Influenza virus hemagglutinin mutants

文档序号：704486 发布日期：2021-04-13 浏览：78次中文

阅读说明：本技术 流感病毒血凝素突变体 (Influenza virus hemagglutinin mutants ) 是由皮尔-奥利弗·拉沃伊奥雷连·洛瑞阿兰·道赛特马克-安德鲁·德奥斯特玛南·科沃托于 2019-06-27 设计创作，主要内容包括：本发明涉及在植物中产生经过修饰的流感病毒蛋白。更具体地说,本发明涉及在植物中产生流感病毒样微粒(VLP)和增加其产量,其中VLP包括经过修饰的流感病毒蛋白,例如经过修饰的流感病毒血凝素(HA)。该HA蛋白可包括与相应的野生型氨基酸序列相比包含至少一个取代的氨基酸序列。还提供了一种对经过修饰的HA蛋白进行编码的核酸。此外,还提供了在植物、植物部分或植物细胞中产生流感病毒样微粒(VLP)的方法和增加植物、植物部分或植物细胞中的流感病毒样微粒(VLP)的产量的方法。(The present invention relates to the production of modified influenza virus proteins in plants. More specifically, the invention relates to the production and increased production of influenza virus-like particles (VLPs) in plants, wherein the VLPs comprise modified influenza virus proteins, such as modified influenza virus Hemagglutinin (HA). The HA protein may comprise an amino acid sequence comprising at least one substitution compared to the corresponding wild-type amino acid sequence. Also provided is a nucleic acid encoding the modified HA protein. In addition, methods of producing influenza virus-like particles (VLPs) in plants, plant parts, or plant cells and methods of increasing the yield of influenza virus-like particles (VLPs) in plants, plant parts, or plant cells are provided.)

1. A nucleic acid comprising a nucleotide sequence encoding a modified H1 influenza virus Hemagglutinin (HA) protein comprising an amino acid sequence comprising at least one substitution as 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 H1a/Michigan/45/15 hemagglutinin.

2. The nucleic acid of claim 1, wherein the at least one substitution of an amino acid corresponding to the amino acid at position 97 of H1a/Michigan/45/15 hemagglutinin is a non-asparagine substitution.

3. The nucleic acid of claim 2, wherein the at least one substitution of an amino acid corresponding to the amino acid at position 97 of H1a/Michigan/45/15 hemagglutinin is an aspartic acid substitution or a conservative aspartic acid substitution.

4. The nucleic acid of claim 1, wherein the at least one substitution of an amino acid corresponding to the amino acid at position 374 of H1a/Michigan/45/15 hemagglutinin is a non-lysine substitution.

5. The nucleic acid of claim 4, wherein the at least one substitution of an amino acid corresponding to the amino acid at position 374 of H1A/Michigan/45/15 hemagglutinin is a glutamic acid substitution or a conservative glutamic acid substitution.

6. The nucleic acid of claim 1, wherein the at least one substitution of an amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin is a non-phenylalanine substitution.

7. The nucleic acid of claim 6, wherein the at least one substitution of an amino acid corresponding to the amino acid at position 390 of H1A/Michigan/45/15 hemagglutinin is an aspartic acid substitution or a conservative aspartic acid substitution.

8. The nucleic acid of claim 1, wherein the at least one substitution of the amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin is a non-leucine substitution.

9. The nucleic acid of claim 8, wherein the at least one substitution of an amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin is a methionine substitution or a conservative methionine substitution.

10. The nucleic acid of claim 8 or 9, further comprising a second substitution at an amino acid corresponding to the amino acid at position 380 of H1a/Michigan/45/15 hemagglutinin, which second substitution is a non-asparagine substitution.

11. The nucleic acid of claim 10, wherein the second substitution of the amino acid corresponding to the amino acid at position 380 of H1a/Michigan/45/15 hemagglutinin is an alanine substitution or a conservative alanine substitution.

12. The nucleic acid of claim 1, comprising:

a first substitution at an amino acid corresponding to the amino acid at position 390 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-phenylalanine substitution, and

a second substitution at an amino acid corresponding to amino acid 429 of H1A/Michigan/45/15 hemagglutinin, which is a non-leucine substitution.

13. The nucleic acid according to claim 12, wherein,

the first substitution is an aspartic acid substitution or a conservative aspartic acid substitution, and

the second substitution is a methionine substitution or a conservative methionine substitution.

14. The nucleic acid of claim 1, comprising:

a first substitution at an amino acid corresponding to the amino acid at position 97 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-asparagine substitution, and

a second substitution at an amino acid corresponding to the amino acid at position 374 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-lysine substitution.

15. The nucleic acid according to claim 14, wherein,

the first substitution is an aspartic acid substitution or a conservative aspartic acid substitution, and

the second substitution is a glutamic acid substitution or a conservative glutamic acid substitution.

16. The nucleic acid of claim 1, comprising:

a first substitution at an amino acid corresponding to the amino acid at position 97 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-asparagine substitution,

a second substitution at an amino acid corresponding to the amino acid at position 390 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-phenylalanine substitution, and

a third substitution at an amino acid corresponding to amino acid 429 of H1A/Michigan/45/15 hemagglutinin, which is a non-leucine substitution.

17. The nucleic acid according to claim 16, wherein,

the first substitution is an aspartic acid substitution or a conservative aspartic acid substitution,

the second substitution is an aspartic acid substitution or a conservative aspartic acid substitution, and

the third substitution is a methionine substitution or a conservative methionine substitution.

18. The nucleic acid of claim 1, comprising:

a first substitution at an amino acid corresponding to the amino acid at position 374 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-lysine substitution,

a second substitution at an amino acid corresponding to the amino acid at position 390 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-phenylalanine substitution, and

a third substitution at an amino acid corresponding to amino acid 429 of H1A/Michigan/45/15 hemagglutinin, which is a non-leucine substitution.

19. The nucleic acid according to claim 18, wherein,

the first substitution is a glutamic acid substitution or a conservative glutamic acid substitution,

the second substitution is an aspartic acid substitution or a conservative aspartic acid substitution, and

the third substitution is a methionine substitution or a conservative methionine substitution.

20. The nucleic acid of claim 1, comprising:

a first substitution at an amino acid corresponding to the amino acid at position 97 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-asparagine substitution,

a second substitution at an amino acid corresponding to the amino acid at position 374 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-lysine substitution,

a third substitution at an amino acid corresponding to the amino acid at position 390 of H1A/Michigan/45/15 hemagglutinin, which substitution is a non-phenylalanine substitution, and

a fourth substitution at an amino acid corresponding to the amino acid at position 429 of H1A/Michigan/45/15 hemagglutinin, which is a non-leucine substitution.

21. The nucleic acid according to claim 20, wherein,

the first substitution is an aspartic acid substitution or a conservative aspartic acid substitution,

the second substitution is a glutamic acid substitution or a conservative glutamic acid substitution,

the third substitution is an aspartic acid substitution or a conservative aspartic acid substitution, and

the fourth substitution is a methionine substitution or a conservative methionine substitution.

22. The nucleic acid of any one of claims 3, 7, 13, 15, 17, 19, or 21, wherein the conservative aspartic acid substitution is glutamic acid, glutamine, or serine.

23. The nucleic acid of claim 5, 15, 19, or 21, wherein the conservative glutamic acid substitution is aspartic acid, glutamine, arginine, asparagine, histidine, or serine.

24. The nucleic acid of claim 9, 13, 17, 19, or 21, wherein the conservative methionine substitution is isoleucine, glutamine, valine, or phenylalanine.

25. The nucleic acid of claim 11, wherein the conservative alanine substitution is serine, glycine, threonine, cysteine, or valine.

26. A modified H1 influenza virus Hemagglutinin (HA) protein encoded by the nucleic acid of any one of claims 1 to 25.

27. A virus-like particle (VLP) comprising a modified H1 influenza virus Hemagglutinin (HA) protein encoded by the nucleic acid of any one of claims 1 to 25.

28. A method of producing an influenza virus-like particle (VLP) in a plant, plant part, or plant cell, the method comprising:

a) introducing the nucleic acid of any one of claims 1-25 into a plant, plant part, or plant cell; and

b) incubating the plant, plant part, or plant cell under conditions that allow expression of the hemagglutinin protein encoded by the nucleic acid, thereby producing the VLP.

29. The method of claim 28, wherein said method further comprises the step c) of harvesting said plant, plant part or plant cell and purifying said VLP.

30. A method of producing an influenza virus-like particle (VLP) in a plant, plant part, or plant cell, the method comprising:

a) providing a plant, plant part, or plant cell comprising the nucleic acid of any one of claims 1 to 25; and

b) incubating the plant, plant part, or plant cell under conditions that allow expression of the hemagglutinin protein encoded by the nucleic acid, thereby producing the VLP.

31. The method of claim 30, wherein said method further comprises the step c) of harvesting said plant, plant part or plant cell and purifying said VLP.

32. A VLP produced by the method of any one of claims 28-31.

33. The VLP of claim 32, further comprising one or more plant-, plant part-, or plant cell-derived lipids, plant-specific N-glycans, modified N-glycans, or a combination thereof.

34. A method of producing an antibody or antibody fragment, comprising administering the VLP of any one of claims 27, 32 or 33 to a subject or host animal, thereby producing the antibody or antibody fragment.

35. An antibody produced by the method of claim 34.

36. A plant, plant part, or plant cell comprising the nucleic acid of any one of claims 1 to 25.

37. A plant, plant part or plant cell comprising the hemagglutinin protein of claim 26 or the VLP of claim 27 or 32.

38. A composition for inducing an immune response comprising an effective dose of the VLP of any one of claims 27, 32 or 33, and a pharmaceutically acceptable carrier, adjuvant, carrier or excipient.

39. A method of inducing immunity to an influenza virus infection in a subject, comprising administering to the subject the VLP of any one of claims 27, 32 or 33.

40. The method of claim 39, wherein the VLP is administered to a subject orally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

41. A method of increasing the yield of influenza Virus Like Particles (VLPs) in a plant, plant part or plant cell, comprising:

a) introducing the nucleic acid of any one of claims 1-25 into the plant, plant part, or plant cell; or providing a plant, plant part or plant cell comprising the nucleic acid of any one of claims 1 to 25; and

b) cultivating the plant, plant part or plant cell under conditions that allow expression of the hemagglutinin protein encoded by the nucleic acid, thereby producing the VLP in higher yield as compared to a plant, plant part or plant cell expressing an unmodified hemagglutinin protein.

42. The method of claim 41, wherein said method further comprises the step c) of harvesting said plant, plant part or plant cell and purifying said VLP.

43. A modified H1 influenza virus Hemagglutinin (HA) protein comprising an amino acid sequence that comprises at least one substitution at one or more amino acids corresponding to the amino acid at position 97, 374, 390 or 429 of an H1a/Michigan/45/15 hemagglutinin compared to the corresponding wild-type amino acid sequence.

Technical Field

The present invention relates to the production of mutant viral proteins in plants. More specifically, the invention relates to the production of influenza virus-like particles and to increasing the yield of influenza virus-like particles in plants.

Background

Influenza viruses are enveloped single-stranded ribonucleic acid viruses of the orthomyxoviridae family. Influenza viruses are highly contagious and may cause mild to severe disease in all age groups.

Vaccination remains the most effective method of preventing influenza infection. Traditionally, vaccination has been accomplished using live attenuated or fully inactivated viruses, which elicit an immune response when administered to a patient. To eliminate the potential risk of live attenuated and fully inactivated viruses regaining replication and infectivity, vaccines comprising recombinant viral proteins are also used to elicit protective immunity against influenza infection.

However, the use of recombinant viral proteins as immunogenic components of vaccines has been subject to a number of limitations. First, without the full 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 not be sufficient 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 major humoral immune response that is ineffective in conferring durable protective immunity.

Influenza a viruses are further classified according to the expression of Hemagglutinin (HA) and Neuraminidase (NA) glycoprotein subtypes on the surface of the virus. There are 18 different hemagglutinin subtypes (H1-H18).

Hemagglutinin is a trimeric lectin that promotes the binding of influenza virions to sialic-acid-containing proteins on the surface of target cells and mediates the release of the viral genome into the target cells. The hemagglutinin protein comprises two structural elements: a head, which is the primary target of seroprotective antibodies; and a handle. An article published by Ha et al in 2002 (EMBO J.21: 865-875; incorporated herein by reference) shows the relative orientation of the individual subdomains of the Stem Domain Cluster (SDC) and the Head Domain Cluster (HDC) in various influenza virus subtypes based on X-ray crystallographic structure.

Hemagglutinin is translated into a single polypeptide HA0 (assembled as a trimer), which HA0 must be cleaved by serine endoproteases between the HA1 (approximately 40kDa) subdomain and the HA2 (approximately 20kDa) subdomain. After cleavage, the two disulfide-bound protein domains adopt the necessary conformation required to achieve viral infectivity. HA1 forms a globular head domain comprising the residual esterase domains E1' and E2 and the Receptor Binding Site (RBS), the least conserved fragment of influenza virus. HA2 is a unidirectional membrane-intrinsic protein with a Fusion Peptide (FP), a soluble extracellular domain (SE), a transmembrane domain (TM) and a cytoplasmic tail domain (CT), these domains being approximately 25, 160, 25 and 10 residues in length, respectively. HA2 forms together with the nitrogen and carbon terminal HA1 residues a stalk domain that includes the transmembrane domain and is relatively conserved.

Various mutations of influenza virus proteins, particularly influenza virus hemagglutinin proteins, have been studied.

For example, Castel N-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 hemagglutinin from influenza A virus (H1N1) pdm09 and determined that D104N, A259T, S124N and E172K mutations resulted in enhanced stability of the predicted influenza virus hemagglutinin protein. In contrast, the S206T, K285E, and E47K mutations had predicted destabilizing effects on hemagglutinin.

In comparing the sequences of the original type A virus (H1N1) pdm [ A/California/7/2009] and the later emerging influenza strain [ A/Brisbane/10/2010], Cotter et al (PLoS Patholog.2014; 10 (1): E1003831) determined that the E47K mutation in the stalk domain of A/California/7/2009 hemagglutinin stabilized the trimer structure, reduced the pH of the membrane fusion, and improved the thermal and acid stability of the virus. Cotter et al also observed that the a/California/7/2009E 47K mutant hemagglutinin was more infectious in ferrets than its wild type counterpart.

Antanasijevic et al (J Biol chem. 2014; 289 (32): 22237-45) investigated the structure-functional properties of the stem-loop domain of H5 hemagglutinin by site-directed mutagenesis at 14 different positions. The a/Vietnam/1203/04(H5N1) mutant was 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 (numbered based on H3) exhibited significantly reduced total hemagglutinin levels, indicating reduced expression and/or assembly of hemagglutinin in the virion. The HA1-D26K, HA2-T49A and HA2-M102L mutants also showed lower hemagglutination titers compared to the wild-type virus. A reduction in the ability of all single mutants to enter A549 lung cells was also observed by Antanasijevic, with the attenuation of the HA1-D26K and HA2-I55A mutants being most pronounced. 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, indicating that this mutation enhances the pattern 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 C179 neutralizing antibodies.

WO2013/177444 to Lu et al and its complement (Proc Natl Acad Sci USA 2014; 111 (1): 125-30) report a method for producing a correctly folded hemagglutinin stem domain 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 stem domain of hemagglutinin, Chloramphenicol Acetyltransferase (CAT) or a folding domain is fused to the carbon terminus of hemagglutinin. To mitigate newly exposed hydrophobicity and/or intermolecular ion pairing leading to aggregation of expressed hemagglutinin stem proteins, five sets 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 appears to be the mutation that has the greatest effect on increasing the solubility of the hemagglutinin stem protein. The M5 mutant was subjected to additional deletions (H38 to C43 and C49 to N61) and C77T mutations to avoid the formation of undesirable disulfide bonds, reduce surface hydrophobicity and pI, and avoid regions with disordered structure.

Hall et al, U.S. application 13/838,796 and its complement (BMC Biotechnology.2014; 14: 111), teach the improvement of the stability and maintenance of the efficacy of recombinant hemagglutinin by mutation of cysteine residues in the carboxy-terminal domain of the hemagglutinin protein, including the Transmembrane (TM) and Cytoplasmic (CT) domains. Specifically, Hall et al demonstrated C539A, C546A, C549A, C524S, and C528A mutations in recombinant Perth/16/2009 hemagglutinin (H3N 2). Mutations of all five cysteine residues or different subsets thereof resulted in comparable hemagglutinin yields, purity, microparticle size, hemagglutination activity and thermostability to the recombinant wild-type hemagglutinin protein. In contrast, the C64S and C76S mutations resulted in a significant decrease in hemagglutinin expression, suggesting a critical role for these residues in proper folding of hemagglutinin. By using a single radial immunodiffusion assay (SRID), holtz et al also showed that mutations of these five cysteine residues increased the efficacy of recombinant hemagglutinin by preventing disulfide cross-linking in the TM and CT domains compared to the wild-type protein. Mutant hemagglutinin proteins retain potency for at least 12 months at 25 ℃, whereas wild-type hemagglutinin proteins show less than 40% potency after only 50 days of purification.

WO2015/020913 teaches that 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 a/Puerto Rico/8/1934(H1N1) are mutated to tyrosine. These mutations promote the formation of a di-tyrosine cross-link, thereby stabilizing or "locking in" the stalk domain of the influenza virus hemagglutinin in its native trimeric conformation.

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 substituted with 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 of the 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.

