Novel influenza antigens

Abstract

An immunogenic composition comprising an influenza A strain hemagglutinin antigen and a polynucleotide encoding the antigen.

Description

[Technical Field]

[0001] The present invention relates to novel influenza virus antigens, nucleotide sequences encoding same, novel immunogenic or vaccine compositions, and uses of the antigens and compositions and methods for producing the antigens and compositions. In particular, the present invention relates to immunogenic compositions comprising a modified form of influenza hemagglutinin (HA) from an influenza A strain, or a nucleotide sequence encoding same, methods for producing same, and uses thereof in the prevention of influenza A virus infection. [Background technology]

[0002] Influenza viruses have a significant impact on global public health, causing millions of cases of severe illness, thousands of deaths, and significant economic losses each year. Influenza viruses belong to the Orthomyxoviridae family, a family of viruses that represent enveloped viruses. Their genomes contain segmented, negative-sense, single-stranded RNA. Influenza viruses are classified into three major types that infect humans: influenza A, influenza B, and influenza C. Influenza A and B are the most clinically important types in humans and are responsible for the annual influenza season. Influenza C infections generally cause mild illness and are not considered to cause human influenza epidemics. Influenza strains are classified according to the host species of origin, the geographic location and year of isolation, serial number, and, for influenza A, by the serological characteristics of the two major surface glycoproteins, HA and neuraminidase (NA), subtypes. It is these surface proteins, particularly HA, that determine the antigenic specificity of influenza subtypes or strains.

[0003] Influenza A and B viruses diverged from each other approximately 2,000 years ago and share structural similarities but low sequence identity in the HA (Ni et al., Biochemistry, 2014, 53: 846-854). Influenza B viruses were first isolated in 1940, and since the 1980s, two genetic lineages have been identified based on the antigenic characteristics of HA: B / Victoria / 2 / 87 (B / Vic) and B / Yamagata / 16 / 88 (B / Yam).

[0004] Influenza B virus strains generally evolve more slowly in terms of genetic and antigenic properties than type A strains. Influenza A viruses are continually evolving and undergoing antigenic drift, and have been responsible for past influenza pandemics.

[0005] Vaccination plays an important role in controlling influenza epidemics and pandemics. Due to antigenic variability, annual vaccination is required to provide immunity against circulating influenza viruses. These are predicted based on virus surveillance data. Current influenza vaccines are primarily strain-specific, making appropriate vaccine strain selection challenging and frequently resulting in suboptimal protection. Current seasonal influenza vaccines are either trivalent (TIV) containing two type A and one type B viruses, or quadrivalent (QIV) containing two type A and two type B viruses. QIV contains both B / Victoria and B / Yamagata type B influenza strains, while TIV contains one type B influenza strain derived from either the Victoria or Yamagata lineage.

[0006] Current immune responses to vaccines largely target the highly variable HA. HA is a trimeric protein, with each monomer containing two polypeptide chains, HA1 and HA2, linked by disulfide bonds and anchored in the viral envelope by a C-terminal transmembrane domain. Each monomer is initially expressed as an inactive HA0 and subsequently cleaved by host proteases into HA1 and HA2 subunits, which are linked via disulfide bonds to form a metastable prefusion state of HA. The triggering event for the conversion of HA from the prefusion conformation to the postfusion conformation is associated with a pH change (decrease) during viral uptake / endocytosis, which leads to membrane fusion and viral internalization. During the conformational transition between the pre-fusion and post-fusion conformations, there are conserved features shared between influenza A and B viruses, but there are substantial differences that affect the detailed mechanism of this process (Ni et al, Biochemistry, 2014, 53: 846-854).

[0007] HA can be functionally divided into two domains: a globular head and a stalk or stem. The globular head is composed of a portion of HA1, while the stalk or stem structure is composed of the N- and C-terminal fragments of HA1 and all of HA2 (Hai et al., J. Virol, 2012 86(10): 5774-5781). The transmembrane domain and cytoplasmic tail are also part of HA2. The HA globular head is the primary target of antibodies against influenza viruses, but it is also highly variable and undergoes constant antigenic drift. In contrast, the HA stem is highly conserved and rarely undergoes antigenic drift, but is not very immunogenic.

[0008] There remains a need for influenza vaccines that are not limited by inherent strain specificity and can confer protection against heterologous strains of influenza. There is also a need for influenza vaccines that do not require existing egg-based production methods. In particular, it would be of great interest to generate vaccines that confer protection against multiple influenza strains, including recent, evolving seasonal strains and potential future seasonal strains. For example, more recently, various approaches have been taken to provide a "universal" influenza A vaccine that protects individuals against heterologous strains, using the conserved stem portion of HA (Yassine et al., Nature Medicine, 2015, 21(9): 1065-1070; Corbett et al., mBio, 2019, 10(1) 10:e02810-18). Summary of the Invention [Problem to be solved by the invention]

[0009] It has been found that by making amino acid substitutions in the coiled-coil region of HA derived from influenza A strains, it is possible to obtain stable trimeric recombinant influenza A strain HA antigens that retain the antigenicity of wild-type influenza strains and can also elicit immune responses against many different influenza A strains.

[0010] It has further been surprisingly found that certain recombinant influenza A strain HA ectodomain constructs expressed as fusions with a heterologous trimerization domain are stable after removal of the trimerization domain. It has also been found that certain recombinant influenza A strain HA ectodomain constructs form stable trimers and can be expressed even without the trimerization domain. These stable trimeric recombinant HA ectodomain antigens that do not contain the trimerization domain (either removed or absent) may potentially be utilized in immunogenic compositions. [Means for solving the problem]

[0011] In one aspect, an immunogenic composition comprising a recombinant influenza A strain hemagglutinin (HA) antigen in a trimeric form, wherein the antigen comprises the ectodomain of HA without the transmembrane and cytoplasmic domains, wherein the ectodomain comprises: (i) a globular head domain; and (ii) a stem domain having a coiled-coil region, the stem domain containing one or more mutations in the coiled-coil region that individually or together stabilize the HA ectodomain in a trimeric pre-fusion form; Including, The recombinant HA optionally comprises a heterologous trimerization domain, together with a pharmaceutically acceptable carrier. Immunogenic compositions are provided.

[0012] In another aspect, an immunogenic composition is provided comprising an isolated polynucleotide, eg, DNA or mRNA, encoding a recombinant HA antigen described herein and a pharmaceutically acceptable carrier.

[0013] In another aspect, there is provided an immunogenic composition as described herein for use in preventing and / or vaccinating against influenza A strain infection or disease.

[0014] In another aspect, there is provided an immunogenic composition for use in preventing and / or vaccinating against influenza infection or disease caused by at least one different influenza A strain, which may be a viral strain from the same or a different influenza A strain subtype as the HA subtype from which the HA ectodomain antigen is derived.

[0015] In another aspect, there is provided a method of preparing an immunogenic composition described herein, comprising: (i) expressing a recombinant HA antigen in a eukaryotic cell from a polynucleotide sequence encoding the HA antigen fused to a heterologous trimerization domain, e.g., a foldon; (ii) purifying recombinant HA trimers from cell supernatants; (iii) removing the trimerization domain; (iv) combining the recombinant HA trimer with a pharmaceutically acceptable carrier. A method is provided that includes:

[0016] In another aspect, there is provided a method of preparing an immunogenic composition as described herein comprising an HA ectodomain comprising one or more mutations in the coiled-coil region that individually or together stabilize the HA ectodomain in a trimeric pre-fusion form, comprising: (i) expressing a recombinant HA antigen from a polynucleotide sequence encoding it, with or without a trimerization domain; (ii) purifying trimeric recombinant HA from cell supernatants; (iii) optionally removing the trimerization domain, if present; (iv) combining the recombinant HA trimer with a pharmaceutically acceptable carrier. A method is provided that includes:

[0017] In another aspect, there is provided a method for preventing and / or vaccinating against influenza A strain infection or disease comprising administering to a person in need thereof, such as a person identified as being at risk of influenza virus infection or disease, an antigen or polynucleotide or immunogenic composition as described above.

[0018] In another aspect, there is provided a method of generating an immune response against an influenza A strain, comprising administering to a human subject a recombinant influenza A strain HA antigen or polynucleotide or immunogenic composition described herein.

[0019] In another embodiment, there is provided the use of a recombinant influenza A strain HA antigen or polynucleotide described herein in the manufacture of an immunogenic composition for generating an immune response against an influenza A strain in a human subject.

[0020] In another aspect, there is provided an immunogenic composition comprising a recombinant influenza A strain HA ectodomain antigen obtained by expressing an influenza A strain HA ectodomain fused to a trimerization domain and subsequent removal of the trimerization domain.

[0021] A brief description of arrays SEQ ID NO: 1 A / Brisbane / 02 / 2018(H1N1)pdm09-like virus (H1N1), also known as Bri18 Bri18 ) derived full-length HA sequence. SEQ ID NO: 2 Full length HA sequence from A / Darwin / 9 / 2021 H3N2, also referred to as A / Darw21 or H3 Darw21 or Dar21. SEQ ID NO: 3 Mut10 amino acid sequence - H1 Brisbane 18 wild type sequence with mutations shown in Table 1. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 4 Mut17 amino acid sequence - H1 Brisbane 18 wild type sequence with the mutations shown in Table 1. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 5 Mut18 amino acid sequence - H1 Brisbane 18 wild type sequence with the mutations shown in Table 1. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 6 Mut23 amino acid sequence - H1 Brisbane 18 wild type sequence with mutations shown in Table 1. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 7 Mut24 amino acid sequence - H1 Brisbane 18 wild type sequence with the mutations shown in Table 1. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 8 Mut27 amino acid sequence - H1 Brisbane 18 wild type sequence with the mutations shown in Table 1. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 9 Foldon sequence SEQ ID NO: 10 H1 Brisbane 18 derived signal peptide SEQ ID NO: 11 Nucleotide sequence encoding Mut10 SEQ ID NO: 12 Nucleotide sequence encoding Mut17 SEQ ID NO: 13 Nucleotide sequence encoding Mut18 SEQ ID NO: 14 Nucleotide sequence encoding Mut23 SEQ ID NO: 15 Nucleotide sequence encoding Mut24 SEQ ID NO: 16 Nucleotide sequence encoding Mut27 SEQ ID NO: 17 Flu622 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 18 Flu629 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 19 Flu632 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 20 Flu638 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 21 Flu639 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 22 Flu643 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 23 Flu650 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 24 Flu672 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 25 Flu679 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 26 Flu680 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 27 Flu681 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 28 Flu682 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 29 Flu683 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 30 Flu685 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 31 Flu686 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 32 Flu687 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 33 Flu688 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 34 Flu689 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 35 Flu690 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 36 Flu691 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 37 Flu692 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 38 Flu693 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 39 Flu695 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 40 Flu696 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 41 Flu697 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 42 Flu707 amino acid sequence - H3 Darw21 wild type sequence with mutations shown in Table 3. Includes signal sequence, foldon, and polyH tail. SEQ ID NO: 43 H3 Darw21-derived signal peptide SEQ ID NO: 44 Nucleotide sequence encoding Flu622 SEQ ID NO: 45 Nucleotide sequence encoding Flu629 SEQ ID NO: 46 Nucleotide sequence encoding Flu632 SEQ ID NO: 47 Nucleotide sequence encoding Flu638 SEQ ID NO: 48 Nucleotide sequence encoding Flu639 SEQ ID NO: 49 Nucleotide sequence encoding Flu643 SEQ ID NO: 50 Nucleotide sequence encoding Flu650 SEQ ID NO: 51 Nucleotide sequence encoding Flu672 SEQ ID NO: 52 Nucleotide sequence encoding Flu679 SEQ ID NO: 53 Nucleotide sequence encoding Flu680 SEQ ID NO: 54 Nucleotide sequence encoding Flu681 SEQ ID NO: 55 Nucleotide sequence encoding Flu682 SEQ ID NO: 56 Nucleotide sequence encoding Flu683 SEQ ID NO: 57 Nucleotide sequence encoding Flu685 SEQ ID NO: 58 Nucleotide sequence encoding Flu686 SEQ ID NO: 59 Nucleotide sequence encoding Flu687 SEQ ID NO: 60 Nucleotide sequence encoding Flu688 SEQ ID NO: 61 Nucleotide sequence encoding Flu689 SEQ ID NO: 62 Nucleotide sequence encoding Flu690 SEQ ID NO: 63 Nucleotide sequence encoding Flu691 SEQ ID NO: 64 Nucleotide sequence encoding Flu692 SEQ ID NO: 65 Nucleotide sequence encoding Flu693 SEQ ID NO: 66 Nucleotide sequence encoding Flu695 SEQ ID NO: 67 Nucleotide sequence encoding Flu696 SEQ ID NO: 68 Nucleotide sequence encoding Flu697 SEQ ID NO: 69 Nucleotide sequence encoding Flu707 SEQ ID NO: 70 H1 (Bri18) wild-type-foldon polypeptide sequence: signal sequence-HA-TEV cleavage site-foldon-His tag SEQ ID NO: 71 H3 (Darw21) wild-type-foldon polypeptide sequence: signal sequence-HA-TEV cleavage site-foldon-His tag SEQ ID NO: 72 Bri18-derived HA sequence shown in Figure 2, excluding the native linker and transmembrane and cytoplasmic regions present in the full-length sequence. SEQ ID NO: 73 HA sequence from Darw21 shown in Figure 2, excluding the native linker and transmembrane and cytoplasmic regions present in the full length sequence. [Brief explanation of the drawings]

[0022] [Figure 1]

[0023] Figure 1 is a diagram of the full-length sequence of the wild-type HA polypeptide of A / Brisbane / 02 / 2018 (H1N1) pdm09-like virus (H1Bri18), also referred to herein as Bri18, in the following order: signal peptide (absent in the mature protein)-HA1 chain-HA1 / HA2 cleavage site-HA2 chain-regions deleted in the constructs described herein within the dotted box, including the transmembrane domain (dark background) and cytoplasmic domain. In both the HA1 and HA2 chains, single amino acid positions that were mutated in various combinations in certain constructs described herein are shaded. [Figure 2] Figure 1 shows a sequence comparison showing the ectodomain HA sequences of H1 strain Bri18 and H3 strain H3-Darw21. The positions of mutations (amino acid substitutions) are indicated by triangles / asterisks / crosses, and regions A, B, and C are identified by different underlining. [Figure 3] Diagram of the H1 / H3 wild-type HA ectodomain-foldon polypeptide sequences showing the linker, TEV cleavage site, foldon and poly-His tail; (a) H1 (Bri18) wild-type HA ectodomain-foldon sequence and (b) H3 (Darw21) wild-type HA ectodomain-foldon sequence. [Figure 4] Schematic diagram of H1 and H3 wild-type and recombinant HA, showing the following order: signal peptide-stem, head, HA1 stem-HA2 stem-linker (native)-transmembrane domain-cytoplasmic tail (wild-type); and signal peptide-stem, head, HA1 stem-HA2 stem-linker (recombinant)-TEV cleavage site-linker (recombinant)-foldon-His tag (recombinant). [Figure 5] Ribbon structures of the H1 and H3 HA ectodomains (Bri18 and Darw21, respectively) are shown as monomers separated from the trimeric molecule to demonstrate the location of mutations in regions A and B of the coiled-coil region of the stem domain. The positions of helices A and B are shown in the H1 and H3 monomers, respectively; both helices are present in the same position in each monomer. [Figure 6]Ribbon structures of H1 and H3 HA ectodomain trimers (Bri18 and Darw21), showing the head and stem domains, including regions A, B, and C of the stem domain. [Figure 7A] Figure 1. Anti-HA functional HI responses induced by HA mut 10 and 23 against homologous and post-pandemic heterologous H1N1 strains 14 days after dose 2. [Figure 7B] This is a continuation of Figure 7A. [Figure 7C] This is a continuation of Figure 7B. [Figure 7D] This is a continuation of Figure 7C. [Figure 7E] This is a continuation of Figure 7D. [Figure 7F] This is a continuation of Figure 7E. [Figure 8A] Figure 1. Anti-HA IgG antibody and functional antibody responses induced by HA mut 10 and 23 against heterologous H1N1 strains at 14 days after dose 2. [Figure 8B] This is a continuation of Figure 8A. [Figure 8C] This is a continuation of Figure 8B. [Figure 8D] This is a continuation of Figure 8C. [Figure 8E] This is a continuation of Figure 8D. [Figure 8F] This is a continuation of Figure 8E. [Figure 9A] Figure 10. Anti-H1 stem-specific CD4 and CD8 T cell responses induced by HA mut 10 and 23 at 14 days after dose 2. [Figure 9B] This is a continuation of Figure 9A. [Figure 9C] This is a continuation of Figure 9B. [Figure 9D] This is a continuation of Figure 9C. [Figure 10A] Figure 1. Anti-HA IgG antibody responses induced by HA mut 10 and 23 against pre-pandemic heterologous H1N1 and heterosubtypic (H2N2, H5N1, and H9N2) strains 14 days after dose 2. [Figure 10B] This is a continuation of Figure 10A. [Figure 10C] This is a continuation of Figure 10B. [Figure 10D] This is a continuation of Figure 10C. [Figure 11-1] Figure 1. Anti-HA functional HI responses induced by HA mut 10 and 23 against pre-pandemic heterologous H1N1 and heterosubtypic (H2N2, H5N1, and H9N2) strains 14 days after dose 2. [Figure 11-2] This is a continuation of Figure 11-1. [Figure 11-3] This is a continuation of Figure 11-2. [Figure 11-4] This is a continuation of Figure 11-3. [Figure 12A] FIG. 11 shows neutralization titers induced by HA mut 10 and 23 against heterosubtypic (H2N2, H5N1, and H9N2) strains at 14 days after dose 2. [Figure 12B] This is a continuation of Figure 12A. [Figure 13A] Figure 14 shows anti-HA IgG antibody responses induced by HA mut 10 and 23 against group A2 (H3N2, H10-stem) or B strains (B / Yam and B / Vic) 14 days after dose 2. [Figure 13B] This is a continuation of Figure 13A. [Figure 13C] This is a continuation of Figure 13B. [Figure 13D] This is a continuation of Figure 13C. DETAILED DESCRIPTION OF THE INVENTION

[0023] Influenza HA ectodomain The recombinant influenza A HA antigen provided herein contains an HA ectodomain, which includes both the head and stem domains of HA. Thus, this HA antigen is not a stem-only (or headless HA stem) antigen. The recombinant influenza HA antigen has advantageous properties, such as providing a soluble HA composition. HA lacking a transmembrane domain is not inserted into the membrane and is therefore soluble and not membrane-bound. Conversely, for nucleic acid delivery, the recombinant HA antigen can be linked to a transmembrane domain, thus providing an antigen that inserts into the membrane in vivo.

[0024] Influenza HA is a homotrimeric surface glycoprotein, with each monomer consisting of two disulfide-linked subunits, HA1 and HA2, derived from the proteolytic cleavage products of a single HA precursor protein, HA0. HA1 comprises all residues N-terminal to the HA1 / HA2 cleavage peptide of the precursor HA0 protein and contains the receptor-binding domain of the HA protein. The HA2 chain comprises all residues C-terminal to the HA1 / HA2 cleavage peptide of the precursor HA0 protein and contains the hydrophobic peptide responsible for insertion into the host cell membrane during membrane fusion, the transmembrane domain spanning the viral membrane, and the cytoplasmic tail. For example, in the case of the H1 strain Bri18, HA1 refers to the region of the HA protein encompassing approximately amino acid residues 1-344 of the HA0 protein, and HA2 refers to the region of the HA protein encompassing approximately amino acid residues 345-566 of the HA0 polypeptide. Residues in the HA2 chain are generally numbered independently of residues in the HA1 chain when referring to the chains independently, e.g., HA2 residues are numbered 1-174 relative to Bri18.

