Influenza virus vaccine and its use

Abstract

Provided herein are Group 2 influenza hemagglutinin stem polypeptides, nucleic acids encoding the polypeptides, vectors containing the nucleic acids, and pharmaceutical compositions comprising the same, as well as methods of their use, particularly in the prevention and / or treatment of influenza virus infection.

Description

[Technical Field]

[0001] The present invention relates to the field of pharmaceuticals. Influenza A hemagglutinin (HA) stem domain polypeptide, nucleic acids encoding the polypeptide, pharmaceutical compositions comprising the polypeptide, and methods of using the same are provided herein.

[0002] This invention was made, at least in part, with the support of the U.S. Government under contract HHSO100201700018C, awarded by the HHS. The U.S. Government has certain rights in this invention. [Background technology]

[0003] Influenza viruses are the primary human pathogens causing a respiratory illness (commonly referred to as "influenza" or "the flu") ranging in severity from asymptomatic infection to primary viral pneumonia that can be fatal. The clinical effects of infection vary depending on the pathogenicity of the influenza strain as well as the host's exposure, medical history, age, and immune status. It is estimated that approximately 1 billion people worldwide are infected with influenza viruses each year, resulting in 3 to 5 million serious cases and an estimated 300,000 to 500,000 influenza-related deaths. The majority of these infections may be caused by influenza A viruses carrying H1 or H3 hemagglutinin subtypes, with a small contribution from influenza B viruses; therefore, representatives of these are usually included in seasonal vaccines. Current immunization efforts rely on the early identification of circulating influenza viruses to enable the timely production of effective seasonal influenza vaccines. In addition to the inherent difficulty in predicting which strains will dominate the following season, antiviral drug resistance and immunity evasion also contribute to the current vaccines' failure to prevent illness and death. Furthermore, the possibility of a pandemic caused by highly pathogenic virus strains that originate from pathogen-carrying animals and reassemble to increase human-to-human transmission poses a significant and real threat to global health.

[0004] The influenza virus is an enveloped RNA virus belonging to the family Orthomyxoviridae. Its genome consists of eight single-stranded RNA segments encoding 11 different proteins, one nucleoprotein (NP), three polymerase proteins (PA, PB1, and PB2), two matrix proteins (M1 and M2), three non-structural proteins (NS1, NS2, and PB1-F2), and two extrinsic glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA).

[0005] Influenza A viruses are widely distributed in nature and can infect various birds and mammals. These viruses are classified based on differences in the antigenic structure of their HA and NA proteins; different combinations of these proteins represent specific viral subtypes, which are further classified into specific influenza virus strains. While all known subtypes can be found in birds, the currently circulating human influenza A subtypes are H1N1 and H3N2. Phylogenetic analysis of influenza A viruses has shown that hemagglutinin is subdivided into two major, so-called lineage groups: the H1, H2, H5, and H9 subtypes of lineage group 1 (group 1 viruses), and the H3, H4, and H7 subtypes of lineage group 2 (group 2 viruses).

[0006] Influenza B virus strains are, strictly speaking, human. Antigenic variations in HA within influenza B virus strains are smaller than those observed in influenza A strains. In humans, two genetically and antigenically distinct lineages of influenza B virus circulate: the B / Yamagata / 16 / 88 (also known as B / Yamagata) lineage and the B / Victoria / 2 / 87 (B / Victoria) lineage. The range of illness caused by influenza B virus is generally milder than that caused by influenza A virus, but severe illness requiring hospitalization is still frequently seen with influenza B infection.

[0007] Antibodies that neutralize influenza viruses are known to primarily target hemagglutinin (HA). Hemagglutinin, or HA, is a trimer glycoprotein immobilized on the viral membrane and has the following dual functions: it is involved in binding to the cell surface receptor sialic acid, and after internalization, it mediates the fusion of the virus with the endosomal membrane, resulting in the release of viral RNA into the cytoplasm of the target cell. HA contains a large head domain and a smaller stem domain. The stem domain is immobilized on the viral membrane by a C-terminal transmembrane domain sequence. This protein is cleaved post-translation to produce two HA polypeptides, HA1 and HA2 (the complete sequence is called HA0) (Figures 1A, 1B). The distal head domain is mainly derived from HA1, and the proximal stem domain is mainly derived from HA2. Cleavage of the HA precursor molecule HA0 is required for activation of viral infectivity, and the distribution of activated proteases in the host is one of the determinants of influenza virus pathogenicity. The HA of mammalian and non-pathogenic avian viruses is cleaved extracellularly, limiting their spread within the host to tissues where they encounter appropriate proteases. On the other hand, the HA of pathogenic viruses is cleaved intracellularly by universally present proteases, giving them the ability to infect various cell types and cause systemic infection.

[0008] The reason seasonal influenza vaccines must be updated annually is the high variability of the virus. In the HA protein, this mutation is particularly evident in the head domain, where antigenic drift and shift have resulted in numerous different variants. This is also the region of immunodominance, so most neutralizing antibodies are directed against this domain and act by interfering with receptor binding. This combination of immunodominance and high variability in the head domain explains why infection with a particular strain does not lead to immunity against other strains. In other words, antibodies induced by the initial infection recognize only a limited number of strains closely associated with the virus of the primary infection.

[0009] Recently, influenza hemagglutinin stem polypeptides lacking the complete influenza hemagglutinin globular head domain or a substantial portion thereof have been reported and have been used to generate immune responses to one or more conserved epitopes of stem domain polypeptides. It is believed that stem polypeptide epitopes are less immunogenic than the highly immunogenic regions of the globular head domain, and therefore the absence of the globular head domain in the stem polypeptide enables the expression of immune responses to one or more epitopes of the stem polypeptide (Steel et al., 2010). Accordingly, Steel et al. created influenza HA stem polypeptides by deleting amino acid residues 53-276 from the HA1 domain of A / Puerto Rico / 8 / 1934 (H1N1) and A / Hong Kong / 1968 (H3N2) strains and substituting the deleted sequences with a short, flexible ligature sequence GGGG. Vaccination of mice with the H3 HK68 construct did not induce antisera that cross-reactive with group 1 HA. Furthermore, as explained in International Publication No. 2013 / 079473, this stem polypeptide is unstable and does not adopt the correct three-dimensional structure when bound to a conserved epitope in the full-length wild-type HA stem region, as evidenced by the previously shown lack of antibody binding.

[0010] Bommakanti et al. (2010) reported a HA2-based polypeptide that included amino acid residues 330 - 501 (HA2), a 7-amino acid linker (GSAGSAG), amino acid residues 16 - 55 of HA1, a 6-amino acid linker GSAGSA, and subsequent residues 290 - 321 of HA1, along with mutations V297T, I300E, Y302T, and C305T in HA1. The design was based on the sequence of H3 HA (A / HongKong / 1968). This polypeptide provided only cross-protection against another influenza virus strain within the H3 subtype (provided against A / Phil / 2 / 82 but not against the H1 subtype (A / PR / 8 / 34)). In a more recent paper by Bommakanti et al. (2012), a stem polypeptide (H1HA0HA6) based on HA from H1N1 A / Puerto Rico / 8 / 1934 was reported. In this polypeptide, the equivalents of amino acid residues 48 - 288 were deleted, and mutations I297T, V300T, I302N, C305S, F392D, F395T, and L402D were created. Both the H3-based polypeptide and the H1-based polypeptide were expressed in E. coli and thus lacked glycans that are part of the native HA protein.

[0011] Corbett et al. (2019) reported on H3 and H7 HA stem trimers of influenza A virus displayed on self-assembling ferritin nanoparticles that induced protective homotypic antibodies in mice. The HA antigen fused to ferritin is also immunogenic but can induce unwanted responses against the carrier nanoparticles, which can lead to a reduction in the HA-directed immune response and its longevity after repeated immunizations. Furthermore, the expression levels and purification challenges of the HA-ferritin fusion protein can prevent the production of large vaccine doses. Also, the accessibility of HA epitopes (such as the binding site for CR8020) close to the surface of such nanoparticles can reduce the immune response against these preferentially conserved HA surfaces.

[0012] Despite the fact that the technology for inactivated influenza virus vaccines grown in eggs has been developed for over 70 years, influenza still remains a major global health burden to this day. Certain antigenic drift of influenza virus hemagglutinin (HA) combined with the immunodominant strain-specific antibody response directed against the variable HA head domain results in conventional vaccine efficacy in the range of 10 - 60% and also leads to the need for seasonal updating of the virus strains included in approved vaccines. Furthermore, current vaccine approaches provide minimal protection against pandemic influenza virus strains.

[0013] The effectiveness of vaccines against H3N2 over the past 10 years has averaged only 33% (Belongia et al (2016)), and recent H3N2 strains have shown increased pathogenicity (Garten et al. (2017)). Moreover, since H7 viruses are one of the greatest pandemic threats from non-seasonal strains, there is a particularly urgent need for better group 2 vaccines.

[0014] Therefore, there is a need for a safe and effective "universal" vaccine that stimulates the production of strong broadly neutralizing antibody responses and provides protection against a wide range of current and future influenza virus strains (both seasonal and pandemic), particularly a vaccine that provides protection against one or more influenza A virus subtypes within phylogenetic group 2 for effective prevention of influenza. Summary of the Invention Means for Solving the Problems

[0015] The present invention provides novel monomeric and polymeric (particularly trimeric) polypeptides derived from group 2 influenza hemagglutinin (HA), which comprises an influenza HA stem domain and lacks a globular head region, and is hereafter referred to herein as influenza hemagglutinin (HA) stem polypeptide or mini-HA. When administered to a subject, particularly a human subject, this polypeptide induces a cellular and / or humoral immune response to at least group 2 influenza viruses. The polypeptides of the present invention present a conserved epitope of the membrane-proximal stem of the group 2 HA molecule to the immune system in the absence of a dominant epitope present in the membrane-distal head domain.

[0016] Therefore, in the HA stem polypeptide of the present invention, a portion of the primary sequence of the HA0 protein, namely the portion constituting the head domain, is deleted, and the remaining amino acid sequence is relinked, either directly or, in some embodiments, by introducing a short, flexible linking sequence ("linker") to restore the continuity of the polypeptide chain. The resulting amino acid sequence is further modified by introducing specific mutations that stabilize the native three-dimensional structure of the residual portion of the HA molecule.

[0017] In a first embodiment, the present invention relates to a monomeric influenza A HA stem polypeptide comprising the HA1 domain and HA2 domain of hemagglutinin (HA) of a group 2 influenza A virus, (i) Deletion of the head region of the HA1 domain, (ii) Modification of the trimerization region of the HA2 domain, (iii) at least two cysteine ​​residues capable of forming at least one monomeric cysteine ​​crosslink It contains an amino acid sequence that includes, The amino acid at position 355 in this amino acid sequence is W. The numbering of amino acid positions in the HA stem polypeptide amino acid sequence follows the H3 numbering system of Winter et al. (1981), which corresponds to the full-length HA numbering system of the reference strain H3N2 A / Aichi / 2 / 68 (SEQ ID NO: 1). Regarding HA stem polypeptides.

[0018] In certain embodiments, the present invention relates to a group 2 influenza A hemagglutinin (HA) stem polypeptide comprising an HA1 domain and an HA2 domain, (i) A deletion in the head region of the HA1 domain, which includes at least an amino acid sequence from the amino acid corresponding to the 50th position to the amino acid corresponding to the 302nd position; (ii) Modification of the trimerization region of the HA2 domain, preferably a modification of the trimerization region in a C helix, wherein the trimerization region includes an amino acid sequence from the amino acid corresponding to the 405th position to the amino acid corresponding to the 419th position; (iii) Cysteine ​​at the amino acid position corresponding to position 310 that can form an intrameric cysteine ​​crosslink when combined with cysteine ​​at position 422, or cysteine ​​at the amino acid position corresponding to position 311 that can form an intrameric cysteine ​​crosslink when combined with cysteine ​​at position 422, or cysteine ​​at the amino acid position corresponding to position 308 that can form an intrameric cysteine ​​crosslink when combined with cysteine ​​at position 418. It contains an amino acid sequence that includes, The amino acid at position 355 in this amino acid sequence is W. The numbering of amino acid positions in the HA stem polypeptide amino acid sequence is based on the H3 numbering system of Winter et al. (mentioned above), which corresponds to the full-length HA numbering system of the reference strain H3N2 A / Aichi / 2 / 68 (SEQ ID NO: 1). Regarding HA stem polypeptides.

[0019] Surprisingly, according to the present invention, the novel group 2 influenza HA stem polypeptide of the present invention can be recombinantly expressed at high levels, is trimer in cell culture supernatant in the absence of further artificial C-terminal trimerizing domains, and / or has a high melting temperature resulting in greater thermal stability. Furthermore, the group 2 HA stem polypeptide of the present invention mimics the stem of full-length group 2 HA by stably presenting epitopes of HA stem-binding antibodies that bind to group 2 HA, such as CR9114 (described in International Publication No. 2013 / 007770) and / or CR8020 (described in International Publication No. 2010 / 130636).

[0020] In a second aspect, the present invention relates to a multimer influenza A hemagglutinin (HA) stem polypeptide comprising at least two HA stem polypeptide monomers described herein.

[0021] In a further embodiment, the present invention provides a nucleic acid molecule encoding a group 2 influenza HA stem polypeptide.

[0022] In yet another aspect, the present invention provides a vector comprising a nucleic acid encoding an influenza HA stem polypeptide, particularly a recombinant adenovirus vector.

[0023] In a further embodiment, the present invention provides a method for inducing an immune response to group 2 influenza HA in a subject in need thereof, comprising administering to the subject an influenza HA stem polypeptide, nucleic acid molecule, and / or vector according to the present invention.

[0024] In another embodiment, the present invention provides a pharmaceutical composition comprising an influenza HA stem polypeptide, a nucleic acid molecule and / or a vector according to the present invention, and a pharmaceutically acceptable carrier.

[0025] In a further embodiment, the present invention provides a group 2 influenza HA stem polypeptide, a nucleic acid molecule encoding the influenza HA stem polypeptide, and / or a vector comprising the nucleic acid molecule, for use in inducing an immune response to influenza viruses, and in particular for use as a vaccine for the prevention of diseases or conditions caused by influenza virus A strains from lineage group 2. [Brief explanation of the drawing]

[0026] [Figure 1] A. Schematic diagram of the polypeptide of the present invention (see figure below); B. Removal of the head region of HA results in the stem polypeptide (mini-HA) of the present invention; C. Three-dimensional representation of the stem-based polypeptide monomer (mini-HA) of the present invention; D. Schematic diagram of the polypeptide of the present invention (in particular, polypeptide UFV180088). [Figure 2] A. Protein expression levels of EXPI-CHO culture supernatant measured by OCTET (anti-C tag); B. SEC analysis of culture supernatant of EXPI-CHO cells expressing construct 180088 (left panel) and SEC-MALS analysis of purified 180088; C. Binding of mAb CR9114 to purified polypeptide by ELISA (EC50 value); D. Temperature stability analysis of purified polypeptide by differential scanning fluorescence measurement. [Figure 3-1] Figure 3: SEC profiles and elution analysis of culture supernatants of EXPI-293 cells expressing several stem polypeptides of the present invention. A. SEC profile of polypeptide with a reversion mutation to a wild-type (WT) residue; dotted line is the stabilized headless mini-HA reference (UFV180141), and black line is the mini-HA mutation to the WT sequence; B. Elution time (left) and trimer peak height (right) of the SEC profile in panel A; C. SEC profile of minimally designed polypeptide and stepwise introduction of selected mutations from UFV180088; dotted line is the minimally designed mini-HA reference (UFV180647), and black line is the mutant mini-HA; D. Elution time and peak height of the SEC profile in panel C. [Figure 3-2](As stated above.) [Figure 4] Stability of trimer stem polypeptides with and without interprotomer disulfide crosslinking. A. SEC analysis of the culture supernatant of Expi293F cells at harvest (left panel) and the culture supernatant of EXPI-CHO cells expressing the purified polypeptide after incubation at 4°C for 1 week (without (UFV180192) and with (UFV180141) cysteine ​​residues introduced at positions 398 and 408 (as shown in the structural mini-HA model at the top of this figure)); B. Temperature stability of the purified polypeptide determined by differential scanning fluorescence measurement; C. SDS-PAGE analysis of protein purity under non-reducing and reducing conditions. As shown, introduced cysteine ​​forms interprotomer disulfide crosslinks, increasing temperature stability. [Figure 5-1] Figure 5: A. Protein expression levels and antibody binding determined by AlphaLISA in the culture supernatant of EXPI-293 expressing different polypeptides at positions 355 and 482. Values ​​are determined by AlphaLISA and normalized to the reference (UFV161333); B. Protein expression, trimer content, and antibody binding determined by AlphaLISA in the culture supernatant of EXPI-293 expressing polypeptides mutated at positions 380 and 432. Values ​​are normalized to the reference (UFV170991); Protein expression, trimer content, and antibody binding determined by AlphaLISA in the culture supernatant of EXPI-293 expressing polypeptides mutated at position 435 of CI. Values ​​are normalized against the reference (UFV170611). C.II. Protein expression levels of constructs UFV171004 (435N) and UFV171197 (435R) expressed in EXPI-CHO culture supernatant, determined by OCTET (left panel) and SEC analysis (right panel). In vitro characterization of purified polypeptides (bottom panel): binding of mAb CR9114 and mAb CT149 (ELISA, EC50 value) and temperature stability (differential scanning fluorescence measurement, Tm50 value (°C)); D. Protein expression levels of polypeptides with a mutation at position 388, determined by OCTET and SEC analysis of EXPI-CHO culture supernatant. [Figure 5-2] (As stated above.) [Figure 5-3] (As stated above.) [Figure 6-1] Figure 6: Schematic diagram of HA head domain (HA1) removal. Expression levels, trimer content, and mAb binding determined by AlphaLISA in the culture supernatant of EXPI-293 cells expressing polypeptide. All data are normalized to reference designs UFV161908(A), UFV160653(B), and UFV160321(C). A. In the reference design (UFV161908), the head domain portion from amino acid 46 to amino acid 306 was removed, and the two HA1 ends were linked by an artificial "GPGS linker". Various alternative cleavage sites are shown for HA head domain removal (HA1 strand up) and direct linking of the HA1 ends (i.e., linking of the N-terminal HA1 segment to the C-terminal HA1 segment after head deletion). In construct UFV170637 (dark gray), the head domain from amino acid position 47 to amino acid 306 was removed, similar to the preferred constructs UFV180088, UFV180089, and UFV180090; B. Direct linking of the HA1 upper / lower chain after removal of the HA head domain; C. Linking of the N-terminus and C-terminus of HA1 by homologous linker sequences derived from the head domain. All constructs have a deletion of the amino acid sequence from amino acid position 46 to amino acid 306. [Figure 6-2] (As stated above.) [Figure 7-1]Figure 7: Expression levels and mAb binding determined by AlphaLISA of the culture supernatant of EXPI-293 expressing a trimer stem polypeptide with mutations that stabilize the B loop. All data are normalized to reference designs UFV161686(A), UFV161333(B, C), and UFV171187(D); A. Optimize and shield the B loop by introducing glycosylation motifs at positions 401-403 for N-linked glycosylation at position 401; Stabilize the B loop by introducing proline residues via point mutation; C. Further shield the B loop by introducing a second glycosylation motif (for N-linked glycosylation at position 393); Combine the addition of DN-linked glycosylation motifs (at positions 401 and 392 or 393) with proline substitution. Protein expression levels determined by OCTET (anti-His2); the SEC profile of the culture supernatant of EXPI-293 cells expressing E. polypeptide indicates that introduction of one glycan (UFV180208) or two glycans and two prolines (UFV180217) is well accepted. Retention time and height of peaks corresponding to the trimer polypeptide are shown in gray. [Figure 7-2] (As stated above.) [Figure 8] Expression levels and mAb binding of EXPI-CHO expressing a trimer stem polypeptide with an N-linked glycan motif at position 38 and a trimer stem polypeptide without this motif. A. Expression levels determined by OCTET (anti-C tag) and antibody binding (EC50 value) determined by ELISA; B. BELISA dilution curves for UFV170282 (solid line) and UFV170278 (dashed line). [Figure 9] Analysis of EXPI-293 culture supernatants expressing polypeptides with altered intraprotomer disulfide crosslink introduction sites. Protein expression and antibody binding determined by AlphaLISA, normalized to reference UFV160595. A. Alternative cysteine ​​was introduced near the location present in reference construct UFV160595; B. Alternative second disulfide crosslink was introduced in the region beneath the first disulfide crosslink. [Figure 10]Analysis of EXPI-293 culture supernatants expressing polypeptides with altered protomer disulfide crosslinking positions. Protein expression and antibody binding determined by AlphaLISA, normalized to the reference design UFV170051. [Figure 11] Analysis of EXPI-293 culture supernatant expressing a soluble trimer polypeptide variant with alternative C-terminal truncation (stepwise to position 515 of UFV171272 and position 499 of UFV171280). A. SEC profile with retention time and height of trimer peaks shown in gray; B. Binding of polypeptide to broad-spectrum neutralizing antibodies CR9114 and CT149, determined by OCTET; Relative KON values ​​of polypeptide compared to reference UFV170991 (black). [Figure 12] In vitro characterization of the polypeptide of the present invention having a residue substitution in the A helix from H3 wild-type (WT) to H1. A. Protein expression levels and antibody binding determined by AlphaLISA of EXPI-293 cell culture supernatant containing the expressed polypeptide. Values ​​are normalized against reference (UFV161454); B. Protein expression levels of three independent EXPI-CHO culture supernatants determined by OCTET (anti-His2, left panel) and SEC-MALS analysis (right panel). In vitro characterization of purified polypeptide (bottom panel): binding to mAb CR9114 and CT149 (ELISA, EC50 value) and temperature stability (differential scanning fluorescence measurement, Tm50 value). [Figure 13] Supernatant analysis of EXPI-293 cells expressing a trimer stem polypeptide with a stem surface mutation to H7 HA. Trimer content and antibody binding determined by AlphaLISA. All data are normalized to reference design UFV172561(A) containing A helix mutations to H1 at positions 379 and 381, or reference design UFV172562(B) containing wild-type H3 A helix residues at positions 379 and 381 in the SEC profile. References are indicated by dotted lines. [Figure 14]SEC analysis of culture supernatants of EXPI-293 cells expressing polypeptides derived from different group 2H3 strains containing relevant design elements for the generation of soluble trimer stem polypeptides. A. SEC profiles of mini-HA polypeptides containing set I design elements and based on A / Hong Kong / 1 / 1968, A / Wisconsin / 67 / 05, or A / Singapore / INFIMH / 16 / 0019 / 2016 (trimer peaks indicated by "T"); B. SEC profiles of polypeptides containing the following further stabilizing mutations in the loop: SEC profiles of polypeptides containing elements of design I (dotted line), design II (gray), and design III (black). [Figure 15] Numbering of amino acid positions in wild-type H3A / Hong Kong / 1 / 1968 (wt A / HK / 1 / 1968) and H3-derived mini-HA designs UFV180088, UFV180089, and UFV180090, according to Winter et al. (1981)'s H3 numbering. [Figure 16] Adenovirus (Ad26.FLU.004) drove the expression and folding of UFV180480 (UFV18088, which has a native transmembrane domain). A) FACS analysis of MRC-5 cells transduced with Ad26.Empty (10,000 VP / cell, negative control) and B) Ad26.FLU.004 (5,000 VP / cell). Transduced MRC-5 cells were stained with CR9114 antibody; C. Western blot analysis of lysates of MRC-5 cells transduced with Ad26.FLU.004 (5,000 VP / cell) or Ad26.Empty (5,000 VP / cell). UFV180088 (200 ng / lane) was loaded as a positive control. All samples were performed under non-reducing (lanes 1-3) or reducing (lanes 4-6) conditions. The expressed mini-HA was detected using the antibody CR9114. [Figure 17-1]Figure 17: In vivo characterization of polypeptides UFV170278 (a polypeptide containing the wild-type motif 38-NAT-40 for N-linked glycosylation) and UFV170282 (a polypeptide in which this glycan motif is knocked out by the point mutation T40I). A. H3 A / Hong Kong / 1 / 1968 FL HA stem-specific antibody titers 4 weeks after the third immunization of mice with the polypeptide of the present invention or PBS. The horizontal line for each group indicates the group median. B. Left panel: Survival rate during the follow-up period after H3N2 A / Hong Kong / 1 / 1968 challenge in mice immunized with the polypeptide of the present invention or PBS shown; panel above UFV170278, panel below UFV170282. Right panel: Relative body weight during the follow-up period after H3N2 A / Hong Kong / 1 / 1968 challenge in mice immunized with the polypeptide of the present invention or PBS; panel above UFV170278, panel below UFV170282. Relative body weight change is expressed relative to day 0. Cumulative weight loss during the follow-up period was determined by calculating the area under the curve (AUC). Error bars indicate 95% confidence intervals. [Figure 17-2] (As stated above.) [Figure 18-1] Figure 18: In vivo characterization of the immunogenicity of the polypeptides UFV180088, UFV180089, and UFV180090 of the present invention in a naive mouse model. A. H3 A / Hong Kong / 1 / 1968 FL HA stem-specific antibody titers after 1 (1×), 2 (2×), or 3 (3×) immunization of mice with the polypeptides of the present invention or PBS. The horizontal line for each group indicates the group median. B. FL HA H3 A / Hong Kong / 1 / 1968, H3 A / Texas / 50 / 2012, and H7 A / Netherlands / 219 / 2003 antibody titers after 1 (1×), 2 (2×), or 3 (3×) immunization of mice with the polypeptides of the present invention or PBS. The horizontal line for each group indicates the group median. The dashed line represents LLOQ, and the white symbols indicate the value in LLOQ. [Figure 18-2] (As stated above.) [Figure 19]Figure 19: In vivo characterization of the polypeptides UFV180088, UFV180089, and UFV180090 of the present invention in an H3N2 lethal naive mouse model. Left panel: Survival rates during the follow-up period after H3N2 A / Hong Kong / 1 / 1968 challenge in mice immunized with the polypeptides of the present invention or PBS shown; top panel for UFV180088, middle panel for UFV180089, and bottom panel for UFV180090. Right panel: Relative body weight during the follow-up period after H3N2 A / Hong Kong / 1 / 1968 challenge in mice immunized with the polypeptides of the present invention or PBS shown. Relative body weight change is expressed relative to day 0. Cumulative weight loss during the follow-up period was determined by calculating the area under the curve (AUC). Error bars indicate 95% confidence intervals. H7N9 lethal naive mouse model. [Modes for carrying out the invention]

