Influenza B virus vaccine and its use

Abstract

Provided herein are isolated variant influenza hemagglutinin polypeptides, methods for providing isolated variant influenza hemagglutinin polypeptides, methods of using isolated variant influenza hemagglutinin polypeptides as diagnostic agents or for isolating antibodies, compositions comprising same, vaccines comprising same, and methods of their use, particularly in the detection, prevention, and / or treatment of influenza.

Description

[Technical Field]

[0001] Introduction The present invention relates to the field of medicine. Provided herein are isolated influenza hemagglutinin polypeptides, methods for providing hemagglutinin type B polypeptides, compositions comprising same, vaccines comprising same, and methods of their use, particularly in the detection, prevention, and / or treatment of influenza type B. [Background technology]

[0002] Influenza A and B viruses are major human pathogens, causing respiratory illnesses (commonly referred to as "influenza" or "flu") ranging in severity from subclinical infection to primary viral pneumonia, which can be fatal. The WHO estimates that annual influenza epidemics result in approximately 1 billion infections, 3-5 million cases of severe illness, and 300,000-500,000 deaths. The severity of pandemic influenza depends on multiple factors, including the virulence of the pandemic virus strain and the level of pre-existing immunity. The most severe influenza pandemic, in 1918, resulted in over 40 million deaths worldwide. Influenza vaccines are formulated annually to match circulating strains, as these strains antigenically evolve due to antigenic drift. Nevertheless, vaccine efficacy is suboptimal and dramatically reduced in the event of an antigenic mismatch between the vaccine and the circulating virus strain. Antiviral agents targeting the influenza virus enzyme neuraminidase have been developed for prophylaxis and therapy. However, the use of these antiviral agents remains limited.An emerging approach to combating influenza is the development of a universal influenza virus vaccine that would provide protection against antigenically distant influenza viruses (1).

[0003] Over the past 30 years, two distinct influenza B lineages have cocirculated in the population to varying degrees each season, and the dominant B lineage in a given season has proven difficult to predict, complicating decisions about which lineages to include in trivalent vaccines (TIVs) (2; US Centers for Disease Control and Prevention, “Seasonal influenza activity surveillance reports 2001–2018,” www.cdc.gov / flu / weekly / pastreports.htm (accessed July 2, 2018); European Centre for Disease Prevention and Control / WHO Regional Office for Europe, “Annual epidemiological reports on seasonal influenza 2001–2018,” ecdc.europa.eu / en / seasonal-influenza / surveillance-and-disease-data / aer (accessed July 2, 2018)). The importance of effective influenza B coverage through vaccination is illustrated by its contribution to the overall burden of seasonal influenza.According to data from the US Centers for Disease Control and reports from several European countries, influenza type B caused 0.8–82% of all laboratory-confirmed influenza cases from 2001–2018, with a seasonal average of 25% ((2); US Centers for Disease Control and Prevention, “Seasonal influenza activity surveillance reports 2001–2018,” www.cdc.gov / flu / weekly / pastreports.htm (accessed July 2, 2018); European Centre for Disease Prevention and Control / WHO Regional Office for Europe, “Annual epidemiological reports on seasonal influenza 2001–2018,” ecdc.europa.eu / en / seasonal-influenza / surveillance-and-disease-data / aer (accessed July 2, 2018) ((3); (4)). Furthermore, influenza type B is the leading cause of overall influenza morbidity and mortality, with attributable hospitalization rates similar to influenza A / H3N2 and higher than A / H1N1 (Thompson et al., JAMA 292:1333-40 (2004)), accounting for 15% of all influenza-related respiratory and cardiovascular deaths in the United States and 34% of pediatric deaths (Ambrose et al., Hum. Vaccin. Immunother. 8:81-8 (2012), (5)).These principles have prompted several health organizations, including the World Health Organization and the US Advisory Committee on Immunization Practices, to recommend the quadrivalent influenza vaccine (QIV), which contains two influenza B antigens (one of each B lineage), as an option for seasonal vaccination (( 6 ); ( 7 ); World Health Organization, “Recommended composition of influenza virus vaccines for use in the 2017-2018 northern hemisphere influenza season,” www.who.int / influenza / vaccines / virus / recommendations / 2018_19_north / en (accessed July 2, 2018)).

[0004] Influenza type B virus strains are found almost exclusively in humans. Antigenic variation in HA within influenza type B virus strains is less than that observed within type A strains. Two genetically and antigenically distinct lineages of influenza type B virus circulate in humans, represented by the B / Yamagata / 16 / 88 (also called B / Yamagata) and B / Victoria / 2 / 87 (B / Victoria) lineages (8). The spectrum of disease caused by influenza type B viruses is generally milder than that caused by influenza type A viruses; however, severe disease requiring hospitalization is still frequently observed with influenza type B virus infection.

[0005] Antibodies that neutralize influenza viruses are known to be primarily directed against hemagglutinin (HA). Hemagglutinin, or HA, is a trimeric glycoprotein anchored to the viral coat and has dual functions: it binds to the cell surface receptor sialic acid and, after internalization, mediates the fusion of the viral membrane with the endosomal membrane, resulting in the release of viral RNA in the cytosol of the cell. HA contains a large head domain and a smaller stem domain. Attachment to the viral membrane is mediated by a C-terminal anchor sequence connected to the stem domain. The protein is posttranslationally cleaved at a designated loop to generate two polypeptides, HA1 and HA2 (the complete sequence is referred to as HA0). The membrane-distal head region is primarily derived from HA1, and the membrane-proximal stem region is primarily derived from HA2.

[0006] For most established vaccine platforms and novel approaches, expression of high-quality antigens in the most relevant conformation is a critical success factor. Influenza vaccine production can be challenging due to the unstable quaternary structure and low expression levels of HA (9). Stress conditions such as heat or prolonged storage can reduce the efficacy of protein-based vaccines, and improving stability can extend vaccine shelf life and alleviate cold-chain issues often encountered in remote or impoverished regions of the world. The pH values ​​that induce HA stability and conformational transitions vary between strains, and few stabilizing mutations have been identified (WO 2021 / 074286). Because the conformational transition of influenza B HA is triggered by low pH, removal of the pH-triggered switch can increase pH stability, which has also been shown to increase general HA stability. While native influenza HA is cleaved into HA1 and HA2 polypeptides, this cleavage does not occur in typical protein expression systems. Therefore, a proteolytic enzyme such as trypsin is added to cleave the precursor HA0 into HA1 and HA2. The cleaved protein is able to fold into a mature, stable trimeric conformation but is sensitive to conformational changes induced by low pH, so hyaluronan cleavage must occur after translocation through low pH cellular compartments.

[0007] Therefore, there is a need to develop strategies that provide a means to obtain high-quality reagents for diagnostics, B cell and mAb isolation, and robust reagents for influenza structural studies. Most importantly, stabilizing HA could improve the developability and efficacy of influenza B virus strains based on various platforms. Summary of the Invention [Means for solving the problem]

[0008] Provided herein are isolated variant influenza hemagglutinin polypeptides, methods of providing the isolated hemagglutinin polypeptides, compositions comprising same, vaccines comprising same, and methods of using the compositions and vaccines.

[0009] Provided herein is an isolated mutant influenza hemagglutinin polypeptide, the isolated mutant influenza hemagglutinin polypeptide comprising at least one stabilizing mutation in at least one of the instability regions (a)-(e) of the polypeptide, wherein the at least one stabilizing mutation comprises a substitution at (a) amino acid position 227, 229, and / or 238 of the head switch, and / or (b) amino acid position 329 and / or 426 of the neck switch, and / or (c) amino acid position 384, 402, 472, and / or 476 of the stem switch, and / or (d) amino acid position 468, 471, 475, or 478 of the repulsive three-fold cluster, and / or (e) amino acid position 235, 430, and / or 433 of the hinge loop, wherein the amino acid positions correspond to those of SEQ ID NO: 1.

[0010] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least two stabilizing mutations in at least one of the instability regions (a)-(e) in the polypeptide.

[0011] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least two stabilizing mutations in two, three, four, or five of the instability regions (a)-(e) in the polypeptide. In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least three stabilizing mutations in three, four, or five of the instability regions (a)-(e) in the polypeptide. In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least four stabilizing mutations in four or five of the instability regions (a)-(e) in the polypeptide. In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least five stabilizing mutations in five of the instability regions (a)-(e) in the polypeptide.

[0012] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 stabilizing mutations.

[0013] In certain embodiments, (a) amino acid position 227 is substituted with an amino acid selected from the group consisting of T, L, R, Q, F, I, and Y, amino acid position 229 is substituted with the amino acid L, and / or amino acid position 238 is substituted with the amino acid F in a head switch, and / or (b) amino acid position 329 is substituted with an amino acid selected from the group consisting of M, W, and F, and / or amino acid position 426 is substituted with an amino acid selected from the group consisting of F, W, Y, and P in a neck switch, and / or (c) amino acid position 384 is substituted with an amino acid selected from the group consisting of F or Y, amino acid position 402 is substituted with the amino acid A, and amino acid position 472 is substituted with an amino acid selected from the group consisting of W, R, F, K, or and / or amino acid position 476 is substituted with amino acid F in the stem switch; and / or (d) amino acid position 468 is substituted with amino acid L, amino acid position 471 is substituted with an amino acid selected from V or Q, amino acid position 475 is substituted with an amino acid selected from the group consisting of Q, N, W, F, and L, or amino acid position 478 is substituted with an amino acid selected from W or R in the hinge loop; and / or (e) amino acid position 235 is substituted with amino acid W, amino acid position 430 is substituted with an amino acid selected from L or Y, and / or amino acid position 433 is substituted with amino acid P in the hinge loop.

[0014] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises the following amino acid sequence: (a) amino acid position 384 is substituted with F and amino acid position 475 is substituted with W, (b) amino acid position 384 is substituted with F and amino acid position 475 is substituted with Q, (c) amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 472 is substituted with K, and amino acid position 476 is substituted with F, (d) amino acid position 384 is substituted with F and (e) amino acid position 384 is substituted with Y, amino acid position 402 is substituted with A, amino acid position 472 is substituted with R, and amino acid position 476 is substituted with F, (f) amino acid position 476 is substituted with F and amino acid position 475 is substituted with W, (g) amino acid position 476 is substituted with F and amino acid position 475 is substituted with Q, (h) amino acid position 2 (i) amino acid position 227 is substituted with T and amino acid position 426 is substituted with Y, (j) amino acid position 227 is substituted with T and amino acid position 426 is substituted with Y and amino acid position 430 is substituted with Y, (k) amino acid position 227 is substituted with T and amino acid position 475 is substituted with W, (l) amino acid position 227 is substituted with T and amino acid position 475 is substituted with Q, (m) amino acid position 227 is substituted with T and amino acid position 384 is substituted with F, (n) amino acid position 227 is substituted with T and amino acid position 476 is substituted with F, (o) amino acid position 227 is substituted with T and amino acid position 426 is substituted with Y and amino acid position 475 is substituted with W, (p) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y and amino acid position 475 is substituted with Q, (q) amino acid position 227 is substituted with T and amino acid position 426 is substituted with Y,(r) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 475 is substituted with Q, and amino acid position 430 is substituted with Y, (s) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, and amino acid position 384 is substituted with F, (t) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, and amino acid position 384 is substituted with (u) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 384 is substituted with F, amino acid position 475 is substituted with W, and amino acid position 430 is substituted with Y; (v) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 384 is substituted with F, and amino acid position 475 is substituted with Q; (w) amino acid position 227 is substituted with T, and (x) amino acid position 227 is substituted with T and amino acid position 235 is substituted with W; (y) amino acid position 475 is substituted with W and amino acid position 426 is substituted with Y; (z) amino acid position 475 is substituted with W and amino acid position 430 is substituted with Y; (aa) amino acid position 475 is substituted with Q and amino acid position 430 is substituted with Y; (bb) amino acid position 475 is substituted with Q and amino acid position 478 is substituted with F, (cc) amino acid position 426 is substituted with Y and amino acid position 384 is substituted with F, (dd) amino acid position 426 is substituted with Y and amino acid position 476 is substituted with F, (ee) amino acid position 426 is substituted with Y and amino acid position 455 is substituted with A, (ff) amino acid position 426 is substituted with Y and amino acid position 329 is substituted with W.(gg) amino acid position 430 is substituted with Y and amino acid position 384 is substituted with F, (hh) amino acid position 430 is substituted with Y and amino acid position 476 is substituted with F, (ii) amino acid position 430 is substituted with Y and amino acid position 426 is substituted with Y, (jj) amino acid position 430 is substituted with Y and amino acid position 426 is substituted with W, (kk) amino acid position 430 is substituted with Y and amino acid position 426 is substituted with F, (ll) amino acid position 433 is substituted with P and amino acid position 430 is substituted with Y, (mm) amino acid position 433 is substituted with P and amino acid position 426 is substituted with Y, (nn) amino acid position 433 is substituted with P and amino acid position 426 is substituted with W, (oo) amino acid position 433 is substituted with P and amino acid position 426 is substituted with F, (pp) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, and amino acid position 475 is substituted with W, (qq) amino acid position 227 is substituted with T (rr) wherein amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 472 is substituted with R, and amino acid position 476 is substituted with F; (rr) wherein amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 468 is substituted with L, and amino acid 471 is substituted with V and amino acid position 472 is substituted with F; (ss) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 468 is substituted with L, amino acid 471 is substituted with V, amino acid position 472 is substituted with W, amino acid position 475 is substituted with F, and amino acid position 478 is substituted with R; (tt) amino acid position 227 is substituted with T and amino acid position 384 is substituted with F;(uu) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 472 is substituted with W, and amino acid position 476 is substituted with F, (uu) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 468 is substituted with L, amino acid 471 is substituted with V, amino acid position 472 is substituted with W, amino acid position 475 is substituted with L, and amino acid position 478 is substituted with R, (v v) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 468 is substituted with L, amino acid position 471 is substituted with Q, amino acid position 472 is substituted with W, amino acid position 475 is substituted with Q, and amino acid position 478 is substituted with R; (ww) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y; (xx) amino acid position 430 is substituted with Y, amino acid position 472 is substituted with K, amino acid position 475 is substituted with Q, and amino acid position 476 is substituted with F; (xx) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 472 is substituted with L, amino acid position 475 is substituted with Q, and amino acid position 476 is substituted with F; (yy) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 471 is substituted with Q, amino acid position 472 is substituted with R, and amino acid position 476 is substituted with F; (zz) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, and amino acid position 426 is substituted with Y;a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 476 with F; (aaa) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, and a substitution at amino acid position 476 with F; or (bbb) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with R, a substitution at amino acid position 475 with W, and a substitution at amino acid position 476 with F;

[0015] In certain embodiments, the mutant influenza hemagglutinin polypeptide further comprises an introduced cleavage site. The introduced cleavage site can be, for example, a furin cleavage site. The furin cleavage site can be introduced, for example, by mutating amino acid positions 359-361 of the polypeptide or by inserting it amino-terminally at amino acid position 362, which corresponds to the amino acid position of SEQ ID NO: 1. In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide further comprises an insertion of the RSV p27 peptide (SEQ ID NO: 2) carboxy-terminally at amino acid position 362.

[0016] In certain embodiments, amino acid position 362 is substituted with Q.

[0017] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide further comprises a deletion of a signal peptide at the amino terminus of the polypeptide, e.g., the signal peptide comprises amino acid positions 1-15 of the polypeptide.

[0018] In certain embodiments, the variant influenza hemagglutinin polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 35, 46-48, 63, 67-75, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 181, 186, 189, 201, 202, 204-208, 216, and 219-222. In certain embodiments, the variant influenza hemagglutinin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 35, 46-48, 63, 67-75, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 181, 186, 189, 201, 202, 204-208, 216, and 219-222, wherein the variant influenza hemagglutinin polypeptide comprises a deletion of a signal peptide at the amino terminus of the polypeptide.

[0019] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide further comprises a carboxy (C)-terminal truncation beginning at amino acid positions 536 through 585, the amino acid positions corresponding to the amino acid positions of SEQ ID NO:1.

[0020] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide further comprises at least one additional glycan motif in the head domain of the polypeptide. The glycan motif can comprise, for example, an amino (N)-linked glycosylation motif substitution at at least one amino acid position selected from the group consisting of (a) 136 or 137, (b) 141, and (c) 151, where the amino acid positions correspond to the amino acid positions of SEQ ID NO: 1. The glycan motif can comprise, for example, an N-linked glycosylation motif substitution at amino acid positions 136 and 141, 136 and 151, 137 and 141, 137 and 151, or 141 and 151. In certain embodiments, the glycan motif comprises an N-linked glycosylation motif substitution at amino acid positions 141 and 151.

[0021] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide further comprises a receptor-binding site mutation in the polypeptide. The receptor-binding site mutation can include, for example, a substitution at an amino acid position selected from the group consisting of (a) 175, (b) 219, (c) 257, and (d) 258, which amino acid positions correspond to amino acid positions in SEQ ID NO: 1. In certain embodiments, (a) 175 is substituted with an amino acid selected from the group consisting of F, W, and Y; (b) 219 is substituted with an amino acid selected from the group consisting of F, W, Y, R, and E; (c) 257 is substituted with an amino acid selected from the group consisting of E, D, V, and F; or (a) 258 is substituted with an amino acid selected from the group consisting of E, D, V, and F. In certain embodiments, (a) 175 is substituted with W, (b) 219 is substituted with E, (c) 257 is substituted with E, or (d) 258 is substituted with E.

