JP2018052953A - Influenza vaccines and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide influenza vaccines using influenza hemagglutinin stem domain.SOLUTION: Disclosed herein is an influenza hemagglutinin stem domain polypeptide comprising: (a) an influenza hemagglutinin HA1 domain comprising HA1 N terminal stem segment covalently linked to an HA1 C terminal segment through a linking sequence of 0-50 amino acid residues; and (b) an influenza hemagglutinin HA2 domain, where the hemagglutinin stem domain polypeptide is resistant to protease cleavage at the junction between HA1 and HA2, one or more amino acids in an amino acid sequence connecting the A helix and helix CD of HA2 are mutated as compared to a wild type influenza HA2 domain, and the HA1 and HA2 domains are derived from an influenza A virus subtype selected from H1, H5 and H3.SELECTED DRAWING: None

Description

  The present invention relates to the field of medicine. Provided herein are influenza hemagglutinin stem domain polypeptides, methods of providing hemagglutinin stem domain polypeptides, compositions comprising the same, vaccines comprising the same and methods for their use in the detection, prevention and / or treatment of influenza in particular Is done.

  Influenza virus is a major human pathogen and is a respiratory disease that varies in severity from asymptomatic infection to primary viral pneumonia (generally referred to as “influenza” or “the flu”) Called). The clinical effect of infection varies depending on the pathogenicity of the influenza strain and the host exposure, history, age, and immune status. Each year, it is estimated that approximately 1 billion people worldwide are infected with influenza virus, resulting in 3-5 million cases of serious illness and an estimated 300,000 to 500,000 influenza-related deaths. The majority of these infections can be attributed to influenza A virus carrying H1 or H3 hemagglutinin subtypes, and the contribution from influenza B virus is small, so all three representatives are included in seasonal vaccines. Current vaccination practices rely on the early identification of circulating influenza viruses to enable timely production of effective seasonal influenza vaccines. Apart from the inherent difficulty of predicting strains that will dominate during the next season, antiviral resistance and immune evasion are also factors that prevent current vaccines from preventing morbidity and mortality. In addition, the potential for epidemics caused by highly pathogenic virus strains arising from pathogen-bearing animals and rearranged to increase human-to-human spread is a significant and realistic threat to global health. It becomes.

  Influenza A virus is widely distributed in nature and can infect a variety of birds and mammals. Influenza viruses are envelope RNA viruses that belong to the family of Orthomyxoviridae. These genomes consist of 11 different proteins, 1 nucleoprotein (NP), 3 polymerase proteins (PA, PB1, and PB2), 2 matrix proteins (M1 and M2), 3 nonstructural proteins (NS1, NS2) And PB1-F2), and 8 single-stranded RNA segments encoding two external glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Viruses are classified based on the difference in antigen structure of HA and NA proteins, and their different combinations represent unique virus subtypes that are further classified into defined influenza virus strains. Although all known subtypes can be found in birds, the currently circulating human influenza A subtypes are H1N1 and H3N2. Phylogenetic analysis demonstrated the subclassification of hemagglutinin into two main groups: H1, H2, H5 and H9 subtypes, especially in phylogenetic group 1 and H3, H4 and H7 subtypes, especially in phylogenetic group 2.

Influenza B virus strains are strictly human. Antigenic variation in HA in influenza B virus strains is smaller than that observed in type A strains. Two genetically and antigenically distinct influenza B virus strains are represented by the B / Yamagata / 16/88 (also referred to as B / Yamagata) and B / Victoria / 2/87 (B / Victoria) strains. As it is circulated in humans (Ferguson et al., 2003). Although the spectrum of disease caused by influenza B virus is generally milder than that caused by influenza A virus, severe illness requiring hospitalization for influenza B infection is still frequently observed.

  It is known that antibodies that neutralize influenza are primarily directed against hemagglutinin (HA). Hemagglutinin or HA is a trimeric glycoprotein that is anchored to the viral coat and has a dual function: it is responsible for binding to the cell surface receptor sialic acid, and after uptake it fuses the virus and endosomal membranes. Mediates and results in the release of viral RNA in the cytosol of the cell. HA contains a large head domain and a smaller stem domain. Attachment to the viral membrane is mediated by a C-terminal anchoring sequence linked to the stem domain. The protein is post-translationally cleaved in a specific loop to yield two polypeptides HA1 and HA2 (the entire sequence is referred to as HA0). The membrane distal head region is primarily derived from HA1, and the membrane proximal stem region is primarily derived from HA2 (FIG. 1).

  The reason that seasonal influenza vaccines must be updated annually is the large variability of the virus. In the hemagglutinin molecule, this mutation is particularly evident in the head domain where antigen drift and shift resulted in a number of different variants. Since this is also an immunodominant zone, most neutralizing antibodies are directed against this domain and act by preventing receptor binding. The combination of head domain immunodominance and large mutations also explains why infection by a particular strain does not result in immunity to other strains: antibodies elicited by the first infection are closely related to the virus of the primary infection It only recognizes a limited number of related stocks.

  Recently, influenza hemagglutinin stem domain polypeptides lacking all or substantially all influenza hemagglutinin globular head domains have been described and used to generate immune responses against one or more conserved epitopes of stem domain polypeptides. ing. The epitope of the stem domain polypeptide is less immunogenic than the highly immunogenic region of the globular head domain, so the absence of a globular head domain in the stem domain polypeptide is one of the stem domain polypeptides. It is believed that expression of immune responses against the above epitopes can be tolerated (Steel et al., 2010). Thus, Steel et al. Deletes amino acid residues 53 to 276 of HA1 from the A / Puerto Rico / 8/1934 (H1N1) and A / HongKong / 1968 (H3N2) strains from the HA primary sequence and replaces it with the short flexible binding sequence GGGG A new molecule was created. Vaccination of mice with the H3HK68 construct did not induce antisera cross-reactive with group 1 HA. Furthermore, as demonstrated in the examples below, stem domain polypeptides are highly unstable and are accurate as evidenced by the lack of binding of antibodies that have been shown to bind to conserved epitopes in the stem region. The three-dimensional structure was not adopted.

  Furthermore, Bommakanti et al. (2010) is a mutation in HA1 of amino acid residues 1-172 of HA2, amino acid residues 7-46 of HA1, amino acid residues 7-46 of HA1, and residues 290-321 of HA1 following the 6-amino acid linker GSAGSA. HA2-based polypeptides have been described for inclusion with V297T, I300E, Y302T and C305T. The design was based on the sequence of H3HA (A / Hong Kong / 1968). This polypeptide only provided cross-protection against another influenza virus strain within the H3 subtype (provided against A / Phil / 2/82 but not the H1 subtype (A / PR / 8 / 34 was not provided).

Thus, stimulating the production of a robust broadly neutralizing antibody response and providing protection against a wide range of current and future influenza virus strains (both seasonal and epidemic), particularly for effective prevention and treatment of influenza Phylogenetic group 1 and / or group 2
There remains a need for safe and effective universal vaccines that provide protection against one or more of the influenza A virus subtypes.

  Provided herein are influenza hemagglutinin stem domain polypeptides, methods of providing stem domain polypeptides, compositions comprising them, vaccines comprising them, and methods of their use.

  In a first aspect, the present invention provides a novel immunogenic polypeptide comprising an influenza hemagglutinin stem domain and lacking a globular head, referred to as an influenza (HA) stem domain polypeptide. A polypeptide can induce an immune response when administered to a subject, particularly a human subject. The polypeptides of the present invention present conserved epitopes of the membrane proximal stem domain HA molecule to the immune system in the absence of dominant epitopes present in the membrane distal head domain. For this purpose, part of the primary sequence of the HA0 protein constituting the head domain is removed and the residual amino acid sequence is introduced directly or, in some embodiments, a short flexible binding sequence (“linker”). By doing so, the continuity of the amino acid chain is restored. The resulting sequence is further modified by introducing defined mutations that stabilize the natural three-dimensional structure of the remaining portion of the HA0 molecule. The immunogenic polypeptide does not include full length HA1 and / or HA2 of influenza virus.

  Influenza hemagglutinin stem domain polypeptides are based on HA of influenza virus strains commonly used for human influenza vaccine production. In particular, the polypeptides are based on HA of H1, H5 and / or H3 subtypes of influenza A virus.

  In certain embodiments, the invention provides: (a) an influenza hemagglutinin HA1 domain comprising an HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a binding sequence of 0-50 amino acid residues; and (b) an influenza hemagglutinin HA2 An influenza hemagglutinin stem domain polypeptide comprising a domain that is resistant to protease cleavage at the junction between HA1 and HA2, and wherein one or more amino acids in the amino acid sequence linking HA2's A helix and helix CD are Provided are hemagglutinin stem domain polypeptides that are mutated relative to the wild-type influenza HA2 domain. Preferably, the HA1 and HA2 domains are derived from an influenza A virus selected from the group consisting of H1, H5 and H3 subtypes.

  The polypeptides of the present invention comprise one or more mutations in the HA2 amino acid sequence linking the C-terminal residue of helix A shown in FIG. 1 to the N-terminal residue of helix CD. In certain embodiments, one or more hydrophobic amino acids in the HA2 amino acid sequence are substituted with hydrophilic amino acids, eg, polar and / or charged amino acids, or the flexible amino acid glycine (G).

In certain embodiments, the HA1 N-terminal stem segment comprises amino acids 1-x of HA1, and the HA1 C-terminal stem segment comprises amino acids y-terminal of HA1 (ie, the C-terminal amino acid of HA1). Thus, in certain embodiments, the deletion of the HA1 segment comprises the amino acid sequence from the amino acid at position x + 1 to the amino acid at position y-1 (including that amino acid). In certain embodiments, the polypeptide does not include a signal sequence. Thus, in certain embodiments, the HA1 N-terminal segment comprises HA1 amino acids p-x, where p is the first amino acid of the mature HA molecule (eg, p = 18 for SEQ ID NO: 1). Those skilled in the art will recognize a signal peptide (eg, amino acids 1-17 of SEQ ID NO: 1).
The polypeptides described herein that do not have can be prepared. In certain embodiments, the polypeptides of the invention contain an intracellular sequence of HA and a transmembrane domain. In other embodiments, the polypeptides of the invention do not include the intracellular sequence of HA and the transmembrane domain. In certain embodiments, intracellular and transmembrane sequences, eg, from position 523, 524, 525, 526, 527, 526, 528, 529, or 530 of the HA2 domain (or their equivalents) to the C-terminus of the HA2 domain. The amino acid sequence has been removed.

  The polypeptide of the present invention does not include full-length HA1.

  In certain embodiments, the polypeptide is glycosylated.

  In certain embodiments, the immunogenic polypeptide is substantially smaller than HA0, and preferably lacks the globular head of all or substantially all HA. Preferably, the immunogenic polypeptide is 360 amino acids or less, preferably 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280, 275, or 270 amino acids in length. In certain embodiments, the immunogenic polypeptide is from about 250 to about 350, preferably from about 260 to about 340, preferably from about 270 to about 330, preferably from about 270 to about 330 amino acids in length.

  In certain embodiments, the polypeptide further comprises one or more additional mutations in the HA1 and / or HA2 domain as compared to the amino acid sequence of the HA on which the HA1 and HA2 domains are based.

The present invention
(A) providing an influenza HA0 amino acid sequence;
(B) removing the cleavage site of HA1 to HA2;
(C) removing from the HA0 sequence the amino acid sequence of the globular head domain, in particular the amino acid sequence starting from x + 1 up to the y-1 position;
(D) introducing one or more mutations in the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD; and (e) 1 in the HA stem domain polypeptide. Also provided are methods of providing influenza hemagglutinin stem polypeptides comprising the general steps of introducing one or more disulfide bridges.

  Polypeptides obtained by such methods are also part of the present invention.

In certain embodiments, the polypeptide is a Group 1 cross-neutralizing antibody CR6261 (disclosed in WO 2008/028946) and / or antibody CR9114 (described below and in co-pending application EP11173953.8). Conserved stem domain epitopes of antibodies that can bind to and neutralize both group 1 and group 2 influenza A viruses, and influenza B viruses. Accordingly, providing an HA stem domain polypeptide that binds to antibody CR6261 and / or antibody CR9114 is another aspect of the invention. In one embodiment, the polypeptide does not bind to CR8057 (described in WO 2010/130636), a monoclonal antibody that binds only to H3 influenza virus. In certain embodiments, the polypeptide binds to antibodies CR8020, CR8043 and / or CR9114. The influenza hemagglutinin stem domain polypeptides provided in the present invention are suitable for use in immunogenic compositions (eg, vaccines) that can generate an immune response against multiple influenza virus A and / or B strains. In one embodiment, the influenza hemagglutinin stem domain polypeptide may generate an immune response against phylogenetic group 1 and / or group 2 influenza A virus strains, particularly both phylogenetic group 1 and group 2 influenza virus strains. In one embodiment, the polypeptide may generate an immune response against the homologous influenza virus strain. In one embodiment, the polypeptide may generate an immune response against heterologous influenza virus strains of the same and / or different subtypes. In a further embodiment, the polypeptide may generate an immune response against both phylogenetic group 1 and group 2 influenza virus strains and influenza B virus strains.

  Polypeptides according to the invention can be used, for example, in stand-alone therapy and / or prevention and / or diagnosis of diseases or conditions caused by influenza viruses, in particular phylogenetic group 1 or 2 influenza A viruses and / or influenza B viruses, or other It can be used in combination with prophylactic and / or therapeutic treatments, for example (existing or future) vaccines, antiviral agents and / or monoclonal antibodies.

  In a further aspect, the present invention provides a nucleic acid molecule encoding an influenza HA stem domain polypeptide. In yet another aspect, the present invention provides a vector comprising a nucleic acid encoding an immunogenic polypeptide.

  In a further aspect, the present invention provides a method of inducing an immune response in a subject, comprising administering to the subject a polypeptide and / or nucleic acid molecule according to the present invention.

  In a further aspect, the present invention provides an immunogenic composition comprising a polypeptide and / or nucleic acid molecule according to the present invention. The immunogenic compositions provided herein can be in any form that allows administration of the composition to a subject, eg, a mouse, ferret or human. In a specific embodiment, the immunogenic composition is suitable for human administration. Polypeptides, nucleic acid molecules and compositions can be used in methods of preventing and / or treating influenza virus diseases and / or for diagnostic purposes. The composition may further comprise a pharmaceutically acceptable carrier or excipient. In certain embodiments, the compositions described herein comprise an adjuvant or are administered in combination with an adjuvant.

  In another aspect, the present invention provides polypeptides, nucleic acids and / or immunogenic compositions for use as vaccines. The invention particularly relates to an immunogenic polypeptide for use as a vaccine in the prevention and / or treatment of a phylogenetic group 1 and / or 2 influenza virus A subtype and / or a disease or condition caused by an influenza B virus, It relates to nucleic acids and / or immunogenic compositions.

  Various embodiments and uses of the polypeptides according to the present invention will become apparent from the following detailed description of the invention.

Figure 2 shows a model of pre-fusion state HA monomers present in natural trimers. HA1 is shown in light grey, and HA2 is shown in dark grey. Helix A (the critical part of the CR6261 epitope) and helix CD (the part of the trimer interface) are shown, and the loops connect their secondary structure elements. Binding of monoclonal antibodies to full-length HA and HA stem domain polypeptides according to the invention analyzed by FACS. A: Percentage of positive cells after staining. B: Average fluorescence intensity. H1-Full-Length (SEQ ID NO: 1), miniHA-cl1 (SEQ ID NO: 3), miniHA-cl1 + 2 (SEQ ID NO: 4), miniHA-cl1 + 3 (SEQ ID NO: 5), miniHA-cl1 + 4 (SEQ ID NO: 6), miniHA-cl1 + 2 + 3 (SEQ ID NO: 7), miniHA-cl1 + 2 + 3 + 4 (SEQ ID NO: 8). Binding of monoclonal antibodies to full-length HA and HA stem domain polypeptides analyzed by FACS. A: Percentage of positive cells after staining. B: Average fluorescence intensity. H1-Full-Length (SEQ ID NO: 1), miniHA (SEQ ID NO: 2), miniHA-cl1 (SEQ ID NO: 3). Binding of serum antibodies to HEK293F expressed full-length HA and polypeptides of the invention. A: Average fluorescence intensity. B: Percentage of positive cells after staining. H1-FL (SEQ ID NO: 1), CL1 (SEQ ID NO: 3), CL1 + 2 (SEQ ID NO: 4) and CL1 + 4 (SEQ ID NO: 6). cM2 is a negative control. Binding of serum antibodies to HEK293F expressed full-length HA and polypeptides of the invention. A: Average fluorescence intensity. B: Percentage of positive cells after staining. H1-FL (SEQ ID NO: 1), CL1 (SEQ ID NO: 3), CL1 + 2 (SEQ ID NO: 4) and CL1 + 4 (SEQ ID NO: 6). cM2 is a negative control. Binding of monoclonal antibodies to full-length HA and HA stem domain polypeptides analyzed by FACS. Top: Percentage of positive cells after staining. Below: Average fluorescence intensity. H1-Full-Length (SEQ ID NO: 1), miniHA-cl1 (SEQ ID NO: 3), H1-mini1-cl11 (SEQ ID NO: 9), H1-mini2-cl11 (SEQ ID NO: 10), H1-mini3-cl11 (SEQ ID NO: 10) 11), H1-mini4-cl11 (SEQ ID NO: 12), H1-mini1-cl11 + 5 (SEQ ID NO: 13), H1-mini2-cl11 + 5 (SEQ ID NO: 14), H1mini3-cl11 + 5 (SEQ ID NO: 15), and H1-mini4- cl11 + 5 (SEQ ID NO: 16). HAA / Brisbane / 59/2007 (SEQ ID NO: 1), miniHA-cluster1 (SEQ ID NO: 3), Mini2-cluster11 (SEQ ID NO: 10), Mini1-cluster11 + 5 (SEQ ID NO: 13), Mini2-cluster11 + 5 (SEQ ID NO: 14) and cM2 Intramuscular immunization with DNA encoding (consensus M2 sequence) or gene gun of DNA encoding HAA / Brisbane / 59/2007 (SEQ ID NO: 1), Mini2-cluster 11 + 5 (SEQ ID NO: 14) and cM2 (consensus M2 sequence) Serum antibody binding to the ectodomain of full-length HA from A / Brisbane59 / 2007 after immunization. Panel A: 28 days after the first immunization. Panel B: 49 days after immunization. HAA / Brisbane / 59/2007 (SEQ ID NO: 1), miniHA-cluster1 (SEQ ID NO: 3), Mini2-cluster11 (SEQ ID NO: 10), Mini1-cluster11 + 5 (SEQ ID NO: 13), Mini2-cluster11 + 5 (SEQ ID NO: 14) and cM2 Intramuscular immunization with DNA encoding (consensus M2 sequence) or gene gun of DNA encoding HAA / Brisbane / 59/2007 (SEQ ID NO: 1), Mini2-cluster 11 + 5 (SEQ ID NO: 14) and cM2 (consensus M2 sequence) Serum antibody binding to the ectodomain of full-length HA from A / Brisbane59 / 2007 after immunization. Panel A: 28 days after the first immunization. Panel B: 49 days after immunization. Binding of monoclonal antibodies to full-length HA and HA stem domain polypeptides analyzed by FACS. Top: Percentage of positive cells after staining. Below: Average fluorescence intensity. H1-Full-Length (SEQ ID NO: 1), miniHA (SEQ ID NO: 2), H1-mini2-cl11 + 5 (SEQ ID NO: 14), H1-mini2-cl1 + 5 (SEQ ID NO: 48), H1-mini2-cl1 + 5 + 6 (SEQ ID NO: 46) H1-mini2-cl11 + 5 + 6 (SEQ ID NO: 47), H1-mini2-cl1 + 5 + 6-trim (SEQ ID NO: 44), H1-mini2-cl1 + 5 + 6-GCN4 (SEQ ID NO: 45). Binding of monoclonal antibodies to full-length HA and HA stem domain polypeptides analyzed by FACS. A: Percentage of positive cells after staining. B: Average fluorescence intensity. Binding of monoclonal antibodies to full-length HA and HA stem domain polypeptides analyzed by FACS. A: Percentage of positive cells after staining. B: Average fluorescence intensity. Binding of monoclonal antibodies to full-length HA and HA stem domain polypeptides analyzed by FACS. A: Percentage of positive cells after staining. B: Average fluorescence intensity. Binding of monoclonal antibodies to full-length HA and HA stem domain polypeptides analyzed by FACS. A: Percentage of positive cells after staining. B: Average fluorescence intensity. Expression of the HongKong / 1/1968 base construct on the cell surface. Expression of the HongKong / 1/1968 base construct on the cell surface. SDS-PAGE (AD) and Western blot (EF) analysis of the purification of some polypeptides of the invention. For Western blots, antibodies directed against the his tag were used for detection. SDS-PAGE (AD) and Western blot (EF) analysis of the purification of some polypeptides of the invention. For Western blots, antibodies directed against the his tag were used for detection. SDS-PAGE (AD) and Western blot (EF) analysis of the purification of some polypeptides of the invention. For Western blots, antibodies directed against the his tag were used for detection. SDS-PAGE (AD) and Western blot (EF) analysis of the purification of some polypeptides of the invention. For Western blots, antibodies directed against the his tag were used for detection. SDS-PAGE (AD) and Western blot (EF) analysis of the purification of some polypeptides of the invention. For Western blots, antibodies directed against the his tag were used for detection. SDS-PAGE (AD) and Western blot (EF) analysis of the purification of some polypeptides of the invention. For Western blots, antibodies directed against the his tag were used for detection. Binding of monoclonal antibodies CR9114 (A), CR8020 (B) and polyclonal anti-H1HA serum (C) to several polypeptides of the invention detected by Elisa. Binding of monoclonal antibodies CR9114 (A), CR8020 (B) and polyclonal anti-H1HA serum (C) to several polypeptides of the invention detected by Elisa. Binding of monoclonal antibodies CR9114 (A), CR8020 (B) and polyclonal anti-H1HA serum (C) to several polypeptides of the invention detected by Elisa. SDS-PAGE (A) and Western blot (B) analysis of glycosylation of polypeptides of the invention. Upon deglycosylation, diffusion bands collect at the expected molecular weight. For Western blots, polyclonal sera directed against H1HA were used for detection. SDS-PAGE (A) and Western blot (B) analysis of glycosylation of polypeptides of the invention. Upon deglycosylation, diffusion bands collect at the expected molecular weight. For Western blots, polyclonal sera directed against H1HA were used for detection. SEC-MALS analysis of the polypeptides of the invention. Traces are labeled with SEQ ID NO. A: Western blot analysis of supernatant of cells expressing SEQ ID NO: 145. For Western blots, antibodies directed against the his tag were used for detection. B: Binding of monoclonal antibodies CR9114 (square), CR6261 (circle), CR8020 (regular triangle) and FI6v3 (inverted triangle) to SEQ ID NO: 145 detected by Elisa. Elution profile of purification of SEQ ID NO: 145 from culture supernatant on His trap column. The polypeptide of the invention elutes in 100 mM (peak A) and 200 mM (peak B) imidazole, respectively. AB: Elution profile of purification of SEQ ID NO: 145 by size exclusion chromatography (Superdex 200). Both peaks A and B contain the polypeptide of the invention. C: Native PAGE analysis of fractions from size exclusion chromatography. The majority of purified proteins run at molecular weights consistent with the monomeric form of the protein. D: SDS PAGE analysis of fractions from size exclusion chromatography. Time course of IgG response to the ectodomain of homologous full-length proteins as a result of the DNA immunization schedule described in this application. IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from the homologous strain H1N1A / Brisbane / 59/2007 (A) and the heterologous strain H1N1A / California / 07/2009 (B). Open circle symbols correspond to values below the detection limit of the assay. IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from seed strain H1N1A / California / 07/2009 (B). Open circle symbols correspond to values below the detection limit of the assay. IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from the homologous strain H1N1A / Brisbane / 59/2007 (A) and the heterologous strain H1N1A / California / 07/2009 (B). Open circle symbols correspond to values below the detection limit of the assay. IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from seed strain H1N1A / California / 07/2009 (B). Open circle symbols correspond to values below the detection limit of the assay. Homologous strains H1N1A / Brisbane / 59/2007 (A), heterologous strains H1N1A / California / 07/2009 (B) heterologous subtype strains H5N1A / Vietnam / 1203/2004 (C) and heterologous subtype strains H3N2A / HongKong / 1 / IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from 1968 (D). Open circle symbols correspond to values below the detection limit of the assay. Homologous strains H1N1A / Brisbane / 59/2007 (A), heterologous strains H1N1A / California / 07/2009 (B) heterologous subtype strains H5N1A / Vietnam / 1203/2004 (C) and heterologous subtype strains H3N2A / HongKong / 1 / IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from 1968 (D). Open circle symbols correspond to values below the detection limit of the assay. Homologous strains H1N1A / Brisbane / 59/2007 (A), heterologous strains H1N1A / California / 07/2009 (B) heterologous subtype strains H5N1A / Vietnam / 1203/2004 (C) and heterologous subtype strains H3N2A / HongKong / 1 / IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from 1968 (D). Open circle symbols correspond to values below the detection limit of the assay. Homologous strains H1N1A / Brisbane / 59/2007 (A), heterologous strains H1N1A / California / 07/2009 (B) heterologous subtype strains H5N1A / Vietnam / 1203/2004 (C) and heterologous subtype strains H3N2A / HongKong / 1 / IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from 1968 (D). Open circle symbols correspond to values below the detection limit of the assay. FACS assay of stem domain polypeptide based on H3HA. Average fluorescence intensity (A) and% positive cells (B) are shown. Individuals against full length hemagglutinin from homologous strains H1N1A / Brisbane / 59/2007 (panel A), heterologous strain H1N1A / California / 07/2009 (panel B) and heterologous subtype strains H5N1A / Vietnam / 1203/2004 (panel C) IgG response 7 weeks after the first immunization for mice. Open circle symbols correspond to values below the detection limit of the assay. Individuals against full length hemagglutinin from homologous strains H1N1A / Brisbane / 59/2007 (panel A), heterologous strain H1N1A / California / 07/2009 (panel B) and heterologous subtype strains H5N1A / Vietnam / 1203/2004 (panel C) IgG response 7 weeks after the first immunization for mice. Open circle symbols correspond to values below the detection limit of the assay. Individuals against full length hemagglutinin from homologous strains H1N1A / Brisbane / 59/2007 (panel A), heterologous strain H1N1A / California / 07/2009 (panel B) and heterologous subtype strains H5N1A / Vietnam / 1203/2004 (panel C) IgG response 7 weeks after the first immunization for mice. Open circle symbols correspond to values below the detection limit of the assay. FACS analysis of binding of mAbs CR6261, CR9114, CR8020 and CR9020, and polyclonal anti-H1 sera to full-length HA and corresponding polypeptides of the invention. Top: Average fluorescence intensity. Below: Percentage of positive cells. The black bar represents the full-length protein, and the striped bar represents the polypeptide of the present invention. The corresponding polypeptide of the present invention derived from full length HA and its sequence has the same background color. Kaplan-Meier survival curve (A), weight change (B), and median clinical score (C) for the influenza challenge experiment described in Example 21. Kaplan-Meier survival curve (A), weight change (B), and median clinical score (C) for the influenza challenge experiment described in Example 21. Kaplan-Meier survival curve (A), weight change (B), and median clinical score (C) for the influenza challenge experiment described in Example 21. Alignment of the H1N1 sequence selected according to Example 22. FACS assay of stem domain polypeptides based on H1HA selected according to Example 22. Average fluorescence intensity is shown. Full length H1HA (SEQ ID NO: 149) in trimeric and monomeric form to immobilized monoclonal antibodies CR6261 (A), CR9114 (B) and CR8020 (C) and s-H1- as determined by biolayer interferometry Kinetics of binding of mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145). Biolayer interferometry following steady state titration of binding of s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) to immobilized CR6261 (A) and CR9114 (B). IgG response to the ectodomain of HA from A / HongKong / 1/1968 at day 49 as measured by ELISA. Experimental details are described in Example 24. A white symbol corresponds to a value below the detection limit of the assay. Kaplan-Meier survival curve (A), body weight change (B) and median clinical score (C) for the influenza challenge experiment described in Example 24. Kaplan-Meier survival curve (A), body weight change (B) and median clinical score (C) for the influenza challenge experiment described in Example 24. Kaplan-Meier survival curve (A), body weight change (B) and median clinical score (C) for the influenza challenge experiment described in Example 24. FACS assay of stem domain polypeptides based on H1HA selected according to Example 25. Average fluorescence intensity is shown. IgG response after 49 days against the ectodomain of HA as measured by HA from A / Wisconsin / 67/2005 (A), A / Hong Kong / 1/1968 (B) and A / Perth / 16/2009 (C) . Experimental details are described in Example 26. Open circle symbols correspond to values below the detection limit of the assay. IgG response after 49 days against the ectodomain of HA as measured by HA from A / Wisconsin / 67/2005 (A), A / Hong Kong / 1/1968 (B) and A / Perth / 16/2009 (C) . Experimental details are described in Example 26. Open circle symbols correspond to values below the detection limit of the assay. IgG response after 49 days against the ectodomain of HA as measured by HA from A / Wisconsin / 67/2005 (A), A / Hong Kong / 1/1968 (B) and A / Perth / 16/2009 (C) . Experimental details are described in Example 26. Open circle symbols correspond to values below the detection limit of the assay. IgG response after 49 days against the ectodomain of HA from A / HongKong / 1/1968 measured by ELISA. Experimental details are described in Example 27. A white symbol corresponds to a value below the detection limit of the assay. FACS assay of stem domain polypeptides based on H1HA selected according to Example 28. Average fluorescence intensity is shown.

Definitions The definitions of terms used in the present invention are listed below.

  Amino acids according to the present invention include 20 natural (or “standard” amino acids) or variants thereof, such as D-proline (D-enantiomer of proline), or any variant not naturally found in proteins, such as norleucine It can be either. Standard amino acids can be divided into groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size and functional group. These properties are important for protein structure and protein-protein interactions. Some amino acids have special properties, for example, cysteine can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, and proline forms periodicity to the polypeptide backbone. However, glycine is more flexible than other amino acids. Table 5 shows the abbreviations and properties of standard amino acids.

  The term “amino acid sequence identity” refers to the degree of identity or similarity between a pair of aligned amino acid sequences, usually expressed as a percentage. The percent identity is the same (ie, the amino acid residue at a given position in the alignment is the same residue) or similar (ie, the amino acid substitution at the given position in the alignment is conservative as discussed below). The percentage of amino acid residues in the candidate sequence (which are substitutions) and the corresponding amino acid residues in the peptide after aligning the sequence and introducing gaps as necessary to achieve maximum percent sequence homology. is there. Sequence homology, such as percent sequence identity and similarity, is determined using sequence alignment techniques well known in the art, for example, by visual inspection and mathematical calculations, or more preferably, the comparison is performed by computer This is done by comparing the sequence information using a program. An exemplary preferred computer program is the Genetics Computer Group (GCG; Madison, Wis.) Wisconsin package version 10.0 program, “GAP” (Devereux et al. (1984)).

A “conservative substitution” refers to the replacement of one class of amino acids with another amino acid of the same class. In certain embodiments, conservative substitutions do not change the structure or function of the polypeptides of the invention, or both. The class of amino acids for conservative substitution purposes is hydrophobic (eg, Met, Ala, Val, Leu), neutral hydrophilic (eg, Cys, Se).
r, Thr), acidic (eg, Asp, Glu), basic (eg, Asn, Gln, His, Lys, Arg), conformational disrupters (eg, Gly, Pro) and aromatics (eg, Trp, Tyr) , Phe).

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

  The term “effective amount” as used herein in the context of administering a therapeutic to a subject refers to the amount of the therapeutic that has a prophylactic and / or therapeutic effect. In certain embodiments, an “effective amount” in the context of administering a therapeutic to a subject is a reduction or amelioration of the severity of an influenza virus infection, associated disease or condition, such as, but not limited to, an influenza virus infection. , Reduction of duration of associated diseases or symptoms, influenza virus infection, prevention of progression of associated diseases or symptoms, influenza virus infection, prevention of onset or recurrence of associated diseases or symptoms, from one subject to another Prevention or reduction of the spread of influenza virus to the patient, reduction of hospitalization and / or duration of hospitalization of the subject, increased survival of subjects with influenza virus infection or associated disease, elimination of influenza virus infection or associated disease, Inhibition or reduction of influenza virus replication, reduced influenza viral titers; refers to a sufficient amount of therapeutic compounds in order to achieve improved and / or improvement of prophylactic or therapeutic effect and / or another therapeutic compound. In certain embodiments, an effective amount does not provide complete protection from influenza virus disease, but results in a lower titer or reduced number of influenza virus compared to an untreated subject. Benefits of reducing influenza virus titer, number or total burden include, but are not limited to, milder infection symptoms, fewer infection symptoms and a reduction in the duration of the disease associated with the infection.

  The term “host” as used herein is intended to refer to an organism or cell into which a vector, eg, a cloning vector or expression vector has been introduced. The organism or cell can be prokaryotic or eukaryotic. Preferably, the host includes isolated host cells, eg, host cells in culture. The term “host cell” merely means that the cell has been modified for (over) expression of a polypeptide of the invention. It should be understood that the term host not only refers to a particular subject organism or cell, but also to the progeny of such an organism or cell. Such progeny may not be virtually identical to the parent organism or cell because certain modifications may occur in subsequent generations due to either mutations or environmental influences, but are still used herein. Within the scope of the term “host”.

  The terms “included” or “as an example” as used herein are considered to be followed by the word “but not limited to”.

The term “infection” as used herein refers to invasion due to the propagation and / or presence of a virus in a cell or subject. In one embodiment, the infection is an “active” infection, ie, the virus is replicating in a cell or subject. Such infection is characterized by the spread of the virus from cells, tissues, and / or organs originally infected by the virus to other cells, tissues, and / or organs. The infection can also be a latent infection, i.e. the virus is not replicating. In certain embodiments, an infection results from the presence of a virus in a cell or subject or refers to a pathological condition due to the invasion of a cell or subject by a virus.