[0027] definition The definitions of terms used in this invention are shown below.

[0028] The amino acids according to the present invention may be any of the 20 natural (or "standard") amino acids or their variants, such as D-proline (the D-enantiomer of proline), or any variant not found in nature in proteins, such as norleucine. Standard amino acids can be classified into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size, and functional groups. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties; for example, cysteine ​​can form covalent disulfide bonds (or disulfide bridges) with other cysteine ​​residues, proline forms rings with the polypeptide backbone, and glycine is more flexible than other amino acids. Table 7 shows the abbreviations and properties of standard amino acids.

[0029] The terms "included" or "including," as used herein, are deemed to be followed by the phrase "but not limited to."

[0030] As used herein, the term “infection” means the entry, replication, and / or presence of a virus in a cell or object. In one embodiment, infection is an “active” infection, i.e., the virus is replicating in the cell or object. Such an infection is characterized by the spread of the virus from the cell, tissue, and / or organ that first infected the cell, tissue, and / or organ to other cells, tissues, and / or organs. Infection can also be a latent infection, i.e., an infection in which the virus is not replicating. In certain embodiments, infection refers to a pathological condition resulting from the presence of a virus in a cell or object, or from the entry of a virus into a cell or object.

[0031] Influenza viruses are generally classified into the following influenza virus types: A, B, and C. The term “influenza virus subtype,” as used herein, refers to an influenza A virus variant characterized by a combination of viral surface proteins, hemagglutinin (H) and neuraminidase (N). According to the present invention, influenza virus subtypes may be referred to by their H number, for example, “influenza virus containing HA of H3 subtype,” “influenza virus of H3 subtype,” or “H3 influenza,” or by a combination of H and N numbers, for example, “influenza virus subtype H3N2,” or “H3N2.” The term “subtype” specifically includes all individual “strains” within each subtype, which usually arise from mutations and exhibit different pathogenic profiles, such as naturally occurring isolates and artificial variants or reassortants. Such strains may also be referred to as various “isolated” of a virus subtype. Therefore, as used herein, the terms “strain” and “isolated” may be used interchangeably. Current nomenclature for human influenza virus strains or isolates includes the virus type (genus), i.e., A, B, or C, the geographical location of the initial isolation, the strain number, and the year of isolation, in addition to the HA and NA antigens, usually shown in parentheses, for example, A / Moscow / 10 / 00(H3N2). Non-human strains also include the host of origin in the nomenclature.

[0032] Subtypes of influenza A viruses can be further classified by referring to their lineage groups. Phylogenetic analysis has shown that hemagglutinin can be subdivided into two main groups: the H1, H2, H5, and H9 subtypes of lineage group 1 (group 1 influenza viruses), and the H3, H4, H7, and H10 subtypes of lineage group 2 (group 2 influenza viruses).

[0033] As used herein, the terms “influenza virus disease” or “influenza” refer to a pathological condition resulting from the presence of influenza viruses (e.g., influenza A or B viruses) in a subject. As used herein, the terms “disease” and “disorder” are used interchangeably. In certain embodiments, this term refers to a respiratory illness caused by infection of a subject with an influenza virus.

[0034] As used herein, the terms “nucleic acid” or “nucleic acid molecule” include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA), as well as analogs of DNA or RNA produced using nucleotide analogs. Nucleic acids may be single-stranded or double-stranded. As will be readily apparent to those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may contain unnatural or derivatized nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more natural nucleotides with analogs, internucleotide modifications, e.g., uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelating agents, alkylating agents, and modifying bonds (e.g., alpha-anomeric nucleic acids, etc.). References to nucleic acid sequences include their complements unless otherwise stated. Therefore, any mention of a nucleic acid molecule with a specific sequence should be understood to include a complementary strand with its complementary sequence. Complementary strands are also useful, for example, in antisense therapy, hybridization probes, and PCR primers.

[0035] As used herein, the numbering of amino acids in HA is based on the H3 numbering as described in Winter et al. (1981). Therefore, the numbering of amino acid residues or amino acid positions refers to the numbering in full-length H3 HA (in particular, the numbering of amino acid positions in A / Aichi / 2 / 68) as described and shown in Figure 2 of Winter et al. (1981). Therefore, the numbering is based on the full-length HA numbering (SEQ ID NO: 1) of the reference strain H3N2 A / Aichi / 2 / 68. The numbering refers in particular to the numbering of amino acid positions in SEQ ID NO: 1. For example, the expression "amino acid at position 392" or "amino acid corresponding to the amino acid at position 392" (these are used interchangeably throughout this application) refers to the amino acid residue at position 392 according to the H3 numbering of Winter et al. (1981). In the polypeptide of the present invention, a portion of the HA1 domain (head domain) is deleted. Therefore, it should be noted that the numbering used herein does not necessarily refer to the actual position of the amino acid in the HA stem polypeptide of the present invention, but rather to the position of the amino acid in a full-length HA molecule (i.e., without head deletion). As those skilled in the art will understand, other influenza virus strains and / or subtypes, as well as equivalent amino acids in the stem polypeptide of the present invention (i.e., amino acids corresponding to the amino acids at specific positions in SEQ ID NO: 1), can be determined by sequence alignment.

[0036] As is known to those skilled in the art, "polypeptide" refers to a polymer of amino acids linked by amide bonds. As used herein, this term may refer to a single polypeptide chain linked by covalent amide bonds. This term may also refer to multiple polypeptide chains associated by non-covalent interactions, such as ionic contact, hydrogen bonding, van der Waals contact, and hydrophobic contact. Those skilled in the art will understand that this term includes polypeptides modified by post-translational processing, such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked and O-linked glycosylation), protease cleavage, and lipid modification (e.g., S-palmitoylation).

[0037] "HA stem polypeptide" refers to a polypeptide derived from natural (or wild-type) hemagglutinin (HA) that does not contain the head domain.

[0038] As used herein, the term “wild-type” refers to HA derived from naturally circulating influenza viruses.

[0039] Influenza viruses have a significant impact on global public health, causing millions of severe illnesses, thousands of deaths, and considerable economic losses each year. Current trivalent or quadrivalent influenza vaccines induce a strong neutralizing antibody response against the vaccine strain and closely related isolates, but rarely extend to more branched strains within a given subtype or to other subtypes. In addition, there are many difficulties in selecting the appropriate vaccine strain, often resulting in suboptimal protection. Furthermore, predicting the subtype of the next epidemic virus, including when and where it will occur, is currently impossible.

[0040] Hemagglutinin (HA) is the major envelope glycoprotein of the influenza virus, a primary target of neutralizing antibodies. Hemagglutinin has two main functions during the entry process. First, hemagglutinin mediates the attachment of the virus to the surface of target cells through interaction with sialic acid receptors. Second, after viral endocytosis, hemagglutinin subsequently causes the fusion of the virus and the endosomal membrane, releasing its genome into the cytoplasm of the target cell. HA contains a large ectodomain of approximately 500 amino acids that is cleaved by host-derived enzymes to produce two polypeptides (HA1 and HA2) bound together by disulfide bonds. The majority of the N-terminal fragment (HA1 domain, approximately 320-330 amino acids) forms a membrane distal spherical "head domain" containing the receptor binding site and most of the antigenic determinants recognized by virus-neutralizing antibodies. The smaller C-terminal region (HA2 domain, approximately 180 amino acids) forms a stem-like structure (stem domain) that anchors the globular domain to the cell membrane or viral membrane. The degree of sequence identity between subtypes is lower for HA1 polypeptide (34%–59% inter-subtype identity) than for HA2 polypeptide (51–80% identity). The majority of the conserved region is the sequence around the protease cleavage site, particularly the 23 amino acids at the N-terminus of HA2, and this sequence is conserved in all influenza A virus subtypes (Lorieau et al., 2010). Part of this region is exposed as a surface loop in the HA precursor molecule (HA0), but becomes isolated when HA0 is cleaved into HA1 and HA2.

[0041] Most neutralizing antibodies bind to loops surrounding the receptor binding site, thereby preventing receptor binding and adhesion. Because these loops are highly mutable, most antibodies targeting these regions are strain-specific, explaining why current vaccines induce such limited strain-specific immunization. For example, fully human monoclonal antibodies against influenza virus hemagglutinin with broad cross-neutralizing efficacy, such as CR6261, have been generated (International Publication No. 2008 / 028946). Functional and structural analysis revealed that these antibodies interfere with membrane fusion processes and are directed to highly conserved epitopes in the stem domain of group 1 influenza HA proteins (Throsby et al., 2008; Ekiert et al., 2009; International Publication No. 2008 / 028946). The identification of CR9114-like antibodies that cross-react with many HA molecules in groups 1 and 2 (described in International Publication No. 2013 / 007770) revealed that the human immune system is capable of inducing extremely broad-spectrum neutralizing antibodies against influenza viruses. However, given the need for annual vaccination schemes, these antibodies are not necessarily induced to a level of protection both after infection with subtype H1 and / or H3 (seasonal) influenza viruses and after vaccination.

[0042] The present invention provides novel HA stem polypeptides that mimic specific epitopes such as antibody CR9114 (including the heavy chain variable region of SEQ ID NO: 7 and the light chain variable region of SEQ ID NO: 8) and / or antibody CR8020 (including the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6). The polypeptides of the present invention can be used in vivo, alone, or in combination with other prophylactic and / or therapeutic measures, to induce influenza virus-binding and / or neutralizing antibodies, preferably cross-binding and / or cross-neutralizing antibodies. "Cross-binding and / or cross-neutralizing antibody" means an antibody that can bind to and / or neutralize at least two, preferably at least three, four, or five different subtypes of influenza A virus of lineage group 2, or an antibody that can bind to and / or neutralize at least one group 1 influenza virus and at least one group 2 influenza virus.

[0043] Influenza HA stem polypeptides that stably present the epitopes of antibodies CR6261 and / or CR9114 have been previously described in International Publication No. 2013 / 079473. At least some of these HA stem polypeptides can stably present the epitopes of CR6261 and / or CR9114 and have been shown to be immunogenic in mice. Further HA stem domain polypeptides that can stably present the epitopes of CR6261 and / or CR9114 are described in International Publication Nos. 2014 / 191435, International Publication Nos. 2016 / 005480, and International Publication Nos. 2016 / 005482. These stem polypeptides are based on the HA of group 1 influenza A virus and induce an immune response only against group 1 influenza A virus.

[0044] The research that led to this invention showed that modifications introduced into the HA stem polypeptide of group 1 did not result in a stable trimer stem polypeptide when using the HA of influenza virus of group 2.

[0045] The present invention provides a Group 2 influenza HA stem polypeptide comprising a novel modification that allows the polypeptide to be fully expressed in mammalian cells, is trimer (e.g., measured by AlphaLISA and SEC), and is thermally stable (e.g., measured by dynamic scanning fluorescence / differential scanning calorimetry (DSF / DSC)). Furthermore, the Group 2 stem polypeptide of the present invention has been shown to induce neutralizing antibodies in vivo. Moreover, the affinity for the polypeptide of the present invention is less than 1 nM for all broad-spectrum neutralizing antibodies (bnAbs) tested (measured by Octet and ELISA), which clearly indicates that the polypeptide mimics the stem of natural full-length HA. Furthermore, this novel HA stem polypeptide does not require an artificial linker, tag, N-terminal trimerizing domain, or C-terminal trimerizing domain.

[0046] Therefore, in the first aspect, the present invention is a monomeric influenza A HA stem polypeptide comprising the HA1 domain and HA2 domain of hemagglutinin (HA) of group 2 influenza A virus, (i) Deletion of the head region of the HA1 domain, (ii) Modification of the trimerization region of the HA2 domain, (iii) at least two cysteine ​​residues capable of forming at least one monomeric cysteine ​​crosslink It contains an amino acid sequence that includes, The amino acid at position 355 in this amino acid sequence is W. The numbering of amino acid positions in the HA stem polypeptide amino acid sequence is based on the H3 numbering system of Winter et al.'s HA nomenclature, which corresponds to the full-length HA numbering of the reference strain H3N2 A / Aichi / 2 / 68 (SEQ ID NO: 1). Regarding HA stem polypeptides.

[0047] Accordingly, the present invention provides an HA stem polypeptide (i.e., a headless HA polypeptide) comprising a modification of the trimerization region in the HA2 domain, preferably a modification in the C-helix, and at least two cysteine ​​residues that form an intrameric disulfide crosslink; the amino acid at position 355 in the amino acid sequence is W; and the numbering of the amino acid positions in the HA stem polypeptide amino acid sequence is H3 numbering according to Winter et al.'s HA nomenclature, based on the full-length HA numbering of reference strain H3N2 A / Aichi / 2 / 68 (SEQ ID NO: 1).

[0048] In certain embodiments, the present invention relates to a monomeric influenza A HA stem polypeptide comprising the HA1 domain and HA2 domain of hemagglutinin (HA) of a group 2 influenza A virus, (i) Deletion of the head region of the HA1 domain, (ii) Modification of the trimerization region of the HA2 domain, (iii) at least two cysteine ​​residues capable of forming at least one monomeric cysteine ​​crosslink It contains an amino acid sequence that includes, It includes a mutation to W at amino acid position 355, The numbering of amino acid positions in the HA stem polypeptide amino acid sequence is based on the H3 numbering system of Winter et al. (mentioned above), which corresponds to the full-length HA numbering system of the reference strain H3N2 A / Aichi / 2 / 68 (SEQ ID NO: 1). We provide HA stem polypeptides.

[0049] In certain embodiments, the amino acid at position 355 is W and the amino acid at position 432 is I, or the amino acid at position 355 is W, the amino acid at position 432 is I, and the amino acid at position 380 is I. In certain embodiments, the polypeptide includes a mutation to W at the amino acid at position 355 and a mutation to I at the amino acid at position 432, or a mutation to W at the amino acid at position 355 and a mutation to I at the amino acid at position 432 and a mutation to I at the amino acid at position 380. According to the present invention, the presence of these amino acids has been shown to increase the trimer level of the polypeptide of the present invention.

[0050] In certain other embodiments, the amino acid at position 355 is a mutation to W, the amino acid at position 378 is a mutation to T, the amino acid at position 379 is a mutation to N, and / or the amino acid at position 381 is a mutation to V. The presence of these amino acids has been shown to increase the expression and binding of broad-spectrum neutralizing antibodies.

[0051] In certain embodiments, the polypeptide further comprises a glycosylation motif (NxT) introduced for N-linked glycosylation at position 401 to shield against potential neoepitopes within the B-loop. Thus, according to the present invention, the polypeptide comprises a glycosylation motif (NxT) at positions 401-403 for N-linked glycosylation at position 401.

[0052] In certain embodiments, the present invention relates to a group 2 influenza A hemagglutinin (HA) stem polypeptide comprising an HA1 domain and an HA2 domain, (i) A deletion in the head region of the HA1 domain, which includes at least an amino acid sequence from the amino acid corresponding to the 50th position to the amino acid corresponding to the 302nd position. (ii) Modification of the trimerization region of the HA2 domain, preferably modification of the trimerization region in a C helix, wherein the trimerization region includes an amino acid sequence from the amino acid corresponding to the 405th position to the amino acid corresponding to the 419th position. (iii) A cysteine ​​at the amino acid position corresponding to position 310 combined with a cysteine ​​at the position corresponding to position 422; or a cysteine ​​at the amino acid corresponding to position 311 combined with a cysteine ​​at the position corresponding to position 422; or a cysteine ​​at the amino acid corresponding to position 308 combined with a cysteine ​​at the position corresponding to position 418, wherein the cysteine ​​residue comprises an amino acid sequence containing a cysteine ​​that can form an intramonomer disulfide crosslink; the amino acid at position 355 of this amino acid sequence is W; and the numbering of the amino acid positions in the HA stem polypeptide amino acid sequence is H3 numbering according to the HA nomenclature of Winter et al. (mentioned above), based on the full-length HA numbering of reference strain H3N2 A / Aichi / 2 / 68 (SEQ ID NO: 1).

[0053] According to the present invention, surprisingly, group 2 influenza HA stem polypeptides having an amino acid sequence in which the amino acid at position 355 is W were found to exhibit higher expression levels in mammalian cells, a greater tendency to trimerize, and / or superior thermal stability compared to previously produced group 2 HA stem polypeptides. Furthermore, the HA stem polypeptides of the present invention induce humoral and / or cellular immune responses to group 2 influenza viruses in vivo.

