INFLUENZA VIRUS-LIKE PARTICLE PRODUCTION IN PLANTS
20220145317 · 2022-05-12
Assignee
Inventors
- Manon Couture (St-Augustin-de-Desmaures, CA)
- Marc-Andre D'Aoust (Quebec, CA)
- Louis-Philippe Vezina (Neuville, CA)
Cpc classification
C12N7/00
CHEMISTRY; METALLURGY
C12N2760/16134
CHEMISTRY; METALLURGY
C12N15/8257
CHEMISTRY; METALLURGY
C12N2760/16122
CHEMISTRY; METALLURGY
C12N9/50
CHEMISTRY; METALLURGY
C12N2760/16251
CHEMISTRY; METALLURGY
C12N15/8258
CHEMISTRY; METALLURGY
C12N2760/16151
CHEMISTRY; METALLURGY
C12N2760/16234
CHEMISTRY; METALLURGY
C12N2760/16222
CHEMISTRY; METALLURGY
International classification
C12N15/82
CHEMISTRY; METALLURGY
Abstract
A method of producing a virus like particle (VLP) in a plant comprising modified H3 hemagglutinin is provided. The method comprises introducing a nucleic acid comprising a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding a modified influenza hemagglutinin (HA) protein into the plant, or portion of the plant, the modified HA protein comprises a modified proteolytic loop. Followed by incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acids, thereby producing the VLP. The modified proteolytic loop may comprise one or more protease cleavage sites exhibiting reduced or abolished cleavage by a protease. Also described is a virus like particle (VLP) produced by the method, and plants expressing the VLP. The virus like particle (VLP) may comprise plant-specific N-glycans, or modified N-glycans.
Claims
1. A modified influenza type A subtype H3 hemagglutinin (HA) comprising a fully deleted proteolytic loop between subunits HA1 and HA2, the fully deleted proteolytic loop characterized as comprising a monobasic cleavage site.
2. The modified influenza HA of claim 1, wherein the modified influenza HA is derived from an unmodified H3 HA comprising 90-100% sequence identity to a sequence defined by SEQ ID NO:19, amino acids 25 to 574 of SEQ ID NO:122, amino acids 25 to 574 of SEQ ID NO:203, or amino acids 25 to 574 of SEQ ID NO:217.
3. The modified influenza HA of claim 1, wherein the monobasic cleavage site is recognized by a Clara-like protease.
4. The modified influenza HA of claim 3, wherein the Clara-like protease is tryptase Clara or trypsin/chymotrypsin.
5. The modified influenza HA of claim 1, wherein the monobasic cleavage site comprises amino acid sequence Q/E-X-R, PEK or a combination thereof.
6. The modified influenza HA of claim 1, wherein the proteolytic loop is fully replaced by a linker sequence.
7. A virus like particle (VLP) comprising the modified influenza HA defined in claim 1.
8. A virus like particle (VLP) comprising the modified influenza HA defined in claim 2.
9. A plant comprising the modified influenza HA of claim 1.
10. A plant comprising the VLP of claim 7.
11. A plant comprising the VLP of claim 8.
12. A composition comprising an effective dose of the VLP of claim 7 for inducing an immune response, and a pharmaceutically acceptable carrier.
13. A vaccine comprising an effective dose of the composition of claim 12 for inducing an immune response.
14. A method of inducing immunity to an influenza virus infection in a subject, comprising administering the composition of claim 12.
15. The method of claim 14, wherein the composition is administered to a subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:
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DETAILED DESCRIPTION
[0114] The following description is of a preferred embodiment.
[0115] The present invention relates to virus-like particles (VLPs) and methods of producing and increasing VLP yield, accumulation and production in plants.
[0116] The present invention provides, in part, a method of producing a virus like particle (VLP) in a plant, or portion of the plant. The method involves introducing a nucleic acid into the plant or portion of the plant. The nucleic acid comprises a regulatory region active in the plant and operatively linked to a nucleotide sequence encoding an influenza hemagglutinin (HA). The HA comprises a modified proteolytic loop or cleavage site. The plant or portion of the plant is incubated under conditions that permit the expression of the nucleic acid, thereby producing the VLP. If desired, the plant or portion of the plant may be harvested and the VLP purified.
[0117] The present invention also provides a VLP produced by this method. The VLP may comprise one or more than one lipid derived from a plant.
[0118] The VLP may be used to prepare a composition comprising an effective dose of the VLP for inducing an immune response, and a pharmaceutically acceptable carrier.
[0119] Also provided herein is a modified hemagglutinin, wherein the proteolytic loop or cleavage site has been modified.
[0120] The present invention also provides plant matter comprising the VLP produced by expressing the nucleic acids described above. The plant matter may be used in inducing immunity to an influenza virus infection in a subject. The plant matter may also be admixed as a food supplement.
[0121] The VLP of the present invention may also be produced by providing a plant or portion of the plant comprising a nucleic acid as defined above, and incubating the plant or portion of the plant under conditions that permit the expression of the nucleic acid, thereby producing the VLP. The VLP may comprise one or more than one lipid derived from a plant. The VLP may be used to prepare a composition comprising an effective dose of the VLP for inducing an immune response, and a pharmaceutically acceptable carrier. The present invention also provides plant matter comprising the VLP produced by expressing the first and second nucleic acids. The plant matter may be used in inducing immunity to an influenza virus infection in a subject. The plant matter may also be admixed as a food supplement.
[0122] The VLP of the present invention comprises one or more modified influenza hemagglutinin (HA). The modified HA may be derived from any HA, for example an H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA as described in WO 2009/009876; WO 2009/076778; WO 2010/003225; WO 2010/003235; WO 2011/03522; which are incorporated herein by reference).
[0123] The current invention includes VLPs comprising HA sequences of influenza strains, where the HA sequences comprise modified polybasic cleavage sites including for example, the modifications as described herein.
HA Protein
[0124] The term “hemagglutinin” or “HA” as used herein refers to a glycoprotein found on the outside of influenza viral particles. HA is a homotrimeric membrane type I glycoprotein, generally comprising a signal peptide, an HA1 domain, and an HA2 domain comprising a membrane-spanning anchor site at the C-terminus and a small cytoplasmic tail. Nucleotide sequences encoding HA are well known and are available—see, for example, the BioDefence Public Health base (Influenza Virus; see URL: biohealthbase.org) or National Center for Biotechnology Information (see URL: ncbi.nlm.nih.gov), both of which are incorporated herein by reference. HA may include any HA, for example an H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA as described in WO 2009/009876; WO 2009/076778; WO 2010/003225; WO 2010/003235; WO 2011/03522; which are incorporated herein by reference). Furthermore, the HA may be based on the sequence of a hemagglutinin that is isolated from one or more emerging or newly-identified influenza viruses. The present invention also includes VLPs that comprise modified HAs obtained from one or more than one influenza subtype.
[0125] The HA monomer may be subdivided in three functional domains—a stem domain, or stem domain cluster (SDC), a globular head domain, or head domain cluster (HDC) and a transmembrane domain cluster (TDC). The SDC and HDC may be collectively referred to as the ‘ectodomain’. A publication by Ha et al. 2002 (EMBO J. 21: 865-875; which is incorporated herein by reference) illustrates the relative orientation of the various subdomains of the SDC and HDC in several influenza subtypes, based on X-ray crystallographic structures.
[0126] HA protein is synthesized as a precursor protein (HA0) of about 75 kDa, which assembles at the surface into an elongated trimeric protein. The precursor protein is cleaved at a conserved activation cleavage site into 2 polypeptide chains, HA1 and HA2 (comprising the transmembrane region), linked by a disulfide bond.
[0127] The term “homotrimer” or “homotrimeric” indicates that an oligomer is formed by three HA protein molecules. Without wishing to be bound by theory, HA protein is synthesized as monomeric precursor protein (HA0) of about 75 kDa, which assembles at the surface into an elongated trimeric protein. Before trimerization occurs, the precursor protein is cleaved at a conserved activation cleavage site (also referred to as fusion peptide) into 2 polypeptide chains, HA1 and HA2 (comprising the transmembrane region), linked by a disulfide bond. The HA1 segment may be 328 amino acids in length, and the HA2 segment may be 221 amino acids in length. Although this cleavage may be important for virus infectivity, it may not be essential for the trimerization of the protein or for immunogenicity. Insertion of HA within the endoplasmic reticulum (ER) membrane of the host cell, signal peptide cleavage and protein glycosylation are co-translational events. Correct refolding of HA requires glycosylation of the protein and formation of 5-6 intra-chain disulfide bonds. The HA trimer assembles within the cis- and trans-Golgi complex, the transmembrane domain playing a role in the trimerization process. The crystal structures of bromelain-treated HA proteins, which lack the transmembrane domain, have shown a highly conserved structure amongst influenza strains. It has also been established that HA undergoes major conformational changes during the infection process, which requires the precursor HA0 to be cleaved into the 2 polypeptide chains HA1 and HA2. The HA protein may be processed (i.e., comprise HA1 and HA2 domains), or may be unprocessed (i.e. comprise the HA0 domain). The unprocessed precursor protein of HA is synthesized as a precursor protein (HA0) of about 75 kDa, which assembles at the surface into an elongated trimeric protein. The precursor protein is cleaved at a conserved cleavage site (also known as a proteolytic loop) into 2 polypeptide chains, HA1 and HA2 (comprising the transmembrane region), linked by a disulfide bond.
[0128] The HA protein as described herein may further be a modified HA (also referred to as “mutant HA”) protein, for example a modified precursor protein (HA0), in which the proteolytic loop or cleavage site is modified.
Modified HA/Cleavage Site
[0129] Following cleavage of HA0, HA becomes sensitive to pH, undergoing irreversible conformational change at the pH of endosome (<pH 6.0). The conformation of the precursor HA0 is stable at low pH, but the cleaved HA1-HA2 form, is metastable (Bullough P A et. al., 1994, Nature. Vol 371:37-43). The pH threshold that induce conformational change in different HA's is approximately pH 5.8-5.9 for the B strains, whereas it is more acidic, pH 5.1 to 5.8, for type A HA's (Beyer W E P et al, 1986, Archives Virol, vol 90: 173). Following cleavage, the amino terminal of HA2 is a nonpolar sequence of 23 amino acids that then become a transmembrane domain spanning cross the host cell membrane (called the fusion peptide;
[0130] In order to optimize the production of vaccine in eggs and maintain an active but attenuated virus, modification of the polybasic cleavage site of H5 (RERRRKKR↓G) has been studied (Horimoto T, et. al, 2006, Vaccine, Vol 24: 3669-3676). Mutants of interest contained a deletion of the 4 first charged amino acids (RERR) and a replacement of amino acids RKKR with TETR that inactivate the polybasic cleavage site but maintained the possibility to process HA0 to HA1-HA2 through the Arginin residue of the TETR motif (see
[0131] As used herein, the term “modified hemagglutinin” or “modified HA”, “mutated hemagglutinin” or “mutated HA” refers to an HA in which the HA has a modification or mutation, for example a substitution, insertion, deletion, or a combination thereof, that results in an altered amino acid sequence in the proteolytic loop or cleavage site of the HA protein.
[0132] The crystal structure of HA0 from A/Hong Kong/68 has been determined (Chen, J., 1998. Cell 95:409-417; incorporated herein by reference). Residues that are exposed to solvent are generally thought of being part of the cleavage site which forms an extended, highly exposed surface loop. A consensus sequence may be determined in this chosen region for example, but not limited to:
[0133] A/H3/HA0 Consensus: NVPEKQTR/GIFGAIAGFIE (SEQ ID NO: 66)
[0134] A/H1/HA0 Consensus: NIPSIQSR/GLFGAIAGFIE (SEQ ID NO: 67)
[0135] Avian H5 Consensus QRESRRKKR/GLFGAIAGFIEG (SEQ ID NO: 1)
[0136] B/HA0 Consensus: PAKLLKER/GFFGAIAGFLE (SEQ ID NO: 68)
Where the cleavage between HA1 and HA2 is indicated by “I” (see Bianchi et al., 2005, Journal of Virology, 79:7380-7388; incorporated herein by reference), and also
[0137] The HA protein may be an influenza type B hemagglutinin or Influenza type A hemagglutinin protein with a modification in the proteolytic loop region, for example a deletion, insertion, substitution or a combination thereof of the proteolytic loop (cleavage site). Without wishing to be bound by theory, modification of the proteolytic loop may ensures that the HA molecule is maintained as an HA0 precursor. Thereby producing a more homogenous and consistent VLP comprising HA0 proteins.
[0138] By “proteolytic loop” or “cleavage site” is meant the consensus sequence of the proteolytic site that is involved in precursor HA0 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 HA0 sequence. Consensus sequence of the influenza HA0 cleavage site may include influenza A consensus hemagglutinin amino acid sequences, including for example consensus H1, consensus H3, consensus H5, or influenza B consensus hemagglutinin amino acid sequences, for example but not limited to B Florida and B Malaysia. Non-limiting examples of sequences of the proteoloytic loop region are shown in
[0139] Residues in the proteolytic loop or cleavage site might be either mutated, for example but not limited to point mutation, substitution, insertion, or deletion. The term “amino acid mutation” or “amino acid modification” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced or abolished cleavage of the proteolytic loop or cleavage site by a protease.
[0140] By “modified proteolytic loop” it is meant that the proteolytic loop may include one or more point mutations, be partially deleted, fully deleted, partially replaced with a linker sequence, fully replaced by a linker sequence, comprise a partial or complete replacement of amino acids within the cleavage site with one or more non-protein amino acids, or a combination thereof. Similarly, by “modified cleavage site”, it is meant that the cleavage site within the proteolytic loop may include one or more point mutations, be partially deleted, fully deleted, partially replaced with a linker sequence, fully replaced by a linker sequence, comprising a partial or complete replacement of amino acids within the cleavage site with one or more non-protein amino acids, or a combination thereof. Modifications to the proteolytic loop, cleavage site, or both, may also involve the deletion, replacement, or substitution of one or more amino acids that are located outside of, or adjacent to, the proteolytic loop or cleavage site sequence. By “linker” it is meant an amino acid sequence comprising one or more amino acids that may be introduced within a proteolytic loop or a cleavage site, or that may replace some or all of the amino acids with the proteolytic loop or cleavage site. A linker may be designed to ensure that any amino acids deletions within the proteolytic loop or cleavage site do not disrupt the expression or subsequent activity of the modified HA.
[0141] By stabilizing the HA protein by modifying or deleting the proteolytic loop, increased product or protein yields may be achieved, when expressing the modified HA in a plant, when compared to a native HA expressed in a plant under the same conditions. Furthermore, by modifying or deleting the proteolytic loop the variability of expression of the expressed modified HA is reduced and the consistency of the produced modified HA is increased, when compared to a native HA expressed in a plant under the same conditions.
[0142] Therefore, the present invention also includes a method of increasing the product yield of a HA protein in a plant. Without wishing to be bound by theory, it is believed that by modifying or deleting the proteolytic loop in an HA protein, improved stability against proteolytic degradation in the plant, stabilization during passage of the HA in the Golgi apparatus secretion process, and during the purification process is provided.
[0143] Furthermore, the present invention also includes a method of increasing the product quality of an HA protein expressed in a plant. By product quality, it is meant for example an increased product yield of an HA expressed in a plant, stability of the product for example increased stability of the HA expressed in a plant, consistency of the product for example the production of a homogenous product for example HA0 or a combination thereof.
[0144] By an increase in product or protein yield, it is meant an increase in relative protein yield by about 20% to about 100%, or any amount therebetween as determined using standard techniques in the art, for example, from about 40% to about 70% or any value therebetween for example about 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 55, 56, 58, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or any amount therebetween, when compared to the product or protein yield of the same HA protein that does not have its proteolytic loop removed.
