression (determined as hemagglutination titrer or activity) were higher for the CPMV HT+, CPMV 160 or CPMV160+ based construct demonstrates significant expression 20 levels.
Table 5a: Relative HA titer (wt HA=1) (see Figure 29A) Fct= 1 H,_, H_.'T —---"-fl 1-0 Ian-1.2 —m- l- —----II- —-"mn 1-0 0-3 n-mn H9HK107399(HT+)(n=2) 1.2 H9HK107399(160+)(n=2) 1.0 0.2 2 0.7 0.1 2 WO 2014/153674 PCT/CA2014/050326 -93, HB Bri6008 (HT) (n = 1) -m-m - HB Mal250604 (160+) (n _ 2) ---m . —---3) - HB M35212 (HT+) (n — 3) "m-.7 . 2) m-- Example 3 sed H7 Hangzhou HA VLP yields when the proteolytic loop is removed (PrL-) compared to the native construct. id="p-246" id="p-246" id="p-246" id="p-246" id="p-246"
[00246] N. benthamiana plants were infiltrated with 2142+1261 and #2152+1261 and the leaves were harvested after a seven—day incubation . Leaf tissue was collected and cut into ~1 cm2 pieces. Pectinase 162L and Pectinase 444L (Biocatalysts), ect CX CG and Multifect CX B (Genencor) were added in a 200 mM Mannitol, 125 mM Citrate, 0.04% sodium bisulfite pH 6.0 buffer. The biomass was 10 digested overnight at room temperature in an l shaker. id="p-247" id="p-247" id="p-247" id="p-247" id="p-247"
[00247] Following ion, the apoplastic fiaction was filtered through a 400 um nylon filter to remove coarse undigested vegetal tissue (<5% of ng biomass). The filtered extract was then centrifuged at room temperature for 15 min at 5000xg to remove protoplasts and intracellular contaminants (proteins, DNA, membranes, vesicles, 15 pigments, etc). Next, the supernatant was depth-filtered (for clarification) using a 1.2um glass fiber filter (Sartopore GF plus/Sartorius Stedim), and a 0.45/0.2pm filter (Sartopore 2/Sartorius Stedim), before being subjected to chromatography. id="p-248" id="p-248" id="p-248" id="p-248" id="p-248"
[00248] The clarified apoplastic fraction was loaded over a cation exchange column (Poros HS Applied Biosystems) equilibrated with an equilibration/elution buffer 20 (50 mM NaPO4, 100 mM NaCl, 0.005% Tween 80 pH 6.0). Once the UV was back to zero, the extract was step-eluted with the equilibration/elution buffer ning increasing concentrations ofNaCl (500 mM). The purified VLPs were concentrated by TFF, diafiltered against formulation buffer (100 mM P04, 150 mM NaCl, 0.01% Tween 80 at pH 7.4) and passed through a 0.22L1m filter. 25 [00249] Hemagglutination assay for H7 was performed based on a method described by Nayak and Reichl (2004). Briefly, successive double dilutions of the test WO 2014/153674 PCT/CA2014/050326 -94, samples (100 uL) were made in V—bottomed 96—well microtiter plates containing 100 uL PBS, leaving 100 uL of diluted sample per well. One hundred microliters of a 0.25% turkey red blood cells suspension (Bio Link Inc., Syracuse, NY) were added to each well, and plates were incubated for 2h at room temperature. The reciprocal of the highest dilution showing complete hemagglutination was recorded as hemagglutination activity. id="p-250" id="p-250" id="p-250" id="p-250" id="p-250"
[00250] Total protein t of ed crude extracts was determined using bovine serum albumin as the nce rd. Relative yields were obtained by comparing the PrL— construct to the native construct used as control. Separation by SDS— PAGE, with denaturing sample loading buffer (0.1M Tris pH 6.8, 0.05% bromophenol 10 blue, 12.5% glycerol, 4% SDS and 5% beta—mercaptoethanol), was performed under reducing conditions and Coomassie Brillant Blue R-250 was used for protein staining. id="p-251" id="p-251" id="p-251" id="p-251" id="p-251"
[00251] Figure 46A shows that the hemagglutination activity in plant extracts is greater for the H7 Hangzhou construct where the proteolytic loop is d (#2152 + #1261, see Example 5.34) compared to the native construct (# 2142 + # 1261, see 15 Example 5.33). id="p-252" id="p-252" id="p-252" id="p-252" id="p-252"
[00252] Figure 46B shows that the relative total protein yield in purified VLP is greater for the H7 Hangzhou construct where the proteolytic loop is removed (#2152 + #1261) compared to the native construct (# 2142 + # 1261). This example demonstrate a good ation between the improvement in the VLP accumulated in plants vs the final 20 yields when performing the complete process. id="p-253" id="p-253" id="p-253" id="p-253" id="p-253"
[00253] Figure 46C shows a SDS—PAGE is, with lane 2 showing the purified H7 ou construct with a removed lytic loop and lane 3 showing the purified native H7 Hangzhou construct. For each lane, 2ug of total protein were loaded on the gel. The purity of the proteins es is similar for both constructs and 25 greater than 90%.
Example 4.1.
Trypsin resistance of mutants H5 Indonesia VLP where the proteolytic loop is modified or removed is greater than native H5 Indonesia.
WO 2014/153674 2014/050326 -95, id="p-254" id="p-254" id="p-254" id="p-254" id="p-254"
[00254] N. benthamiana plants were agroinfiltrated with AGL1/#489, #928, #766 and # 676 as described in Example 1 ). Leaves were collected from the plants 7 days post-infiltration, cut into ~l cm2 pieces. Pectinase l62L (Biocatalysts), ect CX CG and ect CX B (Genencor) were added in a 200 mM Mannitol, 75 mM Citrate, 0.04% sodium bisulf1te pH 6.0 buffer. The biomass was digested overnight at room temperature in an orbital shaker. The digested extracts were coarse— filtered, centrifuged, clarified and purified as described in Example 3 (H7 Hangzhou). id="p-255" id="p-255" id="p-255" id="p-255" id="p-255"
[00255] For each of the native (# 489), PRL— (# 928), TETQ (# 766) and TETR (# 676), H5 Indonesia HA VLP extracts, two samples of HA VLPs were resuspended 10 in buffer (100 mM Na/KPO4, 150 mM NaCl, 0.01% TWEEN 80) at pH 7.4. Trypsin was added in a 1:100 protein ratio. Samples were grabbed after 30, 60 and 120 minutes of incubation at room temperature, then boiled in sample loading buffer to stop the reaction. The non-digested ts (control) and the trypsin-digested extracts analysed by SDS—PAGE gel as described in e 3. 15 [00256] Figure 47A shows an SDS-PAGE analysis of trypsin-digested samples, with lanes 2 through 5 showing the native H5 Indonesia VLP (#489), with lanes 6 through 9 showing PrL- H5 Indonesia VLP (#928), with lanes 10 through 13 showing TETQ H5 Indonesia VLP (#766) and with lanes 14 through 17 showing the TETR H5 Indonesia VLP (#676) at different time points in the digestion (O, 30, 60, and 120 20 minutes). The native H5 Indonesia VPL, with a band ponding to the HAO monomer being detectable at approximately 75 kDa in the non-digested extract in lane 2, was rapidly processed into HA1 and HA2 bands through addition of trypsine, detectable at approximately 50 and 25kDa respectively during the trypsin digestion in lanes 3 through 5. Both the PrL- and the TETQ H5 Indonesia VLPs, stabilized by the 25 removal or modification of the proteolytic site, showed trypsin resistance as the HAO band did not cleave into HA1 and HA2 bands. The TETR H5 sia VLPs were partially stabilized by the modification of the proteolytic site and HAO monomers were d into HA1 and HA2 slower than in the native H5 Indonesia VLPS. id="p-257" id="p-257" id="p-257" id="p-257" id="p-257"
[00257] These data demonstrate the sful protection of the HAO n at 30 its proteolytic site within HA1-HA2, by either deleting the proteolytic loop (prl-) or replacing the proteolytic loop with a linker sequence (TETQ) approach.
WO 53674 PCT/CA2014/050326 -96, id="p-258" id="p-258" id="p-258" id="p-258" id="p-258"
[00258] Example 4.2. Immunogenicity of native H5 Indonesia VLPs is similar to its mutant rparts (PrL-, TETQ and TETR) in mice. id="p-259" id="p-259" id="p-259" id="p-259" id="p-259"
[00259] The native, PrL-, TETR and TETQ H5 Indonesia VLPs extracts were purified as described in e 4.1 ). id="p-260" id="p-260" id="p-260" id="p-260" id="p-260"
[00260] Figure 47B shows immunogenicity (HI titer) of native H5 VLP and its mutant counterparts (prl-, TETQ and TETR) in mice after two doses. BALB/c mice roup) were injected twice intramuscularly, 21 days apart, with 10ug dose of plant based H5 VLP vaccines (native, prl-, TETQ or TETR) based on its HA content.