[0025] The HA head domain is the globular head region of the HA protein, excluding the stem, transmembrane domain, and intracellular region. The HA head consists of approximately 250-300 amino acid residues in the center of the HA1 sequence. The HA head consists of a receptor-binding domain and a vestigial esterase domain. It contains the sialic acid-binding pocket that mediates viral attachment to host cells.

[0026] The influenza HA stem domain is located in the membrane-proximal region of the native HA protein, immediately below the vestigial esterase domain of the HA1 globular head. The influenza HA stem is composed of amino acid residues from both ends of the HA1 chain and the ectodomain portion of the HA2 chain. For example, in the case of the H1 strain Bri18, the HA stem includes residues approximately 18-58 and 293-344 of the HA1 chain and residues 1-222 (or 1-176 if only the ectodomain portion) of the HA2 chain. The stem domain does not contain any transmembrane or cytoplasmic domains.

[0027] In one embodiment, the head domain and stem domain are from the same influenza strain.

[0028] In one embodiment, the recombinant HA ectodomain antigen is a homotrimer, ie, a trimer formed from three identical HA ectodomain monomers.

[0029] In one embodiment, the recombinant HA antigen further comprises a heterotrimerization domain fused, e.g., covalently linked, to the C-terminus. In another embodiment, the recombinant HA antigen is a trimeric antigen from which the trimerization domain, e.g., the heterotrimerization domain, has been removed. In another embodiment, the recombinant HA antigen is expressed without the trimerization domain.

[0030] In one embodiment, the C-terminus of the stem domain is (1) covalently linked to a heterologous trimerization domain; or (2) covalently linked to a carrier protein or nanoparticle; or (3) It is not covalently linked to other amino acid molecules.

[0031] In one embodiment, the recombinant HA ectodomain comprises all or substantially all of the globular head domain, where substantially all of the globular head domain means at least 75%, or at least 85%, or at least 95%, or at least 99% of the length of the amino acid sequence present in the wild-type influenza HA head domain.

[0032] In one embodiment, the recombinant HA ectodomain comprises all or substantially all of the stem domain, where substantially all of the stem domain means at least 75%, or at least 85%, or at least 95%, or at least 99% of the length of the amino acid sequence present in the wild-type influenza HA stem domain.

[0033] In one embodiment, the ectodomain of the recombinant HA comprises all or substantially all, e.g., at least 75%, or at least 85%, or at least 95%, or at least 99%, of the amino acid sequence present in the wild-type influenza HA head domain, and all or substantially all, e.g., at least 75%, or at least 85%, or at least 95%, or at least 99%, of the amino acid sequence present in the wild-type influenza HA stem domain.

[0034] In one embodiment, the recombinant HA ectodomain comprises all or substantially all, eg at least 75%, at least 85%, at least 95%, at least 99%, of the amino acid sequence present in a wild-type influenza HA ectodomain.

[0035] In one embodiment, the recombinant HA antigen comprises amino acid residues 1-520 of influenza A HA, or an immunogenic fragment or derivative thereof having both the head and stem domains.

[0036] H1 influenza strain A / Brisbane / 02 / 2018(H1N1)pdm09-like virus (H1), also referred to herein as Bri18 Bri18The full-length sequence of the HA polypeptide from H3 influenza strain A / Darwin / 9 / 2021 H3N2, also referred to herein as Darw21, is used herein as a reference sequence. This sequence is shown in FIG. 1 and SEQ ID NO: 1. The full-length sequence of the HA polypeptide from H3 influenza strain A / Darwin / 9 / 2021 H3N2, also referred to herein as Darw21, is also used herein as a reference sequence. This sequence is shown in the sequence comparison with Bri18 (ectodomain) in FIG. 2 and SEQ ID NO: 2 (full length). The specific amino acid sequences and positions referenced herein for influenza A strain HA relate to these reference sequences. However, with respect to other influenza A isolates and sequences that may differ in numbering and / or amino acids at certain positions, particularly influenza A strains and sequences from different subtypes, it will be apparent that equivalent sequences and positions in those other isolates and sequences are also included within the scope of the polypeptide and polynucleotide constructs described herein. These equivalent sequences and positions in other influenza A isolates will be apparent to those skilled in the art from the description and figures provided herein.

[0037] The two exemplified strains are from two different subtypes, H1 and H3, which are from two different groups (or clades) of influenza A known as groups 1 and 2, also referred to as groups A1 and A2. The full-length Bri18 HA sequence, shown in Figure 1 and SEQ ID NO: 1, includes the signal sequence, HA1, HA2, transmembrane domain, and cytoplasmic domain. While it is common in the literature to number HAs using H3 influenza as a reference sequence, this convention is not followed here because our starting point is the HA sequence of an H1 strain of influenza. It will be apparent that equivalent positions in HA ectodomains from other strains can be located by reference to sequence and / or structural comparisons. For example, Figure 2 shows a sequence alignment of the HA ectodomain from the Bri18 strain, an H1 strain from influenza A group 1, with the HA ectodomain from the Darw21 strain, an H3 strain from influenza A group 2, and also indicates the locations of specific stabilizing mutations in the two sequences. Figure 5 shows a structural comparison in ribbon form of each monomer of the HA trimers from the same two H1 and H3 strains, indicating the locations of the equivalent stabilizing mutations described herein in the HA from these two strains. Structural comparisons can be performed in 2D format as shown in Figure 5, or in 3D format, or both.

[0038] In one embodiment, the HA ectodomain excludes the transmembrane and cytoplasmic domains and comprises all of the HA1 and all of the HA2 of influenza HA, or an immunogenic fragment or derivative thereof having both the head and stem domains. In a specific embodiment, the HA ectodomain comprises amino acid residues 1-520 of influenza A HA from Bri18, or an equivalent sequence comprising all of the HA1 and all of the HA2 from another influenza A strain HA, or an immunogenic fragment or derivative thereof having both the head and stem domains.

[0039] The recombinant influenza A strain HA antigen described herein has been found to have many useful properties. One advantage relates to the nature of the ectodomain antigen, which preserves the antigenicity of the HA stem while retaining the antigenicity of the HA globular head. Furthermore, mutations that stabilize the coiled-coil structure of the recombinant HA antigen may allow for the removal of the trimerization domain (as shown herein for H1) used in recombinant HA production, or may allow the expression of the recombinant antigen in the absence of the trimerization domain (e.g., for H3). Due to the absence of the HA transmembrane and cytoplasmic regions, the recombinant ectodomain is soluble and easily purified when expressed. The stabilizing mutations identified herein improve the production properties of the recombinant HA, such as yield and heat resistance. Furthermore, by stabilizing the trimeric structure in the native trimeric prefusion form, the antigenic properties of the HA are optimized to generate an immune response that recognizes wild-type influenza virus when encountered in vivo. This immune response has been shown to be directed not only against the influenza strain from which the HA of the recombinant antigen is derived, but also against other influenza strains, including strains from different influenza A subtypes.

[0040] In one embodiment, the influenza HA antigens described herein are capable of binding to antibodies that target at least one epitope, preferably a neutralizing epitope, on wild-type HA. That is, the modifications to wild-type influenza HA in the antigens described herein preserve the conformation of at least one wild-type epitope, preferably a neutralizing epitope. In one embodiment, the recombinant influenza HA antigen retains one or more epitopes present in the wild-type, preferably the CR9114 or FI6 epitope, or more preferably both the CR9114 and FI6 epitopes.

[0041] The recombinant influenza A HA antigens described herein contain mutations, such as amino acid deletions, substitutions (e.g., single amino acid substitutions), or additions, compared to the wild-type HA amino acid sequence. For purposes herein, substitution means the replacement of a wild-type amino acid with any other amino acid at the same amino acid position. In one embodiment, the one or more mutations stabilize the antigen in the correct conformation so that it can elicit an immune response against native HA. In one embodiment, the one or more mutations stabilize the antigen in a trimeric form. In one embodiment, the one or more mutations stabilize the antigen in a pre-fusion form.

[0042] Typically, the recombinant influenza HA antigens described herein contain multiple amino acid substitutions (e.g., single amino acid substitutions) compared to native HA, for example, up to four, up to six, up to eight, or up to ten amino acid substitutions, for example, 1 to 4 or 1 to 6 amino acid substitutions, for example, 1, 2, 3, 4, 5, or 6 amino acid substitutions. Typically, the amino acid substitutions (e.g., single amino acid substitutions) are in HA2. There may also be one or more amino acid substitutions in HA1, particularly one amino acid substitution in HA1. In one embodiment, there is one amino acid substitution in HA1 and one, two, three, or four amino acid substitutions in HA2.

[0043] In one embodiment, the recombinant HA ectodomain antigen does not contain additional elements of HA, such as the transmembrane region or the cytoplasmic region. In one embodiment, the HA antigen is not membrane-bound due to the absence of the transmembrane region or the absence of both the transmembrane region and the cytoplasmic region. In an alternative embodiment for nucleic acid delivery, the HA antigen delivered by a nucleic acid delivery platform, such as mRNA, contains a transmembrane region, e.g., the HA transmembrane region, with or without the cytoplasmic region. The transmembrane region of influenza A strain HA is located from approximately amino acid positions 530-550, and the cytoplasmic tail is located from approximately positions 551-566, in the H1 HA sequence shown herein. See the schematic diagram in Figure 4.

[0044] Prefusion conformation The influenza HA antigen is preferably stabilized in a pre-fusion conformation or state. Stabilization is achieved by stabilizing mutations in the coiled-coil region, e.g., one or more amino acid substitutions, e.g., single amino acid substitutions, that stabilize the HA trimer. Stabilization may be further aided by the presence of a trimerization domain. Mutations in the coiled-coil region, e.g., one or more amino acid substitutions, e.g., single amino acid substitutions, may potentially stabilize HA when the trimerization domain is removed after expression of the recombinant HA antigen, e.g., by enzymatic cleavage. Thus, in one embodiment, the HA antigen is expressed containing a trimerization domain, and the trimerization domain is removed after expression. In a specific embodiment, the HA antigen is an ectodomain antigen that does not contain a trimerization domain; more particularly, the ectodomain antigen is expressed containing a trimerization domain, and the trimerization domain is cleaved therefrom after expression, e.g., by an enzyme.

[0045] Stabilization can be achieved, for example, by helix stabilization, loop optimization, disulfide bond addition, and side chain repacking. HA stabilization can be achieved by introducing mutations (e.g., amino acid substitutions, e.g., single amino acid substitutions) that form or strengthen ionic bonds, salt bridges, or increase hydrophobic packing or cavity filling. Hydrophobic packing promotes and / or drives the association of hydrophobic regions while simultaneously excluding water. Cavity filling fills unoccupied cavity volumes found either buried within the hemagglutinin protein (i.e., within the monomers) or at its interface (i.e., between the monomers of a trimer) by introducing amino acids (e.g., amino acid substitutions, e.g., single amino acid substitutions) that fill such space, enable and / or promote good folding / packing, and avoid the tendency for water to be enclosed or incorporated into the protein's folding. This can be achieved, for example, by replacing amino acids with small side chains, such as (but not limited to) serine, with amino acids with larger side chains. Stabilization of homotrimeric HA antigens can be assessed by characterization studies such as those described in the Examples. In one embodiment, stabilization in the pre-fusion form is assessed by determining the presence of trimeric HA. Alternatively or additionally, stabilization in the pre-fusion form can be assessed by determining the presence of epitopes such as CR9114 and / or FI6 epitopes (e.g., FI6v3), for example, using mAb binding assays.

[0046] The prefusion conformation of HA is discussed in the literature, e.g., Wu & Wilson, Viruses, 2020, 12: 1053, and Ni et al. 2014. Stabilizing mutations are discussed in the literature, e.g., Yassine et al., Nature Medicine, 2015, 21(9): 1065-1070.

[0047] It will be understood that additional modifications that do not negatively affect or further optimize the recombinant influenza HA described herein may be present in either the head region or the stem region, or both, compared to wild-type HA. For example, amino acid insertions, deletions, or substitutions can be made that do not disrupt the pre-fusion conformation of the HA or negatively affect the antigenic properties of the HA described herein. Such amino acid substitutions, deletions, or insertions can be, for example, to alter the properties of one or more epitopes of the HA, either in the globular head or stem region, or both.

[0048] Stabilizing mutations Stabilization can be determined by looking at one or more different parameters after expression and purification, such as the productivity (yield) of the recombinant antigen expression compared to the productivity of the wild-type antigen when recombinantly expressed. Higher productivity is often associated with more stable folding of the recombinant protein. Alternatively or additionally, structural analysis, such as nanoDSF and / or stress testing, can be performed to confirm improved folding stability. Stress testing includes, for example, a heat resistance test, e.g., evaluating the level of degradation and / or aggregation of the recombinant antigen after one week at room temperature or 37°C. Alternatively or additionally, stabilization can be evaluated by the presence of trimers and / or by examining the presence of epitopes present on native HA, such as CR9114 and FI6 (e.g., FI6v3) epitopes. Preferably, the stabilized HA antigen is improved to be equal to or better than wild-type recombinant HA with respect to at least one parameter, preferably two or more parameters, such parameters being selected from the parameters described herein, such as yield after expression, folding stability measured by nanoDSF, stress tests such as thermostability tests, presence of trimers, and presence of CR9114 and / or FI6 (e.g., FI6v3) epitopes measured by binding to CR9114 and / or FI6 (e.g., FI6v3) antibodies.

[0049] The influenza HA trimeric antigens described herein can be stabilized by mutations, preferably site-directed mutations, introduced into the coiled-coil region, e.g., individual amino acid substitutions, additions, or deletions designed to confer improved stability to the HA antigen or trimer. These can be mutations in the coiled-coil core or in the region immediately surrounding the coiled-coil core. In one embodiment, the HA antigen contains one or more stabilizing mutations in the helical structure of the coiled-coil, e.g., helix A or helix B of pre-fusion influenza HA. ​​See Figure 5. In each monomer, helix A is the small helix and helix B is the large helix. In another embodiment, the HA antigen contains one or more stabilizing mutations in one or more of regions A, B, and C. Regions A, B, and C are illustrated for H1 and H3 strains in Figures 2, 4, 5, and 6.

[0050] In one embodiment, the coiled-coil region of the stem domain containing one or more mutations is between 317 and 472, e.g., between 322 and 467 for H1; or between 342 and 473, e.g., between 347 and 468 for H2; or an equivalent range to the coiled-coil region in other HA A strains or subtypes.

[0051] For example, it has been found that mutations (e.g., single amino acid substitutions) at one or more of positions 322, 395, 431, 432, 436, 438, 439, 447, 449, 450, 453, 460, 464, and 467 in HA from the H1 subtype, more particularly the Bri18 strain, can help stabilize the HA ectodomain. Similarly, it has been found that mutations (e.g., single amino acid substitutions) at one or more of positions 347, 396, 399, 418, 428, 437, 440, 448, 451, 454, 465, and 468 in HA from the H3 subtype, more particularly the Darw21 strain, can help stabilize the HA ectodomain. In particular, it has been found that amino acid substitutions at positions 395, and optionally 322, 436, and 447, can have beneficial effects on recombinant HA from H1 strains, such as improved yield. Similarly, it has been found that amino acid substitutions at positions selected from one or more of 396, 399, 418, 437, and 448 can have beneficial effects on recombinant HA from H3 strains. For example, a mutation (e.g., a single amino acid substitution) at position 395 of HA from an H1 subtype, particularly the K395M substitution (an exemplary H1 construct from the Bri18 strain is referred to herein as Mut10), or a mutation (e.g., a single amino acid substitution) at position 396 of HA from an H3 subtype, particularly the K396V / L / I / M substitution, has been found to help stabilize the ectodomain trimer. This mutation (395 / 396 depending on the strain) is in the smaller α-helix adjacent to the coiled-coil, referred to herein as helix A (see the smaller helix in Figure 5, which depicts the HA monomer). Additionally, the K395M substitution and three other substitutions: K322R, W436D, and E447L in subtype H1 (referred to herein as Mut23) have beneficial effects, particularly on protein yield. The exemplary H1 HA ectodomain antigens Mut10 and Mut23 described herein have been shown to stimulate broad anti-HA responses against different influenza strains within the H1 subtype and other subtypes within group 1, e.g., H2, H5, and H9.

[0052] Similarly, a combination of substitutions at some of the equivalent positions in H3, K396V / L / I / M, W437D, and E448V / L / I / M, plus the R399L / I / M / F substitution, stabilizes the ectodomain of this subtype.

[0053] In one embodiment, the influenza HA antigen comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, or fourteen of the following amino acid substitutions compared to wild-type HA: K322R, K395M, G431C, F432C, W436D, Y438D, N439L, E4 and R467M, in particular one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or nine of the following amino acid substitutions compared to wild-type HA: K322R, K395M, W436D, N439L, E447L, E449Q, R450W, D453L, K460I, E464F, and R467M. In one embodiment, the recombinant H1 HA antigen comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight of the following mutations: K322R, G431C, F432C, W436D, Y438D, K460I, E464F, and R467M. In a specific embodiment, the HA is from H1.

[0054] In one embodiment, the recombinant HA ectodomain comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or thirteen of the following mutations (e.g., amino acid substitutions) compared to wild-type HA: K322R, G431C, F432C, W436D, Y438D, N439L, E447L, E449Q, R450W, D453L, K460I, E464F, and R467M. In one embodiment, the recombinant HA ectodomain comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, or eight of the following mutations (e.g., amino acid substitutions) compared to wild-type: K322R, G431C, F432C, W436D, Y438D, K460I, E464F, and R467M. In a specific embodiment, the HA is derived from H1.

[0055] In certain embodiments, the antigen is derived from H1 and comprises a mutation or combination of mutations set forth in Table 1 or Table 2. Additional examples of HA antigens comprising the mutations in Table 1 are described in Table 2 and in the Examples, including, for example, the combinations listed in Table 1 below for the specific HA ectodomain constructs Mut10, Mut17, Mut18, Mut23, Mu24, and Mu27.

[0056] [Table 1]

[0057] [Table 2]

[0058] The mutations described in Tables 1 and 2 for H1, and Table 3 for H3, target various regions or areas of HA, which are located by reference to the annotated amino acid sequence and HA structural diagrams in Figures 2, 4, 5 and 6: (i) Region A, the membrane distal fusion domain region; (ii) Region B, the central fusion domain region; and (iii) Region C, the fusion peptide / membrane-proximal fusion domain region.

[0059] In one embodiment, the influenza antigen comprises one or more region A mutations, for example, substitutions (eg, single amino acid substitutions) at positions selected from one or more of 322 / 323 and 436 / 437.

[0060] In another embodiment, the antigen comprises one or more region B mutations, for example, substitutions (e.g., single amino acid substitutions) at positions selected from one or more of 395 / 396 and 447 / 448. In a particular embodiment, referring to the numbering of H3 strain Darw21 exemplified herein, the antigen comprises a combination of region B mutations, for example, substitutions (e.g., single amino acid substitutions) at each of positions 396, 399, 448, and optionally 437.

[0061] In another embodiment, the antigen comprises one or more region C mutations, eg, substitutions at positions within region C (eg, single amino acid substitutions).

[0062] In further embodiments, the antigen comprises one or more mutations, e.g., single amino acid substitutions, from each of regions A and B, or each of regions A and C, or each of regions B and C, or any combination of the three regions A, B, and C.