[0022] Also provided is an isolated nucleic acid encoding the isolated mutant influenza hemagglutinin polypeptide described herein.

[0023] Also provided are isolated vectors comprising the isolated nucleic acids described herein.

[0024] Also provided is an isolated host cell comprising the vector described herein.

[0025] Also provided are pharmaceutical compositions comprising the isolated mutant influenza hemagglutinin polypeptides, isolated mutant influenza hemagglutinin nucleic acids, and / or isolated vectors described herein and a pharmaceutically acceptable carrier.

[0026] Also provided is a method of inducing an immune response against influenza virus in a subject in need thereof, the method comprising administering to a subject in need thereof a pharmaceutical composition described herein.

[0027] Also provided is a method of producing an isolated variant influenza hemagglutinin polypeptide, comprising culturing an isolated host cell described herein under conditions capable of producing the variant influenza hemagglutinin polypeptide, and recovering the variant influenza hemagglutinin polypeptide from the isolated host cell or culture.

[0028] Also provided are methods of making the pharmaceutical compositions described herein, comprising combining an isolated mutant influenza polypeptide with a pharmaceutically acceptable carrier.

[0029] Various embodiments and uses of the polypeptides according to the present invention will become apparent from the following detailed description of the invention. [Brief explanation of the drawings]

[0030] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1A]Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1B] Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1C]Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1D] Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1E]Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1F] Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1G]Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1H] Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1I]Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1J] Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 1K]Structural and design elements of polypeptides of the invention are shown in Figure 1A. A three-dimensional representation of a polypeptide of the invention (representing the influenza B HA ectodomain; pdb ID 4NRJ (10)). Black spheres indicate the locations of substitutions in the instability region (Figures 1B-1F) and three other regions (Figures 1G-1I). The locations of substitutions for each region are shown in individual panels: head switch (Figure 1B), neck switch (Figure 1C), stem switch (Figure 1D), hinge loop (Figure 1E), repulsive three-fold cluster (Figure 1F), fusion peptide (Figure 1G), receptor binding site (Figure 1H), and base (Figure 1I). Figure 1J. A schematic diagram of one particular polypeptide of the invention, UFV220265 (SEQ ID NO: 73), with the locations of substitutions indicated. φ In this example, the C-terminus is truncated after residue 536 (numbering refers to WT HA; SEQ ID NO: 1). Figure 1K. The polypeptide cleavable during the process; the numbering and mutations as in (FIG. 1J) are shown together with the introduced 27-residue peptide (SEQ ID NO: 2) containing the polybasic cleavage site. [Figure 2-1] Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with substitutions in the receptor binding site (position 167), base (position 392), and fusion peptide (positions 373, 377, 380, and 391), as well as instability regions: head switch (positions 227, 229, 231, 236, 238, and 277), neck switch (positions 329, 332, 426, 453, and 455), stem switch (positions 384, 402, 472, 473, and 476), hinge loop (positions 235, 429, 430, and 433), and repulsive cluster (positions 475 and 478). Analytical size-exclusion chromatography profiles show the amounts of trimer (T) and monomer (M) of stabilized variants in the supernatant of HEK293 cells after transfection compared to wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimer and monomer are indicated by dashed lines. [Figure 2-2]Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with substitutions in the receptor binding site (position 167), base (position 392), and fusion peptide (positions 373, 377, 380, and 391), as well as instability regions: head switch (positions 227, 229, 231, 236, 238, and 277), neck switch (positions 329, 332, 426, 453, and 455), stem switch (positions 384, 402, 472, 473, and 476), hinge loop (positions 235, 429, 430, and 433), and repulsive cluster (positions 475 and 478). Analytical size-exclusion chromatography profiles show the amounts of trimer (T) and monomer (M) of stabilized variants in the supernatant of HEK293 cells after transfection compared to wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimer and monomer are indicated by dashed lines. [Figure 2-3] Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with substitutions in the receptor binding site (position 167), base (position 392), and fusion peptide (positions 373, 377, 380, and 391), as well as instability regions: head switch (positions 227, 229, 231, 236, 238, and 277), neck switch (positions 329, 332, 426, 453, and 455), stem switch (positions 384, 402, 472, 473, and 476), hinge loop (positions 235, 429, 430, and 433), and repulsive cluster (positions 475 and 478). Analytical size-exclusion chromatography profiles show the amounts of trimer (T) and monomer (M) of stabilized variants in the supernatant of HEK293 cells after transfection compared to wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimer and monomer are indicated by dashed lines. [Figure 2-4]Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with substitutions in the receptor binding site (position 167), base (position 392), and fusion peptide (positions 373, 377, 380, and 391), as well as instability regions: head switch (positions 227, 229, 231, 236, 238, and 277), neck switch (positions 329, 332, 426, 453, and 455), stem switch (positions 384, 402, 472, 473, and 476), hinge loop (positions 235, 429, 430, and 433), and repulsive cluster (positions 475 and 478). Analytical size-exclusion chromatography profiles show the amounts of trimer (T) and monomer (M) of stabilized variants in the supernatant of HEK293 cells after transfection compared to wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimer and monomer are indicated by dashed lines. [Figure 2-5] Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with substitutions in the receptor binding site (position 167), base (position 392), and fusion peptide (positions 373, 377, 380, and 391), as well as instability regions: head switch (positions 227, 229, 231, 236, 238, and 277), neck switch (positions 329, 332, 426, 453, and 455), stem switch (positions 384, 402, 472, 473, and 476), hinge loop (positions 235, 429, 430, and 433), and repulsive cluster (positions 475 and 478). Analytical size-exclusion chromatography profiles show the amounts of trimer (T) and monomer (M) of stabilized variants in the supernatant of HEK293 cells after transfection compared to wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimer and monomer are indicated by dashed lines. [Figure 3] Scatter plot representation of the data in Table 1; trimer peak area versus % trimer and dot size representing temperature stability. Wild-type Iowa Flu B HA is highlighted (UFV212130, gray dots). [Figure 4A-1]Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with combinations of substitutions in the head switch, neck switch, stem switch, hinge loop, and repulsive cluster regions. Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized B HA variants in the supernatant of HEK293 cells after transfection, compared with wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimers and monomers are indicated by dashed lines. Figures 4A, 4B, 4C, 4D, and 4E show combinations of mutations in 1, 2, 3, 4, and all five regions, respectively. [Figure 4A-2] Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with combinations of substitutions in the head switch, neck switch, stem switch, hinge loop, and repulsive cluster regions. Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized B HA variants in the supernatant of HEK293 cells after transfection, compared with wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimers and monomers are indicated by dashed lines. Figures 4A, 4B, 4C, 4D, and 4E show combinations of mutations in 1, 2, 3, 4, and all five regions, respectively. [Figure 4B-1] Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with combinations of substitutions in the head switch, neck switch, stem switch, hinge loop, and repulsive cluster regions. Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized B HA variants in the supernatant of HEK293 cells after transfection, compared with wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimers and monomers are indicated by dashed lines. Figures 4A, 4B, 4C, 4D, and 4E show combinations of mutations in 1, 2, 3, 4, and all five regions, respectively. [Figure 4B-2]Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with combinations of substitutions in the head switch, neck switch, stem switch, hinge loop, and repulsive cluster regions. Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized B HA variants in the supernatant of HEK293 cells after transfection, compared with wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimers and monomers are indicated by dashed lines. Figures 4A, 4B, 4C, 4D, and 4E show combinations of mutations in 1, 2, 3, 4, and all five regions, respectively. [Figure 4B-3] Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with combinations of substitutions in the head switch, neck switch, stem switch, hinge loop, and repulsive cluster regions. Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized B HA variants in the supernatant of HEK293 cells after transfection, compared with wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimers and monomers are indicated by dashed lines. Figures 4A, 4B, 4C, 4D, and 4E show combinations of mutations in 1, 2, 3, 4, and all five regions, respectively. [Figure 4C] Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with combinations of substitutions in the head switch, neck switch, stem switch, hinge loop, and repulsive cluster regions. Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized B HA variants in the supernatant of HEK293 cells after transfection, compared with wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimers and monomers are indicated by dashed lines. Figures 4A, 4B, 4C, 4D, and 4E show combinations of mutations in 1, 2, 3, 4, and all five regions, respectively. [Figure 4D]Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with combinations of substitutions in the head switch, neck switch, stem switch, hinge loop, and repulsive cluster regions. Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized B HA variants in the supernatant of HEK293 cells after transfection, compared with wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimers and monomers are indicated by dashed lines. Figures 4A, 4B, 4C, 4D, and 4E show combinations of mutations in 1, 2, 3, 4, and all five regions, respectively. [Figure 4E] Comparison of expression levels of single-chain (uncleaved) Flu B HA variants with combinations of substitutions in the head switch, neck switch, stem switch, hinge loop, and repulsive cluster regions. Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized B HA variants in the supernatant of HEK293 cells after transfection, compared with wild-type Iowa Flu B HA (gray line, UFV212130). The peak heights of the reference trimers and monomers are indicated by dashed lines. Figures 4A, 4B, 4C, 4D, and 4E show combinations of mutations in 1, 2, 3, 4, and all five regions, respectively. [Figure 5-1] Scatter plot representation of the data in Table 2; trimer peak area versus % trimer and dot size representing temperature stability. Wild-type Iowa Flu B HA is highlighted (UFV212130, gray dots in the left center panel). The bottom panel shows the marker legend and provides a description of the stabilizing combinations of unstable regions. [Figure 5-2] Scatter plot representation of the data in Table 2; trimer peak area versus % trimer and dot size representing temperature stability. Wild-type Iowa Flu B HA is highlighted (UFV212130, gray dots in the left center panel). The bottom panel shows the marker legend and provides a description of the stabilizing combinations of unstable regions. [Figure 6A]Figure 6A shows SEC analysis of two wild-type HAs (B / Victoria / 02 / 1987 and B / Guangdong / 120 / 2000) compared with a "repaired" variant HA. The repaired HA contained very rare residue substitutions relative to the consensus shown in Table 4. Figures 6B-6D show a comparison of the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W) from B Iowa, B Ohio, B Brisbane, B Florida, and B Singapore. Figures 6B-6C show the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W). Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized single-chain (uncleaved) Flu B HA (Figure 6B) and cleaved (Figure 6C) variants (stabilized variant group A, black line; variant group B, dashed line; and variant group C, dotted line) in the supernatants of transfected HEK293 cells compared with wild-type (gray line). Figure 6D. Western blot analysis shows the processing of stabilized HA (variant group A) with and without the P27 peptide. Culture supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a blotting membrane to visualize the C-tagged proteins. The major bands correspond to uncleaved (HA0) and cleaved HA (HA2 chain). [Figure 6B-1]Figure 6A shows SEC analysis of two wild-type HAs (B / Victoria / 02 / 1987 and B / Guangdong / 120 / 2000) compared with a "repaired" variant HA. The repaired HA contained very rare residue substitutions relative to the consensus shown in Table 4. Figures 6B-6D show a comparison of the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W) from B Iowa, B Ohio, B Brisbane, B Florida, and B Singapore. Figures 6B-6C show the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W). Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized single-chain (uncleaved) Flu B HA (Figure 6B) and cleaved (Figure 6C) variants (stabilized variant group A, black line; variant group B, dashed line; and variant group C, dotted line) in the supernatants of transfected HEK293 cells compared with wild-type (gray line). Figure 6D. Western blot analysis shows the processing of stabilized HA (variant group A) with and without the P27 peptide. Culture supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a blotting membrane to visualize the C-tagged proteins. The major bands correspond to uncleaved (HA0) and cleaved HA (HA2 chain). [Figure 6B-2]Figure 6A shows SEC analysis of two wild-type HAs (B / Victoria / 02 / 1987 and B / Guangdong / 120 / 2000) compared with a "repaired" variant HA. The repaired HA contained very rare residue substitutions relative to the consensus shown in Table 4. Figures 6B-6D show a comparison of the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W) from B Iowa, B Ohio, B Brisbane, B Florida, and B Singapore. Figures 6B-6C show the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W). Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized single-chain (uncleaved) Flu B HA (Figure 6B) and cleaved (Figure 6C) variants (stabilized variant group A, black line; variant group B, dashed line; and variant group C, dotted line) in the supernatants of transfected HEK293 cells compared with wild-type (gray line). Figure 6D. Western blot analysis shows the processing of stabilized HA (variant group A) with and without the P27 peptide. Culture supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a blotting membrane to visualize the C-tagged proteins. The major bands correspond to uncleaved (HA0) and cleaved HA (HA2 chain). [Figure 6C-1]Figure 6A shows SEC analysis of two wild-type HAs (B / Victoria / 02 / 1987 and B / Guangdong / 120 / 2000) compared with a "repaired" variant HA. The repaired HA contained very rare residue substitutions relative to the consensus shown in Table 4. Figures 6B-6D show a comparison of the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W) from B Iowa, B Ohio, B Brisbane, B Florida, and B Singapore. Figures 6B-6C show the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W). Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized single-chain (uncleaved) Flu B HA (Figure 6B) and cleaved (Figure 6C) variants (stabilized variant group A, black line; variant group B, dashed line; and variant group C, dotted line) in the supernatants of transfected HEK293 cells compared with wild-type (gray line). Figure 6D. Western blot analysis shows the processing of stabilized HA (variant group A) with and without the P27 peptide. Culture supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a blotting membrane to visualize the C-tagged proteins. The major bands correspond to uncleaved (HA0) and cleaved HA (HA2 chain). [Figure 6C-2]Figure 6A shows SEC analysis of two wild-type HAs (B / Victoria / 02 / 1987 and B / Guangdong / 120 / 2000) compared with a "repaired" variant HA. The repaired HA contained very rare residue substitutions relative to the consensus shown in Table 4. Figures 6B-6D show a comparison of the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W) from B Iowa, B Ohio, B Brisbane, B Florida, and B Singapore. Figures 6B-6C show the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W). Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized single-chain (uncleaved) Flu B HA (Figure 6B) and cleaved (Figure 6C) variants (stabilized variant group A, black line; variant group B, dashed line; and variant group C, dotted line) in the supernatants of transfected HEK293 cells compared with wild-type (gray line). Figure 6D. Western blot analysis shows the processing of stabilized HA (variant group A) with and without the P27 peptide. Culture supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a blotting membrane to visualize the C-tagged proteins. The major bands correspond to uncleaved (HA0) and cleaved HA (HA2 chain). [Figure 6D]Figure 6A shows SEC analysis of two wild-type HAs (B / Victoria / 02 / 1987 and B / Guangdong / 120 / 2000) compared with a "repaired" variant HA. The repaired HA contained very rare residue substitutions relative to the consensus shown in Table 4. Figures 6B-6D show a comparison of the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W) from B Iowa, B Ohio, B Brisbane, B Florida, and B Singapore. Figures 6B-6C show the expression levels of single-chain (uncleaved) and cleaved Flu B wild-type HAs and stabilized HAs with different degrees of stabilization: substitution group A (K227T, H384F, Q426Y, G430Y, and E475W), group B (K227T, H384F, and E475W), or group C (H329W and Q426W). Analytical size-exclusion chromatography profiles show the amounts of trimers (T) and monomers (M) of stabilized single-chain (uncleaved) Flu B HA (Figure 6B) and cleaved (Figure 6C) variants (stabilized variant group A, black line; variant group B, dashed line; and variant group C, dotted line) in the supernatants of transfected HEK293 cells compared with wild-type (gray line). Figure 6D. Western blot analysis shows the processing of stabilized HA (variant group A) with and without the P27 peptide. Culture supernatants were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a blotting membrane to visualize the C-tagged proteins. The major bands correspond to uncleaved (HA0) and cleaved HA (HA2 chain). [Figure 7-1] Comparison of expression levels of truncated Flu B HA variants with substitutions in five instability regions (UFV220875) combined with additional substitutions in the stem switch region and repulsive three-fold cluster region. Analytical size exclusion chromatography profiles. [Figure 7-2]Comparison of expression levels of truncated Flu B HA variants with substitutions in five instability regions (UFV220875) combined with additional substitutions in the stem switch region and repulsive three-fold cluster region. Analytical size exclusion chromatography profiles. [Figure 7-3] Comparison of expression levels of truncated Flu B HA variants with substitutions in five instability regions (UFV220875) combined with additional substitutions in the stem switch region and repulsive three-fold cluster region. Analytical size exclusion chromatography profiles. [Figure 8-1] Scatter plot representation of the data in Table 7 for expressed polypeptides (>10 mAU*mL in SEC profile). Trimer peak area versus dot size representing % trimer and temperature stability. [Figure 8-2] Scatter plot representation of the data in Table 7 for expressed polypeptides (>10 mAU*mL in SEC profile). Trimer peak area versus dot size representing % trimer and temperature stability. [Figure 9] An alignment of UFV220265 (SEQ ID NO: 73) and B / Brisbane / 60 / 08 (SEQ ID NO: 1) is shown.

[0031] definition The definitions of terms used in the present invention are given below.