  Influenza viruses are classified into influenza virus types: A, B and C genera. The term “influenza virus subtype” as used herein refers to an influenza A virus variant characterized by a combination of hemagglutinin (H) and neuraminidase (N) virus surface proteins. According to the present invention, influenza virus subtypes have their H numbers, such as “influenza virus containing HA of H3 subtype”, “H3 subtype influenza virus” or “H3 influenza” or the like, and the H number and A combination of N numbers, for example, “influenza virus subtype H3N2” or “H3N2” can be used. The term “subtype” specifically includes all individual “strains”, typically natural isolates and artificial mutations within each subtype that usually arise from mutations and exhibit different pathogenic profiles. Includes body or rear sortant. Such strains can also be referred to as various “isolates” of the virus subtype. Accordingly, the terms “strain” and “isolate” as used herein can be used interchangeably. Current nomenclature for human influenza virus strains or isolates is usually listed in parentheses in the virus type (genus), ie, A, B or C, the geographical location of the first segregation, strain number and year of segregation. Description of HA and NA antigens to be included, for example A / Moscow / 10/00 (H3N2). Non-human strains also include hosts of origin in nomenclature. Influenza A virus subtypes can be further classified by reference to their phylogenetic groups. Phylogenetic analysis demonstrated the subclassification of hemagglutinin into two major groups: H1, H2, H5 and H9 subtypes (“Group 1” influenza viruses) in phylogenetic group 1 and H3 in phylogenetic group 2, among others. H4, H7 and H10 subtypes ("Group 2" influenza virus ").

  The term “influenza virus disease” as used herein refers to a pathological condition resulting from the presence of an influenza virus in a cell or subject, eg, the presence of influenza A or B virus, or the invasion of a cell or subject by an influenza virus. In a specific embodiment, the term refers to a respiratory illness caused by influenza virus.

  As used herein, the term “nucleic acid” includes DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) as well as DNA or RNA analogs produced using nucleotide analogs. Shall. The nucleic acid can be single-stranded or double-stranded. Nucleic acid molecules can be chemically or biochemically modified, or can contain non-natural or derivatized nucleotide bases, which are readily recognized by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more natural nucleotides with analogs, internucleotide modifications, such as uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc. ), Charged bonds (eg, phosphorothioate, phosphorodithioate, etc.), pendant moieties (eg, polypeptides), intercalating agents (eg, acridine, psoralen, etc.), chelating agents, alkylating agents, and modified bonds (eg, Such as alpha anomeric nucleic acids). Reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to include its complementary strand having its complementary sequence. Complementary strands are also useful, for example, for antisense therapy, hybridization probes, and PCR primers.

In certain embodiments, the numbering of amino acids in HA as used herein is the numbering of amino acids in HA0 of wild-type influenza virus, eg, H1N1 influenza strain A / Brisbane / 59/2007 (SEQ ID NO: 1 ) Based on amino acid numbering. Thus, the term “amino acid at position“ x ”in HA” as used herein refers to certain wild-type influenza viruses, eg, A / Brisbane / 59/2007 (SEQ ID NO: 1; the amino acids of the HA2 domain are italicized. Means the amino acid corresponding to the amino acid at position x in HA0. It will be appreciated by those skilled in the art that the corresponding amino acids in other influenza virus strains and / or subtypes can be determined by multiple sequence alignment (see, eg, Table 8). Note that in the numbering system used throughout this application, 1 refers to the N-terminal amino acid of the immature HA0 protein (SEQ ID NO: 1). The mature sequence begins, for example, at position 18 of SEQ ID NO: 1. In certain embodiments, the numbering of the corresponding amino acids is based on the numbering of the amino acids in H3HA0, in particular the amino acid numbering of H3N2 influenza strain A / Wisconsin / 67/2005 (SEQ ID NO: 89). Corresponding amino acids in other H3HA sequences can be determined by alignment. A leader sequence (or signal sequence) that directs transport of the protein during production (eg, corresponding to amino acids 1-17 of SEQ ID NO: 89) is generally not present, for example, in the final polypeptide used in the vaccine. As understood by those skilled in the art. Thus, in one embodiment, the polypeptide according to the invention comprises an amino acid sequence without a leader sequence, ie the amino acid sequence is based on the amino acid sequence of HA0 without a signal sequence.

  "Polypeptide" refers to a polymer of amino acids linked by amide bonds known by those skilled in the art. As used herein, the term can refer to a single polypeptide chain linked by a covalent amide bond. The term can also refer to multiple polypeptide chains that are associated by non-covalent interactions such as ionic, hydrogen bonding, van der Waals and hydrophobic contacts. Those skilled in the art include, for example, post-translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (eg, N-linked glycosylation), protease cleavage, and lipid modification (eg, S-palmitoylation). It is recognized that modified polypeptides are included.

  “Stem domain polypeptide” refers to a polypeptide comprising one or more polypeptide chains that constitute the stem domain of a natural (or wild type) hemagglutinin (HA). Typically, a stem domain polypeptide is a single polypeptide chain (ie, corresponding to the stem domain of a hemagglutinin HA0 polypeptide) or two polypeptide chains (ie, a hemagglutinin HA1 polypeptide associated with a hemagglutinin HA2 polypeptide). Corresponding to the stem domain of the peptide). According to the present invention, a stem domain polypeptide contains one or more mutations compared to a wild type HA molecule, in particular one or more amino acid residues of the wild type HA correspond to a particular wild type HA. It may be substituted by another amino acid that does not occur naturally at the position. The stem domain polypeptide according to the present invention may further comprise one or more of the following binding sequences:

  The term “vector” refers to a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host that is replicated and expressed in some cases. In other words, the vector can transport the nucleic acid molecule to which it is bound. Cloning and expression vectors are contemplated by the term “vector” as used herein. Vectors include, but are not limited to, vectors derived from plasmids, cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) and bacteriophage or plant or animal (eg, human) viruses. . Vectors include origins of replication recognized by the host to be presented and in the case of expression vectors, promoters and other regulatory regions recognized by the host. Certain vectors are capable of autonomous replication in a host into which they are introduced (eg, vectors having a bacterial origin of replication can replicate in bacteria). Other vectors can be integrated into the genome of the host upon introduction into the host, whereby the vector is replicated along the host genome. As used herein, the term “wild type” in the context of a virus refers to an influenza virus that is frequent, naturally circulating, and causing the spread of typical diseases.

DETAILED DESCRIPTION Influenza viruses have a significant impact on global public health, causing millions of seriously ill cases, thousands of deaths, and considerable economic losses each year. Current trivalent influenza vaccines elicit strong neutralizing antibody responses against vaccine strains and closely related isolates, but rarely span more divergent strains within one subtype or other subtypes. Furthermore, the selection of an appropriate vaccine strain presents many difficulties and frequently results in suboptimal protection. Furthermore, the prediction of the next epidemic virus subtype, including where and when it occurs, is currently impossible.

  Hemagglutinin (HA) is the major envelope glycoprotein from influenza A virus, which is the major target for neutralizing antibodies. Hemagglutinin has two main functions during the influx process. First, hemagglutinin mediates viral attachment to the surface of target cells through interaction with sialic acid receptors. Second, after viral endocytosis, hemagglutinin subsequently causes fusion of the virus and endosomal membrane to release its genome into the cytoplasm of the target cell. HA contains a large ectodomain of about 500 amino acids that is cleaved by host-derived enzymes to produce two polypeptides that remain linked by disulfide bonds. The majority of N-terminal fragments (HA1, 320-330 amino acids) form a membrane distal globular domain containing the receptor binding site and most antigenic determinants recognized by virus neutralizing antibodies. The smaller C-terminal part (HA2, approximately 180 amino acids) forms a stem-like structure that anchors the globular domain to the cell or viral membrane. The degree of sequence homology between the subtypes is less for the HA1 polypeptide (34% -59% homology between subtypes) than the HA2 polypeptide (51-80% homology). Most conserved regions are sequences around the cleavage site, particularly the HA2 N-terminal 23 amino acids, which are conserved in all influenza A virus subtypes (Loriau et al., 2010). A portion of this region is exposed as a surface loop in the HA precursor molecule (HA0), but becomes inaccessible when HA0 is cleaved into HA1 and HA2.

  Most neutralizing antibodies bind to the loop surrounding the receptor binding site and prevent receptor binding and attachment. Because these loops are highly mutated, most antibodies that target those regions are strain-specific and why current vaccines induce such limited strain-specific immunity explain. In recent years, however, fully human monoclonal antibodies against influenza virus hemagglutinin have been generated that have broad cross-neutralizing efficacy. Functional and structural analysis revealed that these antibodies interfere with the membrane fusion process and are directed against highly conserved epitopes in the stem domain of influenza HA protein (Throsby et al., 2008; Ekiert). et al. 2009, WO 2008/028946 pamphlet, WO 2010/130636 pamphlet).

  In accordance with the present invention, novel HA stem domain polypeptides containing these epitopes were designed to create universal epitope-based vaccines that induce protection against a wide range of influenza strains. In essence, the highly mutated and immunodominant part, ie, the head domain, is first removed from the full-length HA molecule to create a stem domain polypeptide, also referred to as mini-HA. Thus, the immune response is redirected to the stem domain where the epitope for the broadly neutralizing antibody is located. The exact folding of newly generated molecules using the above broadly neutralizing antibodies was examined to confirm the presence of neutralizing epitopes.

The stem domain polypeptides of the invention can present a conserved epitope of the membrane proximal stem domain HA molecule to the immune system in the absence of a dominant epitope present in the membrane distal head domain. For this purpose, part of the primary sequence of the HA0 protein constituting the head domain is removed and reintroduced directly or, in some embodiments, by introducing a short flexible binding sequence (“linker”). Ligate to restore continuity of the polypeptide chain. The resulting polypeptide sequence is further modified by introducing defined mutations that stabilize the native three-dimensional structure of the remaining portion of the HA0 molecule.

  Accordingly, the present invention comprises (a) an influenza hemagglutinin HA1 domain comprising an HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a binding sequence of 0-50 amino acid residues, and (b) an influenza hemagglutinin HA2 domain. A polypeptide is provided, wherein one or more amino acids in the HA2 domain are mutated. Accordingly, in the polypeptides of the present invention, the HA2 domain contains one or more mutations compared to the HA2 domain of wild-type influenza hemagglutinin on which the HA stem domain polypeptide is based.

  Influenza hemagglutinin stem domain polypeptides are generally based on HA of the influenza A virus subtype used in human influenza virus vaccines. In a preferred embodiment, the stem domain polypeptide is based on the HA of an influenza virus comprising HA of the H1, H5 and / or H3 subtypes.

  The invention specifically includes (a) an influenza hemagglutinin HA1 domain comprising an HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a binding sequence of 0-50 amino acid residues, and (b) an influenza hemagglutinin HA2 domain. Influenza hemagglutinin stem domain polypeptide, resistant to protease cleavage at the junction between HA1 and HA2, wherein one or more amino acids in the amino acid sequence linking HA2's A helix and helix CD are wild-type influenza Hemagglutinin stem domain polypeptides that are mutated compared to the HA2 domain are provided. Preferably, the HA1 and HA2 domains are derived from an influenza A virus subtype selected from the group consisting of H1, H5 and H3.

  Accordingly, the polypeptides of the present invention contain one or more mutations in the HA2 amino acid sequence that link the C-terminal residue of helix A shown in FIG. 1 to the N-terminal residue of helix CD. In certain embodiments, one or more hydrophobic amino acids in the HA2 amino acid sequence are substituted with hydrophilic amino acids, eg, polar and / or charged amino acids, or the flexible amino acid glycine (G).

  The polypeptide of the present invention does not include full-length HA1.

  In certain embodiments, the immunogenic polypeptide is substantially smaller than HA0, and preferably lacks the globular head of all or substantially all HA. Preferably, the immunogenic polypeptide is 360 amino acids or less, preferably 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280, 275, or 270 amino acids in length. In certain embodiments, the immunogenic polypeptide is from about 250 to about 350, preferably from about 260 to about 340, preferably from about 270 to about 330, preferably from about 270 to about 330 amino acids in length.

  In certain embodiments, the polypeptide further comprises one or more additional mutations in the HA1 and / or HA2 domains as compared to the amino acid sequence of the HA from which the HA1 and HA2 domains are derived. Thus, the stability of the stem polypeptide is further increased.

According to the present invention, the “HA1 N-terminal segment” is an influenza hemagglutinin (H
A) refers to a polypeptide segment corresponding to the amino terminal portion of the HA1 domain of a molecule. In certain embodiments, the HA1 N-terminal polypeptide segment comprises amino acids 1 to x of the HA1 domain, and the amino acid on the x position is an amino acid residue within HA1. The term “HA1 C-terminal segment” refers to a polypeptide segment corresponding to the carboxy-terminal portion of the influenza hemagglutinin HA1 domain. In certain embodiments, the HA1 C-terminal polypeptide segment comprises amino acids from the y-position to the C-terminal amino acid (including the amino acids) of the HA1 domain, and the amino acid on the y-position is an amino acid residue within HA1. According to the present invention, y is greater than x, and thus a segment of the HA1 domain is deleted between the HA1 N-terminal segment and the HA1C-terminal segment, ie between the amino acid at position x and the amino acid at position y of HA1. And in some embodiments has been replaced by a binding sequence.

  In certain embodiments, the HA1 N-terminal stem segment comprises amino acids 1-x of HA1 and the HA1 C-terminal stem segment comprises amino acids y-terminal of HA1. Thus, in certain embodiments, the deletion of the HA1 segment comprises the amino acid sequence from the amino acid at position x + 1 to the amino acid at position y-1 (including that amino acid).

  In certain embodiments, the polypeptide does not include a signal sequence. Thus, in certain embodiments, the HA1 N-terminal segment comprises HA1 amino acids p-x, where p is the first amino acid of the mature HA molecule (eg, p = 18 for SEQ ID NO: 1). One skilled in the art can prepare the polypeptides described herein that do not have a signal peptide (eg, amino acids 1-17 of SEQ ID NO: 1). In certain embodiments, the polypeptides of the invention contain an intracellular sequence of HA and a transmembrane domain. In other embodiments, the polypeptides of the invention do not include the intracellular sequence of HA and the transmembrane domain. In certain embodiments, the intracellular and transmembrane sequence, eg, the amino acid at the C-terminus of the HA2 domain from position 523, 524, 525, 526, 527, 526, 528, 529, or 530 (or equivalent) of the HA2 domain. The sequence has been removed.

  According to the present invention, the hemagglutinin stem domain polypeptide is resistant to protease cleavage at the junction between HA1 and HA2. The Arg (R) -Gly (G) sequence spanning HA1 and HA2 is a recognition site for trypsin and trypsin-like proteases and is typically known to those skilled in the art to be cleaved for hemagglutinin activation. is there. Since the HA stem domain polypeptides described herein should not be activated, the influenza hemagglutinin stem domain polypeptides of the present invention are resistant to protease cleavage. Thus, according to the present invention, the protease cleavage site is removed or the protease site spanning HA1 and HA2 is mutated to a sequence that is resistant to protease cleavage.

  In certain embodiments, the C-terminal amino acid residue of the HA1 C-terminal stem segment is any amino acid other than arginine (R) or lysine (K). In certain embodiments, the HA1C terminal amino acid is glutamine (Q), serine (S), threonine (T), asparagine (N), aspartic acid (D) or glutamic acid (E). In certain embodiments, the C-terminal amino acid residue of the HA1 C-terminal stem segment is glutamine (Q).

  In certain embodiments, the polypeptide is glycosylated.

The influenza hemagglutinin stem domain polypeptide can be based on the HA of any native influenza A hemagglutinin virus of the subtype used in human influenza vaccines. The influenza A virus subtype commonly used in influenza vaccines is the H1, H3 or H5 subtype of influenza A virus. “Based” means any natural form of the H1, H3 and / or H5 subtypes where the N-terminal segment and / or the C-terminal segment of the HA1 domain and / or HA2 domain is known or later discovered by those skilled in the art. At least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or the corresponding N-terminal and / or C-terminal segment of the HA1 and / or HA2 domain of influenza hemagglutinin, or Means having 99% amino acid sequence identity. In certain embodiments, the influenza hemagglutinin stem domain polypeptide is based on influenza hemagglutinin of group 1 influenza A viruses. In certain embodiments, the influenza hemagglutinin stem domain polypeptide is based on group 2 influenza A virus influenza hemagglutinin. In some embodiments, the influenza hemagglutinin stem domain polypeptide is a hybrid or chimeric polypeptide comprising or consisting of segments and / or domains from multiple influenza strains or subtypes. For example, an influenza hemagglutinin stem domain polypeptide may comprise HA1 N-terminal and HA1 C-terminal stem segments and / or HA2 domains from different influenza A virus HA subtypes.

  In certain embodiments, the polypeptide is based on H1HA. In certain embodiments, the polypeptide is from an influenza A virus comprising HA of subtype H1, such as from HA from influenza virus A / Brisbane / 59/2007 (H1N1) (SEQ ID NO: 1) or A hemagglutinin stem domain based on It will be appreciated by those skilled in the art that other influenza A viruses, including H1 subtype HA, can also be used in accordance with the present invention. In certain embodiments, the polypeptide comprises an influenza AH1 virus HA-based hemagglutinin stem domain selected from Table 7.

  In certain embodiments, the polypeptide comprises a HA1 N-terminal polypeptide segment comprising amino acids 1 to x of the H1 HA domain, where x is any amino acid from amino acids 46 to 60, for example, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 or 59 amino acids, preferably x is 52, 53, 55 or 59. Preferably, the polypeptide is from the HA1 N-terminal segment without a signal sequence, ie, position 18 of the HA1 domain (eg, for H1HA, eg, SEQ ID NO: 1) or from a corresponding position in other H1 influenza virus strains x The HA1 N-terminal segment containing the amino acid at position is included. Thus, in certain embodiments, the HA1 N-terminal segment comprises the amino acid at the x position from the p position of the HA1 domain (p = 18 for H1HA of SEQ ID NO: 1, or the equivalent position on other H1HA).

  In certain embodiments, the HA1 C-terminal polypeptide segment comprises an amino acid from the y-position of the H1HA1 domain to the C-terminal amino acid (including that amino acid), wherein y is any amino acid from position 290 to position 325 of H1HA1. Preferably, y is 291, 303, 318, or 321. In accordance with the present invention, the HA2 domain contains one or more mutations in the HA2 amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD (FIG. 1). In certain embodiments, one or more hydrophobic amino acids in the HA2 amino acid sequence are replaced with hydrophilic amino acids, eg, polar and / or charged amino acids. In certain embodiments (eg, for H1HA, eg, SEQ ID NO: 1), the HA2 amino acid sequence linking the C-terminal residue of helix A and the N-terminal residue of helix CD is residues 402-418 of influenza HA2. Of the amino acid sequence. In certain embodiments, the HA2 amino acid sequence linking the C-terminal residue of helix A and the N-terminal residue of helix CD is the amino acid sequence MNTQFTAVGKEFN (H / K) LE (K / R) (SEQ ID NO: 17). Including.

  In certain embodiments, x is 59 and y is 291.

  In certain embodiments, x is 52 and y is 321.

  In certain embodiments, x is 53 and y is 303.

  In certain embodiments, x is 55 and y is 318.

In one embodiment, the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD is the amino acid sequence of amino acids 402 to 418 of HA2 of SEQ ID NO: 1. Correspondingly, the polypeptide comprises one or more mutations in the amino acid sequence spanning amino acids 402 to 418 of SEQ ID NO: 1. The amino acid sequence of residues 402 to 418 of serotype H1 influenza HA includes the amino acid sequence MNTQFTAVGKEFN (H / K) LE (K / R) (SEQ ID NO: 17). In certain embodiments, the amino acid sequence of residues 402-418 of influenza HA of serotype H1 comprises the amino acid sequence MNTQX ₁ TAX ₂ GKEX ₃ N (H / K) X ₄ E (K / R).

Accordingly, in certain embodiments, the polypeptide comprises one or more of the mutations in the H1HA2 domain shown in Table 6. In certain embodiments, one or more of the amino acids at positions 406, 409, 413 and 416, ie, one or more of amino acids X ₁ , X ₂ , X ₃ and X ₄ are mutated (numbering is Refers to SEQ ID NO: 1). In certain embodiments, the amino acid at position 406, ie, X ₁ is changed to an amino acid selected from the group consisting of S, T, N, Q, R, H, K, D, E, and G. S is preferable. In certain embodiments, the amino acid on position 409, i.e., _{X 2} is, S, T, N, Q , R, H, K, D, is changed to an amino acid selected from the group consisting of E, and G , Preferably T, Q or G. In certain embodiments, the amino acid on the 413-position, i.e., _{X 3} is, S, T, N, Q , R, H, K, D, E, and changed to an amino acid selected from the group consisting of G, S is preferable. In certain embodiments, the amino acid at position 416, i.e., X ₄ is changed to an amino acid selected from the group consisting of S, T, N, Q, R, H, K, D, E, G; S is preferable. Combinations of these mutations are also possible.

  In certain embodiments, the HA1 N-terminal stem segment comprises amino acid residues 1-59 of HA1, and the HA1 C-terminal stem segment comprises amino acid residues 291-343 of HA1, the amino acid at position 343, ie, R343, It is mutated and is an amino acid other than R, preferably glutamine (Q). In certain embodiments, the HA1 N-terminal segment consists of amino acid residues 1 to 59 of HA1, and the HA1 C-terminal segment consists of amino acid residues 291 to 343 of HA1. It is noted that the amino acid numbering is based on the amino acid numbering in H1HA0, especially the amino acid numbering of H1N1 influenza strain A / Brisbane / 59/2007 (SEQ ID NO: 1). It is noted that the numbering is not always identical since the HA sequences of different influenza subtypes / strains may have insertions or deletions in the head region compared to each other. One skilled in the art can determine the corresponding amino acid positions in the HA sequences of different influenza virus strains and / or subtypes by sequence alignment.

  In certain embodiments, the HA1 N-terminal polypeptide segment does not include a signal sequence. In a preferred embodiment, the HA1 N-terminal segment comprises amino acids 18 to 59 of the HA1 domain. In certain embodiments, the HA1 N-terminal segment consists of amino acids 18-59 of the HA1 domain.

  In some embodiments, the polypeptides of the invention comprise one or more additional mutations in the HA1 domain and / or HA2 domain, ie amino acid substitutions. Thus, in certain embodiments, the HA1 domain further comprises one or more of the following mutations: L58T, V314T and I316T. Again, it is noted that the amino acid numbering is based on the amino acid numbering in H1HA0, in particular the amino acid numbering of the H1N1 influenza strain A / Brisbane / 59/2007 (SEQ ID NO: 1). One skilled in the art can determine the equivalent amino acid in the HA of other influenza H1 viruses and thus can determine the equivalent mutation.

  In a specific embodiment, the HA1 domain includes mutations L58T, V314T, and I316T, and the HA2 domain includes one or more of the following mutations: F406S, V409T, and L416S.

  In certain embodiments, the HA1 domain further comprises mutation K321C, and / or the HA2 domain further comprises one or more of the following mutations: Q405C, F413C, E421C, and Y502S.

  In a specific embodiment, the HA1 domain includes mutations L58T, V314T, I316T, and K321C, and the HA2 domain includes mutations: Q405C, F406S, V409T, and L416S.

  In a specific embodiment, the HA1 domain includes mutations L58T, V314T, and I316T, and the HA2 domain includes mutations: F406S, V409T, F413C, L416S, and E421C.

  In a specific embodiment, the HA1 domain includes mutations L58T, V314T, and I316T, and the HA2 domain includes mutations: F406S, V409T, L416S, and Y502S.

  In a specific embodiment, the HA1 domain includes mutations L58T, V314T, I316T, and K321C, and the HA2 domain includes mutations: Q405C, F406S, V409T, F413C, L416S, and E421C.

  In a specific embodiment, the HA1 domain includes mutations L58T, V314T, I316T, and K321C, and the HA2 domain includes mutations: Q405C, F406S, V409T, F413C, L416S, E421C, and Y502S.

  In other embodiments, the HA2 domain further comprises one or more of mutations M420I and V421I, or equivalent mutations.

  In a specific embodiment, the HA1 domain includes mutations L58T, V314T, and I316T, and the HA2 domain includes one or more of the following mutations: F406S, V409T, L416S, M420I, and V421I.

In certain embodiments, the HA1 N-terminal stem segment comprises amino acid residues 1-52 of HA1, preferably HA1 amino acid residues 18-52, and the HA1 C-terminal stem segment comprises amino acid residues 321-343 of HA1. The amino acid at position 343, ie, R343, is mutated and is an amino acid other than R, preferably glutamine (Q), and the HA2 domain contains the mutations F406S, V409T, L416S, M420I and V421I . In certain embodiments, the HA1 N-terminal stem segment consists of amino acid residues 1-52 of HA1, preferably HA1 amino acid residues 18-52, and the HA1 C-terminal stem segment consists of amino acid residues 321-343 of HA1. .

  In certain embodiments, the HA1 N-terminal stem segment comprises amino acid residues 1 to 53 of HA1, preferably HA1 amino acid residues 18 to 53, and the HA1 C-terminal stem segment comprises amino acid residues 303 to 343 of HA1. The amino acid at position 343, ie, R343, is mutated and is an amino acid other than R, preferably glutamine (Q). In certain embodiments, the HA1 N-terminal stem segment consists of amino acid residues 1-53 of HA1, preferably HA1 amino acid residues 18-53, and the HA1 C-terminal stem segment consists of amino acid residues 303-343 of HA1. . In a specific embodiment, the HA1 domain includes mutations V314T and I316T, and the HA2 domain includes one or more of the following mutations: F406S, V409T, L416S, M420I, and V421I. In a preferred embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 11.

  In certain embodiments, the HA1 N-terminal stem segment comprises amino acid residues 1-55 of HA1, preferably HA1 amino acid residues 18-55, and the HA1 C-terminal stem segment comprises amino acid residues 318-343 of HA1. The amino acid at position 343, ie, R343, is mutated and is an amino acid other than R, and is preferably glutamine (Q). In certain embodiments, the HA1 N-terminal stem segment consists of amino acid residues 1-55 of HA1, preferably HA1 amino acid residues 18-55, and the HA1 C-terminal stem segment consists of amino acid residues 318-343 of HA1. . In one embodiment, the HA2 domain comprises mutations F406S, V409T, L416S, M420I and V421I.

  In certain embodiments, the polypeptide further comprises a mutation R324C in the HA1 domain and T436C in the HA2 domain.

  In a specific embodiment, the HA1 domain includes mutations L58T, V314T, I316T, and R324C, and the HA2 domain includes one or more of the following mutations: F406S, V409T, L416S, M420I, V421I, and T436C. .

  In one embodiment, the HA1 domain includes mutation R324C and the HA2 domain includes mutations F406S, V409T, L416S, M420I, V421I, and T436C.

  In another embodiment, the HA1 domain comprises mutations V314T, I316T and R324C, and the HA2 domain comprises one or more of the following mutations: F406S, V409T, L416S, M420I, V421I and T436C.

  In one embodiment, the HA1 domain includes mutation R324C and the HA2 domain includes mutations F406S, V409T, L416S, M420I, V421I, and T436C.

In certain embodiments, the polypeptides of the invention contain an intracellular sequence of HA and a transmembrane domain. In other embodiments, the intracellular and transmembrane sequences, eg, the C2 terminus of the HA2 domain from position 523, 524, 525, 526, 527, 526, 528, 529, or 530 of the HA2 domain (or their equivalents). The amino acid sequence (numbering according to SEQ ID NO: 1) has been removed. In certain embodiments, the polypeptide is introduced by introducing a sequence known to form a trimeric structure, ie, AYVRKDGEWVLL (SEQ ID NO: 143) ("foldon" sequence), optionally linked via a linker. Further stabilized. The linker may optionally contain a cleavage site for later processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a short linker, eg, a tag sequence linked via EGR, eg, a his tag (HHHHHHH) can be added. In some embodiments, the linker and his tag sequence are added without the presence of a foldon sequence.

  In certain embodiments, the amino acid sequence at the C-terminus of the HA2 domain (numbering according to SEQ ID NO: 1) has been removed from position 530 (or its equivalent) of the HA2 domain. In certain embodiments, the intracellular and transmembrane sequences have been replaced by the amino acid sequence AGRHHHHHHH (SEQ ID NO: 81) or SGRSLVPRGSPGSGYIPEAPRDDGQAYVRKDGEWVLLSFLGHHHHHHHH (SEQ ID NO: 82).

  In certain embodiments, the polypeptide selectively binds to antibodies CR6261 and / or CR9114. In one embodiment, the polypeptide does not bind to antibody CR8057. In one embodiment, CR6261 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21; CR9114 comprises a heavy chain variable comprising the amino acid sequence of SEQ ID NO: 18. A light chain variable region comprising the region and the amino acid sequence of SEQ ID NO: 19. In one embodiment, CR8057 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23.

  As described above, the polypeptide comprises an influenza hemagglutinin HA1 domain comprising a HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a binding sequence of 0-50 amino acid residues. The binding sequence does not occur in native or wild type HA. In certain embodiments, the linker comprises 1 amino acid residue, 2 or fewer amino acid residues, 3 or fewer amino acid residues, 4 or fewer amino acid residues, 5 or fewer amino acid residues, 10 or fewer amino acids A peptide comprising a residue, 15 or fewer amino acid residues, or 20 or fewer amino acid residues, or 30 or fewer amino acid residues, or 40 or fewer amino acid residues or 50 or fewer amino acid residues. In a specific embodiment, the binding sequence is a sequence selected from the group consisting of G, GS, GGG, GSG, GSA, GSGS, GSAG, GGGG, GSAGS, GSGSG, GSAGSA, GSAGSAG, and GSGSSGSG.

The present invention also provides methods for providing the polypeptides of the present invention, in particular methods for providing H1HA stem domain polypeptides according to the present invention, and polypeptides obtained or obtained by these methods. In certain embodiments, the method includes:
(A) providing an influenza HA0 amino acid sequence, particularly an influenza HA0 amino acid sequence of serotype H1;
(B) removing the cleavage site between HA1 and HA2, preferably by mutating the C-terminal amino acid of HA1 to an amino acid other than arginine (R) or lysine (K);
(C) removing the amino acid sequence of the globular head domain from the HA0 sequence (this step deletes the segment of the HA1 domain between the amino acid at the x-position and the amino acid at the y-position, and thus obtained HA1 An N-terminal segment (ranging from the amino acid at position 1 of HA1 to the amino acid at the x-position (including that amino acid)) and a C-terminal segment (ranging from amino acid y of HA1 to the C-terminal amino acid) optionally from 0 to 50 amino acids In certain embodiments, x is an amino acid at any position 46-60 of HA1, preferably an amino acid at position 52, 53, 55, or 59. , Y
Is an amino acid at any position of positions 290-325 of HA1, preferably an amino acid at position 291, 303, 318, or 321. Again, the numbering used refers to SEQ ID NO: 1. A leader sequence (or signal sequence) that directs protein transport during production (eg, corresponding to amino acids 1-17 of SEQ ID NO: 1) is generally not present in, for example, the final polypeptide used in a vaccine. It will be understood by those skilled in the art. Thus, in certain embodiments, a polypeptide according to the invention comprises a HA1 N-terminal segment without a leader sequence);
(D) Preferably in the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD, preferably in particular MNTQFTAVGKEFN (H / K) LE (K / R) (SEQ ID NO: 17) The stability of the pre-fusion conformation of the modified HA is increased by introducing one or more mutations into the amino acid sequence ranging from amino acids 402 to 418 of SEQ ID NO: 1 comprising the amino acid sequence of 17). Destabilizing the structure (mutations preferably include substitution of hydrophobic amino acid residues with hydrophilic amino acid residues);
(E) introducing one or more disulfide bridges into the HA stem domain polypeptide.

  In accordance with the present invention, removal of the cleavage site between HA1 and HA2 can be achieved by mutation of R (in some cases K) to Q at position P1 (eg, cleavage site (in SEQ ID NO: 1)). See Sun et al, 2010 for a description of the nomenclature at position 343.) Mutations to Q are preferred, but S, T, N, D or E are alternatives.

  Removal of the head domain can be accomplished, for example, by deleting amino acids 53 to 320 from SEQ ID NO: 1, or amino acids at corresponding positions in HA from other influenza viruses. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. The remaining part of the sequence can be attached directly or a flexible linker can be introduced. The linker sequence can be 1 to 50 amino acids long. Preferred are flexible linkers of limited length (10 amino acids or less) such as GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like. The length of the deletion, for example, starts the deletion at (x) position (equivalent), for example at position 54, 55, 56, 57 or 58, or increases the length of the deletion Therefore, it can be changed by cutting at position 47, 48, 49, 50, 51, or 52. Similarly, the last amino acid to be deleted is the (y) position (equivalent), for example, at position 315, 316, 317, 318 or 319, or to increase the length of the deletion 321, 322, 323, 324, or 325 (or its equivalent). It is important to understand that changes in the length of a deletion can be partially compensated by matching the length of the linker sequence, i.e., larger deletions can be matched with longer linkers. Yes, and vice versa. These polypeptides are also encompassed by the present invention.

According to the present invention, the solubility of the loop between the A helix and the CD helix is increased. This loop is formed by residues 402 to 418 (equivalent) in H1A / Brisbane / 59/2007 (SEQ ID NO: 1). Therefore, the stability of the pre-fusion conformation of the modified HA is increased and the post-fusion conformation is destabilized. This loop is highly conserved in the H1 sequence, as can be seen in Table 6 below. This can be achieved, for example, by replacing amino acids I, L, F or V in the loop with hydrophilic equivalents. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. Mutation to glycine destabilizes the post-fusion conformation. This is because the high flexibility of this amino acid results in a decrease in the stability of the helix after fusion that can be formed by part of this HA sequence. A consensus sequence that describes the loop of residues 402-418 of influenza HA of serotype H1 is (SEQ ID NO: 17) MNTQFTAVGKEFN (H / K) LE (K / R). In the polypeptides of the present invention, amino acids at positions 406, 409, 413 and / or 416 (or their equivalents determined from sequence alignment) are polar (S, T, N, Q), charged (R, H, K, D, E) or flexible (G) amino acids. Combinations of mutations at these sites are also possible, for example F406S, V409T, L416S. In some cases, mutations to restore consensus amino acids are preferred, eg, V or M is at position 404 (to T), V is at position 408 (to A) or 410 (to G), or I is at position 414. (To N); the incidence of sequences with these specific amino acids is very low. A summary of the above mutations that characterize the polypeptides of the invention is listed in Table 6.

  According to the present invention, one or more disulfide bridges are introduced into the stem domain polypeptide, preferably between amino acids at positions 324 and 436 (or their equivalents) in H1A / Brisbane / 59/2007. . Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using suitable algorithms such as Clustal, Muscle, etc. Engineered disulfide bridges mutate at least one (if the other is already cysteine), but usually two spatially close residues to cysteines, either spontaneously or by active oxidation Created by forming a covalent bond between sulfur atoms.

  Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus the enhanced interaction between monomers in the truncated molecule increases stability. In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced. According to the present invention, a consensus sequence for the formation of a trimeric coiled coil, such as IEAIEKKIEAEEKIE (SEQ ID NO: 83), can be introduced into the polypeptide of the present invention at positions 418 to 433 (or its equivalent). . In one embodiment, the sequence MKQIEDKIEEEIESQQ (SEQ ID NO: 84), derived from GCN4 and known to further trimerize, is introduced at positions 419-433 (or its equivalent). In certain embodiments, the trimer interface is stabilized by modifying M420, L423, V427, G430 to isoleucine.

In certain embodiments, the polypeptides of the invention contain an intracellular sequence of H1HA and a transmembrane domain. In other embodiments, the intracellular and transmembrane sequences, eg, the C2 terminus of the HA2 domain from position 523, 524, 525, 526, 527, 526, 528, 529, or 530 of the HA2 domain (or their equivalents). The amino acid sequence of (numbered by SEQ ID NO: 1) has been removed to produce a soluble polypeptide after expression in cells. In certain embodiments, the polypeptide is further stabilized by introducing a sequence known to form a trimeric structure, ie, AYVRKDGEWVLL (SEQ ID NO: 80), optionally linked via a linker. . The linker may optionally contain a cleavage site for later processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a short linker, eg, a tag sequence linked via EGR, eg, a his tag (HHHHHHH) can be added. In some embodiments, the linker and his tag sequence are added without the presence of a foldon sequence. In certain embodiments, the intracellular and transmembrane sequences are replaced by the amino acid sequence AGRHHHHHHH (SEQ ID NO: 97) or SGRSLVPRGSPGSGYIPEAPRDDGQAYVRKDDGEWVLLSFLGHHHHHHHH (SEQ ID NO: 82).

  Applicants have some specific for group 1 influenza viruses (eg, CR6261 as described in WO 2008/028946) and some specific for group 2 influenza viruses (eg, WO 2010). Has already been identified a broadly neutralizing antibody isolated from primary human B cells from a vaccinated individual that was CR8020) described in US / 130636. Detailed epitope analysis of these monoclonal antibodies revealed the reason for the lack of cross-reactivity of these specific antibodies. In both cases, the presence of glycans in group 1 or group 2 HA molecules on different positions at least partially explained the fact that the antibodies were group specific. The identification of CR9114-like antibodies that cross-react with a number of group 1 and 2 HA molecules described below has revealed that the human immune system is capable of eliciting a very broad range of neutralizing antibodies against influenza virus. However, considering the need for annual vaccination schemes, these antibodies are clearly not induced after infection with subtype H1 and / or H3 (seasonal) influenza viruses and after vaccination, or very low It is only triggered to a degree. Thus, in certain embodiments, the present invention provides a conformationally correct manner of HA stem region such that epitopes that induce broadly neutralizing antibodies are presented to the immune system in the absence of immunodominant variable regions. Provided polypeptides are provided. In different embodiments, the polypeptides of the present invention may comprise group 2 HA molecules (as it is known that glycan patterns differ between H1HA and H3HA, and this difference is known to result in a more group-restricted antibody response. For example, based on H3 (HA). As shown in Example 3 below, CR9114 has a higher in vitro neutralizing ability in the H1 subtype compared to the H3 subtype. Thus, the epitope of CR9114 is hypothesized that H1 is accessible compared to the H3HA molecule, which may be due to glycans on N38 in HA1 that are common to many group 2 HA subtypes. Without being bound by this theory, when the polypeptides of the invention are based on H1, the resulting antibodies are more likely to be damaged by glycans on N38 on group 2 HA molecules, and thus It can be assumed that there is some activity against group 2 influenza viruses. Thus, in some embodiments, the stem domain polypeptide of the present invention comprises an H3HA subtype to allow for the induction of broadly neutralizing antibodies that act with good activity against both group 1 and group 2 influenza viruses. Based on.

  Humans are frequently infected with seasonal influenza viruses that contain H1 or H3 subtypes of HA. Despite exposure to these influenza viruses, it is clear that broadly neutralizing antibodies often do not occur in the natural situation. One reason for this may be that in addition to the presence of variable head regions in the HA, exposure to new subtypes closely related to those already identified does not make the response in any way widespread. Thus, it may be preferable to expose an individual to a more unrelated subtype sequence. Thus, in yet another embodiment, a stem domain polypeptide of the invention contains asparagine (N) (N38) at position 38 in HA1 and is based on a Group 2 subtype HA that is not an H3 subtype .

  In certain embodiments, the polypeptide is based on an influenza A virus subtype. In certain embodiments, the polypeptide is not based on H7HA.

As noted above, the polypeptides of the invention are not only designed based on the parental HA sequence from group 1 influenza vaccine virus subtypes (eg, H1 and H5), but also influenza subtypes from group 2, It may also be based on the HA sequence of group 2 influenza virus subtypes, such as H3, used in particular for influenza vaccines. According to the present invention, polypeptides that conserve the epitopes of CR8020 and CR8043 were constructed. This is because these antibodies can neutralize a wide range of Group 2 strains (WO 2010/130636). In these polypeptides, the beta sheet and its surroundings at the bottom of the stem region should be preserved as much as possible. This is because CR8020 and CR8043 are regions that bind to H3HA.

  In certain embodiments, the HA domain is of the H3 subtype, preferably A / Wisconsin / 67/2005 (SEQ ID NO: 89), or A / HongKong / 1/1968 (SEQ ID NO: 121). It will be appreciated by those skilled in the art that other influenza A viruses, including H3 subtype HA, can also be used in accordance with the present invention.

  In certain embodiments, the polypeptide comprises or consists of a HA1 N-terminal polypeptide segment comprising amino acids from positions 1 to x of the H3HA1 domain, preferably amino acids from position p to x of the HA1 domain, wherein x is Any amino acid at positions 56-69 of H3HA1, for example, positions 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 or 68, preferably x is 61, 62, 63 or 68. In certain embodiments, the HA1 C-terminal polypeptide segment includes amino acids from the y-position of the H3HA1 domain to the C-terminal amino acid (including its amino acids), and y is any amino acid at positions 292-325 (including) of H3HA1 Preferably, y is 293, 306, 318 or 323.

  In certain embodiments, the HA domain is of the H3 subtype, preferably A / Wisconsin / 67/2005 (SEQ ID NO: 89), or A / HongKong / 1/1968 (SEQ ID NO: 121).

  According to the present invention, the head domain has been removed by deleting most of the HA1 sequence and religating the N and C terminal sequences through a short linker. The deletion can vary in length but is spatially close to the last residue of the N-terminal sequence of HA1 and the first residue of the C-terminal sequence to avoid introducing strain through the binding sequence It is preferable. In the H3 sequence, deletions can be introduced at S62-P322, S63-P305 and T64-T317 (corresponding positions). Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. The remaining part of the sequence can be attached directly or a flexible linker can be introduced. The linker sequence can be 1 to 50 amino acids long. Preferred are flexible linkers of limited length (10 amino acids or less) such as GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like. The length of the deletion is, for example, by reducing the number of residues in the deletion by starting at (or equivalent to) positions 63, 64, 65, 66, 67, or increasing the length of the deletion Therefore, it can be changed by cutting at positions 57, 58, 59, 60 or 61. Similarly, the last amino acid to be deleted is at position 317, 318, 319, 320 or 321 (or its equivalent) or to increase the length of the deletion 323, 324, 325, 326, Or it may be in the 327th position (or its equivalent). It is important to understand that changes in the length of a deletion can be partially compensated by matching the length of the linker sequence, i.e., larger deletions can be matched with longer linkers. Yes, and vice versa. These polypeptides are also included in the present invention.

  In some embodiments, x is 61 and y is 323.

  In some embodiments, x is 62 and y is 306.

  In some embodiments, x is 63 and y is 318.

  In certain embodiments, x is in positions 62, 63, 64, 65, 66 (or its equivalent), or positions 56, 57, 58, 59, or 60.

  In certain embodiments, y is in position 306, 318, 319, 320, 321, or 322 (or its equivalent), or position 324, 325, 326, 327, or 328 (or its equivalent).

  In one embodiment, the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD is the amino acid sequence of amino acids 400 to 420 of HA2 of SEQ ID NO: 89; Or corresponding to an amino acid residue at a significant position in other H3 virus strains, the polypeptide has an amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD, It includes 89 amino acids 400-420, or one or more mutations in the amino acid sequence spanning the corresponding amino acid residues in other H3 influenza virus strains.

  In certain embodiments, the amino acid sequence linking the C-terminal residue of helix A of serotype H3 influenza HA to the N-terminal residue of helix CD comprises the amino acid sequence of SEQ ID NO: 104.

  In accordance with the present invention, the polypeptide comprises one or more mutations in the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD. In certain embodiments, the polypeptide comprises one or more of the mutations in Table 8, or a corresponding mutation in other influenza virus strains of the H3 subtype.

  According to the present invention, the cleavage site between HA1 and HA2 has been removed. In certain embodiments, removal of the cleavage site at position 345 (numbering refers to SEQ ID NO: 89) is mutated (R345Q) to prevent the formation of HA1 and HA2 from HA0. Optionally, residues 347-351 (IFGAI, part of the fusion peptide) can be further deleted to minimize exposure of hydrophobic residues to aqueous solvents. The positive charge on cleavage is 100% conserved in H3, so this mutation can be applied in all sequences.

  Deletion of the head domain leaves the B-loop at residues 400-420 currently exposed to aqueous solvents. In H3HA, this loop is highly conserved (eg, Table 9). The consensus sequence is: 401I (E / G) KTNEKFHQIEKEFSVEGR 421 (SEQ ID NO: 104; numbering refers to SEQ ID NO: 89). Some hydrophobic residues are polar (S, T, N, Q) in order to increase the solubility of this loop and to destabilize the post-fusion conformation because of the pre-fusion conformation polypeptide of the invention. Must be modified to charged amino acids (R, H, K, D, E), or flexibility must be increased by mutation to G. Specifically, mutations at positions 401, 408, 411, 415, 418 (numbering refers to SEQ ID NO: 89) contribute to the stability of the polypeptides of the invention.

In order to stabilize the pre-fusion conformation of the polypeptide of the present invention, a covalent bond is introduced between two portions that are distal in the primary sequence but close in the folded pre-fusion conformation. For this purpose, disulfide bridges can be genetically manipulated in the polypeptides of the invention, preferably between positions 326 and 438 (equivalent) in H3A / Wisconsin / 67/2005 (SEQ ID NO: 89). . The corresponding position is determined by an algorithm suitable for those skilled in the art, for example,
It can be easily determined by aligning the sequences using Clustal, Muscle, etc. Engineered disulfide bridges mutate at least one (if the other is already cysteine), but usually two spatially close residues to cysteines, either spontaneously or by active oxidation Created by forming a covalent bond between sulfur atoms. Alternative cysteine bridges can be created by mutation of those residues to cysteine between positions 334 and 393 (equivalent) in H3A / Wisconsin / 67/2005 (SEQ ID NO: 89). In some cases, the cysteine at position 321 (or its equivalent) is modified to glycine to avoid the formation of unwanted disulfide bridges.

  In certain embodiments, the polypeptide comprises one or more of the following mutations: F408S, I411T, F415S, V418G, I401R, K326C, S438C, T334C, I393C, C321G.

  Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus the enhanced interaction between monomers in the truncated molecule increases stability. In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced. The consensus sequence IEAIEKKIEAEIEKKIEAEEK for the formation of trimer coiled coils is introduced at (421-441) (equivalent). To avoid interfering with the formation of disulfide bridges at positions 326-438, an alternative shorter sequence IEAIEKKIEAEEKKI was also used at (equivalent) at positions 421-435. An alternative is to introduce the sequence RMKQIEDKIEIESKQKKIEN derived from GCN4 and known to trimerize at positions 421 to 441 or the shorter sequence RMKQIEDKIEEEIESK at positions 421 to 435.

  The polypeptides of the present invention may contain the intracellular sequence of HA and the transmembrane domain such that the resulting polypeptide is presented on the cell surface upon expression in the cell. In other embodiments, the cytoplasmic and transmembrane sequences from position 522 (or its equivalent) to the C-terminus have been removed so that a secreted (soluble) polypeptide is produced after expression in the cell. Optionally, some additional residues can be included in the soluble protein by deleting sequences from (corresponding to) 523, 524, 525, 526, 527, 528 or 529. The soluble polypeptide is further stabilized by introducing a sequence known to form a trimeric structure, ie, AYVRKDGEWVLL (SEQ ID NO: 143) ("foldon" sequence), optionally linked via a linker. be able to. The linker may optionally contain a cleavage site for later processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a short linker, eg, a tag sequence linked via EGR, eg, a his tag (HHHHHHH) can be added. In some embodiments, the linker and his tag sequence are added without the presence of a foldon sequence.

  According to the present invention, the amino acid sequence of the C-terminal amino acid is removed from position 530 of the HA2 domain (numbered according to SEQ ID NO: 1) and replaced by the following sequence EGRHHHHHHH (SEQ ID NO: 81) Can do.

In certain embodiments, the HA1 N-terminal stem segment does not include a signal sequence. A leader sequence (or signal sequence) that directs transport of the protein during production (eg, corresponding to amino acids 1-17 of SEQ ID NO: 89) is generally not present, for example, in the final polypeptide used in the vaccine. As understood by those skilled in the art. Thus, in certain embodiments, a polypeptide according to the invention comprises an amino acid sequence that does not have a leader sequence.

  According to the invention, the polypeptide is not based on influenza B HA molecules. Strictly speaking, the influenza B virus strain is human. Antigenic mutations in HA within influenza B virus strains are smaller than those observed in type A strains. Two genetically and antigenically distinct influenza B virus strains are represented by the B / Yamagata / 16/88 (also referred to as B / Yamagata) and B / Victoria / 2/87 (B / Victoria) strains. As it is circulated in humans (Ferguson et al., 2003). Although the spectrum of disease caused by influenza B virus is generally milder than that caused by influenza A virus, severe illness requiring hospitalization for influenza B infection is still frequently observed.

  According to the present invention, mimics specific epitopes of CR6261 and CR9114, e.g., elicits cross-neutralizing antibodies when administered in vivo alone or in combination with other prophylactic and / or therapeutic treatments Therefore, polypeptides that can be used as immunogenic polypeptides are provided. “Cross-neutralizing antibodies” have at least 2, preferably at least 3, 4, or 5 different subtypes of phylogenetic group 1 influenza A viruses, and / or at least 2, preferably at least 3 At least all virus strains neutralized by one, four or five phylogenetic group 2 different subtypes of influenza A virus and / or at least two different subtypes of influenza B virus, in particular CR6261 and CR9114 Means an antibody capable of neutralizing

  The polypeptide of the present invention does not include full-length HA1. In certain embodiments, the immunogenic polypeptide is substantially smaller than HA0, and preferably lacks the globular head of all or substantially all HA. Preferably, the immunogenic polypeptide is 360 amino acids or less, preferably 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280, 275, or 270 amino acids in length. In one embodiment, the immunogenic polypeptide is about 250 to about 350, preferably about 260 to about 340, preferably about 270 to about 330, preferably about 270 to about 330 amino acids in length.

  In certain embodiments, the polypeptide selectively binds to antibodies CR6261 and / or CR9114. In one embodiment, the polypeptide does not bind to antibody CR8057. In one embodiment, CR6261 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21; CR9114 comprises a heavy chain variable comprising the amino acid sequence of SEQ ID NO: 18. A light chain variable region comprising the region and the amino acid sequence of SEQ ID NO: 19. In one embodiment, CR8057 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23.

As described above, the polypeptide comprises an influenza hemagglutinin HA1 domain comprising a HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a binding sequence of 0-50 amino acid residues. The binding sequence does not occur in native or wild type HA. In certain embodiments, the linker comprises 1 amino acid residue, 2 or fewer amino acid residues, 3 or fewer amino acid residues, 4 or fewer amino acid residues, 5 or fewer amino acid residues, 10 or fewer amino acids A peptide comprising a residue, 15 or fewer amino acid residues, or 20 or fewer amino acid residues, or 30 or fewer amino acid residues, or 40 or fewer amino acid residues or 50 or fewer amino acid residues. In a specific embodiment, the binding sequence is a sequence selected from the group consisting of G, GS, GGG, GSG, GSA, GSGS, GSAG, GGGG, GSAGS, GSGSG, GSAGSA, GSAGSAG, and GSGSSGSG.

The present invention also provides methods for providing the polypeptides of the present invention, in particular methods for providing the amino acid sequences of HA stem domain polypeptides according to the present invention, and polypeptides obtained or obtained by these methods. In certain embodiments, the method includes:
Providing an influenza HA0 amino acid sequence, eg, an influenza HA0 sequence of serotype H1, H5 or H3;
-Preferably removing the cleavage site between HA1 and HA2 by mutating the C-terminal amino acid of HA1 to an amino acid other than arginine (R) or lysine (K);
-Removing the amino acid sequence of the globular head domain from the HA0 sequence (this step deletes the segment of the HA1 domain between the amino acid at the x-position and the amino acid at the y-position, and thus the N-terminus of the HA1 thus obtained. A 0-50 amino acid binding sequence, optionally in segments (ranging from the amino acid at position 1 of HA1 to the amino acid at position x (including that amino acid) and the C-terminal segment (ranging from amino acid y of HA1 to the C-terminal amino acid) By re-linking through
-Preferably in the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD, in particular MNTQFTAVGKEFN (H / K) LE (K / R) (SEQ ID NO: 17) Alternatively, for H3HA, one or more mutations are introduced into the amino acid sequence spanning amino acids 402 to 418 of H1HA containing the amino acid sequence of I (E / G) KTNEKFHQIEKEFSVEGR421 (SEQ ID NO: 104) before fusion of the modified HA Increasing the stability of the conformation and destabilizing the conformation after fusion (mutations preferably include substitution of hydrophobic amino acid residues with hydrophilic amino acid residues);
-Introducing one or more disulfide bridges into the HA stem domain polypeptide.

  In accordance with the present invention, removal of the cleavage site between HA1 and HA2 can be achieved by mutation of R (in some cases K) to Q at position P1 (eg, cleavage site (in SEQ ID NO: 1)). See Sun et al, 2010 for a description of the nomenclature at position 343.) Mutations to Q are preferred, but S, T, N, D or E are alternatives.

Removal of the head domain can be achieved, for example, by deleting amino acids 53 to 320 from SEQ ID NO: 1. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. The remaining part of the sequence can be attached directly or a flexible linker can be introduced. The linker sequence can be 1 to 50 amino acids long. Preferred are flexible linkers of limited length (10 amino acids or less) such as GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like. The length of the deletion, for example, starts the deletion at (x) position (equivalent), for example at position 54, 55, 56, 57 or 58, or increases the length of the deletion Therefore, it can be changed by cutting at position 47, 48, 49, 50, 51, or 52. Similarly, the last amino acid to be deleted is the (y) position (equivalent), for example, at position 315, 316, 317, 318 or 319, or to increase the length of the deletion 321, 322, 323, 324, or 325 (or its equivalent). It is important to understand that changes in the length of a deletion can be partially compensated by matching the length of the linker sequence, i.e., larger deletions can be matched with longer linkers. Yes, and vice versa. These polypeptides are also encompassed by the present invention.

  According to the present invention, the solubility of the loop between the A helix and the CD helix is increased. This loop is formed by residues 402 to 418 (equivalent) in H1A / Brisbane / 59/2007 (SEQ ID NO: 1). Therefore, the stability of the pre-fusion conformation of the modified HA is increased and the post-fusion conformation is destabilized. This loop is highly conserved in the H1 sequence, as can be seen in Table 6 below. This can be achieved, for example, by replacing amino acids I, L, F or V in the loop with hydrophilic equivalents. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. Mutation to glycine destabilizes the post-fusion conformation. This is because the high flexibility of this amino acid results in a decrease in the stability of the helix after fusion that can be formed by part of this HA sequence. The consensus sequence describing the 402-418 loop of serotype H1 influenza HA is (SEQ ID NO: 17) MNTQFTAVGKEFN (H / K) LE (K / R). In certain polypeptides of the invention, the amino acids at positions 406, 409, 413 and / or 416 (or their equivalents determined from sequence alignment) are polar (S, T, N, Q), charged (R , H, K, D, E) or flexible (G) amino acids. Combinations of mutations at these sites are also possible, for example F406S, V409T, L416S in SEQ ID NO: 10 and SEQ ID NO: 14. In some cases, mutations to restore consensus amino acids are preferred, eg, V or M is at position 404 (to T), V is at position 408 (to A) or 410 (to G), or I is at position 414. (To N); the incidence of sequences with these specific amino acids is very low. A summary of the above mutations that characterize the polypeptides of the invention is listed in Table 6.

  According to the present invention, one or more disulfide bridges are present in the stem domain polypeptide, preferably at positions 324 and 436 (or their equivalents) in H1A / Brisbane / 59/2007: SEQ ID NOs 13-16. Introduced between amino acids. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using suitable algorithms such as Clustal, Muscle, etc. Engineered disulfide bridges mutate at least one (if the other is already cysteine), but usually two spatially close residues to cysteines, either spontaneously or by active oxidation Created by forming a covalent bond between sulfur atoms.

  Polypeptides obtained by the above method are also part of the present invention.

  Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus the enhanced interaction between monomers in the truncated molecule increases stability. In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced. According to the present invention, a consensus sequence for the formation of a trimeric coiled coil, IEAIEKKIEAEEKKIE (SEQ ID NO: 83), can be introduced into the polypeptide of the present invention at positions 418 to 433 (or its equivalent). In one embodiment, the sequence MKQIEDKIEIESKQ (SEQ ID NO: 84), derived from GCN4 and known to further trimerize, is introduced at positions 419-43 (equivalent). In certain embodiments, the trimer interface is stabilized by modifying M420, L423, V427, G430 to isoleucine.

  In certain embodiments, the polypeptide has SEQ ID NO: 3-16, SEQ ID NO: 44-53, SEQ ID NO: 111-114, SEQ ID NO: 119-120, SEQ ID NO: 125, 126, 130, SEQ ID NO: 144-175, and SEQ ID NO: 177. An amino acid sequence selected from the group consisting of ~ 187.

  In certain embodiments, the polypeptide is selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 113, and SEQ ID NO: 130.

  A leader sequence (or signal sequence) that directs protein transport during production (eg, corresponding to amino acids 1-17 of SEQ ID NO: 1) is generally not present, for example, in the final polypeptide used in a vaccine. As understood by those skilled in the art. Thus, in certain embodiments, a polypeptide according to the invention comprises an amino acid sequence that does not have a leader sequence.

  Influenza hemagglutinin stem domain polypeptides can be prepared according to any technique deemed suitable by those of skill in the art, for example, the following technique.

  Accordingly, the immunogenic polypeptides of the present invention are synthesized as DNA sequences by standard methods known in the art, cloned using suitable restriction enzymes and methods known in the art, and subsequently in vitro or in vivo. Can be expressed. Accordingly, the present invention also relates to nucleic acid molecules that encode the above polypeptides. The invention further relates to a vector comprising a nucleic acid encoding a polypeptide of the invention. In certain embodiments, the nucleic acid molecule according to the present invention is part of a vector, eg a plasmid. Such vectors can be easily manipulated by methods well known to those skilled in the art and can be designed to replicate, for example, in prokaryotic and / or eukaryotic cells. In addition, many vectors can be used to transform eukaryotic cells directly or in the form of desired fragments isolated therefrom, and integrate into the genome of such cells in whole or in part. Resulting in a stable host cell containing the desired nucleic acid in the genome. The vector used is suitable for cloning DNA and can be any vector that can be used for transcription of a nucleic acid of interest. When host cells are used, the vector is preferably an integrating vector. Alternatively, the vector can be an episomal replicating vector.

One skilled in the art can select a suitable expression vector and functionally insert a nucleic acid sequence of the invention. To obtain expression of a nucleic acid sequence encoding a polypeptide, a recombinant nucleic acid that encodes a protein or polypeptide in a format in which the sequence capable of driving expression is operably linked to a nucleic acid sequence encoding the polypeptide and expressed. It is well known to those skilled in the art that molecules can be provided. In general, a promoter sequence is placed upstream of the sequence to be expressed. Many expression vectors are available in the art, such as the Invitrogen pcDNA and pEF vector systems, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc. Promoters and / or transcription termination sequences, poly A sequences and the like can be obtained. The resulting expression cassette is useful for producing a polypeptide of interest if the sequence encoding the polypeptide of interest is inserted appropriately with respect to sequences that govern the transcription and translation of the encoded polypeptide. Yes, it is called expression. Sequences that drive expression can include promoters, enhancers, and the like, and combinations thereof. They should be able to function in the host cell and thereby drive the expression of nucleic acid sequences that are operably linked to them. Those skilled in the art will appreciate that a variety of promoters can be used to obtain expression of a gene in a host cell. Promoters can be constitutive or regulatable, can be obtained from a variety of resources, eg, viral, prokaryotic, or eukaryotic resources, or can be engineered. Expression of the nucleic acid of interest can be from a native promoter or derivative thereof, or from a completely heterologous promoter (Kaufman, 2000). Some well-known and well-used promoters for expression in eukaryotic cells are promoters derived from viruses such as adenovirus, such as the E1A promoter, promoters derived from cytomegalovirus (CMV), such as CMV immediate early (IE) promoter (referred to as CMV promoter in the present invention) (for example, pcDNA, obtained from Invitrogen), promoter derived from simian virus 40 (SV40) (Das et al, 1985), and the like. Suitable promoters can also be derived from eukaryotic cells, eg, metallothionein (MT) promoter, elongation factor 1α (EF-1α) promoter (Gill et al., 2001), ubiquitin C or UB6 promoter (Gill et al. 2001), actin promoter, immunoglobulin promoter, heat shock promoter and the like. Testing for promoter function and promoter strength is routine for those skilled in the art and generally includes, for example, cloning of a test gene, eg, lacZ, luciferase, GFP, etc. behind the promoter sequence, and test expression of the test gene. obtain. Of course, a promoter can be altered by deletion, addition, or mutation of its sequence and tested for functionality to find new, attenuated or improved promoter sequences. According to the present invention, strong promoters that confer high transcription levels in optimal eukaryotic cells are preferred.

  The construct can be transfected into a suitable prokaryotic expression system such as a eukaryotic cell (eg, a plant, fungus, yeast or animal cell) or E. coli using methods well known to those skilled in the art. In some cases, a suitable “tag” sequence (such as, but not limited to, a his, myc, strep, or flag tag) or a complete protein (such as, but not limited to, maltose binding protein) Or glutathione S transferase etc.) can be added to the sequences of the invention to allow purification and / or identification of the polypeptide from cells or supernatants. Optionally, the tag can be subsequently removed by proteolytic digestion, including sequences containing specific proteolytic sites.

  Purified polypeptides can be analyzed by spectroscopy known in the art (eg, circular dichroism, Fourier transform infrared and NMR spectroscopy or X-ray crystallography) such as helices and beta sheets. The presence of the desired structure can be investigated. ELISA, Octet, FACS, etc. can be used to investigate the binding of polypeptides of the invention to previously described broadly neutralizing antibodies (CR6261, CR9114, CR8057). Therefore, a polypeptide according to the present invention having an accurate three-dimensional structure can be selected.

  The present invention further relates to immunogenic compositions comprising a therapeutically effective amount of at least one of the polypeptides and / or nucleic acids of the present invention. In certain embodiments, the composition comprises an HA-based hemagglutinin stem domain from (or based on) an HA of one influenza subtype, eg, an HA of an influenza virus comprising HA of the H1 or H7 subtype. Including polypeptides. In certain embodiments, the composition comprises a polypeptide comprising an HA-based hemagglutinin stem domain of two or more different influenza subtypes, for example, the composition comprises an H1 subtype HA-based hemagglutinin stem. It includes both polypeptides comprising domains and polypeptides comprising hemagglutinin stem domains based on H7 subtype HA.

The immunogenic composition preferably further comprises a pharmaceutically acceptable carrier. In the present context, the term “pharmaceutically acceptable” means that at the dosage and concentration at which the carrier is used, it does not cause undesirable or deleterious effects in the subject to which it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (Reming
ton's Pharmaceutical Sciences, 18th edition, A.D. R. Gennaro, Ed. Mack Publishing Company [1990]; Pharmaceutical Formation Development of Peptides and Proteins, S .; Frokkaer and L. Hovgaard, Eds. , Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A .; Kibbe, Ed. , Pharmaceutical Press [2000]). The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can be used, for example, as liquid carriers, particularly for injectable solutions. The exact formulation should suit the mode of administration. The polypeptide and / or nucleic acid molecule is preferably formulated and administered as a sterile solution. Sterile solutions are prepared by sterile filtration or by other methods known per se in the art. The solution can then be filled into lyophilized or pharmaceutical administration containers. The pH of the solution is generally in the range of pH 3.0 to 9.5, such as pH 5.0 to 7.5.

  The invention also relates to a method of inducing an immune response in a subject comprising administering to the subject a polypeptide, nucleic acid molecule and / or immunogenic composition as described above. The subject according to the invention is preferably a mammal that can be infected by an infectious disease-inducing agent, in particular an influenza virus, or otherwise can benefit from the induction of an immune response, such as, for example, Rodents such as mice, ferrets, or household or farm animals, or non-human primates, or humans. Preferably, the subject is a human subject. Accordingly, the present invention particularly relates to group 1 and / or group 2 influenza A viruses, eg influenza viruses comprising HA of the H1, H2, H3, H4, H5, H7 and / or H10 subtypes, and / or influenza B viruses. A method for inducing an immune response against the influenza virus hemagglutinin (HA) of a subject utilizing the polypeptides, nucleic acids and / or immunogenic compositions described herein is provided. In some embodiments, the immune response induced is effective to prevent and / or treat influenza virus infection caused by group 1 and / or group 2 influenza A virus subtypes and / or influenza B virus. . In some embodiments, the immune response induced by the polypeptides, nucleic acids and / or immunogenic compositions described herein is an influenza A virus 2, 3, 4, 5 or 6 subtype and Effective for preventing and / or treating influenza A and / or B virus infection caused by influenza B virus.

It is well known that small proteins and / or nucleic acid molecules do not always effectively induce a strong immune response, so adding an adjuvant can increase the immunogenicity of a polypeptide and / or nucleic acid molecule. May be necessary. In certain embodiments, the immunogenic compositions described herein include or are administered in combination with an adjuvant. Adjuvants for combination administration with the compositions described herein can be administered before, concurrently with, or after administration of the composition. Examples of suitable adjuvants include aluminum salts such as aluminum hydroxide and / or aluminum phosphate; oil-emulsion compositions (or oil-in-water compositions), such as squalene-water emulsions such as MF59 (e.g. , See WO 90/14837); saponin formulations such as QS21 and immune stimulating complex (ISCOM) (eg US Pat. No. 5,057,540; WO 90/03184). Pamphlet, WO 96/11711 pamphlet, WO 2004/004762 pamphlet, WO 2005/002620 pamphlet); bacterial or microbial derivatives, examples of which include monophosphoryl lipid A (MPL) 3-O-deacylated MP (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylated bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, pertussis toxin PT, or tetanus toxoid TT, Matrix M (Isconova). In addition, fusion of polypeptides of the invention or virosome to known immune enhancement techniques, eg, proteins known in the art to improve immune response (eg, tetanus toxoid, CRM197, rCTB, bacterial flagellin, etc.) Inclusion of polypeptides in or combinations thereof can be used. Other non-limiting examples that can be used are, for example, Coffman et al. (2010).

  In one embodiment, the influenza hemagglutinin stem domain polypeptide of the invention is incorporated into a virus-like particle (VLP) vector. VLPs generally comprise viral polypeptides that are typically derived from viral structural proteins. Preferably, the VLP cannot replicate. In certain embodiments, the VLP may lack the entire genome of the virus or may comprise a portion of the genome of the virus. In some embodiments, the VLP cannot infect cells. In some embodiments, VLPs express one or more viral (eg, viral surface glycoprotein) or non-viral (eg, antibody or protein) targeting moieties known to those of skill in the art on their surface.

  In a specific embodiment, the polypeptides of the invention are incorporated into virosomes. Virosomes containing polypeptides according to the present invention can be produced using techniques known to those skilled in the art. For example, virosomes are produced by destroying purified virus, extracting the genome, and rearranging the particles with viral proteins (eg, influenza hemagglutinin stem domain polypeptide) and lipids to form lipid particles containing the viral proteins. be able to.

  The invention also relates to a polypeptide, nucleic acid and / or immunogenic composition as described above for inducing an immune response in a subject against influenza HA, in particular for use as a vaccine. Accordingly, an influenza hemagglutinin stem domain polypeptide, a nucleic acid encoding such a polypeptide, or a vector comprising such a nucleic acid or a polypeptide described herein, against an influenza virus, eg, the stem region of an influenza virus hemagglutinin Can be used to elicit neutralizing antibodies against. In particular, the present invention relates to the above-mentioned polypeptides, nucleic acids for use as vaccines in the prevention and / or treatment of phylogenetic group 1 and / or phylogenetic group 2 influenza A viruses and / or diseases or conditions caused by influenza B viruses. And / or an immunogenic composition. In one embodiment, the vaccine is in the prevention and / or treatment of diseases caused by two, three, four, five, six or more different subtypes of phylogenetic groups 1 and / or 2 and / or influenza B virus. Can be used. The polypeptides of the present invention can be used in vitro after synthesis, or in suitable cell expression systems such as bacteria and eukaryotic cells, or in subjects where it is required in vivo. It can be expressed by expressing a nucleic acid encoding a sex polypeptide. Such nucleic acid vaccines can take any form, such as naked DNA, plasmids, or viral vectors, such as adenoviral vectors.

Administration of polypeptides, nucleic acid molecules, and / or immunogenic compositions according to the present invention can be performed using standard routes of administration. Non-limiting examples include parenteral administration, such as intravenous, intradermal, transdermal, intramuscular, subcutaneous, or mucosal administration, such as intranasal, oral, and the like. One skilled in the art can determine various possibilities for administering the polypeptides, nucleic acid molecules, and / or immunogenic compositions according to the present invention to induce an immune response. In certain embodiments, the polypeptide, nucleic acid molecule, and / or immunogenic composition (or vaccine) is administered more than once, ie, in a so-called allogeneic prime boost regimen. In certain embodiments where the polypeptide, nucleic acid molecule, and / or immunogenic composition is administered more than once, administration of the second dose is, for example, after a time interval of one week or more after administration of the first dose. 2 weeks or more after administration of the first dose, 3 weeks or more after administration of the first dose, 1 month or more after administration of the first dose, 6 weeks or more after administration of the first dose, Administration of a first dose of a polypeptide, nucleic acid molecule, and / or immunogenic composition, such as 2 months or more after administration, 3 months or more after administration of the first dose, 4 months or more after administration of the first dose, etc. It can be implemented up to several years later. It is also possible to administer the vaccine three times or more, such as three times, four times, etc., such that two or more boost doses follow the first priming dose. In other embodiments, the polypeptides, nucleic acid molecules, and / or immunogenic compositions according to the invention are administered only once.