HI titers analysis was done from sera of each animal, 42 dpv (21 days after de 2nd 10 dose) and H5 VLP A/Indonesia/5/2005 (H5N1) was used as antigen. Bars represent relative (%) HI titers ison of each H5 mutants VLP with the H5 VLP native (calculated with the log2 of the HI titer GMT and 95% CI). tical differences between groups for each dose were compared by using a one—way ANOVA followed by a Tukey’s post-hoc analysis on Log2- transformed data (assuming a normal 15 distribution of them). * p < 0.05 was considered significant. No difference between groups for each dose was ed.
Example 5.1: B-2X35S/CPMV-HT/M2 New Caledonia/NOS [Construct number 1261] id="p-261" id="p-261" id="p-261" id="p-261" id="p-261"
[00261] A sequence encoding M2 from influenza A/New Caledonia/20/1999 20 (HlNl) was cloned into 2X35S/CPMV—HT/NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR— based method. A fragment containing the complete M2 coding sequence was ied using primers IF-Sl-Ml+M2ANC.c (Figure 2A, SEQ ID NO: 7) and IF-Sl- C.r (Figure 2B, SEQ ID NO: 8) using synthesized M2 gene (corresponding 25 to nt 1—26 joined to nt 715-982 from GenBank accession number DQ508860; Figure 2C, SEQ ID NO: 9) as template. The PCR product was cloned in CPMV— HT/NOS expression system using In—Fusion cloning system (Clontech, Mountain View, CA). Construct 1191 (Figure 1C) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. 30 Construct number 1191 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in a CPMV HT-based expression cassette. It also incorporates a gene WO 2014/153674 PCT/CA2014/050326 -97, uct for the co—expression of the TBSV P19 ssor of silencing under the alfalfa cyanin gene promoter and terminator. The backbone is a A binary plasmid and the sequence from left to right t-DNA borders is presented in Figure 1D (SEQ ID NO: 4). The resulting construct was given number 1261 (Figure 2D, SEQ ID NO: 10). The amino acid sequence of M2 from influenza A/New Caledonia/20/1999 (H1N1) is presented in Figure 2E (SEQ ID NO: 11). A representation of plasmid 1261 is presented in Figure 11.
Example 5.2: C-2X35S/CPMV-HT/M2 Puerto Rico/NOS gConstruct number 8591 10 [00262] A sequence encoding M2 from influenza A/Puerto Rico/8/1934 (H1N1) was cloned into 2X35S/CPMV—HT/NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter sion cassette using the following PCR— based method. A fragment containing the complete M2 coding sequence was amplified using primers IF-S1-M1+M2ANC.c (Figure 2A, SEQ ID NO: 7) and IF-S1- 15 4-M2ANC.r (Figure 2B, SEQ ID NO: 8), using synthesized M2 gene (corresponding to nt 26—51 joined to nt 740—1007 from Genbank accession number EF467824) (Figure 3A, SEQ ID NO: 12) as template. The PCR product was cloned in 2X35S/CPMV—HT/NOS expression system using In—Fusion cloning system (Clontech, Mountain View, CA). uct 1191 (Figure 1C) was digested with SacII 20 and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 1191 is an acceptor d ed for "In Fusion" cloning of genes of st in a CPMV—HT—based expression te. It also orates a gene construct for the ression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The vector is a 25 pCAMBLA binary plasmid and the sequence from left to right t-DNA borders is presented in Figure 1D (SEQ ID NO: 4). The resulting construct was given number 859 (Figure 3B, SEQ ID NO: 13). The amino acid sequence of M2 from influenza A/Puerto Rico/8/1934 (H1N1) is presented in Figure 3C (SEQ ID NO: 14). A representation of plasmid 859 is presented in Figure 17. 30 Example 5.3: G-2X35S/CPMV-HT/PDISP/HA B Brisbane/NOS into BeYDV+Replicase amplification system (Construct number 10081 WO 2014/153674 PCT/CA2014/050326 -98, id="p-263" id="p-263" id="p-263" id="p-263" id="p-263"
[00263] The preparation of construct 1008 is described in US 61/541,780.
Briefly, a sequence encoding HA from influenza B/Brisbane/60/2008 was cloned into 2X35 S/CPMV—HT/PDISP/NOS comprising the rep1icase amplification system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using a PCR—based method using sized HA B Brisbane gene (corresponding to nt 34- 1791 from Genbank accession number FJ766840). The PCR product was cloned in— frame with a PDI signal peptide in 2X35 S/CPMV—HT/NOS expression cassette into the BeYDV amplification system. Construct 1194 (see Figures 4A, 4B) was digested with SacH and Stul restriction enzyme and the linearized d was used 10 for an assembly reaction to produce construct number 1008 (Figure 4C, Figure 9; SEQ ID NO: 32). id="p-264" id="p-264" id="p-264" id="p-264" id="p-264"
[00264] Construct number 1194 (Figure 4A) is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in frame with an a PDI signal e in a CPMV-HT-based sion cassette into the BeYDV amplification . It 15 also incorporates a gene uct for the co—expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid.
Example 5.4: I-2X35S/CPMV-HT/PDISP-HA B Brisbane with deleted proteolytic 100p into BeYDV+Replicase amplification system [Construct number 20 1059] id="p-265" id="p-265" id="p-265" id="p-265" id="p-265"
[00265] The preparation of construct 1059 is described in US 61/541,780. Briefly, a sequence encoding HA from influenza B/Brisbane/60/2008 with deleted proteolytic loop was cloned into 2X35S/CPMV—HT/PDISP/NOS comprising the BeYDV+rep1icase amplification system in a plasmid containing 25 _pro/P19/Plasto_ter expression cassette using the a PCR-based ligation method (Darveau et al., 1995, Methods in Neuroscience 26: 77—85). In a first round of PCR, a fragment containing HA B Brisbane coding sequence from nt 46 to nt 1065 was ied using synthesized HA B Brisbane gene (corresponding to nt 34-1791 from Genebank accession number FJ766840) as template. A second fragment, containing 30 HA B Brisbane coding sequence from nt 1123 to nt 1758, was amplified using synthesized HA B Brisbane gene (corresponding to nt 1 from Genbank WO 2014/153674 PCT/CA2014/050326 -99, accession number FJ766840) as template. The PCR products from both amplifications were then mixed and used as te for a second round of amplification. The resulting nt (encoding HA B/Brisbane/60/2008 Aa.a. 356-374 with a GG linker between fragments; see Figure 21B) was cloned in-frame with alfalfa PDI signal peptide in 2X35 S/CPMV—HT/NOS expression cassette comprising the BeYDV ication system to produce construct 1194 (Figures 4A, 4B) was digested with SacII and StuI restriction enzyme and the ized plasmid was used for an assembly reaction. The resulting construct was given number 1059 (Figure 5C; SEQ ID NO: 40). 10 [00266]The amino acid sequence of PDISP-HA B/Brisbane/60/2008 with deleted lytic loop is presented in Figure 5D (SEQ ID NO: 41).
Example 5.5: B-2X35S/CPMV-HT/HA B Wisconsin/NOS into BeYDngz+Replicase amplification system (Construct number 14621 id="p-267" id="p-267" id="p-267" id="p-267" id="p-267"
[00267] The preparation of construct 1462 is described in US 61/541,780. 15 Briefly, a sequence encoding HA from influenza B/Wisconsin/1/2010 was cloned into 2X35 S/CPMV—HT/NOS comprising the BeYDV(m)+replicase amplification system in a d containing Plasto_pro/P19/Plasto_ter expression cassette using a PCR— based method. A fragment containing the complete HA B Wisconsin coding sequence was amplified using sized HA B Wisconsin gene (Genbank accession number 20 IN993010) as template. The PCR product was cloned in 2X35S/CPMV—HT/NOS expression cassette into the BeYDV(m) amplification system. Construct 193 (Figure 6D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for an assembly on. id="p-268" id="p-268" id="p-268" id="p-268" id="p-268"
[00268] Construct number 193 (Figure 6D, 6E) is an acceptor d intended 25 for "In " cloning of genes of interest in a CPMV—HT—based expression te into the BeYDV(m) amplification system. It also incorporates a gene uct for the co—expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t—DNA borders is presented in Figure 6E (SEQ ID NO: 30 52). The resulting construct was given number 1462 (Figure 6F, SEQ ID NO: 53).
The amino acid sequence of PDISP/HA from za B/Wisconsin/1/2010 is WO 2014/153674 PCT/CA2014/050326 -1007 presented in Figure 6G (SEQ ID NO: 54). A representation of plasmid 1462 is presented in Figure 6H.
Example 5.6: C-2X35S/CPMV-HT/HA B Wisconsin with deleted proteolytic 100]; into m)+Replicase cation system (Construct number 14672 id="p-269" id="p-269" id="p-269" id="p-269" id="p-269"
[00269] The preparation of construct 1467 is described in US 61/541,780.
