Modified influenza b virus hemagglutinin

Abstract

The present disclosure relates to the production of modified influenza B virus hemagglutinin (HA) proteins. More specifically, the present disclosure relates to the production of influenza virus-like particles (VLPs) in plants and to the enhancement of the production of influenza virus-like particles (VLPs) wherein the VLPs comprise a modified influenza B virus HA protein. The HA protein may comprise an amino acid sequence comprising at least one substitution when compared to the corresponding wild-type amino acid sequence. Nucleic acids encoding the modified B HA proteins are also provided. In addition, methods of producing influenza virus-like particles (VLPs) in a host or host cell and methods of increasing the yield of influenza virus-like particles (VLPs) in a host or host cell are also provided.

Description

Technical Field

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

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

[0003] Influenza viruses are highly pleomorphic 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 penetration of cells. The influenza virus genome consists of eight single-stranded negative-sense RNA segments, of which the fourth largest segment encodes the HA gene.

[0004] The HA molecule exists as a trimer in the virus particle. Each monomer exists as two chains, namely the HA1 and HA2 domains (also called HA1 and HA2 subunits or subdomains), which are connected by a single disulfide bond. The infected host cell produces a precursor glycosylated polypeptide (HA0) with a molecular weight of about 85kDa, which is subsequently cut into HA1 (~40kDa) and HA2 (~20kDa) domains. After cleavage, the two disulfide-bonded protein domains adopt the necessary conformation required for viral infection.

[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 antigenic domain. HA1 contains the vestigial esterase domains E1′ and E2 and the receptor binding site (RBS), where the RBS is the least conserved segment of influenza viruses. HA2 is a single-pass integral membrane protein with a fusion peptide (FP), a soluble HA2 extracellular domain, a transmembrane (TM), and a cytoplasmic tail (CT) (see Figure 1). HA2, together with the N- and C-terminal HA1 residues, forms a stem domain that includes a transmembrane region and is relatively conserved. The stem structure contains the fusion machinery, which undergoes conformational changes in the low pH environment of the late endosome to trigger membrane fusion and penetrate into the cell. The relative conservation of the stem domain may be due to its immunologically subdominant nature (and therefore lack of antibody pressure), but this observation may also be due to a lack of tolerance to changes caused by functional limitations of the fusion machinery. (Kirkpatrick et al., Scientific Reports, Vol. 8, Paper No.: 10432 (2018)).

[0006] Based on antigenic differences, influenza viruses are divided into types A, B, and C. Influenza A and influenza B are the causative microorganisms of seasonal epidemics in humans. In contrast to influenza A, an animal-transmitted pathogen that infects a variety of host species, influenza B mainly infects humans and, to a lesser extent, seals. Unlike influenza A, limited antigenic drift is observed in influenza B viruses, which makes the virus relatively stable. Therefore, the sequence identity between influenza A and B virus HA is low for HA1, about 20%, which is the main target of antigenic variation.

[0007] Although its lack of antigenic diversity impedes pandemic outbreaks, influenza B causes seasonal occurrences of influenza, which can cause severe infections, thousands of deaths, and billions of dollars in losses. Recently, influenza B has attracted increasing attention due to the increased circulation of two distinct lineages of the virus: the Victoria lineage and the Yamagata lineage.

[0008] Various mutations in influenza virus HA proteins, particularly those from influenza A, have been studied.

[0009] For example, Castelán-Vega et al. (Adv Appl Bioinform Chem. 2014; 7: 37-44) used a stability prediction algorithm to compare 7479 full-length amino acid sequences of HA from influenza A (H1N1) pdm09 virus and identified that D104N, A259T, S124N and E172K mutations resulted in predicted enhanced stability of influenza virus HA. In contrast, S206T, K285E and E47K mutations had the expected destabilizing effect on HA.

[0010] Reed et al. 2010 (J. Virol. 83: 3568-3580) produced four recombinant H5N1 viruses containing mutations that changed the acid stability of the HA protein but did not change its expression level, cleavage, receptor binding or membrane fusion efficiency. Two of the mutations (Y231H and N1142K) increased the pH of membrane fusion of the H5N1 HA protein, while the other two mutations (H241Q and K582I) reduced the pH of fusion.

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

[0012] Mutations in the HA protein from influenza B virus have also been studied, albeit in smaller numbers.

[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 virus phenotype of B / Victoria / 504 / 2000 to virus growth. They found that G141E and R162M were most conducive to virus growth; however, only R162M could improve virus growth without antigenic changes.

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

[0015] Kim et al. 2015 (Vaccine, 2015 September; 33) studied mutations that lead to improved growth of cold-adapted influenza B virus. Molecular analysis showed that the following mutations in the HA, NP, and NA genes were required for enhanced viral growth: G156 / N160 in HA, E253, G375 in NP, and T146 in the NA gene.

[0016] Vaccination remains the most effective way to prevent influenza infection. However, the evolving nature of influenza viruses requires ongoing global surveillance and frequent reformulation of influenza vaccines. The World Health Organization (WHO) holds technical consultations each February and September to recommend viruses to include in seasonal influenza vaccines for the northern and southern hemispheres, respectively. These recommendations are based on information provided by the WHO Global Influenza Surveillance and Response System (GISRS).

[0017] Quadrivalent influenza vaccine (QIV) contains the hemagglutinin antigen (HA) of each of the four influenza virus strains recommended by WHO for the upcoming influenza season (typically influenza A(H3N2) and influenza A(H1N1) strains and two influenza B strains, one from each lineage B virus [B / Yamagata and B / Victoria]).

[0018] Typically, vaccination is accomplished using a live attenuated or whole inactivated form of the virus, which elicits an immune response when administered to a patient. In order to eliminate the potential risk of live attenuated and whole inactivated viruses regaining replication competence and becoming infectious, vaccines containing recombinant viral proteins have also been used to elicit protective immunity against influenza infection.

[0019] However, the use of recombinant viral proteins as immunogenic components of vaccines is subject to several limitations. First, in the absence of a complete complement of viral proteins and genetic components required for optimal expression and correct protein folding, the yield of recombinant viral proteins in standard in vitro expression systems may be insufficient for vaccine production purposes. Second, recombinant viral protein vaccines may exhibit poor immunogenicity due to incorrect folding, poor antigen presentation, and / or the generation of a primary humoral immune response that is ineffective in conferring durable protective immunity.

[0020] Virus-like particles (VLPs) are potential candidates for inclusion in immunogenic compositions. VLPs are very similar to mature virus particles, but they do not contain viral genomic material. Therefore, VLPs are non-replicating in nature, which makes them safe for vaccine administration. In addition, VLPs can be engineered to express viral glycoproteins on the VLP surface, which is their most natural physiological configuration. In addition, because VLPs are similar to complete virus particles and are multivalent particle structures, VLPs can be more effective than soluble envelope protein antigens when inducing neutralizing antibodies to glycoproteins.

[0021] VLPs have been produced in plants previously (see, e.g., WO2009 / 076778; WO2009 / 009876; WO 2009 / 076778; WO 2010 / 003225; WO 2010 / 003235; WO2010 / 006452; WO2011 / 03522; WO 2010 / 148511; WO 2013 / 044390 and WO2014153674, each of which is incorporated herein by reference).

[0022] WO2009 / 076778 teaches a method for producing influenza virus VLPs in plants, the method comprising the step of introducing a nucleic acid having a regulatory region active in plants, the regulatory region being operably linked to a nucleotide sequence encoding influenza virus HA from influenza A or B virus.

[0023] WO2009 / 009876 teaches a method for producing influenza virus HA VLPs in plants, wherein influenza virus HA self-assembles into VLPs in plant cells and buds from the plant cell membrane.

[0024] WO2010 / 006452 teaches the production of VLPs comprising modified influenza virus HA proteins, wherein the glycosylation sites at positions 154, 165, 286 or a combination thereof (reference A / Vietnam / 1194 / 04[H5N1] numbering) are eliminated by mutating the residues at the positions to amino acids other than asparagine. WO2010 / 006452 also teaches that the amino acids at positions 156, 167, 288 or a combination thereof may be mutated to residues other than serine or threonine, thereby similarly eliminating the N-linked glycosylation signal triplet "NXS / T". By selectively deleting the glycosylation sites located in the globular head of the HA protein, WO2010 / 006452 demonstrates that the produced HA protein has improved antigenicity and a wider range of cross-reactivity.

[0025] WO2010 / 148511 discloses a method for producing influenza virus VLPs in plants, wherein the VLPs comprise chimeric HA proteins. The chimeric HA proteins comprise 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] WO2014 / 153674 teaches a method for producing influenza virus VLPs in plants, wherein the VLPs comprise modified influenza virus HA with a modified proteolytic loop. The modified proteolytic loop comprises the removal of the proteolytic cleavage site between the HA1 and HA2 domains of the HA0 precursor. Thus, the HA protein is stabilized and protein yield is increased compared to the native HA protein.

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

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

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

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

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

[0032] The modified B HA may comprise a modified HA2 subunit, wherein the modified HA2 subunit may comprise a sequence having 80% to 100% identity to the sequence of SEQ ID NO: 41 or SEQ ID NO: 42. In addition, the modified HA2 subunit may comprise a modified HA2 extracellular domain. The modified HA2 extracellular domain may comprise a sequence having 80% to 100% identity to the sequence of SEQ ID NO: 42. The sequence of the influenza B virus HA protein may have 80% to 100% identity to the sequence of SEQ ID NO: 13, 17, 21, 25, 29, 33 or 37.

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

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

[0035] Also provided is a virus-like particle (VLP), which comprises the modified influenza B virus HA protein described above.

[0036] In another aspect, a method (A) for producing a modified influenza B virus HA protein in a non-human host or host cell is provided, the method comprising:

[0037] a) introducing a nucleic acid comprising a nucleotide sequence encoding the modified influenza virus HA protein into a non-human host or host cell, or providing a non-human host or host cell comprising a nucleic acid comprising a nucleotide sequence encoding the modified influenza virus HA protein, and

[0038] b) cultivating the non-human host or host cell under conditions allowing expression of the nucleic acid, thereby producing the modified influenza B virus HA protein.

[0039] In another aspect, a method (B) for increasing the production of influenza B virus HA protein in a non-human host or host cell is provided, the method comprising:

[0040] a) introducing a nucleic acid comprising a nucleotide sequence encoding the modified influenza virus HA protein into a non-human host or host cell; or providing a non-human host or host cell comprising a nucleic acid comprising a nucleotide sequence encoding the modified influenza virus HA protein; and

[0041] b) cultivating the non-human host or host cell under conditions that allow expression of the modified B HA protein encoded by the nucleic acid, thereby producing the modified B HA at a higher yield compared to a non-human host or host cell expressing an influenza B virus HA protein comprising the parent amino acid sequence of the HA2 extracellular domain.

[0042] The modified influenza B virus HA protein in method (A) or (B) can be further extracted and purified from a non-human host or host cells.

[0043] In another aspect, a modified influenza B virus HA protein produced by method (A) or method (B) is provided.

[0044] In another aspect, a method (C) for producing influenza virus-like particles (VLPs) in a non-human host or a host cell is provided, the method comprising:

[0045] a) providing a non-human host or host cell comprising a nucleic acid containing a nucleotide sequence encoding a modified influenza virus HA protein; or introducing a nucleic acid containing a nucleotide sequence encoding a modified influenza virus HA protein into a non-human host or host cell; and

[0046] b) culturing the non-human host or host cell under conditions that allow expression of the modified influenza B virus HA protein encoded by the nucleic acid, thereby producing VLPs.

[0047] In 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, the method comprising:

[0048] a) introducing a nucleic acid comprising a nucleotide sequence encoding a modified influenza virus HA protein into a non-human host or host cell; or providing a non-human host or host cell comprising a nucleic acid comprising a nucleotide sequence encoding a modified influenza virus HA protein; and

[0049] b) cultivating the non-human host or host cell under conditions that allow expression of the modified B HA protein encoded by the nucleic acid, thereby producing VLPs at a higher yield compared to a non-human host or host cell expressing an influenza B virus HA protein comprising the parent amino acid sequence of the HA2 extracellular domain.

[0050] The method of (C) or (D) may further comprise step c), harvesting the non-human host or host cells, and extracting and purifying VLPs.

[0051] Also provided is a virus-like particle (VLP) produced by the method of (C) or (D). The VLP may further comprise one or more lipids derived from a non-human host or host cell.

[0052] The nucleic acid in the method of (A), (B), (C) or (D) may further comprise a nucleotide sequence encoding a proton channel protein. Alternatively, step a) of the method of (A), (B), (C) or (D) may further comprise introducing a second nucleic acid encoding a proton channel protein; and step b) of the method of (A), (B), (C) or (D) further comprises cultivating a 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 an influenza A virus M2 protein.