[0145] As shown in
[0146] As further shown in
[0147] Several strategies were evaluated in order to inactivate the cleavage of HA0 for both A and B strains. The consensus sequence that is recognized by proteases is enclosed on an extended loop, exposed to the solvent, and closed to the membrane distal part to the protein. In the B strain, this loop contains 2 sequence motifs recognized by proteases and the first N-terminal amino acids of the HA2 domain. A point mutation approach (for examples see Table 2, below) to inactivate the cleavage of HA0 precursor resulted in HA0 production, without an increase accumulation of B strain VLP. Deletion of the sequence motifs comprising the 2 protease cleavage motifs (7 amino acids) abolished the accumulation of the B HA. Removing the entire 18-amino acid loop from the HA protein of the B strain and inserting a linker to conserve structural features (beta strands) of the protein structure was effective (see below;
[0148] Amino acid sequence deletions and insertions include amino acid deletions and insertions of amino acids. A non-limiting example of a deletion in influenza B is the deletion of 17 amino acids (AKLLKERGFFGAIAGFLE) from position 340 to 357 of mature HA protein for example as shown in
[0149] Alternate amino acid mutations for HA from the A strain include amino acid substitutions, insertions and deletions, for example but not limited to a deletion in the proteolytic loop region of H5 Anhui of the amino acid sequence “RERRRKRGLFGAIAGFIE”, a deletion of the amino acid sequence of the proteolytic loop region of H5 Indo comprising “RESRRKKRGLFGAIAGFIE” or a deletion of the amino acid sequence of the proteolytic loop region of H5 Vietnam “RERRRKKRGLFGAIAGFIE”. For H3, the sequence “RNVPEKQTRGIF” may be deleted and replaced by an appropriate linker sequence, for example but not limited to “GS” as shown in
[0150] Furthermore, modifying or altering the proteolytic loop or cleavage site of a HA to reduce or abolish cleavage of the proteolytic loop or cleavage site by a protease, may also comprise non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and/or chemical properties. Non-protein amino acids may also be used for substitution. For example, amino acid substitutions may include replacing a hydrophobic by a hydrophilic amino acid. Amino acid substitutions may include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the protein amino acids.
[0151] Amino acid mutations for HA from the B strain and/or A strains may include amino acid deletions. For example in order to reduce or abolish cleavage of the proteolytic loop or cleavage site by a protease, one or more amino acid are deletion or removal within the proteolytic loop or cleavage site sequence. Non-limiting examples of deletions include removal of amino acids 323 to 341 of native HA H5 protein, for example H5 Anhui (RERRRKRGLFGAIAGFIE), H5 Indo (RESRRKKRGLFGAIAGFIE), or H5 Vietnam (RERRRKKRGLFGAIAGFIE), as shown in
[0152] Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful.
[0153] Therefore the hemagglutinin (HA) sequences of the invention may comprise modified proteolytic loop sequences or cleavage sites, thereby having reduced or abolished cleavage of the proteolytic loop or cleavage site by a protease. The hemagglutinin polypeptide sequences may comprise modified proteolytic loop or modified cleavage site sequences as, for example, set forth in
[0154] Analysis of sequence from H1, H3 and B HAs reveals that H1 possess one monobasic proteolytic site (Clara type monobasic: Q/EXR) that directly precedes the fusion peptide, whereas H3 and B HAs have 2 proteolytic sites, one that is recognized by Clara-like proteases (as found in H1), and another site recognized by trypsine and chymotrypsine-like proteases (P-E/A-K). The consensus sequence for cleavage of these HA is presented in Table 1.
TABLE-US-00001 TABLE 1 Consensus sequence of the proteolytic site for precursor HA.sub.0 cleavage. The sequences recognized by Clara tryptases or trypsine/ chimotrypsine are italicized, and bolded respectively. Several HA strains comprise polybasic Furin type cleavage sites (RKKR; plain text, underlined). H1 NIPSIQSR↓GLF SEQ ID NO: 47 H3 NVPEKQTR↓GIF SEQ ID NO: 48 H5 TGLRNSPQRESRRKKR↓GLF SEQ ID NO: 60 B PAKLLKER↓GFF SEQ ID NO: 59
[0155] In order to avoid a potential proteolytic cleavage of HA0 precursor of the HA, only one proteolytic site may need to be modified from the sequence of H1, whereas, in the case of H3 and B, two monobasic sites may need to be modified.
[0156] For example, a first cleavage site of HA.sub.0 of B/Florida and B/Brisbane may for example be eliminated by replacing the Lys 341 (mature protein numbering) with an Ile (see Table 2). The second monobasic site may be abolished by replacing three amino acids prior to the fusion peptide, KER (344-346), with NIQ. Sequences of several modified proteolytic loops of HA are provided in Table 2.
TABLE-US-00002 TABLE 2 Illustration of examples of mutations to destroy the cleavage of the precursor HA.sub.0. The monobasic site are italicized (Clara-like recognition) and in bold (no underlining; trypsine/chymotrypsine-like). The mutation are shown as bolded and underlined. The arrow represents the site for cleavage for conversion of HA.sub.0 into HA1-HA2. Abolition of precursor Strain Natural sequence cleavage site H5/Indo TGLRNSPQRESRRKKR↓GLF TGLRNSPQ GLF SEQ ID NO: 60 SEQ ID NO: 61 TGLRNSPQ
GLF SEQ ID NO: 62 H1/Brisbane NIPSIQSR↓GLF NIPSIS
GLF SEQ ID NO: 47 SEQ ID NO: 63 H3/Brisbane NVPEKQTR↓GIF NVPE
QT
GIF SEQ ID NO: 48 SEQ ID NO: 64 B/Florida, PAKLLKER↓GFF PA
LL
GFF B/Brisbane SEQ ID NO: 59 SEQ ID NO: 65
[0157] In further examples, the sequences comprising the proteolytic loop in HA0 may be replaced or deleted. For example, an H3 variant containing a deletion of the sequence RNVPEKQT at the C-terminus of HA1 in addition of deletion of the N-terminus amino acids GIFGIA of HA2 is provided in
[0158] In another example, the loop contain the proteolytic cleavage site in, for example H3, may have been replaced by a flexible linker, and the HA2 part may be left intact. A (GSS).sub.3 linker may be designed in order to accommodate the shortened HA1 to HA2. (see
[0159] In another example, HA from influenza B may contain a deletion of sequence ALKLLKER at the C-terminus of HA1 in addition of deletion of the N-terminus amino acids GFFGAIAGFLEG of HA2. The shortened HA1-HA2 may be linked together by a GG linker (see for example
[0160] In another example, HA from influenza B the loop containing the proteolytic site may have been replaced by a flexible linker, and the HA2 part was left intact. A longer GSSS linker may be designed in order to accommodate the shortened HA1 to HA2. (see for example
[0161] As shown in
[0162] In a similar manner, deletion of the proteolytic loop in H5/Indo, and replacement with either a “GG” (Construct 928; see
[0163] As show in
[0164] By an increase in activity, it is meant an increase in hemagglutination capacity by about 2% to about 100%, or any amount therebetween as determined using standard techniques in the art, for example, from about 10% to about 50% or any value therebetween for example about 2, 5, 8, 10, 12, 15, 18, 20, 22, 24, 25, 26, 28, 30, 32, 34, 35, 36, 38, 40, 42, 44, 45, 46, 48, 50, 52, 54, 55, 56, 58, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or any amount therebetween, when compared to the activity of the same HA protein that does not have its proteolytic loop removed.
[0165] The present invention also includes nucleotide sequences encoding modified HA from for example modified H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA, or any nucleotide sequences that hybridize to H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA under stringent conditions, or a nucleotide sequence that hybridizes under stringent hybridisation conditions to a compliment of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA, wherein the nucleotide sequence encodes a hemagglutinin protein that when expressed forms a VLP, and that the VLP induces the production of an antibody. For example, expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP may be used to produce an antibody that is capable of binding HA, including mature HA from B or H3. The VLP, when administered to a subject, induces an immune response. Preferably, the VLP induces the production of an antibody and the VLP, when administered to a subject, induces an immune response.
[0166] For example, expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP may be used to produce an antibody that is capable of binding a virus protein such as for example HA, including but not limited to HA0, HA0 protein with its proteolytic loop deleted or modified, HA1 or HA2 of one or more influenza types or subtypes, such as for example but not limited to subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, type B HA. The VLP, when administered to a subject, induces an immune response.
[0167] Hybridization under stringent hybridization conditions is known in the art (see for example Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 and supplements; Maniatis et al., in Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory, 1982; Sambrook and Russell, in Molecular Cloning: A Laboratory Manual, 3.sup.rd edition 2001; each of which is incorporated herein by reference). An example of one such stringent hybridization conditions may be about 16-20 hours hybridization in 4×SSC at 65° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes. Alternatively, an exemplary stringent hybridization condition could be overnight (16-20 hours) in 50% formamide, 4×SSC at 42° C., followed by washing in 0.1×SSC at 65° C. for an hour, or 2 washes in 0.1×SSC at 65° C. each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church aqueous phosphate buffer (7% SDS; 0.5M NaPO.sub.4 buffer pH 7.2; 10 mM EDTA) at 65° C., with 2 washes either at 50° C. in 0.1×SSC, 0.1% SDS for 20 or 30 minutes each, or 2 washes at 65° C. in 2×SSC, 0.1% SDS for 20 or 30 minutes each.
[0168] Additionally, the present invention includes nucleotide sequences that are characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100% or any amount therebetween, sequence identity, or sequence similarity, with the nucleotide sequence encoding H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16 or type B HA, wherein the nucleotide sequence encodes a hemagglutinin protein (modified HA) with a modified proteolytic loop sequence or cleavage site which has reduced or abolished cleavage of the proteolytic loop or cleavage site by a protease. When nucleotide sequence encoding the modified HA is expressed it forms a VLP, and the VLP induces the production of an antibody. For example, expression of the nucleotide sequence within a plant cell forms a VLP, and the VLP may be used to produce an antibody that is capable of binding HA, including unprocessed HA (HA0) or unprocessed wherein the proteolytic loop has been deleted. The VLP, when administered to a subject, induces an immune response.
[0169] Additionally, the present invention includes nucleotide sequences that are characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100% or any amount therebetween, sequence identity, or sequence similarity, with the nucleotide sequence of SEQ ID NO: 43, 91, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 137, 140, 144, 151, 158, 165, 204, 209, 214, 218, and 222, wherein the nucleotide sequence encodes a modified HA protein that when expressed forms a VLP, and that the VLP induces the production of an antibody that is capable of binding HA, including unprocessed HA (HA0) or unprocessed wherein the proteolytic loop has been deleted or modified. The VLP, when administered to a subject, induces an immune response.
[0170] Furthermore, the present invention includes amino acid sequences that are characterized as having about 70, 75, 80, 85, 87, 90, 91, 92, 93 94, 95, 96, 97, 98, 99, 100% or any amount therebetween, sequence identity, or sequence similarity, with the amino acid sequences of SEQ ID NO: 17, 18, 20, 21, 41, 58, 77, 81, 85, 92, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 134, 143, 147, 154, 161, 168, 194, 199, 205, 210, 215, 219 and 223, wherein the amino acid sequence encodes a modified HA protein that when expressed forms a VLP, and that the VLP induces the production of an antibody that is capable of binding HA, including unprocessed HA (HA0) or unprocessed wherein the proteolytic loop has been deleted or modified. The VLP, when administered to a subject, induces an immune response.
[0171] Sequence identity or sequence similarity may be determined using a nucleotide sequence comparison program, such as that provided within DNASIS (for example, using, but not limited to, the following parameters: GAP penalty 5, #of top diagonals 5, fixed GAP penalty 10, k-tuple 2, floating gap 10, and window size 5). However, other methods of alignment of sequences for comparison are well-known in the art for example the algorithms of Smith & Waterman (1981, Adv. Appl. Math. 2:482), Needleman & Wunsch (J. Mol. Biol. 48:443, 1970), Pearson & Lipman (1988, Proc. Nat'l. Acad. Sci. USA 85:2444), and by computerized implementations of these algorithms (e.g. GAP, BESTFIT, FASTA, and BLAST), or by manual alignment and visual inspection. An example of sequence alignment of HAs from different strains of influenza can be found in
For example, but is not limited to, nucleotide sequences encoding: [0172] a type B HA with a modified proteolytic loop as defined by SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 58, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114 and SEQ ID NO: 168, or nucleotide sequences encoding type B HAs comprising modified proteolytic loop regions as defined in SEQ ID NO: 65, SEQ ID NO: 72, SEQ ID NO:73, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113 and SE ID NO: 165. [0173] an H1 with a modified proteolytic loop include sequences comprising a modified cleavage site as defined by SEQ ID NO: 63. [0174] an H2 with a modified proteolytic loop include sequences comprising a modified cleavage site as defined by SEQ ID NO: 134. [0175] an H3 with a modified proteolytic loop include sequences defined by SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 205, SEQ ID NO: 210, SEQ ID NO: 215, SEQ ID NO: 219 and SEQ ID NO: 223 or comprising a modified cleavage site as defined by SEQ ID NO: 64. [0176] an H5 with a deleted proteolytic loop include sequences comprising a modified cleavage site as defined by SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71. [0177] an H7 with a modified proteolytic loop include sequences comprising a modified cleavage site as defined by SEQ ID NO: 154 or nucleotide sequences encoding type H7 HAs comprising modified proteolytic loop regions as defined in SEQ ID NO: 151. [0178] an H9 with a modified proteolytic loop include sequences comprising a modified cleavage site as defined by SEQ ID NO: 161 or nucleotide sequences encoding type H9 HAs comprising modified proteolytic loop regions as defined in SEQ ID NO: 158.
[0179] The present invention pertains to the use of an HA protein comprising the transmembrane domain and includes HA1 and HA2 domains, for example the HA protein may be HA0, or processed HA comprising HA1 and HA2. The HA protein may be used in the production or formation of VLPs using a plant, or plant cell, expression system.
Amplification Elements and Enhancer Elements/Regulatory Elements
[0180] In another example the modified HA protein may be expressed in an expression system that comprises amplification elements and/or regulatory elements or regions (also referred to herein as enhancer elements). For example an amplification element from a geminivirus such as for example, an amplification element from the bean yellow dwarf virus (BeYDV) may be used to express the modified HA. BeYDV belongs to the Mastreviruses genus adapted to dicotyledonous plants. BeYDV is monopartite having a single-strand circular DNA genome and can replicate to very high copy numbers by a rolling circle mechanism. BeYDV-derived DNA replicon vector systems have been used for rapid high-yield protein production in plants.
[0181] As used herein, the phrase “amplification elements” refers to a nucleic acid segment comprising at least a portion of one or more long intergenic regions (LIR) of a geminivirus genome. As used herein, “long intergenic region” refers to a region of a long intergenic region that contains a rep binding site capable of mediating excision and replication by a geminivirus Rep protein. In some aspects, the nucleic acid segment comprising one or more LIRs, may further comprises a short intergenic region (SIR) of a geminivirus genome. As used herein, “short intergenic region” refers to the complementary strand (the short IR (SIR) of a Mastreviruses). Any suitable geminivirus-derived amplification element may be used herein. See, for example, WO2000/20557; WO2010/025285; Zhang X. et al. (2005, Biotechnology and Bioengineering, Vol. 93, 271-279), Huang Z. et al. (2009, Biotechnology and Bioengineering, Vol. 103, 706-714), Huang Z. et al. (2009, Biotechnology and Bioengineering, Vol. 106, 9-17); which are herein incorporated by reference). If more than one LIR is used in the construct, for example two LIRs, then the promoter, CMPV-HT regions and the nucleic acid sequence of interest and the terminator are bracketed by each of the two LIRs.
[0182] As described herein, co-delivery of bean yellow dwarf virus (BeYDV)-derived vector and a Rep/RepA-supplying vector, by agroinfiltration of Nicotiana benthamiana leaves results in efficient replicon amplification and robust protein production. Western blot analysis of protein extracts from plants transformed with gene constructs driving the expression of modified influenza B HA (from B/Brisbane/60/2008) with or without the proteolytic loop removed (see
[0183] As shown in
[0184] However, the presence of BeyDV is not required when an enhancer element is present in the expression system and when the enhance element is not CPMV-HT. As for example shown in
[0185] Therefore, the mutant (modified) HA protein may be expressed in the absence of an amplification element, such as a geminivirus-based amplification element for example BeYDV, but in the presence of an enhancer element, such for example CPMV 160, CPMV160+ or CPMV HT+.
[0186] The mutant (modified HA) may be expressed in the presence of an enhancer element, such for example CPMV 160, CPMV160+ or CPMV HT+, but in the absence or presence of an amplification element, such for example BeYDV. As shown in
[0187] HA constructs comprising an enhancer element (either CMPV HT+ or CMPV 160+) and a proteolytic loop replaced with a GG linker (deleted proteolytic loop) exhibit increased expression when compared to wild type or HA constructs comprising CPMV HT (
[0188]
[0189] Enhancer elements may be used to achieve high level of transient expression of mutant (modified) HA proteins with modified proteolytic loops. Enhancer elements may be based on RNA plant viruses, including comoviruses, such as Cowpea mosaic virus (CPMV; see, for example, WO2007/135480; WO2009/087391; US 2010/0287670, Sainsbury F. et al., 2008, Plant Physiology; 148: 121-1218; Sainsbury F. et al., 2008, Plant Biotechnology Journal; 6: 82-92; Sainsbury F. et al., 2009, Plant Biotechnology Journal; 7: 682-693; Sainsbury F. et al. 2009, Methods in Molecular Biology, Recombinant Proteins From Plants, vol. 483: 25-39).