[0032] Amino acids according to the present invention can be any of the 20 naturally occurring (or "standard" amino acids) or variants thereof. Standard amino acids can be divided into several groups based on their properties. Important factors include charge, hydrophilicity or hydrophobicity, size, and functional group. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties, such as cysteine, which can form covalent disulfide bonds (or disulfide bridges) to other cysteine ​​residues, proline, which forms rings to the polypeptide backbone, and glycine, which is more flexible than other amino acids. Table 1 shows the abbreviations and properties of the standard amino acids.

[0033] [Table 1]

[0034] The term "amino acid sequence identity" refers to the degree of identity or similarity between a pair of aligned amino acid sequences, usually expressed as a percentage. The percent identity is the percentage of amino acid residues in a candidate sequence that are identical (i.e., amino acid residues at a given position in the alignment are the same residue) or similar (i.e., amino acid substitutions at a given position in the alignment are conservative substitutions, as discussed below) to the corresponding amino acid residues in the peptide, after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percent sequence identity. Sequence homology, including percentages of sequence identity and similarity, is determined using sequence alignment techniques well known in the art, such as visual inspection and mathematical calculation, or, more preferably, comparisons are made by comparing sequence information using a computer program. An exemplary and preferred computer program is the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin Package version 10.0 program, "GAP" (11).

[0035] A "conservative substitution" refers to the replacement of an amino acid of one class with another amino acid of the same class. In certain embodiments, a conservative substitution does not alter the structure or function of a polypeptide, or both. Classes of amino acids for purposes of conservative substitution include hydrophobic (e.g., Met, Ala, Val, Leu), neutral hydrophilic (e.g., Cys, Ser, Thr), acidic (e.g., Asp, Glu), basic (e.g., Asn, Gln, His, Lys, Arg), conformation disruptors (e.g., Gly, Pro), and aromatic (e.g., Trp, Tyr, Phe).

[0036] As used herein, the terms "disease" and "disorder" are used interchangeably to refer to a condition in a subject. In some embodiments, the condition is a viral infection, particularly an influenza virus infection. In specific embodiments, the term "disease" refers to a pathological condition resulting from the presence of a virus in a cell or a subject, or the invasion of a cell or a subject by a virus. In certain embodiments, the condition is a disease in a subject, the severity of which is reduced by inducing an immune response in the subject via administration of an immunogenic composition.

[0037] As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of therapy that has a prophylactic and / or therapeutic effect. In certain embodiments, an "effective amount" in the context of administering a therapy to a subject refers to the amount of therapy that is sufficient to reduce or ameliorate the severity of influenza B virus infection, a disease or symptom associated therewith, for example, but not limited to, reducing the duration of influenza B virus infection, a disease or symptom associated therewith, preventing the progression of influenza virus infection, a disease or symptom associated therewith, preventing the onset or onset or recurrence of influenza B virus infection, a disease or symptom associated therewith, preventing or reducing transmission of influenza virus from one subject to another, reducing hospitalization and / or length of hospitalization in a subject, increasing survival of a subject with influenza B virus infection or a disease associated therewith, eliminating influenza B virus infection or a disease associated therewith, inhibiting or reducing influenza B virus replication, reducing influenza virus titer, and / or achieving an enhanced and / or improved prophylactic or therapeutic effect of another therapy. In certain embodiments, an effective amount does not provide complete protection from influenza B virus disease, but does result in a lower titer or reduced number of influenza B virus compared to untreated subjects. Benefits of a reduced influenza B virus titer, number, or total load include, but are not limited to, a decrease in the severity of symptoms of infection, a reduction in symptoms of infection, and a reduction in the duration of illness associated with infection.

[0038] As used herein, the term "host" is intended to refer to an organism or cell into which a vector, such as a cloning vector or an expression vector, has been introduced. The organism or cell may be a prokaryotic or eukaryotic organism. Preferably, a host comprises an isolated host cell, e.g., a host cell in culture. The term "host cell" simply means that the cell has been modified for (over)expression of a polypeptide of the invention. It should be understood that the term "host" is intended to refer not only to the particular subject organism or cell, but also to the progeny of such an organism or cell. Because certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent organism or cell, but are still included within the scope of the term "host" as used herein.

[0039] As used herein, the terms "including" or "including" shall be considered to be followed by the words "without limitation."

[0040] As used herein, the term "infection" refers to the entry, proliferation, and / or presence of a virus in a cell or a subject. In one embodiment, the infection is an "active" infection, i.e., an infection in which the virus is replicating in a cell or subject. Such an infection is characterized by the spread of the virus from the cell, tissue, and / or organ that the virus initially infected to other cells, tissues, and / or organs. An infection can also be a latent infection, i.e., an infection in which the virus is not replicating. In certain embodiments, infection refers to a pathological state resulting from the presence of a virus in a cell or subject or the invasion of a cell or subject by a virus.

[0041] Influenza viruses are classified into influenza virus types: genera A, B, and C. The term "subtype" specifically includes all individual "strains" within each subtype, which typically arise from mutation and exhibit different pathogenicity profiles, including natural isolates and artificial mutants or reassortants. Such strains may also be referred to as various "isolates" of a viral subtype. Thus, as used herein, the terms "strain" and "isolate" may be used interchangeably. The current nomenclature for human influenza virus strains or isolates includes the virus type (genus), i.e., A, B, or C, the geographic location of the original isolation, the strain number, and the year of isolation.

[0042] As used herein, the term "influenza virus disease" refers to a pathological condition resulting from the presence of an influenza virus, e.g., influenza A or B virus, in a cell or subject, or the invasion of a cell or subject by an influenza virus. In a specific embodiment, the term refers to a respiratory disease caused by an influenza virus.

[0043] As used herein, the term "nucleic acid" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), as well as analogs of DNA or RNA produced using nucleotide analogs. Nucleic acids can be single-stranded or double-stranded. Nucleic acid molecules can be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases, as will be readily understood by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with an analog, internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelators, alkylators, and modified linkages (e.g., alpha-anomeric nucleic acids, etc.). A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Complementary strands are also useful, for example, in antisense therapy, hybridization probes, and PCR primers.

[0044] As used herein, in certain embodiments, the numbering of amino acids in a hemagglutinin is based on the numbering of amino acids in the hemagglutinin of a wild-type influenza virus, e.g., influenza strain B / Brisbane / 60 / 08 (SEQ ID NO: 1). As used herein, the phrase "amino acid position 'x'" therefore refers to the amino acid corresponding to the amino acid at position x in the hemagglutinin of a particular wild-type influenza virus, e.g., B / Brisbane / 60 / 08 (SEQ ID NO: 1). It will be understood by those skilled in the art that the B / Brisbane / 60 / 08 (SEQ ID NO: 1) strain may contain additional or fewer amino acids relative to other influenza strains and / or subtypes, and that equivalent amino acids in other influenza virus strains and / or subtypes may be determined by multiple sequence alignment with the B / Brisbane / 60 / 08 (SEQ ID NO: 1) strain. As an example, Figure 9 shows an alignment of UFV220265 (SEQ ID NO: 73) with wild-type B / Brisbane / 60 / 08 (SEQ ID NO: 1). UFV220265 (SEQ ID NO: 73) contains the following substitution mutations at positions K227T, H384F, Q426Y, G430Y, and E475W of SEQ ID NO: 1. Note that in the numbering system used throughout this application, 1 refers to the N-terminal amino acid of the immature hemagglutinin protein (SEQ ID NO: 1). The mature sequence begins, for example, at position 16 of SEQ ID NO: 1. It will be understood by those skilled in the art that a leader sequence (or signal sequence) that directs transport of a protein during production (e.g., corresponding to amino acids 1-15 of SEQ ID NO: 1) is generally not present in the final polypeptide, i.e., is not used, for example, in a vaccine. In certain embodiments, therefore, a polypeptide according to the present invention comprises an amino acid sequence without a leader sequence, i.e., the amino acid sequence is based on the amino acid sequence of a hemagglutinin without the signal sequence.

[0045] The terms "peptide," "polypeptide," or "protein" can refer to a molecule composed of amino acids and recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide," "polypeptide," and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, e.g., conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids), as well as other modifications known in the art.

[0046] The term "vector" refers to a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host where the second nucleic acid molecule is replicated and, in some cases, expressed. In other words, a vector is capable of transporting a nucleic acid molecule to which it is linked. Cloning vectors and expression vectors are contemplated by the term "vector" as used herein. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), as well as vectors derived from bacteriophages or plant or animal (including human) viruses. A vector contains an origin of replication recognized by the proposed host, and in the case of an expression vector, a promoter and other regulatory regions recognized by the host. Certain vectors are capable of autonomous replication in the host into which they are introduced (e.g., vectors with a bacterial origin of replication can replicate in bacteria). Other vectors can integrate into the host's genome upon introduction into the host, thereby replicating along with the host genome.

[0047] As used herein, the term "wild-type" in the context of viruses refers to influenza viruses that are prevalent, circulate naturally, and cause typical pandemics of disease.

[0048] As used herein, the term "glycan motif" or "N-linked glycosylation motif" refers to a specific amino acid motif in a polypeptide that can be glycosylated via the addition of a glycan molecule. N-linked glycosylation motifs include the specific amino acid motif NxT / S (where x is not P). In polypeptides in which an N-linked glycosylation motif or glycan motif has been substituted, the listed amino acid positions correlate to the asparagine of the NxT / S amino acid motif. As an example, in the polypeptide described below, for positions 136, 137, and 151, N and T were introduced into the polypeptide, N was introduced at positions 136, 137, and 151, and threonine was introduced at positions 138, 139, and 153, respectively; while for position 141, an asparagine (N) was present in the wild-type sequence, and the motif was completed by introducing a threonine at position 143. DETAILED DESCRIPTION OF THE INVENTION

[0049] Influenza viruses have a significant impact on global public health, causing millions of cases of severe illness, thousands of deaths, and considerable economic loss each year.

[0050] Hemagglutinin (HA) is the major envelope glycoprotein from influenza viruses that is the primary target of neutralizing antibodies. Hemagglutinin has two major functions during the entry process. First, hemagglutinin mediates virus attachment to the surface of target cells through interaction with sialic acid receptors. Second, after viral endocytosis, hemagglutinin triggers fusion of the viral membrane with the endosomal membrane, releasing the genome into the target cell cytoplasm. HA contains a large ectodomain of approximately 500 amino acids that is cleaved by host-derived enzymes to generate two polypeptides that remain linked by disulfide bonds. The majority of the N-terminal fragment (HA1, 320-330 amino acids) forms a membrane-distal globular domain that contains the receptor-binding site and most of the determinants recognized by virus-neutralizing antibodies. The smaller C-terminal portion (HA2, approximately 180 amino acids) forms a stem-like structure that anchors the globular domain to the cellular or viral membrane. The degree of sequence homology between HA1 polypeptides is lower than that between HA2 polypeptides. The most conserved region is the sequence surrounding the cleavage site, particularly the amino acids at the HA2 N-terminus, which are conserved among all influenza A and B virus subtypes. Part of this region is exposed as a surface loop in the HA precursor molecule (HA0) but becomes inaccessible upon cleavage of HA0 into HA1 and HA2 (12).

[0051] Isolated mutant hemagglutinin polypeptides To obtain efficient in-process cleavage of recombinant Flu HA protein in a typical mammalian protein expression system, we devised a strategy to add a cleavage site that is cleaved in a low-pH environment next to the HA cleavage site. Mammalian cells contain a furin-like enzyme that is active at low pH. Therefore, Flu HA with an additional engineered furin cleavage site combined with HA-stabilizing substitutions is one potential solution for high-level HA expression in the correct native trimer-cleaved conformation. The introduction of a cleavage site that is cleaved during the production process is another reason to design influenza HA proteins that are stable at low pH.

[0052] In accordance with the present invention, novel isolated mutant hemagglutinin polypeptides have been designed with increased stability that improve the potential for developing influenza B vaccines. Apart from the general increase in stability, the mutant hemagglutinins exhibit a strong increase in stability at low pH. As a result, stabilized HA with an engineered furin site can be cleaved in a low pH environment and expressed as a fully cleaved native HA trimer (a trimer of HA1 and HA2 dimers).

[0053] According to the present invention, the isolated mutant hemagglutinin polypeptide comprises one or more additional mutations, i.e., amino acid substitutions, in at least one, two, three, four, or five of the instability regions (a)-(e), i.e., (a) the head switch (FIG. 1B), (b) within the neck switch (FIG. 1C), (c) within the stem switch (FIG. 1D), (d) the repulsive three-fold cluster (FIG. 1F), and / or (e) the hinge loop (FIG. 1E), compared to the amino acid sequence of the corresponding wild-type influenza virus hemagglutinin polypeptide, i.e., the influenza virus on which the mutant hemagglutinin polypeptide is based.

[0054] The amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 as provided herein. As an example, an amino acid substitution at amino acid position 227 corresponds to an amino acid substitution of lysine (K) at position 227 of SEQ ID NO: 1. As another example, an amino acid substitution at amino acid position 426 corresponds to an amino acid substitution of glutamine (Q) at position 426 of SEQ ID NO: 1. While the specific amino acid positions and residues may vary based on the starting hemagglutinin polypeptide sequence of a particular influenza strain, one skilled in the art can perform a sequence alignment to identify the corresponding amino acid positions and residues corresponding to the positions on SEQ ID NO: 1. The specific amino acid positions and residues may vary due to a heterologous peptide insertion between HA1 and HA2, e.g., the RSV p27 peptide, to optimize furin cleavage. Based on the sequence alignment, the identity of the amino acid positions and residues corresponds to the positions on SEQ ID NO: 1.

[0055] In certain aspects of the invention, provided herein is an isolated mutant influenza hemagglutinin polypeptide, the isolated mutant influenza hemagglutinin polypeptide comprising at least one stabilizing mutation in at least one of the instability regions (a)-(e) in the polypeptide, wherein the at least one stabilizing mutation comprises a substitution at (a) amino acid positions 227, 229, and / or 238 of the head switch, and / or (b) amino acid positions 329 and / or 426 of the neck switch, and / or (c) amino acid positions 384, 402, 472, and / or 476 of the stem switch, and / or (d) amino acid positions 468, 471, 475, or 478 of the repulsive three-fold cluster, and / or (e) amino acid positions 235, 430, and / or 433 of the hinge loop, wherein the amino acid positions correspond to those of SEQ ID NO: 1.

[0056] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least two stabilizing mutations in at least one of the unstable regions (a)-(e) in the polypeptide, i.e., the isolated mutant influenza hemagglutinin polypeptide can comprise two stabilizing mutations in unstable region (a), or two stabilizing mutations in unstable region (b), or two stabilizing mutations in unstable region (c), or two stabilizing mutations in unstable region (d), or two stabilizing mutations in unstable region (e).

[0057] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least two stabilizing mutations in two, three, four, or five of the instability regions (a)-(e) in the polypeptide, i.e., at least two stabilizing mutations can be present in at least two different instability regions. In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least three stabilizing mutations in three, four, or five of the instability regions (a)-(e) in the polypeptide, i.e., at least three stabilizing mutations can be present in at least three different instability regions. In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least four stabilizing mutations in four or five of the instability regions (a)-(e) in the polypeptide, i.e., at least four stabilizing mutations can be present in at least four different instability regions. In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises at least five stabilizing mutations in five of the instability regions (a)-(e) in the polypeptide, i.e., at least five stabilizing mutations can be present in at least five different instability regions.

[0058] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 stabilizing mutations.

[0059] In certain embodiments, (a) amino acid position 227 is substituted with an amino acid selected from the group consisting of T, L, R, Q, F, I, and Y, amino acid position 229 is substituted with the amino acid L, and / or amino acid position 238 is substituted with the amino acid F in a head switch, and / or (b) amino acid position 329 is substituted with an amino acid selected from the group consisting of M, W, and F, and / or amino acid position 426 is substituted with an amino acid selected from the group consisting of F, W, Y, and P in a neck switch, and / or (c) amino acid position 384 is substituted with an amino acid selected from the group consisting of F or Y, amino acid position 402 is substituted with the amino acid A, and amino acid position 472 is substituted with an amino acid selected from the group consisting of W, R, F, K, or and / or amino acid position 476 is substituted with amino acid F in the stem switch; and / or (d) amino acid position 468 is substituted with amino acid L, amino acid position 471 is substituted with an amino acid selected from V or Q, amino acid position 475 is substituted with an amino acid selected from the group consisting of Q, N, W, F, and L, or amino acid position 478 is substituted with an amino acid selected from W or R in the hinge loop; and / or (e) amino acid position 235 is substituted with amino acid W, amino acid position 430 is substituted with an amino acid selected from L or Y, and / or amino acid position 433 is substituted with amino acid P in the hinge loop.