WO2014/191435 similarly teaches a modified influenza virus hemagglutinin comprising an HA1 domain and an HA2 domain, wherein a deletion fragment of the HA1 domain is substituted with a covalent linking sequence of 0 to 50 amino acids, wherein the hemagglutinin is resistant to cleavage at the junction of HA1 and HA2, and wherein one or more of the amino acids at positions 337, 340, 352, 353, 402, 406, 409, 413 and/or 416 have been mutated.

Virus-like particles (VLPs) are potential candidates for inclusion in immunogenic compositions. VLPs are very similar to mature virions, but they do not contain viral genomic material. Thus, VLPs are non-replicative in nature, which makes them safe for administration as vaccines. Furthermore, VLPs can be engineered to express viral glycoproteins on the surface of the VLP, which is their most natural physiological structure. Furthermore, because VLPs resemble intact virions and are multivalent particulate structures, VLPs may be more effective than soluble envelope protein antigens in inducing glycoprotein-neutralizing antibodies.

VLPs have been produced in plants (see, e.g., WO 2009/076778; WO 2009/009876; WO 2009/076778; WO 2010/003225; WO 2010/003235; WO 2010/006452; WO 2011/03522; WO2010/148511 and WO2014153674, which are incorporated herein by reference).

WO2009/076778 teaches a method of producing influenza virus VLPs in a plant, the method comprising introducing a nucleic acid having a regulatory domain active in the plant operably linked to a nucleotide sequence encoding an influenza virus hemagglutinin from an influenza a or b virus.

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.

WO2010/003225 discloses a method of producing influenza virus hemagglutinin VLPs in a plant, the method comprising introducing a nucleic acid having a regulatory domain active in the plant operably linked to a nucleotide sequence encoding an influenza virus hemagglutinin from a/California/04/09(H1N 1).

WO2010/006452 teaches a method of producing VLPs comprising a modified influenza virus hemagglutinin protein, wherein the glycosylation sites at positions 154, 165, 286 or a combination thereof (see 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 amino acids at positions 156, 167, 288 or combinations thereof can be mutated to residues other than serine or threonine, thereby similarly eliminating the nitrogen-linked glycosylation signal triplet "N-X-S/T". By selectively deleting the glycosylation site located in the globular head of the hemagglutinin protein, WO2010/006452 demonstrates that the resulting hemagglutinin protein has improved antigenicity and broader cross-reactivity.

WO2011/035422 teaches a method of preparing plant derived VLPs, the method comprising: obtaining a plant or plant matter comprising VLPs located within apoplast; generating a protoplast/spheroid portion and an apoplast portion; and recovering an apoplast portion comprising plant-derived VLPs.

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

WO2014/153674 teaches a method of producing influenza virus VLPs in plants, wherein the VLPs comprise a modified influenza virus hemagglutinin having a modified proteolytic loop. The modified proteolytic loop comprises a proteolytic cleavage site between the HA1 and HA2 domains that removes the HA0 precursor. Therefore, the hemagglutinin protein is stabilized and the protein yield is improved, compared to the native hemagglutinin protein.

Disclosure of Invention

The present invention relates to the production of modified influenza virus proteins in plants. More specifically, the present invention relates to the production and increased production of influenza virus-like particles (VLPs) in plants, wherein the VLPs comprise a modified influenza virus protein, such as a modified Hemagglutinin (HA) protein.

It is an object of the present invention to provide an improved method for increasing the yield of influenza VLPs in plants.

According to the present invention, there is provided:

a nucleic acid comprising a nucleotide sequence encoding a modified H1 influenza virus Hemagglutinin (HA) protein comprising an amino acid sequence comprising at least one substitution as 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 H1A/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may comprise an amino acid sequence having a non-asparagine substitution at the amino acid corresponding to the amino acid at position 97 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may comprise an amino acid sequence having an aspartic acid substitution or a conservative aspartic acid substitution at the amino acid corresponding to the amino acid at position 97 of H1a/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may comprise an amino acid sequence having a non-lysine substitution at an amino acid corresponding to the amino acid at position 374 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may comprise 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 H1a/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may comprise an amino acid sequence having a non-phenylalanine substitution at an amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may comprise an amino acid sequence having an aspartic acid substitution or a conservative aspartic acid substitution at the amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may comprise an amino acid sequence having a non-leucine substitution at an amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may comprise an amino acid sequence having a methionine substitution or a conservative methionine substitution at the amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may also comprise an amino acid sequence having a non-asparagine substitution at the amino acid corresponding to the amino acid at position 380 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may comprise 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 H1a/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may further comprise an amino acid sequence having a first non-phenylalanine substitution at the amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin and a second non-leucine substitution at the amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may further comprise 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 H1a/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 H1a/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may further comprise an amino acid sequence having a first non-asparagine substitution at the amino acid corresponding to the amino acid at position 97 of H1a/Michigan/45/15 hemagglutinin, a second non-phenylalanine substitution at the amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin, and a third non-leucine substitution at the amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may further comprise an amino acid sequence having a first aspartic acid substitution or a conserved aspartic acid substitution at the amino acid corresponding to the amino acid at position 97 of H1a/Michigan/45/15 hemagglutinin, a second aspartic acid substitution or a conserved aspartic acid substitution at the amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin, and a third methionine substitution or a conserved methionine substitution at the amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may further comprise an amino acid sequence having a first non-lysine substitution at an amino acid corresponding to the amino acid at position 374 of H1a/Michigan/45/15 hemagglutinin, a second non-phenylalanine substitution at an amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin, and a third non-leucine substitution at an amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may further comprise an amino acid sequence having a first glutamic acid substitution or a conservative glutamic acid substitution at an amino acid corresponding to the amino acid at position 374 of H1a/Michigan/45/15 hemagglutinin, a second aspartic acid substitution or a conservative aspartic acid substitution at an amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin, and a third methionine substitution or a conservative methionine substitution at an amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin.

The hemagglutinin protein may further comprise an amino acid sequence having a first non-asparagine substitution at the amino acid corresponding to the amino acid at position 97 of H1a/Michigan/45/15 hemagglutinin, a second non-lysine substitution at the amino acid corresponding to the amino acid at position 374 of H1a/Michigan/45/15 hemagglutinin, a third non-phenylalanine substitution at the amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin, and a fourth non-leucine substitution at the amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin. The hemagglutinin protein may further comprise an amino acid sequence having a first aspartic acid substitution or a conserved aspartic acid substitution at the amino acid corresponding to the amino acid at position 97 of H1a/Michigan/45/15 hemagglutinin, a second glutamic acid substitution or a conserved glutamic acid substitution at the amino acid corresponding to the amino acid at position 374 of H1a/Michigan/45/15 hemagglutinin, a third aspartic acid substitution or a conserved aspartic acid substitution at the amino acid corresponding to the amino acid at position 390 of H1a/Michigan/45/15 hemagglutinin, and a fourth methionine substitution or a conserved methionine substitution at the amino acid corresponding to the amino acid at position 429 of H1a/Michigan/45/15 hemagglutinin.

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

Accordingly, there is provided a modified H1 influenza virus Hemagglutinin (HA) protein comprising an amino acid sequence comprising at least one substitution as compared to the corresponding wild type amino acid sequence, said at least one substitution being at one or more amino acids corresponding to the amino acid at position 97, 374, 390 or 429 of H1a/Michigan/45/15 hemagglutinin.

Further, there is provided a method of producing an influenza Virus Like Particle (VLP) in a plant, plant part or plant cell, the method comprising:

a) introducing the recombinant nucleic acid into a plant, plant part, or plant cell; and is

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 the VLP. The method may further comprise the step c) of harvesting the plant, plant part or plant cell and purifying the VLP.

Also provided is a method of producing an influenza Virus Like Particle (VLP) in a plant, plant part or plant cell, the method comprising:

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

Further, there is provided a method of increasing the yield of influenza Virus Like Particles (VLPs) in a plant, plant part or plant cell, 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 the hemagglutinin protein encoded by the recombinant nucleic acid, thereby producing the VLP in higher yield as compared to a plant, plant part or plant cell expressing an unmodified influenza virus hemagglutinin protein. The method may further comprise the step c) of harvesting the plant, plant part or plant cell and purifying the VLP.

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

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

The VLP may comprise one or more lipids derived from a plant, plant part, or plant cell, plant-specific N-glycans, modified N-glycans, or a combination thereof.

Additionally, a method of producing an antibody or antibody fragment is 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.

Furthermore, a plant, plant part or plant cell comprising the recombinant nucleic acid or the hemagglutinin protein encoded by the recombinant nucleic acid is provided. The hemagglutinin proteins can form VLPs. Thus, there is also provided a plant, plant part or plant cell comprising a VLP comprising a hemagglutinin protein encoded by a recombinant nucleic acid.

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

Further, a modified influenza virus Hemagglutinin (HA) protein is provided, wherein the influenza virus hemagglutinin protein comprises at least one substitution as described herein and is capable of forming a VLP, inducing an immune response, inducing a hemagglutination reaction, or a combination thereof when administered to a subject, the influenza virus hemagglutinin protein comprises a substitution with sequence No. 18, sequence No. 22, sequence No. 24, sequence No. 4, sequence No. 28, sequence No. 32, sequence No. 36, sequence No. 39, sequence No. 41, sequence No. 43, sequence No. 45, sequence No. 47, sequence No. 49, sequence No. 51, sequence No. 53, sequence No. 55, sequence No. 57, sequence No. 59, sequence No. 61, sequence No. 63, sequence No. 65, sequence No. 67, sequence No. 72, sequence No. 77, sequence No. 80, sequence No. 82, sequence No. 84, sequence No. 86, sequence no, An amino acid sequence having about 30% to about 100% sequence identity or sequence similarity to one of the sequences of 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. 143, SEQ ID NO. 145, SEQ ID NO. 147, SEQ ID NO. 149.

This summary does not necessarily describe all features of the invention.

Drawings

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

FIG. 1 shows 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); alignment of amino acid sequences of Hemagglutinin (HA) of A/Massachusetts/06/17(H1N1) (SEQ ID NO: 135). The residues listed are para to amino acids D97, E374, F390, and L429 of hemagglutinin from an influenza H1 strain (H1N1), such as a/California/7/09(H1N 1).

FIG. 2A shows the hemagglutination titers of wild-type A/California/07/09H1, L429M A/California/07/09 mutant H1, N380A A/California/7/09 mutant H1 and F390D A/California/7/09 mutant H1. FIG. 2B shows the hemagglutination titers of wild-type A/California/07/09H1, F390D A/California/07/09 mutant H1, L429M A/California/7/09 mutant H1 and F390D + L429M A/California/07/09 mutant H1, expressed as a percentage relative to wild-type A/California/07/09. FIG. 2C shows post density gradient VLP yields for wild type A/California/07/09H1, N380A A/California/07/09 mutant H1, L429M A/California/07/09 mutant H1, and F390D A/California/07/09 mutant H1.

FIG. 3A shows the hemagglutination titers of wild-type A/California/07/09H1, wild-type A/Michigan/45/15H1, K374E A/Michigan/45/15 mutant H1, and N97D A/Michigan/45/15 mutant H1. FIG. 3B shows wild type A/California/07/09H1, F390D A/California/07/09 mutant H1, wild type A/Michigan/45/15H1, N97D A/Michigan/45/15 mutant H1, K374E A/Michigan/45/15 mutant H1, N380A A/Michigan/A mutant H A, F390A A/Michigan/A mutant H A, L429A A/Michigan/A mutant H A, N390A + K A A/Michigan/A mutant H A, F390A + L429A/Michigan/A mutant H A, F390A + N A A/Michigan/36429H A, and F390A + N A A/Michigan/36429A/36429H A, and L A/A K A A/36429H A, Hemagglutination titers of N97D + F390D + L429M A/Michigan/45/15 mutant H1 and N97D + K374E + F390D + L429M A/Michigan/45/15 mutant H1.

FIG. 4A shows the hemagglutination titers of wild-type A/Michigan/45/15H1, 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 + L429 mutant H429M A/Michigan/45/15H 1; the hemagglutination titer of wild type A/Honduras/17734/16H1, N97D A/Honduras/17734/16 mutant H1, K374E A/Honduras/17734/16 mutant H1, F390D 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 the hemagglutination titer of wild type A/Darwin/11/15H 1, 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 A/Darwin/11/15 mutant H1 and N97D + F390D + L429M A/Darwin/11/15 mutant H1. FIG. 4B shows the hemagglutination 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; the hemagglutination titer of F390D + L429M A/Costa Rica/0513/16 mutant H1, K374E + F390D + L429M A/Costa 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. FIG. 4C shows the 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; the hemagglutination titer of F390D + L429M A/Norway/2147/2017 mutant H1, K374E + F390D + L429M 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.

FIG. 5 shows the hemagglutination titers of wild type A/Indonesia/5/2005H 5, wild type A/Egypt/N04915/2014H5, 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. The numbering is determined according to A/Indonesia/5/2005.

FIG. 6A shows a schematic representation of the carrier 1314 (H1A-Cal-7-2009). FIG. 6B shows a schematic of the carrier 2980 (H1A-Cal-7-09 (F390D)). FIG. 6C shows a schematic representation of the vector 2962(H1A-Cal-7-09 (L429M)). FIG. 6D shows a schematic of the vector 2995 (H1A-Cal-7-09 (F390D + L429M)). FIG. 6E shows a schematic of vector 3640 (H1A-Mich-45-2015). FIG. 6F shows a schematic representation of vector 3774 (H1A-Mich-45-2015 (N97D)). FIG. 6G shows a schematic representation of vector 3771 (H1A-Mich-45-2015 (K374E)). Fig. 6H shows a schematic of vector 3641(H1 a-Mich-45-2015 (F390D)). FIG. 6I shows a schematic representation of vector 3643 (H1A-Mich-45-2015 (L429M)). FIG. 6J shows a schematic of vector 3880 (H1A-Mich-45-2015 (N97D + K374E)). FIG. 6K shows a schematic of vector 3703 (H1A-Mich-45-2015(F390D + L429M)). Fig. 6L shows a schematic of vector 3879(H1 a-Mich-45-2015(N97D + F390D + L429M)). Fig. 6M shows a schematic of vector 3878(H1 a-Mich-45-2015(K374E + F390D + L429M)). FIG. 6N shows a schematic of vector 3881 (H1A-Mich-45-2015 (N97D + K374E + F390D + L429M)). FIG. 6O shows a schematic diagram of the carrier 4091(H1A-Mass-06-2017(F390D + L429M)). FIG. 6P shows a schematic diagram of the carrier 4093(H1A-Mass-06-2017(N97D + F390D + L429M)). FIG. 6Q shows a schematic diagram of the carrier 4092 (H1A-Mass-06-2017 (K374E + F390D + L429M)). FIG. 6R shows a schematic diagram of the carrier 4094 (H1A-Mass-06-2017(N97D + K374E + F390D + L429M)). FIG. 6S shows a schematic diagram of carrier 4715 (H1A-Costa Rica-0513-2016(F390D + L429M)). FIG. 6T shows a schematic representation of vector 4717 (H1A-Costa Rica-0513-2016(N97D + F390D + L429M)). FIG. 6U shows a schematic diagram of carrier 4716(H1A-Costa Rica-0513-2016(K374E + F390D + IA 29M)). FIG. 6V shows a schematic representation of carrier 4718 (H1A-Costa Rica-0513- & 2016(N97D + K374E + F390D + L429M)). FIG. 6W shows a schematic of vector 3944 (H1A-Hond-17734-16). FIG. 6X shows a schematic of vector 3950 (H1A-Hond-17734-16 (N97D)). FIG. 6Y shows a schematic representation of vector 3948 (H1A-Hond-17734-16 (K374E)). FIG. 6Z shows a schematic of vector 3945 (H1A-Hond-17734-16 (F390D)). FIG. 6AA shows a schematic representation of vector 3949 (H1A-Hond-17734-16 (L429M)). FIG. 6BB shows a schematic representation of vector 3946 (H1A-Hond-17734-16 (F390D + L429M)). FIG. 6CC shows a schematic of vector 3951 (H1A-Hond-17734-16 (N97D + F390D + L429M)). FIG. 6DD shows a schematic diagram of carrier 3984 (H1A-Darw-11-15). FIG. 6EE shows a schematic representation of vector 3990 (H1A-Darw-11-15 (N97D)). FIG. 6FF shows a schematic of vector 3988(H1A-Darw-11-15 (K374E)). FIG. 6GG shows a schematic of vector 3985(H1A-Darw-11-15 (F390D)). FIG. 6HH shows a schematic representation of vector 3989(H1A-Darw-11-15 (L429M)). FIG. 6II shows a schematic of vector 3986(H1A-Darw-11-15(F390D + L429M)). FIG. 6JJ shows a schematic representation of vector 3991(H1A-Darw-11-15(N97D + F390D + L429M)). FIG. 6KK shows a schematic representation of vector 3644 (H1A-Mich-45-2015 (N380A)). FIG. 6LL shows a schematic of a vector 3704(H1A-Mich-45-2015(F390D + N380A)). FIG. 6MM shows a schematic representation of carrier 4765(A/Paris/1227/17(F390D + L429M)). FIG. 6NN shows a schematic representation of vector 4766(A/Paris/1227/17(K374E + F390D + L429M)). FIG. 6OO shows a schematic representation of the vector 4767(A/Paris/1227/17(N97D + F390D + L429M)). FIG. 6PP shows a schematic representation of vector 4768(A/Paris/1227/17(N97D + K374E + F390D + L429M)). FIG. 6QQ shows a schematic representation of vector 4775(A/Norway/2147/17(F390D + L429M)). FIG. 6RR shows a schematic representation of vector 4776(A/Norway/2147/17(K374E + F390D + L429M)). FIG. 6SS shows a schematic representation of vector 4777(A/Norway/2147/17(N97D + F390D + L429M)). FIG. 6TT shows a schematic representation of vector 4778(A/Norway/2147/17(N97D + K374E + F390D + L429M)).

