Influenza virus hemagglutinin mutants

Abstract

The present invention relates to producing modified influenza virus proteins in plants. More specifically, the present invention relates to producing influenza virus-like particles (VLPs) in plants and increasing their production, wherein the VLPs include modified influenza virus proteins, such as modified influenza virus hemagglutinin (HA). The HA protein may include an amino acid sequence comprising at least one substitution compared to the corresponding wild-type amino acid sequence. A nucleic acid encoding the modified HA protein is also provided. In addition, a method for producing influenza virus-like particles (VLPs) in plants, plant parts or plant cells and a method for increasing the production of influenza virus-like particles (VLPs) in plants, plant parts or plant cells are also provided.

Description

Technical Field

[0001] The present invention relates to producing mutant viral proteins in plants. More particularly, the present invention relates to producing influenza virus-like particles in plants and increasing the yield of influenza virus-like particles. Background Art

[0002] Influenza viruses are enveloped, single-stranded RNA viruses of the Orthomyxoviridae family. They are highly contagious and can cause mild to severe illness in all age groups.

[0003] Vaccination remains the most effective way to prevent influenza infection. Traditionally, vaccination has been performed using live attenuated viruses or fully inactivated viruses, which elicit an immune response when administered to patients. To eliminate the potential risk of live attenuated and fully inactivated viruses regaining the ability to replicate and infect, vaccines containing recombinant viral proteins are also used to elicit protective immunity against influenza infection.

[0004] However, the use of recombinant viral proteins as immunogenic components of vaccines is subject to numerous limitations. First, without the complete set of viral proteins and genetic components required for optimal expression and proper protein folding, the yield of recombinant viral proteins in standard in vitro expression systems may be insufficient for vaccine production purposes. Second, recombinant viral protein vaccines may exhibit poor immunogenicity due to improper folding, poor antigen presentation, and / or the generation of a primary humoral immune response that is ineffective in conferring long-lasting protective immunity.

[0005] There are four types of influenza viruses: A, B, C and D, of which influenza A and B viruses are the causative organisms of seasonal epidemics of disease in humans.

[0006] Influenza A viruses are further divided based on the expression of hemagglutinin (HA) and neuraminidase (NA) glycoprotein subtypes on the viral surface. There are 18 different hemagglutinin subtypes (H1-H18).

[0007] Hemagglutinin is a trimeric lectin that promotes the binding of influenza virus particles to sialic acid-containing proteins on the surface of target cells and mediates the release of the viral genome into target cells. The hemagglutinin protein consists of two structural elements: the head, which is the primary target of serum protective antibodies, and the stalk. A 2002 article by Ha et al. (EMBO J. 21:865-875; incorporated herein by reference) showed, based on X-ray crystallographic structures, the relative orientation of the individual subdomains of the stalk domain cluster (SDC) and the head domain cluster (HDC) in various influenza virus subtypes.

[0008] Hemagglutinin is translated into a single polypeptide HA0 (assembled into a trimer), which must be cut between the HA1 (approximately 40 kDa) subdomain and the HA2 (approximately 20 kDa) subdomain by a serine endoprotease. After cleavage, the two disulfide-bonded protein domains adopt the necessary conformation required to achieve viral infectivity. HA1 forms a globular head domain comprising residual esterase domains E1' and E2 and a receptor binding site (RBS), which is the least conserved segment of influenza virus. HA2 is a unidirectional membrane-bound protein having a fusion peptide (FP), a soluble extracellular domain (SE), a transmembrane domain (TM), and a cytoplasmic tail domain (CT), with the respective lengths of these domains being approximately 25, 160, 25, and 10 residues. HA2, together with the nitrogen and carbon terminal HA1 residues, forms a stalk domain, which comprises a transmembrane domain and is relatively conserved.

[0009] Various mutations of influenza virus proteins, particularly the influenza virus hemagglutinin protein, have been studied.

[0010] For example, Castelán-Vega et al. (Adv Appl Bioinform Chem. 2014; 7: 37-44) used a stability prediction algorithm to compare 7479 full-length amino acid sequences of the hemagglutinin from influenza A virus (H1N1) pdm09 and determined that the D104N, A259T, S124N, and E172K mutations resulted in predicted increased stability of the influenza virus hemagglutinin protein. In contrast, the S206T, K285E, and E47K mutations were predicted to have a destabilizing effect on the hemagglutinin.

[0011] In comparing the sequences of the original influenza A(H1N1) pdm [A / California / 7 / 2009] and a later emerging influenza strain [A / Brisbane / 10 / 2010], Cotter et al. (PLoS Pathog. 2014;10(1):e1003831) determined that the E47K mutation in the stalk domain of the A / California / 7 / 2009 hemagglutinin stabilizes the trimeric structure, lowers the pH for membrane fusion, and increases the heat and acid stability of the virus. Cotter et al. also observed that the A / California / 7 / 2009 E47K mutant hemagglutinin was more infectious in ferrets than its wild-type counterpart.

[0012] Antanasijevic et al. (J Biol Chem. 2014; 289(32): 22237-45) studied the structure-function properties of the stem-loop domain of the H5 hemagglutinin by site-directed mutagenesis at 14 different positions. A / Vietnam / 1203 / 04 (H5N1) mutants were expressed in HEK 293T cells, and Antanasijevic reported that most mutations in the stem-loop domain did not disrupt expression, proteolytic processing, viral assembly, or receptor binding. However, Antanasijevic observed that the HA1-D26K, HA1-M102L, HA2-V52A, and HA2-I55A mutants (based on H3 numbering) exhibited significantly reduced levels of total hemagglutinin, suggesting that hemagglutinin expression and / or assembly in viral particles was reduced. The HA1-D26K, HA2-T49A, and HA2-M102L mutants also exhibited lower hemagglutination titers compared to wild-type virus. Antanasijevic also observed that all single mutants had reduced ability to enter A549 lung cells, with the HA1-D26K and HA2-I55A mutants showing the most pronounced impairment. Antanasijivec also demonstrated that the HA2-L99A mutant was more sensitive to inhibition of A549 lung cells by the C179 neutralizing antibody than the wild-type virus, suggesting that this mutation enhances the mode of antibody binding and / or neutralization. In contrast, the HA1-I28A, HA1-M31A, HA1-M31L, HA2-I45A, and HA2-I55V mutants were less sensitive to entry inhibition by the C179 neutralizing antibody.

[0013] WO2013 / 177444 by Lu et al. and its companion document (Proc Natl Acad Sci USA. 2014; 111(1): 125-30) report a method for producing correctly folded hemagglutinin stem domains from A / California / 05 / 2009 (H1N1) using an E. coli-based cell-free protein expression system and a simple refolding protocol. To induce trimerization of the hemagglutinin stem domain, chloramphenicol acetyltransferase (CAT) or a folding domain was fused to the carbon terminus of the hemagglutinin. To mitigate newly exposed hydrophobicity and / or intermolecular ion pairing that could lead to aggregation of the expressed hemagglutinin stem protein, five groups of mutations were evaluated: M1 (I69T+I72E+I74T+C77T); M2 (I69T+I72E+I74T+C77T+F164D); M3 (I69T+I72E+I74T+C77T+F164D+L174D); M4 (F164D); and M5 (F164D+L174D). Lu observed that the M5 (F164D+L174D) mutation appeared to have the greatest impact on improving the solubility of the hemagglutinin stem protein. Additional deletions (H38 to C43 and C49 to N61) and the C77T mutation were made to the M5 mutant to prevent the formation of undesirable disulfide bonds, reduce surface hydrophobicity and pI, and avoid regions with disordered structure.

[0014] U.S. application Ser. No. 13 / 838,796 by Holtz et al. and its companion document (BMC Biotechnology. 2014; 14: 111) teach that the stability of recombinant hemagglutinin can be improved and its potency maintained by mutation of cysteine ​​residues in the carboxyl-terminal domain of the hemagglutinin protein, including the transmembrane domain (TM) and the cytoplasmic domain (CT). Specifically, Holtz et al. demonstrated the C539A, C546A, C549A, C524S, and C528A mutations in recombinant Perth / 16 / 2009 hemagglutinin (H3N2). Mutation of all five cysteine ​​residues or different subsets thereof resulted in hemagglutinin yield, purity, particle size, hemagglutination activity, and thermal stability comparable to that of recombinant wild-type hemagglutinin protein. In contrast, the C64S and C76S mutations resulted in a significant decrease in hemagglutinin expression, indicating the critical role of these residues in the correct folding of hemagglutinin. Using a single radial immunodiffusion assay (SRID), Holtz et al. also showed that mutation of these five cysteine ​​residues increased the potency of recombinant hemagglutinin compared to the wild-type protein by preventing disulfide cross-linking in the TM and CT domains. The mutant hemagglutinin proteins maintained their potency for at least 12 months at 25°C, whereas the wild-type hemagglutinin protein exhibited less than 40% potency after only 50 days of purification.

[0015] WO2015 / 020913 teaches the mutation of specific residues at one or more positions selected from the group consisting of positions 403, 406, 411, 422, 429, 432, 433, and 435 of influenza virus A / Puerto Rico / 8 / 1934 (H1N1) to tyrosine. These mutations promote the formation of dityrosine cross-links, thereby stabilizing or "locking" the handle domain of influenza virus hemagglutinin in its native trimeric conformation.

[0016] WO2013 / 079473 discloses a modified influenza virus hemagglutinin without a globular head domain. The polypeptide taught in WO2013 / 079473 comprises an HA1 domain and an HA2 domain, wherein amino acids 53 to 620 (numbered with reference to A / Brisbane / 59 / 2007 [H1N1]) are deleted and replaced by a covalently linked sequence of 0 to 10 amino acids, wherein the carbon-terminal amino acid of the HA1 domain is an amino acid other than arginine or lysine, and wherein one or more amino acids at positions 406, 409, 413, and 416 are mutated to an amino acid selected from the group consisting of serine, threonine, asparagine, glutamine, arginine, histidine, lysine, aspartic acid, glutamic acid, and glycine.

[0017] WO2014 / 191435 similarly teaches a modified influenza virus hemagglutinin comprising an HA1 domain and an HA2 domain, wherein a deleted segment of the HA1 domain is replaced by a covalently linked sequence of 0 to 50 amino acids, wherein the hemagglutinin resists cleavage at the junction of HA1 and HA2, and wherein one or more amino acids at positions 337, 340, 352, 353, 402, 406, 409, 413 and / or 416 have been mutated.

[0018] Virus-like particles (VLP) are potential candidates for inclusion in immunogenic compositions. VLP is very similar to mature virions, but they do not comprise viral genome material. Therefore, VLP is non-replicative in nature, and this makes them safe as vaccine administration. In addition, VLP can be modified to express viral glycoprotein on the surface of VLP, and this is their most natural physiological structure. In addition, because VLP is similar to complete virion and is a multivalent particulate structure, VLP may be more effective than soluble envelope protein antigens in inducing glycoprotein neutralizing antibodies.

[0019] VLPs have been produced in plants (see, for example, WO2009 / 076778; WO2009 / 009876; WO2009 / 076778; WO2010 / 003225; WO2010 / 003235; WO2010 / 006452; WO2011 / 03522; WO2010 / 148511 and WO2014153674, which are incorporated herein by reference).

[0020] WO2009 / 076778 teaches a method for producing influenza virus VLPs in plants, the method comprising introducing a nucleic acid having a regulatory domain active in plants operably linked to a nucleotide sequence encoding an influenza virus hemagglutinin from an influenza A or B virus.

[0021] WO2009 / 009876 teaches a method for producing influenza virus hemagglutinin VLPs in plants, wherein the influenza virus hemagglutinin self-assembles into VLPs in plant cells and in plant cell membrane buds.

[0022] WO2010 / 003225 discloses a method for producing influenza virus hemagglutinin VLPs in plants, the method comprising introducing a nucleic acid having a regulatory domain active in plants, the regulatory domain being operably linked to a nucleotide sequence encoding influenza virus hemagglutinin from A / California / 04 / 09 (H1N1).

[0023] WO2010 / 006452 teaches a method for producing VLPs comprising a modified influenza virus hemagglutinin protein, wherein the glycosylation sites at positions 154, 165, 286, or a combination thereof (referenced to A / Vietnam / 1194 / 04 [H5N1] numbering) have been eliminated by mutating the residues at said positions to amino acids other than asparagine. WO2010 / 006452 also teaches that the amino acids at positions 156, 167, 288, or a combination thereof, can be mutated to residues other than serine or threonine, thereby similarly eliminating the nitrogen-linked glycosylation signal triad "NXS / T." By selectively deleting the glycosylation sites located in the globular head of the hemagglutinin protein, WO2010 / 006452 demonstrates that the resulting hemagglutinin protein has improved antigenicity and broader cross-reactivity.

[0024] WO2011 / 035422 teaches a method for preparing plant-derived VLPs, the method comprising: obtaining a plant or plant material comprising a VLP located within an apoplast; producing a protoplast / spheroid fraction and an apoplast fraction; and recovering the apoplast fraction comprising the plant-derived VLPs.

[0025] WO2010 / 148511 discloses a method for producing influenza virus VLPs in plants, wherein the VLPs comprise a chimeric hemagglutinin protein. The chimeric hemagglutinin protein comprises a stem domain cluster comprising F'1, F'2, and F subdomains; a head domain cluster comprising RB, E1, and E2 subdomains; and a transmembrane domain cluster comprising a transmembrane domain and a carbon-terminal tail domain, wherein at least one subdomain is derived from a first influenza virus strain and the other subdomains are derived from one or more second influenza virus strains.

[0026] WO2014 / 153674 teaches a method for producing influenza virus VLPs in plants, wherein the VLPs comprise a modified influenza virus hemagglutinin with a modified proteolytic loop. The modified proteolytic loop includes a proteolytic cleavage site between the HA1 and HA2 domains of the HA0 precursor, thereby stabilizing the hemagglutinin protein and increasing protein yield compared to the native hemagglutinin protein. Summary of the Invention

[0027] The present invention relates to producing modified influenza virus proteins in plants. More specifically, the present invention relates to producing influenza virus-like particles (VLPs) in plants and increasing the yield of influenza virus-like particles (VLPs), wherein the VLPs include modified influenza virus proteins, such as modified hemagglutinin (HA) proteins.

[0028] It is an object of the present invention to provide an improved method for increasing the production of influenza virus VLPs in plants.

[0029] According to the present invention, there is provided:

[0030] A nucleic acid comprising a nucleotide sequence encoding a modified H1 influenza virus hemagglutinin (HA) protein, the hemagglutinin protein comprising an amino acid sequence comprising at least one substitution compared to the corresponding wild-type amino acid sequence, the at least one substitution being at one or more amino acids corresponding to the amino acid at position 97, 374, 390, or 429 of the H1A / Michigan / 45 / 15 hemagglutinin.

[0031] The hemagglutinin protein may include an amino acid sequence having a non-asparagine substitution at an amino acid corresponding to the amino acid at position 97 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may include an amino acid sequence having an aspartic acid substitution or a conservative aspartic acid substitution at an amino acid corresponding to the amino acid at position 97 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0032] The hemagglutinin protein may include an amino acid sequence having a non-lysine substitution at an amino acid corresponding to the amino acid at position 374 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may include an amino acid sequence having a glutamic acid substitution or a conservative glutamic acid substitution at an amino acid corresponding to the amino acid at position 374 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0033] The hemagglutinin protein may include an amino acid sequence having a non-phenylalanine substitution at an amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may include an amino acid sequence having an aspartic acid substitution or a conservative aspartic acid substitution at an amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0034] The hemagglutinin protein may include an amino acid sequence having a non-leucine substitution at an amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may include an amino acid sequence having a methionine substitution or a conservative methionine substitution at an amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0035] The hemagglutinin protein may further include an amino acid sequence having a non-asparagine substitution at the amino acid corresponding to the amino acid at position 380 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may include an amino acid sequence having an alanine substitution or a conservative alanine substitution at the amino acid corresponding to the amino acid at position 380 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0036] The hemagglutinin protein may also include an amino acid sequence having a first non-phenylalanine substitution at the amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin and a second non-leucine substitution at the amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may also include an amino acid sequence having a first aspartic acid substitution or a conservative aspartic acid substitution at the amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin and a second methionine substitution or a conservative methionine substitution at the amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0037] The hemagglutinin protein may further include an amino acid sequence having a first non-asparagine substitution at the amino acid corresponding to the amino acid at position 97 of the H1 A / Michigan / 45 / 15 hemagglutinin and a second non-lysine substitution at the amino acid corresponding to the amino acid at position 374 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may further include an amino acid sequence having a first aspartic acid substitution or a conservative aspartic acid substitution at the amino acid corresponding to the amino acid at position 97 of the H1 A / Michigan / 45 / 15 hemagglutinin and a second glutamic acid substitution or a conservative glutamic acid substitution at the amino acid corresponding to the amino acid at position 374 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0038] The hemagglutinin protein may also include an amino acid sequence having a first non-asparagine substitution at the amino acid corresponding to the amino acid at position 97 of the H1 A / Michigan / 45 / 15 hemagglutinin, a second non-phenylalanine substitution at the amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin, and a third non-leucine substitution at the amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may also include an amino acid sequence having a first aspartate substitution or a conservative aspartate substitution at the amino acid corresponding to the amino acid at position 97 of the H1 A / Michigan / 45 / 15 hemagglutinin, a second aspartate substitution or a conservative aspartate substitution at the amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin, and a third methionine substitution or a conservative methionine substitution at the amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0039] The hemagglutinin protein may also include an amino acid sequence having a first non-lysine substitution at the amino acid corresponding to the amino acid at position 374 of the H1 A / Michigan / 45 / 15 hemagglutinin, a second non-phenylalanine substitution at the amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin, and a third non-leucine substitution at the amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein may also include an amino acid sequence having a first glutamic acid substitution or a conservative glutamic acid substitution at the amino acid corresponding to the amino acid at position 374 of the H1 A / Michigan / 45 / 15 hemagglutinin, a second aspartic acid substitution or a conservative aspartic acid substitution at the amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin, and a third methionine substitution or a conservative methionine substitution at the amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0040] The hemagglutinin protein can also include an amino acid sequence having a first non-asparagine substitution at the amino acid corresponding to the amino acid at position 97 of the H1 A / Michigan / 45 / 15 hemagglutinin, a second non-lysine substitution at the amino acid corresponding to the amino acid at position 374 of the H1 A / Michigan / 45 / 15 hemagglutinin, a third non-phenylalanine substitution at the amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin, and a fourth non-leucine substitution at the amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin. The hemagglutinin protein can also include an amino acid sequence having a first aspartic acid substitution or a conservative aspartic acid substitution at the amino acid corresponding to the amino acid at position 97 of the H1 A / Michigan / 45 / 15 hemagglutinin, a second glutamic acid substitution or a conservative glutamic acid substitution at the amino acid corresponding to the amino acid at position 374 of the H1 A / Michigan / 45 / 15 hemagglutinin, a third aspartic acid substitution or a conservative aspartic acid substitution at the amino acid corresponding to the amino acid at position 390 of the H1 A / Michigan / 45 / 15 hemagglutinin, and a fourth methionine substitution or a conservative methionine substitution at the amino acid corresponding to the amino acid at position 429 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0041] Also provided are a hemagglutinin protein encoded by the recombinant nucleic acid and a virus-like particle (VLP) comprising the hemagglutinin protein encoded by the recombinant nucleic acid.

[0042] Thus, provided is a modified H1 influenza virus hemagglutinin (HA) protein comprising an amino acid sequence comprising at least one substitution compared to the corresponding wild-type amino acid sequence, wherein the at least one substitution is at one or more amino acids corresponding to the amino acid at position 97, 374, 390 or 429 of the H1 A / Michigan / 45 / 15 hemagglutinin.

[0043] Furthermore, a method of producing influenza virus-like particles (VLPs) in a plant, plant part or plant cell is provided, the method comprising:

[0044] a) introducing the recombinant nucleic acid into a plant, plant part or plant cell; and

[0045] b) incubating the plant, plant part or plant cell under conditions that allow expression of the hemagglutinin protein encoded by the recombinant nucleic acid, thereby producing VLPs. The method may further comprise the step of harvesting the plant, plant part or plant cell and purifying the VLPs c).

[0046] Also provided is a method of producing influenza virus-like particles (VLPs) in a plant, plant part or plant cell, the method comprising:

[0047] a) providing a plant, plant part or plant cell comprising the recombinant nucleic acid described above; and

[0048] b) incubating the plant, plant part or plant cell under conditions that allow expression of the hemagglutinin protein encoded by the recombinant nucleic acid, thereby producing VLPs. The method may further comprise the step of harvesting the plant, plant part or plant cell and purifying the VLPs c).

[0049] In addition, a method for increasing the yield of influenza virus-like particles (VLPs) in plants, plant parts, or plant cells is provided, the method comprising: a) introducing a recombinant nucleic acid into a plant, plant part, or plant cell; or providing a plant, plant part, or plant cell comprising the recombinant nucleic acid; and b) incubating the plant, plant part, or plant cell under conditions that allow expression of a hemagglutinin protein encoded by the recombinant nucleic acid, thereby producing VLPs at a higher yield than a plant, plant part, or plant cell expressing an unmodified influenza virus hemagglutinin protein. The method may further comprise the step of harvesting the plant, plant part, or plant cell and purifying the VLPs.

[0050] The method may further comprise introducing a second nucleic acid encoding a proton channel protein; wherein the plant, plant part or plant cell is cultivated under conditions allowing expression of the proton channel protein encoded by the second nucleic acid. The proton channel protein may be an influenza A virus M2 subtype protein.

[0051] Also provided is a VLP produced by the method described herein.

[0052] The VLP may include one or more lipids derived from plants, plant parts or plant cells, plant-specific N-glycans, modified N-glycans, or a combination thereof.

[0053] In addition, a method for producing an antibody or antibody fragment is also provided, the method comprising administering the VLP to a subject or host animal, thereby producing the antibody or antibody fragment. Also provided is an antibody or antibody fragment produced by the method.

[0054] In addition, a plant, plant part or plant cell comprising a recombinant nucleic acid or a hemagglutinin protein encoded by a recombinant nucleic acid is provided. The hemagglutinin protein can form a VLP. Thus, a plant, plant part or plant cell comprising a VLP comprising a hemagglutinin protein encoded by a recombinant nucleic acid is also provided.

[0055] In addition, a composition for inducing an immune response is provided, comprising an effective dose of the VLPs as described herein and a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient. A method for inducing immunity to influenza virus infection in a subject is also provided, comprising administering the VLPs. The VLPs can be administered to the subject orally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

[0056] In addition, a modified influenza virus hemagglutinin (HA) protein is provided, wherein the influenza virus hemagglutinin protein includes at least one substitution as described herein and is capable of forming VLPs, inducing an immune response when administered to a subject, inducing a hemagglutination reaction, or a combination thereof, the influenza virus hemagglutinin protein includes SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 4, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67 No. 57, No. 59, No. 61, No. 63, No. 65, No. 67, No. 72, No. 77, No. 80, No. 82, No. 84, No. 86, No. 89, No. 91, No. 93, No. 95, No. 97, No. 105, No. 108, No. 124, No. 126, No. 128, No. 140, No. 143, No. 145, No. 147, and No. 149, and an amino acid sequence that has about 30% to about 100% sequence identity or sequence similarity.

[0057] This summary does not necessarily describe all features of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] These and other features of the present invention will become more apparent from the following description given with reference to the accompanying drawings, in which:

[0059] Figure 1 Shown is a sequence alignment of the amino acid sequences of hemagglutinins (HAs) from A / California / 7 / 09 (H1N1) (SEQ ID NO: 130); A / Honduras / 17734 / 16 (H1N1) (SEQ ID NO: 131); A / Darwin / 11 / 15 (H1N1) (SEQ ID NO: 132); A / Costa Rica / 0513 / 16 (H1N1) (SEQ ID NO: 133); A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 134); and A / Massachusetts / 06 / 17 (H1N1) (SEQ ID NO: 135). The listed residues align with amino acids D97, E374, F390, and L429 of hemagglutinins from H1 influenza virus strains (H1N1), such as A / California / 7 / 09 (H1N1).

[0060] Figure 2AHemagglutination titers of wild-type A / California / 07 / 09 H1, L429M A / California / 07 / 09 mutant H1, N380A A / California / 7 / 09 mutant H1, and F390D A / California / 7 / 09 mutant H1 are shown. Figure 2B Shown are the hemagglutination titers of wild-type A / California / 07 / 09 H1, F390D A / California / 07 / 09 mutant H1, L429M A / California / 7 / 09 mutant H1, and F390D+L429M A / California / 07 / 09 mutant H1 as a percentage of wild-type A / California / 07 / 09. Figure 2C Post-density gradient VLP yields are shown for wild-type A / California / 07 / 09 H1, N380A A / California / 07 / 09 mutant H1, L429M A / California / 07 / 09 mutant H1, and F390D A / California / 07 / 09 mutant H1.

[0061] Figure 3A Hemagglutination titers of wild-type A / California / 07 / 09 H1, wild-type A / Michigan / 45 / 15 H1, K374EA / Michigan / 45 / 15 mutant H1, and N97D A / Michigan / 45 / 15 mutant H1 are shown. Figure 3BWild-type A / California / 07 / 09 H1, F390D A / California / 07 / 09 mutant H1, wild-type A / Michigan / 45 / 15 H1, N97D A / Michigan / 45 / 15 mutant H1, K374E A / Michigan / 45 / 15 mutant H1, N380AA / Michigan / 45 / 15 mutant H1, F390D A / Michigan / 45 / 15 mutant H1, L429M A / Michigan / 45 / 15 mutant H1, N97D+K374E A / Michigan / 45 / 15 mutant H1, F390D+L429M A / Michigan / 45 / 15 mutant H1, F390D+N380A Hemagglutination titers of A / Michigan / 45 / 15 mutant H1, L429M+N380A A / Michigan / 45 / 15 mutant H1, K374E+F390D+L429M A / Michigan / 45 / 15 mutant H1, N97D+F390D+L429M A / Michigan / 45 / 15 mutant H1, and N97D+K374E+F390D+L429M A / Michigan / 45 / 15 mutant H1.