[0054] As is known to those skilled in the art, full-length influenza hemagglutinin (HA0) typically comprises an HA1 domain and an HA2 domain. Furthermore, full-length influenza hemagglutinin (HA0) typically comprises a stem domain and a head domain. The stem domain is formed by two segments of the HA1 domain and most or all of the HA2 domain. The two segments of the HA1 domain are separated in the primary sequence by a globular head domain. As described herein, the HA stem polypeptide of the present invention comprises an amino acid sequence that includes several modifications to the HA1 domain and / or HA2 domain compared to the amino acid sequence of wild-type full-length HA polypeptide (HA0), particularly the amino acid sequence of group 2HA. As used throughout this application, the numbering of amino acid positions in the HA stem polypeptide amino acid sequence is the H3 numbering according to Winter et al. (cited above) (i.e., corresponding to the full-length HA numbering of reference strain H3N2 A / Aichi / 2 / 68 (SEQ ID NO: 1)).

[0055] According to the present invention, a stem polypeptide, also called "mini-HA," is created by deleting at least a portion of the immunodominant head of the influenza HA polypeptide with significant changes in the HA1 domain, specifically a portion containing at least the amino acid sequence from position 50 to position 302, from the full-length HA (HA0) protein. The remaining portions of the HA1 domain (i.e., the N-terminal segment and the C-terminal segment of the HA1 domain) are linked directly (i.e., without linkers) or via linkers consisting of 1 to 10 amino acids. Therefore, for example, if the amino acid sequence from position 50 to position 302 is deleted, the amino acid at position 49 (the last amino acid of the N-terminal HA1 segment) is linked to the amino acid at position 303 (the first amino acid of the C-terminal HA1 segment) directly or by substitution with a 1 to 10 amino acid linker in the deleted head region. The deletion of the amino acid sequence from position 50 to position 302 is the minimal deletion in the HA1 domain. According to the present invention, a larger portion of the HA1 domain, for example, the amino acid sequence from amino acid position 47 to amino acid position 306, can also be deleted, as shown in the construct below in Figure 1A.

[0056] In a preferred embodiment, the deletion of the HA1 domain includes at least the amino acid sequence from the 47th amino acid to the 306th amino acid. Thus, in this embodiment, the stem polypeptide includes the N-terminal HA1 segment up to the 46th amino acid and the C-terminal HA1 segment starting from the 307th amino acid (the dark gray portion in Figure 1A).

[0057] In a preferred embodiment, the deletion of the HA1 domain consists of an amino acid sequence from the 47th amino acid to the 306th amino acid.

[0058] In some embodiments, the deletion in the HA1 domain is replaced by a linked sequence of 1 to 10 amino acids.

[0059] Furthermore, as described herein, the HA stem polypeptide of the present invention includes modifications to the trimerization region in the HA2 domain, preferably modifications in the C helix, in order to improve the trimerization of the HA stem polypeptide after deletion of the head region. In certain preferred embodiments, the modifications in the HA2 domain are modifications that enhance the trimerization of the HA stem polypeptide.

[0060] In certain embodiments, the modification involves the introduction of a heterotrimerization domain in the C helix. Generally, the C helix is ​​understood to contain an amino acid sequence (H3 numbering) from amino acid 405 to amino acid 434. In preferred embodiments, the heterotrimerization domain is introduced at a position corresponding to the amino acid sequence from amino acid 405 to amino acid 419 (Figure 1A). Thus, in certain embodiments, the original (wt) amino acid sequence in the HA2 domain from 405 to 419 is replaced by a heterotrimerization sequence of the same length, i.e., the same number of amino acids.

[0061] In certain embodiments, the heterotrimerization domain is a GCN4 sequence.

[0062] In certain preferred embodiments, the modified trimerization region (i.e., including heterologous trimerization domains) 405 KRM405KIEEEIESK 419 (Sequence ID 9) and 405 PMKQIEDKIEEIESK 419 (Sequence ID 10) It contains an amino acid sequence selected from the group consisting of the following.

[0063] In some embodiments, at least one amino acid in the heterotrimerized sequence is mutated to C, enabling the formation of intermonomer cysteine ​​crosslinks (as described below). Therefore, in certain preferred embodiments, the heterotrimerized sequence is 405 RMKCIEDKIEEIESK 419 (Sequence ID 11) and 405PMKCIEDKIEEIESK 419 (SEQ ID NO: 12) comprises an amino acid sequence selected from the group consisting of. In a preferred embodiment, the trimerization region has the amino acid sequence 405 PMKCIEDKIEEIESK 419 (SEQ ID NO: 12).

[0064] In certain embodiments, the modification comprises a change, preferably an optimization, of the 7-residue repeat in the trimerization region, which includes the amino acids from amino acid 405 to amino acid 419 of the C helix. [abcdefg] n The 7-residue repeat represented as generally has hydrophobic residues at a and d, and polar / charged residues at e and g. These motifs are the basis of most coiled-coil structures and are the structural motifs of proteins in which alpha helices are coiled together like the strands of a rope (dimers and trimers being the most common types) (Ciani et al., 2010).

[0065] As a further modification, the HA stem polypeptide according to the present invention comprises at least two cysteine ​​residues that can form (or are capable of forming) monomeric (or intermonomeric) cysteine ​​(or disulfide) crosslinks. The manipulated cysteine ​​crosslinks can be introduced by mutating at least one (if the other is already cysteine), but typically two spatially close residues can be mutated to cysteine, which are then introduced by spontaneous or active oxidation forming a covalent bond between the sulfur atoms of those residues. In preferred embodiments, the polypeptide comprises cysteine ​​at position 310 and cysteine ​​at position 422, or cysteine ​​at the amino acid corresponding to position 311 in combination with cysteine ​​at the position corresponding to position 422; or cysteine ​​at the amino acid position corresponding to position 308 in combination with cysteine ​​at the position corresponding to position 418, which enable the formation of monomeric cysteine ​​crosslinks. In certain embodiments, the polypeptide includes mutations to C in amino acids at positions 310 and / or 422, or mutations to C in amino acids at positions 311 and / or 422, or mutations to C in amino acids at positions 308 and / or 418, wherein the cysteine ​​residues form the intra-monomer cysteine ​​crosslinks. Thus, these cysteine ​​residues form intra-monomer (or intraprotomer) cysteine ​​(or disulfide) crosslinks that stabilize the protein. In preferred embodiments, the polypeptide includes cysteine ​​(mutation) at position 310 and cysteine ​​(natural mutation) at position 422, forming at least one intra-monomer cysteine ​​crosslink.

[0066] The polypeptide according to the present invention generally has at least four natural (i.e., naturally occurring) glycosylation (or glycan) motifs (NxT) for N-linked glycosylation, for example, a glycan motif, at the 8-10 position (8NST). 10 ), 22nd to 24th ( 22 NGT 24 ), 38~40th place ( 38 NAT 40 ) and 483rd to 485th place ( 483 NGT 485) included. In certain embodiments, the polypeptide includes at least one glycan motif introduced at positions 401-403 for N-linked glycosylation at position 401, as described above. In certain embodiments, the polypeptide includes at least one additionally introduced glycosylation motif. Thus, in certain embodiments, at least one additional N-linked glycosylation motif is present and / or introduced at positions 392-394 for N-linked glycosylation at position 392, and / or at positions 393-395 for N-linked glycosylation at position 393. In preferred embodiments, the polypeptide includes a glycosylation motif at positions 401-403 for N-linked glycosylation at position 401, and a glycosylation motif at positions 393-395 for N-linked glycosylation at position 393.

[0067] In further embodiments, the amino acid at position 388 is M. In certain embodiments, the amino acid at position 388 is mutated to M, but other amino acids at this position are also possible, including, but are not limited to, T, V, I, L, F, Y, W, H, K, and R.

[0068] Furthermore, in certain embodiments, the HA polypeptide is - The amino acid at position 31 is E, and the amino acid at position 34 is V. - The amino acid at position 392 is either S or P. - The amino acid at position 395 is either T or P. - The amino acid at position 399 is either S or P. - The amino acid at position 435 is N or R, and / or - Contains an amino acid sequence in which the amino acid at position 439 is Y.

[0069] Therefore, in certain embodiments, the amino acid at position 31 is E and the amino acid at position 34 is V. In certain embodiments, the polypeptide comprises an amino acid sequence including a mutation to E at position 31 and a mutation to V at position 34. According to the present invention, the presence of these amino acid residues (i.e., 31E and 34V) is found to optimize the hydrogen bond network, which is an important contributing factor to the stability of the polypeptide of the present invention. The polypeptide may further comprise an amino acid sequence in which the amino acid at position 392 is S or P (mutation to), the amino acid at position 395 is T or P (mutation to), and / or the amino acid at position 399 is S or P (mutation to). Therefore, the polypeptide of the present invention may comprise one or more mutations in the so-called B loop. This B loop comprises amino acids from position 385 to 404 (see Figure 1C). Mutations in the B loop increase the solubility of the polypeptide by reducing its hydrophobicity. Therefore, in certain preferred embodiments, the polypeptide comprises, compared to the wild-type HA polypeptide, the B loop, - A mutation in the amino acid corresponding to the 392nd position, preferably to S or P, - A mutation in the amino acid corresponding to the amino acid at position 395, preferably to T or P, and - Mutation of the amino acid corresponding to the 399th position to S or P, preferably P. The polypeptide includes at least one additional mutation selected from the group consisting of the following: The polypeptide may further include a mutation to N or R, preferably N, of the amino acid corresponding to the amino acid at position 435, and / or a mutation to Y of the amino acid corresponding to the amino acid at position 439. These mutations are thought to optimize the trimer interface, which contributes to the stability of the trimer in solution.

[0070] Please note again that, as used herein, the amino acid position numbering is based on the H3 numbering by Winter et al. (1981). Also, please note again that, as used herein, the amino acid position numbering is based on the position numbering of the full-length H3 HA polypeptide (HA0). Therefore, as used herein, "amino acid at position 434" refers to the amino acid at position 434 in H3 HA0. Consequently, the numbering does not refer to the actual position of the amino acid in the HA stem polypeptide of the present invention due to head region deletion (see Figure 15).

[0071] According to the present invention, the HA stem polypeptide is a group 2 HA polypeptide. Accordingly, according to the present invention, the modifications described herein are introduced into the HA of an influenza virus of lineage group 2, for example, an influenza virus containing the HA of H3, H7, or H10 subtypes, to produce the HA stem polypeptide of the present invention. In certain embodiments, the HA stem polypeptide is an H3 HA polypeptide. Accordingly, in certain embodiments, the HA stem polypeptide is derived from the HA of an influenza A virus containing the HA of an H3 subtype, such as influenza virus A / Hong Kong / 1 / 68 having the amino acid sequence of SEQ ID NO: 2, or influenza virus A / Wisconsin / 67 / 2005 having the amino acid sequence of SEQ ID NO: 13, or influenza virus A / Singapore / INFMH / 16 / 0019 / 2016 having the amino acid sequence of SEQ ID NO: 14. As those skilled in the art will understand, the polypeptides of the present invention may also be derived from the HA of other H3 influenza A virus strains, including, but not limited to, A / Perth / 16 / 2009 (SEQ ID NO: 15), A / Brisbane / 10 / 2007 (SEQ ID NO: 16), or A / Panama / 2007 / 1999 (SEQ ID NO: 17).

[0072] As described above, the stem polypeptide may or may not include a linking sequence consisting of 1 to 10 amino acid residues that substitute for the deleted HA1 head sequence, thereby linking the two remaining HA1 portions. In certain embodiments, the linking sequence contains 1 to 5 amino acids. In certain embodiments, the linking sequence contains 2, 3, or 4 amino acids. The linking sequence may be a heterologous linking sequence, i.e., an amino acid sequence not present in natural or wild-type HA, such as GGGG and GPSG, but not limited to the following.

[0073] In certain embodiments, the linking sequence is a homologous linking sequence, i.e., an amino acid sequence derived from a corresponding deleted head region such as, but not limited to, NPHR, GDPH, NGGS, GGSN, GSNA, GPGS, GSGF, GSG, GG, GGS, SGS, HPST, IPNI, GLSS, KPGD, DAPI, TPN, and TPNG.

[0074] In preferred embodiments, the polypeptide does not contain a linking sequence.

[0075] As described above, cleavage of the influenza HA0 protein (in HA1 and HA2) is necessary for its activity, and it promotes the entry of the viral genome into target cells by causing fusion of the host endosomal membrane with the viral membrane.

[0076] In certain embodiments, the polypeptides of the present invention contain a natural protease cleavage site. Thus, the Arg(R)-Gly(G) sequence spanning HA1 and HA2 (i.e., amino acid positions 329 and 330) is a recognition site for trypsin and trypsin-like proteases and is known to be normally cleaved for hemagglutinin activation (Figure 1A).

[0077] In certain embodiments, the polypeptide does not contain a protease cleavage site. Therefore, in certain preferred embodiments, the protease cleavage site is removed by mutating the amino acid residue at position 329 to any amino acid other than arginine (R) or lysine (K). In certain embodiments, the amino acid residue at position 329 is not arginine (R). In preferred embodiments, the polypeptide includes a mutation of the amino acid at position 329 to glutamine (Q). Therefore, in certain preferred embodiments, the polypeptide of the present invention includes a cleavage site knockout mutation R329Q to prevent putative cleavage of the molecule during production in vitro or in vivo after administration.

[0078] In other embodiments, the polypeptide includes polybasic cleavage sites, such as furin cleavage sites. Thus, the polypeptide can be cleaved in cells by furin-like proteases to produce cleaved mini-HAs similar to naturally folded and processed HAs.

[0079] In certain embodiments, the polypeptide does not contain a signal sequence. The signal sequence (sometimes called a signal peptide, target signal, localization signal, localization sequence, transport peptide, leader sequence, or leader peptide) is a short peptide (usually 16-30 amino acids long) located at the N-terminus of most newly synthesized proteins destined for the secretory pathway. The signal sequence acts to prompt cells, usually to move proteins toward the cell membrane. Often, the amino acids containing the signal peptide are cleaved from the protein once they reach their final destination. In influenza HA, the signal sequence typically contains the first 16 amino acids of the full-length HA0 amino acid sequence (corresponding to amino acids from position -6 to 10 according to H3 numbering, see Figure 15).

[0080] In certain embodiments, the polypeptide includes (part of) a signal sequence. The polypeptide may include (part of) a wild-type signal sequence, or (part of) an alternative signal sequence, for example, but is not limited to: MKTIIALSYIFCLALG (Sequence ID 18), MKTIIALSYILCLVFA (Sequence ID 19), MKTIIALSYILCLVFT (SEQ ID NO: 20), and MKTIVALSYILCLVFA (Sequence ID 21) It may include a signal sequence selected from the group consisting of the following:

[0081] In a preferred embodiment, the (soluble) polypeptide does not contain a signal sequence.

[0082] In one embodiment, the polypeptide of the present invention is (i) A deletion in the head region of the HA1 domain, consisting of amino acids from position 47 to position 306, where the amino acid at position 46 is directly linked to the amino acid at position 307. (ii) Amino acid sequence that replaces the original amino acid sequence from amino acid position 405 to amino acid position 419 405 PMKCIEDKIEEIESK 419 Introduction of heterologous trimerizing domains including (SEQ ID NO: 12), (iii) A cysteine ​​mutation at the amino acid position corresponding to position 310, combined with a cysteine ​​mutation at the position corresponding to position 422. (iv) Glycosylation motif introduced at positions 393-395 (i.e.) 393 NQT 395 ) and glycosylated motifs introduced at positions 401-403 (i.e.) 401 NAT 403 ) Includes, Furthermore, in the amino acid sequence, (a) The amino acid at position 355 is W (a mutation), (b) The amino acid at position 432 is I (mutation to), and the amino acid at position 380 is I (mutation to), (c) The amino acid at position 378 is T (mutation), and the amino acid at position 379 is N (mutation), and / or the amino acid at position 381 is V (mutation), (d) The amino acid at position 388 is M (a mutation), (e) The amino acid at position 31 is E (mutated to), and the amino acid at position 34 is V (mutated to), (f) The amino acid at position 392 is S (a mutation), (g) The amino acid at position 395 is a mutation to T, (h) The amino acid at position 398 is C (a mutation), (i) The amino acid at position 399 is P (a mutation), (j) The amino acid at position 408 is C (a mutation), The amino acid at position (k)435 is a mutation to N, (l) The amino acid at position 439 is Y (a mutation), and The amino acid at position (m)329 is a mutation to Q, The numbering of amino acid positions in the HA stem polypeptide amino acid sequence corresponds to the H3 numbering of the full-length HA numbering of the reference strain H3N2 A Aichi / 2 / 68 (SEQ ID NO: 1).

[0083] In certain embodiments, the HA1 and HA2 domains are derived from an influenza virus containing H3 subtype HA, preferably influenza virus A / Hong Kong / 1 / 68.

[0084] In certain preferred embodiments, the HA1 and HA2 domains are derived from an influenza virus containing H3 subtype HA, preferably influenza virus A / Hong Kong / 1 / 68, and one or more amino acids in the H3 HA1 and HA2 domains are mutated to the corresponding amino acids of H7 HA.

[0085] Therefore, in certain embodiments, the HA1 and HA2 domains are derived from an influenza virus containing H3 subtype HA, preferably influenza virus A / Hong Kong / 1 / 68~ claim 19, (a) The amino acid at position 25 is K (a mutation), (b) The amino acid at position 367 is Y (a mutation), (c) The amino acid at position 378 is a mutation to T, (d) The amino acid at position 475 is D (a mutation), (e) The amino acid at position 476 is D (a mutation), and / or (f) The amino acid at position 479 is A (a mutation).

[0086] While we do not wish to be bound by theory, it is thought that by altering the surface of a stabilized H3-derived stem polypeptide that possesses desirable characteristics such as expression, folding, and thermal stability against H7 HA, it may be possible to induce an antibody response that may be more protective against more distant H7 viruses without completely switching to an H7-derived stem polypeptide with undesirable behavior (i.e., more difficult to manufacture, with lower expression levels and stability).

[0087] In certain embodiments, the polypeptide includes an HA2 domain comprising a transmembrane (TM) domain and a cytoplasmic (CD) domain (the TM and CD domains include amino acid sequences corresponding to amino acid sequences from the amino acid corresponding to the 514th position to the amino acid corresponding to the 550th position (H3 numbering)). Thus, a membrane-bound mini-HA polypeptide is provided.

[0088] In certain embodiments, to produce secretory soluble stem polypeptides, the polypeptide does not contain a transmembrane domain and a cytoplasmic domain. Therefore, in certain embodiments, the polypeptide includes a truncated HA2 domain, particularly an HA2 domain truncated at the C-terminus. Thus, the truncated HA2 domain according to the present invention is shorter than the full-length HA2 sequence due to the deletion of one or more amino acid residues at the C-terminus of the HA2 domain.

[0089] In certain embodiments, the C-terminal portion of the HA2 domain, beginning with the amino acid corresponding to the 514th amino acid, is deleted, and therefore the complete transmembrane domain and cytoplasmic domain are substantially removed.

[0090] In certain embodiments, a portion of the C-terminal helix is ​​also deleted. According to the present invention, it has been found that even when a fairly large portion of the HA2 domain is deleted, a stable soluble HA stem polypeptide can be provided. Therefore, in certain embodiments, the C-terminal portion of the HA2 domain beginning with an amino acid at positions 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, or 514 is deleted (again, numbered according to the H3 numbering described by Winter et al. (mentioned above)) to produce a soluble polypeptide after expression in cells.

[0091] In a preferred embodiment, the C-terminal portion of the HA2 domain, starting from position 506, is deleted.

[0092] Through optional selection, heterologous amino acid sequences (i.e., amino acid sequences not naturally present in influenza HA) can be ligated to the (truncated) HA2 domain.

[0093] Therefore, in certain embodiments, His-tagged sequences, such as HHHHHH (SEQ ID NO: 22) or HHHHHHH (SEQ ID NO: 23), or the FLAG-tagged DYKDDDDK (SEQ ID NO: 24), or the C-tagged EPEA (SEQ ID NO: 25), or a combination thereof, are linked to the C-terminal amino acid of the (optionally truncated) HA2 domain for detection and / or purification purposes. In certain embodiments, heterologous amino acid sequences, such as His-tagged sequences, may be linked to the (truncated) HA2 domain via a linker. In certain embodiments, the linker may include (part of) proteolytic cleavage sites, such as the amino acid sequences IEGR (SEQ ID NO: 26) or LVPRGS (SEQ ID NO: 27), for enzymatic removal of the His-tagged sequence after purification.

[0094] In certain embodiments, the heterologous amino acid sequence linked to the C-terminal amino acid of the (truncated) HA2 domain is: GYIPEAPRDGQAYVRKDGEWVLLSTFL (Sequence ID 28), AAADYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH(Sequence ID 29), AAADYKDDDDKPGGGGSGGGGSGGGGSHHHHHH(FLAG-GS linker-His tag), (Sequence ID 30), [ka] (Nanoluc-Strep tag, sequence number 31) [ka] (Nanoluc-C tag) (Sequence ID 32) EGRAAAWSHPQFEKGAAWSHPQFEKGAAWSHPQFEK (Strep tag, sequence number 33), EGRAAALPETGGGAAEPEA (Saltase-C tag), (Sequence ID 34), SGRDYKDDDDKPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSWSHPQFEKGAAWSHPQFEKGAAWSHPQFEK(FLAG GS linker-Strep tag), (SEQ ID NO: 35), and EGRAAAEQKLISEEDLGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSWSHPQFEKGAAWSHPQFEKGAAWSHPQFEK(Myc tag - GS linker - Strep tag), (Sequence ID 36) It contains an amino acid sequence selected from the group consisting of the following.

[0095] In certain embodiments, the heterotrimerization domain is linked to the C-terminal amino acid of the (optionally truncated) HA2 domain, for example, the "Fordon" trimerization domain (described in Letarov et al. (1993); S-Guthe et al. (2004)), but is not limited to the following.

