Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
  • C12N7/04—Inactivation or attenuation; Producing viral sub-units
  • C12N7/045—Pseudoviral particles; Non infectious pseudovirions, e.g. genetically engineered

Definitions

• the present invention relates to the expression of proteins of interest in plants.
• the present invention also provides methods and compositions for the production of proteins of interest in plants.
• Plants offer great potential as production systems for recombinant proteins.
• One approach to producing foreign proteins in plants is to generate stable transgenic plant lines. However this is a time consuming and labor intensive process.
• An alternative to transgenic plants is the use of plant virus-based expression vectors. Plant virus-based vectors allow for the rapid, high level, transient expression of proteins in plants.
• RNA plant viruses including
• comoviruses such as Cowpea mosaic virus (CPMV; see, for example,
• Comoviruses are RNA viruses with a bipartite genome.
• the segments of the comoviral RNA genome are referred to as RNA- 1 and RNA-2.
• RNA- 1 encodes the VPg, replicase and protease proteins.
• the replicase is required by the virus for replication of the viral genome.
• the RNA-2 of the como virus cowpea mosaic virus (CPMV) produces a polyprotein of 105 kDa or 95 kDa processed into 4 functional peptides.
• the 5' region of CPMV RNA-2 comprises start codons (AUGs) at positions 115, 161, 512 and 524.
• the start codons at positions 161 and 512 are in the same triplet reading frame. Initiation at the start codon at position 161 results in the synthesis of the 105K polyprotein while initiation at the start codon at position 512 directs the synthesis of the 95K polyprotein. Initiation of translation at the start codon at position 512 in CPMV is more efficient than initiation at position 161, resulting in the production of more 95K polyprotein than 105K polyprotein.
• the start codon at position 115 is not essential for virus replication (Wellink et al., 1993 Biochimie. 75(8):741-7).
• CPMV has served as the basis for the development of vector systems suitable for the production of heterologous polypeptides in plants (Liu et al., 2005 Vaccine 23, 1788-1792; Sainsbury et al., 2007 Virus Expression Vectors (Hefferon, K. ed), pp. 339-555). These systems are based on the modification of RNA-2 but differ in whether full-length or deleted versions are used. Replication of the modified RNA-2 is achieved by co-inoculation with RNA-1. Foreign proteins are fused to the C- terminus of the RNA-2-derived polyproteins.
• RNA-2 molecules are capable of spreading both within and between plants.
• This strategy has been used to express a number of recombinant proteins, such as the Hepatitis B core antigen (HBcAg) and Small Immune Proteins (SIPs), in cowpea plants
• RNA-2 replaced the majority of the coding region of RNA-2 with a sequence of interest to produce a disabled version of CPMV RNA-2 (delRNA- 2).
• the sequence to be expressed was fused to the AUG at position 512 of RNA-2, immediately upstream of the 3' untranslated region (UTR) to create a molecule that mimics RNA-2.
• UTR 3' untranslated region
• Such constructs were capable of replication when introduced into plants in the presence of RNA-1 and a suppressor of silencing, and directed the synthesis of substantial levels of heterologous proteins (Sainsbury et al., 2008 Plant Biotechnol J 6:82-92).
• the sequence to be expressed is positioned between the 5'UTR and the 3' UTR.
• the 5'UTR in the pEAQ series carries the U162C (HT) mutation.
• the present invention relates to the expression of proteins of interest in plants.
• the present invention also provides methods and compositions for the production of proteins of interest in plants.
• the expression enhancer may comprise a nucleotide sequence selected from the group of SEQ ID NO: 24, 27, 68, 69, 70 and 71.
• the stuffer sequence may comprise comprises a length from 0 to about 100 nucleotides, or any length therein between, one or more plant kozak sequences, a multiple cloning site, one or more linker sequences, one or more recombination sites, or a combination thereof.
• the present invention also provides the expression enhancer as defined above, where the kozak sequence is selected from the group of sequences as shown in SEQ ID NO's: 5 - 17.
• the expression enhancer as just defined may comprise a nucleotide sequence selected from the group of SEQ ID NO: 2, 72, 73, 74, 75, 76 and 77.
• a plant expression system comprising a nucleic acid sequence comprising a regulatory region, operatively linked with the expression enhancer CPMVX, CPMVX+, as defined above, the expression enhancer operatively linked with a nucleotide sequence of interest.
• the plant expression system may further comprising a como virus 3' UTR.
• the plant expression system may further comprise a second nucleic acid sequence encoding a suppressor of silencing, for example HcPro or pl9.
• the nucleotide sequence of interest of the plant expression system as defined above may encodes a viral protein or an antibody.
• the viral protein may be an influenza hemagglutinin and may be s selected from the group of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, H16, and an influenza type B hemagglutinin.
• the nucleotide sequence encoding the viral protein or the antibody may comprise a native signal peptide sequence, or a non-native signal peptide, for example the non-native signal peptide may be obtained from Protein disulfide isomerase (PDI).
• PDI Protein disulfide isomerase
• a method of producing a protein of interest in a plant or in a portion of a plant comprising, introducing into the plant or in the portion of a plant the plant expression system comprising CPMVX or CPMVX+, as defined above, and incubating the plant or the portion of a plant under conditions that permit expression of the nucleotide sequence encoding the protein of interest.
• the present invention also provides a plant or portion of a plant transiently transfected or stably transformed with the plant expression system as described above.
• Plant-based expression systems as described herein result in increasing or enhancing expression of a nucleotide sequence encoding a heterologous open reading frame that is operatively linked to the expression enhancer, either CPMVX, or CPMVX+ as defined herein.
• the increase in expression may be determined by comparing the level of expression obtained using the CPMVX based, or CPMVX+ based expression enhancers with the level of expression of the same nucleotide sequence encoding the heterologous open reading frame operatively linked to the prior art enhancer sequence (CPMV HT) comprising an incomplete M protein (as described in Sainsbury F., and Lomonossoff G.P., 2008, Plant Physiol. 148: pp. 1212- 1218; which is incorporated herein by reference).
• An example of a prior art CPMV HT sequence is provided in SEQ ID NO:4.
• the plant based expression systems as described herein may also have a number of properties such as, for example, containing convenient cloning sites for genes or nucleotide sequences of interest, may easily infect plants in a cost-effective manner, may cause efficient local or systemic infection of inoculated plants. In addition, the infection should provide a good yield of useful protein material.
• FIGURE 1A shows a general schematic of an example 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 described herein.
• CPMCX and CPMVX+ are each shown as operatively linked to plant regulatory region at their 5 'ends, and at their 3' ends, in series, a nucleotide sequence of interest (including an ATG initiation site and STOP site), a 3'UTPv, and a terminator sequence.
• An example of construct CPMVX as described herein, is CPMV160.
• FIGURE IB shows examples of several variants of constructs comprising enhancer sequences, as described herein (CPMV160, complete sequence provided as SEQ ID NO:l; CPMV155, complete sequence provided as SEQ ID NO:24; CPMV150, complete sequence provided as SEQ ID NO:27; and
• CPMV114 complete sequence provided as SEQ ID NO:68), operatively linked to plant regulatory region (in these non-limiting examples 2X35 S) at their 5 'ends, and at their 3' ends, a nucleotide sequence of interest, or "GOI", which includes a plant kozak sequence adjacent to the ATG initiation site (elements shown within the square brackets are include for context, and they are not part of the CPMVX or CPMVX+ enhancer sequences).
• GOI nucleotide sequence of interest
• FIGURE 1C shows examples of several variants of constructs comprising enhancer sequences, as described herein (CPMV160+, complete sequence provided as SEQ ID NO:2; CPMV155+, complete sequence provided as SEQ ID NO:72; CPMV150+, complete sequence provided as SEQ ID NO:73; and
• CPMV114+ complete sequence provided as SEQ ID NO: 74
• plant regulatory region in these non-limiting examples 2X35 S
• stuffer fragment in these non-limiting examples, comprising a multiple cloning site and plant kozak sequence
• GOI nucleotide sequence of interest
• FIGURE 2 shows the relative hemagglutination titre (HMG) in crude protein extracts of proteins produced in plants comprising CPMV-HT (prior art) expression constructs, and CPMV160+ based expression constructs, operatively linked with a nucleotide sequence of interest.
• HMG hemagglutination titre
• A/California/07/2009 with a PDI signal peptide (PDI-H1 Cal; construct number 484 5' UTR: CPMV HT; and construct number 1897, 5'UTR: CPMV160+; see Example 5), H3 A/Victoria/361/2011 with a PDI signal peptide (PDI-H3 Vic; construct number 1391, 5'UTR: CPMV HT; and construct number 1800, 5'UTR: CMPV160+; see Examples 1 and 2, respectively), H5 from Influenza A/Indonesia/5/2005 with a native signal peptide (WtSp-H5 Indo; construct number 489, 5'UTR: CMPV HT; and construct number 1880, 5'UTR: CMPV160+; see Example 6), and B/Wisconsin/1/2010 with deleted proteolytic loop and with a native signal peptide (WtSp-B Wis-PrL; construct number 1445, 5'UTR
• FIGURE 3 shows the relative hemagglutination titres (HMG) in crude protein extracts of proteins produced in plants comprising CPMV-HT (prior art) expression constructs, and CPMV160+ based expression constructs.
• HMG hemagglutination titres
• FIGURE 4A shows examples of variants of plant Kozak sequences tested.
• Constructs showing a partial sequence of the CPMVX+, a plant regulatory region, a stuffer fragment, and a nucleotide sequence of interest (GOI).
• the construct comprises a 2X35 S regulatory region, CPMV160+, a stuffer fragment comprising a multiple cloning site and a plant kozak sequence (the 5 'end of a nucleotide sequence of interest is also indicated: "ATG...GOI"; where the GOI is H3 A/Victoria/361).
• Variants of plant kozak sequences are also shown below the sequence (also see Figure 9).
• FIGURE 4B shows HA titers of a nucleotide sequence of interest produced in plants comprising CMPV160+ expression construct and a variant plant Kozak sequence as indicated.
• FIGURE 5 shows the expression of the antibody rituximab (Rituxan) under the control of CPMV-HT (construct numbers 5001 and 5002, see examples 15 and 16) or CPMV160 (construct numbers 2100 and 2109, see example 15 and 16) and with either its native signal peptide or the native signal peptide replaced with the signal peptide of Protein disulfide isomerase (PDI).
• CPMV-HT construct numbers 5001 and 5002, see examples 15 and 16
• CPMV160 construct numbers 2100 and 2109, see example 15 and 16
• PDI Protein disulfide isomerase
• FIGURE 6 shows the sequence components used to prepare construct number 1391(A-2X35S CPMV-HT PDISP H3 Victoria NO S; see example 1).
• Construct number 1391 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 (PDISP/H3 Victoria)).
• PDISP protein disulfide isomerase signal peptide.
• NOS nopaline synthase terminator.
• FIGURE 6B shows primer sequence IF-H3V36111.sl-4r (SEQ ID NO: 17).
• FIGURE 6C shows the sequence of PDISP/H3 Victoria (SEQ ID NO: 18).
• FIGURE 6D shows a schematic representation of construct 1191.
• FIGURE 6E shows construct 1191; from left to right t-DNA borders (underlined), 2X35S CPMV-HT NOS, with Plastocyanine-P19-Plastocyanine silencing inhibitor expression cassette (SEQ ID NO: 19).