CPMV 160 (CPMVX) and CPMV 160+ (CPMVX+)
[0190] In one embodiment the Enhancer Elements are “CPMVX” (also referred as “CPMV 160”) and/or “CPMVX+” (also referred to as “CPMV 160+”) as described in U.S. 61/925,852, which is incorporated herein by reference.
[0191] Expression enhancer “CPMVX” comprises a comovirus cowpea mosaic virus (CPMV) 5′ untranslated region (UTR). The 5′UTR from nucleotides 1-160 of the CPMV RNA-2 sequence (SEQ ID NO: 93), starts at the transcription start site to the first in frame initiation start codon (at position 161), which serve as the 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. It has been shown that removal of AUG 115 in addition to the removal of AUG 161 enhances expression when combined with an incomplete M protein (Sainsbury and Lomonossoff, 2008, Plant Physiology; 148: 1212-1218; WO 2009/087391; which are incorporated herein by reference).
[0192] CPMVX comprises X nucleotides of SEQ ID NO: 93, where X=160, 155, 150, or 114 of SEQ ID NO:93, or a sequence that comprises between 80% to 100% sequence similarity with CPMVX, where X=160, 155, 150, or 114 of SEQ ID NO:93. This expression enhancer is generally referred to as CPMVX (see
[0193] The expression enhancer CPMVX, where X=160, consists of nucleotides 1-160 of SEQ ID NO: 93:
TABLE-US-00003 (SEQ ID NO: 93) 1 tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc 121 gatcttcaac gttgtcagat cgtgcttcgg caccagtaca
[0194] The CPMVX enhancer sequence may further be fused to a stuffer sequence, wherein the CMPVX comprises X nucleotides of SEQ ID NO: 93, where X=160, 155, 150, or 114 of SEQ ID NO: 93, or a sequence that comprises between 80 to 100% sequence similarity with CPMVX, where X=160, 155, 150, or 114 of SEQ ID NO: 93, and the stuffer sequence comprises from 1-100 nucleotides fused to the 3′ end of the CMPVX sequence. For example, 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.
[0195] If the CMPVX sequence comprises a stuffer fragment, then this expression enhancer may be referred to as CPMVX+ (see
[0196] The stuffer sequence may be modified by truncation, deletion, or replacement of the native CMPV 5′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′UTR (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. For example, which is not to be considered limiting, 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 FIG. 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 (
[0197] 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.
[0198] The plant kozak sequence may be any known plant kozak sequences (see for example L. Rangan et. al. Mol. Biotechnol. 2008), including, but not limited to the following plant consensus sequences:
TABLE-US-00004 (SEQ ID NO: 174; plant kingdom) caA(A/C)a (SEQ ID NO: 175; dicots) aaA(A/C)a (SEQ ID NO: 176; arabidopsis) aa(A/G)(A/C)a
The plant kozak sequence may also be selected from the group of:
TABLE-US-00005 (SEQ ID NO: 177) AGAAA (SEQ ID NO: 178) AGACA (SEQ ID NO: 179) AGGAA (SEQ ID NO: 180) AAAAA (SEQ ID NO: 181) AAACA (SEQ ID NO: 182) AAGCA (SEQ ID NO: 183) AAGAA (SEQ ID NO: 184) AAAGAA (SEQ ID NO: 185) AAAGAA (SEQ ID NO: 186; Consensus sequence) (A/-)A(A/G)(A/G)(A/C)A.
[0199] 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 (
CPMV HT+, CPMV HT+ [WT115], CPMV HT+ [511]
[0200] In another embodiment the Enhancer Elements is “CPMV HT+” as described in U.S. 61/971,274, which is incorporated herein by reference. Expression enhancer “CPMV HT+” (see
[0201] A plant expression system comprising a first nucleic acid sequence comprising a regulatory region, operatively linked with one or more than one expression enhancer as described herein (e.g. CPMV HT+, CPMV HT+ [WT115], CPMV HT+ [511]), and a nucleotide sequence encoding a modified HA is also provided. Furthermore, a nucleic acid comprising a promoter (regulatory region) sequence, an expression enhancer (e.g. CPMV HT+ or CPMV HT+ [WT115]) comprising a comovirus 5′UTR and a stuffer sequence with a plant kozak sequence fused to one or more nucleic acid sequences encoding a modified HA are described. The nucleic acid may further comprise a sequence comprising a comovirus 3′ untranslated region (UTR), for example, a plastocyanin 3′ UTR, or other 3′UTR active in a plant, and a terminator sequence, for example a NOS terminator, operatively linked to the 3′end of the nucleotide sequence encoding a modified HA (referred to as nucleotide of interest in
[0202] SEQ ID NO: 173 comprises a “CPMV HT” expression enhancer as known in the prior art (e.g. FIG. 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: 173 with modified nucleotides at position 115 (cgt), and an incomplete M protein with a modified nucleotide at position 162 (acg), and lacks a plant kozak sequence (5′UTR: nucleotides 1-160; incomplete M protein underlined, nucleotides 161-509). SEQ ID NO: 173 also includes a multiple cloning site (italics, nucleotides 510-528) which is not present in the prior art CPMV HT sequence:
TABLE-US-00006 SEQ ID NO: 173 1 tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgcgtgagc 121 gatcttcaac gttgtcagat cgtgcttcgg caccagtaca ttttctt tcactgaagc 181 gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241 ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301 atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361 gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421 atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481 taagcttctg tatattctgc ccaaatttgt cgggccc
[0203] CPMV HT+ with a plant kozak consensus sequence is provided in SEQ ID NO: 187 (nucleotide 1-160, 5′UTR, including modified ATG at positions 115 (GTG) lower case bold and italics; stuffer fragment comprising: an incomplete M protein underlined, nucleotides 161-509, with modified nucleotide at 162 (ACG); a multiple cloning site, italics, nucleotides 510-528; and a consensus plant kozak sequence, caps and bold, nucleotides 529-534).
TABLE-US-00007 (SEQ ID NO: 187) 1 tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgc agc 121 gatcttcaac gttgtcagat cgtgcttcgg caccagtaca
ttttctt tcactgaagc 181 gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241 ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301 atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361 gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421 atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481 taagcttctg tatattctgc ccaaatttgt tcgggcccaa taccgcgg(
) (
)
[0204] SEQ ID NO: 188 (“CPMV HT+ 511”) comprises a segment of the native sequence of the CPMV RNA 2 genome from nucleotides 1-154. The 5′UTR sequence from nucleotides 1-511 of SEQ ID NO: 188 comprises modified “atg” sequences at positions 115 (“g” in place of “a”; italics bold) and 162 (“c” in place of “t”; italics bold), and an incomplete M protein (underlined) from nucleotides 161-511. CPMV HT+ 511 comprises a native M protein kozak consensus sequence (nucleotides 508-511; bold):
TABLE-US-00008 SEQ ID NO: 188 1 tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgc agc 121 gatcttcaac gttgtcagat cgtgcttcgg caccagtaca
ttttctt tcactgaagc 181 gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241 ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301 atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361 gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421 atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481 taagcttctg tatattctgc ccaaatttga a . . .
[0205] Another non-limiting example of a CPMV HT+ enhancer sequence is provided by the sequence of SEQ ID NO: 189 (CPMV HT+ [WT115]). Expression cassettes or vectors comprising CPMV HT+ and including a plant regulatory region in operative association with the expression enhancer sequence of SEQ ID NO: 189, and the transcriptional start site (ATG) at the 3′ end fused to a nucleotide sequence encoding modified HA are also part of the present invention.
[0206] SEQ ID NO: 189 (CPMV HT+ [WT115]) nucleotide 1-160, 5′UTR, with an ATG at position 115-117, lower case bold; stuffer fragment comprising: an incomplete M protein underlined, nucleotides 161-509; with a modified ATG at position 161-153 lower case bold, and underlined, a multiple cloning site, italics, nucleotides 510-528; and a plant kozak sequence, caps and bold, nucleotides 529-534).
TABLE-US-00009 (SEQ ID NO: 189) 1 tattaaaatc ttaataggtt ttgataaaag cgaacgtggg gaaacccgaa ccaaaccttc 61 ttctaaactc tctctcatct ctcttaaagc aaacttctct cttgtctttc ttgc agc 121 gatcttcaac gttgtcagat cgtgcttcgg caccagtaca
ttttctt tcactgaagc 181 gaaatcaaag atctctttgt ggacacgtag tgcggcgcca ttaaataacg tgtacttgtc 241 ctattcttgt cggtgtggtc ttgggaaaag aaagcttgct ggaggctgct gttcagcccc 301 atacattact tgttacgatt ctgctgactt tcggcgggtg caatatctct acttctgctt 361 gacgaggtat tgttgcctgt acttctttct tcttcttctt gctgattggt tctataagaa 421 atctagtatt ttctttgaaa cagagttttc ccgtggtttt cgaacttgga gaaagattgt 481 taagcttctg tatattctgc ccaaatttgt tcgggcccaa taccgcgg
[0207] The plant kozak sequence of SEQ ID NO: 189 may be any plant kozak sequence, including but not limited, to one of the sequences of SEQ ID NO's: 174-186.
“Chimeric Protein”
[0208] The modified HA might further be a chimeric protein. By “chimeric virus protein” or “chimeric virus polypeptide”, also referred to as “chimeric protein” or “chimeric polypeptide”, or “chimeric HA” it is meant a protein or polypeptide that comprises amino acid sequences from two or more than two sources, for example but not limited to, two or more influenza types or subtypes, or influenza's of a different origin, that are fused as a single polypeptide. The chimeric protein or polypeptide may include a signal peptide that is the same as, or heterologous with, the remainder of the polypeptide or protein. The chimeric protein or chimeric polypeptide may be produced as a transcript from a chimeric nucleotide sequence, and following synthesis, and as required, may associate to form a multimeric protein. Therefore, a chimeric protein or a chimeric polypeptide also includes a protein or polypeptide comprising subunits that are associated via disulphide bridges (i.e. a multimeric protein). For example, a chimeric polypeptide comprising amino acid sequences from two or more than two sources may be processed into subunits, and the subunits associated via disulphide bridges to produce a chimeric protein or chimeric polypeptide. A chimeric HA protein may also comprises an antigenic protein or a fragment thereof of a first influenza virus, and a transmembrane domain complex (TDC) from an second virus influenza HA, including a transmembrane domain and cytosolic tail domains (TM/CT). The polypeptide may be a modified HA, and each of the two or more than two amino acid sequences that make up the polypeptide may be obtained from different HA's to produce a chimeric HA, chimeric influenza HA, chimeric modified HA or chimeric modified influenza HA. A chimeric HA may also include an amino acid sequence comprising heterologous signal peptide (a chimeric HA preprotein) that is cleaved after or during protein synthesis. Preferably, the chimeric polypeptide, or chimeric influenza HA is not naturally occurring. A nucleic acid encoding a chimeric polypeptide may be described as a “chimeric nucleic acid”, or a “chimeric nucleotide sequence”. For example a chimeric nucleic acid may comprise a nucleotide sequence encoding the modified HA comprises a chimeric nucleotide sequence encoding, in series, a modified HA ectodomain comprising a modified proteolytic loop, an influenza transmembrane domain, and a cytoplasmic tail, wherein the modified HA ectodomain is from a first influenza strain and the transmembrane domain and the cytoplasmic tail are from a second influenza strain. Examples of chimeric nucleotide acids, wherein the modified HA ectodomain is from a first influenza strain and the transmembrane domain and the cytoplasmic tail are from a second influenza strain are given in Examples 5.14, 5.16, 5.18, 5.19, 5.21, 5.23, 5.42, 5.43, and 5.44. A virus-like particle comprised of chimeric HA may be described as a “chimeric VLP”.
[0209] As described above, the chimeric protein, chimeric polypeptide, or chimeric HA may include a signal peptide that is the same as, or heterologous with, the remainder of the polypeptide or protein. The term “signal peptide” is well known in the art and refers generally to a short (about 5-30 amino acids) sequence of amino acids, found generally at the N-terminus of a polypeptide that may direct translocation of the newly-translated polypeptide to a particular organelle, or aid in positioning of specific domains of the polypeptide chain relative to others. As a non-limiting example, the signal peptide may target the translocation of the protein into the endoplasmic reticulum and/or aid in positioning of the N-terminus proximal domain relative to a membrane-anchor domain of the nascent polypeptide to aid in cleavage and folding of the mature protein, for example a modified HA or chimeric modified HA.
[0210] The HA may also be a chimeric HA or chimeric modified HA, wherein a native transmembrane domain of the HA or modified HA is replaced with a heterologous transmembrane domain. The transmembrane domain of HA proteins is highly conserved (see for example FIG. 1C of WO 2010/148511; which is incorporated herein by reference). The heterologous transmembrane domain may be obtained from any HA transmembrane domain, for example but not limited to the transmembrane domain from H1 California, B/Florida/4/2006 (GenBank Accession No. ACA33493.1), B/Malaysia/2506/2004 (GenBank Accession No. ABU99194.1), H1/Bri (GenBank Accession No. ADE28750.1), H1 A/Solomon Islands/3/2006 (GenBank Accession No. ABU99109.1), H1/NC (GenBank Accession No. AAP34324.1), H2 A/Singapore/1/1957 (GenBank Accession No. AAA64366.1), H3 A/Brisbane/10/2007 (GenBank Accession No. ACI26318.1), H3 A/Wisconsin/67/2005 (GenBank Accession No. AB037599.1), H5 A/Anhui/1/2005 (GenBank Accession No. ABD28180.1), H5 A/Vietnam/1194/2004 (GenBank Accession No. ACR48874.1), H5-Indo (GenBank Accession No. ABW06108.1). The transmembrane domain may also be defined by the following consensus amino acid sequence:
TABLE-US-00010 (SEQ ID NO: 94) ILXIYYSTVAISSLXLXXMLAGXSXWMCS
[0211] Examples of constructs comprising a chimeric HA with a heterologous trans-membrane domain include: construct number 1875 (CPMV-HT+ B Brisbane/60/08 with deleted proteolytic loop+H1TM, with trans-membrane domain and cytoplasmic tail replaced by H1 A/California/07/2009; see example 5.19), construct number 1977 (CPMV-160+ B Brisbane/60/08 with deleted proteolytic loop+H1TM, with trans-membrane domain and cytoplasmic tail replaced by H1 A/California/07/2009; see example 5.14), construct number 1067 (CPMV-HT B Brisbane/60/08 with deleted proteolytic loop+H1TM, with trans-membrane domain and cytoplasmic tail replaced by H1 A/California/07/2009; see example 5.14), construct number 2074 (CPMV HT B Massachusetts/2/2012+H1Tm, with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009; see Example 5.16), construct number 2060 (CPMV HT160+ Massachusetts/2/2012+H1Tm, with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009; see Example 5.16), construct number 2062 (CPMV 160+ B Massachusetts/2/2012+H1Tm, with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009; see Example 5.21), construct number 1860 (CPMV HT+ B Wisconsin/1/2010+H1Tm with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009; see Example 5.23), construct number 1454 (CPMV HT B Wisconsin/1/2010+H1Tm with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009, see Example 5.18) and construct number 1893 (CPMV 160+ B Wisconsin/1/2010+H1Tm with trans-membrane domain and cytoplasmic tail replaced by those of H1 A/California/07/2009) see Example 5.18. Activity of these chimeric modified HA's is shown in
Signal Peptide
[0212] A signal peptide (SP) may be native to the modified HA or chimeric modified HA, or a signal peptide may be heterologous with respect to the primary sequence of the modified HA being expressed. The modified HA may comprise a signal peptide from a first influenza type, subtype or strain with the balance of the HA from one or more than one different influenza type, subtype or strain. For example the native signal peptide of HA subtypes H1, H2, H3, H5, H6, H7, H9 or influenza type B may be used to express the modified HA in a plant system. In some embodiments of the invention, the SP may be of an influenza type B, H1, H3 or H5; or of the subtype H1/Bri, H1/NC, H5/Indo, H3/Bri or B/Flo.
[0213] Furthermore, the modified HA or chimeric modified HA may comprise a native, or a non-native signal peptide; the non-native signal peptide may be of plant origin or obtained from an animal or bacterial polypeptide. The native signal peptide may correspond to that of the HA or modified HA being expressed, additionally, the signal peptide may be from a structural 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. Z11499 also see WO 2009/076778; WO 2010/148511, or WO 2010/003235), 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:
TABLE-US-00011 (SEQ ID NO: 171) ATGGCAACTACTAAAACTTTTTTAATTTTATTTTTTATGATATTAGCAACT ACTAGTTCAACATGTGCT
the amino acid sequence of patatin A signal peptide is:
TABLE-US-00012 (SEQ ID NO: 172) MATTKTFLILFFMILATTSSTCA
[0214] The present invention therefore provides for a modified HA or chimeric modified HA comprising a native, or a non-native signal peptide, and nucleic acids encoding such chimeric modified HA proteins.