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

[0054] In another aspect, a host or host cell comprising the nucleic acid, modified influenza B virus HA protein, VLP or a combination thereof is provided.

[0055] In another aspect, a composition for inducing an immune response is provided, the composition comprising an effective dose of VLP and a pharmaceutically acceptable carrier, adjuvant, vehicle or excipient.

[0056] Also provided is a vaccine for inducing an immune response, the vaccine comprising an effective dose of the modified influenza B virus HA protein, VLP or composition. The vaccine may further comprise an adjuvant.

[0057] Also provided is a method of inducing an immune response in a subject, the method comprising administering a VLP, composition or vaccine to the subject. The VLP, composition or vaccine can be administered orally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously to the subject.

[0058] 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.

[0059] In another aspect, a multivalent immunogenic composition comprising two or more types of VLPs is also provided, wherein at least one type of VLP comprises the above-mentioned modified influenza B virus HA. The composition may also comprise a second type of VLP, wherein the second type of VLP comprises a modified influenza B virus HA. The at least one type of VLP may be a first type of VLP, and wherein the first type of VLP may comprise a modified B HA, the modified B HA being derived from an influenza B virus lineage different from the modified B HA of the second type of VLP. For example, the first type of VLP may comprise a modified BHA 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 also comprise one or more than one type of VLP comprising influenza A virus HA protein. For example, influenza A virus HA may be derived from influenza virus subtype H1 and / or influenza virus subtype H3.

[0060] In another aspect, a tetravalent immunogenic composition is provided, comprising a first type of VLPs comprising a modified influenza B virus HA as described herein, a second type of VLPs comprising a modified influenza B virus HA as described herein, a third type of VLPs comprising an influenza A virus HA, and a fourth type of VLPs comprising an influenza A virus HA, wherein the first type of VLPs comprises a modified B HA derived from an influenza B virus lineage that is different from the modified B HA of the second type of VLPs.

[0061] This summary does not necessarily describe all features of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] These and other features of the present invention will become more apparent from the following description taken in conjunction with the accompanying drawings, in which:

[0063] Figure 1 A schematic diagram of the domain structure of the HA protein (precursor HA0) is shown. HA0 comprises an N-terminal signal sequence (which targets protein synthesis to the ER before being cut and released) and two HA subunits (HA1 and HA2). The domains in the HA2 subunit include a fusion protein (FP), an HA2 extracellular domain, a transmembrane domain (TM) and a cytoplasmic tail (CT). The HA0 precursor protein cannot cause membrane fusion, and requires proteolytic cleavage of the HA1 and HA2 subunits to induce the protein into a form with fusion ability. The cleavage site between the HA1 and HA2 subunits is indicated by an arrow.

[0064] Figure 2A A schematic diagram of vector 4498 is shown, which is used for assembly of a vector plasmid encoding a modified influenza B strain HA protein. Figure 2B A schematic diagram of vector 2879 encoding influenza B strain HA from B / Singapore / INFKK-16-0569 / 2016 is shown. Figure 2C A schematic diagram of vector 8894 encoding a modified influenza B strain HA from B / Singapore / INFKK-16-0569 / 2016 with the L404I mutation is shown. Figure 2D A schematic diagram of vector 7679 encoding influenza B strain HA from B / Washington / 02 / 2019 is shown. Figure 2E A schematic diagram of vector 8881 encoding HA of influenza B strain with L402I mutation from B / Washington / 02 / 2019 is shown. Figure 2F A schematic diagram of vector 8424 encoding influenza B strain HA from B / Rhode Island / 01 / 2019 is shown. Figure 2G A schematic diagram of vector 7787 encoding the influenza B strain HA from B / Rhode Island / 01 / 2019 with the L402I mutation is shown. Figure 2H A schematic diagram of vector 9627 encoding HA of influenza B strain from B / Michigan / 01 / 2021 is shown. Fig.2I A schematic diagram of vector 9628 encoding HA of influenza B strain from B / Michigan / 01 / 2021 with the L402I mutation is shown. Figure 2J A schematic diagram of vector 9629 encoding influenza B strain HA from B / Henan-Xigong / 1118 / 2021 is shown. Figure 2K A schematic diagram of vector 9630 encoding influenza B strain HA from B / Henan-Xigong / 1118 / 2021 with the L402I mutation is shown. Figure 2L A schematic diagram of vector 9866 encoding influenza B strain HA from B / Singapore / WUH4618 / 2021 is shown. Figure 2M A schematic diagram of vector 9867 encoding influenza B strain HA from B / Singapore / WUH4618 / 2021 with the L402I mutation is shown. Figure 2N A schematic diagram of vector 9868 encoding influenza B strain HA from B / Austria / 1359417 / 2021 is shown. Fig.2O A schematic diagram of vector 9869 encoding influenza B strain HA from B / Austria / 1359417 / 2021 with the L402I mutation is shown.

[0065] Figure 3The fold change in production in plants expressing modified influenza B virus HA proteins is shown, where the fold change is calculated relative to the appropriate unmodified (parental) control HA protein (CTL), as follows: B / Singapore / INFKK-16-0569 / 2016 (CTL: construct 2879, L404I: construct 8894), B / Washington / 02 / 2019 (CTL: construct 7679, L404I: construct 8881), B / Rhode Island / 01 / 2019 (CTL: construct 8424, L404I: construct 7787), B / Michigan / 01 / 2021 (CTL: construct 9627, L404I: construct 9628), B / Henan-Xigong / 1118 / 2021 (CTL: construct 9629, L402I: construct 9630), B / Singagore / WUH4618 / 2021 (CTL: construct 9866, L404I: construct 9867) and B / Austria / 1359417 / 2021 (CTL: construct 9868, L402I: construct 9869).

[0066] Figure 4 Shown are the fold changes in the production of drug substance (DS) obtained from any of the hosts expressing modified influenza B strain HA proteins, 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

[0067] The following description is of a preferred embodiment.

[0068] As used herein, the terms "comprising", "having", "including", "containing" and grammatical variations thereof are inclusive or open, and do not exclude other, unlisted elements and / or method steps. When used in conjunction with a product, use or method in this article, the term "substantially consisting of ... " indicates that other elements and / or method steps may exist, but these additions do not substantially affect the manner in which the listed method or use works. When used in conjunction with a product, use or method in this article, the term "consisting of ... " excludes the presence of other elements and / or method steps. In certain embodiments, the product, use or method described herein as comprising certain elements and / or steps may also be substantially composed of those elements and / or steps, and in other embodiments, composed of those elements and / or steps, whether or not these embodiments are specifically mentioned. In addition, the use of the singular includes the plural, and unless otherwise stated, "or" means "and / or". Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. As used herein, the term "about" refers to deviating from a given value by approximately + / -10%. It should be understood that whether or not specifically mentioned, these changes are always included in any given value provided herein. When used herein in conjunction with the term "comprising," use of the word "a" or "an" may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0069] Described herein are modified influenza B virus hemagglutinin (HA) proteins (also referred to as modified B HA proteins, modified influenza B virus HA proteins, modified B HA, modified influenza B virus HA, mutant B HA, influenza mutant B HA, modified B proteins; modified B; influenza viruses or influenza B virus HA comprising modified HA2 subunits or modified HA2 extracellular domains) and methods of producing modified influenza B virus HA proteins in a host or host cell. In addition, modified influenza B virus HA proteins can self-assemble into virus-like particles (VLPs). Therefore, influenza virus VLPs comprising or consisting of modified influenza B virus HA proteins are also provided.

[0070] It has been observed that, when compared to a parental HA that does not include the specific amino acid replacement, modifications such as by replacing specific amino acids in a B HA protein (e.g., a B HA from the Yamagata lineage or the Victoria lineage) result in improved characteristics of the modified B HA protein. The parental HA may also be referred to as an unmodified B HA protein. In some embodiments, the parental or unmodified HA may be a wild-type HA. In other embodiments, as described below, the parental or unmodified HA may comprise other modifications, such as, for example, a deletion or partial deletion of a proteolytic loop and / or replacement of the native transmembrane and TMCT with a cytoplasmic tail domain (TMCT) from an influenza A virus HA.

[0071] With respect to influenza viruses, the term "hemagglutinin" or "HA" as used herein refers to a glycoprotein present on the outside of influenza virus particles. HA is translated into a single protein, namely HA0. HA0 generally comprises a signal peptide (SP), an HA1 domain (also referred to as an HA1 subunit), an HA2 domain (also referred to as an HA2 subunit) comprising a fusion protein (FP), an HA2 extracellular domain and a transmembrane domain (TM), and a cytoplasmic tail (CT), collectively referred to as TM / CT (see Figure 1 ).

[0072] 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-bonded protein domains generate the mature form of the protein subunit as a prerequisite for the conformational changes necessary for fusion and therefore viral infectivity.

[0073] Nucleotide sequences encoding HA and HA amino acid sequences are well known and available - see, for example, BioDefence Public Health base (influenza virus; see URL: biohealthbase.org) or National Center for Biotechnology Information (see URL: ncbi.nlm.nih.gov), both of which are incorporated herein by reference. In addition, influenza strains can be identified and classified by techniques known in the art, such as by hemagglutination inhibition assays, reverse transcriptase PCR, real-time PCR or sequencing (E1 Hefnawi & Sherif (Virology, Vol. 449, 20 January 2014).

[0074] The modified B HA protein may comprise an HA1 domain, an HA2 extracellular domain, a transmembrane domain (TM) and a cytoplasmic tail (CT). The HA1 domain and the HA2 domain may be derived from influenza B virus HA, and the transmembrane domain (TM) and the cytoplasmic tail (CT) may be derived from influenza A virus 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 comprise a natural or non-natural signal peptide.

[0075] The modified influenza B virus HA proteins disclosed herein comprise modifications or mutations that have been found to result in improved B HA characteristics compared to a parent (unmodified) HA protein of the same influenza virus strain or subtype that does not comprise the modifications or mutations (referred to as parent HA, unmodified HA, or control). For example, a modified influenza B virus HA protein may have an amino acid sequence in which at least one amino acid is replaced compared to the corresponding parent amino acid sequence. In some embodiments, a modified B HA protein may have one or more than one replacement in the HA2 extracellular domain when compared to the sequence of the HA2 extracellular domain of a parent B HA.

[0076] For example, a modified influenza B virus hemagglutinin (HA) protein may comprise a modified HA2 subunit (also referred to as HA2), wherein at least one amino acid is modified (e.g., replaced or substituted) compared to a parent sequence, such as, for example, a wild-type amino acid in a sequence. In one aspect, a modified influenza B virus hemagglutinin (HA) protein may comprise a modified HA2 extracellular domain, wherein at least one amino acid is modified (e.g., replaced or substituted) compared to a parent sequence, such as, for example, a wild-type amino acid in a sequence. The amino acid modification may correspond to amino acid position 402 in a sequence alignment with the reference sequence SEQ ID NO: 1 (B / Washington / 09 / 19HA).

[0077] Examples of improved characteristics of the modified B HA protein include, increased HAB protein production when expressed in a host or host cell, compared to a parental B HA protein of the same influenza strain not comprising the modification or mutation; increased VLP production when the modified B HA protein is expressed in a host or host cell, compared to VLP production levels, wherein the B HA protein does not comprise the modification or mutation; increased drug substance (DS) production when obtained from a host or host cell expressing the modified HAB protein, compared to the DS production obtained from a host or host cell expressing the parental (unmodified) HAB protein.

[0078] Modified B virus HA proteins can be produced by introducing changes into the amino acid sequence of influenza B virus HA protein that result in improved HA characteristics as described above. It is routine to isolate nucleic acids encoding such HA molecules, and it is also routine to modify nucleic acids to introduce changes in the amino acid sequence, for example by site-directed mutagenesis.

[0079] The influenza B virus HA protein, mutant B HA protein or modified BHA protein as described herein is modified and comprises one or more mutations, modifications or substitutions in its amino acid sequence, wherein at least the amino acid at position 402 of B / Washington / 09 / 19HA (SEQ ID NO: 1) or the amino acid at position 404 of B / Singapore / INFKK-16-0569 / 2016 (SEQ ID NO: 2) is modified compared to the unmodified (parent) sequence.

[0080] "Corresponds to an amino acid or corresponding to anamino acid" means that the amino acid corresponds to an amino acid in a sequence alignment with an influenza reference strain as described herein.

[0081] The amino acid residue numbering or residue position of HA is based on the numbering of the HA of the influenza reference strain. For example, the reference strain can be B / Washington / 09 / 19 HA (SEQ ID NO: 1), which belongs to the Victoria lineage (see Table 1). The reference strain can also be B / Singapore / INFKK-16-0569 / 2016 (SEQ ID NO: 2), which belongs to the Yamagata lineage (see Table 1).