• FIGURE 6F shows expression cassette number 1391 from 2X35S promoter to NOS terminator.
• the PDISP/H3 Victoria nucleotide sequence is underlined; CPMV 5'UTR in bold;
• FIGURE 6G shows the amino acid sequence of PDISP/H3 Victoria (SEQ ID NO:21).
• FIGURE 6H shows a schematic representation of construct number 1391 (a reference construct).
• FIGURE 7 shows the sequence components used to prepare construct number 1800 (A-2X35S CPMV160+ PDISP H3 Victoria NO S; see example 2).
• PDISP protein disulfide isomerase signal peptide.
• NOS nopaline synthase terminator.
• FIGURE 7A shows primer sequence IF**(SacII)-PDI.sl+4c (SEQ ID NO:22).
• FIGURE 7B shows primer sequence IF-H3V36111.sl-4r (SEQ ID NO:23).
• the sequence of PDISP/H3 Victoria is shown in Figure 6C (SEQ ID NO: 18).
• FIGURE 7C shows a schematic representation of construct 2171 (SacII and Stul restriction enzyme sites used for plasmid linearization are indicated).
• FIGURE 7D shows construct 2171 from left to right t-DNA borders (underlined), 2X35S/CPMV160+/NOS with Plastocyanine-P19- Plastocyanine silencing inhibitor expression cassette, an HI California
• FIGURE 7E shows expression cassette number 1800 from 2X35S promoter to NOS terminator.
• PDISP/H3 Victoria nucleotide sequence is underlined; 5'UTR is shown in bold; plant kozak sequence double underline; a stuffer fragment (multiple cloning site) of 16 base pairs is positioned between the 5'UTR and plant kozak sequence (SEQ ID NO:26).
• the amino acid sequence of PDISP/H3 Victoria is shown in Figure 6G (SEQ ID NO:27).
• FIGURE 8 shows the sequence components used to prepare construct number 1935 (2X35S/CPMV160/ PDISP/H3 Victoria/ NOS; see example 3).
• PDISP protein disulfide isomerase signal peptide.
• NOS nopaline synthase terminator.
• FIGURE 8A shows primer sequence IF-CPMV(fl5'UTR)_SpPDI.c (SEQ ID NO:28).
• FIGURE 8B shows a schematic representation of construct 1190.
• FIGURE 8C shows the nucleic acid sequence of construct 1190 from left to right t-DNA borders (underlined), 2X35S/CPMV160/NOS with Plastocyanine-P19-Plastocyanine silencing inhibitor expression cassette, and a CPMV3'UTR (SEQ ID NO:29).
• FIGURE 8D shows expression cassette number 1935 from 2X35S promoter to NOS terminator. PDISP/H3 Victoria nucleotide sequence is underlined, 5'UTR is shown in bold (SEQ ID NO:30). This cassette does not include a plant kozak sequence or a stuffer fragment (multiple cloning site).
• FIGURE 9 shows sequences comprising variations in a plant kozak sequence used to prepare a selection of "CPMV160+” based constructs (constructs number 1992 to 1999). Variation of sequence between SacII restriction site and ATG of PDISP/H3 Victoria in 2X35S/CPMV160+/ OS expression system, comprising variations in a plant kozak sequence are shown (the sequences are shown as variations from the corresponding sequence from construct 1800; see Example 4). The variant plant kozak sequence are underlined.
• PDISP protein disulfide isomerase signal peptide.
• FIGURE 9A shows the nucleotide sequence of IF-HTl*(-Mprot)-PDI.c (SEQ ID NO: 31; used to prepare construct number 1992).
• FIGURE 9B shows the nucleotide sequence of IF-HT2*(-Mprot)-PDI.c (SEQ ID NO:32; used to prepare construct number 1993).
• FIGURE 9C shows the nucleotide sequence of IF-HT3*(- Mprot)-PDI.c (SEQ ID NO:33; used to prepare construct number 1994).
• FIGURE 9D shows the nucleotide sequence of IF-HT4*(-Mprot)-PDI.c (SEQ ID NO:34; used to prepare construct number 1995).
• FIGURE 9E shows the nucleotide sequence of IF-HT5*(-Mprot)-PDI.c (SEQ ID NO:35; used to prepare construct number 1996).
• FIGURE 9F shows the nucleotide sequence of IF-HT6*(-Mprot)-PDI.c (SEQ ID NO:36 used to prepare construct number 1997).
• FIGURE 9G shows the nucleotide sequence of IF-HT7*(-Mprot)-PDI.c (SEQ ID NO:37; used to prepare construct number 1998).
• FIGURE 9H shows the nucleotide sequence of IF-HT8*(-Mprot)- PDI.c (SEQ ID NO:38; used to prepare construct number 1999).
• FIGURE 91 shows a schematic representation of construct number 1992 comprising a plant kozak sequence (Kozakl) using SEQ ID NO:31 (FIGURE 9A).
• Constructs 1993 -1999 comprise the same features as construct 1992, except that each construct (1993-1999) comprises a modified plant Kozak sequence (Kozakl) as shown in Figures 9B to 9H (SEQ ID NOs: 32 to 38), respectively.
• FIGURE 10 shows sequence components used to prepare construct numbers 484 and 1897 (2X35S/CPMV HT PDISP/Hl California NOS and
• Construct number 484 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 (PDISP/Hl California).
• FIGURE 10A shows the nucleotide sequence of PDISP/Hl California (SEQ ID NO: 39).
• FIGURE 10B shows the amino acid sequence of PDISP/Hl California (SEQ ID NO: 40).
• FIGURE IOC shows a schematic representation of construct number 484 (2X35S/CPMV HT;
• FIGURE 11 shows sequence components used to prepare construct numbers 489, 1880 and 1885 (2X35S/CPMV HT H5 Indonesia NOS; CPMV160+ H5
• Construct number 489 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 (PDISP/Hl California).
• FIGURE 11A shows the nucleotide sequence of native H5 Indonesia (SEQ ID NO: 41).
• FIGURE 11B shows the amino acid sequence of native H5 Indonesia (SEQ ID NO: 42).
• FIGURE 11C shows a schematic representation of construct number 489 (2X35S/CPMV HT; reference constrcut).
• FIGURE 12 shows sequence components used to prepare construct numbers 1240 and 2168 (2X35S/CPMV HT PDISP/H7 Hangzhou NOS and
• Construct number 1240 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 (PDISP/H7 Hangzhou).
• FIGURE 12A shows the nucleotide sequence of PDISP/H7 Hangzhou (SEQ ID NO: 43).
• FIGURE 12B shows the amino acid sequence of PDISP/H7 Hangzhou (SEQ ID NO: 44).
• FIGURE 12C shows a schematic representation of construct number 2140 (2X35S/CPMV HT; reference construct).
• FIGURE 13 shows sequence components used to prepare construct numbers 2130 and 2188 (2X35S/CPMV HT PDISP/H7 Hangzhou+H5 Indonesia TMCT NOS and 2X35S/CPMV160+ PDISP/H7 Hangzhou+H5 Indonesia TMCT NOS, respectively; see Example 8).
• Construct number 2130 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
• PDISP protein disulfide isomerase signal peptide
• NOS nopaline synthase terminator
• TMCT transmembrane domain cytoplasmic tail.
• FIGURE 13A shows the nucleotide sequence of PDISP/H7 Hangzhou+H5 Indonesia TMCT (SEQ ID NO: 45).
• FIGURE 13B shows the amino acid sequence of PDISP/H7
• FIGURE 13C shows a schematic representation of construct number 2130 (2X35S/CPMV HT; reference construct).
• FIGURE 14 shows sequence components used to prepare construct numbers 1039 and 1937 (2X35S/CPMV HT PDISP/HA B Brisbane (PrL-) NOS and
• Construct number 1039 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 (PDISP/HA B Brisbane (PrL-)).
• PDISP protein disulfide isomerase signal peptide
• NOS nopaline synthase terminator
• PrL- deleted proteolytic loop.
• FIGURE 14A shows the nucleotide sequence of PDISP/HA B Brisbane (PrL-) (SEQ ID NO: 47).
• FIGURE 14B shows the amino acid sequence of PDISP/HA B Brisbane (PrL-) (SEQ ID NO: 48).
• FIGURE 14C shows a schematic representation of construct number 1039 (2X35S/CPMV HT; reference construct).
• FIGURE 14D shows a schematic representation of construct number 1937
• FIGURE 15 shows sequence components used to prepare construct numbers 1067 and 1977 (2X35S/CPMV HT PDISP/HA B Brisbane (Prl-)+Hl California TMCT NOS and 2X35S/CPMV160+ PDISP/HA B Brisbane (PrL-)+Hl California TMCT NOS, respectively; see Example 10).
• Construct 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 (PDISP/HA B Brisbane (PrL-)+Hl California TMCT).
• CMPV 5'UTR with mutated start codon at position 161 fused to a sequence encoding an incomplete M protein does not comprise a heterologous kozak sequence between the 5'UTR and the nucleotide sequence of interest (PDISP/HA B Brisbane (PrL-)+Hl California TMCT).
• PDISP protein disulfide isomerase signal peptide
• NOS nopaline synthase terminator
• PrL- deleted proteolytic loop
• TMCT transmembrane domain cytoplasmic tail.
• FIGURE 15A shows the nucleotide sequence of PDISP/HA B Brisbane (PrL-)+Hl California TMCT (SEQ ID NO: 49).
• FIGURE 15B shows the amino acid sequence of PDISP/HA B Brisbane (PrL-)+Hl California TMCT (SEQ ID NO: 50).
• FIGURE 15C shows a schematic representation of construct number 1067 (2X35S/CPMV HT; reference construct).
• FIGURE 15D shows a schematic representation of construct number 1977
• FIGURE 16 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 Example 11).
• Construct number 2072 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 (PDISP/HA B
• PDISP protein disulfide isomerase signal peptide
• NOS nopaline synthase terminator
• PrL- deleted proteolytic loop.
• FIGURE 16A shows the nucleotide sequence of
• FIGURE 16B shows the amino acid sequence of PDISP/HA B Massachusetts (PrL-) (SEQ ID NO: 52).
• FIGURE 16C shows a schematic representation of construct number 2072
• FIGURE 17 shows sequence components used to prepare construct numbers 2074 and 2060 (2X35S/CPMV HT PDISP/HA B Massachusetts (PrL-)+Hl California TMCT NOS and 2X35S/CPMV160+ PDISP/HA B Massachusetts (PrL-)+Hl California TMCT NOS, respectively; see Example 12).
• 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 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-)+Hl California TMCT).
• PDISP protein disulfide isomerase signal peptide
• NOS nopaline synthase terminator
• PrL- deleted proteolytic loop
• TMCT transmembrane domain cytoplasmic tail.
• FIGURE 17A shows the nucleotide sequence of PDISP/HA B Massachusetts (PrL-)+Hl California TMCT (SEQ ID NO: 53).
• FIGURE 17B shows the amino acid sequence of
• FIGURE 17C shows a schematic representation of construct number 2074
• FIGURE 18 shows sequence components used to prepare construct numbers 1445, 1820 and 1975 (2X35S/CPMV HT HA B Wisconsin (PrL-) NOS,
• 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 and the nucleotide sequence of interest (HA B Wisconsin (PrL-)).