[0062] Figure 4AHemagglutination titers of wild-type A / Michigan / 45 / 15 H1, N97D A / Michigan / 45 / 15 mutant H1, K374E A / Michigan / 45 / 15 mutant H1, F390D A / Michigan / 45 / 15 mutant H1, L429M A / Michigan / 45 / 15 mutant H1, F390D+L429M A / Michigan / 45 / 15 mutant H1, and N97+F390D+L429M A / Michigan / 45 / 15 mutant H1 are shown; Hemagglutination titers of A / Honduras / 17734 / 16 mutant H1, L429M A / Honduras / 17734 / 16 mutant H1, F390D+L429M A / Honduras / 17734 / 16 mutant H1, and N97D+F390D+L429M A / Honduras / 17734 / 16 mutant H1; and wild-type A / Darwin / 11 / 15 H1, N97D A / Darwin / 11 / 15 mutant H1, K374E A / Darwin / 11 / 15 mutant H1, F390D A / Darwin / 11 / 15 mutant H1, L429M A / Darwin / 11 / 15 mutant H1, F390D+L429M Hemagglutination titers of A / Darwin / 11 / 15 mutant H1 and N97D+F390D+L429M A / Darwin / 11 / 15 mutant H1. Figure 4BHemagglutination titers of F390D+L429M A / Michigan / 45 / 15, F390D+L429M A / Massachusetts / 06 / 17 mutant H1, K374E+F390D+L429M A / Massachusetts / 06 / 17 mutant H1, N97D+F390D+L429M A / Massachusetts / 06 / 17 mutant H1, and N97D+K374E+F390D+L429M A / Massachusetts / 06 / 17 mutant H1 are shown; Hemagglutination titers of Rica / 0513 / 16 mutant H1, N97D+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1, and N97D+K374E+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1. Figure 4C Hemagglutination titers of F390D+L429M A / Michigan / 45 / 15, F390D+L429M A / Paris / 1227 / 2017 mutant H1, K374E+F390D+L429M A / Paris / 1227 / 2017 mutant H1, N97D+F390D+L429M A / Paris / 1227 / 2017 mutant H1, and N97D+K374E+F390D+L429M A / Paris / 1227 / 2017 mutant H1 are shown; Hemagglutination titers of A / Norway / 2147 / 2017 mutant H1, N97D+F390D+L429M A / Norway / 2147 / 2017 mutant H1, and N97D+K374E+F390D+L429M A / Norway / 2147 / 2017 mutant H1.

[0063] Figure 5Hemagglutination titers of wild-type A / Indonesia / 5 / 2005 H5, wild-type A / Egypt / N04915 / 2014 H5, F393D A / Indonesia / 5 / 2005 mutant H5, F393D A / Egypt / N04915 / 2014 mutant H5, N383A A / Indonesia / 5 / 2005 mutant H5, and N383A A / Egypt / N04915 / 2014 mutant H5 are shown. Numbering is based on A / Indonesia / 5 / 2005.

[0064] Figure 6A A schematic diagram of carrier 1314 (H1 A-Cal-7-2009) is shown. Figure 6B A schematic diagram of vector 2980 (H1 A-Cal-7-09 (F390D)) is shown. Figure 6C A schematic diagram of vector 2962 (H1A-Cal-7-09 (L429M)) is shown. Figure 6D A schematic diagram of vector 2995 (H1 A-Cal-7-09 (F390D+L429M)) is shown. Figure 6E A schematic diagram of carrier 3640 (H1 A-Mich-45-2015) is shown. Figure 6F A schematic diagram of vector 3774 (H1 A-Mich-45-2015 (N97D)) is shown. Figure 6G A schematic diagram of vector 3771 (H1 A-Mich-45-2015 (K374E)) is shown. Figure 6H A schematic diagram of vector 3641 (H1 A-Mich-45-2015 (F390D)) is shown. Figure 6I A schematic diagram of vector 3643 (H1 A-Mich-45-2015 (L429M)) is shown. Figure 6J A schematic diagram of vector 3880 (H1 A-Mich-45-2015 (N97D+K374E)) is shown. Figure 6K A schematic diagram of vector 3703 (H1 A-Mich-45-2015 (F390D+L429M)) is shown. Figure 6L A schematic diagram of vector 3879 (H1 A-Mich-45-2015 (N97D+F390D+L429M)) is shown. Figure 6M A schematic diagram of vector 3878 (H1 A-Mich-45-2015 (K374E+F390D+L429M)) is shown. Figure 6NA schematic diagram of vector 3881 (H1 A-Mich-45-2015 (N97D+K374E+F390D+L429M)) is shown. Figure 6O A schematic diagram of vector 4091 (H1A-Mass-06-2017 (F390D+L429M)) is shown. Figure 6P A schematic diagram of vector 4093 (H1A-Mass-06-2017 (N97D+F390D+L429M)) is shown. Figure 6Q A schematic diagram of vector 4092 (H1 A-Mass-06-2017 (K374E+F390D+L429M)) is shown. Figure 6R A schematic diagram of vector 4094 (H1 A-Mass-06-2017 (N97D+K374E+F390D+L429M)) is shown. Figure 6S A schematic diagram of vector 4715 (H1 A-CostaRica-0513-2016 (F390D+L429M)) is shown. Figure 6T A schematic diagram of vector 4717 (H1 A-CostaRica-0513-2016 (N97D+F390D+L429M)) is shown. Figure 6U A schematic diagram of vector 4716 (H1A-CostaRica-0513-2016 (K374E+F390D+IA29M)) is shown. Figure 6V A schematic diagram of vector 4718 (H1 A-CostaRica-0513-2016 (N97D+K374E+F390D+L429M)) is shown. Figure 6W A schematic diagram of vector 3944 (H1 A-Hond-17734-16) is shown. Figure 6X A schematic diagram of vector 3950 (H1 A-Hond-17734-16(N97D)) is shown. Figure 6Y A schematic diagram of vector 3948 (H1 A-Hond-17734-16 (K374E)) is shown. Figure 6Z A schematic diagram of vector 3945 (H1 A-Hond-17734-16(F390D)) is shown. Figure 6AA A schematic diagram of vector 3949 (H1 A-Hond-17734-16 (L429M)) is shown. Figure 6BB A schematic diagram of vector 3946 (H1 A-Hond-17734-16 (F390D+L429M)) is shown. Figure 6CC A schematic diagram of vector 3951 (H1 A-Hond-17734-16 (N97D+F390D+L429M)) is shown. Figure 6DDA schematic diagram of vector 3984 (H1 A-Darw-11-15) is shown. Figure 6EE A schematic diagram of vector 3990 (H1 A-Darw-11-15(N97D)) is shown. Figure 6FF A schematic diagram of vector 3988 (H1A-Darw-11-15 (K374E)) is shown. Figure 6GG A schematic diagram of vector 3985 (H1A-Darw-11-15 (F390D)) is shown. Figure 6HH A schematic diagram of vector 3989 (H1A-Darw-11-15 (L429M)) is shown. Figure 6II A schematic diagram of vector 3986 (H1A-Darw-11-15 (F390D+L429M)) is shown. Figure 6JJ A schematic diagram of vector 3991 (H1A-Darw-11-15 (N97D+F390D+L429M)) is shown. Figure 6KK A schematic diagram of vector 3644 (H1 A-Mich-45-2015 (N380A)) is shown. Figure 6LL A schematic diagram of vector 3704 (H1A-Mich-45-2015 (F390D+N380A)) is shown. Figure 6MM A schematic diagram of vector 4765 (A / Paris / 1227 / 17 (F390D+L429M)) is shown. Figure 6NN A schematic diagram of vector 4766 (A / Paris / 1227 / 17 (K374E+F390D+L429M)) is shown. Figure 6OO A schematic diagram of vector 4767 (A / Paris / 1227 / 17 (N97D+F390D+L429M)) is shown. Figure 6PP A schematic diagram of vector 4768 (A / Paris / 1227 / 17 (N97D+K374E+F390D+L429M)) is shown. Figure 6QQ A schematic diagram of vector 4775 (A / Norway / 2147 / 17 (F390D+L429M)) is shown. Figure 6RR A schematic diagram of vector 4776 (A / Norway / 2147 / 17 (K374E+F390D+L429M)) is shown. Figure 6SS A schematic diagram of vector 4777 (A / Norway / 2147 / 17 (N97D+F390D+L429M)) is shown. Figure 6TT A schematic diagram of vector 4778 (A / Norway / 2147 / 17 (N97D+K374E+F390D+L429M)) is shown.

[0065] Figure 7AA schematic diagram of vector 2295 (H5 A-Indo-5-05) is shown. Figure 7B A schematic diagram of vector 3680 (H5 A-Indo-5-05 (F393D)) is shown. Figure 7C A schematic diagram of vector 3645 (H5 A-Egypt-N04915-14) is shown. Figure 7D A schematic diagram of vector 3690 (H5 A-Egypt-N04915-14(F392D)) is shown.

[0066] Figure 8 A schematic diagram of vector 1190 (a vector used for fusion cloning into a CPMV 160-based expression cassette) is shown. DETAILED DESCRIPTION

[0067] A preferred embodiment is described below.

[0068] As used herein, the terms "comprising," "having," "including," and "containing," and their grammatical variations, are inclusive or open-ended and do not exclude additional unrecited elements and / or method steps. When used herein in connection with a product, use, or method, the term "consisting essentially of" indicates that additional elements and / or method steps may be present, but that these added items do not materially affect the mode of operation of the method or use. When used herein in connection with a product, use, or method, the term "consisting of" excludes the presence of additional elements and / or method steps. Products, uses, or methods described herein that include certain elements and / or steps may also consist essentially of these elements and / or steps in some embodiments, and consist of these elements and / or steps in other embodiments, regardless of whether these embodiments are specifically mentioned. Furthermore, unless otherwise indicated, the use of the singular includes the plural, and "or" means "and / or." Unless otherwise defined herein, all technical and scientific terms used herein should be understood to have the meanings commonly understood by those of ordinary skill in the art. As used herein, the term "approximately" means approximately + / - 10% of a given value. It should be understood that such differences are always included in any given value provided herein, whether or not specifically mentioned. When used in conjunction with the term "comprising" herein, the use of the word "a" or "an" can mean "one", but also conforms to the meaning of "one or more", "at least one", and "one or more than one".

[0069] The terms "plant," "plant part," "plant part," "plant matter," "plant biomass," "plant material," "plant extract," or "plant leaf" as used herein may include whole plants, tissues, cells, or any part thereof, intracellular plant components, extracellular plant components, liquid or solid extracts of plants, or combinations thereof, that can provide transcriptional, translational, and post-translational machinery for expression of one or more nucleic acids described herein and / or from which expressed proteins or VLPs can be extracted and purified. Plants may include, but are not limited to, herbaceous plants. Additionally, plants may include, but are not limited to, crops, including rapeseed, Brassica plants, corn, Nicotiana plants (tobacco), such as Nicotiana benthamiana, Nicotiana vulgaris, Nicotiana tabacum, Nicotiana safflower, Nicotiana solanaceae, Arabidopsis thaliana, alfalfa, potato, sweet potato (Ipomoea batatus), ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton, corn, rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), safflower (Carthamus tinctorius), and the like.

[0070] As used herein, the term "plant part" refers to any part of a plant, including but not limited to leaves, stems, roots, flowers, fruits, plant cells obtained from leaves, stems, roots, flowers, fruits, plant extracts obtained from leaves, stems, roots, flowers, fruits, or combinations thereof. As used herein, the term "plant extract" refers to the plant-derived products obtained after physical treatment (such as freezing, then extracting in a suitable buffer), mechanical treatment (such as grinding or homogenizing plants or plant parts, then extracting in a suitable buffer), enzyme treatment (such as using cell wall degrading enzymes), chemical treatment (such as using one or more chelating agents or buffers), or a combination thereof. Plant extracts can also be processed to remove unwanted plant components, such as cell wall fragments. Plant extracts can be obtained to help reclaim one or more components from plants, plant parts, or plant cells, such as proteins (including protein complexes, protein superstructures, and / or VLPs), nucleic acids, lipids, carbohydrates, or combinations thereof, from plants, plant parts, or plant cells. If the plant extract comprises protein, it can be referred to as a protein extract. Protein extract can be a plant or protein extract from a crude plant extract of plant tissue, a partially purified protein, or a purified product that comprises one or more proteins, protein complex, protein superstructure and / or VLP. If desired, can use technology well known to those skilled in the art that protein extract or plant extract are partially purified, for example, can extract be carried out salt or pH precipitation, centrifugal, gradient density centrifugation, filtration, chromatography, for example size exclusion chromatography, ion exchange chromatography, affinity chromatography or their combination. Also can use technology well known to those skilled in the art to come purified protein extract.

[0071] As used herein, the terms "construct," "vector," or "expression vector" refer to a recombinant nucleic acid that is used to transfer an exogenous nucleic acid sequence into a host cell (e.g., a plant cell) and direct the expression of the exogenous nucleic acid sequence in the host cell. An "expression cassette" refers to a nucleotide sequence comprising a nucleic acid of interest that is controlled by, and operably linked to, an appropriate promoter or other regulatory element responsible for the transcription of the nucleic acid of interest in the host cell. It will be understood by those skilled in the art that the expression cassette may include a termination (terminator) sequence, which is any sequence that is active in a plant host. For example, the termination sequence may be derived from the RNA-2 genomic segment of a bipartite RNA virus (e.g., cowpea mosaic virus), the termination sequence may be the NOS terminator, or the terminator sequence may be obtained from the 3'UTR of the alfalfa plastocyanin gene.

[0072] The constructs disclosed herein may also include a 3' non-translational domain (UTR). The 3' non-translational domain comprises a polyadenylation signal and any other regulatory signal that can affect mRNA processing or gene expression. The polyadenylation signal is typically characterized by the addition of a polyadenylic acid track at the 3' end of the mRNA precursor. Polyadenylation signals are typically identified by homology to the standard form 5'AATAAA-3', although variations are not uncommon. Non-limiting examples of suitable 3' domains include a 3' transcribed non-translational domain comprising a polyadenylation signal from Agrobacterium tumor-inducing (Ti) plasmid genes, such as nopaline synthase (Nos gene), and plant genes, such as soybean storage protein genes, the small subunit of the ribulose-1,5-bisphosphate carboxylase gene (ssRUBISCO; US4,962,028; incorporated herein by reference), a promoter for regulating plastocyanin expression.

[0073] "Regulatory domain", "regulatory element" or "promoter" refers to a nucleic acid portion that is usually, but not always, upstream of the protein coding region of a gene, which can be composed of DNA or RNA, or of both DNA and RNA. When the regulatory domain is active and operably associated with or operably connected to the nucleotide sequence of interest, this may result in the expression of the nucleotide sequence of interest. Regulatory elements can regulate organ specificity, or control developmental or temporal gene activation. "Regulatory domain" includes promoter elements, core promoter elements that exhibit basal promoter activity, elements that can be induced in response to external stimuli, and elements that regulate promoter activity, such as negative regulatory elements or transcription enhancers. "Regulatory domain" as used herein also includes elements that are active after transcription, such as regulatory elements that regulate gene expression, such as translation and transcription enhancers, translation and transcription repressors, upstream activation sequences, and mRNA instability determinants. The latter several elements can be located near the coding region.

[0074] In the context of the present disclosure, the term "regulatory element" or "regulatory domain" generally refers to a DNA sequence that is usually, but not always, upstream (5') of the coding sequence of a structural gene and that controls the expression of the coding region by enabling recognition of RNA polymerase and / or other factors required to initiate transcription at a specific site. However, it should be understood that other nucleotide sequences located within the intragenic region or at the 3' end of the sequence may also contribute to regulating the expression of the coding region of interest. An example of a regulatory element that enables recognition of RNA polymerase or other transcription factors to ensure initiation at a specific site is a promoter element. Most (but not all) eukaryotic promoter elements contain a TATA box, which is a conserved nucleic acid sequence consisting of adenosine and thymidine nucleotide base pairs that are typically located approximately 25 base pairs upstream of the transcription start site. Promoter elements can include a basal promoter element responsible for initiating transcription, as well as other regulatory elements that modify gene expression.

[0075] There are many types of regulatory domains, including developmentally regulated, inducible, or constitutive regulatory domains. A regulatory domain that is developmentally regulated or controls the differential expression of genes under its control is activated in a particular organ or tissue at a specific time during the development of that organ or tissue. However, some developmentally regulated regulatory domains may become preferentially active in certain organs or tissues at a specific developmental stage, may become active in a developmentally regulated manner, or may maintain a basal level of activity in other organs or tissues within the plant. Examples of tissue-specific regulatory domains (e.g., seed-specific regulatory domains) include the Napin promoter and the Cruciferae promoter (Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau et al., 1994, Plant Cell 14: 125-130). An example of a leaf-specific promoter includes the plastocyanin promoter (see US Pat. No. 7,125,978, which is incorporated herein by reference).

[0076] Inducible regulatory domain is a kind of domain that can directly or indirectly activate the transcription of one or more DNA sequences or genes in response to an inducer.In the absence of an inducer, the DNA sequence or gene will not be transcribed.Usually, the protein factor that specifically binds to the inducible regulatory domain to activate transcription can exist in an inactive form, and then the inducer is directly or indirectly converted into an active form.But, this protein factor may not exist.The inducer can be a chemical reagent, such as protein, metabolite, growth regulator, herbicide or phenolic compound, or is directly applied by heat, cold, salt or toxic elements or indirectly applied by the physiological stress of pathogen or disease factor (such as virus).By applying inducer (such as by spraying, watering, heating or similar methods) to cells or plants outside, the plant cell comprising the inducible regulatory domain can be exposed to the inducer.Inducible regulatory elements can be derived from plants or non-plant genes (such as Gatz, C. and Lenk, IRP, 1998, Trends Plant Sci.3, 352-358). Examples of potential inducible promoters include, but are not limited to, tetracycline-inducible promoters (Gatz, C., 1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 89-108), steroid-inducible promoters (Aoyama, T. and Chua, NH, 1997, Plant J. 2, 397-404), and ethanol-inducible promoters (Salter, MG et al., 1998, Plant Journal 16, 127-132; Caddick, MX et al., 1998, Nature Biotech. 16, 177-180), cytokinin-inducible IB6 and CKI1 genes (Brandstatter, I. and Kieber, JJ, 1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274, 982-985), and the auxin-inducible element DR5 (Ulmasov, T. et al., 1997, Plant Cell 9, 1963-1971).

[0077] Constitutive regulatory domains direct gene expression in different parts of the plant and maintain continuous expression during plant development. Examples of known constitutive regulatory elements include the promoter associated with the CaMV 35S transcript (p35S; Odell et al., 1985, Nature, 313:810-812; incorporated herein by reference), rice actin 1 (Zhang et al., 1991, Plant Cell, 3:1155-1165), actin 2 (An et al., 1996, Plant J., 10:107-121), or tms2 (U.S. Pat. No. 5,428,147), as well as the triosephosphate isomerase 1 (Xu et al., 1994, Plant Physiol. 106:459-467) gene, the maize ubiquitin 1 gene (Cornejo et al., 1993, Plant Mol. Biol. 29:637-646), the Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al., 1995, Plant J. Biol. 29:637-646), and the Arabidopsis thaliana ubiquitin 1 and 6 genes (Holtorf et al., 1995, Plant J. Biol. 29:637-646). Mol. Biol. 29: 637-646), tobacco translation initiation factor 4A gene (Mandel et al., 1995 Plant Mol. Biol. 29: 995-1004), cassava vein mosaic virus promoter pCAS (Verdaguer et al., 1996); ribulose bisphosphate carboxylase small subunit promoter pRBCs (Outhkourov et al., 2003), pUbi (for monocots and dicots).

[0078] As used herein, the term "constitutive" does not necessarily mean that a nucleotide sequence under the control of a constitutive regulatory domain is expressed at the same level in all cell types, but rather that the sequence is expressed in many cell types, even if variations in abundance are often observed.

[0079] The expression constructs described above may be present in a vector. The vector may include border sequences that allow the expression cassette to be transferred and integrated into the genome of an organism or host. The construct may be a plant binary vector, such as a binary transformation vector based on pPZP (Hajdukiewicz et al., 1994). Other exemplary constructs include pBin19 (see Frisch, DA, LW Harris-Haller et al., 1995, Plant Molecular Biology 27:405-409).

[0080] As used herein, the terms "native," "native protein," or "native domain" refer to a protein or domain having the same primary amino acid sequence as a wild-type protein. A native protein or domain can be encoded by a nucleotide sequence that has 100% sequence similarity to the wild-type sequence. A native amino acid sequence can also be encoded by a human codon (hCoD)-optimized nucleotide sequence or a nucleotide sequence that includes increased GC content compared to the wild-type nucleotide sequence, provided that the amino acid sequence encoded by the hCoD-nucleotide sequence exhibits 100% sequence identity to the native amino acid sequence.

[0081] A "human codon-optimized" nucleotide sequence or "hCod" nucleotide sequence refers to a sequence in which appropriate DNA nucleotides are selected so that the synthesis pattern of an oligonucleotide sequence or fragment thereof approximates the codon usage pattern commonly found in oligonucleotide sequences of human nucleotide sequences. "Increased GC content" refers to the selection of appropriate DNA nucleotides for the synthesis of an oligonucleotide sequence or fragment thereof to approximate a codon usage pattern that includes an increased GC content (e.g., from about 1% to about 30%, or any amount therebetween) over the length of the coding portion of the oligonucleotide sequence compared to the corresponding native oligonucleotide sequence. For example, the GC content is about 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30%, or any amount therebetween, over the length of the coding portion of the oligonucleotide sequence. As described below, a human codon-optimized nucleotide sequence or a nucleotide sequence that includes an increased GC content (compared to a wild-type nucleotide sequence) exhibits enhanced expression in a plant, plant part, or plant cell compared to expression of a non-human optimized (or lower GC content) nucleotide sequence.

[0082] Described herein are modified influenza virus hemagglutinin (HA) proteins (also referred to as modified HA proteins, modified influenza virus HA proteins, modified HA, modified influenza virus HA, mutant HA, influenza virus mutant HA, influenza virus HA variants, or HA variants) and methods for producing modified influenza virus hemagglutinin proteins in plants. The modified influenza virus hemagglutinin proteins disclosed herein include modifications or mutations that have been found to result in improved hemagglutinin properties compared to wild-type or unmodified hemagglutinin proteins. For example, the modified influenza virus hemagglutinin protein can have an amino acid sequence with at least one amino acid substitution compared to the corresponding wild-type amino acid sequence.

[0083] Examples of improved properties of the modified hemagglutinin protein include: increased production of the hemagglutinin protein when expressed in plant cells, compared to a wild-type or unmodified hemagglutinin of the same influenza strain or subtype that does not comprise the modification or mutation; increased hemagglutination titer of the modified hemagglutinin protein compared to a wild-type or unmodified hemagglutinin protein; improved integrity, stability, or both of the integrity and stability of VLPs comprising the modified hemagglutinin protein, compared to the integrity or stability, or both of the integrity and stability, of VLPs comprising the modified hemagglutinin protein; increased production of VLPs when expressed in plant cells, compared to the production of wild-type VLPs that do not comprise the modification or mutation; and combinations of these advantages.

[0084] Influenza virus subtypes and strains

[0085] As used herein, the term "influenza virus subtype" refers to influenza A virus variants characterized by various combinations of hemagglutinin (H or HA) and neuraminidase (N) viral surface proteins. According to the present specification, influenza virus subtypes and hemagglutinin (HA) from such virus subtypes may be referred to by their H numbers, for example, "HA of the H1 subtype," "H1 HA," or "H1 influenza virus." The term "subtype" specifically includes all individual "strains" within each subtype, which are typically caused by mutations and may exhibit different pathogenic characteristics. Such strains may also be referred to as various "isolates" of the virus subtype. Therefore, the terms "strain" and "isolate" used herein may be used interchangeably.

[0086] Traditionally, different influenza strains are classified, for example, according to the ability of the influenza virus to agglutinate red blood cells. Specific antibodies to a particular influenza virus strain can bind to the virus, thereby preventing this agglutination. The test for determining the strain type based on this inhibitory effect is commonly referred to as the hemagglutinin inhibition test (HI test or HAI test), and is a well-known standard method for characterizing influenza strains in the art.

[0087] However, hemagglutinin proteins from different strains also exhibit significant sequence similarity at the nucleic acid and amino acid levels. When comparing strains of different subtypes, this level of similarity varies, with some strains exhibiting significantly higher levels of similarity than others (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643). The level of amino acid similarity also varies between strains of different subtypes (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643). This variation is sufficient to define individual subtypes and lineages of different strains, but the DNA and amino acid sequences of different strains are still readily aligned using conventional bioinformatics techniques (Air, Proc. Natl. Acad. Sci. USA, 1981, 78:7643; Suzuki and Nei, Mol. Biol. Evol. 2002, 19:501).

[0088] Multiple nucleotide sequences or corresponding hemagglutinin polypeptide sequences can be aligned to determine a "common portion" or "consensus sequence" of a subtype (see Figure 1 ).

[0089] Based on sequence similarity, influenza virus subtypes can be further classified by reference to their phylogenetic groups. Phylogenetic analysis (Fouchier et al., J Virol. 2005 Mar; 79(5): 2814-22) showed that hemagglutinins can be subdivided into two major groups (Air, Proc. Natl. Acad. Sci. USA, 1981, 78: 7643): i.e., H1, H2, H5, and H9 subtypes in phylogenetic group 1, and H3, H4, and H7 subtypes in phylogenetic group 2.

[0090] Novel influenza virus hemagglutinin proteins, hemagglutinin modifications, hemagglutinin protein variants and mutants are generated by altering the amino acid sequence of the hemagglutinin protein, which alterations result in improved hemagglutinin properties as described above. The isolation of nucleic acids encoding such hemagglutinin molecules is routine, as is the modification of the nucleic acids to introduce changes in the amino acid sequence, for example, by site-directed mutagenesis.

[0091] Described herein are modified influenza virus hemagglutinin proteins and methods for producing modified influenza virus hemagglutinin proteins in plants. Modifications, such as by substitution of specific amino acids in a hemagglutinin protein (e.g., a hemagglutinin from the H1 subtype), have been observed to result in improved properties of the modified hemagglutinin protein compared to wild-type or unmodified hemagglutinin proteins.

[0092] The one or more modifications, mutations or substitutions of the hemagglutinin protein described herein are not located in a known epitope region of the hemagglutinin protein, nor do these modifications, mutations or substitutions add or remove glycosylation sites within the hemagglutinin protein.