[0060] In certain embodiments of the invention, the isolated mutant influenza hemagglutinin polypeptide comprises the following amino acid sequence: (a) amino acid position 384 is substituted with F and amino acid position 475 is substituted with W, (b) amino acid position 384 is substituted with F and amino acid position 475 is substituted with Q, (c) amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 472 is substituted with K, and amino acid position 476 is substituted with F, (d) amino acid position 384 is substituted with F, (e) amino acid position 384 is substituted with Y, amino acid position 402 is substituted with A, amino acid position 472 is substituted with R, and amino acid position 476 is substituted with F, (f) amino acid position 476 is substituted with F and amino acid position 475 is substituted with W, (g) amino acid position 476 is substituted with F and amino acid position 475 is substituted with Q, (h) amino acid position (i) amino acid position 227 is substituted with T and amino acid position 426 is substituted with Y, (j) amino acid position 227 is substituted with T and amino acid position 426 is substituted with Y and amino acid position 430 is substituted with Y, (k) amino acid position 227 is substituted with T and amino acid position 475 is substituted with W, (l) amino acid position 227 is substituted with T and amino acid position 475 is substituted with Q, (m) amino acid position 227 is (n) amino acid position 227 is substituted with T and amino acid position 384 is substituted with F, (n) amino acid position 227 is substituted with T and amino acid position 476 is substituted with F, (o) amino acid position 227 is substituted with T and amino acid position 426 is substituted with Y and amino acid position 475 is substituted with W, (p) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y and amino acid position 475 is substituted with Q, (q) amino acid position 227 is substituted with T and amino acid position 426 is substituted with Y,(r) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 475 is substituted with Q, and amino acid position 430 is substituted with Y, (s) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, and amino acid position 384 is substituted with F, (t) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, and amino acid position 384 is substituted with (u) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 384 is substituted with F, amino acid position 475 is substituted with W, and amino acid position 430 is substituted with Y; (v) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 384 is substituted with F, and amino acid position 475 is substituted with Q; (w) amino acid position 227 is substituted with T, and (x) amino acid position 227 is substituted with T and amino acid position 235 is substituted with W; (y) amino acid position 475 is substituted with W and amino acid position 426 is substituted with Y; (z) amino acid position 475 is substituted with W and amino acid position 430 is substituted with Y; (aa) amino acid position 475 is substituted with Q and amino acid position 430 is substituted with Y; (bb) amino acid position 475 is substituted with Q and amino acid position 478 is substituted with F, (cc) amino acid position 426 is substituted with Y and amino acid position 384 is substituted with F, (dd) amino acid position 426 is substituted with Y and amino acid position 476 is substituted with F, (ee) amino acid position 426 is substituted with Y and amino acid position 455 is substituted with A, (ff) amino acid position 426 is substituted with Y and amino acid position 329 is substituted with W.(gg) amino acid position 430 is substituted with Y and amino acid position 384 is substituted with F, (hh) amino acid position 430 is substituted with Y and amino acid position 476 is substituted with F, (ii) amino acid position 430 is substituted with Y and amino acid position 426 is substituted with Y, (jj) amino acid position 430 is substituted with Y and amino acid position 426 is substituted with W, (kk) amino acid position 430 is substituted with Y and amino acid position 426 is substituted with F, (ll) amino acid position 433 is substituted with P and amino acid position 430 is substituted with Y, (mm) amino acid position 433 is substituted with P and amino acid position 426 is substituted with Y, (nn) amino acid position 433 is substituted with P and amino acid position 426 is substituted with W, (oo) amino acid position 433 is substituted with P and amino acid position 426 is substituted with F, (pp) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, and amino acid position 475 is substituted with W, (qq) amino acid position 227 is substituted with T (rr) wherein amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 472 is substituted with R, and amino acid position 476 is substituted with F; (rr) wherein amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 468 is substituted with L, and amino acid 471 is substituted with V and amino acid position 472 is substituted with F; (ss) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 468 is substituted with L, amino acid 471 is substituted with V, amino acid position 472 is substituted with W, amino acid position 475 is substituted with F, and amino acid position 478 is substituted with R; (tt) amino acid position 227 is substituted with T and amino acid position 384 is substituted with F;(uu) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 472 is substituted with W, and amino acid position 476 is substituted with F, (uu) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 468 is substituted with L, amino acid 471 is substituted with V, amino acid position 472 is substituted with W, amino acid position 475 is substituted with L, and amino acid position 478 is substituted with R, (v v) amino acid position 227 is substituted with T, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 468 is substituted with L, amino acid position 471 is substituted with Q, amino acid position 472 is substituted with W, amino acid position 475 is substituted with Q, and amino acid position 478 is substituted with R; (ww) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y; (xx) amino acid position 430 is substituted with Y, amino acid position 472 is substituted with K, amino acid position 475 is substituted with Q, and amino acid position 476 is substituted with F; (xx) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 472 is substituted with L, amino acid position 475 is substituted with Q, and amino acid position 476 is substituted with F; (yy) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 471 is substituted with Q, amino acid position 472 is substituted with R, and amino acid position 476 is substituted with F; (zz) amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, and amino acid position 426 is substituted with Y;a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 476 with F; (aaa) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, and a substitution at amino acid position 476 with F; or (bbb) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with R, a substitution at amino acid position 475 with W, and a substitution at amino acid position 476 with F;

[0061] In certain embodiments, the mutant influenza hemagglutinin polypeptide further comprises an introduced cleavage site. The introduced cleavage site can be, for example, a furin cleavage site. The furin cleavage site can allow complete cleavage of HA to form the fully processed native trimer. The furin cleavage site can be introduced, for example, by mutating amino acid positions 359-361 of the polypeptide or by inserting it amino-terminally at amino acid position 362, which corresponds to the amino acid position of SEQ ID NO: 1. In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide further comprises modification of the cleavage site to a polybasic cleavage site to allow efficient processing by furin, or insertion of the RSV p27 peptide (SEQ ID NO: 2) carboxy-terminally at amino acid position 362. The cleaved HA can form the fully processed native trimer.

[0062] In certain embodiments, amino acid position 362 is substituted with Q.

[0063] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide further comprises a deletion of a signal peptide at the amino terminus of the polypeptide, e.g., the signal peptide comprises amino acid positions 1-15 of the polypeptide.

[0064] In certain embodiments, the variant influenza hemagglutinin polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 35, 46-48, 63, 67-75, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 181, 186, 189, 201, 202, 204-208, 216, and 219-222. In certain embodiments, the variant influenza hemagglutinin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 35, 46-48, 63, 67-75, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 181, 186, 189, 201, 202, 204-208, 216, and 219-222, wherein the variant influenza hemagglutinin polypeptide comprises a deletion of a signal peptide at the amino terminus of the polypeptide.

[0065] In certain embodiments, the isolated variant influenza hemagglutinin polypeptide is derived from the hemagglutinin of influenza virus type B. Specifically, the isolated variant influenza hemagglutinin polypeptide can be derived from the B / Yamagata / 16 / 1988 (also referred to as B / Yamagata), B / Singapore / INFTT-16-0610 / 2016 (B / Singapore), B / Florida / 04 / 2006 (B / Florida), B / Victoria / 2 / 1987 (B / Victoria), B / Iowa / 06 / 2017 (B / Iowa), B / Ohio / 01 / 2005 (B / Ohio), and / or B / Brisbane / 60 / 2008 (B / Brisbane) lineages.

[0066] In certain embodiments, the isolated mutant influenza hemagglutinin polypeptide further comprises a carboxy (C)-terminal truncation beginning at amino acid positions 536 through 585, the amino acid positions corresponding to the amino acid positions of SEQ ID NO:1.

[0067] Influenza hemagglutinin (HA), in its native form, exists as a trimer on the cellular or viral membrane. In certain embodiments, the intracellular and transmembrane sequences are removed so that a secreted (soluble) polypeptide is produced after intracellular expression. Methods for expressing and purifying the secreted ectodomain of HA have been described (see, e.g., (13), (14, 15), (16, 17), (18)). Those skilled in the art will understand that these methods can also be applied directly to the isolated mutant hemagglutinin polypeptides of the invention to achieve expression of a secreted (soluble) polypeptide. Accordingly, these polypeptides are also encompassed by the present invention.

[0068] Optionally, a his-tag sequence (HHHHHH (SEQ ID NO: 134) or HHHHHHH (SEQ ID NO: 135) or C-tag (SEQ ID NO: 136)) may be linked to the (optionally truncated) isolated mutant hemagglutinin polypeptide for purification purposes, optionally the his-tag sequence being connected via a linker. Optionally, the linker may contain a proteolytic cleavage site for enzymatic removal of the his-tag after purification.

[0069] To facilitate purification of soluble forms, a tag sequence may be added, for example a histidine tag (HHHHHH (SEQ ID NO: 134) or HHHHHHH (SEQ ID NO: 135)) or a FLAG tag (DYKDDDDK) (SEQ ID NO: 137) or a C tag (EPEA) (SEQ ID NO: 136), or a combination thereof, optionally linked via a short linker. The linker may optionally contain (part of) a proteolytic cleavage site, for example IEGR (SEQ ID NO: 138) (factor X) or LVPRGS (SEQ ID NO: 139) (thrombin), for subsequent processing according to protocols well known to those skilled in the art. Processed proteins are also encompassed by the present invention.

[0070] The mutant influenza hemagglutinin polypeptides can be prepared according to any technique deemed suitable by one of skill in the art, including the techniques described below.

[0071] Thus, the immunogenic polypeptides of the present invention can be synthesized as DNA sequences by standard methods known in the art, cloned, and then expressed in vitro or in vivo using appropriate restriction enzymes and methods known in the art. Therefore, the present invention also relates to nucleic acid molecules encoding the above-described polypeptides. The present invention further relates to vectors containing nucleic acids encoding the polypeptides of the present invention. In certain embodiments, the nucleic acid molecules of the present invention are part of a vector, e.g., a plasmid. Such vectors can be easily manipulated by methods well known to those skilled in the art and, for example, can be designed to be replicable in prokaryotic and / or eukaryotic cells. In addition, many vectors can be used to transform eukaryotic cells and integrated, in whole or in part, into the genome of such cells, resulting in stable host cells containing the desired nucleic acid in their genome. The vector used can be any vector suitable for cloning DNA and can be used to transcribe the nucleic acid of interest. When using host cells, the vector is preferably an integrating vector. Alternatively, the vector can be an episomal replicating vector.

[0072] Those skilled in the art can select a suitable expression vector and insert the nucleic acid sequence of the present invention in a functional manner. It is well known to those skilled in the art that, to obtain the expression of a nucleic acid sequence encoding a polypeptide, a sequence capable of driving expression can be operably linked to the nucleic acid sequence encoding the polypeptide to obtain a recombinant nucleic acid molecule encoding a protein or polypeptide in an expressible form. Generally, a promoter sequence is placed upstream of the sequence to be expressed. Many expression vectors, such as Invitrogen's pcDNA and pEF vector series, pMSCV and pTK-Hyg by BD Science, and pCMV-Script by Stratagene, are available in the art and can be used to obtain suitable promoters and / or transcription termination sequences, polyA sequences, etc. When a sequence encoding a polypeptide of interest is properly inserted relative to sequences governing the transcription and translation of the encoded polypeptide, the resulting expression cassette is useful for producing the polypeptide of interest, which is referred to as expression. Sequences driving expression can include promoters, enhancers, etc., and combinations thereof. These can function in host cells, thereby driving the expression of nucleic acid sequences operably linked to them. Those skilled in the art recognize that a variety of promoters can be used to obtain expression of a gene in a host cell. Promoters can be constitutive or regulated, and can be obtained from a variety of sources, including viral, prokaryotic, or eukaryotic sources, or can be artificially designed. Expression of a nucleic acid of interest can be from the native promoter or its derivative, or from a completely heterologous promoter (Kaufman, 2000).Some well-known and frequently used promoters for expression in eukaryotic cells include promoters derived from viruses such as adenovirus, e.g., the E1A promoter, promoters derived from cytomegalovirus (CMV), e.g., the CMV immediate early (IE) promoter (herein referred to as the CMV promoter) (e.g., pcDNA, available from Invitrogen), promoters derived from Simian Virus 40 (SV40), and the like. Suitable promoters can also be derived from eukaryotic cells, e.g., the metallothionein (MT) promoter, the elongation factor 1α (EF-1α) promoter, the ubiquitin C or UB6 promoter, the actin promoter, the immunoglobulin promoter, the heat shock promoter, and the like. Testing promoter function and strength is a matter of routine to those skilled in the art and generally involves cloning a test gene, e.g., lacZ, luciferase, GFP, etc., behind the promoter sequence and testing for expression of the test gene. Of course, promoters can be modified by deleting, adding, or mutating sequences therein and tested for functionality to find new, attenuated, or improved promoter sequences. According to the present invention, strong promoters that result in high transcription levels in the selected eukaryotic cell are preferred.

[0073] Constructs can be transfected into eukaryotic cells (e.g., plant, fungal, yeast, or animal cells) or suitable prokaryotic expression systems (such as E. coli) using methods well known to those skilled in the art. Optionally, a suitable "tag" sequence (e.g., but not limited to, his-, myc-, strep-, or flag tags) or an entire protein (e.g., but not limited to, maltose-binding protein or glutathione S-transferase) can be added to the sequences of the invention to allow for purification and / or identification of the polypeptide from cells or supernatants. Optionally, a sequence containing a specific proteolytic site can be included to allow subsequent removal of the tag by proteolytic digestion.

[0074] Improved HA trimer stability can be assessed by size exclusion chromatography. Increased stability correlates with increased trimer expression levels, decreased monomer expression levels, and increased melting temperatures. Furthermore, purified trimers can be further tested for long-term stability at elevated temperatures or by assessing native trimer content after multiple freeze-thaw cycles.

[0075] The purified polypeptides can be analyzed by spectroscopic methods known in the art (e.g., circular dichroism spectroscopy, Fourier transform infrared spectroscopy, and NMR spectroscopy or X-ray crystallography) to examine the presence of desired structures such as helices and beta sheets. Binding of the polypeptides of the present invention to previously described broadly neutralizing antibodies (CR8071, CR8033) can be examined using ELISA, Octet, FACS, and the like (19). Thus, polypeptides of the present invention having the correct conformation can be selected.

[0076] Pharmaceutical / Immunogenic Compositions and Methods of Use The present invention further relates to immunogenic compositions comprising a therapeutically effective amount of at least one of the polypeptides and / or nucleic acids of the present invention. The immunogenic compositions preferably further comprise a pharmaceutically acceptable carrier. In this context, the term "pharmaceutically acceptable" means that the carrier, at the dosage and concentration employed, does not cause undesirable or harmful effects in the subject to which it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R. Gennaro, Ed., Mack Publishing Company

[1990] ; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis

[2000] ; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press

[2000] ). The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. Physiological saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The correct formulation should suit the mode of administration. The polypeptide and / or nucleic acid molecule is preferably formulated and administered as a sterile solution. Sterile solutions are prepared by sterile filtration or other methods known in the art. The solution can then be lyophilized or filled into pharmaceutical administration containers. The pH of this solution is generally in the range of pH 3.0 to 9.5, for example, pH 5.0 to 7.5.

[0077] The present invention also relates to the influenza variant hemagglutinin polypeptides, nucleic acid molecules, and / or vectors described above for use in inducing an immune response against influenza HA protein. The present invention also relates to methods for inducing an immune response in a subject, comprising administering to the subject the above-described polypeptides, nucleic acid molecules, and / or immunogenic compositions. The subject according to the present invention is preferably a mammal that can be infected with a pathogen that causes an infectious disease, particularly an influenza virus, or that could otherwise benefit from the induction of an immune response; such a subject is, for example, a rodent, such as a mouse, a ferret, or a domestic or farm animal, or a non-human primate, or a human. Preferably, the subject is a human subject. Accordingly, the present invention provides methods for inducing an immune response against influenza B virus hemagglutinin (HA) in a subject, utilizing the polypeptides, nucleic acids, and / or immunogenic compositions described herein.

[0078] It is well known that small proteins and / or nucleic acid molecules do not always efficiently induce strong immune responses, and therefore it may be necessary to increase the immunogenicity of polypeptides and / or nucleic acid molecules by adding an adjuvant. In certain embodiments, the immunogenic compositions described herein comprise or are administered in combination with an adjuvant. Adjuvants for administration in combination with the compositions described herein can be administered before, simultaneously with, or after administration of the compositions. Examples of suitable adjuvants include aluminum salts, such as aluminum hydroxide and / or aluminum phosphate; oil emulsion compositions (or oil-in-water compositions) including squalene-water emulsions such as MF59 (see, e.g., WO 90 / 14837); saponin preparations, such as QS21 and immune stimulating complexes (ISCOMS) (see, e.g., U.S. Pat. No. 5,057,540; WO 90 / 03184; WO 96 / 11711; WO 2004 / 004762; WO 2005 / 002620); bacterial or microbial derivatives (examples of which include monophosphoryl lipid A (MPL), 3-O-deacylated MPL, etc.); Examples of suitable immunopotentiating agents include erythropoietin (e.g., erythropoietin-binding protein (MPL), 3dMPL), CpG-motif-containing oligonucleotides, ADP-ribosylating bacterial toxins or variants thereof, such as E. coli heat-labile enterotoxin LT, cholera toxin CT, pertussis toxin PT, tetanus toxoid TT, matrix M (Isconova), and the like. In addition, known immunopotentiating techniques can be used, such as fusing the polypeptides of the present invention to proteins known in the art for enhancing immune responses (e.g., tetanus toxoid, CRM197, rCTB, bacterial flagellin, etc.), or including the polypeptide in a virosome, or a combination thereof. Other non-limiting examples that can be used are disclosed, for example, by (20).