[0082] Corresponding amino acid positions can be determined by aligning the B HA sequence with the sequence of the HA of their respective reference strains. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by, for example, the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., ed., 1995 Supplement)).

[0083] When referring to a modification, mutant or variant, the wild-type amino acid residue (also simply referred to as "amino acid") is followed by the residue number and the new or substituted amino acid. For example, a substitution of leucine (L) with isoleucine (I) in the residue or amino acid at position 402 is designated L402I.

[0084] Modified B HAs, B HA mutants or variants are named 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.

[0085] Table 1. Corresponding modification positions in HA of different influenza B strains.

[0086]

[0087] *HA0 (without signal peptide)

[0088] **Wild type sequence

[0089] "Modification", "amino acid modification" or "amino acid sequence modification" refers to a 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 can be a wild-type sequence, or the parent sequence can be a sequence that already contains modifications ("parent modifications") compared to the wild-type sequence. "Amino acid substitution" or "replacement" refers to replacing an amino acid in a protein amino acid sequence with a different amino acid compared to the parent sequence. In one embodiment, the modified influenza B virus hemagglutinin (HA) protein comprises a substitution in the HA2 extracellular domain compared to the parent HA2 extracellular domain.

[0090] The terms amino acid, amino acid residue or residue are used interchangeably in the present disclosure. One or more than one amino acid can be replaced by one or more amino acids different from the original amino acid at the position without changing the total length of the protein amino acid sequence. By changing the codon sequence in the nucleotide sequence encoding the protein to a codon sequence of amino acids different from the original amino acid of the parental sequence, substitution or displacement can be experimentally induced. In addition, one or more than one amino acid can be deleted from the amino acid sequence of the protein. The protein produced is a modified influenza B virus HA protein. The modified B HA protein is not naturally occurring.

[0091] The modified B HA includes a non-naturally occurring HA protein that has at least one modification to a parent HA or a naturally occurring HA and has improved properties compared to the parent HA or a 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 and is obtained by replacing one or more amino acid residues of the HA protein with one or more different amino acids.

[0092] Thus, a modified B HA, mutant B HA or recombinant B HA refers to a HA in which the DNA sequence encoding the parent HA is modified to produce a modified or mutated DNA sequence encoding a modification, mutation or substitution of one or more amino acids in the HA amino acid sequence.

[0093] The modified influenza B virus HA protein or mutant influenza B virus HA protein as described herein is modified and contains a mutated or modified residue in a sequence alignment with position 402 of B / Washington / 02 / 2019 (SEQ ID NO: 1). Therefore, influenza B virus HA polypeptides, proteins and / or protein complexes, such as, for example, virus-like particles (VLPs), are provided, wherein the virus-like particles contain modifications or mutations at amino acid positions 402, wherein such amino acid numbering is based on the sequence of B / Washington / 02 / 2019 (SEQ ID NO: 1), or at amino acid positions corresponding to these amino acid positions, For example, as determined by aligning the B HA amino acid sequence with SEQ ID NO: 1. Non-limiting examples of influenza B virus HA amino acid sequences containing these mutations include sequences of SEQ ID NO: 13, 17, 21, 25, 29, 33 or 37.

[0094] Non-limiting examples of strains from which influenza B virus HA may be derived are 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).

[0095] In one aspect of the present disclosure, the modified B HA can have at least a modified residue at position 402, where numbering is relative to the reference strain B / Washington / 02 / 2019 (SEQ ID NO: 1).

[0096] like Figure 3As shown, a modified B HA protein having a residue at position 402 having a change, for example, from leucine (L, Leu) to isoleucine (I, Ile) (hereinafter referred to as L402I) showed up to a 2.3-fold increase in in planta yield compared to a B HA having leucine (L, Leu) at this position (see also Example 3 and Table 4).

[0097] The modified HA from B / Singapore / 0569 / 16 with the L404I substitution showed approximately 2.3-fold increased in planta yield when compared to the parental B / Singapore / 0569 / 16 HA (see Figure 3 ).

[0098] The modified HA from B / Washington / 09 / 19 with the L402I substitution showed approximately a 1.9-fold increase in in planta yield when compared to the parental B / Washington / 09 / 19 HA (see Figure 3 In addition, the modified HA from B / Washington / 09 / 19 with the L402I substitution showed an approximately 2.5-fold improvement in drug substance (DS) yield fold change compared to the unmodified B / Washington / 09 / 19 HA protein (see Figure 4 ).

[0099] The modified HA from B / Rhode Island / 01 / 2019 with the L402I substitution exhibited approximately 1.5-fold increased in planta yield when compared to the unmodified B HA protein (see Figure 3 The yield of drug substance (DS) from the modified B HA from B / Rhode Island / 01 / 2019 was also increased by approximately 1.8-fold compared to the unmodified B / Rhode Island / 01 / 2019 HA protein (see Figure 4 ).

[0100] Increased yield in planta was also observed for modified B HAs from B / Michigan / 01 / 2021 (1.5-fold change), B / Henan-Xigong / 1118 / 2021 (about 1.5-fold change), B / Henan-Xigong / 1118 / 2021 (about 1.2-fold change), B / Singapore / WUH4618 / 2021 (about 1.3-fold change), and B / Austria / 1359417 / 2021 (about 1.3-fold change) (see Figure 3 , Example 3 and Table 4).

[0101] Without wishing to be bound by theory, it has been shown that increased HA production in plants correlates with increased VLP production in plants.

[0102] Thus, in one aspect, the residue at position 402 (according to B / Washington / 09 / 19 numbering) of influenza B virus HA is modified to replace leucine (L, Leu) with isoleucine (I, He).

[0103] For example, the amino acid sequence of the modified B HA protein can have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount therebetween, sequence identity or sequence similarity to the 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 a conservative substitution of isoleucine (I) at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO: 1) with isoleucine (I) or a non-leucine (L) (e.g., valine (V), methionine (M), or phenylalanine (F)), wherein the modified B HA sequence is not naturally occurring, and wherein the HA protein forms a VLP when expressed.

[0104] The present specification also provides nucleic acids comprising a nucleotide sequence encoding a modified B HA having a substitution at position 402 as described above operably linked to a regulatory region active in a plant.

[0105] For example, the nucleotide sequence can have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or any amount therebetween, sequence identity or sequence similarity to a nucleotide sequence encoding a B HA having an 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 a conservative substitution of isoleucine (I) at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO: 1) with isoleucine (I) or a non-leucine (L) (e.g., valine (V), methionine (M), or phenylalanine (F)), wherein the modified B HA sequence is not naturally occurring, and wherein the HA protein forms VLPs when expressed.

[0106] The nucleotide sequence may have about 70, 75, 80, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount of sequence identity or sequence similarity with the nucleotide sequence of SEQ ID NO: 12, 16, 20, 24, 28, 32 or 36, wherein the nucleotide codon encoding amino acid residue 402 of the modified BH encodes a conservative substitution of isoleucine (I) or a non-leucine (L) (e.g., valine (V), methionine (M) or phenylalanine (F)) at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO: 1), and wherein the modified B HA sequence is not naturally occurring.

[0107] Influenza A viruses are divided into two 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."

[0108] Influenza B viruses are not divided into subtypes, but into two co-circulating lineages that differ in phylogeny and antigenicity, named after the viruses B / Yamagata / 16 / 88 (Yamagata lineage) and B / Victoria / 2 / 87 (Victoria lineage). Similar to influenza A viruses, influenza B viruses can then be further divided into specific branches and subbranches. Compared with influenza A viruses, influenza B viruses generally change more slowly in genetic and antigenic properties.

[0109] Traditionally, different influenza strains are classified based on the ability of, for example, influenza virus to agglutinate red blood cells (RBC or erythrocytes). Antibodies specific to a particular influenza strain can be bound to the virus and therefore prevent such agglutination. Determinations based on such inhibition to determine strain type are commonly referred to as hemagglutinin inhibition assays (HI assays or HAI assays) and they are standard and well-known methods for characterizing influenza strains in the art.

[0110] However, HA proteins from different strains also show significant sequence similarity at the nucleic acid and amino acid levels. The level of similarity between strains from different B lineages can vary. This variation is sufficient to establish individual lineages and evolutionary lineages of different strains, but the DNA and amino acid sequences of different strains are easily compared using conventional bioinformatics techniques (Langat, Pinky et al. PLoSpathogens 2017Dec; vol. 13(12)).

[0111] Multiple nucleotide sequences or corresponding polypeptide sequences of hemagglutinin (HA) can be aligned to determine the "consensus" or "consensus sequence" of a subtype or lineage (see Gravel et al. iScience 24, Nov. 2021). For example, the consensus sequence of the B HA2 domain is shown in the sequence of 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 of the B HA2 extracellular domain can be determined.

[0112] Therefore, influenza B virus HA proteins having modified HA2 extracellular domains are also provided, wherein the HA2 extracellular domain comprises one or more than one mutation, modification or substitution in its amino acid sequence, wherein at least the amino acid corresponding to the amino acid at position 58 of SEQ ID NO: 40 has been modified compared to the unmodified (parent) sequence. For example, leucine (L) is modified at position 58 to a non-leucine, such as isoleucine (I) (L58I).

[0113] Thus, the modified B virus HA protein described herein may comprise a modified HA2 subunit (or HA2 domain) comprising the following fusion peptide (italics) and HA2 extracellular domain sequence:

[0114]

[0115] in

[0116] X1 = I at position 58

[0117] X2 = L or I at position 78

[0118] X3 = S at position 129 or not present

[0119] X4 = E or D at position 132

[0120] X5 = N or D at position 158

[0121] X6 = A or D at position 159.

[0122] Thus, in one aspect, a modified influenza B virus HA protein is provided, the influenza B virus HA protein comprising a modified HA2 domain, wherein the modified HA2 domain comprises at least one substitution when compared to the sequence of a parent (unmodified) or wild-type HA2 domain. The modified HA2 domain may have an amino acid sequence having about 80, 82, 83, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount of sequence identity or sequence similarity thereto with the amino acid sequence of SEQ ID NO: 41, wherein the sequence comprises a non-leucine, such as, for example, isoleucine, at position 85. The modified influenza B virus HA protein may comprise an HA2 domain comprising or consisting of the sequence of SEQ ID NO: 41.

[0123] Conservative substitution

[0124] As described herein, residues in a B HA protein can be identified and modified, substituted or mutated to produce a modified B HA protein or a B HA protein variant. The substitution or mutation of a particular position is not limited to the amino acid substitutions described herein or given in the Examples. For example, a B HA variant may contain conservative substitutions or conservative substitutions of the amino acid substitutions.

[0125] As used herein, the term "conservative substitution" or "conservative substitution" and grammatical variants thereof refer to the presence of an amino acid residue in the HA protein sequence that is different from the substitution or residue but belongs to the same class of amino acids (i.e., a non-polar residue replaces a non-polar residue, an aromatic residue replaces an aromatic residue, a polar uncharged residue replaces a polar uncharged residue, and a charged residue replaces a charged residue). In addition, conservative substitutions may encompass residues having the same sign as the residue replacing the wild-type residue and generally having an interface hydropathicity value of similar magnitude to the residue replacing the wild-type residue.

[0126] As used herein, the term "non-polar residue" refers to glycine (G, Gly), alanine (A, Ala), valine (V, Va1), leucine (L, Leu), isoleucine (I, Ile) and proline (P, Pro); the term "aromatic residue" refers to phenylalanine (F, Phe), tyrosine (Y, Tyr) and tryptophan (W, Trp); the term "polar uncharged residue" refers to serine (S, Ser), threonine (T, Thr), cysteine ​​(C, Cys), methionine (M, Met), asparagine (N, Asn) and glutamine (Q, Gln); the term "charged residue" refers to the negatively charged amino acids aspartic acid (D, ASP) and glutamic acid (E, Glu) and the positively charged amino acids lysine (K, Lys), arginine (R, Arg) and histidine (H, His). Other classifications of amino acids can be shown below:

[0127] Amino acids with hydrophobic side chains (aliphatic): alanine (A, Ala), isoleucine (I, Ile), leucine (L, Leu), methionine (M, Met), and valine (V, Val);

[0128] Amino acids with hydrophobic side chains (aromatic): phenylalanine (F, Phe), tryptophan (W, Trp), tyrosine (Y, Tyr);

[0129] Amino acids with polar neutral side chains: asparagine (N, Asn), cysteine ​​(C, Cys), glutamine (Q, Gln), serine (S, Ser), and threonine (T, Thr);

[0130] Amino acids with charged side chains (acidic): aspartic acid (D, Asp), glutamic acid (E, Glu);

[0131] • Amino acids with charged side chains (basic): arginine (R, Arg); histidine (H, His); lysine (K, Lys), glycine (G, Gly) and proline (P, Pro).

