Modified influenza B virus hemagglutinin

Abstract

The present disclosure relates to the production of modified influenza B hemagglutinin (HA) proteins. More specifically, the disclosure relates to the production and increased production of influenza virus-like particles (VLPs) in plants, where the VLPs comprise modified influenza B HA proteins. The HA proteins can comprise an amino acid sequence that includes at least one substitution when compared to the corresponding wild-type amino acid sequence. Nucleic acids encoding the modified B HA proteins are further provided. Also provided are methods of producing influenza virus-like particles (VLPs) and methods of increasing influenza virus-like particle (VLP) production in a host or host cell.

Description

[Technical Field]

[0001] The present disclosure relates to modified influenza B virus hemagglutinin (HA) proteins and virus-like particles comprising modified influenza B hemagglutinin (HA) proteins. The present disclosure further relates to the production of modified influenza B hemagglutinin (HA) proteins and virus-like particles in a host or host cell. [Background technology]

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

[0003] Influenza viruses are highly polymorphic particles composed of two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA mediates viral attachment to host cells and viral-cell membrane fusion during viral entry. The influenza virus genome consists of eight single-stranded negative-sense RNA segments, the fourth largest of which encodes the HA gene.

[0004] HA molecules exist as trimers in virions. Each monomer exists as two chains, the HA1 domain and the HA2 domain (also called the HA1 and HA2 subunits or subdomains), linked by a single disulfide bond. Infected host cells produce a glycosylated precursor polypeptide (HA0) with a molecular weight of approximately 85 kDa, which is subsequently cleaved into the HA1 (approximately 40 kDa) and HA2 (approximately 20 kDa) domains. After cleavage, the two disulfide-linked protein domains adopt the essential conformation required for viral infectivity.

[0005] The membrane-distal globular head constitutes the majority of the HA1 structure and contains the sialic acid-binding pocket for viral entry and the major antigen domain. HA1 contains the vestigial esterase domains E1' and E2, as well as the receptor-binding site (RBS), which is the least conserved segment among influenza viruses. HA2 is a single-pass integral membrane protein with a fusion peptide (FP), a soluble HA2 ectodomain, a transmembrane (TM), and a cytoplasmic tail (CT) (see Figure 1). Together with the N- and C-terminal HA1 residues, HA2 forms the stalk domain, which includes the transmembrane region and is relatively conserved. The stalk structure contains the fusion machinery, which undergoes a conformational change in the low pH environment of the late endosome, leading to membrane fusion and cell entry. While the relative conservation of the stalk domain may be due to its immunodominant nature (and the resulting lack of antibody pressure), this observation may also result from a lack of tolerance to change due to functional constraints of the fusion machinery (Kirkpatrick et al. Scientific Reports volume 8, Article number: 10432 (2018)).

[0006] Influenza viruses are classified into types A, B, and C based on antigenic differences. Influenza types A and B are the causative agents of seasonal epidemics in humans. In contrast to influenza A, which is a zoonotic pathogen that infects multiple host species, influenza B primarily infects humans and occasionally seals. Unlike influenza A, influenza B viruses exhibit limited drift, making them relatively stable. Thus, for HA1, the primary target of antigenic variation, the sequence identity between influenza A and B viruses is low, at approximately 20%.

[0007] Although this lack of antigenic diversity prevents pandemic outbreaks, influenza B contributes to seasonal outbreaks of influenza, which can lead to severe infections costing thousands of lives and billions of dollars. Influenza B has become a growing concern in recent years due to the increased circulation of two distinct lineages of the virus, the Victoria lineage and the Yamagata lineage.

[0008] Various mutations in the influenza HA protein, specifically the influenza A HA protein, have been studied.

[0009] For example, Castelan-Vega et al. (Adv Appl Bioinform Chem. 2014;7:37-44) compared the 7,479 full-length amino acid sequences of influenza A(H1N1)pdm09 virus HA using a stability prediction algorithm and identified the D104N, A259T, S124N, and E172K mutations as causing a predicted increase in influenza HA stability. In contrast, the S206T, K285E, and E47K mutations had a predicted destabilizing effect on HA.

[0010] Reed et al. (2010) (J. Virol. 83:3568-3580) generated four recombinant H5N1 viruses containing mutations that altered the acid stability of the HA protein without altering the levels of expression, cleavage, receptor binding, or membrane fusion efficiency. Two of the mutations increased the pH of membrane fusion of the H5N1 HA protein (Y231H and N1142K), and the remaining two mutations decreased the pH of fusion (H241Q and K582I).

[0011] In Zaraket et al. 2013 (J Virol. 2013 May; 87(9)), they investigated how mutations that alter the activation pH of the HA protein affect the fitness of avian H5N1 influenza viruses in mammalian models. In Zaraket et al. 2013 (J Virol. 2013 Sept; 87(17)), they investigated how lowering the HA activation pH (increasing acid stability) affects the properties of highly pathogenic H5N1 influenza viruses in mammalian hosts.

[0012] Although the number is small, mutations in the influenza B HA protein were also investigated.

[0013] For example, Lugovtsev et al. (2007) (Virology, 2007 September; 365(2)) used reverse genetics to analyze the contribution of amino acid substitutions previously identified in the high-growth phenotype of B / Victoria / 504 / 2000 to viral growth. Lugovtsev et al. found that G141E and R162M were most favorable for viral growth, but that only R162M was capable of improving viral growth without antigenic changes.

[0014] Chen et al. 2007 (Vaccine, 2007 January;26(13)) investigated the effect of the 196 / 197 glycosylation site on influenza B virus growth and antigenicity.

[0015] Kim et al. 2015 (Vaccine, 2015 September; 33) investigated the mutations responsible for the growth enhancement in cold-adapted influenza B viruses. Molecular analysis revealed that the following mutations in the HA, NP, and NA genes were required for the enhanced virus growth: G156 / N160 in HA, E253, G375 in NP, and T146 in the NA gene.

[0016] Vaccination remains the most effective method for preventing influenza infection. However, the constantly evolving nature of influenza viruses necessitates ongoing global surveillance and frequent reformulation of influenza vaccines. The World Health Organization (WHO) convenes global meetings each February and September to make recommendations regarding the viruses to be included in seasonal influenza vaccines for the Northern and Southern Hemispheres, respectively. These recommendations are based on information provided by the WHO's Global Influenza Surveillance and Response System (GISRS).

[0017] Quadrivalent influenza vaccines (QIV) contain the hemagglutinin antigen (HA) of each of the four influenza strains recommended by WHO for the upcoming influenza season (usually influenza A(H3N2) and A(H1N1) strains and two influenza B strains, one each of the B virus lineages [B / Yamagata and B / Victoria]).

[0018] Traditionally, vaccination is carried out using live attenuated or whole inactivated forms of the virus that induce an immune response when administered to a patient. To eliminate the potential risk of live attenuated and whole inactivated viruses regaining the ability to replicate and become infectious, vaccines containing recombinant viral proteins have also been used to induce protective immunity against influenza infection.

[0019] However, the use of recombinant viral proteins as immunogenic components of vaccines suffers from several limitations. First, in the absence of the full complement of viral proteins and genetic components required for optimal expression and proper protein folding, recombinant viral protein production in standard in vitro expression systems may be insufficient for vaccine manufacturing purposes. Second, recombinant viral protein vaccines may exhibit poor immunogenicity due to improper folding, poor antigen presentation, and / or the generation of a primarily humoral immune response that is ineffective in conferring long-lasting protective immunity.

[0020] Virus-like particles (VLPs) are potential candidates for inclusion in immunogenic compositions. VLPs closely resemble mature virions but do not contain viral genomic material. Therefore, VLPs are non-replicative in nature, which makes them safe for administration as vaccines. In addition, VLPs can be genetically engineered to express viral glycoproteins on their surface, which is their most natural physiological configuration. Furthermore, because VLPs resemble native virions and are multivalent particulate structures, VLPs may be more effective at inducing neutralizing antibodies against glycoproteins rather than soluble envelope protein antigens.

[0021] VLPs have previously 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, WO 2010 / 148511, WO 2013 / 044390 and WO 2014153674, the contents of which are incorporated by reference herein).

[0022] WO 2009 / 076778 teaches a method for producing influenza VLPs in plants, comprising the step of introducing a nucleic acid having a regulatory region active in the plant operably linked to a nucleotide sequence encoding influenza HA of influenza A or B.

[0023] WO 2009 / 009876 teaches a method for producing influenza HA VLPs in plants, where the influenza HA self-assembles into VLPs in plant cells and buds from the plant cell membrane.

[0024] WO 2010 / 006452 teaches the production of VLPs containing modified influenza HA proteins in which glycosylation sites at positions 154, 165, 286, or combinations thereof (see A / Vietnam / 1194 / 04 [H5N1] numbering) have been eliminated by mutating the residues at those positions to amino acids other than asparagine. WO 2010 / 006452 further teaches that amino acids at positions 156, 167, 288, or combinations thereof can be mutated to residues other than serine or threonine, similarly eliminating the N-linked glycosylation signal triplet "NXS / T." By selectively deleting glycosylation sites located in the globular head of the HA protein, WO 2010 / 006452 demonstrates that the resulting HA protein has increased antigenicity and broader cross-reactivity.

[0025] WO 2010 / 148511 discloses a method for producing influenza VLPs in plants, the VLPs comprising a chimeric HA protein comprising a stem domain cluster having F'1, F'2 and F subdomains, a head domain cluster having RB, E1 and E2 subdomains, and a transmembrane domain cluster having a transmembrane domain and a C-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.

[0026] WO 2014 / 153674 teaches a method for producing influenza VLPs in plants, the VLPs comprising a modified influenza HA with a modified proteolytic loop, which involves removing the proteolytic cleavage site between the HA1 and HA2 domains of the HAO precursor, thereby stabilizing the HA protein and increasing protein yield compared to native HA protein.

[0027] WO 2013 / 044390 teaches the production of virus-like particles (VLPs) by co-expressing influenza HA and proton channel proteins in plants. Summary of the Invention

[0028] The present invention relates to the production of modified influenza B hemagglutinin (HA) proteins. The present invention also relates to virus-like particles (VLPs) comprising modified influenza B HA proteins. The modified B HA proteins and VLPs comprising modified B HA exhibit improved characteristics when compared to VLPs comprising unmodified B HA or unmodified B HA proteins. The present invention further relates to the production and increased production of influenza virus-like particles (VLPs) in a host or host cell, wherein the VLPs comprise modified influenza B HA proteins.

[0029] It is an object of the present invention to provide improved methods for increasing influenza VLP production in a host or host cell, such as a plant or plant cell.

[0030] In accordance with the present invention, there is provided a modified influenza B virus hemagglutinin (HA) protein comprising a modified HA2 ectodomain, wherein the modified HA2 ectodomain comprises an amino acid sequence having at least one amino acid substitution compared to a parent HA2 ectodomain amino acid sequence, the at least one substitution corresponding to amino acid position 402 in a sequence alignment with a reference sequence of SEQ ID NO: 1 (B / Washington / 09 / 19HA). The parent HA2 ectodomain amino acid sequence can be a wild-type amino acid sequence of an influenza B virus.

[0031] The substitution can be a non-leucine substitution. For example, the substitution can be isoleucine or a conservative substitution for isoleucine. A conservative substitution for isoleucine can be methionine, phenylalanine, or valine.

[0032] The modified B HA may comprise a modified HA2 subunit, which may have a sequence that may have 80% to 100% identity to the sequence of SEQ ID NO: 41 or SEQ ID NO: 42. Furthermore, the modified HA2 subunit may comprise a modified HA2 ectodomain. The modified HA2 ectodomain may have a sequence that may have 80% to 100% identity to the sequence of SEQ ID NO: 42. The sequence of the influenza B HA protein may have 80% to 100% identity to the sequence of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37.

[0033] The modified influenza B HA may comprise a plant-specific N-glycan, a modified N-glycan, or a combination thereof. Furthermore, the modified influenza B HA protein may be a chimeric B HA protein, which comprises a transmembrane and cytoplasmic tail (TM / CT) derived from an influenza A HA protein. In addition, the modified influenza B HA protein may have a modified proteolytic cleavage site. Thus, a modified influenza B HA protein is also provided, in which the proteolytic cleavage site is modified.

[0034] Further provided is a nucleic acid comprising a nucleotide sequence encoding the modified influenza HA protein described above.

[0035] Also provided are virus-like particles (VLPs) comprising the modified influenza B HA proteins described above.

[0036] In another aspect, a method (A) of producing a modified influenza B HA protein in a non-human host or host cell is provided, comprising: a) introducing into a non-human host or host cell a nucleic acid comprising a nucleotide sequence encoding the modified influenza HA protein as described above, or providing a non-human host or host cell comprising a nucleic acid comprising a nucleotide sequence encoding the modified influenza HA protein as described above; and b) incubating the non-human host or host cells under conditions that allow expression of the nucleic acid, thereby producing the modified influenza B HA protein.