Co-Expression with Channel Protein
[0215] The mutant (modified) HA may be produced in a plant by co-expressing a first nucleic acid encoding the modified HA with a second nucleic acid encoding a channel protein, for example but not limited to a proton channel protein. The first and second nucleic acids may be introduced to the plant in the same step, or they may be introduced to the plant sequentially. The first and second nucleic acids may be introduced in the plant in a transient manner, or in a stable manner. Furthermore, a plant that expresses a first nucleic acid encoding the modified HA may transformed with a channel protein, for example but not limited to a proton channel protein, (second nucleic acid) so that both the first and the second nucleic acids are co-expressed in the plant. Alternatively, a plant that expresses a channel protein, for example but not limited to a proton channel protein, (second nucleic acid) may be transformed with a first nucleic acid encoding the modified HA so that both the first and the second nucleic acids are co-expressed in the plant. Additionally, a first plant expressing the first nucleic acid encoding modified HA, may be crossed with a second plant expressing the second nucleic acid encoding the channel protein for example but not limited to a proton channel protein, to produce a progeny plant that co-expresses the first and second nucleic acids encoding the modified HA and the channel protein, for example but not limited to a proton channel protein, respectively.
[0216] Without wishing to be bound by theory, the pH of a cellular compartment comprising modified HA, including the Golgi apparatus, may be important for the folding, stability and/or proteolysis of HA. Proton channel proteins, such as for example influenza M2 and BM2 protein may regulate the pH in cellular compartments. For example, M2 regulates the potentiation of membrane fusion by buffering intracellular compartments both in late and early stages of influenza viral replication.
[0217] By co-expressing a channel protein, for example but not limited to a proton channel protein, along with a modified HA, the pH within the Golgi apparatus may increase, and result in an increase in stability, reduction of degradation, or a combination thereof, and increase expression levels and yield of modified HA and/or VLPs.
[0218] By co-expressing a modified HA along with a channel protein, for example but not limited to a proton channel protein, in a plant, increased yield of HA and/or VLPs are observed, when compared to a plant that expressed the modified without co-expression of the channel protein, for example but not limited to a proton channel protein (see
[0219] Furthermore, the efficacy of M2 from influenza A/Puerto Rico/8/1934 to increase accumulation of the modified influenza B HA and H3 was compared to that of M2 from influenza A/New Caledonia/20/1999. For the modified influenza B HA, the comparison was undertaken by western blot analysis of protein extracts from plants transformed with constructs 1059, 1059+1261 and 1059+859. The results obtained demonstrated that the co-expression of M2 from influenza A/Puerto Rico/8/1934 (encoded by construct no. 859) was as efficient as the co-expression of M2 from influenza A/New Caledonia/20/1999 (encoded by construct no. 1261) for increasing accumulation of the modified influenza B HA (
[0220] As used herein, the terms “M2,” “M2 protein,” “M2 sequence” and “M2 domain” refer to all or a portion of an M2 protein sequence isolated from, based upon or present in any naturally occurring or artificially produced influenza virus strain or isolate. Thus, the term M2 and the like include naturally occurring M2 sequence variants produced by mutation during the virus life-cycle or produced in response to a selective pressure (e.g., drug therapy, expansion of host cell tropism or infectivity, etc.), as well as recombinantly or synthetically produced M2 sequences. Non-limiting example of sequences that may be used with the present invention include M2 from A/Puerto Rico/8/1934 and M2 from A/New Caledonia/20/1999.
Immune Response
[0221] An “immune response” generally refers to a response of the adaptive immune system. The adaptive immune system generally comprises a humoral response, and a cell-mediated response. The humoral response is the aspect of immunity that is mediated by secreted antibodies, produced in the cells of the B lymphocyte lineage (B cell). Secreted antibodies bind to antigens on the surfaces of invading microbes (such as viruses or bacteria), which flags them for destruction. Humoral immunity is used generally to refer to antibody production and the processes that accompany it, as well as the effector functions of antibodies, including Th2 cell activation and cytokine production, memory cell generation, opsonin promotion of phagocytosis, pathogen elimination and the like. The terms “modulate” or “modulation” or the like refer to an increase or decrease in a particular response or parameter, as determined by any of several assays generally known or used, some of which are exemplified herein.
[0222] A cell-mediated response is an immune response that does not involve antibodies but rather involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. Cell-mediated immunity is used generally to refer to some Th cell activation, Tc cell activation and T-cell mediated responses. Cell mediated immunity is of particular importance in responding to viral infections.
[0223] For example, the induction of antigen specific CD8 positive T lymphocytes may be measured using an ELISPOT assay; stimulation of CD4 positive T-lymphocytes may be measured using a proliferation assay. Anti-influenza antibody titres may be quantified using an ELISA assay; isotypes of antigen-specific or cross reactive antibodies may also be measured using anti-isotype antibodies (e.g. anti-IgG, IgA, IgE or IgM). Methods and techniques for performing such assays are well-known in the art.
[0224] Cross-reactivity HA1 titres may also be used to demonstrate the efficacy of an immune response to other strains of virus related to the vaccine subtype. For example, serum from a subject immunized with a vaccine composition of a first strain (e.g. VLPs of A/Indonesia 5/05) may be used in an HA1 assay with a second strain of whole virus or virus particles (e.g. A/Vietnam/1194/2004), and the HA1 titer determined.
[0225] Cytokine presence or levels may also be quantified. For example a T-helper cell response (Th1/Th2) will be characterized by the measurement of IFN-γ and IL-4 secreting cells using by ELISA (e.g. BD Biosciences OptEIA kits). Peripheral blood mononuclear cells (PBMC) or splenocytes obtained from a subject may be cultured, and the supernatant analyzed. T lymphocytes may also be quantified by fluorescence-activated cell sorting (FACS), using marker specific fluorescent labels and methods as are known in the art.
[0226] A microneutralization assay may also be conducted to characterize an immune response in a subject, see for example the methods of Rowe et al., 1973. Virus neutralization titers may be obtained several ways, including: 1) enumeration of lysis plaques (plaque assay) following crystal violet fixation/coloration of cells; 2) microscopic observation of cell lysis in culture; 3) ELISA and spectrophotometric detection of NP virus protein (correlate with virus infection of host cells).
[0227] The term “virus like particle” (VLP), or “virus-like particles” or “VLPs” refers to structures that self-assemble and comprise virus proteins for example an influenza HA protein or modified HA protein such as for example an HA0 protein, wherein the proteolytic loop has been modified. VLPs are generally morphologically and antigenically similar to virions produced in an infection, but lack genetic information sufficient to replicate and thus are non-infectious. In some examples, VLPs may comprise a single protein species, or more than one protein species. For VLPs comprising more than one protein species, the protein species may be from the same species of virus, or may comprise a protein from a different species, genus, subfamily or family of virus (as designated by the ICTV nomenclature). In other examples, one or more of the protein species comprising a VLP may be modified from the naturally occurring sequence, such as for example a modified HA as described herein. VLPs may be produced in suitable host cells including plant and insect host cells. Following extraction from the host cell and upon isolation and further purification under suitable conditions, VLPs may be purified as intact structures.
[0228] Furthermore, VLPs may be produced that comprise a combination of HA subtypes. For example, VLPs may comprise one or more than one HA or one or more than one modified HA from the subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, subtype B HA or a combination thereof. Selection of the combination of HAs or modified HAs may be determined by the intended use of the vaccine prepared from the VLP. For example a vaccine for use in inoculating birds may comprise any combination of HA subtypes or modified HA subtypes, while VLPs useful for inoculating humans may comprise subtypes one or more than one of subtypes or modified subtype of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, subtype B HA. However, other HA subtype or modified HA subtype combinations may be prepared depending upon the use of the VLP. In order to produce VLPs comprising combinations of HA subtypes or modified subtype HAs, the desired HA subtype or modified HA subtype may be co-expressed within the same cell, for example a plant cell.
[0229] The VLPs produced from influenza derived proteins, in accordance with the present invention do not comprise M1 protein. The M1 protein is known to bind RNA (Wakefield and Brownlee, 1989) which is a contaminant of the VLP preparation. The presence of RNA is undesired when obtaining regulatory approval for the VLP product, therefore a VLP preparation lacking RNA may be advantageous.
[0230] The VLPs produced as described herein do not typically comprise neuramindase (NA). However, NA may be co-expressed with HA should VLPs comprising HA and NA be desired.
[0231] The invention also includes, but is not limited to, virus derived VLPs that obtain a lipid envelope from the plasma membrane of the cell in which the VLP proteins are expressed. For example, if the VLP is expressed in a plant-based system, the VLP may obtain a lipid envelope from the plasma membrane of the cell.
[0232] Generally, the term “lipid” refers to a fat-soluble (lipophilic), naturally-occurring molecules. The term is also used more specifically to refer to fatty-acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids), as well as other fat-soluble sterol-containing metabolites or sterols. Phospholipids are a major component of all biological membranes, along with glycolipids, sterols and proteins. Examples of phospholipids include phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and the like. Examples of sterols include zoosterols (e.g., cholesterol) and phytosterols. Over 200 phytosterols have been identified in various plant species, the most common being campesterol, stigmasterol, ergosterol, brassicasterol, delta-7-stigmasterol, delta-7-avenasterol, daunosterol, sitosterol, 24-methylcholesterol, cholesterol or beta-sitosterol. As one of skill in the art would understand, the lipid composition of the plasma membrane of a cell may vary with the culture or growth conditions of the cell or organism from which the cell is obtained.
[0233] Cell membranes generally comprise lipid bilayers, as well as proteins for various functions. Localized concentrations of particular lipids may be found in the lipid bilayer, referred to as ‘lipid rafts’. Without wishing to be bound by theory, lipid rafts may have significant roles in endo and exocytosis, entry or egress of viruses or other infectious agents, inter-cell signal transduction, interaction with other structural components of the cell or organism, such as intracellular and extracellular matrices.
[0234] In plants, influenza VLPs bud from the plasma membrane therefore the lipid composition of the VLPs reflects their origin. The VLPs produced according to the present invention comprise HA of one or more than one type or subtype of influenza, complexed with plant derived lipids. Plant lipids can stimulate specific immune cells and enhance the immune response induced. Plant membranes are made of lipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and also contain glycosphingolipids, saponins, and phytosterols. Additionally, lipid rafts are also found in plant plasma membranes—these microdomains are enriched in sphingolipids and sterols. In plants, a variety of phytosterols are known to occur, including stigmasterol, sitosterol, 24-methylcholesterol and cholesterol (Mongrand et al., 2004).
[0235] PC and PE, as well as glycosphingolipids can bind to CD1 molecules expressed by mammalian immune cells such as antigen-presenting cells (APCs) like dendritic cells and macrophages and other cells including B and T lymphocytes in the thymus and liver (Tsuji M., 2006). CD1 molecules are structurally similar to major histocompatibility complex (MEW) molecules of class I and their role is to present glycolipid antigens to NKT cells (Natural Killer T cells). Upon activation, NKT cells activate innate immune cells such as NK cells and dendritic cells and also activate adaptive immune cells like the antibody-producing B cells and T-cells.
[0236] A variety of phytosterols may be found in a plasma membrane—the specific complement may vary depending on the species, growth conditions, nutrient resources or pathogen state, to name a few factors. Generally, beta-sitosterol is the most abundant phytosterol.
[0237] The phytosterols present in an influenza VLP complexed with a lipid bilayer, such as a plasma-membrane derived envelope may provide for an advantageous vaccine composition. Without wishing to be bound by theory, plant-made VLPs complexed with a lipid bilayer, such as a plasma-membrane derived envelope, may induce a stronger immune reaction than VLPs made in other expression systems, and may be similar to the immune reaction induced by live or attenuated whole virus vaccines.
[0238] The VLP as described herein may be complexed with a plant-derived lipid bilayer. In some embodiments the plant-derived lipid bilayer may comprise the envelope of the VLP. The plant derived lipids may comprise lipid components of the plasma membrane of the plant where the VLP is produced, including, but not limited to, phosphatidylcholine (PC), phosphatidylethanolamine (PE), glycosphingolipids, phytosterols or a combination thereof. A plant-derived lipid may alternately be referred to as a ‘plant lipid’. Examples of phytosterols are known in the art, and include, for example, stigmasterol, sitosterol, 24-methylcholesterol and cholesterol—see, for example, Mongrand et al., 2004.
[0239] VLPs may be assessed for structure and size by, for example, hemagglutination assay, electron microscopy, or by size exclusion chromatography.
[0240] For size exclusion chromatography, total soluble proteins may be extracted from plant tissue by homogenizing (Polytron) sample of frozen-crushed plant material in extraction buffer, and insoluble material removed by centrifugation. Precipitation with PEG may be used. The soluble protein is quantified, and the extract passed through a size exclusion matrix, for example but not limited to Sephacryl™. Following chromatography, fractions may be further analyzed by immunoblot to determine the protein complement of the fraction.
[0241] Without wishing to be bound by theory, the capacity of HA to bind to RBC from different animals is driven by the affinity of HA for sialic acids α2,3 or α2,3 and the presence of these sialic acids on the surface of RBC. Equine and avian HA from influenza viruses agglutinate erythrocytes from all several species, including turkeys, chickens, ducks, guinea pigs, humans, sheep, horses and cows; whereas human HAs will bind to erythrocytes of turkey, chickens, ducks, guinea pigs, humans and sheep (see also Ito T. et al, 1997, Virology, vol 227, p 493-499; and Medeiros R et al, 2001, Virology, vol 289 p. 74-85).
[0242] Correct folding of the expressed virus protein may be important for stability of the protein, formation of multimers, formation of VLPs, function of the virus protein and recognition of the virus protein by an antibody, among other characteristics. Folding and accumulation of a protein may be influenced by one or more factors, including, but not limited to, the sequence of the protein, the relative abundance of the protein, the degree of intracellular crowding, the pH in a cell compartment, the availability of cofactors that may bind or be transiently associated with the folded, partially folded or unfolded protein, the presence of one or more chaperone proteins, or the like.
[0243] Heat shock proteins (Hsp) or stress proteins are examples of chaperone proteins, which may participate in various cellular processes including protein synthesis, intracellular trafficking, prevention of misfolding, prevention of protein aggregation, assembly and disassembly of protein complexes, protein folding, and protein disaggregation. Examples of such chaperone proteins include, but are not limited to, Hsp60, Hsp65, Hsp 70, Hsp90, Hsp100, Hsp20-30, Hsp10, Hsp100-200, Hsp100, Hsp90, Lon, TF55, FKBPs, cyclophilins, ClpP, GrpE, ubiquitin, calnexin, and protein disulfide isomerases (see, for example, Macario, A. J. L., Cold Spring Harbor Laboratory Res. 25:59-70. 1995; Parsell, D. A. & Lindquist, S. Ann. Rev. Genet. 27:437-496 (1993); U.S. Pat. No. 5,232,833). As described herein, chaperone proteins, for example but not limited to Hsp40 and Hsp70 may be used to ensure folding of a virus protein.
[0244] Examples of Hsp70 include Hsp72 and Hsc73 from mammalian cells, DnaK from bacteria, particularly mycobacteria such as Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis (such as Bacille-Calmette Guerin: referred to herein as Hsp71). DnaK from Escherichia coli, yeast and other prokaryotes, and BiP and Grp78 from eukaryotes, such as A. thaliana (Lin et al. 2001 (Cell Stress and Chaperones 6:201-208). A particular example of an Hsp70 is A. thaliana Hsp70 (encoded by Genbank ref: AY120747.1). Hsp70 is capable of specifically binding ATP as well as unfolded polypeptides and peptides, thereby participating in protein folding and unfolding as well as in the assembly and disassembly of protein complexes.
[0245] Examples of Hsp40 include DnaJ from prokaryotes such as E. coli and mycobacteria and HSJ1, HDJ1 and Hsp40 from eukaryotes, such as alfalfa (Frugis et al., 1999. Plant Molecular Biology 40:397-408). A particular example of an Hsp40 is M. sativa MsJ1 (Genbank ref: AJ000995.1). Hsp40 plays a role as a molecular chaperone in protein folding, thermotolerance and DNA replication, among other cellular activities.