[0063] In the following embodiments, the numbering refers to influenza A strains with amino acid positions arranged according to the H1 sequence Bri18 exemplified herein.

[0064] In one embodiment, the recombinant HA ectodomain comprises a K395M mutation in the absence of other mutations in the coiled-coil region (e.g., Mut10).

[0065] In one embodiment, the recombinant HA ectodomain comprises the K322R, K395M, W436D and E447L mutations in the absence of other mutations in the coiled-coil region (e.g., Mut23 for H1).

[0066] In one embodiment, the recombinant HA ectodomain comprises a K395M mutation in the absence of other mutations in the coiled-coil region, and one additional mutation selected from K322R, W436D, and E447L.

[0067] In one embodiment, the recombinant HA ectodomain comprises a K395M mutation in the absence of other mutations in the coiled-coil region, and two additional mutations selected from K322R, W436D and E447L.

[0068] In one embodiment, the recombinant HA ectodomain comprises the K322R and K395M mutations in the absence of other mutations in the coiled-coil region.

[0069] In one embodiment, the recombinant HA ectodomain comprises the K395M and W436D mutations in the absence of other mutations in the coiled-coil region.

[0070] In one embodiment, the recombinant HA ectodomain comprises the K395M and E447L mutations in the absence of other mutations in the coiled-coil region.

[0071] In one embodiment, the recombinant HA ectodomain comprises the K322R, K395M and W436D mutations in the absence of other mutations in the coiled-coil region.

[0072] In one embodiment, the recombinant HA ectodomain comprises the K322R, K395M and E447L mutations in the absence of other mutations in the coiled-coil region.

[0073] In one embodiment, the recombinant HA ectodomain comprises the K395M, W436D and E447L mutations in the absence of other mutations in the coiled-coil region.

[0074] In the following embodiments, the numbering refers to influenza A strains with amino acid positions arranged according to the H3 sequence Darw21 exemplified herein.

[0075] In one embodiment, the recombinant HA ectodomain comprises the V418P mutation in the absence of other mutations in the coiled-coil region (e.g., Flu639 for H3).

[0076] In one embodiment, the recombinant HA ectodomain comprises a V418P mutation and a W437D mutation in the absence of other mutations in the coiled-coil region (eg, Flu707 for H3).

[0077] In one embodiment, the recombinant HA ectodomain comprises K396I / L, R399F / L, and E448L / I mutations, with or without a W437D mutation, in the absence of other mutations in the coiled-coil region (e.g., Flu632, Flu680, Flu689 for H3). In certain embodiments, the recombinant HA ectodomain comprises K396I, R399F, and E448L mutations in the absence of other mutations in the coiled-coil region (e.g., Flu632 for H3); or K396L, R399L, W437D, and E448L mutations in the absence of other mutations in the coiled-coil region (e.g., Flu680 for H3); or K396L, R399F, W437D, and E448L mutations in the absence of other mutations in the coiled-coil region (e.g., Flu689 for H3).

[0078] In certain embodiments, the antigen comprises a mutation or combination of mutations set forth in Table 3. Examples of HA antigens comprising the mutations of Table 3 are described in the Examples and include, for example, the combinations listed in Table 3 below for the specific HA ectodomain constructs Flu622, Flu629, Flu632, Flu638, Flu639, Flu643, Flu650, Flu672, Flu679, Flu680, Flu681, Flu682, Flu683, Flu685, Flu686, Flu687, Flu688, Flu689, Flu690, Flu691, Flu692, Flu693, Flu695, Flu696, Flu697, and Flu707.

[0079] [Table 3]

[0080] It will be apparent that equivalent positions for substitutions and other mutations in the HA ectodomain from influenza strains other than Bri18 and Darw21, including, but not limited to, other H1 or other H3 strains, are within the scope of the present invention. These equivalent positions are located by reference to sequence comparisons with the Bri18 HA or Darw21 sequences presented herein, as well as the sequence comparisons illustrated in Figure 2. Equivalent positions of stabilizing mutations in the HA ectodomain from other influenza strains can alternatively or additionally be identified using structural comparisons. The ribbon diagram illustrating the comparison of H1 and H3 (Bri18 and Darw21) monomers illustrated in Figure 5, in which the structural locations of stabilizing mutations are indicated, is one such structural comparison. Other structural comparisons can include 3D comparisons. Equivalent positions can be determined using sequence comparisons and then confirmed using structural comparisons, or vice versa.

[0081] In one embodiment, the mutations (e.g., single amino acid substitutions) introduced into the ectodomain HA described herein do not create disulfide bonds.

[0082] The influenza HA antigen can be included in a construct that includes additional polypeptide sequences. The additional polypeptide sequences can include, for example, one or more signal peptides. In some embodiments, the signal peptide is not present in the final construct of the HA antigen or composition or method or use herein.

[0083] It will be apparent that the specific mutations described in the tables herein can be converted into conservative amino acid substitutions, i.e., the substituted amino acid itself is conservatively replaced with an amino acid having similar properties that achieves similar stabilization.

[0084] Conservative amino acid substitutions, as referred to herein, refer to the replacement of an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties, and generally do not substantially alter the functional properties of the protein in which the substitution is made.

[0085] Examples of groups of conservative amino acid substitutions include: (1) Basic side chains: arginine, histidine, and lysine; (2) Acidic side chains: aspartate (aspartic acid) and glutamate (glutamic acid); (3) aromatic side chains: phenylalanine, tryptophan, and tyrosine; (4) amide-containing side chains: asparagine and glutamine; (5) sulfur-containing side chains: cysteine ​​and methionine; (6) Aliphatic side chains: glycine, alanine, valine, leucine, isoleucine; and (7) Aliphatic-hydroxyl side chains: serine and threonine Examples include:

[0086] Examples of conservative amino acid substitution groups include: Alanine-valine, Arginine-lysine, Aspartic acid-glutamic acid, Asparagine-glutamine, isoleucine-leucine-valine, and Phenylalanine-Tyrosine Examples include:

[0087] The percent sequence identity / degree of similarity can be adjusted to take into account conservative substitutions (see Pearson (1994) Methods Mol. Biol. 24: 307-331).

[0088] In certain embodiments, the HA antigen comprises, or more suitably consists of, a construct set forth in Tables 1, 2, and 3, e.g., a polypeptide sequence selected from SEQ ID NOs: 3-8 and 17-42, with or without a signal peptide, or a sequence having at least 85% identity, or at least 87% identity, or at least 90% identity, such as 95% or more, such as 98% or more, for example 99% or more sequence identity, to any one of the amino acid sequences of SEQ ID NOs: 3-8 and 17-42, with or without a signal peptide. In further embodiments, the HA antigen comprises the polypeptide sequence of SEQ ID NOs: 3-8 or 17-42, with or without a signal peptide, in which particular elements, e.g., a His tag or a His tag and a trimerization domain, are absent.

[0089] For example, the HA antigen may comprise one of the following antigens, each of which may not include a signal peptide in its final form: (1) an HA ectodomain antigen comprising mutations as shown for Mut10 in Table 1, preferably SEQ ID NO: 3, with or without a trimerization domain and a His tag; (2) an HA ectodomain antigen comprising a mutation as shown for Mut17 in Table 1, preferably SEQ ID NO: 4, with or without a trimerization domain and a His tag; (3) an HA ectodomain antigen comprising a mutation as shown for Mut18 in Table 1, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 5; (4) an HA ectodomain antigen comprising a mutation as shown for Mut23 in Table 1, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 6; (5) an HA ectodomain antigen comprising a mutation as shown for Mut24 in Table 1, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 7; (6) an HA ectodomain antigen comprising a mutation as shown for Mut27 in Table 1, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 8; (7) an HA ectodomain antigen comprising mutations as shown for Flu622 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 17; (8) an HA ectodomain antigen comprising mutations as shown for Flu629 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 18; (9) an HA ectodomain antigen comprising mutations as shown for Flu632 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 19; (10) an HA ectodomain antigen comprising mutations as shown for Flu638 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 20; (11) an HA ectodomain antigen comprising mutations as shown for Flu639 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 21; (12) an HA ectodomain antigen comprising mutations as shown for Flu643 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 22; (13) an HA ectodomain antigen comprising mutations as shown for Flu650 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 23; (14) an HA ectodomain antigen comprising mutations as shown for Flu672 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 24; (15) an HA ectodomain antigen comprising mutations as shown for Flu679 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 25; (16) An HA ectodomain antigen comprising mutations as shown for Flu680 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 26; (17) An HA ectodomain antigen comprising mutations as shown for Flu681 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 27; (18) An HA ectodomain antigen comprising mutations as shown for Flu682 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 28; (19) An HA ectodomain antigen comprising mutations as shown for Flu683 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 29; (20) an HA ectodomain antigen comprising a mutation as shown for Flu685 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 30; (21) An HA ectodomain antigen comprising mutations as shown for Flu686 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 31; (22) An HA ectodomain antigen comprising mutations as shown for Flu687 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 32; (23) an HA ectodomain antigen comprising mutations as shown for Flu688 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 33; (24) An HA ectodomain antigen comprising mutations as shown for Flu689 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 34; (25) an HA ectodomain antigen comprising mutations as shown for Flu690 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 35; (26) An HA ectodomain antigen comprising mutations as shown for Flu691 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 36; (27) An HA ectodomain antigen comprising mutations as shown for Flu692 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 37; (28) An HA ectodomain antigen comprising mutations as shown for Flu693 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 38; (29) An HA ectodomain antigen comprising the mutations as shown for Flu695 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 39; (30) An HA ectodomain antigen comprising a mutation as shown for Flu696 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 40; (31) An HA ectodomain antigen comprising mutations as shown for Flu697 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 41; (32) An HA ectodomain antigen comprising mutations as shown for Flu707 in Table 3, with or without a trimerization domain and a His tag, preferably SEQ ID NO: 42;

[0090] In further embodiments, particularly for nucleic acid delivery, the HA antigen comprises a polypeptide sequence of SEQ ID NOs: 3-8 and 17-42, with or without a signal peptide, or a sequence having at least 85% identity, or at least 87% identity, or at least 90% identity, e.g., 95% or more, e.g., 98% or more, e.g., 99% or more sequence identity, to any one of the amino acid sequences of SEQ ID NOs: 3-8 and 17-42, with or without a signal peptide, which does not comprise a trimerization domain, does not comprise a His tag, and further comprises a transmembrane domain (which may be homologous or heterologous, and is optionally trimeric), and optionally a cytoplasmic domain. In a specific embodiment for nucleic acid delivery, the HA antigen comprises a polypeptide sequence selected from (1) to (32) above, with or without a signal peptide, and further comprising a transmembrane domain (homologous or heterologous) and optionally a cytoplasmic region, without a trimerization domain or His tag. In embodiments for nucleic acid delivery, the transmembrane domain can be an HA transmembrane domain, e.g., a transmembrane domain derived from an influenza strain from which the antigen is derived, i.e., a homologous transmembrane domain. The cytoplasmic domain can be an HA cytoplasmic domain, e.g., a cytoplasmic domain derived from an influenza strain from which the antigen is derived, i.e., a homologous cytoplasmic domain. The purpose of the transmembrane and cytoplasmic domains is to function as a membrane anchor for the HA antigen.

[0091] Particles / nanoparticles, e.g., ferritin nanoparticles The influenza HA antigens described herein can be displayed on the surface of nanoparticles, a strategy known as nanoparticularization. In one embodiment, the influenza HA ectodomain antigen is displayed on the surface of self-assembling protein nanoparticles, preferably ferritin nanoparticles, more preferably insect or bacterial ferritin nanoparticles, most preferably Helicobacter pylori ferritin nanoparticles (such as those disclosed in Corbett et al. 2019, WO 2013 / 044203, WO 2015 / 183969, and WO 2018 / 045308). It will be apparent that alternative protein nanoparticles known in the art, such as, but not limited to, lumazine and encapsulin, or other protein nanoparticles, including artificially constructed protein nanoparticles, can also be used.

[0092] In certain embodiments, the influenza HA or ectodomain is fused to a heterologous polypeptide, such as ferritin. When ferritin is expressed as a fusion with an HA antigen, it can function as a trimerization domain for the formation and / or stabilization of HA trimers. Preferably, the heterologous polypeptide (such as ferritin) and the influenza HA or ectodomain monomer are linked by a linker.

[0093] Particle technology is well known, and the fusion strategy described herein, such as ferritin fusion, is merely one example. Other suitable examples include conjugation, which can be achieved chemically or by using other approaches to protein ligation, such as the Streptococcus pyogenes-derived system known as SpyTag / SpyCatcher. Multi-component nanoparticle technology, in which more than one, for example, two or more different influenza antigens are displayed, can also be applied. Examples include fusion with heterologous polypeptides, such as insect ferritin, or combining different antigens in nanoparticles by chemical conjugation. Insect ferritin can be genetically engineered to display two different trimeric antigens in a predetermined ratio and geometric pattern.

[0094] In further embodiments, the influenza HA ectodomain is in the form of a rosette structure, such as those described in WO 2017 / 149054. In certain embodiments, the influenza HA ectodomain is fused to a hydrophobic signal, such as a transmembrane domain (which may be a homologous or heterologous transmembrane domain), and a heterologous trimerization domain for the purpose of forming the rosette structure in vivo or in vitro.

[0095] immunogenic fragment The influenza HA ectodomain antigens described herein also encompass immunogenic fragments of the HA ectodomain region, wherein the fragments comprise the mutations described herein. In one embodiment, the influenza HA ectodomain is a polypeptide consisting of an influenza HA ectodomain antigen described herein, or an immunogenic fragment thereof comprising the head and stem domains and one or more mutations (e.g., amino acid substitutions) described herein.

[0096] Immunogenic fragments of the influenza HA ectodomain useful in the present invention comprise, e.g., consist of, a fragment of the influenza ectodomain that is capable of raising a neutralizing antibody and / or T cell response (such as a CD4 or CD8 T cell response) against influenza virus, preferably a protective immune response (e.g., partially or completely reducing the severity of one or more symptoms and / or the time that a subject experiences one or more symptoms following infection, reducing the likelihood of developing an established infection following antigen challenge, and / or slowing disease progression (e.g., extending survival)).

[0097] Suitably, an immunogenic fragment of an influenza HA ectodomain comprises one or more epitopes, for example one, two or three or more epitopes, from the full-length influenza HA stem or ectodomain.

[0098] HA epitope Certain epitopes on influenza HA are known to be neutralizing epitopes for influenza viruses, including the CR9114 stem epitope, the FI6 (e.g., FI6v3) stem epitope, the 5A7 stem epitope, the CR8033 epitope, and the CR071 epitope (CR9114, CR8033, and CR071 are described, for example, in Dreyfus et al., Science, 2012, 337(6100): 1343-8; FI6 is described, for example, in Corti et al., Science, 2011, 333(6044): 850-6; and 5A7 is described, for example, in Yasugi et al., PLOS Pathogens, 2013, 9(2): e1003150).

[0099] In one embodiment, the CR9114 epitope is present in an influenza HA antigen described herein. In another embodiment, the FI6 (e.g., FI6v3) epitope is present in an influenza HA antigen described herein. In another embodiment, both the CR9114 and FI6 (e.g., FI6v3) epitopes are present in an HA antigen. When epitopes are present in a recombinant HA antigen, this means that mAbs targeting those epitopes are able to bind to the recombinant antigen in a suitable assay.

[0100] Recombinant HA antigen A recombinant HA antigen can comprise or be encoded by one or more nucleic acids derived from an artificially constructed nucleic acid, for example, a nucleic acid can comprise or be encoded by a cloned nucleic acid formed by ligating heterologous nucleic acid.

[0101] The recombinant HA antigen comprises the hemagglutinin-derived sequences HA1 and HA2 of the ectodomain, and may contain other non-hemagglutinin-derived sequences, e.g., one or more linker sequences, each of which may be a flexible linker sequence, or a cleavage site such as a furin cleavage site. A linker sequence may be present, for example, between the HA1 and HA2 regions of the recombinant HA. The linker sequence may promote independent folding of the HA domain. The linker sequence may be an amino acid sequence synthesized as part of a recombinant fusion protein. A linker, particularly a short linker, when present, may be present between the ectodomain and the trimerization domain and / or between the trimerization domain and the His tag. In other embodiments, a chemical linker is used to link synthetically or recombinantly produced subsequences. Such flexible linkers are known to those skilled in the art. A cleavage site, such as a furin cleavage site, may be present between the HA1 and HA2 regions of the HA. The furin cleavage site may be used to enable activation, for example, from vector technology. This can improve the representativity and immunogenicity of the antigen.

[0102] In addition to, or as an alternative to, a flexible linker, the recombinant HA antigen can further comprise a polypeptide subsequence derived from a protein unrelated to hemagglutinin, such as a sequence with affinity for a known antibody to facilitate affinity purification and / or detection. Such detection and purification-facilitating domains include, but are not limited to, metal-chelating peptides such as polyhistidine tracts and histidine-tryptophan modules, which enable purification with immobilized metals, and protein A domains, which enable purification with immobilized immunoglobulins. Examples include gD tags, Avi tags, c-Myc epitopes, polyhistidine tags, fluorescent proteins (e.g., GFP), heterologous fusion sequences encoding β-galactosidase proteins or glutathione S-transferase, or any other sequence useful for detecting or purifying fusion proteins expressed in or on cells. A preferred additional polypeptide sequence is a polyhistidine tag, such as a 4-, 6-, 8-, or 10-histidine tag, particularly a 6-histidine tag. The inclusion of a cleavable linker sequence between the purification domain (e.g., a polyhistidine tag) and the HA antigen can be useful to facilitate purification. For example, an enzyme cleavage site, such as a TEV cleavage site or a thrombin cleavage site, can be included between the additional polypeptide and the remainder of the recombinant HA sequence.

[0103] Alternatively or additionally, a cleavable linker sequence, e.g., an enzymatic cleavage site, e.g., a TEV cleavage site or a thrombin cleavage site, can be included between the trimerization domain and the remainder of the recombinant HA sequence. This may allow the trimerization domain to be removed in the final recombinant HA. Thus, the recombinant HA described herein can comprise or consist of (in that order) an HA ectodomain, a heterologous trimerization domain, a purification tag (e.g., a polyhistidine tag), and optionally, a cleavable linker sequence between i) the purification tag and the remainder of the recombinant HA and / or ii) between the trimerization domain and the HA ectodomain.

[0104] In some embodiments, the His tag and optionally the trimerization domain (eg, foldon or GCN4), if present, are cleaved after expression of the protein and are therefore not present in the final HA antigen.

[0105] For example, the recombinant HA antigen described herein can comprise or consist of i) an amino acid sequence comprising the ectodomain of HA, and ii) the heterologous trimerization domain (foldon) of SEQ ID NO: 9, or a derivative thereof that maintains the ability to induce trimerization of recombinant HA monomers. In particular, the recombinant HA ectodomain antigen can comprise, e.g., consist of, any one of the amino acid sequences of SEQ ID NO: 3, 4, 5, 6, 7, or 8, more preferably a sequence having at least 80% identity, such as 90% or more, such as 95% or more, such as 98% or more, for example 99% or more sequence identity to SEQ ID NO: 3 or 6. All of the amino acid sequences of SEQ ID NOs: 3 to 8 comprise a foldon sequence and a poly-His tag, either or both of which are optionally removed to provide the final HA antigen. Alternatively, the recombinant HA ectodomain antigen may comprise, for example consist of, any one of the amino acid sequences of SEQ ID NOs: 17 to 42, more suitably a sequence having at least 80% identity, such as 90% or more, for example 95% or more, such as 98% or more, for example 99% or more sequence identity to SEQ ID NOs: 19, 21, 26, 34 or 42. All of the amino acid sequences of SEQ ID NOs: 17 to 42 comprise a foldon sequence and a poly-His tag, either or both of which are optionally removed to provide the final HA antigen.