[0037] In another aspect, a method (B) is provided for increasing the production of a modified influenza B HA protein in a non-human host or host cell, comprising: a) introducing into a non-human host or host cell a nucleic acid comprising a nucleotide sequence encoding the modified influenza HA protein as described above, or providing a non-human host or host cell comprising a nucleic acid comprising a nucleotide sequence encoding the modified influenza HA protein as described above; and b) incubating the non-human host or host cells under conditions that allow expression of the modified B HA protein encoded by the nucleic acid, thereby producing the modified B HA in higher yields compared to a non-human host or host cell that expresses an influenza B HA protein comprising the HA2 ectodomain parent amino acid sequence.

[0038] The modified influenza B HA protein in method (A) or (B) may be further extracted and purified from the non-human host or host cells.

[0039] In another aspect, there is provided a modified influenza B HA protein produced by method (A) or method (B).

[0040] In another aspect, a method (C) for producing influenza virus-like particles (VLPs) in a non-human host or host cell is provided, comprising: a) providing a non-human host or host cell comprising a nucleic acid comprising a nucleotide sequence encoding a modified influenza HA protein, or introducing into a non-human host or host cell a nucleic acid comprising a nucleotide sequence encoding a modified influenza HA protein; and b) incubating the non-human host or host cells under conditions that allow expression of the modified influenza B HA protein encoded by the nucleic acid, thereby producing VLPs.

[0041] In yet another aspect, a method (D) for increasing the production of influenza virus-like particles (VLPs) in a non-human host or host cell is provided, comprising: a) introducing into a non-human host or host cell a nucleic acid comprising a nucleotide sequence encoding a modified influenza HA protein, or providing a non-human host or host cell comprising a nucleic acid comprising a nucleotide sequence encoding a modified influenza HA protein; and b) incubating the non-human host or host cells under conditions that allow expression of the modified B HA protein encoded by the nucleic acid, thereby producing VLPs in higher yields compared to a non-human host or host cells that expresses an influenza B HA protein that comprises the HA2 ectodomain parent amino acid sequence.

[0042] Method (C) or (D) may further comprise step c), i.e., harvesting the non-human host or host cells and extracting and purifying the VLPs.

[0043] Further provided are virus-like particles (extracted VLPs) produced by the method of (C) or (D). The VLPs may further comprise a non-human host cell or one or more lipids derived from the host cell.

[0044] The nucleic acid of method (A), (B), (C), or (D) may further comprise a nucleotide sequence encoding a proton channel protein. Alternatively, step a) of method (A), (B), (C), or (D) may further comprise introducing a second nucleic acid encoding a proton channel protein, and step b) of method (A), (B), (C), or (D) may further comprise incubating the non-human host or host cell under conditions that allow expression of the proton channel protein encoded by the second nucleic acid. The proton channel protein may be influenza A M2 protein.

[0045] In another aspect, a method of producing an antibody or antibody fragment is provided, the method comprising administering the VLP described above to a subject, or host animal, thereby producing the antibody or antibody fragment. Further provided is an antibody produced by the above method.

[0046] In another aspect, a host or host cell comprising a nucleic acid, a modified influenza B HA protein, a VLP, or a combination thereof is provided.

[0047] In a further aspect, there is provided a composition for eliciting an immune response comprising an effective amount of a VLP and a pharmaceutically acceptable carrier, adjuvant, vehicle or excipient.

[0048] Also provided is a vaccine for eliciting an immune response, the vaccine comprising an effective amount of a modified influenza B HA protein, VLP, or composition described above. The vaccine may further comprise an adjuvant.

[0049] Further provided are methods for inducing an immune response in a subject, the methods comprising administering the VLPs, composition or vaccine to the subject. The VLPs, composition or vaccine may be administered to the subject orally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

[0050] The non-human host or host cell may include a plant, a plant part, a plant cell, a fungus, a fungal cell, an insect, an insect cell, an animal or an animal cell.

[0051] In another aspect, a multivalent immunogenic composition is also provided comprising two or more types of VLPs, at least one of which comprises the modified influenza B HA described above. The composition may further comprise a second type of VLP, wherein the second type of VLP comprises the modified influenza B HA. At least one of the VLPs may be a first type of VLP, wherein the first type of VLP comprises a modified B HA derived from an influenza B lineage different from the modified B HA of the second type of VLP. For example, the first type of VLP may comprise a modified B HA derived from the B / Victoria lineage, and the modified B HA in the second type of VLP may be derived from the B / Yamagata lineage. The composition may further comprise one or more types of VLPs comprising an influenza A HA protein. For example, the influenza A HA may be derived from influenza subtype H1 and / or influenza subtype H3.

[0052] In yet another aspect, a tetravalent immunogenic composition is provided comprising a first type of VLPs comprising a modified influenza B HA as described herein, a second type of VLPs comprising a modified influenza B HA as described herein, a third type of VLPs comprising an influenza A HA, and a fourth type of VLPs comprising an influenza A HA, wherein the first type of VLPs comprises a modified B HA derived from a different influenza B strain than the modified B HA of the second type of VLPs.

[0053] This summary of the invention does not necessarily describe all features of the invention.

[0054] These and other features of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0055] [Figure 1] A schematic diagram of the domain structure of the HA protein (precursor HAO) is shown. HAO contains an N-terminal signal sequence (which targets protein synthesis to the ER before cleavage and release) and two HA subunits (HA1 and HA2). Domains in the HA2 subunit include the fusion protein (FP), HA2 ectodomain, transmembrane domain (TM), and cytoplasmic tail (CT). The HAO precursor protein is incapable of causing membrane fusion; proteolytic cleavage of the HA1 and HA2 subunits is required to render the protein into a fusion-competent form as starting material. The cleavage site between the HA1 and HA2 subunits is indicated by an arrow.

[0056] [Figure 2A] 1 shows a schematic diagram of vector 4498 required for assembly of a vector plasmid encoding a modified influenza strain B HA protein. [Figure 2B] Schematic diagram of vector 2879 encoding influenza B strain HA of B / Singapore / INFKK-16-0569 / 2016. [Figure 2C] FIG. 1 shows a schematic diagram of vector 8894 encoding a modified influenza B strain HA of B / Singapore / INFKK-16-0569 / 2016 with the L404I mutation. [Figure 2D] Schematic diagram of vector 7679 encoding influenza B strain HA of B / Washington / 02 / 2019 is shown. [Figure 2E] Schematic diagram of vector 8881 encoding influenza B strain HA of B / Washington / 02 / 2019 with the L402I mutation. [Figure 2F] A schematic diagram of vector 8424 encoding influenza B strain HA of B / Rhode Island / 01 / 2019 is shown. [Figure 2G] A schematic diagram of vector 7787 encoding influenza B strain HA of B / Rhode Island / 01 / 2019 with the L402I mutation is shown. [Figure 2H] A schematic diagram of vector 9627 encoding influenza B strain HA of B / Michigan / 01 / 2021 is shown. [Figure 2I] A schematic diagram of vector 9628 encoding influenza B strain HA of B / Michigan / 01 / 2021 with the L402I mutation is shown. [Figure 2J] A schematic diagram of vector 9629 encoding influenza B strain HA of B / Henan-Xigong / 1118 / 2021 is shown. [Figure 2K] A schematic diagram of vector 9630 encoding influenza B strain HA of B / Henan-Xigong / 1118 / 2021 carrying the L402I mutation is shown. [Figure 2L] A schematic diagram of vector 9866 encoding influenza B strain HA of B / Singapore / WUH4618 / 2021 is shown. [Figure 2M] A schematic diagram of vector 9867 encoding influenza B strain HA of B / Singapore / WUH4618 / 2021 with the L402I mutation is shown. [Figure 2N] Schematic diagram of vector 9868 encoding influenza B strain HA of B / Austria / 1359417 / 2021. [Figure 2O] Schematic diagram of vector 9869 encoding influenza B strain HA of B / Austria / 1359417 / 2021 with the L402I mutation.

[0057] [Figure 3]Figure 1 shows the fold change in production in plants expressing modified influenza B HA proteins. Fold changes were calculated relative to the appropriate unmodified (parental) control protein (CTL) as follows: B / Singapore / INFKK-16-0569 / 2016 (CTL: construct 2879, L404I: construct 8894), B / Washington / 02 / 2019 (CTL: construct 7679, L402I: construct 8881), B / Rhode Island / 01 / 2019 (CTL: construct 8424, L402I: construct 8881). Construct 7787), B / Michigan / 01 / 2021 (CTL: Construct 9627, L402I: Construct 9628), B / Henan-Xigong / 1118 / 2021 (CTL: Construct 9629, L402I: Construct 9630), B / Singapore / WUH4618 / 2021 (CTL: Construct 9866, L402I: Construct 9867) and B / Austria / 1359417 / 2021 (CTL: Construct 9868, L402I: Construct 9869).

[0058] [Figure 4] Shown is the fold change in drug substance (DS) production obtained from either host expressing the modified influenza B strain HA protein, calculated relative to the appropriate parental control HA protein (CTL) as follows: B / Washington / 02 / 2019 (CTL: Construct 7679, L402I: Construct 8881) and B / Rhode Island / 01 / 2019 (CTL: Construct 8424, L402I: Construct 7787). DETAILED DESCRIPTION OF THE INVENTION

[0059] The following description is of a preferred embodiment.

[0060] As used herein, the terms "comprising," "having," "including," "containing," and grammatical variations thereof are inclusive or open-ended and do not exclude additional, unspecified elements and / or method steps. The term "consisting essentially of," when used in connection with a product, use, or method herein, means that additional elements and / or method steps may be present, but these additions do not materially affect the manner in which the specified method or use functions. The term "consisting of," when used in connection with a product, use, or method herein, excludes the presence of additional elements and / or method steps. Products, uses, or methods described herein as including certain elements and / or steps may also consist essentially of these elements and / or steps in some embodiments, and in other embodiments, consist of these elements and / or steps, regardless of whether these embodiments are specifically recited. Additionally, the use of the singular includes the plural, and "or" means "and / or" unless otherwise specified. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. As used herein, the term "about" refers to approximately a + / - 10% variation from a given value. It should be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to. When used herein in conjunction with the term "comprising," the use of the terms "a" or "an" can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0061] Described herein are modified influenza B hemagglutinin (HA) proteins (influenza or influenza B HA comprising modified B HA protein, modified influenza B HA protein, modified B HA, modified influenza B HA, mutant B HA, influenza mutant B HA, modified B protein, modified B, modified HA2 subunit, or modified HA2 ectodomain) and methods of producing the modified influenza B HA proteins in a host or host cell. Furthermore, the modified influenza B HA proteins can self-assemble into virus-like particles (VLPs). Accordingly, influenza VLPs comprising or consisting of modified influenza B HA proteins are also provided.

[0062] For example, modifications, e.g., by substitution of specific amino acids, in a B HA protein, such as a B HA of the Yamagata or Victoria lineage, result in improved properties of the modified B HA protein when compared to a parent HA that does not contain the specific amino acid substitution. The parent HA may also be referred to as an unmodified B HA protein. In some embodiments, the parent or unmodified HA may be a wild-type HA. In other embodiments, the parent or unmodified HA may include other modifications, such as, for example, a deletion or partial deletion of the proteolytic loop and / or a substitution of the native transmembrane and cytoplasmic tail domain (TMCT) of influenza A HA, as described below.

[0063] With respect to influenza virus, the term "hemagglutinin" or "HA," as used herein, refers to a glycoprotein found on the outside of influenza virus particles. HA is translated as a single protein, HA0. HA0 generally comprises a signal peptide (SP), an HA1 domain (also referred to as the HA1 subunit), an HA2 domain (also referred to as the HA2 subunit) containing the fusion protein (FP), an HA2 ectodomain, and a transmembrane domain (TM) and a cytoplasmic tail (CT), collectively referred to as TM / CT (see Figure 1).

[0064] For viral activation, HA0 (assembled as a trimer) must be cleaved at a specific site between the HA1 and HA2 domains of the protein. After cleavage, the two disulfide-linked protein domains fuse, thus producing the mature form of the protein subunit, a prerequisite for the conformational change required for viral infectivity.

[0065] The nucleotide sequence encoding HA, as well as the HA amino acid sequence, are well known and available. See, for example, the BioDefence Public Health base (Influenza Virus, see URL: biohealthbase.org) or the Center for Biotechnology Information (see URL: ncbi.nlm.nih.gov), the contents of both of which are incorporated herein by reference. Furthermore, influenza strains can be identified and typed by techniques known in the art, for example, by hemagglutination inhibition assay, reverse transcriptase PCR, real-time PCR, or sequencing (ElHefnawi & Sherif (Virology, Volume 449, 20 January 2014)).