[0246] Among Hsps, Hsp70 and its co-chaperone, Hsp40, are involved in the stabilization of translating and newly synthesized polypeptides before the synthesis is complete. Without wishing to be bound by theory, Hsp40 binds to the hydrophobic patches of unfolded (nascent or newly transferred) polypeptides, thus facilitating the interaction of Hsp70-ATP complex with the polypeptide. ATP hydrolysis leads to the formation of a stable complex between the polypeptide, Hsp70 and ADP, and release of Hsp40. The association of Hsp70-ADP complex with the hydrophobic patches of the polypeptide prevents their interaction with other hydrophobic patches, preventing the incorrect folding and the formation of aggregates with other proteins (reviewed in Hartl, F U. 1996. Nature 381:571-579).
[0247] Native chaperone proteins may be able to facilitate correct folding of low levels of recombinant protein, but as the expression levels increase, the abundance of native chaperones may become a limiting factor. High levels of expression of virus protein in the agroinfiltrated leaves may lead to the accumulation of virus protein in the cytosol, and co-expression of one or more than one chaperone proteins such as Hsp70, Hsp40 or both Hsp70 and Hsp40 may reduce the level of misfolded or aggregated proteins, and increase the number of proteins exhibiting tertiary and quaternary structural characteristics that allow for formation of virus-like particles.
[0248] Therefore, the present invention also provides for a method of producing virus protein VLPs in a plant, wherein a first nucleic acid encoding a virus protein is co-expressed with a second nucleic acid encoding a channel protein, for example but not limited to a proton channel protein, and a third nucleic acid encoding a chaperone. The first, second and third nucleic acids may be introduced to the plant in the same step, or may be introduced to the plant sequentially.
[0249] The VLP produced within a plant may induce a virus protein comprising plant-specific N-glycans. Therefore, this invention also provides for a VLP comprising virus protein having plant specific N-glycans.
[0250] Furthermore, modification of N-glycan in plants is known (see for example WO 2008/151440; WO 2010/006452; or U.S. 60/944,344; which are incorporated herein by reference) and virus protein having modified N-glycans may be produced. Virus protein comprising a modified glycosylation pattern, for example with reduced fucosylated, xylosylated, or both, fucosylated and xylosylated, N-glycans may be obtained, or virus protein having a modified glycosylation pattern may be obtained, wherein the protein lacks fucosylation, xylosylation, or both, and comprises increased galatosylation. Furthermore, modulation of post-translational modifications, for example, the addition of terminal galactose may result in a reduction of fucosylation and xylosylation of the expressed virus protein when compared to a wild-type plant expressing virus protein.
[0251] For example, which is not to be considered limiting, the synthesis of virus protein having a modified glycosylation pattern may be achieved by co-expressing the protein of interest along with a nucleotide sequence encoding beta-1.4galactosyltransferase (GalT), for example, but not limited to mammalian GalT, or human GalT however GalT from another sources may also be used. The catalytic domain of GalT may also be fused to a CTS domain (i.e. the cytoplasmic tail, transmembrane domain, stem region) of N-acetylglucosaminyl transferase (GNT1), to produce a GNT1-GalT hybrid enzyme, and the hybrid enzyme may be co-expressed with virus protein. The virus protein may also be co-expressed along with a nucleotide sequence encoding N-acetylglucosaminyltrasnferase III (GnT-III), for example but not limited to mammalian GnT-III or human GnT-III, GnT-III from other sources may also be used. Additionally, a GNT1-GnT-III hybrid enzyme, comprising the CTS of GNT1 fused to GnT-III may also be used.
[0252] Therefore the present invention also includes VLP's comprising one or more virus protein having modified N-glycans.
[0253] Non-limiting example of sequences that may be used with the present invention to produce modified HA's also include those described in WO 2009/009876; WO 2009/076778; WO 2010/003225; WO 2010/148511; WO 2010/003235; WO 2010/006452 (which are herein incorporated by reference), for example, but not limited to:
[0254] H1 protein encoded by the nucleic acid molecule for example from A/Brisbane/59/2007 (H1N1), A/New Caledonia/20/99 (H1N1), A/Solomon Islands 3/2006 (H1N1), /PuertoRico/8/34 (H1N1), A/Brisbane/59/2007 (H1N1), strain;
[0255] H2 protein encoded by the nucleic acid molecule may be from the A/Singapore/1/57 (H2N2) strain;
[0256] 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/Perth/16/2009 (H3N2), A/Switzerland/9715293/2013, or A/Texas/50/2012 (H3N2) strain;
[0257] H5 protein encoded by the nucleic acid molecule may be from the A/Anhui/1/2005 (H5N1), A/Indonesia/5/2005 (H5N1), or A/Vietnam/1194/2004 (H5N1) strain;
[0258] H6 protein encoded by the nucleic acid molecule may be from the A/Teal/HongKong/W312/97 (H6N1) strain;
[0259] H7 protein encoded by the nucleic acid molecule may also be from the A/Hangzhou/1/13 (H7N9) or A/Equine/Prague/56 (H7N7) strain;
[0260] H9 protein encoded by the nucleic acid molecule may be from the A/HongKong/1073/99 (H9N2) strain;
[0261] HA protein from B subtype encoded by the nucleic acid may be from the B/Florida/4/2006, B/Massachusetts/2/12, B/Malaysia/2506/2004, B/Wisconsin/1/2010, or B/Brisbane/60/2008 strain.
TABLE-US-00013 TABLE 3 Examples of constructs that have been prepared as described herein: Constr. Expression Ampl. # Enhancer Element Description Example 1261 CPMV-HT A/New Caledonia/20/1999 (H1N1) Example 5.1 859 CPMV-HT A/Puerto Rico/8/1934 (H1N1) Example 5.2 1008 CPMV-HT BeYDV B/Brisbane/60/2008 Example 5.3 1059 CPMV-HT BeYDV B/Brisbane/60/2008 with deleted Example 5.4 proteolytic loop 1462 CPMV-HT BeYDV B/Wisconsin/1/2010 Example 5.5 1467 CPMV-HT BeYDV B/Wisconsin/1/2010 with deleted Example 5.6 proteolytic loop 1039 CPMV-HT B/Brisbane/60/2008 with deleted Example 5.7 proteolytic loop 1029 CPMV-HT B/Brisbane/60/2008 Example 5.11 1829 CPMV-HT+ B/Brisbane/60/2008 with deleted Example 5.12 proteolytic loop 1937 CPMV- B/Brisbane/60/2008 with deleted Example 5.13 160+ proteolytic loop 1067 CPMV-HT B/Brisbane/60/2008 with deleted Example 5.14 proteolytic loop +H1 California TMCT 1977 CPMV- B/Brisbane/60/2008 with deleted Example 5.14 160+ proteolytic loop +H1 California TMCT 1875 CPMV-HT+ B/Brisbane/60/2008 with deleted Example 5.19 proteolytic loop +H1 California TMCT 676 CPMV-HT H5 A/Indonesia/5/2005 with TETR Example 5.8 cleavage site 766 CPMV-HT H5 A/Indonesia/5/2005 with TETQ Example 5.9 cleavage site 928 CPMV-HT H5 A/Indonesia/5/2005 with deleted Example 5.10 proteolytic loop 489 CPMV-HT H5 A/Indonesia/5/2005 (native) Example 5.24 2220 CPMV-HT+ H2 A/Singapore/1/57 (native) Example 5.27 2221 CPMV-HT+ H2 A/Singapore/1/57 with deleted Example 5.28 proteolytic loop 2222 CPMV- H2 A/Singapore/1/57 (native) Example 5.29 160+ 2223 CPMV- H2 A/Singapore/1/57 with deleted Example 5.29 160+ proteolytic loop 2019 CPMV-HT+ H3 A/Perth/16/09 (native) Example 5.30 2039 CPMV-HT+ H3 A/Perth/16/09 with deleted Example 5.31 proteolytic loop 2139 CPMV- H3 A/Perth/16/09 (native) Example 5.30 160+ 2159 CPMV- H3 A/Perth/16/09 with deleted Example 5.31 160+ proteolytic loop 1819 CPMV-HT+ H3 A/Victoria /361/11 (native) Example 5.26 2230 CPMV-HT+ H3 A/Victoria /361/11 with deleted Example 5.32 proteolytic loop 1800 CPMV- H3 A/Victoria /361/11 (native) Example 5.25 160+ 2250 CPMV- H3 A/Victoria /361/11 with deleted Example 5.32 160+ proteolytic loop 2142 CPMV-HT+ H7 A/Hangzhou/1/13 (native) Example 5.33 2152 CPMV-HT+ H7 A/Hangzhou/1/13 with deleted Example 5.34 proteolytic loop 2224 CPMV-HT+ H9 A/Hong Kong/1073/99 (native) Example 5.35 2225 CPMV-HT+ H9 A/Hong Kong/1073/99 with deleted Example 5.36 proteolytic loop 2226 CPMV- H9 A/Hong Kong/1073/99 (native) Example 5.35 160+ 2227 CPMV- H9 A/Hong Kong/1073/99 with deleted Example 5.36 160+ proteolytic loop 2013 CPMV- B/Malaysia /2506/04 (native) Example 5.37 160+ 2014 CPMV- B/Malaysia /2506/04 with deleted Example 5.38 160+ proteolytic loop 2070 CPMV-HT B/Massachusetts/2/12 (native) Example 5.39 2072 CPMV-HT B/Massachusetts/2/12 with deleted Example 5.15 proteolytic loop 2080 CPMV-HT+ B/Massachusetts/2/12 (native) Example 5.39 2052 CPMV-HT+ B/Massachusetts/2/12 with deleted Example 5.20 proteolytic loop 2090 CPMV- B/Massachusetts/2/12 (native) Example 5.39 160+ 2050 CPMV- B/Massachusetts/2/12 with deleted Example 5.15 160+ proteolytic loop 2074 CPMV HT HA B/Massachusetts/2/12 (PrL−) +H1 Example 5.16 California TMCT 2060 CPMV- HA B/Massachusetts/2/12 (PrL−) +H1 Example 5.16 160+ California TMCT 2062 CPMV-HT+ HA B/Massachusetts/2/12 (PrL−) +H1 Example 5.21 California TMCT 1445 CPMV HT B/Wisconsin/1/2010 with deleted Example 5.17 proteolytic loop (PrL−) 1839 CPMV-HT+ B/Wisconsin/1/2010 with deleted Example 5.22 proteolytic loop (PrL−) 1820 CPMV160+ B/Wisconsin/1/2010 with deleted Example 5.17 proteolytic loop (PrL−) 1975 CPMV160 B/Wisconsin/1/2010 with deleted Example 5.17 proteolytic loop (PrL−) 1454 CPMV-HT HA B Wisconsin with deleted proteolytic Example 5.18 loop (PrL−) +H1 California TMCT 1893 CPMV- HA B Wisconsin with deleted proteolytic Example 5.18 160+ loop (PrL−) +H1 California TMCT 1860 CPMV-HT+ HA B Wisconsin with deleted proteolytic Example 5.23 loop (PrL−) +H1 California TMCT 2102 CPMV-HT+ B Florida with deleted proteolytic loop Example 5.40 (PrL−) 2104 CPMV-HT+ BeYDV B Florida with deleted proteolytic loop Example 5.40 (PrL−) 2016 CPMV-HT+ B Florida +H1 California TMCT with Example 5.41 deleted proteolytic loop (PrL−) 2108 CPMV-HT+ BeYDV B Florida +H1 California TMCT with Example 5.41 deleted proteolytic loop (PrL−) 2801 CPMV160 H3 A/Switzerland/9715293/2013 Example 5.43 (native) 2841 CPMV160 H3 A/Switzerland/9715293/2013 with Example 5.43 deleted proteolytic loop 2840 CPMV160 H3 A/Switzerland/9715293/2013 +H1 Example 5.44 California TMCT 2843 CPMV160 H3 A/Switzerland/9715293/2013 +H1 Example 5.44 California TMCT with deleted proteolytic loop 2320 CPMV160+ H3 A/Victoria /361/11 +H1 California Example 5.42 TMCT 2340 CPMV160+ H3 A/Victoria /361/11 +H1 California Example 5.42 TMCT with deleted proteolytic loop 2451 CPMV160 H3 A/Texas/50/2012 (native) Example 5.43 2453 CPMV160 H3 A/Texas/50/2012 with deleted Example 5.43 proteolytic loop 2452 CPMV160 H3 A/Texas/50/2012 +H1 California Example 5.44 TMCT 2454 CPMV160 H3 A/Texas/50/2012 +H1 California Example 5.44 TMCT with deleted proteolytic loop
TABLE-US-00014 TABLE 4 Description of sequences SEQ ID NO: Description Figure 1 Avian H5 proteolytic loop consensus sequence 2 IF-H5A-I-05.si + 3c 1A 3 IF-H5dTm.r 1B 4 Construct 1191 1D 5 Cassette 489 1E 6 Amino acid sequence 1F H5 A/Indonesia/5/2005 (H5N1) 7 IF-S1- 2A M1 + M2ANC.c 8 IF-S1-4-M2ANC.r 2B 9 Synthetic M2 (nt 1- 2C 26 joined to 715-982 from DQ508860) 10 Cassette 1261 2D 11 M2 from influenza 2E A/New Caledonia/20/1999 (H1N1) 12 Synthetic M2 (nt 26- 3A 51 joined to nt 740- 1007 from EF467824) 13 Cassette 859 3B 14 Amino acid sequence 3C M2 influenza A/Puerto Rico/8/1934 (H1N1) 15 Cassette 1039 8A 16 Amino acid sequence 21A B/Brisbane/60/2008 17 Amino acid sequence 21B delta-proteolytic loop of type B HA (with linker GG) 18 Amino acid sequence 21C replacing cleavage site of B HA with linker 19 Amino acid sequence 21D H3 A/Perth/16/2009 20 Amino acid sequence 21E delta-proteolytic loop H3 (with linker GS) 21 Amino acid sequence 21F Replacing cleavage site of H3 with linker 22 H1 New Cal linker 15 region 23 H1 Brisbane linker 15 region 24 H1 Sol Islands linker 15 region 25 H2A Singapore 15 linker region 26 H3A Brisbane linker 15 region 27 H3A WCN linker 15 region 28 H5 Anhui linker 15 region 29 H5 Indo linker 15 region 30 H5 Vietnam linker 15 region 31 Construct 1194 4B 32 Cassette 1008 4C 33 H6 Teal HK linker 15 region 34 H7 Eq Prague linker 15 region 35 H9A HK linker 15 region 36 B Florida linker 15 region 37 B Malaysia 15 38 1039 + 1059.r 5A 39 1039 + 1059.c 5B 40 Cassette 1059 5C 41 Amino acid sequence 5D PDISP/HA influenza B/Brisbane/60/2008 (deleted proteolytic loop) 42 Nucleotide sequence 1G H5 A/Indonesia/5/2005 (H5N1) 43 nucleotide sequence 5E PDISP/HA influenza B/Brisbane/60/2008 (deleted proteolytic loop) 44 H5/Indo cleavage 19 site natural sequence 45 H5/Indo modified 19 cleavage site (TETR) 46 H5/Indo modified 19 cleavage site (TETQ) 47 H1 cleavage site Table 1 48 H3 cleavage site Table 1 49 IF-HAB110.S1 + 3c 6A 50 IF-HAB110.s1-4r 6B 51 Synthetic HA B 6C Wisconsin 52 Construct 193 6E 53 Cassette 1462 6F 54 Amino acid sequence 6G HA influenza B/Wisconsin/1/2010 55 HAB110(PrL−).r 7A 56 HAB110(PrL−).c 7B 57 Cassette 1467 7C 58 Amino acid sequence 7D HA influenza B/Wisconsin/1/2010 (deleted PL) 59 B cleavage site Table 1 60 H5/Indo natural Table 2 cleavage site 61 H5/Indo modified Table 2 cleavage site 62 H5/Indo modified Table 2 cleavage site 63 H1/Brisbane Table 2 modified cleavage site 64 H3/Brisbane Table 2 modified cleavage site 65 B/Florida, Table 2 B/Brisbane modified cleavage site 66 A/H3/HA0 Consensus 67 A/H1/HA0 Consensus 68 B/HA0 Consensus 69 H5 Anhui proteolytic 18C loop deletion 70 H5 Indo proteolytic 18C loop deletion 71 H5 Vietnam 18C proteolytic loop deletion 72 B Florida proteolytic 18C loop deletion 73 B Malaysia 18C proteolytic loop deletion 74 MutCleavage- 23A H5(Indo).r 75 MutCleavage- 23B H5(Indo).c 76 Cassette 676 23C 77 Amino acid sequence 23D influenza A/Indonesia/5/2005 (H5N1) TETR cleavage site mutant. 