FIG. 7A shows a schematic representation of vector 2295 (H5A-Indo-5-05). FIG. 7B shows a schematic representation of vector 3680 (H5A-Indo-5-05 (F393D)). FIG. 7C shows a schematic representation of vector 3645 (H5A-Egypt-N04915-14). FIG. 7D shows a schematic representation of vector 3690 (H5A-Egypt-N04915-14 (F392D)).

Fig. 8 shows a schematic of vector 1190 (a vector for fusion cloning into a CPMV 160-based expression cassette).

Detailed Description

A preferred embodiment is described below.

As used herein, the terms "comprising," "having," "including," and "containing" and grammatical variations thereof are inclusive or open-ended and do not exclude additional unrecited elements and/or method steps. The term "consisting essentially of", when used herein in connection with a product, use, or method, means that additional elements and/or method steps may be present, but that such added items do not materially affect the manner of action of the method or use. The term "consisting of" when used herein in connection with a product, use, or method excludes the presence of additional elements and/or method steps. A product, use, or method described herein as comprising certain elements and/or steps may also consist essentially of those elements and/or steps in certain embodiments and consist of those elements and/or steps in other embodiments, whether or not those embodiments are specifically mentioned. Furthermore, unless otherwise specified, the use of the singular includes the plural, and "or" means "and/or". Unless otherwise defined herein, all technical and scientific terms used herein are to be understood as having the meaning commonly understood by one of ordinary skill in the art. The term "about" as used herein means approximately +/-10% from a given value. It is to be understood that such a difference is always included in any given value provided herein, whether or not it is specifically referred to. The use of the words "a" or "an" when used herein in conjunction with the term "comprising" may mean "one," but also conform to the meaning of "one or more," at least one, "and" one or more than one.

The term "plant", "plant part", "plant matter", "plant biomass", "plant material", "plant extract" or "plant leaf" as used herein may include an intact plant, tissue, cell or any part thereof, intracellular plant components, cell explant components, liquid or solid extracts of plants, or combinations thereof, capable of providing transcription, translation and post-translation mechanisms for expression of one or more nucleic acids described herein and/or from which expressed proteins or VLPs may be extracted and purified. Plants may include, but are not limited to, herbs. Furthermore, the plant may include, but is not limited to, a crop plant including rape, brassica, maize, nicotiana (tobacco), etc., such as burley tobacco, daylily, nicotiana, Carthamus tinctorius, solanaceae, arabidopsis thaliana, alfalfa, potato, sweet potato (Ipomoea batatas), ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton, maize, rye (Secale), Sorghum (Sorghum bicolor, Sorghum vulgare), safflower (Carthamus tinctorius).

The term "plant part" as used herein refers to any part of a plant, including, but not limited to, a leaf, stem, root, flower, fruit, plant cells obtained from a leaf, stem, root, flower, fruit, plant extracts obtained from a leaf, stem, root, flower, fruit, or combinations thereof. The term "plant extract" as used herein refers to a plant-derived product obtained after physical treatment (e.g., freezing and then extracting in an appropriate buffer), mechanical treatment (e.g., grinding or homogenizing the plant or plant part and then extracting in an appropriate buffer), enzymatic treatment (e.g., using a cell wall degrading enzyme), chemical treatment (e.g., using one or more chelating agents or buffers), or a combination thereof. Plant extracts may also be processed to remove unwanted plant components, such as cell wall fragments. Plant extracts may be obtained to aid in the recovery of one or more components from a plant, plant part, or plant cell, such as proteins (including protein complexes, protein superstructures, and/or VLPs), nucleic acids, lipids, carbohydrates, or combinations thereof, from the plant, plant part, or plant cell. If the plant extract includes protein, it may be referred to as a protein extract. The protein extract may be a crude plant extract, a partially purified plant or protein extract from plant tissue, or a purified product comprising one or more proteins, protein complexes, protein superstructures, and/or VLPs. If desired, the protein extract or plant extract may be partially purified using techniques known to those skilled in the art, for example, the extract may be subjected to salt or pH precipitation, centrifugation, gradient density centrifugation, filtration, chromatography, such as size exclusion chromatography, ion exchange chromatography, affinity chromatography, or a combination thereof. Protein extracts may also be purified using techniques known to those skilled in the art.

The terms "construct," "vector," or "expression vector" as used herein refer to a recombinant nucleic acid 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 under the control of, and operably linked to, an appropriate promoter or other regulatory element responsible for transcription of the nucleic acid of interest in a host cell. It will be appreciated by those skilled in the art that the expression cassette may include a termination (terminator) sequence, which is any sequence active in a plant host. For example, the termination sequence may be derived from an RNA-2 genomic fragment of a bipartite RNA virus (e.g., cowpea mosaic virus), the termination sequence may be a NOS terminator, or the terminator sequence may be obtained from the 3' UTR of the alfalfa plastid blue protein gene.

Constructs of the disclosure may also include a 3' untranslated domain (UTR). The 3' untranslated domain comprises a polyadenylation signal as well as any other regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by the addition of a polyadenylation track at the 3' end of the mRNA precursor. Polyadenylation signals are usually recognized by homology to the standard 5 'AATAAA-3', although variations are not uncommon. Non-limiting examples of suitable 3 'domains are the 3' transcribed non-translated domains comprising the polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes, such as nopaline synthase (Nos gene) and plant genes, such as the soybean storage protein gene, the small subunit of the ribulose-1, 5-bisphosphate carboxylase gene (ssRUBISCO; U.S. Pat. No. 4,962,028; incorporated herein by reference), which are promoters for regulating plastid blue protein expression.

"regulatory domain", "regulatory element" or "promoter" refers to a nucleic acid portion, usually, but not always, upstream of the protein coding region of a gene, which may consist of DNA or RNA, or both DNA and RNA. When the regulatory domain is active and is operably associated or operably linked to the nucleotide sequence of interest, this may result in expression of the nucleotide sequence of interest. The regulatory elements are capable of regulating organ specificity, or controlling development or temporal gene activation. "regulatory domain" includes promoter elements, core promoter elements that exhibit basal promoter activity, elements that are inducible in response to an external stimulus, and elements that regulate promoter activity, such as negative regulatory elements or transcriptional enhancers. "regulatory domain" as used herein also includes elements which are active after transcription, for example regulatory elements which regulate gene expression, such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability determinants. The latter several elements may be located near the coding region.

In the context of the present disclosure, the term "regulatory element" or "regulatory domain" generally refers to a DNA sequence, usually but not always upstream (5') of a structural gene coding sequence, that controls the expression of a coding region by effecting recognition by RNA polymerase and/or other factors required to initiate transcription at a particular site. However, it will be appreciated that other nucleotide sequences located within an intragenic region or at the 3' end of a sequence may also be useful in regulating the expression of a coding region of interest. One example of a regulatory element that effects recognition of RNA polymerase or other transcription factor to ensure priming at a particular site is a promoter element. Most (but not all) eukaryotic promoter elements contain a TATA box, a conserved nucleic acid sequence consisting of adenosine and thymidine nucleotide base pairs, usually located about 25 base pairs upstream of the transcription start site. Promoter elements may include the basic promoter elements responsible for initiating transcription, as well as other regulatory elements that modify gene expression.

There are various types of regulatory domains, including developmentally regulated, inducible or constitutive regulatory domains. At a specific time during development of a specific organ or tissue of that organ, a regulatory domain is activated in that organ or tissue which is developmentally regulated or under its control controls differential expression of a gene. However, certain developmentally regulated regulatory domains may become preferentially active in certain organs or tissues at particular developmental stages, they may also become active in a developmentally regulated manner, or maintain basal levels of activity in other organs or tissues within a plant. Examples of tissue-specific regulatory domains (e.g., seed-specific regulatory domains) include the Napin promoter and the crucifer promoter (Rask et al, 1998, J.plant Physiol.152: 595-599; Bilodeau et al, 1994, Plant Cell 14: 125-130). An example of a leaf-specific promoter includes the plastocyanin promoter (see US 7,125,978, incorporated herein by reference).

An inducible regulatory domain is a domain that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequence or gene will not be transcribed. In general, a protein factor that specifically binds to an inducible regulatory domain to activate transcription can exist in an inactive form and then be converted directly or indirectly to an active form by an inducer. However, the protein factor may not be present. The inducer may be a chemical agent, such as a protein, metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress applied directly by heat, cold, salt or toxic elements or indirectly through the action of a pathogen or disease agent (e.g., a virus). Plant cells comprising an inducible regulatory domain may be exposed to an inducer by externally applying the inducer to the cells or plants (e.g., by spraying, watering, heating, or the like). Inducible regulatory elements can be derived from Plant or non-Plant genes (e.g., Gatz, C. and Lenk, I.R.P., 1998, Trends Plant Sci.3, 352-358). Examples of potentially inducible promoters include, but are not limited to, the tetracycline-inducible promoter (Gatz, C., 1997, Ann.Rev.plant physiol.plant mol.biol.48, 89-108), the steroid-inducible promoter (Aoyama, T. and Chua, N.H., 1997, Plant J.2, 397-) and the ethanol-inducible promoter (Salter, M.G. et al, 1998, Plant Journal 16, 127-.

Constitutive regulatory domains direct the expression of genes at different parts of a plant and are expressed continuously during plant development. Examples of known constitutive regulatory elements include promoters associated with the CaMV 35S transcript (p 35S; 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.5,428,147), and triosephosphate isomerase 1(Xu et al, 1994, Plant Physiol.106: 459-467) genes, maize ubiquitin 1 gene (Cornejo et al, 1993, Plant mol.biol.29: 637-646), Arabidopsis 1 and 6 genes (Holtorf et al, 1995, Plant mol.biol.29: translation initiation factor A), tobacco vein promoter 4A (Arabidopsis thaliana et al, 1995, Mo., 26-1004-promoter); the promoters of the small subunit of rubisco, PRBCs (Outhkourov et al, 2003), pUbi (for both monocotyledonous and dicotyledonous plants).

The term "constitutive" as used herein does not necessarily mean that a nucleotide sequence is expressed at the same level in all types of cells under the control of a constitutive regulatory domain, but rather that the sequence is expressed in many types of cells, even if changes in abundance are often observed.

The expression construct as described above may be present in a vector. The vector may include border sequences that allow for transfer and integration of the expression cassette into the genome of the organism or host. The construct may be a plant binary vector, such as a pPZP-based binary transformation vector (Hajdukiewicz et al, 1994). Other exemplary constructs include pBin19 (see Frisch, D.A., L.W.Harris-Haller et al, 1995, Plant Molecular Biology 27: 405-.

The term "native", "native protein" or "native domain" as used herein refers to a protein or domain having the same main amino acid sequence as the wild type. A native protein or domain may be encoded by a nucleotide sequence that has 100% sequence similarity to the wild-type sequence. The native amino acid sequence may also be encoded by a human codon (hCod) optimized nucleotide sequence or a nucleotide sequence comprising an 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 with the native amino acid sequence.

A "human codon-optimized" nucleotide sequence or "hCod" nucleotide sequence refers to the selection of appropriate DNA nucleotides such that the manner of synthesis of the oligonucleotide sequence or fragment thereof approximates the codon usage pattern that is 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 the 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 as 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 between these values over the length of the coding portion of the oligonucleotide sequence. As described below, a human codon-optimized nucleotide sequence or a nucleotide sequence comprising increased GC content (as compared to the wild-type nucleotide sequence) exhibits enhanced expression in a plant, plant part or plant cell as compared to the expression of a non-human optimized (or lower GC content) nucleotide sequence.

Described herein are a modified influenza virus Hemagglutinin (HA) protein (also known as modified HA protein, modified influenza virus HA protein, modified HA, modified influenza virus HA, mutant HA, influenza virus HA variant, or HA variant) and a method of producing a modified influenza virus hemagglutinin protein in a plant. The modified influenza virus hemagglutinin proteins disclosed herein comprise modifications or mutations that have been found to result in improved hemagglutinin properties compared to wild-type hemagglutinin or unmodified hemagglutinin proteins. For example, the modified influenza virus hemagglutinin protein may have an amino acid sequence with at least one amino acid substitution compared to the corresponding wild-type amino acid sequence.

Examples of improved properties of modified hemagglutinin proteins include: when expressed in a plant cell, increased hemagglutinin protein production compared to a wild-type or unmodified hemagglutinin of the same influenza strain or subtype which does not comprise the modification or mutation; the modified hemagglutinin protein has an increased hemagglutinin titer compared to a wild-type or unmodified hemagglutinin protein; the integrity, stability or both of the VLP comprising the modified hemagglutinin protein is increased compared to the integrity or stability or both of the VLP comprising wild-type hemagglutinin not comprising the modification or mutation; when expressed in a plant cell, the yield of VLPs is increased compared to the yield of wild-type VLPs not comprising the modification or mutation; and combinations of these advantages.

Influenza virus subtypes and strains

The term "influenza virus subtype" as used herein refers to influenza a virus variants characterized by various combinations of hemagglutinin (H or HA) and ceramidase (N) virus surface proteins. According to the present description, influenza virus subtypes and Hemagglutinin (HA) from such virus subtypes may be referred to by their H number, e.g., "HA of H1 subtype", "H1 HA", or "H1 influenza virus". The term "subtype" specifically includes all individual "strains" within each subtype, which are usually caused by mutations and may exhibit different pathogenic characteristics. Such strains may also be referred to as various "isolates" of the viral subtype. Thus, the terms "strain" and "isolate" as used herein are used interchangeably.

Traditionally, different influenza strains are classified, for example, according to the ability of influenza viruses to agglutinate erythrocytes. Antibodies specific for a particular influenza strain can bind to the virus, thereby preventing such agglutination. Assays for determining the type of strain based on this inhibition are commonly referred to as the hemagglutinin inhibition assay (HI assay or HAI assay) and are well known standard methods in the art for characterizing influenza strains.

However, hemagglutinin proteins from different strains also show significant sequence similarity at the nucleic acid and amino acid levels. This level of similarity is found to be different when strains of different subtypes are compared, with some strains showing significantly higher levels of similarity than others (Air, proc. natl. acad. sci. usa, 1981, 78: 7643). The level of amino acid similarity differs between strains of different subtypes (Air, Proc. Natl. Acad. Sci. USA, 1981, 78: 7643). This variation is sufficient to determine the individual subtypes and evolutionary lineages of the different strains, but the DNA and amino acid sequences of the 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).

Multiple nucleotide sequences or corresponding hemagglutinin polypeptide sequences can be aligned to determine a "consensus portion" or "consensus sequence" for a subtype (see figure 1).

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

Novel influenza virus hemagglutinin proteins, hemagglutinin modifications, hemagglutinin protein variants and mutants are produced by changes in the amino acid sequence of the hemagglutinin protein which result in improved hemagglutinin properties as described above. Isolation of nucleic acids encoding such hemagglutinin molecules is routine, as are nucleic acid modifications used to introduce changes in the amino acid sequence, e.g., by site-directed mutagenesis.

Described herein are a modified influenza virus hemagglutinin protein and a method of producing a modified influenza virus hemagglutinin protein in a plant. It has been observed that modification, for example by substituting specific amino acids in the hemagglutinin protein (e.g. hemagglutinin from the H1 subtype), results in improved properties of the modified hemagglutinin protein compared to the wild-type hemagglutinin protein or the unmodified hemagglutinin protein.

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

The hemagglutinin protein, mutant hemagglutinin protein or modified hemagglutinin protein described herein is modified and comprises one or more mutations, modifications or substitutions in its amino acid sequence at any one or more amino acids corresponding to the amino acid at position 97, 374, 380, 390 or 429 of a/Michigan/45/15 hemagglutinin (seq id no 134; see fig. 1) or a/California/07/09 hemagglutinin (seq id no 130; see fig. 1).

"corresponding to an amino acid" or "corresponding to an amino acid" refers to one amino acid corresponding to another amino acid in a sequence alignment with an influenza virus reference strain as described below.

The amino acid residue number or residue position of hemagglutinin is consistent with the numbering of hemagglutinin for a reference strain of influenza virus. For example, in the case of the H1 influenza virus, the reference strain may be A/Michigan/45/15 hemagglutinin (SEQ ID NO: 134; see FIG. 1) or A/California/07/09 hemagglutinin (SEQ ID NO: 130; see FIG. 1). The corresponding amino acid position can be determined by aligning the sequence of a hemagglutinin (e.g., H1 hemagglutinin) with the sequence of the hemagglutinin of its corresponding reference strain. Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignments for comparison can be made, for example, by Smith and Waterman at adv.appl.math.2: 482(1981), by Needleman and Wunsch, J.mol.biol.48: 443(1970) by Pearson and Lipman at proc.nat' l.acad.sci.usa 85: 2444(1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package of Genetics Computer Group, Inc. at science 575, Madison, Wis.), or by manual alignment and visual inspection (see, for example, the molecular biology laboratory Manual, supplementary 1995, Ausubel et al. An amino acid sequence alignment of multiple influenza a virus hemagglutinin domains is shown in figure 1, and these examples should not be considered limiting.