  The polypeptide, nucleic acid molecule, and / or immunogenic composition can also be administered in a heterologous prime-boost regimen, either as a prime or boost.

  The present invention further provides methods of preventing and / or treating influenza virus disease in a subject utilizing the polypeptides, nucleic acids and / or compositions described herein. In a specific embodiment, a method of preventing and / or treating influenza virus disease in a subject administers an effective amount of the above-described polypeptide, nucleic acid and / or immunogenic composition to a subject in need thereof. Including that. A therapeutically effective amount is a polypeptide, nucleic acid, and / or as defined herein that is effective in preventing, ameliorating and / or treating a disease or condition resulting from infection with a group 1 or 2 influenza A virus, and / or influenza B virus. Or refers to the amount of the composition. Prevention includes inhibiting or reducing the spread of influenza virus or inhibiting or reducing the onset, development or progression of one or more symptoms associated with infection with influenza virus. An improvement as used herein may refer to a reduction in symptoms of visible or sensory disease symptoms, viremia, or any other measurable flu infection.

  Those in need of treatment include those who are already suffering from the pathology resulting from infection with group 1 or group 2 influenza A virus, or influenza B virus, and those who should be prevented from infection with influenza virus. Thus, a polypeptide, nucleic acid and / or composition of the invention is not infected with an unmedicated subject, i.e. has no disease caused by an influenza virus infection or has not been infected by an influenza virus infection and is still infected. The subject can be administered to a subject who has not been or has been infected and / or has been infected by an influenza virus.

  In one embodiment, prevention and / or treatment can be targeted in a group of patients susceptible to influenza virus infection. Such patient groups include, but are not limited to, for example, elderly (eg, ≧ 50 years old, ≧ 60 years old, preferably ≧ 65 years old), young (eg, ≦ 5 years old, ≦ 1 year old) , Hospitalized patients and patients who have been treated with antiviral compounds but have shown an inappropriate antiviral response.

  In another embodiment, the polypeptide, nucleic acid and / or immunogenic composition is one or more other active agents in the subject, such as existing or future influenza vaccines, monoclonal antibodies and / or antiviral agents, and It can be administered in combination with / or antibacterial agents and / or immunomodulators. One or more other active agents may be beneficial in the treatment and / or prevention of influenza virus diseases, or may improve symptoms or pathologies associated with influenza virus diseases. In some embodiments, the one or more other active agents are analgesics, antipyretic drugs, or therapeutics that relieve or assist breathing.

  Dosage regimens of the polypeptides and / or nucleic acid molecules of the invention can be adjusted to provide the optimum desired response (eg, a therapeutic response). A suitable dosage range may be, for example, 0.1-100 mg / kg body weight, preferably 1-50 mg / kg body weight, preferably 0.5-15 mg / kg body weight. The exact dosage of the polypeptide and / or nucleic acid molecule to be used will depend on, for example, the route of administration and the severity of the infection or the disease caused thereby, and should be determined according to the judgment of the physician and the circumstances of each subject. . For example, the effective dose will vary depending on the patient's target site, physiological condition (eg, age, weight, health), regardless of whether the treatment is prophylactic or therapeutic. Usually, the patient is a human but non-human mammals such as transgenic mammals can also be treated. The therapeutic dose is optimally titered to optimize safety and efficacy.

  The polypeptides of the invention can also be used to confirm the binding of monoclonal antibodies identified as potential therapeutic candidates. Furthermore, the polypeptides of the present invention can be used as diagnostic tools to test an individual's immune status, for example, by checking for the presence of antibodies in the serum of such individuals that can bind to the polypeptide of the present invention. Can be used to Thus, the present invention is also an in vitro diagnostic method for detecting the presence of an influenza infection in a patient, comprising a) contacting a biological sample obtained from said patient with a polypeptide according to the present invention; and b) an antibody -Relates to a method comprising detecting the presence of an antigen complex.

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

  The invention is further illustrated in the following examples and figures. The examples in no way limit the scope of the invention.

Example 1
Identification of novel group 1 and group 2 cross-neutralizing antibodies: CR9114 Peripheral blood was collected from normal healthy donors by venipuncture into EDTA anticoagulant sample tubes. The scFv phage display library was obtained essentially as described in WO 2008/028946, which is incorporated herein by reference. Selections include influenza A subtype H1 (A / New Caledonia / 20/99), H3 (A / Wisconsin / 67/2005), H4 (A / Duck / Hong Kong / 24/1976), H5 (A / Chicken / Vietnam) / 28/2003), H7 (A / Netherlands / 219/2003) and H9 (A / Hong Kong / 1073/99) recombinant hemagglutinin (HA). Two consecutive selection rounds were performed prior to the isolation of individual single chain phage antibodies. After the second round of selection, monoclonal phage antibodies were prepared using individual E. coli colonies. Selection supernatants containing single chain phage antibodies obtained in the above screening were validated for specificity in ELISA, ie binding to different HA antigens. To this end, baculovirus expressing recombinant H1 (A / New Caledonia / 20/99), H3 (A / Wisconsin / 67/2005), H5 (A / Vietnam / 1203/04) H7 (A / Netherlands / 219) / 2003), and B (B / Ohio / 01/2005) HA (Protein Sciences, CT, USA) were coated on Maxisorp ™ ELISA plates. Of the obtained single-stranded phage antibodies, the single-stranded phage antibody SC09-114 was shown to specifically bind to recombinant influenza AH1, H3, H5, H7 and influenza BHA. The binding and specificity of SC09-114 was validated by FACS analysis. To this end, full-length recombinant influenza A subtypes H1 (A / New Caledonia / 20/1999), H3 (A / Wisonsin / 67/2005) and H7 (A / Netherlands / 219/2003) HA are used in PER. It was expressed on the surface of C6 cells. SC09-114 has been shown to specifically bind to influenza A subtypes H1, H3 and H7HA. The scFv heavy and light chain variable regions were cloned as previously described (WO 2008/028946). The resulting expression construct encoding human IgG1 heavy and light chains is transiently expressed in combination with 293T cells to obtain a supernatant containing human IgG1 antibody CR9114, which is subjected to standard purification procedures. Produced using. Table 1 lists the amino acid sequences of the CDRs of the heavy and light chains of CR9114. The nucleotide and amino acid sequences of the heavy and light chain variable regions are listed below.

Example 2
CR9114 Cross-Binding Reactivity CR9114 was validated for binding specificity, ie binding to different HA antigens, in an ELISA. To this end, baculovirus-expressing recombinant H1 (A / New Caledonia / 20/1999), H3 (A / Wisconsin / 67/2005), H5 (A / Vietnam / 1203/04, H7 (A / Netherlands / 219) / 2003) and H9 (A / HongKong / 1073/99) HA (Protein Sciences, CT, USA) were coated on Maxisorp ™ ELISA plates, as an unrelated IgG CR4098 was used. It was shown to have heterogeneous subtype cross-linking activity against all recombinant HAs, see Table 2.

  In addition, the antibodies were tested for heterotypic binding by FACS analysis. To this end, full-length recombinant influenza A subtypes H1 (A / New Caledonia / 20/1999), H3 (A / Wisonsin / 67/2005) and H7 (A / Netherlands / 219/2003) HA are used in PER. It was expressed on the surface of C6 cells. Cells were incubated with CR9114 for 1 hour, followed by 3 washing steps with PBS + 0.1% BSA. Bound antibody was detected using PE conjugated anti-human antibody. As a control, untransfected PER. C6 cells were used. CR9114 showed cross-binding activity against influenza A subtypes H1, H3 and H7HA, but wild type PER. C6 cells were not shown. See Table 2.

Example 3
Cross-neutralizing activity of CR9114 An additional in vitro virus neutralization assay (VNA) was performed to determine if CR9114 was able to block multiple influenza A strains. VNA was performed on MDCK cells (ATCC CCL-34). MDCK cells were transformed into MDCK cell culture medium (MEM medium (complete MEM medium) supplemented with antibiotics, 20 mM Hepes and 0.15% (w / v) sodium bicarbonate) in 10% (v / v) fetal bovine. Cultured in serum supplemented). H1 used in the assay (A / WSN / 33, A / New Caledonia / 20/1999, A / Solomon Islands / IVR-145 (A / Solomon Islands / 3/2006 high growth rear sortant), A / Brisbane / 59 / 2007, A / NYMC / X-181 (A / California / 07/2009 high growth rear sortant), H2 (A / Env / MPU3156 / 05), H3 (A / Hong Kong / 1/68, A / Joannesburg / 33 / 94, A / Panama / 2000/1999, A / Hiroshima / 52/2005, A / Wisconsin / 67/2005 and A / Brisbane / 10/2007), H4 (A / WF / HK / MPA89 / 06), H5 (PR8-H5N1-HK97 (6: 2 rear sortant of A / HongKong / 156/97 and A / PR / 8/34) and A / Eurasian Wigeon / MPF461 / 07), H6 (A / Eurasian Wigeon) / MPD411 / 07), H7 (NIBRG-60 (6: 2 rear sortant of A / Mallard / Netherlands / 12/2000) and PR8-H7N7-NY (A / New York / 107/2003 (H7N7) and A / PR / 8/34 7: 1 rear sortant)), H8 (A / Eurasian Wigeon / MPH571 / 08) H9 (A / HongKong / 1073/99 and A / Chick / HK / SSP176 / 09), H10 (A / Chick / ermany / N / 49) and H14 (PR8-H14N5 (A / mallard / Astrakhan / 263/1982 (H14N5) and of A / PR / 8/34 6 : 2 reassortant)) all strains 5.7 × ¹⁰ 3 TCID50 The titer was calculated according to the method of Spearman and Karber: IgG preparation (200 μg / ml) was made up in quadruplicate in complete MEM medium. In a well, serially diluted 2-fold (1: 2 to 1: 512) 25 μl of each IgG dilution was mixed with 25 μl of virus suspension (100 TCID50 / 25 μl) for 1 hour at 37 ° C. The suspension was then incubated in a confluent MDCK culture in 50 μl complete MEM medium. Transferred in duplicate onto 96 well plates containing product. Prior to use, MDCK cells were seeded at 3 × 10 ⁴ cells per well in MDCK cell culture medium, grown until cells reached confluence, washed with 300-350 μl PBS, pH 7.4, and finally 50 μl of complete MEM medium was added to each well. Inoculated cells were cultured at 37 ° C. for 3-4 days and observed daily for expression of cytopathic effect (CPE). CPE was compared to a positive control.

  CR9114 has been shown to have heterologous subtype cross-neutralizing activity against representative strains of all tested influenza A subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9 and H10 viruses. See Table 3.

Example 4
Design of Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes of CR6261 and CR9114 Based on H1HA A fully human monoclonal antibody against influenza virus hemagglutinin with broad cross-neutralizing efficacy was previously identified. CR6261 (described in WO 2008/028946) was shown to have broad cross-neutralizing activity against phylogenetic group 1 influenza A viruses. Furthermore, it has been shown that CR9114 described above can bind to and neutralize both influenza A viruses and influenza B viruses of phylogenetic groups 1 and 2. Functional and structural analysis revealed that these antibodies interfere with the membrane fusion process and are directed against highly conserved epitopes in the stem domain of the influenza HA protein (Throsby et al. (2008). Ekiert et al. (2009) WO 2008/028946, and co-pending application EP 11173953.8).

  In studies that have led to the present invention, a new molecule containing HA stem domains containing these epitopes can be designed and used, for example, as a vaccine to induce protection against a wide range of influenza strains. A sex polypeptide was produced. In essence, the highly mutated immunodominant portion, ie, the head domain, is first removed from the full length HA molecule to create an HA stem domain polypeptide, also referred to as “mini-HA”. Thus, the immune response is redirected to the stem domain where the epitope for the broadly neutralizing antibody is located. Antibodies CR6261 and CR9114 are used to check the correct folding of newly generated molecules and to confirm the presence of neutralizing epitopes.

  Thus, the polypeptides of the present invention present conserved epitopes of the membrane proximal stem domain HA molecule to the immune system in the absence of dominant epitopes present in the membrane distal head domain. For this purpose, a portion of the primary sequence of the HA0 protein that constitutes the head domain is removed and religated directly or by introducing a short flexible binding sequence ("linker") for chain continuity. To recover. The resulting sequence is further modified by introducing defined mutations that stabilize the natural three-dimensional structure of the remaining portion of the HA0 molecule.

  The function of the HA molecule in the virus is to bind to the cell surface receptor sialic acid and mediate viral and endosomal membrane fusion after uptake into the endosome, resulting in the release of viral RNA into the cell. An essential step in the fusion process is the large conformational change of the HA molecule that rearranges the secondary structure elements of the molecule so that the fusion peptide is exposed. As a result, there are two conformations of HA molecules (before and after fusion) that are very different with respect to tertiary structure. Since the viral HA protein is primarily exposed to the immune system in the pre-fusion state, it is important to confirm that the polypeptide of the present invention adopts this conformation. This requirement can be met by stabilizing the pre-fusion conformation and simultaneously destabilizing the post-fusion conformation. This stabilization / destabilization is essential. This is because the pre-fusion conformation is metastable and the adoption of the post-fusion conformation results in a stable conformation, ie, a low energy minimum (Chen et al, 1995).

  In this example, HA from H1N1A / Brisbane / 59/2007 (SEQ ID NO: 1) is considered as a primary (or wild type) sequence to produce a polypeptide of the invention.

  In the first step, the polypeptides of the invention are constructed by removing the HA1 sequence at positions 59-291 (numbering refers to the position in the HA0 sequence shown in SEQ ID NO: 1. In certain embodiments. The HA1 portion includes amino acids 18-343 and the HA2 portion includes amino acid residues 344-565; because SEQ ID NO: 1 includes a signal peptide and the HA1 portion begins at position 18. is there). This results in the removal of residues 60 to 290 of HA0. These residues were replaced by GGGG binding sequences. Next, the accessible surface area of each residue in both the pre- and post-fusion conformations was calculated by Brugel (Delhaise et al., 1984). The degree of exposure and embedding of each residue was determined as described in Samantha et al (2002) focusing on the residues exposed in the pre-fusion conformation and embedded in the post-fusion conformation. Further analysis of these residues shows that some of these amino acid residues can be mutated so that the mutation has no effect on the pre-fusion conformation and de-stabilizes the post-fusion conformation. showed that. These residues generally have hydrophobic side chains and are involved in the formation of coiled coils in the post-fusion conformation. Mutations of these amino acid residues to hydrophilic amino acids disrupt the coiled coil properties (contacts between helices in the coiled coil are generally hydrophobic) and thus destabilize the post-fusion conformation.

According to this assumption, several mutations in the HA2 part of the sequence: Phe406 to Ser (F406S), Val409 to Thr (V409T), Leu416 to Ser (L416S) and Tyr502 to Ser (Y502S). ) Mutation was introduced. These are mutations that remove hydrophobic residues from the surface of HA. It should be noted that mutation of L416 to either S or T also introduces a consensus N-glycosylation site (the consensus sequence is NX, which is S / T). Further increase the solubility of this region, and mutate Leu58 to Thr (L58T), Val314 to Thr (V314T), and Ile316 to Thr (I316T); , Present in the portion of the sequence corresponding to HA1 after cleavage of the native HA0 chain, the last two mutations maintain the beta branch of the side chain but remove hydrophobic residues from the surface. As shown in Figure 1, some of these mutations were introduced into all variants, and others were tested in separate polypeptides to find mutations. It was investigated whether or not affect the non-desired to have.

  In order to increase the stability of the polypeptide, two disulfide bridges were investigated to anchor the HA in a pre-fusion conformation. Disulfide bridges are present at a spatially appropriate distance from each other (in the full length HA molecule) and are formed between residues that already have a Cbeta atom in the correct position to form the disulfide bridge. The proposed first disulfide bridge exists between position 321 (HA1 domain) and position 405 (HA2 domain). Within the HA2 domain, a disulfide bridge was created between positions 413 and 421.

  Since cleavage of HA at position R343 is an essential step because conformational changes can occur in the polypeptides of the invention, the cleavage site is introduced by introducing a mutation of Arg (R) into Gln (Q). Was removed. Another solution according to the invention is to change Arg to Gln and delete residues 345 to 350 of a small portion of the fusion peptide of HA2. Removal of these (hydrophobic) sequences further stabilizes the polypeptide.

In certain embodiments, the polypeptides of the invention contain an intracellular sequence of HA and a transmembrane domain. In other embodiments, the cytoplasmic sequence of HA2 from position 523, 524, 525, 526, 526, 527, 528, 529, or 530 (or their equivalent) to the C-terminus of HA2 (numbered by SEQ ID NO: 1) And the transmembrane sequence was removed so that, for example, secreted (soluble) polypeptides that can be used in vaccines are produced after expression in cells. The soluble polypeptide was further stabilized by introducing a sequence known to form a trimeric structure, ie, AYVRKDGEWVLL (SEQ ID NO: 143), optionally linked via a linker. The linker may optionally contain a cleavage site for later processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a short linker, eg, a tag sequence linked via EGR, eg, a his tag (HHHHHHH) can be added. In some embodiments, the linker and his tag sequence are added without the presence of a foldon sequence. According to the present invention, the amino acid sequence of the C-terminal amino acid is removed from position 530 of the HA2 domain (numbering according to SEQ ID NO: 1) and the following sequence:
Replaced by EGRHHHHHHH (SEQ ID NO: 81) containing a short linker and a his tag, or SGRSLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLLSTFLGHHHHHHH (SEQ ID NO: 82) containing a thrombin cleavage site, trimerization domain, and his tag.

The mutations were classified into clusters according to their function and localization in the three-dimensional structure of the HA stem polypeptide. All polypeptides contain H1HA sequences 1-59 and 291-565 and R343Q mutations with the following additional mutations: L58T, V314T, I316T, F406S, V409T, L416S (SEQ ID NO: 3; named cluster1) To do. In addition, variants with additional mutations were created:
Cluster 2: K321C, Q405C (SEQ ID NO: 4)
Cluster3: F413C, E421C (SEQ ID NO: 5)
Cluster 4: HA2 Y502S (SEQ ID NO: 6).

  In addition, two variants containing the cluster1 sequence and further mutations of cluster2 and 3 (SEQ ID NO: 7) or cluster2, 3 and 4 (SEQ ID NO: 8) were generated.

In general, the genes encoding the above protein sequences were synthesized using methods known to those skilled in the art and cloned into the expression vector pcDNA2004. For comparison reasons, the sequence described by Steel et al (2010) based on the full-length sequence (SEQ ID NO: 1) and the H1N1A / Puerto Rico / 8/1934 sequence (H1-PR8-dH1; SEQ ID NO: 24) Included in the experiment.

The expression vector (1 μg / ml) was transfected into HEK293F (Invitrogen) suspension cells (10 ⁶ cells / ml, 30 ml) using 40 μl 293transfectin as a transfection agent and allowed to grow for another 2 days. Cells were harvested and aliquoted (0.3 ml, approximately 3 ^* 10 ⁵ cells), and aliquots were raised against polyclonal serum or HA-specific monoclonal antibody (5 microgram / ml) against H1HA to examine expression. ) And the secondary antibody used for staining. Cells were then analyzed for expression of the membrane-attached HA stem domain polypeptide of the present invention by fluorescence-coupled cell sorting (FACS) using polyclonal sera raised against H1HA to examine expression. . A panel of monoclonal antibodies of known specificity that binds to full-length proteins (eg, CR6261 and CR9114) is used to examine the presence of conserved epitopes and the correct folding of full-length HA and the mini-HA polypeptides of the invention by inference It was. The results are expressed as percentage of positive cells and average fluorescence intensity and are shown in FIG.

  The results show that all constructs are expressed on the cell surface. This is because the reaction with polyclonal serum (anti-H1 poly) results in more than 75% of all analyzed cells being positive compared to about 4% for untransfected cells. This is confirmed by similar mean fluorescence intensity (MFI) values for all constructs after treatment with polyclonal sera. All control experiments in the absence of IgG using only labeled anti-human IgG or irrelevant mAb are negative. Both A / Brisbane / 59/2007 and A / Puerto Rico / 8/1934 full-length HA proteins are monoclonal antibodies CR6261, CR6254, CR6328 (all known to bind to and neutralize H1HA; Throsby et al. (2008), WO 2008/028946 pamphlet), CR9114 (above), CR8001 (binding to H1HA but not neutralizing H1; described in WO2010 / 130636 pamphlet) CR8057 (binding only to some H3 strains, also described in WO2010 / 130636 pamphlet) and CR6307 (Throsby et al. (2008), WO2008 / 028946 pamphlet) Less recognized.

  Considering the discontinuity and conformational features of the CR6261 epitope (Ekiert et al. 2009), it can be concluded that both full-length proteins exist in their native three-dimensional conformation. For the newly designed polypeptides of the present invention tested in this experiment, binding by the same recognition pattern by the panel of monoclonal antibodies: CR6261, CR6254, CR6328, CR9114 and CR8001 was observed, but binding by CR6307 and CR8057 Was not observed. This is most evident in data based on the percentage of positive cells, but is also observed in average fluorescence intensity data. The best results are obtained for miniHA-cluster1 with respect to both% positive cells and average fluorescence intensity.

The addition of further modifications, such as the disulfide bridges (clusters 2 and 3) and the cluster4 Y502S mutation (or combinations thereof), resulted in a reduction in the percentage of positive cells and lower average intensity. However, the construct described by Steel et al. (2010) (SEQ ID NO: 24), which lacks further modifications, is not recognized above background levels by any of the antibodies used in this experiment. It is later concluded that this protein is not displayed in the natural three-dimensional conformation it has in HA.

  The above results show the importance of the cluster1 mutation that increases the hydrophilic character of the loop formed by residues 402 to 418 and its surrounding area that link the A helix and long backbone helix (CD) of the HA molecule. Point to. To further confirm the beneficial effects of cluster1 mutations on the stability and folding of the polypeptides of the invention, miniHA (SEQ ID NO: 2; according to Steel, but based on A / Brisbane) and miniHA_cluster1 (invention) In a separate experiment (Figure 3).

  About 60% of the cells transfected with miniHA-cluster1 are positive after binding of CR6261, CR6254, CR6328 and CR9114, whereas transfection with miniHA (by Steel but A / Brisbane based polypeptide; sequence Number 2) yields values very close to the background level (1-3%). We have found that mutations in cluster1 contribute well to the proper folding and stability of the polypeptides according to the invention compared to the unmodified mini protein lacking these mutations (SEQ ID NO: 2). Conclude.

  Steel et al. Created a new molecule by deleting amino acid residues 53 to 276 of H1A / Puerto Rico / 8/1934 and H3HK68 strain HA1 from the primary sequence and replacing it with a short flexible linker. As shown in this example, this is a highly unstable molecule that does not adjust the exact conformation, as evidenced by the lack of binding of the antibody already shown to bind to a conserved epitope in the stem region. Bring. Inaccurate folding is usually caused by large areas of solvent exposure shielded by globular heads in full-length HA molecules. Since this area is natural and hydrophobic, the molecule is no longer stable and therefore needs adaptation.

  The exchange of hydrophobic residues for hydrophilic residues made in the polypeptides of the invention attenuates this effect and stabilizes the HA stem domain polypeptide. Further stabilization of the native three-dimensional fold of the stem domain is achieved by introducing disulfide bridges at appropriate positions to closely link residues that are spatially close in the natural tertiary structure but separated in the primary structure Is done.

Example 5
Immunogenicity of the HA stem domain polypeptide of Example 4 To evaluate the immunogenicity of the stem domain polypeptide, full length H1 (SEQ ID NO: 1), miniHA-cluster1 (SEQ ID NO: 3) from A / Brisbane / 59/2007 ), Mice were immunized with expression vectors encoding miniHA-cluster1 + 2 (SEQ ID NO: 4) and miniHA-cluster1 + 4 (SEQ ID NO: 6). For reasons of comparison, Steel et al. The miniHA design (mini-PR8; SEQ ID NO: 24) according to (2010) and the corresponding full-length protein HA from A / Puerto Rico / 8/1934 were also included in the experiment. An expression vector encoding cM2 was also included as a negative control.

Groups of 4 mice (BALB \ c) were immunized intramuscularly on days 1, 21, and 42 with 50 μg construct + 50 μg adjuvant (pUMCV1-GM-CSF). On day 49, the final blood collection was performed and serum was collected. Serum was analyzed by FACS assay. HEK293F (Invitrogen) suspension cells (10 ⁶ cells / ml, 30 ml) were transfected with an expression vector (1 microgram / ml) using 40 microliters of 293transfectin as a transfection agent for another 2 days. Allowed to grow. Cells were harvested and aliquoted (0.3 ml, approximately 3 ^* 10 ⁵ cells) and aliquots were treated with construct-specific serum, stained with secondary antibodies, and analyzed by fluorescence-coupled cell sorting. The results are shown in FIG.

  As expected, as demonstrated by% positive cells and MFI, cM2-specific serum (negative control) recognizes cM2, but does not recognize full-length HA or stem domain polypeptides. In contrast, full-length HA-specific serum stains cells expressing the corresponding full-length HA (SEQ ID NO: 1), but at lower levels (about 80% for the full length, about 40% positive cells, full length But also miniHA-cluster1 (SEQ ID NO: 3), miniHA-cluster1 + 2 (SEQ ID NO: 4) and miniHA-cluster1 + 4 (SEQ ID NO: 6). Vice versa; sera specific for miniHA-cluster1 (SEQ ID NO: 3), miniHA-cluster1 + 2 (SEQ ID NO: 4) and miniHA-cluster1 + 4 (SEQ ID NO: 6) express the corresponding construct and full-length HA (SEQ ID NO: 1) Recognize cells to do. The results are summarized in Table 4 below.

  In contrast to the above results for miniHA-cluster1 (SEQ ID NO: 3), miniHA-cluster1 + 2 (SEQ ID NO: 4) and miniHA-cluster1 + 4 (SEQ ID NO: 6), sera obtained from mice immunized with full-length PR8 were H1-PR8-dH1 (SEQ ID NO: 24) did not bind well to the transfected cells. The percentage of positive cells was approximately 20% compared to 40-50% for miniHA-cluster1 (SEQ ID NO: 3) and miniHA-cluster1 + 2 (SEQ ID NO: 4). The results are also reflected in what is observed at an average fluorescence intensity slightly above the background level.

  Taken together, the data show that the polypeptides of the invention can induce an immune response directed against full-length HA. In particular, modifications in the region of residues 402-418 (numbering according to SEQ ID NO: 1) are important for creating stable molecules.

Example 6
Preparation of Second Generation Stem Domain Polypeptides The average fluorescence intensity of the stem domain polypeptides described in Example 4 is in all cases lower than that observed for the corresponding full length protein; MiniHA-cluster 1 (SEQ ID NO: 3) has an intensity that is on the order of 10% of the average intensity of the full-length construct after binding to the monoclonal antibody. This indicates that the expression and / or folding of the stem domain polypeptide on the cell surface is lower than that observed for the full-length protein and the design can be further improved. The results obtained from the first generation showed that improvement of the first generation construct was possible and therefore the second design round was started.

The polypeptide described in Example 4 has the same deletion of the HA0 chain, ie the deletion of residues L60 to K290 (mini1; numbering is full length HA0 from H1N1A / Brisbane / 59/2007; SEQ ID NO: 1 Point to the inside position). This approach creates a long unstructured loop that is no longer attached to the head domain. It was estimated that this loop did not contribute to total protein stability and could be shortened considerably without affecting the folding of other parts of the polypeptide. Three additional deletions were designed and replaced with the GGGG linker sequence as described above and combined with the cluster1 mutation described above. Deletions are S53 to P320 (mini2), H54 to I302 (mini3), and G56 to G317 (mini4). Additional modifications identical to cluster1 above (L58T, V314T, I316T, F406S, V409T, L
416S) was introduced. Some of the residues belonging to this cluster are part of the deleted sequence and can therefore no longer be modified (see below). In addition, two additional mutations were created in long helix C that formed a trimeric coiled coil in the pre-fusion state. Trimeric coiled coils are well known in the art to be stabilized by Ile at positions 420a and d of the 7 residue repeat sequence characteristic of this structural motif (Suzuki et al., (2005); Woolson et al. al. (2005)). This finding was applied by introducing Ile at positions 420 (M420I) and 427 (V427I). The combination of these two mutations and the mutation of cluster1 is named cluster11; for classification, the combinations are listed below:
Mini1: deletion L60 to K290 cluster 11: M420I, V427I, L58T, V314T, I316T, F406S, V409T, L416S
Mini2: deletion S53 to P320 cluster11: M420I, V427I, F406S, V409T, L416S
Mini3: deletion H54 to I302 cluster11: M420I, V427I, V314T, I316T, F406S, V409T, L416S
Mini4: deletion G56 to G317 cluster11: M420I, V427I, F406S, V409T, L416S

To further stabilize the pre-fusion state of the stem domain polypeptide, an additional disulfide bridge was introduced between positions 324 and 436 (cluster5: R324C, T436C) and combined with different deletion mutants. The following combinations were synthesized and tested for binding in the FACS assay described above:
Mini1-cluster 11 (SEQ ID NO: 9)
Mini2-cluster 11 (SEQ ID NO: 10)
Mini3-cluster 11 (SEQ ID NO: 11)
Mini4-cluster 11 (SEQ ID NO: 12)
Mini1-cluster 11 + 5 (SEQ ID NO: 13)
Mini2-cluster 11 + 5 (SEQ ID NO: 14)
Mini3-cluster 11 + 5 (SEQ ID NO: 15)
Mini4-cluster 11 + 5 (SEQ ID NO: 16)

  For comparison reasons, miniHA-cluster 1 (SEQ ID NO: 3) was also included in the experiment. The results are shown in FIG.

  In all cases, the stem domain polypeptide is present on the cell surface after transfection of the expression vector into HEK293F cells as evidenced by the percentage of positive cells (> 90%) after treatment with polyclonal anti-H1 serum. did.

  All HA stem domain polypeptides in this experiment were recognized by CR6261, CR6254, CR6328 and CR9114 but not by CR8057; the last one is predicted. This is because this mAb is specific for H3HA. However, there is a clear difference in the percentage of cells positive for different antibodies and the MFI. The best antibody characterized is CR6261, with known epitope details. The epitope is discontinuous and conformational, so this antibody binding can be viewed as a stringent test of the correct folding of the HA stem domain polypeptide. CR9114 is broadly neutralizing and covers strains from both groups 1 and 2 (Table 3). Details are not known for the CR6328 and CR6254 epitopes, but based on the higher values found for% positive cells and MFI, and the smaller spread of the data, the binding of these antibodies is less sensitive than the CR6261 It is considered a folding probe.

Comparing the percentage of positive cells (considering data for all antibodies), one can rank Mini1 to 4 constructs (maximum to minimum%)
About combination with cluster11 Mini2>Mini1>Mini4> Mini3 and combination with cluster11 + 5 Mini2> Mini1 = Mini4> Mini3

  This ranking is also reflected in the data for MFI, leading to the conclusion that the deletion of the Mini2 construct, S53 to P320, results in the highest level of protein displayed on the cell surface in the correct conformation from this set.

  When MiniHA-cluster1 (SEQ ID NO: 3) is compared to mini1-cluster11 (SEQ ID NO: 9), the additional mutations M420I, V427I are not believed to result in additional stabilization of the construct; Results in lower percentage of positive cells and MFI values, but the difference is small.

  Introduction of disulfide bridges R324C, T436C (cluster 5) results in an increase in correctly folded proteins on the cell surface for mini2-cluster 11 (SEQ ID NO: 10) and mini4-cluster 11 (SEQ ID NO: 12), but mini1-cluster 11 (SEQ ID NO: 9) and mini3-cluster 11 (SEQ ID NO: 11) provide minimal improvement or no improvement. Overall best results are obtained for mini2-cluster 11 + 5 (SEQ ID NO: 14). This is particularly evident from the MFI value, which is about 50% of the value for the full length construct for this construct.

  In certain embodiments, the polypeptides of the invention contain an intracellular sequence of HA and a transmembrane domain. In other embodiments, HA2 cytoplasmic and transmembrane sequences (numbers according to SEQ ID NO: 1) from positions 523, 524, 525, 526, 526, 527, 528, 529, or 530 (or their equivalents) of HA2. The sequence known to form a trimer structure is removed, ie, AYVRKDGEWVLL (SEQ ID NO: 143) is optionally replaced by introduction, optionally linked via a linker. The linker may optionally contain a cleavage site for later processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a short linker, eg, a tag sequence linked via EGR, eg, his tag HHHHHHH can be added. According to the present invention, the amino acid sequence of the C-terminal amino acid was removed from position 530 of the HA2 domain (numbering according to SEQ ID NO: 1) and replaced by SEQ ID NO: 81 or SEQ ID NO: 82.

Example 7
Immunogenicity of the second generation HA stem domain polypeptide To evaluate the immunogenicity of the stem domain polypeptide of Example 6, the full length H1 (SEQ ID NO: 1) from A / Brisbane / 59/2007, miniHA-cluster1 ( Mice were immunized with expression vectors encoding SEQ ID NO: 3), Mini2-cluster 11 (SEQ ID NO: 10), Mini1-cluster 11 + 5 (SEQ ID NO: 13), Mini2-cluster 11 + 5 (SEQ ID NO: 14). An expression vector encoding cM2 was also included as a negative control.

Groups of 4 mice (BALB \ c) were immunized intramuscularly on days 1, 21, and 42 with 50 μg construct + 50 μg adjuvant (pUMCV1-GM-CSF). On day 49, the final blood collection was performed and serum was collected. Full length HA0 (SEQ ID NO: 1), negative control c
M2 and Mini2-cluster 11 + 5 (SEQ ID NO: 14) were also administered to separate groups of mice by gene gun using about 10 μg construct + about 2 μg adjuvant (pUMCV1-GM-CSF) and the same immunization scheme. Serum was analyzed by Elisa using the recombinant ectodomain of full-length HA from A / Brisbane / 59/2007 strain (obtained from Protein Sciences Corporation, Meriden, CT, USA) as an antigen. Briefly, 96 well plates were coated overnight at 4 ° C. with 50 ng HA and then incubated with block buffer (100 μl PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. The plate is washed with PBS + 0.05% Tween-20 and 100 μl of a 2-fold dilution series in block buffer starting with a 20-fold dilution of serum is added. HRP-conjugated goat anti-mouse IgG was used to detect bound antibody using standard protocols well established in the art. The mAb 3AH1InA134 (Hytest, Turku, Finland) was used to compare the titer to the standard curve to derive Elisa units / ml (EU / ml).