• Construct number 1975 includes a CPMV 5'UTR comprising 160 nucleotides, and does not include a stuffer fragment (multiple cloning site), or a plant 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 18A shows the nucleotide sequence of HA B Wisconsin (PrL-) (SEQ ID NO: 55).
• FIGURE 18B shows the amino acid sequence of HA B Wisconsin (PrL-) (SEQ ID NO: 56).
• FIGURE 18C shows a schematic representation of construct number 1445 (2X35S/CPMV HT; reference construct).
• FIGURE 18D shows a schematic representation of construct number 1820 (2X35S/CPMV160+; a CPMVX+ based construct).
• FIGURE 19 shows sequence components used to prepare construct numbers 1454 and 1893 (2X35S/CPMV HT HA B Wisconsin (PrL-)+Hl California TMCT NOS and 2X35S/CPMV160+ HA B Wisconsin (PrL-)+Hl California TMCT NOS, respectively; see Example 14).
• 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 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-)+Hl California TMCT).
• NOS nopaline synthase terminator
• PrL- deleted proteolytic loop
• TMCT transmembrane domain cytoplasmic tail.
• FIGURE 19A shows the nucleotide sequence of HA B Wisconsin (PrL-)+Hl California TMCT (SEQ ID NO: 57).
• FIGURE 19B shows the amino acid sequence of PDISP/HA B Wisconsin (PrL-)+Hl California TMCT (SEQ ID NO: 58).
• FIGURE 19C shows a schematic representation of construct number 1454 (2X35S/CPMV HT; reference construct).
• FIGURE 20 shows sequence components used to prepare construct numbers
• Construct number 5001 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 (HC rituximab (Rituxan)).
• HC heavy chain
• NOS nopaline synthase terminator.
• FIGURE 20A shows the nucleotide sequence of HC rituximab (Rituxan; SEQ ID NO: 59).
• FIGURE 20B shows the amino acid sequence of HC rituximab (Rituxan; SEQ ID NO: 60).
• FIGURE 20C shows a schematic
• FIGURE 20D shows a schematic representation of construct number 2100
• FIGURE 21 shows sequence components used to prepare construct numbers
• Construct number 5001 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 (PDISP/HC Rituzan).
• PDISP protein disulfide isomerase signal peptide
• HC heavy chain
• NOS nopaline synthase terminator.
• FIGURE 21 A shows the nucleotide sequence of PDISP/HC rituximab (Rituxan; SEQ ID NO: 61).
• FIGURE 21B shows the amino acid sequence of PSISP/HC rituximab (Rituxan; SEQ ID NO: 62).
• FIGURE 21C shows a schematic representation of construct number 5002 (2X35S/CPMV HT; reference construct).
• FIGURE 22 shows sequence components used to prepare construct numbers
• Construct number 5021 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 (LC rituximab (Rituxan)).
• LC light chain; NOS: nopaline synthase terminator.
• FIGURE 22A shows the nucleotide sequence of LC rituximab (Rituxan; SEQ ID NO: 63).
• FIGURE 22B shows the amino acid sequence of LC rituximab (Rituxan; SEQ ID NO: 64).
• FIGURE 22C shows a schematic representation of construct number 5021 (2X35S/CPMV HT; reference construct).
• FIGURE 23 shows sequence components used to prepare construct numbers
• Construct number 5001 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 (PDISP/LC rituximab (Rituxan)).
• PDISP protein disulfide isomerase signal peptide
• HC heavy chain
• NOS nopaline synthase terminator.
• FIGURE 23A shows the nucleotide sequence of PDISP/LC rituximab (Rituxan; SEQ ID NO: 65).
• FIGURE 23B shows the amino acid sequence of PSISP/LC rituximab (Rituxan; SEQ ID NO: 66).
• FIGURE 23C shows a schematic representation of construct number 5022 (2X35S/CPMV HT; reference construct).
• FIGURE 23D shows a schematic representation of construct number 2129
• the present invention relates to the expression of proteins of interest in plants.
• the present invention also provides methods and compositions for the production of proteins of interest in plants.
• This expression enhancer is generally referred to as CPMVX (see Figure 1A).
• the stuffer sequence may 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.
• CPMV114+ when X-160, 155, 150, or 114, respectively.
• the stuffer sequence may be modified by truncation, deletion, or replacement of the native CMPV5'UTR sequence that is located 3 'to nucleotide 160.
• the modified stuffer sequence may be removed, replaced, truncated or shortened when compared to the initial or unmodified (i.e. native) stuffer sequence associated with the 5'UTPv (as described in Sainsbury F., and Lomonossoff G.P., 2008, Plant Physiol. 148: pp. 1212-1218).
• the stuffer sequence may comprise a one or more restriction sites (polylinker, multiple cloning site, one or more cloning sites), one or more plant kozak sequences, one or more linker sequences, one or more recombination sites, or a combination thereof.
• a stuffer sequence may comprise in series, a multiple cloning site of a desired length fused to a plant kozak sequence.
• the stuffer sequence does not comprise a nucleotide sequence from the native 5'UTR sequence that is positioned 3' to nucleotide 160 of the native CPMV 5'UTR, for example nucleotides 161 to 512 as shown in Figure 1 of Sainsbury F., and Lomonossoff G.P. (2008, Plant Physiol. 148: pp. 1212-1218; which is incorporated herein by reference), or nucleotides 161-509 of SEQ ID NO:4. That is, the incomplete M protein present in the prior art CPMV HT sequence ( Figure 1 ; of Sainsbury F., and Lomonossoff G.P., 2008) is removed from the 5'UTR in the present invention.
• the expression enhancer CPMVX, or CPMVX+ may be operatively linked at the 5 'end of the enhancer sequence with a regulatory region that is active in a plant, and operatively linked to a nucleotide sequence of interest at the 3 'end of the expression enhancer ( Figure 1A), in order to drive expression of the nucleotide sequence of interest within a plant host.
• Expression systems to produce one or more proteins of interest in a plant using either CMPVX or CPMVX+ are also provided.
• the expression systems described herein comprise an expression cassette comprising CPMVX, or a sequence that comprises 80% sequence similarity with CPMVX, and optionally, a stuffer sequence fused to CMPVX (CPMVX+).
• the expression cassette comprising CMPVX or CMPVX+ may further comprise a regulatory region that is active in a plant that is operatively linked to the 5 'end of the expression enhancer.
• a nucleotide sequence of interest may be operatively linked to the 3 'end of the expression cassette so that when introduced within a plant, expression of the nucleotide sequence of interest within a plant host is achieved.
• Plant cells, plant tissues, whole plants, inoculum, nucleic acids, constructs comprising nucleotide sequences of interest encoding proteins of interest, expression cassettes or expression systems comprising CPMVX or CPMVX+ as described herein, and methods of expressing a protein of interest in plants are also provided.
• the expression enhancer CMPVX may also be referred to as CPMV160; CPMV155; CPMV150; CPMV114, when X- 160, 155, 150, or 114, respectively.
• nucleotide sequence of interest may be fused (operatively linked) to the enhancer sequence comprising a plant regulatory region, using a variety of approaches. For example, which are not to be considered limiting:
• a nucleotide sequence of interest that is operatively linked to CPMV160 may not require a plant kozak sequence fused to its 5' end, as nucleotides 150-160, or 155- 160, of SEQ ID NO:l comprise a kozak-like sequence.
• a plant kozak sequence may be included in constructs comprising CPMV160 if desired (see Figure IB: "+/- plant kozak").
• X-155, 150, or 114 then including a plant kozak sequence that is fused to the 5 'end of the nucleotide sequence of interest in constructs comprising CPMV155, CPMV150, or CPMV114 is recommended for optimal expression of the nucleotide sequence of interest.
• the nucleotide sequence of interest that is fused to CPMVX+ would not include a multiple cloning site or plant kozak sequence (the resulting construct would .be analogous to those as presented in Figure IB).
• the nucleotide sequence of interest may include at its 5 ' end a corresponding sequence to permit fusion with the multiple cloning sire of the expression enhancer, and a plant kozak sequence immediately upstream from the ATG initiation site of the nucleotide sequence of interest (see figure 1C).
• the construct may, or may not, comprise a multiple cloning site located between the 5 'UTR and the plant kozak sequence.
• the construct may further comprise a 3 ' untranslated region (UTR) sequence, for example, a comovirus 3'UTR, or a plastocyanin 3' UTR, and a terminator sequence, for example a NOS terminator, operatively linked to the 3'end of the nucleotide sequence of interest (see Figure 1A).
• UTR 3 ' untranslated region
• terminator sequence for example a NOS terminator
• a plant expression system comprising a nucleic acid comprising a regulatory region, operatively linked with one or more than one expression enhancer as described herein (e.g. CPMVX), and a nucleotide sequence of interest, is also provided. Furthermore, a nucleic acid comprising a promoter (regulatory region) sequence, operatively linked with an expression enhancer comprising a CPMV 5 'UTR and a modified or deleted stuffer sequence (e.g. CPMVX+) and a nucleotide sequence of interest is described.
• the nucleic acid may further comprise a sequence encoding a 3'UTR, for example a comovirus 3' UTR, or a plastocyanin 3' UTR, and a terminator sequence, for example a NOS terminator, so that the nucleotide sequence of interest is inserted upstream from the 3 'UTR.
• a 3'UTR for example a comovirus 3' UTR, or a plastocyanin 3' UTR
• a terminator sequence for example a NOS terminator
• operatively linked it is meant that the particular sequences interact either directly or indirectly to carry out an intended function, such as mediation or modulation of expression of a nucleic acid sequence.
• the interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences.
• "Expression enhancer(s)”, “enhancer sequence(s)” or “enhancer element(s)”, as referred to herein include sequences derived from, or that share sequence similarity with, portions of the CPMV 5 'UTR from the R A-2 genome segment.
• An enhancer sequence can enhance expression of a downstream heterologous open reading frame (ORF) to which they are attached.
• ORF heterologous open reading frame
• the term "5 'UTR” or “5' untranslated region” or “5' leader sequence” refers to regions of an mR A that are not translated.
• the 5 'UTR typically begins at the transcription start site and ends just before the translation initiation site or start codon (usually AUG in an mRNA, ATG in a DNA sequence) of the coding region.
• the length of the 5 'UTR may be modified by mutation for example substitution, deletion or insertion of the 5 'UTR.
• the 5 'UTR may be further modified by mutating a naturally occurring start codon or translation initiation site such that the codon no longer functions as start codon and translation may initiate at an alternate initiation site.
• the 5 'UTR from nucleotides 1-160 of the CPMV R A -2 sequence starts at the transcription start site to the first in frame initiation start codon (at position 161), which serve as the initiation site for the production of the longer of two carboxy coterminal proteins encoded by a wild-type comovirus genome segment. Furthermore a 'third' initiation site at (or corresponding to) position 115 in the CPMV RNA-2 genomic sequence may also be mutated, deleted or otherwise altered.
• a stuffer sequence for example
• the stuffer sequence comprises from 0-500 nucleotides fused to the 3' end of the CMPVX sequence.
• the stuffer sequence comprises an multiple cloning site (MCS), or an MCS linked to a plant kozak sequence, and does not include an M protein.
• MCS multiple cloning site
• the CMPVX sequence comprises a stuffer fragment (without an M protein)
• this expression enhancer may be referred to as "CPMVX+" (see Figures 1A and 1C), as "CMPVX comprising a stuffer sequence and a plant kozak sequence", or as "CMPVX comprising an MCS along with a plant kozak sequence".