In referring to modifications, mutants or variants, the wild type amino acid residue (also referred to simply as "amino acid") is followed by the residue number and a new or substituted amino acid. For example, the aspartic acid (D, Asp) substitution residue or asparagine (N, Asn) at position 97 in an amino acid is designated N97D (see table 1).

Modified hemagglutinin, hemagglutinin mutants or variants (e.g., modified H1 hemagglutinin) are named in the same manner by using the one letter amino acid code for the wild type residue followed by the one letter amino acid code for its position and replacement residues. Multiple mutants are represented by individual single mutants of the component separated by slashes (/) or plus signs (+). 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 H1 hemagglutinin and aspartic acid (D, Asp) replaces phenylalanine (F, Phe) at position.

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 parentheses.

¹A/Michigan/45/15 or A/California/07/09

²A/Indonesia/05/05

The modified influenza virus Hemagglutinin (HA) protein may comprise an amino acid sequence having at least one amino acid substitution compared to the corresponding wild-type amino acid sequence.

"amino acid substitution" or "substitution" refers to the replacement of an amino acid in a protein 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 may be replaced by one or more amino acids that differ from the original or wild-type amino acid at that position without altering 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 of a different amino acid compared to the original or wild-type amino acid. The resulting protein is a modified protein, such as a modified influenza virus hemagglutinin protein. The modified influenza hemagglutinin protein is not naturally occurring.

Modified hemagglutinin includes non-naturally occurring hemagglutinin proteins, has at least one modification to the naturally occurring hemagglutinin, and has improved properties compared to the naturally occurring hemagglutinin protein from which the amino acid sequence of the modified hemagglutinin is derived. Modified hemagglutinin proteins have amino acid sequences that are not found in nature, which are derived by replacing one or more amino acid residues of the hemagglutinin protein with one or more different amino acids.

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

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 the amino acid sequence found in nature. In the case of conserved residues, such modifications, substitutions or replacements should also not result in a naturally occurring hemagglutinin sequence.

Conservative substitutions

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

The term "conservative substitution" and grammatical variations thereof as used herein refers to the presence of an amino acid residue in the sequence of the hemagglutinin protein that is different from the substitution or the residue in question but belongs to the same class of amino acids (i.e., a non-polar residue that replaces a non-polar residue, an aromatic residue that replaces an aromatic residue, a polar non-charged residue that replaces a polar non-charged residue, a charged residue that replaces a charged residue). Furthermore, conservative substitutions may encompass residues having an interfacial hydrophilicity value of the same sign and approximately similar magnitude as the residue that replaces the wild-type residue.

The term "nonpolar residue" as used herein 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), as well as the positively charged amino acids lysine (K, Lys), arginine (R, Arg) and histidine (H, His). Other classifications of amino acids are as follows:

amino acids with hydrophobic side chains (aliphatic): alanine (a, Ala), isoleucine (I, Ile), leucine (L, Leu), methionine (M, Met), and valine (V, Val);

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

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

amino acids with charged side chains (acidic): aspartic acid (D, Asp), glutamic acid (E, Glu);

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

Conservative amino acid substitutions may have a similar effect on the resulting activity of the hemagglutinin protein variant or modified hemagglutinin protein as the original substitution or modification. More information on conservative substitutions can be found, for example, in the articles of Ben Bassat et al (J.Bacteriol, 169: 751-.

The correlation of polypeptide sequences is usually determined using Blosum matrices. Blosum matrices are created using large, reliable alignments databases (BLOCKS databases) that account for alignments of pairs of sequences that have a correlation below a certain threshold percentage of identity (Henikoff et al, Proc. Natl. Acad. Sci. USA, 89: 10915-. For the highly conservative target frequency of the BLOSUM90 matrix, a 90% identity threshold is used. For the BLOSUM65 matrix, a 65% identity threshold is used. Scores above zero in the Blosum matrix are considered "conservative substitutions" at a selected percentage identity. Exemplary conservative amino acid substitutions are shown in table 2 below.

TABLE 2 exemplary conservative amino acid substitutions

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

Modification of H1 hemagglutinin

Described herein are a modified influenza H1 hemagglutinin protein and a method of producing a modified influenza H1 hemagglutinin protein in a plant. It has been observed that modification of specific amino acids in hemagglutinin proteins from the H1 subtype results in improved properties of the modified H1 hemagglutinin protein compared to the wild-type H1 hemagglutinin protein or the unmodified H1 hemagglutinin protein.

To improve the properties of the H1 hemagglutinin protein, a total of 42 single, double and/or triple modifications were tested. As described and exemplified herein, only the modification or combination of modifications at a particular position improved the properties of the H1 hemagglutinin protein. Modifications at 32 positions or combinations of these positions have an adverse effect on the properties of the H1 hemagglutinin protein (data not shown).

Examples of improved properties of the H1 hemagglutinin mutant protein include: when expressed in a plant cell, the production or accumulation of hemagglutinin protein is increased compared to a wild-type or unmodified H1 hemagglutinin of the same influenza strain or subtype which does not comprise the modification or mutation; the hemagglutinin protein, modified or mutated, has an increased hemagglutinin titer compared to the wild-type or unmodified H1 hemagglutinin protein; the integrity, stability or both of the VLP comprising the modified H1 hemagglutinin protein is increased compared to the integrity or stability or both of the VLP comprising wild-type hemagglutinin not comprising the mutation; when expressed in a plant cell, the yield of VLPs is increased compared to the yield of wild-type VLPs not comprising the modification or mutation; and combinations of these advantages.

The modified H1 hemagglutinin protein or the mutant H1 hemagglutinin protein described herein is modified and comprises one or more mutations or modifications at any one or more residues para to the sequence 97, 374, 380, 390 and/or 429 of a/California/07/09 hemagglutinin (seq id No. 130; see fig. 1). Accordingly, there is provided an H1 influenza virus hemagglutinin polypeptide, protein and/or protein complex, e.g. a Virus Like Particle (VLP), which VLP comprises a modification or mutation at one or more of amino acid positions 97, 374, 380, 390 and/or 429 (such amino acid numbering being based on the sequence of a/California/07/09 hemagglutinin (seq id No. 130) as shown in figure 1) or at an amino acid position corresponding to such amino acid position, e.g. as determined by alignment of the H1 hemagglutinin amino acid sequence with seq id No. 130. Non-limiting examples of influenza H1 hemagglutinin amino acid sequences comprising one or more of such mutations include sequence nos. 131, 132, 133, 134, 135, 138 and 139.

Modified H1 hemagglutinin proteins described herein include H1 hemagglutinin protein having an amino acid sequence with about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity or sequence similarity to the amino acid sequence encoding hemagglutinin from H1 (seq id No. 130, 131, 132, 133, 134, 135, 138 or 139) or any amount between these values, wherein the amino acid sequence has one or more mutations or modifications at any one or more residues that are para to the position 97, 374, 380, 390 and 429 sequences of a/California/07/09 hemagglutinin (seq id No. 130), and wherein the hemagglutinin protein forms a VLP when expressed.

Further, the H1 hemagglutinin protein may 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 of sequence identity or sequence similarity between these values to the nucleotide sequence encoding hemagglutinin from H1 (seq id No. 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 that are para to the a/California/07/09 hemagglutinin position 97, 374, 380, 390 and 429 sequences, and wherein the nucleotide sequence encodes a hemagglutinin protein that forms a VLP when expressed.

Non-limiting examples of strains from which H1 hemagglutinin may be derived are 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/Hondaus/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).

The modified or mutated H1 hemagglutinin may be mono-, di-, tri-or tetra-substituted at residue 97, 374, 380, 390 or 429. In a monosubstituted H1 hemagglutinin, one residue may be mutated at position 97, 374, 380, 390 or 429. In a disubstituted H1 hemagglutinin, two residues may be substituted, for example the residues at positions 380 and 429, 97 and 374 or 390 and 429 may be substituted. In the trisubstituted H1 hemagglutinin mutant, three residues may be substituted. For example, the residues at positions 97, 390 and 429 or the residues at positions 97, 374 and 429 may be substituted. In the tetrasubstituted H1 hemagglutinin mutant, four residues may be substituted. For example, the residues at positions 97, 374, 390 and 429 may be substituted. (all H1 hemagglutinin numbering was determined from sequence alignment with reference strain A/California/07/09 hemagglutinin).

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):

monosubstituted H1 hemagglutinin mutant: N97D, K374E, F390D or L429M;

disubstituted H1 hemagglutinin mutants: N390A/L429M, N97D/K374E or N380/1429M;

trisubstituted H1 hemagglutinin mutants: N97D/F390D/L429M or K374E/F390D/1429M;

tetrasubstituted H1 hemagglutinin mutant: N97D/K374E/F390D/L429M.

One or more of the mutations described herein specifically increase production of influenza virus hemagglutinin protein and VLP production in a plant. Mutations at other positions have been observed to significantly reduce or have no significant effect on the amount of accumulation of influenza virus hemagglutinin protein in plant cells or VLP production.

Monosubstituted H1 hemagglutinin

Modification at position 97

In one aspect of the disclosure, the modified H1 hemagglutinin may have a modified residue at least at position 97. This residue is not involved in the receptor binding of hemagglutinin and has been shown to be located in one of the residual esterase (VE) subdomains of the globular head of hemagglutinin. X-ray crystallography analysis indicated that this residue was buried in the hemagglutinin trimer. Thus, this residue is not part of the antigenic site, is not involved in antigenic changes, and is not recognized by broadly neutralizing antibodies.

In influenza A (H1N1) pdm09 virus, this residue is predicted to be involved in the stability of hemagglutinin (see Castel a N-Vega et al, 2014). However, Castel N-Vega et al indicated that single point mutations appear to have little effect on hemagglutinin stability, and cited Yang et al (structural stability of hemagglutinin for pdm09 influenza A (H1N1), 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 H1N1 virus (which can be broken down into monomers) circulating early in 2009 and the recombinant trimeric hemagglutinin protein of the most recent virus (which can be expressed as trimers). Yang et al found that A/Texas/1/2011(Tex 11) had a unique Asp97Asn (D97N) substitution in hemagglutinin compared to the four other A (H1N1) pdm09 strains studied for sequence differences. However, the H1 hemagglutinin influenza virus strain evolved thereafter had asparagine (N, Asn) at position 97 (see fig. 1, H1 sequence alignment), indicating that the H1 hemagglutinin virus strain has the evolutionary advantage of asparagine (N, Asn) at this position. Thus, it was unexpected that a modification from asparagine (N, Asn) at position 97 to non-asparagine resulted in the improvement of the H1 hemagglutinin protein properties described herein.

As shown in fig. 3A, 3B, 4A and 4B, H1 hemagglutinin with a residue at position 97, for example, changing from asparagine (N, Asn) to aspartic acid (AspD) (hereinafter referred to as N97D) showed an increase in hemagglutination titer of up to 1200% compared to H1 hemagglutinin with asparagine (N, Asn) at this position (see table 5A). FIG. 3A shows that hemagglutinin from A/Michigan/45/15 with the N97D substitution exhibited an approximately 1100% increase in hemagglutination titer compared to A/Michigan/45/15 HA wild-type (also known as H1 Michigan). The hemagglutination titer from hemagglutinin with the N97D substitution a/Honduras/17734/16 was increased by approximately 375% compared to wild-type a/Honduras/17734/16 hemagglutinin (see fig. 4A and 4B). Furthermore, as shown in FIGS. 4A and 4B, the hemagglutination titer from A/Darwin/11/15 with the N97D substitution was increased by about 300% compared to the wild-type A/Darwin/11/15 hemagglutinin.

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

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

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

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 of sequence identity or sequence similarity therebetween to a nucleotide sequence encoding 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), such as a glutamic acid (E, Glu), glutamine (Q, gin), or serine (S, Ser), at position 97, wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms a VLP upon expression.

For example, the modified H1 hemagglutinin may have one or more modifications; wherein at least residue 97 of H1 hemagglutinin is modified as described herein. For example, the modified H1 hemagglutinin may be a mono-, di-, tri-, or tetra-substituted H1 hemagglutinin in which at least the residue at position 97 is modified. In a non-limiting example, the modified H1 hemagglutinin may have a substitution residue at position 97 and one or more substituents at positions 374, 390, 429 or a combination thereof, wherein the modified H1 hemagglutinin sequence is not naturally occurring.

Furthermore, a method of producing a VLP in a plant is provided, the VLP comprising a modified H1 hemagglutinin having a substitution at position 97as 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

Furthermore, there is provided a method of increasing yield of a VLP in a plant, the VLP comprising a modified H1 hemagglutinin having a substitution at position 97as 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present specification also provides a VLP comprising a H1 hemagglutinin having a substitution at position 97. The VLP may be produced by the methods provided by the present disclosure. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Modification at position 374

In one aspect of the disclosure, the residue at position 374 in H1 hemagglutinin may be substituted (numbering according to a/California/07/09 hemagglutinin numbering). This residue is located in the stem of H1 hemagglutinin.

Cotter et al (PLoS Patholog.2014; 10 (1): E1003831) determined that the E47K (HA2 numbering) mutation in the stem domain of A/California/7/2009 hemagglutinin stabilized the trimeric structure, reduced the pH of the membrane fusion, and improved the thermal and acid stability of the virus. Position 47 of the HA2 handle domain of H1N1 in Cotter corresponds to position 374 in this disclosure (HA 0 numbering of A/California/7/2009). Cotter et al also observed that the a/California/7/2009E 47K 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) which showed that by changing glutamic acid to lysine at position 374 and introducing a basic side chain change at position 374, it was possible to form a new salt bridge across the monomer interface with Glu 21 on the adjacent chain, thereby improving stability at lower pH. Poplar et al found that the presence of lysine at position 374 enhanced the ability of mutant Tex09 ectodomain trimers to resist heat and acidity changes to a level comparable to the ability of Wash11 recHA.

However, it was surprisingly found that the replacement of lysine (K, Lys) with glutamic acid (E, Glu) at position 374(K374E) of the hemagglutinin of H1Michigan influenza virus (a/Michigan/45/15(H1N1)) resulted in an increase of approximately 1200% of the hemagglutination titer compared to plant extracts expressing wild-type H1 hemagglutinin (see fig. 3A, 3B, 4A, 4B and table 5A).

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

The 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 that is active in plants.

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 of sequence identity or sequence similarity therebetween with the nucleotide sequence encoding hemagglutinin from H1Michigan (A/Michigan/45/15, SEQ ID NO: 136), wherein the nucleotide sequence encodes a hemagglutinin protein having a glutamic acid (E, Glu) or 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 a VLP upon expression.

For example, the modified H1 hemagglutinin may have one or more modifications; wherein at least residue 374 of H1 hemagglutinin is modified as described herein. For example, the modified H1 hemagglutinin may be a mono-, di-, tri-, or tetra-substituted H1 hemagglutinin in which at least the residue at position 374 is modified. In a non-limiting example, the modified H1 hemagglutinin may 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 is not naturally occurring.

Further, the present specification provides a method of producing a VLP in a plant, the VLP 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 a modified H1 hemagglutinin having a substitution at position 374 and operably linked to a regulatory domain that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

Furthermore, there is provided a method of increasing yield of a VLP in a plant, the VLP 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 a modified H1 hemagglutinin having a substitution at position 374 and operably linked to a regulatory domain that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present specification also provides a VLP comprising a H1 hemagglutinin having a substitution at position 374. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Modification at position 390

In one aspect of the disclosure, the residue at position 390 in H1 hemagglutinin may be substituted (numbering according to a/California/07/09 hemagglutinin numbering).

WO2013/177444 to Lu et al and its complement (Proc Natl Acad Sci USA 2014; 111 (1): 125-30) report a method for producing a correctly folded hemagglutinin stem domain 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 stem domain of hemagglutinin, Chloramphenicol Acetyltransferase (CAT) or a folding domain is fused to the carbon terminus of hemagglutinin. To mitigate newly exposed hydrophobicity and/or intermolecular ion pairing leading to aggregation of expressed hemagglutinin stem proteins, five sets 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 points out that the soluble yield of the mutants is low and insoluble inclusion bodies are formed. Lu also indicates that mutants M3 and M5 produced much less aggregates than other variants of the wild type, thus further developing mutant M5(F164D + L174D). Lu observed that the M5(F164D + L174D) mutation appears to be the mutation that has the greatest effect on increasing the solubility of the hemagglutinin stem protein. The position 164 of Lu corresponds to the position 390 in this disclosure. The M4(F164D) mutation in Lu does not show advantages over the other mutations tested, and is actually inferior to the mutants M3(I69T + I72E + I74T + C77T + F164D + L174D) and M5(F164D + L174D).

When phenylalanine (F, Phe) at position 390 and leucine (L, Leu) at position 400 (H1 hemagglutinin numbering) corresponding to phenylalanine at position 164 and leucine at position 174 of M5(F164D + L174D) of Lu were altered in H1 hemagglutinin of the present disclosure, no increase in VLP production was observed, and the H1F 390D + L400D mutant exhibited complete loss of hemagglutination activity (data not shown). Therefore, the equivalent mutation of the M5 mutant in Lu did not result in an improvement of the properties of hemagglutinin expressed in plants.

It was unexpectedly found that when the hydrophobic amino acid at position 390 in H1 hemagglutinin was substituted with a charged amino acid, an increase of approximately 60% in VLP yield was observed from plants expressing H1 hemagglutinin with substitution at position 390 after iodixanol gradient purification compared to plants infiltrated with the wild type construct (see fig. 3B, 4A, 4B, tables 5A, 5B). In addition, as shown in table 5C, the overall process yield increased to 226%. However, equivalent modifications in hemagglutinin from H5 (F393D) resulted in a decrease in hemagglutination titers (see fig. 5, table 6).