[0106] For example, the recombinant HA antigen described herein can comprise i) an amino acid sequence comprising an HA antigen, and ii) a heterologous trimerization domain, e.g., a foldon as set forth in SEQ ID NO: 9, or a derivative thereof that maintains the ability to induce trimerization of recombinant HA monomers. In particular, the recombinant HA ectodomain antigen can comprise or consist of any one of the amino acid sequences set forth in SEQ ID NOs: 3-8 and 17-42. All of these sequences contain a foldon, which is optionally removed to provide the final HA antigen.

[0107] The recombinant HA antigen described herein presented in the nanoparticles can comprise, for example, i) an amino acid sequence comprising an HA ectodomain antigen, excluding the foldon, such as any of the amino acid sequences set forth in SEQ ID NOS: 3-8 and 17-42; and optionally ii) a heterologous polypeptide capable of forming nanoparticles, such as ferritin. In one embodiment, the recombinant HA antigen is an ectodomain antigen described herein fused to H. pylori ferritin.

[0108] The recombinant HA antigens described herein, delivered as nucleic acids such as mRNA, can comprise i) an amino acid sequence comprising an HA ectodomain antigen, and optionally ii) a transmembrane domain (either homologous, i.e., derived from the same influenza strain, or heterologous, i.e., derived from another influenza strain or another source, and optionally trimeric), such that when the antigen is expressed, the antigen is membrane-tethered, or a heterologous polypeptide capable of forming a nanoparticle, such as ferritin, such that when expressed, the antigen is presented on the surface of the nanoparticle.

[0109] Polynucleotides encoding HA antigens The polynucleotide construct encoding the recombinant HA antigen can include a signal sequence. Typically, the signal sequence is appropriate for the host cell in which the recombinant HA is expressed. In one embodiment, a native signal peptide sequence is used. In another embodiment, a heterologous signal peptide sequence is used.

[0110] Thus, a polynucleotide encoding a recombinant influenza HA antigen described herein can comprise a sequence encoding an HA ectodomain, a heterotrimerization domain (e.g., a foldon), a purification tag (e.g., a polyhistidine tag), and a signal peptide (e.g., SEQ ID NO: 10 or 43), for example, in the following order: signal peptide (e.g., SEQ ID NO: 10 or 43), HA ectodomain, a heterotrimerization domain (e.g., a foldon), a purification tag (e.g., a polyhistidine tag). In another example, a polynucleotide encoding a recombinant HA antigen described herein comprises a sequence encoding (in that order) a signal peptide (e.g., SEQ ID NO: 10 or 43), an HA ectodomain, a cleavable linker sequence (e.g., a TEV cleavage site), a heterotrimerization domain (e.g., a foldon), and a purification tag (e.g., a polyhistidine tag).

[0111] The polynucleotide sequence encoding the recombinant influenza HA antigen may comprise i) a polynucleotide sequence encoding the HA ectodomain and ii) SEQ ID NO: 9, which encodes a foldon or a derivative of foldon that maintains the ability to induce trimerization of expressed recombinant HA monomers.

[0112] In certain embodiments, the polynucleotide sequence encoding the recombinant HA antigen described herein comprises or consists of a sequence encoding the polypeptide of any one of SEQ ID NOs: 3-8 or 17-42, such as the polynucleotide sequence of SEQ ID NOs: 11-16 or 44-69.

[0113] In another embodiment, the polynucleotide sequence encoding the recombinant influenza HA antigen comprises a sequence encoding an ectodomain and a transmembrane domain. In one embodiment, the transmembrane domain is a native influenza HA transmembrane or a functional derivative that anchors the antigen in the membrane. In one embodiment, the polynucleotide sequence is formulated for delivery as a nucleic acid vaccine.

[0114] In another embodiment, the polynucleotide sequence encoding the recombinant influenza HA antigen comprises a sequence encoding an ectodomain and a sequence encoding a polypeptide capable of forming nanoparticles. In one embodiment, the polypeptide capable of forming nanoparticles is ferritin. In one embodiment, the polynucleotide sequence is formulated for delivery as a nucleic acid vaccine.

[0115] Nucleic acid-based vaccines are contemplated herein for any of the influenza HA antigens described. The nucleic acid can be RNA (i.e., RNA-based vaccines or mRNA delivery platforms) or DNA (i.e., DNA-based vaccines, e.g., plasmid DNA vaccines), including, for example, viral vectors. The sequence of the nucleic acid molecule can be modified, for example, to increase the efficacy of nucleic acid expression or replication, or to provide additional stability or resistance to degradation, or to reduce reactogenicity, or to activate the interferon pathway, which affects antigen expression.

[0116] Messenger RNA (mRNA) can direct a subject's cellular machinery to produce proteins. As used herein, the term mRNA includes conventional mRNA or mRNA analogs, such as those containing modified backbones or modified bases (e.g., pseudouridine, etc.). mRNA can have a 5' cap or not. mRNA can encode two or more antigens. For example, an mRNA encoding an HA antigen as described herein can encode only the HA antigen, or it can encode a second HA antigen or an additional protein. If an additional protein is encoded, the mRNA can be polycistronic.

[0117] mRNA can be non-replicative or replicative, also known as self-amplifying. Self-amplifying mRNA molecules can be alphavirus-derived mRNA replicons. mRNA amplification can also be achieved by providing non-replicative mRNA encoding antigens together with separate mRNA encoding replication machinery.

[0118] Self-replicating RNA molecules are well known in the art and can be produced, for example, by using replication elements from alphaviruses and by replacing structural viral proteins with nucleotide sequences encoding the protein of interest.

[0119] The mRNA can also be codon optimized, hi some embodiments, the mRNA can be codon optimized for expression in human cells.

[0120] Various carrier systems have been described that encapsulate or complex mRNA to enhance mRNA delivery and the resulting expression of the encoded antigen compared to unencapsulated or uncomplexed mRNA. The present invention can utilize any suitable carrier system. Specific carrier systems include lipid nanoparticles (LNPs), which are non-virion liposomal particles that can encapsulate mRNA; cationic nanoemulsion (CNE) delivery systems, in which cationic oil-in-water emulsions can be used to deliver mRNA to the interior of cells; and lipidoid-coated iron oxide nanoparticles (LIONs), which can deliver mRNA to cells and can be assisted by the application of an external magnetic field after administration to a subject. In one embodiment, the mRNA encoding the HA antigen described herein is encapsulated or complexed in a carrier system selected from LNPs, CNEs, and LIONs.

[0121] Trimerization domain A preferred trimerization domain induces trimerization and enhances the stability of the recombinant HA antigen monomer. Preferably, the trimerization domain is or is derived from the natural trimerization domain of the T4 bacteriophage fibritin "foldon." A foldon sequence that forms a β-propeller structure comprising the C-terminus of the fibritin domain of T4 bacteriophage can be used. For example, the trimerization domain can comprise or consist of the foldon amino acid sequence set forth in SEQ ID NO: 9 or a derivative of this sequence that maintains the ability to induce trimerization of the recombinant monomer.

[0122] Another suitable trimerization domain is the leucine zipper trimerization motif derived from yeast transcriptional activator GCN4. Further suitable trimerization domains include chloramphenicol acetyltransferase (CAT). The trimerization domain is located at the C-terminus of the HA ectodomain, i.e., at the stem end of HA. Additional trimerization domains include human-derived trimerization domains, such as a trimer tag, or an HIV-derived trimerization domain. Typically, the trimerization domain is fused to the HA sequence via a short linker region. The region between the trimerization domain and the HA sequence can contain a cleavable linker sequence, allowing the HA sequence to be separated from the trimerization domain at a later stage. That is, the HA sequence can be linked, optionally via a linker sequence, to a heterologous sequence containing a protease cleavage site, a trimerization domain, and a purification tag, such as a histidine tag, to aid in purification (e.g., in that order). Such heterologous trimerization domains can be linked to the HA sequence by techniques known in the art, such as molecular cloning.

[0123] Preparation of recombinant HA antigen The use of recombinant DNA technology to produce influenza vaccines offers several advantages. These include the potential avoidance of the steps of adapting and passage of infectious viruses in eggs, and the production of more highly purified proteins under safer and strictly controlled conditions. Furthermore, there is no need to include a viral inactivation step. Any suitable cloning and expression system can be used to recombinantly produce the recombinant HA antigen.

[0124] Nucleotide sequences encoding the recombinant HA antigens of the present invention can be synthesized and / or cloned and expressed according to techniques well known to those skilled in the art. See, for example, Sambrook, et al. Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989). In some embodiments, polynucleotide sequences will be codon-optimized for a particular recipient host cell using standard methodologies. For example, a DNA construct encoding a hemagglutinin sequence can be codon-optimized for expression in other hosts, such as bacteria, mammalian, or insect cells. Suitable host cells include bacterial cells, such as E. coli, fungal cells, such as yeast, insect cells, such as Drosophila S2, Spodoptera Sf9, Sf00+, or Hi-5, and animal cells, such as CHO.

[0125] Suitably, the HA antigens described herein are expressed in eukaryotic cells, e.g., mammalian cells, e.g., human cells such as HEK293T cells, non-human mammalian cells such as CHO cells, or insect cells, optionally further comprising purifying / isolating the recombinant HA from the cells.

[0126] Hemagglutinin sequences can be generated by standard recombinant methods known in the art, such as polymerase chain reaction (PCR) or reverse transcription PCR, reverse genetic engineering, or the DNA can be synthesized. For PCR, primers can be generated using hemagglutinin nucleotide sequences available in public databases.

[0127] Sequence identity Identity with respect to a sequence is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with a reference amino acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity.

[0128] Sequence identity can be determined by standard methods commonly used to compare the similarity of two polypeptide amino acid positions. Using computer programs such as BLAST, FASTA, MUSCLE, or CLUSTALO, two polypeptides are aligned for optimal matching of their respective amino acids (over the entire length of one or both sequences, or over a predetermined portion of one or both sequences). The program provides default opening and gap penalties, and a scoring matrix such as PAM250 (a standard scoring matrix; see Dayhoff et al. (1978) A model of evolutionary change in proteins, in Atlas of Protein Sequence and Structure, vol. 5, supp. 3) can be used with the computer program. For example, percent identity can then be calculated as follows: the total number of identical matches is multiplied by 100, and then divided by the sum of the length of the longer sequence in the matched span and the number of gaps introduced into the shorter sequence to align the two sequences (e.g., divided by the length of the alignment).

[0129] Influenza strains Influenza virus "types" refer to influenza A, B, and C. The designation of a virus as a specific type is related to differences in the sequence of its respective M1 (matrix) protein or NP (nucleoprotein). Influenza A viruses are further divided into groups 1 and 2. These groups, also called clades, are further divided into subtypes, which refer to classifications based on the sequence of the virus's HA protein. Examples of currently commonly recognized subtypes are H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, and H18. Of these, influenza A subtypes in group 1 are H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. The influenza A subtypes in group 2 are H3, H4, H7, H10, H14 and H15. Finally, the term "strain" refers to viruses within a subtype that differ from each other by having small genetic variations in their genomes.

[0130] The HA ectodomain sequence of the recombinant HA antigens described herein can be from any influenza A subtype, e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18. In one embodiment, the HA sequence of the HA antigen is from a strain selected from group 1 influenza A subtypes, including H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18. In a specific embodiment, the HA sequence is from an H1 strain from the human population, e.g., Bri18. In a further embodiment, the HA sequence of the HA antigen is from a strain selected from group 2 influenza A subtypes, including H3, H4, H7, H10, H14, and H15.

[0131] In one embodiment, the HA sequence is from a naturally occurring HA from a non-pandemic strain. In one embodiment, the HA sequence can be from a naturally occurring HA from a circulating influenza B virus or a virus strain recommended by the WHO for seasonal influenza vaccines. For example, a circulating or vaccine-recommended influenza virus can be a virus strain identified by the WHO as a circulating or vaccine-recommended seasonal influenza virus strain, or a virus strain identified by the WHO in a previous season as a circulating or vaccine-recommended seasonal strain. In one embodiment, the HA sequence is from a virus strain identified by the WHO as having the potential to cause an epidemic in the following influenza season. In one embodiment, the HA sequence is from a virus strain that is a novel influenza virus strain to which a large portion of the human population has no immunity. In one embodiment, the HA ectodomain sequence is from a strain with the potential to cause a pandemic. Typically, the WHO identifies and publishes such strains.

[0132] Additional antigens The present invention can include multiple antigenic components, for example, with the goal of generating a broad immune response against influenza viruses. As a result, there can be two or more antigens, there can be two or more polynucleotides encoding one antigen, there can be one polynucleotide encoding two or more antigens, or there can be a mixture of antigens and polynucleotides encoding antigens. Polysaccharides, such as polysaccharide conjugates, can also be present.

[0133] The term antigen refers to a polypeptide capable of eliciting an immune response. Preferably, an antigen comprises at least one B or T cell epitope. The immune response elicited can be an antigen-specific B cell response, which generates neutralizing antibodies. The immune response elicited can be an antigen-specific T cell response, which can be a systemic and / or local response. An antigen-specific T cell response can include a CD4+ T cell response, such as a response comprising CD4+ T cells expressing multiple cytokines, e.g., IFNγ, TNFα, and / or IL2. Alternatively or additionally, an antigen-specific T cell response can include a CD8+ T cell response, such as a response comprising CD8+ T cells expressing multiple cytokines, e.g., IFNγ, TNFα, and / or IL2.

[0134] Vaccine Delivery It will be apparent that the recombinant HA antigens described herein can be delivered in any suitable vaccine delivery mode, for example, in the form of protein or in the form of nucleic acid, including DNA (including viral delivery platforms such as adenovirus) or nucleic acid delivery platforms, such as RNA, including mRNA, optionally formulated in a carrier system. Suitable delivery systems include viral vectors, such as adenovirus vectors. In either case, the delivery mode will include a pharmaceutically acceptable diluent or carrier. Optionally, one or more adjuvants can be used.

[0135] immunogenic composition In one aspect, there is provided an immunogenic composition comprising an influenza HA antigen or polynucleotide as described herein and a pharmaceutically acceptable carrier.

[0136] In one embodiment, an immunogenic composition comprising an HA antigen as described herein further comprises an adjuvant. Preferably, the adjuvant is an oil-in-water emulsion adjuvant. Oil-in-water emulsion adjuvants are well known in the art and are described in more detail below.

[0137] In one embodiment, an immunogenic composition comprising an HA polynucleotide encoding an HA antigen as described herein further comprises a polynucleotide carrier or delivery system.

[0138] In one embodiment, the immunogenic composition is monovalent, i.e., it contains an influenza HA antigen from only one influenza A strain or a polynucleotide encoding same. In an alternative embodiment, the composition is multivalent, i.e., it contains influenza virus antigens from multiple virus strains. For example, the composition can be bivalent, trivalent, or tetravalent, e.g., it can contain two or three seasonal virus strains along with a recombinant HA antigen described herein. For example, the composition can include an antigen or polynucleotide described herein, further together with one strain A antigen or polynucleotide, and optionally one or two strain B antigens or polynucleotides.

[0139] In one embodiment, the immunogenic composition is an improved seasonal influenza vaccine in which the HA antigen is capable of inducing an immune response against at least one other influenza strain from the same or a different subtype, e.g., an influenza A hemagglutinin subtype. In a further embodiment, the immunogenic composition is capable of inducing an immune response against two, three, or four or more different strains, including one or more from each of two different subtypes, such as H1 and H3. In one embodiment, the immunogenic composition comprises an HA antigen from H1 that is capable of inducing an immune response against one or more other H1 strains. In one embodiment, the immunogenic composition comprises an HA antigen from H1 that is capable of inducing a heterosubtypic immune response against one or more group 1 subtypes, such as H2, H5, or H9. In one embodiment, the immunogenic composition comprises an HA antigen from H1 that is capable of inducing a heterosubtypic immune response against one or more group 2 subtypes, such as H10.

[0140] In one embodiment, an immunogenic composition is provided comprising: (i) an influenza HA ectodomain antigen in trimeric form, the antigen comprising one or more stabilizing mutations described herein in the coiled-coil region; and (ii) a squalene-based adjuvant.

[0141] Adjuvants In one embodiment, the immunogenic composition of the present invention includes an adjuvant. In particular, the adjuvant can be an emulsion, such as an oil-in-water emulsion. Optionally, other immunostimulants can be present in the oil-in-water emulsion. In a specific embodiment, the oil-in-water emulsion contains a metabolizable non-toxic oil, such as squalene or squalane, optionally a tocol, such as a tocopherol, particularly α-tocopherol, and an emulsifier (or surfactant), such as the nonionic surfactant polyoxyethylene sorbitan monooleate (TWEEN-80™ or Polysorbate 80™). Surfactant mixtures can be used, such as a polyoxyethylene sorbitan monooleate / sorbitan trioleate (SPAN85™) mixture or a polyoxyethylene sorbitan monooleate / t-octylphenoxypolyethoxyethanol (TRITON X-100™) mixture.

[0142] In one embodiment, the oil-in-water emulsion has one of the following compositions: - 0.5 to 11 mg squalene, 0.05 to 5% polyoxythylene sorbitan monooleate (TWEEN-80™ or Polysorbate 80™) and optionally 2 to 12% alpha-tocopherol; or - about 5% squalene, about 0.5% polyoxyethylene sorbitan monooleate (TWEEN-80™ or Polysorbate 80™) and about 0.5% sorbitan trioleate (SPAN85™). This adjuvant is called MF59.

[0143] Squalene emulsion adjuvants are described in more detail below.

[0144] An alternative adjuvant that can be used includes an immunologically active saponin fraction (e.g., QS21) derived from the bark of Quillaja Saponaria Molina presented in the form of liposomes and lipopolysaccharides (e.g., 3D-MPL), optionally further containing a sterol (cholesterol). In one embodiment, the adjuvant comprises or consists of a saponin (e.g., QS21) presented in the form of liposomes, lipid A derivatives such as 3D-MPL, and a sterol (e.g., cholesterol). The liposomes preferably contain a neutral lipid, such as phosphatidylcholine, dioleoylphosphatidylcholine (DOPC), or dilaurylphosphatidylcholine. The liposomes can also contain a charged lipid, which increases the stability of the liposome-QS21 structure relative to liposomes composed of saturated lipids. An example of such an adjuvant is AS01, which contains 3D-MPL and QS21 in a quenched form containing cholesterol and can be prepared as described in WO 96 / 33739. Either the AS01B or AS01E form of this adjuvant can be used. The AS01B adjuvant contains liposomes, which also contain dioleoylphosphatidylcholine (DOPC), cholesterol, and 3D-MPL (in an amount of approximately 1000 micrograms of DOPC, 250 micrograms of cholesterol, and 50 micrograms of 3D-MPL per vaccine dose), QS21 (50 micrograms / dose), NaCl phosphate buffer, and water up to a volume of 0.5 mL.

[0145] The AS01E adjuvant contains the same ingredients as AS01B, but at a lower concentration of approximately 500 micrograms DOPC, 125 micrograms cholesterol, 25 micrograms 3D-MPL and 25 micrograms QS21, NaCl phosphate buffer and water up to a volume of 0.5 mL.