• nucleotide 160 of SEQ ID NO: 1 CPMV 160
• a nucleotide sequence of interest with or without a 5 'plant kozak sequence located at the 5' end adjacent to an initiation sequence ATG
• AGT initiation sequence
• the construct comprising CPMV160 may further comprise a regulatory region operatively linked to the 5 'end of the expression enhancer, and a sequence encoding a 3'UTR, for example a como virus 3' untranslated region (UTR) or a plastocyanin 3' UTR, and a terminator sequence, for example a NOS terminator, fused to the 3' end of the nucleotide sequence of interest.
• a 3'UTR for example a como virus 3' untranslated region (UTR) or a plastocyanin 3' UTR
• a terminator sequence for example a NOS terminator
• CPMV160 may not require the addition of a plant kozak sequence to the 5' end of the nucleotide sequence of interest, since the sequence at positions 150-155, 155-160, or 150-160 of SEQ ID NO: 1 may function as an active (native) kozak sequence in a plant.
• a non- limiting example of such an enhancer is CPMV160+ (see figure 1C) comprising the sequence of SEQ ID NO:2 (5 'UTR: nucleotide 1-160; multiple cloning site in italics nucleotides 161-176; plant kozak sequence in caps and bold, nucleotides 177-181):
• constructs using SEQ ID NO:2 as an expression enhancer include constructs 1800, 1897, 1880, 2168, 2188, 1937, 1977, 2050, 2060, 1975, 1893, 2100, 2109, 2120, 2129 (see Examples 3, and 5-18, respectively).
• any multiple cloning site (MCS), or an MCS of different length (either shorter or longer) may used in place of the sequence at nucleotides 161-176 of SEQ ID NO:2.
• the plant kozak sequence of SEQ ID NO:2 may be any plant kozak sequence, including but not limited, one of the sequences selected from SEQ ID NO's:5-17 (also see Figure 4A; the construct of Figure 4 includes SEQ ID NO:2, with variations of the plant kozak sequence as indicated, and comprises a plant regulatory region attached to the 5 ' end of the 5 'UTR, and the transcription initiation site, ATG, of a nucleotide sequence of interest, located 3' to the plant kozak sequence).
• the expression enhancer CPMVX may include an "A" in position 115
• CMPVX, 115A comprises the sequence of the wild-type CPMV RNA2 genome (see WO 2009/087391, which is incorporated herein by reference, for the complete sequence of the wild type CPMV RNA-2 genome segment).
• An example of an expression enhancer CPMVX, 115A is
• CPMV 160, 115 A as defined by SEQ ID NO: 69 (the "A” is shown in bold and underline):
• a non-limiting example of an expression enhancer CPMVX+, 115A is "CPMV 160+, 115 A", as defined by SEQ ID NO: 75 (the "A" is shown in bold and underline):
• any MCS, or an MCS of different length may used in place of the MCS sequence of SEQ ID NO:75, and the plant kozak sequence may be any plant kozak sequence.
• the expression enhancer consists of nucleotide 1-155 of SEQ ID NO:l (CPMV155):
• nucleotide sequence of interest with a plant kozak sequence located at the 5 ' end, adjacent an initiation sequence (ATG), may be fused to the 3' end of the 5'UTR (after nucleotide 155 of SEQ ID NO:l), so that the overall construct resembles that as shown in Figure IB (CPMV155).
• the construct comprising CPMV155 may further comprise a regulatory region operatively linked to the 5 'end of the expression enhancer, and a sequence encoding a 3'UTR, for example a comovirus 3' untranslated region (UTR) or a plastocyanin 3 ' UTR, and a terminator sequence, for example a NOS terminator, fused to the 3' end of the nucleotide sequence of interest.
• the nucleotide sequence of interest comprises a plant kozak sequence at its 5' end, since the native kozak sequence or a portion of this sequence
• the expression enhancer may comprise CPMV155+, comprising the sequence of SEQ ID NO: 72 (5'UTR: nucleotide 1-155; multiple cloning site in italics nucleotides 156-171; plant kozak sequence in caps and bold, nucleotides 172-176):
• any MCS including an MCS's of different length, may used in place of the MCS sequence of SEQ ID NO: 72, and the plant kozak sequence may be any plant kozak sequence.
• the expression enhancer CPMV155 may include an "A” in position 115 (115A), so that "CMPV155, 115 A” comprises the sequence of the wild-type CPMV RNA2 genome (see WO 2009/087391, which is incorporated herein by reference), as defined by SEQ ID NO: 70 ("A” is bolded and underlined):
• the expression enhancer CPMV155+ may also include an "A” in position 115 (115A), so that "CMPV155+, 115a” comprises the sequence of the wild-type CPMV RNA2 genome (WO 2009/087391, which is incorporated herein by reference), as defined by SEQ ID NO: 76 (the "A” is shown in bold and underline):
• any MCS, or an MCS of different length may used in place of the MCS sequence of SEQ ID NO:76, and the plant kozak sequence may be any plant kozak sequence.
• the expression enhancer consists of nucleotide 1-150 of SEQ ID NO:l (CPMV150):
• nucleotide sequence of interest with a plant kozak sequence located at the 5 ' end, adjacent an initiation sequence (ATG), may be fused to the 3' end of the 5'UTR (after nucleotide 150 of SEQ ID NO:l), so that the overall construct resembles that as shown in Figure IB (CPMV150).
• the construct comprising CPMV150 may further comprise a regulatory region operatively linked to the 5 'end of the expression enhancer, and a sequence encoding a 3'UTR, for example a comovirus 3' untranslated region (UTR) or a plastocyanin 3 ' UTR, and a terminator sequence, for example a NOS terminator, fused to the 3' end of the nucleotide sequence of interest.
• the nucleotide sequence of interest comprises a plant kozak sequence at its 5' end, since the native kozak sequence at position 150-160 of SEQ ID NO:l, is removed.
• the expression enhancer may comprise CPMV150+, comprising the sequence of SEQ ID NO:73 (5'UTR: nucleotide 1-150; multiple cloning site in italics nucleotides 156-166; plant kozak sequence in caps and bold, nucleotides 167-171):
• any MCS including an MCS's of different length, may used in place of the MCS sequence of SEQ ID NO:73, and the plant kozak sequence may be any plant kozak sequence.
• the expression enhancer CPMV150 may include an "A” in position 115 (115A), so that "CMPV150, 115 A” comprises the sequence of the wild-type CPMV RNA2 genome (see WO 2009/087391, which is incorporated herein by reference) as defined by SEQ ID NO: 71 (the "A” is shown in bold and underline):
• the expression enhancer CPMV 150+ may also include an "A” in position 115 (115A), so that "CMPV150+, 115 A” comprises the sequence of the wild-type CPMV RNA2 genome (WO 2009/087391, which is incorporated herein by reference), as defined by SEQ ID NO: 77 (the "A” is shown in bold and underline):
• any MCS, or an MCS of different length may used in place of the MCS sequence of SEQ ID NO:77, and the plant kozak sequence may be any plant kozak sequence.
• the expression enhancer consists of nucleotide 1-114 of SEQ ID NO:l:
• nucleotide sequence of interest with a plant kozak sequence located at the 5' end, adjacent an initiation sequence (ATG), may be fused to the 3' end of the 5'UTR (after nucleotide 114 of SEQ ID NO:l), so that the overall construct resembles that as shown in Figure IB (CPMVl 14).
• the construct comprising CPMVl 114 may further comprise a regulatory region operatively linked to the 5 'end of the expression enhancer, and a sequence encoding a 3'UTR, for example a comovirus 3' untranslated region (UTR) or a plastocyanin 3 ' UTR, and a terminator sequence, for example a NOS terminator, fused to the 3' end of the nucleotide sequence of interest.
• a 3'UTR for example a comovirus 3' untranslated region (UTR) or a plastocyanin 3 ' UTR
• a terminator sequence for example a NOS terminator
• the nucleotide sequence of interest comprises a plant kozak sequence at its 5' end, since there is kozak-like sequence 5' to nucleotide 114 of SEQ ID NO:l.
• the expression enhancer may comprise CPMVl 14+, comprising the sequence of SEQ ID NO: 74 (5'UTR: nucleotide 1-114; multiple cloning site in italics nucleotides 115-130; plant kozak sequence in caps and bold, nucleotides 131-135):
• any MCS including an MCS's of different length, may used in place of the MCS sequence of SEQ ID NO:73, and the plant kozak sequence may be any plant kozak sequence.
• the expression enhancer may also comprise nucleotides 1-160 of SEQ ID NO: 1, fused with a plant kozak sequence located downstream from position 160 of SEQ ID NO: 1.
• the plant kozak sequence may be located immediately adjacent to nucleotide 160 of SEQ ID NO:l, or the expression enhancer may comprise a stuffer fragment of about 0 to about 500 nucleotides, or any amount therebetween, located immediately adjacent to nucleotide 160 of SEQ ID NO: 1 (CPMVX+) and the plant kozak sequence linked to 3' end of the stuffer fragment.
• the stuffer fragment may comprise a multiple cloning site (MCS) of from about 4 to 100 nucleotides or any amount therebetween, and a nucleotide sequence of interest comprising a plant kozak sequence and a corresponding cloning site at its 5' end may be operatively linked to the CMPVX expression enhancer using the MCS, or the stuffer fragment may comprise a multiple cloning site of from about 4 to 100 nucleotides fused to a plant kozak sequence, and a nucleotide sequence of interest may be fused to the expression enhancer immediately downstream of the plant kozak sequence.
• the stuffer fragment does not comprise a sequence encoding an M protein.
• FIG. 1C An example, which is not to be considered limiting, of a construct, comprising in series, a plant regulatory region fused to a CPMV 5'UTR consisting of nucleotides 1-160 of SEQ ID NO:l, that is fused to a stuffer fragment is CPMV 160+ as shown in Figure 1C (in Figure 1C, the ATG start site of the nucleotide sequence of interest "GOI", is also shown for clarity).
• the stuffer fragment is fused to the 3' end of the CPMV 1-160 sequence and comprises, in series, a multiple cloning site fused to a plant kozak sequence (in this example which is not to be considered limiting, the plant kozak sequence is: AGAAA).
• the stuffer fragment does not comprise any sequence encoding an M protein If the CPMV 160+ construct is fused to a nucleotide sequence of interest (as shown in Figure 1C), then the plant kozak sequence is located 5' to the nucleotide sequence of interest, and adjacent to the ATG initiation site of the nucleotide sequence of interest.
• the multiple cloning site may comprise one or more than one suitable restriction sites, and the sequence of the multiple cloning site is not limited to the example shown in Figure 1 C.
• the plant kozak sequence may be any plant kozak sequence and not limited to the sequence shown in Figure 1 C. Construct numbers 1800, 1897, 1880, 2168, 2188, 1937, 1977, 2050, 2060, 1975, 1893, 2100, 2109, 2120, 2129 (see Examples 3, and 5-18, respectively) are examples of
• FIG. 1C Also shown in Figure 1C are example of expression enhancers CPMV155+, CPMV150+, and CPMV114+ each comprising nucleotides 1-155, 1-150, or 1-114 of SEQ ID NO: 1, respectively, fused to a stuffer fragment in a similar manner as that described for CPMV 160+, above.
• the ATG start site of the nucleotide sequence of interest (GOI) is also shown for each of CPMV155+, CPMV150+, and CPMV 114+.