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

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

For example, the modified H1 hemagglutinin protein may have an amino acid sequence with 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 therebetween to the amino acid sequence of hemagglutinin from H1a/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, gin), or serine (S, Ser), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms a VLP upon expression.

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

For example, the modified H1 hemagglutinin may have one or more modifications; wherein at least residue 390 of H1 hemagglutinin is modified as described herein. For example, the modified H1 hemagglutinin may be a mono-, di-, tri-, or tetra-substituted H1 hemagglutinin in which at least the residue at position 390 is modified. In a non-limiting example, the modified H1 hemagglutinin may have a substituted residue at position 390 and one or more substituents at positions 97, 374, 380, 429 or a combination thereof.

Furthermore, a method of producing a VLP in a plant is provided, the VLP 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

Furthermore, there is provided a method of increasing yield of a VLP in a plant, the VLP 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present specification also provides a VLP comprising a H1 hemagglutinin having a substitution at position 390. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Modification at position 429

In one aspect of the disclosure, the residue at position 429 in H1 hemagglutinin may be modified (numbering according to H1A/Michigan/45/15 (SEQ ID NO: 134)).

Antanasijevic et al (J Biol chem. 2014; 289 (32): 22237-45) investigated the structure-functional properties of the stem-loop domain of H5 hemagglutinin by site-directed mutagenesis at 14 different positions. The HA1-D26K, HA1-M102L, HA2-V52A and HA2-I55A mutants (numbered based on H3) were observed by Antanasijevic to exhibit significantly reduced total hemagglutinin levels, indicating reduced expression and/or assembly of hemagglutinin in the virion. The HA1-D26K, HA2-T49A and HA2-M102L mutants also showed lower hemagglutination titers compared to the wild-type virus. Position 102 in the hemagglutinin of H5 of Antanasijevic corresponds to position 429 in the hemagglutinin of H1 of the present specification.

When H1 hemagglutinin was modified to introduce changes at V19I (HA1-I28V), L20M (HA1-M31L), T368A (HA2-T41A), N380A (HA2-N53A) or L429M (HA2-M102L), T368A was found to cause complete loss of activity, V19I and L20M had lower activity, while N380A and L429M showed higher activity than H1a/California wild-type hemagglutinin.

Thus, most of the residues determined by Antanasijevic to be of significance for the expression and/or assembly or hemagglutination titer of hemagglutinin from H5 do not appear to be converted to have similar significance in hemagglutinin from H1.

However, as shown in FIGS. 2A, 2B, 2C, 3B, 4A and 4B, when residue 429 in H1 hemagglutinin was mutated from leucine (L, Leu) to methionine (M, Met), the modified H1 hemagglutinin showed an increase in hemagglutinin titer (100-160%) compared to the wild-type H1 hemagglutinin (see Table 5A). Furthermore, as shown in figure 2C, plants expressing H1 hemagglutinin with substitutions at position 429 showed an approximately 30% increase in VLP yield after sucrose gradient purification compared to plants infiltrated with the wild type construct (see table 5B). In addition, as shown in table 5C, the overall process yield increased to 260%.

Furthermore, a 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 increase in hemagglutination titer of about 60% compared to the unmodified H1 hemagglutinin (see fig. 2B).

Thus, in one aspect, the residue at position 429 of H1 hemagglutinin (numbered according to H1A/Michigan/45/15 amino acid sequence (seq id no 134)) can be modified to replace the 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. For example, the H1 hemagglutinin protein may be modified to contain 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).

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

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

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 of sequence identity or sequence similarity to the nucleotide sequence encoding hemagglutinin from H1A/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 is not naturally occurring, and wherein the hemagglutinin protein forms a VLP upon expression.

For example, the modified H1 hemagglutinin may 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 may be a mono-, di-, tri-, or tetra-substituted H1 hemagglutinin in which at least the residue at position 429 is modified. In a non-limiting example, the modified H1 hemagglutinin may have a substituted residue at position 429 and one or more substituents at positions 97, 374, 380, 390, or a combination thereof.

Furthermore, a method of producing a VLP in a plant is provided, the VLP 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing a VLP.

Furthermore, there is provided a method of increasing yield of a VLP in a plant, the VLP 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing a VLP.

The present specification also provides a VLP comprising an H1 hemagglutinin having a substitution at position 429. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Disubstituted H1 hemagglutinin

Also provided is a H1 hemagglutinin protein comprising at least one double substitution or double modification. Thus, the H1 hemagglutinin protein has at least two modifications from the wild-type H1 hemagglutinin protein. For example, the H1 hemagglutinin may 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)).

Modification of positions 380 and 429

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

As shown in fig. 3B, the hemagglutination titer of H1 hemagglutinin having a residue at position 380 modified from asparagine to alanine and a residue at position 429 modified from leucine to methionine was approximately 800% higher compared to wild-type H1 hemagglutinin (see table 5A).

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 to replace asparagine (N, Asn) with a non-asparagine at position 380 and leucine (L, Leu) with a non-leucine at position 429 to produce a modified H1 hemagglutinin with a non-naturally occurring sequence. For example, the H1 hemagglutinin may be modified by replacing the polar amino acid with a hydrophobic amino acid at position 380 and the leucine (L, Leu) with another hydrophobic amino acid other than leucine at position 429 to produce a modified H1 hemagglutinin with a non-naturally occurring sequence.

For example, the H1 hemagglutinin protein may be modified to include an alanine or conservative alanine substitution at position 380. The conservative alanine substitution may 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 may be modified to include 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).

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 that is active in plants.

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 therebetween to the nucleotide sequence of seq id No. 136, wherein the nucleotide codon encoding amino acid residue 380 of modified H1 hemagglutinin encodes an alanine or conservative alanine substitution (e.g., serine (S, Ser), glycine (G, Gly), threonine (T, Thr), cysteine (C, Cys), or valine (V, Val)), and the nucleotide codon encoding amino acid residue of modified H1 hemagglutinin encodes a methionine (M, Met) or a conservative methionine substitution other than leucine (L, Leu) (e.g., 429 (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 a VLP upon expression.

Furthermore, a method of producing a VLP in a plant is provided, the VLP comprising a modified H1 hemagglutinin with 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

Furthermore, a method of increasing yield of a VLP in a plant comprising a modified H1 hemagglutinin having substitutions at positions 380 and 429 as described above is provided. 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present specification also provides a VLP comprising an H1 hemagglutinin having substitutions at positions 380 and 429. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Modifications at positions 390 and 429

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

As shown in FIGS. 2B, 3B, 4A, 4B and Table 5A, the hemagglutination titer of H1 hemagglutinin having a residue at position 390 modified from phenylalanine to aspartic acid and a residue at position 429 modified from leucine to methionine was increased by approximately 400-1200% compared to the wild-type H1 hemagglutinin. Furthermore, as shown in table 5C, the overall process yield increased to 633%.

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)) may be modified to replace phenylalanine (F, Phe) with a non-phenylalanine at position 390 and leucine (L, Leu) with a non-leucine at position 429 to produce a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin may be modified by replacing a hydrophobic amino acid with a charged amino acid at position 390 and a hydrophobic amino acid other than leucine at position 429 (L, Leu) to produce a modified H1 hemagglutinin having a non-naturally occurring sequence.

For example, the H1 hemagglutinin protein may be modified to include an aspartic acid (D, Asp) or conservative aspartic acid (D, Asp) substitution at position 390. The conservative substitution may be, for example, asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). In addition, the H1 hemagglutinin protein may be modified to include 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).

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 that is active in plants.

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 modified H1 hemagglutinin encodes an aspartic acid (D, Asp) or 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 modified H1 hemagglutinin encodes 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), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms a VLP when expressed.

For example, the modified H1 hemagglutinin may have one or more modifications; wherein at least residues 390 and 429 of H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin may be a di-, tri-, or tetra-substituted H1 hemagglutinin in which at least the residues at positions 390 and 429 are modified. In a non-limiting example, the modified H1 hemagglutinin may have substituted residues at positions 390 and 429 and one or more substituents at positions 97, 374, 380 or a combination thereof.

Furthermore, a method of producing a VLP in a plant is provided, the VLP comprising a modified H1 hemagglutinin with 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 being operably linked to a regulatory domain that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

Furthermore, a method of increasing yield of a VLP in a plant comprising a modified H1 hemagglutinin having substitutions at positions 390 and 429 as described above is provided. 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 being operably linked to a regulatory domain that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present specification also provides a VLP comprising an H1 hemagglutinin having substitutions at positions 390 and 429. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Modifications at positions 97 and 374

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

As shown in fig. 3B and table 5A, H1 hemagglutinin with a residue modified from asparagine to aspartic acid at position 97 and a residue modified from lysine to glutamic acid at position 374 exhibited an approximately 1200% increase in hemagglutination titer compared to wild-type H1 hemagglutinin.

Thus, in one aspect, the residues at positions 97 and 374 of the H1 hemagglutinin may be modified (numbering according to H1A/Michigan/45/15 (seq id No. 134)), asparagine (N, Asn) at position 97 replaced with non-asparagine, and lysine (K, Lys) at position 374 replaced with non-lysine to produce a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin may be modified by replacing the charged amino acid with a polar amino acid at position 97 and another charged amino acid other than lysine (K, Lys) at position 374 to produce a modified H1 hemagglutinin having a non-naturally occurring sequence.

For example, the modified H1 hemagglutinin protein may 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 of sequence identity or sequence similarity therebetween to the amino acid sequence of hemagglutinin from H1a/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) at position 97, such as glutamic acid (E, Glu), glutamine (Q, gin) or serine (S, Ser), and the amino acid sequence has 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, gin), 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 proteins form VLPs upon expression.

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 that is active in plants.

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 therebetween to the nucleotide sequence of seq id No. 136, wherein the nucleotide codon encoding amino acid residue 97 of modified H1 hemagglutinin encodes a substitution of aspartic acid (D, Asp) or a conserved aspartic acid (D, Asp) other than asparagine (N, Asn), such as glutamic acid (E, Glu), glutamine (Q, gin) or serine (S, Ser), and the amino acid codon encoding amino acid residue of modified H1 hemagglutinin encodes a substitution of glutamic acid (E, Glu) or a conserved glutamic acid (E, Glu) 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 proteins form VLPs upon expression.

For example, the modified H1 hemagglutinin may have one or more modifications; wherein at least residues 97 and 374 of H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin may be a di-, tri-, or tetra-substituted H1 hemagglutinin in which at least the residues at positions 97 and 374 are modified. In a non-limiting example, the modified H1 hemagglutinin may have substituted residues at positions 97 and 374, and one or more substituents at positions 380, 390 and 429, or a combination thereof.

Furthermore, a method of producing a VLP in a plant is provided, the VLP comprising a modified H1 hemagglutinin with 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

Furthermore, there is provided a method of increasing yield of a VLP in a plant, the VLP 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present specification also provides a VLP comprising a H1 hemagglutinin having substitutions at positions 97 and 374. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Trisubstituted H1 hemagglutinin

Modifications at positions 97, 390 and 429

Also provided is a H1 hemagglutinin protein comprising at least one trisubstitution or trisubstitution modification. Thus, the H1 hemagglutinin protein has at least three modifications from the wild-type H1 hemagglutinin protein. For example, the H1 hemagglutinin may 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)).

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

For example, as shown in figures 3B, 4A, and 4B, H1 hemagglutinin having a modification from asparagine to aspartic acid at position 90, a modification from phenylalanine to aspartic acid at position 390, and a modification from leucine to methionine at position 429 showed an approximately 2600% increase in hemagglutination titer compared to wild-type H1 hemagglutinin. In addition, as shown in table 5C, the overall process yield increased to 647%.

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

For example, the H1 hemagglutinin protein may be modified to contain 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). The modified hemagglutinin protein may further comprise an aspartic acid (D, Asp) or conservative aspartic acid (D, Asp) substitution at position 390. The conservative aspartic acid substitution can be, for example, an asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser) substitution. In addition, the H1 hemagglutinin protein may be modified to include 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).

For example, the modified H1 hemagglutinin protein may 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 of sequence identity or sequence similarity therebetween to the amino acid sequence of hemagglutinin from H1a/Michigan/45/15(H1N1), which has an aspartic acid (D, Asp) or conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn) at position 97, such as glutamic acid (E, Glu), glutamine (Q, gin) or serine (S, Ser), which has an aspartic acid (D, Asp) or 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 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 a VLP when expressed.

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 that is active in plants.

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 of sequence identity or sequence similarity therebetween to a nucleotide sequence encoding hemagglutinin from H1a/Michigan/45/15(H1N1) having an aspartic acid (D, Asp) or conservative substitution of an aspartic acid (D, Asp) other than asparagine (N, Asn) at position 97 (e.g., glutamic acid (E, Glu), glutamine (Q, gin) or serine (S, Ser) at position 390 (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, gln) or serine 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 is not naturally occurring, and wherein the hemagglutinin protein forms a VLP upon expression.

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 therebetween to the nucleotide sequence of seq id No. 136, wherein the nucleotide codon encoding amino acid residue 97 of modified H1 hemagglutinin encodes a substitution of aspartic acid (D, Asp) or a conserved aspartic acid (D, Asp) other than asparagine (N, Asn), such as glutamic acid (E, Glu), glutamine (Q, gin) or serine (S, Ser), and the nucleotide codon encoding amino acid residue 390 of modified H1 hemagglutinin encodes a substitution of aspartic acid (D, Asp) or a conserved aspartic acid (D, Asp) (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 a methionine (M, Met) or a conserved methionine (M, Met) substitution (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)) other than leucine (L, Leu) at position 429, wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms a VLP upon expression.

For example, the modified H1 hemagglutinin may have one or more modifications; wherein at least residues 97, 390 and 429 of H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin may be a tri-or tetra-substituted H1 hemagglutinin in which at least the residues at positions 97, 390 and 429 are modified. In a non-limiting example, a modified H1 hemagglutinin may have substituted residues at positions 97, 390, 429 and 374.

Furthermore, a method of producing a VLP in a plant is provided, the VLP 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

Furthermore, a method of increasing yield of a VLP in a plant comprising a modified H1 hemagglutinin having substitutions at positions 97, 390 and 429 as described above is provided. 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present specification also provides a VLP comprising a H1 hemagglutinin having substitutions at positions 97, 390 and 429. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Modifications at positions 374, 390 and 429

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

For example, as shown in figure 3B, H1 hemagglutinin having a lysine to glutamic acid modification at position 374, a phenylalanine to aspartic acid modification at position 390, and a leucine to methionine modification at position 429 showed an approximately 2500% increase in hemagglutinin titer compared to wild-type H1 hemagglutinin. Furthermore, as shown in table 5C, the overall process yield increased to 689%.

Thus, in one aspect, the H1 hemagglutinin may be modified at residues at positions 374, 390, and 429 (numbered as H1A/Michigan/45/15 (seq id no 134)), lysine (K, Lys) at position 374 replaced with a non-lysine, phenylalanine (F, Phe) at position 390 replaced with a non-phenylalanine, and leucine at position 429 replaced with a non-leucine to produce a modified H1 hemagglutinin having a non-naturally occurring sequence. For example, the H1 hemagglutinin may be modified by replacing lysine with a charged amino acid other than lysine at position 374, a hydrophobic amino acid with a charged amino acid at position 390, and 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.

For example, the H1 hemagglutinin protein may be modified to contain 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, gin), arginine (R, Arg), asparagine (N, Asn), histidine (H, His), or serine (S, Ser). The modified hemagglutinin protein may further comprise an aspartic acid (D, Asp) or conservative aspartic acid (D, Asp) substitution at position 390. The conservative substitution may be, for example, asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser). In addition, the H1 hemagglutinin protein may be modified to include 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).

For example, the modified H1 hemagglutinin protein may 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 of sequence identity or sequence similarity therebetween to the amino acid sequence of hemagglutinin from H1A/Michigan/45/15(H1N1) (seq id no 134), wherein the amino acid sequence has 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, gin), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser), the amino acid sequence has aspartic acid (D, Asp) or a conservative aspartic acid (D) at position 390, asp) substitution, such as asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, gin) or serine Ser), and the amino acid sequence has a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu), such as isoleucine (I, Ile), glutamine (Q, gin), 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 a VLP upon expression.

The present specification 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 that is active in plants.

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 of sequence identity or sequence similarity therebetween to a nucleotide sequence encoding hemagglutinin from H1a/Michigan/45/15(H1N1) having a glutamic acid (E, Glu) or conservative glutamic acid (E, Glu) substitution other than lysine (K, Lys) at position 374 (e.g., aspartic acid (D, Asp), glutamine (Q, gin), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser)), aspartic acid (D, Asp) or a conservative aspartic acid (D, Asp) substitution at position 390 (e.g., asparagine (N, asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine Ser)), and a conservative methionine (M, Met) substitution (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val), or phenylalanine (F, Phe)) other than leucine (L, Leu) at position 429 (e.g., a modified H1 hemagglutinin protein, wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms a VLP when expressed.

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 374 of modified H1 hemagglutinin encodes a 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 390 of 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 Ser)) and the nucleotide codon encoding amino acid residue 429 of the modified H1 hemagglutinin encodes a methionine (M, Met) or a conserved 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 proteins form VLPs upon expression.