78 H5I505_TETQ.r 24A 79 H5I505_TETQ.c 24B 80 Cassette 766 25C 81 Amino acid sequence 25D influenza A/Indonesia/5/2005 (H5N1) TETQ cleavage site mutant. 82 H5I505(PrL−).r 26A 83 H5I505(PrL−).c 26B 84 Cassette 928 26C 85 Amino acid sequence 26D influenza A/Indonesia/5/2005 (H5N1) with deleted proteolytic loop. 86 IF-S2 + S4-B Bris.c 30A 87 IF-S1a4-B Bris.r 30B 88 Synthesized HA B 30C Brisbane gene 89 Construct 1029 30D 90 Amino acid sequence 30E of PDISP/HA from influenza B/Brisbane/60/2008 91 Nucleotide sequence 31A of PDISP/HA B Brisbane (PrL−). 92 Amino acid sequence 31B of PDISP/HA B Brisbane (PrL−) 93 Nucleotide sequence of CPMVX/CPMVX+ 94 consensus amino acid sequence of transmembrane domain 95 Nucleotide sequence 33A of PDISP/HA B Brisbane (PrL−) +H1 California TMCT. 96 Amino acid sequence 33B of PDISP/HA B Brisbane (PrL−) +H1 California TMCT. 97 Nucleotide sequence 34A of PDISP/HA B Massachusetts (PrL) 98 Amino acid sequence 34B of PDISP/HA B Massachusetts (PrL) 99 Nucleotide sequence 35A of PDISP/HA B Massachusetts (PrL) +H1 California TMCT. 100 Amino acid sequence 35B of PDISP/HA B Massachusetts (PrL−) +H1 California TMCT. 101 Nucleotide sequence 36A of HA B Wisconsin (PrL−). 102 Amino acid sequence 36B of HA B Wisconsin (PrL−). 103 Nucleotide sequence 37A of HA B Wisconsin (PrL−) +H1 California TMCT 104 Amino acid sequence 37B of HA B Wisconsin (PrL−) +H1 California TMC. 105 Nucleotide sequence 38A of PDISP/HA B Brisbane (PrL−) +H1 California TMCT. 106 Amino acid sequence 38B of PDISP/HA B Brisbane (PrL−) +H1 California TMCT. 107 Nucleotide sequence 39A of PDISP/HA B Massachussetts (PrL−). 108 Amino acid sequence 39B of PDISP/HA B Massachussetts (PrL−). 109 Nucleotide sequence 40A of PDISP/HA B Massachussetts (PrL−) +H1 California TMCT. 110 Amino acid sequence 40B of PDISP/HA B Massachussetts (PrL−) +H1 California TMCT. 111 Nucleotide sequence 41A of HA B Wisconsin (PrL−). 112 Amino acid sequence 41B of HA B Wisconsin (PrL−). 113 Nucleotide sequence 42A of HA B Wisconsin (PrL−) +H1 California TMCT 114 Amino acid sequence of HA 42B B Wisconsin (PrL−) +H1 California TMC. 115 Nucleotide sequence of 43A native H5 Indonesia. 116 Amino acid sequence of 43B native H5 Indonesia 117 IF**(SacII)-PDI.s1 + 4c 44A 118 IF-H3V36111.s1-4r 44B 119 Nucleotide sequence of 44C PDISP/H3 Victoria. 120 Construct 2171 44E 121 Construct 1800 44F 122 Amino acid sequence of 44G PDISP/H3 Victoria 123 IF(SacII)-Kozac_PDI.c 45A 124 IF-H3V36111.s1-4r 45B 125 Construct 2181 45D 126 Construct 1819 45E 127 IF**-H2S157.s1-6r 48A 128 Nucleotide sequence of 48B PDISP/H2 Singapore. 129 Expression cassette number 48C 2220 130 Amino acid sequence of 48D PDISP/H2 Singapore 131 H2S157(Prl−).r 49A 132 H2S157(Prl−).c 49B 133 Expression cassette number 49C 2221 134 Amino acid sequence of 49D PDISP/H2 Singapore with deleted proteolytic loop 135 Expression cassette number 50A 2222 136 Expression cassette number 50B 2223 137 Nucleotide sequence of 51A PDISP/H3 Perth 138 IF**-H3P1609.S1-6r 51B 139 Amino acid sequence of 51C PDISP/H3 Perth 140 Nucleotide sequence of 52A PDISP/H3 Perth with deleted proteolytic loop 141 H3P1609(Prl−)#2.r 52B 142 H3P1609(Prl−)#2.c 52C 143 Amino acid sequence of 52D PDISP/H3 Perth with deleted proteolytic loop 144 Nucleotide sequence of 53A PDISP/H3 Victoria with deleted proteolytic loop 145 H3V36111(Prl−).r 53B 146 H3V36111(Prl−).c 53C 147 Amino acid sequence of 53D PDISP/H3 Victoria with deleted proteolytic loop 148 Nucleotide sequence of 54A PDISP/H7 Hangzhou 149 IF*-H7H113.s1-6r 54B 150 Amino acid sequence of 54C PDISP/H7 Hangzhou 151 Nucleotide sequence of 55A PDISP/H7 Hangzhou with deleted proteolytic loop 152 H7H113(PrL−).r 55B 153 H7H113(PrL−).c 55C 154 Amino acid sequence of 55D PDISP/H7 Hangzhou with deleted proteolytic loop 155 Nucleotide sequence of 56A PDISP/H9 Hong Kong 156 IF**-H9HK107399.S1-6r 56B 157 Amino acid sequence of 56C PDISP/H9 Hong Kong 158 Nucleotide sequence of 57A PDISP/H9 Hong Kong with deleted proteolytic loop 159 H9HK107399(Prl−).r 57B 160 H9HK107399(Prl−).c 57C 161 Amino acid sequence of 57D PDISP/H9 Hong Kong with deleted proteolytic loop 162 Nucleotide sequence of 58A PDISP/HA B Malaysia 163 IF**-HBM250604.S1-6r 58B 164 Amino acid sequence of 58C PDISP/HA B Malaysia 165 Nucleotide sequence of 59A PDISP/HA B Malaysia with deleted proteolytic loop 166 HBM250604(PrL−).r 59B 167 HBM250604(PrL−).c 59C 168 Amino acid sequence of 59D PDISP/HA B Malaysia with deleted proteolytic loop 169 Nucleotide sequence of 60A PDISP/HA B Massachusetts 170 Amino acid sequence of 60B PDISP/HA B Massachusetts 171 nucleotide sequence of PatA SP 172 amino acid sequence of patatin A signal peptide 173 CPMV HT sequence 174 Plant consensus kozak sequence—plant kingdom 175 Plant consensus kozak sequence—dicots 176 Plant consensus kozak sequence—arabidopsis 177 Plant consensus kozak sequence 178 Plant consensus kozak sequence 179 Plant consensus kozak sequence 180 Plant consensus kozak sequence 181 Plant consensus kozak sequence 182 Plant consensus kozak sequence 183 Plant consensus kozak sequence 184 Plant consensus kozak sequence 185 Plant consensus kozak sequence 186 Kozak consensus sequence 187 Nucleotide sequence of CPMV HT+ 188 Nucleotide sequence of CPMV HT+ 511 189 Nucleotide sequence ofCPMV HT+[WT115] 190 HBF406(PrL−).r 61A 191 HBF406(PrL−).c 61B 192 IF*-HBF406.s1-6r 61C 193 Nucleotide sequence of 61D PDISP/HA B Florida with deleted proteolytic loop 194 Amino acid sequence of 61E PDISP/HA B Florida with deleted proteolytic loop 195 Expression cassette number 61F 2102 196 Expression cassette number 61H 2104 197 IF-H1cTMCT.S1-4r 62A 198 Nucleotide sequence of 62B PDISP/HA B Florida +H1Cal TMCT with deleted proteolytic loop 199 Amino acid sequence of 62C PDISP/HA B Florida +H1Cal TMCT with deleted proteolytic loop 200 Expression cassette number 62D 2106 201 Expression cassette number 62F 2108 202 Nucleotide sequence of 64E PDISP/HA A/Switz/9715293/13 203 Amino acid sequence of 64F PDISP/HA A/Switz/9715293/13 204 Nucleotide sequence of 64H PDISP/HA A/Switz/9715293/13 (PrL−) 205 Amino acid sequence of 641 PDISP/HA A/Switz/9715293/13 (PrL−) 206 IF-H1TMCT + H3_Swi.r 63B 207 Nucleotide sequence of 65A PDISP/HA A/Switz/9715293/13- H1cTMCT 208 Amino acid sequence of 65B PDISP/HA A/Switz/9715293/13- H1cTMCT 209 Nucleotide sequence of 65D PDISP/HA A/Switz/9715293/13 (PrL−)- H1cTMCT 210 Amino acid sequence of 65E PDISP/HA A/Switz/9715293/13 (PrL−)- H1cTMCT 211 IF-H3_Swi_13.c 64A 212 Nucleotide sequence of 63A PDISP/HA A/Vic/361/11- H1cTMCT 213 Amino acid sequence of 63C PDISP/HA A/Vic/361/11- H1cTMCT 214 Nucleotide sequence of 63E PDISP/HA A/Vic/361/11 (PrL−)-H1cTMCT 215 Amino acid sequence of 63F PDISP/HA A/Vic/361/11 (PrL−)-H1cTMCT 216 Nucleotide sequence of 64K PDISP/HA A/Tex/50/12 217 Amino acid sequence of 64L PDISP/HA A/Tex/50/12 218 Nucleotide sequence of 64N PDISP/HA A/Tex/50/12 (PrL−) 219 Amino acid sequence of 640 PDISP/HA A/Tex/50/12 (PrL−) 220 Nucleotide sequence of 65G PDISP/HA A/Tex/50/12- H1cTMCT 221 Amino acid sequence of 65H PDISP/HA A/Tex/50/12- H1cTMCT 222 Nucleotide sequence of 65J PDISP/HA A/Tex/50/12 (PrL−)-H1cTMCT 223 Amino acid sequence of 65K PDISP/HA A/Tex/50/12 (PrL−)-H1cTMCT 224 Cloning vector 1590 from 64B left to right T-DNA 225 Construct 2801 from 2X35S 64C prom to NOS term
EXAMPLES
Example 1
Agrobacterium Transfection
[0262] 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 μM acetosyringone, 50 μg/ml kanamycin and 25 μg/ml of carbenicillin pH5.6 to an OD.sub.600 between 0.6 and 1.6. Agrobacterium suspensions were centrifuged before use and re-suspended in infiltration medium (10 mM MgCl.sub.2 and 10 mM MES pH 5.6).
Preparation of Plant Biomass, Inoculum and Agroinfiltration
[0263] The terms “biomass” and “plant matter” as used herein are meant to reflect any material derived from a plant. Biomass or plant matter may comprise an entire plant, tissue, cells, or any fraction thereof. Further, biomass or plant matter may comprise intracellular plant components, extracellular plant components, liquid or solid extracts of plants, or a combination thereof. Further, 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.
[0264] 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.
[0265] Agrobacteria transfected with each construct were grown in a YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 μM acetosyringone, 50 μg/ml kanamycin and 25 μg/ml of carbenicillin pH5.6 until they reached an OD.sub.600 between 0.6 and 1.6. Agrobacterium suspensions were centrifuged before use and resuspended in infiltration medium (10 mM MgCl.sub.2 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.
Leaf Harvest and Total Protein Extraction
[0266] Following incubation, the aerial part of plants was harvested, frozen at −80° C. and crushed into pieces. Total soluble proteins were extracted by homogenizing (Polytron) each sample of frozen-crushed plant material in 3 volumes of cold 50 mM Tris pH 8.0, 0.5 M NaCl, 0.1% Triton X-100 and 1 mM phenylmethanesulfonyl fluoride. After homogenization, the slurries were centrifuged at 10,000 g for 10 min at 4° C. and these clarified crude extracts (supernatant) kept for analyses.
Protein Analysis and Immunoblotting
[0267] The total protein content of clarified crude extracts was determined by the Bradford assay (Bio-Rad, Hercules, Calif.) 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, Ind.) 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 16-18 h at 4° C.
[0268] 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 μg/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). Horseradish peroxidase-enzyme conjugation of human IgG antibody was carried out by using the EZ-Link Plus® Activated Peroxidase conjugation kit (Pierce, Rockford, Ill.).
TABLE-US-00015 TABLE 4 Electrophoresis conditions, antibodies, and dilutions for immunoblotting of expressed proteins. Electro- HA Influenza phoresis Primary Secondary subtype strain condition antibody Dilution antibody Dilution B B/ Non- TGA, 1:20000 Rabbit 1:10 000 Brisbane/ reducing AS397 anti- 60/2008 sheep (JIR 313- 035-045) B B/ Non- NIBSC 1:2000 Rabbit 1:10 000 Wisconsin/ reducing 07/356 anti- 1/2010 sheep (JIR 313- 035-045) B B/ Non- NIBSC 1:2000 Rabbit 1:10 000 Malaysia/ reducing 07/184 anti- 2506/2004 sheep (JIR 313- 035-045) H3 A/Perth/ Non- TGA, 1:20000 Rabbit 1:10 000 16/2009 reducing AS400 anti- (H3N2) sheep (JIR 313- 035-045) H3 A/ Non- TGA, 1:20000 Rabbit 1:10 000 Victoria/ reducing AS400 anti- 361/2011 sheep (JIR 313- 035-045) H1 A/ Reducing Sino, 1 μg/ml Goat 1:7 500 California/ 11055- anti- 07/2009 MMO1 mouse (H1N1) (JIR 115- 035-146) H5 A/ Reducing CBER, 1:4000 Rabbit 1:10 000 Indonesia/ S-7858 anti- 05/2005 sheep (H5N1) (JIR 313- 035-045) JIR: Jackson ImmunoResearch, West Grove, PA, USA; CBER: Center for Biologics Evaluation and Research, Rockville, MD, USA. Sino: Sino Biological inc., Beijing, China. TGA: Therapeutic Goods Administration, Australia. NIBSC: National Institute for Biological Standards and Control, United Kingdom
Hemagglutination Assay
[0269] Hemagglutination assay was based on a method described by Nayak and Reichl (2004). Briefly, serial double dilutions of the test samples (100 μL) were made in V-bottomed 96-well microtiter plates containing 100 μL PBS, leaving 100 μL of diluted sample per well. One hundred microliters of a 0.25% turkey red blood cells suspension (Bio Link Inc., Syracuse, N.Y.) were added to each well, and plates were incubated for 2 h at room temperature. The reciprocal of the highest dilution showing complete hemagglutination was recorded as HA activity. In parallel, a recombinant HA standard (A/Vietnam/1203/2004 H5N1) (Protein Science Corporation, Meriden, Conn.) was diluted in PBS and run as a control on each plate.
VLP Extraction by Cell Wall Digestion
[0270] Leaf tissue was collected from the Nicotiana benthamiana plants and cut into ˜1 cm.sup.2 pieces. The leaf pieces were soaked in 500 mM mannitol for 30 minutes at room temperature (RT). The mannitol solution was then removed and changed with the enzyme mix (mixture of cellulases from Trichoderma viride (Onozuka R-10; 3% v/v) and a mixture of pectinases from Rhizopus sp. (MACEROZYME™; 0.75% v/v; both from Yakult Pharmaceuticals) in protoplasting solution (500 mM mannitol, 10 mM CaCl.sub.2) and 5 mM MES/KOH (pH 5.6)). The ratio used was 20 g of leaf pieces per 100 mL solution. This preparation was spread evenly into a shallow vessel (˜11×18 cm) and incubated for 16 hours on a rotary shaker at 40 rpm and 26° C.
[0271] Alternately, VLP extraction may be performed as follows: plants were agroinfiltrated with AGL1/#489, 928, 676 and 766. Leaf tissue was collected from the N. benthamiana plants at day 7 post-infiltration and cut into ˜1 cm.sup.2 pieces. Pectinase 162L (Biocatalysts), Multifect CX CG and Multifect CX B (Genencor) were added to a 200 mM Mannitol, 75 mM Citrate, 0.04% sodium bisulfite pH 6.0 buffer; digestion buffer. The biomasses were digested in duplicate overnight at room temperature in an orbital shaker.