[0146] In one embodiment, the influenza HA ectodomain antigen is physically associated with an adjuvant, for example, liposomes of AS01 or an emulsion of a squalene-containing adjuvant such as AS03.

[0147] Squalene emulsion adjuvant As used herein, the term "squalene emulsion adjuvant" means a squalene-containing oil-in-water emulsion adjuvant.

[0148] Squalene is a branched-chain unsaturated terpenoid ([(CH3)2C[=CHCH2CH2C(CH3)]2=CHCH2-]2;C 30 H50; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS Registry Number 7683-64-9). Squalene is readily available from commercial sources or can be obtained by methods known in the art. Squalene exhibits good bioavailability and is easily metabolized.

[0149] The squalene emulsion adjuvant may include one or more tocopherols, preferably where the weight ratio of squalene to tocopherol is 20 or less (i.e., 20 weight units or less of squalene per weight unit of tocopherol, or alternatively, at least 1 weight unit of tocopherol per 20 weight units of squalene).

[0150] Although any of α, β, γ, δ, ε, and / or ξ-tocopherol can be used, α-tocopherol (also referred to herein as alpha-tocopherol) is typically used. Both D-alpha-tocopherol and D / L-alpha-tocopherol can be used. Tocopherols are readily available from commercial sources or can be obtained by methods known in the art. In some embodiments, the squalene emulsion adjuvant contains alpha-tocopherol, particularly D / L-alpha-tocopherol.

[0151] Squalene emulsion adjuvants typically have droplet sizes below the micron level. A droplet size of 200 nm or less is beneficial because it facilitates sterilization by filtration. There is evidence that droplet sizes in the 80-200 nm range are of particular interest for reasons of efficacy, manufacturing consistency, and stability (Klucker, 2012; Shah, 2014; Shah, 2015; Shah, 2019). Preferably, squalene emulsion adjuvants have an average droplet size of less than 1 μm, particularly less than 500 nm, and particularly less than 200 nm. Preferably, squalene emulsion adjuvants have an average droplet size of at least 50 nm, particularly at least 80 nm, particularly at least 100 nm, for example at least 120 nm. The squalene emulsion adjuvant may have an average droplet size of 50 to 200 nm, for example 80 to 200 nm, particularly 120 to 180 nm, especially 140 to 180 nm, for example about 160 nm.

[0152] Uniformity of droplet size is desirable. A polydispersity index (PdI) of greater than 0.7 indicates that the sample has a very wide size distribution, with a reported value of 0 meaning no size variation, while values ​​below 0.05 are rarely observed. Preferably, the squalene emulsion adjuvant has a polydispersity of 0.5 or less, particularly 0.3 or less, for example 0.2 or less.

[0153] As used herein, droplet size refers to the average diameter of the oil droplets in an emulsion and can be determined in various ways, for example, using instruments such as the Accusizer™ and Nicomp™ series instruments available from Particle Sizing Systems (Santa Barbara, USA), the Zetasizer™ instrument from Malvern Instruments (UK), or the particle size distribution analyzer instrument from Horiba (Kyoto, Japan), using dynamic light scattering and / or single particle optical detection techniques. See Light Scattering from Polymer Solutions and Nanoparticle Dispersions, Schartl, 2007. Dynamic light scattering (DLS) is the preferred method by which droplet size is determined. A preferred method for defining the average droplet diameter is the Z-average, i.e., the intensity-weighted average hydrodynamic size of the aggregate collection of droplets measured by DLS. The Z-average is derived from a cumulant analysis of the measured correlation curve, where a single particle size (droplet diameter) is assumed and a single exponential fit is applied to the autocorrelation function. Thus, references herein to the average droplet size should be taken as an intensity-weighted average, ideally the Z-average. PdI values ​​are readily provided by the same instruments that measure the average diameter.

[0154] To maintain a stable submicron emulsion, one or more emulsifiers (i.e., surfactants) are generally required. Surfactants can be classified by their "HLB" (Griffin's hydrophilic / lipophilic balance), with an HLB in the range of 1-10 generally indicating that the surfactant is more soluble in oil than in water, while an HLB in the range of 10-20 indicates that the surfactant is more soluble in water than in oil. HLB values ​​are readily available for many surfactants of interest or can be determined experimentally; for example, polysorbate 80 has an HLB of 15.0, and TPGS has an HLB of 13-13.2. Sorbitan trioleate has an HLB of 1.8. When two or more surfactants are mixed, the HLB of the resulting mixture is typically calculated by a weighted average, e.g., a 70 / 30 wt% mixture of polysorbate 80 and TPGS has an HLB of (15.0 x 0.70) + (13 x 0.30), or 14.4. A 70 / 30 wt% mixture of polysorbate 80 and sorbitan trioleate has an HLB of (15.0 x 0.70) + (1.8 x 0.30), or 11.04.

[0155] Surfactants are typically metabolizable (biodegradable) and biocompatible, and would be suitable for use as pharmaceuticals. Surfactants can include ionic (cationic, anionic, or zwitterionic) and / or nonionic surfactants. In many cases, it is desirable to use only nonionic surfactants, for example, due to their pH independence. Thus, the present invention can use surfactants, including, but not limited to, the following surfactants:

[0156] - polyoxyethylene sorbitan ester surfactants (commonly called Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, in particular polysorbate 80;

[0157] - copolymers of ethylene oxide (EO), propylene oxide (PO) and / or butylene oxide (BO) sold under the trade names DOWFAX™, Pluronic™ (for example F68, F127 or L121 grades) or Synperonic™, such as linear EO / PO block copolymers, for example Poloxamer 407, Poloxamer 401 and Poloxamer 188;

[0158] - Octoxynol, Octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol), in which the number of repeating ethoxy (oxy-1,2-ethanediyl) groups can vary, is of particular interest; - (Octylphenoxy)polyethoxyethanol (IGEPAL CA-630 / NP-40); - phospholipids, e.g., phosphatidylcholine (lecithin);

[0159] - polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as polyoxyethylene-4-lauryl ether (Brij 30, Emulgen 104P), polyoxyethylene-9-lauryl ether and polyoxyethylene 12 cetyl / stearyl ether (Eumulgin® B1, cetereth-12 or polyoxyethylene cetostearyl ether);

[0160] - sorbitan esters (commonly known as Span), such as sorbitan trioleate (Span 85), sorbitan monooleate (Span 80) and sorbitan monolaurate (Span 20); Alternatively, tocopherol derivative surfactants, such as alpha-tocopherol-polyethylene glycol succinate (TPGS).

[0161] Numerous examples of pharmaceutically acceptable surfactants are known in the art; see, for example, Handbook of Pharmaceutical Excipients, 6th edition, 2009. The selection of surfactants for use in squalene emulsion adjuvants and methods for optimizing their selection are exemplified in Klucker, 2012. Generally, the surfactant component has an HLB of 10-18, e.g., 12-17, particularly 13-16. This can typically be achieved using a single surfactant or, in some embodiments, a mixture of surfactants. Surfactants of particular interest include poloxamer 401, poloxamer 188, polysorbate 80, sorbitan trioleate, sorbitan monooleate, and polyoxyethylene 12 cetyl / stearyl ether, either alone, in combination with each other, or in combination with other surfactants. Of particular interest are polysorbate 80, sorbitan trioleate, sorbitan monooleate, and polyoxyethylene 12 cetyl / stearyl ether, either alone or in combination with one another. A particular surfactant of interest is polysorbate 80. A particular combination of surfactants of interest is polysorbate 80 and sorbitan trioleate. A further combination of surfactants of interest is sorbitan monooleate and polyoxyethylene cetostearyl ether.

[0162] In certain embodiments, the squalene emulsion adjuvant comprises one surfactant, such as polysorbate 80. In some embodiments, the squalene emulsion adjuvant comprises two surfactants, such as polysorbate 80 and sorbitan trioleate or sorbitan monooleate and polyoxyethylene cetostearyl ether. In other embodiments, the squalene emulsion adjuvant comprises three or more surfactants, e.g., three surfactants.

[0163] When tocopherol is present, the weight ratio of squalene to tocopherol may be 20 or less, for example 10 or less. Preferably, the weight ratio of squalene to tocopherol is 0.1 or more. Typically, the weight ratio of squalene to tocopherol is 0.1 to 10, particularly 0.2 to 5, particularly 0.3 to 3, for example 0.4 to 2. Preferably, the weight ratio of squalene to tocopherol is 0.72 to 1.136, particularly 0.8 to 1, particularly 0.85 to 0.95, for example 0.9.

[0164] When a surfactant is present, typically the weight ratio of squalene to surfactant is 0.73 to 6.6, especially 1 to 5, especially 1.2 to 4. Suitably the weight ratio of squalene to surfactant is 1.71 to 2.8, especially 2 to 2.4, especially 2.1 to 2.3, for example 2.2.

[0165] The amount of squalene in a single human dose of a squalene emulsion adjuvant is typically at least 1.2 mg. Generally, the amount of squalene in a single human dose of a squalene emulsion adjuvant is 50 mg or less. The amount of squalene in a single human dose of a squalene emulsion adjuvant may be 1.2 to 20 mg, particularly 1.2 to 15 mg. The amount of squalene in a single human dose of a squalene emulsion adjuvant may be 1.2 to 2 mg, 2 to 4 mg, 4 to 8 mg, or 8 to 12.1 mg. For example, the amount of squalene in a single human dose of a squalene emulsion adjuvant may be 1.21 to 1.52 mg, 2.43 to 3.03 mg, 4.87 to 6.05 mg, or 9.75 to 12.1 mg.

[0166] When tocopherol is present, the amount of tocopherol in a single dose, such as a human dose, of a squalene emulsion adjuvant is typically at least 1.3 mg. Generally, the amount of tocopherol in a single dose, such as a human dose, of a squalene emulsion adjuvant is 55 mg or less. The amount of tocopherol in a single dose, such as a human dose, of a squalene emulsion adjuvant may be 1.3 to 22 mg, particularly 1.3 to 16.6 mg. The amount of tocopherol in a single dose, such as a human dose, of a squalene emulsion adjuvant may be 1.3 to 2 mg, 2 to 4 mg, 4 to 8 mg, or 8 to 13.6 mg. For example, the amount of tocopherol in a single dose, such as a human dose of a squalene emulsion adjuvant may be 1.33 to 1.69 mg, 2.66 to 3.39 mg, 5.32 to 6.77 mg, or 10.65 to 13.53 mg.

[0167] When a surfactant is present, the amount of surfactant in a single dose, such as a human dose, of a squalene emulsion adjuvant is typically at least 0.4 mg. Generally, the amount of surfactant in a single dose, such as a human dose, of a squalene emulsion adjuvant is 18 mg or less. The amount of surfactant in a single dose, such as a human dose, of a squalene emulsion adjuvant may be 0.4 to 9.5 mg, particularly 0.4 to 7 mg. The amount of surfactant in a single dose, such as a human dose, of a squalene emulsion adjuvant may be 0.4 to 1 mg, 1 to 2 mg, 2 to 4 mg, or 4 to 7 mg. For example, the amount of surfactant in a single dose, such as a human dose, of a squalene emulsion adjuvant may be 0.54 to 0.71 mg, 1.08 to 1.42 mg, 2.16 to 2.84 mg, or 4.32 to 5.68 mg.

[0168] In certain embodiments, the squalene emulsion adjuvant can consist essentially of squalene, a surfactant, and water. In certain other embodiments, the squalene emulsion adjuvant can consist essentially of squalene, a tocopherol, a surfactant, and water. The squalene emulsion adjuvant can contain additional components, such as buffers and / or tonicity adjusters, e.g., modified phosphate buffered saline (disodium phosphate, potassium dihydrogen phosphate, sodium chloride, and potassium chloride), if desired or needed depending on the intended final form and vaccination strategy.

[0169] High-pressure homogenization (HPH or microfluidization) can be applied to obtain squalene emulsion adjuvants containing tocopherol, which exhibit uniform small droplet size and long-term stability (see EP 0868918 and WO 2006 / 100109). Briefly, an oil phase composed of squalene and tocopherol can be formulated under a nitrogen atmosphere. An aqueous phase, typically composed of water for injection or phosphate-buffered saline and polysorbate 80, is prepared separately. The oil and aqueous phases are combined, for example, in a ratio of 1:9 (oil phase volume: aqueous phase volume), and then subjected to homogenization and microfluidization, for example, by passing once through an in-line homogenizer and three times through a microfluidizer (at approximately 15,000 psi). The resulting emulsion can then be sterile filtered, for example, through two successive 0.5 / 0.2 μm filters (i.e., 0.5 / 0.2 / 0.5 / 0.2). See WO 2011 / 154444. Desirably, the operation is carried out under an inert atmosphere, for example, nitrogen. Positive pressure can be applied. See WO 2011 / 154443.

[0170] International patent application WO 2020160080 and Lodaya R, et al.: J Control Release (2019) 316:12-21 describe a self-emulsifying adjuvant system (SEAS), a squalene emulsion adjuvant with tocopherol, and its manufacture.

[0171] Vaccination regimens, dosing, and efficacy criteria Preferably, the immunogenic compositions described herein will be in a standard 0.5 mL injectable dose in most cases and contain 15 μg or less of influenza virus strain-derived hemagglutinin antigenic component as measured by single radial immunodiffusion (SRD) (J.M. Wood et al.: J. Biol. Stand. 5 (1977) 237-247; J.M. Wood et al., J. Biol. Stand. 9 (1981) 317-330). Preferably, the vaccine dose volume will be 0.25 mL to 1 mL, particularly the standard 0.5 mL or 0.7 mL vaccine dose volume. Some adaptation of the dose volume will routinely be made depending on the HA concentration in the original bulk sample and also on the delivery route, with relatively low doses administered by the intranasal or intradermal route. Immunogenic compositions for use in accordance with the present invention may contain a small amount of HA antigen, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 μg of HA per influenza virus strain, or an amount not exceeding 15 μg of HA per virus strain. The small amount of HA may be as low as practically feasible, provided that it allows for the formulation of a vaccine that meets international, e.g., EU or FDA, standards for efficacy. A suitable small amount of HA is 1-7.5 μg of HA per influenza virus strain, preferably 3.5-5 μg, e.g., 3.75 or 3.8 μg, of HA per influenza virus strain, typically about 5 μg of HA per influenza virus strain. Another suitable amount of HA is 0.1-5 μg of HA per influenza virus strain, preferably 1.0-2 μg of HA per influenza virus strain, e.g., 1.9 μg of HA per influenza virus strain.

[0172] The influenza medicaments (e.g., immunogenic compositions) described herein preferably meet certain international standards for vaccines, which are applied internationally to measure the effectiveness of influenza vaccines.

[0173] Serological variables will be evaluated according to the European Medicines Agency's criteria for human use (CHMP / BWP / 214 / 96, Committee for Medicinal Products for Human Use (CPMP)) or as amended.

[0174] Approaches to establishing strong and lasting immunity often involve repeated immunization, i.e., boosting the immune response by administering one or more additional doses. Such additional administration can be with the same immunogenic composition (homologous boost) or with a different immunogenic composition (heterologous boost). The present invention can be applied as either a prime immunization or a boost, as part of a homologous or heterologous prime / boost regimen.

[0175] Therefore, the administration of recombinant HA antigen can be part of a multi-dose administration regimen.For example, recombinant HA antigen can be provided as the initial immunization dose in a multi-dose regimen, particularly in a 2- or 3-dose regimen, particularly in a 2-dose regimen.Recombinant HA antigen can be provided as the booster dose in a multi-dose regimen, particularly in a 2- or 3-dose regimen, for example in a 2-dose regimen.

[0176] The priming and boosting doses can be homologous or heterologous. As a result, the recombinant HA antigen can be provided as the priming dose and boosting dose in a homologous multiple-dose regimen, particularly a 2- or 3-dose regimen, particularly a 2-dose regimen. Alternatively, the recombinant HA antigen can be provided as the priming dose or boosting dose in a heterologous multiple-dose regimen, particularly a 2- or 3-dose regimen, particularly a 2-dose regimen, and the boosting dose can be different (e.g., a different HA antigen; or alternative antigen presentation, such as a protein or viral vector-type antigen, with or without an adjuvant).

[0177] The time between doses can be from 2 weeks to 6 months, for example, from 3 weeks to 3 months. Periodic booster doses over longer periods, such as every 2 to 10 years, can also be provided.

[0178] Treatment method In a further embodiment, the immunogenic composition comprising the HA antigen or polynucleotide is for use in medicine, for example for use in preventing or vaccinating against influenza, for example to be administered to a person (e.g., a subject) at risk of influenza infection.

[0179] In yet a further embodiment, the immunogenic composition comprising the antigen or polynucleotide is for use in the prevention of influenza caused by a hemagglutinin subtype different from the subtype on which the HA antigen is based. For example, an HA antigen from H1 could be used to protect against influenza caused by a non-H1 influenza A strain virus, e.g., from a group 1 subtype such as H2, H5, or H9, or vice versa.

[0180] In a further aspect, there is provided a method for preventing and / or treating influenza disease comprising administering a recombinant HA antigen or immunogenic composition as described herein to a person in need thereof, e.g., a person (e.g., a subject) at risk of influenza infection, e.g., an elderly person (50 years of age or older, particularly 65 years of age or older).

[0181] In one embodiment of the above method or use, less than 15 micrograms, for example 3.75 to 10 micrograms, of HA is administered per dose.

[0182] In one aspect, the present invention provides a recombinant HA antigen as described herein for use in a vaccination regimen for the prevention of influenza, at a dose of less than 10 micrograms, or less than 8 micrograms, or 1 to 7.5 micrograms, or 1 to 5 micrograms of recombinant HA, wherein the hemagglutinin sequence is from or derived from an influenza virus strain identified by an international organization, such as the WHO, that monitors influenza virus outbreaks as being associated with an outbreak or having the potential to be associated with a future outbreak.

[0183] Administration route The compositions of the present invention can be administered by any suitable delivery route, such as intradermal, mucosal (e.g., intranasal), oral, intramuscular (IM), or subcutaneous. Other delivery routes are known in the art.

[0184] The intramuscular delivery route is particularly preferred for immunogenic compositions, especially adjuvanted immunogenic compositions. The composition can be presented in a single-dose container, or alternatively, a multi-dose container. In this example, an antimicrobial preservative such as thiomersal can be present to prevent contamination during use. A thiomersal concentration of 5 μg / 0.5 mL dose (i.e., 10 μg / mL) or 10 μg / 0.5 mL dose (i.e., 20 μg / mL) is preferably present. A suitable IM delivery device, such as a needleless liquid jet injection device, e.g., Biojector 2000 (Bioject, Portland, OR), could be used. Alternatively, a pen injector device, such as one used for home delivery of epinephrine, could be used to allow self-administration of the vaccine. The use of such a delivery device may be particularly suitable for mass immunization campaigns.

[0185] Intradermal delivery is another suitable route.Any suitable device, for example, a short needle device, can be used for intradermal delivery.Such devices are well known in the art.Intradermal vaccines can also be administered by a device that limits the effective penetration length of the needle into the skin, for example, those described in International Publication No. 99 / 34850 and European Patent No. 1092444, which are incorporated herein by reference, and their functional equivalents.A jet injection device is also suitable, which delivers liquid vaccines to the dermis through a liquid jet injector or through a needle that pierces the stratum corneum and generates a jet that reaches the dermis.A ballistic powder / particle delivery device is also suitable, which uses compressed gas to accelerate the vaccine in powder form through the outer layer of the skin to the dermis.In addition, a conventional syringe can be used in the classic Mantoux method of intradermal administration.