[0093] The hemagglutinin protein, mutant hemagglutinin protein or modified hemagglutinin protein described herein is modified and has an amino acid sequence that is different from that of A / Michigan / 45 / 15 hemagglutinin (SEQ ID NO: 134; see Figure 1 ) or A / California / 07 / 09 hemagglutinin (SEQ ID NO: 130; see Figure 1 ) comprises one or more mutations, modifications or substitutions at any one or more amino acids corresponding to the amino acid at position 97, 374, 380, 390 or 429 of the present invention.

[0094] "Corresponding to an amino acid" or "corresponding to an amino acid" means that one amino acid corresponds to another amino acid in a sequence alignment with a reference strain of influenza virus as described below.

[0095] The amino acid residue numbers or residue positions of the hemagglutinin are consistent with the numbering of the hemagglutinin of a reference influenza virus strain. For example, in the case of H1 influenza virus, the reference strain may be the A / Michigan / 45 / 15 hemagglutinin (SEQ ID NO: 134; see Figure 1 ) or A / California / 07 / 09 hemagglutinin (SEQ ID NO: 130; see Figure 1 The corresponding amino acid positions can be determined by aligning the sequence of a hemagglutinin (e.g., H1 hemagglutinin) with the sequence of a hemagglutinin of a corresponding reference strain. Sequence alignment methods for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Park, Madison, Wisconsin, USA), or by manual alignment and visual inspection (see, for example, Ausubel et al., Molecular Biology: A Laboratory Manual, 1995 Supplement). Figure 1The amino acid sequence alignments of multiple influenza A virus hemagglutinin domains are shown in , and these examples should not be considered limiting.

[0096] When referring to a modification, mutant, or variant, the wild-type amino acid residue (also referred to simply as "amino acid") is followed by the residue number and the new or substituted amino acid. For example, an aspartic acid (D, Asp) substitution residue or asparagine (N, Asn) at position 97 in the amino acid sequence is designated N97D (see Table 1).

[0097] Modified hemagglutinins, hemagglutinin mutants or variants (e.g., modified H1 hemagglutinins) are named in the same manner using the single-letter amino acid code for the wild-type residue followed by the single-letter amino acid code for its position and the substituted residue. Multiple mutants are represented by the individual component single mutants separated by a slash ( / ) or a plus sign (+). Thus, for example, the H1 hemagglutinin mutant N380A / L429M is a double substitution mutant in which alanine (A, Ala) replaces asparagine (N, Asp) at residue position 380 and methionine (M, Met) replaces leucine (L, Leu) at residue position 429; the H1 hemagglutinin mutant protein N380A / F390D is a double substitution variant in which alanine (A, Ala) replaces asparagine (N, Asn) at position 380 in the H1 hemagglutinin and aspartic acid (D, Asp) replaces phenylalanine (F, Phe) at position 429.

[0098] Table 1. Positions of modifications in hemagglutinin and corresponding amino acid / residue positions in H1 and H5 influenza reference strains. Exemplary modifications are shown in brackets.

[0099]

[0100] 1 A / Michigan / 45 / 15 or A / California / 07 / 09

[0101] 2 A / Indonesia / 05 / 05

[0102] The modified influenza virus hemagglutinin (HA) protein may include an amino acid sequence having at least one amino acid substitution compared to the corresponding wild-type amino acid sequence.

[0103] "Amino acid substitution" or "substitution" refers to the replacement of an amino acid in a protein's amino acid sequence with a different amino acid. The terms "amino acid," "amino acid residue," or "residue" are used interchangeably in this disclosure. One or more amino acids can be replaced with one or more amino acids that differ from the original or wild-type amino acid at that position without changing the overall length of the protein's amino acid sequence. Such substitutions or replacements can be experimentally induced by changing the codon sequence encoding the protein in the nucleotide sequence to a codon sequence encoding an amino acid that differs from the original or wild-type amino acid. The resulting protein is a modified protein, such as a modified influenza virus hemagglutinin protein. Modified influenza hemagglutinin proteins do not occur naturally.

[0104] Modified hemagglutinins include non-naturally occurring hemagglutinin proteins having at least one modification to a naturally occurring hemagglutinin and having improved properties compared to the naturally occurring hemagglutinin protein from which the amino acid sequence of the modified hemagglutinin is derived. The modified hemagglutinin protein has an amino acid sequence not found in nature, which is derived by replacing one or more amino acid residues of the hemagglutinin protein with one or more different amino acids.

[0105] Thus, a modified hemagglutinin, mutant hemagglutinin, or recombinant hemagglutinin refers to a hemagglutinin in which the DNA sequence encoding the naturally occurring hemagglutinin has been modified to produce a modified or mutated DNA sequence encoding a modification, mutation, or substitution of one or more amino acids in the hemagglutinin amino acid sequence.

[0106] Some residues identified for modification, mutation, or substitution correspond to conserved residues, while others do not. In the case of non-conserved residues, substitution of one or more amino acids is limited to substitutions that result in a modified hemagglutinin having an amino acid sequence that does not correspond to an amino acid sequence found in nature. In the case of conserved residues, such modification, substitution, or replacement should also not result in a naturally occurring hemagglutinin sequence.

[0107] Conservative substitution

[0108] As described herein, residues in the hemagglutinin protein can be identified and modified, substituted, or mutated to produce modified hemagglutinin proteins or hemagglutinin protein variants. Substitutions or mutations at specific positions are not limited to the amino acid substitutions described herein or given in the examples. For example, a hemagglutinin variant may comprise conservative substitutions of the amino acid substitutions described.

[0109] As used herein, the term "conservative substitution" and its grammatical variations refer to the presence of an amino acid residue in the sequence of the hemagglutinin protein that is different from the substitution or residue but belongs to the same class of amino acids (i.e., a non-polar residue replacing a non-polar residue, an aromatic residue replacing an aromatic residue, a polar uncharged residue replacing a polar uncharged residue, a charged residue replacing a charged residue). In addition, conservative substitutions can encompass residues having an interfacial hydrophilicity value of the same sign and approximately similar magnitude as the residue replacing the wild-type residue.

[0110] As used herein, the term "non-polar residue" refers to glycine (G, Gly), alanine (A, Ala), valine (V, Val), leucine (L, Leu), isoleucine (I, Ile) and proline (P, Pro); the term "aromatic residue" refers to phenylalanine (F, Phe), tyrosine (Y, Tyr) and tryptophan (W, Trp); the term "polar uncharged residue" refers to serine (S, Ser), threonine (T, Thr), cysteine ​​(C, Cys), methionine (M, Met), asparagine (N, Asn) and glutamine (Q, Gln); the term "charged residue" refers to the negatively charged amino acids aspartic acid (D, Asp) and glutamic acid (E, Glu), and the positively charged amino acids lysine (K, Lys), arginine (R, Arg) and histidine (H, His). Other classifications of amino acids are as follows:

[0111] Amino acids with hydrophobic side chains (aliphatic): alanine (A, Ala), isoleucine (I, Ile), leucine (L, Leu), methionine (M, Met), and valine (V, Val);

[0112] Amino acids with hydrophobic side chains (aromatic): phenylalanine (F, Phe), tryptophan (W, Trp), tyrosine (Y, Tyr);

[0113] Amino acids with polar neutral side chains: asparagine (N, Asn), cysteine ​​(C, Cys), glutamine (Q, Gln), serine (S, Ser), and threonine (T, Thr);

[0114] Amino acids with charged side chains (acidic): aspartic acid (D, Asp), glutamic acid (E, Glu);

[0115] Amino acids with charged side chains (basic): arginine (R, Arg); histidine (H, His); lysine (K, Lys), glycine (G, Gly) and proline (P, Pro).

[0116] Conservative amino acid substitutions may have a similar effect on the activity of the resulting hemagglutinin protein variant or modified hemagglutinin protein as the original substitution or modification. More information on conservative substitutions can be found, for example, in papers by Ben Bassat et al. (J. Bacteriol, 169: 751-757, 1987), O'Regan et al. (Gene, 77: 237-251, 1989), Sahin-Toth et al. (Protein ScL, 3: 240-247, 1994), Hochuli et al. (Bio / Technology, 6: 1321-1325, 1988), and in widely used genetics and molecular biology textbooks.

[0117] The Blosum matrix is ​​often used to determine the relatedness of polypeptide sequences. The Blosum matrix was created using a large, trusted database of alignments (the BLOCKS database), which takes into account alignments of paired sequences whose relatedness is below a certain identity threshold percentage (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992). For highly conserved target frequencies for the BLOSUM90 matrix, a 90% identity threshold was used. For the BLOSUM65 matrix, a 65% identity threshold was used. Scores above zero in the Blosum matrix are considered "conservative substitutions" at the selected percentage identity. Table 2 below shows exemplary conservative amino acid substitutions.

[0118] Table 2. Exemplary conservative amino acid substitutions

[0119]

[0120]

[0121] The nucleotide sequence encoding the modified hemagglutinin protein can be optimized for human codon usage, increased GC content, or a combination thereof.The modified hemagglutinin protein can be expressed in a plant, plant part, or plant cell.

[0122] Modification of H1 hemagglutinin

[0123] Described herein is a modified H1 influenza virus hemagglutinin protein and a method for producing the modified H1 influenza virus hemagglutinin protein in plants. It has been observed that modification of specific amino acids in a hemagglutinin protein from an H1 subtype results in improved properties of the modified H1 hemagglutinin protein compared to wild-type H1 hemagglutinin protein or unmodified H1 hemagglutinin protein.

[0124] A total of 42 single, double, and / or triple modifications were tested to improve the properties of the H1 hemagglutinin protein. As described herein and shown in the examples, only modifications at specific positions or combinations of modifications improved the properties of the H1 hemagglutinin protein. Modifications at 32 positions or combinations of these positions had a negative effect on the properties of the H1 hemagglutinin protein (data not shown).

[0125] Examples of improved properties of the H1 hemagglutinin mutant proteins include: increased production or accumulation of the hemagglutinin protein when expressed in plant cells, compared to wild-type or unmodified H1 hemagglutinin of the same influenza strain or subtype that does not comprise the modification or mutation; increased hemagglutination titer of the modified or mutated hemagglutinin protein compared to wild-type or unmodified H1 hemagglutinin protein; improved integrity, stability, or both of the integrity and stability of VLPs comprising the modified H1 hemagglutinin protein, compared to the integrity or stability, or both of the integrity and stability, of VLPs comprising the wild-type hemagglutinin that does not comprise the mutation; increased yield of VLPs when expressed in plant cells, compared to the yield of wild-type VLPs that do not comprise the modification or mutation; and combinations of these advantages.

[0126] The modified H1 hemagglutinin protein or mutant H1 hemagglutinin protein described herein is modified and is used in conjunction with A / California / 07 / 09 hemagglutinin (SEQ ID NO: 130; see Figure 1 ) comprises one or more mutations or modifications at any one or more residues in the sequence opposite positions 97, 374, 380, 390 and / or 429. Thus, an H1 influenza virus hemagglutinin polypeptide, protein and / or protein complex, such as a virus-like particle (VLP), is provided, the VLP comprising a modification or mutation at one or more of amino acid positions 97, 374, 380, 390 and / or 429 (such amino acid numbering is based on the example of FIG. 1 ). Figure 1 The sequence of the A / California / 07 / 09 hemagglutinin shown in FIG130 (SEQ ID NO: 130) may include modifications or mutations at amino acid positions corresponding to such amino acid positions, as determined, for example, by alignment of the H1 hemagglutinin amino acid sequence with SEQ ID NO: 130. Non-limiting examples of H1 influenza virus hemagglutinin amino acid sequences that include one or more such mutations include SEQ ID NOs: 131, 132, 133, 134, 135, 138, and 139.

[0127] The modified H1 hemagglutinin proteins described herein include H1 hemagglutinin proteins having an amino acid sequence that has about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any amount in between, sequence identity or sequence similarity to an amino acid sequence encoding a hemagglutinin from H1 (SEQ ID NOs: 130, 131, 132, 133, 134, 135, 138, or 139), wherein the amino acid sequence has one or more mutations or modifications at any one or more residues aligned with positions 97, 374, 380, 390, and 429 of A / California / 07 / 09 hemagglutinin (SEQ ID NO: 130), and wherein the hemagglutinin protein forms VLPs when expressed.

[0128] Additionally, the H1 hemagglutinin protein can be encoded by a nucleotide sequence having about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any amount in between, sequence identity or sequence similarity to a nucleotide sequence encoding a hemagglutinin from H1 (SEQ ID NOs: 130, 131, 132, 133, 134, 135, 138, or 139), wherein the H1 amino acid sequence has one or more mutations or modifications at any one or more residues sequenced opposite positions 97, 374, 380, 390, and 429 of the A / California / 07 / 09 hemagglutinin, and wherein the nucleotide sequence encodes a hemagglutinin protein that forms VLPs when expressed.

[0129] Non-limiting examples of strains from which H1 hemagglutinin can be derived include A / California / 07 / 09 (H1N1, SEQ ID NO: 130), A / Michigan / 45 / 15 (H1N1, SEQ ID NO: 134), A / Massachusetts / 06 / 17 (H1N1, SEQ ID NO: 135), A / Costa Rica / 0513 / 16 (H1N1, SEQ ID NO: 133), A / Honduras / 17734 / 16 (H1N1, SEQ ID NO: 131), A / Darwin / 11 / 15 (H1N1, SEQ ID NO: 132), A / Paris / 1227 / 2017 (SEQ ID NO: 138), or A / Norway / 2147 / 2017 (SEQ ID NO: 139).

[0130] The modified or mutant H1 hemagglutinin can be monosubstituted, disubstituted, trisubstituted, or tetrasubstituted at residues at positions 97, 374, 380, 390, or 429. In a monosubstituted H1 hemagglutinin, one residue can be mutated at position 97, 374, 380, 390, or 429. In a disubstituted H1 hemagglutinin, two residues can be substituted, for example, residues at positions 380 and 429, 97 and 374, or 390 and 429. In a trisubstituted H1 hemagglutinin mutant, three residues can be substituted. For example, residues at positions 97, 390, and 429, or residues at positions 97, 374, and 429 can be substituted. In a tetrasubstituted H1 hemagglutinin mutant, four residues can be substituted. For example, residues at positions 97, 374, 390, and 429 can be substituted. (All H1 hemagglutinin numbering is based on sequence alignment with the reference strain A / California / 07 / 09 hemagglutinin).

[0131] Non-limiting examples of modified H1 hemagglutinin proteins include the following modifications or mutations in the hemagglutinin sequence compared to the H1 hemagglutinin wild-type sequence (numbering according to A / California / 07 / 09 hemagglutinin):

[0132] Single-substituted H1 hemagglutinin mutants: N97D, K374E, F390D, or L429M;

[0133] Double-substituted H1 hemagglutinin mutants: N390A / L429M, N97D / K374E, or N380 / L429M;

[0134] Triple-substituted H1 hemagglutinin mutants: N97D / F390D / L429M or K374E / F390D / L429M;

[0135] • Tetrasubstituted H1 hemagglutinin mutant: N97D / K374E / F390D / L429M.

[0136] One or more mutations described herein specifically increase the yield and VLP yield of influenza virus hemagglutinin protein in plants. It has been observed that mutations at other positions significantly reduce the accumulation amount or VLP yield of influenza virus hemagglutinin protein in plant cells, or have no significant effect on it.

[0137] Monosubstituted H1 hemagglutinin

[0138] Modification at position 97

[0139] In one aspect of the present disclosure, the modified H1 hemagglutinin may have a modified residue at least at position 97. This residue is not involved in hemagglutinin receptor binding and has been shown to be located within one of the residual esterase (VE) subdomains of the globular head of hemagglutinin. X-ray crystallographic analysis has shown that this residue is buried within the hemagglutinin trimer. Therefore, this residue is not part of the antigenic site, does not participate in antigenic variation, and is not recognized by broadly neutralizing antibodies.

[0140] In influenza A(H1N1)pdm09 virus, this residue was predicted to be involved in the stability of the hemagglutinin (see Castelán-Vega et al., 2014). However, Castelán-Vega et al. noted that single point mutations appear to have little effect on the stability of the hemagglutinin and cited a paper by Yang et al. (Structural stability of influenza A(H1N1)pdm09 virus hemagglutinin, J. Virol. 2014;88(9):4828-4838). Yang et al. used size exclusion chromatography analysis of the recombinant hemagglutinin ectodomain to compare the differences between the recombinant trimeric hemagglutinin protein of the early 2009 H1N1 virus (which can be broken down into monomers) and the recombinant trimeric hemagglutinin protein of the more recent virus (which can be expressed as a trimer). Yang et al. found that A / Texas / 1 / 2011 (Tex 11) had a unique Asp97Asn (D97N) substitution in the hemagglutinin compared to four other A(H1N1)pdm09 strains studied for sequence differences. However, H1 hemagglutinin influenza strains that have evolved since then have asparagine (N, Asn) at position 97 (see Figure 1 , H1 sequence alignment), which suggests that H1 hemagglutinin strains have an evolutionary advantage of asparagine (N, Asn) at this position. Therefore, it is unexpected that the modification of asparagine (N, Asn) at position 97 to a non-asparagine residue results in the improved properties of the H1 hemagglutinin protein described herein.

[0141] like Figure 3A 、 3B As shown in Figures 4A and 4B, the H1 hemagglutinin having a residue at position 97 changed from, for example, asparagine (N, Asn) to aspartic acid (AspD) (hereinafter referred to as N97D) showed an increase in hemagglutination titer of up to 1200% compared to the H1 hemagglutinin having asparagine (N, Asn) at this position (see Table 5A). Figure 3AIt was shown that the hemagglutinin from A / Michigan / 45 / 15 with the N97D substitution exhibited an approximately 1100% increase in hemagglutination titer compared to the A / Michigan / 45 / 15 HA wild type (also known as H1 Michigan). The hemagglutinin from A / Honduras / 17734 / 16 with the N97D substitution exhibited an approximately 375% increase in hemagglutination titer compared to the wild type A / Honduras / 17734 / 16 hemagglutinin (see Figure 4A and 4B ). In addition, if Figure 4A and 4B As shown, the hemagglutination titer of the hemagglutinin from A / Darwin / 11 / 15 carrying the N97D substitution was increased by approximately 300% compared to the wild-type A / Darwin / 11 / 15 hemagglutinin.

[0142] Thus, in one aspect, the residue at position 97 of the H1 hemagglutinin (numbered according to the A / California / 07 / 09 hemagglutinin numbering) can be modified to replace the charged amino acid with a polar amino acid at position 97, thereby generating a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin protein can be modified to include aspartic acid (D, Asp) or any other polar amino acid, such as glutamine (Q, Gln), histidine (H, His), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine ​​(C, Cys), or tryptophan (W, Trp), at position 97.

[0143] The H1 hemagglutinin can be modified to replace the asparagine at position 97 with a non-asparagine at position 97. For example, the hemagglutinin protein can be mutated to include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) at position 97. Such conservative substitutions can be, for example, glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). In addition, the H1 hemagglutinin can be modified to replace a non-aspartic acid (D, Asp) at position 97 with an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn). Such conservative aspartic acid substitutions can be, for example, glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser).

[0144] For example, the modified H1 hemagglutinin protein can have an amino acid sequence 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 to the amino acid sequence of the hemagglutinin from H1 (SEQ ID NO: 134), wherein the amino acid sequence has an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) at position 97, such as glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms a VLP when expressed.

[0145] The present specification also provides a nucleic acid comprising a nucleotide sequence encoding the modified H1 hemagglutinin having a substitution at position 97 as described above and operably linked to a regulatory domain active in plants.

[0146] For example, the nucleotide sequence may have 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 to the nucleotide sequence encoding the hemagglutinin from H1 (SEQ ID NO: 134), wherein the nucleotide sequence encodes a modified H1 hemagglutinin protein having an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) at position 97 (e.g., glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser)), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms a VLP when expressed.

[0147] The nucleotide sequence may have 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 to the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 97 of the modified H1 hemagglutinin encodes aspartic acid (D, Asp) at position 97 or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn), such as glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser), and wherein the modified H1 hemagglutinin sequence does not occur in nature.

[0148] For example, the modified H1 hemagglutinin can have one or more modifications, wherein at least residue 97 of the H1 hemagglutinin is modified as described herein. For example, the modified H1 hemagglutinin can be a monosubstituted, disubstituted, trisubstituted, or tetrasubstituted H1 hemagglutinin, wherein at least the residue at position 97 is modified. In a non-limiting example, the modified H1 hemagglutinin can have a substituted residue at position 97 and one or more substituents at positions 374, 390, 429, or a combination thereof, wherein the modified H1 hemagglutinin sequence does not occur in nature.

[0149] In addition, a method of producing VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having a substitution at position 97 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having a substitution at position 97 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0150] In addition, a method for increasing the production of VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having a substitution at position 97 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding the modified H1 hemagglutinin having a substitution at position 97 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0151] The present disclosure also provides a VLP comprising an H1 hemagglutinin having a substitution at position 97. The VLP can be produced by the method provided by the present disclosure. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0152] Modification at position 374

[0153] In one aspect of the disclosure, the residue at position 374 in the H1 hemagglutinin (numbered according to A / California / 07 / 09 hemagglutinin numbering) can be substituted. This residue is located in the stem of the H1 hemagglutinin.

[0154] Cotter et al. (PLoS Pathog. 2014; 10(1): e1003831) determined that the E47K (HA2 numbering) mutation in the stem domain of the A / California / 7 / 2009 hemagglutinin stabilized the trimeric structure, lowered the pH for membrane fusion, and increased the thermal and acid stability of the virus. Position 47 of the HA2 stem domain of H1N1 in Cotter's study corresponds to position 374 (HA0 numbering of A / California / 7 / 2009) in the present disclosure. Cotter et al. also observed that the A / California / 7 / 2009 E47K mutant hemagglutinin was more infectious in ferrets than its wild-type counterpart. Yang et al. also obtained similar results in 2014 (J. Virol. May 2014 vol. 88 NO., 94828-4838). They showed that by changing glutamic acid to lysine at position 374 and introducing a basic side chain change at position 374, it is possible to form a new salt bridge across the monomer interface with Glu 21 on the adjacent chain, thereby improving stability at lower pH. Yang et al. found that the presence of lysine at position 374 enhanced the ability of the mutant Tex09 ectodomain trimer to resist heat and acidity changes to a level comparable to that of Wash11 recHA.

[0155] However, it was unexpectedly discovered that, for example, substitution of lysine (K, Lys) with glutamic acid (E, Glu) at position 374 (K374E) of the hemagglutinin of the H1 Michigan influenza virus (A / Michigan / 45 / 15 (H1N1)) resulted in an approximately 1200% increase in hemagglutination titer compared to plant extracts expressing wild-type H1 hemagglutinin (see Figure 3A 、 3B , 4A, 4B and Table 5A).

[0156] Thus, in one aspect, the residue at position 374 of the H1 hemagglutinin (numbered according to the A / California / 07 / 09 hemagglutinin numbering) can be modified to replace a non-glutamic acid with a glutamic acid at position 374, thereby generating a modified H1 hemagglutinin having a non-naturally occurring sequence. The H1 hemagglutinin can be modified to replace the non-glutamic acid with a glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys), such as aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser). In addition, a non-lysine can be substituted for lysine (K, Lys) at position 374 to generate a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) (e.g., aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser)) can be used to replace lysine (K, Lys) at position 374.

[0157] For example, the modified H1 hemagglutinin protein can have an amino acid sequence 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 to the amino acid sequence of the hemagglutinin from H1 Michigan (A / Michigan / 45 / 15, SEQ ID NO: 134), wherein the amino acid sequence has glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) at position 374, such as aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0158] The present specification also provides a nucleic acid comprising a nucleotide sequence encoding an H1 hemagglutinin having a substitution at position 374 and operably linked to a regulatory domain active in plants.

[0159] For example, the nucleotide sequence can have 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 to a nucleotide sequence encoding a hemagglutinin from H1 Michigan (A / Michigan / 45 / 15, SEQ ID NO: 136), wherein the nucleotide sequence encodes a hemagglutinin protein having glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) at position 374 (e.g., aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Ash), histidine (H, His), or serine (S, Ser)), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms VLPs when expressed.

[0160] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount of sequence identity or sequence similarity with the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 374 encodes glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) (e.g., aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser)), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms VLPs when expressed.

[0161] For example, the modified H1 hemagglutinin can have one or more modifications, wherein at least residue 374 of the H1 hemagglutinin is modified as described herein. For example, the modified H1 hemagglutinin can be a monosubstituted, disubstituted, trisubstituted, or tetrasubstituted H1 hemagglutinin, wherein at least the residue at position 374 is modified. In a non-limiting example, the modified H1 hemagglutinin can have a substituted residue at position 374 and one or more substituents at positions 97, 390, 429, or a combination thereof, wherein the modified H1 hemagglutinin sequence does not occur in nature.

[0162] In addition, the present description provides a method for producing VLPs in plants, the VLPs comprising a modified H1 hemagglutinin having a substitution at position 374. The method comprises introducing into a plant or plant part a nucleic acid encoding the modified H1 hemagglutinin having a substitution at position 374 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0163] In addition, a method for increasing the production of VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having a substitution at position 374 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding the modified H1 hemagglutinin having a substitution at position 374 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0164] This specification also provides a VLP comprising an H1 hemagglutinin having a substitution at position 374. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0165] Modification at position 390

[0166] In one aspect of the disclosure, the residue at position 390 in the H1 hemagglutinin (numbered according to A / California / 07 / 09 hemagglutinin numbering) can be substituted.

[0167] WO2013 / 177444 by Lu et al. and its companion document (Proc Natl Acad Sci USA. 2014; 111(1): 125-30) report a method for producing correctly folded hemagglutinin stem domains from A / California / 05 / 2009 (H1N1) using an E. coli-based cell-free protein expression system and a simple refolding protocol. To induce trimerization of the hemagglutinin stem domain, chloramphenicol acetyltransferase (CAT) or a folding domain was fused to the carbon terminus of the hemagglutinin. To mitigate the newly exposed hydrophobicity and / or intermolecular ion pairing that can lead to aggregation of the expressed hemagglutinin stem protein, five groups of mutations were evaluated: M1 (I69T+I72E+I74T+C77T); M2 (I69T+I72E+I74T+C77T+F164D); M3 (I69T+I72E+I74T+C77T+F164D+L174D); M4 (F164D); and M5 (F164D+L174D). Lu noted that the mutants produced very low soluble yields and formed insoluble inclusion bodies. Lu also noted that mutants M3 and M5 produced significantly fewer aggregates than other wild-type variants, leading to the development of mutant M5 (F164D+L174D). Lu observed that the M5 (F164D + L174D) mutation appeared to have the greatest impact on improving the solubility of the hemagglutinin stem protein. Lu's position 164 corresponds to position 390 in the present disclosure. Lu's M4 (F164D) mutation did not appear to be superior to the other mutations tested and was, in fact, inferior to the mutants M3 (I69T + I72E + I74T + C77T + F164D + L174D) and M5 (F164D + L174D).