  The Elisa results after 28 and 49 days are shown in FIGS. 6A and B, respectively. Sera obtained from mice immunized with DNA encoding Mini2-cluster11 + 5 (SEQ ID NO: 14) were immunized 28 and 49 days after immunization using a gene gun and 49 days after immunization intramuscularly. Shows clear binding to domain full length HA. For Mini2-cluster11 (SEQ ID NO: 10) and Mini1-cluster11 + 5 (SEQ ID NO: 13), a response was detected for one of the four mice, whereas no binding was detected for miniHA-cluster1 (SEQ ID NO: 3). It was.

  Taken together, the data show that the polypeptides of the invention can induce an immune response directed against full-length HA. In particular, modifications in the region of residues 402-418 (numbering according to SEQ ID NO: 1), deletions S53 to P320, are important for creating stable molecules in combination with disulfide bridges R324C, T436C.

Example 8
Preparation of Third Generation Stem Domain Polypeptides A third round of design was performed to further improve the design of stem domain polypeptides. An additional mutation that increases the hydrophilicity of the surface that is embedded in full length HA but not in the stem domain polypeptide was introduced at position 413 and named F413G (numbered by SEQ ID NO: 1), cluster6. . This cluster was identified as mini-2 deletion (S53 to P320), disulfide bridge of cluster5 (R324C, T436C) and cluster1 (ie F406S, V409T, L416S; SEQ ID NO: 46) or cluster11 (M420I, V427I, F406S, V409T). L416S; SEQ ID NO: 47) in combination with any of the mutations. Combinations of the mini-2 deletion (S53 to P320) with cluster1 (F406S, V409T, L416S) and cluster5 (R324C, T436C) are also included in this experiment for reference (SEQ ID NO: 48).

Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus the enhanced interaction between monomers in the truncated molecule increases stability. In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced. According to the present invention, the consensus sequence for the formation of a trimeric coiled coil, IEAIEKKIEAEEKKIE (SEQ ID NO: 83), is located in the polypeptide of the present invention at positions 418 to 433 in H1A / Brisbane / 59/2007 (SEQ ID NO: 44). ) (
(Numbering according to SEQ ID NO: 1) (equivalent). An alternative is to introduce the sequence MKQIEDKIEIESKQ (SEQ ID NO: 84), derived from GCN4 and known to trimerize, at positions 419-433 (SEQ ID NO: 45).

  In the case of the stem domain polypeptides described by SEQ ID NO: 44 to SEQ ID NO: 48, all proteins are found in HEK293F cells as evidenced by the percentage of positive cells (> 90%) after treatment with polyclonal anti-H1 serum. Was present on the cell surface after transfection of the expression vector. The results are shown in FIG.

  All HA stem domain polypeptides in this experiment were recognized by CR6261, CR6328 and CR9114 except miniHA (SEQ ID NO: 2) but not recognized by CR8020; the last is predicted. This is because this mAb is specific for H3HA. The percentage of positive cells is approximately 80% for stem domain polypeptides using CR6261, CR6328 and CR9114 for staining, excluding miniHA which is only recognized by polyclonal anti-H1 serum. Again, this indicates a lack of proper folding of this particular construct. However, there is a clear difference in MFI for different antibodies. The best antibody characterized is CR6261, with known epitope details. The epitope is discontinuous and conformational, so this antibody binding can be viewed as a stringent test of the correct folding of the HA stem domain polypeptide. CR9114 is broadly neutralizing and covers strains from both groups 1 and 2 (Table 3). Details of the epitope of CR6328 are not known, but competition with CR6261 is observed in binding studies for full-length HA.

  Regardless of the monoclonal antibody used in the experiment, H1-mini2-cl11 + 5 (SEQ ID NO: 14), H1-mini2-cl1 + 5 (SEQ ID NO: 48), H1-mini2-cl1 + 5 + 6 (SEQ ID NO: 46) and H1-mini2-cl11 + 5 + 6 ( The MFI for SEQ ID NO: 47) is very similar. Inclusion of a consensus trimerization domain (SEQ ID NO: 44) residue results in an MFI of 3-4 compared to the equivalent sequence without the trimerization domain (ie, H1-mini2-cluster1 + 5 + 6; SEQ ID NO: 46). Although reduced by a factor of 2, the results are still clearly better than in the absence of stem polypeptide modification after the deletion of the head domain (see miniHA results). Addition of a GCN4 trimerization sequence (SEQ ID NO: 45) increases MFI to a level comparable to the full-length protein.

Example 9
Design of Additional Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes of CR6261 and CR9114 Polypeptides of the present invention designed according to the above procedure can be further modified to increase stability. Such modifications can be introduced to improve the formation of trimeric forms of the polypeptides of the invention as compared to monomeric and / or dimeric species. As mentioned above, native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus the enhanced interaction between monomers in the truncated molecule increases stability. In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced.

According to the present invention, the consensus sequence IEAIEKKIEAEEKKIE (SEQ ID NO: 83) for the formation of a trimeric coiled coil is located in positions 418 to 433 in H1A / Brisbane / 59/2007 (SEQ ID NO: 44) in the polypeptide of the present invention. ) (Numbering according to SEQ ID NO: 1) (equivalent). Alternatively, IEAIEKKIEAEEKKI (SEQ ID NO: 85) can be introduced at 419-433 (SEQ ID NO: 49), or IEAIEKKIEAEIEKK (SEQ ID NO: 86) can be introduced at 420-433 (SEQ ID NO: 50). An alternative is to introduce the sequence MKQIEDKIEIESKQ (SEQ ID NO: 84), derived from GCN4 and known to trimerize, at positions 419-433 (SEQ ID NO: 45). Alternatively, MKQIEDKIEIESK (SEQ ID NO: 87) can be introduced at positions 420-433 (SEQ ID NO: 51), or RMKQIEDKIEIESKQK (SEQ ID NO: 88) can be introduced at positions 417-433 (SEQ ID NO: 52). Similarly, the trimer interface is enhanced by modifying M420, L423, V427, G430 to isoleucine (SEQ ID NO: 53).

  All peptides were shown to bind to CR9114 and CR6261.

  In certain embodiments, the polypeptides of the invention do not contain signal sequences and / or intracellular sequences of HA and transmembrane domains as described above.

Example 10
Design of Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes of CR6261 and CR9114 Based on H7HA The above procedure for designing polypeptides of the present invention was also applied to H7. In this example, the design of a polypeptide of the invention based on serotype H7 is described. Although the H7 influenza virus A / Mallard / Netherlands / 12/2000 HA (SEQ ID NO: 31) was used as the parental sequence, those skilled in the art will appreciate that other H7 sequences could be used as well. This is because this sequence is particularly conserved in the stem region.

  The first modification of the sequence is to mutate R to Q (R339Q) to remove the cleavage site in position 339 (numbering refers to SEQ ID NO: 31 to prevent the formation of HA1 and HA2 from HA0. In some cases, residues 341 to 345 (LFGAI, part of the fusion peptide) can be further deleted to minimize exposure of hydrophobic residues to aqueous solvents. 100% conserved in H7, so this mutation can be applied in all sequences.

  The second modification is to remove the head domain by deleting most of the HA1 sequence and religating the N and C terminal sequences through a short linker. Deletions can vary in length but are spatially close to the last residue of the N-terminal sequence of HA1 and the first residue of the C-terminal sequence to avoid introducing strain through the binding sequence It is preferable. In the H7 sequence, a deletion can be introduced at R53 to P315 (mini2; SEQ ID NO: 33) (corresponding position) in H7A / Mallard / Netherlands / 12/2000 (SEQ ID NO: 31). Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. The remaining part of the sequence can be attached directly or a flexible linker can be introduced. The linker sequence can be 1 to 50 amino acids long. Preferred are flexible linkers of limited length (10 amino acids or less) such as GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like.

SEQ ID NO: 40 describes such a polypeptide of the invention lacking T54-C314 (mini5; SEQ ID NO: 40). The above deletion ensures that the unstructured region formed by residues 280-310 is also removed; this is beneficial for the total stability of the polypeptides of the invention. Similar effects were observed for the polypeptides of the invention derived from the H1 sequence (see above).

  Deletion of the head domain leaves the residue 394-414 loop currently exposed to aqueous solvents. In H7HA, this loop is highly conserved (see Table 7). The consensus sequence is: LI (E / D / G) KTNQQFELIDNEF (N / T / S) E (I / V) E (Q / K) (SEQ ID NO: 32).

  Some hydrophobic residues are polar (S, T, N, Q) to increase the solubility of this loop and destabilize the post-fusion conformation for the polypeptide of the present invention in a pre-fusion conformation. , Modified to charged amino acids (R, H, K, D, E) or increased flexibility by mutation to G. Specifically, mutations at positions 402, 404, 405, 409, 412 (numbering refers to SEQ ID NO: 31) contribute to the stability of the polypeptides of the invention.

  For F402 and F409, mutations to S are preferred, but other polar (T, N, Q), charged (R, H, K, D, E) and highly flexible amino acids (G) have the same effect Have For position 404 (96% L), a mutation to N or S is preferred; the latter amino acid occurs infrequently but naturally, in which case no mutation at this position is necessary. Other polar (T, Q), charged (R, H, K, D, E) and highly flexible amino acids (G) have the same effect. For position 405 (99% I), a mutation to T or D is preferred. D also occurs naturally, in which case no mutation at this position is necessary. Other polar (S, N, Q), charged (R, H, K,) and highly flexible amino acids (G) have the same effect. For position 412 (I or V), mutation to N is preferred, but other polar (S, T, Q), charged (R, H, K, D, E) or flexible (G) residues are possible It is. Thus, the polypeptide contains at least one of the above mutations. For example, combinations of two or more mutations were also applied as shown in SEQ ID NOs: 34-39 and 41-43.

  In order to stabilize the pre-fusion conformation of the polypeptide of the present invention, a covalent bond was introduced between two parts that are distal in the primary sequence but close in the folded pre-fusion conformation. For this purpose, disulfide bridges are preferably present in the polypeptides of the invention, preferably at positions 319 and 432 in H7A / Mallard / Netherlands / 12/2000 (SEQ ID NO: 36-39, 42, 43). Genetic manipulation was performed between them. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using suitable algorithms such as Clustal, Muscle, etc. Engineered disulfide bridges mutate at least one (if the other is already cysteine), but usually two spatially close residues to cysteines, either spontaneously or by active oxidation Created by forming a covalent bond between sulfur atoms.

  As mentioned above, native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus the enhanced interaction between monomers in the truncated molecule increases stability. In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced. It is well known in the art that trimer coiled coils are stabilized by Ile at positions a and d of the 7 residue repeat sequence characteristic of this structural motif. Here, this finding was applied by introducing Ile at positions 419, 423, 426 and 430 (SEQ ID NO: 38, 43) (or equivalent). Alternatively, the consensus sequence EAIEKKIEAI for the formation of trimer coiled coils is introduced at positions 417 to 426 (SEQ ID NO: 39) (or equivalent).

  These sequences (SEQ ID NOs: 33-43) were subjected to the fluorescence binding cell sorting assay described above. However, none of the monoclonal antibodies CR8020, CR8043, CR9114, or CR8957 could detect the binding. We conclude that these sequences do not present epitopes of these antibodies, and consequently the proteins present on the cell membrane are not folded into their natural three-dimensional structure.

Example 11
Design of Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes of CR8020, CR8043 and CR9114 Based on H3HA In the first step, the sequence representing the polypeptide of the present invention is converted to the parental sequence H3A / Wisconsin / 67/2005. HA (SEQ ID NO: 89) was used to construct analogously to that described by Steel et al. (Steel et al 2010). The head of HA is removed from amino acid D69 by deletion of a portion of HA1 at amino acid K292.

  These residues can be replaced by 3 or 4 Gly. The 4Gly linker was tested by Steel et al. And gave good expression results, which were employed here to create mini-H3 (SEQ ID NO: 90). In order to prevent cleavage of the polypeptide chain, a normal post-translational processing step for the full-length HA protein, the cleavage site at position 345 (arginine) was mutated to glutamine (R345Q).

  Next, the accessible surface area of each residue of both the constructed mini-HA and the post-fusion conformation was calculated by Brugel. The extent of exposure and implantation of each residue was determined as described by Samantha et al. (Samantha et al., 2002). The focus was on residues that were exposed in the pre-fusion conformation and embedded in the post-fusion conformation. Further analysis of these residues shows that some of these can be modified so that the mutation has no effect on the pre-fusion conformation and destabilizes the post-fusion conformation. In general, these residues have a hydrophobic chain and are involved in the formation of coiled coils in the post-fusion conformation. Mutation of these residues to include hydrophilic side chains disrupts coiled-coil properties (contacts between helices in coiled coils are generally hydrophobic) and thus destabilizes the post-fusion conformation Let Residues that are exposed in the pre-fusion conformation, embedded in the fusion conformation and predicted to have a destabilizing effect on the post-fusion conformation after mutation are L397, I401 and L425 (according to SEQ ID NO: 89) Numbering). Here, L397K and I401T are included.

  The loop (B-loop, residues 401-420) connecting helix A (residues 383-400) with the central helix CD (residues 421-470) changes conformation upon adoption of the post-fusion state; It becomes helical and is part of an elongated trimeric coiled coil. In order to stabilize the pre-fusion loop conformation of this linker and / or to destabilize the post-fusion conformation, it should be sufficient to mutate all residues involved in the formation of the coiled-coil core. It was estimated that there was. For N405, several mutations are designed, in particular residues carrying negative charges (Asp and Glu, N405D, N405E). This is because this extra charge reinforces the ionic network observed in the pre-fusion conformation. A mutation to neutral Ala (N405A) is also included in this study. We also mutated Phe408 to Thr, His409 to Ser, and Val418 to Ser (numbered by SEQ ID NO: 89; F408T, H409S, V418S) and newly exposed after removal of the head domain The solubility of the resulting surface was further increased.

Five disulfide bridges were designed to fix the HA in a pre-fusion conformation. These bridges exist at a spatially appropriate distance from each other and are formed between residues that already have Cβ atoms in the correct positions to form disulfide bridges. These are introduced between positions 320 and 406 (A320C, E406C; numbering according to SEQ ID NO: 89), positions 326 and 438 (K326C, S438C) and positions 415 and 423 (F415C, Q423C). The first two are bridges between the HA1 and HA2 portions of the chain, while the last one covalently links the tops of the B loops together. The K326C, S438C disulfide bridge is accompanied by a mutation (D435A) in Asa435 to Ala. The disulfide bridges F347C / N461C and S385C / L463C were taken from the paper by Bomakanti et al (2010) and were also used in the study.

  Several additional mutations are designed to remove newly exposed hydrophobic residues from the solvent. Ile at position 67 (numbering according to SEQ ID NO: 89) is mutated to Thr (I67T). This mutation maintains the beta branch of the side chain but removes hydrophobic residues from the surface. The same can be said for the mutation of Ile298 to Thr (I298T). Another mutation is introduced at position 316 isoleucine in the native sequence. Intuitively, it is proposed to mutate this residue to Thr to maintain beta branching but remove hydrophobicity from the surface. However, this mutation results in the introduction of an extra N-glycosylation site (position 314 is Asn) and thus introduces a mutation to Gln (I316Q).

  Gly495 was also mutated to Glu (G495E). This mutation is designed to introduce an ionic bridge. This is because there is a positive charge around. Some H3 strains that naturally have Glu in this position have already been provided.

  An important residue of HA is position 345 (Arg). This is because this residue is the position where protease cleavage occurs to allow protein fusion. This Arg to Gln mutation (R345Q) prevents cleavage from occurring, thereby immobilizing the protein in the pre-fusion state.

The above mutations were clustered as follows:
Cluster1: I67T, I98T, I316Q, F408T, H409S, V418S
Cluster2: A320C, E406C
Cluster3 K326C, D435A, S438C
Cluster4 L397K, I401T
Cluster5 N405D or N405E or N405A
Cluster6 F415C, Q423C
Cluster7 G495E
Cluster8 F347C, S385C, N461C, L463C

To obtain the polypeptides of the present invention, the clusters were combined with deletions D69 to K292 and R345Q mutations according to the following scheme.
H3 Mini-HA cluster 1 (SEQ ID NO: 91)
H3 Mini-HA cluster 1 + 2 (SEQ ID NO: 92)
H3 Mini-HA cluster 1 + 3 (SEQ ID NO: 93)
H3 Mini-HA cluster 1 + 4 (SEQ ID NO: 94)
H3 Mini-HA cluster1 + 5N405A (SEQ ID NO: 95)
H3 Mini-HA cluster1 + 5N405D (SEQ ID NO: 96)
H3 Mini-HA cluster1 + 5N405E (SEQ ID NO: 97)
H3 Mini-HA cluster 1 + 6 (SEQ ID NO: 98)
H3 Mini-HA cluster 1 + 7 (SEQ ID NO: 99)
H3 Mini-HA cluster 1 + 2 + 3 + 4 + 5 + 6 + 7-N405E (SEQ ID NO: 100)
H3 Mini-HA cluster1 + 2 + 3 + 4 + 5 + 6 + 7-N405A (SEQ ID NO: 101) H3 Mini-HA cluster1 + 2 + 3 + 4 + 5 + 6 + 7-N405D (SEQ ID NO: 102)
H3 Mini-HA cluster 1 + 8 (SEQ ID NO: 103)

  In general, the genes encoding the above protein sequences were synthesized using methods known to those skilled in the art and cloned into the expression vector pcDNA2004. For comparison reasons, the full length HA sequence of H3A / Wisconsin / 67/2005 and the full length HA sequence of H1A / Brisbane / 59/2007 containing the cleavage site mutation R343Q were included in the experiment.

The expression vector (1 μg / ml) was transfected into HEK293F (Invitrogen) suspension cells (10 ⁶ cells / ml, 30 ml) using 40 μl 293transfectin as a transfection agent and allowed to grow for another 2 days. Cells were harvested and aliquoted (0.3 ml, approximately 3 ^* 10 ⁵ cells), and aliquots were raised against polyclonal serum generated against H3HA (Protein Sciences Corp, Meriden, CT, USA) for expression or Treated with either HA-specific monoclonal antibody (5 microgram / ml) and secondary antibody used for staining. Cells were then analyzed by fluorescence-coupled cell sorting (FACS) for expression of the membrane-attached HA stem domain polypeptide of the invention using polyclonal sera raised against H3HA or H1HA to examine expression. A panel of monoclonal antibodies of known specificity that binds to full-length proteins (CR8020, CR8043 and CR9114) is used to examine the presence of conserved epitopes and the correct folding of full-length HA and the mini-HA polypeptides of the invention by inference It was. Monoclonal antibodies CR6261 (known not to bind to H3HA) and CR8057 (binding to the head domain of HA from A / Wisconsin / 67/2005) were also included in the experiment. The results are expressed as the percentage of positive cells and are shown in FIG.

  The results show that all constructs are expressed on the cell surface. This is because 80-90% of all analyzed cells were positive for the H3 base sequence and more than 50% for the full-length H1 sequence compared to less than 4% for untransfected cells compared to H3 polyclonal serum. This is to bring about something. Using anti-H1 polyclonal, 60-70% of all cells are positive, except for the full-length H1 sequence approaching 100%. All control experiments in the absence of IgG using only labeled anti-human or anti-rabbit IgG are negative. Both A / Wisconsin / 67/2005 and A / Brisbane / 59/2007 full-length HA proteins are recognized by the monoclonal antibody CR9114, which is known to be able to neutralize both strains. A / Wisconsin / 67/2005 full-length HA further binds to CR8020, CR8043, and CR8057 (described in WO2010 / 130636, which binds only to some H3 strains), but CR6261 (Throsby et al (2008), WO2008 / 028946 pamphlet). The reverse is true for the full length HA from A / Brisbane / 59/2007; it binds to CR6261 but not to CR8020, CR8043 and CR8057.

The polypeptides set forth in SEQ ID NO: 91 to SEQ ID NO: 103 are in each case CR8020, as evidenced by the lack of signal above background in FIG.
Cannot bind to CR8043 and CR9114. It was therefore concluded that these sequences do not present epitopes of these antibodies and that consequently the proteins present on the cell membrane are not folded into their natural three-dimensional structure.

Example 12
Design of Additional Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes of CR8020, CR8043 and CR9114 Based on H3HA In this example, the design of additional polypeptides of the invention based on serotype H3 is described. HA of H3 influenza viruses A / Wisconsin / 67/2005 (SEQ ID NO: 89) and A / Hong Kong / 1/1968 (SEQ ID NO: 121) were used as parental sequences.

  Modification of the first sequence removes the cleavage site in position 345 (numbering refers to SEQ ID NO: 89 by preventing R from forming HA1 and HA2 by mutating R to Q (R345Q) In some cases, residues 347-351 (IFGAI, part of the fusion peptide) can be further deleted to minimize exposure of hydrophobic residues to aqueous solvents. 100% conserved in H3, so this mutation can be applied in all sequences.

  The second modification is to remove the head domain by deleting most of the HA1 sequence and religating the N and C terminal sequences through a short linker. Deletions can vary in length but are spatially close to the last residue of the N-terminal sequence of HA1 and the first residue of the C-terminal sequence to avoid introducing strain through the binding sequence It is preferable. In the H3 sequence, deletions can be introduced in S62-P322 (mini2; SEQ ID NO: 105), S63-P305 (mini3; SEQ ID NO: 119) and T64-T317 (mini4; SEQ ID NO: 120 (corresponding position). Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle, etc. The remaining portions of the sequences can be directly combined, Alternatively, a flexible linker can be introduced, the linker sequence can be from 1 to 50 amino acids long, flexible linkers of limited length (10 amino acids or less), eg GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG Or an analogue is preferred. The length is increased, for example, by reducing the number of residues in the deletion by starting the deletion at positions 63, 64, 65, 66, 67 (or its equivalent) or by increasing the length of the deletion Thus, it can be changed by cutting at positions 57, 58, 59, 60 or 61. Similarly, the last amino acid to be deleted is at (the equivalent of) positions 317, 318, 319, 320 or 321. Or at position 323, 324, 325, 326, or 327 (or equivalent) to increase the length of the deletion. It is important to understand that it can be partially compensated by matching, i.e. larger deletions can be matched with longer linkers and vice versa. It is. These polypeptides are also encompassed by the present invention.

Deletion of the head domain leaves the B-loop at residues 400-420 currently exposed to aqueous solvents. In H3HA, this loop is highly conserved (see Table 9). The consensus sequence is: 401I (E / G) KTNEKFHQIEKEFSVEGR 421 (SEQ ID NO: 104; numbering refers to SEQ ID NO: 89). Some hydrophobic residues are polar (S, T, N, Q) to increase the solubility of this loop and destabilize the post-fusion conformation for the polypeptide of the present invention in a pre-fusion conformation. Must be modified to charged amino acids (R, H, K, D, E), or flexibility must be increased by mutation to G. Specifically, mutations at positions 401, 408, 411, 415, 418 (numbering refers to SEQ ID NO: 89) contribute to the stability of the polypeptides of the invention.

  For F408 and F415, mutations to S are preferred, but other polar (T, N, Q), charged (R, H, K, D, E) and highly flexible amino acids (G) have the same effect Have For position 411 (I), mutations to T are preferred; other polar (S, N, Q), charged (R, H, K, D, E) and highly flexible amino acids (G) are identical This has an effect and is therefore included in the present invention. For position 418 (V), a mutation to G is preferred. Other polarities (S, T, N, Q), charges (R, H, K, D, E) have the same effect and are therefore included in the present invention. For position 401 (I), mutations to R are preferred, but other polar (S, T, N, Q), charged (H, K, D, E) or flexible (G) residues are possible. . Accordingly, the polypeptides of the present invention contain at least one of the above mutations. For example, combinations of two or more mutations are possible as shown in SEQ ID NOs: 123-127 and 129-131.

  In order to stabilize the pre-fusion conformation of the polypeptide of the present invention, a covalent bond is introduced between two portions that are distal in the primary sequence but close in the folded pre-fusion conformation. For this purpose, disulfide bridges are genetically engineered in the polypeptides of the invention, preferably between positions 326 and 438 (equivalent) in H3A / Wisconsin / 67/2005 (SEQ ID NO: 89). Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using suitable algorithms such as Clustal, Muscle, etc. Engineered disulfide bridges mutate at least one (if the other is already cysteine), but usually two spatially close residues to cysteines, either spontaneously or by active oxidation Created by forming a covalent bond between sulfur atoms. Alternative cysteine bridges can be created by mutation of these residues to cysteine between positions 334 and 393 (equivalent) in H3A / Wisconsin / 67/2005 (SEQ ID NO: 89). In some cases, the cysteine at position 321 (or its equivalent) is modified to glycine to avoid the formation of unwanted disulfide bridges.

  Native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus the enhanced interaction between monomers in the truncated molecule increases stability. In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced. A consensus sequence for the formation of a trimer coiled coil, IEAIEKKIEAEIEKIIEIEKK, is introduced at positions 421-441 (or its equivalent). To avoid interfering with the formation of disulfide bridges at positions 326-438, an alternative shorter sequence IEAIEKKIEAEEKKI was also used at (equivalent) at positions 421-435. An alternative is to introduce the sequence RMKQIEDKIEIESKQKKIEN derived from GCN4 and known to trimerize at positions 421 to 441 or the shorter sequence RMKQIEDKIEEEIESK at positions 421 to 435.

The polypeptides of the present invention may contain the intracellular sequence of HA and the transmembrane domain such that the resulting polypeptide is presented on the cell surface upon expression in the cell. In other embodiments, the cytoplasmic and transmembrane sequences from position 522 (or its equivalent) to the C-terminus have been removed so that a secreted (soluble) polypeptide is produced after expression in the cell. Optionally, some additional residues can be included in the soluble protein by deleting sequences from (corresponding to) 523, 524, 525, 526, 527, 528 or 529. The soluble polypeptide is further stabilized by introducing a sequence known to form a trimeric structure, ie, AYVRKDGEWVLL (SEQ ID NO: 143) ("foldon" sequence), optionally linked via a linker. be able to. The linker may optionally contain a cleavage site for later processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a short linker, eg, a tag sequence linked via EGR, eg, a his tag (HHHHHHH) can be added. In some embodiments, the linker and his tag sequence are added without the presence of a foldon sequence.

  An important residue of HA is position 345 (Arg). This is because this residue is the position where protease cleavage occurs to allow protein fusion. This Arg to Gln mutation (R345Q) prevents cleavage from occurring, thereby immobilizing the protein in the pre-fusion state.

The above mutations were clustered as follows:
Cluster9 F408S, I411T, F415S
Cluster10 V418G
Cluster11 I401R
Cluster12 K326C, S438C
Cluster13 T334C, I393C
Cluster14 C321G
RMKQIEDKIEEESKQKKIEN in GCN4 421 to 441
Or RMKQIEEDKIEEIESK in positions 421 to 435
IEAIEKKIEAEEKKIIEIEKK in the 441th position from tri 421
Or IEAIEKKIEAEEKKI at positions 421 to 435

Using the full-length HA sequence from H3N2A / Wisconsin / 67/2005 as a starting point, the above cluster was combined with the S62-P322 deletion (mini2; SEQ ID NO: 105) to obtain the polypeptide of the invention.
SEQ ID NO: 105: H3-mini2
SEQ ID NO: 106: H3-mini2-cl9 + 10
SEQ ID NO: 107: H3-mini2-cl9 + 11
Sequence number 108: H3-mini2-cl9 + 10 + 11
SEQ ID NO: 109: H3-mini2-cl9 + 10 + 11-tri (tri sequence at positions 421 to 441)
SEQ ID NO: 110: H3-mini2-cl9 + 10 + 11-GCN4 (GCN4 sequence at positions 421 to 441)
Sequence number 111: H3-mini2-cl9 + 10 + 11 + 12
SEQ ID NO: 112: H3-mini2-cl9 + 10 + 12
SEQ ID NO: 113: H3-mini2-cl9 + 10 + 11 + 12-GCN4 (short GCN4 sequence at positions 421 to 435)
SEQ ID NO: 114: H3-mini2-cl9 + 10 + 11 + 12-tri (short tri sequence at positions 421 to 435)
SEQ ID NO: 115: H3-mini2-cl9 + 13
Sequence number 116: H3-mini2-cl9 + 10 + 11 + 13
SEQ ID NO: 117: H3-mini2-cl9 + 10 + 11 + 13-GCN4 (GCN4 sequence at positions 421 to 441)
SEQ ID NO: 118: H3-mini2-cl9 + 10 + 11 + 13-tri (tri sequence at positions 421 to 441)

In addition, deletions S63-P305 (mini3) and T64-T317 (mini4) were combined with clusters 9, 10, 11 and 14 to create polypeptides of the invention.
SEQ ID NO: 119: H3-mini3-cl9 + 10 + 11 + 12 + 14
Sequence number 120: H3-mini4-cl9 + 10 + 11 + 12 + 14

Using the sequence of full length HA from H3N2A / HongKong / 1/1968 as a starting point, the above cluster was combined with the S62-P322 deletion to obtain the polypeptide of the invention.
SEQ ID NO: 121: H3 Full length A / Hong Kong / 1/1968 SEQ ID NO: 122: HK68H3m2-cl9
SEQ ID NO: 123: HK68H3m2-cl9 + 10
Sequence number 124: HK68H3m2-cl9 + 10 + 11
SEQ ID NO: 125: HK68H3m2-cl9 + 10 + 12
SEQ ID NO: 126: HK68H3m2-cl9 + 10 + 11 + 12
SEQ ID NO: 127: HK68H3m2-cl9 + 10 + 11 + 13
SEQ ID NO: 128: HK68H3m2-cl9 + 10 + 11 + 12-tri (short tri sequence at positions 421 to 435)
SEQ ID NO: 129: HK68H3m2-cl9 + 10 + 11 + 13-tri (tri sequence at positions 421 to 441)
SEQ ID NO: 130: HK68H3m2-cl9 + 10 + 11 + 12-GCN4 (short GCN4 sequence at positions 421 to 435)
SEQ ID NO: 131: HK68H3m2-cl9 + 10 + 11 + 13-GCN4 (the GCN4 sequence at positions 421 to 441).

  In general, the genes encoding the above protein sequences were synthesized using methods known to those skilled in the art and cloned into the expression vector pcDNA2004. For comparison reasons, full length HA sequences of H3A / Wisconsin / 67/2005, and / or H3A / HongKong / 1/1968 were included in the experiment.

The expression vector (1 μg / ml) was transfected into HEK293F (Invitrogen) suspension cells (10 ⁶ cells / ml, 30 ml) using 40 μl 293transfectin as a transfection agent and allowed to grow for another 2 days. Cells were harvested and aliquoted (0.3 ml, approximately 3 ^* 10 ⁵ cells), and aliquots were raised against polyclonal serum generated against H3HA (Protein Sciences Corp, Meriden, CT, USA) for expression or Treated with either HA-specific monoclonal antibody (5 microgram / ml) and secondary antibody used for staining. Cells were then analyzed by fluorescence-coupled cell sorting (FACS) for expression of the membrane-attached HA stem domain polypeptide of the invention using polyclonal sera raised against H3HA or H1HA to examine expression. A panel of monoclonal antibodies of known specificity that binds to full-length proteins (CR8020, CR8043 and CR9114) is used to examine the presence of conserved epitopes and the correct folding of full-length HA and the mini-HA polypeptides of the invention by inference It was. Monoclonal antibodies CR6261 (known not to bind to H3HA) and CR8057 (binding to the head domain of HA from A / Wisconsin / 67/2005) were also included in the experiment. The results are expressed as a percentage of positive cells, and the A / Wisconsin / 67/2005 H3HA base sequence is shown in FIG. 9 and the A / HongKong / 1/1968 H3HA base sequence is shown in FIG.

The results show that all A / Wisconsin / 67/2005 base constructs (FIG. 9) are expressed on the cell surface. This is because the reaction with H3 polyclonal serum results in more than about 80% of all analyzed cells being positive compared to less than 5% for untransfected cells. All control experiments in the absence of IgG using only labeled anti-human or anti-rabbit IgG are negative. A / Wisconsin / 67/2005 full-length HA is known to be able to bind to this protein. Monoclonal antibodies CR8020, CR8043, CR8057 (binding only to some H3 strains, described in WO2010 / 130636) And CR 9114 but not by mAb CR6261. In contrast, most of the stem domain polypeptides are not recognized by CR8020, CR8043, or CR9114, with some obvious exceptions. Polypeptides containing the cluster12 mutation were recognized by CR8020 and / or CR8043. H3-mini2-cl9 + 10 + 11 + 12 (SEQ ID NO: 111), H3-mini2-cl9 + 10 + 12 (SEQ ID NO: 112), H3-mini2-cl9 + 10 + 11 + 12-GCN4 (SEQ ID NO: 113) and H3-mini2-cl9 + 10 + 11 + 12-tri (SEQ ID NO: 114) are CR8020 (Percentage of positive cells in the range of about 10-60%) and recognition by CR8043 (40-70%) (indicated by arrows). Of the four positive constructs, H3-mini2-cl9 + 10 + 11 + 12-GCN4 (SEQ ID NO: 113) shows the maximum response in this assay. Identical results are obtained from the average fluorescence intensity shown in panel B (Figure 9). H3-mini2-cl9 + 10 + 11 + 12 (SEQ ID NO: 111), H3-mini2-cl9 + 10 + 12 (SEQ ID NO: 112), H3-mini2-cl9 + 10 + 11 + 12-GCN4 (SEQ ID NO: 113) and H3-mini2-cl9 + 10 + 11 + 12-tri (SEQ ID NO: 114) are CR8020 And average fluorescence intensity well above background after exposure and staining with CR8043, with a maximum response for H3-mini2-cl9 + 10 + 11 + 12-GCN4 (SEQ ID NO: 113). None of the HA-based polypeptides from A / Wisconsin / 67/2005 can recognize CR9114.