[0132] Conservative amino acid substitutions may have similar effects on the activity of the resulting HA protein variant or modified HA protein as the original substitution or modification. Additional information on conservative substitutions can be found in, for example, 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.

[0133] Blosum matrices are commonly used to determine the relevance of polypeptide sequences. A large database of credible alignments (BLOCKS database) is used to create a Blosum matrix, in which counts are compared to paired sequence alignments associated with less than a certain threshold percentage identity (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992). For the highly conservative target frequencies of the BLOSUM90 matrix, a 90% identity threshold was used. For the BLOSUM65 matrix, a 65% identity threshold was used. Zero and above scores in the Blosum matrix are considered to be "conservative substitutions" under a selected identity percentage. The following table shows exemplary conservative amino acid substitutions: Table 2.

[0134] Table 2. Exemplary conservative amino acid substitutions.

[0135]

[0136]

[0137] 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, for example, in a plant, plant part, or plant cell.

[0138] As described above, the parent sequence can be a wild-type sequence, or the parent sequence can be a sequence that already contains modifications ("parent modifications") when compared to the wild-type sequence. The parent modification can be an amino acid deletion or substitution. For example, the parent modification can include modifications such as the deletion of a proteolytic cleavage site (also known as a proteolytic loop). For example, the cleavage site and / or the fusion peptide or a portion of the fusion peptide can be deleted to prevent cleavage of the HA protein. For example, the C-terminus of the H1 domain, which includes the cleavage side and the fusion peptide, and the N-terminus of the HA2 domain may have been modified. For example, the C-terminus of the H1 domain may include one or more amino acid deletions. In addition, the N-terminus of the fusion peptide domain of HA2 may include one or more deletions of amino acids 1 to 23 of SEQ ID NO: 41. For example, amino acids 1-11 of SEQ ID NO: 41 may be deleted. Thus, also provided are modified B virus HA proteins comprising a modified HA2 ectodomain, wherein the sequence of the modified HA2 ectodomain comprises amino acids 12 to 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 may have an amino acid sequence having about 80, 82, 83, 85, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or any amount of sequence identity or sequence similarity thereto to the amino acid sequence of SEQ ID NO: 42, wherein the sequence comprises a non-leucine, such as, for example, isoleucine, at position 35. The modified influenza B virus HA protein may comprise an HA2 ectodomain comprising or consisting of the sequence of SEQ ID NO: 42.

[0139] Parent modifications may also include modifications of the transmembrane and cytoplasmic tail (TMCT). For example, the native TMCT in the parent sequence may be replaced with a TMCT from an influenza virus HA different from the parent HA.

[0140] Therefore, compared with wild-type B HA, the modified B HA protein may include further modifications, such as deletions or replacements. For example, the proteolytic cleavage site may be deleted or modified in the modified B HA protein to prevent the proteolytic cleavage of the HA0 precursor to the HA1 and HA2 subunits. The cleavage site is an important surface loop in the influenza virus HA protein and can be determined, for example, by sequence alignment or structural analysis of the HA protein (see, for example, Bertram et al. Reviews in Medical Virology, Vol. 20, September 2010). Influenza virus HA proteins comprising modified proteolytic cleavage sites and methods for producing influenza virus HA proteins comprising modified proteolytic cleavage sites are described, for example, in PCT patent applications WO2013 / 044390 and WO 2014 / 153674, which are incorporated herein by reference.

[0141] In addition, the natural transmembrane and cytoplasmic tail domain (TMCT) of influenza B virus HA can be replaced by the TMCT of influenza A virus HA. Therefore, the modified B HA can include non-natural TMCT. For example, the modified B HA can have a natural TMCT replaced by the TMCT of influenza virus 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 a non-natural TMCT from influenza virus HA H1 or H5. For example, the replacement of TMCT in influenza virus HA is described in PCT patent application WO 2010 / 148511, which is incorporated herein by reference.

[0142] When expressed in a host or host cell, the modified influenza B virus HA may include a signal peptide that directs localization. The signal peptide may be a native (relative to the protein) signal or leader sequence, or a heterologous signal sequence.

[0143] Thus, as described herein, modified influenza B virus proteins can be produced as precursor proteins comprising modified influenza B virus proteins and heterologous amino acid signal peptide sequences. For example, modified influenza B virus protein precursors can include a signal peptide from protein disulfide isomerase (PDI SP; nucleotides 32-103 of accession number Z11499).

[0144] The modified influenza B virus HA protein as described herein can be further introduced into virus-like particles (VLP). The term "virus-like particle" (VLP) or "multiple virus-like particles" or "VLP" refers to a virus-like structure, which is usually similar to the virus particles produced in the infection in morphology and antigenicity, but lacks genetic information sufficient to replicate, and is therefore non-infectious. VLP is a self-assembled structure and comprises one or more structural proteins, such as, for example, modified influenza B virus HA protein. Therefore, VLP may comprise modified influenza B virus HA protein. VLP may further comprise influenza virus protein, wherein the influenza virus protein is composed of modified influenza B virus HA protein.

[0145] VLP can be produced in suitable host or host cell (including plant and plant cell).After extraction from host or host cell and separation and further purification under suitable conditions, VLP can be recovered as intact structure.

[0146] Any suitable method can be used, for example chemical or biochemical extraction to purify or extract VLP. VLP is relatively sensitive to drying, heat, pH, surfactant and detergent. Therefore, use the method that maximizes output, minimizes the pollution of cellular protein to VLP part, keeps the integrity of protein or VLP, and when necessary, relevant lipid envelope or film, makes cell wall loosen to release protein or VLP method may be useful. Minimize or eliminate detergent or surfactant such as for example SDS or Triton TM The use of X-100 may be beneficial to improve the VLP extraction yield.The structure and size of the VLPs may then be evaluated, for example, by electron microscopy or by size exclusion chromatography.

[0147] For enveloped viruses, such as influenza, it may be advantageous for the lipid layer or membrane to be retained by the virus. The composition, quality, and quantity of lipids may vary from system to system (e.g., enveloped viruses produced by plants will include plant lipids or phytosterols in the envelope) and may help improve the immune response.

[0148] Without wishing to be bound by theory, plant-produced VLPs comprising plant-derived lipids may elicit a stronger immune response than VLPs produced in other production systems, and the immune response induced by these plant-produced VLPs may be stronger than that elicited by live or attenuated whole virus vaccines.

[0149] 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 present plant lipids, may be beneficial for the production of VLPs in plants.

[0150] The VLPs produced in plants may comprise a modified influenza B virus HA protein comprising plant-specific N-glycans. Therefore, the present disclosure also provides VLPs comprising modified influenza B virus HA proteins having plant-specific N-glycans. In addition, VLPs comprising plant lipids and modified influenza B virus HA proteins having plant-specific N-glycans are provided.

[0151] Furthermore, methods are provided for producing VLPs comprising a modified B HA as described above in a host or host cell, such as, for example, a plant.

[0152] The method may include introducing a nucleic acid encoding a modified B HA, wherein the modified B HA has a substitution at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO: 1) operably linked to a regulatory region active in a host or host cell, and culturing the host or host cell under conditions that allow expression of the nucleic acid, thereby producing VLPs. The method may also include introducing a nucleic acid encoding an influenza B virus HA protein, wherein the influenza B virus HA protein comprises a modified HA2 extracellular domain described herein operably linked to a regulatory region active in a host or host cell, and culturing the host or host cell under conditions that allow expression of the nucleic acid, thereby producing VLPs.

[0153] In addition, a method for increasing the production of VLPs in a host or host cell is provided, wherein the VLPs comprise a modified B HA having a substitution at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO: 1) as described above. The method comprises introducing a nucleic acid encoding a modified B HA, wherein the modified B HA has a substitution at position 402 (numbering corresponding to reference strain B / Washington / 09 / 19, SEQ ID NO: 1) operably linked to a regulatory region active in the host or host cell, and culturing the host or host cell under conditions allowing expression of the nucleic acid, thereby producing VLPs.

[0154] In addition, a method for increasing the yield of VLPs in a host or host cell is provided, wherein the VLPs comprise an influenza B virus HA protein comprising a modified HA2 extracellular domain as described above. The method comprises introducing a nucleic acid encoding an influenza B virus HA protein comprising a modified HA2 extracellular domain operably linked to a regulatory region active in the host or host cell, and cultivating the host or host cell under conditions that allow expression of the nucleic acid, thereby producing VLPs.

[0155] The present specification also provides VLPs comprising a B HA having a replacement at position 402 and / or comprising an influenza B virus HA protein containing a modified HA2 extracellular domain as described herein. VLPs can be produced by methods as provided in the present disclosure. VLPs comprising modified B HA show improved characteristics compared to VLPs comprising unmodified BHA proteins.

[0156] Also provided herein is a method for improving the generation or yield of the VLP comprising the modified influenza B virus HA in plants. For example, the method may include introducing the nucleic acid encoding the modified influenza B virus HA as described herein into plants, plant parts or plant cells. The nucleic acid encoding the modified influenza B virus HA may be optimized for human codon usage, increased GC content or its combination. One or more modified influenza B virus HA proteins may be expressed in plants, plant parts or plant cells to produce VLPs comprising one or more modified influenza B virus HA proteins. Alternatively, the method may include providing a plant, plant part or plant cell comprising a nucleic acid encoding the modified influenza B virus HA protein to produce VLPs comprising one or more modified influenza B virus HA proteins.

[0157] The method for producing the VLP of the influenza B virus HA comprising modification may also include the step of introducing the second nucleic acid sequence into plant, plant part or plant cell, wherein the second nucleic acid encodes the proton channel protein co-expressed with the influenza B virus HA of modification.For example, the proton channel protein can be influenza A subtype M2 protein, such as A / New Caledonia / 20 / 99M2.The coexpression of proton channel protein can cause the accumulation of the influenza B virus HA protein of modification and / or the VLP comprising the influenza virus HA protein of modification to increase, as described in for example WO 201 3 / 044390, which is incorporated herein by reference.

[0158] "Coexpression" refers to introducing and expressing two or more nucleotide sequences in plant, plant part or plant cell, each of which encodes a protein of interest or a fragment of the protein of interest. Two or more nucleotide sequences can be introduced into plant, plant part or plant cell in a vector, so that each of the two or more nucleotide sequences is controlled by a separate regulatory region (for example, comprising a dual construct). Alternatively, two or more nucleotide sequences can be introduced into plant, plant part or plant cell in a separate vector (for example, comprising a single construct), and each vector comprises a suitable regulatory region for expressing the corresponding nucleic acid. For example, two nucleotide sequences, respectively on a separate vector and introduced into a separate Agrobacterium tumefaciens host, can be coexpressed by mixing the suspension of every kind of Agrobacterium tumefaciens host with required volume (for example, identical volume, or the ratio of every kind of Agrobacterium tumefaciens host) before vacuum infiltration. In this way, the co-infiltration of a plurality of Agrobacterium tumefaciens suspensions allows the coexpression of a plurality of transgenics.

[0159] The present disclosure also provides a drug substance (DS), comprising as a desired product the modified influenza B virus HA protein as described above, the drug substance being substantially free of product-related impurities, wherein the impurities are not immunologically active. The preferred drug substance is further substantially free of process-related impurities.

[0160] In the context of this application, the term "drug substance" refers to a product or active ingredient suitable for use as i) an active ingredient of a medicament or drug product, ii) an active pharmaceutical ingredient of a medicament or drug product, iii) a substantially purified active principle of a medicament or drug product, or iv) a substantially purified active ingredient of a medicament or drug product. The medicament or drug product may be a vaccine.

[0161] Thus, a drug substance (DS) comprising an immunologically active modified influenza B virus HA protein is further provided. The yield of DS obtained from host cells expressing the modified influenza B virus HA is higher than that obtained from hosts expressing unmodified influenza B virus HA protein (see Figure 4 ). Thus, also provided are methods for increasing the yield of DS obtained from a host or host cell expressing a modified influenza B virus HA protein compared to the yield of DS obtained from a host expressing an unmodified (parent) S protein.

[0162] Therefore, there is further provided a drug substance (DS) comprising an immunologically active modified influenza B virus HA protein.

[0163] The modified influenza B virus HA protein can self-assemble into virus-like particles (VLPs). Therefore, a DS comprising a VLP containing a modified influenza B virus HA protein is also provided.