Briefly, a sequence encoding HA from influenza B/Wisconsin/1/2010 with deleted proteolytic loop was cloned into 2X35S/CPMV—HT/NOS comprising the BeYDV(m)+replicase amplification system in a plasmid containing _pro/P19/Plasto_ter expression cassette using a PCR-based ligation method 10 (Darveau et al. 1995, Methods in Neuroscience 26: 77—85). In a first round of PCR, a nt containing HA B Wisconsin coding sequence from nt 1 to nt 1062 was ied using primers IF-HAB110.S1+3c (Figure 6A, SEQ ID NO: 49) and HAB110(PrL—).r (Figure 7A, SEQ ID NO: 55) HA B Wisconsin , using synthesized gene (Genbank accession number JN993010) (Figure 6C, SEQ ID NO: 51) as 15 te. A second fragment, containing HA B Wisconsin coding sequence from nt 1120 to nt 1755, was amplified using primers HAB110(PrL-).c (Figure 7B, SEQ ID NO: 56) and and IF—HAB110.s1—4r (Figure 6B, SEQ ID NO: 50), using synthesized HA B Wisconsin gene (Genbank accession number JN993010) (Figure 6C, SEQ ID NO: 51) as template. The PCR products from both amplifications were then mixed 20 and used as template for a second round of amplification using 110.S1+3c (Figure 6A, SEQ ID NO: 49) and IF-HAB110.s1-4r (Figure 6B, SEQ ID NO: 50) as primers. The resulting fragment (encoding HA onsin/1/2010 Aa.a. 340—358 with a GG linker n fragments) was cloned in 2X35S/CPMV—HT/NOS expression cassette comprising the BeYDV(m) amplification system using In-Fusion 25 cloning system (Clontech, Mountain View, CA). Construct 193 (Figure 6D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. id="p-270" id="p-270" id="p-270" id="p-270" id="p-270"
[00270] Construct number 193 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in a CPMV—HT—based expression cassette into the 30 BeYDV(m) amplification system. It also incorporates a gene uct for the co— expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin WO 2014/153674 PCT/CA2014/050326 -1017 gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t—DNA borders is presented in Figure 6E (SEQ ID NO: 52). The resulting construct was given number 1467 (Figure 7C, SEQ ID NO: 57).
The amino acid sequence of HA from Influenza B/Wisconsin/1/2010 with deleted proteolytic loop is presented in Figure 7D (SEQ ID NO: 58). A representation of plasmid 1467 is presented in Figure 7E.
Example 5.7: A-2X3SS/CPMV-HT/PDISP-HA B Brisbane with d proteolytic loop (Construct number 1039) ] The ation of construct 1192 is described in US 61/541,780. 10 Briefly, a sequence encoding HA from influenza B/Brisbane/60/2008 with deleted proteolytic loop was cloned into 2X35S/CPMV—HT/PDISP/NOS in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR— based ligation method (Darveau et al., 1995, Methods in Neuroscience 26: . In a first round of PCR, a fragment containing HA B Brisbane coding sequence from nt 15 46 to nt 1065 was amplified using synthesized HA B Brisbane gene (corresponding to nt 34—1791 from Genebank accession number 40) as template. A second nt, containing HA B Brisbane coding sequence from nt 1123 to nt 1758, was amplified using synthesized HA B Brisbane gene (corresponding to nt 34-1791 from Genbank accession number FJ766840) as template. The PCR products from both 20 amplifications were then mixed and used as template for a second round of amplification. The resulting fragment (encoding HA B/Brisbane/60/2008 Aa.a. 356— 374 with a GG linker n nts) was cloned in-frame with alfalfa PDI signal peptide in 2X35 S/CPMV—HT/NOS expression cassette. Construct 1192 was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In- 25 Fusion ly reaction.
] Construct number 1192 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in frame with an alfalfa PDI signal peptide in a CPMV—HT—based expression cassette. It also incorporates a gene construct for the co— sion of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin 30 gene er and terminator. The backbone is a pCAMBIA binary plasmid The resulting uct was given number 1039 (Figure 8B). The amino acid sequence of WO 2014/153674 PCT/CA2014/050326 -1027 PDISP—HA bane/60/2008 with deleted proteolytic loop is presented in Figure 5D (SEQ ID NO: 41). A representation of plasmid 1039 is presented in Figure 8A (SEQ ID NO: 15).
Example 5.8: A-2X35S/CPMV-HT/H5 from A/Indonesia/5/2005 with TETR cleavage site mutation gConstruct number 676! id="p-273" id="p-273" id="p-273" id="p-273" id="p-273"
[00273] A sequence encoding H5 from A/Indonesia/5/2005 with TETR cleavage site mutation was cloned into 2X35 S/CPMV-HT/ NOS in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR— based ligation method (Darveau et al., 1995, Methods in cience 26: 77—85). In 10 a first round of PCR, a fragment containing H5 from nesia/5/2005 coding sequence from nt 1 to nt 1015 was ied using primers IF-H5A-I—05.s1+3c (Figure 1A, SEQ ID NO: 2) and MutCleavage-H5(Indo).r (Figure 23A, SEQ ID NO: 74), using synthesized H5 from A/Indonesia/5/2005 (Figure 1G, SEQ ID NO: 42) as template. A second fragment, containing H5 from nesia/5/2005 coding 15 sequence from nt 1038 to nt 1707, was amplified using primers MutCleavage— H5(Indo).c (Figure 23B, SEQ ID NO : 75) and Tm.r (Figure 1B, SEQ ID NO: 3), using sized H5 from A/Indonesia/5/2005 (Figure 1G, SEQ ID NO: 42) as template. The PCR products from both amplifications were then mixed and used as template for a second round of amplification using -I-05.s1+3c (Figure 1A, 20 SEQ ID NO: 2) and IF-H5dTm.r (Figure 1B, SEQ ID NO: 3) as primers. The ing fragment (encoding H5 from A/Indonesia/5/2005 Aa.a. 339—346 with a TETR linker between fragments) was cloned in 2X35 S/CPMV—HT/NOS expression cassette using In—Fusion cloning system (Clontech, Mountain View, CA). Construct 1191 (Figure ID) was digested with SacII and StuI restriction enzyme and the 25 linearized plasmid was used for the In—Fusion assembly reaction. Construct number 1191 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in frame in a CPMV—HT—based expression cassette. It also incorporates a gene construct for the co—expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary 30 plasmid and the ce from left to right t-DNA borders is presented in Figure 1D (SEQ ID NO: 4). The resulting uct was given number 676 (Figure 23C, SEQ ID NO: 76). The amino acid ce of H5 from A/Indonesia/5/2005 TETR cleavage WO 2014/153674 PCT/CA2014/050326 -1037 site mutant is presented in Figure 23D (SEQ ID NO: 77). A schematic representation of plasmid 676 is presented in Figure 23E.
Example 5.9: B-2X35S/CPMV-HT/H5 from A/Indonesia/5/2005 with TETQ cleavage site mutation gConstruct number 7662 ] A sequence encoding H5 from nesia/5/2005 with TETQ cleavage site mutation was cloned into 2X35 S/CPMV-HT/ NOS in a d containing Plasto_pro/P19/Plasto_ter expression te using the following PCR— based ligation method (Darveau et al., 1995, Methods in Neuroscience 26: 77—85). In a first round of PCR, a fragment containing H5 from A/Indonesia/5/2005 coding 10 sequence from nt 1 to nt 1015 was amplified using primers IF-H5A-I—05.s1+3c (Figure 1A, SEQ ID NO: 2) and H51505_TETQ.r (Figure 24A, SEQ ID NO: 78), using synthesized H5 from A/Indonesia/5/2005 (Figure 1G, SEQ ID NO: 42) as te. A second fragment, containing H5 from nesia/5/2005 coding sequence from nt 1038 to nt 1707, was amplified using s H51505_TETQ.c 15 (Figure 24B, SEQ ID NO : 79) and Tm.r (Figure 1B, SEQ ID NO: 3), using synthesized H5 from A/Indonesia/5/2005 (Figure 1G, SEQ ID NO: 42) as template.
The PCR products from both ications were then mixed and used as template for a second round of ication using IF-H5A-I—05.s1+3c (Figure 1A, SEQ ID NO: 2) and IF-H5dTm.r (Figure 1B, SEQ ID NO: 3) as primers. The resulting fragment 20 (encoding H5 from A/Indonesia/5/2005 Aa.a. 339—346 with a TETQ linker between fragments) was cloned in 2X35 S/CPMV—HT/NOS expression cassette using In— Fusion cloning system (Clontech, Mountain View, CA). Construct 1191 (Figure ID) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In—Fusion assembly on. Construct number 1191 is an acceptor 25 plasmid intended for "In Fusion" cloning of genes of interest in frame in a CPMV- HT—based expression cassette. It also incorporates a gene construct for the co— expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the ce from left to right t—DNA borders is presented in Figure 1D (SEQ ID NO: 4). 30 The resulting construct was given number 766 (Figure 24C, SEQ ID NO: 80). The amino acid sequence of H5 from A/Indonesia/5/2005 TETQ cleavage site mutant is WO 2014/153674 PCT/CA2014/050326 —1047 presented in Figure 24D (SEQ ID NO: 81). A schematic representation of plasmid 766 is presented in Figure 24E.