[0096] In certain embodiments, the HA stem polypeptide of the present invention comprises an amino acid sequence selected from SEQ ID NOs: 40-44, 46-64, 66, 67, 69-97, 156-164, 169-181, and 189-212.

[0097] In a preferred embodiment, the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 40-42, 207, and 210-212, preferably 210-212, and more preferably 210.

[0098] In certain embodiments, the polypeptide is glycosylated when expressed in a suitable cell (e.g., a mammalian cell). The polypeptides of the present invention generally contain four innate glycosylation motifs (NxT) as described above. Also, as described above, according to the present invention, in certain embodiments, the polypeptide contains at least one introduced glycosylation motif introduced at positions 401-403 for N-linked glycosylation at position 401. The polypeptide preferably contains an additional glycosylation motif introduced at positions 393-395 for N-linked glycosylation at position 393.

[0099] In further embodiments, the present invention provides a polymeric, preferably trimeric, HA stem polypeptide. To obtain a stable trimeric HA stem polypeptide, the polypeptide of the present invention preferably contains at least two cysteine ​​residues that can form (or form) monomeric (also referred to as protomeric) cysteine ​​crosslinks. Therefore, in certain embodiments, the polypeptide contains cysteine ​​at position 396 in combination with cysteine ​​at position 408, or cysteine ​​at position 397 in combination with cysteine ​​at position 408, or cysteine ​​at position 398 in combination with cysteine ​​at position 408, or cysteine ​​at position 398 in combination with cysteine ​​at position 405.

[0100] In certain embodiments, the polypeptide includes a mutation to C at the 396th amino acid and a mutation to C at the 408th amino acid, or a mutation to C at the 397th amino acid and a mutation to C at the 408th amino acid, or a mutation to C at the 398th amino acid and a mutation to C at the 408th amino acid, or a mutation to C at the 398th amino acid and a mutation to C at the 405th amino acid, forming inter-monomer cysteine ​​crosslinks between the cysteine ​​at the 396th position of the first monomer and the cysteine ​​at the 408th position of the second monomer, or between the cysteine ​​at the 397th position of the first monomer and the cysteine ​​at the 408th position of the second monomer, or between the cysteine ​​at the 398th position of the first monomer and the cysteine ​​at the 408th position of the second monomer, or between the cysteine ​​at the 398th position of the first monomer and the cysteine ​​at the 405th position of the second monomer. Note that in some embodiments, the amino acid at position 405 or 408 is located within a heterotrimeric sequence.

[0101] In a preferred embodiment, the polypeptide comprises a cysteine ​​at position 398 and a cysteine ​​at position 408, and an inter-monomer cysteine ​​crosslink is formed between the cysteine ​​at position 398 of the first monomer and the amino acid at position 408 of the second monomer.

[0102] The present invention further provides nucleic acid molecules encoding the influenza HA stem polypeptide of the present invention. As will be understood by those skilled in the art, as a result of the degeneracy of the genetic code, numerous different nucleic acid molecules may encode the same polypeptide. Also, naturally, those skilled in the art can use common techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded in the described polynucleotides to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed. Thus, unless otherwise stated, “nucleic acid molecules encoding an amino acid sequence” include all nucleotide sequences that are degenerate of each other and encode the same amino acid sequence.

[0103] In certain embodiments, the nucleic acid molecule encoding the influenza HA stem polypeptide is codon-optimized for expression in mammalian cells, such as human cells. Methods for codon optimization are known and have already been described (e.g., in International Publication No. 96 / 09378).

[0104] In certain embodiments, the nucleic acid molecule encoding the influenza HA stem polypeptide includes a nucleic acid sequence selected from SEQ ID NO: 208 and SEQ ID NO: 209.

[0105] Influenza hemagglutinin stem domain polypeptides can be prepared by any method deemed suitable to those skilled in the art, such as the method described below. Accordingly, the polypeptides of the present invention can be synthesized as DNA sequences by standard methods known in the art, cloned in vitro or in vivo using suitable restriction enzymes and methods known in the art, and subsequently expressed.

[0106] The present invention further relates to a vector comprising a nucleic acid molecule encoding the polypeptide of the present invention. In certain embodiments, the nucleic acid molecule according to the present invention is therefore part of a vector, for example, a plasmid. Such a vector can be readily manipulated by methods well known to those skilled in the art and can be designed, for example, to replicate in prokaryotic and / or eukaryotic cells. The vector used may be any vector that is suitable for DNA cloning and can be used for transcription of the nucleic acid of interest. If a host cell is used, the vector is preferably an embedding vector. Alternatively, the vector may be an episome replication vector. Those skilled in the art can select a suitable expression vector and functionally insert the nucleic acid sequence of the present invention. It is well known to those skilled in the art that, in order to obtain expression of a nucleic acid sequence encoding a polypeptide, an expression-driving sequence can be functionally bound to the nucleic acid sequence encoding the polypeptide, resulting in a recombinant nucleic acid molecule encoding a protein or polypeptide in an expressible format. Expression-driving sequences may include promoters, enhancers, and combinations thereof. These must function in host cells and thereby be able to drive the expression of the nucleic acid sequence functionally bound to them. Those skilled in the art recognize that various promoters can be used to obtain gene expression in host cells. Promoters can be constitutive or regulatory and can be obtained from various sources (e.g., viral, prokaryotic, or eukaryotic sources) or artificially designed. The expression of the nucleic acid of interest may be from a natural promoter or its derivative, or from a completely heterogeneous promoter (Kaufman, 2000). Some well-known promoters commonly used for expression in eukaryotic cells include promoters derived from viruses, e.g., adenoviruses, e.g., the E1A promoter; promoters derived from cytomegalovirus (CMV), e.g., the CMV pre-early (IE) promoter (referred to as the CMV promoter in this invention) (e.g., obtained from pcDNA, Invitrogen); and promoters derived from Simian virus 40 (SV40) (Das et al, 1985).Suitable promoters may also be derived from eukaryotic cells, including metallothionein (MT) promoters, elongation factor 1α (EF-1α) promoters (Gill et al., 2001), ubiquitin C or UB6 promoters (Gill et al., 2001), actin promoters, immunoglobulin promoters, and heat shock promoters. Testing promoter function and promoter strength is standard practice for those skilled in the art and generally includes, for example, cloning of test genes, such as lacZ, luciferase, or GFP after the promoter sequence, and expression testing of test genes. Naturally, promoters can be modified by deletion, addition, or mutation of their sequence, and their functionality can be tested to find new, weaker, or improved promoter sequences. According to the present invention, a potent promoter that confers a high transcription level in optimal eukaryotic cells is preferred.

[0107] The construct can be transfected into eukaryotic cells (e.g., plant, fungal, yeast, or animal cells) or suitable prokaryotic expression systems such as E. coli using methods well known to those skilled in the art. In some cases, as described above, suitable “tag” sequences (e.g., his tag, myc tag, strep tag, saltase, c tag, or flag tag, etc.) or complete proteins (e.g., maltose-binding protein or glutathione S-transferase, etc., etc., not limited to the above) can be added to the sequence of the present invention to enable the purification and / or identification of polypeptides from cells or supernatants. Optionally, the tag can be removed later by proteolytic digestion, including sequences containing specific proteolytic sites.

[0108] In a preferred embodiment, the polypeptide is produced in mammalian cells.

[0109] The purified polypeptide can be analyzed by spectroscopic methods known in the art (e.g., circular dichromatic spectroscopy, Fourier transform infrared spectroscopy, and NMR spectroscopy or X-ray crystallography) to investigate the presence of desired structures such as helices and beta sheets. The binding of the polypeptide to broad-spectrum neutralizing antibodies such as CR8020 and / or CR9114 can be investigated using ELISA, AlphaLISA, label-free biolayer interferometry (Octet), and FACS. Therefore, polypeptides according to the present invention with accurate three-dimensional structures can be selected. Trimer content can be analyzed, for example, by SDS gel electrophoresis under non-reducing conditions, size exclusion chromatography in the presence of antibody Fab fragments of broad-spectrum neutralizing antibodies such as CR8020 and / or CR9114, and AlphaLISA using differently labeled antibodies. The stability of the polypeptide can be evaluated as described above after temperature stress, freeze-thaw cycles, increased protein concentration, or agitation. Furthermore, the melting temperature of the polypeptide can be evaluated by differential scanning fluorescence (DSF) and / or differential scanning calorimetry (DSC).

[0110] In certain embodiments, the nucleic acid is inserted into a recombinant vector which can be used as a vaccine component. Preferably, the recombinant vector is a human adenovirus, for example, human adenovirus serotype 26 (Ad26). Thus, the present invention also provides a recombinant adenovirus vector comprising a nucleic acid molecule encoding the HA stem polypeptide according to the present invention. In preferred embodiments, the nucleic acid molecule encoding the stem polypeptide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 208 and SEQ ID NOs: 209.

[0111] The preparation of recombinant adenovirus vectors is well known in the art. The term “recombinant” in relation to adenovirus, as used herein, means artificially modified, for example, having actively cloned modified ends and / or containing heterologous genes, i.e., not being a naturally occurring wild-type adenovirus. In certain embodiments, the adenovirus vector according to the present invention lacks at least one essential gene function in the E1 region of the adenovirus genome necessary for viral replication, e.g., the E1a region and / or the E1b region. In certain embodiments, the adenovirus vector according to the present invention lacks at least a portion of the non-essential E3 region. In certain embodiments, the vector lacks at least one essential gene function in the E1 region and at least a portion of the non-essential E3 region. An adenovirus vector may be “multiple deletion,” meaning that the adenovirus vector lacks one or more essential gene functions in each of two or more regions of the adenovirus genome. For example, the aforementioned El-deficient or E1, E3-deficient adenovirus vectors may further lack at least one essential gene in the E4 region and / or at least one essential gene in the E2 region (e.g., the E2A region and / or the E2B region). Adenovirus vectors, methods for constructing the same and methods for propagating the same are well known in the art and are described, for example, in U.S. Patent Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and 6,113,913.

[0112] In certain embodiments, the adenovirus is a human adenovirus of serotype 26 or 35.

[0113] The present invention further provides pharmaceutical compositions comprising polypeptides, nucleic acids, and / or vectors according to the present invention, and pharmaceutically acceptable carriers. The present invention particularly relates to pharmaceutical compositions comprising therapeutically effective amounts of the polypeptides, nucleic acids, and / or vectors of the present invention. The pharmaceutical compositions further comprise pharmaceutically acceptable carriers. In this context, the term “pharmaceutically acceptable” means that, at the doses and concentrations in which the carriers are used, they do not cause any undesirable or adverse effects in the subjects to whom they are administered. Such pharmaceutically acceptable carriers or excipients are well known in the art (see, for example, Remington: The Science and Practice of Pharmacy - 22nd edition, Loyd V. Ed. Allen, Pharmaceutical Press

[2013] ; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis

[2000] ; Remington: Essentials of Pharmaceutics, Linda Felton, Pharmaceutical Press

[2013] , and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press

[2000] ). The term “carrier” refers to a diluent, excipient, or vehicle together with which polypeptides, nucleic acids, and / or vectors are administered. Physiological saline, as well as aqueous dextrose and glycerol solutions, can be used, for example, as liquid carriers, particularly for injection.

[0114] The polypeptide or nucleic acid molecules of the present invention can also be administered in combination with or conjugated to nanoparticles, such as polymers, liposomes, viromosomes, or virus-like particles. The polypeptide or nucleic acid molecules can be combined with nanoparticles, capsidated with nanoparticles, or conjugated to nanoparticles (e.g., covalently or by adsorption).

[0115] The present invention further relates to polypeptides, nucleic acids, and / or vectors described herein for use as pharmaceuticals.

[0116] The present invention relates in particular to polypeptides, nucleic acids, and / or vectors described herein for use in inducing an immune response to influenza viruses, particularly against group 2 influenza viruses.

[0117] The present invention also provides a method for inducing an immune response to influenza A virus in a subject requiring such response, comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule and / or vector described herein. The subject according to the present invention is preferably a mammal that can be infected with the influenza virus or otherwise can benefit from the induction of an immune response, such subjects being, for example, rodents such as mice, ferrets, or domestic or farm animals, or non-human primates, or humans. Preferably, the subject is a human subject.

[0118] In certain embodiments, the present invention provides a method for inducing an immune response to a group 2 influenza A virus. The immune response may include humoral (i.e., induction of influenza virus neutralizing antibodies) and / or cellular immune responses. In certain embodiments, the present invention provides a method for inducing an immune response to at least one, two, three, four, five, or six subtypes of a group 2 influenza virus. In certain embodiments, the present invention provides a method for inducing an immune response to an influenza virus containing HA of the H3 subtype.

[0119] In certain embodiments, the induced immune response is effective in preventing influenza virus infection caused by group 2 influenza A viruses, such as influenza A viruses containing H3 subtype HA and / or influenza A viruses containing H7 subtype HA. In certain embodiments, the induced immune response is effective in preventing influenza virus infection caused by influenza A viruses containing H3 subtype HA. In certain embodiments, the induced immune response is effective in preventing influenza virus infection caused by influenza A viruses containing H3 and H7 subtype HA.

[0120] The present invention further relates to polypeptides, nucleic acids, and / or vectors described herein for use as influenza vaccines, particularly for use as vaccines against influenza caused by group 2 influenza virus strains.

[0121] In certain embodiments, the polypeptides, nucleic acid molecules, and / or vectors of the present invention are administered in combination with an adjuvant. The adjuvant may be administered before, simultaneously with, or after the administration of the polypeptides, nucleic acid molecules, and / or vectors of the present invention. Examples of suitable adjuvants include aluminum salts, e.g., aluminum hydroxide and / or aluminum phosphate; oil-emulsion compositions (or oil-in-water compositions), e.g., squalene-water emulsions, e.g., MF59 (see, for example, International Publication No. 90 / 14837); saponin formulations, e.g., QS21 and immunostimulatory complexes (ISCOM), etc. (see, for example, U.S. Patent No. 5,057,540, International Publication No. 90 / 03184, International Publication No. 96 / 11711) See International Publication No. 2004 / 004762 and International Publication No. 2005 / 002620; examples include bacterial or microbial derivatives (such as monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL) (optionally incorporated into liposomes), CpG motif-containing oligonucleotides, ADP-ribosylated bacterial toxins or their variants, e.g., E. coli (E. coli) heat-unstable enterotoxin LT, cholera toxin CT, pertussis toxin PT, or tetanus toxoid TT), Matrix M, or combinations thereof. Furthermore, known immunoenhancing techniques, such as the fusion of the polypeptides of the present invention to proteins known in the art to enhance immune responses (e.g., tetanus toxoid, CRM197, rCTB, or bacterial flagellin), or the incorporation of the polypeptides into visomes, or combinations thereof, can be used.

[0122] Administration of polypeptides, nucleic acid molecules, and / or vectors according to the present invention can be carried out using standard administration routes. Non-limiting examples include parenteral administration, e.g., intravenous, intradermal, transdermal, intramuscular, subcutaneous, or mucosal administration, e.g., intranasal, oral. Those skilled in the art will be able to determine the various possibilities for inducing an immune response by administering polypeptides, nucleic acid molecules, and / or vectors according to the present invention.

[0123] In certain embodiments, polypeptides, nucleic acid molecules, and / or vectors are administered in two or more doses, i.e., in so-called homogeneous prime boost regimens. The second dose can be administered up to several years after the first dose, for example, one week after the first dose, two weeks after the first dose, three weeks after the first dose, one month after the first dose, six weeks after the first dose, two months after the first dose, three months after the first dose, or four months or more after the first dose. It is also possible to administer polypeptides, nucleic acid molecules, and / or vectors three or more times (e.g., three, four times, etc.) so that two or more boost doses follow the first prime dose.

[0124] Polypeptides, nucleic acid molecules, and / or vectors can also be administered as a prime or booster in a heterologous prime-boost regimen.

[0125] The present invention provides a method for preventing influenza virus disease in a subject requiring such prevention, further comprising administering to the subject a therapeutically effective amount of the HA polypeptide, immunogenic fragment, nucleic acid molecule, and / or vector described herein. A therapeutically effective amount refers to the amount of polypeptide, nucleic acid molecule, or vector effective in preventing, relieving, and / or treating a disease or condition resulting from influenza infection. Prevention includes inhibiting or reducing the spread of the influenza virus, or inhibiting or reducing the onset, manifestation, or progression of one or more symptoms associated with influenza virus infection. Remission, as used herein, may refer to the reduction of visible or perceptible symptoms of the disease, viremia, or any other measurable signs of influenza infection.

[0126] Subjects requiring treatment include those already suffering from conditions resulting from influenza virus infection, and those for whom influenza virus infection should be prevented. Therefore, the polypeptides, nucleic acids, and / or vectors of the present invention can be administered to untreated subjects, i.e., subjects who do not have diseases caused by influenza virus infection, or who have never been infected with influenza virus and are not currently infected, or subjects who are already infected with influenza virus.

[0127] In one embodiment, prevention can be targeted to patient groups susceptible to influenza virus infection. Such patient groups include, but are not limited to, the elderly (e.g., ≥50, ≥60, preferably ≥65), younger individuals (e.g., ≤5, ≤1), hospitalized patients, immunocompromised subjects, and patients who have been treated with antiviral compounds but have not shown an adequate antiviral response.

[0128] The polypeptides, nucleic acid molecules, and / or vectors of the present invention may be administered to a subject in combination with one or more other active agents, such as surrogate influenza vaccines, monoclonal antibodies, antiviral agents, antibacterial agents, and / or immunomodulators. One or more other active agents may be beneficial for the treatment and / or prevention of influenza viral disease, or may improve symptoms or conditions associated with influenza viral disease. In some embodiments, one or more other active agents are analgesics, antipyretics, or therapeutic agents that relieve or assist respiration.

[0129] The present invention will be further illustrated by the following embodiments and drawings. The embodiments are not intended to limit the scope of the present invention. [Examples]

[0130] Example 1: HA stem-based polypeptide - Structure and design elements of preferred polypeptides of the present invention, UFV180088, UFV180089 and UFV180090 Polypeptide UFV180088, representing the stem (or stalk) of uncleaved influenza virus hemagglutinin (HA0) derived from H3 influenza virus A / Hong Kong / 1 / 68, was prepared by deleting at least a portion of the head domain derived from HA1, particularly the region containing amino acids from position 47 to 306 (Figures 1A and 1B). Note that the H3 numbering by Winter et al. (previously cited) is used for the amino acid position numbering in this invention. The main structural elements of the polypeptide (mini-HA) of this invention, including the A helix, B loop, and C, D, and E helices, are shown in Figure 1C.

[0131] When expressed as a soluble ectodomain, the polypeptide of the present invention is truncated at the C-terminus after the last helix (ending at position 499). UFV180088 was cleaved at position 506; that is, the C-terminal portion of the HA sequence starting from amino acid 506 was deleted.

[0132] The polypeptide UFV180088 of the present invention was made resistant to protease cleavage by a mutation, for example, glutamine (Q), at the natural monobasic cleavage site amino acid arginine (R) at position 329 (i.e., the C-terminal amino acid of the HA1 domain, see Figure 1), as described in this example. In contrast to the natural full-length HA, the polypeptide of the present invention containing the mutation R329Q can no longer be cleaved and is not subject to the associated structural changes that would embed a hydrophobic fusion peptide within the protein.

[0133] Removal of the head domain leaves behind a portion of the molecule already shielded from the aqueous solvent to which it is exposed. For this reason, the stem polypeptide was stabilized by mutating several amino acid residues in the B loop, namely the region containing amino acids 385-404 (Figure 1C), compared to the parent wild-type full-length HA derived from A / Hong Kong / 1 / 1968. In particular, the amino acid at position 388 was mutated to M, and the amino acid at position 392 was mutated to S.

[0134] Furthermore, to reduce the helix tendency of the B-loop, proline was introduced, particularly at position 399. Finally, to shield potential neoepitopes within the B-loop, one or two N-linked glycosylation motifs (i.e., NxT) were introduced into the B-loop, specifically at positions 393–395 for N-linked glycosylation at position 393, and at positions 401–403 for N-linked glycosylation at position 401.

[0135] Furthermore, to promote the stable trimerization of polypeptides derived from soluble HA stems, the trimerization domain sequence derived from GCN4 is used. 405 PMKCIEDKIEEIESK 419 (SEQ ID NO: 12) was introduced into the HA2 domain, specifically the C helix, substituting amino acids from position 405 to 419 of the original (i.e., wild-type) amino acid sequence.

[0136] Furthermore, cysteine ​​(if not already present) was introduced at positions 398 and 408 (position 408 being located within the introduced GCN4 sequence), forming an interprotomer disulfide bridge between the cysteine ​​at position 398 of the first monomer and the cysteine ​​at position 408 of the adjacent monomer, thereby covalently bonding the monomer to the trimer stem polypeptide.

[0137] To further stabilize and increase the expression of polypeptide UFV180088, and to ensure accurate folding similar to that of the wild-type full-length HA stem, further mutations were introduced into the polypeptide, particularly at positions 31 (D31E), 34 (I34V), 310 (K310C), 355 (H355W), 378 (N378T), 379 (379N), 380 (K380I), 381 (L381V), 422 (S422C), 432 (E432I), 435 (H435R), and 439 (L439Y) (Figure 1D).

[0138] Variants of polypeptide UFV180088, namely UFV180089 and UFV180090, were prepared. These polypeptides contained further mutations compared to UFV180088. Thus, UFV180089 contained additional mutations (compared to UFV180088) L367Y, N475D, A476D, and E479A. UFV180090 contained additional mutations (compared to UFV 180088) L25K, L367Y, A476D, and E479A, but did not contain mutations G379N and L381V.