[0168] When the phenylalanine (F, Phe) at position 390 and the leucine (L, Leu) at position 400 corresponding to the phenylalanine at position 164 and the leucine at position 174 of Lu (F164D + L174D) were changed in the H1 hemagglutinin of the present disclosure (H1 hemagglutinin numbering), no increase in VLP production was observed, and the H1 F390D + L400D mutant showed a complete loss of hemagglutination activity (data not shown). Therefore, equivalent mutations to the M5 mutant in Lu did not result in improved properties of hemagglutinins expressed in plants.

[0169] Unexpectedly, it was found that when the hydrophobic amino acid at position 390 in the H1 hemagglutinin was substituted with a charged amino acid, an approximately 60% increase in VLP production was observed from plants expressing the H1 hemagglutinin having the substitution at position 390 after iodixanol gradient purification compared to plants infiltrated with the wild-type construct (see Figure 3B 、 4A, 4B, Tables 5A, 5B). In addition, as shown in Table 5C, the overall yield increased to 226%. However, the equivalent modification in the hemagglutinin from H5 (F393D) resulted in a decrease in hemagglutination titer (see Figure 5 , Table 6).

[0170] Thus, in one aspect, the residue at position 390 of the H1 hemagglutinin (numbered according to the A / California / 07 / 09 hemagglutinin numbering) can be modified to replace the hydrophobic amino acid at position 390 with a charged amino acid, thereby generating a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin protein can be modified to include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390. The conservative aspartic acid substitution can be, for example, an asparagine (N, Asn), a glutamic acid (E, Glu), a glutamine (Q, Gln), or a serine (S, Ser) substitution.

[0171] The H1 hemagglutinin can be modified to replace the non-aspartic acid at position 390 with aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution. The conservative aspartic acid substitution can be, for example, an asparagine (N, Asn), a glutamic acid (E, Glu), a glutamine (Q, Gln), or a serine (S, Ser). In addition, the H1 hemagglutinin can be modified to replace the phenylalanine (F, Phe) at position 390 with a non-phenylalanine. For example, the hemagglutinin protein can be modified to include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390. The conservative aspartic acid substitution can be, for example, an asparagine (N, Asn), a glutamic acid (E, Glu), a glutamine (Q, Gln), or a serine (S, Ser).

[0172] For example, the modified H1 hemagglutinin protein can have an amino acid sequence that has 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 to the amino acid sequence of the hemagglutinin from H1 A / Michigan / 45 / 15 (SEQ ID NO: 134), wherein the amino acid sequence has an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390, such as asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms a VLP when expressed.

[0173] The present disclosure also provides a nucleic acid comprising a nucleotide sequence encoding a modified H1 hemagglutinin having a substitution at position 390 as described above and operably linked to a regulatory domain active in plants.

[0174] For example, the nucleotide sequence can have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount of sequence identity or sequence similarity with the nucleotide sequence encoding the hemagglutinin from H1 A / Michigan / 45 / 15 (SEQ ID NO: 136), wherein the nucleotide sequence encodes a modified H1 hemagglutinin protein having an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser)) at position 390, wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0175] The nucleotide sequence may have 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 to the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 390 of the modified H1 hemagglutinin encodes an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser)) at position 390, wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0176] For example, the modified H1 hemagglutinin can have one or more modifications, wherein at least residue 390 of the H1 hemagglutinin is modified as described herein. For example, the modified H1 hemagglutinin can be a monosubstituted, disubstituted, trisubstituted, or tetrasubstituted H1 hemagglutinin, wherein at least the residue at position 390 is modified. In non-limiting examples, the modified H1 hemagglutinin can have a substituted residue at position 390 and one or more substituents at positions 97, 374, 380, 429, or a combination thereof.

[0177] In addition, a method for producing VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having a substitution at position 390 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having a substitution at position 390 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0178] In addition, a method for increasing the production of VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having a substitution at position 390 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding the modified H1 hemagglutinin having a substitution at position 390 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0179] This specification also provides a VLP comprising an H1 hemagglutinin having a substitution at position 390. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0180] Modification at position 429

[0181] In one aspect of the present disclosure, residue 429 (numbered according to H1 A / Michigan / 45 / 15 (SEQ ID NO: 134)) in H1 hemagglutinin may be modified.

[0182] Antanasijevic et al. (J Biol Chem. 2014; 289 (32): 22237-45) studied the structure-function properties of the stem-loop domain of the H5 hemagglutinin by site-directed mutagenesis at 14 different positions. Antanasijevic observed that the HA1-D26K, HA1-M102L, HA2-V52A, and HA2-I55A mutants (based on H3 numbering) exhibited significantly reduced levels of total hemagglutinin, indicating that the expression and / or assembly of hemagglutinin in viral particles was reduced. Compared with the wild-type virus, the HA1-D26K, HA2-T49A, and HA2-M102L mutants also exhibited lower hemagglutination titers. Position 102 in the hemagglutinin of Antanasijevic's H5 corresponds to position 429 in the H1 hemagglutinin of the present specification.

[0183] When the H1 hemagglutinin was modified to introduce changes at V19I (HA1-I28V), L20M (HA1-M31L), T368A (HA2-T41A), N380A (HA2-N53A), or L429M (HA2-M102L), it was found that T368A resulted in a complete loss of activity, V19I and L20M had lower activities, while N380A and L429M exhibited higher activity than the H1 A / California wild-type hemagglutinin.

[0184] Thus, most of the residues identified by Antanasijevic as important for the expression and / or assembly or hemagglutination titer of the hemagglutinin from H5 do not appear to translate to similar importance in the hemagglutinin from H1.

[0185] However, if Figure 2A 、 2B As shown in Figures 2C, 3B, 4A and 4B, when residue 429 in the H1 hemagglutinin was mutated from leucine (L, Leu) to methionine (M, Met), the modified H1 hemagglutinin exhibited an increase in hemagglutination potency (100-160%) compared to the wild-type H1 hemagglutinin (see Table 5A). Figure 2C As shown in Table 5B, plants expressing H1 hemagglutinin with a substitution at position 429 showed approximately 30% increased VLP production after sucrose gradient purification compared to plants infiltrated with the wild-type construct (see Table 5B). Furthermore, as shown in Table 5C, the overall yield increased to 260%.

[0186] In addition, compared with unmodified H1 hemagglutinin (see Figure 2B ), the disubstituted H1 hemagglutinin in which the phenylalanine at position 390 was modified to aspartic acid and the leucine at position 429 was modified to methionine showed an approximately 60% improvement in hemagglutination potency compared to .

[0187] Thus, in one aspect, the residue at position 429 of the H1 hemagglutinin (numbered according to the H1A / Michigan / 45 / 15 amino acid sequence (SEQ ID NO: 134)) can be modified by replacing the leucine (L, Leu) at position 429 with another hydrophobic amino acid other than leucine, thereby generating a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin protein can be modified to include a methionine (M, Met) at position 429, or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe).

[0188] In addition, the H1 hemagglutinin can be modified to replace the leucine at position 429 with a non-leucine. For example, the hemagglutinin protein can be mutated to include a methionine (M, Met) or a conservative methionine (M, Met) substitution at position 429 other than leucine (L, Leu). The conservative methionine substitution can be, for example, isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe). In addition, the hemagglutinin protein can be modified to replace a non-methionine at position 429 with a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu). The conservative methionine substitution can be, for example, isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe).

[0189] For example, the modified H1 hemagglutinin protein can have an amino acid sequence that has 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 to the amino acid sequence of the hemagglutinin from H1 Michigan (A / Michigan / 45 / 15 (H1N1), SEQ ID NO: 134), wherein the amino acid sequence has a methionine (M, Met) substitution at position 429 or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe). The modified H1 hemagglutinin sequence is not naturally occurring, and the hemagglutinin protein forms a VLP when expressed.

[0190] The present disclosure also provides a nucleic acid comprising a nucleotide sequence encoding a modified H1 hemagglutinin having a substitution at position 429 as described above and operably linked to a regulatory domain active in plants.

[0191] For example, the nucleotide sequence can have 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 to the nucleotide sequence encoding the hemagglutinin from H1 A / Michigan / 45 / 15 (SEQ ID NO: 136), wherein the nucleotide sequence encodes a modified H1 hemagglutinin protein having a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) at position 429 (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms a VLP when expressed.

[0192] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity or sequence similarity, or any amount therebetween, to the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 429 of the modified H1 hemagglutinin encodes a methionine (M, Met) at position 429 or a conservative methionine (M, Met) substitution other than leucine (L, Leu) (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0193] For example, the modified H1 hemagglutinin can have one or more modifications, wherein at least residue 429 of the H1 hemagglutinin is modified as described herein. For example, the modified H1 hemagglutinin can be a monosubstituted, disubstituted, trisubstituted, or tetrasubstituted H1 hemagglutinin, wherein at least the residue at position 429 is modified. In non-limiting examples, the modified H1 hemagglutinin can have a substituted residue at position 429 and one or more substituents at positions 97, 374, 380, 390, or a combination thereof.

[0194] In addition, a method for producing VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having a substitution at position 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having a substitution at position 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0195] In addition, a method for increasing the production of VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having a substitution at position 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding the modified H1 hemagglutinin having a substitution at position 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0196] This specification also provides a VLP comprising an H1 hemagglutinin having a substitution at position 429. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0197] Disubstituted H1 hemagglutinin

[0198] Also provided is an H1 hemagglutinin protein comprising at least one double substitution or double modification. Thus, the H1 hemagglutinin protein has at least two modifications from a wild-type H1 hemagglutinin protein. For example, the H1 hemagglutinin can have any two combinations of the following modified residues: 97, 374, 380, 390, and 429 (numbered according to A / Michigan / 45 / 15 (SEQ ID NO: 134)).

[0199] Modifications at positions 380 and 429

[0200] In one aspect of the present specification, the modified H1 hemagglutinin may have modified residues at least at positions 380 and 429.

[0201] like Figure 3B As shown, the hemagglutination potency of the H1 hemagglutinin having the residue at position 380 modified from asparagine to alanine and the residue at position 429 modified from leucine to methionine was increased by approximately 800% compared to the wild-type H1 hemagglutinin (see Table 5A).

[0202] Thus, in one aspect, the residues at positions 380 and 429 of the H1 hemagglutinin (numbered according to H1A / Michigan / 45 / 15 (SEQ ID NO: 134)) can be modified by replacing asparagine (N, Asn) at position 380 with a non-asparagine residue and replacing leucine (L, Leu) at position 429 with a non-leucine residue to produce a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin can be modified by replacing a polar amino acid at position 380 with a hydrophobic amino acid and replacing leucine (L, Leu) at position 429 with another hydrophobic amino acid other than leucine to produce a modified H1 hemagglutinin having a non-naturally occurring sequence.

[0203] For example, the H1 hemagglutinin protein can be modified to include an alanine or a conservative alanine substitution at position 380. The conservative alanine substitution can be, for example, serine (S, Ser), glycine (G, Gly), threonine (T, Thr), cysteine ​​(C, Cys), or valine (V, Val). In addition, the H1 hemagglutinin protein can be modified to include a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe) at position 429.

[0204] For example, the modified H1 hemagglutinin protein can have an amino acid sequence that has about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount of sequence identity or sequence similarity to the amino acid sequence of the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 134), wherein the amino acid sequence has an alanine or conservative alanine substitution at position 380, such as serine (S, Ser), glycine (G, Gly), threonine (T, T). hr), cysteine ​​(C, Cys) or valine (V, Val), and the amino acid sequence has a methionine (M, Met) at position 429 or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0205] The present specification also provides a nucleic acid comprising a nucleotide sequence encoding a modified H1 hemagglutinin having substitutions at positions 380 and 429 as described above and operably linked to a regulatory domain active in plants.

[0206] For example, the nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity or sequence similarity, or any amount therebetween, to the nucleotide sequence encoding the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 136), wherein the nucleotide sequence has an alanine or conservative alanine substitution (e.g., serine (S, Ser), glycine (G, Gly), threonine (T, Thr), cysteine ​​(C), or a cysteine ​​residue at position 380. The invention also provides a method for encoding a modified H1 hemagglutinin protein having an amino acid sequence of amino acid residues 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 70, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95

[0207] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount thereof of sequence identity or sequence similarity to the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 380 of the modified H1 hemagglutinin is replaced by alanine or a conservative alanine substitution (e.g., serine (S, Ser), glycine (G, Gly), threonine (T, Thr), cysteine ​​(C, Cys), or the like). ) or valine (V, Val)), and the nucleotide codon encoding amino acid residue 429 of the modified H1 hemagglutinin encodes methionine (M, Met) or a conservative methionine substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0208] In addition, a method for producing VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 380 and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 380 and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0209] In addition, a method for increasing the production of VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 380 and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 380 and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0210] This specification also provides a VLP comprising an H1 hemagglutinin having substitutions at positions 380 and 429. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0211] Modifications at positions 390 and 429

[0212] In one aspect of the present disclosure, the modified H1 hemagglutinin may have modified residues at least at positions 390 and 429.

[0213] like Figure 2B 、 3B As shown in Figures 4A, 4B and Table 5A, the hemagglutination titer of the H1 hemagglutinin having the residue modified from phenylalanine to aspartic acid at position 390 and the residue modified from leucine to methionine at position 429 was increased by approximately 400-1200% compared to the wild-type H1 hemagglutinin. In addition, as shown in Table 5C, the overall process yield was increased to 633%.

[0214] Thus, in one aspect, the residues at positions 390 and 429 of the H1 hemagglutinin (numbered according to H1A / Michigan / 45 / 15 (SEQ ID NO: 134)) can be modified by replacing phenylalanine (F, Phe) at position 390 with a non-phenylalanine and replacing leucine (L, Leu) at position 429 with a non-leucine, to produce a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin can be modified by replacing a hydrophobic amino acid at position 390 with a charged amino acid and replacing leucine (L, Leu) at position 429 with another hydrophobic amino acid other than leucine, to produce a modified H1 hemagglutinin having a non-naturally occurring sequence.

[0215] For example, the H1 hemagglutinin protein can be modified to include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390. The conservative substitution can be, for example, asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). In addition, the H1 hemagglutinin protein can be modified to include a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe) at position 429.

[0216] For example, the modified H1 hemagglutinin protein can have an amino acid sequence that has 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 to the amino acid sequence of the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 134), wherein the amino acid sequence has an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid ( E, Glu), glutamine (Q, Gln) or serine (S, Ser)), and the amino acid sequence has a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) at position 429 (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0217] The present specification also provides a nucleic acid comprising a nucleotide sequence encoding a modified H1 hemagglutinin having substitutions at positions 390 and 429 as described above and operably linked to a regulatory domain active in plants.

[0218] For example, the nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity or sequence similarity, or any amount therebetween, to the nucleotide sequence encoding the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 136), wherein the nucleotide sequence has an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid (E, Glu)) at position 390. ), glutamine (Q, Gln) or serine (S, Ser)) and has a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) at position 429 (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe)), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0219] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount thereof of sequence identity or sequence similarity to the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 390 of the modified H1 hemagglutinin is substituted with aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gl) n) or serine (S, Ser)), and the nucleotide codon encoding amino acid residue 429 of the modified H1 hemagglutinin encodes methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms VLPs when expressed.

[0220] For example, the modified H1 hemagglutinin can have one or more modifications, wherein at least residues 390 and 429 of the H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin can be a disubstituted, trisubstituted, or tetrasubstituted H1 hemagglutinin, wherein at least the residues at positions 390 and 429 are modified. In non-limiting examples, the modified H1 hemagglutinin can have substituted residues at positions 390 and 429, and one or more substituents at positions 97, 374, 380, or a combination thereof.

[0221] In addition, a method for producing VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 390 and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 390 and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0222] In addition, a method for increasing the production of VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 390 and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 390 and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0223] This specification also provides a VLP comprising an H1 hemagglutinin having substitutions at positions 390 and 429. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0224] Modifications at positions 97 and 374

[0225] In one aspect of the present disclosure, the modified H1 hemagglutinin may have modified residues at least at positions 97 and 374.

[0226] like Figure 3B As shown in Table 5A, the H1 hemagglutinin having the residue at position 97 modified from asparagine to aspartic acid and the residue at position 374 modified from lysine to glutamic acid exhibited approximately 1200% improvement in hemagglutination potency compared to the wild-type H1 hemagglutinin.

[0227] Thus, in one aspect, the residues at positions 97 and 374 of the H1 hemagglutinin (numbered according to H1A / Michigan / 45 / 15 (SEQ ID NO: 134)) can be modified by replacing asparagine (N, Asn) at position 97 with a non-asparagine residue and replacing lysine (K, Lys) at position 374 with a non-lysine residue to produce a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin can be modified by replacing the charged amino acid at position 97 with a polar amino acid and replacing the charged amino acid at position 374 with another charged amino acid other than lysine (K, Lys) to produce a modified H1 hemagglutinin having a non-naturally occurring sequence.

[0228] For example, the H1 hemagglutinin protein can be modified to include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) at position 97. Such conservative substitutions can be, for example, glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). In addition, the H1 hemagglutinin protein can be modified to include a glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys), such as aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser) at position 374.

[0229] For example, the modified H1 hemagglutinin protein can have an amino acid sequence that has about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount of sequence identity or sequence similarity to the amino acid sequence of the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 134), wherein the amino acid sequence has an aspartic acid (D, Asp) substitution at position 97 or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn), such as glutamic acid (E, Glu), glutamic acid (Glu), or glutamic acid (Glu). The invention relates to a method for producing a VLP comprising: expressing a modified H1 hemagglutinin sequence comprising: a glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) at position 374, such as aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms VLPs when expressed.

[0230] The present specification also provides a nucleic acid comprising a nucleotide sequence encoding a modified H1 hemagglutinin having substitutions at positions 97 and 374 as described above and operably linked to a regulatory domain active in plants.

[0231] For example, the nucleotide sequence may have 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 to the nucleotide sequence encoding the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 136), wherein the nucleotide sequence has an aspartic acid (D, Asp) at position 97 or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) (e.g., glutamic acid (E, Glu), glutamine (Q, The invention also provides a method for encoding a modified H1 hemagglutinin protein having a glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) at position 374 (e.g., aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser)), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0232] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount thereof of sequence identity or sequence similarity to the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 97 of the modified H1 hemagglutinin is substituted with aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) other than asparagine (N, Asn) (e.g., glutamic acid (E, Glu), glutamine (Q, Gln) or serine (S, S) er)), and the amino acid codon encoding amino acid residue 374 of the modified H1 hemagglutinin encodes glutamate (E, Glu) or a conservative glutamate (E, Glu) substitution other than lysine (K, Lys), such as aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms VLPs when expressed.

[0233] For example, the modified H1 hemagglutinin can have one or more modifications, wherein at least residues 97 and 374 of the H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin can be a disubstituted, trisubstituted, or tetrasubstituted H1 hemagglutinin, wherein at least the residues at positions 97 and 374 are modified. In a non-limiting example, the modified H1 hemagglutinin can have substituted residues at positions 97 and 374, and one or more substituents at positions 380, 390, and 429, or a combination thereof.

[0234] In addition, a method for producing VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 97 and 374 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 97 and 374 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0235] In addition, a method for increasing the production of VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 97 and 374 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 97 and 374 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0236] This specification also provides a VLP comprising an H1 hemagglutinin having substitutions at positions 97 and 374. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0237] Trisubstituted H1 hemagglutinin

[0238] Modifications at positions 97, 390, and 429

[0239] Also provided is an H1 hemagglutinin protein comprising at least one tri-substitution or tri-modification. Thus, the H1 hemagglutinin protein has at least three modifications from a wild-type H1 hemagglutinin protein. For example, the H1 hemagglutinin can have any three combinations of the following modified residues: 97, 374, 390, and 429 (numbered according to A / Michigan / 45 / 15 (SEQ ID NO: 134)).

[0240] In one aspect of the present specification, the modified H1 hemagglutinin may have modified residues at least at positions 97, 390, and 429.

[0241] For example, Figure 3B 、 4A As shown in Tables 4 and 4B, the H1 hemagglutinin having a residue modified from asparagine to aspartic acid at position 90, a residue modified from phenylalanine to aspartic acid at position 390, and a residue modified from leucine to methionine at position 429 exhibited an approximately 2600% increase in hemagglutination titer compared to the wild-type H1 hemagglutinin. In addition, as shown in Table 5C, the overall process yield was increased to 647%.

[0242] Thus, in one aspect, the H1 hemagglutinin residues at positions 97, 390, and 429 (numbered according to H1A / Michigan / 45 / 15 (SEQ ID NO: 134)) can be modified by replacing asparagine (N, Asn) at position 97 with a non-asparagine, replacing phenylalanine (F, Phe) at position 390 with a non-phenylalanine, and replacing leucine at position 429 with a non-leucine (L, Leu) to produce a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin can be modified by replacing a polar amino acid at position 97 with a charged amino acid, replacing a hydrophobic amino acid at position 390 with an amino acid to be charged, and replacing leucine at position 429 with another hydrophobic amino acid other than leucine to produce a modified H1 hemagglutinin having a non-naturally occurring sequence.

[0243] For example, the H1 hemagglutinin protein can be modified to include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 97 in addition to asparagine (N, Asn). The conservative aspartic acid substitution can be, for example, glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). The modified hemagglutinin protein can also include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390. The conservative aspartic acid substitution can be, for example, asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). In addition, the H1 hemagglutinin protein can be modified to include methionine (M, Met) at position 429 or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe).

[0244] For example, the modified H1 hemagglutinin protein can have an amino acid sequence that has 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 to the amino acid sequence of the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 134), wherein the amino acid sequence has an aspartic acid (D, Asp) substitution at position 97 or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn), such as glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser), and the amino acid sequence has an aspartic acid (D, Asp) substitution at position 97, such as glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser), and the amino acid sequence has an aspartic acid (D, Asp) substitution at position 390. The invention relates to a method for producing a VLP comprising: producing an H1 hemagglutinin protein comprising: a) an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution, such as asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln) or serine (Ser), at position 429, and b) an amino acid sequence comprising: a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe), at position 429, wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms VLPs when expressed.

[0245] The present disclosure also provides a nucleic acid comprising a nucleotide sequence encoding a modified H1 hemagglutinin having substitutions at positions 97, 390, and 429 as described above and operably linked to a regulatory domain active in plants.

[0246] For example, the nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity or sequence similarity, or any amount therebetween, to the nucleotide sequence encoding the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 136), wherein the nucleotide sequence has an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) at position 97 (e.g., glutamic acid (E, Glu), glutamine (Q, Gln) or serine (S, Ser)), at position 390. having an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine Ser)) and having a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)) at position 429, wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms VLPs when expressed.

[0247] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount of sequence identity or sequence similarity with the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 97 of the modified H1 hemagglutinin encodes aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) (e.g., glutamic acid (E, Glu), glutamine (Q, Gln) or serine (S, Ser)), and the nucleotide codon encoding amino acid residue 390 of the modified H1 hemagglutinin encodes aspartic acid (D, Asp). The invention relates to a method for producing a modified H1 hemagglutinin protein comprising: encoding an amino acid residue 429 of the present invention and encoding a methionine (M, Met) at position 429 or a conservative methionine (M, Met) substitution other than leucine (L, Leu) such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0248] For example, the modified H1 hemagglutinin can have one or more modifications, wherein at least residues 97, 390, and 429 of the H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin can be a trisubstituted or tetrasubstituted H1 hemagglutinin, wherein at least the residues at positions 97, 390, and 429 are modified. In a non-limiting example, the modified H1 hemagglutinin can have substituted residues at positions 97, 390, 429, and 374.

[0249] In addition, a method for producing VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 97, 390, and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 97, 390, and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0250] In addition, a method for increasing the production of VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 97, 390, and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 97, 390, and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0251] This specification also provides a VLP comprising an H1 hemagglutinin having substitutions at positions 97, 390, and 429. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0252] Modifications at positions 374, 390, and 429

[0253] In one aspect of the present disclosure, the modified H1 hemagglutinin may have modified residues at least at positions 374, 390, and 429.

[0254] For example, Figure 3B As shown in Table 5, H1 hemagglutinin having a residue modified from lysine to glutamic acid at position 374, a residue modified from phenylalanine to aspartic acid at position 390, and a residue modified from leucine to methionine at position 429 exhibited an approximately 2500% increase in hemagglutination titer compared to wild-type H1 hemagglutinin. In addition, as shown in Table 5C, the overall process yield was increased to 689%.

[0255] Thus, in one aspect, the H1 hemagglutinin residues at positions 374, 390, and 429 (numbered according to H1A / Michigan / 45 / 15 (SEQ ID NO: 134)) can be modified by replacing lysine (K, Lys) at position 374 with a non-lysine, replacing phenylalanine (F, Phe) at position 390 with a non-phenylalanine, and replacing leucine at position 429 with a non-leucine, to produce a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin can be modified by replacing lysine at position 374 with a charged amino acid other than lysine, replacing a hydrophobic amino acid at position 390 with a charged amino acid, and replacing leucine (L, Leu) at position 429 with another hydrophobic amino acid other than leucine, to produce a modified H1 hemagglutinin having a non-naturally occurring sequence.

[0256] For example, the H1 hemagglutinin protein can be modified to include glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys), such as aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser) at position 374. The modified hemagglutinin protein can also include aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390. The conservative substitution can be, for example, asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). In addition, the H1 hemagglutinin protein can be modified to include methionine (M, Met) at position 429 or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe).

