NZ715583B2

NZ715583B2 - Influenza virus vaccines and uses thereof

Info

Publication number: NZ715583B2
Application number: NZ715583A
Authority: NZ
Inventors: Zhaoqing Ding; Antonietta Impagliazzo; Jan Wilem Meijberg; Katarina Radosevic; Jehangir Wadia; Michelle Wagner; Robert Anthony Williamson
Original assignee: Janssen Vaccines & Prevention Bv
Priority date: 2013-05-30
Filing date: 2014-05-27
Publication date: 2021-05-27

Abstract

The present invention provides influenza hemagglutinin stem domain polypeptides comprising (a) an influenza hemagglutinin HA1 domain that comprises an HAl N-terminal stem segment, comprising the amino acids from position 1 to position x, preferably from position p to position x, of the HA1 domain, covalently linked by a linking sequence of 0-50 amino acid residues to an HA1 C- terminal stem segment, comprising the amino acids from position y to and including the C-terminal amino acid of the HA1 domain and (b) an influenza hemagglutinin HA2 domain, wherein the hemagglutinin stem domain polypeptide is resistant to protease cleavage at the junction between HA1 and HA2, and wherein one or more amino of the amino acids at positions 337, 340, 352, 353, 402, 406, 409, 413 and/or 416 have been mutated as compared to the corresponding positions in wild-type influenza HA. ovalently linked by a linking sequence of 0-50 amino acid residues to an HA1 C- terminal stem segment, comprising the amino acids from position y to and including the C-terminal amino acid of the HA1 domain and (b) an influenza hemagglutinin HA2 domain, wherein the hemagglutinin stem domain polypeptide is resistant to protease cleavage at the junction between HA1 and HA2, and wherein one or more amino of the amino acids at positions 337, 340, 352, 353, 402, 406, 409, 413 and/or 416 have been mutated as compared to the corresponding positions in wild-type influenza HA.

Description

INFLUENZA VIRUS VACCINES AND USES THEREOF INTRODUCTION The invention relates to the field of medicine. Described herein are influenza hemagglutinin stem domain polypeptides, methods for providing hemagglutinin stem domain polypeptides, compositions comprising the same, vaccines comprising the same and methods of their use, in particular in the detection, prevention and/or treatment of influenza.

BACKGROUND Influenza viruses are major human pathogens, causing a respiratory disease (commonly referred to as "influenza" or "the flu") that ranges in severity from sub- clinical infection to primary viral pneumonia which can result in death. The clinical effects of infection vary with the virulence of the influenza strain and the exposure, history, age, and immune status of the host. Every year it is estimated that approximately 1 billion people worldwide undergo infection with influenza virus, leading to severe illness in 3-5 million cases and an estimated 300,000 to 500,000 of influenza related deaths. The bulk of these infections can be attributed to influenza A viruses carrying H1 or H3 hemagglutinin subtypes, with a smaller contribution from Influenza B viruses, and therefore representatives of all three are included in the seasonal vaccine. The current immunization practice relies on early identification of circulating influenza viruses to allow for timely production of an effective seasonal influenza vaccine. Apart from the inherent difficulties in predicting the strains that will be dominant during the next season, antiviral resistance and immune escape also play a role in failure of current vaccines to prevent morbidity and mortality. In addition to this the possibility of a pandemic caused by a highly virulent viral strain originating from animal reservoirs and reassorted to increase human to human spread, poses a significant and realistic threat to global health.

Influenza A viruses are widely distributed in nature and can infect a variety of birds and mammals. Influenza viruses are enveloped RNA viruses that belong to the family of Orthomyxoviridae. Their genomes consist of eight single-stranded RNA segments that code for 11 different proteins, one nucleoprotein (NP), three polymerase proteins (PA, PB1, and PB2), two matrix proteins (M1 and M2), three non-structural proteins (NS1, NS2, and PB1- F2), and two external glycoproteins: hemagglutinin (HA) and neuraminidase (NA). The viruses are classified on the basis of differences in antigenic structure of the HA and NA proteins, with their different combinations representing unique virus subtypes that are further classified into specific influenza virus strains. Although all known subtypes can be found in birds, currently circulating human influenza A subtypes are H1N1 and H3N2.