[0139] Example 2: Expression, purification, and in vitro characterization of the trimer polypeptide of the present invention Protein expression in mammalian cells DNA fragments encoding the polypeptides UFV180088, UFV180089, and UFV180090 (described in Example 1) of the present invention were synthesized (Genscript) and cloned into a pcDNA2004 expression vector (pcDNA3 plasmid with an in-house modified enhanced CMV promoter). ExpiFectamine TM Polypeptides were produced in ExpiCHO suspension cells by transiently transfecting each industrial-grade DNA using transfection reagents (Gibco, ThermoFisher Scientific) according to the manufacturer's protocol. TM Cells were cultured in expression medium. Following the manufacturer's protocol, ExpiFectamine CHO enhancer and ExpiCHO feed (Gibco, ThermoFisher Scientific) were added to the cell culture one day after transfection. The culture supernatant containing the secreted polypeptide was collected between days 7 and 11, clarified by centrifugation, and then filtered through a 0.2 μm bottle-top filter (Corning).

[0140] Protein purification Polypeptides were purified using a two-step protocol. First, the recovered and clarified culture supernatant was loaded onto a HiScale 16 / 20 column (GE Healthcare) packed with affinity resin (Capture Select) consisting of a C-tag specific single-domain antibody immobilized on agarose-based beads (ThermoFisher Scientific). This resin is highly specific to the C-tag, i.e., the four-residue acid peptide (EPEA (SEQ ID NO: 25)) fused to the C-terminus of the polypeptide. The amount of the target polypeptide in the recovered culture supernatant was determined by OCTET before purification (see the section on culture supernatant and purified protein analysis). C-tagged proteins were eluted using Tris buffer containing 2M MgCl2. Based on the UV signal (A280), the eluted fractions were pooled and filtered through a Millex-GV 0.22 μm filter membrane (Merck Millipore). Next, the collected elution peaks were applied to a Superdex 200pg 26 / 60 column (GE Healthcare) equilibrated in electrophoresis buffer (20 mM Tris, 150 mM NaCl, pH 7.8) to remove potential multimeric and / or monomeric protein impurities. The trimer fractions were pooled and their purity was evaluated by analytical SEC-MALS.

[0141] Analysis of culture supernatant and purified protein As described above, the level of expressed stem polypeptides in the recovered culture supernatant was evaluated before purification using biolayer interferometry with the OCTET platform (ForteBio). In short, CaptureSelect TM A biotin anti-C tag conjugate (ThermoFisher Scientific) was immobilized on a streptavidin (SA) biosensor (ForteBio), and a standard curve was established by evaluating the binding shift of a clear reference batch dilution series of purified homologous polypeptides. Subsequently, the binding shift of pre-diluted recovered culture supernatants (10- and 30-fold dilutions with kinetic buffer (ForteBio)) containing the polypeptide of the present invention was measured, and the polypeptide concentration was calculated using the established standard curve.

[0142] The trimer content of polypeptides in the culture supernatant and purified polypeptides was evaluated by size exclusion chromatography-multiangle light scattering (SEC-MALS) analysis using a high-performance liquid chromatography (HPLC) Infinity 1260 series setup (Agilent). 40 μg of each purified polypeptide was flowed through a TSK gel G3000SWxl column (Sigma-Aldrich) at 1 mL / min, and the molar mass of the eluted substance was measured using a miniDAWN Treos multiangle light scattering detector and an Optilab T-rEx differential refractive index detector (Wyatt Technology). The data were analyzed using the Astra6 software package (Wyatt Technology), and molecular weight calculations were derived from the refractive index signals.

[0143] The correct folding of the purified polypeptide of the present invention is determined by ELISA (antibody-conjugated EC). 50 The values ​​were evaluated. For this purpose, stem polypeptides were coated with a concentration of 10 nM and incubated with a dilution series of monoclonal antibody (mAb) CR9114 (described in International Publication 2013 / 007770) using 70 nM as the starting concentration. Antibody binding was determined by incubation with a secondary anti-human Fc HRP antibody (mouse anti-human IgG, Jackson ImmunoResearch) and visualized by adding a POD substrate. Readout was performed using an EnSight® multimode plate reader (PerkinElmer). EC 50 The values ​​were calculated using the Spotfire suite (Tibco Software Inc.).

[0144] The thermal stability of the purified polypeptide was determined by differential scanning fluorescence (DSF) by monitoring the fluorescence emission of Sypro Orange Dye (ThermoFisher Scientific) added to a 6 μg polypeptide solution. As the temperature was gradually increased from 25°C to 95°C (60°C / hour), the polypeptide unfolded, the fluorescent dye bound to the exposed hydrophobic residues, and a characteristic change in emission occurred. The melting curve was measured using a ViiA7 real-time PCR instrument (Applied BioSystems), and the Tm curve was analyzed using the Spotfire suite (Tibco Software Inc.). 50 The value was calculated. Tm 50 The value represents the temperature at which 50% of the protein unfolds, and therefore serves as a measure of the polypeptide's temperature stability.

[0145] Results and Conclusions Polypeptide expression levels and trimer content were determined in two independent 70 mL ExpiCHO transfections on day 9 post-transfection (Figure 2A). All polypeptides were well expressed. Polypeptide UFV180088, derived from H3N2 A / Hong Kong / 1 / 68, was expressed at a level of approximately 700 mg / L in the culture supernatant. Polypeptides UFV180089 and UFV180090, which are similar in design to polypeptide UFV180088 but with further modifications to the surface amino acids (i.e., modifying the surface to be more closely similar with H7 HA) L367Y, N475D, A476D, and E479A, as well as modified L25K, L367Y, N379G, V381L, A476D, and E479A, were expressed at levels of approximately 500 mg / L and approximately 350 mg / L, respectively.

[0146] Analysis of the crude cell culture supernatant by analytical SEC (Figure 2B, left panel) showed the presence of a distinct population of soluble trimer polypeptides (retention time approximately 8.3 minutes). Similar analysis also showed that a two-step purification protocol yielded very pure trimer polypeptides (Figure 2B, right panel). Furthermore, the trimer polypeptides were correctly folded and showed an epitope for the broad-spectrum neutralizing monoclonal antibody CR9114, which has an EC of less than 1 nM. 50 This was revealed by ELISA analysis, which showed strong CR9114 binding with a value (Figure 2C). Furthermore, the temperature at which 50% of the polypeptide unfolded was determined by DSF. All polypeptides were temperature stable, and the Tm values ​​for UFV180088, UFV180089, and UFV180090 were 66.6°C, 64.6°C, and 60.9°C, respectively. 50 The values ​​are shown (Figure 2D).

[0147] In conclusion, the polypeptide of the present invention described in this embodiment was purified from the cell culture supernatant as a well-expressed and properly folded trimer polypeptide.

[0148] Example 3: Characterization of single-point mutations in the polypeptide of the present invention (SEC profile) design To evaluate the contribution of the mutations introduced into the trimer polypeptide of the present invention (schematically shown in Figure 1), amino acids were modified to include polypeptide UFV180141 (containing all the features of UFV180088, i.e., deletion of the head region from amino acid 47 to amino acid 306 (i.e., deletion 47-306) and introduction of the trimerization region at positions 405-419 of the HA2 domain (note that the introduced GCN4 sequence is slightly different compared to UFV180088: i.e. 405 RMKCIEDKIEEIESK 419(SEQ ID NO: 11), containing cysteine ​​at the amino acid position corresponding to position 310 in combination with cysteine ​​at the position corresponding to position 422 (forming an intraprotomeral disulfide bridge), containing Q at position 329 (protease cleavage resistance), the amino acid at position 355 is W; the amino acid at position 378 is T, the amino acid at position 379 is N, the amino acid at position 381 is V; containing a glycan motif at positions 401-403), containing cysteine ​​at the position corresponding to position 398 in combination with cysteine ​​at the position corresponding to position 408 (in the GCN4 sequence) to form an interprotomeral disulfide bridge, containing M at position 388, E at position 31 and V at position 34, I at positions 380 and 432, S at position 392, T at position 395, S at position 399, N at position 435 and Y at position 439) from the skeletal strain A / Hong The original amino acids in Kong / 1 / 1968 (Table 1, Figure 3A) were reversed. Similar to UFV180088, polypeptide UFV180141 was truncated after the amino acid at position 506. However, UFV180141 lacks an additional glycosylation motif at positions 393-395, a B-loop stabilizing proline at position 405, and has 399S instead of 399P. Furthermore, UFV180088 contains the C-terminal tag EPEA (SEQ ID NO: 25), while UFV180141 contains a different C-terminal tag.

[0149] The exception was the C408Q mutation, which did not revert to wild-type H3 but rather reverted to the introduced GCN4 trimerization domain sequence (introduced at positions 405-419). The impact of the absence of specific mutations was evaluated by SEC analysis.

[0150] Another method for evaluating the beneficial effects of selected mutations is the stepwise introduction of those mutations into a minimally designed polypeptide, namely UFV180647, which includes the following features: deletion of the head region from amino acid 47 to amino acid 306 (i.e., deletion 47-306), introduction of the trimerization domain of the HA2 domain, namely at positions 405-419. 405 RMKCIEDKIEEIESK 419The introduction of (SEQ ID NO: 11); cysteine ​​at the position corresponding to position 422 is combined with cysteine ​​at the position corresponding to position 310 (forming an intraprotomer disulfide crosslink), Q (protease cleavage resistance) is at position 329, the amino acid at position 355 is W; the amino acid at position 378 is T, the amino acid at position 379 is N, and the amino acid at position 381 is V; a glycan motif is included at positions 401-403, cysteine ​​at the position corresponding to position 398 is combined with cysteine ​​at the position corresponding to position 408 (forming an interprotomer disulfide crosslink), and M is at position 388. The mutant constructs were analyzed by Analytic SEC and compared with the minimally designed polypeptide (Table 2, Figure 3C).

[0151] [Table 1]

[0152] [Table 2]

[0153] Protein expression in mammalian Expi293F cells DNA fragments encoding the polypeptides listed in Tables 1 and 2 were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG linker-His tag were produced on a microscale (200 μL) in the eukaryotic suspension cell line Expi293F for screening and purification purposes. In short, industrial-grade DNA was transiently transfected into cells using the ExpiFectamine293 transfection kit (Gibco, ThermoFisher Scientific) at a cell density of 2.5E+06 vc / mL in a 96 half-deep well plate (System Duetz), and incubated in a shaking flask containing Expi293 expression medium (Gibco, ThermoFisher Scientific) at 37°C, 250 rpm, 8% CO2, and 75% humidity. The cell culture supernatant containing the secreted polypeptide was collected on day 3, clarified by centrifugation (10 min, 400 × g), and then filtered (96-well filter plate, 0.22 μm PVDF membrane, Corning).

[0154] Analysis of the culture supernatant The content of the polypeptide of the present invention in the recovered Expi293 cell culture was evaluated by analytical SEC using an Agilent Infinity 1260 series high-performance liquid chromatography (HPLC) setup. 100 μL of culture supernatant was injected onto a TSK gel G3000SWxl column (Sigma-Aldrich) at a flow rate of 1 mL / min, and elution was monitored by UV detection (Figure 3A). Alternatively, the sample was analyzed by ultra-high-performance liquid chromatography (UHPLC) using a Vanquish system (ThermoFisher Scientific) equipped with a BEH 200A column (Waters, injection volume 40 μL, flow rate 0.35 mL / min), and the eluted fraction was monitored by a Helios light scattering detector (Wyatt Technologies, Figure 3C). The SEC profile was analyzed using the Astra 6 software package (Wyatt Technology). The elution time and trimer peak (height and shape) of the SEC profile are visualized in Figures 3B and 3D.

[0155] Results and Conclusions For example, a subset of revertant mutations, such as UFV180193 (W355H revertant), UFV180194 (GCN4 deletion), UFV180195 (intramolecular disulfide crosslink deletion), and UFV180199 (I432e revertant), appeared to be detrimental to stem polypeptide expression, while other revertant mutations were tolerated (Figures 3A and 3B).

[0156] Polypeptides lacking the stabilizing mutations introduced into UFV180088 (Table 2) showed a significant improvement in the expression level of the minimal trimer stem polypeptide when the mutations of the present invention were added incrementally (towards the design of UFV180088). For example, as shown in Figure 3C, the trimer peak of polypeptide UFV1801034 shifted its elution time and increased in height upon introduction of the mutations K380I and E432I (Figure 3D). Incremental addition of stabilizing mutations in the B loop (UFV181042: F392S, H393N, I395T, F399P, R405P) further increased the expression of the trimer polypeptide.

[0157] This example demonstrates that at least the following amino acid positions (e.g., mutations): namely, mutations to W at position 355, and / or mutations to T at position 378, mutations to N at position 379, mutations to V at position 381, and / or mutations to I at position 432, or mutations to I at positions 432 and 380, as well as glycosylation motifs at positions 401-403 for N-linked glycosylation at position 401, are beneficial for obtaining high levels of the desired soluble trimer polypeptide. Adding stabilizing mutations to the B loop (e.g., F392S, H393N, I395T, and F399P) further increases the expression of the trimer polypeptide.

[0158] Example 4: Interprotomer disulfide crosslinking; Stability of the polypeptide of the present invention design To evaluate the contribution of introduced cysteine ​​(schematically shown in Figure 4) to the B-loop and C-helix forming interprotomer disulfide crosslinks in the polypeptide of the present invention, cysteine ​​was reverted to the wild-type residue (glutamic acid) located at the A / Hong Kong / 1 / 1968 (position 398) of the respective skeleton strains, and to glutamine (residue 408) located in the introduced GCN4 trimerization domain sequence (405-419). The effects of omitting cysteine ​​and subsequent disulfide crosslinks were evaluated by analytical SEC, DSF, and SDS-PAGE.

[0159] Protein expression, purification, and characterization DNA fragments encoding polypeptides UFV180192 and UFV180141 were produced on a microscale in Expi293F cells as described in Example 3, and on an intermediate scale in ExpiCHO cells as described in Example 2 (approximately 60 mL, collected on day 8). The culture supernatant was analyzed by analytical SEC on the day of collection (microscale) or after incubation of the collected culture supernatant at 4°C for 1 week (intermediate scale), as in Example 2. From the collected culture supernatant (intermediate scale), the his-tagged polypeptide was purified using a two-step protocol with the AKTA Avant 25 system (GE Healthcare Life Sciences). In the first step, immobilized metal affinity chromatography was performed using a pre-packed cOmplete His-tagged purification column (Roche), washed with 1 mM imidazole, and eluted with 300 mM imidazole. In the second stage, size exclusion chromatography was performed using an SRT-10C SEC-300 column (Sepax Technologies), and the trimer peak fraction was recovered. The thermal stability of the purified protein was measured by DSF (as in Example 2), and the purity of the protein was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing and reducing conditions by running a 10% bis-tris gel using a Bolt system according to the manufacturer's instructions for use (Invitrogen).

[0160] Results and Conclusions Analysis of cell culture supernatants by SEC showed that both polypeptides with and without introduced cysteine ​​in the B-loop (position 398) and C-helix (position 408) (UFV180141) expressed trimer polypeptides in solution (Figure 4A; left panel). On the recovery day 3 days after small-scale transfection, polypeptide UFV180192 was more abundant than peptide UFV180141. Nevertheless, on day 8, higher-scale recovery after storing supernatant containing UFV180192 at 4°C for 1 week showed a second non-trimer peak (retention time of approximately 8.5 minutes), indicating structural instability of the construct lacking interprotomer disulfide crosslinking (Figure 4A; right panel). The difference in stability was confirmed by DSF evaluation of the purified polypeptides. Polypeptides lacking interprotomer disulfide crosslinks (UFV180192) unfolded at 50.8°C, while polypeptides with introduced cysteine ​​at positions 398 and 408 unfolded at 67.7°C (Figure 4B). SDS-PAGE analysis of the purified polypeptides showed that polypeptides without cysteine ​​at positions 398 and 408 migrated, as expected, with a monomer height of approximately 40 kDa under both non-reducing and reducing conditions. In contrast, polypeptides with two introduced cysteine ​​showed a major band with a monomer height of approximately 120 kDa under non-reducing conditions and approximately 40 kDa under reducing conditions, as expected for covalent trimers (Figure 4C).

[0161] The data presented in this example demonstrate that the covalent bonding of monomers by introducing two cysteine ​​groups at positions 398 and 408 to form an interprotomer disulfide bond results in a considerably more stable soluble trimer polypeptide.

[0162] Example 5: Alternative substitution of positions 355, 380, 342, 435, and 388. design To optimize the polypeptide of the present invention, alternative substitutions were tested at positions 355 (Figure 5A), 380 and 432 (Figure 5B), 435 (Figure 5C), and 388 (Figure 5D). Polypeptide expression and folding were evaluated in the supernatant of Expi293F cell culture.

[0163] Protein expression in mammalian cells DNA fragments encoding polypeptides were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG-linker-His tag for screening purposes were produced in Expi293F eukaryotic cells on a microscale (200 μL) as described in Example 3, with the exception of the polypeptides shown in Figures 5CII and 5D (on intermediate scales of 50 mL and 30 mL, respectively) produced in the ExpiCHO eukaryotic cell line as described in Example 2.

[0164] Analysis of the culture supernatant The expression, folding, and trimer content of the polypeptide of the present invention were evaluated by an amplified luminescence proximity homogeneity assay (AlphaLISA, Figures 5A, 5B, 5CI) according to the manufacturer's instructions for use (PerkinElmer). This lysis-binding equilibrium assay is based on the successful binding of both the donor beads and acceptor beads to the polypeptide. When in proximity, laser irradiation of the donor beads at 680 nm generates a stream of singlet oxygen, inducing a chemical event in the nearby acceptor beads, resulting in chemiluminescence at 615 nm. Expression levels were measured by an expression-AlphaLISA setup by simultaneously adding nickel donor beads (anti-His tag) and anti-FLAG tag acceptor beads to the cell culture supernatant. This expression-AlphaLISA setup recognizes the C-terminal Flag-Linker-His tag regardless of polypeptide folding. Correct polypeptide folding was evaluated in Binding-AlphaLISA by simultaneously adding nickel donor beads, human IgG CR9114 (2nM) or CT149 (1nM), and anti-human IgG acceptor beads to the cell culture supernatant. A signal can only be obtained if the polypeptide folds correctly and is capable of binding to influenza virus HA-specific IgG.

[0165] The content of trimer polypeptides present in the culture supernatant was determined using a trimer-AlphaLISA setup. This depends on human IgG such as CT149 or CR9114, which specifically binds to monomeric HA. When a 1:1 mixture of differently labeled IgG CT149 or CR9114 is added to HA, the AlphaLISA signal can only be detected if a multimer is present that allows binding to at least two antibodies, rather than a monomer that allows binding to only a single antibody. Trimer-AlphaLISA was performed by simultaneously adding streptavidin donor beads and anti-DIG IgG acceptor beads to the culture supernatant in the presence of biotinylated and DIG-labeled CT149 IgG or CR9114 IgG (0.5 nM each, 1:1 ratio).

[0166] Detection beads were added to all AlphaLISA setups at a concentration of 10 μg / mL. The culture supernatant was tested at different dilutions to avoid the hook effect, according to the manufacturer's instructions. After incubation in the dark at room temperature for 2 hours, data was read out using an EnSight® multimode plate reader (PerkinElmer). All data were normalized to their respective reference constructs set to 100%.

[0167] The levels of expressed polypeptides in cell culture supernatant were evaluated by biolayer interferometry using the OCTET platform according to the manufacturer's instructions for use (ForteBio). In short, a standard curve was established by measuring the binding shift of a dilution series of a clear, purified, homogeneous polypeptide reference batch using an anti-HIS (HIS2) biosensor (ForteBio). Subsequently, the binding shift of pre-diluted (in dynamic buffer, ForteBio) cell culture supernatant containing the polypeptide of the present invention was measured, and the polypeptide concentration was calculated using the established standard curve (Figures 5CII, 5D). The content of the polypeptide of the present invention in the culture recovery was evaluated by analytical SEC in HPLC (Figure 5CII) and UHPLC (Figure 5D, 10 μL injection volume) as described in Example 4. After purification of the selected polypeptides UFV171004 and UFV171197, binding to monoclonal antibodies CR9114 and CT149 was evaluated by ELISA. Finally, the thermal stability of these purified polypeptides was determined by DSF (as described in Example 2, Figure 5CII).

[0168] Results and Conclusions Introducing tryptophan (W) at position 355 of polypeptide UFV161739 significantly increased expression and antibody binding to CR9114 and CT149 compared to a construct with wild-type histidine at position 355 (UFV161333). Co-introduction of 355W and isoleucine (I) at position 482 (UFV161800) further increased expression and antibody binding compared to a construct with only the single mutant 355W. Co-introduction of phenylalanine (F) at position 355 and 482I was well tolerated, but the increase in expression and antibody binding was small compared to a control construct with wild-type residues at positions 355 and 482, respectively (Figure 5A).

[0169] Multiple amino acid residues and their combinations were evaluated at positions 380 and 432. Overall, the amino acid substitutions tested were well tolerated, and their impact on expression levels, trimer content, and antibody binding was very small (Figure 5B). Co-introduction of isoleucine at positions 380 and 432 in the polypeptide (UFV171004) resulted in the highest trimer yield (215%) compared to the reference molecule.