Example 5.10: C-2X3SS/CPMV-HT/H5 from A/Indonesia/5/2005 with deleted proteolytic loop gConstruct number 9281 id="p-275" id="p-275" id="p-275" id="p-275" id="p-275"
[00275] A sequence encoding H5 from A/Indonesia/5/2005 with deleted proteolytic loop was cloned into 2X35S/CPMV—HT/ NOS in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the ing PCR-based ligation method presented by Darveau et a1. (Methods in Neuroscience 26: 77—85 (1995)). In a first round of PCR, a fragment containing H5 from A/Indonesia/5/2005 coding 10 sequence from nt 1 to nt 1011 was ied using primers IF-H5A-I—05.s1+3c (Figure 1A, SEQ ID NO: 2) and H5I505(PrL-).r (Figure 25A, SEQ ID NO: 82), using synthesized H5 from A/Indonesia/5/2005 (Figure 1G, SEQ ID NO: 42) as template. A second fragment, containing H5 from A/Indonesia/5/2005 coding sequence from nt 1075 to nt 1707, was amplified using primers H5I505(PrL-).c e 25B, SEQ ID 15 NO : 83) and IF—H5dTm.r (Figure 1B, SEQ ID NO: 3), using synthesized H5 from A/Indonesia/5/2005 (Figure 1G, SEQ ID NO: 42) as template. The PCR products from both amplifications were then mixed and used as template for a second round of amplification using IF-H5A-I-05.s1+3c (Figure 1A, SEQ ID NO: 2) and IF-H5dTm.r (Figure 1B, SEQ ID NO: 3) as primers. The resulting fragment ing H5 from 20 A/Indonesia/5/2005 Aa.a. 33 8—358 with a GG linker between fragments) was cloned in 2X35S/CPMV—HT/NOS sion cassette using In—Fusion cloning system (Clontech, Mountain View, CA). Construct 1191 (Figure 1D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the ion assembly on. Construct number 1191 is an acceptor plasmid intended for "In 25 " cloning of genes of interest in frame in a CPMV-HT-based expression cassette. It also orates a gene construct for the co—expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator.
The backbone is a pCAMBIA binary plasmid and the sequence from left to right t- DNA s is presented in Figure 1D (SEQ ID NO: 4). The resulting construct was 30 given number 928 (Figure 25C, SEQ ID NO: 84). The amino acid sequence of H5 from A/Indonesia/5/2005 with d proteolytic loop is presented in Figure 25D (SEQ ID NO: 85). A representation of plasmid 928 is presented in Figure 25E.
WO 2014/153674 PCT/CA2014/050326 -1057 Exam le 5.11 - F-2X35S/CPMV-HT/PDISP/HA B Brisbane/NOS Construct number 10291 id="p-276" id="p-276" id="p-276" id="p-276" id="p-276"
[00276] A sequence encoding HA from influenza B/Brisbane/60/2008 was cloned into 2X35S/CPMV—HT/PDISP/NOS expression system in a plasmid ning Plasto_pro/P19/Plasto_ter expression cassette using the ing PCR— based . A fragment containing HA B Brisbane coding ce without his wild type signal peptide was amplified using primers S4-B Bris.c e 30A, SEQ ID NO: 86) and IF-S1a4-B Bris.r e 30B, SEQ ID NO: 87), using synthesized HA B Brisbane gene (corresponding to nt 34-1791 from Genbank 10 accession number FJ766840) (Figure 30C, SEQ ID NO: 88) as template. The PCR product was cloned in-frame with alfalfa PDI signal e in CPMV- HT/NOS expression system using In—Fusion cloning system (Clontech, Mountain View, CA). Construct 1192 was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In—Fusion assembly reaction. Construct 15 number 1192 is an acceptor plasmid intended for "In Fusion" cloning of genes of st in frame with an alfalfa PDI signal peptide in a CPMV-HT-based expression cassette. It also incorporates a gene uct for the co—expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator.
The backbone is a pCAMBIA binary plasmid and the sequence from left to right t- 20 DNA borders. The resulting construct was given number 1029 (Figure 30D, SEQ ID NO: 89). The amino acid sequence of PDISP/HA from influenza B/Brisbane/60/2008 is presented in Figure 30E (SEQ ID NO: 90). A representation of plasmid 1029 is presented in Figure 30F.
Example 5.12 - 2X3SS/CPMV HT gconstruct no 10392 and HT+ gconstruct no 25 18292 for PDISP/HA B Brisbane gPrL-l id="p-277" id="p-277" id="p-277" id="p-277" id="p-277"
[00277] A coding sequence corresponding to HA from Influenza B/Brisbane/60/2008 with deleted proteolytic loop (PrL-) in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Brisbane (PrL-; Figure 31A, SEQ ID NO: 91) was cloned into original HT and 30 modified HT+ using the same PCR—based method described in Examples 5.7 and 5.11, but with modified PCR primers specifically ed for PDISP/HA B Brisbane (PrL-). The amino acid ce of mature HA B Brisbane (PrL-) fused with PDISP WO 2014/153674 PCT/CA2014/050326 -1067 is presented in Figure 31B (SEQ ID NO: 92). Representations of plasmid 1039 and 1829 are presented in Figure 8B and 31D.
Example 5.13 - 2X3SS/CPMV HT (construct no 1039) and 2X3SS/CPMV160+ sconstruct no 1937! for PDISP/HA B Brisbane gPrL-L id="p-278" id="p-278" id="p-278" id="p-278" id="p-278"
[00278] A coding sequence corresponding to HA from Influenza B/Brisbane/60/2008 with d proteolytic loop (PrL—) (see US provisional application 806,227 Filed March 28, 2013, which is incorporated herein by reference, for additional information re: deleted proteolytic loop regions in HA sequences) in which the native signal peptide has been replaced by that of alfalfa 10 protein disulfide isomerase (PDISP/HA B Brisbane (PrL-)) (Figure 32A, SEQ ID NO: 93) was cloned into al CPMV—HT and CPMV160+ using the same PCR—based method as described in Example 5.7 and in e 5.11, but with modified PCR primers specifically designed for PDISP/HA B Brisbane . The amino acid sequence of mature HA B Brisbane (PrL-) fused with PDISP is ted in Figure 15 31B (SEQ ID NO: 92). entations of plasmid 1039 and 1937 are presented in Figure 8B and Figure 32C.
Example 15.14 - 2X3SS/CPMV HT gconstruct no 10672 and CPMV160+ ruct no 1977) for PDISP/HA B Brisbane gPrL-[+H1 California TMCT id="p-279" id="p-279" id="p-279" id="p-279" id="p-279"
[00279] A chimer hemagglutinin coding sequence corresponding to the 20 ectodomain of HA from Influenza B/Brisbane/60/08 with deleted proteolytic loop (PrL-) (see US provisional application No.61/806,227 Filed March 28, 2013, which is incorporated herein by reference, for additional information re: deleted proteolytic loop regions in HA sequences) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from influenza fornia/7/2009 and with the signal peptide of 25 alfalfa protein disulfide isomerase /HA B Brisbane (PrL-)+H1 California TMCT) (Figure 33A, SEQ ID NO: 95) was cloned into original CPMV-HT and CPMV160+ using the same PCR—based method as described in Examples 5.7 and 5.11, but with modified PCR primers specifically designed for PDISP/HA B Brisbane (PrL-)--H1 California TMCT. The amino acid sequence of mature HA B Brisbane 30 (PrL-)--H1 California TMCT fused with PDISP is presented in Figure 33B (SEQ ID NO: 96). Representations of plasmid 1067 and 1977 are presented in Figure 33C and Figure 33D.
WO 2014/153674 PCT/CA2014/050326 -1077 Example 5.15 - 2X35S/CPMV HT gconstruct no 20722 and 2X35S/CPMV160+ goonstruct no 2050) for PDISP/HA B Massachussetts gPrL-l id="p-280" id="p-280" id="p-280" id="p-280" id="p-280"
[00280] A coding sequence corresponding to HA from Influenza B/Massachussetts/2/2012 with deleted proteolytic loop (PrL—) (see US provisional application No.6l/806,227 Filed March 28, 2013 for additional information re: deleted proteolytic loop regions in HA sequences, which is incorporated herein by reference) in which the native signal peptide has been ed by that of alfalfa protein disulfide isomerase /HA B Massachussetts (PrL-)) (Figure 34A, SEQ ID NO: 97) was cloned into original CPMV—HT and CPMV160+ using the same 10 PCR—based method as described in Examples 5.7 and 5.11, but with modified PCR primers specifically designed for PDISP/HA B Massachussetts (PrL-). The amino acid sequence of mature HA B Massachussetts (PrL—) fused with PDISP is presented in Figure 34B (SEQ ID NO: 98). entations of plasmid 2072 and 2050 are presented in Figure 34C and Figure 34D. 15 e 5.16 - 2X35S/CPMV HT (construct no 2074) and 2X35S/CPMV160+ goonstruct no 2060! for PDISP/HA B Massachussetts gPrL-)+H1 California TMCT id="p-281" id="p-281" id="p-281" id="p-281" id="p-281"
[00281] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/Massachussetts/2/2012 with deleted proteolytic 20 loop (PrL—) (see US provisional application No.6l/806,227 Filed March 28, 2013 for additional information re: deleted proteolytic loop s in HA sequences, which is incorporated herein by nce) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from za A/California/7/2009 and with the signal peptide of alfalfa n disulfide isomerase (PDISP/HA B Massachussetts 25 (PrL-)+Hl California TMCT) (Figure 35A, SEQ ID NO: 99) was cloned into original CPMV—HT and 0+ using the same PCR—based method as bed in Examples 5.7 and 5.11, but with modified PCR primers ically designed for PDISP/HA B Massachussetts (PrL—)+Hl California TMCT. The amino acid sequence of mature HA B Massachussetts (PrL—)+Hl rnia TMCT fused with PDISP is 30 presented in Figure 35B (SEQ ID NO: 100). Representations of plasmid 2074 and 2060 are presented in Figure 35C and 35D.