[0066] The modified B HA protein may comprise an HA1 domain, an HA2 ectodomain, a transmembrane domain (TM), and a cytoplasmic tail (CT). The HA1 and HA2 domains may be derived from influenza B HA, and the transmembrane domain (TM) and cytoplasmic tail (CT) may be derived from influenza A HA. The modified HA protein may further comprise a cleavage site and a fusion peptide. In some embodiments, the cleavage site and / or the fusion peptide may be modified. As further described below, the modified B HA may be produced as a precursor protein and may include a native or non-native signal peptide.

[0067] The modified influenza B HA proteins disclosed herein contain modifications or mutations that have been found to result in improved B HA properties compared to a parent (unmodified) HA protein of the same strain or subtype of influenza that does not contain one or more modifications or mutations, referred to as the parent HA, unmodified HA, or control. For example, a modified influenza B HA protein can have an amino acid sequence that has at least one amino acid substitution when compared to the corresponding parent amino acid sequence. In some embodiments, a modified B HA protein can have one or more substitutions in the HA2 ectodomain when compared to the sequence of the HA2 ectodomain of the parent B HA.

[0068] For example, a modified influenza B virus hemagglutinin (HA) protein can include a modified HA2 subunit (also referred to as HA2) in which at least one amino acid has been modified (e.g., by substitution or replacement) compared to a parent sequence, e.g., the wild-type amino acid in the sequence. In one aspect, a modified influenza B virus hemagglutinin (HA) protein can include a modified HA2 ectodomain in which at least one amino acid has been modified (e.g., by substitution or replacement) compared to a parent sequence, e.g., the wild-type amino acid in the sequence. The amino acid modification can correspond to amino acid position 402 in a sequence alignment with the reference sequence of SEQ ID NO: 1 (B / Washington / 09 / 19HA).

[0069] Examples of improved properties of the modified B HA proteins include increased HA B protein production when expressed in a host or host cell compared to the parent B HA protein of the same influenza strain that does not contain the modification or mutation; increased VLP production when the modified B HA protein that does not contain the modification or mutation is expressed in a host or host cell compared to the level of VLP production; and increased drug substance (DS) yield when the DS is obtained from a host or host cell expressing the modified HA B protein compared to the DS yield obtained from a host or host cell expressing the modified HA B protein and combinations thereof.

[0070] Modified B virus HA proteins can be generated by introducing changes into the amino acid sequence of the influenza B HA protein that result in the improved properties of the HA described above. Isolation of nucleic acids encoding such HA molecules is routine, as is modification of the nucleic acid to introduce changes in the amino acid sequence, for example, by site-directed mutagenesis.

[0071] The influenza B HA proteins, mutant B HA proteins or modified B HA proteins described herein are modified to include one or more mutations, modifications or substitutions in their amino acid sequence, such that at least one amino acid corresponding to amino acid 402 of B / Washington / 09 / 19 HA (SEQ ID NO: 1) or corresponding to amino acid 404 of B / Singapore / INFKK-16-0569 / 2016 (SEQ ID NO: 2) is modified when compared to the unmodified (parent) sequence.

[0072] "Corresponding to an amino acid" or "corresponding to an amino acid" means that the amino acid corresponds to an amino acid in a sequence alignment with the influenza reference strains.

[0073] The number of amino acid residues in HA or the residue position in HA is related to the numbering of the HA of the influenza reference strain. For example, the reference strain may be B / Washington / 09 / 19 HA (SEQ ID NO: 1) belonging to the Victoria lineage (see Table 1). The reference strain may also be B / Singapore / INFKK-16-0569 / 2016 (SEQ ID NO: 2) belonging to the Yamagata lineage (see Table 1).

[0074] Corresponding amino acid positions can be determined by aligning the sequence of B HA with the sequence of the HA of each reference strain. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0075] When 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 the new or substituted amino acid. For example, a substitution of leucine (L) for isoleucine (I) at residue or amino acid position 402 is referred to as L402I.

[0076] Modified B HA, B HA mutants or variants are designated in the same manner, using the single letter amino acid code for the unmodified (parent) or wild-type residue, followed by its position, and the single letter amino acid code for the substituted residue.

[0077] [Table 1]

[0078] "Modification," "amino acid modification," or "amino acid sequence modification" refers to the mutation, substitution, replacement, or deletion of one or more amino acid residues in a sequence compared to the original parent (unmodified) sequence. The parent sequence may be a wild-type sequence, or the parent sequence may be a sequence that already contains a modification (the "parent modification") when compared to the wild-type sequence. An "amino acid substitution" or "substitution" refers to the replacement of an amino acid in the amino acid sequence of a protein with a different amino acid compared to the parent sequence. In one embodiment, the modified influenza B virus hemagglutinin (HA) protein contains a substitution in the HA2 ectodomain compared to the parent HA2 ectodomain.

[0079] The terms amino acid, amino acid residue, or residue are used interchangeably in this disclosure. One or more amino acids can be substituted with one or more amino acids different from the original amino acid at that position without changing the overall amino acid sequence of the protein. Substitution or replacement can be experimentally induced by changing the codon sequence of the nucleotide sequence encoding the protein to a codon sequence for a different amino acid compared to the original amino acid in the parent sequence. Furthermore, one or more amino acids can be deleted from the amino acid sequence of the protein. The resulting protein is a modified influenza B HA protein. The modified B HA protein does not occur in nature.

[0080] Modified B HA includes non-naturally occurring HA proteins that have at least one modification to a parent or naturally occurring HA and have improved properties compared to the parent or naturally occurring HA protein from which the amino acid sequence of the modified B HA is derived. The modified B HA protein has an amino acid sequence not found in nature, achieved by substituting one or more amino acid residues of the HA protein with one or more different amino acids.

[0081] Thus, a modified B HA, mutant B HA or recombinant B HA has a DNA sequence encoding the parent HA that has been modified to produce a modified or mutated DNA sequence that encodes a modification, mutation or substitution of one or more amino acids in the HA amino acid sequence.

[0082] The modified or variant influenza B HA proteins described herein are modified to include a mutation or modified residue in a sequence alignment of B / Washington / 02 / 2019 (SEQ ID NO: 1) at position 402. Thus, influenza B HA polypeptides, proteins and / or protein complexes, e.g., virus-like particles (VLPs), are provided that include, for example, a modification or mutation at amino acid position 402, where the numbering of such amino acids is based on the sequence of B / Washington / 02 / 2019 (SEQ ID NO: 1), or the amino acid position corresponding to such amino acid position, as determined by alignment of the B HA amino acid sequence to, e.g., SEQ ID NO: 1. Non-limiting examples of influenza B HA amino acid sequences that include such mutations include the sequences of SEQ ID NOs: 13, 17, 21, 25, 29, 33, or 37.

[0083] Non-limiting examples of strains from which influenza B HA can be derived include wt HA B / Singapore / INFKK-16-0569 / 2016 (EPI592707) (SEQ ID NO:2), wt HA B / Washington / 02 / 2019 (EPI1368874) (SEQ ID NO:1), wt HA B / Rhode Island / 01 / 2019 (EPI1383242) (SEQ ID NO:3), wt HA B / Michigan / 01 / 2021 (EPI1843974) (SEQ ID NO:4), wt HA B / Henan-Xigong / 1118 / 2021 (EPI1878454) (SEQ ID NO:5), wt HA B / Austria / 1359417 / 2021 (EPI1845793) (SEQ ID NO:6) or wt HA B / Singapore / WUH4618 / 2021 (EPI1883660) (SEQ ID NO: 7) (see also Table 2).

[0084] In one aspect of the disclosure, the modified B HA may be modified at least at residue 402, where the numbering is with respect to the reference strain B / Washington / 02 / 2019 (SEQ ID NO: 1).

[0085] As shown in Figure 3, modified B HA proteins having a residue at position 402 changed, for example, from leucine (L, Leu) to isoleucine (I, Ile), referred to herein as L402I, showed up to a 2.3-fold increase in production in plants when compared to B HA having leucine (L, Leu) at this position (see also Example 3 and Table 4).

[0086] The modified HA of B / Singapore / 0569 / 16 with the L404I substitution exhibited approximately a 2.3-fold increase in production in planta when compared to the parental B / Singapore / 0569 / 16 HA (see Figure 3).

[0087] The modified HA of B / Washington / 09 / 19 with the L402I substitution exhibited approximately a 1.9-fold increase in production in planta when compared to the parent B / Washington / 09 / 19 HA (see Figure 3). Furthermore, the modified HA of B / Washington / 09 / 19 with the L402I substitution exhibited approximately a 2.5-fold increase in fold change in drug substance (DS) production when compared to the unmodified B / Washington / 09 / 19 HA protein (see Figure 4).

[0088] The modified HA of B / Rhode Island / 01 / 2019 with the L402I substitution exhibited approximately a 1.5-fold increase in production in plants when compared to the unmodified B HA protein (see Figure 3). The modified B HA of B / Rhode Island / 01 / 2019 also exhibited approximately a 1.8-fold increase in drug substance (DS) production when compared to the unmodified B / Rhode Island / 01 / 2019 HA protein (see Figure 4).

[0089] Increased in planta production was also observed for the modified B HAs B / Michigan / 01 / 2021 (1.5-fold change), B / Henan-Xigong / 1118 / 2021 (approximately 1.5-fold change), B / Henan-Xigong / 1118 / 2021 (approximately 1.2-fold change), B / Singapore / WUH4618 / 2021 (approximately 1.3-fold change), and B / Austria / 1359417 / 2021 (approximately 1.3-fold change) (see Figure 3, Example 3, and Table 4).

[0090] Without wishing to be bound by theory, increased HA production in plants has been shown to correlate with increased VLP production in plants.

[0091] In one aspect, it is therefore provided that residue 402 of influenza B HA (numbering according to B / Washington / 09 / 19 numbering) can be modified to substitute leucine (L, Leu) for isoleucine (I, Ile).

[0092] For example, the modified B HA protein can have an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or any amount of sequence identity or similarity therebetween to the amino acid sequence of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, or SEQ ID NO:37, wherein the amino acid sequence has an isoleucine (I) or a conservative substitution of an isoleucine (I) other than leucine (L), such as for example, a valine (V), a methionine (M), or a phenylalanine (F) at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO:1), wherein the modified B HA sequence is not naturally occurring, and wherein the HA protein forms a VLP upon expression.

[0093] Also provided herein is a nucleic acid comprising a nucleotide sequence encoding a modified B HA having a substitution at position 402 as described above, operably linked to a regulatory region that is active in plants.

[0094] For example, the nucleotide sequence can have an amino acid sequence having about 70%, about 75%, about 80%, about 85%, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or any amount of sequence identity or similarity therebetween to a nucleotide sequence encoding a B HA having the amino acid sequence of SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33, or SEQ ID NO:37, wherein the amino acid sequence has an isoleucine (I) or a conservative substitution of isoleucine (I) but not leucine (L), such as for example, valine (V), methionine (M), or phenylalanine (F) at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO:1), wherein the modified B HA sequence is not naturally occurring, and the HA protein forms a VLP upon expression.

[0095] The nucleotide sequence has about 70%, about 75%, about 80%, about 85%, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or any amount of sequence identity or similarity therebetween to the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:32, or SEQ ID NO:36, and the nucleotide codon encoding amino acid residue 402 of the modified BH encodes isoleucine (I) or a conservative substitution of isoleucine (I) for leucine (L), such as valine (V), methionine (M), or phenylalanine (F), at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO:1), and the modified BH HA sequence is not naturally occurring.

[0096] Influenza A viruses are divided into subtypes based on two proteins on the surface of the virus, hemagglutinin (H) and neuraminidase (N). Influenza A subtypes can be further divided into different genetic "clades" and "subclades."

[0097] Influenza B viruses are not divided into subtypes, but instead are classified into two co-circulating phylogenetically and antigenically distinct lineages, later named viruses B / Yamagata / 16 / 88 (Yamagata lineage) and B / Victoria / 2 / 87 (Victoria lineage). Like influenza A viruses, influenza B viruses can be further divided into specific clades and subclades. Influenza B viruses generally change more slowly with respect to their genetic and antigenic characteristics than influenza A viruses.

[0098] Traditionally, different strains of influenza have been classified based on their ability to agglutinate, for example, red blood cells (RBCs, or erythrocytes). Antibodies specific to a particular influenza strain can bind to the virus, thereby preventing such agglutination. Assays that determine strain type based on such inhibition are typically known as hemagglutinin inhibition assays (HI assays or HAI assays), which are standard and well-known methods in the art for characterizing influenza strains.