[0257] For example, the modified H1 hemagglutinin protein can have an amino acid sequence that has 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 to the amino acid sequence of the hemagglutinin from H1A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 134), wherein the amino acid sequence has glutamic acid (E, Glu) at position 374 or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys), such as aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, The invention relates to a method for producing a hemagglutinin protein comprising: providing a glutamic acid sequence comprising: an aspartic acid sequence (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390, such as asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln) or serine (Ser), and providing a methionine (M, Met) substitution at position 429, such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0258] The present invention also provides a nucleic acid comprising a nucleotide sequence encoding a modified H1 hemagglutinin having substitutions at positions 374, 390 and 429 as described above and operably linked to a regulatory domain active in plants.

[0259] For example, the nucleotide sequence may have about 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or any amount thereof, of sequence identity or sequence similarity to the nucleotide sequence encoding the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 136), wherein the nucleotide sequence has glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) at position 374 (e.g., aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser)). , encoding a modified H1 hemagglutinin protein having an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (Ser)) at position 390, and a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)) at position 429, wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0260] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount thereof of sequence identity or sequence similarity to the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 374 of the modified H1 hemagglutinin encodes glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) (e.g., aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser)), and ...), The nucleotide codon encoding amino acid residue 90 encodes aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine Ser)), and the nucleotide codon encoding amino acid residue 429 of the modified H1 hemagglutinin encodes methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0261] For example, the modified H1 hemagglutinin can have one or more modifications, wherein at least residues 374, 390, and 429 of the H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin can be a trisubstituted or tetrasubstituted H1 hemagglutinin, wherein at least the residues at positions 374, 390, and 429 are modified. In a non-limiting example, the modified H1 hemagglutinin can have substituted residues at positions 374, 390, 429, and 97.

[0262] In addition, a method for producing VLPs in plants is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 374, 390, and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 374, 390, and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0263] In addition, a method for increasing the production of VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 374, 390, and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 374, 390, and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0264] This specification also provides a VLP comprising an H1 hemagglutinin having substitutions at positions 374, 390, and 429. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0265] Tetrasubstituted H1 hemagglutinin

[0266] Modifications at positions 97, 374, 390, and 429

[0267] Also provided is an H1 hemagglutinin protein comprising at least one tetrasubstituted or tetramodified protein. Thus, the H1 hemagglutinin protein comprises at least four modifications from a wild-type H1 hemagglutinin protein. For example, the H1 hemagglutinin protein may comprise modifications at positions 97, 374, 390, and 429 (numbered according to H1A / Michigan / 45 / 15 (SEQ ID NO: 134)).

[0268] Therefore, in one aspect of the present specification, the modified H1 hemagglutinin may have modified residues at least at positions 97, 374, 390, and 429.

[0269] For example, Figure 3B As shown, H1 hemagglutinin having a residue modified from asparagine to aspartic acid at position 97, a residue modified from lysine to glutamic acid at position 374, a residue modified from phenylalanine to aspartic acid at position 390, and a residue modified from leucine to methionine at position 429 exhibited an approximately 3300% increase in hemagglutination potency compared to wild-type H1 hemagglutinin.

[0270] Thus, in one aspect, residues at positions 97, 374, 390, and 429 of the H1 hemagglutinin (numbering according to the A / California / 07 / 09 hemagglutinin) can be modified by replacing the asparagine (N, Asn) at position 97 with a non-asparagine, the lysine at position 374 with a non-lysine, the phenylalanine (F, Phe) at position 390 with a non-phenylalanine, and the leucine at position 429 with a non-leucine to generate a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin can be modified by replacing a polar amino acid with a charged amino acid at position 97, replacing lysine with a charged amino acid other than lysine at position 374, replacing a hydrophobic amino acid with a charged amino acid at position 390, and replacing leucine (L, Leu) with another hydrophobic amino acid other than leucine at position 429 to produce a modified H1 hemagglutinin having a non-naturally occurring sequence.

[0271] For example, the H1 hemagglutinin protein can be modified to include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) at position 97. The conservative aspartic acid substitution can be, for example, glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). In addition, the H1 hemagglutinin protein can be modified to include a glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys), such as aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser) at position 374. The modified hemagglutinin protein can also include an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390. The conservative aspartate substitution can be, for example, an asparagine (N, Asn), a glutamic acid (E, Glu), a glutamine (Q, Gln), or a serine (S, Ser) substitution. In addition, the H1 hemagglutinin protein can be modified to include a methionine (M, Met) at position 429 or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe).

[0272] For example, the modified H1 hemagglutinin protein can have an amino acid sequence 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 to the amino acid sequence of the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 134), wherein the amino acid sequence has an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn), such as glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser) at position 97, and the amino acid sequence has a glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys), such as aspartic acid (D, Asp), glutamine (Q, Gln), or serine (S, Ser) at position 374. , Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser), the amino acid sequence has an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390, such as asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln) or serine (S, Ser), and the amino acid sequence has a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) at position 429, such as isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F, Phe), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms VLPs when expressed.

[0273] The present invention also provides a nucleic acid comprising a nucleotide sequence encoding a modified H1 hemagglutinin having substitutions at positions 97, 374, 390 and 429 as described above and operably linked to a regulatory domain active in plants.

[0274] For example, the nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount thereof, sequence identity or sequence similarity to the nucleotide sequence encoding the hemagglutinin from H1 A / Michigan / 45 / 15 (H1N1) (SEQ ID NO: 136), wherein the nucleotide sequence has aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) at position 97 (e.g., glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser)), glutamic acid (E, Glu) or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) at position 374 (e.g., aspartic acid (D, Asp), glutamine (Q, Gln), or serine (S, Ser)). The invention also provides a method for encoding a modified H1 hemagglutinin protein having an aspartic acid (R, Arg), an asparagine (N, Asn), a histidine (H, His), or a serine (S, Ser) at position 390, an aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), a glutamic acid (E, Glu), a glutamine (Q, Gln), or a serine (S, Ser)) at position 390, and a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)) at position 390, wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms a VLP when expressed.

[0275] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount thereof of sequence identity or sequence similarity to the nucleotide sequence of SEQ ID NO: 136, wherein the nucleotide codon encoding amino acid residue 97 of the modified H1 hemagglutinin is for aspartic acid (D, Asp) or a conserved aspartic acid (D, Asp) other than asparagine (N, Asn). The nucleotide codon encoding amino acid residue 374 of the modified H1 hemagglutinin encodes a glutamic acid (E, Glu) substitution at position 374 or a conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) (e.g., aspartic acid (D, Asp), glutamine (Q, Gln), arginine (R, Ar)). g), asparagine (N, Asn), histidine (H, His), or serine (S, Ser)), the nucleotide codon encoding amino acid residue 390 of the modified H1 hemagglutinin encodes aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser)), and the nucleotide codon encoding amino acid residue 429 of the modified H1 hemagglutinin encodes methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)), wherein the modified H1 hemagglutinin sequence does not occur in nature, and wherein the hemagglutinin protein forms VLPs when expressed.

[0276] For example, the modified H1 hemagglutinin can have one or more modifications wherein at least residues 97, 374, 390, and 429 of the H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin can be a tetrasubstituted H1 hemagglutinin wherein residues at positions 97, 374, 390, and 429 are modified.

[0277] In addition, a method of producing VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 97, 374, 390, and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 97, 374, 390, and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0278] In addition, a method for increasing the production of VLPs in a plant is provided, the VLPs comprising a modified H1 hemagglutinin having substitutions at positions 97, 374, 390, and 429 as described above. The method comprises introducing into a plant or plant part a nucleic acid encoding a modified H1 hemagglutinin having substitutions at positions 97, 374, 390, and 429 and operably linked to a regulatory domain active in a plant, and incubating the plant or plant part under conditions permitting expression of the nucleic acid, thereby producing VLPs.

[0279] This specification also provides a VLP comprising an H1 hemagglutinin having substitutions at positions 97, 374, 390, and 429. The VLP can be produced by the method provided herein. Compared to a VLP comprising an unmodified H1 hemagglutinin protein, the VLP comprising the modified H1 hemagglutinin protein exhibits improved properties.

[0280] Also provided herein is a method for increasing the yield or harvest of VLPs comprising mutant influenza virus hemagglutinins in plants. For example, the method may comprise introducing a nucleic acid encoding a mutant influenza virus hemagglutinin as described herein into a plant, plant part, or plant cell. The nucleic acid encoding the mutant influenza virus hemagglutinin may be optimized for human codon usage, increased GC content, or a combination thereof. One or more mutant influenza virus hemagglutinin proteins may be expressed in a plant, plant part, or plant cell to produce VLPs comprising one or more mutant influenza virus hemagglutinin proteins. Alternatively, the method may comprise providing a plant, plant part, or plant cell comprising a nucleic acid encoding a mutant influenza virus hemagglutinin protein to produce VLPs comprising one or more mutant influenza virus hemagglutinin proteins.

[0281] The method for producing VLPs comprising mutant influenza virus hemagglutinin may further comprise the step of introducing a second nucleic acid sequence into a plant, plant part or plant cell, wherein the second nucleic acid encodes a proton channel protein co-expressed with the mutant influenza virus hemagglutinin. For example, the proton channel protein may be an influenza A virus M2 subtype protein, such as A / New Caledonia / 20 / 99 M2. The co-expression of the proton channel protein may result in an increase in the accumulation of mutant influenza virus hemagglutinin protein and / or VLPs comprising the mutant influenza virus hemagglutinin protein, as described in, for example, WO 2013 / 044390, which is incorporated herein by reference.

[0282] In addition, the mutant influenza virus hemagglutinin may further comprise a modified proteolytic loop or cleavage site as described in WO 2013 / 044390 and WO 2014 / 153674, which are incorporated herein by reference.

[0283] " coexpression " refers to introducing and expressing two or more nucleotide sequences in plant, plant or vegetable cell, each in these two or more nucleotide sequences encodes a fragment of protein of interest or protein of interest.Can introduce described two or more nucleotide sequences to plant, plant part or vegetable cell in a carrier, make each in described two or more nucleotide sequences all be under the control of independent regulatory domain (for example comprising double construct).Perhaps, can introduce described two or more nucleotide sequences in independent carrier (for example comprising single construct), and each carrier comprises the suitable regulatory domain for the expression of corresponding nucleic acid.For example, be positioned at respectively on the independent carrier and be introduced into two kinds of nucleotide sequences in the independent agrobacterium tumefaciens host by before vacuum infiltration the suspension of the aspiration of each agrobacterium tumefaciens host (for example equal amounts, perhaps can change the ratio of each agrobacterium tumefaciens host) be mixed and realize coexpression.In this way, the co-infiltration of multiple agrobacterium tumefaciens host suspension allows the coexpression of multiple transgenics.

[0284] The nucleic acid encoding the mutant influenza virus hemagglutinin as described herein may also include a sequence that enhances expression of the mutant influenza virus hemagglutinin in a plant, plant part, or plant cell. The sequence that enhances expression may include a cowpea mosaic virus (CPMV) enhancer element operably associated with the nucleic acid encoding the mutant influenza virus hemagglutinin protein.

[0285] The nucleic acid comprising a nucleotide sequence encoding a modified influenza virus hemagglutinin (HA) as described herein may also include a sequence that enhances expression of the hemagglutinin protein in a plant, plant part, or plant cell. The sequence that enhances expression may include a CPMV enhancer element or a plant-derived expression enhancer operably bound to the nucleic acid encoding the modified influenza virus hemagglutinin protein. The sequence encoding the modified influenza virus hemagglutinin (HA) may also be optimized for human codon usage, increased GC content, or a combination thereof.

[0286] As used herein, the term "CPMV enhancer element" refers to a nucleotide sequence encoding a 5'UTR or a modified CPMV sequence known in the art that regulates the Cowpea Mosaic Virus (CPMV) RNA2 polypeptide. For example, CPMV enhancer elements or CPMV expression enhancers include nucleotide sequences described in WO2015 / 14367; WO2015 / 103704; WO2007 / 135480; WO2009 / 087391; Sainsbury F. and Lomonossoff GP (2008, Plant Physiol. 148: pp. 1212-1218), all of which are incorporated herein by reference. The CPMV enhancer sequence can enhance the expression of a downstream heterologous open reading frame to which it is attached. The CPMV expression enhancer may comprise CPMV HT, CPMVX (wherein X=160, 155, 150, 114), such as CPMV 160, CPMVX+ (wherein X=160, 155, 150, 114), such as CPMV 160+, CPMV-HT+, CPMVHT+[WT115], or CPMV HT+

[511] (WO2015 / 143567; WO2015 / 103704, which are incorporated herein by reference). The CPMV expression enhancer can be used in a plant expression system comprising a regulatory domain operably linked to the CPMV expression enhancer sequence and a nucleotide sequence of interest.

[0287] As used herein, the term "CPMV enhancer element" refers to a nucleotide sequence encoding a 5'UTR or a modified CPMV sequence known in the art that regulates the Cowpea Mosaic Virus (CPMV) RNA2 polypeptide. For example, CPMV enhancer elements or CPMV expression enhancers include nucleotide sequences described in WO2015 / 14367; WO2015 / 103704; WO2007 / 135480; WO2009 / 087391; Sainsbury F. and Lomonossoff GP (2008, Plant Physiol. 148: pp. 1212-1218), all of which are incorporated herein by reference. The CPMV enhancer sequence can enhance the expression of a downstream heterologous open reading frame to which it is attached. The CPMV expression enhancer may comprise CPMV HT, CPMVX, CPMVX+, CPMV-HT+, CPMVHT+[WT115], or CPMV HT+

[511] (WO2015 / 14367; WO2015 / 103704, which are incorporated herein by reference). The CPMV expression enhancer may be used in a plant expression system comprising a regulatory domain operably linked to the CPMV expression enhancer sequence and a nucleotide sequence of interest.

[0288] The term "5' UTR" or "5' untranslated region" or "5' leader sequence" refers to a region of an mRNA that is not translated. The 5' UTR typically begins at the transcription start site and ends before the translation start site or the start codon of the coding region. The 5' UTR may regulate the stability and / or translation of the mRNA transcript.

[0289] As used herein, the term "plant-derived expression enhancer" refers to a nucleotide sequence obtained from a plant that encodes a 5'UTR. An example of a plant-derived expression enhancer is described in U.S. Provisional Patent Application 2006 / 010086 (filed on March 14, 2018, which is incorporated herein by reference) or in a paper by Diamos AG et al. (2016, Front Plt Sci. 7:1-15; which is incorporated herein by reference). The plant-derived expression enhancer can be selected from nbMT78, nbATL75, nbDJ46, nbCHP79, nbEN42, atHSP69, atGRP62, atPK65, atRP46, nb30S72, nbGT61, nbPV55, nbPPI43, nbPM64, and nbH2A86 as described in US 62 / 643,053. Plant-derived expression enhancers can be used in plant expression systems comprising a regulatory domain operably linked to the plant-derived expression enhancer sequence and a nucleotide sequence of interest.

[0290] "Operably linked" refers to specific sequences that interact directly or indirectly to perform a predetermined function, such as mediation or regulation of nucleic acid sequence expression. For example, the interaction of operably linked sequences can be mediated by a protein that interacts with the operably linked sequences.

[0291] When one or more mutant influenza virus hemagglutinin proteins are expressed in plants, plant parts or plant cells, the one or more mutant influenza virus hemagglutinin proteins self-assemble into VLPs. Plants, plant parts or plant cells can be harvested under appropriate extraction and purification conditions to maintain the integrity of the VLPs, and VLPs comprising one or more mutant influenza virus hemagglutinins can be purified.

[0292] The present invention also provides a use of a mutant influenza virus hemagglutinin or a VLP comprising a mutant influenza virus hemagglutinin as described herein in inducing immunity to influenza infection in a subject. Disclosed herein is an antibody or antibody fragment prepared by administering a mutant influenza virus hemagglutinin or a VLP comprising a mutant influenza virus hemagglutinin to a subject or host animal. Also provided is a composition for inducing an immune response in a subject, comprising an effective dose of a mutant influenza virus hemagglutinin or a VLP virus comprising a mutant influenza virus hemagglutinin as described herein, and a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient. Also provided is a vaccine for inducing an immune response in a subject, wherein the vaccine comprises an effective dose of a mutant influenza virus hemagglutinin.

[0293] Also provided herein is a method of inducing immunity to influenza infection in a subject, comprising administering to the subject a mutant influenza virus hemagglutinin or a VLP comprising the mutant influenza virus hemagglutinin orally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

[0294] As used herein, the term "influenza virus" refers to an enveloped virus strain of the Orthomyxoviridae family characterized by having a negative-stranded single-stranded RNA genome. Depending on the specific strain, the influenza virus genome consists of eight gene segments encoding 12-14 proteins.

[0295] There are four types of influenza viruses: A, B, C, and D. Influenza A and B are the causative organisms of seasonal epidemics of disease in humans. Influenza A viruses are further classified based on the expression of hemagglutinin and neuraminidase (NA) glycoprotein subtypes.

[0296] The term "hemagglutinin" or "HA" as used herein refers to a trimeric lectin that promotes the binding of influenza virus particles to sialic acid-containing proteins on the surface of target cells and mediates the release of the viral genome into target cells. There are 18 different hemagglutinin subtypes (H1-H18). The hemagglutinin protein consists of two structural elements: a head, which is the main target of serum protective antibodies; and a stalk. Hemagglutinin is translated into a single polypeptide HA0 (assembled as a trimer), which must be cut between the HA1 (approximately 40 kDa) and HA2 (approximately 20 kDa) subdomains by a serine endoprotease. After cleavage, the two disulfide-bonded protein domains adopt the necessary conformation required to achieve viral infectivity.

[0297] The influenza A virus hemagglutinin proteins or modified influenza A virus hemagglutinin proteins disclosed herein include any known hemagglutinin protein derived from any known influenza A strain, but also include modifications of known influenza A strains developed over time. For example, the influenza virus hemagglutinin may be derived from A / California / 07 / 09 (H1N1), A / Michigan / 45 / 15 (H1N1), A / Massachusetts / 06 / 17 (H1N1), A / Costa Rica / 0513 / 16 (H1N1), A / Honduras / 17734 / 16 (H1N1), or A / Darwin / 11 / 15 (H1N1). The influenza A virus hemagglutinin may include a strain-derived hemagglutinin, wherein the hemagglutinin has about 30-100% amino acid sequence identity, or any amount therebetween, with any hemagglutinin derived from the influenza A virus strains listed above, where the influenza virus hemagglutinin protein comprises at least one substitution as described herein and is capable of forming VLPs, inducing an immune response when administered to a subject, inducing hemagglutination, or a combination thereof.

[0298] For example, the influenza virus hemagglutinin protein can have 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100%, or any amount therebetween, amino acid sequence identity to any hemagglutinin from the influenza A virus strains listed above, and include at least one substitution as described herein, and be capable of forming VLPs, inducing immunity, inducing hemagglutination, or a combination thereof when administered to a subject. Figure 1 The amino acid sequence alignments of several influenza A virus hemagglutinin domains are shown in , and these examples should not be considered limiting.

[0299] When referring to specific sequences, the terms "percent similarity," "sequence similarity," "percent identity," or "sequence identity" are used, for example, as described in the University of Wisconsin GCG software program or by manual alignment and visual inspection (see, for example, Ausubel et al., Molecular Biology: A Laboratory Manual, 1995 Supplement). Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the algorithm of Smith and Waterman (1981, Adv. Appl. Math. 2:482), by the alignment algorithm of Needleman and Wunsch (1970, J. Mol. Biol. 48:443), by the search similarity method of Pearson and Lipman (1988, Proc. Natl. Acad. Sci. USA 85:2444), or by computerized implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package by Genetics Computer Group, Inc., Madison, Wisconsin (575).

[0300] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, described in Altschul et al. (1977, Nuc. Acids Res. 25:3389-3402) and Altschul et al. (1990, J. Mol. Biol. 215:403-410), respectively. BLAST and BLAST 2.0 can be used to determine percent sequence identity for nucleic acids and proteins of the invention using the parameters described herein. For example, the BLASTN program (for nucleotide sequences) can use a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands as defaults. For amino acid sequences, the BLASTP program can use as defaults a wordlength of 3, expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89: 10915) with alignments (B) of 50, expectation (E) of 10, M of 5, N of -4, and a comparison of both strands. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see URL: ncbi.nlm.nih.gov / ).

[0301] As used herein, the term "virus-like particle," VLP, or variations thereof, refers to influenza virus particles that include one or more influenza virus hemagglutinin proteins and self-assemble into a non-replicating, non-infectious viral capsid structure devoid of all parts of the influenza genome.

[0302] Production of influenza virus hemagglutinin protein in plants

[0303] In the case where the influenza A virus hemagglutinin protein comprises at least one substitution as described herein and is capable of forming VLPs, inducing an immune response when administered to a subject, inducing hemagglutination, or a combination thereof, the influenza A virus hemagglutinin protein comprises a polypeptide comprising a polypeptide having a sequence similar to that of A / California / 07 / 09 (H1N1, sequence number 130), A / Michigan / 45 / 15 (H1N1, sequence number 134), A / Massachusetts / 06 / 17 (H1N1, sequence number 135), A / Costa The influenza A virus hemagglutinin sequences of Rica / 0513 / 16 (H1N1, SEQ ID NO: 133), A / Honduras / 17734 / 16 (H1N1, SEQ ID NO: 131), A / Darwin / 11 / 15 (H1N1, SEQ ID NO: 132), A / Paris / 1227 / 2017 (SEQ ID NO: 138), and A / Norway / 2147 / 2017 (SEQ ID NO: 139) have about 30 to about 10

[0015] The present invention also provides any hemagglutinin protein having an amino acid sequence that has between about 0%, about 40 to about 100%, about 50 to about 100%, about 60 to about 100%, about 70 to about 100%, about 80 to about 100%, about 85 to about 100%, about 90 to about 100%, about 95 to about 100%, or about 97 to about 100%, about 98 to about 100%, or any amount therebetween, of sequence identity or sequence similarity to any hemagglutinin protein.

[0304] In addition, the modified influenza virus hemagglutinin (HA) protein, when comprising at least one substitution as described herein and capable of forming VLPs, inducing an immune response when administered to a subject, inducing hemagglutination, or a combination thereof, includes sequences with sequences of SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 4, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 100%, about 90% to about 100%, about 95% to about 100%, about 97% to about 100%, about 98% to about 100%, or any amount therebetween of an amino acid sequence having sequence identity or sequence similarity to one of the sequences of SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 105, SEQ ID NO: 108, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, or SEQ ID NO: 148.

[0305] As described herein, one or more specific mutations or modifications in influenza virus hemagglutinin result in increased accumulation of hemagglutinin protein and increased VLP production in plants compared to wild-type influenza virus hemagglutinin.

[0306] Examples of mutant influenza A virus hemagglutinin proteins having enhanced ability to produce influenza virus hemagglutinin and / or VLPs in plants include, but are not limited to:

[0307] F390D A / California / 07 / 09 mutant H1 (Construct No. 2980, SEQ ID NO: 18): L429M A / California / 07 / 09 mutant H1 (Construct No. 2962, SEQ ID NO: 22); F390D+L429M A / California / 07 / 09 mutant H1 (Construct No. 2995, SEQ ID NO: 24); N380A A / Michigan / 45 / 15 mutant H1 (Construct No. 3644, SEQ ID NO: 105); F390D+N380A A / Michigan / 45 / 15 mutant H1 (Construct No. 3704, SEQ ID NO: 108); N97D A / Michigan / 45 / 15 mutant H1 (Construct No. 3774, SEQ ID NO: 28); K374E A / Michigan / 45 / 15 mutant H1 (Construct No. 3771, SEQ ID NO: 32); F390D A / Michigan / 45 / 15 mutant H1 (Construct No. 3641, SEQ ID NO: 36); L429M A / Michigan / 45 / 15 mutant H1 (Construct No. 3643, SEQ ID NO: 39); N97D+K374E A / Michigan / 45 / 15 mutant H1 (Construct No. 3880, SEQ ID NO: 41); F390D+L429M A / Michigan / 45 / 15 mutant H1 (Construct No. 3703, SEQ ID NO: 43); N97D+F390D+L429M A / Michigan / 45 / 15 mutant H1 (Construct No. 3879, SEQ ID NO: 45); K374E+F390D+L429M A / Michigan / 45 / 15 mutant H1 (Construct No. 3878, Sequence No. 47); N97D+K374E+F390D+L429M A / Michigan / 45 / 15 mutant H1 (Construct No. 3881, Sequence No. 49); F390D+L429M A / Massachusetts / 06 / 17 mutant H1 (Construct No. 4091, Sequence No. 51); N97D+F390D+L429M A / Massachusetts / 06 / 17 mutant H1 (Construct No. 4093, Sequence No. 53); K374E+F390D+L429M A / Massachusetts / 06 / 17 mutant H1 (Construct No. 4092, SEQ ID NO: 55); N97D+K374E+F390D+L429M A / Massachusetts / 06 / 17 mutant H1 (Construct No. 4094, SEQ ID NO: 57); F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 (Construct No. 4715, SEQ ID NO: 59);N97D+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 (Construct No. 4717, SEQ ID NO. 61); K374E+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 (Construct No. 4716, SEQ ID NO. 63); N97D+K374E+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 (Construct No. 4718, SEQ ID NO. 65); N97D A / Honduras / 17734 / 16 mutant H1 (Construct No. 3950, SEQ ID NO. 68); K374E A / Honduras / 17734 / 16 mutant H1 (Construct No. 3948, SEQ ID NO. 72); F390D A / Honduras / 17734 / 16 mutant H1 (Construct No. 3945, SEQ ID NO: 75); L429M A / Honduras / 17734 / 16 mutant H1 (Construct No. 3949, SEQ ID NO: 80); F390D+L429M A / Honduras / 17734 / 16 mutant H1 (Construct No. 3946, SEQ ID NO: 82); N97D+F390D+L429M A / Honduras / 17734 / 16 mutant H1 (Construct No. 3951, SEQ ID NO: 84); N97D A / Darwin / 11 / 15 mutant H1 (Construct No. 3990, SEQ ID NO: 86); K374E A / Darwin / 11 / 15 mutant H1 (Construct No. 3988, SEQ ID NO: 89); F390D A / Darwin / 11 / 15 mutant H1 (Construct No. 3985, SEQ ID NO. 91); L429M A / Darwin / 11 / 15 mutant H1 (Construct No. 3989, SEQ ID NO. 93); F390D+L429M A / Darwin / 11 / 15 mutant H1 (Construct No. 3986, SEQ ID NO. 95); N97D+F390D+L429M A / Darwin / 11 / 15 mutant H1 (Construct No. 3991, SEQ ID NO. 97); F390D+L429M A / Paris / 1227 / 2017 mutant H1 (Construct No. 4765, SEQ ID NO. 124); K374E+F390D+L429M A / Paris / 1227 / 2017 mutant H1 (Construct No. 4766, SEQ ID NO. 126); N97D+F390D+L429M A / Paris / 1227 / 2017 mutant H1 (Construct No. 4767, SEQ ID NO. 128); N97D+K374E+F390D+L429M A / Paris / 1227 / 2017 mutant H1 (Construct No. 4768, SEQ ID NO. 140);F390D+L429M A / Norway / 2147 / 2017 mutant H1 (Construct No. 4775, SEQ ID NO. 142); K374E+F390D+L429M A / Norway / 2147 / 2017 mutant H1 (Construct No. 4776, SEQ ID NO. 144); N97D+F390D+L429M A / Norway / 2147 / 2017 mutant H1 (Construct No. 4777, SEQ ID NO. 146); and N97D+K374E+F390D+L429M A / Norway / 2147 / 2017 mutant H1 (Construct No. 4778, SEQ ID NO. 148).