[0164] In another aspect, a pharmaceutical product (also referred to as a pharmaceutical preparation or pharmaceutical composition) is also provided. The pharmaceutical product can be formulated into a finished dosage form, such as a solution, a capsule or a tablet. The pharmaceutical product comprises a drug substance. The pharmaceutical product can also comprise other ingredients, such as, for example, a pharmaceutically available carrier and / or excipient, such as a buffer system, an adjuvant, a preservative, a tonicity agent, a chelating agent, an antiadhesive agent, a vehicle, etc. The pharmaceutically available carrier and excipient are well known in the art. Therefore, a pharmaceutical product, a pharmaceutical preparation or a pharmaceutical composition comprising a pharmaceutically available carrier and / or excipient and VLP is also provided, wherein the VLP comprises a modified influenza B virus HA protein or the VLP comprises a viral protein, wherein the viral protein is composed of a modified influenza B virus HA protein.

[0165] When one or more than one modified influenza B virus HA protein is expressed in a host or host cell, the one or more than one modified influenza B virus HA protein self-assembles into VLPs. The host or host cell can be harvested under suitable extraction and purification conditions to maintain the integrity of the VLPs, and the VLPs comprising one or more than one mutant influenza virus HA can be purified.

[0166] The present disclosure also provides the use of modified influenza B virus HA as described herein, VLPs or DS comprising modified influenza B virus HA for inducing an immune response in a subject or inducing immunity to influenza infection. An antibody or antibody fragment is also disclosed herein, which is prepared by administering modified influenza B virus HA, VLPs or DS comprising modified influenza B virus HA to a subject or host animal. A composition is also provided, comprising an effective dose of modified influenza B virus HA as described herein, VLPs or DS comprising modified influenza B virus HA, and a pharmaceutically available carrier, adjuvant, vehicle or excipient for inducing an immune response in a subject. A vaccine for inducing an immune response in a subject is also provided, wherein the vaccine comprises an effective dose of modified influenza B virus HA.

[0167] Compositions are also provided, comprising an effective dose of a modified influenza B virus HA protein as described herein, a VLP or DS comprising a modified influenza B virus HA protein, and a pharmaceutically acceptable carrier, adjuvant, vehicle or excipient for inducing an immune response in a subject. A vaccine for inducing an immune response to influenza virus in a subject is also provided, wherein the vaccine comprises an effective dose of a modified influenza B virus HA protein, a VLP or DS comprising a modified influenza B virus HA protein.

[0168] The composition or vaccine may include VLPs containing influenza virus HA protein, wherein the HA protein is derived from the same influenza virus type, subtype, pedigree, subgenus or strain, or the composition or vaccine may include multiple VLP types, wherein each VLP type includes HA protein, wherein the HA protein can be derived from different influenza types, subtypes, pedigrees, subgenus or strains, i.e., the composition or vaccine may include a mixture of different influenza VLPs. For example, the composition or vaccine may include the first VLP and the second VLP, wherein the first VLP includes the first influenza virus HA protein from the first influenza subtype, pedigree or strain, and the second VLP includes the second influenza virus HA protein from the second influenza subtype, pedigree or strain. In addition, the composition may also include the 3rd VLP, wherein the 3rd VLP includes the 3rd influenza virus HA protein from the 3rd influenza subtype, pedigree or strain, and / or the composition or vaccine may include the 4th VLP, wherein the 4th VLP includes the 4th influenza virus HA protein from the 4th influenza subtype, pedigree, subgenus or strain.

[0169] The composition or vaccine may also include VLPs comprising HA proteins from more than one type of HA subtype, lineage or strain. For example, the VLP may include a first modified B HA protein and a second HA protein from a first B HA lineage or strain, wherein the second HA protein is derived from HA from a second B lineage or strain, or the second HA is derived from HA from an influenza A subtype or strain. In addition, the VLP may include a third HA protein, wherein the third HA is derived from a third B lineage or strain, or the third HA is derived from HA from an influenza A subtype or strain, and / or the VLP may include a fourth HA, wherein the fourth HA is derived from a fourth B lineage or strain, or the fourth HA is derived from HA from an influenza A subtype or strain.

[0170] Therefore, the present specification also provides monovalent (monovalent, univalent) or multivalent (multivalent, polyvalent) compositions or vaccines. Monovalent compositions or vaccines can immunize subjects to a single type of influenza virus strain, while multivalent compositions or vaccines can immunize subjects to more than one influenza virus strain. For example, the composition or vaccine can be a bivalent composition or vaccine, which can immunize subjects to two different types of influenza virus families, subgroups, types, subtypes, lineages or strains after administration. In addition, the composition or vaccine can be a trivalent composition, or the vaccine or composition can be a tetravalent (tetravalent, quadrivalent) composition or vaccine. In addition, for different types of viruses, vaccines can also be multivalent. For example, a vaccine can immunize a subject to one or more than one influenza virus strain (a first type of virus) and to a second type of virus (e.g., coronavirus).

[0171] Therefore, a multivalent immunogenic composition comprising two or more types of VLPs is also provided, wherein at least one type of VLP (the first type of VLP) comprises a modified B HA protein as described herein (the first modified B HA). The multivalent immunogenic composition may also comprise a second type of VLP, which also comprises a modified B HA protein as described herein (the second modified B HA), wherein the first and second types of VLPs comprise modified B HA proteins derived from different influenza B viruses. For example, the first and second VLPs may comprise modified B HA proteins belonging to different influenza B virus lineages, respectively. The multivalent immunogenic composition may further comprise one or more than one type of VLP, which comprises an influenza A virus HA protein. For example, the influenza A virus HA may be derived from influenza virus subtype H1 and / or influenza virus subtype H3.

[0172] Also provided is a tetravalent immunogenic composition, the composition comprising a first type of VLP containing a modified influenza B virus HA as described herein, a second type of VLP containing a modified influenza B virus HA as described herein, a third type of VLP containing an influenza A virus HA, and a fourth type of VLP containing an influenza A virus HA, wherein the first type of VLP comprises a modified B HA derived from an influenza B virus pedigree different from the modified B HA of the second type of VLP. For example, the modified B HA in the first type of VLP can be derived from the B / Victoria pedigree, and the modified B HA in the second type of VLP can be derived from the B / Yamagata pedigree. In addition, the influenza A virus HA of the third type of VLP can be derived from an influenza A virus subtype different from the influenza A virus HA of the fourth type of VLP. For example, the influenza A virus HA in the third type of VLP can be derived from influenza virus H3, and the influenza A virus HA in the fourth type of VLP can be derived from influenza virus H1.

[0173] 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.

[0174] Adjuvant systems for enhancing a subject's immune response to a vaccine antigen are well known and can be used in combination with a vaccine or pharmaceutical composition as described herein. A variety of adjuvants can be used. Common adjuvants used in humans are aluminum hydroxide, aluminum phosphate, and calcium phosphate. There are also some adjuvants based on oil emulsions (oil-in-water or water-in-oil emulsions, such as Freund's incomplete adjuvant (FIA), Montanide TM , Adjuvant 65 and Lipovant TM ), products from bacteria (or their synthetic derivatives), endotoxins, fatty acids, paraffin oil or vegetable oils, cholesterol and fatty amines or natural organic compounds, such as, for example, squalene. Non-limiting adjuvants that can be used include, for example, oil-in-water emulsions of squalene oil (e.g., MF-59 or AS03), adjuvants consisting of the synthetic TLR4 agonist pyranosyl lipid A (GLA) incorporated into a stable emulsion (SE) (GLA-SE) or CpG1018 (a toll-like receptor (TLR9) agonist adjuvant).

[0175] Thus, the vaccine or pharmaceutical composition may include one or more than one adjuvant. For example, the vaccine or pharmaceutical composition may include aluminum hydroxide, aluminum phosphate, calcium phosphate, oil-in-water or water-in-oil emulsions, emulsions containing squalene (e.g., MF-59 or AS03), emulsions containing GLA-SE, or CpG 1018.

[0176] Also provided herein are methods for inducing an immune response to influenza virus infection or inducing immunity to influenza virus infection in a subject, the method comprising administering to the subject orally, intranasally, intramuscularly, intraperitoneally, intravenously or subcutaneously a modified influenza B virus HA or a VLP comprising the modified influenza B virus HA.

[0177] Influenza B virus HA proteins or modified influenza B virus HA proteins as disclosed herein include any known HA protein derived from any known influenza B virus strain, but also include modifications of known influenza B virus strains developed over time. For example, influenza virus HA can be derived from B / Washington / 02 / 2019 (EPI1368874), B / Singapore / INFKK-16-0569 / 2016 (EPI592707), B / Rhode Island / 01 / 2019 (EPI1383242), B / Michigan / 01 / 2021 (EPI1843974), B / Henan-Xigong / 1118 / 2021 (EPI1878454), B / Austria / 1359417 / 2021 (EPI1845793) or B / Singapore / WUH4618 / 2021 (EPI1883660). The influenza B virus HA may include an HA derived from a strain, wherein the HA has an amino acid sequence identity of about 30-100% or any amount therebetween with any HA derived from the influenza B virus strains listed above, so long as the influenza virus HA protein comprises at least one substitution as described herein, and is capable of forming VLPs, inducing an immune response when administered to a subject, inducing hemagglutination, or a combination thereof.

[0178] For example, the influenza virus HA protein can have 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100% or any amount of amino acid sequence identity (sequence similarity, percent identity, percent similarity) to any HA derived from the influenza B virus strains listed above, and comprises at least one substitution as described herein, and is capable of forming VLPs, inducing an immune response when administered to a subject, inducing hemagglutination, or a combination thereof.

[0179] When referring to specific sequences, the terms "percent similarity", "sequence similarity", "percent identity" or "sequence identity" are used, for example, as described in the University of Wisconsin GCG software program or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds., 1995 Supplement). Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed using, for example, the algorithm of Smith & Waterman (1981, Adv. Appl. Math. 2:482), the alignment algorithm of Needleman & Wunsch (1970, J. Mol. Biol. 48:443), by the search similarity method of Pearson & Lipman (1988, Proc. Natl. Acad. Sci. USA 85:2444), by computerized 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, Wisconsin).

[0180] Examples of algorithms suitable 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 with the parameters described herein to determine percent sequence identity for nucleic acids and proteins of the invention. For example, the BLASTN program (for nucleotide sequences) can use the default values ​​of word length (W) 11, expectation (E) 10, M=5, N=-4, and comparison of both strands. For amino acid sequences, the BLASTP program can use a word length of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, Proc. Natl. Acad. Sci. USA 89: 10915), alignments (B) of 50, an expectation value (E) of 10, M = 5, N = -4, and default values ​​for 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 / ).

[0181] Influenza B virus HA proteins 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%, 95 to about 100%, or about 97 to about 100%, about 98 to about 100%, or any amount therebetween, sequence identity or sequence similarity to an influenza B virus HA sequence from 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) and wt HA B / Singapore / WUH4618 / 2021 (EPI1883660) (SEQ ID NO: 7), as long as the influenza virus HA protein contains at least one substitution as described herein and is capable of forming VLPs, inducing an immune response when administered to a subject, inducing hemagglutination, or a combination thereof.

[0182] In addition, modified influenza virus HA proteins 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%, 95% to about 100%, or about 97% to about 100%, about 98% to about 100%, or any amount of sequence identity or sequence similarity therebetween 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, as long as the influenza virus HA protein comprises at least one substitution as described herein and is capable of forming VLPs, inducing an immune response when administered to a subject, inducing hemagglutination, or a combination thereof.

[0183] As described herein, one or more than one specific mutation or modification in influenza B virus HA results in increased HA protein accumulation and increased VLP production in plants compared to unmodified influenza virus HA.

[0184] Examples of modified influenza B virus HA proteins with improved influenza virus HA and / or VLP production in plants include, but are not limited to, the following:

[0185] L404I B / Singapore / INFKK-16-0569 / 2016 mutant HA (construct #8894, SEQ ID NO: 13),

[0186] L402I B / Washington / 02 / 2019 mutant HA (construct #8881, SEQ ID NO: 17),

[0187] L402I B / Rhode Island / 01 / 2019 mutant HA (Construct #7787, SEQ ID NO: 21),

[0188] L402I B / Michigan / 01 / 2021 mutant HA (Construct #9628, SEQ ID NO: 25),

[0189] L402I B / Henan-Xigong / 1118 / 2021 mutant HA (construct #9630, SEQ ID NO: 29),

[0190] L402I B / Singapore / WUH4618 / 2021 mutant HA (Construct #9867, SEQ ID NO: 33) and

[0191] • L402I B / Austria / 1359417 / 2021 mutant HA (Construct #9869, SEQ ID NO: 37).