[0099] However, the HA proteins of different viral strains also show significant sequence similarity at both the nucleic acid and amino acid levels. The level of similarity can vary between strains of different B lineages. While this variation is sufficient to define distinct lineages and the evolutionary lineage of different strains, the DNA and amino acid sequences of different strains are still easily aligned using conventional bioinformatics techniques (Langat, Pinky et al. PLoS pathogens 2017 Dec; vol. 13(12)).

[0100] Multiple nucleotide sequences, or corresponding polypeptide sequences of hemagglutinin (HA), can be aligned to determine a "consensus" or "consensus sequence" for a subtype or strain (see Gravel et al., iScience 24, Nov. 2021). For example, the consensus sequence for the B HA2 domain is found in SEQ ID NO: 40 (the fusion peptide (FP) is italicized, and the TMCT domain is underlined). By excluding the sequence of the FP and the sequence of the TMCT domain, the consensus sequence for the B HA2 ectodomain can be determined.

[0101] Accordingly, also provided is an influenza B HA protein having a modified HA2 ectodomain, wherein the HA2 ectodomain comprises one or more mutations, modifications, or substitutions in its amino acid sequence, wherein at least one amino acid corresponding to amino acid position 58 of SEQ ID NO: 40 is modified when compared to the unmodified (parent) sequence. For example, a leucine (L) is modified to a non-leucine, such as an isoleucine (I) at position 58 (L58I).

[0102] Thus, as described herein, a modified B virus HA protein can comprise a modified HA2 subunit (or HA2 domain) comprising the following fusion peptide (in italics) and HA2 ectodomain sequence:

number

[0103] In one aspect, a modified influenza B virus HA protein is provided that comprises a modified HA2 domain, wherein the modified HA2 domain comprises at least one substitution when compared to the parent (unmodified) sequence or wild-type HA2 domain. The modified HA2 domain can have an amino acid sequence having about 80%, about 82%, about 83%, about 85%, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or any amount therebetween, sequence identity or similarity to the amino acid sequence of SEQ ID NO:41, wherein the sequence includes a non-leucine, such as isoleucine, at position 58. The modified influenza B virus HA protein can comprise an HA2 domain that comprises or consists of the sequence of SEQ ID NO:41.

[0104] conservative substitution As described herein, residues of the B HA protein can be identified and modified, substituted, or mutated to produce modified B HA proteins or B HA protein variants. Substitutions or mutations at specific positions are not limited to the amino acid substitutions described herein or shown in the examples. For example, B HA variants can contain conservative substitutions or conservative substitutions of the amino acid substitutions described.

[0105] As used herein, the terms "conservative substitution" or "conservative substitution," and grammatical variations thereof, refer to the presence of an amino acid residue in the sequence of a different HA protein but within the same class of amino acids as the described substitution or described residue (i.e., a non-polar residue substituting a non-polar residue, an aromatic residue substituting an aromatic residue, a polar uncharged residue substituting a polar uncharged residue, a charged residue substituting a charged residue). In addition, conservative substitutions can include residues having an interfacial hydropathicity index value of the same sign and generally similar magnitude as the residue substituting the wild-type residue.

[0106] As used herein, the term "non-polar residue" refers to glycine (G, Gly), alanine (A, Ala), valine (V, Val), leucine (L, Leu), isoleucine (I, Ile), and proline (P, Pro); the term "aromatic residue" refers to phenylalanine (F, Phe), tyrosine (Y, Tyr), and tryptophan (W, Trp); the term "polar uncharged residue" refers to serine (S, Ser), threonine (T, Thr), cysteine ​​(C, Cys), methionine (M, Met), asparagine (N, Asn), and glutamine (Q, Gln); and the term "charged residue" refers to the negatively charged amino acids aspartic acid (D, Asp) and glutamic acid (E, Glu), and the positively charged amino acids lysine (K, Lys), arginine (R, Arg), and histidine (H, His). Other classifications of amino acids include: · 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): can be arginine (R, Arg); histidine (H, His); lysine (K, Lys), glycine (G, Gly) and proline (P, Pro).

[0107] Conservative amino acid substitutions are likely to have a similar effect on the activity of the resulting HA protein variant or modified HA protein as the original substitution or modification. Further information on conservative substitutions can be found, for example, in Ben Bassat et al. (J. Bacteriol, 169:751-757, 1987), O'Regan et al. (Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein ScL, 3:240-247, 1994), Hochuli et al. (Bio / Technology, 6:1321-1325, 1988), and widely used genetics and molecular biology textbooks.

[0108] The Blosum matrix is ​​commonly used to determine the relatedness of polypeptide sequences. It is generated using a large database of reliable alignments (the BLOCKS database), which counts pairwise sequence alignments that are related below a certain threshold percentage identity (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992). For highly conserved target frequencies in the BLOSUM90 matrix, a threshold of 90% identity was used. A threshold of 65% identity was used for the BLOSUM65 matrix. A score of zero or greater in the Blosum matrix is ​​considered a "conservative substitution" at a selected percentage identity. The following table shows exemplary conservative amino acid substitutions: Table 2. [Table 2]

[0109] The nucleotide sequence encoding the modified B HA protein can be optimized for human codon usage, increased GC content, or a combination thereof. The modified HA protein can be expressed in a host or host cell, such as a plant, plant part, or plant cell.

[0110] As described above, the parent sequence may be a wild-type sequence, or the parent sequence may be a sequence that already includes a modification ("parent modification") when compared to the wild-type sequence. The parent modification may be an amino acid deletion or substitution. For example, the parent modification may include a modification such as the deletion of a proteolytic cleavage site (also referred to as a proteolytic loop). For example, the cleavage site and / or the fusion peptide or a portion of the fusion peptide may be deleted to prevent cleavage of the HA protein. For example, the C-terminus of the H1 domain and the N-terminus of the HA2 domain, which include the cleavage site and fusion peptide, may be modified. For example, the C-terminus of the H1 domain may include one or more deletions of amino acids. Furthermore, the N-terminus of the fusion peptide domain of HA2 may include one or more deletions of amino acids 1-23 of SEQ ID NO: 41. For example, amino acids 1-11 of SEQ ID NO: 41 may be deleted. Accordingly, modified influenza B virus HA proteins are also provided that comprise a modified HA2 ectodomain, wherein the sequence of the modified HA2 ectodomain comprises amino acids 12-181 of SEQ ID NO: 41, or wherein the sequence of the modified HA2 ectodomain comprises the sequence of SEQ ID NO: 42. Thus, the modified HA2 ectodomain can have an amino acid sequence that shares about 80%, about 82%, about 83%, about 85%, about 87%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, or any amount therebetween, sequence identity or similarity with the amino acid sequence of SEQ ID NO: 42, wherein the sequence includes a non-leucine, such as isoleucine, at position 35. The modified influenza B virus HA protein can comprise an HA2 ectodomain that comprises or consists of the sequence of SEQ ID NO: 42.

[0111] The parental modifications may also include modifications of the transmembrane and cytoplasmic tail domain (TMCT), for example, the native TMCT in the parental sequence may be replaced with the TMCT of an influenza HA that is different from the parental HA.

[0112] Thus, the modified B HA protein may contain further modifications, such as deletions or substitutions, compared to wild-type B HA. For example, the proteolytic cleavage site may be deleted or modified in the modified B HA protein to prevent proteolytic cleavage of the HA precursor into HA1 and HA2 subunits. The cleavage site is a prominent surface loop in the influenza HA protein and can be determined, for example, by sequence alignment or structural analysis of the HA protein (see, e.g., Bertram et al., Reviews in Medical Virology, Volume 20, September 2010). Influenza HA proteins containing modified proteolytic cleavage sites and methods for producing influenza HA proteins containing modified proteolytic cleavage sites are described, for example, in PCT Publication Nos. WO 2013 / 044390 and WO 2014 / 153674, the contents of which are incorporated herein by reference.

[0113] Furthermore, the native transmembrane and cytoplasmic tail domain (TMCT) of influenza B HA can be replaced with the TMCT of influenza A HA. Thus, the modified B HA can contain a non-native TMCT. For example, the modified B HA can have the native TMCT replaced with the TMCT of influenza H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. In a preferred embodiment, the TMCT in the modified B HA is the non-native TMCT of influenza HA H1 or H5. TMCT substitutions in influenza HA are described, for example, in PCT Application WO 2010 / 148511, the contents of which are incorporated herein by reference.

[0114] The modified influenza B HA may include a signal peptide that directs localization when expressed in a host or host cell. The signal peptide may be the native (for the protein) signal or leader sequence or a heterologous signal sequence.

[0115] Thus, as described herein, modified influenza B proteins can be produced as precursor proteins that include a modified influenza B protein and a heterologous amino acid signal peptide sequence. For example, a modified influenza B protein precursor can include the signal peptide of protein disulfide isomerase (PDI SP; nucleotides 32-103 of Accession No. Z11499).

[0116] The modified influenza B HA protein described herein can further be incorporated into a virus-like particle (VLP). The term "virus-like particle" (VLP) or "virus-like particle" or "VLP" refers to a virus-like structure that is generally morphologically and antigenically similar to the virions produced in an infection, but lacks sufficient genetic information for replication and is therefore non-infectious. VLPs are structures that self-assemble and comprise one or more structural proteins, such as a modified influenza B HA protein. Thus, the VLP can comprise a modified influenza B HA protein. The VLP can further comprise an influenza virus protein, where the influenza virus protein consists of a modified influenza B HA protein.

[0117] VLPs can be produced in suitable hosts or host cells, including plants and plant cells. After extraction from the host or host cells, and upon isolation and further purification under suitable conditions, the VLPs can be recovered as native structures.

[0118] VLPs can be purified or extracted using any suitable method, such as chemical or biochemical extraction. VLPs are relatively sensitive to drying, heat, pH, surfactants, and detergents. Therefore, it may be useful to maximize production yield, minimize contamination of the VLP fraction with cellular proteins, maintain the integrity of the protein or VLP and, if necessary, the associated lipid envelope or membrane, and use methods that loosen the cell wall to release the protein or VLP. For example, minimizing or eliminating the use of detergents or surfactants, such as SDS or Triton™ X-100, may be beneficial to improve the yield of VLP extraction. VLPs can then be evaluated for structure and size, for example, by electron microscopy or size exclusion chromatography.

[0119] For enveloped viruses, such as influenza viruses, it can be advantageous for the lipid layer or membrane to be retained by the virus. The composition, quality, and quantity of lipids can vary depending on the system (e.g., plant-produced enveloped viruses can contain plant lipids or phytosterols in the envelope), and this can contribute to an improved immune response.

[0120] Without wishing to be bound by theory, plant-made VLPs containing plant-derived lipids may induce a stronger immune response than VLPs produced in other manufacturing systems, and the immune response induced by these plant-made VLPs may be stronger when compared to the immune response induced by live or attenuated whole virus vaccines.

[0121] Furthermore, the ability of plant N-glycans to promote the capture of glycoprotein antigens by antigen-presenting cells, in addition to the potential adjuvant effect of the presence of plant lipids, may be advantageous for the production of VLPs in plants.

[0122] VLPs produced in plants can contain modified influenza B HA proteins that contain plant-specific N-glycans. Accordingly, the present disclosure also provides VLPs that contain modified influenza B HA proteins with plant-specific N-glycans. Furthermore, VLPs are provided that contain plant lipids and modified influenza B HA proteins with plant-specific N-glycans.

[0123] Additionally provided are methods for producing VLPs comprising the above-described modified B HA in a host or host cell, such as a plant.

[0124] The method can include introducing a nucleic acid encoding a modified B HA having a substitution at position 402 (reference strain B / Washington / 09 / 19, numbering corresponding to SEQ ID NO:1) operably linked to a regulatory region active in the host or host cell, and incubating the host or host cell under conditions allowing expression of the nucleic acid, thereby producing VLPs. The method can also include introducing a nucleic acid encoding an influenza B virus HA protein comprising a modified HA2 ectodomain described herein operably linked to a regulatory region active in the host or host cell, and incubating the host or host cell under conditions allowing expression of the nucleic acid, thereby producing VLPs.

[0125] Additionally, there is provided a method for increasing the production of VLPs comprising a modified B HA having the above-described substitution at position 402 (reference strain B / Washington / 09 / 19, numbering corresponding to SEQ ID NO:1) into a host or host cell, which method comprises introducing a nucleic acid encoding a modified B HA having a substitution at position 402 (reference strain B / Washington / 09 / 19, numbering corresponding to SEQ ID NO:1) operably linked to a regulatory region active in the host or host cell, and incubating the host or host cell under conditions allowing expression of the nucleic acid, thereby producing VLPs.

[0126] Also provided is a method for increasing the production of VLPs comprising an influenza B virus HA protein comprising a modified HA2 ectodomain as described above in a host or host cell, comprising introducing a nucleic acid encoding an influenza B virus HA protein comprising a modified HA2 ectodomain operably linked to a regulatory region active in the host or host cell, and incubating the host or host cell under conditions allowing expression of the nucleic acid, thereby producing VLPs.