• the stuffer fragment is fused to the 3' end of the CPMV enhancer sequence comprises, in series, a multiple cloning site fused to a plant kozak sequence.
• the stuffer fragment does not comprise any sequence encoding an M protein.
• the multiple cloning site may comprise one or more than one suitable restriction sites, and the sequence of the multiple cloning site is not limited to the examples shown in Figure 1 C.
• the plant kozak sequence may be any plant kozak sequence and not limited to the sequence shown in Figure 1C (AGAAA).
• the expression enhancer may also comprise the expression enhancer
• nucleic acid sequence encoding a protein of interest (nucleotide sequence of interest) to be joined to the enhancer will comprises, in series from the 5' end to the 3' end of the nucleotide sequence of interest, a multiple cloning site (complimentary with that of the stuffer fragment; the stuffer fragment does not comprise any sequence encoding an M protein.) fused to a plant kozak sequence located upstream from and adjacent to an ATG initiation site (transcriptional start site) of the nucleotide sequence of interest.
• the expression enhancer may further comprise one or more "kozak consensus sequence” or "kozak sequence”.
• Kozak sequences play a major role in the initiation of translation.
• the rate of translation can be optimized by ensuring that any mRNA instability sequences are eliminated from the transgene construct, and that the translational start site or initiation site matches the Kozak consensus for plants (Gutierrrez, R.A. et al., 1999, Trends Plant Sci. 4, 429-438; Kawaguchi, R. and Bailey-Serres, J., 2002, Curr. Opin. Plant Biol. 5, 460 ⁇ 165).
• the most highly conserved position in this motif is the purine (which is most often an A) three nucleotides upstream of the ATG codon, which indicates the start of translation (Kozak, M, 1987, J. Mol. Biol. 20:947-950, herein incorporated by reference).
• Plant Kozak consensus 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 may be fused to the nucleotide sequence of interest as described herein.
• the plant kozak sequence may be any known plant kozak sequences (see for example L. Rangan et. al.
• the plant kozak sequence may also be selected from the group of (see Figure 4):
• AGAAA SEQ ID NO: 8
• AAAAA SEQ ID NO: 11
• AAACA SEQ ID NO: 12
• AAGCA (SEQ ID NO: 13)
• AAGAA SEQ ID NO: 14
• AAAGAA SEQ ID NO: 15
• AAAGAA SEQ ID NO: 16
• the expression enhancer may further comprise one or more "restriction site(s)” or “restriction recognition site(s)", “multiple cloning site”, “MCS”, “cloning site(s)” “polylinker sequence” or “polylinker' to facilitate the insertion of the nucleotide of interest into the plant expression system.
• Restrictions sites are specific sequence motifs that are recognized by restriction enzymes as are well known in the art.
• the expression enhancer may comprise one or more restriction sites or cloning sites that are located downstream (3') of the 5'UTR.
• the one or more restriction sites or cloning sites may further be located up-stream (5') of one or more kozak sequences, and located between a 5' UTR and a kozak sequence.
• the polylinker sequence (multiple cloning site) may comprise any sequence of nucleic acids that are useful for adding and removing nucleic acid sequences, including a nucleotide sequence encoding a protein of interest, to the 3 ' end of the 5 'UTR.
• a polylinker sequence may comprise from 4 to about 100 nucleic acids, or any amount therebetween.
• CPMVX may also comprise any plant kozak sequence including but not limited to, one of the sequences of SEQ ID NO's:5- 17.
• the 5'UTR for use in the expression enhancer described herein may be derived from a bipartite RNA virus, e.g. from the RNA-2 genome segment of a bipartite RNA virus such as a comovirus, provided that it exhibits 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the sequence as set forth in either SEQ ID NO's: 1 and 2.
• the enhancer sequence may have from about 80% to about 100% identity to the sequence of SEQ ID NO's: 1 and 2, or any amount therebetween, from about 90% to about 100% identity to the sequence of SEQ ID NO's: 1 and 2, or any amount therebetween, about 95% to about 100%, identity to the sequence of SEQ ID NO's: 1 and 2, or any amount therebetween, or about 98% to about 100%, identity to the sequence of SEQ ID NO's: 1 and 2, or any amount therebetween wherein the expression enhancer, when operatively linked to a plant regulatory region and a plant kozak sequence as described herein, increases the level of expression of a nucleotide sequence of interest that is operatively linked to the expression enhancer when compared to the level of expression of the nucleotide sequence of interest fused to the CMPV HT (SEQ ID NO:4; prior art enhancer sequence comprising an incomplete M protein as described in Sainsbury F., and Lomonossoff G.P., 2008, Plant Physiol. 148:
• SEQ ID NO:4 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 nucleotides 1-160 of SEQ ID NO:4 with modified nucleotides at positions 115 (cgt) and 162 (acg), and an incomplete M protein, and lacks a plant kozak sequence (5'UTR: nucleotides 1-160; incomplete M protein underlined, nucleotides 161 - 509).
• SEQ ID NO:4 also includes a multiple cloning site (italics, nucleotides 510-528) which is not present in the prior art CPMV HT sequence:
• Constructs comprising CPMV HT are used herein as reference constructs, so that the expression levels of a nucleotide sequence of interest, or a product encoded by the nucleotide sequence of interest produced using a construct comprising CPMVX or CPMVX+, may be compared.
• Constructs 1391, 484, 489, 2140, 2130, 1039, 1067, 2072, 2074, 1445, 1454, 5001, 5002, 5021 and 5022 (see Examples 1 and 5-18, respectively) comprise the reference construct CPMV HT.
• HI A/California/07/2009 (“PDI-H1 Cal”, or "HI A/California/07/2009”): CPMV160+ based construct number 1897, CPMV HT based construct number 484 (see Example 5);
• H3 A/Victoria/361/2011 (“PDI-H3 Vic", or "H3 A/Victoria/361/2011”): CPMV160+ based construct number 1800; CPMV HT based construct number 1391 (see Examples 1 and 2, respectively);
• B Brisbane/60/08 with deleted proteolytic loop and with a PDI signal peptide (“B Brisbane/60/08"): CPMV160+ based construct number 1937; CMPV HT based construct number 1039 (see Example 9);
• B Brisbane/60/08+HlTm with deleted proteolytic loop fused to the transmembrane domain and cytoplasmic tail and with a PDI signal peptide ("B Brisbane/60/08+HlTm"): CPMV160+ based construct number 1977; CMPV HT based construct 1067 (see Example 10),
• B Massachusetts/2/2012 2012 with deleted proteolytic loop and with a PDI signal peptide (“B Massachusetts/2/2012 2012"): CPMV160+ based construct number 2050; CPMV HT based construct number 2072 ( see Example 11),
• B Massachusetts/2/2012+HlTm with deleted proteolytic loop fused to the transmembrane domain and cytoplasmic tail and with a PDI signal peptide (“B Massachusetts/2/2012+HlTm"): CPMV 160+ based construct number 2060; CPMV HT based construct 2074 (see Example 12),
• B Wisconsin/1 /2010+HlTm with deleted proteolytic loop fused to the transmembrane domain and cytoplasmic tail and with the native signal peptide (“B Wisconsin/l/2010+HlTm"): CPMV160+ based construct number 1893; CPMV HT based construct 1454 (see Example 14);
• Rituximab under the control of CPMV-HT with a native or PDI signal peptide ("CPMV-HT/wild-type SP" and "CPMV-HT/PDISP”; construct numbers 5001 and 5002, respectively, see examples 15 and 16), or CPMV160+ ("CPMV160+/wile-typeSP” and "CPMV160+/PDISP”; construct numbers 2100 and 2109, respectively, see example 15 and 16).
• the expression (determined as hemagglutination activity or rituximab (Rituxan) expression as the case may be) is increased in the CMPV 160+ based construct when compared to that for the prior art CPMV based construct.
• the nucleotide sequences of interest encoded chimeric or modified proteins, for example comprising heterologous signal peptides (e.g. PDI), heterologous transmembrane domain cytoplasmic tail sequences (TDCT), and/or modified sequences including a deleted proteolytic loop (PrL-).
• percent similarity when referring to a particular sequence are used for example as set forth in the University of Wisconsin GCG software program, or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 supplement).
• Optimal alignment of sequences for comparison can be conducted, using for example the algorithm of Smith & Waterman, (1981, Adv. Appl. Math. 2:482), by the alignment algorithm of Needleman & Wunsch, (1970, J. Mol. Biol. 48:443), by the search for similarity method of Pearson & Lipman, (1988, Proc. Nat'l. Acad. Sci. USA 85:2444), by computerized implementations of these algorithms (for example: GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.).
• BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
• BLASTN program for nucleotide sequences
• W wordlength
• E expectation
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology
• a nucleotide sequence interest that encodes a protein requires the presence of a "translation initiation site” or “initiation site” or “translation start site” or “start site” or “start codon” located upstream of the gene to be expressed.
• initiation sites may be provided either as part of an enhancer sequence or as part of a nucleotide sequence encoding the protein of interest.
• Expression cassette refers to a nucleotide sequence comprising a nucleic acid of interest under the control of, and operably (or operatively) linked to, an appropriate promoter or other regulatory elements for transcription of the nucleic acid of interest in a host cell.
• 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 comprises the sequence variability of related sequences based on analysis of alignment of multiple sequences, 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 HI, consensus H3, consensus H5, or influenza B consensus hemagglutinin amino acid sequences, for example but not limited to B Florida, B Malaysia, B Wisconsin and B Massachusetts.
• Non limiting examples of sequences of the proteoloytic loop region are shown in Figure 15 and 18B of US provisional application No.61/806,227 (filed March 28, 2013, which is incorporated herein by reference; also see Bianchi et al., 2005, Journal of Virology, 79:7380-7388; incorporated herein by reference).
• Residues in the proteolytic loop or cleavage site might be either mutated, for example but not limited to point mutation, substitution, insertion, or deletion.
• amino acid mutation or “amino acid modification” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and
• nucleic acid construct comprising an expression enhancer sequence operatively linked to a nucleotide sequence of interest encoding a protein of interest.
• plant expression systems comprising an enhancer sequence as described herein .
• plant expression system comprising a plant regulatory region, in operative association with an enhancer sequence that is operatively linked to a nucleotide sequence of interest, the nucleotide sequence of interest encoding a protein of interest.
• the enhancer sequence may be selected from any one of SEQ ID NO's:l, 2, 24, 27, 68, 69 and 70-77, or a .nucleotide sequence that exhibits 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the sequence as set forth in any one of SEQ ID NO's:l, 2, 24, 27, 68, 69 and 70-77, wherein the expression enhancer, when operatively linked to a plant regulatory region and a plant kozak sequence as described herein, increases the level of expression of a nucleotide sequence of interest that is operatively linked to the expression enhancer when compared to the level of expression of the nucleotide sequence of interest fused to the CMPV HT (SEQ ID NO: 4; prior art enhancer sequence comprising an incomplete M protein as described in Sainsbury F., and Lomonossoff G.P., 2008, Plant Physiol. 148: pp. 1212-1218; which is incorporated here
• the enhancer sequence of the present invention may be used to express a protein of interest in a host organism for example a plant.
• the protein of interest may also be heterologous to the host organism in question and introduced into the plant cells using transformation techniques know in the art.