For example, the modified H1 hemagglutinin may have one or more modifications; wherein at least residues 374, 390 and 429 of H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin may be a tri-or tetra-substituted H1 hemagglutinin in which at least the residues at positions 374, 390 and 429 are modified. In a non-limiting example, a modified H1 hemagglutinin may have substituted residues at positions 374, 390, 429 and 97.

Furthermore, a method of producing a VLP in a plant is provided, the VLP 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

Furthermore, a method of increasing yield of a VLP in a plant comprising a modified H1 hemagglutinin having substitutions at positions 374, 390 and 429 as described above is provided. 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 that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing the VLP.

The present specification also provides a VLP comprising a H1 hemagglutinin having substitutions at positions 374, 390 and 429. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

Tetrasubstituted H1 hemagglutinin

Modifications at positions 97, 374, 390 and 429

Also provided is a H1 hemagglutinin protein comprising at least one tetrasubstituted or tetrasubstituted modification. Thus, the H1 hemagglutinin protein has at least four modifications from the wild-type H1 hemagglutinin protein. For example, the H1 hemagglutinin may have modifications at positions 97, 374, 390, and 429 (numbering according to H1A/Michigan/45/15 (SEQ ID NO: 134)).

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

For example, as shown in figure 3B, H1 hemagglutinin having a residue at position 97 modified from asparagine to aspartic acid, a residue at position 374 modified from lysine to glutamic acid, a residue at position 390 modified from phenylalanine to aspartic acid, and a modification at position 429 from leucine to methionine, exhibited an increase in hemagglutinin titer of approximately 3300% compared to wild-type H1 hemagglutinin.

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

For example, the H1 hemagglutinin protein may be modified to contain 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). Furthermore, the H1 hemagglutinin protein may be modified to contain 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, gin), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser). The modified hemagglutinin protein may further comprise an aspartic acid (D, Asp) or conservative aspartic acid (D, Asp) substitution at position 390. The conservative aspartic acid substitution can be, for example, an asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln), or serine (S, Ser) substitution. In addition, the H1 hemagglutinin protein may be modified to include 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).

For example, the modified H1 hemagglutinin protein may 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 of sequence identity or sequence similarity therebetween to the amino acid sequence of hemagglutinin from H1a/Michigan/45/15(H1N1), which has an aspartic acid (D, Asp) or conservative aspartic acid (D, Asp) substitution other than asparagine (N, Asn), such as glutamic acid (E, Glu), glutamine (Q, gin) or serine (S, Ser), at position 97, 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), the amino acid sequence having an aspartic acid (D, Asp) or 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 a VLP upon expression.

The present specification 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 that is active in plants.

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 of sequence identity or sequence similarity therebetween to the nucleotide sequence encoding hemagglutinin from H1a/Michigan/45/15(H1N1) having an aspartic acid (D, Asp) or conservative aspartic acid (D, Asp) substitution at position 97 (e.g., glutamic acid (E, Glu), glutamine (Q, gin) or serine (S, Ser) other than asparagine (N, Asn)), a glutamic acid (E, Glu) or conservative glutamic acid (E, Glu) substitution 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)), a modified H1 hemagglutinin protein having an aspartic acid (D, Asp) or conservative aspartic acid (D, Asp) substitution at position 390 (e.g., asparagine (N, Asn), glutamic acid (E, Glu), glutamine (Q, Gln) or serine (S, Ser)), and having a methionine (M, Met) or a conservative methionine (M, Met) substitution other than leucine (L, Leu) at position 390 (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 a VLP upon expression.

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 to the nucleotide sequence of seq id No. 136, or any amount therebetween, wherein the nucleotide codon encoding amino acid residue 97 of modified H1 hemagglutinin encodes a substitution of aspartic acid (D, Asp) or a conserved aspartic acid (D, Asp) other than asparagine (N, Asn), such as glutamic acid (E, Glu), glutamine (Q, gin), or serine (S, Ser), for glutamic acid (E, Glu) at position 374 or a conserved glutamic acid (E, Glu) other than lysine (K, Lys), for the nucleotide codon encoding amino acid residue of modified H35374 84 hemagglutinin (E, Glu), asp), glutamine (Q, Gln), arginine (R, Arg), asparagine (N, Asn), histidine (H, His) or serine (S, Ser)), the nucleotide codon encoding amino acid residue 390 of the modified H1 hemagglutinin encodes an aspartic acid (D, Asp) or 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 a methionine (M, Met) or conservative methionine (M, Met) substitution (e.g., isoleucine (I, Ile), glutamine (Q, Gln), valine (V, Val) or phenylalanine (F) in addition to leucine (L, Leu), phe)), wherein the modified H1 hemagglutinin sequence is not naturally occurring, and wherein the hemagglutinin protein forms a VLP upon expression.

For example, the modified H1 hemagglutinin may have one or more modifications; wherein at least residues 97, 374, 390 and 429 of H1 hemagglutinin are modified as described herein. For example, the modified H1 hemagglutinin may be a tetrasubstituted H1 hemagglutinin in which the residues at positions 97, 374, 390 and 429 are modified.

Furthermore, a method of producing a VLP in a plant is provided, the VLP 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 being operably linked to a regulatory domain that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing a VLP.

Furthermore, a method of increasing yield of a VLP in a plant comprising a modified H1 hemagglutinin having substitutions at positions 97, 374, 390 and 429 as described above is provided. 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 being operably linked to a regulatory domain that is active in plants, and incubating the plant or plant part under conditions that allow expression of the nucleic acid, thereby producing a VLP.

The present specification also provides a VLP comprising a H1 hemagglutinin having substitutions at positions 97, 374, 390 and 429. The VLP may be produced by the methods provided herein. The VLP protein comprising a modified H1 hemagglutinin exhibits improved properties compared to VLPs comprising an unmodified H1 hemagglutinin protein.

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

The method of producing a VLP comprising a mutant influenza virus hemagglutinin may further comprise the step of introducing into a plant, plant part, or plant cell a second nucleic acid sequence encoding a proton channel protein that is co-expressed with the mutant influenza virus hemagglutinin. For example, the proton channel protein may be an influenza A virus subtype M2 protein, such as A/New Caledonia/20/99M 2. Co-expression of the proton channel protein may result in an increased accumulation of mutant influenza virus hemagglutinin proteins and/or VLPs comprising the mutant influenza virus hemagglutinin proteins, for example as described in WO 2013/044390, which is incorporated herein by reference.

In addition, the mutant influenza virus hemagglutinin may further comprise a modified proteolytic loop or cleavage site as described in WO 2013/044390 and WO2014/153674, which are incorporated herein by reference.

"Co-expression" refers to the introduction and expression in a plant, plant or plant cell of two or more nucleotide sequences, each of which encodes a protein of interest or a fragment of a protein of interest. The two or more nucleotide sequences can be introduced into a plant, plant part, or plant cell within a vector such that each of the two or more nucleotide sequences is under the control of an independent regulatory domain (e.g., including a double construct). Alternatively, the two or more nucleotide sequences may be introduced in separate vectors (e.g., comprising a single construct), and each vector comprises the appropriate regulatory domains for expression of the respective nucleic acid. For example, two nucleotide sequences, each located on a separate vector and introduced into a separate Agrobacterium tumefaciens host, can be co-expressed by mixing a desired amount of suspension (e.g., equal amounts, or varying the ratio of each Agrobacterium tumefaciens host) of each Agrobacterium tumefaciens host prior to vacuum infiltration. In this manner, the co-infiltration of multiple Agrobacterium tumefaciens host suspensions allows for the co-expression of multiple transgenes.

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

Nucleic acids comprising a nucleotide sequence encoding a modified influenza virus Hemagglutinin (HA) as described herein may further comprise a sequence that enhances expression of the hemagglutinin protein in a plant, plant part or plant cell. The expression enhancing sequence may comprise a CPMV enhancer element or a plant derived expression enhancer operably associated with 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.

The term "CPMV enhancer element" as used herein refers to a nucleotide sequence encoding the 5' UTR or modified CPMV sequences known in the art that modulate the cowpea mosaic virus (CPMV) RNA2 polypeptide. For example, a CPMV enhancer element or CPMV expression enhancer is included in WO 2015/14367; WO 2015/103704; WO 2007/135480; WO 2009/087391; the nucleotide sequences described in Sainsbury F. and Lomonossoff G.P (2008, Plant Physiol.148: p.1212-1218), which are incorporated herein by reference. The CPMV enhancer sequence may enhance expression of the downstream heterologous open reading frame to which it is attached. CPMV expression enhancers may include CPMV HT, CPMVX (where X is 160, 155, 150, 114), such as CPMV160, CPMVX + (where X is 160, 155, 150, 114), such as CPMV160 +, CPMV-HT +, CPMV HT + [ WT115], or CPMV HT + [511] (WO 2015/143567; WO2015/103704, which are incorporated herein by reference). The CPMV expression enhancer is useful in plant expression systems that include a regulatory domain operably linked to a CPMV expression enhancer sequence and a nucleotide sequence of interest.

The term "CPMV enhancer element" as used herein refers to a nucleotide sequence encoding the 5' UTR or modified CPMV sequences known in the art that modulate the cowpea mosaic virus (CPMV) RNA2 polypeptide. For example, a CPMV enhancer element or CPMV expression enhancer is included in WO 2015/14367; WO 2015/103704; WO 2007/135480; WO 2009/087391; the nucleotide sequences described in Sainsbury F. and Lomonossoff G.P (2008, Plant Physiol.148: p.1212-1218), which are incorporated herein by reference. The CPMV enhancer sequence may enhance expression of the downstream heterologous open reading frame to which it is attached. The CPMV expression enhancer may comprise CPMV HT, CPMVX +, CPMV-HT +, CPMV HT + [ WT115] or CPMV HT + [511] (WO 2015/14367; WO2015/103704, which are incorporated herein by reference). The CPMV expression enhancer is useful in plant expression systems that include a regulatory domain operably linked to a CPMV expression enhancer sequence and a nucleotide sequence of interest.

The term "5 ' UTR" or "5 ' untranslated region" or "5 ' leader" refers to an mRNA domain that is not translated. The 5' UTR usually starts at the start site of transcription and ends before the start codon of the translation start site or coding region. The 5' UTR can modulate the stability and/or translation of mRNA transcripts.

The term "plant-derived expression enhancer" as used herein refers to a nucleotide sequence obtained from a plant that encodes the 5' UTR. An example of a plant-derived expression enhancer is described in U.S. provisional patent application 2006/010086 (filed 3/14/2018, which is incorporated herein by reference) or in the paper by Diamos a.g. et al (2016, Front Plt sci.7: 1-15; which is incorporated herein by reference). Plant derived expression enhancers may 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 are useful in plant expression systems that include a regulatory domain operably linked to a plant-derived expression enhancer sequence and a nucleotide sequence of interest.

"operably linked" refers to the direct or indirect interaction of particular sequences to perform a predetermined function, such as mediation or modulation of expression of a nucleic acid sequence. For example, the interaction of operably linked sequences may be mediated by a protein that interacts with the operably linked sequences.

When one or more mutant influenza virus hemagglutinin proteins are expressed in a plant, plant part or plant cell, the one or more mutant influenza virus hemagglutinin proteins self-assemble into a VLP. 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 hemagglutinin can be purified.

The invention also provides a use of a mutant influenza virus hemagglutinin or a VLP comprising a mutant influenza virus hemagglutinin as described herein, for inducing immunity to an influenza infection in a subject. Also disclosed herein is an antibody or antibody fragment prepared by administering to a subject or host animal a mutant influenza virus hemagglutinin or a VLP comprising a mutant influenza virus hemagglutinin. Also provided is a composition for inducing an immune response in a subject, the composition 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.

Also provided herein is a method of inducing immunity to influenza infection in a subject, the method comprising administering to the subject a mutant influenza virus hemagglutinin or a VLP comprising a mutant influenza virus hemagglutinin by oral, intranasal, intramuscular, intraperitoneal, intravenous, or subcutaneous administration.

The term "influenza virus" as used herein refers to an enveloped virus strain of the orthomyxoviridae family characterized by having a negative single-stranded RNA genome. Depending on the particular strain, the influenza genome consists of eight gene segments encoding 12-14 proteins.

There are four types of influenza viruses: type a, type b, type c and type d, wherein influenza a and type b viruses are the causative microorganisms of seasonal disease epidemics in the human population. Influenza a viruses are further classified according to the expression of hemagglutinin and Neuraminidase (NA) glycoprotein subtypes.

The term "hemagglutinin" or "HA" as used herein refers to a trimeric lectin that promotes the binding of influenza virions to sialic-acid containing proteins on the surface of target cells and mediates the release of the viral genome into the target cells. There are 18 different hemagglutinin subtypes (H1-H18). The hemagglutinin protein comprises two structural elements: a head, which is the primary target of seroprotective antibodies; and a handle. Hemagglutinin is translated into a single polypeptide HA0 (assembled as a trimer), which HA0 must be cleaved by serine endoproteases between the HA1 (approximately 40kDa) subdomain and the HA2 (approximately 20kDa) subdomain. After cleavage, the two disulfide-bound protein domains adopt the necessary conformation required to achieve viral infectivity.

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 to known influenza a strains that have progressed over time. For example, influenza 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/Hondaus/17734/16 (H1N1) or A/Darwin/11/15(H1N 1). An influenza a virus hemagglutinin may comprise a hemagglutinin from a strain, wherein where the influenza virus hemagglutinin protein comprises at least one substitution as described herein and is capable of forming a VLP, induces an immune response, induces a hemagglutinin, or a combination thereof when administered to a subject, the hemagglutinin has about 30-100% amino acid sequence identity to any hemagglutinin derived from an influenza a virus strain listed above or any amount therebetween.

For example, an influenza virus hemagglutinin protein may 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 derived from the influenza a virus strains listed above, and include at least one substitution as described herein and be capable of forming a VLP, inducing immunity when administered to a subject, inducing hemagglutination or a combination thereof. An amino acid sequence alignment of multiple influenza a virus hemagglutinin domains is shown in figure 1, and these examples should not be considered limiting.

The terms "percent similarity", "sequence similarity", "percent identity" or "sequence identity" when referring to a particular sequence are used, for example, as described in the university of wisconsin GCG software program or by means of manual alignment and visual inspection (see, for example, the 1995 supplementary edition "molecular biology laboratory manual", authored by Ausubel et al). Methods of sequence alignment for comparison are well known in the art. Optimal sequence alignments 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 similarity search method of Pearson and Lipman (1988, Proc. Natl. Acad. Sci. USA 85: 2444), by computerized implementations of these algorithms (e.g., GAP, STBEFIT, FASTA and TFASTA in the packages of Genetics Computer groups, Inc. of Madison, Wis.).

One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1977, Nuc. acids Res.25: 3389-. Percent sequence identity for nucleic acids and proteins of the invention is determined using BLAST and BLAST 2.0 and the parameters described herein. For example, the BLASTN program (for nucleotide sequences) may use 11 words long (W), 10 expect values (E), M-5, N-4, and two strand comparisons as defaults. For amino acid sequences, the BLASTP program may use as defaults the alignment (B) ═ 50, expectation (E) ═ 10, M ═ 5, N ═ 4, and two strand comparisons of word length ═ 3, expectation (E) ═ 10, and BLOSUM62 scoring matrices (see Henikoff and Henikoff, 1989, proc. natl. acad. sci. usa 89: 10915). Software for performing BLAST analysis is publicly available through the national center for Biotechnology information (see URL: ncbi. nlm. nih. gov /).

The term "virus-like particle", VLP or variant thereof, as used herein refers to an influenza virus particle that includes one or more influenza virus hemagglutinin proteins and self-assembles into a non-replicating, non-infectious viral capsid structure without all parts of the influenza genome.

Production of influenza virus hemagglutinin protein in plants

Where the influenza a virus hemagglutinin protein comprises at least one substitution as described herein and is capable of forming a VLP, induces an immune response upon administration to a subject, induces hemagglutination, or a combination thereof, the influenza a virus hemagglutinin protein comprises a hemagglutinin comprising about 30 to about 100% of the sequence of influenza a virus hemagglutinin from 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), and a/Norway/2147/2017 (seq id No. 139), 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.

Further, the modified influenza virus Hemagglutinin (HA) protein comprises at least one substitution as described herein and is capable of forming a VLP, inducing an immune response, inducing a hemagglutination reaction, or a combination thereof when administered to a subject, with sequence No. 18, sequence No. 22, sequence No. 24, sequence No. 4, sequence No. 28, sequence No. 32, sequence No. 36, sequence No. 39, sequence No. 41, sequence No. 43, sequence No. 45, sequence No. 47, sequence No. 49, sequence No. 51, sequence No. 53, sequence No. 55, sequence No. 57, sequence No. 59, sequence No. 61, sequence No. 63, sequence No. 65, sequence No. 68, sequence No. 72, sequence No. 76, sequence No. 80, sequence No. 82, sequence No. 84, sequence No. 86, sequence No. 89, sequence No. 91, sequence No. 93, sequence, One of the sequences of SEQ ID NO 126, SEQ ID NO 128, SEQ ID NO 140, SEQ ID NO 142, SEQ ID NO 144, SEQ ID NO 146, SEQ ID NO 148 has an amino acid sequence of about 30% to about 100%, 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%, about 97% to about 100%, about 98% to about 100%, or any amount therebetween, of sequence identity or sequence similarity.

As described herein, one or more specific mutations or modifications in an influenza virus hemagglutinin results in increased accumulation of hemagglutinin proteins and increased VLP production in plants as compared to a wild-type influenza virus hemagglutinin.