[0127] The present disclosure further provides VLPs comprising a B HA with a substitution at position 402 as described herein and / or a VLP comprising an influenza B virus HA protein comprising a modified HA2 ectodomain. The VLPs can be produced by the methods provided by this disclosure. VLPs comprising the modified B HA exhibit improved characteristics when compared to VLPs comprising an unmodified B HA protein.

[0128] Also provided herein are methods for increasing the production or yield of VLPs comprising modified influenza B HA in plants. For example, the methods, as described herein, can include introducing a nucleic acid encoding a modified influenza B HA into a plant, plant part, or plant cell. The nucleic acid encoding the modified influenza B HA can be optimized for human codon usage, increased GC content, or a combination thereof. One or more modified influenza B HA proteins can be expressed in the plant, plant part, or plant cell to produce VLPs comprising the one or more modified influenza B HA proteins. Alternatively, the methods can include providing a plant, plant part, or plant cell comprising a nucleic acid encoding a modified influenza B HA protein to produce VLPs comprising one or more modified influenza B HA proteins.

[0129] The method for producing VLPs comprising a modified influenza B HA can further include introducing a second nucleic acid sequence into the plant, plant part, or plant cell, wherein the second nucleic acid encodes a proton channel protein co-expressed with the modified influenza B HA. For example, the proton channel protein can be an influenza A subtype M2 protein, such as A / New Caledonia / 20 / 99 M2. Co-expression of the proton channel protein can result in increased accumulation of the modified influenza B HA protein and / or VLPs comprising the modified influenza HA protein, for example, as described in WO 2013 / 044390, the contents of which are incorporated herein by reference.

[0130] "Co-expression" refers to the introduction and expression of two or more nucleotide sequences, each of which encodes a protein of interest or a fragment of a protein of interest in a plant, plant part, or plant cell. Each of the two or more nucleotide sequences can be introduced into a plant, plant part, or plant cell within a single vector such that they are under the control of separate regulatory regions (e.g., a dual construct). Alternatively, each of the two or more nucleotide sequences can be introduced into a plant, plant part, or plant cell within separate vectors (e.g., comprising a single construct), each vector containing the appropriate regulatory regions for expression of the corresponding nucleic acid. For example, two nucleotide sequences, each on separate vectors and introduced into separate Agrobacterium tumefaciens hosts, can be co-expressed by mixing suspensions of the respective A. tumefaciens hosts in the desired volumes (e.g., equal volumes, or the ratio of each A. tumefaciens host can be varied) prior to vacuum immersion. In this way, co-infection of multiple A. tumefaciens cells allows for simultaneous expression of multiple transgenes.

[0131] The present disclosure further provides a drug substance (DS) comprising the above-described modified influenza B HA protein as a desired product, wherein the drug substance is substantially free of product-related impurities, which impurities are not immunoreactive. Preferred drug substances are also substantially free of process-related impurities.

[0132] Within the context of this application, the term "drug substance" refers to i) an active ingredient of a medicament or formulation, ii) an active pharmaceutical ingredient of a medicament or formulation, iii) a bulk purified active ingredient of a medicament or formulation, or iv) a product or active ingredient that is suitable for use as a bulk purified active ingredient of a medicament or formulation. The medicament or formulation may be a vaccine.

[0133] Thus, further provided is a drug substance (DS) comprising an immunologically active modified influenza B HA protein. Higher yields of DS are obtained from host cells expressing the modified influenza B HA compared to DS obtained from a host expressing an unmodified influenza B HA protein (see Figure 4). Thus, methods for increasing the yield of DS obtained from a host or host cells expressing a modified influenza B HA protein compared to the yield of DS obtained from a host expressing the unmodified (parent) S protein are also provided.

[0134] Accordingly, there is further provided a drug substance (DS) comprising an immunologically active modified influenza B HA protein.

[0135] The modified influenza B HA protein can self-assemble into a virus-like particle (VLP). Accordingly, a DS comprising a VLP comprising the modified influenza B HA protein is also provided.

[0136] In a further aspect, formulations (also referred to as pharmaceutical formulations or pharmaceutical compositions) are also provided. The formulations may be formulated as a final dosage form, for example, a solution, a capsule, or a tablet. The formulations include a drug substance. The formulations may further include other components, such as a pharmaceutically acceptable carrier and / or excipients, such as a buffer system, an adjuvant, a preservative, one or more tonicity agents, one or more chelating agents, an antiadherent agent, a vehicle, etc. Pharmaceutically acceptable carriers and excipients are well known within the art. Thus, also provided are formulations, pharmaceutical formulations, or pharmaceutical compositions comprising a pharmaceutically acceptable carrier and / or excipient and a VLP, wherein the VLP comprises a modified influenza B HA protein, or wherein the VLP comprises a viral protein and the viral protein consists of the modified influenza B HA protein.

[0137] When one or more modified influenza B HA proteins are expressed in a host or host cells, the one or more modified influenza B HA proteins self-assemble into VLPs. The host or host cells can be harvested under suitable extraction and purification conditions to maintain the integrity of the VLPs, and VLPs comprising one or more mutant influenza HAs can be purified.

[0138] The present disclosure also provides use of the modified influenza B HA, VLP comprising the modified influenza B HA, or DS described herein for inducing an immune response in a subject or for inducing immunity to influenza infection in a subject. Also disclosed herein is an antibody or antibody fragment purified by administering the modified influenza B HA, VLP comprising the modified influenza B HA, or DS to a subject or host animal. Further provided is a composition comprising an effective amount of the modified influenza B HA, VLP comprising the modified influenza B HA, or DS described herein, and a pharmaceutically acceptable carrier, adjuvant, vehicle, or excipient for inducing an immune response in a subject. Also provided is a vaccine for inducing an immune response in a subject, the vaccine comprising an effective amount of the modified influenza B HA.

[0139] Further provided are compositions comprising an effective amount of a modified influenza B HA protein, a VLP or a DS comprising the modified influenza B HA protein, and a pharmaceutically acceptable carrier, adjuvant, vehicle or excipient, as described herein, for inducing an immune response in a subject. Also provided are vaccines for inducing an immune response against influenza in a subject, the vaccines comprising an effective amount of a modified influenza B HA, a VLP or a DS comprising the modified influenza B HA protein.

[0140] A composition or vaccine can include VLPs comprising an influenza HA protein, where the HA protein is from the same influenza type, subtype, lineage, subgenus, or strain, or the composition or vaccine can include multiple VLP types, each VLP type comprising an HA protein, where the HA protein may be from a different influenza type, subtype, lineage, subgenus, or strain. That is, a composition or vaccine can include a mixture of different influenza VLPs. For example, a composition or vaccine can include a second VLP comprising a first influenza HA protein of a first influenza subtype, lineage, or strain and a second influenza HA protein of a second influenza subtype, lineage, or strain. Furthermore, a composition can also include a third VLP comprising a third influenza HA protein of a third influenza subtype, lineage, or strain, and / or a fourth VLP comprising a fourth influenza HA protein of a fourth influenza subtype, lineage, subgenus, or strain.

[0141] The composition or vaccine may further comprise a VLP comprising HA proteins of two or more HA subtypes, strains, or strains. For example, the VLP may comprise a first modified B HA protein and a second HA protein of a first B HA strain or strain, where the second HA protein is derived from the HA of a second B strain or strain, or where the second HA is derived from the HA of an influenza A subtype or strain. Furthermore, the VLP may comprise a third HA protein, where the third HA is derived from a third B strain or strain, or where the third HA is derived from the HA of an influenza A subtype or strain, and / or the VLP may comprise a fourth HA, where the fourth HA is derived from a fourth B strain or strain, or where the fourth HA is derived from the HA of an influenza A subtype or strain.

[0142] Accordingly, this description also provides for compositions or vaccines that are monovalent (univalent) or multivalent (polyvalent). A monovalent composition or vaccine can immunize a subject against a single influenza strain, while a multivalent composition or vaccine can immunize a subject against two or more influenza strains. For example, a composition or vaccine can be a bivalent composition or vaccine, but upon administration, immunize a subject against two different types of influenza family, subgroup, type, subtype, lineage, or strain. Furthermore, a composition or vaccine can be a trivalent composition, or the vaccine or composition can be a tetravalent or quadrivalent composition or vaccine. Furthermore, a vaccine can also be multivalent against different types of viruses. For example, a vaccine can immunize a subject against one or more influenza strains (a first type of virus) and against a second type of virus (e.g., a coronavirus).

[0143] Thus, multivalent immunogenic compositions comprising two or more types of VLPs are also provided, wherein at least one type of VLP (a first type of VLP) comprises a modified B HA protein (a first modified B HA) as described herein. The multivalent immunogenic composition may further comprise a second type of VLP that also comprises a modified B HA protein (a second modified B HA) as described herein, wherein the first type of VLP and the second type of VLP comprise modified B HA proteins derived from different influenza B viruses. For example, the first VLP and the second VLP may each comprise a modified B HA protein belonging to a different influenza B lineage. The multivalent immunogenic composition may further comprise one or more types of VLPs comprising an influenza A HA protein. For example, the influenza A HA may be derived from influenza subtype H1 and / or influenza subtype H3.

[0144] Also provided are tetravalent immunogenic compositions comprising a first type of VLPs comprising a modified influenza B HA described herein, a second type of VLPs comprising a modified influenza B HA described herein, a third type of VLPs comprising an influenza A HA, and a fourth type of VLPs comprising an influenza A HA, wherein the first type of VLPs comprises a modified B HA derived from a different influenza B strain than the modified B HA of the second type of VLPs. For example, the modified B HA in the first type of VLPs can be derived from the B / Victoria strain, and the modified B HA in the second type of VLPs can be derived from the B / Yamagata strain. Furthermore, the influenza A HA in the third type of VLPs can be derived from a different influenza A subtype than the influenza A HA of the fourth type of VLPs. For example, the influenza A HA in the third type of VLPs can be derived from influenza H3, and the influenza A HA in the fourth type of VLPs can be derived from influenza H1.

[0145] The monovalent or multivalent composition or vaccine may further comprise a pharmaceutically acceptable carrier, adjuvant, vehicle or excipient for eliciting an immune response in a subject.

[0146] Adjuvant systems for enhancing a subject's immune response to vaccine antigens are well known and can be used with the vaccines or pharmaceutical compositions described herein. There are many types of adjuvants that can be used. Common adjuvants for human use are aluminum hydroxide, aluminum phosphate, and calcium phosphate. There are also several adjuvants based on oil emulsions (oil-in-water or water-in-oil emulsions such as Freund's incomplete adjuvant (FIA), Montanide™, Adjuvant 65, and Lipovant™), bacterial products (or their synthetic derivatives), endotoxins, fatty acids, paraffin or vegetable oils, cholesterol and fatty amines, or natural organic compounds such as squalene. Non-limiting adjuvants that may be used include, for example, an oil-in-water emulsion of squalene oil (e.g., MF-59 or AS03), an adjuvant composed of a synthetic TLR4 to glucopyranosyl lipid A (GLA) (GLA-SE) incorporated into a stable emulsion (SE), or the toll-like receptor (TLR9) agonist adjuvant CpG1018.

[0147] Thus, the vaccine or pharmaceutical composition may include one or more adjuvants, for example, aluminum hydroxide, aluminum phosphate, calcium phosphate, an oil-in-water or water-in-oil emulsion, an emulsion containing squalene (e.g., MF-59 or AS03), an emulsion containing GLA-SE, or CpG1018.

[0148] Also provided herein are methods for inducing an immune response in a subject or for inducing immunity to influenza infection, comprising administering a modified influenza B HA or a VLP comprising a modified influenza B HA to a subject orally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.

[0149] Influenza B HA proteins or modified influenza B HA proteins as disclosed herein include any known HA protein from any known influenza B strain, but also include modifications to known influenza B strains that have developed over time. For example, the influenza HA can be from B / Washington / 02 / 2019 (EPI 1368874), B / Singapore / INFKK-16-0569 / 2016 (EPI 592707), B / Rhode Island / 01 / 2019 (EPI 1383242), B / Michigan / 01 / 2021 (EPI 1843974), B / Henan-Xigong / 1118 / 2021 (EPI 1878454), B / Austria / 1359417 / 2021 (EPI 1845793), or B / Singapore / WUH4618 / 2021 (EPI 1883660). Influenza B HA includes HA from a strain, wherein the HA has about 30-100%, or any amount therebetween, amino acid sequence identity to any HA from an influenza B strain listed above, where the influenza HA protein comprises at least one substitution described herein, is capable of being formed into a VLP, and upon administration to a subject, elicits an immune response, elicits hemagglutination, or a combination thereof.