[0186] Another suitable route of administration is the subcutaneous route. Any suitable device, such as a classic needle, can be used for subcutaneous delivery. Preferably, a needle-free jet injector service is used. Such devices are well known in the art. Preferably, the device is pre-filled with a liquid vaccine formulation.

[0187] Alternatively, the vaccine is administered intranasally. Typically, the vaccine is administered locally to the nasopharyngeal region, preferably without being inhaled into the lungs. It is desirable to use an intranasal delivery device that delivers the vaccine formulation to the nasopharyngeal region without or substantially without penetrating the lungs.

[0188] A suitable device for intranasal administration of a vaccine according to the present invention is a spray device. Suitable commercially available nasal spray devices include Accuspray™ (Becton Dickinson). Nebulizers produce a fine spray that can be easily inhaled into the lungs and therefore does not efficiently reach the nasal mucosa. Therefore, nebulizers are not preferred.

[0189] A suitable spray device for intranasal use is one whose performance does not depend on the pressure applied by the user. These devices are known as pressure threshold devices. Liquid is released from the nozzle only when a threshold pressure is applied. These devices make it easier to achieve a spray with uniform droplet size. Pressure threshold devices suitable for use according to the present invention are known in the art and are described, for example, in International Publication No. 91 / 13281 and European Patent Nos. 311863B and 516636, which are incorporated herein by reference. Such devices are commercially available from Pfeiffer and are also described in Bommer, R. Pharmaceutical Technology Europe, September 1999.

[0190] Alternatively, epidermal or transdermal vaccination routes are also contemplated herein.

[0191] array SEQ ID NO: 1 A / Brisbane / 02 / 2018 (H1N1)pdm09-like virus (H1N1), also known as Bri18 Bri18 ) derived full-length HA sequence. [ka] SEQ ID NO: 2 Full-length HA sequence from A / Darwin / 9 / 2021 H3N2, also called Darw21. [ka] SEQ ID NOs: 3-8 show mutations in bold compared to the H1 Brisbane 18 WT sequence: SEQ ID NO: 3 Mut10 [ka] SEQ ID NO: 4 Mut17 [ka] SEQ ID NO: 5 Mut18 [ka] [ka] SEQ ID NO: 6 Mut23 [ka] SEQ ID NO:7 Mut24 [ka] SEQ ID NO:8 Mut27 [ka] SEQ ID NO: 9 Foldon sequence [ka] SEQ ID NO: 10 Bri 18 signal peptide [ka] SEQ ID NOs: 11 to 16 show nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 3 to 8. SEQ ID NO: 11 Mut10 [ka] [ka] SEQ ID NO: 12 Mut17 [ka] SEQ ID NO: 13 Mut18 [ka] SEQ ID NO: 14 Mut23 [ka] [ka] SEQ ID NO: 15 Mut24 [ka] SEQ ID NO: 16 Mut27 [ka] [ka] SEQ ID NOs: 17-42 show HA polypeptide antigens containing mutations compared to the H1 Brisbane 18 WT sequence. SEQ ID NO: 17 [ka] SEQ ID NO: 18 [ka] SEQ ID NO: 19 [ka] SEQ ID NO: 20 [ka] SEQ ID NO: 21 [ka] SEQ ID NO: 22 [ka] SEQ ID NO: 23 [ka] SEQ ID NO: 24 [ka] SEQ ID NO: 25 [ka] SEQ ID NO: 26 [ka] SEQ ID NO: 27 [ka] [ka] SEQ ID NO: 28 [ka] SEQ ID NO: 29 [ka] SEQ ID NO: 30 [ka] SEQ ID NO: 31 [ka] SEQ ID NO: 32 [ka] SEQ ID NO: 33 [ka] SEQ ID NO: 34 [ka] SEQ ID NO: 35 [ka] SEQ ID NO: 36 [ka] SEQ ID NO: 37 [ka] SEQ ID NO: 38 [ka] [ka] SEQ ID NO: 39 [ka] SEQ ID NO: 40 [ka] SEQ ID NO: 41 [ka] SEQ ID NO: 42 [ka] SEQ ID NO: 43 Darw21 signal peptide [ka] SEQ ID NOs: 44 to 69 show nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 17 to 42. SEQ ID NO: 44 [ka] [ka] SEQ ID NO: 45 [ka] SEQ ID NO: 46 [ka] SEQ ID NO: 47 [ka] SEQ ID NO: 48 [ka] SEQ ID NO: 49 [ka] [ka] SEQ ID NO:50 [ka] SEQ ID NO:51 [ka] SEQ ID NO:52 [ka] [ka] SEQ ID NO:53 [ka] SEQ ID NO:54 [ka] SEQ ID NO: 55 [ka] [ka] SEQ ID NO:56 [ka] SEQ ID NO:57 [ka] SEQ ID NO:58 [ka] SEQ ID NO:59 [ka] SEQ ID NO: 60 [ka] [ka] SEQ ID NO: 61 [ka] SEQ ID NO: 62 [ka] SEQ ID NO: 63 [ka] [ka] SEQ ID NO: 64 [ka] SEQ ID NO: 65 [ka] SEQ ID NO: 66 > Flu695 [ka] SEQ ID NO: 67 [ka] SEQ ID NO: 68 [ka] [ka] SEQ ID NO: 69 [ka] SEQ ID NO: 70 H1 WT foldon sequence: signal sequence-HA-TEV cleavage site-foldon-His tag [ka] SEQ ID NO: 71 H3 WT foldon sequence: signal sequence-HA-TEV cleavage site-foldon-His tag [ka] SEQ ID NO:72 Bri18-derived HA ectodomain sequence shown in Figure 2 [ka] [ka] SEQ ID NO: 73 The Darw21-derived HA ectodomain sequence shown in Figure 2 [ka] [Example]

[0192] [Example 1] Design and construction of influenza A strain HA constructs (a) H1 influenza A strain (group 1) Recombinantly expressed H1 hemagglutinin has been shown to be suboptimal, with difficulties maintaining its trimeric conformation. To mitigate the risk of losing the ectodomain trimeric conformation and improve both manufacturability and antigenic characteristics, we performed a thorough analysis of the A / Brisbane / 02 / 2018 (H1N1) HA 3D modeled structure using MOE to identify potential interesting positions in HA2 and HA1 helix B (see Figures 1, 2, and 3) and their surrounding environment for introducing mutations to improve trimeric conformational stability.

[0193] Using the 3D modeled structure of the A / Brisbane / 02 / 2018 (H1N1) strain HA, we identified several positions to mutate to attempt to stabilize the trimeric conformation.

[0194] Mutations to hydrophobic residues at positions 450, 453, 460, 464, and 467 can promote hydrophobic exclusion and favor trimerization, leading to the formation of a trimeric coiled-coil structure stabilized by these hydrophobic interactions.

[0195] For example, mutations at positions 322, 436, and 438 to polar charged residues can promote favorable protomer interactions.

[0196] Positions 395 and 477; in HA2 helix A and HA2 helix B, respectively, can be targeted to increase HA1 / HA2 interaction by either hydrophobic reinforcement or cavity filling / hydrogen bond introduction.

[0197] Mutations were introduced either alone as single amino acid substitutions or in combination for possible additive effects. The mutation combinations used in the ectodomain constructs are shown in Tables 1 and 2.

[0198] (b) H3 influenza A strains (group 2) A thorough analysis of the A / Darwin / 9 / 2021 H3N2 HA 3D modeled structure was performed using MOE to identify potential interesting positions in HA2 and HA1 helix B (see Figures 2, 3, and 4) and their surrounding environment for introducing mutations to improve trimer conformational stability.

[0199] Using the 3D modeled structure of the A / Darwin / 9 / 2021 H3N2 strain HA, we identified several positions to mutate to attempt to stabilize the trimer conformation.

[0200] Mutations to hydrophobic residues at positions 347, 440, 451, 454, 455, and 468 can promote hydrophobic exclusion and favor trimerization, leading to the formation of a trimeric coiled-coil structure stabilized by these hydrophobic interactions.

[0201] Position 418: Found at the end of the interloop region connecting helices A and B, mutation to proline at these positions may prevent post-fusion conformation formation through steric hindrance introduced by the specific side chain derived from proline.

[0202] Positions 369, 399, and 448; in HA2 helix A for the first position and in HA2 helix B for the latter two positions, respectively, can be targeted to increase HA1 / HA2 interactions by either hydrophobic reinforcement or cavity filling / hydrogen bond introduction.

[0203] For example, mutation of position 437 to a polar charged residue can promote favorable protomer interactions.

[0204] Mutations were introduced either alone as single amino acid substitutions or in combination for possible additive effects. The mutation combinations used in the ectodomain constructs are shown in Table 3.

[0205] [Example 2] Cloning, protein expression and purification Cloning The gene was codon-optimized for human protein expression, synthesized, and cloned into the pmaxCloning™ vector (Lonza, catalog number VDC-1040) by GENEWIZ using EcoRI / NotI restriction sites. The pmaxCloning™ vector backbone contains the cytomegalovirus immediate-early promoter (PCMV IE) for protein expression, a chimeric intron for enhanced gene expression, and a pUC origin of replication for propagation in E. coli. A bacterial promoter (P) provides kanamycin resistance gene expression in E. coli. A multiple cloning site (MCS) is located between the CMV promoter and the SV40 polyadenylation signal (SV40polyA).

[0206] Each construct contained a sequence encoding the influenza hemagglutinin (HA) ectodomain of SEQ ID NO: 1 or 2 with mutations as shown in Tables 1, 2, or 3. All constructs were fused at the C-terminus to a TEV cleavage site followed by a foldon followed by a 6xHis tag (except mut10, which lacks a TEV cleavage site).

[0207] Expression Expi293F™ cells (ThermoFisher, Cat. No. A14528) were used for recombinant protein expression. Cell culture and transfection were performed according to the manufacturer's instructions. Small-scale cultures (3 mL cultures in 24-deep well plates) were used for candidate screening, and medium-scale cultures (125 mL) were performed for selected top candidates.

[0208] The day before transfection, cell density and viability were assessed using a TC20™ automated cell counter (Bio-Rad). Cells were counted at 2 × 10 6Cells were seeded at a density of 3 x 10 cells / mL in fresh pre-warmed Expi293™ Expression Medium (ThermoFisher, Cat. No. A1435102) and cultured at 37°C, 110 rpm in a humidified 8% CO2 incubator. On the day of transfection, cell density and viability were assessed (viability ≥ 95%), and cells were resuspended in fresh pre-warmed Expi293™ Expression Medium at a density of 3 x 10 cells / mL. 6 The cells were diluted to a final density of 1000 cells / mL. Transfection was performed using the ExpiFectamine™ 293 Transfection Kit (Thermofisher, Cat. No. A14524), which contains a transfection enhancer and ExpiFectamine 293 transfection reagent. Briefly, the plasmid DNA and transfection reagent were separately diluted in OptiMEM medium (Thermofisher, Cat. No. 31985062) and incubated at RT for 5 minutes (1 μg of plasmid DNA was used per mL of cell culture). Then, both mixtures were combined and incubated at RT for an additional 20 minutes. The ExpiFectamine™ 293 / plasmid DNA complex solution was then carefully added to the cells. The cells were cultured at 37°C and 110 rpm in a humidified 8% CO2 incubator. One day after transfection (18–22 hours after transfection), ExpiFectamine™ 293 transfection enhancers 1 and 2 were added. Four days after transfection, cells were harvested by centrifugation at 5,000 × g for 10 minutes at 4°C. The cell pellet was discarded, and Complete™ protease inhibitor cocktail (Roche, catalog number 11697498001) was added to the supernatant. Protein expression was checked by SDS-PAGE and Western blot before purification (data not shown).

[0209] purification Purification for HTP expression (2.5 mL cultures in a deep 24-well format) was performed by adding 200 μL of Nickel Sepharose Excel (GE) slurry pre-equilibrated in Buffer A (20 mM bicine, 500 mM NaCl, 20 mM imidazole, pH 8.3) containing 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) (Sigma) and 20 mM bicine pH 8.3. After overnight rocking at 900 rpm, samples were transferred to a 96DW Thompson filter plate and washed three times with 1 mL of Buffer A under negative pressure. Proteins were eluted by centrifugation (800 g for 10 min) twice with 110 μL of buffer B (20 mM bicine, 500 mM NaCl, 500 mM imidazole, pH 8.3), desalted on a PD multitrap G-25, and analyzed by SDS-PAGE.

[0210] Purification of medium-scale expression (125 mL culture) was performed using a gravity-flow column packed with 3 mL of Nickel Sepharose Excel (GE) pre-equilibrated in buffer A (20 mM bicine, 500 mM NaCl, 20 mM imidazole, pH 8.3). After sample loading, the resin was washed with 15 CV of buffer A, and the protein was eluted with 4 CV of buffer B (20 mM bicine, 500 mM NaCl, 500 mM imidazole, pH 8.3). The protein was then concentrated using a Vivaspin 20 with a 10 kDa cutoff at 3000 g and 4 °C. The concentrated sample was loaded onto a Superdex200 increase 10 / 300 (GE) or Superdex200 16 / 600 (GE) column equilibrated in buffer C (20 mM bicine, 150 mM NaCl, pH 8.3) using a flow rate of 0.75 mL / min. Fractions corresponding to the protein of interest were pooled together, filtered at 0.22 μM, and stored at −80°C.

[0211] Protein concentration was determined by RCDC assay (Biorad) and purity was determined by SDS-PAGE.

[0212] Characterization UPLC - Ultra High Performance Liquid Chromatography The stability of trimeric assembly of semi-purified HA constructs from high-throughput screening (HTS) experiments was assessed by HPLC-SEC-UV. Briefly, 10 μL of each preparation was injected onto a 4.6 × 150 mm BEH column (Acquity) with a 200A pore size at 0.3 mL / min. UV at 280 nm was recorded during a 10-minute run. The column was maintained at 30°C throughout the experiment, and the samples were maintained at 8°C. The elution times of the HA peaks were compared with calibration standards (Waters BEH200 SEC Protein Standard Mix, ref. Waters 186006518). Based on retention time, peak areas in defined regions of elution for aggregates, oligomers, trimers, and monomers were recorded, respectively.

[0213] BLI - BioLayer Interference All IgG binding measurements were performed using an Octet Red instrument (Pall-ForteBio, Menlo Park, USA). All measurements were performed in 1x kinetics buffer (KB) (Pall-ForteBio, Menlo Park, USA). Mutant proteins were prepared by diluting the protein solution in 1x KB to a concentration of 263 nM and immobilized on a Ni-NTA sensor chip for 180 s. Unbound ligand was washed away by incubating the sensor chip in buffer solution for 60 s. Binding was monitored upon immersion in FI6v3 or CR9114 solutions (4000 nM to 62.5 nM) in KB for 300 s. Dissociation was monitored upon immersion in 1x KB buffer for 60 s.

[0214] Differential scanning fluorimetry Thermal unfolding of 0.5 mg / mL HA was monitored between 20 and 95°C by either endogenous Trp fluorescence (measured at 330 and 350 nm with an excitation of 290 nm on a Nano DSF NT-Plex instrument (Nanotemper Technologies, Munich, Germany)) or added Sypro Orange fluorescence (measured at 640 nm with an emission of 498 nm on a LightCycler 480 (Roche, Basel, Switzerland)). The temperature was increased at a rate of 1°C / min and 0.3°C / s, respectively. The change in fluorescence intensity (SYPRO) or the ratio of the intensities at 330 nm and 350 nm (Trp) was used to calculate the change with each temperature increase and qualitatively assess the position of the major transition.

[0215] Differential scanning calorimetry (DSC) Unfolding was monitored at 0.4 mg / mL HA using a MicroCal PAEQ-n automated DSC (Malvern Panalytical, France) between 20 and 95 °C at 5 °C intervals. Data were analyzed with integrated software to determine melting temperatures.

[0216] AUC Analytical sedimentation velocity ultracentrifugation (SV-AUC) was performed to determine the molecular weight and stoichiometry of the proteins by measuring the rate at which molecules migrate through a buffer in response to centrifugal force.

[0217] SV-AUC was performed using a Beckman-Coulter Optima AUC analytical ultracentrifuge with an AN-60Ti rotor, and protein was 0.5 mg / mL and frozen at -80°C prior to the experiment. The rotor speed selected for the run was 20,000 rpm, the temperature was maintained at 20°C, and the absorbance profile at 280 nm was recorded every minute.

[0218] Protein-specific and solvent densities were calculated using the software SEDNTERP1 (Sedimentation Interpretation Program Version 1.11). Data sets were analyzed by the Sedfit 15.01b program using a continuous size distribution c(s) model.

[0219] Circular dichroism spectroscopy Far-UV CD spectra were measured on a Chirascan spectrometer in 4 mM bicine, pH 8.3, and 30 mM NaCl at a concentration of 0.2 mg / mL for the HA candidate. Spectra were acquired between 190 and 260 nm using a 0.5 mm pathlength cell with a 1 nm bandwidth. The temperature was held at 25°C. Thermal melting to 95°C was performed by increasing the temperature by 1°C per minute, holding for 2 minutes, followed by acquisition of spectra at every 5°C increase.

[0220] [Example 3] Results of A / Brisbane / 02 / 2018(H1N1) ectodomain constructs

[0221] [Table 4]

[0222] A first batch of Bri18 HA-based constructs was designed, prepared, and evaluated according to the preceding examples. In total, 33 ectodomain constructs were examined (see Table 2). Readouts from the characterization studies described in Example 2 for six lead constructs (see Table 1) are presented in this example. Readouts were combined to generate the summary display of data in Table 4. AUC and DSF were performed after TEV cleavage. Unless otherwise noted, experiments were performed in the presence of a trimerization domain.

[0223] All constructs except Mut27 maintained their trimeric form, as confirmed by AUC. The aberrant elution by UPLC was attributed to nonspecific interactions with the BEH column matrix. Consistent with this, Mut27 exhibited reduced antibody recognition. Mut18 and Mut24 also exhibited poor antibody recognition. Mut10, Mut17, and Mut23 maintained antibody recognition and increased trimer stability. Three unfolding events were observed during HA unfolding with nanoDSF. The highest melting temperature (approximately 80°C) was attributed to the dissociation of the foldon compared with the stability of the foldon alone (not shown). Tm1 in Table 4 is considered to indicate unfolding of the HA headgroup, based on the loss of the HA headgroup upon acidification, while Tm2 is considered to indicate unfolding of the stem. Both Tm1 and Tm2 increased by at least 2°C for the three mutants, indicating overall stabilization of the HA trimer by the mutations. When foldons were truncated from control and Mut10, Mut17, and Mut23, the mutants showed an increased trimeric population as determined by AUC and increased thermal stability as determined by nanoDSF compared to the control (not shown).