[0308] Inducing immunity to influenza infection

[0309] "Immune response" generally refers to the reaction of the adaptive immune system of a subject. The adaptive immune system generally includes humoral reactions and cell-mediated reactions. Humoral reactions are immune features mediated by secretory antibodies produced in B lymphocyte lineage cells (B cells). Secreted antibodies bind to antigens on the surface of invading microorganisms (such as viruses or bacteria), marking them for destruction. Humoral immunity is generally used to refer to antibody production and its accompanying processes, as well as the effector functions of antibodies, including Th2 cell activation and cytokine production, memory cell production, opsonin-promoting phagocytosis, pathogen elimination, etc. The term "modulation" refers to the increase or decrease of a specific reaction or parameter determined by any one of a variety of well-known or commonly used tests, some of which are shown in this article.

[0310] A cell-mediated response is an immune response that does not involve antibodies but rather involves the activation of macrophages, natural killer (NK) cells, and antigen-specific cytotoxic T lymphocytes, as well as the release of various cytokines in response to the antigen. Cell-mediated immunity is often used to refer to certain Th cell activation, Tc cell activation, and T cell-mediated responses. Cell-mediated immunity may be particularly important in responses to viral infections.

[0311] For example, the induction of antigen-specific CD8 positive T lymphocytes can be measured using an ELISPOT assay; the stimulation of CD4 positive T lymphocytes can be measured using a proliferation assay. Anti-influenza virus hemagglutinin antibody titers can be quantified using an enzyme-linked immunosorbent assay (ELISA); the isotype of antigen-specific or cross-reactive antibodies can also be determined 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.

[0312] The presence or level of cytokines can also be quantified. For example, T helper cell responses (Th1 / Th2) are characterized by measuring IFN- and IL-4 secreting cells using ELISA (e.g., BD Biosciences OptEIA kit). Peripheral blood mononuclear cells (PBMCs) or splenocytes obtained from a subject can be cultured and the supernatant analyzed. T lymphocytes can also be quantitatively analyzed by fluorescence activated cell sorting (FACS) using marker-specific fluorescent markers and methods known in the art.

[0313] Microneutralization assays can also be performed to characterize the immune response of a subject, for example, see the method published by Rowe et al. in 1973. Virus neutralization titers can be quantified in a variety of ways, including: counting the number of lytic plaques after crystallographic fixation / staining of cells (plaque analysis); microscopic observation of lysis of in vitro cultured cells; and 2) enzyme-linked immunosorbent assay and spectrophotometric detection of influenza virus.

[0314] As used herein, the term "epitope" refers to the structural portion of an antigen to which an antibody specifically binds.

[0315] For example, an immune response can be observed in Balb / C mice in response to the administration of wild-type influenza virus hemagglutinin protein or VLP or mutant influenza virus hemagglutinin protein or VLP produced from plants. Serum samples of blood collected from animals can be analyzed by enzyme-linked immunosorbent assay (ELISA) for H1-specific total IgG and IgA antibodies. In the serum of each treatment group, mice immunized with wild-type influenza virus hemagglutinin or mutant influenza virus hemagglutinin protein produced from plants may exhibit hemagglutinin-specific IgG antibody titers.

[0316] Plant Expression

[0317] The constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and the like. For reviews of these techniques, see, for example, Weissbach and Weissbach, Methods in Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2nd ed. (1988); and Miki and Iyer, 1996. Plants Basic knowledge of gene transferPlant Metabolism, 2nd ed., DT. DT. Dennis, DH Turpin, DD Lefebrvre, DB Layzell (eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). Other methods include direct DNA uptake, use of liposomes, electroporation (e.g., using protoplasts), microinjection, microparticles or whiskers, and vacuum infiltration. See, for example, Bilang et al. (1991, Gene 100:247-250), Scheid et al. (1991, Mol. Gen. Genet. 228:104-112), Guerche et al. (1987, Plant Science 52:111-116), Neuhause et al. (1987, Theor. Appl Genet. 75:30-36), Klein et al. (2987, Nature 327:70-73); Freeman et al. (1984, Plant Cell Physiol. 29:1353), Howell et al. (1985, Science 227:1229-1231), DeBlock et al. (1989, Plant Physiology 91: 694-701), Methods in Plant Molecular Biology (Weissbach and Weissbach, eds., Academic Press Inc., 1988), Methods in Plant Molecular Biology (Schuler and Zielinski, eds., Academic Press Inc., 1989), WO 92 / 09696, WO 94 / 00583, EP 331083, EP 175966, Liu and Lomonossoff (2002, J Virol Meth, 105: 343-348), EP 290395; WO8706614; U.S. Patents 4,945,050; 5,036,006; and 5,100,792, U.S. Patent Application 08 / 438,666 filed May 10, 1995, and 07 / 951,715 filed September 25, 1992 (all of which are incorporated herein by reference).

[0318] Transient expression methods can be used to express the constructs of the present invention (see D'Aoust et al., 2009, Methods in Mol. Biol., Vol. 483, pp. 41-50; Liu and Lomonossoff, 2002, J. M. Virol., 105: 343-348; these documents are incorporated herein by reference). Alternatively, vacuum-based transient expression methods such as those described in Kapila et al. (1997, Plant Sci., 122, 101-108; these documents are incorporated herein by reference) or WO 00 / 063400, WO 00 / 037663 (these documents are incorporated herein by reference) can be used. These methods may include, but are not limited to, agroinoculation or agroinfiltration, syringe infiltration methods, but, as described above, other transient methods may also be used. By agroinoculation, agroinfiltration, or syringe infiltration, the Agrobacterium mixture containing the desired nucleic acid enters the intercellular spaces of a tissue, such as leaves, aerial parts (including stems, leaves, and flowers), other parts of a plant (stems, roots, flowers), or the entire plant. After crossing the epidermis, Agrobacterium infects and transfers copies of the t-DNA into the cell. The t-DNA is episomally transcribed and the mRNA is translated, resulting in the production of the protein of interest in the infected cell. However, the delivery of the t-DNA into the nucleus is transient.

[0319] Transgenic plants, plant cells or seeds comprising the gene constructs of the present invention are also considered part of the present invention and can be used as platform plants suitable for transient protein expression as described herein. Methods for regenerating whole plants from plant cells are also known in the art (e.g., see Guerineau and Mullineaux (1993, Plant Transformation and Expression Vectors, Oxford Plant Molecular Biology Labfax, Oxford, 1993). RRD edition), BIOS Science Press, pages 121-148). Generally, the transformed plant cells are cultured in an appropriate culture medium that may contain a selective agent (e.g., an antibiotic), wherein a selective marker is used to facilitate identification of the transformed plant cells. Once callus tissue is formed, the formation of buds can be promoted by using appropriate plant hormones according to known methods, and the buds can be transferred to a rooting medium to regenerate the plant. These plants can then be used to form repeated generations from seeds or using vegetative propagation techniques. Transgenic plants can also be produced without using tissue culture. Methods for the stable transformation and regeneration of these organisms are established in the art and are known to those skilled in the art. Available techniques can be found in the works of Vasil et al. (Plant Cell Culture and Somatic Cell Genetics, Laboratory Procedures and Their Applications, Academic Press, 1984) and Weissbach and Weissbach (Methods in Plant Molecular Biology, Academic Press, 1989). The method for obtaining the transformed and regenerated plants is not crucial to the present invention.

[0320] In one embodiment, the invention provides the method for the conversion of plant, plant part or plant cell to the nucleic acid construct of the present invention.If preparation is carried out transformation or co-transformation by two or more nucleic acid constructs, then this nucleic acid construct can be introduced into the agrobacterium in a single transfection event so that nucleic acid is gathered and bacterial cell is transfected.Or, construct can be introduced successively.In this case, as described above, the first construct is introduced into the agrobacterium so that cell is grown under the selective condition (for example, under the condition of having antibiotic) that only the bacterium of single transformation can grow.After this first selection step, as described above, the second nucleic acid construct is introduced into the agrobacterium, and cell is grown under the dual selective condition that only the bacterium of double transformation can grow.Then, can use the bacterium of double transformation to transform plant, plant part or plant cell as described herein, or can carry out further conversion step to include the 3rd nucleic acid construct.

[0321] Alternatively, if a plant, plant part or plant cell is to be transformed or co-transformed with two or more nucleic acid constructs, the nucleic acid constructs can be introduced into the plant by co-infiltrating a mixture of Agrobacterium cells with the plant, plant part or plant cell, wherein each Agrobacterium cell can contain one or more constructs to be introduced into the plant. In order to vary the relative expression level of the nucleotide sequence of interest in the construct in the plant, plant part or plant cell, the concentration of each Agrobacterium population containing the desired construct can be varied during the infiltration step.

[0322] Table 3: Serial Number and Serial Description

[0323]

[0324]

[0325]

[0326]

[0327]

[0328] The present invention will be further illustrated in the following examples.

[0329] Example 1: Influenza virus hemagglutinin construct

[0330] Influenza virus hemagglutinin constructs were generated using techniques well known in the art. For example, wild-type A-California-07-09 hemagglutinin, F390D A-California-07-09 hemagglutinin, and F390D+L429M A-California-07-09 hemagglutinin were cloned as described below. Similar techniques were used to obtain other H1 mutants. The hemagglutinin sequence primers, templates, and products are described in Example 3 (Production of Influenza Virus Hemagglutinin and VLPs in Plants) and Table 4.

[0331] Table 4 below summarizes wild-type and mutant hemagglutinin proteins, primers, templates, and products.

[0332] A. Modification of H1 hemagglutinin

[0333] 2X35S / CPMV 160 / PDISP-HA0 H1 A-California-07-09 / NOS (Construct No. 1314)

[0334] The following PCR-based method was used to clone the sequence encoding the mature HA0 of the influenza virus hemagglutinin from A / California / 07 / 09 fused to the alfalfa PDI secretion signal peptide into the 2X35S / CPMV 160 / NOS expression system. The PDISP-H1 A / California / 7 / 09 gene sequence (SEQ ID NO: 1) was used as a template and primers IF-CPMV (f15′UTR)_SpPDI.c (SEQ ID NO: 13) and IF-H1cTMCT.S1-4r (SEQ ID NO: 14) were used to amplify the fragment containing the PDISP-A / California / 07 / 09 coding sequence. The PCR product was cloned into the 2X35S / CPMV 160 / NOS expression system using the Fusion Cloning System (Clontech, Mountain View, CA, USA). Construct 1190 ( Figure 8 ), and a linearized plasmid was used for the fusion assembly reaction. Construct 1190 is a receptor plasmid for "fusion" cloning of the gene of interest in an expression cassette based on 2X35S / CPMV160 / NOS. It also combines a gene construct for co-expressing the TBSV P19 silencing suppressor under the action of the alfalfa plastocyanin gene promoter and terminator. The backbone is the pCAMBIA binary plasmid, and the t-DNA border sequence from left to right is shown in sequence number 98. The resulting construct is numbered 1314 (sequence number 99). The amino acid sequence of mature HA0 from A / California / 07 / 09 fused with the alfalfa PDI secretion signal peptide (PDISP) is shown in sequence number 2. In Figure 6A A schematic diagram of plasmid 1314 is shown in FIG.

[0335] 2X35S / CPMV 160 / PDISP-HA0 H1 A-California-07-09(F390D) / NOS (Construct No. 2980)

[0336] The following PCR-based method was used to clone the sequence encoding the mature HAO of influenza virus hemagglutinin from A / California / 07 / 09 (F390D) fused to the alfalfa PDI secretion signal peptide into the 2X35S / CPMV 160 / NOS expression system. In the first round of PCR, the PDISP-H1 A / California / 7 / 09 gene sequence (SEQ ID NO: 1) was used as a template, and primers IF-CPMV (f15'UTR)_SpPDI.c (SEQ ID NO: 13) and H1Cal (F390D).r (SEQ ID NO: 15) were used to amplify a fragment containing the PDISP-H1 A / California / 07 / 09 fragment with the mutated F390D amino acid. Using the PDISP-H1 A / California / 07 / 09 gene sequence (SEQ ID NO: 1) as a template, a second fragment containing the F390D mutation and the remainder of H1 A / California / 07 / 09 were amplified using H1Cal(F390D).c (SEQ ID NO: 16) and IF-H1cTMCT.S1-4r (SEQ ID NO: 14). The PCR products of the two amplifications were then mixed and used as templates for a second round of amplification using IF-CPMV(f15′UTR)_SpPDI.c (SEQ ID NO: 13) and IF-H1cTMCT.S1-4r (SEQ ID NO: 14) as primers. The final PCR product was cloned into the 2X35S / CPMV 160 / NOS expression system using the Fusion Cloning System (Clontech, Mountain View, CA, USA). Construct 1190 ( Figure 8 ), and a linearized plasmid was used for the fusion assembly reaction. Construct 1190 is a receptor plasmid for "fusion" cloning of the gene of interest in an expression cassette based on 2X35S / CPMV 160 / NOS. It also combines a gene construct for co-expressing the TBSVP19 silencing suppressor under the action of the alfalfa plastocyanin gene promoter and terminator. The backbone is the pCAMBIA binary plasmid, and the t-DNA border sequence from left to right is shown in sequence number 98. The resulting construct is numbered 2980 (sequence number 100). The amino acid sequence of the mutated PDISP-HA from A / California / 07 / 09 (F390D) is shown in sequence number 18. In Figure 6B A schematic diagram of plasmid 2980 is shown in FIG.

[0337] 2X35S / CPMV 160 / PDISP-HA0 H1 A-California-07-09(F390D+L429M) / NOS(Build Body No. 2995)

[0338] The following PCR-based method was used to clone the sequence encoding the mature HA0 of influenza virus hemagglutinin from A / California / 07 / 09 (F390D+L429M) fused to the alfalfa PDI secretion signal peptide into the 2X35S / CPMV160 / NOS expression system. In the first round of PCR, the PDISP-H1 A / California / 7 / 09 (F390D) gene sequence (SEQ ID NO: 17) was used as a template, and primers IF-CPMV (f15′UTR)_SpPDI.c (SEQ ID NO: 13) and H1Cal (L429M).r (SEQ ID NO: 19) were used to amplify a fragment of PDISP-H1 A / California / 07 / 09 containing the mutated F390D and L429M amino acids. Using the PDISP-H1 A / California / 7 / 09 (F390D) gene sequence (SEQ ID NO: 17) as a template, H1Cal(L429M).c (SEQ ID NO: 20) and IF-H1cTMCT.S1-4r (SEQ ID NO: 14) were used to amplify the second fragment containing the L429M mutation and the remainder of H1A / California / 07 / 09. The PCR products of the two amplifications were then mixed and used as templates for a second round of amplification using IF-CPMV(f15′UTR)_SpPDI.c (SEQ ID NO: 13) and IF-H1cTMCT.S1-4r (SEQ ID NO: 14) as primers. The final PCR product was cloned into the 2X35S / CPMV160 / NOS expression system using the Fusion Cloning System (Clontech, Mountain View, CA, USA). Construct 1190 ( Figure 8 ), and a linearized plasmid is used for the fusion assembly reaction. Construct 1190 is a receptor plasmid for "fusion" cloning of the gene of interest in an expression cassette based on 2X35S / CPMV 160 / NOS. It also combines a gene construct for co-expressing the TBSV P19 silencing suppressor under the action of the alfalfa plastocyanin gene promoter and terminator. The backbone is the pCAMBIA binary plasmid, and the t-DNA border sequence from left to right is shown in sequence number 98. The resulting construct is numbered 2995 (sequence number 101). The amino acid sequence of the mutant PDISP-HA from A / California / 07 / 09 (F390D+L429M) is shown in sequence number 24. Figure 6D A schematic diagram of plasmid 2995 is shown in FIG.

[0339] Example 2: Method

[0340] Agrobacterium tumefaciens transfection

[0341] Agrobacterium tumefaciens strain AGL1 was transfected with wild-type influenza virus hemagglutinin or mutant influenza virus hemagglutinin expression vectors by electroporation using the method described by D'Aoust et al. (Plant Biotech. J. 6: 930-40). Transfected Agrobacterium was 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 carbenicillin at pH 5.6 until the OD was 0. 600 Before use, the Agrobacterium suspension was centrifuged and resuspended in infiltration medium (10 mM MgCl2 and 10 mM MES, pH 5.6).

[0342] Plant biomass, inoculum preparation and agroinfiltration

[0343] Nicotiana benthamiana plants were grown from seeds in flat plots filled with commercial peat moss substrate. The plants were grown in a greenhouse under a 16 / 8 photoperiod and a temperature of 25°C daytime and 20°C nighttime. Three weeks after sowing, individual plantlets were selected, transplanted into pots, and grown in the greenhouse under the same environmental conditions for an additional three weeks.

[0344] Agrobacterium transfected with each wild-type influenza virus hemagglutinin or mutant influenza virus hemagglutinin expression vector was 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 carbenicillin at pH 5.6 until they reached an OD between 0.6 and 1.6. 600 . The Agrobacterium suspension was centrifuged before use and resuspended in infiltration medium (10 mM MgCl2 and 10 mM MES, pH 5.6) and stored at 4°C overnight. On the day of infiltration, the culture batch was diluted to 2.5 times the culture volume and kept warm before use. In an airtight stainless steel tank with a vacuum of 20-40 Torr, a whole Nicotiana benthamiana plant was inverted in the bacterial suspension for 2 minutes. The plants were returned to the greenhouse and incubated for 6 or 9 days until harvest.

[0345] Leaf harvest and total protein extraction

[0346] Protein was extracted from fresh biomass cut into approximately 1 cm² pieces by overnight enzymatic extraction at room temperature using an orbital shaker. The slurry was then filtered through a large pore nylon filter to remove undigested coarse plant tissue.

[0347] In order to obtain " full process output ", slurry is centrifuged to remove protoplasts and intracellular pollutants. Make supernatant clarification by depth filtration.Then the clarified part is loaded on the cation exchange column, adopts the progressive elution step that improves sodium chloride concentration gradually.With TFF concentrated purified VLP, with preparation buffer its diafiltration, and make it pass through filter.By the protein content of the VLP of BCA test analysis purification, and by hemagglutination assay analysis activity.By comparing the protein output of the new construct with the protein output of the natural construct as a control, obtain relative yield.

[0348] In order to obtain " rear density gradient yield ", slurry is centrifuged to remove protoplasts and intracellular pollutants. The supernatant is further centrifuged to remove extra debris. With glass fiber filter, supernatant is carried out depth filtration clarification. Then the clarified part is loaded on the discontinuous iodixanol density gradient device. Carry out separation density gradient centrifugation as follows: prepare 38 milliliters of test tubes that comprise discontinuous iodixanol density gradient in Tris buffer (35%, 30%, 25%, 20%, 15%, 10% and 5% continuous layer), and cover with clarification extract. Under 120000g, gradient solution is centrifuged 2 hours (4 ℃). After centrifugation, discard the initial 5 milliliters collected from bottom to top, and collect the next 5 milliliters for carrying out protein content analysis (BCA), activity measurement (hemagglutination test) and the intensity measurement (densitometric determination) of HAO band on simplified SDS-PAGE. By comparing the HAO band intensity of the new construct with the HAO band intensity of the natural construct used as a control, relative yield is obtained.

[0349] Hemagglutination test

[0350] The hemagglutination assay was based on the method described by Nayak and Reichl (2004). Briefly, a serial dilution of the test sample (100 μl) was performed in a V-bottom 96-well microtiter plate containing 100 μl of PBS, leaving 100 μl of the diluted sample in each well. 100 μl of a 0.25% turkey (for H1) red blood cell suspension (from BioLink Inc., New York, USA) was added to each well, and the plate was incubated for 2 hours at room temperature. The reciprocal of the highest dilution indicating complete hemagglutination was recorded as the hemagglutinin activity. Simultaneously, a recombinant hemagglutinin standard (A / Vietnam / 1203 / 2004H5N1) (from Protein Science Corporation, Meriden, Connecticut, USA) was diluted in PBS and placed on each plate as a control.

[0351] Protein analysis and immunoblotting

[0352] Immunoblotting was performed by first incubation with the primary mAb diluted 1 / 500 in 2% skim milk in 0.1% TBS-Tween 20. Peroxidase-conjugated goat anti-mouse antibody (Jackson Immunoresearch, cat# 115-035-146) diluted 1 / 10,000 was used as the secondary antibody, and chemiluminescent detection was performed in 2% skim milk in 0.1% TBS-Tween 20 immunoreactive complex using luminol as a substrate (Roche Diagnostics Corporation). Horseradish peroxidase-enzyme conjugation of human IgG antibodies was performed using an activated peroxidase conjugation kit (Pierce, Rockford, IL).

[0353] Example 3: Production of influenza virus hemagglutinin protein in plants

[0354] A. Modification of H1 hemagglutinin

[0355] Influenza virus hemagglutinin constructs were generated using techniques well known in the art (see Example 1). Table 4 below summarizes wild-type and mutant hemagglutinin proteins, primers, templates, and products. The sequences used are listed in Example 4 and the sequence listing.

[0356] F390D A / California / 07 / 09 mutant H1

[0357] F390D A / California / 07 / 09 mutant H1 was constructed by mutating the phenylalanine residue at position 390 of wild-type A / California / 07 / 09 H1 to aspartic acid (Construct No. 2980). Figure 2A and 2B As shown, purified extracts from N. benthamiana plants infiltrated with construct No. 2980 exhibited approximately a 60% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / California / 07 / 09H1 (construct No. 1314). Figure 2C As shown, N. benthamiana infiltrated with construct no. 2980 exhibited approximately 60% increased VLP production following iodixanol gradient purification compared to plants infiltrated with the wild-type construct.

[0358] L429M A / California / 07 / 09 mutant H1

[0359] L429M A / California / 07 / 09 mutant H1 was constructed by mutating the leucine residue at position 429 of wild-type A / California / 07 / 09 H1 to methionine (Construct No. 2962). Figure 2A and 2B As shown, purified extracts from N. benthamiana plants infiltrated with construct No. 2962 exhibited approximately 20% increased hemagglutination titers compared to extracts from N. benthamiana plants infiltrated with wild-type A / California / 07 / 09H1 (construct No. 1314). Figure 2C As shown, N. benthamiana infiltrated with construct no. 2962 exhibited approximately 30% increased VLP production following iodixanol gradient purification compared to plants infiltrated with the wild-type construct.

[0360] F390D+L429M A / California / 07 / 09 mutant H1

[0361] The F390D+L429M A / California / 07 / 09 mutant H1 was constructed by introducing a double mutation into the A / California / 07 / 09 H1 wild-type sequence, wherein the phenylalanine at position 390 was mutated to aspartic acid and the leucine at position 429 was mutated to methionine (Construct No. 2995). Figure 2B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 2995 exhibited approximately 60% increased hemagglutination titers compared to extracts from N. benthamiana plants infiltrated with wild-type A / California / 07 / 09H1 (Construct No. 1314).

[0362] N97D A / Michigan / 45 / 15 mutant H1

[0363] The N97D A / Michigan / 45 / 15 mutant H1 was constructed by mutating the asparagine residue at position 97 of wild-type A / Michigan / 45 / 15 H1 to aspartic acid (Construct No. 3774). Figure 3A and 3B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3774 exhibited approximately 1100% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0364] K374E A / Michigan / 45 / 15 mutant H1

[0365] K374E A / Michigan / 45 / 15 mutant H1 was constructed by mutating the lysine residue at position 374 of wild-type A / Michigan / 45 / 15 H1 to glutamic acid (Construct No. 3771). Figure 3A and 3B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3771 exhibited approximately 1100% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0366] F390D A / Michigan / 45 / 15 mutant H1

[0367] The F390D A / Michigan / 45 / 15 mutant H1 was constructed by mutating the phenylalanine residue at position 390 of the wild-type A / Michigan / 45 / 15 H1 to aspartic acid (Construct No. 3641). Figure 3B As shown, purified extracts from N. benthamiana plants infiltrated with construct No. 3641 exhibited similar hemagglutination activity compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (construct No. 3640). However, the overall yield of purified VLPs with H1 was increased by 172% compared to wild-type (see Table 5C).