[0079] In one embodiment, the influenza variant hemagglutinin polypeptides of the invention are incorporated into a viral-like particle (VLP) vector. VLPs generally comprise viral polypeptides, typically derived from viral structural proteins. Preferably, the VLP is unable to replicate. In certain embodiments, the VLP can lack the entire viral genome or can comprise a portion of the viral genome. In some embodiments, the VLP is unable to infect cells. In some embodiments, the VLP expresses on its surface one or more viral (e.g., viral surface glycoproteins) or non-viral (e.g., antibodies or proteins) targeting moieties known to those of skill in the art.

[0080] In certain embodiments, the polypeptides of the present invention are incorporated into virosomes. Virosomes containing the polypeptides of the present invention can be produced using techniques known to those skilled in the art. For example, virosomes can be produced by disrupting purified viruses, extracting the genome, and reassembling particles with viral proteins (e.g., mutant influenza hemagglutinin polypeptides described herein) and lipids to form lipid particles containing viral proteins.

[0081] The present invention also specifically relates to the above-described polypeptides, nucleic acids, and / or immunogenic compositions for inducing an immune response in a subject against influenza HA, for use as a vaccine. Thus, the influenza variant hemagglutinin polypeptides described herein, nucleic acids encoding such polypeptides, or vectors comprising such nucleic acids or polypeptides can be used to elicit protective antibodies against influenza virus. The present invention relates to the above-described polypeptides, nucleic acids, and / or immunogenic compositions for use as vaccines in the prevention and / or treatment of diseases or conditions caused by influenza virus.

[0082] The polypeptides of the invention can be synthesized and used in vitro or in suitable cellular expression systems, including bacterial and eukaryotic cells, or alternatively, can be expressed in vivo in a subject in need thereof by expressing a nucleic acid encoding the immunogenic polypeptide. Such nucleic acid vaccines can take any form, including naked DNA, mRNA, self-replicating RNA, circular RNA, plasmids, or viral vectors, including adenoviral vectors.

[0083] Administration of the polypeptides, nucleic acid molecules, and / or immunogenic compositions according to the present invention can be carried out using standard administration routes. Non-limiting examples include intravenous, intradermal, transdermal, intramuscular, subcutaneous, etc., or parenteral administration, such as mucosal administration, e.g., intranasal, oral, etc. Those skilled in the art can determine various possibilities for administering the polypeptides, nucleic acid molecules, and / or immunogenic compositions according to the present invention to induce an immune response. In certain embodiments, the polypeptides, nucleic acid molecules, and / or immunogenic compositions (or vaccines) are administered more than once, i.e., in a so-called allogeneic prime-boost regimen. In certain embodiments in which the polypeptide, nucleic acid molecule, and / or immunogenic composition is administered more than once, the administration of the second dose can be performed after an interval of up to several years after the administration of the first dose of the polypeptide, nucleic acid molecule, and / or immunogenic composition, such as, for example, one week or more after the administration of the first dose, two weeks or more after the administration of the first dose, three weeks or more after the administration of the first dose, one month or more after the administration of the first dose, six weeks or more after the administration of the first dose, two months or more after the administration of the first dose, three months or more after the administration of the first dose, four months or more after the administration of the first dose, etc. It is also possible to administer the vaccine more than twice, e.g., three times, four times, etc., with an initial prime administration followed by more than one boost administration. In other embodiments, the polypeptide, nucleic acid molecule, and / or immunogenic composition according to the invention is administered only once.

[0084] The polypeptides, nucleic acid molecules, and / or immunogenic compositions can also be administered as either the prime or boost in a heterologous prime-boost regimen.

[0085] The present invention further provides methods for preventing and / or treating influenza virus disease in a subject using the polypeptides, nucleic acids, and / or compositions described herein. In certain embodiments, the method for preventing and / or treating influenza virus disease in a subject comprises administering to a subject in need thereof an effective amount of a polypeptide, nucleic acid molecule, and / or immunogenic composition, as described above. A therapeutically effective amount refers to an amount of a polypeptide, nucleic acid, and / or composition as defined herein that is effective for preventing, ameliorating, and / or treating a disease or condition resulting from infection with an influenza virus. Prevention includes inhibiting or reducing transmission of influenza virus or inhibiting or reducing the onset, development, or progression of one or more symptoms associated with infection with an influenza virus. As used herein, amelioration may refer to a reduction in visible or perceptible disease symptoms, viremia, or any other measurable sign of influenza infection.

[0086] Those in need of treatment include those already suffering from a condition resulting from influenza virus infection, as well as those in whom influenza virus infection is to be prevented. Thus, the polypeptides, nucleic acids, and / or compositions of the invention can be administered to naive subjects, i.e., subjects who do not have a disease caused by influenza virus infection or who are not infected and are not currently infected with influenza virus infection, or subjects who are already infected and / or have been infected with influenza virus.

[0087] In one embodiment, prophylaxis and / or treatment may be targeted to patient populations susceptible to influenza virus infection, including, but not limited to, elderly patients (e.g., 50 years of age or older, 60 years of age or older, preferably 65 years of age or older), young patients (e.g., 5 years of age or younger, 1 year of age or younger), hospitalized patients, and patients who have been treated with antiviral compounds but have demonstrated an inadequate antiviral response.

[0088] In another embodiment, the polypeptide, nucleic acid, and / or immunogenic composition can be administered to a subject in combination with one or more other active agents, such as an existing or future influenza vaccine, a monoclonal antibody, and / or an antiviral agent, and / or an antibacterial agent, and / or an immunomodulatory agent. The one or more other active agents may be beneficial in the treatment and / or prevention of influenza virus disease or may ameliorate a symptom or condition associated with influenza virus disease. In some embodiments, the one or more other active agents are pain relievers, fever reducers, or therapies that relieve or assist breathing.

[0089] The polypeptides of the present invention can also be used to confirm the binding of monoclonal antibodies identified as potential therapeutic candidates. In addition, the polypeptides of the present invention can be used as a diagnostic tool to test the immune status of an individual, for example, by demonstrating whether antibodies capable of binding to the polypeptides of the present invention are present in the serum of such an individual. Thus, the present invention also relates to an in vitro diagnostic method for detecting the presence of influenza infection in a patient, comprising the steps of: a) contacting a biological sample obtained from the patient with a polypeptide according to the present invention; and b) detecting the presence of an antibody-antigen complex.

[0090] The polypeptides of the present invention can also be used to identify new or improve existing binding molecules, such as monoclonal antibodies and antiviral agents.

[0091] The present invention is further illustrated in the following examples and figures, which are not intended to limit the scope of the invention in any way.

[0092] Embodiment The present invention also provides the following non-limiting embodiments.

[0093] Embodiment 1 is directed to a polypeptide comprising at least one stabilizing mutation in at least one of the instability regions (a)-(e) in the polypeptide, (a) amino acid positions 227, 229, and / or 238, and / or (b) amino acid positions 329 and / or 426, and / or (c) amino acid positions 384, 402, 472, and / or 476, and / or (d) amino acid positions 468, 471, 475, and / or 478, and / or (e) at least one stabilizing mutation, including a substitution mutation at amino acid positions 235, 430, and / or 433; An isolated mutant influenza hemagglutinin polypeptide, the amino acid positions of which correspond to the amino acid positions of SEQ ID NO:1.

[0094] Embodiment 2 is an isolated mutant influenza hemagglutinin polypeptide of embodiment 1, comprising at least two stabilizing mutations in at least one of the instability regions (a)-(e) in the polypeptide.

[0095] Embodiment 3 is an isolated mutant influenza hemagglutinin polypeptide of embodiment 1, comprising at least two stabilizing mutations in two, three, four, or five of the instability regions (a) through (e) in the polypeptide.

[0096] Embodiment 3a is an isolated mutant influenza hemagglutinin polypeptide of embodiment 1, comprising at least three stabilizing mutations in three, four, or five of the instability regions (a) through (e) in the polypeptide.

[0097] Embodiment 3b is an isolated mutant hemagglutinin polypeptide according to embodiment 1, comprising at least four stabilizing mutations in four or five of the instability regions (a) to (e) in the polypeptide.

[0098] Embodiment 3c is an isolated mutant hemagglutinin polypeptide according to embodiment 1, comprising at least five stabilizing mutations in five of the instability regions (a) to (e) in the polypeptide.

[0099] Embodiment 4 is an isolated mutant influenza hemagglutinin polypeptide of claim 1 comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 stabilizing mutations.

[0100] Embodiment 5 is (a) amino acid position 227 is substituted with an amino acid selected from the group consisting of T, L, R, Q, F, I, and Y, amino acid position 229 is substituted with amino acid L, and / or amino acid position 238 is substituted with amino acid F; and / or (b) a substitution at amino acid position 329 with an amino acid selected from the group consisting of M, W, and F, and / or a substitution at amino acid position 426 with an amino acid selected from the group consisting of F, W, Y, and P; and / or (c) amino acid position 384 is substituted with an amino acid selected from F or Y, amino acid position 402 is substituted with an amino acid A, amino acid position 472 is substituted with an amino acid selected from W, R, F, K, or L, and / or amino acid position 476 is substituted with an amino acid F; and / or (d) amino acid position 468 is substituted with an amino acid L, amino acid position 471 is substituted with an amino acid selected from V or Q, amino acid position 475 is substituted with an amino acid selected from the group consisting of Q, N, W, F, and L, and / or amino acid position 478 is substituted with an amino acid selected from W or R; and / or (e) The isolated variant influenza hemagglutinin polypeptide of embodiment 1, wherein amino acid position 235 is substituted with the amino acid W, amino acid position 430 is substituted with an amino acid selected from L or Y, and / or amino acid position 433 is substituted with the amino acid P.

[0101] Embodiment 6 is (a) a substitution at amino acid position 384 with F and a substitution at amino acid position 475 with W; (b) a substitution at amino acid position 384 with F and a substitution at amino acid position 475 with Q; (c) a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 472 with K, and a substitution at amino acid position 476 with F; (d) a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 472 with R, and a substitution at amino acid position 476 with F; (e) a substitution at amino acid position 384 with Y, a substitution at amino acid position 402 with A, a substitution at amino acid position 472 with R, and a substitution at amino acid position 476 with F; (f) a substitution at amino acid position 476 with F and a substitution at amino acid position 475 with W; (g) a substitution at amino acid position 476 with F and a substitution at amino acid position 475 with Q; (h) a substitution at amino acid position 227 with T and a substitution at amino acid position 426 with Y; (i) a substitution at amino acid position 227 with T and a substitution at amino acid position 430 with Y; (j) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, and a substitution at amino acid position 430 with Y; (k) a substitution at amino acid position 227 with T and a substitution at amino acid position 475 with W; (l) a substitution at amino acid position 227 with T and a substitution at amino acid position 475 with Q; (m) a substitution at amino acid position 227 with T and a substitution at amino acid position 384 with F; (n) a substitution at amino acid position 227 with T and a substitution at amino acid position 476 with F; (o) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, and a substitution at amino acid position 475 with W; (p) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, and a substitution at amino acid position 475 with Q; (q) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 475 with W, and a substitution at amino acid position 430 with Y; (r) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 430 with Y; (s) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, and a substitution at amino acid position 384 with F; (t) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 384 with F, and a substitution at amino acid position 475 with W; (u) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 384 with F, a substitution at amino acid position 475 with W, and a substitution at amino acid position 430 with Y; (v) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 384 with F, and a substitution at amino acid position 475 with Q; (w) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 384 with F, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 430 with Y; (x) a substitution at amino acid position 227 with T and a substitution at amino acid position 235 with W; (y) a substitution at amino acid position 475 with W and a substitution at amino acid position 426 with Y; (z) a substitution at amino acid position 475 with W and a substitution at amino acid position 430 with Y; (aa) a substitution at amino acid position 475 with Q and a substitution at amino acid position 478 with R; (bb) a substitution at amino acid position 475 with Q and a substitution at amino acid position 478 with F; (cc) a substitution at amino acid position 426 with Y and a substitution at amino acid position 384 with F; (dd) a substitution at amino acid position 426 with Y and a substitution at amino acid position 476 with F; (ee) a substitution at amino acid position 426 with Y and a substitution at amino acid position 455 with A; (ff) a substitution at amino acid position 426 with Y and a substitution at amino acid position 329 with W; (gg) a substitution at amino acid position 430 with Y and a substitution at amino acid position 384 with F; (hh) a substitution at amino acid position 430 with Y and a substitution at amino acid position 476 with F; (ii) a substitution at amino acid position 430 with Y and a substitution at amino acid position 426 with Y; (jj) a substitution at amino acid position 430 with Y and a substitution at amino acid position 426 with W; (kk) a substitution at amino acid position 430 with Y and a substitution at amino acid position 426 with F; (ll) a substitution at amino acid position 433 with P and a substitution at amino acid position 430 with Y; (mm) a substitution at amino acid position 433 with P and a substitution at amino acid position 426 with Y; (nn) a substitution at amino acid position 433 with P and a substitution at amino acid position 426 with W; (oo) a substitution at amino acid position 433 with P and a substitution at amino acid position 426 with F; (pp) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, and a substitution at amino acid position 475 with W; (qq) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with R, and a substitution at amino acid position 476 with F; (rr) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 468 with L, a substitution at amino acid position 471 with V, a substitution at amino acid position 472 with F, a substitution at amino acid position 475 with F, and a substitution at amino acid position 478 with R; (ss) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 468 with L, a substitution at amino acid position 471 with V, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with F, and a substitution at amino acid position 478 with R; (tt) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, and a substitution at amino acid position 476 with F; (uu) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 468 with L, a substitution at amino acid position 471 with V, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with L, and a substitution at amino acid position 478 with R; (vv) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 468 with L, a substitution at amino acid position 471 with Q, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 478 with R; (ww) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with K, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 476 with F; (xx) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with L, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 476 with F; (yy) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 471 with Q, a substitution at amino acid position 472 with R, and a substitution at amino acid position 476 with F; (zz) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 476 with F; (aaa) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, and a substitution at amino acid position 476 with F; or (bbb) The isolated mutant influenza hemagglutinin polypeptide of embodiment 5, wherein amino acid position 227 is substituted with T, amino acid position 384 is substituted with F, amino acid position 402 is substituted with A, amino acid position 426 is substituted with Y, amino acid position 430 is substituted with Y, amino acid position 472 is substituted with R, amino acid position 475 is substituted with W, and amino acid position 476 is substituted with F.

[0102] Embodiment 7 is the isolated mutant influenza hemagglutinin polypeptide of any one of embodiments 1 to 6, wherein the mutant influenza hemagglutinin polypeptide further comprises an introduced cleavage site.

[0103] Embodiment 8 is the isolated mutant influenza hemagglutinin polypeptide of embodiment 7, wherein the introduced cleavage site is a furin cleavage site.

[0104] Embodiment 9 is the isolated mutant influenza hemagglutinin polypeptide of embodiment 8, wherein a furin cleavage site is introduced by mutating amino acid positions 359-361 of the polypeptide, or wherein a furin cleavage site is introduced by insertion amino-terminally at amino acid position 362, which amino acid position corresponds to the amino acid position of SEQ ID NO:1.

[0105] Embodiment 10 is an isolated mutant influenza hemagglutinin polypeptide of embodiment 8 or 9, further comprising an insertion of the RSV p27 peptide (SEQ ID NO: 2) carboxy-terminal to amino acid position 362.

[0106] Embodiment 11 is an isolated mutant influenza hemagglutinin polypeptide of any one of embodiments 1 to 9, wherein the amino acid at position 362 is substituted with Q.

[0107] Embodiment 12 is the isolated mutant influenza hemagglutinin polypeptide of any one of embodiments 1 to 11, further comprising a deletion of the signal peptide at the amino terminus of the polypeptide.

[0108] Embodiment 13 is the isolated mutant influenza hemagglutinin polypeptide of embodiment 12, wherein the signal peptide comprises amino acid positions 1-15 of the polypeptide.

[0109] Embodiment 14 is the isolated variant influenza hemagglutinin polypeptide of any one of embodiments 1 to 13, wherein the variant influenza hemagglutinin polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 35, 46-48, 63, 67-75, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 181, 186, 189, 201, 202, 204-208, 216, and 219-222.

[0110] Embodiment 15 is the isolated variant influenza hemagglutinin polypeptide of embodiment 14, wherein the variant influenza hemagglutinin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 35, 46-48, 63, 67-75, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 181, 186, 189, 201, 202, 204-208, 216, and 219-222.

[0111] Embodiment 16 is the isolated mutant influenza hemagglutinin polypeptide of any one of embodiments 1 to 15, wherein the mutant influenza hemagglutinin polypeptide further comprises a carboxy (C)-terminal truncation starting at amino acid positions 536 to 585, the amino acid positions corresponding to the amino acid positions of SEQ ID NO: 1.

[0112] Embodiment 17 is an isolated nucleic acid encoding the isolated mutant influenza hemagglutinin polypeptide of any one of embodiments 1-16.

[0113] Embodiment 18 is an isolated vector comprising the isolated nucleic acid of embodiment 17.

[0114] Embodiment 19 is an isolated host cell comprising the vector of embodiment 18.

[0115] Embodiment 20 is a pharmaceutical composition comprising an isolated mutant influenza hemagglutinin polypeptide of any one of embodiments 1 to 16 and a pharmaceutically acceptable carrier.

[0116] Embodiment 21 is a pharmaceutical composition comprising the isolated nucleic acid of embodiment 17.