• a heterologous gene in an organism may replace an endogenous equivalent gene, i.e. one which normally performs the same or a similar function, or the inserted sequence may be additional to the endogenous gene or other sequence.
• the enhancer sequence operatively linked to a nucleotide sequence of interest may also be operatively linked to promoter, or plant regulatory region, and a 3'UTR and terminator sequences.
• the enhancer sequence may be defined by, for example, any one of SEQ ID NO's:l, 2, 24, 27, 68, 69 and 70-77, or a .nucleotide sequence that exhibits 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the sequence as set forth in any one of SEQ ID NO's:l, 2, 24, 27, 68, 69 and 70-77.
• the nucleotide sequence of interest is located between the enhancer sequence and the termination sequence (see Figure 1 A).
• Either the expression enhancer or the nucleotide sequence of interest may comprise a plant kozak sequence.
• the invention further provides an expression cassette comprising in series, a promoter or plant regulatory region, operatively linked to an expression enhancer sequence as described herein which is fused with a nucleotide sequence of interest, a 3'UTR sequence, and a terminator sequence.
• the enhancer sequence may be defined by, for example, any one of SEQ ID NO's:l, 2, 24, 27, 68, 69 and 70-77, or a .nucleotide sequence that exhibits 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the sequence as set forth in any one of SEQ ID NO's:l, 2, 24, 27, 68, 69 and 70-77.
• Either the expression enhancer or the nucleotide sequence of interest may comprise a plant kozak sequence.
• the termination (terminator) sequence may be any sequence that is active the plant host, for example the termination sequence may be derived from the R A-2 genome segment of a bipartite RNA virus, e.g. a comovirus, or the termination sequence may be a NOS terminator.
• the constructs of the present invention can further comprise a 3' untranslated region (UTR).
• a 3' untranslated region contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression.
• the polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3' end of the mRNA precursor.
• Polyadenylation signals are commonly recognized by the presence of homology to the canonical form 5' AATAAA-3' although variations are not uncommon.
• suitable 3 ' regions are the 3 ' transcribed non-translated regions containing a polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and plant genes such as the soybean storage protein genes, the small subunit of the ribulose-1, 5-bisphosphate carboxylase gene (ssRUBISCO; US 4,962,028; which is incorporated herein by reference), the promoter used in regulating plastocyanin expression (Pwee and Gray 1993; which is incorporated herein by reference).
• the termination (terminator) sequence may be obtained from the 3'UTR of the alfalfa plastocyanin gene.
• nucleotide (or nucleic acid) sequence of interest or “coding region of interest” it is meant any nucleotide sequence, or coding region (these terms may be used interchangeably) that is to be expressed within a host organism, for example a plant, to produce a protein of interest.
• a nucleotide sequence of interest may encode, but is not limited to, native or modified proteins, an industrial enzyme or a modified industrial enzyme, an agricultural protein or a modified agricultural protein, a helper protein, a protein supplement, a pharmaceutically active protein, a nutraceutical, a value-added product, or a fragment thereof for feed, food, or both feed and food use.
• the protein of interest may comprise a native, or a non-native signal peptide; the non-native signal peptide may be of plant origin.
• the signal peptide may be a protein disulfide isomerase signal peptide (PDI).
• PDI protein disulfide isomerase signal peptide
• the native signal peptide may correspond to that of the protein of interest being expressed.
• the nucleotide sequence of interest, or coding region of interest may also include a nucleotide sequence that encodes a pharmaceutically active protein, for example growth factors, growth regulators, antibodies, antigens, and fragments thereof, or their derivatives useful for immunization or vaccination and the like.
• a pharmaceutically active protein for example growth factors, growth regulators, antibodies, antigens, and fragments thereof, or their derivatives useful for immunization or vaccination and the like.
• Such proteins include, but are not limited to a protein that is a human pathogen, a viral protein, for example but not limited to VLP-forming antigens, one or more proteins from Respiratory syncytial virus (RSV), Rotavirus, influenza virus, human immunodeficiency virus (HIV), Rabies virus, human papiloma virus (HPV), Enterovirus 71 (EV71), or interleukins, for example one or more than one of IL-1 to IL-24, IL-26 and IL-27, cytokines, Erythropoietin (EPO), insulin, G-CSF, GM-CSF, hPG-CSF, M-CSF or combinations thereof, interferons, for example, interferon-alpha, interferon-beta, interferon-gama, blood clotting factors, for example, Factor VIII, Factor IX, or tPA hGH, receptors, receptor agonists, antibodies for example but not limited to rituxim
• the protein of interest may also include an influenza hemagglutinin (HA; see WO 2009/009876, which is incorporated herein by reference).
• HA is a homotrimeric membrane type I glycoprotein, generally comprising a signal peptide, an HAl domain, and an HA2 domain comprising a membrane-spanning anchor site at the C-terminus and a small cytoplasmic tail.
• An HA protein may be of a type A influenza, a type B influenza, or is a subtype of type A influenza HA selected from the group of HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15, and H16.
• the HA may be from a type A influenza, selected from the group HI, H2, H3, H5, H6, H7 and H9. Fragments of the HAs listed above may also be considered a protein of interest.
• domains from an HA type or subtype listed above may be combined to produce chimeric HA's (see for example WO2009/076778 which is incorporated herein by reference).
• HA proteins examples include A/New
• H1N1 Caledonia/20/99
• H5N1 A/Indonesia/5/2006
• H5N1 A/chicken/New York/1995
• A/herring gull/DE/677/88 H2N8
• A/Texas/32/2003 A/mallard/MN/33/00
• A/Brisbane 10/2007 H3N2
• A/Wisconsin/67/2005 H3N2
• B/Malaysia/2506/2004 B/Florida/4/2006
• A/Singapore/ 1/57 H2N2
• A/Anhui/1/2005 H5N1
• the HA protein may be an HI, H2, H3, H5, H6, H7 or H9 subtype.
• the HI protein may be from the A/New Caledonia/20/99 (HlNl), A/PuertoRico/8/34 (HlNl), A/Brisbane/59/2007 (HlNl), A/Solomon Islands 3/2006 (HlNl), A/California/04/2009 (HlNl) or A/California/07/2009 (HlNl) strain.
• the H3 protein may also be from the A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2), A/Victoria/361/2011 (H3N2), A/Texas/50/2012 (H3N2), A/Hawaii/22/2012 (H3N2), A/New York/39/2012 (H3N2), or A/Perth/16/2009 (H3N2) strain.
• the H2 protein may be from the A/Singapore/1/57 (H2N2) strain.
• the H5 protein may be from the A/Anhui/1/2005 (H5N1),
• the H6 protein may be from the A/Teal/HongKong/W312/97 (H6N1) strain.
• the H7 protein may be from the A/Equine/Prague/56 (H7N7) strain, or H7 A/Hangzhou/1/2013, A/Anhui/1/2013 (H7N9), or A/Shanghai/2/2013 (H7N9) strain.
• the H9 protein is from the A/HongKong/1073/99 (H9N2) strain.
• the HA protein may be from an influenza virus may be a type B virus, including B/Malaysia/2506/2004,
• Non-limiting examples of amino acid sequences of the HA proteins from HI, H2, H3, H5, H6, H7, H9 or B subtypes include sequences as described in WO 2009/009876, WO 2009/076778, WO 2010/003225 (which are incorporated herein by reference).
• the influenza virus HA protein may be H5 Indonesia.
• the HA may also be a chimeric HA, wherein a native transmembrane domain of the HA is replaced with a heterologous transmembrane domain.
• the transmembrane domain of HA proteins is highly conserved (see for example Figure 1C of WO 2010/148511; which is incorporated herein by reference).
• heterologous transmembrane domain may be obtained from any HA transmembrane domain, for example but not limited to the transmembrane domain from HI
• transmembrane domain may also be defined by the following consensus amino acid sequence: iLXiYystvAiSslXIXXmlagXsXwmcs (SEQ ID NO:78)
• the HA may comprise a native, or a non-native signal peptide; the non-native signal peptide may be of plant origin.
• the native signal peptide may correspond to that of the hemagglutinin being expressed, or may correspond to a second hemagglutinin.
• the signal peptide may be from a structural protein or hemagglutinin of a virus other than 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. Zl 1499), or the patatin signal peptide (PatA SP; located nucleotides 1738 - 1806 of GenBank Accession number A08215).
• the nucleotide sequence of PatA SP for this accession number is:
• the present invention also provides nucleic acid molecules comprising sequences encoding an HA protein.
• the nucleic acid molecules may further comprise one or more regulatory regions operatively linked to the sequence encoding an HA protein.
• the nucleic acid molecules may comprise a sequence encoding an HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15, H16 or HA from type B influenza.
• the HA protein encoded by the nucleic acid molecule may be an HI, H2, H3, H5, H6, H7, H9 subtype an HA from type B.
• the HI protein encoded by the nucleic acid may be from the A/New Caledonia/20/99 (H1N1), A/PuertoRico/8/34 (H1N1), A/Brisbane/59/2007 (H1N1), A/Solomon Islands 3/2006 (H1N1), A/California/04/2009 (H1N1) or A/California/07/2009 (H1N1) strain.
• the H3 protein encoded by the nucleic acid molecule may be from the A/Brisbane 10/2007 (H3N2), A/Wisconsin/67/2005 (H3N2), A/Victoria/361/2011 (H3N2), A/Texas/50/2012 (H3N2), A/Hawaii/22/2012 (H3N2), A/New York/39/2012 (H3N2), or A/Perth/16/2009 (H3N2) strain.
• the H2 protein encoded by the nucleic acid molecule may be from the A/Singapore/1/57 (H2N2) strain.
• the H5 protein encoded by the nucleic acid molecule A/Anhui/1/2005 (H5N1), A/Vietnam/ 1194/2004 (H5 1), or A/Indonesia/5/2005 strain.
• the H6 protein encoded by the nucleic acid molecule may be from the A/Teal/HongKong/W312/97 (H6N1) strain.
• the H7 protein encoded by the nucleic acid molecule may be from the A/Equine/Prague/56 (H7N7) strain, or H7 A/Hangzhou/1/2013, A/Anhui/1/2013 (H7N9), or
• the H9 protein encoded by the nucleic acid molecule may be from the A/HongKong/1073/99 (H9N2) strain.
• the HA protein encoded by the nucleic acid molecule may be from an influenza virus type B virus, including B/Malaysia/2506/2004, B/Florida/4/2006, B/Brisbane/60/08,
• B/Massachusetts/2/2012-like virus (Yamagata lineage), or B/Wisconsin/1/2010 (Yamagata lineage).
• Non-limiting examples of amino acid sequences of the HA proteins from HI, H2, H3, H5, H6, H7, H9 or B subtypes include sequences as described in WO 2009/009876, WO 2009/076778, WO 2010/003225 (which are incorporated herein by reference).
• the influenza virus HA protein may be H5 Indonesia.
• Table 1 Examples of constructs that have been prepared as described herein:
• nucleic acid sequence of interest encodes a product that is directly or indirectly toxic to the plant, then such toxicity may be reduced by selectively expressing the nucleotide sequence of interest within a desired tissue or at a desired stage of plant development.
• the coding region of interest or the nucleotide sequence of interest may be expressed in any suitable plant host which is either transformed or comprises the nucleotide sequences, or nucleic acid molecules, or genetic constructs, or vectors of the present invention.