[0368] L429M A / Michigan / 45 / 15 mutant H1

[0369] L429M A / Michigan / 45 / 15 mutant H1 was constructed by mutating the leucine residue at position 429 of wild-type A / Michigan / 45 / 15 H1 to aspartic acid (Construct No. 3643). Figure 3B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3643 exhibited approximately a 500% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0370] N97D+K374E A / Michigan / 45 / 15 mutant H1

[0371] The N97D+K374E A / Michigan / 45 / 15 mutant H1 was constructed by introducing a double mutation into the wild-type sequence of A / Michigan / 45 / 15, wherein the asparagine at position 97 was replaced by an aspartic acid residue and the lysine at position 374 was replaced by a glutamic acid residue (Construct No. 3880). Figure 3B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3880 exhibited approximately 1100% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0372] F390D+L429M A / Michigan / 45 / 15 mutant H1

[0373] The F390D+L429M A / Michigan / 45 / 15 mutant H1 was constructed by introducing a double mutation into the wild-type sequence of A / Michigan / 45 / 15, wherein the phenylalanine at position 390 was mutated to an aspartic acid residue and the leucine at position 429 was replaced by methionine (Construct No. 3703). Figure 3B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3703 exhibited approximately 1100% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0374] N97D+F390D+L429M A / Michigan / 45 / 15 mutant H1

[0375] The N97D+F390D+L429M A / Michigan / 45 / 15 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Michigan / 45 / 15, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 3879). Figure 3B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3879 exhibited approximately a 2300% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0376] K374E+F390D+L429M A / Michigan / 45 / 15 mutant H1

[0377] K374E+F390D+L429M A / Michigan / 45 / 15 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Michigan / 45 / 15, wherein the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 3878). Figure 3B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3878 exhibited approximately a 2500% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0378] N97D+K374E+F390D+L429M A / Michigan / 45 / 15 mutant H1

[0379] The N97D+K374E+F390D+L429M A / Michigan / 45 / 15 mutant H1 was constructed by introducing four mutations into the wild-type sequence of A / Michigan / 45 / 15, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 3881). Figure 3B As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3881 exhibited approximately a 3300% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0380] N97D+F390D+L429M A / Massachusetts / 06 / 17 mutant H1

[0381] The N97D+F390D+L429M A / Massachusetts / 06 / 17 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Massachusetts / 06 / 17, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4093). Figure 4BAs shown, purified extracts of N. benthamiana plants infiltrated with construct No. 4093 exhibited approximately 40% increased hemagglutination titers compared to extracts of N. benthamiana plants infiltrated with the double mutant construct F390D+L429M A / Massachusetts / 06 / 17 mutant H1 (construct No. 4091).

[0382] K374E+F390D+L429M A / Massachusetts / 06 / 17 mutant H1

[0383] K374E+F390D+L429M A / Massachusetts / 06 / 17 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Massachusetts / 06 / 17, wherein the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4092). Figure 4B As shown, purified extracts of N. benthamiana plants infiltrated with construct No. 4092 exhibited approximately 50% increased hemagglutination titers compared to extracts of N. benthamiana plants infiltrated with the double mutant construct F390D+L429M A / Massachusetts / 06 / 17 mutant H1 (construct No. 4091).

[0384] N97D+K374E+F390D+L429M A / Massachusetts / 06 / 17 mutant H1

[0385] The N97D+K374E+F390D+L429M A / Massachusetts / 06 / 17 mutant H1 was constructed by introducing four mutations into the wild-type sequence of A / Massachusetts / 06 / 17, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4094). Figure 4B As shown, purified extracts of N. benthamiana plants infiltrated with construct No. 4094 exhibited approximately 70% increased hemagglutination titers compared to extracts of N. benthamiana plants infiltrated with the double mutant construct F390D+L429M A / Massachusetts / 06 / 17 mutant H1 (construct No. 4091).

[0386] N97D+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1

[0387] The N97D+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Costa Rica / 0513 / 16, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4717). Figure 4B As shown, purified extracts of N. benthamiana plants infiltrated with construct No. 4717 exhibited approximately 100% increase in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with the double mutant construct F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 (construct No. 4715).

[0388] K374E+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1

[0389] K374E+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Costa Rica / 0513 / 16, wherein the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4716). Figure 4B As shown, purified extracts of N. benthamiana plants infiltrated with construct No. 4716 exhibited approximately 10% increase in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with the double mutant construct F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 (construct No. 4715).

[0390] N97D+K374E+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1

[0391] The N97D+K374E+F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 was constructed by introducing four mutations into the wild-type sequence of A / Costa Rica / 0513 / 16, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4718). Figure 4BAs shown, purified extracts of N. benthamiana plants infiltrated with construct No. 4718 exhibited approximately 140% increase in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with the double mutant construct F390D+L429M A / Costa Rica / 0513 / 16 mutant H1 (construct No. 4715).

[0392] N97D A / Honduras / 17734 / 16 mutant H1

[0393] The N97D A / Honduras / 17734 / 16 mutant H1 was constructed by mutating the asparagine residue at position 97 of wild-type A / Honduras / 17734 / 16 H1 to aspartic acid (Construct No. 3950). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3950 exhibited approximately a 300% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Honduras / 17734 / 16H1 (Construct No. 3944).

[0394] K374E A / Honduras / 17734 / 16 mutant H1

[0395] K374E A / Honduras / 17734 / 16 mutant H1 was constructed by mutating the lysine residue at position 374 of wild-type A / Honduras / 17734 / 16 H1 to glutamic acid (Construct No. 3948). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3948 exhibited approximately 100% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Honduras / 17734 / 16H1 (Construct No. 3944).

[0396] F390D A / Honduras / 17734 / 16 mutant H1

[0397] The F390D A / Honduras / 17734 / 16 mutant H1 was constructed by mutating the phenylalanine residue at position 390 of wild-type A / Honduras / 17734 / 16 H1 to aspartic acid (Construct No. 3945). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3945 exhibited approximately 30% increased hemagglutination titers compared to extracts from N. benthamiana plants infiltrated with wild-type A / Honduras / 17734 / 16 H1 (Construct No. 3944).

[0398] L429M A / Honduras / 17734 / 16 mutant H1

[0399] L429M A / Honduras / 17734 / 16 mutant H1 was constructed by mutating the leucine residue at position 429 of wild-type A / Honduras / 17734 / 16 H1 to methionine (Construct No. 3949). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3949 exhibited approximately a 400% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Honduras / 17734 / 16 H1 (Construct No. 3944).

[0400] F390D+L429M A / Honduras / 17734 / 16 mutant H1

[0401] The F390D+L429M A / Honduras / 17734 / 16 mutant H1 was constructed by introducing a double mutation into the wild-type sequence of A / Honduras / 17734 / 16, wherein the phenylalanine at position 390 was mutated to an aspartic acid residue and the leucine at position 429 was replaced by methionine (Construct No. 3946). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3946 exhibited approximately a 300% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Honduras / 17734 / 16 H1 (Construct No. 3944).

[0402] N97D+F390D+L429M A / Honduras / 17734 / 16 mutant H1

[0403] The N97D+F390D+L429M A / Honduras / 17734 / 16 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Honduras / 17734 / 16, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 3951). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3951 exhibited approximately a 600% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Honduras / 17734 / 16 H1 (Construct No. 3944).

[0404] N97D A / Darwin / 11 / 15 mutant H1

[0405] The N97D A / Darwin / 11 / 15 mutant H1 was constructed by mutating the asparagine residue at position 97 of the wild-type A / Darwin / 11 / 15 H1 to aspartic acid (Construct No. 3990). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3990 exhibited approximately a 200% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Darwin / 11 / 15 H1 (Construct No. 3984).

[0406] K374E A / Darwin / 11 / 15 mutant H1

[0407] K374E A / Darwin / 11 / 15 mutant H1 was constructed by mutating the lysine residue at position 374 of wild-type A / Darwin / 11 / 15 H1 to glutamic acid (Construct No. 3988). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3988 exhibited approximately a 300% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Darwin / 11 / 15 H1 (Construct No. 3984).

[0408] F390D A / Darwin / 11 / 15 mutant H1

[0409] The F390D A / Darwin / 11 / 15 mutant H1 was constructed by mutating the phenylalanine residue at position 390 of the wild-type A / Darwin / 11 / 15 H1 to aspartic acid (Construct No. 3985). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3985 exhibited approximately 50% increased hemagglutination titers compared to extracts from N. benthamiana plants infiltrated with wild-type A / Darwin / 11 / 15 H1 (Construct No. 3984).

[0410] L429M A / Darwin / 11 / 15 mutant H1

[0411] L429M A / Darwin / 11 / 15 mutant H1 was constructed by mutating the leucine residue at position 429 of wild-type A / Darwin / 11 / 15 H1 to methionine (Construct No. 3989). Figure 4AAs shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3989 exhibited approximately a 500% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Darwin / 11 / 15 H1 (Construct No. 3984).

[0412] F390D+L429M A / Darwin / 11 / 15 mutant H1

[0413] The F390D+L429M A / Darwin / 11 / 15 mutant H1 was constructed by introducing a double mutation into the wild-type sequence of A / Darwin / 11 / 15, wherein the phenylalanine at position 390 was mutated to an aspartic acid residue and the leucine at position 429 was replaced by methionine (Construct No. 3986). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3986 exhibited approximately a 300% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Darwin / 11 / 15 H1 (Construct No. 3984).

[0414] N97D+F390D+L429M A / Darwin / 111 / 15 mutant H1

[0415] The N97D+F390D+L429M A / Darwin / 11 / 15 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Darwin / 11 / 15, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 3991). Figure 4A As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3991 exhibited approximately 1100% increase in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild type A / Darwin / 11 / 15 H1 (Construct No. 3984).

[0416] F390D+L429M A / Paris / 1227 / 2017 mutant H1

[0417] F390D+L429M A / Paris / 1227 / 2017 mutant H1 was constructed by introducing a double mutation into the wild-type sequence of A / Paris / 1227 / 2017, wherein the phenylalanine at position 390 was mutated to an aspartic acid residue and the leucine at position 429 was replaced by methionine (Construct No. 4765). Figure 4CAs shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 4765 exhibited approximately 117% increased hemagglutination titers compared to extracts from N. benthamiana plants infiltrated with F390D+L429M A / Massachusetts / 06 / 17 mutant H1 (Construct No. 4091).

[0418] K374+F390D+L429M A / Paris / 1227 / 2017

[0419] K374E+F390D+L429M A / Paris / 1227 / 2017 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Paris / 1227 / 2017, wherein the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4766). Figure 4C As shown, purified extracts of N. benthamiana plants infiltrated with Construct No. 4766 exhibited approximately 127% increase in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with F390D+L429M A / Paris / 1227 / 2017 mutant H1 (Construct No. 4765).

[0420] N97D+F390D+L429M A / Paris / 1227 / 2017

[0421] The N97D+F390D+L429M A / Paris / 1227 / 2017 mutant H1 was constructed by introducing three mutations into the wild-type sequence of A / Paris / 1227 / 2017, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4767). Figure 4C As shown, purified extracts of N. benthamiana plants infiltrated with Construct No. 4767 exhibited approximately 171% increase in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with F390D+L429M A / Paris / 1227 / 2017 mutant H1 (Construct No. 4765).

[0422] N97D+K374E+F390D+L429M A / Paris / 1227 / 2017

[0423] The N97D+K374E+F390D+L429M A / Paris / 1227 / 2017 mutant H1 was constructed by introducing four mutations into the wild-type sequence of A / Paris / 1227 / 2017, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4768). Figure 4C As shown, purified extracts of N. benthamiana plants infiltrated with Construct No. 4768 exhibited approximately 230% increase in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with F390D+L429M A / Paris / 1227 / 2017 mutant H1 (Construct No. 4765).

[0424] F390D+L429M A / Norway / 2147 / 2017 mutant H1

[0425] F390D+L429M A / Norway / 2147 / 2017 mutant H1 was constructed by introducing a double mutation into the wild-type sequence of A / Norway / 2147 / 2017, wherein the phenylalanine at position 390 was mutated to an aspartic acid residue and the leucine at position 429 was replaced by methionine (Construct No. 4775). Figure 4C As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 4775 exhibited approximately 112% increased hemagglutination titers compared to extracts from N. benthamiana plants infiltrated with F390D+L429MA / Massachusetts / 06 / 17 mutant H1 (Construct No. 4091).

[0426] K374+F390D+L429M A / Norway / 2147 / 2017

[0427] K374E+F390D+L429M A / Norway / 2147 / 2017 mutant H1 was constructed by introducing triple mutations into the wild-type sequence of A / Norway / 2147 / 2017, wherein the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4776). Figure 4C As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 4776 exhibited approximately 128% increased hemagglutination titers compared to extracts from N. benthamiana plants infiltrated with F390D+L429M A / Norway / 2147 / 2017 mutant H1 (Construct No. 4775).

[0428] N97D+F390D+L429M A / Norway / 2147 / 2017

[0429] The N97D+F390D+L429M A / Norway / 2147 / 2017 mutant H1 was constructed by introducing three mutations into the wild-type sequence of A / Norway / 2147 / 2017, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4777). Figure 4C As shown, purified extracts of N. benthamiana plants infiltrated with Construct No. 4777 exhibited approximately 200% increase in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with F390D+L429M A / Norway / 2147 / 2017 mutant H1 (Construct No. 4775).

[0430] N97D+K374E+F390D+L429M A / Norway / 2147 / 2017

[0431] The N97D+K374E+F390D+L429M A / Norway / 2147 / 2017 mutant H1 was constructed by introducing four mutations into the wild-type sequence of A / Norway / 2147 / 2017, wherein the asparagine at position 97 was mutated to an aspartic acid residue, the lysine at position 374 was mutated to a glutamic acid residue, the phenylalanine at position 390 was mutated to an aspartic acid residue, and the leucine at position 429 was replaced by a methionine residue (Construct No. 4778). Figure 4C As shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 4778 exhibited approximately 213% increased hemagglutination titers compared to extracts from N. benthamiana plants infiltrated with F390D+L429M A / Norway / 2147 / 2017 mutant H1 (Construct No. 4775).

[0432] N380A A / Michigan / 45 / 15

[0433] The N380A A / Michigan / 45 / 15 mutant H1 was constructed by mutating the asparagine residue at position 380 of the wild-type A / Michigan / 45 / 15 H1 to an alanine residue (Construct No. 3644). Figure 3BAs shown, purified extracts from N. benthamiana plants infiltrated with Construct No. 3644 exhibited approximately 80% reduction in hemagglutination titer compared to extracts from N. benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640).

[0434] N380A+F390D A / Michigan / 45 / 15

[0435] A N380A+F390D A / Michigan / 45 / 15 mutant H1 was also constructed, in which a double mutation was introduced into the wild-type A / Michigan / 45 / 15 H1 by replacing the asparagine at position 380 with an alanine residue and the phenylalanine at position 390 with an aspartic acid residue (Construct No. 3704). Figure 3B As shown, purified extracts of Nicotiana benthamiana plants infiltrated with construct No. 3704 exhibited approximately a 50% reduction in hemagglutination titer compared to extracts of Nicotiana benthamiana plants infiltrated with wild-type A / Michigan / 45 / 15 H1 (Construct No. 3640). Given the reduction in hemagglutination titer observed with the N380A A / Michigan / 45 / 15 mutant H1, these results suggest that mutation of the asparagine residue at position 380 has a negative effect on the expression of influenza virus hemagglutinin proteins in plants and / or the stability of influenza virus hemagglutinin proteins.

[0436] One or more mutations described herein specifically increase the yield and VLP yield of influenza virus hemagglutinin protein in plants. It has been observed that mutations at other positions significantly reduce the accumulation amount or VLP yield of influenza virus hemagglutinin protein in plant cells, or have no significant effect on it.

[0437] It was also observed that the increased hemagglutination titers achieved with influenza virus hemagglutinin proteins comprising one or more mutations described herein were specific for H1 influenza virus hemagglutinin. Similar enhancements were not observed in plants infiltrated with constructs encoding mutant influenza virus hemagglutinins derived from non-H1 strains.

[0438] For example, F393D A / Indonesia / 5 / 2005 mutant H5 (Construct No. 3680) was constructed by mutating the phenylalanine residue at position 393 of wild-type A / Indonesia / 5 / 2005 H5 to aspartic acid. Figure 5 As shown, purified extracts of N. benthamiana plants infiltrated with construct no. 3680 exhibited approximately 98% reduction in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with wild type A / Indonesia / 5 / 2005 H5 (construct no. 2295).

[0439] Similarly, purified extracts of N. benthamiana plants infiltrated with F392D A / Egypt / N04915 / 2014 mutant H5 (Construct No. 3690) exhibited approximately 99% reduction in hemagglutination titer compared to extracts of N. benthamiana plants infiltrated with wild-type A / E04915 / 2014 H5 (Construct No. 3645) (see Figure 5 , Table 6).

[0440] One or more mutations described herein specifically increase the yield and VLP yield of influenza virus hemagglutinin protein in plants. It has been observed that mutations at other positions significantly reduce the accumulation amount or VLP yield of influenza virus hemagglutinin protein in plant cells, or have no significant effect on it.

[0441]

[0442]

[0443]

[0444] Example 3: Hemagglutination titer, post-density gradient yield, and overall process yield

[0445] The measured hemagglutination titers are summarized in Table 5A. The hemagglutination titers were measured as described in Example 2. The relative hemagglutination titers were obtained by comparing the hemagglutination titers of the mutant or modified hemagglutinin proteins with the hemagglutination titers of the wild-type hemagglutinin (Table 5A).

[0446] Table 5B summarizes the measured post-density gradient yields. Post-density gradient yields were measured as described in Example 2. Relative yields were obtained by comparing the hemagglutinin band intensity of the mutant or modified hemagglutinin proteins with the hemagglutinin band intensity of the wild-type hemagglutinin (Table 5B).

[0447] Table 5C summarizes the measured overall yields. Overall yields were obtained as described in Example 2. Relative yields were obtained by comparing the protein yields of mutant or modified hemagglutinin proteins with the protein yield of wild-type hemagglutinin (Table 5C).

[0448]

[0449]

[0450] Example 4: Sequence

[0451] The following sequences were used (see Table 4):

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487] All cited documents are incorporated herein by reference.