[0192] The genetic constructs comprising one or more than one modified B HA proteins of the present specification can be expressed in any suitable host or host cell transformed by the 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, for example, mammals. Thus, the host or host cell can be selected from plants or plant cells, fungi or fungal cells, bacteria or bacterial cells, insects or insect cells, and animals or animal cells. The mammal or animal may 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, a part of a plant, or a plant cell.

[0193] As used herein, the term "plant", "part of a plant", "plant part", "plant matter", "plant biomass", "plant material", "plant extract" or "plant leaf" may include a complete plant, tissue, cell or any part thereof, an intracellular plant component, an extracellular plant component, a liquid or solid extract of a plant or a combination thereof, which can provide transcription, translation and post-translational machinery for expressing one or more than one nucleic acid described herein, and / or from which expressed proteins or VLPs can be extracted and purified. Plants may include, but are not limited to, herbaceous plants. In addition, plants can include, but are not limited to, crop plants, including, for example, rapeseed, Brassica spp., corn, Nicotiana spp. (tobacco), e.g., Nicotiana benthamiana, Nicotiana rustica, Nicotiana, tabacum, Nicotiana alata, Arabidopsis thaliana, alfalfa, potato, sweet potato (Ipomoea batatus), ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton, corn, rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), safflower (Carthamus tinctorius).

[0194] As used herein, the term "plant part" refers to any plant part, including but not limited to leaves, stems, roots, flowers, fruits, plant cells derived from leaves, stems, roots, flowers, fruits, plant extracts or their combinations derived from leaves, stems, roots, flowers, fruits. As used herein, the term "plant extract" refers to the product of plant origin obtained after physically (for example, by freezing, then extracting in a suitable buffer), mechanically (for example, by grinding or homogenizing a plant or plant part, then extracting in a suitable buffer), enzymatically (for example, using a cell wall degrading enzyme), chemically (for example, using one or more chelating agents or buffers) or by its combination to process plants, parts of plants, plant cells or their combinations. Plant extracts can be further processed to remove undesirable plant components, for example, cell wall fragments. Plant extracts can be obtained to help reclaim one or more components from plants, parts of plants or plant cells, for example, reclaiming proteins (including protein complexes, protein superstructures and / or VLPs), nucleic acids, lipids, carbohydrates or their combinations from plants, parts of plants or plant cells. If the plant extract comprises protein, it can be referred to as a protein extract. Protein extract can be a crude plant extract, a partially purified plant or protein extract or a purified product, which comprises one or more proteins, protein complexes, protein superstructures and / or VLPs from plant tissue. If desired, protein extract or plant extract can be partially purified using technology well known to those skilled in the art. For example, extract can be subjected to salt or pH precipitation, centrifugation, gradient density centrifugation, filtration, chromatography, for example, size exclusion chromatography, ion exchange chromatography, affinity chromatography or its combination. Technical purification of protein extracts well known to those skilled in the art can also be used.

[0195] The nucleic acid encoding the modified influenza B virus HA as described herein may also include a sequence that enhances the expression of the modified influenza B virus HA in plants, parts of plants or plant cells. The sequence that enhances expression may include, for example, an expression enhancer of plant origin or an expression enhancer of plant virus origin. The expression enhancer may be effectively combined with the nucleic acid encoding the modified influenza hemagglutinin (HA) protein. The sequence encoding the modified influenza hemagglutinin (HA) may also be optimized for human codon usage, increased GC content or a combination thereof.

[0196] As used herein, the term "plant-derived expression enhancer" refers to a nucleotide sequence derived from a plant, encoding a nucleotide sequence of 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 regulatory region operably linked to the plant-derived expression enhancer sequence and a nucleotide sequence of interest.

[0197] Additionally, expression enhancing sequences may include expression enhancers from plant viruses, for example, the cowpea mosaic virus (CPMV) enhancer element.

[0198] As used herein, the term "CPMV enhancer element" refers to a nucleotide sequence encoding a 5'UTR or a modified CPMV sequence as known in the art that regulates the cowpea mosaic virus (CPMV) RNA2 polypeptide. For example, a CPMV enhancer element or CPMV expression enhancer, including WO2015 / 14367; WO2015 / 103704; WO2007 / 135480; WO2009 / 087391; Sainsbury F., and Lomonossoff GP (2008, Plant Physiol. 148: pp. 1212-1218), each of which is incorporated herein by reference. CPMV enhancer sequences can enhance the expression of downstream heterologous open reading frames (ORFs) to which they are connected. The CPMV expression enhancer may include CPMV HT, CPMVX (wherein X=160, 155, 150, 114), for example, CPMV 160, CPMVX+ (wherein X=160, 155, 150, 114), for example, CPMV 160+, CPMV-HT+, CPMV HT+[WT115] or CPMV HT+

[511] (WO2015 / 143567; WO2015 / 103704, which are incorporated herein by reference). In a preferred embodiment, the CPMV expression enhancer is CPMV 160. 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 described in the present disclosure).

[0199] "Operably linked" means that specific sequences interact directly or indirectly to implement a predetermined function, such as mediation or regulation of nucleic acid sequence expression. For example, the interaction of operably linked sequences can be mediated, for example, by a protein that interacts with the operably linked sequences.

[0200] As used herein, the term "construct", "vector" or "expression vector" refers to a recombinant nucleic acid used to transfer an exogenous nucleic acid sequence to a host cell (e.g., a plant cell) and direct the expression of the exogenous nucleic acid sequence in the host cell. An "expression cassette" refers to a nucleotide sequence comprising a promoter or other regulatory element suitable for transcription of the nucleic acid of interest in a host cell and operably (or operably) connected to the promoter or other regulatory element. As will be appreciated by those skilled in the art, an expression cassette may include a termination (terminator) sequence, which is any sequence active in a plant host. For example, the termination sequence may be derived from a bipartite RNA virus, e.g., the RNA-2 genomic segment of the cowpea mosaic virus, the termination sequence may be a NOS terminator, or the terminator sequence may be derived from the 3'UTR of the alfalfa plastocyanin gene.

[0201] The constructs disclosed herein may also include a 3' untranslated region (UTR). The 3' untranslated region contains polyadenylation signals and any other regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is generally characterized by affecting the addition of polyadenylic acid chains to the 3' end of the mRNA precursor. Polyadenylation signals are generally identified by the presence of homology to the typical form 5'AATAAA-3', although variations are not uncommon. Non-limiting examples of suitable 3' regions are non-translated regions of 3' transcription of polyadenylation signals containing Agrobacterium (Agrobacterium) tumor induction (Ti) plasmid genes, such as nopaline synthase (Nos gene) and plant genes, such as soybean storage protein genes, ribulose-1,5-bisphosphate carboxylase gene small subunit (ssRUBISCO; US 4,962,028; the patent is incorporated herein by reference), and promoters used in regulating plastocyanin expression.

[0202] "Regulatory region", "regulatory element" or "promoter" means a nucleic acid portion that is usually but not always upstream of the protein coding region of a gene, which can be composed of DNA or RNA, or composed of both DNA and RNA. When the regulatory region is active and operably combined with or operably connected to the nucleotide sequence of interest, this may result in the expression of the nucleotide sequence of interest. The regulatory element may be able to mediate organ specificity, or control development or temporal gene activation. "Regulatory region" includes promoter elements, core promoter elements that show basal promoter activity, inducible elements that respond to external stimuli, elements that mediate promoter activity, such as negative regulatory elements or transcription enhancers. As used herein, "regulatory region" also includes elements that are active after transcription, for example, regulatory elements that regulate gene expression, such as translation and transcription enhancers, translation and transcription repressors, upstream activation sequences, and mRNA instability determinants. Several of these latter elements can be located near the coding region.

[0203] In the context of the present disclosure, the term "regulatory element" or "regulatory region" generally refers to a DNA sequence that is usually, but not always, upstream (5') of the coding sequence of a structural gene, which controls the expression of the coding region by providing recognition for RNA polymerase and / or other factors required to initiate transcription at a specific site. However, it should be understood that other nucleotide sequences located within introns or at the 3' end of the sequence may also help to regulate the expression of the coding region of interest. An example of a regulatory element that provides recognition for RNA polymerase or other transcription factors to ensure initiation at a specific site is a promoter element. Most (but not all) eukaryotic promoter elements contain a TATA box, which is a conserved nucleic acid sequence consisting of adenosine and thymidine nucleotide base pairs that are usually located about 25 base pairs upstream of the transcription start site. The promoter element may include a basic promoter element responsible for transcription initiation as well as other regulatory elements that alter gene expression.

[0204] There are several types of regulatory regions, including developmentally regulated, inducible or constitutive regulatory regions. At a specific time during the development of certain organs or tissues, a regulatory region that activates developmentally regulated or controls the differential expression of genes controlled by it in the organ or tissue. However, some developmentally regulated regulatory regions may be preferentially active in certain organs or tissues at specific developmental stages, they may also be active in a developmentally regulated manner, or they may also be at a basal level in other organs or tissues in the plant. Examples of tissue-specific regulatory regions (e.g., seed-specific regulatory regions) include napin promoters and cruciferin promoters (Rask et al., 1998, J. Plant Physiol. 152: 595-599; Bilodeau et al., 1994, Plant Cell 14: 125-130). Examples of leaf-specific promoters include plastocyanin promoters (see US7,125,978, which is incorporated herein by reference).

[0205] Inducible regulatory region is a regulatory region that can directly or indirectly activate one or more DNA sequences or genes in response to an inducing agent. In the absence of an inducing agent, the DNA sequence or gene will not be transcribed. Usually, the protein factor that specifically binds to the inducible regulatory region to activate transcription can exist in an inactive form, and is then directly or indirectly converted into an active form by the inducing agent. However, the protein factor can also be non-existent. The inducing agent can be a chemical reagent, such as a protein, a metabolite, a growth regulator, a herbicide or a phenolic compound, or is directly applied by heat, cold, salt or toxic elements or indirectly applied by pathogens or disease factors, such as physiological stress indirectly applied by the effect of viruses. By applying an inducing agent to cells or plants outside, such as by spraying, watering, heating or similar methods, the plant cells comprising the inducible regulatory region can be exposed to the inducing agent. The inducible regulatory element can be derived from a plant or non-plant gene (for example Gatz, C. and Lenk, IRP, 1998, Trends Plant Sci.3, 352-358). Examples of potential inducible promoters include, but are not limited to, tetracycline-inducible promoters (Gatz, C., 1997, Ann. Rev. Plant Physiol. Plant Mol. Biol. 48, 89-108), steroid-inducible promoters (Aoyama, T. and Chua, NH, 1997, Plant J. 2, 397-404) and ethanol-inducible promoters (Salter, MG et al., 1998, Plant Journal 16, 127-132; Caddick, MX et al., 1998, Nature Biotech. 16, 177-180), cytokinin-inducible IB6 and CKI1 genes (Brandstatter, I. and Kieber, JJ, 1998, Plant Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274, 982-985) and the auxin-inducible element DR5 (Ulmasov, T et al., 1997, Plant Cell 9, 1963-1971).

[0206] Constitutive regulatory regions direct gene expression in different parts of the plant and continue to be expressed during plant development. Examples of known constitutive regulatory elements include the promoter associated with the CaMV 35S transcript (p35S; Odell et al., 1985, Nature, 313:810-812; incorporated herein by reference), the rice actin 1 (Zhang et al., 1991, Plant Cell, 3:1155-1165), actin 2 (An et al., 1996, Plant J., 10:107-121) or tms 2 (US Pat. No. 5,428,147) and triosephosphate isomerase 1 (Xu et al., 1994, Plant Physiol. 106:459-467) genes, the maize ubiquitin 1 gene (Cornejo et al., 1993, Plant Mol. Biol. 29:637-646), the Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al., 1995, Plant J. Biol. 29:637-646), the rice ... Mol.Biol.29:637-646), tobacco translation initiation factor 4A gene (Mandel et al., 1995 Plant Mol.Biol.29:995-1004), cassava vein mosaic virus promoter pCAS (Verdaguer et al., 1996); ribulose bisphosphate carboxylase small subunit promoter pRbcS: (Outchkourov et al., 2003), pUbi (for monocots and dicots).

[0207] The term "constitutive" as used herein does not necessarily mean that a nucleotide sequence under the control of a constitutive regulatory region is expressed at the same level in all cell types, but rather that the sequence is expressed in a wide range of cell types, even if variations in abundance are often observed.

[0208] The expression construct as described above may be present in a vector. The vector may include border sequences that allow the expression cassette to be transferred and integrated into the genome of an organism or host. The construct may be a plant binary vector, for example, a binary transformation vector based on pPZP (Hajdukiewicz et al., 1994). Other example constructs include pBin19 (see Frisch, DA, LW Harris-Haller et al. 1995, Plant Molecular Biology 27: 405-409).