[0150] For example, an influenza HA protein can comprise 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 of amino acid sequence identity (sequence similarity, percent identity, percent similarity) to any HA from an influenza B strain listed above, contain at least one substitution as described herein, be capable of being formed into a VLP, and elicit an immune response, elicit hemagglutination, or a combination thereof, when administered to a subject.

[0151] The terms "percent similarity," "sequence similarity," "percent identity," or "sequence identity," when referring to particular sequences, are used, for example, as described in the University of Wisconsin GCG software program, or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 supplement). Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, using the algorithm of Smith & Waterman, (1981) Adv. Appl. Math. 2:482, by the alignment algorithm of Needleman & Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444, or by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.).

[0152] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977, Nuc. Acids Res. 25:3389-3402) and Altschul et al. (1990, J. Mol. Biol. 215:403-410), respectively. BLAST and BLAST 2.0 are used to determine percent sequence identity for the nucleic acids and proteins of the invention using the parameters described herein. For example, the BLASTN program (for nucleotide sequences) can use as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. For amino acid sequences, the BLASTP program may use as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89:10915) with an alignment (B) of 50, an expectation (E) of 10, M=5, N=4, and a comparison of both strands. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see URL: ncbi.nlm.nih.gov / ).

[0153] Influenza B HA proteins include wt HA B / Singapore / INFKK-16-0569 / 2016 (EPI592707) (SEQ ID NO: 2), wt HA B / Washington / 02 / 2019 (EPI1368874) (SEQ ID NO: 1), wt HA B / Rhode Island / 01 / 2019 (EPI1383242) (SEQ ID NO: 3), wt HA B / Michigan / 01 / 2021 (EPI1843974) (SEQ ID NO: 4), wt HA B / Henan-Xigong / 1118 / 2021 (EPI1878454) (SEQ ID NO: 5), wt HA The influenza B HA sequences of B / Austria / 1359417 / 2021 (EPI1845793) (SEQ ID NO: 6), and wt HA B / Singapore / WUH4618 / 2021 (EPI1883660) (SEQ ID NO: 7) include any HA protein comprising an amino acid sequence having 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%, or about 97 to about 100%, about 98 to about 100%, or any amount therebetween.

[0154] Furthermore, when the modified influenza HA protein contains at least one substitution as described herein, is capable of forming a VLP, elicits an immune response upon administration to a subject, induces hemagglutination, or a combination thereof, it includes any HA protein comprising an amino acid sequence having 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, with the sequence of SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO: 37.

[0155] As described herein, one or more specific mutations or modifications in influenza B HA result in increased accumulation of HA protein and increased VLP production in plants when compared to unmodified influenza HA.

[0156] Examples of modified influenza B HA proteins with enhanced influenza HA and / or VLP production in plants include the following: L404I B / Singapore / INFKK-16-0569 / 2016 mutant HA (construct #8894, SEQ ID NO: 13), L402I B / Washington / 02 / 2019 mutant HA (construct #8881, SEQ ID NO: 17), L402I B / Rhode Island / 01 / 2019 mutant HA (construct #7787, SEQ ID NO: 21), L402I B / Michigan / 01 / 2021 mutant HA (construct #9628, SEQ ID NO: 25), L402I B / Henan-Xigong / 1118 / 2021 mutant HA (construct #9630, SEQ ID NO: 29), L402I B / Singapore / WUH4618 / 2021 mutant HA (construct #9867, SEQ ID NO: 33) and L402I B / Austria / 1359417 / 2021 mutant HA (construct #9869, SEQ ID NO: 37),

[0157] One or more modified genetic constructs comprising the modified B HA proteins herein can be expressed in any suitable host or host cell transformed with a nucleic acid, or nucleotide sequence, or construct, or vector of the present disclosure. The host or host cell can be from any source, including plants, fungi, bacteria, insects, and animals, such as mammals. Thus, the host or host cell can be selected from a plant or plant cell, a fungus or fungal cell, a bacterium or bacterial cell, an insect or insect cell, and an animal or animal cell. The mammal or animal need not be human. Thus, the host or host cell can be a non-human host or host cell. In a preferred embodiment, the host or host cell is a plant, plant part, or plant cell.

[0158] The terms "plant," "plant part," "plant part," "plant matter," "plant biomass," "plant material," "plant extract," or "plant leaf," as used herein, can include a whole plant, tissue, cell, or any part thereof, intracellular plant components, extracellular plant components, liquid or solid extracts of a plant, or combinations thereof, which are capable of providing the transcriptional, translational, and post-translational machinery for expressing one or more nucleic acids described herein and / or from which the expressed proteins or VLPs can be extracted and purified. Plants can include, but are not limited to, herbaceous plants. Furthermore, plants may include, but are not limited to, crops including, for example, canola, rapeseed, corn, Nicotiana (tobacco), e.g., Nicotiana benthamiana, Nicotiana rustica, Nicotiana tabacum, Nicotiana alata, Arabidopsis thaliana, alfalfa, potato, sweet potato (Ipomoea batatus), carrot, pea, oat, rice, soybean, wheat, barley, sunflower, cotton, maize, rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), safflower (Carthamus tinctorius).

[0159] As used herein, the term "plant part" refers to any part of a plant, including, but not limited to, leaves, stems, roots, flowers, fruits, plant cells obtained from leaves, stems, roots, flowers, fruits, plant extracts obtained from leaves, stems, roots, flowers, fruits, or combinations thereof. As used herein, the term "plant extract" refers to a plant-derived product obtained after treating a plant, plant part, plant cell, or combinations thereof physically (e.g., by freezing followed by extraction in a suitable buffer), mechanically (e.g., by grinding or homogenizing the plant or plant part followed by extraction in a suitable buffer), enzymatically (e.g., using cell wall-degrading enzymes), chemically (e.g., one or more chelating agents or buffers), or a combination thereof. The plant extract may be further processed to remove undesirable plant components, such as cell wall debris. A plant extract can be obtained to facilitate 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 a combination thereof, from a plant, plant part, or plant cell. When a plant extract contains proteins, it can be referred to as a protein extract. The protein extract can be a crude plant extract, a partially purified plant or protein extract, or a purified product containing one or more proteins, protein complexes, protein superstructures, and / or VLPs from plant tissue. If desired, the protein extract or plant extract can be partially purified using techniques known to those skilled in the art. For example, the extract can 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. The protein extract can also be purified using techniques known to those skilled in the art.

[0160] The nucleic acid encoding the modified influenza B HA described herein can further comprise a sequence that enhances expression of the modified influenza B HA in a plant, plant part, or plant cell. Expression-enhancing sequences include, for example, a plant-derived expression enhancer or a plant virus-derived expression enhancer. The expression enhancer can be operably linked to the nucleic acid encoding the modified influenza hemagglutinin (HA) protein. The modified influenza hemagglutinin (HA)-encoding sequence can also be optimized for human codon usage, increased GC content, or a combination thereof.

[0161] As used herein, the term "plant-derived expression enhancer" refers to a nucleotide sequence obtained from a plant, the nucleotide sequence encoding a 5'UTR. Examples of plant-derived expression enhancers are described in WO 2019 / 173924 or WO 2020 / 181354. The plant-derived expression enhancer can be used in a plant expression system comprising a plant-derived expression enhancer sequence and a regulatory region operably linked to a nucleotide sequence of interest.

[0162] Furthermore, expression-enhancing sequences may include expression enhancers derived from plant viruses, such as the cowpea mosaic virus (CPMV) enhancer element.

[0163] As used herein, the term "CPMV enhancer element" refers to a nucleotide sequence encoding a 5' UTR that controls the Cowpea Mosaic Virus (CPMV) RNA2 polypeptide or a modified CPMV sequence as known in the art. For example, a CPMV enhancer element or CPMV expression enhancer includes a nucleotide sequence as described in WO 2015 / 14367, WO 2015 / 103704, WO 2007 / 135480, WO 2009 / 087391, Sainsbury F., and Lomonossoff GP, (2008, Plant Physiol. 148:1212-1218), each of which is incorporated herein by reference. CPMV enhancer sequences can enhance expression of downstream heterologous open reading frames (ORFs) to which they are linked. The CPMV expression enhancer can include CPMV HT, CPMVX (where X=160, 155, 150, 114), e.g., CPMV160, CPMVX+ (where X=160, 155, 150, 114), e.g., CPMV160+, CPMV-HT+, CPMV HT+[WT115], or CPMV HT+

[0511] (WO 2015 / 143567, WO 2015 / 103704, the contents of which are incorporated herein by reference). In a preferred embodiment, the CPMV expression enhancer is CPMV160. The CPMV expression enhancer can be used in a plant expression system comprising a regulatory region operably linked to a CPMV expression enhancer sequence and a nucleotide sequence of interest, e.g., a nucleotide sequence encoding a modified B HA of the present disclosure.

[0164] "Operably linked" means that subsequences interact, either directly or indirectly, to perform their intended function, such as mediating or regulating the expression of a nucleic acid sequence. The interaction of operably linked sequences can be mediated, for example, by proteins that interact with the operably linked sequences.

[0165] As used herein, the terms "construct," "vector," or "expression vector" refer to a recombinant nucleic acid for introducing an exogenous nucleic acid sequence into a host cell (e.g., a plant cell) and directing expression of the exogenous nucleic acid sequence in the host cell. An "expression cassette" refers to a nucleotide sequence containing a nucleic acid of interest operably linked to and under the control of an appropriate promoter or other regulatory elements for transcription of the nucleic acid in the host cell. As one skilled in the art will appreciate, an expression cassette can include a termination (terminator) sequence, which can be any sequence active in a plant host. For example, the termination sequence can be derived from the RNA-2 genome segment of a segmented RNA virus, such as a comovirus; the termination sequence can be a NOS terminator; or the terminator sequence can be obtained from the 3'UTR of the alfalfa plastocyanin gene.

[0166] The constructs of the present disclosure may further comprise a 3' untranslated region (UTR). The 3' untranslated region contains a polyadenylation signal and other regulatory signals capable of inducing mRNA processing or gene expression. Polyadenylation signals are typically characterized by the addition of a polyadenylic acid track to the 3' end of a pre-mRNA. Polyadenylation signals are generally recognized by their homology to the canonical form 5'AATAAA-3', although variations commonly occur. Non-limiting examples of suitable 3' regions are Agrobacterium tumor-inducing (Ti) plasmid genes such as nopaline synthase (Nos gene) and plant genes such as soybean storage protein genes, the small subunit of ribulose-1, 5-bisphosphate carboxylase gene (ssRUBISCO, U.S. Pat. No. 4,962,028, the contents of which are incorporated herein by reference), and the 3' transcribed, untranslated region containing the polyadenylation signal of promoters used in regulating plastocyanin expression.

[0167] "Regulatory region," "regulatory element," or "promoter" typically refers to a portion of nucleic acid, although not necessarily, upstream of the protein-coding region of a gene, which may be composed of either DNA or RNA, or both. When a regulatory region is active and operably associated with or operably linked to a nucleotide sequence of interest, it can cause expression of the nucleotide sequence of interest. Regulatory elements may be capable of mediating organ specificity or regulating developmental or chronological gene activation. "Regulatory region" includes elements that mediate promoter activity, such as promoter elements, core promoter elements exhibiting basal promoter activity, sequences inducible in response to external stimuli, negative regulatory elements, or transcriptional enhancers. As used herein, "regulatory region" also includes regulatory elements that regulate gene expression after transcription, such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activating sequences, and mRNA instability determinants. Some of these latter elements may be located proximal to the coding region.

[0168] In the context of this disclosure, the term "regulatory element" or "regulatory region" typically refers to a sequence of DNA, usually, though not necessarily, upstream (5') to the coding sequence of a structural gene, that controls expression of the coding region by providing recognition for RNA polymerase and / or other factors required for transcription to initiate at a specific site. However, it should be understood that other nucleotide sequences located within introns, or sequences 3', also contribute to regulating expression of a coding region of interest. One example of a regulatory element that recognizes RNA polymerase or other transcription factors to ensure initiation at a specific site is a promoter element. Most, but not all, eukaryotic promoter elements contain a TATA box, a conserved nucleic acid sequence composed of adenosine and thymidine nucleotide base pairs usually located approximately 25 bases upstream of the transcription start site. A promoter element can include a basal promoter element, responsible for transcription initiation, and other regulatory elements that modify gene expression.

[0169] There are several types of regulatory regions, including developmentally regulated, inducible, or constitutively expressed. Developmentally regulated regulatory regions, or the differential expression of genes under their control, are activated at specific times during organ or tissue development within a specific period or tissue of an organ. However, some developmentally regulated regulatory regions may be preferentially active within a specific organ or tissue at a specific developmental stage; they may also be active in a developmentally regulated manner or at a basal level in other organs or tissues within the plant. Examples of tissue-specific, e.g., seed-specific, regulatory regions include the napin promoter and the cruciferin promoter (Rask et al., 1998, J. Plant Physiol. 152:595-599; Bilodeau et al., 1994, Plant Cell 14:125-130). An example of a leaf-specific promoter is the plastocyanin promoter (U.S. Pat. No. 7,125,978, the contents of which are incorporated herein by reference).