• suitable hosts include, but are not limited to, Arabidopsis, agricultural crops including for example canola, Brass ica spp., maize, Nicotiana spp., (tobacco) for example, Nicotiana benthamiana, alfalfa, potato, sweet potato (Ipomoea batatus), ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton, corn, rye (Secale cereale), sorghum ⁇ Sorghum bicolor, Sorghum vulgare), safflower (Carthamus tinctorius).
• Arabidopsis agricultural crops including for example canola, Brass ica spp., maize, Nicotiana spp., (tobacco) for example, Nico
• biomass and plant matter refer to any material derived from a plant.
• Biomass or plant matter may comprise an entire plant, or part of plant including the leaf, root, stem, flower, seed, it may also include any tissue of the plant, any cells of the plant, or any fraction of the plant, part or the plant, tissue or cell.
• biomass or plant matter may comprise intracellular plant components, extracellular plant components, liquid or solid extracts of plants, or a combination thereof.
• biomass or plant matter may comprise plants, plant cells, tissue, a liquid extract, or a combination thereof, from plant leaves, stems, fruit, roots or a combination thereof.
• a portion of a plant may comprise plant matter or biomass.
• regulatory region By “regulatory region” “regulatory element” or “promoter” it is meant a portion of nucleic acid typically, but not always, upstream of the protein coding region of a gene, which may be comprised of either DNA or RNA, or both DNA and RNA. When a regulatory region is active, and in operative association, or operatively linked, with a gene of interest, this may result in expression of the gene of interest.
• a regulatory element may be capable of mediating organ specificity, or controlling developmental or temporal gene activation.
• a “regulatory region” includes promoter elements, core promoter elements exhibiting a basal promoter activity, elements that are inducible in response to an external stimulus, elements that mediate promoter activity such as negative regulatory elements or transcriptional enhancers.
• regulatory region also includes elements that are active following transcription, for example, regulatory elements that modulate gene expression such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability determinants.
• regulatory element typically refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site.
• upstream 5'
• RNA polymerase RNA polymerase
• regulatory region typically refers to a sequence of DNA, usually, but not always, upstream (5') to the coding sequence of a structural gene, which controls the expression of the coding region by providing the recognition for RNA polymerase and/or other factors required for transcription to start at a particular site.
• a regulatory element that provides for the recognition for RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a promoter element.
• eukaryotic promoter elements contain a TATA box, a conserved nucleic acid sequence comprised of adenosine and thymidine nucleotide base pairs usually situated approximately 25 base pairs upstream of a transcriptional start site.
• a promoter element may comprise a basal promoter element, responsible for the initiation of transcription, as well as other regulatory elements (as listed above) that modify gene expression.
• regulatory regions There are several types of regulatory regions, including those that are developmentally regulated, inducible or constitutive.
• a regulatory region that is developmentally regulated, or controls the differential expression of a gene under its control, is activated within certain organs or tissues of an organ at specific times during the development of that organ or tissue.
• some regulatory regions that are developmentally regulated may preferentially be active within certain organs or tissues at specific developmental stages, they may also be active in a
• tissue-specific regulatory regions for example see- specific a regulatory region, include the napin promoter, and the cruciferin promoter (Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau et al., 1994, Plant Cell 14: 125-130).
• An example of a leaf-specific promoter includes the plastocyanin promoter (see US 7,125,978, which is incorporated herein by reference).
• An inducible regulatory region is one that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed.
• the protein factor that binds specifically to an inducible regulatory region to activate transcription may be present in an inactive form, which is then directly or indirectly converted to the active form by the inducer. However, the protein factor may also be absent.
• the inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus.
• a plant cell containing an inducible regulatory region may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods.
• Inducible regulatory elements may be derived from either plant or non-plant genes (e.g. Gatz, C. and Lenk, I.R.P., 1998, Trends Plant Sci. 3, 352-358; which is incorporated by reference).
• Examples, of potential inducible promoters include, but not limited to, tetracycline-inducible promoter (Gatz, C.,1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 89-108; which is incorporated by reference), steroid inducible promoter (Aoyama, T.
• a constitutive regulatory region directs the expression of a gene throughout the various parts of a plant and continuously throughout plant
• Examples of known constitutive regulatory elements include promoters associated with the CaMV 35S transcript. (p35S; Odell et al., 1985, Nature, 313: 810- 812), the rice actin 1 (Zhang et al, 1991, Plant Cell, 3: 1155-1165), actin 2 (An et al, 1996, Plant J., 10: 107-121), or tms 2 (U.S. 5,428,147, which is incorporated herein by reference), and triosephosphate isomerase 1 (Xu et. al., 1994, Plant Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene (Cornejo et al, 1993, Plant Mol. Biol.
• regulatory regions comprising enhancer sequences with demonstrated efficiency in leaf expression, have been found to be effective in transient expression.
• attachment of upstream regulatory elements of a photosynthetic gene by attachment to the nuclear matrix may mediate strong expression.
• up to -784 from the translation start site of pea plastocyanin (US 7,125,978, which is incorporated herein by reference) may be used mediate strong reporter gene expression.
• constitutive does not necessarily indicate that a nucleotide sequence under control of the constitutive regulatory region is expressed at the same level in all cell types, but that the sequence is expressed in a wide range of cell types even though variation in abundance is often observed.
• the expression constructs as described above may be present in a vector.
• the vector may comprise border sequences which permit the transfer and integration of the expression cassette into the genome of the organism or host.
• the construct may be a plant binary vector, for example a binary transformation vector based on pPZP (Hajdukiewicz, et al. 1994).
• Other example constructs include pBinl9 (see Frisch, D. A., L. W. Harris-Haller, et al. 1995, Plant Molecular Biology 27: 405- 409).
• constructs of this invention may be further manipulated to include selectable markers.
• selectable markers include enzymes that provide for resistance to chemicals such as an antibiotic for example, gentamycin, hygromycin, kanamycin, or herbicides such as
• phosphinothrycin glyphosate, chlorosulfuron, and the like.
• enzymes providing for production of a compound identifiable by colour change such as GUS (beta-glucuronidase), or luminescence, such as luciferase or GFP, may be used.
• a vector may also include a expression enhancer as described herein.
• the expression enhancer may be positioned on a T-DNA which also contains a suppressor of gene silencing and NPTII.
• the polylinker may also encode one or two sets of 6 x Histidine residues to allow the inclusion of N- or C-terminal His-tags to the protein of interest to facilitate protein purification.
• Post-transcriptional gene silencing may be involved in limiting expression of transgenes in plants, and co-expression of a suppressor of silencing from the potato virus Y (HcPro) may be used to counteract the specific degradation of transgene mRNAs (Brigneti et al., 1998, EMBO J. 17, 6739-6746, which is incorporated herein by reference).
• Alternate suppressors of silencing are well known in the art and may be used as described herein (Chiba et al., 2006, Virology 346:7-14; which is incorporated herein by reference), for example but not limited to, TEV- pl/HC-Pro (Tobacco etch virus-pl/HC-Pro), BYV -p21, pl9 of Tomato bushy stunt virus (TBSV pl9; the construction of pl9 is described in described in WO
• capsid protein of Tomato crinkle virus (TCV -CP), 2b of Cucumber mosaic virus; CMV-2b), p25 of Potato virus X (PVX-p25), pl l of Potato virus M (PVM-pl l), pl l of Potato virus S (PVS- pl 1), pl6 of Blueberry scorch virus, (BScV -pl6), p23 of Citrus tristeza virus (CTV- p23), p24 of Grapevine leafroll-associated virus-2, (GLRaV-2 p24), plO of Grapevine virus A, (GVA-plO), pl4 of Grapevine virus B (GVB-pl4), plO of Heracleum latent virus (HLV-plO), or pl6 of Garlic common latent virus (GCLV-pl6).
• TMV -CP capsid protein of Tomato crinkle virus
• CMV-2b capsid protein of Tomato crinkle virus
• PVX-p25 capsid protein
• one or more suppressors of silencing for example, but not limited to, HcPro, TEV -pl/HC-Pro, BYV-p21, TBSV pl9, TCV-CP, CMV-2b, PVX- p25, rgscam, B2 protein from FHV, the small coat protein of CPMV, and coat protein from TCV, PVM-pl l, PVS-pl l, BScV-pl6, CTV-p23, GLRaV-2 p24, GBV-pl4, HLV-plO, GCLV-pl6, or GVA-plO may be co-expressed along with the comovirus- based expression cassette, geminivirus-derived amplification element, and the nucleic acid sequence encoding the protein of interest to further ensure high levels of protein production within a plant.
• constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro-injection, electroporation, etc.
• Ti plasmids Ri plasmids
• plant virus vectors direct DNA transformation, micro-injection, electroporation, etc.
• Weissbach and Weissbach Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1988); and Mild and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. DT. Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Addison Wesly, Langmans Ltd. London, pp.
• Transient expression methods may be used to express the constructs of the present invention (see D'Aoust et al., 2009, Methods in molecular biology, Vol 483, pages41-50; Liu and Lomonossoff, 2002, Journal of Virological Methods, 105:343-348; which is incorporated herein by reference).
• a vacuum- based transient expression method as described by Kapila et al., (1997, Plant Sci. 122, 101-108; which is incorporated herein by reference), or WO 00/063400, WO 00/037663 (which are incorporated herein by reference) may be used.
• Agro-inoculation or Agro-infiltration, syringe infiltration
• transient methods may also be used as noted above.
• Agro-inoculation, Agro-infiltration, or syringe infiltration a mixture of Agrobacteria comprising the desired nucleic acid enter the intercellular spaces of a tissue, for example the leaves, aerial portion of the plant (including stem, leaves and flower), other portion of the plant (stem, root, flower), or the whole plant.
• the Agrobacteria After crossing the epidermis the Agrobacteria infect and transfer t-DNA copies into the cells.
• the t-DNA is episomally transcribed and the mRNA translated, leading to the production of the protein of interest in infected cells, however, the passage of t- DNA inside the nucleus is transient.
• transgenic plants, plant cells or seeds containing the gene construct of the present invention that may be used as a platform plant suitable for transient protein expression described herein.
• Methods of regenerating whole plants from plant cells are also known in the art (for example see Guerineau and Mullineaux (1993, Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148).
• transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells.
• the nucleic acid construct may be introduced into the Agrobacterium in a single transfection event the nucleic acids are pooled, and the bacterial cells transfected as described. Alternately, the constructs may be introduced serially. In this case, a first construct is introduced to the
• Agrobacterium as described, the cells grown under selective conditions (e.g. in the presence of an antibiotic) where only the singly transformed bacteria can grow.
• a second nucleic acid construct is introduced to the Agrobacterum as described, and the cells grown under doubly-selective conditions, where only the doubly -transformed bacteria can grow.
• the doubly-transformed bacteria may then be used to transform a plant, plant portion or plant cell as described herein, or may be subjected to a further transformation step to accommodate a third nucleic acid construct.
• the nucleic acid construct may be introduced into the plant by co-infiltrating a mixture of Agrobacterium cells with the plant, plant portion, or plant cell, each Agrobacterium cell may comprise one or more constructs to be introduced within the plant.
• concentration of the various Agrobacteria populations comprising the desired constructs may be varied.
• the present disclosure further provides a transgenic plant comprising the expression system as defined herein, wherein the heterologous nucleic acid of interest in the cassette is expressed at an enhanced level when compared to other analogous expression systems that lack one or more components of the expression system as described herein, for example CMPV HT (SEQ ID NO:4).