[0209] The constructs disclosed herein can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, and the like. For a review of these techniques, see, for example, Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular Biology, 2nd edition (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2nd edition. DT. Dennis, DH Turin, DD Lefebvre, DB Layzell (eds.), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997). Other methods include direct DNA uptake, use of liposomes, electroporation, for example, use of protoplasts, microinjection, microparticles or whiskers, and vacuum infiltration.See, e.g., Bilang et al. (1991, Gene 100:247-250), Scheid et al. (1991, Mol. Gen. Genet. 228:104-112), Guerche et al. (1987, Plant Science 52:111-116), Neuhause et al. (1987, Theor. Appl Genet. 75:30-36), Klein et al. (2987, Nature 327:70-73); Freeman et al. (1984, Plant Cell Physiol. 29:1353), Howell et al. (1985, Science 227:1229-1231), DeBlock et al. (1989, Plant Physiology 91:694-701), Methods 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), WO 92 / 09696, WO 94 / 00583, EP 331083, EP 175966, Liu and Lomonossoff (2002, J Virol Meth, 105:343-348), EP 290395; WO 8706614; U.S. Patent Nos. 4,945,050; 5,036,006; and 5,100,792, U.S. Patent Application Serial Nos. 08 / 438,666, filed May 10, 1995, and 07 / 951,715, filed September 25, 1992 (all of which are incorporated herein by reference).

[0210] Transient expression methods can be used to express the constructs described in the present disclosure (see D'Aoust et al., 2009, Methods in molecular biology, volume 483, pages 41-50; Liu and Lomonossoff, 2002, Journal of Virological Methods, 105: 343-348; the above documents are incorporated herein by reference). Alternatively, vacuum-based transient expression methods can be used, such as Kapila et al. (1997, Plant Sci. 122, 101-108; the document is incorporated herein by reference) or WO 00 / 063400, WO 00 / 037663 (incorporated herein by reference). These methods can include (for example, but not limited to) methods of agrobacterium inoculation or agrobacterium infiltration, syringe infiltration, but other transient methods described above can also be used. By agrobacterium inoculation, agrobacterium infiltration or syringe infiltration, a mixture of agrobacterium containing desired nucleic acid enters the intercellular space of tissue, for example, leaves, aerial parts (including stems, leaves and flowers) of plants, other parts of plants (stems, roots, flowers) or whole plants. After passing through the epidermis, agrobacterium infects and transfers t-DNA copies into cells. The t-DNA is transcribed as an episome and its mRNA is translated, resulting in the production of the protein of interest in infected cells, however, it is instantaneous for t-DNA to enter the nucleus.

[0211] Transgenic plants, plant cells or seeds containing the genetic constructs of the present disclosure are also considered to be part of the present disclosure and can be used as platform plants suitable for transient protein expression as described herein. Methods for regenerating whole plants from plant cells are also known in the art (e.g., see Guerineau and Mullineaux (1993, Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (CroyRRD ed) Oxford, BIOS Scientific Publishers, pp. 121-148). Generally, the transformed plant cells are cultured in an appropriate culture medium, which may contain a selection agent, such as an antibiotic, wherein a selectable marker is used to facilitate recognition of the transformed plant cells. Once callus tissue is formed, shoot formation may be promoted by the use of appropriate plant hormones according to known methods, and the shoots are transferred to a rooting medium for plant regeneration. The plants can then be used to establish repeated generations from seeds or using vegetative propagation techniques. Transgenic plants can also be produced without the use of tissue culture. Methods for stable transformation and regeneration of these organisms are established in the art and are known to those skilled in the art. In Vasil et al. (Cell Culture and Somatic Cell Genetics of Plants, Vols. I, II and III, Laboratory Procedures and Their Applications, Academic Available techniques are reviewed in Weissbach and Weissbach (Methods for Plant Molecular Biology, Academic Press, 1984) and Weissbach and Weissbach (Methods for Plant Molecular Biology, Academic Press, 1989). The method used to obtain transformed and regenerated plants is not critical to the present invention.

[0212] If plant, plant part or vegetable cell will be transformed or co-transformed by two or more nucleic acid constructs, then nucleic acid construct can be introduced into Agrobacterium in a single transfection event, thereby mixed nucleic acid and transfected bacterial cells. Alternatively, construct can be introduced successively. In this case, as described, the first construct is introduced into Agrobacterium, and cells are grown under the selective conditions (for example, in the presence of antibiotic) where only the bacterium of single transformation can grow therein. After this first selection step, as described, the second nucleic acid construct is introduced into Agrobacterium, and cells are grown under the dual selective conditions where only the bacterium of double transformation can grow therein. Then, the bacterium of double transformation can be used to transform plant as herein described, part of plant or vegetable cell, or further transformation step can be carried out to accommodate the 3rd nucleic acid construct.

[0213] Alternatively, if a plant, plant part or plant cell is to be transformed or co-transformed with two or more nucleic acid constructs, the nucleic acid constructs can be introduced into the plant by co-infiltration of a mixture of Agrobacterium cells and the plant, plant part or plant cell, each Agrobacterium cell can contain one or more constructs to be introduced into the plant. In order to change the relative expression level of the nucleotide sequence of interest within the construct in the plant, plant part or plant cell, the concentration of the various Agrobacterium populations containing the desired constructs can be varied during the infiltration step.

[0214]

[0215]

[0216] The present invention will be further illustrated in the following examples.

[0217] Example 1: Influenza virus HA construct

[0218] Influenza virus HA constructs were produced using techniques well known in the art. For example, wild-type B / Singapore / INFKK-16-0569 / 2016 was cloned as described below. Other modified influenza B virus HAs were obtained using similar techniques, and HA sequence primers, templates, and products are described in Example 3 (producing influenza virus HA and VLPs in plants) and Table 5.

[0219] Table 5 below provides a summary of parental (unmodified) and modified HA proteins, primers, templates, and products.

[0220] Different strains of influenza B virus HA with M2 in the 2X35S-CPMV 160-NOS terminator (construct no. 2879, 8894, 7679, 8881, 8424, 7787, 9627, 9628, 9629, 9630, 9866, 9867, 9868 and 9869)

[0221] The sequence encoding HA0 of influenza virus HA from B / Singapore / INFKK-16-0569 / 2016, in which the native signal peptide has been replaced by the signal peptide of alfalfa protein disulfide isomerase (PDISP / HAB / Singapore / INFKK-16-0569 / 2016), was cloned into the 2X35S / CPMV160 / NOS expression system with M2 (CPMV160) from influenza strain A / New / Caledonia / 20 / 1999 using the following PCR-based method. The 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-HlcTMCT.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 in the 2X35S / CPMV160 / NOS expression system using the In-Fusion cloning system (Clontech, Mountain View, California). The construct number 4498 ( Figure 2A ) and the linearized plasmid was used for the In-Fusion assembly reaction. Construct No. 4498 is a recipient plasmid intended for "In Fusion" cloning of genes of interest in an expression cassette based on 2X35S / CPMV160 / NOS. It also introduces a gene construct for co-expressing the TBSV P19 silencing suppressor gene under the alfalfa plastocyanin gene promoter and terminator and for co-expressing M2 from influenza strain A / New / Caledonia / 20 / 1999 using the same promoter and terminator. The backbone is the pCAMBIA binary plasmid, and the sequence of the t-DNA border from left to right is as follows Figure 2A The resulting construct was numbered 2879 (SEQ ID NO: 39), and a schematic diagram of plasmid 2879 is shown in Figure 2B The amino acid sequence of the mature HA0 of influenza virus HA from B / Singapore / INFKK-16-0569 / 2016 fused to PDISP is shown in the sequence of SEQ ID NO: 11. In Example 3, the introduction of modifications into the B HA protein is described.

[0222] Example 2: Method

[0223] Agrobacterium tumefaciens transfection

[0224] Agrobacterium tumefaciens strain AGL1 was transfected by electroporation with parental (unmodified) influenza virus HA or mutant influenza virus HA expression vectors using the method described by D'Aoust et al., 2008 (Plant Biotech. J. 6: 930-40). Transfected Agrobacterium was grown in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 μM acetosyringone, 50 μg / ml kanamycin and 25 μg / ml carbenicillin at pH 5.6 until OD 0. 600 Between 0.6 and 1.6. Agrobacterium suspensions were centrifuged before use and resuspended in infiltration medium (10 mM MgCl2 and 10 mM MES pH 5.6).

[0225] Preparation of plant biomass, inoculation and Agrobacterium infiltration

[0226] Nicotiana benthamiana plants were grown from seeds in flats filled with commercial sphagnum moss substrate. Plants were grown in a greenhouse under 16 / 8 photoperiod and 25°C day / 20°C night temperature conditions. Three weeks after sowing, individual plantlets were picked, transplanted into pots, and grown in a greenhouse under the same environmental conditions for another three weeks.

[0227] Agrobacterium transfected with each parental influenza virus HA or mutant influenza virus HA expression vector was grown in YEB medium supplemented with 10 mM 2-(N-morpholino)ethanesulfonic acid (MES), 20 μM acetosyringone, 50 μg / ml kanamycin, and 25 μg / ml carbenicillin, pH 5.6, until they reached an OD between 0.6 and 1.6. 600 The Agrobacterium suspension was centrifuged before use, resuspended in infiltration medium (10 mM MgCl2 and 10 mM pH 5.6 MES) and stored at 4°C overnight. On the day of infiltration, the culture batch was diluted 2.5 times the culture volume and warmed before use. Whole plants of Nicotiana benthamiana were inverted in the bacterial suspension in an airtight stainless steel tank under a vacuum of 20-40 Torr for 2 minutes. The plants were returned to the greenhouse for a 6-day or 9-day incubation period until harvest.

[0228] Leaf harvest and total protein extraction

[0229] The cells were cut into 1 cm pieces by enzymatic extraction overnight at room temperature using an orbital shaker. 2 Protein is extracted from fresh biomass of 100% flaked cellulose. The slurry is then filtered through a large pore nylon filter to remove coarse undigested plant tissue.

[0230] In planta, clarified crude extracts were evaluated for yield and analyzed using a capillary-based electrophoresis method (ProteinSimple, BioTechne) and a WES analysis system. Briefly, soluble proteins from crude extracts were separated by molecular weight in a capillary and immobilized on a matrix. Anti-HA antibodies (Novusbiological, catalog # NB100-56578) were used for detection according to the manufacturer's instructions. Fold changes in yield were measured to evaluate changes in HA protein. Figure 3 In planta fold changes in production of modified HA normalized to the appropriate parental HA are shown.

[0231] After small-scale clarification and purification to remove impurities, drug substance (DS) yield fold changes were assessed by densitometric analysis of Coomassie-stained proteins on SDS gels, and immunologically relevant products were included in the quantification and purity measurements. Figure 4 Shown in FIG. 5 are fold changes (%) in drug substance (DS) production of mutant HA normalized to the appropriate parental HA, as further described in the present application.

[0232] Example 3: Production of Modified Influenza B Virus HA and VLPs in Plants

[0233] Modification of AB HA

[0234] Modified influenza B virus HA constructs were generated using techniques well known in the art (see Example 1). Table 5 below provides a summary of parent (unmodified) and modified HA proteins, primers, templates, and products. The sequences used are provided in Example 5 and the sequence listing.

[0235] B / Singapore / INFKK-16-0569 / 2016

[0236] The L404IB / 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). Figure 3 As shown, purified extracts from N. benthamiana plants agroinfiltrated with construct #8894 showed approximately 2.3-fold increase in yield in planta compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Singapore / INFKK-16-0569 / 2016 (construct #2879).

[0237] B / Washington / 09 / 19

[0238] The L402I B / Washington / 09 / 19 mutant HA (Construct #8881) was constructed by mutating the leucine residue at position 402 of the parental B / Washington / 09 / 19 HA to isoleucine. Figure 3 As shown, purified extracts from N. benthamiana plants agroinfiltrated with construct #8881 showed approximately a 1.9-fold increase in yield in planta compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Washington / 09 / 19 (construct #7679).

[0239] B / Rhode Island / 01 / 2019

[0240] The L402I B / Rhode Island / 01 / 2019 mutant HA (Construct #7787) was constructed by mutating the leucine residue at position 402 of the parent B / Rhode Island / 01 / 2019 to isoleucine. Figure 3 As shown, purified extracts from N. benthamiana plants agroinfiltrated with construct #7787 showed approximately a 1.5-fold increase in yield in planta compared to extracts from N. benthamiana plants agroinfiltrated with the parental B / Rhode Island / 01 / 2019HA (construct #8424).