[0170] An inducible regulatory region is one that can directly or indirectly activate transcription of one or more DNA sequences or genes in response to an inducer. In the absence of the inducer, the DNA sequence or gene is not transcribed. Typically, the protein factor that specifically binds to the inducible regulatory region to activate transcription can exist in an inactive form, which is subsequently converted directly or indirectly to an active form by the inducer. However, the protein factor may also be absent. The inducer can be a protein, a metabolite, a growth regulator, a chemical such as a herbicide or a phenolic compound, or a physiological stress imposed directly by heat, cold, salt, or a toxic element, or indirectly through the action of a pathogen or disease agent such as a virus. Plant cells containing an inducible regulatory region can be exposed to an inducer by externally applying the inducer to the cells or plant, such as by spraying, irrigation, heat, or similar methods. Inducible regulatory elements can be derived from either plant or non-plant genes (see, for example, Gatz, C. and Lenk, IRP, 1998, Trends Plant Sci. 3, 352-358). Examples of potential inducible promoters include 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, NH, 1997, Plant J. 2, 397-404), and the ethanol-inducible promoter (Salter, MG, et al., 1998, Plant Journal 16, 127-132; Caddick, MX, et al., 1998, Nature Biotech. 16, 177-180), the cytokinin-inducible IB6 and CKI1 genes (Brandstatter, I. and Kieber, JJ, 1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274, 982-985) and the auxin-inducible element DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971).

[0171] Constitutive regulatory regions direct the expression of genes throughout various parts of a plant and sequentially throughout plant development. Examples of known constitutive regulatory elements include the CaMV35S transcript (p35S; Odell et al., 1985, Nature, 313:810-812, the contents of which are incorporated herein by reference), rice actin 1 (Zhang et al., 1991, Plant Cell, 3:1155-1165), actin 2 (An et al., 1996, Plant J., 10:107-121) or tms2 (U.S. Pat. No. 5,428,147), and triosephosphate isomerase 1 (Xu et al., 1994, Plant Physiol. 106:459-467) genes, maize ubiquitin 1 gene (Cornejo et al., 1993, Plant J. Mol. Biol. 29:637-646), Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al., 1995, Plant Mol. Biol. 29:637-646), tobacco translation initiation factor 4A gene (Mandel et al., 1995 Plant Mol. Biol. 29:995-1004), the cassava vein mosaic virus promoter pCAS (Verdaguer et al., 1996), the small subunit of ribulose bisphosphate carboxylase pRbcS (Outchkourov et al., 2003), and promoters associated with pUbi (monocots and dicots).

[0172] The term "constitutive expression," as used herein, does not necessarily indicate that a nucleotide sequence under the control of a constitutive expression regulatory region is expressed at the same level in all cell types, although variations in abundance are often observed.

[0173] The above expression constructs can be present in a vector. The vector can include border sequences that allow the expression cassette to be introduced and integrated into the genome of an organism or host. The construct can be a plant binary transformation vector, such as a pPZP-based binary transformation vector (Hajdukiewicz, et al. 1994). Other exemplary constructs include pBin19 (see Frisch, DA, LW Harris-Haller, et al. 1995, Plant Molecular Biology 27:405-409).

[0174] The constructs of the present disclosure can be introduced into plant cells using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of these techniques, see, e.g., Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geison and Corey, Plant Molecular Biology, 2nd Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants In Plant Metabolism, 2nd Ed. D.T. Dennis, D.H. Turpin, D.D. Lefebvre, D.B. Layzell (eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). Other methods include direct DNA uptake, the use of liposomes, electroporation, eg, the use of protoplasts, microinjection, microprojectiles or whiskers, and vacuum immersion.For example, Bilang, et al. (1991, Gene100:247-250), Scheid et al. (1991, Mol. Gen. Genet.228:104-112), Guerche et al. (1987, Plant Science 52:111-116), Neuhause et al. (1987, Theor. Appl. Genet.75:30-36), Klein et al. (2987, Nature 327:70-73); Freeman et al. (1984, Plant Cell Physiol.29:1353), Howell et al. (1985, Science 227:1229-1231), DeBlock et al. (1989, Plant Physiology) 91:694-701), Methods for Plant Molecular Biology (Weissbach and Weissbach, eds., Academic Press Inc., 1988), Methods in Plant Molecular Biology (Schuler and Zielinski, eds., Academic Press Inc., 1989), International Publication No. 92 / 09696, International Publication No. 94 / 00583, European Patent No. 331083, European Patent No. 175966, Liu and Lomonossoff(2002, J Virol Meth., 105:343-348), European Patent No. 290395; International Publication No. 8706614; U.S. Patent Nos. 4,945,050; 5,036,006; and 5,100,792; U.S. Patent Application Publication No. 08 / 438,666, filed May 10, 1995; and U.S. Patent Application Publication No. 07 / 951,715, filed September 25, 1992, the contents of which are all incorporated herein by reference.

[0175] To express the constructs of the present disclosure, transient expression methods can be used (see D'Aoust et al., 2009, Methods in molecular biology, Vol. 483, pages 41-50; Liu and Lomonossoff, 2002, Journal of Virological Methods, 105:343-348, the contents of which are incorporated herein by reference). Alternatively, vacuum-based transient expression methods can be used, as described in Kapila et al. (1997, Plant Sci. 122, 101-108, the contents of which are incorporated herein by reference), or in WO 00 / 063400 and WO 00 / 037663 (incorporated herein by reference). These methods can include, but are not limited to, agroinoculation or agroinfiltration, and syringe infiltration. However, other transient methods can also be used, as described above. With agroinoculation, agroinfiltration, or syringe infiltration, a mixture of Agrobacteria containing the desired nucleic acid enters the intercellular spaces of tissues, such as leaves, the above-ground parts of the plant (including stems, leaves, and flowers), other parts of the plant (stems, roots, flowers), or the entire plant. After crossing the epidermis, the Agrobacteria infect and transfer t-DNA copies into cells. The t-DNA is episomally transcribed and the mRNA is translated, thereby resulting in the production of the protein of interest in the infected cell, but the passage of the t-DNA inside the nucleus is transient.

[0176] Transgenic plants, plant cells, or seeds containing the genetic constructs of the present disclosure, which may be used as suitable platform plants for the transient expression of proteins described herein, are also considered part of the present disclosure. Methods for regenerating whole plants from plant cells are also known in the art (see, for example, Guerineau and Mullineaux (1993, Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148). Generally, transformed plant cells are cultured on an appropriate medium containing a selection agent, such as an antibiotic, where a selection marker is used to facilitate identification of transformed plant cells. Once callus is formed, shoot formation can be promoted using appropriate plant hormones according to known methods, and the shoots are transferred to rooting medium for plant regeneration. The plants can then be used to establish repeated generations from seed or using vegetative propagation techniques. Transgenic plants can also be generated without the use of tissue culture. Methods for stable transformation and regeneration of these organisms are established in the art and known to those skilled in the art. Available techniques are described in detail in Vasil et al. (Cell Culture and Somatic Cell Genetics of Plants, Vol. I, II and III, Laboratory Procedures and Plant Molecular Biology and Its Applications, Academic Press, 1984) and Weissbach and Weissbach (Methods for Plant Molecular Biology, Academic Press, 1989). The method for obtaining transformed and regenerated plants is not critical to the present invention.

[0177] When 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 Agrobacterium in a single transfection event, such that the nucleic acids are pooled and the bacterial cells are transfected. Alternatively, the constructs can be introduced sequentially. In this case, a first construct is introduced into Agrobacterium as described, and the cells are grown under selective conditions (e.g., in the presence of an antibiotic) that allow only singly transformed bacteria to grow. After this first selection step, a second nucleic acid construct is introduced into Agrobacterium as described, and the cells are grown under double selective conditions that allow only doubly transformed bacteria to grow. The doubly transformed bacteria can then be used to transform plants, plant parts, or plant cells as described herein, or can be subjected to a further transformation step to accommodate a third nucleic acid construct.

[0178] Alternatively, if a plant, plant part, or plant cell is to be transformed or co-transformed with more than one nucleic acid construct, the nucleic acid constructs are introduced into the plant by co-soaking a mixture of Agrobacterium cells and the plant, plant part, or plant cell, each Agrobacterium cell containing one or more constructs to be introduced into the plant. During the soaking step, the concentrations of the different Agrobacteria populations containing the desired constructs can be varied to vary the relative expression levels within the plant, plant part, or plant cell of the nucleotide sequences of interest within the constructs. [Table 3-1] [Table 3-2] [Example]

[0179] The invention is further illustrated in the following examples.

[0180] Example 1: Influenza HA construct Influenza HA constructs were produced using techniques well known in the art. For example, wild-type B / Singapore / INFKK-16-0569 / 2016 was cloned as shown below. Other modified influenza B HAs were obtained using similar techniques, with the HA sequence primers, templates, and products shown in Example 3 (Influenza HA and VLP production in plants) and Table 5.

[0181] A summary of the parental (unmodified) and modified HA proteins, primers, templates and products is provided in Table 5 below. Influenza B HA of different strains carrying M2 in the 2X35S-CPMV 160-NOS term (construct numbers 2879, 8894, 7679, 8881, 8424, 7787, 9627, 9628, 9629, 9630, 9866, 9867, 9868, and 9869) The sequence encoding the HAO from influenza HA of B / Singapore / INFKK-16-0569 / 2016, in which the native signal peptide was replaced by the signal peptide of alfalfa protein disulfide isomerase (PDISP / HA B / Singapore / INFKK-16-0569 / 2016), was cloned into the 2X35S / CPMV160 / NOS expression system (CPMV160) with M2 from influenza strain A / New / Caledonia / 20 / 1999 using the following PCR-based method: A fragment containing the PDISP / HA B / Singapore / INFKK-16-0569 / 2016 coding sequence was amplified using primers IF-SpPDI.c (SEQ ID NO:8) and IF-H1cTMCT.s1-4r (SEQ ID NO:9) and the PDISP / HA B / Singapore / INFKK-16-0569 / 2016 sequence (SEQ ID NO:10) as a template. The PCR product was cloned into the 2X35S / CPMV160 / NOS expression system using the In-Fusion cloning system (Clontech, Mountain View, CA). Construct number 4498 (Figure 2A) was digested with SacII and StuI restriction enzymes, and the linearized plasmid was used for the In-Fusion assembly reaction. Construct number 4498 is an acceptor plasmid intended for "In-Fusion" cloning of a gene of interest into a 2X35S / CPMV160 / NOS-based expression cassette. It also introduces gene constructs for co-expression of a silenced TBSV P19 suppressor under the alfalfa plastocyanin gene promoter and terminator, as well as for co-expression of influenza strain A / New / Caledonia / 20 / 1999 M2, which uses the same promoter and terminator. The backbone is the pCAMBIA binary plasmid, and the sequences of the t-DNA borders from left to right are shown in Figure 2A (SEQ ID NO: 38). The resulting construct is designated number 2879 (SEQ ID NO: 39) and a representation of plasmid 2879 is shown in Figure 2B. The amino acid sequence of mature HA0 from influenza HA of B / Singapore / INFKK-16-0569 / 2016 fused to PDISP is shown in sequence SEQ ID NO: 11.The introduction of modifications into the B HA protein is described in Example 3.

[0182] Example 2: Method Agrobacterium tumefaciens gene transfer Agrobacterium tumefaciens strain AGL1 was transfected by electroporation with parental (unmodified) influenza HA or mutant influenza HA expression vectors using the method described by D'Aoust et al., 2008 (Plant Biotech. J. 6:930-40). Transfected Agrobacterium were grown in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 μM acetosyringone, 50 μg / mL kanamycin, and 25 μg / mL carbenicillin (pH 5.6) until an OD of 0.6–1.6 was reached. 600 The Agrobacterium suspension was centrifuged before use and resuspended in soaking medium (10 mM MgCl2 and 10 mM MES, pH 5.6).

[0183] Plant biomass, inoculants and agroinfiltration N. benthamiana plants were grown from seeds in flats filled with commercially available peat moss substrate. Plants were grown in a greenhouse under a 16 / 8 photoperiod and a temperature regime of 25°C day / 20°C night. Three weeks after sowing, plants were individually removed, repotted, and grown in the greenhouse under the same environmental conditions for an additional three weeks.