[0272] Following enzyme-assisted extraction, leaf debris was removed by filtration (nylon filter of 250 or 400 μm mesh). The coarse filtered extract was centrifuged at 5000×g for 5 minutes. Supernatant was submitted to detection of HA expression (hemagglutination activity (see
Example 2
Effect of Modified Proteolytic Loop on Accumulation of HA
[0273] As shown in
[0274] Similar results were observed in the accumulation level of B/Wisconsin comprising a modified proteolytic loop (Construct No: 1467), which is greater than that observed for the native B/Wisconsin HA (Construct No: 1462;
[0275] Expression of H5/Indo comprising a modified proteolytic loop was also observed with modifications including a proteolytic loop comprising a GG linker (Construct No: 928; SEQ ID NO:85), a TETR linker (Construct No: 676; SEQ ID NO:77), or a TETQ linker (Construct No: 766; SEQ ID NO: 8;
Effect of Influenza M2 Co-Expression on the Accumulation Level of HA
[0276] The co-expression of M2 was evaluated for its impact on the accumulation level of a modified influenza B HA. Construct No. 1059 encodes an influenza B HA in which the proteolytic loop is replaced by a 2 amino acid linker (GG in place of aa 341-359). The results from western blot analysis presented in
[0277] Co-expression of M2 with type A HA comprising a modified proteolytic loop also resulted in HA expression. For example, co-expression of modified H3, with the proteolytic loop replaced with a GS linker or a (GSS).sub.3 linker (see
[0278] The efficacy of M2 from influenza A/Puerto Rico/8/1934 to increase accumulation of the modified influenza B HA and H3 was compared to that of M2 from influenza A/New Caledonia/20/1999. For the modified influenza B HA, the comparison was undertaken by western blot analysis of protein extracts from plants transformed with constructs 1059, 1059+1261 and 1059+859. The results obtained demonstrated that the co-expression of M2 from influenza A/Puerto Rico/8/1934 (encoded by construct no. 859) was as efficient as the co-expression of M2 from influenza A/New Caledonia/20/1999 (encoded by construct no. 1261) for increasing accumulation of the modified influenza B HA (
Effect of Influenza M2 Co-Expression on the Accumulation Level of Different Strains of B HA
[0279] Western blot analysis of protein extracts from plants transformed with gene constructs driving the expression of influenza B HA (from B/Wisconsin/1/2010) (constructs no. 1462) in the presence or absence of M2-expression construct (construct no. 1261) showed that M2 co-expression results in increased accumulation of influenza B HA (
[0280] The co-expression of M2 was also evaluated for its impact on the accumulation level of a modified influenza B HA. Construct no. 1467 encodes an influenza B HA in which the proteolytic loop is replaced by a 2 amino acid linker (GG in place of aa 341-359). The results from western blot analysis presented in
Effect of Amplification Element BeYDV and Modified Proteolytic Loop on Accumulation of HA
[0281] Western blot analysis of protein extracts from plants transformed with gene constructs driving the expression of modified influenza B HA (from B/Brisbane/60/2008) with or without the proteolytic loop removed (see
Effect of Modified Proteolytic Loop on Relative HA Titer and Hemagglutination
[0282] With reference to
TABLE-US-00016 TABLE 5a Relative HA titer (wt HA = 1) (see FIG. 29A) Fct = 1 wt PrL− Description (n = ) GM MD n = GM MD n = H2 Sin157 (HT+) (n = 2) 1.0 0.4 2 1.2 0.1 2 H2 Sin157 (160+) (n = 2) 1.0 0.3 2 1.9 0.9 2 H3 Per1609 (HT+) (n = 4) 1.0 0.0 4 4.9 4.2 4 H3 Per1609 (160+) (n = 4) 1.0 0.3 4 6.1 3.6 4 H3 Vic36111 (HT+) (n = 6) 1.0 0.3 6 0.9 0.3 6 H3 Vic36111 (160+) (n = 6) 1.0 0.3 6 1.2 0.4 6 H5 Indo505 (HT) (n = 3) 1.0 0.1 3 1.2 0.3 3 H7 Han113 (HT+) (n = 5) 1.0 0.2 5 1.0 0.1 5 H9 HK107399 (HT+) (n = 2) 1.0 0.4 2 1.2 0.2 2 H9 HK107399 (160+) (n = 2) 1.0 0.2 2 0.7 0.1 2 HB Bri6008 (HT) (n = 1) 1.0 0.6 1 0.6 0.1 1 HB Mal250604 (160+) (n = 2) 1.0 0.3 2 8.4 2.4 2 HB Mas212 (HT) (n = 3) 1.0 0.1 3 1.4 1.3 3 HB Mas212 (HT+) (n = 3) 1.0 0.6 3 6.7 3.5 3 HB Mas212 (160+) (n = 2) 1.0 0.8 2 11.5 4.2 2
Increased HA Titer when the Proteolytic Loop is Removed (PrL-) Compared to the Native Construct
[0283]
TABLE-US-00017 TABLE 5B HA titer (see FIG. 29B) Native PrL− HA Type GM n = GM n = H3 A/Switz 1214 4 2487 4 H3 A/Switz-H1cTm 1758 4 3854 4 H3 A/Vic 501 5 601 5 H3 A/Vic-H1cTm 675 4 1174 4 H3 A/Tex 825 3 1159 3 H3 A/Tex-H1cTm 851 3 2078 3
Example 3
[0284] Increased 117 Hangzhou HA VLP Yields when the Proteolytic Loop is Removed (PrL-) Compared to the Native Construct.
[0285] N. benthamiana plants were infiltrated with AGL1/#2142+1261 and #2152+1261 and the leaves were harvested after a seven-day incubation period. Leaf tissue was collected and cut into ˜1 cm.sup.2 pieces. Pectinase 162L and Pectinase 444L (Biocatalysts), Multifect CX CG and Multifect CX B (Genencor) were added in a 200 mM Mannitol, 125 mM Citrate, 0.04% sodium bisulfite pH 6.0 buffer. The biomass was digested overnight at room temperature in an orbital shaker.
[0286] Following digestion, the apoplastic fraction was filtered through a 400 μm nylon filter to remove coarse undigested vegetal tissue (<5% of starting biomass). The filtered extract was then centrifuged at room temperature for 15 min at 5000×g to remove protoplasts and intracellular contaminants (proteins, DNA, membranes, vesicles, pigments, etc). Next, the supernatant was depth-filtered (for clarification) using a 1.2 μm glass fiber filter (Sartopore GF plus/Sartorius Stedim), and a 0.45/0.2 μm filter (Sartopore 2/Sartorius Stedim), before being subjected to chromatography.
[0287] The clarified apoplastic fraction was loaded over a cation exchange column (Poros HS Applied Biosystems) equilibrated with an equilibration/elution buffer (50 mM NaPO4, 100 mM NaCl, 0.005% Tween 80 pH 6.0). Once the UV was back to zero, the extract was step-eluted with the equilibration/elution buffer containing increasing concentrations of NaCl (500 mM). The purified VLPs were concentrated by TFF, diafiltered against formulation buffer (100 mM PO4, 150 mM NaCl, 0.01% Tween 80 at pH 7.4) and passed through a 0.22 μm filter.
[0288] Hemagglutination assay for H7 was performed based on a method described by Nayak and Reichl (2004). Briefly, successive double dilutions of the test samples (100 μL) were made in V-bottomed 96-well microtiter plates containing 100 μL PBS, leaving 100 μL of diluted sample per well. One hundred microliters of a 0.25% turkey red blood cells suspension (Bio Link Inc., Syracuse, N.Y.) were added to each well, and plates were incubated for 2 h at room temperature. The reciprocal of the highest dilution showing complete hemagglutination was recorded as hemagglutination activity.
[0289] Total protein content of clarified crude extracts was determined using bovine serum albumin as the reference standard. Relative yields were obtained by comparing the PrL-construct to the native construct used as control. Separation by SDS-PAGE, with denaturing sample loading buffer (0.1M Tris pH 6.8, 0.05% bromophenol blue, 12.5% glycerol, 4% SDS and 5% beta-mercaptoethanol), was performed under reducing conditions and Coomassie Brillant Blue R-250 was used for protein staining.
[0290]
[0291]
[0292]
Example 4.1
[0293] Trypsin Resistance of Mutants 115 Indonesia VLP where the Proteolytic Loop is Modified or Removed is Greater than Native 115 Indonesia.
[0294] N. benthamiana plants were agroinfiltrated with AGL1/#489, #928, #766 and #676 as described in Example 1 (above). Leaves were collected from the plants 7 days post-infiltration, cut into ˜1 cm2 pieces. Pectinase 162L (Biocatalysts), Multifect CX CG and Multifect CX B (Genencor) were added in a 200 mM Mannitol, 75 mM Citrate, 0.04% sodium bisulfite pH 6.0 buffer. The biomass was digested overnight at room temperature in an orbital shaker. The digested extracts were coarse-filtered, centrifuged, clarified and purified as described in Example 3 (H7 Hangzhou).
[0295] For each of the native (#489), PRL- (#928), TETQ (#766) and TETR (#676), H5 Indonesia HA VLP extracts, two samples of HA VLPs were resuspended in buffer (100 mM Na/KPO.sub.4, 150 mM NaCl, 0.01% TWEEN 80) at pH 7.4. Trypsin was added in a 1:100 protein ratio. Samples were grabbed after 30, 60 and 120 minutes of incubation at room temperature, then boiled in sample loading buffer to stop the reaction. The non-digested extracts (control) and the trypsin-digested extracts analysed by SDS-PAGE gel as described in Example 3.
[0296]
[0297] These data demonstrate the successful protection of the HA0 protein at its proteolytic site within HA1-HA2, by either deleting the proteolytic loop (prl-) or replacing the proteolytic loop with a linker sequence (TETQ) approach.
Example 4.2. Immunogenicity of Native 115 Indonesia VLPs is Similar to its Mutant Counterparts (PrL-, TETQ and TETR) in Mice
[0298] The native, PrL-, TETR and TETQ H5 Indonesia VLPs extracts were purified as described in Example 4.1 (above).
[0299]
Example 5.1: B-2X35S/CPMV-HT/M2 New Caledonia/NOS (Construct Number 1261)
[0300] A sequence encoding M2 from influenza A/New Caledonia/20/1999 (H1N1) was cloned into 2X355/CPMV-HT/NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the complete M2 coding sequence was amplified using primers IF-S1-M1+M2ANC.c (
Example 5.2: C-2X35S/CPMV-HT/M2 Puerto Rico/NOS (Construct Number 859)
[0301] A sequence encoding M2 from influenza A/Puerto Rico/8/1934 (H1N1) was cloned into 2X355/CPMV-HT/NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing the complete M2 coding sequence was amplified using primers IF-S1-M1+M2ANC.c (
Example 5.3: G-2X35S/CPMV-HT/PDISP/HA B Brisbane/NOS into BeYDV+Replicase Amplification System (Construct Number 1008)
[0302] The preparation of construct 1008 is described in U.S. 61/541,780. Briefly, a sequence encoding HA from influenza B/Brisbane/60/2008 was cloned into 2X355/CPMV-HT/PDISP/NOS comprising the BeYDV+replicase amplification system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using a PCR-based method using synthesized HA B Brisbane gene (corresponding to nt 34-1791 from Genbank accession number FJ766840). The PCR product was cloned in-frame with alfalfa PDI signal peptide in 2X35S/CPMV-HT/NOS expression cassette into the BeYDV amplification system. Construct 1194 (see
[0303] Construct number 1194 (
Example 5.4: I-2X35S/CPMV-HT/PDISP-HA B Brisbane with Deleted Proteolytic Loop into BeYDV+Replicase Amplification System (Construct Number 1059)
[0304] The preparation of construct 1059 is described in U.S. 61/541,780. Briefly, a sequence encoding HA from influenza B/Brisbane/60/2008 with deleted proteolytic loop was cloned into 2X355/CPMV-HT/PDISP/NOS comprising the BeYDV+replicase amplification system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using a PCR-based ligation method (Darveau et al., 1995, Methods in Neuroscience 26: 77-85). In a first round of PCR, a fragment containing HA B Brisbane coding sequence from nt 46 to nt 1065 was amplified using synthesized HA B Brisbane gene (corresponding to nt 34-1791 from Genebank accession number FJ766840) as template. A second fragment, containing HA B Brisbane coding sequence from nt 1123 to nt 1758, was amplified using synthesized HA B Brisbane gene (corresponding to nt 34-1791 from Genbank accession number FJ766840) as template. The PCR products from both amplifications were then mixed and used as template for a second round of amplification. The resulting fragment (encoding HA B/Brisbane/60/2008 Δa.a. 356-374 with a GG linker between fragments; see
[0305] The amino acid sequence of PDISP-HA B/Brisbane/60/2008 with deleted proteolytic loop is presented in
Example 5.5: B-2X35S/CPMV-HT/HA B Wisconsin/NOS into BeYDV(m)+Replicase Amplification System (Construct Number 1462)
[0306] The preparation of construct 1462 is described in U.S. 61/541,780. Briefly, a sequence encoding HA from influenza B/Wisconsin/1/2010 was cloned into 2X355/CPMV-HTNOS comprising the BeYDV(m)+replicase amplification system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using a PCR-based method. A fragment containing the complete HA B Wisconsin coding sequence was amplified using synthesized HA B Wisconsin gene (Genbank accession number JN993010) as template. The PCR product was cloned in 2X355/CPMV-HT/NOS expression cassette into the BeYDV(m) amplification system. Construct 193 (
[0307] Construct number 193 (
Example 5.6: C-2X35S/CPMV-HT/HA B Wisconsin with Deleted Proteolytic Loop into BeYDV(m)+Replicase Amplification System (Construct Number 1467)
[0308] The preparation of construct 1467 is described in U.S. 61/541,780. Briefly, a sequence encoding HA from influenza B/Wisconsin/1/2010 with deleted proteolytic loop was cloned into 2X35S/CPMV-HT/NOS comprising the BeYDV(m)+replicase amplification system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using a PCR-based ligation method (Darveau et al. 1995, Methods in Neuroscience 26: 77-85). In a first round of PCR, a fragment containing HA B Wisconsin coding sequence from nt 1 to nt 1062 was amplified using primers IF-HAB110.S1+3c (
[0309] Construct number 193 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV-HT-based expression cassette into the BeYDV(m) amplification system. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in
Example 5.7: A-2X35S/CPMV-HT/PDISP-HA B Brisbane with Deleted Proteolytic Loop (Construct Number 1039)
[0310] The preparation of construct 1192 is described in U.S. 61/541,780. Briefly, a sequence encoding HA from influenza B/Brisbane/60/2008 with deleted proteolytic loop was cloned into 2X35S/CPMV-HT/PDISP/NOS in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based ligation method (Darveau et al., 1995, Methods in Neuroscience 26: 77-85). In a first round of PCR, a fragment containing HA B Brisbane coding sequence from nt 46 to nt 1065 was amplified using synthesized HA B Brisbane gene (corresponding to nt 34-1791 from Genebank accession number FJ766840) as template. A second fragment, containing HA B Brisbane coding sequence from nt 1123 to nt 1758, was amplified using synthesized HA B Brisbane gene (corresponding to nt 34-1791 from Genbank accession number FJ766840) as template. The PCR products from both amplifications were then mixed and used as template for a second round of amplification. The resulting fragment (encoding HA B/Brisbane/60/2008 Δa.a. 356-374 with a GG linker between fragments) was cloned in-frame with alfalfa PDI signal peptide in 2X355/CPMV-HT/NOS expression cassette. Construct 1192 was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction.