  FIG. 10 shows that all A / Hong Kong / 1/1968 base constructs (FIG. 10) are expressed on the cell surface. This is because the reaction with H3 polyclonal serum for most constructs results in about 40-60% of all analyzed cells being positive compared to less than 5% for untransfected cells. is there. All control experiments in the absence of IgG using only labeled anti-human or anti-rabbit IgG are negative. Although the percentage of positive cells for the full-length protein from A / HongKong / 1/1968 after treatment with polyclonal serum is low (approximately 10%), the strong signal resulting from the binding of CR8020, CR8043 and CR9114 indicates that the protein is It is present on the surface. CR8057 does not recognize the A / HongKong / 1/1968 base sequence, only the full-length protein from A / Wisconsin / 67/2005. 4 constructs (containing the cluster12 mutation): HK68 H3m2-cl9 + 10 + 12 (SEQ ID NO: 125), HK68H3m2-cl9 + 10 + 11 + 12 (SEQ ID NO: 126), HK68H3m2-cl9 + 10 + 11 + 12-tri (sequence containing truncated tri sequences at positions 421-435) No. 128) and HK68H3m2-cl9 + 10 + 11 + 12-GCN4 (SEQ ID NO: 130 containing a short GCN4 sequence at positions 421-435), CR8020 and Recognized by CR8043. Maximum signal (MFI) is obtained for HK68H3m2-cl9 + 10 + 11 + 12-GCN4 (SEQ ID NO: 130); this stem domain polypeptide construct also shows detectable binding to CR9114.

  In summary, the inventors have shown that the stem domain polypeptides of the present invention can be obtained for group 2 serotypes, particularly H3 subtype influenza A virus, according to the method described above.

Example 13
Design, Expression and Partial Purification of Soluble Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes In certain embodiments, the polypeptides of the invention are such that the resulting polypeptide is presented on the cell surface upon expression in the cell. Contains the intracellular sequence of HA and the transmembrane domain. In other embodiments, the cytoplasmic and transmembrane sequences at C-terminal from position 523, 524, 525, 526, 527, 528, 529 or 530 (numbered according to SEQ ID NO: 1) (equivalent) of HA2 Expression in is removed, for example, to result in a secreted (soluble) polypeptide that can be used in a vaccine. A soluble polypeptide is introduced by linking a sequence known to form a trimeric structure (also known as “foldon”), ie, AYVRKDGEWVLL (SEQ ID NO: 143), optionally via a linker (eg, GSGYIPEAPRDGQAYVRKDGEWVLLSTFL). By doing so, it can be further stabilized. The linker may optionally contain a cleavage site for processing after purification according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a short linker, eg, a tag sequence linked via EGR, eg, a histidine tag (6 or 7 consecutive histidines) can be added. In some embodiments, the linker and histidine tag are added without the presence of a foldon sequence.

  According to the present invention, the amino acid sequence of the C-terminal amino acid is removed from position 530 of the HA2 domain of the full-length HA from H1N1A / Brisbane / 59/2007 (numbering according to SEQ ID NO: 1) and includes a short linker and a histidine tag Was replaced by the sequence EGRHHHHHHH (SEQ ID NO: 81). This exchange is performed as SEQ ID NO: 44: H1-mini2-cluster1 + 5 + 6-trim (resulting in SEQ ID NO: 144: s-H1-mini2-cluster1 + 5 + 6-trim), SEQ ID NO: 45: H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145: s -Yields H1-mini2-cluster1 + 5 + 6-GCN4), SEQ ID NO: 46: mini2-cluster1 + 5 + 6 (A / Brisbane / 59/2007) (sequence number 146: yields s-H1-mini2-cluster1 + 5 + 6), SEQ ID NO: 47: mini2- cluster11 + 5 + 6 (A / Brisbane / 59/2007) (resulting in SEQ ID NO: 147: s-H1-mini2-cluster11 + 5 + 6), SEQ ID NO: 48: mini2-cluster1 + 5 (A / Brisbane / 59/2007): was applied to (SEQ ID NO: 148 s-H1-mini2-cluster1 + 5 the results).

  Similarly, for comparison reasons, the exchange was applied to SEQ ID NO: 1 H1 Full length (A / Brisbane / 59/2007), and arginine 343 was modified to glutamine (R343Q mutation) to obtain SEQ ID NO: 149: s -The HA cleavage site was damaged by generating H1 Full length R343Q. In addition, two polypeptides of the invention were generated using different linkers between the N-terminal and C-terminal portions of HA1: s-H1-mini2-cluster1 + 5 + 6-nl (SEQ ID NO: 150) and s-H1-mini2- cluster1 + 5 + 6-nl2 (SEQ ID NO: 151).

In general, genes encoding the above protein sequences were synthesized using methods known to those skilled in the art and cloned into the expression vector pcDNA2004neo. Expression vectors were transfected into HEK293F (Invitrogen) suspension cells using \ 293transfectin as a transfection agent according to protocols well known in the art and allowed to grow for an additional 7 days. Cells were separated from the culture medium by centrifugation and discarded, while the supernatant containing the soluble polypeptide of the invention was collected for further processing. The supernatant was purified by immobilized metal affinity chromatography on a Ni-NTA column to bind the His-tagged polypeptide of the present invention to the resin and collect the flow-through. Columns are 3-10 column volumes of 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 10 mM imidazole (“wash solution”), 5-15 column volumes of 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 100 mM. Of imidazole (“stringent wash”) and eluted with 20 mM sodium phosphate pH 7.4, 500 mM NaCl, 500 mM imidazole. In each case, the buffer composition or volume used was adapted to increase purity or yield, or a linear gradient was used instead of a step gradient. Fractions were fully collected and analyzed on SDS-PAGE and Western blot using polyclonal anti-H1HA serum for detection (see FIGS. 11a-h). The results show a clear concentration of the polypeptide of the invention in the eluate compared to the starting material.

  In order to confirm proper folding and functionality of the purified polypeptides of the invention, the preparation was tested for the binding of monoclonal antibody CR9114. For this purpose, a monoclonal antibody capable of binding to a His tag (6 or 7 consecutive histidines) at the C-terminus of the protein is placed on a standard 96-well plate and 100 microliters of a 1 μg / ml antibody solution is placed in each well. Coated by applying and incubating overnight at 4 ° C. After removal of excess solution and washing, the plate was blocked with 150 microliters of 2% skim milk solution for 1 hour at room temperature. After removal of the blocking agent and washing, 100 μl of a 1 μg / ml solution of the polypeptide of the present invention and the corresponding full-length protein (SEQ ID NO: 149) ectodomain were added and incubated at room temperature for 2 hours. After removal of excess polypeptide of the invention, mAb CR9114, mAb CR8020 (negative control) or polyclonal sera raised against H1HA (positive control) in rabbits at concentrations varying between 2 and 20 μg / ml Added and incubated for 2 hours at room temperature. Binding was detected via HRP-conjugated anti-human antibody using protocols well known in the art.

  The results (FIG. 12) show that monoclonal antibody CR9114 is bound to the purified soluble polypeptide and full-length ectodomain of the present invention (FIG. 12a), whereas monoclonal antibody CR8020 is not bound (FIG. 12b). Polyclonal anti-H1 serum also binds very similar to the polypeptides of the invention and the full-length ectodomain (FIG. 12c). Thus, given that the broadly neutralizing epitope of CR9114 is conserved in the polypeptides of the invention, and considering the discontinuity and conformational nature of this epitope, the stem domain is properly folded and the natural full-length HA It is concluded that a three-dimensional structure that is equivalent or very similar to the three-dimensional structure is adopted.

  The polypeptide preparations of the present invention were heterogeneous in size as determined from SDS-PAGE and Western blot results. We hypothesized that this variation was due to variations in protein glycosylation patterns between individual protein molecules. To confirm this, a small aliquot of the protein preparation was treated with 3 units of N-glycosidase F (an enzyme that removes N-linked carbohydrate moieties from asparagine residues) at 37 ° C. for 18 hours, SDS-PAGE and Western blot. Was analyzed. The results (FIGS. 13a and 13b) show that treatment with N-glycosidase collects the diffusion band of the polypeptide of the invention into a single band at the predicted molecular weight calculated from the amino acid sequence. This is clear evidence that the observed size heterogeneity actually results from variations in glycosylation patterns.

The polypeptide preparations of the invention were further characterized by HP-SEC. To this end, about 40 μg of the polypeptide of the invention is added to a Tosoh TSK coupled to a multi-angle light scattering detector in a volume of 43-63 μl (0.64-0.93 mg / ml polypeptide concentration). -Applied to a gel G2000 SWxl column. The results are shown in FIG. The main peak (residence time about 8 minutes) arises from the polypeptide of the present invention and is well separated from larger species, indicating that further purification can be achieved. Based on multi-angle light scattering detector data, the main peak corresponds to a molecular species having a molecular weight of 50-80 kilodaltons depending on the polypeptide of the invention being tested (see Table 9). In light of the size heterogeneity and variations of the above polypeptide glycosylation and the dependence of the results on the hydrodynamic shape of the molecule, these numbers should be construed as merely representations.

Example 14
Expression and Partial Purification of Soluble Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes of Monoclonal Antibodies CR9114, CR6261 and FI6v3 To obtain a highly pure preparation of the polypeptide of the present invention, HEK293F cells were transfected with s-H1-mini2- The expression vector pcDNA2004 containing the gene encoding cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) was transfected. It will be appreciated by those skilled in the art that leader sequences (or signal sequences) that direct protein transport during production (corresponding to amino acids 1-17 of SEQ ID NO: 145) are not present in the secreted final polypeptide. To this end, HEK293F cells (Invitrogen) are spun down at 300 g for 5 min and resuspended in 300 mL pre-warmed Freestyle ™ medium via SF1000 to give 1.0 ^* 10 ⁶ vc / mL. Sowing. The culture was incubated for 1 hour at 37 ° C., 10% CO ₂ , 110 rpm in a multitron incubator. After 1 hour, plasmid DNA was pipetted in 9.9 mL Optimem medium to a concentration of 1.0 μg / mL in a 300 mL culture volume. In parallel, 440 μL of 293fectin® was pipetted in 9.9 mL of Optimem medium and incubated for 5 minutes at room temperature. After 5 minutes, the plasmid DNA / Optimem mixture was added to the 293fectin® / Optimem mixture and incubated at room temperature for 20 minutes. After incubation, the plasmid DNA / 293fectin® mixture was added dropwise to the cell suspension. Transfected cultures were incubated in a multitron incubator at 37 ° C., 10% CO ₂ and 110 rpm. On day 7, the cells were separated from the culture medium by centrifugation (3000 g for 30 minutes), while the supernatant containing the soluble polypeptide of the invention was filtered on a 0.2 μm bottle top filter for further processing.

  To confirm the presence of the polypeptide of the invention, a small aliquot of the supernatant was analyzed by Western blot using a monoclonal antibody directed against the his tag for detection (FIG. 15a). Several bands are observed at an apparent molecular weight of 37-50 kDa, which approaches or exceeds the calculated molecular weight based on the amino acid composition of the protein. Size heterogeneity is caused by variations in glycosylation patterns. This is because earlier experiments have shown that treating this protein with N-glycosidase F to remove attached N-linked glycans from the protein results in a collection of bands at the expected molecular weight.

The presence of broadly neutralizing epitopes on the polypeptides of the invention was confirmed by ELISA using broadly neutralizing antibodies CR6261, CR9114 and FI6v3 as probes. For comparison reasons, monoclonal antibody CR8020 is also included in the experiment as a negative control; this antibody can bind to HA molecules from group 2 viruses (eg, H3 and H7HA), but HA molecules from group 1 (eg, H1 and H5HA) cannot bind. For this purpose, a monoclonal antibody capable of binding to a His tag (6 or 7 consecutive histidines) at the C-terminus of the protein is placed on a standard 96 well plate with 100 microliters of a 1 μg / ml antibody solution in each well. And coated by incubating overnight at 4 ° C. After removal of excess solution and washing, the plate was blocked with 150 microliters of 2% skim milk solution for 1 hour at room temperature. After removal of the blocking agent and washing, 100 microliters of supernatant was added and incubated for 2 hours at room temperature. After removal of excess polypeptide of the invention, mAb CR9114 was added and added in a 1: 2 dilution series starting at a concentration of 5 μg / ml and incubated for 2 hours at room temperature. Binding was detected via HRP-conjugated anti-human antibody using protocols well known in the art. While clear binding of CR9114, FI6v3 and to a lesser extent CR6261 to the polypeptides of the invention is observed, no response is observed for CR8020, indicating that the observed binding is specific for the test monoclonal antibody. This is shown (FIG. 15b).

  For purification purposes, 250 ml culture supernatant was applied to a 5 ml His trap column, washed with 75 ml wash buffer (20 mM TRIS, 500 mM NaCl, pH 7.8), and stepped gradient imidazole (wash buffer). Elution with 10, 50, 100, 200, 300 and 500 mM in the solution). The chromatogram (Figure 16) shows multiple peaks, and the polypeptide of the invention elutes at 100 mM imidazole (peak A) and 200 mM imidazole (peak B). Both peaks were collected, concentrated and applied to a size exclusion column (Superdex 200) for further purification. The elution profile is shown in FIGS. 17a and 17b. Fractions were collected and analyzed on SDS-PAGE (FIGS. 17c and d). Fraction 3 from both peaks A and B contains the highly pure polypeptide of the present invention. The final yield was about 10 μg / ml culture supernatant. The purified batch is endotoxin free (administered at 5 mg / kg; administered at <1 EU / mg; Chromogenic LAL); bioburden is less than 1 CFU / 50 μg.

Example 15
Design of Stem Domain Polypeptide Containing Conserved Stem Domain Epitope of CR9114 Based on Influenza BHA The above procedure for designing the polypeptide of the present invention was also applied to influenza B. In this example, the HA sequences collected from both known strains, ie, B / Florida / 4/2006 (B / Yamagata strain) and B / Malaysia / 2506/2004 (B / Victoria strain) virus sequences. Based on the polypeptides of the invention are described. Those skilled in the art will appreciate that other influenza BHA sequences can be used. This is especially because the stem region is well conserved. Thus, polypeptides derived from other influenza BHA sequences according to the following are also encompassed by the present invention.

  The first modification of the HA sequence of B / Florida / 4/2006 is 361 (numbering refers to SEQ ID NO: 132) by mutating R (or K in the case of a limited number) to Q (R361Q). ) To prevent the formation of HA1 and HA2 from HA0. Optionally, residues 363 to 367 (GFGAI, part of the fusion peptide) can be further deleted to minimize exposure of hydrophobic residues to aqueous solvents. The positive charge at cleavage is 100% conserved in HA from influenza B, so this mutation can be applied in all sequences.

The second modification is to remove the head domain by deleting most of the HA1 sequence and religating the N and C terminal sequences through a short linker. Deletions can vary in length but are spatially close to the last residue of the N-terminal sequence of HA1 and the first residue of the C-terminal sequence to avoid introducing strain through the binding sequence It is preferable. In the B sequence, a deletion can be introduced at P51 to I336 (m2; SEQ ID NO: 133) (corresponding position) in B / Florida / 4/2006 (SEQ ID NO: 132). Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. The remaining part of the sequence can be attached directly or a flexible linker can be introduced. The linker sequence can be 1-50 amino acids long. Preferred are flexible linkers of limited length (10 amino acids or less) such as GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like.

  SEQ ID NO: 133 describes such a polypeptide of the invention containing the deletions P51 to I332 (m2; SEQ ID NO: 133). The above deletion ensures that the unstructured region formed by residues between P51 and N58 as well as E306 to I337 is also removed; this is beneficial to the overall stability of the polypeptides of the invention. . Similar effects were observed for the polypeptides of the invention derived from the H1 sequence (see above).

  Deletion of the head domain leaves the residue 416-436 loop currently exposed to aqueous solvents. In BHA, this loop is highly conserved (see Table 10). The consensus sequence is: LSELEVKNLQRLSGAMDELHN.

  Some hydrophobic residues are polar (S, T, N, Q) in order to increase the solubility of this loop and destabilize the post-fusion conformation for the polypeptide of the present invention in a pre-fusion conformation. Must be modified to charged amino acids (R, H, K, D, E), or increased flexibility by mutation to G. Specifically, mutations at positions 421, 424, 427, 434 (numbering refers to SEQ ID NO: 132) contribute to the stability of the polypeptides of the invention.

  For V421 and L427, mutations to T are preferred, but other polar (S, N, Q), charged (R, H, K, D, E) and highly flexible amino acids (G) have the same effect. Have. For position 424, a mutation to S is preferred. Other polar (N, T, Q), charged (R, H, K, D, E) and highly flexible amino acids (G) have the same effect. For L434, a mutation to G is preferred. Other polarities (S, T, N, Q) and charges (R, H, K, D, E) have the same effect. A polypeptide containing at least one of the above mutations was made. For example, combinations of two or more mutations are possible as shown in SEQ ID NOs: 134-136.

  In order to stabilize the pre-fusion conformation of the polypeptide of the present invention, a covalent bond was introduced between two parts that are distal in the primary sequence but close in the folded pre-fusion conformation. For this purpose, disulfide bridges are genetically engineered in polypeptides, preferably between K340 and S454 positions in HA from B / Fluorida / 4/2006 (SEQ ID NOs 134-136). Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using suitable algorithms such as Clustal, Muscle, etc. Engineered disulfide bridges mutate at least one (if the other is already cysteine), but usually two spatially close residues to cysteines, either spontaneously or by active oxidation Created by forming a covalent bond between sulfur atoms.

  In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced. A sequence that supports the formation of a trimeric coiled coil derived from GCN4, RRMKQIEDKIEEILSKI, is introduced at positions 436-452 (SEQ ID NO: 135) (or its equivalent), or RMKQIEDKIEEILSKI is introduced at positions 436-451 (SEQ ID NO: 136).

The same procedure was performed on HA from B / Malaysia / 2506/2004 (SEQ ID NO: 137) to provide the polypeptide. H from B / Florida / 4/2006
Compared to A, this HA has an additional asparagine residue inserted at position 178, which can be easily grasped in the alignment of the two sequences. Consequently, a cleavage site is present at position 362 and the corresponding mutation to prevent cleavage is R362Q. In this case, the deletion for removing the head region is, for example, P51 to I337 (m2; SEQ ID NO: 138). Again, the remaining part of the sequence can be directly attached, or a flexible linker can be introduced. The linker sequence can be 1-50 amino acids long. Preferred are flexible linkers of limited length (10 amino acids or less) such as GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like.

  SEQ ID NO: 138 describes such a polypeptide containing the deletions P51 to I332 (m2; SEQ ID NO: 138). The above deletion ensures that the unstructured region formed by residues P51-N58 as well as E307-I338 is also removed; this is beneficial to the overall stability of the polypeptides of the invention. Similar effects were observed for the polypeptides of the invention derived from the H1 sequence (see above).

  Deletion of the head domain leaves the loop of residues L420-H436 currently exposed to aqueous solvents. In BHA, this loop is highly conserved (see Table x). The consensus sequence is: LSELEVKNLQRLSGAMDELHN.

  In order to increase the solubility of this loop in the pre-fusion conformation and destabilize the post-fusion conformation, some hydrophobic residues can be polar (S, T, N, Q), charged amino acids (R, H, It must be modified to K, D, E) or increased by flexibility to G. Specifically, mutations at positions 422, 425, 428, 435 (numbering refers to SEQ ID NO: 137) were tested.

  For positions V422 and L428, mutations to T are preferred, but other polar (S, N, Q), charged (R, H, K, D, E) and highly flexible amino acids (G) have the same effect Have For position 425, a mutation to S is preferred. Other polar (N, T, Q), charged (R, H, K, D, E) and highly flexible amino acids (G) have the same effect. For L435, a mutation to G is preferred. Other polarities (S, T, N, Q) and charges (R, H, K, D, E) have the same effect. A polypeptide containing at least one of the above mutations was made. For example, combinations of two or more mutations are possible as shown in SEQ ID NOs: 139-141.

  In order to stabilize the pre-fusion conformation of the polypeptide of the present invention, a covalent bond is introduced between two portions that are distal in the primary sequence but close in the folded pre-fusion conformation. For this purpose, a disulfide bridge is gened in the polypeptide of the invention, preferably between the K341 and S455 (or equivalent) in HA from B / Malaysia / 2506/2004 (SEQ ID NO: 139-141). Manipulate. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using suitable algorithms such as Clustal, Muscle, etc. Engineered disulfide bridges mutate at least one (if the other is already cysteine), but usually two spatially close residues to cysteines, either spontaneously or by active oxidation Created by forming a covalent bond between sulfur atoms.

As mentioned above, native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus the enhanced interaction between monomers in the truncated molecule increases stability. In the stem domain, trimerization is mediated by the formation of a trimeric coiled-coil motif. By enhancing this motif, a more stable trimer can be produced. A sequence that supports the formation of a trimer coiled coil derived from GCN4, RRMKQIEDKIEEILSKI, is introduced at positions 437-453 (SEQ ID NO: 135) (or equivalent), or RMKQIEDKIEILSKI is introduced at positions 437-452 (SEQ ID NO: 136).

  Influenza B hemagglutinin based polypeptides SEQ ID NOs 133-136 and 138-141 were tested for the presence of an epitope of CR9114 by fluorescence-coupled cell sorting as described above. However, no binding of mAb CR9114 was observed for these constructs.

Example 16
Immunogenicity of 3rd generation HA stem domain polypeptides To assess the immunogenicity of stem domain polypeptides, mice were isolated from A / Brisbane / 59/2007 full length H1 (SEQ ID NO: 1), Mini3-cluster 11 (SEQ ID NO: 11), Mini2-cluster11 + 5 (SEQ ID NO: 14), mini2-cluster1 + 5 (SEQ ID NO: 48), mini2-cluster1 + 5 + 6 (SEQ ID NO: 46), mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) and mini2-cluster1 + 5 + 6-nl (SEQ ID NO: 152) Mice were immunized with an expression vector encoding). An expression vector encoding cM2 was also included as a negative control.

  Groups of 4 mice (BALB \ c) were immunized intramuscularly on days 1, 21, and 42 with 50 μg construct + 50 μg adjuvant (pUMCV1-GM-CSF). On day 49, the final blood collection was performed and serum was collected. Serum was analyzed by Elisa using the recombinant ectodomain of full length HA from A / Brisbane / 59/2007 and A / California / 07/2009 strains (obtained from Protein Sciences Corporation, Meriden, CT, USA) as an antigen. did. Briefly, 96 well plates were coated overnight at 4 ° C. with 50 ng HA and then incubated with block buffer (100 μl PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. The plate is washed with PBS + 0.05% Tween-20 and 100 μl of a 2-fold dilution series in block buffer starting with a 20-fold dilution of serum is added. The bound antibody uses HRP-conjugated goat anti-mouse IgG to detect the bound antibody using standard protocols well established in the art. The mAb 3AH1InA134 (Hytest, Turku, Finland) is used to compare the titer to a standard curve to derive Elisa units / ml (EU / ml).

  The time course of the IgG response to the ectodomain of the homologous full-length protein induced by the above immunization schedule is shown in FIG. A high response can already be observed for mice immunized with DNA encoding the full-length protein (SEQ ID NO: 1) after 4 weeks. The response is increased by boost injection as shown by the increase in titer at 7 weeks. Polypeptides mini2-cluster11 + 5 (SEQ ID NO: 14), mini2-cluster1 + 5 (SEQ ID NO: 48), mini2-cluster1 + 5 + 6 (SEQ ID NO: 46), mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) and mini2-cluster1 + 5 + 6-nl (sequence) Immunization with DNA encoding number 152) results in an intermediate titer that is further increased upon booster immunization, as evidenced by the titer at week 7. Immunization with DNA encoding Mini3-cluster 11 (SEQ ID NO: 11) and negative control cM2 does not result in a detectable response in this assay.

  FIG. 19 shows IgG at 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from the homologous strain H1N1A / Brisbane / 59/2007 (panel A) and the heterologous strain H1N1A / California / 07/2009. Indicates a response. DNA encoding the polypeptide of the present invention mini2-cluster11 + 5 (SEQ ID NO: 14), mini2-cluster1 + 5 (SEQ ID NO: 48), mini2-cluster1 + 5 + 6 (SEQ ID NO: 46), mini2-cluster1 + 5 + 6-GCN4 (polypeptide encoding the polypeptide of the present invention (SEQ ID NO: 14) Antibodies induced by DNA encoding SEQ ID NO: 45) and mini2-cluster1 + 5 + 6-nl (SEQ ID NO: 152) bind equally well to the ectodomain of hemagglutinin from homologous and heterologous strains. In contrast, immunization with DNA encoding the full-length protein (SEQ ID NO: 1) results in high titers against homologous hemagglutinin (the titer observed for immunization with DNA encoding the polypeptide of the invention). But more than an order of magnitude higher) results in lower titers against the heterologous hemagglutinin ectodomain. Immunization with DNA encoding Mini3-cluster 11 (SEQ ID NO: 11) and negative control cM2 does not result in a detectable response to any of the hemagglutinin ectodomains in this assay.

  In summary, the polypeptides of the present invention mini2-cluster11 + 5 (SEQ ID NO: 14), mini2-cluster1 + 5 (SEQ ID NO: 48), mini2-cluster1 + 5 + 6 (SEQ ID NO: 46), mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) and mini2-cluster1 + 5 + 6- The antibody raised against nl (SEQ ID NO: 152) can recognize full-length hemagglutinin. These epitopes are localized on the hemagglutinin stem domain and need to be conserved among the full-length hemagglutinins from H1N1A / Brisbane / 59/2007 and H1N1A / California / 07/2009.

Example 17
Immunogenicity of the third generation HA stem domain polypeptide mini2-cluster1 + 5 + 6-GCN4 To further evaluate the immunogenicity of the stem domain polypeptide of the present invention, the mouse was expressed by an expression vector encoding mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45). Were primed and boosted twice with a purified protein s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) at 3-week intervals. For comparison reasons, separate groups were immunized at intervals of 3 weeks with expression vectors encoding full length H1 (SEQ ID NO: 1) from mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) and A / Brisbane / 59/2007. Immunized 3 times. An expression vector encoding cM2 was also included as a negative control.

Groups of 4 mice (BALB \ c) were treated on day 1 with 1000 μg construct encoding mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) +100 μg adjuvant (pUMCV1-GM-CSF) and 21 and 42 On the day, 10 μg Matrix-M was adjuvanted with s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145; 100 μg purified protein) and immunized intramuscularly (im). One group received the second and third immunizations subcutaneously, while the other group received the second and third immunizations intramuscularly. A third group was further primed on day 1 as described above with 100 μg construct encoding mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) +100 μg adjuvant (pUMCV1-GM-CSF) on days 21 and 41 S-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145; 100 μg of purified protein) was boosted with immunization with Montanide ISA-720 (1: 1 v / v). For comparison, on days 1, 21, and 42, a group of 4 mice (BALB \ c) was transferred to mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45), full length H1 from A / Brisbane / 59/2007 (sequence Number 1) or 100 μg construct encoding cM2 was immunized intramuscularly and adjuvanted with 100 μg adjuvant (pUMCV1-GM-CSF).

  On day 49, the final blood collection was performed and serum was collected. Serum was obtained from recombinant full-length HA (Protein Science Corpus US, Protein Science Corp. US, H1N1A / California / 07/2009, H5N1A / Vietnam / 1203/2004 and H3N2A / HongKong, 1968 strains). Was obtained as an antigen and analyzed by ELISA. Briefly, 96 well plates were coated overnight at 4 ° C. with 50 ng HA and then incubated with block buffer (100 μl PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. The plate is washed with PBS + 0.05% Tween-20 and 100 μl of a 2-fold dilution series in block buffer starting with a 20-fold dilution of serum is added. The bound antibody uses HRP-conjugated goat anti-mouse IgG to detect the bound antibody using standard protocols well established in the art. Titers are compared to a standard curve composed of serial dilutions of mouse monoclonal antibodies that bind to the HA antigen and expressed as ELISA units per ml (EU / ml). FIG. 20 shows the homologous strain H1N1A / Brisbane / 59/2007 (panel A), heterologous strain H1N1A / California / 07/2009 (panel B) heterologous subtype strain H5N1A / Vietnam / 1203/2004 (panel C) and heterologous subtype Shown is the IgG response 7 weeks after the first immunization for individual mice against the ectodomain of full-length hemagglutinin from strain H3N2A / HongKong / 1/1968 (panel D). Antibodies induced by immunization with DNA encoding the polypeptide mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) of the present invention are H1N1A / Brisbane / 59/2007, H1N1A / California / 07/2009 and to a lesser extent H5N1A / Vietnam / 1203/2004 HA can be recognized. Antibodies elicited by immunization with DNA encoding full-length H1 (SEQ ID NO: 1) from A / Brisbane / 59/2007 recognize the homologous protein very well, but with lower titers in FIGS. 17B and C As evidenced, the heterologous HA from H1N1A / California / 07/2009 and the heterogeneous subtype HA from H5N1A / Vietnam / 1203/2004 are less recognized. A group of mice immunized with DNA encoding mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45; prime) and then booster immunized with s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) protein are homologous H1N1A It shows high titers against ectodomains of HA derived from / Brisbane / 59/2007, heterologous H1N1A / California / 07/2009 and heterosubtype H5N1A / Vietnam / 1203/2004.

FIG. 20D shows the IgG response at week 7 against the ectodomain of HA from H3N2A / HongKong / 1/1968. Unlike mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) and s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) derived from HA of strain H1N1A / Brisbane / 59/2007 belonging to influenza group 1, H3N2A / HongKong / 1/1968 belongs to influenza group 2 and is therefore phylogenetically far from the parent sequence used to design the polypeptides of the invention used in this experiment. Three immunizations with DNA encoding mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45) or full-length HA (SEQ ID NO: 1) from H1N1A / Brisbane / 59/2007 resulted in IgG levels detectable by ELISA against this antigen. Will not bring. In contrast, immunization with DNA encoding mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45), followed by two booster immunizations with the purified protein s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) results in H3N2A High potency against HA from / HongKong / 1/1968. This result shows that both the immunization route used (ie intramuscular and subcutaneous) is added to the protein boost immunization (Matrix-M or Montanide).
Obtained independently of ISA-720).

  In summary, immunization with a polypeptide of the invention results in IgG capable of recognizing HA from a wide range of influenza strains, eg, homologous, heterologous, and heterologous subtype strains from influenza group 1 and strains from influenza group 2. Can trigger. In contrast, immunization with full-length HA results in high titers of homologous strains against HA, reduced titers against heterologous and heterologous subtype strains, and IgG levels below the detection limit for strains from influenza group 2.

Example 18
Design of Additional Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes of CR6261 and CR9114 Polypeptides of the present invention designed according to the above procedure can be further modified to increase stability. Such modifications can be introduced to improve the formation of trimeric forms of the polypeptides of the invention as compared to monomeric and / or dimeric species. As mentioned above, native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that hold the trimer together are located in the head domain. Thus, after removal of the head, the tertiary structure is destabilized, and thus enhanced interaction between the monomers in the truncated molecule increases the stability of the trimeric form. In the stem domain, trimerization is mediated by the formation of trimers. By enhancing the coiled-coil motif in the stem domain, a more stable trimeric form can be achieved.

  According to the present invention, the consensus sequence IEAIEKKIEAEEKKIE (SEQ ID NO: 83) for the formation of a trimeric coiled coil is present in the polypeptide of the present invention at positions 418-433 in H1A / Brisbane / 59/2007 (SEQ ID NO: 44). ) (Numbering by SEQ ID NO: 1) (equivalent). Alternatively, IEAIEKKIEAEEKKI (SEQ ID NO: 85) can be introduced at 419-433 (SEQ ID NO: 49), or IEAIEKKIEAEIEKK (SEQ ID NO: 86) can be introduced at 420-433 (SEQ ID NO: 50). An alternative is to introduce the sequence MKQIEDKIEIESKQ (SEQ ID NO: 84), derived from GCN4 and known to trimerize, at positions 419-433 (SEQ ID NO: 45). Alternatively, MKQIEDKIEIESK (SEQ ID NO: 87) can be introduced at positions 420-433 (SEQ ID NO: 51), or RMKQIEDKIEIESKQK (SEQ ID NO: 88) can be introduced at positions 417-433 (SEQ ID NO: 52). Similarly, the trimer interface can be enhanced by modifying M420, L423, V427, G430 to isoleucine (SEQ ID NO: 53).

  In certain embodiments, the polypeptides of the invention contain an intracellular sequence of H1HA and a transmembrane domain. In other embodiments, the C2 cytoplasmic sequence of HA2 from position 523, 524, 525, 526, 526, 527, 528, 529, or 530 (numbered by SEQ ID NO: 1) (or their equivalent) of HA2. And the transmembrane sequence is removed so that a secreted (soluble) polypeptide is produced. The soluble polypeptide can be further stabilized as described above.

Description of linker variants Generally, the genes encoding the above protein sequences (SEQ ID NO: 44-46; SEQ ID NO: 49-53 and SEQ ID NO: 152-157) were synthesized using methods known to those skilled in the art and expressed in the expression vector pcDNA2004. For comparison reasons, an expression vector encoding the full-length sequence (SEQ ID NO: 1) and cM2 was included in the experiment.

HEK293F (Invitrogen) suspension cells (10 ⁶ cells / ml, 30 ml) were transfected with the expression vector (1 μg / ml) using 40 μl of 293-transfectin as a transfection agent and grown for another 2 days. . Cells were harvested; aliquoted (0.3 ml, approximately 3 ^* 10 ⁵ cells) and aliquots were raised against polyclonal serum or HA-specific monoclonal antibody (5 microgram / ml) against H1HA to examine expression. ) And the secondary antibody used for staining. Cells were then analyzed by fluorescence-coupled cell sorting (FACS) for expression of the membrane-attached HA stem domain polypeptide of the invention using polyclonal sera raised against H1HA to examine expression. Presence of conserved epitopes using a panel of monoclonal antibodies of known specificity that bind to full-length proteins (CR6261, CR9114, CR9020 and CR8020), as well as precise folding of full-length HA and the mini-HA polypeptides of the invention by inference I investigated. The results are expressed as percentage of positive cells and average fluorescence intensity and are shown in FIG.

  The results show that all test variants are expressed on the cell surface as evidenced by a positive response from polyclonal anti-H1 serum. The H3HA specific antibody CR8020 does not recognize any of the constructs included in the experiment, whereas CR9020 that binds to the head domain of H1HA clearly recognizes only the full-length protein. All polypeptides of the present invention, and full-length proteins, are recognized by CR6261 and CR9114, indicating that the corresponding epitope is present in the polypeptides of the present invention in the same conformation as the wild-type protein. Of the polypeptides of the invention having additional trimer motifs contained in the helix CD (see FIG. 1), SEQ ID NOs: 45, 51 and 52 containing the GCN4 derived sequences SEQ ID NOs: 84, 87 and 88 are: Give a response (MFI) equal to or better than SEQ ID NOs: 44, 49 and 50, which contain the consensus trimer sequences of SEQ ID NOs: 83, 85 and 86, respectively.