[0241] B / Michigan / 01 / 2021

[0242] The L402I B / Michigan / 01 / 2021 mutant HA (Construct #9628) was constructed by mutating the leucine residue at position 402 of the parental B / Michigan / 01 / 2021 HA to isoleucine. Figure 3 As shown, purified extracts from N. benthamiana plants agroinfiltrated with construct #9628 showed approximately a 1.5-fold increase in yield in planta compared to extracts from N. benthamiana plants agroinfiltrated with the parental B / Michigan / 01 / 2021 HA (construct #9627).

[0243] B / Henan-Xigong / 1118 / 2021

[0244] The L402I B / Henan-Xigong / 1118 / 2021 mutant HA (construct #9630) was constructed by mutating the leucine residue at position 402 of the parental B / Henan-Xigong / 1118 / 2021 HA to isoleucine. Figure 3As shown, purified extracts from N. benthamiana plants agroinfiltrated with construct #9630 showed approximately a 1.2-fold increase in yield in plants compared to extracts from N. benthamiana plants agroinfiltrated with the parental B / Henan-Xigong / 1118 / 2021 HA (construct #9629).

[0245] B / Singapore / WUH4618 / 2021

[0246] The L402I B / Singapore / WUH4618 / 2021 mutant HA (Construct #9867) was constructed by mutating the leucine residue at position 402 of the parental B / Singapore / WUH4618 / 2021 HA to isoleucine. Figure 3 As shown, purified extracts from N. benthamiana plants agroinfiltrated with construct #9867 showed approximately a 1.3-fold increase in yield in planta compared to extracts from N. benthamiana plants agroinfiltrated with the parent B / Singapore / WUH4618 / 2021 (construct #9866).

[0247] B / Austria / 1359417 / 2021

[0248] The L402I B / Austria / 1359417 / 2021 mutant HA (Construct #9869) was constructed by mutating the leucine residue at position 402 of the parent B / Austria / 1359417 / 2021 to isoleucine. Figure 3 As shown, purified extracts of N. benthamiana plants agroinfiltrated with construct #9869 showed approximately a 1.3-fold increase in yield in planta compared to extracts of N. benthamiana plants agroinfiltrated with the parental B / Austria / 1359417 / 2021 HA (construct #9868).

[0249] One or more than one modification described herein specifically increases the production of influenza virus HA protein and VLP yield in plants.

[0250] Example 4: In vivo production and drug substance (DS) production

[0251] A summary of in planta and drug substance (DS) yields is given in Table 4. In planta yields were measured as described in Example 2. In planta yield fold changes were obtained by comparing the yield of the mutant or modified HA protein with that of the appropriate parental HA (see Figure 3 ).

[0252] The fold change in DS production was obtained by comparing the production of mutant or modified HA proteins with that of the appropriate unmodified (parental) HA (see Figure 4 ).

[0253] Table 4: Increased in planta and drug substance (DS) production of modified B HA protein (L402I) compared to unmodified B HA protein

[0254]

[0255]

[0256] Example 5: Sequence

[0257] The following sequences were used (see also Table 4):

[0258] wt HA B / Washington / 02 / 2019 AA(SEQ ID NO: 1)

[0259]

[0260] wt HA B / Singapore / INFKK-16-0569 / 2016 AA(SEQ ID NO: 2)

[0261]

[0262]

[0263] wt HA B / Rhode Island / 01 / 2019 AA(SEQ ID NO: 3)

[0264]

[0265] wt HA B / Michigan / 01 / 2021 AA(SEQ ID NO: 4)

[0266]

[0267]

[0268] wt HA B / Henan-Xigong / 1118 / 2021 AA(SEQ ID NO: 5)

[0269]

[0270] wt HA B / Austria / 1359417 / 2021 AA(SEQ ID NO: 6)

[0271]

[0272]

[0273] wt HA B / Singapore / WUH4618 / 2021 AA(SEQ ID NO:7)

[0274]

[0275] IF-SpPDI.c DNA(SEQ ID NO:8)

[0276]

[0277] IF-H1cTMCT.s1-4r DNA(SEQ ID NO:9)

[0278]

[0279] B / Singapore / INFKK-16-0569 / 2016(PrL-)DNA(SEQ ID NO:10)

[0280]

[0281]

[0282] B / Singapore / INFKK-16-0569 / 2016(PrL-)AA(SEQ ID NO:11)

[0283]

[0284]

[0285] B / Singapore / INFKK-16-0569 / 2016(PrL-)+L404IDNA(SEQ ID NO:12)

[0286]

[0287]

[0288] B / Singapore / INFKK-16-0569 / 2016(PrL-)+L404I AA(SEQ ID NO:13)

[0289]

[0290] B / Washington / 02 / 2019(PrL-)DNA(SEQ ID NO:14)

[0291]

[0292] B / Washington / 02 / 2019(PrL-)AA(SEQ ID NO:15)

[0293]

[0294] B / Washington / 02 / 2019(PrL-)+L402IDNA(SEQ ID NO:16)

[0295]

[0296]

[0297] B / Washington / 02 / 2019(PrL-)+L402I AA(SEQ ID NO:17)

[0298]

[0299]

[0300] B / Rhode Island / 01 / 2019(PrL-)DNA(SEQ ID NO:18)

[0301]

[0302] B / Rhode Island / 01 / 2019(PrL-)AA(SEQ ID NO:19)

[0303]

[0304] B / Rhode Island / 01 / 2019(PrL-)+L402IDNA(SEQ ID NO:20)

[0305]

[0306]

[0307] B / Rhode Island / 01 / 2019(PrL-)+L402I AA(SEQ ID NO:21)

[0308]

[0309]

[0310] B / Michigan / 01 / 2021(PrL-)DNA(SEQ ID NO:22)

[0311]

[0312]

[0313] B / Michigan / 01 / 2021(PrL-)AA(SEQ ID NO:23)

[0314]

[0315] B / Michigan / 01 / 2021(PrL-)+L402I DNA(SEQ ID NO:24)

[0316]

[0317]

[0318] B / Michigan / 01 / 2021(PrL-)+L402I AA(SEQ ID NO:25)

[0319]

[0320]

[0321] B / Henan-Xigong / 1118 / 2021(PrL-)DNA(SEQ ID NO:26)

[0322]

[0323]

[0324] B / Henan-Xigong / 1118 / 2021(PrL-)AA(SEQ ID NO:27)

[0325]

[0326] B / Henan-Xigong / 1118 / 2021(PrL-)+L402IDNA(SEQ ID NO:28)

[0327]

[0328]

[0329] B / Henan-Xigong / 1118 / 2021(PrL-)+L402I AA(SEQ ID NO:29)

[0330]

[0331]

[0332] B / Singapore / WUH4618 / 2021(PrL-)DNA(SEQ ID NO:30)

[0333]

[0334]

[0335] B / Singapore / WUH4618 / 2021(PrL-)AA(SEQ ID NO:31)

[0336]

[0337] B / Singapore / WUH4618 / 2021(PrL-)+L402IDNA(SEQ ID NO:32)

[0338]

[0339]

[0340] B / Singapore / WUH4618 / 2021(PrL-)+L402I AA(SEQ ID NO:33)

[0341]

[0342]

[0343] B / Austria / 1359417 / 2021(PrL-)DNA(SEQ ID NO:34)

[0344]

[0345]

[0346] B / Austria / 1359417 / 2021(PrL-)AA(SEQ ID NO:35)

[0347]

[0348] B / Austria / 1359417 / 2021(PrL-)+L402IDNA(SEQ ID NO: 36)

[0349]

[0350]

[0351] B / Austria / 1359417 / 2021(PrL-)+L402I AA(SEQ ID NO: 37)

[0352]

[0353]

[0354] Cloning vector 4498 T-DNA from left to right (SEQ ID NO: 38)

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361] Construct 2879, 2X35S promoter to NOS terminator DNA (SEQ ID NO: 39)

[0362]

[0363]

[0364]

[0365] Influenza B virus HA2 consensus sequence (SEQ ID NO: 40) (fusion peptide (FP) italicized, TM / CT underlined)

[0366]

[0367] in

[0368] X(78)=L or I

[0369] X(129) = S or does not exist

[0370] X(132)=E or D

[0371] X(158) = N or D

[0372] X(159)A or D

[0373] X(204)=L or I

[0374] X(208)=I or V

[0375] Influenza B virus HA2 fusion peptide and HA2 extracellular domain consensus sequence with modification (X1) at position 402 (SEQ ID NO: 41)

[0376]

[0377] Influenza B virus HA2 extracellular domain consensus sequence with modification (X1) at position 402 (SEQ ID NO: 42)

[0378]

[0379] in:

[0380] X1=I

[0381] X2=L or I

[0382] X3=S or does not exist

[0383] X4=E or D

[0384] X5=N or D

[0385] X6=A or D

[0386] wt H5 A / Indonesia / 5 / 2005 AA(SEQ ID NO: 43)

[0387]

[0388]

[0389] All citations are incorporated herein by reference.

[0390] The invention has been described with respect to one or more embodiments. However, it will be apparent to those skilled in the art that certain changes and modifications may 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 extracellular domain, wherein: The modified HA2 extracellular domain comprises an amino acid sequence having at least one amino acid substitution compared to the parent HA2 extracellular domain amino acid sequence, wherein the at least one substitution corresponds to amino acid position 402 in the sequence alignment with the reference sequence SEQ ID NO: 1 (B / Washington / 09 / 19HA).

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

3. The modified influenza B virus HA protein according to claim 1 or 2, wherein The substitution is non-leucine.

4. The modified influenza B virus HA protein according to claims 1 to 3, wherein The substitution is for isoleucine or is a conservative substitution of isoleucine.

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

6. The modified influenza B virus HA protein according to claim 1, wherein The sequence of the modified HA2 extracellular domain has 80% to 100% identity to the sequence of SEQ ID NO:

42.

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

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

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

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

11. A method for producing a modified influenza B virus HA protein in a non-human host or a host cell, comprising: a) introducing the nucleic acid according to claim 9 into the non-human host or host cell, or providing the non-human host or host cell comprising the nucleic acid according to claim 9, and b) cultivating the non-human host or host cell under conditions allowing expression of the nucleic acid, thereby producing the modified influenza B virus HA protein.

12. A method for increasing the yield of influenza B virus HA protein in a non-human host or host cell, comprising: a) introducing the nucleic acid according to claim 9 into the non-human host or host cell; or providing the non-human host or host cell comprising the nucleic acid according to claim 9; and b) cultivating the non-human host or host cell under conditions that allow expression of the modified B HA protein encoded by the nucleic acid, thereby producing the modified B HA at a higher yield compared to a non-human host or host cell expressing an influenza B virus HA protein comprising the HA2 extracellular domain parent amino acid sequence.

13. The method according to claim 11 or 12, wherein: The modified influenza B virus HA protein is further extracted and purified from the non-human host or host cell.

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

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

16. A method for increasing the yield of influenza virus-like particles (VLPs) in a non-human host or a host cell, comprising: a) introducing the nucleic acid according to claim 9 into the non-human host or host cell; or providing the non-human host or host cell comprising the nucleic acid according to claim 9; and b) cultivating the non-human host or host cell under conditions that allow expression of the modified B HA protein encoded by the nucleic acid, thereby producing the VLPs in a higher yield compared to a non-human host or host cell expressing an influenza B virus HA protein comprising the HA2 extracellular domain parent amino acid sequence.

17. The method according to claim 15 or 16, wherein: The method further comprises step c), harvesting the non-human host or host cell, and extracting and purifying the VLP.

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

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

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

21. An antibody produced by the method according to claim 20.

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

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

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

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

26. A method for eliciting an immune response to influenza virus infection in a subject, the method comprising administering to the subject a VLP according to any one of claims 10, 18 or 19, a composition according to claim 23, or a vaccine according to claim 24 or 25.

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

28. The method according to any one of claims 11 or 15-17, wherein The non-human host or host cell includes 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 types of VLPs, wherein: At least one type of VLP comprises the modified influenza B virus HA according to any one of claims 1-8.

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 the modified influenza B virus HA according to any one of claims 1-8.

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

32. The multivalent immunogenic composition according to any one of claims 29 to 31, wherein The composition also comprises one or more than one type of VLPs comprising influenza A virus HA protein.

33. A tetravalent immunogenic composition comprising a first type of VLP comprising a modified influenza B virus HA according to any one of claims 1-8, a second type of VLP comprising a modified influenza B virus HA according to any one of claims 1-8, a third type of VLP comprising an influenza A virus HA and a fourth type of VLP comprising an influenza A virus HA, wherein: The first type of VLPs comprises a modified B HA derived from an influenza B virus lineage that is different from the modified B HA of the second type of VLPs.

Images