WO 53674 PCT/CA2014/050326 -1087 Example 5.17 - 2X35S/CPMV HT [construct no 1445 [, 2X3SS/CPMVHT+ 1construct no 1820] and CPMV160+ [construct no 1975] for HA B Wisconsin gPrL-[ id="p-282" id="p-282" id="p-282" id="p-282" id="p-282"
[00282] A coding sequence corresponding to HA from za B/Wisconsin/1/2010 with deleted proteolytic loop (PrL—) (see US provisional ation No.61/806,227 Filed March 28, 2013 for additional information re: deleted proteolytic loop regions in HA sequences, which is incorporated herein by reference) with his native signal peptide (HA B Wisconsin (PrL-)) (Figure 36AA, SEQ ID NO: 101) was cloned into original CPMV—HT, CPMVHT+, and CPMV160 10 using the same PCR—based method as described in es 5.7 and 5.11, but with modified PCR primers specifically designed for HA B Wisconsin (PrL-). The amino acid sequence of HA B Wisconsin (PrL-) with his native signal peptide is presented in Figure 36B (SEQ ID NO: 102). Representations of plasmid 1445, 1820 and 1975 are presented in Figures 36C, 36D and 36E, respectively. 15 Example 5.18 - 2X35S/CPMV HT [construct no 1454] and 2X3SS/CPMV160+ 1construct no 1893] for HA B Wisconsin gPrL-[+H1 California TMCT id="p-283" id="p-283" id="p-283" id="p-283" id="p-283"
[00283] A chimer lutinin coding sequence corresponding to the ectodomain of HA from Influenza B/ Wisconsin /2/2012 with d proteolytic loop (PrL—) (see US provisional application No.61/806,227 Filed March 28, 2013 for 20 additional information re: deleted lytic loop regions in HA sequences, which is incorporated herein by nce) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from influenza A/California/7/2009 with the native signal peptide of HA B Wisconsin (HA B Wisconsin (PrL-)+H1 California TMCT) (Figure 37A, SEQ ID NO: 103) was cloned into al CPMV-HT and CPMV160+ 25 using the same PCR—based method as described in Examples 5.7 and 5.11, but with modified PCR primers specifically designed for HA B Wisconsin (PrL—)+H1 rnia TMCT. The amino acid sequence of HA B sin (PrL—)+H1 California TMCT is presented in Figure 37 (SEQ ID NO: 104). Representations of plasmid 1454 and 1893 are presented in Figure 37C and 37D. 30 Example 5.19: 2X35S/CPMV HT [construct no 1067] and HT+ [construct no 1875] for PDISP/HA B ne gPrL-[+H1 California TMCT id="p-284" id="p-284" id="p-284" id="p-284" id="p-284"
[00284] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/Brisbane/60/08 with deleted proteolytic loop WO 2014/153674 PCT/CA2014/050326 -1097 (PrL-) fused to the embrane domain and cytoplasmic tail (TMCT) of H1 from influenza A/California/7/2009 and with the signal peptide of alfalfa protein disulfide ase (PDISP/HA B Brisbane (PrL-)+H1 California TMCT) (Figure 38A, SEQ ID NO: 105) was cloned into original HT and modified HT+ using the same PCR- based method as described in Example 5.26, but with modified PCR primers specifically designed for PDISP/HA B Brisbane (PrL-)+H1 rnia TMCT. The amino acid sequence of mature HA B Brisbane +H1 California TMCT fused with PDISP is presented in Figure 38B (SEQ ID NO: 106). Representations of plasmid 1067 and 1875 are presented in Figure 33C and 39C. 10 Example 5.20: 2X3SS/CPMV HT sconstruct no 2072) and HT+ gconstruct no 20522 for PDISP/HA B Massachussetts gPrL-l id="p-285" id="p-285" id="p-285" id="p-285" id="p-285"
[00285] A coding sequence ponding to HA from Influenza B/Massachussetts/2/2012 with deleted proteolytic loop (PrL—) in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase 15 (PDISP/HA B Massachussetts (PrL—)) (Figure 39A, SEQ ID NO: 107) was cloned into original HT and modified HT+ using the same PCR—based method as as described in e 5.26, but with modified PCR primers specifically designed for PDISP/HA B Massachussetts (PrL-). The amino acid sequence of mature HA B Massachussetts (PrL-) fused with PDISP is presented in Figure 39B (SEQ ID NO: 20 108). Representations of plasmid 2072 and 2052 are presented in Figure 34C and Figure 39C. e 5.21: 2X3SS/CPMV HT sconstruct no 2074) and HT+ gconstruct no 20622 for PDISP/HA B Massachussetts gPrL-[+H1 California TMCT id="p-286" id="p-286" id="p-286" id="p-286" id="p-286"
[00286] A chimer hemagglutinin coding sequence corresponding to the 25 ectodomain of HA from Influenza B/Massachussetts/2/2012 with deleted proteolytic loop (PrL-) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from influenza A/California/7/2009 and with the signal peptide of alfalfa protein disulfide isomerase /HA B Massachussetts (PrL-)+H1 rnia TMCT) (Figure 40A, SEQ ID NO: 109) was cloned into original HT and modified HT+ using 30 the same PCR—based method as as described in Example 5.26, but with ed PCR s specifically designed for PDISP/HA B Massachussetts (PrL-)+H1 California TMCT. The amino acid sequence of mature HA B Massachussetts (PrL—)+H1 WO 2014/153674 PCT/CA2014/050326 - 1107 California TMCT fused with PDISP is presented in Figure 40B (SEQ ID NO: 110).
Representations of plasmid 2074 and 2062 are presented in Figure 35C and Figure 40C.
Example 5.22: 2X3SS/CPMV HT ruct no 1445] and HT+ ruct no 1839] for HA B Wisconsin gPrL-[ id="p-287" id="p-287" id="p-287" id="p-287" id="p-287"
[00287] A coding sequence corresponding to HA from Influenza B/Wisconsin/1/2010 with d proteolytic loop (PrL-) with his native signal peptide (HA B Wisconsin (PrL-)) (Figure 41A, SEQ ID NO: 111) was cloned into original HT and modified HT+ using the same PCR—based method as described in 10 e 5.26, but with modified PCR primers specifically ed for HA B Wisconsin (PrL-). The amino acid sequence of HA B Wisconsin (PrL-) with his native signal peptide is ted in Figure 41B (SEQ ID NO: 112). Representations of plasmid 1445 and 1839 are presented in Figure 36C and 41C.
Example 5.23: 2X3SS/CPMV HT [construct no 1454] and HT+ [construct no 15 1860] for HA B sin gPrL-[+H1 California TMCT id="p-288" id="p-288" id="p-288" id="p-288" id="p-288"
[00288] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/ Wisconsin /2/2012 with deleted proteolytic loop (PrL-) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from influenza A/California/7/2009 with the native signal peptide of HA B Wisconsin (HA 20 B Wisconsin (PrL-)+H1 California TMCT) (Figure 42A, SEQ ID NO: 113) was cloned into original HT and modified HT+ using the same PCR—based method as described in Example 5.26 but with modified PCR primers specifically designed for HA B Wisconsin +H1 California TMCT. The amino acid sequence of HA B sin (PrL-)+H1 California TMCT is presented in Figure 42B (SEQ ID NO: 25 114). Representations of plasmid 1454 and 1860 are presented in Figure 37C and 42C.
Example 5.24 - 2X3SS/CPMV HT [construct no 489], 2X3SS/CPMV160+ 1construct no 1880] and 2X3SS/CPMV160 [construct no 1885] for H5 Indonesia id="p-289" id="p-289" id="p-289" id="p-289" id="p-289"
[00289] A coding sequence corresponding to native H5 from Influenza 30 A/Indonesia/5/2005 (Figure 43A, SEQ ID NO: 115) was cloned into original CPMV— HT, CPMV160+ and CPMV160 using the same PCR—based method as described in Example 5.25 but with ed PCR primers specifically designed for H5 Indonesia.