  Variations in the composition of the linker connecting amino acids 52 and 321 (numbering refers to SEQ ID NO: 1) in the polypeptides of the invention do not result in significant changes in the recognition of monoclonal antibodies CR6261 and CR9114. The maximum change is observed when GGGG in SEQ ID NO: 46 is replaced by HNGK (resulting in SEQ ID NO: 152), which results in a somewhat lower response to CR6261, but does not affect the response to CR9114. Removal of the linker and introduction of amino acids 53 to 56 (SHNG) of SEQ ID NO: 1, i.e. the linker is SEQ ID NO: 46 (resulting in SEQ ID NO: 153), SEQ ID NO: 45 (resulting in SEQ ID NO: 154) or SEQ ID NO: 50 (SEQ ID NO: The production of the polypeptide of the present invention that does not have (in 155) does not affect the response in the FACS assay, indicating that the linker sequence is not critical.

  SEQ ID NO: 156 is derived from SEQ ID NO: 46 by introducing mutations I337N, I340N and F352Y, while SEQ ID NO: 157 contains an additional mutation at position 353, namely I353N. These mutations do not result in an improved response to CR6261 and CR9114 in the FACS assay shown in FIG.

In certain embodiments, the polypeptides of the invention contain an intracellular sequence of HA and a transmembrane domain. In other embodiments, HA2 523, 524, 525, 526, 5
Removing the cytoplasmic and transmembrane sequences at the C-terminus of HA2 from positions 26, 527, 528, 529, or 530 (numbered by SEQ ID NO: 1) (or their equivalents) to form a trimeric structure Is optionally replaced by introducing a known sequence, ie, AYVRKDGEVVLL (SEQ ID NO: 143), optionally linked via a linker. The linker may optionally contain a cleavage site for later processing according to protocols well known to those skilled in the art. To facilitate purification of the soluble form, a short linker, eg, a tag sequence linked via EGR, eg, His tag HHHHHHH, can be added. In accordance with the present invention, the amino acid sequence was replaced by SEQ ID NO: 81 or SEQ ID NO: 82 by removing the amino acid sequence of the C-terminal amino acid from position 530 of the HA2 domain (numbered by SEQ ID NO: 1).

Example 19
Immunogenicity of third generation HA stem domain polypeptides To evaluate the immunogenicity of stem domain polypeptides, the full length H1 from A / Brisbane / 59/2007 (SEQ ID NO: 1), Mini3-cluster11 (SEQ ID NO: 11) Mini2-cluster 11 + 5 (SEQ ID NO: 14), mini2-cluster 1 + 5 + 6 (SEQ ID NO: 46), mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45), mini2-cluster1 + 5 + 6-nl (SEQ ID NO: 152), mini2-cluster1 + 5 + 6-nl2 (SEQ ID NO: 15) ), Mini2-cluster1 + 5 + 6-nl2s-GCN4 (SEQ ID NO: 154), mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 51), mini2- Mice were immunized with expression vectors encoding cluster1 + 5 + 6-GCN4t3 (SEQ ID NO: 52), mini2-cluster1 + 5 + 6 + 12 (SEQ ID NO: 156) and mini2-cluster1 + 5 + 6 + 12 + 13 (SEQ ID NO: 157). An expression vector encoding cM2 was also included as a negative control.

Groups of 4 mice (BALB \ c) were immunized intramuscularly at days 1, 21, and 42 with 100 μg construct + 100 μg adjuvant (pUMCV1-GM-CSF). On day 49, the final blood collection was performed and serum was collected. Serum was obtained from recombinant full-length HA (Protein
Sciences Corporation, obtained from Meriden, CT, USA) was used as an antigen and analyzed by ELISA. Briefly, 96 well plates were coated overnight at 4 ° C. with 50 ng HA and then incubated with block buffer (100 μl PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. The plate is washed with PBS + 0.05% Tween-20 and 100 μl of a 2-fold dilution series in block buffer starting with a 50-fold dilution of serum is added. Bound antibody is detected using standard protocols well established in the art using HRP-conjugated goat anti-mouse IgG. Titers are compared to a standard curve composed of serial dilutions of mouse monoclonal antibodies that bind to the HA antigen and expressed as ELISA units per ml (EU / ml).

FIG. 22 shows the full length from the homologous strain H1N1A / Brisbane / 59/2007 (panel A), heterologous strain H1N1A / California / 07/2009 (panel B) and heterologous subtype strain H5N1A / Vietnam / 1203/2004 (panel C). Shown is the IgG response 7 weeks after the first immunization for individual mice to hemagglutinin. The polypeptides Mini2-cluster11 + 5 (SEQ ID NO: 14), mini2-cluster1 + 5 + 6 (SEQ ID NO: 46), mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45), mini2-cluster1 + 5 + 6-nl (SEQ ID NO: 152), mini2-cluster1 + 5 + 6-n2 of the present invention (SEQ ID NO: 153), mini
2-cluster1 + 5 + 6-nl2s-GCN4 (SEQ ID NO: 154), mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 51), mini2-cluster1 + 5 + 6-GCN4t3 (SEQ ID NO: 52), mini2-cluster1 + 5 + 6 + 12 (SEQ ID NO: 15 + 6lu) and 12 + 13lu The antibody induced by immunization with DNA encoding 157) binds equally well to the ectodomain of hemagglutinin from homologous H1N1A / Brisbane / 59/2007 and heterologous H1N1A / California / 07/2009 strains (FIG. 22A). And B). Maximum titers are observed for mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45), mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 51) and mini2-cluster1 + 5 + 6-nl2s-GCN4 (SEQ ID NO: 154). In contrast, immunization with DNA encoding the full-length protein (SEQ ID NO: 1) results in high titers against homologous hemagglutinin (the titer observed for immunization with DNA encoding the polypeptide of the invention). Is more than an order of magnitude higher), but results in lower titers (greater than an order of magnitude) against the heterologous hemagglutinin ectodomain. Immunization with DNA encoding Mini3-cluster11 (SEQ ID NO: 11) and negative control cM2 does not produce a detectable response to any of the hemagglutinin ectodomains in this assay.

  The titer against the ectodomain of heterologous subtype hemagglutinin from H5N1A / Vietnam / 1203/2004 (FIG. 22C) shows a clear response for mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 51). The observable titers are also shown for 2 out of 4 mice after immunization with DNA encoding mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45), and mini2-cluster1 + 5 + 6-nl (SEQ ID NO: 152), mini2 -Cluster1 + 5 + 6-nl2 (SEQ ID NO: 153), mini2-cluster1 + 5 + 6-nl2s-GCN4 (SEQ ID NO: 154) are obtained for 1 of 4 mice. Surprisingly, we also find detectable titers after immunization with DNA encoding Mini3-cluster 11 (SEQ ID NO: 11) and Mini2-cluster 11 + 5 (SEQ ID NO: 14). The first construct did not induce any detectable antibody titer against homologous and heterologous H1HA, while the last induced only a mild response (FIGS. 22A and B). Comparison of the sequence and H5HA of all constructs in this experiment shows a putative linear epitope located at the membrane distal end of the long CD helix (see FIG. 1). The mutation of methionine to isoleucine at position 175 in SEQ ID NO: 11 and position 156 in SEQ ID NO: 14 is the linear sequence ERRRIENLNKK (positions 172 to 181 in SEQ ID NO: 11; positions 153 to 162 in SEQ ID NO: 14) Bring. This sequence is also present in HA from H5N1A / Vietnam / 1203/2004, but not in HA from H1N1A / Brisbane / 59/2007 and H1N1A / California / 07/2009, where the corresponding sequence Are ERRMENLNKK and EKRIENLNKK, respectively.

In summary, the polypeptides of the present invention mini2-cluster11 + 5 (SEQ ID NO: 14), mini2-cluster1 + 5 + 6 (SEQ ID NO: 46), mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45), mini2-cluster1 + 5 + 6-nl (SEQ ID NO: 152), mini2- cluster1 + 5 + 6-nl2 (SEQ ID NO: 153), mini2-cluster1 + 5 + 6-nl2s-GCN4 (SEQ ID NO: 154), mini2-cluster1 + 5 + 6-GCN4t3 (SEQ ID NO: 52), mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 5+), mini2-cluster1 + 5 + 6-GCN4t1 (2 ++) 156) and mini2-cluster1 + 5 + 6 + 12 + 13 (SEQ ID NO: 1) Antibodies raised against 7) may recognize the full length hemagglutinin. The epitopes of these antibodies should be localized on the hemagglutinin stem domain and are conserved between full length hemagglutinins from H1N1A / Brisbane / 59/2007 and H1N1A / California / 07/2009. mini2-cluster1 + 5 + 6-GCN4t2 (SEQ ID NO: 51), and to a lesser extent, mini2-cluster11 + 5 (SEQ ID NO: 14), mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 45), mini2-cluster1 + 5 + 6-nl (SEQ ID NO: 152), mini2-cluster6 + Antibodies elicited through immunization with DNA encoding -nl2 (SEQ ID NO: 153), mini2-cluster1 + 5 + 6-nl2s-GCN4 (SEQ ID NO: 154) and mini2-cluster1 + 5 + 6 + 12 (SEQ ID NO: 156) are also H5N1A / Vietnam / 1203 Can recognize the ectodomain of HA from / 2004. In the polypeptide of the present invention, Mini3-cluster 11 (SEQ ID NO: 11) can induce an antibody that recognizes the ectodomain of HA from H5N1A / Vietnam / 1203/2004.

Example 20
General Method for Designing Stem Domain Polypeptides Containing Conserved Stem Domain Epitopes of CR6261 and CR9114 Based on the above results, the general practice of producing the polypeptides of the invention from influenza virus HA0 sequences, particularly from influenza HA sequences of serotype H1 Define the method. The method includes the following steps:
1. Removal of cleavage sites of HA1-HA2. This removal can be accomplished by mutation of R (in some cases K) to Q at position P1 (eg, Sun for a description of the nomenclature for the cleavage site (position 343 in SEQ ID NO: 1)). see et al, 2010. Mutations to Q are preferred, but S, T, N, D or E are alternatives.
2. Removal of the head domain by deleting amino acids 53-320 from SEQ ID NO: 1, or corresponding positions in HA from other influenza viruses. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. The remaining portions of the sequence can be joined directly or by introducing a flexible linker. The linker sequence can be 1-50 amino acids long. Preferred are flexible linkers of limited length (10 amino acids or less) such as GGG, GGGG, GSA, GSAG, GSAGSA, GSAGSAG or the like. The length of the deletion may be 47, 48, 49, for example by starting the deletion at position 54, 55, 56, 57 or 58 (or its equivalent) or to increase the length of the deletion. It can also be changed by cutting at position 50, 51 or 52. Similarly, the last amino acid to be deleted is at position 315, 316, 317, 318 or 319 (or its equivalent), or 321, 322, 323, 324, or to increase the length of the deletion, or It can be at 325 (equivalent to). It is important to understand that changes in the length of a deletion can be partially compensated by matching the length of the linker sequence, i.e., larger deletions can be matched with longer linkers. Yes, and vice versa. These polypeptides are also encompassed by the present invention.
3. Before fusion of the modified HA by increasing the solubility of the loop (between A helix and CD helix) formed by residues 402-418 (equivalent to) residues in H1A / Brisbane / 59/2007 (SEQ ID NO: 1) To increase the stability of the conformation and to destabilize the conformation after fusion. This loop is highly conserved in the H1 sequence, as can be seen in Table 6 below. This can be achieved, for example, by replacing amino acid I, L, F or V residues in the loop with hydrophilic equivalents. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using a suitable algorithm, such as Clustal or Muscle. Mutation to glycine destabilizes the post-fusion conformation. This is because the high flexibility of this amino acid results in a decrease in the stability of the helix after fusion that can be formed by part of this HA sequence. A consensus sequence that describes the loop of residues 402-418 of influenza HA of serotype H1 is (SEQ ID NO: 17) MNTQFTAVGKEFN (H / K) LE (K / R). In the polypeptides of the present invention, amino acids at positions 406, 409, 413 and / or 416 (or their equivalents determined from sequence alignment) are polar (S, T, N, Q), charged (R, H, K, D, E) or flexible (G) amino acids. It should be noted that mutation of either L416 to either S or T also introduces a consensus N-glycosylation site (the consensus sequence is NX (S / T)). Asparagine glycosylation at this position further increases the solubility of this region. Combinations of mutations at these sites are also possible, for example F406S, V409T, L416S in SEQ ID NO: 10 and SEQ ID NO: 14. In some cases, mutations to restore consensus amino acids are preferred, eg, V or M is at position 404 (to T), V is at position 408 (to A) or 410 (to G), or I is at position 414. (To N); the incidence of sequences with these specific amino acids is very low. A summary of the above mutations that characterize the polypeptides of the invention is listed in Table 6.
4). Introducing a disulfide bridge into the polypeptide of the invention, preferably between the amino acids at positions 324 and 436 in H1A / Brisbane / 59/2007 (equivalent); SEQ ID NOs: 13-16. Corresponding positions can be readily determined by those skilled in the art by aligning the sequences using suitable algorithms such as Clustal, Muscle, etc. Engineered disulfide bridges mutate at least one (if the other is already cysteine), but usually two spatially close residues to cysteines, either spontaneously or by active oxidation Created by forming a covalent bond between sulfur atoms.

  Using the general method according to the present invention described above, the polypeptide of the present invention can be transformed into H1N1A / California / 04/2009 (SEQ ID NO: 159), H1N1A / California / 07/2009 (SEQ ID NO: 56), H1N1A / Puerto Rico / Based on the HA0 sequence of 8/1934 (SEQ ID NO: 78) and H1N1A / Texas / 36/1991 (SEQ ID NO: 64). In addition, this method was applied to another subtype that was part of Group 1, namely HA from H5, using HA from H5N1A / Vietnam / 1203/2004 (SEQ ID NO: 158).

H1mini-HAA / California / 07/2009 (SEQ ID NO: 160) is created from H1FLHAA / California / 07/2009 (SEQ ID NO: 56) by:
1. Removing the cleavage site: mutation R344Q (numbering refers to SEQ ID NO: 56. 2. P321 is deleted from residues K53 and the GGGG linker is D52 and K322 (numbering refers to SEQ ID NO: 56) 3. Introduce in between 3. A helix-CD helix loop (residues 403-419 in SEQ ID NO: 56) with a serine residue at position 407,417 (F407S, L417S; numbering is SEQ ID NO: 2. introducing threonine at position 410 (V410T; numbering refers to SEQ ID NO: 56) and glycine residue at position 414 (F414G; numbering refers to SEQ ID NO: 2); Introducing a bridge by mutating residues lysine 325 and threonine 437 to cysteine (K325) , T437C; numbering refers to SEQ ID NO: 56)
5. An additional stabilizing element was introduced by replacing 419KRIENLNKKKVDDGFLD434 (numbering refers to SEQ ID NO: 56) with the sequence RMKQIEDKIEIESKQ.

A mini-HA sequence based on the full-length HA (SEQ ID NO: 159) from A / California / 04/2009 can be generated in the same manner, H1mini-HAA / California / 07/2009 (SEQ ID NO: 160) Is the same as

Similarly, H1mini-HAA / Puerto Rico / 8/1934 (SEQ ID NO: 161) is created from H1FLHAA / Puerto Rico / 8/1934 (SEQ ID NO: 78) by:
1. Removing the cleavage site: mutation R343Q (numbering refers to SEQ ID NO: 78) 2. 2. Delete P320 from residue K53 and introduce a GGGG linker between D52 and K321 (numbering refers to SEQ ID NO: 78). Serine residue at position 406, 416 (F406S, L416S; numbering refers to SEQ ID NO: 78) threonine 409 in the A helix to CD helix loop (residues 402-418 in SEQ ID NO: 78) 3. Introduce a position (V409T; numbering refers to SEQ ID NO: 78) and a glycine residue at position 413 (F413G; numbering refers to SEQ ID NO: 78). Disulfide bridges are introduced by mutating residues arginine 324 and threonine 436 to cysteine (R324C, T436C; numbering refers to SEQ ID NO: 78)
5. An additional stabilizing element was introduced by replacing 418KRMENNLNKVDDGFLD433 (numbering refers to SEQ ID NO: 78) with the sequence RMKQIEDKIEIESKQ.

  The additional difference between H1mini-HAA / Puerto Rico / 8/1934 (SEQ ID NO: 161) and H1FLHAA / Puerto Rico / 8/1934 (SEQ ID NO: 78) is position 397, full length protein (SEQ ID NO: 78) Serine but threonine in the polypeptide of the invention of SEQ ID NO: 161 (S397T mutation). This is a natural mutation in the A / Puerto Rico / 8/1934 sequence and therefore sequences containing this mutation are also included in the present invention.

H1mini-HAA / Texas / 36/1991 (SEQ ID NO: 162) is generated from H1FLHAA / Texas / 36/1991 (SEQ ID NO: 64) by:
1. Removing the cleavage site: mutation R344Q (numbering refers to SEQ ID NO: 64) 2. 2. Delete P321 from residue S53 and introduce a GGGG linker between D52 and K322 (numbering refers to SEQ ID NO: 64). In the loop of A helix to CD helix (residues 403 to 419 in SEQ ID NO: 64), a serine residue at position 407,417 (F407S, L417S; numbering refers to SEQ ID NO: 64), threonine 3. Introducing at position 410 (V410T; numbering refers to SEQ ID NO: 64) and a glycine residue at position 414 (F414G; numbering refers to SEQ ID NO: 64) Disulfide bridges are introduced by mutating residues arginine 325 and threonine 437 to cysteine (R325C, T437C; numbering refers to SEQ ID NO: 64)
5. An additional stabilizing element was introduced by replacing 419RRMENNLNKVDDGFLD434 (numbering refers to SEQ ID NO: 64) with the sequence RMKQIEDKIEIESKQ.

H5mini-HAA / Vietnam / 1203/2004 (SEQ ID NO: 163) is created from H5FLHAA / Vietnam / 1203/2004 (SEQ ID NO: 158) by:
1. Removal of cleavage site. Since H5FLHAA / Vietnam / 1203/2004 (SEQ ID NO: 158) contains a polybasic cleavage site (341RRRKKR346), single site mutations are not sufficient to prevent protein cleavage. Instead, 341RRRRKK345 is deleted and the R346Q mutation is introduced.
2. 2. Delete P319 from residue K52 and introduce a GGGG linker between K51 and K320 (numbering refers to SEQ ID NO: 158). Serine residues at positions 409 and 419 (F409S, L419S; numbering refers to SEQ ID NO: 158) in the A helix to CD helix loop (residues 405 to 421 in SEQ ID NO: 158), threonine 3. Introducing at position 412 (V412T; numbering refers to SEQ ID NO: 158) and a glycine residue at position 416 (F416G; numbering refers to SEQ ID NO: 158) Disulfide bridges are introduced by mutating residues lysine 323 and threonine 439 to cysteine (K323C, T439C; numbering refers to SEQ ID NO: 158)
5. An additional stabilizing element was introduced by replacing 421RRIENLNKKEDGFLDV437 (numbering refers to SEQ ID NO: 158) with the sequence RMKQIEDKIEIESKQI.

  Genes encoding the protein sequences of SEQ ID NOs: 56, 160, 78, 161, 162, 158 and 163 were synthesized and cloned into the expression vector pcDNA2004 using methods generally known to those skilled in the art. For comparison reasons, the full length HA sequence of H3A / HongKong / 1/1968 (SEQ ID NO: 121) and the full length HA sequence of H1A / Brisbane / 59/2007 (SEQ ID NO: 1) with an additional cleavage site mutation R343Q Included in the experiment.

The expression vector (1 μg / ml) was transfected into HEK293F (Invitrogen) suspension cells (10 ⁶ cells / ml, 30 ml) using 40 μl 293transfectin as a transfection agent and allowed to grow for another 2 days. Cells were harvested; aliquoted (0.3 ml, approximately 3 ^* 10 ⁵ cells), and aliquots were raised against polyclonal serum or HA specific against H1HA (Sino Biological Inc. Beijing, China) to examine expression Treatment with either of the monoclonal antibodies (5 microgram / ml) and the secondary antibody used for staining. The cells were then analyzed by fluorescence coupled cell sorting (FACS) for expression of the membrane-attached HA stem domain polypeptide of the present invention on the cell surface. The presence of conserved epitopes using a panel of monoclonal antibodies of known specificity (CR6261, CR9114) that binds to the stem domain in the full-length protein, as well as correct folding of full-length HA and the mini-HA polypeptides of the invention by inference I investigated. Monoclonal antibodies CR8020 (known not to bind to H1 and H5HA) and CR9020 (binding to the head domain of HA from H1A / Brisbane / 59/2007) were also included in the experiment. Results are expressed as percentage of positive cells and mean fluorescence intensity (MFI) and are shown in FIG.

  Treatment of transfected cells with polyclonal anti-H1 serum results in 20-80% positive cells for full length HA (black bars) and 40-50% positive cells for mini-HA. The negative controls FLH3A / HongKong / 1/1968 and cM2 show very few positive cells. This is reflected by the mean fluorescence intensity (lower panel) showing a clearly detectable signal for all H1 full length HA proteins. The signal for full-length H5HA remains low; however, this can be explained by fewer transfected cells in combination with reduced recognition by polyclonal H1 serum. Negative controls FLA / HongKong / 1/1968 and cM2 show the intensity at the background level.

CR6261 and CR911 known to be strong group 1 stem binders
Both 4 recognize all group 1 full-length HA and mini-HA proteins as indicated by the large number of positive cells (about 50 to about 95%) and high MFI. This is strong evidence that neutralizing epitopes of these antibodies are present in the mini-HA protein, indicating a three-dimensional structure that is strongly common to the native structure of the HA stem domain in full-length HA. As expected, the negative control CR8020 (specific for group 2 HA) did not bind to H1 and H5 full-length HA nor to H1 and H5mini-HA, indicating that CR6261 / CR9114 neutralization to the mini-HA protein. FIG. 5 shows that the observed binding of the antibody does not result from a specific protein-protein interaction. The binding between full length H3HA from A / HongKong / 1/1968 and CR9114 or CR8020 is clearly observed from both the percentage of positive cells and MFI, consistent with earlier observations and the function of these monoclonal antibodies Prove sex. Similarly, the negative control antibody CR9020 (HA head binder for A / Brisbane / 59/2007) recognizes neither mini-HA nor full-length HA protein except for HA from A / Brisbane / 59/2007. Further supports the specificity of binding observed between CR6261 and CR9114.

  In summary, four novel HA-derived polypeptides of the present invention that were shown to contain epitopes recognized by neutralizing CR6261 and CR9114 antibodies in the absence of the HA head domain were generated.

Example 21
Protection against lethal influenza challenge in mice by the polypeptides of the invention Determine whether the polypeptides of the invention can induce an immune response that protects mice from other deaths upon exposure to lethal influenza viruses. In order to do this, an influenza challenge experiment was conducted. Mice were treated with an expression vector encoding SEQ ID NO: 78, 161, 45 and 6, and full length HA (SEQ ID NO: 1) from A / Brisbane / 59/2007 containing an additional R343Q mutation to remove the cleavage site. Immunized intramuscularly. An expression vector encoding cM2 was included as a negative control. Immunization was performed according to the following test protocol using 50 μg expression construct + 50 μg adjuvant (pUMCV1-GM-CSF).

Test protocol day 1 Blood collection.
Day 0 Vaccine administration (intramuscular).
Day 21 Vaccine administration (intramuscular).
Day 28 Blood sampling.
Day 42 Vaccine administration (intramuscular).
Day 47 Blood sampling.
Day 48 Measurement of body weight, body temperature, clinical score and mortality.
Day 49 Challenge with a lethal dose of influenza virus infection (nasal).
Day 49 Residual inoculum is utilized for virus back titration.
Day 49-70 Daily measurements of body weight, body temperature, clinical score and mortality.
Animals with a clinical score ≧ 3 are monitored twice daily.
Animals with a clinical score ≧ 4 or body temperature ″ 32 ° C. are immediately removed from the study, regardless of which animal first falls.
Day 70 Euthanasia of all mice.
Groups 1-6: Challenge with PR8 (A / Puerto Rico 8/34, H1N1) Group 1: SEQ ID NO: 78
Group 2: SEQ ID NO: 161
Group 3: SEQ ID NO: 1R343Q
Group 4: SEQ ID NO: 45
Group 5: SEQ ID NO: 6
Group 6: 10 mice per group of empty vectors. 60 mice in total. BALB / c.

Materials and methods:
Virus strains and sources:
Influenza virus strain PR8 (A / Puerto Rico 8/34, H1N1) was supplied by Virapur (San Diego). Stock solution 1 × 10e8 pfu / ml batch # E2004B.
Storage conditions: -75 ° C ± 10 ° C. Freezer: -86 ° C UCT freezer. Thermo Form. Fisher Scientific.

animal:
Mice, BALB / c (no specific pathogens; SPF), females, about 17-19 grams 6-8 weeks old on test day 0. Identified by “Ear Identification”, supplied by Charles River Laboratories. All animals were acclimated and maintained for 11 days before the start of the experiment.

Methods for reconstitution of DNA-administered inoculum The appropriate DNA formulations listed above were prepared, aliquoted and stored at -20 ° C. One aliquot per construct was thawed to room temperature just prior to injection, withdrawn into a syringe and injected. The remainder of each aliquot was discarded after completion of all injections in each immunization round.

Dose Levels and Methods of Administration Mice were treated with 9.75 mg Xylasol (Graeb E Dr. AG (www.graeub.com); catalog number: 763.02) and 48.75 mg Ketasol (Graeub E Dr. AG (kg). anesthesia by intraperitoneal injection using: catalog number: 668.51). Using a 0.5 ml syringe with a G29 needle, 50 μl of the DNA solution was injected intramuscularly (im) into each quadriceps of the hind paw and a total injection volume of 100 μl per mouse. Gave rise to The remainder of each aliquot was discarded after completion of all injections in each immunization round.

Virus administration:
Method of inoculum reconstitution Viral material was stored at -75 ° C ± 10 ° C and thawed prior to administration. Once thawed, the material was diluted in cold PBS (4 ° C.) corresponding to 5LD50 / 50 μl for A / PR / 8/34 challenge. Diluted virus was kept on ice until administration to mice.

Dose levels and methods of administration Animals were anesthetized by intraperitoneal injection with 9.75 mg Xylasol and 48.75 mg Ketasol per kg body weight, each animal receiving 50 μl of virus solution intranasally. Unused material was returned to the laboratory for reverse titer measurement.

Blood collection and serum preparation Blood samples were collected for the number of days specified in the above test protocol (intermediate blood collection: 100-150 μl via retro-orbital cannulation, final blood collection via cardiac puncture: approximately 300
˜500 μl). Serum was isolated from this blood by centrifugation at 14000 g for 5 minutes and stored at −20 ° C. on dry ice until transport.

Clinical scoring Clinical signs after viral challenge are scored in a scoring system (1 point for healthy mice; 2 points for mice showing signs of discomfort, eg, slight nap, slight changes in gait and increased gait; 3 points for mice showing signs of strong napping, abdominal contraction, gait changes, inactivity period, increased respiration rate and occasional blistering sound (bullet sound / sounding noise); Scoring was performed according to 4 points for mice dying and 5 points for dead mice. The animals were investigated twice daily as long as they received a score of 3. Scoring was performed by a single investigator, with mice having symptoms partially represented by two scores scored +/− 0.5.

Weighing All animals were weighed daily starting from day 48 (confirmation number 2216). The animals were also weighed in the case of death before the end of the test, ie when removed from the test. Body weight was recorded in grams (g).

Virus reverse titration The dose of virus administered was determined by titrating 8 replicate samples from the inoculum remaining after the animal inoculation was completed. TCID50 measurement was used for virus reverse titration measurement according to the protocol outlined in “Current Protocols in Immunology, Animal Models of Infectious Disease 19.11.7”.

result:
The test was conducted in accordance with the defined test protocol without technical difficulties. Reverse titration of the inoculum of influenza virus strain PR8 (A / Puerto Rico 8/34, H1N1) resulted in the following TCID50: PR8 (A / Puerto Rico8 / 34, H1N1): 3.2 × 10 4 TCID50 / ml .

  FIG. 24A shows the Kaplan-Meier survival curve for this experiment. Immunization with DNA encoding polypeptides SEQ ID NOs: 45, 6 and 161 of the present invention resulted in 50, 40 and 40% survival rates of mice infected with lethal doses of influenza, respectively, It is shown that immunization with the polypeptide of can actually induce a protective immune response. In contrast, all mice immunized with an empty vector control die from infection 8 days after virus challenge. Immunization with DNA encoding the full-length HA (SEQ ID NO: 78) of the same species as the challenge strain completely protects all animals from lethal challenge (ie, 100% survival) while additional cleavage site mutations ( R343Q; numbering refers to SEQ ID NO: 1) immunization with DNA encoding full length HA (SEQ ID NO: 1) from heterologous strain A / Brisbane / 59/2007 contains 90% survival of infected animals Bring rate.

The results obtained from the survival curves are also reflected in the mean body weight change and the median clinical score for each group shown in FIGS. 24B and 24C. Animals immunized with polypeptides SEQ ID NOs: 45, 6, and 161 of the present invention show a weight loss of up to 25-30%, whereas animals that survive 9 days after infection show weight gain. A drop in clinical score from 4 to 3 is also observed for animals immunized with SEQ ID NO: 45. Animals immunized with DNA encoding full length HA (SEQ ID NO: 78) of the same species as the challenge strain show no weight loss, while additional cleavage site mutations (R343Q; numbering refers to SEQ ID NO: 1) Animals immunized with DNA encoding full-length HA (SEQ ID NO: 1) from the containing heterologous strain A / Brisbane / 59/2007 experience weight loss and clinical symptoms, but surviving animals are fully covered. Maximum weight loss and clinical symptoms are observed for the control group immunized with the empty vector, consistent with the lack of survival of those animals.

  Taken together, the polypeptide SEQ ID NOs: 6, 45 and 78 of the present invention can induce a protective response to lethal challenge by H1N1A / Puerto Rico / 8/1934 in mice. It is noted that the polypeptide SEQ ID NOs 6 and 45 of the present invention are derived from a HA molecule heterologous to the challenge strain, while SEQ ID NO 78 is derived from a homologous influenza strain. Thus, the polypeptides of the invention can induce protection against both homologous and heterologous influenza infections.

Example 22
Selection of a panel of representative H1N1HA sequences and design of polypeptides of the invention based on those sequences. This method is found in H1N1 virus to show the wide applicability of the design method described in Example 20 Applied to a panel of selected HA0 sequences covering the majority of native sequence variations. Selection of a panel of representative HA sequences from a pool of known human H1N1 HA sequences in this example has the purpose of selecting the fewest strains with the greatest representativeness. To achieve this goal, we quantified all differences between the human H1N1 influenza virus HA sequences present in the influenza virus sequence database, investigated the structure of these differences, and identified uniform subgroups. From each of these groups, the most representative sequence contributing to the panel was selected.

  The initial step of this procedure is a quantification of the difference between each pair or sequence in the sequence database considered. The inverse PAM250 (rPAM250) matrix (Xu, 2004) is used to quantify the difference at each amino acid position. The total difference for the pair is then quantified using Euclidean addition. All pairwise differences are used to form a symmetric n × n matrix of differences (where n is equal to the number of virus strains considered).

  Structure the difference matrix using principal coordinate analysis (PCA) (Higgins, 1992). PCA is based on dimensional reduction. The input matrix is considered as a distribution in n-dimensional space (n is equal to the number of strains considered). The variability is then analyzed and structured such that a minimum dimension is required to cover most (or all) variability. The result is an m-dimensional coordinate system (m is the number of dimensions to cover most or all of the variability), the maximum variation is on the first axis and then decreases. Place all considered arrays in that coordinate system. If only two or three dimensions are required, the results can be plotted completely in 2D or 3D graphs, respectively, where differences between strains can be visualized. If more dimensions are needed, an additional 3D plot can be constructed from the first three axes, but the graph does not cover all variability. This is because some of the mutations exist in the fourth and higher dimensions.

The sequences in m-dimensional space are then clustered using both hierarchical and k-means clustering. Average ligation within groups is used to obtain groups with similar internal variability, avoiding the majority of single strain clusters. Clustering starts at 1 (all strains in one cluster) and runs at all levels up to n (each strain forming its own cluster). From each cluster, the most central strain is selected as the most representative. The most central strain set then forms a panel of representative strains for that level clustering. For each level of clustering, the coverage (or percentage of explained mutations) is compared to the sum of the square distances to the center of the coordinate system of each stock, and the sum of the square distances to each center of each stock. Is estimated by computer processing. Then, the minimum level of coverage to be achieved is set to be the minimum required size of the representative panel.

  In addition, if the Xu rPAM250 matrix had a gap on one of the two sequences (due to insertion or deletion), a small value was assigned for the difference. In addition, the weight factor was included in the procedure to explain the large difference in the number of isolates throughout the year. This mutation was considered partly a true developmental mutation and partly driven by different levels of surveillance / recognition. Therefore, the weighting factor was set in divided by the square root of the number of observations for a particular year. This weight factor was taken into account when constructing an m-dimensional space during cluster analysis and center point selection and in estimating the level of mutations covered.

  In this example, a construction sequence consisting of a portion of HA encoding a polypeptide of the invention is used. Two different sets of construction sequences were created. In the first set, a signal sequence (eg, amino acids 1-18), amino acids 53-320 (HA head domain), a transmembrane sequence (amino acids 530 to the C-terminal amino acid) (numbering refers to SEQ ID NO: 1) Or the native sequence was considered excluding the equivalent of these positions in other sequences. In the second set, the amino acids at positions 406, 409, 416, 324, 436, 413 (or their equivalent positions) were not considered. This is because they are modified according to the general method described in Example 20. In addition, positions 419-433 (equivalent) reflecting the addition of GCN4-based stabilizing sequences in the polypeptides of the invention described in Example 9 were not considered in the second set.

  Using the method described above, 7 HA sequences covering 75% of the sequence variations were selected from construction sequence set 1 and 8 HA sequences covering 74% of the sequence variations were selected from construction sequence set 2. The stocks are listed in Table 10. Since 3 of the selected sequences appear in both sets, 13 unique HA sequences remain. Using these sequences, the polypeptides of the present invention were designed according to the method described in Example 20. In addition, the stabilized GCN4 sequence MKQIEDKIEEESKQ (SEQ ID NO: 84) is introduced in its counterpart at positions 419-433 (numbering refers to SEQ ID NO: 1) as described in Example 9. The polypeptides of the invention designed based on the HA sequences of H1N1A / Memphis / 20/1978 and H1N1A / USSR / 92/1977 are identical, A / Wisconsin / 629-D01415 / 2009 and H1N1A / Sydney / DD3-55 This is the same as the polypeptide of the present invention designed based on the / 2010 HA sequence. Therefore, a total of 10 unique polypeptides of the present invention were designed and the alignment of these sequences is shown in FIG.