[0224] [Example 4] A / Darwin / 9 / 2021 H3N2 ectodomain construct results A high-throughput screen was performed on the Darw21 construct. The constructs were characterized using the following readouts: Productivity: The amount of purified protein (expressed in mg / L) was measured colorimetrically. Previous analyses suggested that higher productivity is often accompanied by more stable folding. We used this parameter as a selection criterion for full-length ectodomain constructs. - UPLC-SEC: to distinguish between soluble forms of a protein (either monomers, trimers, higher oligomers or soluble aggregates), each form appearing as a (partially) separated peak on the elution profile. - BLI: a biosensor technique used to quantify the binding of structure-specific immunological tools to immobilized mutants. Here, CR9114 and FI3v6 monoclonal antibodies were used to probe the stem region of the protein compared to a control. Binding was recorded as the increase in the thickness (expressed in nm) of the sensing surface when the antibody ligand bound to the immobilized HA protein mutant. In previous experiments, we observed increased binding to the more stable HA trimer. - Differential scanning fluorimetry: a protein folding fingerprinting technique used to confirm the stability of the protein fold by measuring the unfolding temperature (Tm, expressed in °C) compared to that of a reference Darw21 sequence. This readout is used to confirm that the mutation pattern does not disrupt the protein folding. The readout was based on the fluorescence of the endogenous protein or by following the change in fluorescence of an exogenously added hydrophobic SYPRO Orange probe. - Differential scanning calorimetry: a protein folding fingerprinting technique used to confirm the stability of the protein fold by measuring the unfolding temperature (Tm, expressed in °C) compared to that of a reference Darw21 sequence. Heat capacity readouts are used to confirm that the mutation pattern does not disrupt protein folding. - Circular dichroism spectroscopy: a protein folding fingerprinting technique used to confirm the stability of a protein's structure and folding by measuring its secondary structure composition and its denaturation compared to that of a reference Darw21 sequence. This readout is used to confirm that mutation patterns do not alter protein folding.

[0225] One hundred and thirty-five constructs were expressed, purified, characterized for their oligomeric state (by UPLC), and antigenicity by recognition with the CR9114 and FI3v6 antibodies. Twenty-six constructs that produced HA trimers with robust antibody binding at levels comparable to or greater than the Darwin21 control, or with clearly increased affinity, were selected for detailed characterization.

[0226] After selection of 26 candidates, medium-scale expression and purification was performed both to confirm the productivity of each selected candidate and to provide more protein material for characterization purposes. At medium scale, the following characterization assays were performed: - UPLC for assessment of trimer conformation after foldon cleavage. This readout provides an analysis of the intrinsic oligomerization state of the HA trimer. NanoDSF (measuring changes in intrinsic fluorescence) and DSF (measuring changes in fluorescence of exogenously added SYPRO Orange) were used to assess the intrinsic stability of the HA trimer and to assess the stability of the trimer after foldon cleavage. DSC (measuring changes in heat capacity) and CD spectroscopy (measuring changes in secondary structure) were performed on the top constructs in addition to previous experiments to help assign unfolding to specific domains of the HA trimer. -BLI was repeated on the foldon-containing samples using CR9114 and FI6v3 as probes for the conformation of the stem region of the construct compared to the reference.

[0227] The results are shown in Table 5. Overall, nanoDSF and DSF measurements showed a major transition with equivalent melting temperatures. Five candidates increased their melting temperatures by 2–5°C compared to the control: Flu632, Flu639, Flu643, Flu691, and Flu689. All of these candidates, except Flu639 and Flu643, increased their affinity for FI6v3 by approximately 10-fold or more. Several other candidates increased FI6v3 affinity by 5-fold or less without increasing their melting temperatures.

[0228] The top five candidates were further analyzed for stability and antibody affinity, including Flu690, which had the highest affinity for FI6v3 (approximately 100-fold) and CR9114 (10-fold), but was not thermostabilized; and Flu687, which had a 5-fold increase in affinity for FI6v3, but neither increased the affinity nor the melting temperature for CR9114.

[0229] Control unfolding of H3 Darwin21 showed a single transition when followed by Trp fluorescence. In contrast, two transitions were observed when unfolding was followed by SyproOrange, which binds to exposed hydrophobic residues, and differential scanning calorimetry. Circular dichroism spectroscopy following thermal unfolding also showed two transitions due to the two-step unfolding of the helical portion. However, the β-sheet structure appeared to unfold in a single cooperative transition.

[0230] Flu632 and Flu691 displayed fluorescence and calorimetry profiles similar to the control, but the melting temperatures of each transition were shifted by 5°C higher, suggesting stabilized trimerized helices. Two transitions were observed in both the helix and sheet portions, indicating strong cooperativity in the unfolding of the entire structure, possibly suggesting a heterogeneous population. The same feature was observed in Flu689, but the mutation only increased the melting transition by 2°C, making it the only stabilizing construct to show a ~10-fold increase in CR9114 affinity.

[0231] Differential scanning fluorimetry with Trp or SyproOrange and circular dichroism spectroscopy following thermal unfolding indicated that Flu639 and Flu643 increased the stability of the helical and sheet segments in the structure, increasing the cooperativity of unfolding, with only one transition observed at a melting temperature 5°C higher than the control. These data are consistent with a homogeneously stabilized HA trimer, although this construct did not exhibit higher affinity for the antibody than the control.

[0232] [Table 5]

[0233] [Example 5] Transfection and purification of Mut10 hemagglutinin ectodomain and Fab FI6v3 Plasmids encoding the Mut10 hemagglutinin ectodomain (HA) or the FI6v3 Fab domain were engineered with a C-terminal hexahistidine tag and transiently expressed in 500 mL of Expi293F™ GnTI- cells (ThermoFisher Scientific). Culture supernatants containing Mut10 HA were harvested 5 days posttransfection and filtered through a 0.22 μm filter before being purified by nickel affinity chromatography using an AKTA Avant system. The supernatants were loaded onto a HisTrap column (Cytiva Life Sciences) pre-equilibrated with an equilibration buffer consisting of 25 mM HEPES, pH 7.5, and 150 mM NaCl. The captured protein was eluted with a step gradient of 25 mM HEPES, pH 7.5, 150 mM NaCl, and 500 mM imidazole. The step gradient was performed as follows: starting with 5 column volumes of 4% elution buffer, 5 column volumes of 4–25% elution buffer, 5 column volumes of 25% step elution buffer, 5 column volumes of a 25%–50% gradient, 5 column volumes of 50% elution buffer, 5 column volumes of a 50%–100% gradient, and finally 5 column volumes of a 100% step elution. Protein was eluted with 4% and 18% elution buffer, corresponding to 20 mM and 90 mM imidazole, respectively. The 4% and 18% imidazole fractions were collected, concentrated to 1 mL using a 10 kDa Amicon Ultra Centrifugal Concentrator filter unit (EMD Millipore), filtered through a 0.2 μm filter, and loaded at 1 mL / min onto a HiLoad 16 / 600 Superdex 200 pg column (Cytiva Life Sciences) pre-equilibrated with equilibration buffer. The chromatogram showed two peaks, the first eluting between 40 and 50 mL, and the second, larger peak eluting between 55 and 68 mL. The peak fractions were analyzed by SDS-PAGE to assess purity and identify the target protein. The target protein eluted between 55 and 69 mL in the second peak, and was determined to be >95% pure.Fractions were collected and stored in equilibration buffer at −80° C. until further use.

[0234] For FI6v3 Fab, culture supernatants were harvested 5 days posttransfection and filtered through a 0.22 μm filter before purification via nickel affinity chromatography on an AKTA Avant system. The culture supernatant was loaded onto a HisTrap column (Cytiva Life Sciences) equilibrated with an equilibration buffer consisting of 25 mM HEPES pH 7.5, 150 mM NaCl. The captured protein was eluted in a step gradient with an elution buffer consisting of 25 mM HEPES pH 7.5, 150 mM NaCl, and 500 mM imidazole. The step gradient was performed in the same manner as above, with minor modifications. The step gradient was performed as follows: starting with 5 column volumes of 4% step elution buffer, followed by 5 column volumes of 4–25% elution buffer, 5 column volumes of 25% step elution buffer, a 5 column volume gradient from 25% to 100%, and finally a 5 column volume 100% step elution. The protein was eluted with 4% and 6%-22% elution buffer, corresponding to 20 mM and 30-110 mM imidazole, respectively. The 4% and 6%-22% imidazole fractions were collected and concentrated to 1 mL using a 10 kDa Amicon Ultra Centrifugal Concentrator filter unit (EMD Millipore), then filtered through a 0.22 μm filter and loaded at 1 mL / min onto a HiLoad 16 / 600 Superdex 200 pg column (Cytiva Life Sciences) pre-equilibrated with equilibration buffer consisting of 25 mM HEPES pH 7.5, 150 mM NaCl. The chromatogram showed two peaks: the first eluted between 46 and 70 mL, and the second, larger peak eluted between 91 and 114 mL. The peak fractions were analyzed by SDS-PAGE to assess purity and identify the target protein. The target protein eluted between 91 and 114 mL in the second peak and was determined to be >95% pure. Fractions were collected and stored in equilibration buffer at -80°C until further use.

[0235] Crystallization of the Mut10:FI6v3 complex Both proteins were concentrated to 10 mg / mL using a 10 kDa Amicon Ultra Centrifugal Concentrator filter unit (EMD Millipore). Mut10 and FI6v3 Fab were mixed at a molar ratio of 1:1.2 and incubated overnight at 4 °C to ensure complex formation. High-throughput crystal screening was performed in 96-well, 2-well sitting drop plates (Art Robbins Instruments) using a Gryphon Robotics Instrument (Art Robbins Instruments) at a protein-to-buffer ratio of 1:1. The crystal screen was incubated at 20 °C using a Formulatrix Rock Imager 1000 (Formulatrix), and droplets were imaged on a Fibonacci schedule (0, 1, 2, 3, 5 days, etc.). Several crystal hits were obtained, and the conditional hits are listed in Table 6. Crystals were harvested, cryoprotected in 20% ethylene glycol, flash-frozen in liquid nitrogen, and shipped to the Advanced Photon Source at Argonne National Labs for data collection. The best diffracting crystals were found to be obtained from conditions containing 10% w / v 2-propanol, 0.1 M HEPES pH 7.5, and 20% w / v PEG 4000 (crystals appeared after 8 days and grew to full size by day 13). Diffraction data were processed with HKL2000 to obtain a resolution of 2.9 Å in space group I213.

[0236] Molecular replacement and refinement The structures of the influenza A virus H1 A / Michigan / 45 / 2015 ectodomain (PDB ID 7KNA) and the F16v3 antibody heavy and light chains (PDB ID 3ZTJ) were modified using PHENIX Sculptor and used as search models for molecular replacement in PHENIX Phaser for the Mut10 HA ectodomain and F16v3 Fab, respectively [1-3]. One molecule of the Mut10:F16v3 Fab complex occupies the asymmetric unit, and applying symmetry reproduces the predicted HA trimer with three copies of F16v3 Fab bound (Figure 1). The model was refined through iterative refinement using PHENIX Refinement and Coot [4-6]. Final validation was performed using MOLPROBITY, with the statistics shown in Table 6 [7].

[0237] Structural analysis and comparison of the Mut10:F16v3 complex The Mut10 structure contains a single K395M mutation buried between two α-helices at residues 382–402 and 419–470 of the HA2 polypeptide chain, resulting in the substitution of a positively charged lysine residue with an uncharged, nonpolar methionine residue. Despite this mutation, the structure demonstrates that F16v3 binding is conserved at the epitope. Superposition of a single HA protomer from Mut10 with a single HA protomer from H1 HA (PDB ID 3ZTN) yielded an RMSD of 0.52 across 399 Cα atoms, confirming nearly identical structural conservation between the two structures.

[0238] [Table 6]

[0239] [Table 7]

[0240] References for Example 5 1. Bunkoeczi, G. and R. J. Read. "Improvement of molecular-replacement models with sculptor." Acta Crystallogr D Biol Crystallogr 67 (2011): 303-12. 2. Adams, P. D., P. V. Afonine, G. Bunkoeczi, V. B. Chen, I. W. Davis, N. Echols, J. J. Headd, L. W. Hung, G. J. Kapral, R. W. Grosse-Kunstleve, et al. "Phenix: A comprehensive python-based system for macromolecular structure solution." Acta Crystallogr D Biol Crystallogr 66 (2010): 213-21. 3. Liebschner, D., P. V. Afonine, M. L. Baker, G. Bunkoeczi, V. B. Chen, T. I. Croll, B. Hintze, L. W. Hung, S. Jain, A. J. McCoy, et al. "Macromolecular structure determination using x-rays, neutrons and electrons: Recent developments in phenix." Acta Crystallogr D Struct Biol 75 (2019): 861-77. 4. Afonine, P. V., B. K. Poon, R. J. Read, O. V. Sobolev, T. C. Terwilliger, A. Urzhumtsev and P. D. Adams. "Real-space refinement in phenix for cryo-em and crystallography." Acta Crystallogr D Struct Biol 74 (2018): 531-44. 5. Emsley, P., B. Lohkamp, ​​WG Scott and K. Cowtan. "Features and development of coot." Acta Crystallogr D Biol Crystallogr 66 (2010): 486-501. 6. Emsley, P. and M. Crispin. "Structural analysis of glycoproteins: Building n-linked glycans with coot." Acta Crystallogr D Struct Biol 74 (2018): 256-63. 7. Williams, CJ, JJ Headd, NW Moriarty, MG Prisant, LL Videau, LN Deis, V. Verma, DA Keedy, BJ Hintze, VB Chen, et al. "Molprobity: More and better reference data for improved all-atom structure validation." Protein Sci 27 (2018): 293-315.

[0241] [Example 6] Mouse immunogenicity studies Research A The immunogenicity of influenza HA full-length ectodomain trimer protein containing the foldon and based on the A / Brisbane / 02 / 2018 H1 sequence was evaluated in naive CB6F1 mice. Female CB6F1 mice were immunized intramuscularly twice, 28 days apart, with: (a) Mut10, 0.2 μg / dose, adjuvanted with AS03A, −1 / 10 human dose (HD); a total of 20 animals were used in this group (split into two independent studies). (b) Mut23, 0.2 μg / dose, adjuvanted with AS03A, −1 / 10 human dose (HD); a total of 20 animals were used in this group (split into two independent studies). (c) QIV 2019 / 2020 (a commercially available quadrivalent influenza vaccine from GlaxoSmithKline (GSK) containing inactivated split influenza (Flu) virions of virus strains A / Brisbane / 02 / 2018 H1N1, A / Kansas / 14 / 2017 H3N2, B / Colorado / 06 / 2017 (B / Victoria), and B / Phuket / 3073 / 2013 (B / Yamagata)), 2.66 μg / strain / dose, unadjuvanted; a total of 8 animals were used in this group (split into two independent studies). (d) QIV 2019 / 2020, 0.27 μg / strain / dose, adjuvanted with AS03A, −1 / 10 human dose (HD); a total of 8 animals were used in this group (split into 2 independent studies). (e) NaCl as a placebo control group; a total of 4 animals were used in this group (divided into 2 independent studies).

[0242] Due to volume limitations, some assays were performed on pooled serum samples instead of individual serum samples, as highlighted in each graph.

[0243] Spleen and serum samples were collected on day 42 (corresponding to 14 days after the second immunization) and analyzed as described in Examples 8-13 using the assay protocol described in Example 7.

[0244] Research B Another study was performed to evaluate the immunogenicity of influenza HA full-length ectodomain constructs in primed CB6F1 mice. Female CB6F1 mice were primed intranasally with inactivated influenza virus A / California / 7 / 2009 H1N1 on day 0 and immunized intramuscularly on days 28 and 56 with: (a) Mut10, 0.2 μg / dose, adjuvanted with AS03A, −1 / 10 human dose (HD); a total of 20 animals were used in this group. (b) Mut23, 0.2 μg / dose, adjuvanted with AS03A, −1 / 10 human dose (HD); a total of 20 animals were used in this group. (c) QIV 2019 / 2020 (a commercial quadrivalent influenza vaccine from GSK containing inactivated split influenza virions of virus strains A / Brisbane / 02 / 2018 H1N1, A / Kansas / 14 / 2017 H3N2, B / Colorado / 06 / 2017 (B / Victoria), and B / Phuket / 3073 / 2013 (B / Yamagata)), 2.66 μg / strain / dose, unadjuvanted; a total of 8 animals were used in this group. (d) QIV 2019 / 2020, 0.27 μg / strain / dose, adjuvanted with AS03A, −1 / 10 human dose (HD); a total of 8 animals were used in this group. (e) A control group primed with PBS but not immunized; a total of 4 animals were used in this group. (f) NaCl (unprimed) placebo control group; a total of 4 animals were used in this group.

[0245] Spleen and serum samples were collected on day 70 (corresponding to 14 days after the second immunization) and analyzed as described in Examples 8-13 using the assay protocol described in Example 7.

[0246] [Example 7] Assay Protocol IgGserologyELISA Mouse IgG antibodies were quantified by ELISA using whole or split Flu virus or recombinant HA (stem-only protein) as coating antigen, diluted (50 μL / well) to reach concentrations of 0.5, 1, or 4 μg / mL depending on the antigen tested and adsorbed overnight at 4°C to 96-well microtiter plates (Maxisorb Immunoplate, Nunc, 439454). Plates were then incubated with 100 μL / well of PBS + 10% milk (saturation buffer) for 1 hour at 37°C. Twelve two-fold dilutions of serum (diluted in PBS + 1% BSA + 0.1% Tween 20, further referred to as dilution buffer) were added (50 μL / well) to the coated plates and incubated for 90 minutes at 37°C. Plates were then washed four times with PBS + 0.1% Tween 20. Peroxidase-conjugated goat anti-mouse IgG (Jackson, 115-035-003) diluted 1 / 250 in dilution buffer was added to each well (50 μL / well) and incubated for 1 h at 37°C. After an additional washing step, the plate was incubated with OPDA substrate (Sigma, P4664) for 20 min at RT. The reaction was stopped with H2SO42N, and the optical density was read at 490-620 nm. Titers were expressed as the ELISA 50% endpoint titer, corresponding to the dilution of the sample corresponding to an optical density of 1.5 (50% of the high plateau). In the absence of detectable binding activity, the corresponding sample was assigned an arbitrary titer corresponding to half of the first serum dilution (1:100), i.e., 50.

[0247] Hemagglutination Inhibition Assay (HI) The principle of the HAI assay is based on the ability of specific anti-influenza antibodies to inhibit red blood cell (RBC) agglutination by influenza virus hemagglutinin (HA). Serum was first treated with receptor-destroying enzyme (Sigma, catalog no. C-8772) at a concentration of 2% to remove nonspecific inhibitors (incubation at 37°C for 18 hours), heat-inactivated at 56°C for 30 minutes, and then treated with chicken RBCs at a concentration of 5% (incubation at +4°C for 1 hour). After pretreatment, two-fold dilutions of the decanted serum were incubated with 4 hemagglutinating units of whole influenza virus for 30 minutes at room temperature. Chicken RBCs were then added at a concentration of 0.5%, and the inhibition of hemagglutination was scored. Titers were expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination. Because the initial serum dilution was 1:20, a titer of 10 was used for samples below the detection limit.