WO 2014/153674 PCT/CA2014/050326 -1117 The amino acid sequence of native H5 from Influenza A/Indonesia/5/2005 is presented in Figure 43B (SEQ ID NO: 116). Representation of plasmid 489 is presented in Figure 43C.
Example 5.25 - 2X35S/CPMV160+/PDISP/H3 Victoria/ NOS (Construct number 1800! id="p-290" id="p-290" id="p-290" id="p-290" id="p-290"
[00290] A sequence encoding H3 from Influenza A/Victoria/361/2011 in which the native signal peptide has been replaced by that of alfalfa n disulfide isomerase (PDISP/H3 Victoria) was cloned into 2X35 160+/NOS expression system (CPMV160+) using the following PCR-based . A fragment containing 10 the PDISP/H3 Victoria coding sequence was amplified using primers IF**(SacII)— PDI.s1+4c (Figure 44A, SEQ ID NO: 117) and IF-H3V361 1 l.s1-4r (Figure 44B, SEQ ID NO: 118), using PDISP/H3 Victoria sequence (Figure 44C, SEQ ID NO: 119) as template. The PCR product was cloned in 2X35 S/CPMV160+/NOS expression system using In-Fusion g system ech, Mountain View, CA). uct 15 number 2171 (Figure 44D) was ed with SacII and StuI ction enzyme and the linearized plasmid was used for the In—Fusion assembly reaction. Construct number 2171 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in a CPMV160+ based expression cassette. It also orates a gene construct for the ression of the TBSV P19 ssor of silencing under the 20 alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in Figure 44E (SEQ ID NO: 120). The resulting construct was given number 1800 (Figure 44F, SEQ ID NO: 121). The amino acid sequence of mature H3 from Influenza A/Victoria/361/2011 fused with PDISP is presented in Figure 44G (SEQ ID 25 NO: 122). A representation of plasmid 1800 is presented in Figure 44H.
Exam 1e 5.26: CPMV-HT+/ PDISP/H3 Victoria/ NOS Construct number 18192 id="p-291" id="p-291" id="p-291" id="p-291" id="p-291"
[00291] A sequence encoding H3 from Influenza A/Victoria/361/2011 in which the native signal peptide has been replaced by that of alfalfa protein disulfide 30 isomerase (PDISP/H3 Victoria) was cloned into 2X35 S-CPMV—HT+/NOS expression using the following PCR-based . A fragment containing the PDISP/H3 Victoria coding sequence was amplified using primers IF(SacII)-Kozac_PDI.c (Figure WO 2014/153674 PCT/CA2014/050326 -1127 45A, SEQ ID NO: 123) andIF—H3V36111.s1—4r (Figure 45B, SEQ ID NO: 124), using PDISP/H3 Victoria sequence (Figure 44C, SEQ ID NO: 119) as template. The PCR product was cloned in CPMV—HT+/NOS sion system using In— Fusion cloning system (Clontech, in View, CA). Construct number 2181 e 45D) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 2181 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in a CPMV- HT+ based expression cassette. It also incorporates a gene construct for the co— expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin 10 gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t—DNA borders is presented in Figure 45E (SEQ ID NO: 125). The resulting construct was given number 1819 (Figure 45E, SEQ ID NO: 126).
The amino acid sequence of mature H3 from Influenza A/Victoria/361/2011 fused with PDISP is ted in Figure 44G (SEQ ID NO: 122). A representation of 15 plasmid 1819 is presented in Figure 45F.
Exam 1e 5.27 2X3SS/CPMV HT+/ PDISP/H2 Sin a ore/ NOS Construct number 22201 ] A sequence encoding H2 from Influenza A/Singapore/1/1957 in which the native signal peptide has been replaced by that of alfalfa protein disulfide 20 isomerase (PDISP/H2 Singapore) was cloned into 2X35S/CPMV HT+/NOS sion system using the following PCR—based method. A fragment containing the PDISP/H2 Singapore coding sequence was amplified using primers IF(SacII)— Kozac_PDI.c (described for construct 1819 in Example 5.26) and IF**—H2S157.s1—6r (Figure 48A, SEQ ID NO: 127), using PDISP/H2 Singapore sequence e 48B, 25 SEQ ID NO : 128) as te. The PCR product was cloned in 2X35S/CPMV HT+/NOS expression system using In—Fusion cloning system (Clontech, Mountain View, CA). Construct number 2181 (described for construct 1819 in Example 5.26) was digested with SacII and StuI ction enzyme and the linearized plasmid was used for the In—Fusion assembly reaction. Construct number 2181 is an or 30 plasmid ed for "In Fusion" cloning of genes of interest in a CPMV HT+ based expression cassette. It also incorporates a gene construct for the co—expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and WO 53674 PCT/CA2014/050326 -1137 terminator. The backbone is a pCAMBLA binary plasmid and the ce from left to right t—DNA borders is presented in Figure 45D. The resulting construct was given number 2220 (Figure 48C, SEQ ID NO: 129). The amino acid sequence of mature H2 from Influenza A/Singapore/1/1957 fused with PDISP is presented in Figure 48D (SEQ ID NO: 130). A representation of plasmid 2220 is presented in Figure 48E.
Example 5.28 2X3SS/CPMV HT+/ PDISP/H2 Singapore with deleted proteolytic loop/ NOS gConstruct number 22211 id="p-293" id="p-293" id="p-293" id="p-293" id="p-293"
[00293] A sequence encoding H2 from Influenza A/Singapore/1/1957 with deleted proteolytic loop in which the native signal peptide has been replaced by that 10 of alfalfa protein ide isomerase (PDISP/H2 Singapore with deleted proteolytic loop) was cloned into 2X35S/CPMV S expression system using the following PCR-based ligation method presented by Darveau et al. ds in Neuroscience 26: 77—85 (1995)). In a first round of PCR, a fragment containing H2 from Influenza A/Singapore/1/1957 coding sequence from nt 1 to nt 1032 was 15 ied with primers IF(SacII)-Kozac_PDI.c ibed for construct 1819 in Example 5.26) and H2S157(Prl-).r (Figure 49A, SEQ ID NO: 131), using PDISP/H2 ore sequence (Figure 48B, SEQ ID NO: 128) as te. A second fragment, containing H2 from Influenza A/Singapore/1/1957 coding sequence from nt 1084 to nt 1716, was amplified with primers H2S157(Prl-).c (Figure 49B, SEQ ID NO : 132) 20 and IF**-H2S157.s1-6r (Figure 48A, SEQ ID NO: 127) using PDISP/H2 Singapore sequence e 48B, SEQ ID NO :128) as template. The PCR products from both amplifications were then mixed and used as template for a second round of amplification using IF(SacII)-Kozac_PDI.c (described for construct 1819 in Example 5.26) and IF**-H2S157.s1-6r (Figure 48A, SEQ ID NO: 127) as primers. The PCR 25 product (comprising PDISP/H2 Singapore coding sequence with aa 321 to 337 replaced by a GG linker) was cloned in CPMV HT+/NOS expression system using In—Fusion g system (Clontech, in View, CA). Construct number 2181 (described for construct 1819 in Example 5.26) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly 30 reaction. Construct number 2181 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in a CPMV HT+ based expression cassette. It also incorporates a gene construct for the co—expression of the TBSV P19 suppressor of WO 53674 PCT/CA2014/050326 —1147 silencing under the alfalfa Plastocyanin gene er and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in Figure 45D ibed for construct 1819, in Example 5.26). The resulting construct was given number 2221 e 49C, SEQ ID NO: 133). The amino acid sequence of mature H2 from Influenza A/Singapore/1/1957 with d lytic loop fused with PDISP is presented in Figure 49D (SEQ ID NO: 134). A representation of plasmid 2221 is presented in Figure 49E.
Exam le 5.29 PDISP/H2 Sin a ore Construct number 2222 and PDISP/H2 Sin a ore with deleted roteol tic 100 Construct number 2223 in 10 CPMV OS expression system id="p-294" id="p-294" id="p-294" id="p-294" id="p-294"
[00294] Sequences encoding H2 from Influenza A/Singapore/1/1957 with or without proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H2 Singapore and PDISP/H2 Singapore with d proteolytic loop) were cloned into 2X35 S/CPMV 160+/NOS expression 15 system using the same PCR—based method as construct 2220 and 2221, respectively, but using modified forward primer IF**(SacII)—PDI.sl+4c (described for construct 1800 in Example 5.25) for amplification and a different acceptor plasmid. Resulting PCR products were cloned in CPMV 160+/NOS expression system using In— Fusion cloning system ech, Mountain View, CA). Construct number 2171 20 (described for construct 1800 in Example 5.25) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 2171 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in a CPMV 160+ based expression cassette. It also incorporates a gene construct for the co—expression of the TBSV P19 suppressor of 25 silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in (described for uct 1800 in Example 5.25). The resulting constructs were given number 2222 for PDISP/H2 Singapore (Figure 50A, SEQ ID NO: 135) and 2223 for PDISP/H2 Singapore with d proteolytic loop (Figure 50B, SEQ ID 30 NO: 136). Representations of plasmid 2222 and 2223 are presented in figure 50C and 50D respectively.