• the present disclosure further comprises a method for generating a protein of interest, comprising the steps of providing a plant, or plant part, that expresses the expression system as described herein, harvesting, at least, a tissue in which the protein of interest has been expressed and optionally, isolating the protein of interest from the tissue.
• the invention provides:
• an expression enhancer comprising a comovirus 5'UTR selected from any one of SEQ ID NO's:l, 2, 24, 27, 68, 69 and 70-77, or a .nucleotide sequence that exhibits 100%, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the sequence as set forth in any one of SEQ ID NO's:l, 2, 24, 27, 68, 69 and 70-77, wherein the expression enhancer, when operatively linked to a plant regulatory region and a plant kozak sequence as described herein, increases the level of expression of a nucleotide sequence of interest that is operatively linked to the expression enhancer when compared to the level of expression of the nucleotide sequence of interest fused to the CMPV HT (SEQ ID NO: 4; prior art enhancer sequence comprising an incomplete M protein as described in Sainsbury F., and Lomonossoff G.P., 2008, Plant Physiol. 148:
• one or more expression systems comprising a comovirus-based expression enhancer or expression cassette as defined above, a promoter (regulatory region), optionally a polylinker, a kozak sequence, a nucleic acid encoding a protein of interest, and a terminator.
• a host organism such as a plant using one or more expression systems or vectors as described herein.
• a host organism such as a plant using one or more expression systems or vectors as described herein.
• Consensus dicot kozak sequence 46 Amino acid sequence of
• Nucleotide sequence of construct 65 Nucleotide sequence of 2171 PDISP/LC rituximab
• Example 1 2X35 S/CPMV-HT/PDISP/H3 Victoria/ OS (Construct number 1391)
• a fragment containing the PDISP/H3 Victoria coding sequence was amplified using primers IF- PDI.S1+3C ( Figure 6A, SEQ ID NO: 67) and IF-H3V36111.sl-4r ( Figure 6B, SEQ ID NO: 17), using PDISP/H3 Victoria sequence ( Figure 6C, SEQ ID NO :18) as template.
• the PCR product was cloned in 2X35S/CPMV-HT/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, CA). Construct number 1191 ( Figure 6D) was digested with SacII and Stul restriction enzyme and the 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 a CPMV-HT-based expression cassette. It also incorporates a gene construct for the co- expression of the TBSV PI 9 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: 19).
• the resulting construct was given number 1391 ( Figure 6F, SEQ ID NO: 20).
• the amino acid sequence of mature H3 from Influenza A/Victoria/361/2011 fused with PDISP is presented in Figure 6G (SEQ ID NO: 21).
• a representation of plasmid 1391 is presented in Figure 6H.
• Example 2 2X35S/CPMV160+/PDISP/H3 Victoria/ NOS (Construct number 1800)
• a fragment containing the PDISP/H3 Victoria coding sequence was amplified using primers IF**(SacII)- PDI.sl+4c ( Figure 7A, SEQ ID NO: 22) and IF-H3V3611 l.sl-4r ( Figure 7B, SEQ ID NO: 23), using PDISP/H3 Victoria sequence ( Figure 7C, SEQ ID NO: 24) as template.
• the PCR product was cloned in 2X35S/CPMV160+/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, CA).
• Construct number 2171 ( Figure 7D) 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 CPMV160+ based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV PI 9 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 7E (SEQ ID NO: 25). The resulting construct was given number 1800 ( Figure 7F, SEQ ID NO: 26).
• a fragment containing the PDISP/H3 Victoria coding sequence was amplified using primers IF-CPMV(fl5'UTR)_SpPDI.c ( Figure 8A, SEQ ID NO: 28) and IF-H3V36111.sl-4r ( Figure 7B, SEQ ID NO: 23), using PDISP/H3 Victoria sequence ( Figure 7C, SEQ ID NO : 24) as template.
• the PCR product was cloned in 2X35S/CPMV160/NOS expression system using In- Fusion cloning system (Clontech, Mountain View, CA).
• Construct number 1190 ( Figure 8B) was digested with SacII and Stul restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction.
• Construct number 1190 is an acceptor plasmid intended for "In Fusion" cloning of genes of interest in a
• CPMV160-based expression cassette It also incorporates a gene construct for the co- expression of the TBSV PI 9 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 8C (SEQ ID NO: 29).
• the resulting construct was given number 1935 (Figure 8D, SEQ ID NO: 30).
• the amino acid sequence of mature H3 from Influenza A/Victoria/361/2011 fused with PDISP is presented in Figure 7G (SEQ ID NO: 27).
• a representation of plasmid 1935 is presented in Figure 8E.
• Example 4 Variation of sequence between SacII restriction site and ATG of PDISP/H3 Victoria in 2X35S/CPMV160+/NOS expression system
• a coding sequence corresponding to native H5 from Influenza A/Indonesia/5/2005 was cloned into original CPMV- HT, CPMV160+ and CPMV160 using the same PCR-based method as construct 1391 (see Example 1), 1800 (see Example 2) and 1935 (see Example 3), respectively but with modified PCR primers specifically designed for H5 Indonesia.
• the amino acid sequence of native H5 from Influenza A/Indonesia/5/2005 is presented in Figure 1 IB (SEQ ID NO: 42).
• Representations of plasmid 489, 1880 and 1885 are presented in Figure 11C to Figure 1 IE.
• FIG. 12B A/Hangzhou/1/2013 fused with PDISP is presented in Figure 12B (SEQ ID NO:44).
• Representations of plasmid 2140 and 2168 are presented in Figure 12C and 12D.
• a chimer hemagglutinin coding sequence corresponding to the 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 HI from influenza A/California/7/2009 and with the signal peptide of alfalfa protein disulfide isomerase (PDISP/HA B Brisbane (PrL-)+Hl California TMCT) ( Figure 15 A, SEQ ID NO: 49) was cloned into original CPMV-HT and CPMV160+ using the same PCR-based method as construct 1391 (see Example 1) and 1800 (see Example 2), respectively, but with modified PCR primers specifically designed for PDISP/HA B Brisbane (
• Example 12 2X35S/CPMV HT (construct no 2074)
• a chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/ Wisconsin /2/2012 with deleted proteolytic loop (PrL-) (see US provisional application 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) fused to the transmembrane domain and cytoplasmic tail (TMCT) of HI from influenza A/California/7/2009 with the native signal peptide of HA B Wisconsin (HA B Wisconsin (PrL-)+Hl California TMCT) ( Figure 19A, SEQ ID NO: 57) was cloned into original CPMV-HT and CPMV160+ using the same PCR-based method as construct 1391 (see Example 1), and 1800 (see Example 2), respectively, but with modified PCR primers specifically designed for HA B Wisconsin (PrL-)+Hl California TMCT.
• a coding sequence corresponding to the heavy chain of monoclonal IgGl antibody Rituximab (HC rituximab (Rituxan); Figure 20A, SEQ ID NO: 59) was cloned into original CPMV-HT and CPMV160+ using the same PCR-based method as construct 1391 (see Example 1), and 1800 (see Example 2), respectively but with modified PCR primers specifically designed for HC rituximab (Rituxan).
• the amino acid sequence of HC rituximab (Rituxan) is presented in Figure 20B (SEQ ID NO:60).
• Representations of plasmid 5001 and 2100 are presented in Figure 20C and Figure 20D.
• a coding sequence corresponding to the heavy chain of monoclonal IgGl antibody Rituximab in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HC rituximab (Rituxan); Figure 21 A, SEQ ID NO: 61) was cloned into original CPMV-HT and CPMV160+ using the same PCR-based method as construct 1391 and 1800, respectively but with modified PCR primers specifically designed for PDISP/HC rituximab (Rituxan).
• the amino acid sequence of mature HC rituximab (Rituxan) fused with PDISP is presented in Figure 21B (SEQ ID NO: 62).
• Representations of plasmid 5002 and 2109 are presented in Figure 21C and Figure 21D.
• LC rituximab (Rituxan; Figure 22A, SEQ ID NO: 63) was cloned into original CPMV-HT and CPMV160+ using the same PCR-based method as construct 1391 and 1800, respectively but with modified PCR primers specifically designed for LC rituximab (Rituxan).
• the amino acid sequence of LC rituximab (Rituxan) is presented in Figure 22B (SEQ ID NO: 64).
• Representations of plasmid 5021 and 2120 are presented in Figure 22C and Figure 22D.
• a coding sequence corresponding to the light chain of monoclonal IgGl antibody Rituximab in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/LC rituximab (Rituxan; Figure 23A, SEQ ID NO: 65) was cloned into original CPMV-HT and CPMV160+ using the same PCR-based method as construct 1391 and 1800, respectively but with modified PCR primers specifically designed for PDISP/LC rituximab (Rituxan).
• the amino acid sequence of mature LC rituximab (Rituxan) fused with PDISP is presented in Figure 23B (SEQ ID NO: 66).
• Representations of plasmid 5022 and 2129 are presented in Figure 23C and Figure 23D.
• Agrobacterium strain AGL1 was transfected by electroporation with the DNA constructs using the methods described by D'Aoust et al 2008 (Plant Biotechnology Journal 6:930-940). Transfected Agrobacterium were grown in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 ⁇ acetosyringone, 50 ⁇ g/ml kanamycin and 25 ⁇ g/ml of carbenicillin pH5.6 to an OD 6 oo between 0.6 and 1.6. Agrobacterium suspensions were centrifuged before use and resuspended in infiltration medium (10 mM MgC ⁇ and 10 mM MES pH 5.6).
• MES 2-(N-morpholino)ethanesulfonic acid
• 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 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.
• Agrobacteria transfected with each construct were grown in a YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 ⁇ acetosyringone, 50 ⁇ g/ml kanamycin and 25 ⁇ g/ml of carbenicillin pH5.6 until they reached an OD 6 oo between 0.6 and 1.6.
• Agrobacterium suspensions were centrifuged before use and resuspended in infiltration medium (10 mM MgCl2 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 culture volumes and allowed to warm before use.
• Whole plants of N. benthamiana were placed upside down in the bacterial suspension in an 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.
• Example 20 Protein analysis and immunoblotting
• the total protein content of clarified 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, Indianapolis, IN) for immunodetection. Prior to immunoblotting, the membranes were blocked with 5% skim milk and 0.1% Tween-20 in Tris-buffered saline (TBS-T) for 16-18h at 4°C.
• PVDF polyvinylene difluoride
• Immunoblotting was performed with a first incubation with a primary antibody (Table 4 presents the antibodies and conditions used for the detection of each HA), in 2 ⁇ g/ml in 2% skim milk in TBS-Tween 20 0.1%. Secondary antibodies 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).
• Table 4 Electrophoresis conditions, antibodies, and dilutions for immunoblotting of expressed proteins.
• NIBSC National Institute for Biological Standards and Control, United Kingdom
• Hemagglutination assay was based on a method described by Nayak and Reichl (2004). Briefly, serial double dilutions of the test samples (100 ⁇ ) were made in V-bottomed 96-well microtiter plates containing 100 ⁇ ⁇ PBS, leaving 100 ⁇ , of diluted sample per well. One hundred microliters of a 0.25% turkey red blood cells suspension (Bio Link Inc., Syracuse, NY; for all B strains, HI, H5 and H7) or 0.5% guinea pig red blood cells suspension (for H3) 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.

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