[0117] Embodiment 22 is a pharmaceutical composition comprising the isolated vector of embodiment 18.

[0118] Embodiment 23 is a method for inducing an immune response against influenza virus in a subject in need thereof, comprising administering to the subject in need thereof a pharmaceutical composition according to any one of embodiments 20 to 22.

[0119] Embodiment 24 is a method of producing an isolated variant influenza hemagglutinin polypeptide, the method comprising culturing the isolated host cell of embodiment 19 under conditions capable of producing the variant influenza hemagglutinin polypeptide, and recovering the variant influenza hemagglutinin polypeptide from the cell or culture.

[0120] Embodiment 25 is a method of making the pharmaceutical composition of embodiment 20, comprising combining an isolated mutant influenza polypeptide with a pharmaceutically acceptable carrier. [Example]

[0121] Example 1: Positioning of stabilizing substitutions in polypeptide-HA The structure and arrangement of changes in the sequence of the polypeptide (HA0) representing the ectodomain of influenza virus hemagglutinin are shown in Figure 1A for the HA trimer (left) and monomer (right). Figure 1B shows the same substitutions for five different instability regions (head switch, neck switch, stem switch, hinge loop, and repulsive cluster) and three additional regions (fusion peptide, receptor binding site, and base). When expressed as a soluble ectodomain, the polypeptide is truncated at the carboxy (C) terminus; for example, after position 536 of SEQ ID NO: 1, for UFV220265 (SEQ ID NO: 73), the polypeptide is only 534 amino acids long; note that SEQ ID NO: 1 contains two additional amino acids (Figure 2A) when compared to UFV220265 (Figure 9), which omits the native C-terminal transmembrane and cytoplasmic domains (amino acids 550-585). Note that for numbering of amino acid positions, the wild-type HA B / Brisbane / 60 / 08 (SEQ ID NO: 1) numbering was used and the signal peptide (residues 1-15) was included.

[0122] Amino acid substitutions in the HA polypeptide at the positions shown in Figure 1 were evaluated to stabilize HA, increase expression, and ensure correct folding and trimerization similar to the native conformation of wild-type full-length HA on the surface of the virion.

[0123] Example 2: Characterization of stabilizing mutations design To improve the stability of WT Flu B HA, stabilizing amino acid substitutions were introduced into five destabilizing regions across HA: the head switch, neck switch, stem switch, hinge loop, and "repulsive cluster," as well as three additional regions: the fusion peptide, base, and receptor-binding site. Most of these regions are pH-sensitive switches (pH switches), which become destabilized when their histidines become protonated at lower pH. The repulsive cluster does not contain histidines but is closely linked to the pH-sensitive stem switch.

[0124] Culture supernatant analysis Plasmids encoding the wild-type (wt) Flu B Iowa HA protein ectodomain and variants with amino acid substitutions were synthesized and codon-optimized with Genscript. In all plasmids, the transmembrane region and cytoplasmic tail were replaced with a three-residue linker and C-tag (SEQ ID NO: 136). Constructs were cloned into pCDNA2004 by standard methods well known in the art, including site-directed mutagenesis and PCR, and constructs were sequenced. Proteins were expressed in Expi293F cells. Expi293F cells were cultured in ExpiFectamine (Life Technologies). Transiently transfected cells were cultured for 3 days at 37°C and 10% CO2 using a chromatographic technique (Technologies, Carlsbad, CA) according to the manufacturer's instructions. The culture supernatant was collected, and cells and cell debris were removed by centrifugation at 300 g for 5 minutes. The clarified supernatant was then sterile filtered using a 0.22 μm vacuum filter and stored at 4°C until use. Analytical SEC (Figure 2) on the day of collection was performed using an ultra-high-performance liquid chromatography system (Vanquish, Thermo Fisher Scientific; Waltham, MA) and a μDAWN light scattering detector (Wyatt) coupled to an Optilab μT-rEX refractive index detector (Wyatt Technology Corporation; Santa Barbara, CA) in combination with an in-line Nanostar DLS reader (Wyatt Technology Corporation). The clarified crude cell culture supernatant was then transferred to a UnixC column equipped with a corresponding guard column (Sepax) equilibrated with running buffer (150 mM sodium phosphate, 50 mM NaCl, pH 7.0). The flow was applied to a SEC-300 15 cm column (Sepax catalog number 231300-4615) at 0.3 mL / min. When analyzing supernatant samples, the μMALS detector was taken offline and analytical SEC data was analyzed using the Chromeleon software package.The stability of the different proteins in the supernatants was also determined by measuring their melting temperatures (Tm) using differential scanning fluorimetry (DSF) (Figure 3 and Table 2). For this purpose, SYPRO Orange 5000x (S6650, Invitrogen; Waltham, MA) was diluted (1:250) in PBS to obtain a 20x working solution. For each reaction, 15 μL of supernatant was mixed with 5 μL of SYPRO 20x in a MicroAmp Fast Optical 96-well plate (4346906, ThermoFisher). PBS was used as a negative control. The plate was covered with MicroAmp Optical Adhesive Film (4311971, ThermoFisher) and then read on a ViiA7 Real-time PCR machine. The melting temperatures of all HA variants were expressed as the temperature at which 50% of the protein was melted (Tm). 50 ) (Table 2).

[0125] Results and Conclusions Comparison of expression levels of wt Flu B HA and HA variants with stabilizing substitutions by analytical size exclusion chromatography (SEC) of clarified cell culture supernatants for trimer and monomer content is shown in Figure 2, and melting temperatures (Tm 50 ) are shown in Table 2 and Figure 3.

[0126] [Table 2-1]

[0127] [Table 2-2] Improved temperature stability (Tm) compared to the reference (UFV212130) 50 Polypeptides having an increase in saturation (>0.5°C) and / or improved trimer % (≥5%) are *◆Values ​​obtained in a different experiment in which the reference polypeptide UFV212130 showed values ​​similar to those listed here.

[0128] The relative stabilizing ability of different amino acid substitutions is shown in the scatter plot in Figure 3, which shows the trimer expression level (trimer peak area under the curve) versus the percentage of trimers. The diameter of the markers corresponds to the Tm values ​​listed in Table 2. 50 All three values ​​correlate with the quality and stability of the HA trimer.

[0129] Stabilizing amino acid substitutions were found in all five regions: -Head switch (K227L, K227T, K227R, K227Q, K227F, K227I, K227Y, T229L, T277V, T236N, H238F, and H235T) -Neck Switch (Q426F, Q426W, Q426Y, and S455A) -Stem Switch (H384F, H384Y, H384W, H476Y, H476F, and S472R) -Repulsive Cluster (E475Q, E475N, E475W, E475F, E475L, L478W, and L478R) - Hinge Loop (V235K, V235W, G430L, G430Y, and D433P)

[0130] SEC analysis of culture supernatants from Expi293F cells expressing polypeptides containing these substitutions showed increased trimer expression titers and % trimer compared to wild-type HA. Furthermore, DSF analysis showed that improved expression correlated with increased melting temperatures, and all three values ​​correlated with the quality and stability of the HA trimer.

[0131] Example 3: Characterization of combinations of stabilizing mutations design Next, variants with selected mutation combinations were constructed to assess whether their effects on trimer expression, trimer ratio, and melting temperature were additive. Selected stabilizing amino acid substitutions from each region were combined and compared with the single-chain mutations obtained from Figure 3.

[0132] Culture supernatant analysis The DNA fragment encoding the polypeptide was synthesized and the polypeptide was produced in Expi293F cells as described in Example 2. Peak areas (trimer and trimer %) and temperature stability values ​​were obtained by SEC and DSF as described in Example 2.

[0133] Results and Conclusions Figures 4A-4E show SEC profiles of clarified cell culture supernatants of Expi293F cells expressing the polypeptides. A compilation of all the mutation combination data is listed in Table 3 and shown in the scatter plot of Figure 5 showing trimer expression levels versus trimer percentage. The diameter of the markers corresponds to the Tm values ​​listed in Table 3. 50 is scaled based on

[0134] [Table 3-1]

[0135] [Table 3-2]

[0136] [Table 3-3] * Polypeptides with improved thermal stability (Tm 50 ♦ Values ​​obtained in a different experiment in which the reference polypeptide UFV212130 showed values ​​similar to those listed here.

[0137] Most variants with selected mutation combinations exhibited approximately 2-3 fold higher expression titers and higher trimer percentages compared to the WT HA reference polypeptide (UFV212130), with the exception of the combination E475F + L478W (UFV220313). The improved expression correlates well with the observed increase in melting temperature compared to single-mutant polypeptides, regardless of the type of combination (single- or multi-region). The top three expressing polypeptides contain four substitutions in the stem switch: UFV220455 (substitutions H384F, S402A, S472W, and H476F), UFV220521 (substitutions H384Y, S402A, S472R, and H476F), and UFV220522 (substitutions H384Y, S402A, S472W, and H476F). For these variants, the relative trimer percentages are 93.6, 95.6, and 92.8%, respectively. Compared with these three variants, the polypeptide with substitutions in two regions showed lower expression titers, but the trimer % was improved. Combining substitutions from multiple regions improved the expression level and trimer % (up to 100%), as well as the protein temperature stability. The polypeptide UFV220265, which contains substitutions in all five of the unstable regions, showed the highest expression level (9.7 mAU). * mL) and the highest temperature stability (Tm of 71.5°C) 50 ) were shown. Taken together, substitutions in different regions are additive and improve trimer titer and protein stability.

[0138] Example 4: Universal Single Chain design Next, we tested the generality of the stabilizing substitutions by introducing them into soluble HAs from both the Yamagata (B / Singapore / INFTT-16-0610 / 2016 and B / Florida / 04 / 2006) and Victoria strains (B / Iowa / 06 / 2017, B / Brisbane / 60 / 08, and B / Ohio / 01 / 05). Several sequences obtained from the GISAID initiative and / or influenza virus databases contain extremely rare, strain-specific residues. These rare residues may have been acquired, for example, by egg adaptation, sequencing errors, or other reasons, and need to be optimized or "repaired" to obtain native sequences that can fold correctly and efficiently (9, 21). For the two HAs that were not expressed, several extremely rare residues were substituted into the consensus Flu B HA (Table 4). Figure 6A shows that two HA sequences (B / Victoria / 02 / 1987; SEQ ID NOs: 224 and 225 and B / Guangdong / 120 / 2000; SEQ ID NOs: 226 and 227) repaired with the substitutions listed in Table 4 show high expression of HA in analytical SEC. Next, for the other three HAs (B / Ohio / 01 / 2005, B / Florida / 04 / 2006, and B / Singapore / INFTT-16-0610 / 2016), two very rare mutations were observed, and therefore, these were "repaired" back to the consensus residues (Table 4).

[0139] [Table 4]

[0140] Different levels of HA stabilization were achieved by selecting different stabilizing substitutions. A fully stabilized version was designed by introducing five stabilizing substitutions from five different instability regions (K227T + H384F + Q426Y + G430Y + E475W). Less stabilized versions were created by introducing either three stabilizing substitutions in two pH switch regions (K227T in the head switch and H384F in the stem switch) and the repulsive cluster region (E475W), or two stabilizing substitutions from the neck switch region (H329W, Q426W).

[0141] Culture supernatant analysis For wild-type and stabilized HA, clarified cell culture supernatants of Expi293F cells expressing the polypeptides were obtained as described in Example 2. The relative expression and percentage of trimer were calculated by integrating the peak areas of trimer and monomer in SEC as described in Example 2. The thermal stability was obtained by measuring the melting temperature by DSF as described in Example 2.

[0142] Results and Conclusions Figure 6A shows that it is important to confirm that HA shows any expression to demonstrate the universal nature of the stabilizing substitutions. Thus, Figure 6A shows that HA expression can be obtained by substituting very rare mutations into consensus residues without introducing any stabilizing substitutions. Apparently, some HAs contained either sequencing errors or, for example, egg adaptation, and HA expression was detected only when the erroneous mutations were restored to wild-type. Therefore, B / Victoria / 02 / 1987 and B / Guangdong / 120 / 2000 were repaired according to Table 4 to ensure that the stabilizing substitutions had their effect on well-folded HA without inherent sequence errors. Figure 6B shows the SEC profile of clarified cell culture supernatant of Expi293F cell expression of the polypeptides. Peak areas (total trimer expression and trimer %) and temperature stability values ​​are listed in Table 5.

[0143] [Table 5] ◆ Includes two consensus repair mutations * The stabilized polypeptide contains the mutations: A. K227T, H384F, Q426Y, G430Y, and E475W, B. K227T, H384F, and E475W, C. H329W and Q426W.

[0144] Compared to the WT HA polypeptide, all stabilized variants showed higher expression titers, increased percent trimers, and improved temperature stability. For stabilized variant C (stabilized neck switch region only), some monomeric polypeptides were observed, whereas variants B (stabilized head and stem pH switches + repulsive cluster) and A (stabilized in all five regions) were expressed exclusively as trimeric polypeptides. Variant A polypeptide stabilized in all five regions exhibited a melting temperature approximately 3–6°C higher than variant B polypeptide lacking the Q426Y and G430Y mutations, demonstrating the importance of these two substitutions. Collectively, these findings indicate that the stabilizing mutations are universally applicable to HA from both the Yamagata and Victoria lineages.

[0145] Example 5: Characterization of stabilizing mutations in truncated HA design HA requires proteolytic cleavage to undergo maturation. The cleaved HA folds into a native trimeric conformation, where the fusion peptide participates in trimerization (22, 23, and 24). This maturation process can increase HA stability at neutral pH but decreases stability at low pH because low pH induces conformational changes required for membrane fusion. HA cleavage does not occur during expression in known mammalian cell expression systems. Therefore, we designed a cleavable variant of the polypeptide that can be posttranslationally cleaved in mammalian cells by inserting a 27-residue peptide derived from respiratory syncytial virus (RSV) at the HA0 cleavage site (Figure 1K). Furin-like proteases present in mammalian cells cleave the P27 peptide from the RSV fusion protein when inserted between the furin cleavage site or a weak furin cleavage site and the fusion peptide (Figure 1K). Because furin acts at low pH, this furin-based maturation process can only be successful if HA is sufficiently stabilized and maintained in a prefusion conformation. As in Example 4, for each strain, three variants with different levels of stabilization were tested: fully stabilized (variant A) and less stabilized (variants B and C), and all constructs contain a cleavable p27 peptide.

[0146] Culture supernatant analysis The HA variants described in Example 4 were modified by inserting RSV p27 before the fusion peptide, and the plasmids were transfected into Expi293F cells as described in Example 2, except that 20% of a furin-encoding vector was cotransfected to ensure adequate intracellular furin levels due to the p27 peptide-containing design. Peak areas (trimer and trimer %) and temperature stability values ​​obtained by SEC and DSF were obtained as described in Example 2. Additionally, culture supernatants were analyzed by Western blot to analyze the processing of HA0 in HA1 and HA2. Briefly, protein samples for denaturing gel electrophoresis were prepared by mixing 10 μL of clarified supernatant with lithium dodecyl sulfate (LDS) sample buffer and incubated at 95°C for 10 minutes under reducing conditions. Samples were run on 4-12% Bolt Bis-Tris Gels (Invitrogen) at 200 V for 30 minutes. After rinsing with demineralized water, the gel was blotted onto a PVDF membrane using iBlot 2 (Invitrogen). The membrane was then blocked for 60 min in Intercept Blocking Buffer (Li-Cor), washed with TBS-T (3 × 5 min), incubated with streptavidin CW800 (Li-Cor) for 45 min, washed with TBS-T (2 × 5 min), followed by a final wash with PBS. C-tagged polypeptides were visualized by scanning with an Odyssey scanner (Li-Cor Biosciences; Lincoln, NE).

[0147] Results and Conclusions Figures 6C-6D show SEC profiles and WB of clarified cell culture supernatants of Expi293F cells expressing the polypeptides. Peak areas (total trimer expression and % trimer) and temperature stability values ​​are listed in Table 6.

[0148] [Table 6] 1. All HA is cleavable and contains the P27 peptide, 2. The stabilized polypeptide contains the mutations: A. K227T, H384F, Q426Y, G430Y, and E475W, B. K227T, H384F, and E475W, C. H329W and Q426W.

[0149] Only the A variant of HA, containing all five stabilizing substitutions in all five instability regions, showed high trimeric expression, whereas only low levels of monomer were observed for non-stabilized or semi-stabilized HA by SEC (Figure 6B). The melting temperatures of the cleaved, fully stabilized variants were similar to those of the uncleaved variants (Table 5). In reduced Western blots, a band corresponding to HA2 was observed in the culture supernatant of Exi293F cells cotransfected with furin and expressing the fully stabilized cleavable variant, indicating processing of the introduced furin site (Figure 6C). In summary, a cleaved, trimeric, temperature-stable HA polypeptide was produced, but complete stabilization (K227T, H384F, Q426Y, G430Y, and E475W) was required for cleavage at low pH.

[0150] Example 6 design Further combinations of stabilizing substitutions in the stem switch region and repulsive three-fold cluster were tested in furin-cleavable Flu B HA (B / Iowa / 06 / 2017), as described in Example 5. Alternative stabilizing substitutions in the stem switch and repulsive cluster were evaluated in a furin-cleavable HA parent design containing three substitutions: K227T, Q426Y, and G430Y (Table 7).