WO 2014/153674 PCT/CA2014/050326 -1157 Example 5.30 2X3SS/CPMV HT+ (construct no 2019] and 160+ (construct no 2139] for PDISP/H3 Perth id="p-295" id="p-295" id="p-295" id="p-295" id="p-295"
[00295] A coding sequence corresponding to H3 from Influenza A/Perth/16/2009 in which the native signal peptide has been replaced by that of alfalfa n disulfide isomerase (PDISP/H3 Perth) (Figure 51A, SEQ ID NO: 137) was cloned into modified CPMV HT+ and 160+ using the same In —based approach as construct 2220 and 2222, respectively, but with ed PCR primer specifically designed for PDISP/H3 Perth (Figure 51B, SEQ ID NO: 138). The amino acid sequence of mature H3 from Influenza A/Perth/16/2009 fused with PDISP is 10 presented in Figure 51C (SEQ ID NO: 139). Representations of plasmid 2019 and 2139 are presented in Figure 51D and Figure 51E.
Example 5.31 2X3SS/CPMV HT+ (construct no 2039] and 160+ (construct no 2159] for PDISP/H3 Perth with d proteolytic loop id="p-296" id="p-296" id="p-296" id="p-296" id="p-296"
[00296] A coding sequence corresponding to H3 from Influenza 15 A/Perth/16/2009 with deleted proteolytic loop in which the native signal peptide has been replaced by that of a protein disulfide isomerase (PDISP/H3 Perth with deleted proteolytic 100p) (Figure 52, SEQ ID NO: 140) was cloned into modified CPMV HT+ and 160+ using the same In Fusion-based approach as uct 2221 and 2223, respectively, but with modified PCR primers specifically designed for 20 PDISP/H3 Perth with deleted proteolytic loop (Figure 51B (SEQ ID NO: 138), 52B (SEQ ID NO: 141) and 53C (SEQ ID NO: 142). The amino acid ce of mature H3 from Influenza A/Perth/16/2009 with deleted proteolytic loop fused with PDISP is presented in Figure 52D (SEQ ID NO: 143). entations of plasmid 2039 and 2159 are presented in Figure 52E and Figure 52F. 25 Example 5.32 2X3SS/CPMV HT+ (construct no 2230] and 160+ (construct no 2250] for PDISP/H3 Victoria with deleted proteolytic loop id="p-297" id="p-297" id="p-297" id="p-297" id="p-297"
[00297] A coding sequence ponding to H3 from Influenza A/Victoria/361/2011 with deleted proteolytic loop in which the native signal peptide has been replaced by that of alfalfa n disulfide isomerase (PDISP/H3 Victoria 30 with deleted proteolytic 100p) (Figure 53, SEQ ID NO: 144) was cloned into modified WO 2014/153674 PCT/CA2014/050326 -1167 CPMV HT+ and 160+ using the same In Fusion-based approach as uct 2221 and 2223 (see Examples 5.28 and 5.29), respectively, but with modified PCR primer specifically designed for PDISP/H3 ia with deleted proteolytic loop (Figures 53B (SEQ ID NO: 145) and 53C (SEQ ID NO: 146). The amino acid sequence of mature H3 from Influenza A/Victoria/361/2011 with deleted proteolytic loop fused with PDISP is ted in Figure 53D (SEQ ID NO: 147). Representations of plasmid 2230 and 2250 are presented in Figure 53E and Figure 53F.
Example 5.33 2X35S/CPMV HT+/PDISP/H7 Hangzhou/NOS gConstruct no 2142) 10 [00298] A coding sequence corresponding to H7 from Influenza A/Hangzhou/1/2013 in which the native signal e has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H7 Hangzhou) (Figure 54A, SEQ ID NO: 148) was cloned into modified CPMV HT+ using the same In Fusion-based approach as construct 2220 but with modified PCR primer specifically designed for H7 15 Hangzhou (Figure 54B, SEQ ID NO: 149). The amino acid sequence of mature H7 from Influenza A/Hangzhou/1/2013 fused with PDISP is presented in Figure 54C (SEQ ID NO: 150). Representation of plasmid 2142 is presented in Figure 54E.
Example 5.34 2X358/CPMV HT+/PDISP/H7 Hangzhou with deleted proteolytic loop/NOS ruct no 2152) 20 [00299] A coding ce corresponding to H7 from Influenza A/Hangzhou/1/2013 with deleted proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/ H7 Hangzhou with deleted proteolytic loop) (Figure 55A, SEQ ID NO: 151) was cloned into modified CPMV HT+ using the same In Fusion-based approach as construct 2221 but 25 with modified PCR primers specifically designed for PDISP/ H7 Hangzhou with deleted proteolytic loop (Figure 54B (SEQ ID NO: 149), Figure 55B (SEQ ID NO: 152) and Figure 55C (SEQ ID NO: 153)). The amino acid sequence of mature H7 from za A/Hangzhou/1/2013 with d proteolytic loop fused with PDISP is presented in Figure 55D (SEQ ID NO: 154). Representation of plasmid 2152 is 30 presented in Figure 55E.
WO 2014/153674 PCT/CA2014/050326 -1177 Example 5.35 2X3SS/CPMV HT+ [construct no 2224] and 160+ ruct no 2226] for PDISP/H9 Hong Kong id="p-300" id="p-300" id="p-300" id="p-300" id="p-300"
[00300] A coding sequence corresponding to H9 from Influenza A/Hong K0ng/1073/1999 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H9 Hong Kong) (Figure 56A, SEQ ID NO: 155) was cloned into modified CPMV HT+ and 160+ using the same In Fusion—based approach as construct 2220 and 2222, respectively, but with modified PCR primer specifically designed for PDISP/H9 Hong Kong (Figure 56B, SEQ ID NO: 156). The amino acid sequence of mature H9 from Influenza A/Hong 073/1999 fused 10 with PDISP is presented in Figure 56C (SEQ ID NO: 157). Representations of plasmid 2224 and 2226 are presented in Figures 56D and Figure 56E.
Example 5.36 2X3SS/CPMV HT+ [construct no 2225] and 160+ [construct no 2227 2 for PDISP/H9 Hong Kong with d proteolytic loop id="p-301" id="p-301" id="p-301" id="p-301" id="p-301"
[00301] A coding sequence corresponding to H9 from Influenza A/Hong 15 K0ng/1073/1999 with d proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase /H9 Hong Kong with deleted proteolytic 100p) (Figure 57A, SEQ ID NO: 158) was cloned into modified CPMV HT+ and 160+ using the same In Fusion-based approach as construct 2221 and 2223, respectively, but with modified PCR primers specifically 20 designed for PDISP/H9 Hong Kong with deleted lytic 100p (Figure 56B (SEQ ID NO: 156), Figure 57B (SEQ ID NO: 159) and Figure 57C (SEQ ID NO: 160)).
The amino acid sequence of mature H9 from Influenza A/Hong K0ng/1073/1999 with deleted proteolytic 100p fused with PDISP is presented in Figure 57D (SEQ ID NO: 161). Representations of plasmid 2225 and 2227 are presented in Figure 57E and 25 Figure 57F.
Example 5.37 2X3SS/CPMV 160+/PDISP/HA B Malaysia/NOS [Construct no 2013] id="p-302" id="p-302" id="p-302" id="p-302" id="p-302"
[00302] A coding sequence corresponding to HA from za ysia/2506/2004 in which the native signal e has been replaced by that of 30 alfalfa protein disulfide isomerase /HA B Malaysia) (Figure 58A, SEQ ID WO 2014/153674 PCT/CA2014/050326 -1187 NO: 162) was cloned into modified CPMV 160+ using the same In Fusion—based ch as construct 2222 but with modified PCR primer specifically designed for PDISP/HA B Malaysia (Figure 58B, SEQ ID NO: 163). The amino acid sequence of mature HA from Influenza B/Malaysia/2506/2004 fused with PDISP is presented in Figure 58C (SEQ ID NO: 164). Representation of plasmid 2013 is presented in Figure 58D.