Phylogenetic analysis has demonstrated a subdivision of hemagglutinins into two main groups: inter alia the H1, H2, H5 and H9 subtypes in phylogenetic group 1 and inter alia the H3, H4 and H7 subtypes in phylogenetic group 2.

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

It is known that antibodies that neutralize the influenza virus 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 mediates the fusion of the viral and endosomal membrane leading to release of the viral RNA in the cytosol of the cell.

HA comprises a large head domain and a smaller stem domain. Attachment to the viral membrane is mediated by a C-terminal anchoring sequence connected to the stem domain. The protein is post-translationally cleaved in a designated loop to yield two polypeptides, HA1 and HA2 (the full sequence is referred to as HA0). The membrane distal head region is mainly derived from HA1 and the membrane proximal stem region primarily from HA2 (.

The reason that the seasonal influenza vaccine must be updated every year is the large variability of the virus. In the hemagglutinin molecule this variation is particularly manifested in the head domain where antigenic drift and shift have resulted in a large number of different variants. Since this is also the area that is immunodominant, most neutralizing antibodies are directed against this domain and act by interfering with receptor binding. The combination of immunodominance and large variation of the head domain also explains why infection with a particular strain does not lead to immunity to other strains: the antibodies elicited by the first infection only recognize a limited number of strains closely related to the virus of the primary infection.

Recently, influenza hemagglutinin stem domain polypeptides, lacking all or substantially all of the influenza hemagglutinin globular head domain, have been described and used to generate an immune response to one or more conserved epitopes of the stem domain polypeptide. It is believed that epitopes of the stem domain polypeptide are less immunogenic than the highly immunogenic regions of a globular head domain, thus the absence of a globular head domain in the stem domain polypeptide might allow an immune response against one or more epitopes of the stem domain polypeptide to develop (Steel et al., 2010). Steel et al. thus have created a new molecule by deleting amino acid residue 53 to 276 of HA1 of the A/Puerto Rico/8/1934 (H1N1) and A/Hong Kong/1968 (H3N2) strains from the HA primary sequence, and replacing this by a short flexible linking sequence GGGG. Vaccination of mice with the H3 HK68 construct did not elicit antisera that were cross-reactive with group 1 HAs. In addition, as shown in , the stem domain polypeptides were highly unstable and did not adopt the correct conformation as proven by the lack of binding of antibodies that were shown to bind to conserved epitopes in the stem region.

In addition, Bommakanti et al. (2010) described an HA2 based polypeptide comprising amino acid residues 1-172 of HA2, a 7-amino acid linker (GSAGSAG), amino acid residues 7-46 of HA1, a 6-amino acid linker GSAGSA, followed by residues 290-321 of HA1, with the mutations V297T, I300E, Y302T and C305T in HA1. The design was based on the sequence of H3 HA (A/Hong Kong/1968). The polypeptide did only provide cross-protection against another influenza virus strain within the H3 subtype (A/Phil/2/82 but not against an H1 subtype (A/PR/8/34). In a more recent paper by Bommakanti et al (2012) a stem domain sequence based on HA from H1N1 A/Puerto Rico/8/1934 (H1HA0HA6) is described. In this polypeptide the equivalent of residues 55 to 302 have been deleted and mutations I311T, V314T, I316N, C319S, F406D, F409T, and L416D have been made. Both the H3 and HA based polypeptides were expressed in E. coli and therefore lack the glycans that are aprt of the naturally occurring HA proteins.

When expressed in E.coli the polypeptide is recovered mainly as high molecular weight aggregates and a minor monomeric fraction. The polypeptide binds CR6261 with two apparent dissociation constants of 9 and 0.2 μM. The authors show that mice can survive a challenge with 1 LD90 of the homologous H1N1 A/Puerto Rico/8/1934 virus after immunization (twice, 4 week interval) with 20 μg of protein adjuvanted with 100 μg of CpG7909. The authors also describe circularly permutated polypeptides comparable to those described above for A/Hong Kong/1/1968 derived polypeptides. These polypeptides are derived from HA’s from H1N1 A/Puerto Rico/8/1934, H1N1 A/North Carolina/20/99 or H1N1 A/California/07/2009 and can provide partial protection in a mild challenge (1LD90) model in mice of H1N1 A/Puerto Rico/8/1934 (i.e. within the same subtype). Sera from guinea pigs immunized with these polypeptides did not exhibit detectable levels of neutralization when tested in a neutralization assay.