[0151] Culture supernatant analysis Plasmids encoding the cleavable polypeptides were transfected into Expi293F cells as described in Example 5. Peak areas (trimer and % trimer) and temperature stability values ​​were obtained by SEC and DSF as described in Example 2.

[0152] Results and Conclusions Figure 7 shows the SEC profile of clarified cell culture supernatant of Expi293F cells expressing the polypeptide. The peak areas (total trimer expression and % trimer) and temperature stability values ​​are listed in Table 7 and shown in the scatter plot of Figure 8, which indicates a prominent trimer peak (>2 mAu * The expression level of trimers versus the percentage of trimers for the polypeptides shown (Tm mL) are shown. Markers are those listed in Table 7. 50 is scaled based on

[0153] [Table 7] * All except UFV212130 contain K227T, Q426Y, and G430Y mutations. Values ​​for low-expressing proteins (OD280<10mAu peak height in SEC profile) are not shown (listed as not applicable).

[0154] For variants lacking the stabilizing substitution H384F (UFV221167 and UFV221171), no or only negligible levels of trimeric and monomeric polypeptides were observed in SEC (Figure 7), whereas all variants containing this substitution were expressed exclusively as trimers (Table 7). Polypeptide UFV221176 was shown to be the most temperature-stable variant, and polypeptide UFV221162 was expressed at the highest level. The second most stable polypeptide (UFV221175) and the third most stable polypeptide (UFV221174) differed in substitutions at position 472, which were L and K, respectively, whereas this position was mutated to arginine in the most stable polypeptide. For these polypeptides, stability correlates with expression level: 7.5, 6.2, and 5.4 for UFV221176, UFV221175, and UFV221174, respectively. In summary, additional substitutions at positions 402, 472, 475, and 476 further improved expression titer, trimer %, and protein stability.

[0155] References (1) F.Krammer et al., Influenza. Nat Rev Dis Primers 4, 3 (2018). (2) C.S. Ambrose, M.J. Levin, The rationale for quadrivalent influenza vaccines. Hum Vaccin Immunother 8, 81 - 88 (2012). (3) F. Dijkstra, G.A. Donker, B. Wilbrink, A.B. Van Gageldonk - Lafeber, M.A. Van Der Sande, Long time trends in influenza - like illness and associated determinants in The Netherlands. Epidemiol Infect 137, 473 - 479 (2009). (4) V. Peltola, T. Ziegler, O. Ruuskanen, Influenza A and B virus infections in children. Clin Infect Dis 36, 299 - 305 (2003). (5) W.W. Thompson et al., Influenza - associated hospitalizations in the United States.JAMA 292, 1333 - 1340 (2004). (6) L.A. Grohskopf et al., Prevention and Control of Seasonal Influenza with Vaccines: Recommendations of the Advisory Committee on Immunization Practices - United States, 2017 - 18 Influenza Season. MMWR Recomm Rep 66, 1 - 20 (2017). (7)L.A.Grohskopf,L.Z.Sokolow,A.M.Fry,E.B.Walter,D.B.Jernigan,Update:ACIP Recommendations for the Use of Quadrivalent Live Attenuated Influenza Vaccine(LAIV4)-United States, 2018-19 Influenza Season.MMWR Morb Mortal Wkly Rep 67,643-645(2018)。 (8)N.M.Ferguson,A.P.Galvani,R.M.Bush,Ecological and immunological determinants of influenza evolution.Nature 422,428-433(2003)。 (9)F.J.Milder et al.,Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions.Proc Natl Acad Sci U S A 119(2022)。 (10)F.Ni,I.N.Mbawuike,E.Kondrashkina,Q.Wang,The roles of hemagglutinin Phe-95 in receptor binding and pathogenicity of influenza B virus.Virology 450-451,71-83(2014)。 (11)J.Devereux,P.Haeberli,O.Smithies, A comprehensive set of sequence analysis programs for the VAX.Nucleic Acids Res 12,387-395(1984)。 (12)J.L.Lorieau,J.M.Louis,A.Bax,The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid: water interface.Proc Natl Acad Sci U S A 107,11341-11346(2010)。 (13)T.A.Dopheide,C.W. Ward, The location of the bromelain cleavage site in a Hong Kong influenza virus Haemagglutinin.J Gen Virol 52,367-370(1981)。 (14)D.C.Ekiert et al.,Antibody recognition of a highly conserved influenza virus epitope.Science 324,246-251(2009)。 (15)D.C.Ekiert et al.,A highly conserved neutralizing epitope on group 2 influenza A viruses.Science 333,843-850(2011)。 (16)J.Stevens et al.,Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus.Science 312,404-410(2006)。 (17)J.Stevens et al.,Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus.Science 303,1866-1870(2004)。 (18)I.A.Wilson,J.J.Skehel,D.C.Wiley,Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution.Nature 289,366-373(1981)。 (19)C.Dreyfus et al.,Highly conserved protective epitopes on influenza B viruses.Science 337,1343-1348(2012)。

[0156] (20)R.L.Coffman,A.Sher,R.A.Seder,Vaccine adjuvants:putting innate immunity to work.Immunity 33,492-503(2010)。 (21)L.Rutten et al.,A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers.Cell Rep 23,584-595(2018)。 (22)H.L.Turner et al.,Potent anti-influenza H7 human monoclonal antibody induces separation of hemagglutinin receptor-binding head domains.PLoS Biol 17,e3000139(2019)。 (23)M.G.Joyce et al.,Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses.Cell 166,609-623(2016)。 (24)S.Bangaru et al.,A Site of Vulnerability on the Influenza Virus Hemagglutinin Head Domain Trimer Interface.Cell 177,1136-1152 e1118(2019). (25)US Centers for Disease Control and Prevention, “Seasonal influenza activity surveillance reports 2001-2018” www.cdc.gov / flu / weekly / pastreports.htm (accessed on July 2,2018) (26)European Center for Disease Prevention and Control / WHO Regional Office for Europe, “Annual epidemiological reports on seasonal influenza 2001-2018,” ecdc.europa.eu / en / seasonal-influenza / surveillance-and-disease-data / aer (accessed on July 2,2018) (27) World Health Organization, “Recommended composition of influenza virus vaccines for use in the 2017-2018 northern hemisphere influenza season,” www.who.int / influenza / vaccines / virus / recommendations / 2018_19_north / en (accessed on July 2,2018) (28) International Publication No. 2008 / 028946 (29) International Publication No. 2010 / 130636 (30) International Publication No. 2013 / 007770 (31) International Publication No. 2015 / 148806

[0157] array SEQ ID NO: 1: Full length B / Brisbane / 60 / 08

[0158] [ka]

[0159] SEQ ID NO: 2: RSV p27 peptide

[0160] [ka]

[0161] Sequence number 3 UFV212130 Soluble (ectodomain) B / IOWA / 06 / 2017

[0162] [ka]

[0163] SEQ ID NO: 4: UFV212175

[0164] [ka]

[0165] SEQ ID NO: 5: UFV212136

[0166] [ka]

[0167] SEQ ID NO: 6: UFV212137

[0168] [ka]

[0169] SEQ ID NO: 7: UFV212138

[0170] [ka]

[0171] SEQ ID NO: 8: UFV212199

[0172] [ka]

[0173] SEQ ID NO: 9: UFV212200

[0174] [ka]

[0175] SEQ ID NO: 10: UFV212173

[0176] [ka]

[0177] SEQ ID NO: 11: UFV212208

[0178] [ka]

[0179] SEQ ID NO: 12: UFV212209

[0180] [ka]

[0181] SEQ ID NO: 13: UFV212139

[0182] [ka]

[0183] SEQ ID NO: 14: UFV212140

[0184] [ka]

[0185] SEQ ID NO: 15: UFV212174

[0186] [ka]

[0187] SEQ ID NO: 16: UFV212141

[0188] [ka]

[0189] SEQ ID NO: 17: UFV212201

[0190] [ka]

[0191] SEQ ID NO: 18: UFV212170

[0192] [ka]

[0193] SEQ ID NO: 19: UFV212171

[0194] [ka]

[0195] SEQ ID NO: 20: UFV212172

[0196] [ka]

[0197] SEQ ID NO: 21: UFV212196

[0198] [ka]

[0199] SEQ ID NO: 22: UFV212216

[0200] [ka]

[0201] SEQ ID NO: 23: UFV212215

[0202] [ka]

[0203] SEQ ID NO: 24: UFV212212

[0204] [ka]

[0205] SEQ ID NO: 25: UFV212143

[0206] [ka]

[0207] SEQ ID NO: 26: UFV212188

[0208] [ka]

[0209] SEQ ID NO: 27: UFV212217

[0210] [ka]

[0211] SEQ ID NO: 28: UFV212203

[0212] [ka]

[0213] SEQ ID NO: 29: UFV212155

[0214] [ka]

[0215] SEQ ID NO: 30: UFV212156

[0216] [ka]

[0217] SEQ ID NO: 31: UFV212157

[0218] [ka]

[0219] SEQ ID NO: 32: UFV212211

[0220] [ka]

[0221] SEQ ID NO: 33: UFV212144

[0222] [ka]

[0223] SEQ ID NO: 34: UFV212145

[0224] [ka]

[0225] SEQ ID NO: 35: UFV212163

[0226] [ka]

[0227] SEQ ID NO: 36: UFV212164

[0228] [ka]

[0229] SEQ ID NO: 37: UFV212166

[0230] [ka]

[0231] SEQ ID NO: 38: UFV212193

[0232] [ka]

[0233] SEQ ID NO: 39: UFV212194

[0234] [ka]

[0235] SEQ ID NO: 40: UFV212165

[0236] [ka]

[0237] SEQ ID NO: 41: UFV212202

[0238] [ka]

[0239] SEQ ID NO: 42: UFV212161

[0240] [ka]

[0241] SEQ ID NO: 43: UFV212162

[0242] [ka]

[0243] SEQ ID NO: 44: UFV212148

[0244] [ka]

[0245] SEQ ID NO: 45: UFV212149

[0246] [ka]

[0247] SEQ ID NO: 46: UFV212150

[0248] [ka]

[0249] SEQ ID NO: 47: UFV212151

[0250] [ka]

[0251] SEQ ID NO: 48: UFV212152

[0252] [ka]

[0253] SEQ ID NO: 49: UFV212153

[0254] [ka]

[0255] SEQ ID NO: 50: UFV212181

[0256] [ka]

[0257] SEQ ID NO: 51: UFV212182

[0258] [ka]

[0259] SEQ ID NO: 52: UFV220244

[0260] [ka]

[0261] SEQ ID NO: 53: UFV220245

[0262] [ka]

[0263] SEQ ID NO: 54: UFV220246

[0264] [ka]

[0265] SEQ ID NO: 55: UFV220247

[0266] [ka]

[0267] SEQ ID NO: 56: UFV220248

[0268] [ka]

[0269] SEQ ID NO: 57: UFV220249

[0270] [ka]

[0271] SEQ ID NO: 58: UFV220250

[0272] [ka]

[0273] SEQ ID NO: 59: UFV220251

[0274] [ka]

[0275] SEQ ID NO: 60: UFV220252

[0276] [ka]

[0277] SEQ ID NO: 61: UFV220253

[0278] [ka]

[0279] SEQ ID NO: 62: UFV220254

[0280] [ka]

[0281] SEQ ID NO: 63: UFV220255

[0282] [ka]

[0283] SEQ ID NO: 64: UFV220256

[0284] [ka]

[0285] SEQ ID NO: 65: UFV220257

[0286] [ka]

[0287] SEQ ID NO: 66: UFV220258

[0288] [ka]

[0289] SEQ ID NO: 67: UFV220259

[0290] [ka]

[0291] SEQ ID NO: 68: UFV220260

[0292] [ka]

[0293] SEQ ID NO: 69: UFV220261

[0294] [ka]

[0295] SEQ ID NO: 70: UFV220262

[0296] [ka]

[0297] SEQ ID NO: 71: UFV220263

[0298] [ka]

[0299] SEQ ID NO: 72: UFV220264

[0300] [ka]

[0301] SEQ ID NO: 73: UFV220265

[0302] [ka]

[0303] SEQ ID NO: 74: UFV220266

[0304] [ka]

[0305] SEQ ID NO: 75: UFV220267

[0306] [ka]

[0307] SEQ ID NO: 76: UFV220268

[0308] [ka]

[0309] SEQ ID NO: 77: UFV220269

[0310] [ka]

[0311] SEQ ID NO: 78: UFV220270

[0312] [ka]

[0313] SEQ ID NO: 79: UFV220271

[0314] [ka]

[0315] SEQ ID NO: 80: UFV220272

[0316] [ka]

[0317] SEQ ID NO: 81: UFV220273

[0318] [ka]

[0319] SEQ ID NO: 82: UFV220274

[0320] [ka]

[0321] SEQ ID NO: 83: UFV220275

[0322] [ka]

[0323] SEQ ID NO: 84: UFV220276

[0324] [ka]

[0325] SEQ ID NO: 85: UFV220277

[0326] [ka]

[0327] SEQ ID NO: 86: UFV220278

[0328] [ka]

[0329] SEQ ID NO: 87: UFV220279

[0330] [ka]

[0331] SEQ ID NO: 88: UFV220280

[0332] [ka]

[0333] SEQ ID NO: 89: UFV220281

[0334] [ka]

[0335] SEQ ID NO: 90: UFV220282

[0336] [ka]

[0337] SEQ ID NO: 91: UFV220283

[0338] [ka]

[0339] SEQ ID NO: 92: UFV220284

[0340] [ka]

[0341] SEQ ID NO: 93: UFV220285

[0342] [ka]

[0343] SEQ ID NO: 94: UFV220286

[0344] [ka]

[0345] SEQ ID NO: 95: UFV220287

[0346] [ka]

[0347] SEQ ID NO: 96: UFV220288

[0348] [ka]

[0349] SEQ ID NO: 97: UFV220289

[0350] [ka]

[0351] SEQ ID NO: 98: UFV220290

[0352] [ka]

[0353] SEQ ID NO: 99: UFV220291

[0354] [ka]

[0355] SEQ ID NO: 100: UFV220292

[0356] [ka]

[0357] SEQ ID NO: 101: UFV220293

[0358] [ka]

[0359] SEQ ID NO: 102: UFV220294

[0360] [ka]

[0361] SEQ ID NO: 103: UFV220295

[0362] [ka]

[0363] SEQ ID NO: 104: UFV220296

[0364] [ka]

[0365] SEQ ID NO: 105: UFV220297

[0366] [ka]

[0367] SEQ ID NO: 106: UFV220298

[0368] [ka]

[0369] SEQ ID NO: 107: UFV220299

[0370] [ka]

[0371] SEQ ID NO: 108: UFV220300

[0372] [ka]

[0373] SEQ ID NO: 109: UFV220301

[0374] [ka]

[0375] SEQ ID NO: 110: UFV220302

[0376] [ka]

[0377] SEQ ID NO: 111: UFV220303

[0378] [ka]

[0379] SEQ ID NO: 112: UFV220304

[0380] [ka]

[0381] SEQ ID NO: 113: UFV220305

[0382] [ka]

[0383] SEQ ID NO: 114: UFV220306

[0384] [ka]

[0385] SEQ ID NO: 115: UFV220307

[0386] [ka]

[0387] SEQ ID NO: 116: UFV220308

[0388] [ka]

[0389] SEQ ID NO: 117: UFV220309

[0390] [ka]

[0391] SEQ ID NO: 118: UFV220310

[0392] [ka]

[0393] SEQ ID NO: 119: UFV220311

[0394] [ka]

[0395] SEQ ID NO: 120: UFV220312

[0396] [ka]

[0397] SEQ ID NO: 121: UFV220313

[0398] [ka]

[0399] SEQ ID NO: 122: UFV220314

[0400] [ka]

[0401] SEQ ID NO: 123: UFV220315

[0402] [ka]

[0403] SEQ ID NO: 124: UFV220316

[0404] [ka]

[0405] SEQ ID NO: 125: UFV220317

[0406] [ka]

[0407] SEQ ID NO: 126: UFV220318

[0408] [ka]

[0409] SEQ ID NO: 127: UFV220319

[0410] [ka]

[0411] SEQ ID NO: 128: UFV220320

[0412] [ka]

[0413] SEQ ID NO: 129: UFV220450

[0414] [ka]

[0415] SEQ ID NO: 130: UFV220454

[0416] [ka]

[0417] SEQ ID NO: 131: UFV220455

[0418] [ka]

[0419] SEQ ID NO: 132: UFV220521

[0420] [ka]

[0421] SEQ ID NO: 133: UFV220522

[0422] [ka]

[0423] SEQ ID NO: 134: HIS-tag

[0424] [ka]

[0425] SEQ ID NO: 135: HIS-tag

[0426] [ka]

[0427] SEQ ID NO: 136: C-tag

[0428] [ka]

[0429] SEQ ID NO: 137: FLAG tag

[0430] [ka]

[0431] SEQ ID NO: 138: Factor X proteolytic cleavage site

[0432] [ka]

[0433] SEQ ID NO: 139: Thrombin proteolytic cleavage site

[0434] [ka]

[0435] SEQ ID NO: 140: UFV220877

[0436] [ka]