Examples of mutant influenza a virus hemagglutinin proteins having the ability to enhance production of influenza virus hemagglutinin and/or VLPs in plants include, but are not limited to:

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 SEQ ID NO: 47); N97D + K374E + F390D + L429M A/Michigan/45/15 mutant H1 (construct No. 3881, SEQ ID NO. 49); F390D + L429M A/Massachusetts/06/17 mutant H1 (construct No. 4091, SEQ ID NO: 51); N97D + F390D + L429M A/Massachusetts/06/17 mutant H1 (construct No. 4093, SEQ ID 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).

Induction of immunity to influenza infection

An "immune response" generally refers to a response of the adaptive immune system of a subject. The adaptive immune system typically includes both humoral and cell-mediated responses. Humoral responses are immune profiles mediated by secreted antibodies produced in cells of the B lymphocyte lineage (B cells). The secreted antibodies bind to antigens on the surface of invading microorganisms (e.g., viruses or bacteria), which marks them as being destroyed. Humoral immunity is commonly used to refer to antibody production and its attendant processes, as well as effector functions of antibodies, including Th2 cell activation and cytokine production, memory cell production, opsonin promoting phagocytosis, pathogen elimination, and the like. The term "modulate" refers to an increase or decrease in a particular reaction or parameter as determined by any of a number of well known or commonly used assays, some of which are set forth herein.

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

For example, induction of antigen-specific CD8 positive T lymphocytes can be measured using the ELISPOT assay; 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 (ELISA) assay; 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 making such assays are well known in the art.

The presence or level of cytokines may 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 suite). Peripheral Blood Mononuclear Cells (PBMC) or spleen cells obtained from a subject can be cultured and the supernatant analyzed. Quantitative analysis of T lymphocytes can also be performed by Fluorescence Activated Cell Sorting (FACS) using label specific fluorescent labels and methods known in the art.

Microneutralization assays may also be performed to characterize the immune response of a subject, see, for example, the method published by Rowe et al in 1973. Viral neutralization titers can be quantified in a number of ways, including: counting the number of lytic plaques after violent fixation/staining of the crystals on the cells (plaque analysis); microscopic observation of cell lysis in vitro culture; and 2) detecting the influenza virus by enzyme-linked immunosorbent assay and spectrophotometry.

The term "epitope" as used herein refers to a structural portion of an antigen to which an antibody specifically binds.

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

Plant expression

The constructs of the present invention can be introduced into plant cells using titanium plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, and the like. For reviews on these techniques, reference may be made, for example, to Weissbach and Weissbach, methods of plant molecular biology, academic Press, New York VIII, pp.421-463 (1988); geierson and Corey, second edition plant molecular biology (1988); and of Miki and IyerIn plants Basic knowledge of Gene transfer(s). Plant metabolism, second edition, DT. Dennis, DH Turpin, DD Lefebrvre, DB Layzell (eds), Addison Wesly, Langmans Ltd. London, pp.561-. Other methods include direct DNA uptake, use of liposomes, electroporation (e.g., using protoplasts), microinjection, microparticles or whiskers, and vacuum infiltration. See, for example, the article by Bilang et al (1991, Gene 100: 247-; freeman et al (1984) Plant Cell Physiology 29: 1353), Howell et al (1985) Science 227: 1229-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 Lomonosoff (2002, J Virol Meth, 105: 343-; WO 8706614; U.S. Pat. nos. 4,945,050; 5,036,006; and 5,100,792, U.S. patent application 08/438,666 filed 5/10 in 1995, and 07/951,715 filed 9/25 in 1992 (all of which are incorporated herein by reference).

Transient expression methods may be used to express constructs of the invention (see D' Aoust et al, 2009, methods in molecular biology 483, pages 41-50; Liu and Lomonosoff, 2002, J. methods in virology, 105: 343-). 348; which are incorporated herein by reference). Alternatively, vacuum-based transient expression methods such as those described by Kapila et al (1997, Plant Sci.122, 101-108; incorporated herein by reference) or WO 00/063400, WO 00/037663 (these documents are incorporated herein by reference) may be used. These methods may include, for example, but are not limited to, agricultural inoculation or agricultural infiltration, syringe infiltration methods, however, as noted above, other transient methods may also be used. The agrobacterium mixture comprising the desired nucleic acid enters the intercellular spaces of the tissue, such as the leaves, aerial parts of the plant (including stems, leaves and flowers), other parts of the plant (stems, roots, flowers) or the entire plant, by agricultural inoculation, agricultural infiltration or syringe infiltration. After passage through the epidermis, the Agrobacterium infects and transfers the t-DNA copies into the cells. t-DNA is transcribed episomally, while mRNA is translated, which results in the production of the protein of interest in the infected cell, however, the transport of t-DNA within the nucleus is transient.

Transgenic plants, plant cells or seeds comprising the genetic constructs of the invention are also considered part of the invention, and may 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 (see, for example, Guerineau and Mullineau (1993, "plant transformation and expression vectors", incorporated by Labfax in Oxford plant molecular biology (Croy RRD edition), BIOS scientific Press, page 121-148.) generally, transformed plant cells are cultured in a suitable medium which may contain a selective agent (e.g., an antibiotic) wherein a selectable marker is used to facilitate identification of the transformed plant cells, once callus tissue is formed, shoot formation may be promoted by the use of appropriate plant hormones according to known methods and shoots are transferred to rooting medium for regeneration of the plant. And are known to those skilled in the art. Techniques which can be used are found in the work by Vasil et al (plant cell culture and somatic genetics, laboratory procedures and their applications, academic Press, 1984) and the work by Weissbach and Weissbach (methods in plant molecular biology, academic Press, 1989). The method of obtaining transformed and regenerated plants is not critical to the present invention.

If a plant, plant part or plant cell is to be transformed or co-transformed by two or more nucleic acid constructs, the nucleic acid constructs can be introduced into agrobacterium in a single transfection event, allowing nucleic acids to pool and the bacterial cell to be transfected. Alternatively, the constructs may be introduced sequentially. In this case, the first construct is introduced into Agrobacterium as described above, allowing the cells to grow under selective conditions (e.g.in the presence of an antibiotic) in which only a single transformed bacterium can grow. After this first selection step, a second nucleic acid construct is introduced into the Agrobacterium as described above and the cells are grown under conditions of double selectivity, in which only the doubly transformed bacteria are able to grow. The plant, plant part, or plant cell may then be transformed using the double-transformed bacteria as described herein, or a further transformation step may be performed to incorporate the third nucleic acid construct.

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, where each agrobacterium cell can comprise one or more constructs to be introduced into the plant. In order to alter 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 comprising the desired construct may be altered during the infiltration step.

Table 3: sequence number and sequence description

The invention will be further illustrated in the following examples.

Example 1: influenza virus hemagglutinin constructs

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 follows. Other H1 mutants were obtained using similar techniques, hemagglutinin sequence primers, templates and products are illustrated in example 3 (production of influenza virus hemagglutinin and VLPs in plants) and table 4.

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

Modification of H1 hemagglutinin

2X35S/CPMV 160/PDISP-HA 0H 1A-California-07-09/NOS (construct No. 1314)

The following PCR-based method was used to clone the sequence encoding mature HA0 from influenza virus hemagglutinin from A/California/07/09 fused to the alfalfa PDI secretion signal peptide into the 2X35S/CPMV160/NOS expression system. A fragment containing the coding sequence of PDISP-A/CaliforniｃA/07/09 was amplified using the PDISP-H1A/CaliforniｃA/7/09 gene sequence (SEQ ID NO: 1) as ｃA template, using primers IF-CPMV (f 15' UTR) _ SpPDI.c (SEQ ID NO: 13) and IF-H1cTMCT.S1-4r (SEQ ID NO: 14). The PCR products were cloned into the 2X35S/CPMV160/NOS expression system using a fusion cloning system (Clontech, mountain View, Calif.). Construct 1190 (fig. 8) was digested with SacII and StuI restriction enzymes and linearized plasmid was used for the fusion assembly reaction. Construct 1190 is a recipient plasmid for "fusion" cloning of a gene of interest in a 2X35S/CPMV160/NOS based expression cassette. It also incorporates gene constructs for co-expression of a TBSV P19 silencing suppressor under the action of the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and the t-DNA border sequence from left to right is shown in SEQ ID NO. 98. The resulting construct was numbered 1314 (SEQ ID NO: 99). The amino acid sequence of mature HA0 from a/California/07/09 fused to alfalfa PDI secretion signal peptide (PDISPs) is shown in seq id No. 2. A schematic of plasmid 1314 is shown in fig. 6A.

2X35S/CPMV 160/PDISP-HA 0H 1A-California-07-09 (F390D)/NOS (construct No.) 2980)

The sequence encoding mature HA0 of influenza virus hemagglutinin from a/California/07/09(F390D) fused to the alfalfa PDI secretion signal peptide was cloned into the 2X35S/CPMV160/NOS expression system using the following PCR-based method. In the first round of PCR, a fragment containing PDISP-H1A/California/07/09 having a mutated F390D amino acid was amplified using the primers IF-CPMV (F15' UTR) _ SpPDI.c (SEQ ID NO: 13) and H1Cal (F390D). r (SEQ ID NO: 15) using the PDISP-H1A/California/7/09 gene sequence (SEQ ID NO: 1) as a template. A second fragment containing the F390D mutation and the remainder of H1A/California/07/09 were amplified using the PDISP-H1A/California/07/09 gene sequence (SEQ ID NO: 1) as a template, 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 (f 15' 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 a fusion cloning system (Clontech, mountain View, Calif.). Construct 1190 (fig. 8) was digested with SacII and StuI restriction enzymes and linearized plasmid was used for the fusion assembly reaction. Construct 1190 is a recipient plasmid for "fusion" cloning of a gene of interest in a 2X35S/CPMV160/NOS based expression cassette. It also incorporates gene constructs for co-expression of a TBSV P19 silencing suppressor under the action of the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and the t-DNA border sequence from left to right is shown in SEQ ID NO. 98. The resulting construct was numbered 2980 (SEQ ID NO: 100). The amino acid sequence of the mutated PDISP-HA from A/California/07/09(F390D) is shown in SEQ ID NO. 18. A schematic of plasmid 2980 is shown in fig. 6B.

Construction of 2X35S/CPMV 160/PDISP-HA 0H 1A-California-07-09 (F390D + L429M)/NOS Body number 2995)

The sequence encoding mature HA0 from influenza virus hemagglutinin of A/California/07/09(F390D + L429M) fused to the alfalfa PDI secretion signal peptide was cloned into the 2X35S/CPMV160/NOS expression system using the following PCR-based method. In the first round of PCR, a fragment of PDISP-H1A/California/07/09 comprising F390D and L429M amino acids having mutations was amplified using the gene sequence (SEQ ID NO: 17) of PDISP-H1A/California/7/09 (F390D) as a template using primers IF-CPMV (F15' UTR) _ SpPDI.c (SEQ ID NO: 13) and H1Cal (L429M). r (SEQ ID NO: 19). A second fragment containing the L429M mutation and the remainder of H1A/California/07/09 were amplified using the gene sequence PDISP-H1A/California/7/09 (F390D) (SEQ ID NO: 17) as a template, using H1Cal (L429M). c (SEQ ID NO: 20) 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 (f 15' 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 a fusion cloning system (Clontech, mountain View, Calif.). Construct 1190 (fig. 8) was digested with SacII and StuI restriction enzymes and linearized plasmid was used for the fusion assembly reaction. Construct 1190 is a recipient plasmid for "fusion" cloning of a gene of interest in a 2X35S/CPMV160/NOS based expression cassette. It also incorporates gene constructs for co-expression of a TBSV P19 silencing suppressor under the action of the alfalfa plastocyanin gene promoter and terminator. The backbone is a pCAMBIA binary plasmid, and the t-DNA border sequence from left to right is shown in SEQ ID NO. 98. The resulting construct was numbered 2995 (SEQ ID NO: 101). The amino acid sequence of the mutated PDISP-HA from A/California/07/09(F390D + L429M) is shown in SEQ ID NO. 24. A schematic of plasmid 2995 is shown in fig. 6D.

Example 2: method of producing a composite material

Agrobacterium tumefaciens transfection

Agrobacterium tumefaciens strain AGL1 was transfected by electroporation with either wild-type influenza virus hemagglutinin or mutant influenza virus hemagglutinin expression vectors using the methods described by D' Aoust et al (Plant Biotech.J.6: 930-40). The transfected Agrobacterium was grown in YEB medium at pH 5.6 with the addition of 10 mM 2- (N-morpholino) ethanesulfonic acid (MES), 20 μm acetosyringone, 50. mu.g/ml kanamycin and 25. mu.g/ml carbenicillin, until OD₆₀₀Reaching between 0.6 and 1.6. The Agrobacterium suspension was centrifuged before use and resuspended in osmotic medium (10 mM MgCl)₂And 10 mmol MES, pH 5.6).

Plant biomass, preparation of inoculum and Agrobacterium infiltrationInto

Nicotiana benthamiana plants were grown from seeds in flat ground filled with a commercial peat moss substrate. The plants were grown in the greenhouse under 16/8 photoperiods and at a temperature of 25 ℃ during the day/20 ℃ at night. Three weeks after sowing, each plantlet was selected, transplanted into a pot, and grown for another three weeks in a greenhouse under the same environmental conditions.

Agrobacterium transfected with each of the wild-type influenza virus hemagglutinin or mutant influenza virus hemagglutinin expression vectors was grown in YEB medium supplemented with 10 mM 2- (N-morpholino) ethanesulfonic acid (MES), 20 μm acetosyringone, 50. mu.g/ml kanamycin and 25. mu.g/ml carbenicillin, pH 5.6. Until they reach an OD of between 0.6 and 1.6₆₀₀. The Agrobacterium suspension was centrifuged before use and resuspended in osmotic medium (10 mM MgCl)₂And 10 mmole MES, pH 5.6) and stored overnight at 4 ℃. On the day of infiltration, culture batches were diluted to 2.5 culture volumes and incubated prior to use. Whole benthic tobacco was inverted in bacterial suspension for 2 minutes in an air tight stainless steel jar with a vacuum of 20-40 torr. The plants were returned to the greenhouse and incubated for 6 or 9 days until harvest.

Leaf harvesting and Total protein extraction

Proteins were extracted from fresh biomass cut into approximately 1 square centimeter pieces by overnight enzymatic extraction using an orbital shaker at room temperature. The slurry was then filtered through a macroporous nylon filter to remove undigested rough plant tissue.

To obtain "whole process yield," the slurry is centrifuged to remove protoplasts and intracellular contaminants. The supernatant was clarified by depth filtration. The clarified fraction is then loaded onto a cation exchange column, using a step-wise elution step with gradually increasing sodium chloride concentration. Purified VLPs were concentrated with TFF, diafiltered with formulation buffer and passed through a filter. Purified VLPs were analyzed for protein content by BCA assay and activity by hemagglutination assay. Relative yields were obtained by comparing the protein yield of the new construct with the protein yield of the native construct used as a control.

To obtain a "post density gradient yield," the slurry is centrifuged to remove protoplasts and intracellular contaminants. The supernatant was further centrifuged to remove additional debris. The supernatant was clarified by depth filtration using a glass fiber filter. The clarified fraction was then loaded on a discontinuous iodixanol density gradient device. Separation density gradient centrifugation was performed as follows: a 38 ml tube containing a discontinuous iodixanol density gradient in Tris buffer (35%, 30%, 25%, 20%, 15%, 10% and 5% continuous layers) was prepared and covered with a clear extract. The gradient solution was centrifuged at 120000g for 2 hours (4 ℃). After centrifugation, the first 5 ml collected from bottom to top was discarded, while the next 5 ml was collected for protein content analysis (BCA), activity measurement (hemagglutination assay) and intensity measurement of the HA0 band (density assay) on simplified SDS-PAGE. Relative yields were obtained by comparing the HA0 band intensity of the new construct with the HA0 band intensity of the native construct used as a control.

Hemagglutination assay

The hemagglutination assay was based on the method described by Nayak and Reichl (2004). Briefly, serial double dilutions of test samples (100 microliters) were performed in V-bottomed 96-well microtiter plates containing 100 microliters of PBS, leaving 100 microliters of diluted samples in each well. To each well was added 100 microliters of a 0.25% turkey (for H1) red blood cell suspension (Bio Link Inc., new york, usa) and the well plates were incubated at room temperature for 2 hours. The reciprocal of the highest dilution indicating complete hemagglutination was recorded as the hemagglutinin activity. Meanwhile, recombinant hemagglutinin standard (A/Vietnam/1203/2004H5N1) (Protein Science Corporation, Metridon, Connecticut, USA) was diluted in PBS and used as a control on each well plate.

Protein analysis and immunoblotting

Immunoblotting was performed by a first incubation in 0.1% TBS-Tween 20 using a primary mAb diluted to 1/500 in 2% skim milk. Peroxidase-conjugated mountain using dilution 1/10000Goat anti-mouse antibody (Jackson Immunoresearch, cat #115-035-146) was used as a secondary antibody for chemiluminescent detection in 2% skim milk in 0.1% TBS-Tween 20 immunoreactive complex, using luminol as a matrix (Roche Diagnostics Corporation). Use ofAn activated peroxidase coupling kit (Pierce from rockford, il) for horseradish peroxidase-enzyme coupling of human IgG antibodies.