[0311] Construct number 1192 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in frame with an alfalfa PDI signal peptide in a CPMV-HT-based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid. The resulting construct was given number 1039 (
Example 5.8: A-2X35S/CPMV-HT/H5 from A/Indonesia/5/2005 with TETR Cleavage Site Mutation (Construct Number 676)
[0312] A sequence encoding H5 from A/Indonesia/5/2005 with TETR cleavage site mutation was cloned into 2X355/CPMV-HT/NOS in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based ligation method (Darveau et al., 1995, Methods in Neuroscience 26: 77-85). In a first round of PCR, a fragment containing H5 from A/Indonesia/5/2005 coding sequence from nt 1 to nt 1015 was amplified using primers IF-HSA-I-05.s1+3c (
Example 5.9: B-2X35S/CPMV-HT/H5 from A/Indonesia/5/2005 with TETQ Cleavage Site Mutation (Construct Number 766)
[0313] A sequence encoding H5 from A/Indonesia/5/2005 with TETQ cleavage site mutation was cloned into 2X355/CPMV-HT/NOS in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based ligation method (Darveau et al., 1995, Methods in Neuroscience 26: 77-85). In a first round of PCR, a fragment containing H5 from A/Indonesia/5/2005 coding sequence from nt 1 to nt 1015 was amplified using primers IF-HSA-I-05.s1+3c (
Example 5.10: C-2X35S/CPMV-HT/H5 from A/Indonesia/5/2005 with Deleted Proteolytic Loop (Construct Number 928)
[0314] A sequence encoding H5 from A/Indonesia/5/2005 with deleted proteolytic loop was cloned into 2X355/CPMV-HT/NOS in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based ligation method presented by Darveau et al. (Methods in Neuroscience 26: 77-85 (1995)). In a first round of PCR, a fragment containing H5 from A/Indonesia/5/2005 coding sequence from nt 1 to nt 1011 was amplified using primers IF-HSA-I-05.s1+3c (
Example 5.11—F-2X35S/CPMV-HT/PDISP/HA B Brisbane/NOS (Construct Number 1029)
[0315] A sequence encoding HA from influenza B/Brisbane/60/2008 was cloned into 2X355/CPMV-HT/PDISP/NOS expression system in a plasmid containing Plasto_pro/P19/Plasto_ter expression cassette using the following PCR-based method. A fragment containing HA B Brisbane coding sequence without his wild type signal peptide was amplified using primers IF-S2+S4-B Bris.c (
Example 5.12-2X35S/CPMV HT (Construct No 1039) and HT+ (Construct No 1829) for PDISP/HA B Brisbane (PrL-)
[0316] A coding sequence corresponding to HA from Influenza B/Brisbane/60/2008 with deleted proteolytic loop (PrL-) in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Brisbane (PrL-;
Example 5.13-2X35S/CPMV HT (Construct No 1039) and 2X35S/CPMV160+ (Construct No 1937) for PDISP/HA B Brisbane (PrL-)
[0317] A coding sequence corresponding to HA from Influenza B/Brisbane/60/2008 with deleted proteolytic loop (PrL-) (see U.S. provisional application No. 61/806,227 Filed Mar. 28, 2013, which is incorporated herein by reference, for additional information re: deleted proteolytic loop regions in HA sequences) in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Brisbane (PrL-)) (
Example 15.14-2X35S/CPMV HT (Construct No 1067) and 2X35S/CPMV160+ (Construct No 1977) for PDISP/HA B Brisbane (PrL-)+H1 California TMCT
[0318] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/Brisbane/60/08 with deleted proteolytic loop (PrL-) (see U.S. provisional application No. 61/806,227 Filed Mar. 28, 2013, which is incorporated herein by reference, for additional information re: deleted proteolytic loop regions in HA sequences) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from influenza A/California/7/2009 and with the signal peptide of alfalfa protein disulfide isomerase (PDISP/HA B Brisbane (PrL-)+H1 California TMCT) (
Example 5.15-2X35S/CPMV HT (Construct No 2072) and 2X35S/CPMV160+ (Construct No 2050) for PDISP/HA B Massachusetts (PrL-)
[0319] A coding sequence corresponding to HA from Influenza B/Massachusetts/2/2012 with deleted proteolytic loop (PrL-) (see U.S. provisional application No. 61/806,227 Filed Mar. 28, 2013 for additional information re: deleted proteolytic loop regions in HA sequences, which is incorporated herein by reference) in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Massachussetts (PrL-)) (
Example 5.16-2X35S/CPMV HT (Construct no 2074) and 2X35S/CPMV160+ (Construct No 2060) for PDISP/HA B Massachusetts (PrL-)+H1 California TMCT
[0320] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/Massachussetts/2/2012 with deleted proteolytic loop (PrL-) (see U.S. provisional application No. 61/806,227 Filed Mar. 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 H1 from influenza A/California/7/2009 and with the signal peptide of alfalfa protein disulfide isomerase (PDISP/HA B Massachussetts (PrL-)+H1 California TMCT) (
Example 5.17-2X35S/CPMV HT (Construct No 1445), 2X35S/CPMVHT+ (Construct No 1820) and CPMV160+ (Construct No 1975) for HA B Wisconsin (PrL-)
[0321] A coding sequence corresponding to HA from Influenza B/Wisconsin/1/2010 with deleted proteolytic loop (PrL-) (see U.S. provisional application No. 61/806,227 Filed Mar. 28, 2013 for additional information re: deleted proteolytic loop regions in HA sequences, which is incorporated herein by reference) with his native signal peptide (HA B Wisconsin (PrL-)) (
Example 5.18-2X35S/CPMV HT (Construct No 1454) and 2X35S/CPMV160+ (Construct No 1893) for HA B Wisconsin (PrL-)+H1 California TMCT
[0322] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/Wisconsin/2/2012 with deleted proteolytic loop (PrL-) (see U.S. provisional application No. 61/806,227 Filed Mar. 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 H1 from influenza A/California/7/2009 with the native signal peptide of HA B Wisconsin (HA B Wisconsin (PrL-)+H1 California TMCT) (
Example 5.19: 2X35S/CPMV HT (Construct No 1067) and HT+ (Construct No 1875) for PDISP/HA B Brisbane (PrL-)+H1 California TMCT
[0323] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/Brisbane/60/08 with deleted proteolytic loop (PrL-) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from influenza A/California/7/2009 and with the signal peptide of alfalfa protein disulfide isomerase (PDISP/HA B Brisbane (PrL-)+H1 California TMCT) (
Example 5.20: 2X35S/CPMV HT (Construct No 2072) and HT+ (Construct No 2052) for PDISP/HA B Massachusetts (PrL-)
[0324] A coding sequence corresponding to HA from Influenza B/Massachussetts/2/2012 with deleted proteolytic loop (PrL-) in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Massachusetts (PrL-)) (
Example 5.21: 2X35S/CPMV HT (Construct No 2074) and HT+ (Construct No 2062) for PDISP/HA B Massachusetts (PrL-)+H1 California TMCT
[0325] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/Massachusetts/2/2012 with deleted proteolytic loop (PrL-) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from influenza A/California/7/2009 and with the signal peptide of alfalfa protein disulfide isomerase (PDISP/HA B Massachusetts (PrL-)+H1 California TMCT) (
Example 5.22: 2X35S/CPMV HT (Construct No 1445) and HT+ (Construct No 1839) for HA B Wisconsin (PrL-)
[0326] A coding sequence corresponding to HA from Influenza B/Wisconsin/1/2010 with deleted proteolytic loop (PrL-) with his native signal peptide (HA B Wisconsin (PrL-)) (
Example 5.23: 2X35S/CPMV HT (Construct No 1454) and HT+ (Construct No 1860) for HA B Wisconsin (PrL-)+H1 California TMCT
[0327] A chimer hemagglutinin coding sequence corresponding to the ectodomain of HA from Influenza B/Wisconsin/2/2012 with deleted proteolytic loop (PrL-) fused to the transmembrane domain and cytoplasmic tail (TMCT) of H1 from influenza A/California/7/2009 with the native signal peptide of HA B Wisconsin (HA B Wisconsin (PrL-)+H1 California TMCT) (
Example 5.24-2X35S/CPMV HT (Construct No 489), 2X35S/CPMV160+ (Construct No 1880) and 2X35S/CPMV160 (Construct No 1885) for H5 Indonesia
[0328] A coding sequence corresponding to native H5 from Influenza A/Indonesia/5/2005 (
Example 5.25-2X35S/CPMV160+/PDISP/H3 Victoria/NOS (Construct Number 1800)
[0329] A sequence encoding H3 from Influenza A/Victoria/361/2011 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Victoria) was cloned into 2X355/CPMV160+/NOS expression system (CPMV160+) using the following PCR-based method. A fragment containing the PDISP/H3 Victoria coding sequence was amplified using primers IF**(SacII)-PDI.s1+4c (
Example 5.26: 2X35S/CPMV-HT+/PDISP/H3 Victoria/NOS (Construct Number 1819)
[0330] A sequence encoding H3 from Influenza A/Victoria/361/2011 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Victoria) was cloned into 2X355-CPMV-HT+/NOS expression using the following PCR-based method. A fragment containing the PDISP/H3 Victoria coding sequence was amplified using primers IF(SacII)-Kozac_PDI.c (
Example 5.27 2X35S/CPMV HT+/PDISP/H2 Singapore/NOS (Construct Number 2220)
[0331] A sequence encoding H2 from Influenza A/Singapore/1/1957 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H2 Singapore) was cloned into 2X355/CPMV HT+/NOS expression system using the following PCR-based method. A fragment containing the PDISP/H2 Singapore coding sequence was amplified using primers IF(SacII)-Kozac_PDI.c (described for construct 1819 in Example 5.26) and IF**-H2S157.s1-6r (
Example 5.28 2X35S/CPMV HT+/PDISP/H2 Singapore with Deleted Proteolytic Loop/NOS (Construct Number 2221)
[0332] A sequence encoding H2 from Influenza A/Singapore/1/1957 with deleted proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H2 Singapore with deleted proteolytic loop) was cloned into 2X355/CPMV HT+/NOS expression system using the following PCR-based ligation method presented by Darveau et al. (Methods in Neuroscience 26: 77-85 (1995)). In a first round of PCR, a fragment containing H2 from Influenza A/Singapore/1/1957 coding sequence from nt 1 to nt 1032 was amplified with primers IF(SacII)-Kozac_PDI.c (described for construct 1819 in Example 5.26) and H.sub.2S157(Prl-).r (
Example 5.29 PDISP/H2 Singapore (Construct Number 2222) and PDISP/H2 Singapore with Deleted Proteolytic Loop (Construct Number 2223) in 2X355/CPMV 160+/NOS Expression System
[0333] Sequences encoding H2 from Influenza A/Singapore/1/1957 with or without proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H2 Singapore and PDISP/H2 Singapore with deleted proteolytic loop) were cloned into 2X355/CPMV 160+/NOS expression system using the same PCR-based method as construct 2220 and 2221, respectively, but using modified forward primer IF**(SacII)-PDI.s1+4c (described for construct 1800 in Example 5.25) for amplification and a different acceptor plasmid. Resulting PCR products were cloned in 2X355/CPMV 160+/NOS expression system using In-Fusion cloning system (Clontech, Mountain View, Calif.). Construct number 2171 (described for construct 1800 in Example 5.25) was digested with SacII and StuI restriction enzyme and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 2171 is an acceptor plasmid intended for “In Fusion” cloning of genes of interest in a CPMV 160+ based expression cassette. It also incorporates a gene construct for the co-expression of the TBSV P19 suppressor of silencing under the alfalfa Plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid and the sequence from left to right t-DNA borders is presented in (described for construct 1800 in Example 5.25). The resulting constructs were given number 2222 for PDISP/H2 Singapore (
Example 5.30 2X355/CPMV HT+ (Construct No 2019) and 160+ (Construct No 2139) for PDISP/H3 Perth
[0334] A coding sequence corresponding to H3 from Influenza A/Perth/16/2009 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Perth) (
Example 5.31 2X35S/CPMV HT+ (Construct No 2039) and 160+ (Construct No 2159) for PDISP/H3 Perth with Deleted Proteolytic Loop
[0335] A coding sequence corresponding to H3 from Influenza A/Perth/16/2009 with deleted proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Perth with deleted proteolytic loop) (
Example 5.32 2X35S/CPMV HT+ (Construct No 2230) and 160+ (Construct No 2250) for PDISP/H3 Victoria with Deleted Proteolytic Loop
[0336] A coding sequence corresponding to H3 from Influenza A/Victoria/361/2011 with deleted proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Victoria with deleted proteolytic loop) (
Example 5.33 2X35S/CPMV HT+/PDISP/H7 Hangzhou/NOS (Construct No 2142)
[0337] A coding sequence corresponding to H7 from Influenza A/Hangzhou/1/2013 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H7 Hangzhou) (
Example 5.34 2X35S/CPMV HT+/PDISP/H7 Hangzhou with Deleted Proteolytic Loop/NOS (Construct No 2152)
[0338] A coding sequence corresponding to H7 from Influenza A/Hangzhou/1/2013 with deleted proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H7 Hangzhou with deleted proteolytic loop) (
Example 5.35 2X35S/CPMV HT+ (Construct No 2224) and 160+ (Construct No 2226) for PDISP/H9 Hong Kong
[0339] A coding sequence corresponding to H9 from Influenza A/Hong Kong/1073/1999 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H9 Hong Kong) (
Example 5.36 2X35S/CPMV HT+ (Construct No 2225) and 160+ (Construct No 2227) for PDISP/H9 Hong Kong with Deleted Proteolytic Loop
[0340] A coding sequence corresponding to H9 from Influenza A/Hong Kong/1073/1999 with deleted proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H9 Hong Kong with deleted proteolytic loop) (
Example 5.37 2X35S/CPMV 160+/PDISP/HA B Malaysia/NOS (Construct No 2013)
[0341] A coding sequence corresponding to HA from Influenza B/Malaysia/2506/2004 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Malaysia) (
Example 5.38 2X35S/CPMV 160+/PDISP/HA B Malaysia with Deleted Proteolytic Loop/NOS (Construct No 2014)
[0342] A coding sequence corresponding to HA from Influenza B/Malaysia/2506/2004 with deleted proteolytic loop in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Malaysia with deleted proteolytic loop) (
Example 5.39 2X35S/CPMV HT (Construct No 2070), HT+ (Construct No 2080) and 160+ (-Mprot) (Construct No 2090) for PDISP/HA B Massachusetts
[0343] A coding sequence corresponding to HA from Influenza B/Massachusetts/2/2012 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Massachusetts) (
Example 5.40 2X35S/CPMV HT+ (Construct No 2102), HT+ with BeYDV (Construct No 2104) for PDISP/HA B Florida with Deleted Proteolytic Loop
[0344] A coding sequence corresponding to HA from Influenza B/Florida with the proteolytic loop deleted and in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Florida) (
Example 5.41 2X35S/CPMV HT+ (Construct No 2106), HT+ with BeYDV (Construct No 2108) for PDISP/HA B Florida+111 California TMCT with Deleted Proteolytic Loop
[0345] A coding sequence corresponding to HA from Influenza B/Florida+H1 California TMCT with the proteolytic loop deleted and in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/HA B Florida+H1 California TMCT) (
Example 5.42 2X35S/CPMV 160+ for PDISP/HA 113 Victoria+111 California TMCT with (Construct No 2320) and without (Construct No 2340) Proteolytic Loop
[0346] A coding sequence corresponding to HA from Influenza H3 Victoria/361/11+H1 California TMCT in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Victoria+H1 California TMCT,
Example 5.43 H3 A/Switzerland/9715293/2013 and A/Texas/50/2012 with (Constructs No 2801 and 2451, Respectively) and without (Constructs No 2841 and 2453, Respectively) Proteolytic Loop in 2X35S-CPMV 160-NOS Term
[0347] A coding sequence corresponding to H3 from Influenza A/Switzerland/9715293/2013 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Switzerland) was cloned into 2X355/CPMV160/NOS expression system (CPMV160) using the following PCR-based method. A fragment containing the PDISP/H3 Switzerland coding sequence was amplified using primers IF-H3_Swi_13.c (
[0348] Similarly, a sequence corresponding to H3 from Influenza A/Switzerland/9715293/2013 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase and the proteolytic loop removed (PDISP/H3 Switzerland (Prl-),
[0349] A coding sequence corresponding to H3 from Influenza A/Texas/50/2012 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Texas) was cloned into 2X355/CPMV160/NOS expression system (CPMV160) using the following PCR-based method. A fragment containing the PDISP/H3 Texas coding sequence was amplified using primers IF-H3 Swi 13.c (
[0350] Similarly, a sequence corresponding to H3 from Influenza A/Texas/50/2012 in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase and the proteolytic loop removed (PDISP/H3 Texas (Prl-),
Example 5.44 H3 A/Switzerland/9715293/2013+H1 California TMCT and A/Texas/50/2012+H1 California TMCT with (Constructs No 2840 and 2452, Respectively) and without (Constructs No 2843 and 2454, Respectively) Proteolytic Loop in 2X35S-CPMV 160-NOS Term
[0351] A coding sequence corresponding to H3 from Influenza A/Switzerland/9715293/2013+H1 California TMCT in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Switzerland+H1 California TMCT,
[0352] Similarly, a sequence corresponding to H3 from Influenza A/Switzerland/9715293/2013+H1 California TMCT in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase and the proteolytic loop removed (PDISP/H3 Switz+H1 California TMCT (Prl-),
[0353] A coding sequence corresponding to H3 from Influenza A/Texas/50/2012+H1 California TMCT in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase (PDISP/H3 Texas+H1 California TMCT) was cloned into 2X355/CPMV160/NOS expression system (CPMV160) using the following PCR-based method. A fragment containing the PDISP/H3 Texas coding sequence was amplified using primers IF-H3 Swi 13.c (
[0354] Similarly, a sequence corresponding to H3 from Influenza A/Texas/50/2012+H1 California TMCT in which the native signal peptide has been replaced by that of alfalfa protein disulfide isomerase and the proteolytic loop removed (PDISP/H3 Texas+H1 California TMCT (Prl-),
[0355] All citations are hereby incorporated by reference.
[0356] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.