[0170] At position 435, four different amino acid substitutions (K, N, Q, R) were shown to be well tolerated. Each polypeptide and the reference polypeptide UFV170611 containing wild-type histidine at position 435 showed comparable AlphaLISA levels in terms of expression, trimer content, and binding to monoclonal antibodies CR9114 and CT149 (Figure 5CI). Further characterization of polypeptides with substitutions 435N (UFV171004) and 435R (UFV171197) confirmed similar expression levels (OCTET), SEC profiles, and binding sensitivities to mAb CR9114 and CT149 (ELISA). However, polypeptides with asparagine (N) at position 435 showed approximately 4°C higher thermal stability compared to polypeptides with arginine (R) substitution at this position (Figure 5CII).

[0171] Polypeptides with amino acid substitutions (M, V, I, L, F, Y, W, H, K, and R) at position 388, located at the top of the A-helix, were evaluated for expression level (Octet) and trimer content (analytical SEC). Expression levels were ranked from 475 mg / L for methionine substitution (UFV180088) to a less desirable expression level of 192 mg / L for arginine substitution. All SEC profiles showed trimer polypeptides, indicating that all evaluated residues at position 388 were acceptable and did not affect the overall structure (Figure 5D). All polypeptides with amino acid substitutions at position 388 eluted with shorter retention times compared to the reference polypeptide (UFV180088) due to differences in the length of the C-terminal tags (Flag-linker-His tag and C-tag, respectively).

[0172] In summary, single alternative amino acid substitutions are possible and well tolerated in various optimization regions of the polypeptide of the present invention, with minimal impact on expression levels, trimer content, and protein folding.

[0173] Example 6: Clarification of HA1 head loss design The stem polypeptide of the present invention preferably contains a deletion from amino acids 47 to 306 (schematically shown in Figure 1). In this example, alternative deletions and linkers derived from the head domain between the HA1 terminals that result after the deletion were searched for (schematically shown in Figure 6). First, the alternative deletion sites on the HA1 upper chain were evaluated (Table 3). The HA1 terminals present in the reference design (UFV161908, which has a deletion from amino acids 47 to 306 and is substituted with a GPGS linker) are shown in gray.

[0174] [Table 3]

[0175] Secondly, alternative deletion sites in the HA1 upper chain and alternative deletion sites in the HA1 lower chain were combined (Table 4).

[0176] [Table 4]

[0177] Thirdly, we investigated the use of homologous linkers containing amino acid sequences derived from the removed head domain to ligate the HA1 terminus (Table 5).

[0178] [Table 5]

[0179] Protein expression and culture supernatant analysis in mammalian cells DNA fragments encoding the polypeptide of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG-linker-His tag for screening purposes were produced in microscale (200 μL) in Expi293F eukaryotic cells as described in Example 3. Polypeptide expression, trimer content, and folding (mAb binding of CR9114 or CT149) were evaluated by AlphaLISA as described in Example 5. All data were normalized to their respective reference constructs UFV161908 (Figure 6A), UFV160653 (Figure 6B), and UFV160321 (Figure 6C), set to 100%.

[0180] Results and Conclusions Alteration of the head domain deletion site in the HA1 upper chain had minimal impact on expression levels, trimer yield, and protein folding (i.e., antibody binding) (Figure 6A). In contrast, expression of polypeptides containing alternative deletion sites in both the HA1 upper and lower chains reduced expression levels by approximately 50% and antibody CT149 binding by approximately 70% (Figure 6B). This indicates that deletion sites within the tested range, particularly those on the HA1 lower chain, affect both polypeptide expression and folding.

[0181] An alternative to directly ligating the remaining HA1 ends after head domain removal (i.e., the N-terminal and C-terminal HA1 fragments) is ligation via homologous ligation sequences. As shown in Table 5, for this purpose, the HA1 upper chain (residue 45) was ligated to the HA1 lower chain (residue 307) by a short sequence derived from the corresponding H3 HA head domain. This resulted in polypeptides with expression levels altered by 33% to 223% compared to the reference (Figure 6C). Similarly, antibody CT149 binding showed a wide range, with values ​​observed in the range of 57% to 350% compared to the reference. The well-expressed polypeptides also showed high antibody binding for the majority.

[0182] Overall, the generation of correctly folded stem polypeptides by deleting the HA head domain does not depend on a single precise deletion site. Direct ligation of the HA1 terminus successfully generated variations in deletion sites in both the upper and lower chains of HA1. Alternatively, ligation of the HA1 terminus by introducing homologous amino acid linkers derived from the head domain was also possible, allowing for the selection of numerous peptides with different sequence compositions and lengths (at least 2-5 amino acids) to religate the HA1 terminus after head domain removal.

[0183] Example 7: Optimization of the B-loop in the polypeptide of the present invention design After deletion of the HA1 head domain, the B loop of the HA stem polypeptide (Figures 1B and C, including amino acids 385-404) is exposed. To partially shield this region from the immune system and further stabilize the loop, glycosylation motifs and proline residues were introduced (Figure 7). The glycosylation motif (NxT) was introduced at positions 401-403 by mutations E401N and E403T for N-linked glycosylation at position 401, and tested in combination with an N-linked glycan motif at positions 398-400 by a mutation S398N (which produces an NxT motif), or an N-linked glycan motif at positions 392-394 by mutations S392N and Q394T, or an N-linked glycan motif at positions 393-395 by mutations H393N and I395T (both which produce an NxT motif). Proline substitutions that can reduce the helix tendency of B-loop sequences were introduced by single-point mutations at any position between 385 and 406, and by double-point mutations at 392 and 396 or 398.

[0184] Protein expression and culture supernatant analysis in mammalian cells DNA fragments encoding the polypeptide of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG-linker-His tag for screening purposes were produced on a microscale (200 μL) in the Expi293F eukaryotic cell line as described in Example 3. Polypeptide expression, trimer content, and folding (binding of mAb CR9114, CT149 (described by Wu et al. (2015)), and SD15013 (containing the amino acid sequence of SEQ ID NO: 39)) were evaluated by AlphaLISA as described in Example 5 (binding of SD15013 was evaluated using anti-His acceptor beads and streptavidin donor beads in the presence of 2 nM SD15013). Cell culture supernatants were analyzed by analytical SEC on the day of collection as described in Example 2.

[0185] All data was normalized to the respective reference constructs UFV161686 (Figure 8A), UFV161333 (Figures 7B and C), and UFV171187 (Figure 7D), which were set to 100%.

[0186] Results and Conclusions Introducing a single glycosylation motif into the B-loop by mutating the residues at positions 401, 402, and 403 to N, A, and T, respectively, for N-linked glycosylation at position 401, resulted in a twofold increase in polypeptide expression levels. Similarly, significant increases in binding were observed with antibodies CR9114 and CT149 (5-fold and 7-fold, respectively), and with the single-domain SD15013 (10-fold) (Figure 7A).

[0187] The introduction of proline into the B-loop did not affect polypeptide expression levels, with values ​​changing from 94% to 128% compared to the reference construct (Figure 7B). In contrast, the addition of proline residues at positions 386, 387, 388, and 389 was detrimental to antibody binding. Minimal binding of CR9114, CT149, and SD15013 showed that when proline was introduced to the N-terminus of the B-loop, it negatively affected polypeptide folding. Introducing a single proline at any of positions 390–405, or two prolines at positions 392 and 396 or 392 and 398, increased CR9114 binding by approximately 40%, while CT149 binding remained relatively similar or decreased (approximately 65% ​​compared to the reference). SD15013 showed the greatest spread in these constructs, with relative binding ranging from approximately 50% to approximately 150% compared to the reference. The introduction of two prolines was well tolerated, and the binding of CR9114, CT149, and SD15013 was generally found to be at the average antibody binding value for single proline introduction. An exception was observed for the binding of SD15013 to UFV161708, showing 147% binding, but decreased binding was observed in single mutations at positions 392 and 396 (64% and 75% compared to the reference).

[0188] The introduction of a second glycosylation motif into the B loop was well tolerated (Figure 7C), and both polypeptides with the additional motif at position 393 (UFV161715) or with the additional motif at position 398 (UFV161721) both showed relatively similar expression levels compared to the reference, with increased CR9114 binding (approximately 145%), no effect on CT149 binding (approximately 90%), and a slight decrease in SD15013 binding (approximately 60%).

[0189] Simultaneous introduction of a proline residue and / or a second glycosylation motif (positions 392-395) resulted in polypeptides with approximately twice the expression of a reference molecule containing a single glycosylation motif at positions 401-403 and no proline (Figure 7D). Antibody binding (CR9114 and CT149) was largely unaffected, but a decrease in SD15013 was observed (approximately 48-88% compared to the reference). Given the low likelihood of simultaneous glycosylation of N398 and N401 due to their proximity, additional glycans at N393 or N392 are preferred.

[0190] SEC-MALS analysis of EXPI-293 cell culture supernatants containing the polypeptide of the present invention having one N-linked glycan motif (UFV180208) or two N-linked glycan motifs and two prolines (UFV180217) shows distinct peaks corresponding to each trimer polypeptide (Figure 7E).

[0191] Mutations in the morphology of the B-loop, including stabilizing proline residues and additional N-linked glycosylation motifs, were well tolerated. Although differences in expression and antibody binding were observed (most prominent with SD15013 binding), the introduction of proline, except for the N-terminal region of the B-loop (positions 392–389) and the second N-linked glycosylation motif, was possible without affecting protein folding and trimerization.

[0192] Example 8: N-linked glycosylated motif at position 38 of the polypeptide of the present invention design To evaluate the effects on the expression and folding of a conserved glycosylation motif close to the CR9114 epitope (position 38), a polypeptide containing the wild-type motif 38-NAT-40 for N-linked glycosylation (UFV170282) was compared with a polypeptide in which the motif was knocked out by the point mutation T40I (UFV170278).

[0193] Protein expression, culture supernatant analysis, purification, and characterization in mammalian cells The DNA fragment encoding the polypeptide of the present invention was synthesized, expressed, and purified as described in Example 2. The level of the expressed polypeptide in the culture supernatant was evaluated using a 25-fold diluted cell culture supernatant containing immobilized mAb CT149 and the polypeptide of the present invention, using biolayer interferometry on the OCTET platform as described in Example 2. The binding strength of the antibody to the purified polypeptide was evaluated using ELISA (EC) as described in Example 2. 50 It was evaluated by ).

[0194] Results and Conclusions Removal of the N-linked glycosylation motif at position 38 (UFV170278) resulted in an approximately 50% decrease in expression level compared to the polypeptide containing the motif (UFV170282). However, both polypeptides maintained sufficient expression, with values ​​exceeding 255 mg per liter of culture supernatant. ELISA measurement revealed that the antibody binding strength for CR9114 and CT149 was EC2. 50 As is evident from the value of approximately 1 nM, there was no significant difference between the two polypeptides (Figures 8A and 8B).

[0195] In conclusion, removal of the N-linked glycosylation motif at position 38 was well tolerable, although a decrease in expression levels was observed, and did not appear to affect polypeptide folding.

[0196] Example 9: Alternative position of intramolecular disulfide bridge between HA1 and HA2 design The protomer of the trimer stem polypeptide of the present invention is preferably stabilized by introducing a disulfide bridge that covalently bonds the HA1 lower chain (position 310) to the C helix (position 422) of the HA2 chain. Alternative options for this intraprotomer disulfide bridge were evaluated by slightly shifting the precise positions of each cysteine ​​(positions 311 / 422 and 308 / 418). In addition to the 310 / 422 disulfide bridge, a second pair of cysteines was evaluated to link HA1 (position 26) to the C-terminal portion (position 433) of the HA2 C helix.

[0197] Protein expression and culture supernatant analysis in mammalian cells A DNA fragment encoding the polypeptide of the present invention was synthesized as described in Example 2. The polypeptide was expressed in Expi-293 cells as described in Example 2, except that the experimental scale was changed from a microscale (200 μL) to an intermediate scale (30 mL) of culture growth. The expression level and folding (binding of mAb CR9114 and CT149) of the polypeptide of the present invention were evaluated by AlphaLISA as described in Example 5, using CR9114 and CT149 concentrations of 2.5 nM and 1.25 nM, respectively.

[0198] Results and Conclusions Rearrangement of cysteine ​​from residue 310 to 311 or 308 (in combination with rearrangement of cysteine ​​from position 422 to 418) was well tolerated and had only minimal effect on polypeptide expression levels and folding, as evidenced by antibody binding (Figure 9A). Introducing a second disulfide crosslink in the region below the 310-422 disulfide crosslink was possible in principle, as indicated by the unaffected expression levels; however, the dramatic reduction in CR9114 and CT149 binding (2% and 48% relative to the reference, respectively) negatively impacted the folding of the desired conserved stem epitope (Figure 9B).

[0199] Example 10: Alternative location of interprotomer disulfide bridges design The protomers of the HA stem polypeptide are preferably covalently linked by interprotomeral disulfide crosslinks at the top of the trimer HA protein (Figure 4A). Two cysteine ​​residues are introduced, one in the B loop (position 398) and the other in the C helix (position 408), both of which pair with sterically adjacent cysteines in adjacent protomers within the trimer; that is, cysteine ​​398 of protomer 1 forms a disulfide bond with cysteine ​​408 of protomer 2, cysteine ​​398 of protomer 2 forms a disulfide bond with cysteine ​​408 of protomer 3, and cysteine ​​398 of protomer 3 forms a disulfide bond with cysteine ​​408 of protomer 1. Alternative options for this interprotomeral disulfide bond were explored by slightly shifting the precise location of the point mutation to cysteine ​​upward or downward (Figure 10).

[0200] Protein expression and culture supernatant analysis in mammalian cells DNA fragments encoding the polypeptide of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG-linker-His tag for screening purposes were produced on a microscale (200 μL) in the Expi293F eukaryotic cell line as described in Example 3. The expression, trimer content, and folding (binding of mAb CR9114 or CT149) of the polypeptide of the present invention were evaluated by AlphaLISA as described in Example 5.

[0201] Results and Conclusions Polypeptides with introduced interprotomeral disulfides showed approximately 1.8 times lower expression compared to polypeptides without these cysteines. However, the difference in trimerization was dramatic (Figure 10). Polypeptides with interprotomeral disulfides were expressed at the same level and all showed similarly high levels of trimerization and binding affinity to antibodies CR9114 and CT149. This demonstrates the critical importance of interprotomeral disulfide crosslinking for trimerization and correct folding of conserved stem epitopes. Furthermore, slight changes in the position of the interprotomeral disulfide crosslinks were well tolerated.

[0202] Example 11: Alternative truncation at the C-terminus design Influenza virus hemagglutinin is a membrane protein located on the surface of the virus particle, with its C-terminal portion embedded in the viral membrane. In the soluble version of the polypeptide of the present invention, the ectodomain can be truncated at different positions within the innate linker sequence (positions 500-513) that links the C-terminal alpha-helix of the ectodomain to the transmembrane (TM) domain and the cytoplasmic domain.

[0203] Alternative C-terminal cleavage locations were evaluated (Table 6).

[0204] [Table 6]

[0205] Analysis of cell culture supernatant The DNA fragments encoding the polypeptides listed in Table 6 were synthesized as described in Example 3 and expressed in suspended EXPI-293 cell cultures as described in Example 4.

[0206] The cell culture supernatant was collected and the trimeric polypeptide level was analyzed by analytical SEC using HPLC as described in Example 4A. The correct folding of the expressed polypeptide of the present invention was evaluated in the cell culture supernatant by using the OCTET platform by the biolayer interferometry method as described in Example 6. Briefly, the supernatant diluted 5-fold with kinetic buffer (ForteBio) was evaluated for binding to biotinylated human monoclonal antibody CR9114 or CT149 (10 μg / mL) loaded on a streptavidin biosensor (ForteBio). Curve fitting was performed for the first 100 seconds of the association step to calculate the K ON value and the curve was fitted to a 1:1 model. A MOCK sample was included as a negative control.

[0207] Results and Conclusions C-terminal truncation between residues 501 and 513 was well tolerated and had little effect on trimer formation, expression levels, and antibody binding. The truncated polypeptide showed a similar trimer peak pattern in SEC analysis (Figure 11A) and, in Octet analysis, good or improved K ON values for CR9114 and CT149 binding (Figure 11B). When the truncation (after position 499 in UFV171280) reached the C-terminal helix of the ectodomain, a clear decrease in trimer expression and antibody binding levels occurred, as shown by SEC and OCTET analyses.

[0208] Example 12: Substitution Mutations in the A Helix of the Polypeptide of the Present Invention Design The positioning and folding of the C-terminal portion of the A-helix of the polypeptide of the present invention are crucial for the accurate representation of the conserved stem epitope. To find the optimal structure, three residues of the A-helix (378, 379, and 381) were mutated with residues derived from either group 1HA (H1 A / Brisbane / 59 / 07) or group 2HA (H3 A / Hong Kong / 1 / 1968) at this position. Furthermore, the putative A-helix stabilizing mutant G379A was evaluated.

[0209] Protein expression, culture supernatant analysis, purification, and characterization in mammalian cells DNA fragments encoding the polypeptide of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG-linker-His tag for screening purposes were produced on a microscale (200 μL) in the Expi293F eukaryotic cell line as described in Example 3. Polypeptide expression, trimer content, and folding (by binding of 2.5 nM mAb CR9114 or CT149) were evaluated by AlphaLISA as described in Example 5. Furthermore, the polypeptide was expressed on an intermediate scale (50 mL) in EXPI-CHO cells as described in Example 4, and the expression level was determined by biolayer interferometry as described in Example 5. The crude cell culture supernatant was analyzed by SEC-MALS by high-performance liquid chromatography (HPLC) as described in Example 3. The polypeptide was purified by a two-step protocol of affinity chromatography and size exclusion chromatography as described in Example 4. The antigenicity of the purified polypeptide was evaluated by ELISA (EC9114 and CT149 antibody-conjugated ELISA). 50 The temperature at which 50% of the polypeptide unfolds was evaluated by the value and determined by DSF, as described in Example 2.

[0210] Results and Conclusions The gradual increase in the amount of H1-derived residues at positions 378, 379, and 381 affected polypeptide expression levels. Polypeptide UFV161448, containing all three H1-like residues, was the least expressed polypeptide (84%), while UFV161451, containing H1-like residues at positions 379 and 381, was the most expressed polypeptide (163%). Polypeptides containing putative A-helix stabilizing mutant 379A (UFV161459 and UFV161458) expressed the worst levels (42% and 75%, respectively). Correct polypeptide folding was assessed using AlphaLISA. The relative signals for both CR9114 and CT149 binding showed significant spread. Polypeptide UFV161453 (379N) showed minimal binding of 61% and 24% to CR9114 and CT149, respectively. Polypeptide UFV161448, containing all mutations to H1 (378T, 379N, and 381V), showed the highest binding rates to CR9114 and CT149, at 1706% and 841%, respectively (Figure 12A).

[0211] The effects of A-helix mutations were further studied, particularly in the more stabilized trimer stem polypeptides UFV171004 and (UFV171116), including the H355W mutation and alternative interprotomer disulfide crosslinks (at 397 / 408). In three independent intermediate scales of ExpiCHO production, UFV171004 (containing H1 residues at positions 378, 379, and 381 of the A-helix) was expressed at slightly higher levels than polypeptide UFV171116 (containing H3 residues at those positions).

[0212] Similarly, minimal differences were observed in SEC-MALS analysis of the culture supernatant, and in both constructs, the peaks corresponding to the trimer fraction (retention time of approximately 8 minutes) overlapped in shape and height. Binding of antibodies CR9114 and CT149 as a measure of correct protein folding was determined by ELISA, and strong binding (EC) was confirmed. 50It showed a <0.01nM) temperature stability. Temperature stability determined by DSF showed a significant difference between the two polypeptides. 50% of polypeptide UFV171116 unfolded at 66.2°C, while polypeptide UFV171004 unfolded at 68.0°C. 50 The values ​​are shown (Figure 12B).

[0213] In summary, introducing an H1 residue into the A helix of H3-based stem polypeptides resulted in polypeptides that were more thermally stable and showed increased CR9114 and CT149 binding. The difference in antibody binding was not very dramatic, particularly in constructs UFV171004 and UFV171116, which contained the stabilizing mutant H355W.

[0214] Example 13: Introduction of surface mutation to H7 design The trimer stem polypeptide of the present invention is based on HA derived from H3 influenza virus A / Hong Kong / 1 / 1968, as described in the previous examples. Although highly conserved in the stems of H3 and H7 hemagglutinin hemagglutinins of group 2, a small number of surface residues in the region of the conserved stem epitope differ. In this example, selected residues located on the polypeptide surface were stepwise mutated from H3 residues (present in reference UFV172561 and UFV172562) to the corresponding H7 residues. These residues include positions in the β2 / β3 loop (residues 25 and 27), A helix residues (residue 367), and lower polypeptide residues (residues 475, 476, and 479).

[0215] Protein expression and culture supernatant analysis in mammalian cells DNA fragments encoding the polypeptide of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG-linker-His tag for screening purposes were produced on a microscale (200 μL) in the Expi293F eukaryotic cell line as described in Example 3. The expression, trimer content, and folding (binding of mAb CR9114 or CT149) of the polypeptide of the present invention were evaluated by AlphaLISA as described in Example 5. All AlphaLISA data were normalized to the respective reference constructs UFV172561 and UFV172562, set to 100%. The first reference construct contained mutations to the H1 residues at positions 379 and 381, and the second reference contained wild-type H3 residues at positions 379 and 381. Furthermore, the culture supernatant was analyzed by analytical SEC on the day of collection as described in Example 2.