[0488] The invention has been described with reference to one or more embodiments. However, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the scope of the invention as defined in the appended claims. Sequence Listing <110> ARAMIS BIOTECHNOLOGIES INC. <120> Influenza virus hemagglutinin mutants <130> BM000910 <140> 201980038608.4 <141> 2019-06-27 <150> US 62 / 690,780 <151> 2018-06-27 <160> 148 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Cal DNA <400> 1 atggcgaaaa acgttgcgat tttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg ctgacacatt atgtataggt tatcatgcga acaatcaac agacactgta 120 gacagtac agaagaa tgtaaccta acacactctg ttaccttct agaaaag 180 cataacggga aactatgca actaagagggg gtagccccat tgcattgggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagctcatgg 300 tcctacattg tggaacacc tagttcagac aatggaacgt gttacccagg agatttcatc 360 gatttagagg agctagaga gcaattgagc tcagtgtcat catttgaag gtttgagata 420 ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt 480 cctcatgctg gagcaaaag cttctacaa aatttaat ggctagttaa aaaaggaaat 540 tcatacccaa agctcagcaa atcctacatt atgataag ggaagaagt cctcgtgcta 600 tggggcattc accatccatc tactagtgct gaccacaa gtctctca gatgcagat 660 gcatatgttt ttgtggggtc atcagatac agcaagagt tcaagccgga atagcaata 720 agacccaag tgagggatca agaagggaga atgaactatt actggacact agtagagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgata tgcattcgca 840 atggaaagaa atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat 900 acaacttgtc aaacacccaa gggtgctata aacaccagcc tcccattca gatacat 960 ccgatcacaa ttggaaatg tccaaatat gtaaaaagca caaattgag actggccaca 1020 ggattgagga atatcccgtc tattcaatct agaggactt tggggccat tgccggttc 1080 attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgacctgaag agcacacaga atgccattga cgagattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagt tcacagcagt aggtaaag 1260 ttcaaccacc tggaaaaag atagagaat ttaaaaaa aagttgatga tggttttcctg 1320 gatatttgga cttacaatgc cgaactgttg gttctattgg aaatgaag aactttggac 1380 taccacgatt caatgtgaa gaacttatat gaaaggta gaagccagct aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 aggagaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 2 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Figure AA <400> 2 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Only Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val 530 535 540 Ala Ser Ser Leu Val Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp 545,550,555,560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 3 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 My DNA <400> 3 atggcgaaaa acgttgcgat ttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaattcaac agacactgta 120 gacacagtac tagaaaagaa tgtaacagta acacactctg ttaaccttct ggaagacaag 180 cataacggaa aactatgcaa actaagaggg gtagccccat tgcatttggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagttcatgg 300 tcctacattg tggaaacatc taattcagac aatggaacgt gttacccagg agatttcatc 360 aattatgagg agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata 420 ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt 480 cctcacgctg gagcaaaaag cttctacaaa aacttgatat ggctagttaa aaaaggaat tcatacccaa agcttaacca atcctacatt aatgataaag ggaaagaagt cctcgtgctg tggggcattc accatccatc tactactgct gaccaacaaa gtctctatca gaatgcagat gcatatgttt ttgtggggac atcaagatac agcaagaagt tcaagccgga aatagcaaca 720 agacccaaag tgagggatca agagggaga atgaactatt actggacact agtagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcaca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc agacacccga gggtgctata aacaccagcc tcccatttca gaatacat ccgatcacaa ttggaaaatg tccaaagtat gtaaaaagca caaattgag actggccaca ggattgagga atgttccgtc tattcaatct agaggcctat tcggggccat tgccggcttc attgaagggg ggtggacagg gatggtag ggatggtacg gttatcacca tcaaaatgag caggggtcag price cgacctgag agcacacaaa atgccattga caagattact aaaaagtaa attctgttat tgaaagatg atacacagt tcacagcagt gggtaaag 1260 ttcaaccacc tggaaaaag atagagaat ctaataaaa aagttgatga tggttttcctg 1320 gatatttgga cttacaatgc cgaactgttg gttctattgg aaatgaag aactttggac 1380 tatcacgatt caatgtgaa gaacttgtat gaaaaaaccagtt aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 agagaaaaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagcttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 4 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Mich AA <400> 4 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Ser Asn Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asn Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Asn Gln Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Thr Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Thr Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Thr 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Thr Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Glu Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485,490,495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys 515,520,525 The Glu Ser of Thr Arg and Tyr Gln and the Glu Ser of Thr Val 530 535 540 To Be To Be A Leo Val To Be A Leo Gly Ala Ile To Be Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565,570 <210> 5 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Mass DNA <400> 5 atggcgaaaa acgttgcgat tttcggctta ttgttttctc ttctgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaatcac agacactgta 120 gacacagtac tgtacagta acacactctg ttaccttct gggagacag 180 cataacggaa actagcaa actaagagggg gtagccccat tgcattgggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagatcatgg tcctacattg tggaacatc taattcagac aatggaacgt gttacccagg agatttcatc aattatgagg agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt cctcacgctg gagcaaaaag cttctacaaa aacttgatat ggctagttaa aaaaggaat tcatacccaa agcttaacca atcctacatt aatgataaag ggaaagaagt cctcgtgctg tggggcattc accatccatc tactactgct gaccaacaaa gtctctatca gaatgcagat gcatatgttt ttgtggggac atcaagatac agcaagaagt tcaagccgga aatagcaaca 720 agacccaaag tgagggatca agagggaga atgaactatt actggacact agtagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcaca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc agacacccga gggtgctata aacaccagcc tcccatttca gaatgtgcat ccgatcacaa ttggaaatg tccaagtat gtaaaaagca caaattgag actggccaca 1020 ggattgagga atgttccgtc tattcaatct agaggcctat tcggggccat tgccggctc 1080 attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgacctgaag agcacacaaa atgccattga cavattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagt tcacagcagt gggtaaag 1260 ttcaaccacc tggaaaaag atagagaat ctaataaaa aagttgatga tggttttcctg 1320 gatatttgga cttacaatgc cgaactgttg gttctattgg aaatgaag aactttggac 1380 tatcacgatt caatgtgaa gaacttgtat gaaaaaaccagtt aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 agagaaaaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagcttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 6 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Mass AA <400> 6 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Arg Ser Trp Ser Tyr Ile Val Glu Thr Ser Asn Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asn Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Asn Gln Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Thr Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Thr Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Thr 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Thr Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Glu Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Val His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val 530 535 540 Ala Ser Ser Leu Val Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 7 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 CostaR DNA <400> 7 atggcgaaaa acgttgcgat ttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaattcaac agacactgta 120 gacacagtac tagaaaagaa tgtaacagta acacactctg ttaaccttct ggaagacaag 180 cataacggaa aactatgcaa actaagaggg gtagccccat tgcatttggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagttcatgg 300 tcctacattg tggaaacatc taattcagac aatggaacgt gttacccagg agatttcatc 360 aattatgagg agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata 420 ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt 480 cctcacgctg gagcaaaaag cttctacaaa aacttgatat ggctagttaa aaaaggaaat 540 tcatacccaa agcttaacca atcctacgtg aatgataaag ggaaagaagt cctcgtgctg 600 tggggcattc accatccacc cactactgct gaccaacaaa gtctctatca gaatgcagat 660 gcatatgttt ttgtggggac atcaaagtac agcaagaagt tcaagccgga aatagcaaca 720 agacccaaag tgagggatca agagggaga atgaactatt actggacact agtagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcaca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc agacacccga gggtgctata aacaccagcc tcccatttca gaatacat ccgatcacaa ttggaaaatg tccaaagtat gtaaaaagca caaattgag actggccaca ggattgagga atgttccgtc tattcaatct agaggcctat tcggggccat tgccggcttc attgaagggg ggtggacagg gatggtag ggatggtacg gttatcacca tcaaaatgag caggggtcag price cgacctgag agcacacaaa atgccattga caagattact aacaaagtaa attctgttat tgaaaagatg aatacacagt tcacagcagt gggtaaagag ttcaaccacc tggaaaaaag aatagagaat ctaaataaaa aagttgatga tggtttcctg gacatttgga cttacaatgc cgaactgttg gttctattgg aaaatgaaag aactttggac tatcacgatt caaatgtgaa gaacttgtat gaaaaagtaa gaaaccagtt aaaaaacaat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca aatgcgataa cacgtgcatg 1500 gaaagtgtca aaaatgggac ttatgactac caaaatact cagaggaagc aaaattaaac 1560 agagaaaaaa tagatggggt aaagctggaa tcaacaagga tttaccagat tttggcgatc 1620 tattcaactg tcgccagttc attggtactg gtagtctccc tgggggcaat cagcttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 8 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 CostaR AA <400> 8 Put Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Ser Asn Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asn Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Asn Gln Ser Tyr Val Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Pro Thr 195 200 205 Thr Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Thr Ser Lys Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Thr 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Thr Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Glu Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys 515 520 525 Leu Glu Make Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Make Thr Val 530 535 540 Here Dear Dear Leu Val Leu Val Val Dear Leu Gly Here Ile Dear Phe Trp 545,550,555,560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 9 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Dog DNA <400> 9 atggcgaaaa acgttgcgat ttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaattcaac agacactgta 120 gacacagtac tagaaaagaa tgtaacagta acacactctg ttaaccttct agaagacaag 180 cataacggga aactatgcaa actaagaggg gtacccccat tgcatttggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaaccac tctccacagc aagttcatgg 300 tcctacattg tggaaacatc tagttcagac aatggaacgt gttacccagg agatttcatc 360 aattatgagg agctaaggga gcaattgagc tcagtgtcat catttgagag atttgagata ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac agcagcatgt cctcacgctg gggcaaaaag cttctacaaa aatttaat ggctagttaa aaaaggaaat tcatacccaa agctcagcca atcctacatt aatgataaag gaaaagaagt cctcgtgctg tggggcattc accatccatc tactactgct gaccaacaaa gtctctatca gaatgcagat gcatatgttt ttgtggggac atcaagatac agcaagaagt tcaagccgga aatagcaata 720 agacccaaag tgagggatca agagggaga atgaactatt actggacact agtagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcaca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc agacacccga gggtgctata aacaccagcc tcccatttca gaatacat ccgatcacaa ttgggaaatg tccaaagtat gtaaaaagca caaattgag actggccaca ggattgagga atgtcccatc tattcaatct agaggcctat tcggggcgat tgccggcttc 1080. attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgaccagaag agcacacaa gtgccattga caaaattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagt tcacagcagt gggtaaag 1260 ttcaccact tggaaaaag atagagaat ttaaaaa aagttgatga tggttttccctg 1320 gatatttgga cttacaatgc cgaactgctg gttctattgg aaatgaaag aactttggac 1380 taccacgact aaatgtgaa gaacttgtat gaaaggta gaaaccagtt aaaaaacaat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 agagaaaaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagcttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 10 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Hond AA <400> 10 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Pro Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Pro Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asn Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Gln Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Thr Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Thr Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Thr Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Glu Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Gln Lys Ser Thr Gln Ser Ala Ile Asp Lys Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val 530 535 540 Ala Ser Ser Leu Val Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 11 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Bull DNA <400> 11 atggcgaaaa acgttgcgat tttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaatcac agacactgta 120 gacagtac agaagaa tgtaaccta acacactctg ttaccttct agaaaag 180 cacaacggga aactatgca actaagagggg gtagccccat tgcattgggg taaatgtaac 240 attgctggct ggatcctggg aaacccagag tgtgaatcac tctccacagc aagttcatgg 300 tcctacattg tggaaacatc tagttcagac aatggaacgt gttacccagg agatttcatc 360 aattatgagg agctagaga gcaattgagc tcagtgtcat catttgaag atttgagata 420 ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt 480 cctcacgctg gagcaaaag cttctacaa atttaat ggctaactaa aaaaggaat 540 tcatacccaa agctcagcca atcctacatt atgataaag ggaaagaat cctcgtgctg 600 tggggcattc accatccatc tactactgct gaccacaa gtctctca gatgcagat 660 gcatatgtttt ttgtggggac atcagatac agcaagaagt tcaagccgga atagcaata 720 agacccaag tgagggatca agaaggaga atgaactatt actggacact agtagagacca 780 ggtgacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcaca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc agacacccga gggtgctata aacaccagcc tcccatttca gaatacat ccgatcacaa ttggaaaatg tccaaagtat gtgaaaagca caaattgag actggccaca ggattgagga atgtcccatc tattcaatct agaggcctat tcggggccat tgccggcttc attgaagggg ggtggacagg gatggtag ggatggtacg gttatcacca tcaaaatgag caggggtcag ggtatgcagc cgacctgaag agcacacaaa atgccattga caaattact aacaaagtaa attctgttat tgaaaagatg aatacacagt tcacagcagt gggtaaagag ttcaaccact tggaaaaaag aatagagaat ttaaataaaa aagttgatga tggtttcctg gacatttgga cttacaatgc cgaactgctg gttctattgg aaaatgaaag aactttggac taccacgatt caatgtga gaacttgtat gaaaggta gaaccagtt aaaaaacaat gccaaggaa ttggaaatgg ttgctttgaa ttttaccaca aatgcgataa cacgtgcatg gaagtgtca aaatgggac ttatgactac ccaaatact caggggaagc aaaattaac 1560 agagaaaaaa tagaggggt aaagctggaa tcaacaaa tttaccaat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagcttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 12 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Part AA <400> 12 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Only Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Ser Ser Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asn Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Thr 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Gln Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Ile Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Thr Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Thr Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Thr Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Glu Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Gly Glu Ala Lys Leu Asn Arg Glu Lys Ile Glu Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val 530 535 540 Ala Ser Ser Leu Val Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 13 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer IF-CPMV(fl5'UTR)_SpPDI.c <400> 13 tcgtgcttcg gcaccagtac aatggcgaaa aacgttgcga ttttcggct 49 <210> 14 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Primer IF-H1cTMCT.S1-4r <400> 14 actaaagaaa ataggccttt aaatacatat tctacactgt agagac 46 <210> 15 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Cal(F390D).r <400> 15 ctctttacct actgctgtgt cctgtgtatt catcttttca ataacagaat tta 53 <210> 16 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Cal(F390D).c <400> 16 ttattgaaaa catgaataca cagtaggtaa aggttcac 50 <210> 17 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Cal-F390D DNA sequence <400> 17 atggcgaaaa acgttgcgat ttcggctta ttgttttctc ttctgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaatcaac agacactgta 120 gacagtac agaagaa tgtaaccta acacactctg ttaccttct agaaaag 180 cataacggga aactatgca actaagagggg gtagccccat tgcattgggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagctcatgg 300 tcctacattg tggaacacc tagttcagac aatggaacgt gttacccagg agatttcatc 360 gatttagagg agctagaga gcaattgagc tcagtgtcat catttgaag gtttgagata 420 ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt 480 cctcatgctg gagcaaaag cttctacaa aatttaat ggctagttaa aaaaggaaat 540 tcatacccaa agctcagcaa atcctacatt atgataag ggaagaagt cctcgtgcta 600 tggggcattc accatccatc tactagtgct gaccacaa gtctctca gatgcagat 660 gcatatgttt ttgtggggtc atcagatac agcaagagt tcaagccgga atagcaata 720 agacccaag tgagggatca agaagggaga atgaactatt actggacact agtagagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgata tgcattcgca 840 atggaaagaa atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat 900 acaacttgtc aaacacccaa gggtgctata aacaccagcc tcccattca gatacat 960 ccgatcacaa ttggaaatg tccaaatat gtaaaaagca caaattgag actggccaca 1020 ggattgagga atatcccgtc tattcaatct agaggactt tggggccat tgccggttc 1080 attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgacctgaag agcacacaga atgccattga cgagattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagg acaccagt aggtaagg 1260 ttcaaccacc tggaaaaag atagagaat ttaaaaaa aagttgatga tggttttccctg 1320 gatatttgga cttacaatgc cgaactgttg gttctattgg aaatgaag aactttggac 1380 taccacgatt caatgtgaa gaacttatat gaaaggta gaagccagct aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 aggagaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagttctctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 18 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Cal-F390D AA sequence <400> 18 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Asp Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val 530 535 540 Ala Ser Ser Leu Val Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 19 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Cal(L429M).r <400> 19 gttctttcat tttccattag aaccaacagt tcggcattgt aagtccaa 48 <210> 20 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Cal(L429M).c <400> 20 cgaactgttg gttctaatgg aaaatgaaag aactttggac taccacga 48 <210> 21 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Cal-L429M DNA sequence <400> 21 atggcgaaaa acgttgcgat tttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaattcaac agacactgta 120 gacacagtac tagaaaagaa tgtaacagta acacactctg ttaaccttct agaagacaag 180 cataacggga aactatgcaa actaagaggg gtagccccat tgcatttggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagctcatgg 300 tcctacattg tggaaacacc tagttcagac aatggaacgt gttacccagg agatttcatc 360 gattatgagg agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata 420 ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt 480 cctcatgctg gagcaaaaag cttctacaaa aatttaatat ggctagttaa aaaaggaaat 540 tcatacccaa agctcagcaa atcctacatt atgataag ggaagaagt cctcgtgcta 600 tggggcattc accatccatc tactagtgct gaccacaa gtctctca gatgcagat 660 gcatatgttt ttgtggggtc atcagatac agcaagagt tcaagccgga atagcaata 720 agacccaag tgagggatca agaagggaga atgaactatt actggacact agtagagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgata tgcattcgca 840 atggaaagaa atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat 900 acaacttgtc aaacacccaa gggtgctata aacaccagcc tcccattca gatacat 960 ccgatcacaa ttggaaatg tccaaatat gtaaaaagca caaattgag actggccaca 1020 ggattgagga atatcccgtc tattcaatct agaggactt tggggccat tgccggttc 1080 attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgacctgaag agcacacaga atgccattga cgagattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagt tcacagcagt aggtaaag 1260 ttcaaccacc tggaaaaag atagagaat ttaaaaaa aagttgatga tggttttccctg 1320 gatatttgga cttacaatgc cgaactgttg gttctaatgg aaatgaag aactttggac 1380 taccacgatt caatgtgaa gaacttatat gaaaggta gaagccagct aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 aggagaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagttctctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 22 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Cal-L429M AA sequence <400> 22 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys 515,520,525 The Glu Ser of Thr Arg and Tyr Gln and the Glu Ser of Thr Val 530 535 540 To Be To Be A Leo Val To Be A Leo Gly Ala Ile To Be Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565,570 <210> 23 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Cal-F390D+L429M DNA sequence <400> 23 atggcgaaaa acgttgcgat tttcggctta ttgttttctc ttctgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaatcac agacactgta 120 gacagtac agaagaa tgtaaccta acacactctg ttaccttct agaaaag 180 cataacggga aactatgca actaagagggg gtagccccat tgcattgggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagctcatgg 360. tcctacattg tggaaacacc tagttcagac aatggaacgt gttacccagg agatttcatc gattatgagg agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt cctcatgctg gagcaaaaag cttctacaaa aatttaat ggctagttaa aaaaggaat tcatacccaa agctcagcaa atcctacatt aatgataaag ggaaagagt cctcgtgcta tggggcattc accatccatc tactagtgct gaccaacaaa gtctctatca gaatgcagat gcatatgttt ttgtggggtc atcaagatac agcaagaagt tcaagccgga aatagcaata 720 agacccaaag tgagggatca agagggaga atgaactatt actggacact agtagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcgca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc aaacacccaa gggtgctata aacaccagcc tcccatttca gaatacat ccgatcacaa ttggaaatg tccaaatat gtaaaaagca caaattgag actggccaca 1020 ggattgagga atatcccgtc tattcaatct agaggactt tggggccat tgccggttc 1080 attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgacctgaag agcacacaga atgccattga cgagattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagg acaccagt aggtaagg 1260 ttcaaccacc tggaaaaag atagagaat ttaaaaaa aagttgatga tggttttcctg 1320 gatatttgga cttacaatgc cgaactgttg gttctaatgg aaatgaag aactttggac 1380 taccacgatt caatgtgaa gaacttatat gaaaggta gaagccagct aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 aggagaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 24 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Cal-F390D+L429M AA sequence <400> 24 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Pro Ser Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Ser Lys Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Ser Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Ser Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Ile 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Ala Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Lys Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Ile Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Asp Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Ser Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Glu Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val 530 535 540 Ala Ser Ser Leu Val Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 25 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Mich(N97D).r <400> 25 tagctcctca taatcgatga aatctcctgg gtaacacgtt ccattgtctg aa 52 <210> 26 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> H1Mich(N97D).c <400> 26 cccaggagat ttcatcgatt atgaggagct aagagagcaa ttgagctcag 50 <210> 27 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Mich-N97D DNA <400> 27 atggcgaaaa acgttgcgat tttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaattcaac agacactgta 120 gacacagtac tagaaaagaa tgtaacagta acacactctg ttaaccttct ggagacaag 180 cataacggaa aactatgcaa actaagaggg gtagccccat tgcatttggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagttcatgg tcctacattg tggaacatc taattcagac aatggaacgt gttacccagg agatttcatc gattatgagg agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt cctcacgctg gagcaaaaag cttctacaaa aacttgatat ggctagttaa aaaaggaat tcatacccaa agcttaacca atcctacatt aatgataaag ggaaagaagt cctcgtgctg tggggcattc accatccatc tactactgct gaccaacaaa gtctctatca gaatgcagat gcatatgttt ttgtggggac atcaagatac agcaagaagt tcaagccgga aatagcaaca 720 agacccaaag tgagggatca agagggaga atgaactatt actggacact agtagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcaca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc agacacccga gggtgctata aacaccagcc tcccatttca gaatacat ccgatcacaa ttggaaatg tccaagtat gtaaaaagca caaattgag actggccaca 1020 ggattgagga atgttccgtc tattcaatct agaggcctat tcggggccat tgccggctc 1080 attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgacctgaag agcacacaaa atgccattga cavattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagt tcacagcagt gggtaaag 1260 ttcaaccacc tggaaaaag atagagaat ctaataaaa aagttgatga tggttttcctg 1320 gatatttgga cttacaatgc cgaactgttg gttctattgg aaatgaag aactttggac 1380 tatcacgatt caatgtgaa gaacttgtat gaaaaaaccagtt aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 agagaaaaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagcttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 28 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Mich-N97D AA <400> 28 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Ser Asn Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asp Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Asn Gln Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Thr Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Thr Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Thr 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Thr Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Glu Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val 530 535 540 Ala Ser Ser Leu Val Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 29 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Mich(K374E).r <400> 29 tttgttagta atctcgtcaa tggcattttg tgtgctcttc aggtcggctg catatcc 57 <210> 30 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Mich(K374E).c <400> 30 acaaaatgcc attgacgaga ttactaacaa agtaaattct gttatgaaa 50 <210> 31 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Mich-K374E DNA <400> 31 atggcgaaaa acgttgcgat tttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaattcaac agacactgta 120 gacacagtac tagaaaagaa tgtaacagta acacactctg ttaaccttct ggagacaag 180 cataacggaa aactatgcaa actaagaggg gtagccccat tgcatttggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagttcatgg 300 tcctacattg tggaacatc taattcagac aatggaacgt gttacccagg agatttcatc aattatgagg agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt cctcacgctg gagcaaaaag cttctacaaa aacttgatat ggctagttaa aaaaggaat tcatacccaa agcttaacca atcctacatt aatgataaag ggaaagaagt cctcgtgctg tggggcattc accatccatc tactactgct gaccaacaaa gtctctatca gatgcagat gcatatgttt ttgtggggac atcaagatac agcaagaagt tcaagccgga aatagcaaca 720 agacccaaag tgagggatca agagggaga atgaactatt actggacact agtagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcaca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc agacacccga gggtgctata aacaccagcc tcccatttca gaatacat ccgatcacaa ttggaaaatg tccaaagtat gtaaaaagca caaattgag actggccaca ggattgagga atgttccgtc tattcaatct agaggcctat tcggggccat tgccggctc 1080 attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgacctgaag agcacacaaa atgccattga cgagattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagt tcacagcagt gggtaaag 1260 ttcaaccacc tggaaaaag atagagaat ctaataaaa aagttgatga tggttttcctg 1320 gatatttgga cttacaatgc cgaactgttg gttctattgg aaatgaag aactttggac 1380 tatcacgatt caatgtgaa gaacttgtat gaaaaaaccagtt aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 agagaaaaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagcttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 32 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Mich-K374E AA <400> 32 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Ser Asn Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asn Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Asn Gln Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Thr Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Thr Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Thr 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Thr Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Glu Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Glu Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Phe Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leu Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485 490 495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys 515 520 525 Leu Glu Ser Thr Arg Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val 530 535 540 Ala Ser Ser Leu Val Leu Val Val Ser Leu Gly Ala Ile Ser Phe Trp 545 550 555 560 Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile 565 570 <210> 33 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Mich(F390D).r <400> 33 cccactgctg tgtcctgtgt attcatcttt tcaataacag aatttactt 49 <210> 34 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Primer H1Mich(F390D).c <400> 34 aaagatgaat acacaggaca cagcagtggg taaagagttc aaccacctg 49 <210> 35 <211> 1722 <212> DNA <213> Artificial Sequence <220> <223> PDI-H1 Mich-F390D DNA <400> 35 atggcgaaaa acgttgcgat tttcggctta ttgttttctc ttcttgtgtt ggttccttct 60 cagatcttcg cggacacatt atgtataggt tatcatgcga acaattcaac agacactgta 120 gacacagtac tagaaaagaa tgtaacagta acacactctg ttaaccttct ggagacaag 180 cataacggaa aactatgcaa actaagaggg gtagccccat tgcatttggg taaatgtaac 240 attgctggct ggatcctggg aaatccagag tgtgaatcac tctccacagc aagttcatgg 300 tcctacattg tggaaacatc taattcagac aatggaacgt gttacccagg agatttcatc 360 aattatgagg agctaagaga gcaattgagc tcagtgtcat catttgaaag gtttgagata ttccccaaga caagttcatg gcccaatcat gactcgaaca aaggtgtaac ggcagcatgt cctcacgctg gagcaaaaag cttctacaaa aacttgatat ggctagttaa aaaaggaat tcatacccaa agcttaacca atcctacatt aatgataaag ggaaagaagt cctcgtgctg tggggcattc accatccatc tactactgct gaccaacaaa gtctctatca gaatgcagat gcatatgttt ttgtggggac atcaagatac agcaagaagt tcaagccgga aatagcaaca 720 agacccaaag tgagggatca agagggaga atgaactatt actggacact agtagccg 780 ggagacaaaa taacattcga agcaactgga aatctagtgg taccgagata tgcattcaca atggaaaga atgctggatc tggtattatc atttcagata caccagtcca cgattgcaat acaacttgtc agacacccga gggtgctata aacaccagcc tcccatttca gaatacat ccgatcacaa ttggaaaatg tccaaagtat gtaaaaagca caaattgag actggccaca ggattgagga atgttccgtc tattcaatct agaggcctat tcggggccat tgccggcttc attgaagggg ggtggacagg gatggtagat ggatggtacg gttatcacca tcaaatgag 1140 caggggtcag gatatgcagc cgacctgaag agcacacaaa atgccattga cavattact 1200 aaaaagtaa attctgttat tgaaagatg atacacagg acaccagt gggtaaag 1260 ttcaaccacc tggaaaaag atagagaat ctaataaaa aagttgatga tggttttcctg 1320 gatatttgga cttacaatgc cgaactgttg gttctattgg aaatgaag aactttggac 1380 tatcacgatt caatgtgaa gaacttgtat gaaaaaaccagtt aaaaaacat 1440 gccaaggaaa ttggaaacgg ctgctttgaa ttttaccaca atgcgataa cacgtgcatg 1500 gaaagtgtca aaatgggac ttatgactac cchaaatact cagaggaagc aaaattaac 1560 agagaaaaaa tagaggggt aaagctggaa tcaagga tttaccagat ttggcgatc 1620 tattcaactg tcgccagttc attgtactg gtagtctccc tggggcaat cagcttctgg 1680 atgtgctcta atgggtctct acagtgtaga atatgtattt aa 1722 <210> 36 <211> 573 <212> PRT <213> Artificial Sequence <220> <223> PDI-H1 Mich-F390D AA <400> 36 Met Ala Lys Asn Val Ala Ile Phe Gly Leu Leu Phe Ser Leu Leu Val 1 5 10 15 Leu Val Pro Ser Gln Ile Phe Ala Asp Thr Leu Cys Ile Gly Tyr His 20 25 30 Ala Asn Asn Ser Thr Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val 35 40 45 Thr Val Thr His Ser Val Asn Leu Leu Glu Asp Lys His Asn Gly Lys 50 55 60 Leu Cys Lys Leu Arg Gly Val Ala Pro Leu His Leu Gly Lys Cys Asn 65 70 75 80 Ile Ala Gly Trp Ile Leu Gly Asn Pro Glu Cys Glu Ser Leu Ser Thr 85 90 95 Ala Ser Ser Trp Ser Tyr Ile Val Glu Thr Ser Asn Ser Asp Asn Gly 100 105 110 Thr Cys Tyr Pro Gly Asp Phe Ile Asn Tyr Glu Glu Leu Arg Glu Gln 115 120 125 Leu Ser Ser Val Ser Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Thr 130 135 140 Ser Ser Trp Pro Asn His Asp Ser Asn Lys Gly Val Thr Ala Ala Cys 145 150 155 160 Pro His Ala Gly Ala Lys Ser Phe Tyr Lys Asn Leu Ile Trp Leu Val 165 170 175 Lys Lys Gly Asn Ser Tyr Pro Lys Leu Asn Gln Ser Tyr Ile Asn Asp 180 185 190 Lys Gly Lys Glu Val Leu Val Leu Trp Gly Ile His His Pro Ser Thr 195 200 205 Thr Ala Asp Gln Gln Ser Leu Tyr Gln Asn Ala Asp Ala Tyr Val Phe 210 215 220 Val Gly Thr Ser Arg Tyr Ser Lys Lys Phe Lys Pro Glu Ile Ala Thr 225 230 235 240 Arg Pro Lys Val Arg Asp Gln Glu Gly Arg Met Asn Tyr Tyr Trp Thr 245 250 255 Leu Val Glu Pro Gly Asp Lys Ile Thr Phe Glu Ala Thr Gly Asn Leu 260 265 270 Val Val Pro Arg Tyr Ala Phe Thr Met Glu Arg Asn Ala Gly Ser Gly 275 280 285 Ile Ile Ile Ser Asp Thr Pro Val His Asp Cys Asn Thr Thr Cys Gln 290 295 300 Thr Pro Glu Gly Ala Ile Asn Thr Ser Leu Pro Phe Gln Asn Ile His 305 310 315 320 Pro Ile Thr Ile Gly Lys Cys Pro Lys Tyr Val Lys Ser Thr Lys Leu 325 330 335 Arg Leu Ala Thr Gly Leu Arg Asn Val Pro Ser Ile Gln Ser Arg Gly 340 345 350 Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Thr Gly Met 355 360 365 Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn Glu Gln Gly Ser Gly 370 375 380 Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala Ile Asp Lys Ile Thr 385 390 395 400 Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr Gln Asp Thr Ala 405 410 415 Val Gly Lys Glu Phe Asn His Leu Glu Lys Arg Ile Glu Asn Leu Asn 420 425 430 Lys Lys Val Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu 435 440 445 Leu Leu Val Leu Leu Glu Asn Glu Arg Thr Leu Asp Tyr His Asp Ser 450 455 460 Asn Val Lys Asn Leo Tyr Glu Lys Val Arg Asn Gln Leu Lys Asn Asn 465 470 475 480 Ala Lys Glu Ile Gly Asn Gly Cys Phe Glu Phe Tyr His Lys Cys Asp 485,490,495 Asn Thr Cys Met Glu Ser Val Lys Asn Gly Thr Tyr Asp Tyr Pro Lys 500 505 510 Tyr Ser Glu Glu Ala Lys Leu Asn Arg Glu Lys Ile Asp Gly Val Lys 515,520,525 The Glu Ser of Thr Arg and Tyr Gln a...

Claims

1. A modified H1 influenza virus hemagglutinin HA protein comprising amino acid substitutions compared to a corresponding wild-type H1 influenza HA protein sequence, wherein the amino acid substitutions consist of amino acid substitutions corresponding to the following positions: i) positions 390 and 429 of the reference sequence SEQ ID NO: 134; ii) positions 390, 429 and 97 of the reference sequence SEQ ID NO: 134; iii) positions 390, 429 and 374 of the reference sequence SEQ ID NO: 134; or iv) positions 390, 429, 97 and 374 of the reference sequence SEQ ID NO: 134; wherein: The amino acid corresponding to position 97 of the reference sequence SEQ ID NO: 134 is substituted with aspartic acid; The amino acid corresponding to position 374 of the reference sequence SEQ ID NO: 134 is substituted with glutamic acid; The amino acid corresponding to position 390 of the reference sequence SEQ ID NO: 134 is substituted with aspartic acid; and The amino acid substitution corresponding to position 429 of the reference sequence SEQ ID NO: 134 is methionine.

2. A nucleic acid encoding the modified H1 influenza virus hemagglutinin HA protein as claimed in claim 1.

3. A virus-like particle (VLP), comprising the modified H1 influenza virus hemagglutinin (HA) protein according to claim 1.

4. A method for producing influenza virus-like particles (VLPs) in plants, plant parts or plant cells, the method comprising: a) introducing the nucleic acid according to claim 2 into a plant, a plant part or a plant cell; as well as b) cultivating the plant, plant part or plant cell under conditions allowing expression of the hemagglutinin protein encoded by the nucleic acid, thereby producing the VLP.

5. The method according to claim 4, wherein the method further comprises the step c) of harvesting the plant, plant part or plant cell and purifying the VLP.

6. A method for producing influenza virus-like particles (VLPs) in plants, plant parts or plant cells, the method comprising: a) providing a plant, plant part or plant cell comprising the nucleic acid according to claim 2; as well as b) cultivating the plant, plant part or plant cell under conditions allowing expression of the hemagglutinin protein encoded by the nucleic acid, thereby producing VLPs.

7. The method according to claim 6, wherein the method further comprises the step c) of harvesting the plant, plant part or plant cell and purifying the VLP.

8. A VLP produced by the method of any one of claims 4 to 7.

9. The VLP of claim 8, further comprising one or more lipids derived from plants, plant parts or plant cells, plant-specific N-glycans, modified N-glycans, or combinations thereof.

10. A composition for inducing an immune response, comprising an effective dose of the VLP according to any one of claims 3, 8 or 9, and a pharmaceutically acceptable carrier.

11. The composition of claim 10, wherein the pharmaceutically acceptable carrier comprises an adjuvant, a vehicle or an excipient.

12. Use of the VLPs of any one of claims 3, 8 or 9 in the manufacture of a medicament for inducing immunity to influenza virus infection in a subject.

13. The use according to claim 12, wherein the VLP is formulated for administration to a subject orally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

14. A method for increasing the yield of influenza virus-like particles (VLPs) in plants, plant parts or plant cells, comprising: a) introducing the nucleic acid according to claim 2 into the plant, plant part or plant cell; or providing a plant, plant part or plant cell comprising the nucleic acid according to claim 2; and b) cultivating the plant, plant part or plant cell under conditions allowing expression of the hemagglutinin protein encoded by the nucleic acid, thereby producing the VLPs at a higher yield compared to a plant, plant part or plant cell expressing an unmodified hemagglutinin protein.

15. The method according to claim 14, wherein the method further comprises the step c) of harvesting the plant, plant part or plant cell and purifying the VLP.

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