[0248] FACS-based in vitro influenza neutralization assay MDCK cells were seeded at 30,000 cells / well in 96-well cell culture plates (Nunc, catalog no. 167008) the day before the test. To remove nonspecific inhibitors, serum was pretreated with 2% receptor-destroying enzyme (Sigma, catalog no. C-8772) (incubated at 37°C for 18 hours) and heat-inactivated at 56°C for 30 minutes. Neutralization assays were performed using the following infectious medium: Ultra-MDCK medium (BioWhittaker, catalog no. BE12-749Q) supplemented with 1% penicillin-streptomycin (Invitrogen, catalog no. 15140-122) and 2 μg / mL TPCK-treated trypsin (Sigma, catalog no. T1426). Four three-fold serial dilutions of serum were prepared in a single batch in a 96-well plate. The serum dilutions were mixed with an equal volume of influenza virus diluted in infectious medium to reach an MOI of 0.2 for H1N1 strains, corresponding to 6000 TCID50 / well, and an MOI of 0.033 for H5N1 strains, corresponding to approximately 1000 TCID50 / well. The plates were incubated at 35°C for 2 hours. Six wells served as virus-only controls, and two wells served as cell-only controls. After incubation, the medium was removed from the plates containing the cells, and the serum-virus mixture was transferred to these plates. After a centrifugation step (2000 rpm for 1 hour), the plate contents were removed and replaced with 200 μL of fresh medium. The plates were incubated at 35°C and 5% CO2 for 16 hours. After incubation, the presence of virus was detected using the following fluorescent staining method. Cells were washed with PBS and detached by trypsinization. The trypsin activity was blocked by adding PBS + 1% FBS. The cells were harvested into V-bottom plates and stained. After a washing step with PBS+1% FBS, cells were fixed with Cytofix / cytoperm reagent (BD, Cat. No. 51-2090KZ) for 20 min at 4°C.After a washing step with Permwash buffer (BD, catalog no. 51-2091KZ), infected cells were stained with FITC-conjugated anti-Flu A nucleoprotein monoclonal antibody (Thermofisher, catalog no. MA1-7322) for 30 minutes at 4°C. After a washing step with Permwash buffer, cells were resuspended in PBS, and plates were analyzed by flow cytometry using a BD Fortessa flow cytometer and FlowJo software. Percentage of neutralization was determined for each well based on the virus-only control, considered 0% neutralization. The 50% neutralization titer was calculated for each sample using linear regression. Because the lowest serum dilution was 1:50, a titer of 17 was used for samples below the limit of detection.

[0249] Antibody-dependent cellular cytotoxicity (ADCC) reporter bioassay (Promega) To determine ADCC functionality, a mouse FcgRIII kit from Promega was used with the following protocol. Serial dilutions of serum were prepared in 96-well plates. Target cells (Expi293 cells transfected in-house to express the hemagglutinin stem antigen from the A / Michigan / 45 / 2015 H1N1 strain) were added to each well (24,000 cells / well). Effector cells (Jurkat cells from the kit, transfected with an enzymatic pathway that induces bioluminescence when activated by antigen-antibody-FcgRIII complexes) were also added to each well (60,000 cells / well) and incubated at 37°C for 6 hours. Subsequently, luciferase activity was measured using a luminescence plate reader after adding Bio-Glow substrate (provided in the kit). Results were expressed as the area under the curve (AUC).

[0250] Intracellular cytokine staining (ICS) Flu-specific T cell responses were assessed by ICS using splenocytes harvested 14 days after the second immunization. Splenocytes were restimulated in vitro (6 h) with a pool of 15mers covering the Flu H1-stem sequence (based on the A / Michigan / 45 / 2015 H1N1 sequence). Splenocyte T lymphocyte isolation: Spleens were harvested and placed in Roswell Park Memorial Institute 1640 medium supplemented with glutamine, penicillin / streptomycin, sodium pyruvate, non-essential amino acids, and 2-mercaptoethanol. A cell suspension was prepared from each spleen using a tissue grinder. The spleen cell suspension was filtered twice (through a 100 μm cell strainer). The filter was rinsed with 35 mL (for the first wash) or 12 mL (for the second wash) of PBS EDTA 2 mM. After centrifugation (335 g, 10 min, RT), cells were resuspended in complete medium (Roswell Park Memorial Institute 1640 medium supplemented with glutamine, penicillin / streptomycin, sodium pyruvate, non-essential amino acids, 2-mercaptoethanol, and 5% heat-inactivated fetal bovine serum; hereafter referred to as complete medium). In vitro stimulation: Fresh splenocytes were plated at approximately 1 million cells per well in a round-bottom 96-well plate. Subsequently, cells were stimulated for 6 h at 37°C, 5% CO2 with anti-CD28 (clone 37.51) and anti-CD49d (clone 9C10(MFR4.B)) at 1 μg / mL, with or without 1 μg / mL of a 15-mer overlapping peptide pool covering the Flu H1-stem sequence (based on the A / Michigan / 45 / 2015 H1N1 sequence). After 2 hours of stimulation at 37°C, Brefeldin A diluted 1 / 1000 in complete medium was added for an additional 4 hours at 37°C. Plates were then transferred to 4°C overnight. Staining of splenocytes: Cells were stained and analyzed using a 6-color ICS assay. Cells were transferred to a V-bottom 96-well plate and centrifuged at 189g for 5 minutes at 4°C.After a washing step with 250 μL of PBS and 1% fetal bovine serum, the cells were resuspended in 50 μL of Flow Buffer (PBS 1x, 1% fetal bovine serum) containing anti-CD16 / 32 (clone 2.4G2) diluted 1 / 50 for 10 min at 4°C. Subsequently, 50 μL of Flow Buffer containing anti-CD4-V450 (clone RM4-5) and anti-CD8-PerCp-Cy5.5 (clone 53-6.7) antibodies diluted 1 / 200 (diluted 1 / 100) and Live / dead-PO (1 / 1000) was added for 30 min at 4°C. The cells were centrifuged (189 g, 5 min, 4°C) and washed with 200 μL of Flow Buffer. The spleen cells were fixed and permeabilized with 200 μL of Cytofix / Cytoperm solution for 20 min at 4°C. The cells were centrifuged (500g, 5 min, 4°C) and washed with 200 μL of Perm / Wash buffer. After another centrifugation step (500g, 5 min, 4°C), the cells were stained with anti-IL2-FITC (clone JES6-5H4, 1 / 400 dilution), anti-IFNγ-APC (clone XMG1.2, 1 / 200 dilution), and anti-TNFα-PE (clone MP6-XT22, 1 / 700 dilution) antibodies in 50 μL of Perm / Wash buffer for 1 h at 4°C. This cytokine panel was selected based on the Th1 profile and pro-inflammatory cytokines known to be induced by the AS01 adjuvant system. The cells were washed twice with Perm / Wash buffer and resuspended in 220 μL of PBS. The stained cells were analyzed by flow cytometry using a BD Fortessa flow cytometer and FlowJo software. All antibodies and buffers used in ICS were from BD Biosciences.

[0251] [Example 8] HA mut 10 and HA mut 23 induce functional HI antibody responses against homologous and post-pandemic heterologous H1N1 strains 14 days after dose 2. The HI responses induced by HA mut 10 and HA mut 23 against homologous (A / Brisbane / 2 / 2018) and post-pandemic heterologous (A / Michigan / 45 / 2015 and anti-A / California / 7 / 2009) H1N1 strains in naive (Study A) and primed (Study B) mouse models, measured 14 days after the second immunization, are shown in Figure 7. Individual titer values ​​are shown along with the geometric mean titer (GMT) and 95% confidence interval (95CI).

[0252] conclusion In addition to inducing a homologous HI response (against A / Brisbane / 2 / 2018), administration of HA mut 10 and HA mut 23 induced cross-reactive anti-HA responses against heterologous post-pandemic H1N1 strains (A / Michigan / 45 / 2015 and A / California / 7 / 2009) in both naive (Study A) and primed (Study B) animals. The variability of HI responses induced by HA mut 10 and HA mut 23 was significantly reduced when assessed in the primed model (Study B). Measured HI titers were similar (naive and primed animals) or lower (naive animals) for HA mut 10 and HA mut 23 compared with QIV (±AS03).

[0253] [Example 9] HA mut10 and HA mut23 induce anti-HA stem-binding and functional antibody responses, as well as neutralizing antibody responses, against post-pandemic heterologous H1N1 strains 14 days after dose 2 To characterize the induced antibody responses, anti-H1 stem-binding antibodies were measured by ELISA 14 days after the second immunization in naive animals. Results from Study A are shown in Figure 8. Pooled serum titers are shown along with the geometric mean titer (GMT) and 95% confidence interval (95CI).

[0254] ADCC activity against the A / Michigan / 45 / 2015 stem was measured 14 days after the second immunization by ADCC reporter bioassay (Promega). Results from Study A are shown in Figure 8. Pooled serum AUC (area under the curve) values ​​are shown along with the median.

[0255] Neutralizing antibodies against the A / Singapore / GP1908 / 2015 H1N1 and A / California / 7 / 2009 H1N1 strains were measured 14 days after the second immunization in Studies A and B. Results from this assay are shown in Figure 8. Individual titer values ​​are shown along with the geometric mean titer (GMT) and 95% confidence interval (95CI).

[0256] conclusion Because HI antibody responses are directed exclusively against the head of the HA molecule, we performed anti-stem ELISA to examine whether HA mut 10 and HA mut 23 could also induce stem-specific antibodies. The stem-only HA used was expressed on nanoparticles to ensure antigen stability while avoiding the use of foldons, the presence of which could lead to the detection of non-influenza-specific but foldon-specific Ab responses.

[0257] The levels of stem-specific antibody titers detected in naive mice immunized with HAmut10 and HAmut23 were significantly higher than those induced by animals immunized with QIV. Furthermore, in contrast to antibodies induced by QIV, anti-stem antibodies induced by HAmut23 showed functional ADCC titers.

[0258] Neutralizing antibodies were also induced by HA mut 10 and 23 at similar or slightly higher levels in naive and primed animals compared with animals immunized with adjuvanted or unadjuvanted QIV, respectively.

[0259] Taken together, these data highlight the ability of HA mut 10 and / or 23 to induce both stem- and head-specific antibody responses against heterologous H1 strains with functional competence comparable to or greater than that of QIV.

[0260] [Example 10] HA mut 10 and HA mut 23 induce low H1-stem specific CD4 T cell responses at 14 days after dose 2, but not CD8 T cell responses. Anti-H1 stem-specific CD4 and CD8 T cells were measured 14 days after the second immunization in naive (Study A) and primed (Study B) mouse models. Results from Study A and Study B are shown in Figure 9. The frequencies of H1 stem-specific CD4 or CD8 T cells expressing IFNγ and / or IL2 and / or TNFα are shown along with the median values.

[0261] conclusion H1 stem (A / Michigan / 45 / 2015)-specific CD4 T cells expressing IFNγ and / or IL2 and / or TNFα were detected in low percentages in both naive (Study A) and primed (Study B) mouse models, whereas induction of CD8 T cells was not detected in either model.

[0262] [Example 11] HA mut 10 and HA mut 23 induce broad heterologous and heterosubtypic HA-binding antibody responses against pre-pandemic heterologous H1N1 strains and heterosubtypic (H2N2, H5N1, and H9N2) strains 14 days after dose 2. Anti-HA IgG binding antibodies induced by HA mut 10 and HA mut 23 in a naive (Study A) mouse model against pre-pandemic heterologous H1N1 and heterosubtypic (H2N2, H5N1, and H9N2) strains were measured by ELISA 14 days after the second immunization and are shown in Figure 10. Pooled (Figures 10A, C, D) and individual (Figure 10B) titers are shown, along with the geometric mean titers (GMTs) and 95% confidence intervals (95CI).

[0263] conclusion High levels of cross-reactive IgG antibody titers directed against a prepandemic H1N1 strain (A / New Caledonia / 20 / 99) and a heterosubtypic H9N2 strain (A / Hong Kong / 1073 / 99) were detected in naive mice immunized with HA mut 10 and HA mut 23 (Figure 10). Lower levels of cross-reactive antibodies were also detected against heterosubtypic H5N1 strains (A / Vietnam / 1194 / 2004 H5N1) and H2N2 strains (A / Singapore / 1 / 57 H2N2). Because whole virus was used as the coating antigen, the antibody responses measured in QIV-immunized animals were not specific to the HA antigen but also included responses to all other split flu components. Therefore, comparison with QIV-immunized mice is not relevant in this assay.

[0264] [Example 12] HA mut 10 and HA mut 23 cross-reactive functional antibody responses to heterosubtypic (H5N1) strains 14 days after dose 2 The HI responses induced by HA mut 10 and HA mut 23 against pre-pandemic heterologous H1N1 and heterosubtypic (H2N2, H5N1, and H9N2) strains in naive (Study A) and primed (Study B) mouse models, measured 14 days after the second immunization, are shown in Figure 11. Pooled serum titers are shown along with the geometric mean titers (GMTs) and 95% confidence intervals (95CI).

[0265] Neutralizing antibodies against A / Vietnam / 1194 / 2004 H5N1 were measured 14 days after the second immunization in Study A and Study B, and the results of this assay are shown in Figure 12. Values ​​were geometric mean titers (GMT) and 95% confidence intervals (95CI).

[0266] conclusion In contrast to the cross-reactive IgG responses observed, HI responses were not induced by HA mut 10 and / 23 against pre-pandemic heterologous H1N1 strains (A / New Caledonia / 20 / 99) and heterosubtypic H9N2 strains (A / Hong Kong / 1073 / 99), heterosubtypic H5N1 strains (A / Vietnam / 1194 / 2004 H5N1), and H2N2 strains (A / Singapore / 1 / 57 H2N2) 14 days after dose 2.

[0267] However, although higher in the primed model compared with the naive model, neutralizing antibodies were also induced by HA mut 10 and 23 at similar or slightly higher levels in both naive and primed animals compared with animals immunized with adjuvanted or unadjuvanted QIV, respectively.

[0268] Because the HI response is directed exclusively against the head of the HA molecule, these data suggest the induction of a stem-based cross-functional antibody response against heterosubtypic strains from group A1.

[0269] [Example 13] HA mut 10 and HA mut 23 induce limited cross-reactive antibody responses against group A2 (H3N2, H10-stem) or lineage B (B / Yam and B / Vic) strains 14 days after dose 2. Anti-HA IgG binding antibodies induced by HA mut 10 and HA mut 23 in a naive (Study A) mouse model against group A2 (H3N2, H10-stem) or lineage B (B / Yam and B / Vic) strains, measured by ELISA 14 days after the second immunization, are shown in Figure 13. Individual titers are shown along with the geometric mean titer (GMT) and 95% confidence interval (95CI).

[0270] conclusion Although no cross-reactive IgG antibody titers were detected against H3N2 strains (A / Hong Kong / 2671 / 2019 H3N2), low levels of cross-reactive anti-stem IgG were detected against H10 (A / Jianxi-Donghu / 346 / 2013 stem). This highlights the ability of HA mut 10 and / or 23 to induce cross-reactive antibodies against the stem portion of the HA in the A2 group to levels comparable to or greater than those of the QIV group. No cross-reactive antibodies were detected against the B strains tested. Because split flu virus was used as the coating antigen (except for the evaluation of the H10 stem), the antibody responses measured in QIV-immunized animals were not specific to the HA antigen but also included responses to all other split flu components. Therefore, comparison with QIV-immunized mice is not relevant in this assay.

Claims

1. 1. An immunogenic composition comprising a recombinant influenza A strain hemagglutinin (HA) antigen in trimeric form together with a pharmaceutically acceptable carrier, wherein the antigen comprises the ectodomain of HA without the transmembrane and cytoplasmic domains, wherein the ectodomain comprises: (i) a globular head domain; and (ii) a stem domain having a coiled-coil region, the stem domain containing one or more mutations in the coiled-coil region that individually or together stabilize the HA ectodomain in a trimeric pre-fusion form; Including, An immunogenic composition, wherein the recombinant HA optionally comprises a heterologous trimerization domain.

2. The immunogenic composition of claim 1, wherein the recombinant HA has one or more amino acid substitutions in the coiled-coil region compared to the wild type.

3. The coiled-coil region of the stem domain containing one or more amino acid substitutions is 3. The immunogenic composition of claim 1 or 2, wherein the amino acid sequence is from amino acid positions (a) 317 to 472, for example 322 to 467, for H1; or (b) 342 to 473, for example 347 to 468, for H2; or an equivalent range of the coiled-coil region in other HA A strains or subtypes.

4. Recombinant HA, (a) HA group A1 subtype, e.g., H1, positions 322, 395, 431, 432, 436, 438, 439, 447, 449, 450, 453, 460, 464, and 467; or (b) Group A2 subtypes, e.g., H3 at positions 347, 396, 399, 418, 428, 437, 440, 448, 451, 454, 465, and 468 The immunogenic composition of any one of claims 1 to 3, having one or more amino acid substitutions at one or more positions selected from:

5. 5. The immunogenic composition of any one of claims 1 to 4, wherein there are one or more amino acid substitutions at one or more positions selected from positions 395, 436 and 447 of an H1 subtype, or positions 396, 437 and 448 of an H3 subtype, or equivalent positions of other influenza A subtypes.

6. 6. The immunogenic composition of any one of claims 1 to 5, wherein the recombinant HA comprises R at position 422 of H1 or R at position 423 of H3.

7. 7. The immunogenic composition of claim 1, wherein the recombinant HA has at least one stabilizing amino acid substitution selected from K322R, K395M, G431C, F432C, W436D, Y438D, N439L, E447L, E449Q, R450W, D453L, K460I, E464F and R467M for the H1 subtype, or at least one stabilizing amino acid substitution selected from I347F, K396L / I / V / M, R399L / M / F / I / L, V418P, W437D, N440I, E448M / I / L / M / V, H451L, D454A, E465M and K468M for the H3 subtype.

8. 8. The immunogenic composition of claim 1, wherein the one or more amino acid substitutions comprise or consist of amino acid substitutions at positions 395 / 396, such as K395 / 396L / I / V / M.

9. The immunogenic composition of claim 8, wherein the recombinant HA further comprises an amino acid substitution at positions 436 / 437, such as W436 / 437D, and / or an amino acid substitution at positions 447 / 448, such as E447 / 448M / I / L / M / V.

10. 10. The immunogenic composition of any one of claims 1 to 9, wherein the recombinant HA has an amino acid sequence with at least 85%, or at least 87%, or at least 90% identity to an amino acid sequence selected from SEQ ID NOs: 3 to 8 and 17 to 42, with or without a signal sequence.

11. 11. The immunogenic composition of any one of claims 1 to 10, wherein the HA comprises a foldon.

12. 12. The immunogenic composition of any one of claims 1 to 11, wherein the HA ectodomain does not include the transmembrane domain and the cytoplasmic domain, but includes all of the HA1 region and all of the HA2 region.

13. The stem domain is (1) covalently linked to a heterotrimerization domain; or (2) covalently linked to a carrier protein or nanoparticle; or (3) not covalently linked to other amino acid molecules; 13. The immunogenic composition of any one of claims 1 to 12.

14. An immunogenic composition comprising an isolated polynucleotide, such as DNA or mRNA, encoding a recombinant HA antigen of the immunogenic composition of any one of claims 1 to 13, and a pharmaceutically acceptable carrier.

15. The immunogenic composition of claim 14, wherein the polynucleotide is in a nucleic acid delivery platform.

16. 16. The immunogenic composition of claim 14 or 15, wherein the HA antigen comprises a transmembrane domain, such as the HA transmembrane domain, with or without the HA cytoplasmic domain.

17. 14. A method for preparing the immunogenic composition of any one of claims 1 to 13, comprising: (i) expressing a recombinant HA antigen in a eukaryotic cell from a polynucleotide sequence encoding the HA antigen fused to a heterologous trimerization domain, e.g., a foldon; (ii) purifying recombinant HA trimers from cell supernatants; (iii) removing the trimerization domain; (iv) combining the recombinant HA trimer with a pharmaceutically acceptable carrier. A method comprising:

18. 1. A method for preparing an immunogenic composition comprising an HA ectodomain containing one or more mutations in the coiled-coil region that individually or together stabilize the HA ectodomain in a trimeric pre-fusion form, comprising: (i) expressing a recombinant HA antigen, with or without a trimerization domain, from a polynucleotide sequence encoding it; (ii) purifying trimeric recombinant HA from cell supernatants; (iii) optionally, removing the trimerization domain, if present; (iv) combining the recombinant HA trimer with a pharmaceutically acceptable carrier. A method comprising:

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