[0216] Results and Conclusions Introducing β2 / β3 loops, A helix (containing H1-like residues at positions 379 and 381), and H7-like residues to the base of the stem resulted in a slight decrease in expression and trimer yield, and the variation in antibody binding to the trimer stem polypeptide as determined by AlphaLISA (±20%) was relatively small. Similarly, SEC analysis of crude cell culture supernatant showed a decrease in polypeptide expression levels, including surface mutations to H7 (Figure 13A).

[0217] A similar effect is observed when surface mutations are introduced into skeletal variants containing H3-like residues at positions 379 and 381 (Figure 13B).

[0218] In summary, the surface of the H3 HA-derived stem polypeptide of the present invention can be modified toward H7, particularly in the presence of an H1-like residue on the upper part of the A-helix.

[0219] Example 14: General application to the Group 2 mini-HA approach design The design elements necessary for the production of the trimer stem polypeptide of the present invention are expressed in the H3 HA skeleton (A / Hong Kong / 1 / 1968) and have also been introduced into two alternative H3 skeletons, namely A / Wisconsin / 67 / 2005 and A / Singapore / INFIMH / 16 / 0019 / 2016. The design elements are introduced incrementally; set I polypeptide contains the minimum set of mutations, set II further contains partial B-loop stabilizing mutations, and set III contains all additional B-loop stabilizing mutations.

[0220] Protein expression and culture supernatant analysis in mammalian cells DNA fragments encoding the polypeptide of the present invention were synthesized as described in Example 2. Polypeptides containing a C-terminal FLAG linker-His tag for screening purposes were produced on a microscale (200 μL) in the Expi293F eukaryotic cell line, and the crude cell culture supernatant was analyzed by Analytical SEC on the day of collection as described in Example 3.

[0221] Results and Conclusions As observed in SEC-MALS analysis, the introduction of the Set I design element resulted in trimer stem polypeptides in all three skeletons, although the trimer peak was most pronounced in the polypeptide derived from A / Wisconsin / 67 / 2005 and minimal in the polypeptide derived from A / Hong Kong / 1 / 1968 (Figure 14A). Further stabilizing mutations in the B loop (Sets II and III) resulted in a significant increase in polypeptide expression levels and trimer content (Figure 14B). In summary, these results confirm that soluble trimer mini-HA can be obtained by introducing the polypeptide modifications of the present invention into the other Group 2 skeletons.

[0222] Example 15: Adenovirus-driven in vitro expression of precisely folded trimer group 2 mini-HA on the cell membrane of human lung fibroblasts. In this example, the adenovirus 26 (Ad26.FLU.004)-driven expression on the cell surface of human lung fibroblasts (MRC-5) and the folding of trimeric UFV180480 (UFV18088 with a native transmembrane domain) were evaluated. MRC-5 cells were transduced in medium (5,000 VP / cell). After 2 days, the cells were lysed in lysis buffer to evaluate the expression of trimeric UFV180480 by Western blot analysis, or the cells were harvested by trypsin treatment and the cell surface expression of correctly folded UFV180480 was evaluated using flow cytometry. In either case, Ad26.Empty lacking the transgene encoding UFV180480 was included as a negative control.

[0223] Protein lysates from Ad26.FLU.004-transduced cells, along with the protein UFV180088 acting as a positive control, were processed for SDS-PAGE under reducing conditions (to ensure complete unfolding of the trimeric group 2 mini-HA protein) or non-reducing conditions (to ensure the trimeric nature of the mini-HA protein), and the proteins were transferred to a nitrocellulose blot. Expression was tested by probing the blot with the group 2 mini-HA structure-specific biotinylated antibody CR9114, and expression was visualized using HRP-conjugated streptavidin.

[0224] To test the cell membrane-associated expression of UFV180480 by flow cytometry, Ad26.FLU.004-transduced cells were trypsinized and resuspended in flow cytometry buffer. Non-permeabilized cells were probed with CR9114, and after extensive washing, probed with a PE-conjugated anti-human antibody to visualize UFV180480.

[0225] Results and Conclusions Cell surface expression of UFV180480 was analyzed using flow cytometry. Compared to Ad26.Empty transduced cells (Figure 16A), the majority (approximately 96.3%) of Ad26.FLU.004 transduced MRC-5 cells showed high levels of UFV180480 on the cell surface (Figure 16B).

[0226] Flow cytometry analysis of UFV180480 expression in MRC-5 cells did not distinguish between monomeric and trimeric UFV180480 expression. Therefore, Western blot analysis was performed. Based on molecular weight and comparison with trimeric UFV180088, Ad26.FLU.004 was shown to drive trimeric UFV180480 expression in MRC-5 cells (Figure 16C). Based on the amino acid sequence of monomeric UFV180480, the molecular weight was estimated to be 34.4 kDa, which gives rise to a trimeric UFV180480 size of approximately 103.2 kDa. Lysates (5,000 VP / cell) from Ad26.FLU.004 transdescent cells, rather than Ad26.Empty transdescent cells, showed a band at approximately 103.2 kDa and exhibited slightly higher electrophoresis compared to the trimer UFV180088 (expected MW 89.1 kDa) (compare lanes 1-3 in Figure 16C). No band was observed at approximately 34 kDa, indicating that the majority of UFV180480 was in its trimer form (Figure 16C, lane 1). When treated under reducing (i.e., completely unfolded) conditions, none of the samples showed any specific band (Figure 16C, lanes 4-6).

[0227] Therefore, according to the present invention, it was confirmed that UFV180480 is expressed in vitro by transduction of Ad26.FLU.004. The presence of the trimer protein on the cell surface of human cells was also confirmed.

[0228] Example 16: Polypeptides UFV170278 and UFV170282 of the present invention are immunogenic and induce protection in a lethal H3N2 A / Hong Kong / 1 / 1968 naive mouse challenge model. In this example, the in vivo immunogenicity and protective effects (based on survival rate at the end of the follow-up period) of dose ranges of UFV170278 (with a conservation motif for N-linked glycan at position 38) and UFV170282 (without a conservation motif for N-linked glycan at position 38) with 2% (v / v) Adjuplex adjuvant were evaluated compared to pseudo-immunized (PBS) animals.

[0229] Ten female BALB / c mice (6-8 weeks old) were immunized by intramuscular injection three times at 3-week intervals with a dose range of soluble trimer UFV170278 or UFV170282 with 2% (v / v) Adjuplex as an adjuvant. The dose range consisted of four 10-fold dilutions starting from 30 mcg and going down to 0.03 μg. As a negative control, 18 mice were immunized three times with PBS. Four weeks after the last immunization, blood was collected from the mice and the immune response was analyzed. One day later, the mice were given 12.5 × LD50. 50 Mice were challenged with the mouse-adapted H3N2 A / Hong Kong / 1 / 1968 challenge virus and monitored for 3 weeks (survival, body weight, clinical score). Survival rate at the end of the follow-up period was the primary outcome parameter.

[0230] result All doses of UFV170278 and UFV170282 induced significantly higher H3 A / Hong Kong / 1 / 1968 HA stem-specific antibody titers (measured by CR9114 competitive assay) compared to the PBS group titer, demonstrating that UFV170278 and UFV170282 are immunogenic (P<0.001; ANOVA, post-hoc t-test, stepwise study (starting from highest dose), and 2x Bonferroni correction for constructs) (see Figure 17A).

[0231] Furthermore, compared to the PBS group, Adjuplex adjuvant-added UFV170278 and UFV170282 provided significant protection at all doses (P<0.001; Fisher's exact test, stepwise study (starting from the highest dose), and 2x Bonferroni correction for constructs) (see Figure 17B). Weight loss (defined by area under the curve) was significantly reduced at all doses compared to the PBS group (P<0.001; ANOVA, 2x Bonferroni correction for constructs, and stepwise study starting from the highest dose) (see Figure 17B).

[0232] conclusion Therefore, according to the present invention, UFV170278 and UFV170282 are immunogenic and provide protection in the lethal H3N2 A / Hong Kong / 1 / 1968 mouse challenge model.

[0233] Example 17: Polypeptides UFV180088, UFV180089, and UFV180090 of the present invention are immunogenic in the naive mouse challenge model. In this example, the in vivo immunogenicity of UFV180088, UFV180089, and UFV180090 with 2% (v / v) Adjuplex adjuvant in their respective dose ranges was evaluated compared to pseudo-immunized (PBS) animals.

[0234] Ten female BALB / c mice (6-8 weeks old) were immunized with 3 mcg of soluble trimer UFV180088, UFV180089, or UFV180090 by intramuscular injection one, two, or three times at 3-week intervals. The final immunization was administered on the same day. As a negative control, 18 mice were immunized three times with PBS. All immunizations were administered with 2% (v / v) Adjuplex as an adjuvant. Four weeks after the final immunization, blood samples were collected from the mice and the immune response was analyzed.

[0235] result All constructs induced significantly higher H3 A / Hong Kong / 1 / 1968 HA stem-specific antibody titers (measured by CR9114 competitive assay) after two or three immunizations compared to the PBS group titer, thus demonstrating that UFV180088, UFV180089, and UFV180090 are immunogenic (P<0.001; Wilcoxon, 3x Bonferroni correction for multiple comparisons and stepwise studies (starting from highest dose)) (see Figure 18A). No constructs induced significant stem-specific antibodies after a single immunization.

[0236] All constructs induced significantly higher H3 A / Hong Kong / 1 / 1968 and H3 A / Texas / 50 / 2012 HA-specific antibody titers (measured by FL HA-binding ELISA) compared to the PBS group after two or three immunizations (P<0.01; Wilcoxon, 3x Bonferroni correction for multiple comparisons and stepwise studies (starting from highest dose)) (see Figure 18B). Furthermore, single immunization with UFV180088 and UFV180089 induced significantly higher H3 A / Texas / 50 / 2012 HA-specific antibody titers compared to the PBS group (P<0.01).

[0237] All constructs induced significantly higher H7A / Netherlands / 219 / 2003 HA-specific antibody titers (measured by FL HA binding assay) compared to the PBS group titers after three immunizations (P<0.001; Wilcoxon, 3x Bonferroni correction for multiple comparisons and stepwise testing (starting from highest dose)) (see Figure 18B). Two immunizations with UFV180089 and UFV180090 induced significantly higher titers compared to the PBS group titers (P<0.001 and P<0.01, respectively), but no significantly higher titers were detected after a single immunization.

[0238] conclusion Therefore, according to the present invention, UFV180088, UFV180089, and UFV180090 were demonstrated to be immunogenic in a naive mouse model. All constructs induced significant HA stem-specific antibody titers and antibodies that bound to multiple phylogenetically distinct H3 (from strains isolated in different years) and H7 HA proteins.

[0239] Example 18: Polypeptides UFV180088, UFV180089, and UFV180090 of the present invention induce protection against a lethal challenge by H3N2 A / Hong Kong / 1 / 1968 in a naive mouse challenge model. In this example, the in vivo protective effect (based on survival rate at the end of the follow-up period) of the dose ranges of UFV180088, UFV180089, and UFV180090 with 2% (v / v) Adjuplex adjuvant was evaluated compared to pseudo-immunized (PBS) animals.

[0240] Ten female BALB / c mice (6-8 weeks old) were immunized with 3 mcg of soluble trimer UFV180088, UFV180089, or UFV180090 by intramuscular injection one, two, or three times at 3-week intervals. The final immunization was administered on the same day. As a negative control, 18 mice were immunized three times with PBS. All immunizations were administered with 2% (v / v) Adjuplex as an adjuvant. Four weeks after the final immunization, mice were given 25 × LD50. 50 Mice were challenged with the mouse-adapted H3N2 A / Hong Kong / 1 / 1968 challenge virus and monitored for 3 weeks (survival, body weight, clinical score). Survival rate at the end of the follow-up period was the primary outcome parameter.

[0241] result UFV180088, UFV180089, and UFV180090 were shown to provide significant protection compared to the PBS group after two or three immunizations (P<0.001; Fisher's exact test, 2x Bonferroni correction for constructs, and stepwise study starting from the highest dose) (see Figure 19). No constructs induced significant protection after one immunization. Weight loss (defined by area under the curve) was significantly reduced at all doses compared to the PBS group, except after one immunization with UFV180089 (P<0.05; ANOVA, 2x Bonferroni correction for constructs, and stepwise study starting from the highest dose) (see Figure 19).

[0242] conclusion Therefore, according to the present invention, UFV180088, UFV180089, and UFV180090 are immunogenic and provide protection in the lethal H3N2 A / Hong Kong / 1 / 1968 mouse challenge model.

[0243] [Table 7]

[0244] References Belongia et al. (2016), Lancet Infect.Dis.16:942-951. Bommakanti et al. (2010), Proc. Natl. Acad. Sci. USA 107(31):13701-13706. Bommakanti et al. (2012), J Virol 86:13434. Ciani et al. (2010), Proc. Natl. Acad. Sci. USA 107(46):19850-19855. Corbett et al. (2019), mBio 10(1):e2810-2818. Ekiert et al. (2009), Science 324(5924):246-51. Das et al.(1985),Prog Nucleic Acid Res Mol Biol 32:217-236. Garten et al.(2017),MMWR Morb.Mortal Wkly Rep.67:634-642. Gill et al.(2001),Gene Therapy 8:1539-1546. Kaufmann(2000),Mol Biotechnol 16:151-160. Letarov et al.(1993),Biochemistry Moscow 64:817-823. Lorieau et al.(2010),Proc.Natl.Acad.Sci.USA,107:11341. S-Guthe et al.(2004)J.Mol.Biol.337:905-915. Steel et al.(2010),mBio 1(1):1-9. Throsby et al.(2008),Plos One 12(3):1-15. Winter et al.(1981)Nature 292,72-75. Wu et al.(2015)Nature Communications 6(7708):1-11. Furthermore, the present invention provides the following: [1] A monomeric influenza A HA stem polypeptide comprising the HA1 domain and HA2 domain of hemagglutinin (HA) of group 2 influenza A virus, - Deletion of the head region of the HA1 domain, - Modification of the trimerization region of the HA2 domain, - At least two cysteine ​​residues capable of forming at least one monomeric cysteine ​​crosslink It contains an amino acid sequence that includes, The amino acid at position 355 of the aforementioned amino acid sequence is W. The numbering of amino acid positions in the HA stem polypeptide amino acid sequence is H3 numbering, which corresponds to the full-length HA numbering of the reference strain H3N2 A / Aichi / 2 / 68 (SEQ ID NO: 1), and is an HA stem polypeptide. [2] The polypeptide described in [1], wherein the amino acid at position 432 is I, or the amino acid at position 432 is I and the amino acid at position 380 is I. [3] The polypeptide as described in [1] or [2], wherein the amino acid at position 378 is T, the amino acid at position 379 is N, and / or the amino acid at position 381 is V. [4] The polypeptide according to [1], [2], or [3], further comprising a glycosylation motif introduced at positions 401-403 for N-linked glycosylation at position 401. [5] The polypeptide according to any one of [1] to [4], wherein the deletion in the head region of the HA1 domain includes a deletion comprising at least an amino acid sequence from the amino acid corresponding to the 50th position to the amino acid corresponding to the 302nd position. [6] The polypeptide according to [5], wherein the deletion in the head region of the HA1 domain comprises at least an amino acid sequence from the 47th amino acid to the 306th amino acid. [7] The polypeptide according to any one of [1] to [6], wherein the trimerization region of the HA2 domain includes an amino acid sequence from the amino acid corresponding to the 405th position to the amino acid corresponding to the 419th position. [8] The modification of the trimerizing domain is the polypeptide according to any one of [1] to [7], including the introduction of a heterologous trimerizing domain. [9] The polypeptide described in [8], wherein the heterotrimerizing domain is a GCN4 sequence.

[10] The modification of the trimerizing domain comprises a modification of a 7-residue repeat sequence in the C helix, as described in any one of [1] to [8].

[11] The modified trimerized region of the HA2 domain is an amino acid sequence 405 RMKQIEDKIEEIESK 419 (Sequence ID 9) or 405 PMKQIEDKIEEIESK 419 A polypeptide as described in any one of items [1] to [9], including (Sequence ID 10).

[12] A polypeptide according to any one of [1] to

[11] , comprising cysteine ​​at the amino acid position corresponding to position 310 in combination with cysteine ​​at the position corresponding to position 422, or cysteine ​​at the amino acid position corresponding to position 311 in combination with cysteine ​​at the position corresponding to position 422, or cysteine ​​at the amino acid position corresponding to position 308 in combination with cysteine ​​at the position corresponding to position 418, wherein the cysteine ​​can form monomeric cysteine ​​crosslinks.

[13] The polypeptide according to

[12] , comprising a cysteine ​​at the amino acid position corresponding to position 310 in combination with a cysteine ​​at the position corresponding to position 422, wherein the cysteine ​​forms the at least one monomeric cysteine ​​crosslink.

[14] The polypeptide is one of the following, with amino acid M at position 388: [1] to

[13] .

[15] A polypeptide according to any one of [1] to

[14] , comprising at least one additionally introduced glycosylated motif.

[16] The polypeptide according to

[15] , wherein the at least one additionally introduced glycosylation motif is located at positions 392-394 for N-linked glycosylation at position 392, and / or at positions 393-395 for N-linked glycosylation at position 393.

[17] A polypeptide as described in any one of [1] to

[16] , wherein one or more amino acids in the B loop are mutated to P.

[18] - The amino acid at position 31 is E, and the amino acid at position 34 is V. - The amino acid at position 392 is either S or P. - The amino acid at position 395 is either T or P. - The amino acid at position 399 is either S or P. - The amino acid at position 435 is N or R, and / or - The amino acid at position 439 is Y. A polypeptide as described in any one of the items [1] to

[17] .

[19] The HA stem polypeptide monomer is the polypeptide according to any one of [1] to

[18] , wherein the HA stem polypeptide monomer does not contain a protease cleavage site between the HA1 domain and the HA2 domain.

[20] The polypeptide described in

[19] , wherein the amino acid at position 329 is not arginine (R), but preferably glutamine (Q).

[21] The HA stem polypeptide monomer comprises a natural cleavage site or a polybasic cleavage site, as described in any one of [1] to

[18] .

[22] The polypeptide according to any one of [1] to

[21] , wherein the HA1 domain and HA2 domain are derived from an influenza virus containing H3 subtype HA, preferably from influenza virus A / Hong Kong / 1 / 68.

[23] The polypeptide described in

[22] , wherein one or more amino acids in the H3 HA and HA2 domains are mutated to the corresponding amino acids of H7 HA.

[24] The amino acid at position 25 is K. The amino acid at position 367 is Y. The amino acid at position 378 is T. The amino acid at position 475 is D. The amino acid at position 476 is D, and / or The amino acid at position 479 is A.

[23] Polypeptides as described above.

[25] A polypeptide described in any one of the following sections [1] to

[24] , comprising (part of) a signal sequence.

[26] A polypeptide according to any one of the following paragraphs [1] to

[25] , comprising a truncated HA2 domain.

[27] The polypeptide according to

[26] , wherein at least the C-terminal portion of the HA2 domain starting with the amino acid corresponding to the 516th amino acid is deleted.

[28] The polypeptide according to

[26] or

[27] , wherein the C-terminal portion of the HA2 domain starting with the amino acid corresponding to the 506th amino acid is deleted.

[29] The polypeptide according to any one of [1] to

[28] , wherein the deletion in the head region of the HA1 domain is replaced by a linked sequence consisting of 1 to 10 amino acids.

[30] A polypeptide according to any one of [1] to

[29] , comprising cysteine ​​at the position corresponding to the 396th position in combination with cysteine ​​at the position corresponding to the 408th position, or cysteine ​​at the position corresponding to the 397th position in combination with cysteine ​​at the position corresponding to the 408th position, or cysteine ​​at the position corresponding to the 398th position in combination with cysteine ​​at the 405th position.

[31] The polypeptide according to

[30] , comprising cysteine ​​at the position corresponding to position 398 in combination with cysteine ​​at the position corresponding to position 408.

[32] A multimer influenza A hemagglutinin (HA) stem polypeptide comprising at least two HA stem polypeptide monomers as described in any one of items [1] to

[31] .

[33] A multimeric influenza A hemagglutinin (HA) stem polypeptide comprising at least two HA stem polypeptide monomers as described in

[30] or

[31] , wherein the first HA stem polypeptide monomer is linked to the second monomer by an intermeral disulfide crosslink between the cysteine ​​at position 396, 397, or 398 of the first monomer and the cysteine ​​at position 408 of the second monomer, or the first HA stem polypeptide monomer is linked to the second monomer by an intermeral disulfide crosslink between the cysteine ​​at position 398 of the first monomer and the cysteine ​​at position 405 of the second monomer.

[34] A multimer influenza A hemagglutinin (HA) stem polypeptide as described in

[33] , wherein a first HA stem polypeptide monomer is linked to a second monomer by an inter-monomer disulfide crosslink between the cysteine ​​at position 398 of the first monomer and the cysteine ​​at position 408 of the second monomer.

[35] The polypeptide is a trimer, a polymer polypeptide as described in any one of

[30] to

[34] .

[36] A nucleic acid encoding an HA stem polypeptide monomer as described in any one of items [1] to

[31] .

[37] A vector containing the nucleic acid molecule described in

[36] .

[38] A recombinant adenovirus vector, as described in

[37] .

[39] A pharmaceutical composition comprising a monomeric HA stem polypeptide as described in any one of [1] to

[31] , a polymeric influenza HA stem polypeptide as described in any one of

[32] to

[35] , a nucleic acid as described in

[36] , and / or a vector as described in

[37] or

[38] , and a pharmaceutically acceptable carrier.

[40] A monomeric HA stem polypeptide as described in any one of [1] to

[31] , a multimeric influenza HA stem polypeptide as described in any one of

[32] to

[35] , a nucleic acid as described in

[36] , and / or a vector as described in

[37] or

[38] , for use in inducing an immune response against influenza virus.