  Polypeptide SEQ ID NO: 164-SEQ ID NO: 173 of the present invention, and corresponding full-length HA SEQ ID NO: 45 having polypeptide sequence ID 45 of the present invention based on the HA sequence of A / Brisbane / 59/2007 and an additional cleavage site R343Q An expression vector containing DNA encoding 1 in the expression vector pcDNA2004 was used for transfection of HEK293F cells and the cells were analyzed by FACS as described above. In addition to human monoclonal antibodies CR6261, CR9114 and CR8020, a mouse monoclonal antibody C179 known to neutralize influenza AH1 and H2 strains (Okuna et al., 1993) was included in the experiment. The results are shown in FIG.

All polypeptides of the invention and the full-length sequence of A / Brisbane / 59/2007 are expressed on the cell surface and are recognized by the broadly neutralizing antibodies CR6261, CR9114 and C179, but not by CR8020. The last is known to bind only to HA from influenza A group 2. The binding of antibodies CR6261, CR9114 and C179 indicates that the broadly neutralizing epitope is well conserved in the polypeptides of the invention. In view of the sequence variations covered in these sequences, this is a clear indication of the general applicability of our design method to generate polypeptides of the invention containing broadly neutralizing epitopes. It is proof.

Example 23
Characterization of the polypeptide s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) of the invention The purified polypeptide s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) of the invention is described in Example 13. Obtained as follows. To confirm the presence of CR6261 and CR9114 conformational epitopes, the binding of these antibodies to purified proteins was tested by biolayer interferometry (Octet Red ³⁸⁴ , Forte Bio). For this purpose, biotinylated CR6261, CR9114 and CR8020 are immobilized on a streptavidin-coated sensor, the sensor is first exposed to a solution of the purified polypeptide of the present invention (250 nM) and the association rate is then measured, followed by a wash solution The dissociation rate was measured by exposure to. For comparison reasons, the experiment was repeated with both the trimer and monomeric forms using the full-length protein (SEQ ID NO: 149). The results are shown in FIG.

  Immobilized CR6261 is a monomeric and trimeric form of full-length HA ectodomain from H1N1A / Brisbane / 59/2007 as evidenced by a clear response after exposure to these proteins in solution Both are recognized (FIG. 27A). The response observed for the trimeric protein is greater than that observed for monomers with the same sequence (about 1.3 nm and 0.9), and smaller size monomers compared to the trimer This is an effect caused by (at least in part). Binding of CR6261 to s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) results in a maximum response of about 0.25 nm. Upon exposure to the wash solution, complex dissociation is observed for all three analytes, the fastest release observed for the polypeptide s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) of the present invention, Slowest for the trimeric form of the ectodomain of full-length HA (SEQ ID NO: 149) from H1N1A / Brisbane / 59/2007.

  Similar to CR6261, immobilized CR9114 is both a trimer and monomeric form of the full-length ectodomain from H1N1A / Brisbane / 59/2007, as well as the polypeptide s-H1-mini2-cluster1 + 5 + 6- Recognizes GCN4 (SEQ ID NO: 145). Responses were as follows for CR6261 (trimer, monomeric full length HA (SEQ ID NO: 149) and stem domain polypeptide s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) for all three analytes, respectively. 5, 1.4 and 0.8 nm), and in all three cases, release of the antigen is minimal or undetectable upon exposure of the complex to the wash buffer. For CR8020, no response was observed for any of the analytes, consistent with the influenza group 2 stem domain specificity of this antibody.

To further characterize the binding of CR6261 and CR9114 to the purified stem domain polypeptide, titrations were performed. For this purpose, immobilized CR6261 containing sensors were exposed to s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) solution at concentrations of 500, 250, 125, 63, 31, 16 and 8 nM, respectively, after 14000 s. The final response of was recorded. The response was plotted as a function of stem domain polypeptide concentration and a fit to a steady state 1: 1 binding model was performed, resulting in a dissociation constant K _d of approximately 190 nM for the CR6261 / stem domain polypeptide complex, FIG. . Similarly, sensors modified with immobilized CR9114 were s-H1-mini2 at concentrations of 80, 40, 20, 10, 5, 2.5 and 1.3 nM, respectively.
Exposure to -cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) and the final response after 10800 s was recorded. Fitting the final response as a function of stem domain polypeptide concentration resulted in a K _d value of 5.4 nM for the CR9114 / stem domain polypeptide complex (FIG. 28B).

  In summary, the polypeptide s-H1-mini2-cluster1 + 5 + 6-GCN4 (SEQ ID NO: 145) of the present invention can bind to the broadly neutralizing monoclonal antibodies CR6261 and CR9114, which correspond to the corresponding in this stem domain polypeptide. Supports the presence of neutralizing epitopes.

Example 24
Protection against lethal influenza challenge in mice by polypeptides of the invention Determine whether a polypeptide of the invention can induce an immune response that protects mice against death otherwise upon exposure to lethal influenza virus Therefore, an influenza challenge experiment was conducted. Mice were immunized intramuscularly with expression vectors encoding H3 full length A / Hong Kong / 1/1968 (SEQ ID NO: 121), HK68H3m2-cl9 + 10 + 11 (SEQ ID NO: 124) and HK68H3m2-cl9 + 10 + 11 + 12-GCN4 SEQ ID NO: 130. Immunization was performed using 50 μg expression construct + 50 μg adjuvant (pUMCV1-GM-CSF) according to the following test protocol.

Test protocol day 1 Blood collection.
Day 0 Vaccine administration (intramuscular).
Day 21 Vaccine administration (intramuscular).
Day 28 Blood sampling.
Day 42 Vaccine administration (intramuscular).
Day 47 Blood sampling.
Day 48 Measurement of body weight, body temperature, clinical score and mortality.
Day 49 Challenge with a lethal dose of influenza virus infection (nasal).
Day 49 Residual inoculum is used for virus reverse titer measurement.
Day 49-70 Daily measurements of body weight, body temperature, clinical score and mortality.
Animals with a clinical score ≧ 3 are monitored twice daily.
Animals with a clinical score ≧ 4 or body temperature ″ 32 ° C. are immediately removed from the study, regardless of which animal first falls.
Day 70 Euthanasia of all mice.
Groups 7-10: Challenge with HK68 (A / HongKong / 1/68, H3N2) Group 7: SEQ ID NO: 121
Group 8: SEQ ID NO: 130
Group 9: SEQ ID NO: 124
Group 10: 10 mice per group of empty vectors. 40 mice in total. BALB / c.

Materials and methods:
Virus strains and sources Influenza virus strain HK68 (A / HongKong / 1/68)
J. et al. Provided by Katz (Center for Disease Control and Prevention, Atlanta, GA, USA) and then grown by Virapur (San Diego). The virus was passaged multiple times in the mouse lung to improve pathogenicity in the mouse. A suitable reference for this virus is Frace
et al. , Vaccine 1999; 17: 2237. Stock solution 3 × 10e8
pfu / ml. Batch # F1109A.
Storage conditions: -75 ° C ± 10 ° C. Freezer: -86 ° C UCT freezer. Thermo Form. Fisher Scientific.

animal:
Mice, BALB / c (no specific pathogens; SPF), females, about 17-19 grams 6-8 weeks old on test day 0. Identified by “Ear Identification”, supplied by Charles River Laboratories. All animals were acclimated and maintained for 11 days before the start of the experiment.

Methods for reconstitution of DNA-administered inoculum The appropriate DNA formulations listed above were prepared, aliquoted and stored at -20 ° C. One aliquot per construct was thawed to room temperature just prior to injection, withdrawn into a syringe and injected. The remainder of each aliquot was discarded after completion of all injections in each immunization round.

Dose Levels and Methods of Administration Mice were treated with 9.75 mg Xylasol (Graeb E Dr. AG (www.graeub.com); catalog number: 763.02) and 48.75 mg Ketasol (Graeub E Dr. AG (kg). anesthesia by intraperitoneal injection using: catalog number: 668.51). Using a 0.5 ml syringe with a G29 needle, 50 μl of the DNA solution was injected intramuscularly (im) into each quadriceps of the hind paw and a total injection volume of 100 μl per mouse. Gave rise to The remainder of each aliquot was discarded after completion of all injections in each immunization round.

Virus administration:
Method of inoculum reconstitution Viral material was stored at -75 ° C ± 10 ° C and thawed prior to administration. Once thawed, the material was diluted in cold PBS (4 ° C.) corresponding to 10 LD50 / 50 μl for the A / HK / 1/68 challenge. Diluted virus was kept on ice until administration to mice.

Dose levels and methods of administration Animals were anesthetized by intraperitoneal injection with 9.75 mg Xylasol and 48.75 mg Ketasol per kg body weight, each animal receiving 50 μl of virus solution intranasally. Unused material was returned to the laboratory for reverse titer measurement.

Blood collection and serum preparation Blood samples were collected on the days specified in the above test protocol (intermediate blood collection: 100-150 μl via retro-orbital cannulation, final blood collection via cardiac puncture: approximately 300-500 μl). Serum was isolated from this blood by centrifugation at 14000 g for 5 minutes and stored at −20 ° C. on dry ice until transport.

Clinical scoring Clinical signs after viral challenge are scored in a scoring system (1 point for healthy mice; 2 points for mice showing signs of discomfort, eg, slight nap, slight changes in gait and increased gait; 3 points for mice showing signs of strong napping, abdominal contraction, gait changes, inactivity period, increased respiration rate and occasional blistering sound (bullet sound / sounding noise); Scoring was performed according to 4 points for mice dying and 5 points for dead mice. The animals were investigated twice daily as long as they received a score of 3. Scoring was performed by a single investigator, with mice having symptoms partially represented by two scores scored +/− 0.5.

Weighing All animals were weighed daily starting from day 48 (confirmation number 2216). The animals were also weighed in the case of death before the end of the test, ie when removed from the test. Body weight was recorded in grams (g).

Virus reverse titration The dose of virus administered was determined by titrating 8 replicate samples from the inoculum remaining after the animal inoculation was completed. TCID50 measurement was used for virus reverse titration measurement according to the protocol outlined in “Current Protocols in Immunology, Animal Models of Infectious Disease 19.11.7”.

result:
The test was conducted in accordance with the defined test protocol without technical difficulties. Reverse titer measurement of the inoculum of influenza virus strain HK68 (A / HongKong / 1/68, H3N2) resulted in the following TCID50: HK68 (A / HongKong / 1/68): 1 × 10 ³ TCID50 / ml .

  FIG. 29 shows the IgG response to the ectodomain of HA from A / HongKong / 1/1968 at day 49 as measured by ELISA. Immunization of the polypeptides of the invention with DNA encoding SEQ ID NO: 124 and SEQ ID NO: 130 induces a clearly detectable response against the H3HAHK68 ectodomain, whereas no response is detected for the empty vector negative control. As expected, a maximum response is observed for immunization with H3 full length A / HongKong / 1/1968 (SEQ ID NO: 121).

  FIG. 30A shows the Kaplan-Meier survival curve for this experiment. Immunization with DNA encoding the polypeptide SEQ ID NOs: 124 and 130 of the present invention resulted in 40 and 20% survival rates of mice infected with lethal doses of influenza, respectively, which is due to the polypeptides of the present invention. It shows that immunization can actually induce a protective immune response. In contrast, all mice immunized with the empty vector control all die from infection 10 days after virus challenge. Immunization with DNA encoding the full length HA (SEQ ID NO: 121) of the same species as the challenge strain completely protects all animals from lethal challenge (ie 100% survival).

  The results obtained from the survival curves are also reflected in the mean weight change and the median clinical score for each group shown in FIGS. 30B and 30C. Animals immunized with DNA encoding the polypeptide SEQ ID NOs: 124 and 130 of the present invention show a weight loss of up to 20%, whereas animals that survive 9 days after infection show weight gain. Mice immunized with DNA encoding full length HA (SEQ ID NO: 121) do not show weight loss. Maximum weight loss and clinical symptoms are observed for the control group immunized with the empty vector, consistent with the lack of survival of those animals.

  Taken together, the polypeptide SEQ ID NOs 124 and 130 of the present invention are immunogenic and can induce a protective response to lethal challenge with H3N2A / HongKong / 1/1968 in mice.

Example 25
Design and characterization of stem domain polypeptides based on H3HA An additional set of constructs was designed to further improve the stem domain polypeptides described in Example 12. Two cysteine mutations that allow the formation of stabilized disulfide bridges at positions 53 and 334 (T53C, G334C; cluster 16) and 39 and 51 (G39C-E51C; cluster 17) (numbering refers to SEQ ID NO: 121) Two additional pairs were designed. In addition, two sequences were designed to be inserted between positions 420-421, ie at the N-terminal side of the long CD helix (see FIG. 1). The insert sequences were designed so that they facilitate the formation of intermonomer disulfide bridges between individual monomers in the trimer molecule. Two different sequences were designed: NATGGCCGG (Cluster 18) and GSGKCCGG (Cluster 19). The cluster18 sequence also includes sequences that introduce glycosylation sites (ie, NAT) into the stem domain polypeptide. In some cases, the glycosylation site is also introduced by mutation to NAT at positions 417-419. Using the full-length HA sequence from A / Hong Kong / 1/1968 as a starting point, the above modifications were combined with the S62-P322 deletion to yield the following stem domain polypeptides:
SEQ ID NO: 174: H3HKmini2a-linker + cl9 + 10 + 11 + 12 + GCN4T-CG7-1
SEQ ID NO: 175: HK68H3mini2a-linker + cl9_ + 10 + 12 + 18 + GCN4T
Sequence number: 176: HK68H3mini2a-linker + cl9_ + 10 + 12 + 16 + CG7-GCN4T
SEQ ID NO: 177: HK68H3mini2a-linker + cl9_ + 10 + 12 + 19 + GCN4T
SEQ ID NO: 178: HK68H3mini2a-linker + cl9_ + 10 + 12 + 17 + CG7-GCN4T
Sequence number: 179: H3HK68mini2a-linker2 + cl9_ + 10 + 12 + GCN4T

  In general, the genes encoding the above protein sequences were synthesized using methods known to those skilled in the art and cloned into the expression vector pcDNA2004. Cell surface expression and monoclonal antibody binding was analyzed by fluorescence-coupled cell sorting as described above. For comparison reasons, the full length HA of H3N2A / HongKong / 1/1968 (SEQ ID NO: 121), which further contains the R345Q mutation in the cleavage site, and SEQ ID NO: 130 (HK68H3m2-cl9 + 10 + 11 + 12-GCN4) and the negative control cM2 Was also included in the experiment.

  FIG. 31 shows the results of this experiment. All constructs are expressed on the cell surface as evidenced by the response observed for polyclonal anti-H3 serum (MFI, panel A; percentage of positive cells, panel B). Binding of CR8043 and CR8020 was observed for SEQ ID NOs: 174, 175, 176, 177, 178, 179, 121 and 130, indicating that mutations in cluster16 contribute to the stabilization of the conformational epitope of those antibodies. Indicates not to. For mAb CR9114, binding above background can only be observed for SEQ ID NO: 174: H3HKmini2a-linker + cl9 + 10 + 11 + 12 + GCN4T-CG7-1 and to a lesser extent SEQ ID NO: 177: HK68H3mini2a-linker + cl9_ + 10 + 12 + 17 + CG7-CG7-CG7-CG Both sequences contain an additional glycosylation site in the B loop, indicating the stabilizing effect of this modification on the conformational neutralizing epitope of CR9114.

In summary, the inventors have shown that the stem domain polypeptides of the invention can be obtained for group 2 serotypes, particularly the H3 subtype, according to the method described above. Further stabilization of these stem domain polypeptides can be achieved by introducing glycosylation sites in the B loop. These sequences are also encompassed by the present invention.

Example 26
Immunogenicity of HA stem domain polypeptides based on H3HA To assess the immunogenicity of stem domain polypeptides, the full length H3 from A / Wisconsin / 67/2005 (SEQ ID NO: 89), SEQ ID NO: 105: H3- mini2, SEQ ID NO: 108: H3-mini2-cl9 + 10 + 11, SEQ ID NO: 112: H3-mini2-cl9 + 10 + 12, SEQ ID NO: 111: H3-mini2-cl9 + 10 + 11 + 12, SEQ ID NO: 114: H3-mini2-cl9 + 10 + 11 + 12-tri, SEQ ID NO: 113: H3- encodes mini2-cl9 + 10 + 11 + 12-GCN4, SEQ ID NO: 119: H3-mini3-cl9 + 10 + 11 + 12 + 14, SEQ ID NO: 120: H3-mini4-cl9 + 10 + 11 + 12 + 14 Mice were immunized with the expression vector. An expression vector encoding cM2 was also included as a negative control.

  Groups of 4 mice (BALB \ c) were immunized intramuscularly on days 1, 21, and 42 with 50 μg construct + 50 μg adjuvant (pUMCV1-GM-CSF). On day 49, the final blood collection was performed and serum was collected. The negative control plasmid cM2 was administered by gene gun using about 10 μg construct + about 2 μg adjuvant (pUMCV1-GM-CSF) and the same immunization scheme. Serum was analyzed by ELISA using recombinant full length HA from A / Wisconsin / 67/2005 and A / Hong Kong / 1/1968 (obtained from Protein Sciences Corporation, Meriden, CT, USA) as antigen. Briefly, 96 well plates were coated overnight at 4 ° C. with 50 ng HA and then incubated with block buffer (100 μl PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. The plate is washed with PBS + 0.05% Tween-20 and 100 μl of a 2-fold dilution series in block buffer starting with a 50-fold dilution of serum is added. Bound antibody is detected using standard protocols well established in the art using HRP-conjugated goat anti-mouse IgG. Titers are compared to a standard curve composed of serial dilutions of mouse monoclonal antibodies that bind to the HA antigen and expressed as ELISA units per ml (EU / ml). The results of an ELISA using HA from A / Wisconsin / 67/2005, A / Hong Kong / 1/1968 and A / Perth / 16/2009 after 49 days are shown in FIGS. 32A, B and C, respectively. Sera obtained from mice immunized with DNA encoding the stem domain polypeptide included in this experiment were homologous full-length HA from A / Wisconsin / 67/2005 and to a similar extent A / Hong Kong / 1/1968. Can recognize heterologous full-length HA from In contrast, sera obtained from mice immunized with full-length HA from A / Wisconsin / 67/2005 (SEQ ID NO: 89) were obtained from A / Hong Kong / 1/1968 and A / Perth / 16/2009. It shows a higher response to homologous HA than to heterologous HA.

  Taken together, the data show that polypeptides of the invention derived from H3HA can induce an immune response directed against full-length HA.

Example 27
Immunogenicity of HA stem domain polypeptides based on A / Hong Kong / 1/1968 H3HA To evaluate the immunogenicity of stem domain polypeptides, the full-length H3 from A / Hong Kong / 1/1968 (SEQ ID NO: 121 ), SEQ ID NO: 124: HK68H3m2-cl9 + 10 + 11, SEQ ID NO: 125: HK68H3m2-cl9 + 10 + 12, SEQ ID NO: 1.
Mice were immunized with an expression vector encoding 26: HK68H3m2-cl9 + 10 + 11 + 12, SEQ ID NO: 128: HK68H3m2-cl9 + 10 + 11 + 12-tri, SEQ ID NO: 130: HK68H3m2-cl9 + 10 + 11 + 12-GCN4. An expression vector encoding cM2 was also included as a negative control.

  Groups of 4 mice (BALB \ c) were immunized intramuscularly at days 1, 21, and 42 with 100 μg construct + 100 μg adjuvant (pUMCV1-GM-CSF). On day 49, the final blood collection was performed and serum was collected. The negative control plasmid cM2 was administered by gene gun using about 10 μg construct + about 2 μg adjuvant (pUMCV1-GM-CSF) and the same immunization scheme. Serum was analyzed by ELISA using recombinant full length HA from A / Hong Kong / 1/1968 (obtained from Protein Sciences Corporation, Meriden, CT, USA) as an antigen. Briefly, 96 well plates were coated overnight at 4 ° C. with 50 ng HA and then incubated with block buffer (100 μl PBS, pH 7.4 + 2% skim milk) for 1 hour at room temperature. The plate is washed with PBS + 0.05% Tween-20 and 100 μl of a 2-fold dilution series in block buffer starting with a 50-fold dilution of serum is added. Bound antibody is detected using standard protocols well established in the art using HRP-conjugated goat anti-mouse IgG. Titers are compared to a standard curve composed of serial dilutions of mouse monoclonal antibodies that bind to the HA antigen and expressed as ELISA units per ml (EU / ml).

  The results of an ELISA using HA from A / Hong Kong / 1/1968 after 49 days are shown in FIG. Sera obtained from mice immunized with DNA encoding the stem domain polypeptide included in this experiment can recognize allogeneic full-length HA from A / Hong Kong / 1/1968. As expected, immunization with DNA encoding full-length HA results in high antibody titers against the homologous HA protein, whereas immunization with a negative control expression vector encoding cM2 is A / HongKong / 1 / It does not induce antibodies that recognize 1968 full-length HA.

  Taken together, the data show that polypeptides of the invention derived from H3HA can induce an immune response directed against full length H3HA.

Example 28
Design of alternative stem domain polypeptides based on H1HA that can elicit antibodies that neutralize group 1 and group 2 influenza viruses Examples 4 and 6 show that H1 stably exposes epitopes of broadly neutralizing CR6261 antibodies. Disclosed are sequence-based polypeptides. Given the fact that CR6261 neutralizes influenza virus from phylogenetic group 1 exclusively, polypeptides designed against this epitope cannot elicit a strong response to phylogenetic group 2 influenza virus. Another approach to designing polypeptides according to the present invention that induce such broad cross-neutralizing antibodies uses H1HA sequence variants that are more closely similar to H3HA sequences with respect to the structural and biochemical characteristics of key amino acids in the epitope. It is to be. Based on a comparison between the structure of group-specific antibodies and molecules (CR6261, F10 and HB36) and crystallized pan influenza antibody FI6 (Corti et al, 2011), we have group 1-group 2T49N (HA2) mutations. It has been found that mutations can only be provided by FI6 without the introduction of steric collisions. Asparagine at position 49 of HA2 is present in two group 1 viruses in the NCBI influenza database: A / swine / Hubei / S1 / 2009 (ACY06623) and A / swine / Haseluene / IDT2617 / 2003 (ABV60697). Thus, in one embodiment, the H1 sequences that form the basis of the invention disclosed in Examples 4, 6 and 9 are those of their N49-containing HA sequences. Alternatively, the sequences according to the invention described in Examples 4, 6 and 9 have an additional mutation at position 49 in HA2 to change T to N amino acids. Table 7 shows a sequence alignment of exemplary H1HA sequences that can be used as starting sequences for the polypeptides of the invention.

  SEQ ID NO: 180 is derived from SEQ ID NO: 45 by mutation T392N (numbering refers to SEQ ID NO: 1). T392 in SEQ ID NO: 1 corresponds to threonine at position 49 in HA2, as described above. Using methods well known to those skilled in the art, the gene encoding this polypeptide of the invention was synthesized and cloned into the expression vector pcDNA2004. The presence of CR9114 and CR6261 neutralizing epitopes was confirmed by fluorescence-coupled cell sorting as described above. The results are shown in FIG. The MFI for SEQ ID NO: 180 is equivalent to the MFI observed for SEQ ID NO: 45 and SEQ ID NO: 1 for CR6261 and CR9114 binding, while CR9020 (known to bind to the head region of the full-length HA molecule) Only number 1 is recognized and CR8020 (specific for HA of influenza A group 2) does not recognize SEQ ID NO 180, SEQ ID NO 1 or SEQ ID NO 45. The negative control cM2 is not recognized by any of the monoclonal antibodies used in this experiment.

  In summary, SEQ ID NO: 180 containing mutation T392N contains neutralizing epitopes for CR6261 and CR9114.

Example 29
Design of Additional Polypeptides of the Invention Lacking Transmembrane Sequences The native form of influenza HA exists as a trimer on cell or viral membranes. In certain embodiments, intracellular and transmembrane sequences are removed such that a secreted (soluble) polypeptide is produced after expression in the cell. Methods for expressing and purifying the secreted ectodomain of HA have been described (see, for example, Dopheide et al 2009; Ekiert et al 2009, 2011; Stevens et al 2004, 2006; Wilson et al 1981). Those skilled in the art will appreciate that these methods can also be applied directly to the stem domain polypeptides of the present invention to achieve expression of secreted (soluble) polypeptides. Accordingly, these polypeptides are also encompassed by the present invention. For example, in the case of a polypeptide of the invention derived from the HA sequence of a group 1 influenza virus, the soluble polypeptide of the invention is from residue 514 (equivalent) to the C-terminus (numbering refers to SEQ ID NO: 1) Can be generated by deletion of the polypeptide sequence. Alternatively, additional residues can be included in the polypeptides of the invention, for example by deleting sequences from residues 515, 516, 517, 518, 519, 520, 521 or 522. Optionally, a his tag sequence (HHHHHH or HHHHHHH) can be added, optionally linked via a linker, for purification purposes. Optionally, the linker may contain a proteolytic cleavage site to remove the his tag after purification. Soluble polypeptides can be further stabilized by introducing sequences known to form trimer structures, such as foldon sequences. Polypeptides obtained as described above are also encompassed by the present invention.

SEQ ID NOs: 181-185 show the sequences of the soluble polypeptides of the present invention derived from the HA sequence of H1N1A / Brisbane / 59/2007. Similarly, SEQ ID NOs: 186-187 show the sequences of the soluble polypeptides of the present invention derived from the H3N2A / HongKong / 1/1968 HA sequence. One skilled in the art will appreciate that equivalent sequences can be designed for polypeptides of the invention derived from other HA sequences of other influenza A vaccine strains, eg, H1, H3, H5 subtypes. It will also be apparent to those skilled in the art that six C-terminal histidines are attached for purification purposes. Because there are other purification methods that do not use this tag, the six histidine sequences are optional, and sequences lacking this purification tag are also encompassed by the present invention.

References

Various embodiments and uses of the polypeptides according to the present invention will become apparent from the following detailed description of the invention.
The present invention also provides the following.
[1] (a) an influenza hemagglutinin HA1 domain comprising an HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a binding sequence of 0 to 50 amino acid residues, and (b) an influenza hemagglutinin stem comprising an influenza hemagglutinin HA2 domain A domain polypeptide, wherein the hemagglutinin stem domain polypeptide is resistant to protease cleavage at the junction between HA1 and HA2, and wherein one or more amino acids in the amino acid sequence linking the A helix and helix CD of HA2 Is mutated compared to the wild-type influenza HA2 domain, said HA1 and HA2 domains being derived from an influenza A virus subtype selected from the group consisting of H1, H5 and H3 Incoming influenza hemagglutinin stem domain polypeptide.
[2] The polypeptide according to [1], which does not contain a full-length HA1 domain.
[3] The polypeptide according to [1] or [2], which is glycosylated.
[4] (a) HA1 N-terminal segment comprising amino acids 1-x of HA1 covalently bonded to the HA1 C-terminal stem segment comprising amino acids y to terminus of HA1 by a binding sequence of 0 to 50 amino acid residues, and (b) The polypeptide according to [1], [2] or [3], comprising an influenza hemagglutinin HA2 domain.
[5] An influenza hemagglutinin-based influenza hemagglutinin HA1 and / or HA2 domain from an influenza A virus containing HA of the H1 subtype, wherein x = 46-60 any amino acid, y = 290-325 The polypeptide according to any one of [1] to [4], which is an arbitrary amino acid.
[6] The influenza hemagglutinin HA1 and / or HA2 domain based on influenza hemagglutinin from an influenza A virus comprising an HA subtype of HA selected from the group consisting of the influenza A viruses of Table 7 Polypeptide.
[7] Influenza hemagglutinin-based influenza hemagglutinin HA1 and / or HA2 domain from influenza A virus containing HA of H3 subtype, wherein x = 56-69 any amino acid, y = 292-325 The polypeptide according to any one of [1] to [4], which is an arbitrary amino acid.
[8] The C-terminal amino acid residue of the HA1 C-terminal stem segment is any amino acid other than arginine (R) or lysine (K), preferably glutamine (Q), [1] to [7] The polypeptide according to any one of the above.
[9] The polypeptide according to any one of [1] to [8], comprising one or more additional mutations in the HA1 domain and / or the HA2 domain.
[10] The polypeptide according to any one of [1] to [9], comprising one or more disulfide bridges.
[11] The polypeptide according to any one of [1] to [10], which does not include a signal sequence.
[12] HA1 N-terminal segment comprising HA1 amino acids p to x covalently linked to the HA1 C-terminal stem segment comprising amino acids y to terminus of HA1 by a binding sequence of 0 to 50 amino acid residues, and (b) influenza hemagglutinin HA2 The polypeptide of [11], comprising a domain and p = first amino acid of mature HA.
[13] The polypeptide according to any one of [1] to [12], which does not include intracellular and transmembrane sequences of HA.
[14] does not include the C-terminal part of the influenza H1HA2 domain spanning from amino acid residues 520, 521, 522, 523, 524, 525, 526, 527, 528, 529 or 530 of H1HA2 to the C-terminal amino acid, [13] The described polypeptide.
[15] The polypeptide according to any one of [1] to [14], which selectively binds to antibody CR6261 and / or CR9114 and does not bind to antibody CR8057.
[16] The polypeptide according to any one of [1] to [15], which selectively binds to antibody CR8020, CR843 and / or CR9114 and does not bind to antibody CR8057.
[17] (a) providing an influenza HA0 amino acid sequence;
(B) removing the cleavage site of HA1 to HA2;
(C) removing the amino acid sequence of the globular head domain from the HA0 sequence;
(D) introducing one or more mutations in the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD; and
(E) introducing one or more disulfide bonds into the HA stem domain polypeptide.
A method of providing an influenza hemagglutinin stem domain polypeptide comprising:
[18] An influenza hemagglutinin stem domain polypeptide obtained by the method according to [17].
[19] A nucleic acid encoding the polypeptide according to any one of [1] to [16].
[20] An immunogenic composition comprising the polypeptide according to any one of [1] to [16] and / or the nucleic acid molecule according to [19].
[21] The polypeptide according to any one of [1] to [16], the nucleic acid according to [19], and / or the nucleic acid according to [20], which is used as a pharmaceutical, particularly as a vaccine. An immunogenic composition.

Claims (21)

  (A) an influenza hemagglutinin HA1 domain comprising an HA1 N-terminal stem segment covalently linked to the HA1 C-terminal stem segment by a binding sequence of 0-50 amino acid residues, and (b) an influenza hemagglutinin stem domain polypeptide comprising an influenza hemagglutinin HA2 domain Wherein the hemagglutinin stem domain polypeptide is resistant to protease cleavage at the junction between HA1 and HA2, and one or more amino acids in the amino acid sequence linking HA2's A helix and helix CD are wild-type Mutated compared to the influenza HA2 domain, said HA1 and HA2 domains are derived from an influenza A virus subtype selected from the group consisting of H1, H5 and H3 Influenza hemagglutinin stem domain polypeptide.   2. The polypeptide of claim 1, wherein the polypeptide does not comprise a full length HA1 domain.   The polypeptide according to claim 1 or 2, which is glycosylated.   (A) HA1 N-terminal segment comprising HA1 amino acids 1-x covalently linked to the HA1 C-terminal stem segment comprising amino acids y-terminal of HA1 by a binding sequence of 0-50 amino acid residues, and (b) influenza hemagglutinin HA2 4. A polypeptide according to claim 1, 2 or 3, comprising a domain.   Influenza hemagglutinin-based influenza hemagglutinin HA1 and / or HA2 domain from influenza A virus containing H1 subtype HA, x = 46-60 any amino acid, y = 290-325 any amino acid The polypeptide according to any one of claims 1 to 4, which is   6. The polypeptide of claim 5, comprising an influenza hemagglutinin HA1 and / or HA2 domain based on influenza hemagglutinin from an influenza A virus comprising an HA subtype of HA selected from the group consisting of the influenza A viruses of Table 7. .   Influenza hemagglutinin-based influenza hemagglutinin HA1 and / or HA2 domain from influenza A virus containing H3 subtype HA, any amino acid x = 56-69 and any amino acid y = 292-325 The polypeptide according to any one of claims 1 to 4, which is   The C-terminal amino acid residue of the HA1 C-terminal stem segment is any amino acid other than arginine (R) or lysine (K), preferably glutamine (Q). The described polypeptide.   9. A polypeptide according to any one of the preceding claims comprising one or more further mutations in the HA1 domain and / or the HA2 domain.   10. A polypeptide according to any one of claims 1 to 9, comprising one or more disulfide bridges.   The polypeptide according to any one of claims 1 to 10, which does not contain a signal sequence. HA1 N-terminal segment comprising HA1 amino acids p to x covalently linked to the HA1 C-terminal stem segment comprising amino acids y to terminus of HA1 by a binding sequence of 0 to 50 amino acid residues, and (b) comprising an influenza hemagglutinin HA2 domain 12. The polypeptide of claim 11, wherein p = first amino acid of mature HA.   The polypeptide according to any one of claims 1 to 12, which does not contain intracellular and transmembrane sequences of HA.   14. The poly of claim 13, not including the C-terminal portion of the influenza H1 HA2 domain spanning from the amino acid residues 520, 521, 522, 523, 524, 525, 526, 527, 528, 529 or 530 of H1HA2 to the C-terminal amino acid. peptide.   15. A polypeptide according to any one of claims 1 to 14, which selectively binds to antibody CR6261 and / or CR9114 and does not bind to antibody CR8057.   16. A polypeptide according to any one of claims 1-15, which selectively binds to antibody CR8020, CR843 and / or CR9114 and does not bind to antibody CR8057. (A) providing an influenza HA0 amino acid sequence;
(B) removing the cleavage site of HA1 to HA2;
(C) removing the amino acid sequence of the globular head domain from the HA0 sequence;
(D) introducing one or more mutations in the amino acid sequence linking the C-terminal residue of helix A to the N-terminal residue of helix CD; and (e) 1 in the HA stem domain polypeptide. A method of providing an influenza hemagglutinin stem domain polypeptide comprising introducing one or more disulfide bonds.   An influenza hemagglutinin stem domain polypeptide obtained by the method of claim 17.   A nucleic acid encoding the polypeptide according to any one of claims 1 to 16.   An immunogenic composition comprising the polypeptide of any one of claims 1 to 16, and / or the nucleic acid molecule of claim 19.   21. A polypeptide according to any one of claims 1 to 16, a nucleic acid according to claim 19, and / or an immunogenic composition according to claim 20, for use as a medicament, in particular as a vaccine. .