[0437] SEQ ID NO: 141: UFV220880 soluble (ectodomain) B / Ohio / 01 / 2005

[0438] [ka]

[0439] SEQ ID NO: 142: UFV220882

[0440] [ka]

[0441] SEQ ID NO: 143: UFV220884

[0442] [ka]

[0443] SEQ ID NO: 144: UFV220886

[0444] [ka]

[0445] SEQ ID NO: 145: UFV220888 soluble (ectodomain) B / Brisbane / 60 / 2008

[0446] [ka]

[0447] SEQ ID NO: 146: UFV220890

[0448] [ka]

[0449] SEQ ID NO: 147: UFV220892

[0450] [ka]

[0451] SEQ ID NO: 148: UFV220894

[0452] [ka]

[0453] SEQ ID NO: 149: UFV220896 soluble (ectodomain) B / Florida / 04 / 2006

[0454] [ka]

[0455] SEQ ID NO: 150: UFV220898

[0456] [ka]

[0457] SEQ ID NO: 151: UFV220900

[0458] [ka]

[0459] SEQ ID NO: 152: UFV220902

[0460] [ka]

[0461] SEQ ID NO: 153: UFV220904 soluble (ectodomain) B / Singapore / INFTT-16-0610 / 2016

[0462] [ka]

[0463] SEQ ID NO: 154: UFV220906

[0464] [ka]

[0465] SEQ ID NO: 155: UFV220908

[0466] [ka]

[0467] SEQ ID NO: 156: UFV220910

[0468] [ka]

[0469] SEQ ID NO: 157: UFV220875

[0470] [ka]

[0471] SEQ ID NO: 158: UFV220876

[0472] [ka]

[0473] SEQ ID NO: 159: UFV220878

[0474] [ka]

[0475] SEQ ID NO: 160: UFV220879

[0476] [ka]

[0477] SEQ ID NO: 161: UFV220881

[0478] [ka]

[0479] SEQ ID NO: 162: UFV220883

[0480] [ka]

[0481] SEQ ID NO: 163: UFV220885

[0482] [ka]

[0483] SEQ ID NO: 164: UFV220887

[0484] [ka]

[0485] SEQ ID NO: 165: UFV220889

[0486] [ka]

[0487] SEQ ID NO: 166: UFV220891

[0488] [ka]

[0489] SEQ ID NO: 167: UFV220893

[0490] [ka]

[0491] SEQ ID NO: 168: UFV220895

[0492] [ka]

[0493] SEQ ID NO: 169: UFV220897

[0494] [ka]

[0495] SEQ ID NO: 170: UFV220899

[0496] [ka]

[0497] SEQ ID NO: 171: UFV220901

[0498] [ka]

[0499] SEQ ID NO: 172: UFV220903

[0500] [ka]

[0501] SEQ ID NO: 173: UFV220905

[0502] [ka]

[0503] SEQ ID NO: 174: UFV220907

[0504] [ka]

[0505] SEQ ID NO: 175: UFV220909

[0506] [ka]

[0507] SEQ ID NO: 176: UFV220911

[0508] [ka]

[0509] SEQ ID NO: 177

[0510] [ka]

[0511] SEQ ID NO: 178: UFV221029

[0512] [ka]

[0513] SEQ ID NO: 179: UFV221030

[0514] [ka]

[0515] SEQ ID NO: 180: UFV221031

[0516] [ka]

[0517] SEQ ID NO: 181: UFV221032

[0518] [ka]

[0519] SEQ ID NO: 182: UFV221033

[0520] [ka]

[0521] SEQ ID NO: 183: UFV221034

[0522] [ka]

[0523] SEQ ID NO: 184: UFV221035

[0524] [ka]

[0525] SEQ ID NO: 185: UFV221036

[0526] [ka]

[0527] SEQ ID NO: 186: UFV221037

[0528] [ka]

[0529] SEQ ID NO: 187: UFV221038

[0530] [ka]

[0531] SEQ ID NO: 188: UFV221039

[0532] [ka]

[0533] SEQ ID NO: 189: UFV221040

[0534] [ka]

[0535] SEQ ID NO: 190: UFV221041

[0536] [ka]

[0537] SEQ ID NO: 191: UFV221184

[0538] [ka]

[0539] SEQ ID NO: 192: UFV221185

[0540] [ka]

[0541] SEQ ID NO: 193: UFV221186

[0542] [ka]

[0543] SEQ ID NO: 194: UFV221042

[0544] [ka]

[0545] SEQ ID NO: 195: UFV221043

[0546] [ka]

[0547] SEQ ID NO: 196: UFV221044

[0548] [ka]

[0549] SEQ ID NO: 197: UFV221045

[0550] [ka]

[0551] SEQ ID NO: 198: UFV221046

[0552] [ka]

[0553] SEQ ID NO: 199: UFV221159

[0554] [ka]

[0555] SEQ ID NO: 200: UFV221160

[0556] [ka]

[0557] SEQ ID NO: 201: UFV221161

[0558] [ka]

[0559] SEQ ID NO: 202: UFV221162

[0560] [ka]

[0561] SEQ ID NO: 203: UFV221163

[0562] [ka]

[0563] SEQ ID NO: 204: UFV221164

[0564] [ka]

[0565] SEQ ID NO: 205: UFV221165

[0566] [ka]

[0567] SEQ ID NO: 206: UFV221166

[0568] [ka]

[0569] SEQ ID NO: 207: UFV221167

[0570] [ka]

[0571] SEQ ID NO: 208: UFV221168

[0572] [ka]

[0573] SEQ ID NO: 209: UFV221169

[0574] [ka]

[0575] SEQ ID NO: 210: UFV221170

[0576] [ka]

[0577] SEQ ID NO: 211: UFV221171

[0578] [ka]

[0579] SEQ ID NO: 212: UFV221172

[0580] [ka]

[0581] SEQ ID NO: 213: UFV221173

[0582] [ka]

[0583] SEQ ID NO: 214: UFV221174

[0584] [ka]

[0585] SEQ ID NO: 215: UFV221175

[0586] [ka]

[0587] SEQ ID NO: 216: UFV221176

[0588] [ka]

[0589] SEQ ID NO: 217: UFV221177

[0590] [ka]

[0591] SEQ ID NO: 218: UFV221178

[0592] [ka]

[0593] SEQ ID NO: 219: UFV221179

[0594] [ka]

[0595] SEQ ID NO: 220: UFV221180

[0596] [ka]

[0597] SEQ ID NO: 221: UFV221181

[0598] [ka]

[0599] SEQ ID NO: 222: UFV221182

[0600] [ka]

[0601] SEQ ID NO: 223: UFV221183

[0602] [ka]

Claims

1. 1. An isolated mutant influenza hemagglutinin polypeptide, comprising at least one stabilizing mutation in at least one of instability regions (a)-(e) in said polypeptide, (a) amino acid positions 227, 229, and / or 238, and / or (b) amino acid positions 329 and / or 426, and / or (c) amino acid positions 384, 402, 472, and / or 476, and / or (d) amino acid positions 468, 471, 475, and / or 478, and / or (e) amino acid positions 235, 430, and / or 433 and at least one stabilizing mutation, including a substitution mutation at An isolated mutant influenza hemagglutinin polypeptide, wherein said amino acid positions correspond to the amino acid positions of SEQ ID NO:

1.

2. 2. The isolated mutant influenza hemagglutinin polypeptide of claim 1, comprising at least two stabilizing mutations in at least one of the instability regions (a)-(e) in the polypeptide.

3. 2. The isolated mutant influenza hemagglutinin polypeptide of claim 1, comprising at least two stabilizing mutations in two, three, four, or five of the instability regions (a) through (e) in the polypeptide.

4. 2. The isolated mutant influenza hemagglutinin polypeptide of claim 1, comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 stabilizing mutations.

5. (a) a substitution at amino acid position 227 with an amino acid selected from the group consisting of T, L, R, Q, F, I, and Y, a substitution at amino acid position 229 with the amino acid L, and / or a substitution at amino acid position 238 with the amino acid F; and / or (b) amino acid position 329 is substituted with an amino acid selected from the group consisting of M, W, and F, and / or amino acid position 426 is substituted with an amino acid selected from the group consisting of F, W, Y, and P; and / or (c) amino acid position 384 is substituted with an amino acid selected from F or Y, amino acid position 402 is substituted with an amino acid A, amino acid position 472 is substituted with an amino acid selected from W, R, F, K, or L, and / or amino acid position 476 is substituted with an amino acid F; and / or (d) amino acid position 468 is substituted with an amino acid L, amino acid position 471 is substituted with an amino acid selected from V or Q, amino acid position 475 is substituted with an amino acid selected from the group consisting of Q, N, W, F, and L, and / or amino acid position 478 is substituted with an amino acid selected from W or R; and / or (e) amino acid position 235 is substituted with the amino acid W, amino acid position 430 is substituted with an amino acid selected from L or Y, and / or amino acid position 433 is substituted with the amino acid P; 5. The isolated mutant influenza hemagglutinin polypeptide of any one of claims 1 to 4.

6. (a) a substitution at amino acid position 384 with F and a substitution at amino acid position 475 with W; (b) a substitution at amino acid position 384 with F and a substitution at amino acid position 475 with Q; (c) a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 472 with K, and a substitution at amino acid position 476 with F; (d) a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 472 with R, and a substitution at amino acid position 476 with F; (e) a substitution at amino acid position 384 with Y, a substitution at amino acid position 402 with A, a substitution at amino acid position 472 with R, and a substitution at amino acid position 476 with F; (f) a substitution at amino acid position 476 with F and a substitution at amino acid position 475 with W; (g) a substitution at amino acid position 476 with F and a substitution at amino acid position 475 with Q; (h) a substitution at amino acid position 227 with T and a substitution at amino acid position 426 with Y; (i) a substitution at amino acid position 227 with T and a substitution at amino acid position 430 with Y; (j) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, and a substitution at amino acid position 430 with Y; (k) a substitution at amino acid position 227 with T and a substitution at amino acid position 475 with W; (l) a substitution at amino acid position 227 with T and a substitution at amino acid position 475 with Q; (m) a substitution at amino acid position 227 with T and a substitution at amino acid position 384 with F; (n) a substitution at amino acid position 227 with T and a substitution at amino acid position 476 with F; (o) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, and a substitution at amino acid position 475 with W; (p) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, and a substitution at amino acid position 475 with Q; (q) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 475 with W, and a substitution at amino acid position 430 with Y; (r) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 430 with Y; (s) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, and a substitution at amino acid position 384 with F; and / or (t) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 384 with F, and a substitution at amino acid position 475 with W; (u) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 384 with F, a substitution at amino acid position 475 with W, and a substitution at amino acid position 430 with Y; (v) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 384 with F, and a substitution at amino acid position 475 with Q; (w) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 384 with F, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 430 with Y; (x) a substitution at amino acid position 227 with T and a substitution at amino acid position 235 with W; (y) a substitution at amino acid position 475 with W and a substitution at amino acid position 426 with Y; (z) a substitution at amino acid position 475 with W and a substitution at amino acid position 430 with Y; (aa) a substitution at amino acid position 475 with Q and a substitution at amino acid position 478 with R; (bb) a substitution at amino acid position 475 with Q and a substitution at amino acid position 478 with F; (cc) a substitution at amino acid position 426 with Y and a substitution at amino acid position 384 with F; (dd) a substitution at amino acid position 426 with Y and a substitution at amino acid position 476 with F; (ee) a substitution at amino acid position 426 with Y and a substitution at amino acid position 455 with A; (ff) a substitution at amino acid position 426 with Y and a substitution at amino acid position 329 with W; (gg) a substitution at amino acid position 430 with Y and a substitution at amino acid position 384 with F; (hh) a substitution at amino acid position 430 with Y and a substitution at amino acid position 476 with F; (ii) a substitution at amino acid position 430 with Y and a substitution at amino acid position 426 with Y; (jj) a substitution at amino acid position 430 with Y and a substitution at amino acid position 426 with W; (kk) a substitution at amino acid position 430 with Y and a substitution at amino acid position 426 with F; (ll) a substitution at amino acid position 433 with P and a substitution at amino acid position 430 with Y; (mm) a substitution at amino acid position 433 with P and a substitution at amino acid position 426 with Y; (nn) a substitution at amino acid position 433 with P and a substitution at amino acid position 426 with W; (oo) a substitution at amino acid position 433 with P and a substitution at amino acid position 426 with F; (pp) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, and a substitution at amino acid position 475 with W; (qq) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with R, and a substitution at amino acid position 476 with F; (rr) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 468 with L, a substitution at amino acid position 471 with V, a substitution at amino acid position 472 with F, a substitution at amino acid position 475 with F, and a substitution at amino acid position 478 with R; (ss) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 468 with L, a substitution at amino acid position 471 with V, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with F, and a substitution at amino acid position 478 with R; (tt) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, and a substitution at amino acid position 476 with F; (uu) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 468 with L, a substitution at amino acid position 471 with V, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with L, and a substitution at amino acid position 478 with R; (vv) a substitution at amino acid position 227 with T, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 468 with L, a substitution at amino acid position 471 with Q, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 478 with R; (ww) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with K, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 476 with F; (xx) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with L, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 476 with F; (yy) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 471 with Q, a substitution at amino acid position 472 with R, and a substitution at amino acid position 476 with F; (zz) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, a substitution at amino acid position 475 with Q, and a substitution at amino acid position 476 with F; (aaa) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with W, and a substitution at amino acid position 476 with F; or (bbb) a substitution at amino acid position 227 with T, a substitution at amino acid position 384 with F, a substitution at amino acid position 402 with A, a substitution at amino acid position 426 with Y, a substitution at amino acid position 430 with Y, a substitution at amino acid position 472 with R, a substitution at amino acid position 475 with W, and a substitution at amino acid position 476 with F; 6. The isolated mutant influenza hemagglutinin polypeptide of claim 5.

7. 7. The isolated mutant influenza hemagglutinin polypeptide of any one of claims 1 to 6, wherein the mutant influenza hemagglutinin polypeptide further comprises an introduced cleavage site.

8. 8. The isolated mutant influenza hemagglutinin polypeptide of claim 7, wherein the introduced cleavage site is a furin cleavage site.

9. 9. The isolated mutant influenza hemagglutinin polypeptide of claim 8, wherein the furin cleavage site is introduced by mutating amino acid positions 359-361 of the polypeptide, or wherein the furin cleavage site is introduced amino-terminally at amino acid position 362, which amino acid position corresponds to the amino acid position of SEQ ID NO:

1.

10. 10. The isolated mutant influenza hemagglutinin polypeptide of claim 8 or 9, further comprising an insertion of the RSV p27 peptide carboxy-terminal to amino acid position 362 (SEQ ID NO: 2).

11. 10. The isolated mutant influenza hemagglutinin polypeptide of any one of claims 1 to 9, wherein the amino acid at position 362 is substituted with Q.

12. 12. The isolated mutant influenza hemagglutinin polypeptide of any one of claims 1 to 11, further comprising a deletion of a signal peptide at the amino terminus of the polypeptide.

13. 13. The isolated mutant influenza hemagglutinin polypeptide of claim 12, wherein said signal peptide comprises amino acid positions 1-15 of said polypeptide.

14. 14. The isolated variant influenza hemagglutinin polypeptide of any one of claims 1-13, wherein the variant influenza hemagglutinin polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 35, 46-48, 63, 67-75, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 181, 186, 189, 201, 202, 204-208, 216, 219-222.

15. 15. The isolated mutant influenza hemagglutinin polypeptide of claim 14, wherein the mutant influenza hemagglutinin polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, 35, 46-48, 63, 67-75, 142, 146, 150, 154, 158, 162, 166, 170, 174, 178, 181, 186, 189, 201, 202, 204-208, 216, 219-222, and wherein the mutant influenza hemagglutinin polypeptide comprises a deletion of the signal peptide at the amino terminus of the polypeptide.

16. 16. The isolated mutant influenza hemagglutinin polypeptide of any one of claims 1 to 15, wherein the mutant influenza hemagglutinin polypeptide further comprises a carboxy (C)-terminal truncation beginning at amino acid position 536 through amino acid position 585, wherein said amino acid positions correspond to the amino acid positions of SEQ ID NO:

1.

17. 17. An isolated nucleic acid encoding the isolated mutant influenza hemagglutinin polypeptide of any one of claims 1-16.

18. 18. An isolated vector comprising the isolated nucleic acid of claim 17.

19. 20. An isolated host cell comprising the vector of claim 18.

20. 17. A pharmaceutical composition comprising the isolated mutant influenza hemagglutinin polypeptide of any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

21. A pharmaceutical composition comprising the isolated nucleic acid of claim 17.

22. A pharmaceutical composition comprising the isolated vector of claim 18.

23. 23. A method for inducing an immune response against influenza virus in a subject in need thereof, comprising administering to said subject in need thereof the pharmaceutical composition of any one of claims 20 to 22.

24. 20. A method for producing an isolated mutant influenza hemagglutinin polypeptide, comprising culturing the isolated host cell of claim 19 under conditions capable of producing said mutant influenza hemagglutinin polypeptide, and recovering said mutant influenza hemagglutinin polypeptide from said cell or culture.

25. 21. A method of making the pharmaceutical composition of claim 20, comprising combining an isolated mutant influenza polypeptide with a pharmaceutically acceptable carrier.

Images