[41] A monomeric HA stem polypeptide as described in any one of [1] to

[31] , a multimeric influenza HA stem polypeptide as described in any one of

[32] to

[35] , a nucleic acid as described in

[36] , and / or a vector as described in

[37] or

[38] , for use as a vaccine.

[0245] array Sequence ID 1 CAA24269.1 Hemagglutinin (Influenza A virus (A / Aichi / 2 / 1968(H3N2) (excluding signal sequence)) [ka] Sequence ID 1: H3 Full length (A / Hong Kong / 1 / 68) [ka] CR6261 VH protein (SEQ ID NO: 3) EVQLVESGAEVKKPGSSVKVSCKASGGPFRSYAISWVRQAPGQGPEWMGGIIPIFGTTKYAPKFQGRVTITADDFAGTVYMELSSLRSEDTAMYYCAKHMGYQVRETMDVWGKGTTVTVSS CR6261 VL protein (SEQ ID NO: 4) QSVLTQPPSVSAAPGQKVTISSCSGSSSNIGNDYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEANYYCATWDRRPTAYVVFGGGTKLTVL CR8020 VH protein (SEQ ID NO: 5) QVQLQQSGAEVKTPGASVKVSCKASGYTFTSFGVSWIRQAPGQGLEWIGWISAYNGDTYYAQKFQARVTMTTDTSTTTAYMEMRSLRSDDTAVYYCAREPPLFYSSWSLDNWGQGTLVTVSS CR8020 VL protein (SEQ ID NO: 6) EIVLTQSPGTLSLSPGERATLSCRASQSVSMNYLAWFQQKPGQAPRLLIYGASRRATGIPDRISGSGSGTDFLTISRLEPADFAVYYCQQYGTSPRTFGQGAKVEIK CR9114 VH protein (SEQ ID NO: 7) QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGLDWMGGISPIFGSTAYAQKFQGRVTISADIFSNTAYMELNSLTSEDTAVYFCARHGNYYYYSGMDVWGQGTTVTVSS CR9114 VL protein (SEQ ID NO: 8) SYVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPKLLIYSNDQRPSVVPDRFSGSKSGTSASLAISGLQSEDEAEYYCAAWDDSLKGAVFGGGTQLTVL FL HA (SEQ ID NO: 13) from Wisconsin / 67 / 2005 [ka] FL HA (SEQ ID NO: 14) from A / Singapore / INFMH / 16 / 0019 / 2016 [ka] FL HA (SEQ ID NO: 15) from A / Perth / 16 / 2009 [ka] FL HA (SEQ ID NO: 16) from A / Brisbane / 10 / 2007 [ka] FL HA (SEQ ID NO: 17) from A / Panama / 2007 / 1999 [ka] CT149 VH protein (SEQ ID NO: 37) [ka] CT149 VL protein (SEQ ID NO: 38) EVVLTQSPGTLALPPGERATLSCRASHRVGSTYIAWYQQKSGQAPRRLIYGASNRATDIPDRFSGSGSGTDFTLTIRRLEPEDSAVYYCQQFSVSPWTFGQGTRVEIK SD15013 (Sequence ID 39) [ka] Sequence ID 40: UFV180088 [ka] Sequence ID 41: UFV180089 [ka] Sequence ID 42: UFV180090 [ka] Sequence ID 43: UFV180141 [ka] Sequence ID 44: UFV180192 [ka] Sequence ID 45: UFV180193 [ka] Sequence ID 46: UFV180194 [ka] Sequence ID 47: UFV180195 [ka] Sequence ID 48: UFV180196 [ka] Sequence ID 49: UFV180197 [ka] Sequence ID 50: UFV180198 [ka] Sequence ID 51: UFV180199 [ka] Sequence ID 52: UFV180200 [ka] Sequence ID 53: UFV180201 [ka] Sequence ID 54: UFV180202 [ka] Sequence ID 55: UFV180203 [ka] Sequence ID 56: UFV180204 [ka] Sequence ID 57: UFV180205 [ka] Sequence ID 58: UFV180206 [ka] Sequence ID 59: UFV180207 [ka] Sequence ID 60: UFV181034 [ka] Sequence ID 61: UFV181036 [ka] Sequence ID 62: UFV181038 [ka] Sequence ID 63: UFV181040 [ka] Sequence ID 64: UFV181042 [ka] Sequence ID 65: UFV161333 [ka] Sequence ID 66: UFV161739 [ka] Sequence ID 67: UFV161800 [ka] Sequence ID 68: UFV161804 [ka] Sequence ID 69: UFV170991 [ka] Sequence ID 70: UFV171004 [ka] Sequence ID 71: UFV171190 [ka] Sequence ID 72: UFV171191 [ka] Sequence ID 73: UFV171192 [ka] Sequence ID 74: UFV171193 [ka] Sequence ID 75: UFV171194 [ka] Sequence ID 76: UFV171195 [ka] Sequence ID 77: UFV170611 [ka] Sequence ID 78: UFV170612 [ka] Sequence ID 79: UFV170613 [ka] Sequence ID 80: UFV170614 [ka] Sequence ID 81: UFV161908 [ka] Sequence ID 82: UFV171197 [ka] Sequence ID 83: UFV180666 [ka] Sequence ID 84: UFV180667 [ka] Sequence ID 85: UFV180668 [ka] Sequence ID 86: UFV180669 [ka] Sequence ID 87: UFV180670 [ka] Sequence ID 88: UFV180671 [ka] Sequence ID 89: UFV180672 [ka] Sequence ID 90: UFV180673 [ka] Sequence ID 91: UFV180674 [ka] Sequence ID 92: UFV180675 [ka] Sequence ID 93: UFV170636 [ka] Sequence ID 94: UFV170637 [ka] Sequence ID 95: UFV170638 [ka] Sequence ID 96: UFV170639 [ka] Sequence ID 97: UFV170640 [ka] Sequence ID 98: UFV160653 [ka] Sequence ID 99: UFV160764 [ka] Sequence ID 100: UFV160765 [ka] Sequence ID 101: UFV160766 [ka] Sequence ID 102: UFV160674 [ka] Sequence ID 103: UFV160675 [ka] Sequence ID 104: UFV160767 [ka] Sequence ID 105: UFV160768 [ka] Sequence ID 106: UFV160769 [ka] Sequence ID 107: UFV160770 [ka] Sequence ID 108: UFV160771 [ka] Sequence ID 109: UFV160772 [ka] Sequence ID 110: UFV160321 [ka] Sequence ID 111: UFV160403 [ka] Sequence ID 112: UFV160404 [ka] Sequence ID 113: UFV160405 [ka] Sequence ID 114: UFV160406 [ka] Sequence ID 115: UFV160407 [ka] Sequence ID 116: UFV160408 [ka] Sequence ID 117: UFV160409 [ka] Sequence ID 118: UFV160410 [ka] Sequence ID 119: UFV160411 [ka] Sequence ID 120: UFV160412 [ka] Sequence ID 121: UFV160413 [ka] Sequence ID 122: UFV160414 [ka] Sequence ID 123: UFV160415 [ka] Sequence ID 124: UFV160416 [ka] Sequence ID 125: UFV160417 [ka] Sequence ID 126: UFV160418 [ka] Sequence ID 127: UFV160419 [ka] Sequence ID 128: UFV160420 [ka] Sequence ID 129: UFV161686 [ka] Sequence ID 130: UFV161722 [ka] Sequence ID 131: UFV161723 [ka] Sequence ID 132: UFV161688 [ka] Sequence ID 133: UFV161689 [ka] Sequence ID 134: UFV161690 [ka] Sequence ID 135: UFV161691 [ka] Sequence ID 136: UFV161692 [ka] Sequence ID 137: UFV161693 [ka] Sequence ID 138: UFV161694 [ka] Sequence ID 139: UFV161695 [ka] Sequence ID 140: UFV161696 [ka] Sequence ID 141: UFV161697 [ka] Sequence ID 142: UFV161698 [ka] Sequence ID 143: UFV161699 [ka] Sequence ID 144: UFV161700 [ka] Sequence ID 145: UFV161701 [ka] Sequence ID 146: UFV161702 [ka] Sequence ID 147: UFV161703 [ka] Sequence ID 148: UFV161704 [ka] Sequence ID 149: UFV161705 [ka] Sequence ID 150: UFV161706 [ka] Sequence ID 151: UFV161707 [ka] Sequence ID 152: UFV161708 [ka] Sequence ID 153: UFV161709 [ka] Sequence ID 154: UFV161715 [ka] Sequence ID 155: UFV161721 [ka] Sequence ID 156: UFV171187 [ka] Sequence ID 157: UFV171120 [ka] Sequence ID 158: UFV171121 [ka] Sequence ID 159: UFV170994 [ka] Sequence ID 160: UFV170995 [ka] Sequence ID 161: UFV180208 [ka] Sequence ID 162: UFV180217 [ka] Sequence ID 163: UFV170278 [ka] Sequence ID 164: UFV170282 [ka] Sequence ID 165: UFV160595 [ka] Sequence ID 166: UFV161196 [ka] Sequence ID 167: UFV161198 [ka] Sequence ID 168: UFV161169 [ka] Sequence ID 169: UFV170062 [ka] Sequence ID 170: UFV170051 [ka] Sequence ID 171: UFV170428 [ka] Sequence ID 172: UFV170440 [ka] Sequence ID 173: UFV171272 [ka] Sequence ID 174: UFV171273 [ka] Sequence ID 175: UFV171274 [ka] Sequence ID 176: UFV171275 [ka] Sequence ID 177: UFV171276 [ka] Sequence ID 178: UFV171277 [ka] Sequence ID 179: UFV171278 [ka] Sequence ID 180: UFV171279 [ka] Sequence ID 181: UFV171280 [ka] Sequence ID 182: UFV161454 [ka] Sequence ID 183: UFV161453 [ka] Sequence ID 184: UFV161459 [ka] Sequence ID 185: UFV161451 [ka] Sequence ID 186: UFV161458 [ka] Sequence ID 187: UFV161450 [ka] Sequence ID 188: UFV161448 [ka] Sequence ID 189: UFV171116 [ka] Sequence ID 190: UFV172561 [ka] Sequence ID 191: UFV172563 [ka] Sequence ID 192: UFV172564 [ka] Sequence ID 193: UFV172571 [ka] Sequence ID 194: UFV172562 [ka] Sequence ID 195: UFV172588 [ka] Sequence ID 196: UFV172583 [ka] Sequence ID 197: UFV172585 [ka] Sequence ID 198: UFV180642 [ka] Sequence ID 199: UFV180645 [ka] Sequence ID 200: UFV180647 [ka] Sequence ID 201: UFV181106 [ka] Sequence ID 202: UFV181107 [ka] Sequence ID 203: UFV181109 [ka] Sequence ID 204: UFV181117 [ka] Sequence ID 205: UFV181118 [ka] Sequence ID 206: UFV181120 [ka] Sequence ID 207: UFV180480 (UFV180088 + natural transmembrane™ domain) [ka] Nucleotide sequence encoding sequence number 208:UFV180088 [ka] Sequence ID 209: Nucleotide sequence encoding UFV180480 (UFV180088 + TM domain) [ka] Sequence ID 210 (minimum sequence of UFV180088) [ka] Sequence ID 211 (minimum sequence of UFV180089) [ka] Sequence ID 212 (minimum sequence of UFV180090) [ka]

Claims

1. A monomeric influenza A HA stem polypeptide comprising the HA1 domain and HA2 domain of hemagglutinin (HA) of group 2 influenza A virus, wherein the following modifications are made - A deletion in the head region of the HA1 domain, comprising at least an amino acid sequence from the 47th amino acid to the 306th amino acid, - Modification of the trimerization region of the HA2 domain, - At least two cysteine ​​residues capable of forming at least one monomeric cysteine ​​crosslink It contains the amino acid sequence of wild-type HA of group 2 influenza A virus, This includes a further mutation of the amino acid at position 355 of the aforementioned amino acid sequence to W, The numbering of amino acid positions in the HA stem polypeptide amino acid sequence is based on the monomeric influenza A HA stem polypeptide shown in Sequence ID No.

1.

2. A monomeric influenza A HA stem polypeptide comprising, in addition to the monomeric influenza A HA stem polypeptide described in Claim 1, a mutation to the amino acid at position 432 to I, or a mutation to the amino acid at position 432 to I and a mutation to the amino acid at position 380 to I.

3. A monomeric influenza A HA stem polypeptide comprising a mutation to T at the 378th amino acid, a mutation to N at the 379th amino acid, and / or a mutation to V at the 381st amino acid, relative to the monomeric influenza A HA stem polypeptide described in Claim 1 or 2.

4. A monomeric influenza A HA stem polypeptide, further comprising a glycosylation motif introduced at positions 401 to 403 for N-linked glycosylation at position 401, relative to the monomeric influenza A HA stem polypeptide according to claim 1, 2, or 3.

5. The monomeric influenza A HA stem polypeptide according to any one of claims 1 to 4, wherein the trimerization region of the HA2 domain includes an amino acid sequence from the amino acid corresponding to the 405th position to the amino acid corresponding to the 419th position.

6. The modification of the trimerization region includes the introduction of a heterologous trimerization domain, wherein the monomeric influenza A HA stem polypeptide is as described in any one of claims 1 to 5.

7. The monomeric influenza A HA stem polypeptide according to claim 6, wherein the heterologous trimerizing domain is a GCN4 sequence.

8. The monomeric influenza A HA stem polypeptide according to any one of claims 1 to 6, wherein the modification of the trimerizing region includes modification of a 7-residue repeat sequence in the C helix.

9. The trimerization region of the HA2 domain is an amino acid sequence ４０５ RMKQIEDKIEEIESK ４１９ (Sequence No. 9) or ４０５ PMKQIEDKIEEIESK ４１９ A monomeric influenza A HA stem polypeptide according to any one of claims 1 to 7, comprising (SEQ ID NO: 10).

10. A monomeric influenza A HA stem polypeptide according to any one of claims 1 to 9, comprising cysteine ​​at the amino acid position corresponding to position 310 in combination with cysteine ​​at the position corresponding to position 422, or cysteine ​​at the amino acid position corresponding to position 311 in combination with cysteine ​​at the position corresponding to position 422, or cysteine ​​at the amino acid position corresponding to position 308 in combination with cysteine ​​at the position corresponding to position 418, wherein the cysteine ​​can form an intramonomeric cysteine ​​crosslink.

11. The monomeric influenza A HA stem polypeptide according to claim 10, comprising a cysteine ​​at the amino acid position corresponding to position 310 in combination with a cysteine ​​at the position corresponding to position 422, wherein the cysteine ​​forms the at least one monomeric cysteine ​​crosslink.

12. A monomeric influenza A HA stem polypeptide comprising a mutation to M at the 388th amino acid relative to the monomeric influenza A HA stem polypeptide according to any one of claims 1 to 11.

13. A monomeric influenza A HA stem polypeptide comprising at least one additionally introduced glycosylated motif to the monomeric influenza A HA stem polypeptide according to any one of claims 1 to 12.

14. The monomeric influenza A HA stem polypeptide according to claim 13, wherein the at least one additionally introduced glycosylation motif is located at positions 392-394 for N-linked glycosylation at position 392 and / or at positions 393-395 for N-linked glycosylation at position 393.

15. A monomeric influenza A HA stem polypeptide, wherein one or more amino acids in the B loop are mutated to P, compared to the monomeric influenza A HA stem polypeptide described in any one of claims 1 to 14.

16. With respect to the monomeric influenza A HA stem polypeptide according to any one of claims 1 to 15, - Including mutations to amino acid E at position 31 and to amino acid V at position 34, - Including a mutation to S or P of the amino acid at position 392, - Including a mutation to T or P at the amino acid position 395, - Including mutations to S or P of the amino acid at position 399, - Includes mutations to the N or R of the amino acid at position 435, and / or - Including a mutation to Y at amino acid position 439, Monomeric influenza A HA stem polypeptide.

17. A monomeric influenza A HA stem polypeptide, wherein a mutation is introduced into the monomeric influenza A HA stem polypeptide according to any one of claims 1 to 16 such that there is no protease cleavage site between the HA1 domain and the HA2 domain.

18. The monomeric influenza A HA stem polypeptide according to claim 17, wherein the amino acid at position 329 is not arginine (R).

19. The monomeric influenza A HA stem polypeptide according to claim 18, wherein the amino acid at position 329 is glutamine (Q).

20. A monomeric influenza A HA stem polypeptide according to any one of claims 1 to 16, comprising a natural cleavage site, or comprising a polybasic cleavage site introduced to the monomeric influenza A HA stem polypeptide according to any one of claims 1 to 16.

21. The monomeric influenza A HA stem polypeptide according to any one of claims 1 to 20, wherein the HA1 domain and HA2 domain are derived from an influenza virus containing HA of the H3 subtype.

22. The monomeric influenza A HA stem polypeptide according to claim 21, wherein the HA1 domain and HA2 domain are derived from influenza A / Hong Kong / 1 / 68 virus.

23. A monomeric influenza A HA stem polypeptide, wherein one or more amino acids in the HA1 domain and HA2 domain are mutated to the corresponding amino acids of H7 HA, compared to the monomeric influenza A HA stem polypeptide described in Claim 21 or 22.

24. The amino acid at position 25 is K. The amino acid at position 367 is Y. The amino acid at position 378 is T. The amino acid at position 475 is D. The amino acid at position 476 is D, and / or The amino acid at position 479 is A. The monomeric influenza A HA stem polypeptide according to claim 23.

25. A monomeric influenza A HA stem polypeptide according to any one of claims 1 to 24, comprising a signal sequence or a portion of a signal sequence.

26. A monomeric influenza A HA stem polypeptide comprising a truncated HA2 domain relative to the monomeric influenza A HA stem polypeptide according to any one of claims 1 to 25.

27. The monomeric influenza A HA stem polypeptide according to claim 26, wherein at least the C-terminal portion of the HA2 domain starting with the amino acid corresponding to the 516th amino acid is deleted.

28. The monomeric influenza A HA stem polypeptide according to claim 26 or 27, wherein the C-terminal portion of the HA2 domain starting with the amino acid corresponding to the 506th amino acid is deleted.

29. The monomeric influenza A HA stem polypeptide according to any one of claims 1 to 28, wherein the deletion in the head region of the HA1 domain is replaced by a linking sequence consisting of 1 to 10 amino acids.

30. A monomeric influenza A HA stem polypeptide comprising, in combination with the monomeric influenza A HA stem polypeptide according to any one of claims 1 to 29, a mutation to cysteine ​​at the position corresponding to the 408th position in combination with a mutation to cysteine ​​at the 396th position, or a mutation to cysteine ​​at the 408th position in combination with a mutation to cysteine ​​at the 397th position, or a mutation to cysteine ​​at the 408th position in combination with a mutation to cysteine ​​at the 398th position, or a mutation to cysteine ​​at the 405th position in combination with a mutation to cysteine ​​at the 398th position.

31. The monomeric influenza A HA stem polypeptide according to claim 30, comprising a mutation to cysteine ​​at the position corresponding to 398 in combination with a mutation to cysteine ​​at the position corresponding to 408.

32. A multimer influenza A HA stem polypeptide comprising at least two monomeric influenza A HA stem polypeptides as described in any one of claims 1 to 31.

33. A multimer influenza A HA stem polypeptide comprising at least two monomeric influenza A HA stem polypeptides as described in claim 30 or 31, wherein the first monomeric influenza A HA stem polypeptide is linked to the second monomeric influenza A HA stem polypeptide by an intermeral disulfide crosslink between the cysteine ​​at position 396, 397, or 398 of the first monomeric influenza A HA stem polypeptide and the cysteine ​​at position 408 of the second monomeric influenza A HA stem polypeptide, or the first monomeric influenza A HA stem polypeptide is linked to the second monomeric influenza A HA stem polypeptide by an intermeral disulfide crosslink between the cysteine ​​at position 398 of the first monomeric influenza A HA stem polypeptide and the cysteine ​​at position 405 of the second monomeric influenza A HA stem polypeptide.

34. The multimer influenza A HA stem polypeptide according to claim 33, wherein the first monomeric influenza A HA stem polypeptide is linked to the second monomeric influenza A HA stem polypeptide by an inter-monomer disulfide crosslink between the cysteine ​​at position 398 of the first monomeric influenza A HA stem polypeptide and the cysteine ​​at position 408 of the second monomeric influenza A HA stem polypeptide.

35. A trimer, a multimer influenza A HA stem polypeptide according to any one of claims 32 to 34.

36. A nucleic acid encoding a monomeric influenza A HA stem polypeptide according to any one of claims 1 to 31.

37. A vector comprising the nucleic acid molecule described in claim 36.

38. The vector according to claim 37, which is a recombinant adenovirus vector.

39. A pharmaceutical composition comprising a monomeric influenza A HA stem polypeptide according to any one of claims 1 to 31, a polymeric influenza A HA stem polypeptide according to any one of claims 32 to 35, a nucleic acid according to claim 36, and / or a vector according to claim 37 or 38, and a pharmaceutically acceptable carrier.

40. A monomeric influenza A HA stem polypeptide according to any one of claims 1 to 31, a polymeric influenza A HA stem polypeptide according to any one of claims 32 to 35, a nucleic acid according to claim 36, and / or a vector according to claim 37 or 38, for use in inducing an immune response against influenza virus.

41. A monomeric influenza A HA stem polypeptide according to any one of claims 1 to 31, a polymeric influenza A HA stem polypeptide according to any one of claims 32 to 35, a nucleic acid according to claim 36, and / or a vector according to claim 37 or 38, for use as a vaccine.

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