Example 3: production of influenza virus hemagglutinin protein in plants

Modification of H1 hemagglutinin

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

F390D A/California/07/09 mutant H1

The F390D a/California/07/09 mutant H1 was constructed by mutating the phenylalanine residue at position 390 of wild type a/California/07/09H1 to aspartic acid (construct No. 2980). As shown in fig. 2A and 2B, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 2980 exhibited an approximately 60% increase in hemagglutination titer compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/California/07/09H1 (construct No. 1314). Furthermore, as shown in figure 2C, the burley tobacco infiltrated with construct No. 2980 exhibited an approximately 60% increase in VLP production after iodixanol gradient purification compared to plants infiltrated with the wild-type construct.

L429M A/California/07/09 mutant H1

L429M A/California/07/09 mutant H1 was constructed by mutating the leucine residue at position 429 of wild-type A/California/07/09H1 to methionine (construct No. 2962). As shown in fig. 2A and 2B, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 2962 exhibited an approximately 20% increase in hemagglutination titer compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/California/07/09H1 (construct No. 1314). Furthermore, as shown in figure 2C, the burley tobacco infiltrated with construct no 2962 exhibited an approximately 30% increase in VLP production after iodixanol gradient purification compared to plants infiltrated with the wild-type construct.

F390D + L429M A/California/07/09 mutant H1

F390D + L429M a/California/07/09 mutant H1 was constructed by introducing a double mutation into the a/California/07/09H1 wild-type sequence, where phenylalanine at position 390 was mutated to aspartic acid and leucine at position 429 was mutated to methionine (construct No. 2995). As shown in fig. 2B, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 2995 exhibited an increase in hemagglutination titer of approximately 60% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/California/07/09H1 (construct No. 1314).

N97D A/Michigan/45/15 mutant H1

N97D a/Michigan/45/15 mutant H1 was constructed by mutating the asparagine residue at position 97 of wild type a/Michigan/45/15H1 to aspartic acid (construct No. 3774). As shown in fig. 3A and 3B, purified extracts of nicotiana benthamiana infiltrated with construct No. 3774 exhibited an approximately 1100% increase in hemagglutination titer compared to extracts of nicotiana benthamiana infiltrated with wild-type a/Michigan/45/15H1 (construct No. 3640).

K374E A/Michigan/45/15 mutant H1

K374E A/Michigan/45/15 mutant H1 was constructed by mutating the lysine residue at position 374 of wild type A/Michigan/45/15H1 to glutamic acid (construct No. 3771). As shown in fig. 3A and 3B, purified extracts of nicotiana benthamiana infiltrated with construct No. 3771 exhibited an approximately 1100% increase in hemagglutination titer compared to extracts of nicotiana benthamiana infiltrated with wild-type a/Michigan/45/15H1 (construct No. 3640).

F390D A/Michigan/45/15 mutant H1

F390D a/Michigan/45/15 mutant H1 was constructed by mutating the phenylalanine residue at position 390 of wild-type a/Michigan/45/15H1 to aspartic acid (construct No. 3641). As shown in fig. 3B, purified extracts of nicotiana benthamiana infiltrated with construct No. 3641 exhibited similar hemagglutination titer activity as compared to the nicotiana benthamiana extract infiltrated with wild-type a/Michigan/45/15H1 (construct No. 3640). However, the overall process yield of purified VLPs with H1 was found to increase to 172% compared to wild type (see table 5C).

L429M A/Michigan/45/15 mutant H1

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

N97D + K374E A/Michigan/45/15 mutant H1

N97D + K374E a/Michigan/45/15 mutant H1 was constructed by introducing a double mutation into the wild-type sequence of wild-type a/Michigan/45/15 in which 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). As shown in fig. 3B, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 3880 exhibited an increase in hemagglutination titer of about 1100% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Michigan/45/15H1 (construct No. 3640).

F390D + L429M A/Michigan/45/15 mutant H1

F390D + L429M a/Michigan/45/15 mutant H1 was constructed by introducing a double mutation into the wild type sequence of wild type a/Michigan/45/15, in which 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). As shown in fig. 3B, purified extracts of nicotiana benthamiana infiltrated with construct No. 3703 exhibited an approximately 1100% increase in hemagglutination titer compared to extracts of nicotiana benthamiana infiltrated with wild-type a/Michigan/45/15H1 (construct No. 3640).

N97D + F390D + L429M A/Michigan/45/15 mutant H1

N97D + F390D + L429M a/Michigan/45/15 mutant H1 was constructed by introducing three mutations into the wild-type sequence of wild-type a/Michigan/45/15, in which asparagine at position 97 was mutated to an aspartic acid residue, phenylalanine at position 390 was mutated to an aspartic acid residue, and leucine at position 429 was replaced by a methionine residue (construct No. 3879). As shown in fig. 3B, purified extracts of nicotiana benthamiana infiltrated with construct No. 3879 exhibited an increase in hemagglutination titer of about 2300% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Michigan/45/15H1 (construct No. 3640).

K374E + F390D + L429M A/Michigan/45/15 mutant H1

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

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

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

N97D + F390D + L429M A/Massachusetts/06/17 mutant H1

N97D + F390D + L429M a/Massachusetts/06/17 mutant H1 was constructed by introducing three mutations into the wild-type sequence of wild-type a/Massachusetts/06/17, wherein asparagine at position 97 was mutated to an aspartic acid residue, phenylalanine at position 390 was mutated to an aspartic acid residue, and leucine at position 429 was replaced by a methionine residue (construct No. 4093). As shown in FIG. 4B, purified extracts of Nicotiana benthamiana infiltrated with construct No. 4093 exhibited approximately a 40% increase in hemagglutination titer compared to Nicotiana benthamiana extracts infiltrated with double mutant construct F390D + L429M A/Massachusetts/06/17 mutant H1 (construct No. 4091).

K374E + F390D + L429M A/Massachusetts/06/17 mutant H1

K374E + F390D + L429M a/Massachusetts/06/17 mutant H1 was constructed by introducing three mutations into the wild-type sequence of wild-type 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). As shown in FIG. 4B, purified extracts of Nicotiana benthamiana plants infiltrated with construct No. 4092 exhibited approximately a 50% increase in hemagglutination titer compared to Nicotiana benthamiana plant extracts infiltrated with double mutant construct F390D + L429M A/Massachusetts/06/17 mutant H1 (construct No. 4091).

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

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

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

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

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

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

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

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

N97D A/Honduras/17734/16 mutant H1

N97D A/Honduras/17734/16 mutant H1 was constructed by mutating the asparagine residue at position 97 of wild type A/Honduras/17734/16H1 to aspartic acid (construct No. 3950). As shown in fig. 4A, purified extracts of the nicotiana benthamiana plant infiltrated with construct No. 3950 exhibited an increase in hemagglutination titer of about 300% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Honduras/17734/16H1 (construct No. 3944).

K374E A/Honduras/17734/16 mutant H1

K374E A/Honduras/17734/16 mutant H1 was constructed by mutating the lysine residue at position 374 of wild type A/Honduras/17734/16H1 to glutamic acid (construct No. 3948). As shown in fig. 4A, purified extracts of nicotiana benthamiana plants infiltrated using construct No. 3948 exhibited approximately 100% increase in hemagglutination titer compared to extracts of nicotiana benthamiana plants infiltrated using wild-type a/Honduras/17734/16H1 (construct No. 3944).

F390D A/Honduras/17734/16 mutant H1

F390D a/Honduras/17734/16 mutant H1 was constructed by mutating the phenylalanine residue at position 390 of wild type a/Honduras/17734/16H1 to aspartic acid (construct No. 3945). As shown in fig. 4A, purified extracts of nicotiana benthamiana plants infiltrated using construct No. 3945 exhibited approximately a 30% increase in hemagglutination titer compared to the nicotiana benthamiana plant extract infiltrated using wild-type a/Honduras/17734/16H1 (construct No. 3944).

L429M A/Honduras/17734/16 mutant H1

L429M A/Honduras/17734/16 mutant H1 was constructed by mutating the leucine residue at position 429 of wild-type A/Honduras/17734/16H1 to methionine (construct No. 3949). As shown in fig. 4A, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 3949 exhibited an increase in hemagglutination titer of about 400% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Honduras/17734/16H1 (construct No. 3944).

F390D + L429M A/Honduras/17734/16 mutant H1

F390D + L429M a/Honduras/17734/16 mutant H1 was constructed by introducing a double mutation into the wild type sequence of wild type 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). As shown in fig. 4A, purified extracts of the nicotiana benthamiana plant infiltrated with construct No. 3946 exhibited an increase in hemagglutination titer of about 300% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Honduras/17734/16H1 (construct No. 3944).

N97D + F390D + L429M A/Honduras/17734/16 mutant H1

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

N97D A/Darwin/11/15 mutant H1

N97D A/Darwin/11/15 mutant H1 was constructed by mutating the asparagine residue at position 97 of wild type A/Darwin/11/15H 1 to aspartic acid (construct No. 3990). As shown in fig. 4A, purified extracts of nicotiana benthamiana plants infiltrated using construct No. 3990 exhibited approximately 200% increase in hemagglutination titer compared to extracts of nicotiana benthamiana plants infiltrated using wild-type a/Darwin/11/15H 1 (construct No. 3984).

K374E A/Darwin/11/15 mutant H1

K374E A/Darwin/11/15 mutant H1 was constructed by mutating the lysine residue at position 374 of wild type A/Darwin/11/15H 1 to glutamic acid (construct No. 3988). As shown in fig. 4A, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 3988 exhibited an increase in hemagglutination titer of about 300% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Darwin/11/15H 1 (construct No. 3984).

F390D A/Darwin/11/15 mutant H1

F390D a/Darwin/11/15 mutant H1 was constructed by mutating the phenylalanine residue at position 390 of wild type a/Darwin/11/15H 1 to aspartic acid (construct No. 3985). As shown in fig. 4A, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 3985 exhibited an approximately 50% increase in hemagglutination titer compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Darwin/11/15H 1 (construct No. 3984).

L429M A/Darwin/11/15 mutant H1

L429M A/Darwin/11/15 mutant H1 was constructed by mutating the leucine residue at position 429 of wild-type A/Darwin/11/15H 1 to methionine (construct No. 3989). As shown in fig. 4A, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 3989 exhibited an increase in hemagglutination titer of about 500% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Darwin/11/15H 1 (construct No. 3984).

F390D + L429M A/Darwin/11/15 mutant H1

F390D + L429M a/Darwin/11/15 mutant H1 was constructed by introducing a double mutation into the wild type sequence of wild type 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). As shown in fig. 4A, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 3986 exhibited an increase in hemagglutination titer of about 300% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Darwin/11/15H 1 (construct No. 3984).

N97D + F390D + L429M A/Darwin/111/15 mutant H1

N97D + F390D + L429M a/Darwin/11/15 mutant H1 was constructed by introducing three mutations into the wild-type sequence of wild-type a/Darwin/11/15, wherein asparagine at position 97 was mutated to an aspartic acid residue, phenylalanine at position 390 was mutated to an aspartic acid residue, and leucine at position 429 was replaced by a methionine residue (construct No. 3991). As shown in fig. 4A, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 3991 exhibited an increase in hemagglutination titer of about 1100% compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Darwin/11/15H 1 (construct No. 3984).

F390D + L429M A/Paris/1227/2017 mutant H1

F390D + L429M a/Paris/1227/2017 mutant H1 was constructed by introducing a double mutation into the wild type sequence of wild type a/Paris/1227/2017 in which 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). As shown in FIG. 4C, the purified extract of Nicotiana benthamiana infiltrated with construct No. 4765 exhibited an increase in hemagglutination titer of approximately 117% compared to the extract of Nicotiana benthamiana infiltrated with F390D + L429M A/Massachusetts/06/17 mutant H1 (construct No. 4091).

K374+F390D+L429M A/Paris/1227/2017

K374E + F390D + L429M a/Paris/1227/2017 mutant H1 was constructed by introducing three mutations into the wild-type sequence of wild-type 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). As shown in fig. 4C, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 4766 exhibited an increase in hemagglutination titer of about 127% compared to the nicotiana benthamiana plant extract infiltrated with F390D + L429M a/Paris/1227/2017 mutant H1 (construct No. 4765).

N97D+F390D+L429M A/Paris/1227/2017

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

N97D+K374E+F390D+L429M A/Paris/1227/2017

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

F390D + L429M A/Norway/2147/2017 mutant H1

F390D + L429M a/Norway/2147/2017 mutant H1 was constructed by introducing a double mutation into the wild type sequence of wild type 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). As shown in FIG. 4C, purified extracts of Nicotiana benthamiana infiltrated with construct No. 4775 exhibited an approximately 112% increase in hemagglutination titer compared to Nicotiana benthamiana extracts infiltrated with F390D + L429M A/Massachusetts/06/17 mutant H1 (construct No. 4091).

K374+F390D+L429M A/Norway/2147/2017

K374E + F390D + L429M a/Norway/2147/2017 mutant H1 was constructed by introducing three mutations into the wild-type sequence of wild-type 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). As shown in fig. 4C, purified extracts of nicotiana benthamiana infiltrated with construct No. 4776 exhibited approximately 128% increase in hemagglutination titer compared to extracts of nicotiana benthamiana infiltrated with F390D + L429M a/Norway/2147/2017 mutant H1 (construct No. 4775).

N97D+F390D+L429M A/Norway/2147/2017

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

N97D+K374E+F390D+L429M A/Norway/2147/2017

N97D + K374E + F390D + L429M a/Norway/2147/2017 mutant H1 was constructed by introducing four mutations into the wild-type sequence of wild-type a/Norway/2147/2017, wherein asparagine at position 97 was mutated to an aspartic acid residue, lysine at position 374 was mutated to a glutamic acid residue, phenylalanine at position 390 was mutated to an aspartic acid residue, and leucine at position 429 was replaced by a methionine residue (construct No. 4778). As shown in fig. 4C, the purified extract of the nicotiana benthamiana plant infiltrated with construct No. 4778 exhibited an approximately 213% increase in hemagglutination titer compared to the nicotiana benthamiana plant extract infiltrated with F390D + L429M a/Norway/2147/2017 mutant H1 (construct No. 4775).

N380A A/Michigan/45/15

N380A a/Michigan/45/15 mutant H1 was constructed by mutating the asparagine residue at position 380 of wild-type a/Michigan/45/15H1 to an alanine residue (construct No. 3644). As shown in fig. 3B and 3C, purified extracts of nicotiana benthamiana infiltrated with construct No. 3644 exhibited approximately 80% reduction in hemagglutination titer compared to extracts of nicotiana benthamiana infiltrated with wild-type a/Michigan/45/15H1 (construct No. 3640).

N380A+F390D A/Michigan/45/15

N380A + F390D a/Michigan/45/15 mutant H1 was also constructed in which a double mutation was introduced into wild-type a/Michigan/45/15H1 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). As shown in fig. 3B, purified extracts of nicotiana benthamiana infiltrated with construct No. 3704 exhibited approximately a 50% reduction in hemagglutination titer compared to the nicotiana benthamiana plant extract infiltrated with wild-type a/Michigan/45/15H1 (construct No. 3640). Given the reduced hemagglutination titer observed with N380A a/Michigan/45/15 mutant H1, these results indicate that mutation of the asparagine residue at position 380 has a deleterious effect on expression of influenza virus hemagglutinin protein in plants and/or stability of influenza virus hemagglutinin protein.

It was also observed that the increased hemagglutination titer achieved with influenza virus hemagglutinin proteins comprising one or more of the mutations described herein was specific for H1 influenza virus hemagglutinin. Similar enhancement was not observed in plants infiltrated with constructs encoding mutant influenza virus hemagglutinin from strains other than H1.

For example, F393D A/Indonesia/5/2005 mutant H5 was constructed by mutating the phenylalanine residue at position 393 of wild-type A/Indonesia/5/2005H 5 to aspartic acid (construct No. 3680). As shown in figure 5, purified extracts of nicotiana benthamiana plants infiltrated using construct No. 3680 exhibited approximately 98% reduction in hemagglutination titer compared to the nicotiana benthamiana plant extract infiltrated using wild-type a/Indonesia/5/2005H 5 (construct No. 2295).

Similarly, purified extracts of b.benthamiana infiltrated with F392D a/Egypt/N04915/2014 mutant H5 (construct No. 3690) exhibited a decrease in hemagglutination titer of about 99% compared to extracts of b.benthamiana plants infiltrated with wild-type a/E04915/2014H5 (construct No. 3645) (see figure 5, table 6).

Example 3: hemagglutination titer, post density gradient yield and overall process yield

Table 5A summarizes the measured hemagglutination titers. Hemagglutination titers were measured as described in example 2. Relative hemagglutination titers were obtained by comparing the hemagglutination titer of mutant or modified hemagglutinin proteins with that of wild-type hemagglutinin (table 5A).

Table 5B summarizes the post density gradient yield measured. The post density gradient yield was measured as described in example 2. Relative yields were obtained by comparing the hemagglutinin band intensity of mutant or modified hemagglutinin proteins with that of wild-type hemagglutinin (table 5B).

Table 5C summarizes the measured overall process yields. The overall process yield was obtained as described in example 2. Relative yields were obtained by comparing the protein yield of mutant or modified hemagglutinin proteins with the protein yield of wild-type hemagglutinin (table 5C).

Example 4: sequence of

The following sequence was used (see table 4):

all cited documents are incorporated herein by reference.

The invention has been described with reference to one or more embodiments. 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 following claims.

386页详细技术资料下载

上一篇：一种医用注射器针头装配设备

下一篇：分解含反式脂肪酸油脂的新型脂肪酶

Influenza virus hemagglutinin mutants

相关技术

网友询问留言