Example 5.38 2X3SS/CPMV 160+/PDISP/HA B ia with deleted proteolytic loop/NOS (Construct no 2014) id="p-303" id="p-303" id="p-303" id="p-303" id="p-303"
[00303] A coding sequence corresponding to HA from Influenza 10 B/Malaysia/2506/2004 with deleted proteolytic loop in which the native signal peptide has been replaced by that of a protein disulfide isomerase (PDISP/HA B Malaysia with deleted proteolytic loop) (Figure 59A, SEQ ID NO: 165) was cloned into modified CPMV 160+ using the same In Fusion—based approach as construct 2223 but with modified PCR s specifically designed for PDISP/HA B ia 15 with deleted proteolytic loop e 58B (SEQ ID NO: 163), Figure 59B (SEQ ID NO: 166), Figure 59C (SEQ ID NO: 167). The amino acid sequence of mature HA from Influenza B/Malaysia/2506/2004 with deleted proteolytic loop fused with PDISP is presented in Figure 59D (SEQ ID NO: 168). Representation of plasmid 2014 is presented in Figure 59E. 20 Example 5.39 2X358/CPMV HT (construct no 20702, HT+ (construct no 20801 and 160+ (-Mprot[ ruct no 2090) for PDISP/HA B Massachusetts id="p-304" id="p-304" id="p-304" id="p-304" id="p-304"
[00304] A coding sequence corresponding to HA from Influenza B/Massachusetts/2/2012 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/ HA B Massachusetts) (Figure 60A, 25 SEQ ID NO: 169) was cloned into original HT, modified HT+ and 160+ using the same In Fusion-based ch as construct 2072, 2220 and 2222, respectively, but with modified PCR primers specifically designed for PDISP/ HA B Massachusetts.
The amino acid sequence of mature HA from Influenza B/Massachusetts/2/2012 fused with PDISP is presented in Figure 60B (SEQ ID NO: 170). entations of 30 plasmid 2070, 2080 and 2090 are presented in Figure 60C, Figure 60D and Figure 60E.
WO 2014/153674 PCT/CA2014/050326 -1197 Exam le 5.40 2X35S/CPMV HT+ construct no 2102 HT+ with BeYDV gconstruct no 21042 for PDISP/HA B Florida with deleted proteolytic loop id="p-305" id="p-305" id="p-305" id="p-305" id="p-305"
[00305] A coding sequence corresponding to HA from Influenza B/Florida with the proteolytic loop deleted and in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/ HA B Florida) (Figure 61D, SEQ ID NO: 193) was cloned into modified HT+ the same In Fusion—based approach as described above, but with modified PCR primers specifically ed for PDISP/ HA ida (see Figures Fig 61A (SEQ ID N0:l90), Fig 61B (SEQ ID NO:191) and Fig 61C (SEQ ID NO: 192). The nucleotide sequence of the resulting 10 expression te 2102 is given in Fig 61F (SEQ ID NO: 195). Similarly, a coding sequence corresponding to HA from Influenza B/Florida with the proteolytic loop deleted and in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase was cloned into modified HT+ together with amplification element BeYDV. The tide sequence of the ing expression cassette 2104 is 15 given in Fig 61F (SEQ ID NO: 196). The amino acid sequence of mature HA fiom Influenza B/Florida with deleted proteolytic loop fused with PDISP is presented in Figure 61E (SEQ ID NO: 194). Representations of plasmid 2102 and 2104 are presented in Figure 61G and 611.
Example 5.41 2X3SS/CPMV HT+ ruct no 2106), HT+ with BeYDV 20 1construct no 2108! for PDISP/HA B Florida +H1 California TMCT with d lytic loop id="p-306" id="p-306" id="p-306" id="p-306" id="p-306"
[00306] A coding sequence corresponding to HA from Influenza B/Florida +H1 California TMCT with the proteolytic 100p deleted and in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase / 25 HA B Florida +H1 California TMCT) (Figure 62B, SEQ ID NO: 198) was cloned into modified HT+ the same In Fusion—based ch as described above, but with modified PCR primers specifically designed for PDISP/ HA B/Florida+Hl California TMCT (see Figures Fig 61A (SEQ ID N01197). The nucleotide sequence of the resulting expression cassette 2106 is given in Fig 62D (SEQ ID NO: 200). Similarly, 30 a coding sequence corresponding to HA from za B/Florida +H1 California TMCT with the proteolytic 100p deleted and in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase was cloned into d HT+ together with amplification element BeYDV. The nucleotide sequence of the WO 2014/153674 PCT/CA2014/050326 - 1207 resulting expression cassette 2108 is given in Fig 62F (SEQ ID NO: 201). The amino acid sequence of mature HA from Influenza B/Florida+H1 California TMCT with deleted proteolytic loop fused with PDISP is ted in Figure 62C (SEQ ID NO: 199). Representations of plasmid 2106 and 2108 are presented in Figure 62E and 62G. id="p-307" id="p-307" id="p-307" id="p-307" id="p-307"
[00307] All citations are hereby incorporated by reference. id="p-308" id="p-308" id="p-308" id="p-308" id="p-308"
[00308] The present invention has been described with regard to one or more ments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the 10 ion as defined in the claims.

Claims (13)

WHAT IS CLAIMED IS:
1. A nucleic acid comprising a regulatory region active in a plant and an expression enhancer active in a plant, in the absence of a Bean Yellow Dwarf Virus (BeYDV) amplification element, the regulatory region and the expression enhancer operatively linked 5 to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) selected from influenza type B HA and influenza type A subtype H3 and H7 HA, the ed HA sing a fully deleted proteolytic loop between subunits HA1 and HA2, the fully deleted proteolytic loop comprising a monobasic or a multi-basic cleavage site, wherein the expression enhancer is not T. 10
2. The nucleic acid of claim 1, n the expression enhancer is selected from the group consisting of CPMVX, , CPMV-HT+, CPMV HT+[WT115] and CPMV HT+ [511], wherein: CPMVX comprises X tides of SEQ ID NO:93, where X=160, 155, 150, or 114 of SEQ ID NO:93, or a sequence that comprises between 80% to 100% sequence 15 rity with CPMVX, where X=160, 155, 150, or 114 of SEQ ID NO:93, CPMVX+ ses X nucleotides of SEQ ID NO:93, where X=160, 155, 150, or 114 of SEQ ID NO:93, or a sequence that comprises between 80 to 100% sequence similarity with CPMVX, where X=160, 155, 150, or 114 of SEQ ID NO:93, and a stuffer sequence ses from 1-100 nucleotides fused to the 3’ end of the CMPVX sequence, 20 CPMV-HT+ comprises a comovirus 5’ untranslated region (UTR) and a modified, lengthened, or truncated stuffer sequence, CPMV HT+[WT115] comprises the sequence of SEQ ID NO:189, and CPMV HT+ [511] comprises the sequence of SEQ ID NO:188.
3. The nucleic acid of claim 1, wherein the modified HA is maintained as a HA0 25 precursor.
4. The nucleic acid of claim 1, wherein the proteolytic loop is fully replaced by a linker sequence.
5. The nucleic acid of claim 4, wherein the linker sequence has the amino acid sequence GG, TETQ or TETR.
6. The nucleic acid of claim 1, n the nucleotide sequence encoding the influenza hemagglutinin (HA) protein has at least 80% ce ty to an amino acid sequence 5 selected from the group consisting of amino acids 25 to 576 of SEQ ID NOs: 98 and 100, amino acids 25 to 559 of SEQ ID NO: 143, amino acids 25 to 559 of SEQ ID NO: 147, amino acids 25 to 549 of SEQ ID NO: 154, amino acids 25 to 577 of SEQ ID NO: 168, and amino acids 25 to 576 of SEQ ID NOs: 194 and 199.
7. The nucleic acid of claim 1, wherein the modified HA comprises a native HA signal 10 peptide or a non-native HA signal peptide.
8. The nucleic acid of claim 1, wherein the nucleotide sequence encoding the modified HA comprises a chimeric nucleotide sequence encoding, in series, a modified HA ectodomain comprising a fully deleted proteolytic loop, an influenza transmembrane domain, and a cytoplasmic tail, wherein the modified HA ectodomain is from a first influenza strain 15 and the transmembrane domain and the cytoplasmic tail are from a second nza strain.
9. The nucleic acid of claim 1, wherein the modified HA is influenza type A subtype H3 and the fully deleted proteolytic loop comprises the sequence of SEQ ID NO: 48.
10. The nucleic acid of claim 1, wherein the modified HA is influenza type B and the fully d proteolytic loop comprises the sequence of SEQ ID NO: 59. 20 11. The nucleic acid of claim 1, wherein the modified HA is nza type A subtype
11. H7 and the fully deleted proteolytic loop comprises the ce of amino acids 340 to 357 of SEQ ID NO: 150.
12. The c acid of claim 1, n the monobasic cleavage site is recognized by a Clara-like protease. 25
13. The nucleic acid of claim 12, wherein the Clara-like protease is tryptase Clara or trypsin/chymotrypsin.
NZ712752A 2013-03-28 2014-03-28 Influenza virus-like particle production in plants NZ712752B2 (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US201361806227P 2013-03-28 2013-03-28
US61/806,227 2013-03-28
US201461925852P 2014-01-10 2014-01-10
US61/925,852 2014-01-10
US201461971274P 2014-03-27 2014-03-27
US61/971,274 2014-03-27
PCT/CA2014/050326 WO2014153674A1 (en) 2013-03-28 2014-03-28 Influenza virus-like particle production in plants

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NZ712752A NZ712752A (en) 2021-06-25
NZ712752B2 true NZ712752B2 (en) 2021-09-28

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