[0184] Agrobacteria transfected with the parental influenza HA or mutant influenza HA expression vectors, respectively, were grown at an OD between 0.6 and 1.6. 600Agrobacterium was grown in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 μM acetosyringone, 50 μg / mL kanamycin, and 25 μg / mL carbenicillin (pH 5.6) until a bacterial colony was reached. The Agrobacterium suspension was centrifuged before use, resuspended in soaking medium (10 mM MgCl2 and 10 mM MES (pH 5.6)), and stored overnight at 4°C. On the day of soaking, the culture batch was diluted to 2.5 times the culture volume and warmed before use. Whole N. benthamiana plants were placed upside down in the bacterial suspension in a sealed stainless steel tank under a vacuum of 20–40 Torr for 2 minutes. The plants were returned to the greenhouse for a 6- or 9-day incubation period before harvest.

[0185] Leaf harvest and total protein extraction Use an orbital shaker to shake the sample about 1 cm 2 Proteins were extracted from freshly cut biomass by enzymatic extraction overnight at room temperature, and the slurry was then filtered through a large-pore nylon filter to remove coarse undigested plant tissue.

[0186] In planta production was assessed in sorted crude extracts and analyzed using capillary-based electrophoresis (Protein Simple, BioTechne) technology and a WES analysis system. Briefly, soluble proteins from the crude extracts were separated by molecular weight in a capillary and immobilized on a matrix. An anti-HA antibody (Novus biological, catalog number NB100-56578) was used for detection according to the manufacturer's instructions. Fold changes in production were measured to assess changes in HA protein. In planta production fold changes for modified HAs normalized to the appropriate parent HA are shown in Figure 3.

[0187] Drug Substance (DS) production fold change was assessed after small-scale sorting and purification to remove impurities by densitometric analysis of Coomassie-stained proteins on SDS gels, and immunologically relevant products were included in the quantification and purity determination. Drug Substance (DS) production fold change (%) is shown for the variant HAs normalized to the appropriate parental HA in Figure 4, as further described herein.

[0188] Example 3: Modified influenza B HA and VLP production in plants Modification of AB HA Modified influenza B HA constructs were produced using techniques well known in the art (see Example 1). A summary of the parental (unmodified) and modified HA proteins, primers, templates and products is provided below in Table 5. The sequences used are provided in Example 5 and in the Sequence Listing.

[0189] B / Singapore / INFKK-16-0569 / 2016 The L404I B / Singapore / INFKK-16-0569 / 2016 mutant HA was constructed by mutating the leucine residue at position 404 of the parent B / Singapore / INFKK-16-0569 / 2016 to isoleucine (construct #8894). As shown in Figure 3, purified extracts from N. benthamiana plants agroinfiltrated with construct #8894 exhibited approximately a 2.3-fold increase in in planta yield compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Singapore / INFKK-16-0569 / 2016 (construct #2879).

[0190] B / Washington / 09 / 19 The L402I B / Washington / 09 / 19 mutant HA was constructed by mutating the leucine residue at position 402 of the parent B / Washington / 09 / 19 HA to isoleucine (construct #8881). As shown in Figure 3, purified extracts from N. benthamiana plants agroinfiltrated with construct #8881 exhibited approximately a 1.9-fold increase in in planta yield compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Washington / 09 / 19 (construct #7679).

[0191] B / Rhode Island / 01 / 2019 The L402I B / Rhode Island / 01 / 2019 mutant HA was constructed by mutating the leucine residue at position 402 of the parent B / Rhode Island / 01 / 2019 to isoleucine (construct #7787). As shown in Figure 3, purified extracts from N. benthamiana plants agroinfiltrated with construct #7787 exhibited approximately a 1.5-fold increase in in planta yield compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Rhode Island / 01 / 2019 HA (construct #8424).

[0192] B / Michigan / 01 / 2021 The L402I B / Michigan / 01 / 2021 mutant HA was constructed by mutating the leucine residue at position 402 of the parent B / Michigan / 01 / 2021 HA to isoleucine (construct #9628). As shown in Figure 3, purified extracts from N. benthamiana plants agroinfiltrated with construct #9628 exhibited approximately a 1.5-fold increase in in planta yield compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Michigan / 01 / 2021 HA (construct #9627).

[0193] B / Henan Province-Xigong / 1118 / 2021 The L402I B / Henan-Xigong / 1118 / 2021 mutant HA was constructed by mutating the leucine residue at position 402 of the parent B / Henan-Xigong / 1118 / 2021 HA to isoleucine (construct #9630). As shown in Figure 3, purified extracts from N. benthamiana plants agroinfiltrated with construct #9630 exhibited approximately a 1.2-fold increase in in planta yield compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Henan-Xigong / 1118 / 2021 HA (construct #9629). B / Singapore / WUH4618 / 2021 The L402I B / Singapore / WUH4618 / 2021 mutant HA was constructed by mutating the leucine residue at position 402 of the parent B / Singapore / WUH4618 / 2021 HA to isoleucine (construct #9867). As shown in Figure 3, purified extracts from N. benthamiana plants agroinfiltrated with construct #9867 exhibited approximately a 1.3-fold increase in in planta yield compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Singapore / WUH4618 / 2021 (construct #9866).

[0194] B / Austria / 1359417 / 2021 The L402I B / Austria / 1359417 / 2021 mutant HA was constructed by mutating the leucine residue at position 402 of the parent B / Austria / 1359417 / 2021 HA to isoleucine (construct #9869). As shown in Figure 3, purified extracts from N. benthamiana plants agroinfiltrated with construct #9869 exhibited approximately a 1.3-fold increase in in planta yield compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Austria / 1359417 / 2021 HA (construct #9868).

[0195] One or more of the modifications described herein specifically increase influenza HA protein production and VLP production in plants.

[0196] Example 4: In plant yield and drug substance (DS) yield A summary of in planta yields and drug substance (DS) yields is shown in Table 4. In planta yields were measured as described in Example 2. In planta production fold changes were obtained by comparing the production of the mutated or modified HA proteins with the appropriate parental HA (see Figure 3).

[0197] DS production fold changes were obtained by comparing the production of mutant or modified HA proteins with the appropriate unmodified (parent) HA (see Figure 4). [Table 4] [Table 5] Example 5: Arrays The following sequences were used (see also Table 4): wt HA B / Washington / 02 / 2019AA (SEQ ID NO: 1)

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[0198] The present invention has been described with respect to one or more embodiments, however, it will be apparent to those skilled in the art that certain variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

1. A modified influenza B virus hemagglutinin (HA) protein comprising a modified HA2 ectodomain, wherein the modified HA2 ectodomain comprises an amino acid sequence having at least one amino acid substitution compared to a parent HA2 ectodomain amino acid sequence, and the at least one substitution corresponds to amino acid position 402 in a sequence alignment with the reference sequence of SEQ ID NO: 1 (B / Washington / 09 / 19HA).

2. 2. The modified influenza B HA protein of claim 1, wherein the parent HA2 ectodomain amino acid sequence is the wild-type amino acid sequence of an influenza B virus.

3. 3. The modified influenza B HA protein of claim 1 or 2, wherein the substitution is a non-leucine substitution.

4. The modified influenza B HA protein of claim 1 , wherein the substitution is a substitution for isoleucine or a conservative substitution for isoleucine.

5. 5. The modified influenza B HA protein of claim 4, wherein the conservative substitution for isoleucine is methionine, phenylalanine, or valine.

6. 2. The modified influenza B HA protein of claim 1, wherein the sequence of the modified HA2 ectodomain comprises 80% to 100% identity with the sequence of SEQ ID NO:

42.

7. 2. The modified influenza B HA protein of claim 1, wherein the sequence of the influenza B HA protein comprises 80% to 100% identity with the sequence of SEQ ID NO: 13, 17, 21, 25, 29, 33, or 37.

8. The modified influenza B HA protein of any one of claims 1 to 7, wherein the HA comprises a plant-specific N-glycan, a modified N-glycan, or a combination thereof.

9. A nucleic acid comprising a nucleotide sequence encoding a modified influenza HA protein according to any one of claims 1 to 8.

10. A virus-like particle (VLP) comprising a modified influenza B HA protein according to any one of claims 1 to 8.

11. 1. A method for producing a modified influenza B HA protein in a non-human host or host cell, comprising: a) introducing the nucleic acid of claim 9 into the non-human host or the host cell, or providing the non-human host or the host cell containing the nucleic acid of claim 9; and b) incubating said non-human host or said host cells under conditions that allow expression of said nucleic acid, thereby producing said modified influenza B HA protein; A method comprising:

12. 1. A method for increasing the production of influenza B HA protein in a non-human host or host cell, comprising: a) introducing the nucleic acid of claim 9 into the non-human host or the host cell, or providing the non-human host or the host cell containing the nucleic acid of claim 9; and b) incubating the non-human host or host cells under conditions that allow expression of the modified B HA protein encoded by the nucleic acid, thereby producing the modified B HA in increased yields relative to a non-human host or host cells that expresses an influenza B HA protein comprising the HA2 ectodomain parent amino acid sequence; A method comprising:

13. 13. The method of claim 11 or 12, wherein the modified influenza B HA protein is further extracted and purified from the non-human host or the host cells.

14. A modified influenza B HA protein produced by the method of any one of claims 11 to 13.

15. 1. A method for producing influenza virus-like particles (VLPs) in a non-human host or host cell, comprising: a) providing the non-human host or the host cell comprising the nucleic acid of claim 9 or introducing the nucleic acid of claim 9 into the non-human host or the host cell; and b) incubating said non-human host or said host cells under conditions that allow expression of said modified influenza B HA protein encoded by said nucleic acid, thereby producing said VLPs; A method comprising:

16. 1. A method for increasing the production of influenza virus-like particles (VLPs) in a non-human host or host cell, comprising: a) introducing the nucleic acid of claim 9 into the non-human host or the host cell, or providing the non-human host or the host cell containing the nucleic acid of claim 9; and b) incubating said non-human host or said host cells under conditions that allow expression of said modified B HA protein encoded by said nucleic acid, thereby producing said VLPs in higher yields compared to a non-human host or host cells that expresses an influenza B HA protein comprising the HA2 ectodomain parent amino acid sequence; A method comprising:

17. 17. The method of claim 15 or 16, wherein the method further comprises step c), i.e., harvesting the non-human host or the host cells, and extracting and purifying the VLPs.

18. A VLP produced by the method of any one of claims 15 to 17.

19. 19. The VLP of claim 10 or 18, further comprising one or more lipids derived from the non-human host or the host cell.

20. A method for producing an antibody or antibody fragment, comprising administering a VLP described in any one of claims 10, 18 or 19 to a subject or host animal, thereby producing the antibody or antibody fragment.

21. 21. An antibody produced by the method of claim 20.

22. A host or host cell comprising the nucleic acid of claim 9, a modified influenza B HA protein of any one of claims 1 to 8, a VLP of any one of claims 10, 18 or 19, or a combination thereof.

23. 20. A composition for eliciting an immune response comprising an effective amount of a VLP according to any one of claims 10, 18 or 19, and a pharmaceutically acceptable carrier, adjuvant, vehicle or excipient.

24. 24. A vaccine for inducing an immune response, said vaccine comprising an effective amount of a modified influenza B HA protein according to any one of claims 1 to 8, a VLP according to claim 10, 18 or 19, or a composition according to claim 23.

25. 25. The vaccine of claim 24, further comprising an adjuvant.

26. A method for inducing an immune response against influenza infection in a subject, the method comprising administering to the subject a VLP described in claim 10, 18 or 19, a composition described in claim 23, or a vaccine described in claim 24 or 25.

27. 27. The method of claim 26, wherein the VLP, composition or vaccine is administered to the subject orally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously.

28. 18. The method of any one of claims 11 or 15 to 17, wherein the non-human host or the host cell comprises a plant, a plant part, a plant cell, a fungus, a fungal cell, an insect, an insect cell, an animal or an animal cell.

29. A multivalent immunogenic composition comprising two or more VLPs, wherein at least one VLP comprises a modified influenza B HA according to any one of claims 1 to 8.

30. 30. The multivalent immunogenic composition of claim 29, wherein the composition further comprises a second type of VLP, wherein the second type of VLP comprises a modified influenza B HA according to any one of claims 1 to 8.

31. 31. The multivalent immunogenic composition of claim 30, wherein the at least one type of VLP is a first type of VLP, and the first type of VLP comprises a modified B HA derived from an influenza B strain that is different from the modified B HA of the second type of VLP.

32. The multivalent immunogenic composition of any one of claims 29 to 31, wherein the composition further comprises one or more VLPs comprising an influenza A HA protein.

33. 10. A tetravalent immunogenic composition comprising a first type of VLPs comprising a modified influenza B HA of any one of claims 1 to 8, a second type of VLPs comprising a modified influenza B HA of any one of claims 1 to 8, a third type of VLPs comprising an influenza A HA, and a fourth type of VLPs comprising an influenza A HA, wherein the first type of VLPs comprise a modified B HA derived from a different influenza B strain than the modified B HA of the second type of VLPs.

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