There thus still exists a need for a safe and effective universal vaccine that stimulates the production of a robust, broadly neutralizing antibody response and that offers protection against a broad set of current and future influenza virus strains (both seasonal and pandemic), in particular providing protection against one or more influenza A virus subtypes within phylogenetic group 1 and/or group 2, for effective prevention and therapy of influenza. It is an object of the present invention to go some way towards meeting this need and/or at least to provide the public with a useful choice.

SUMMARY Described herein are influenza hemagglutinin stem domain polypeptides, methods for providing stem domain polypeptides, compositions comprising the same, vaccines comprising the same and methods of their use.

In a first aspect, the present invention provides an influenza hemagglutinin (HA) stem domain polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 66 and SEQ ID NO: 73.

In a second aspect the present invention provides a nucleic acid molecule encoding a polypeptide of the first aspect.

In a third aspect the present invention provides a vector comprising the nucleic acid molecule of the second aspect.

In a fourth aspect the present invention provides a composition comprising the polypeptide of the first aspect, and/or a nucleic acid molecule according to the second aspect and/or a vector according to the third aspect.

In a fifth aspect the present invention provides a use of an influenza HA stem domain polypeptide according to the first aspect, a nucleic acid according to the second aspect, and/or a composition according to the fourth aspect in the manufacture of a medicament.

In a sixth aspect the present invention provides a use of an influenza HA stem domain polypeptide according to the first aspect, a nucleic acid molecule according to the second aspect and/or a composition according to the fourth aspect in the manufacture of a medicament for inducing an immune response against influenza HA protein in a patient in need thereof.

In a seventh aspect the present invention provides a use of an influenza HA stem domain polypeptide according to the first aspect, a nucleic acid molecule according to the second aspect and/or a composition according to the fourth aspect in the manufacture of a medicament for the prophylaxis and/or treatment of influenza infection in a patient in need thereof.

Also described are novel immunogenic polypeptides comprising an influenza hemagglutinin stem domain and lacking the globular head, referred to as influenza hemagglutinin (HA) stem domain polypeptides. The polypeptides are capable of inducing an immune response when administered to a subject, in particular a human subject. The polypeptides described herein present conserved epitopes of the membrane proximal stem domain HA molecule to the immune system in the absence of dominant epitopes that are present in the membrane distal head domain. To this end, part of the primary sequence of the HA0 protein making up the head domain is removed and the remaining amino acid sequence is reconnected, either directly or, in some embodiments, by introducing a short flexible linking sequence (‘linker’) to restore the continuity of the amino acid chain. The resulting sequence is further modified by introducing specific mutations that stabilize the native 3-dimensional structure of the remaining part of the HA0 molecule. The immunogenic polypeptides do not comprise the full-length HA1 and/or HA2 of an influenza virus.

According to the present disclosure, the polypeptides are based on HA of influenza A viruses of the H1 subtype.

The present disclosure describes influenza hemagglutinin stem domain polypeptides comprising (a) an influenza hemagglutinin HA1 domain that comprises an HAl N- terminal stem segment, covalently linked by a linking sequence of 0-50 amino acid residues to an HA1 C- terminal stem segment, and (b) an influenza hemagglutinin HA2 domain, wherein the hemagglutinin stem domain polypeptides are resistant to protease cleavage at the junction between HA1 and HA2,and wherein one or more amino acids in the amino acid sequence connecting the A helix and the helix CD of HA2 have been mutated as compared to a wild-type influenza HA2 domain..

In certain embodiments, the polypeptides described herein comprise one or more mutations on position 337, 340, 352 or 353 of SEQ ID NO: 1, or equivalent positions in other influenza viruses of the H1 subtype. A mutation means that an amino acid on a specific position has been substituted by another amino acid which is not present on the corresponding position in the wild-type influenza HA, i.e. the HA of the influenza virus on which the stem polypeptide is based.

In certain embodiments, the polypeptides described herein further comprise one or more mutations in the HA2 amino acid sequence connecting the C-terminal residue of helix A to the N-terminal residue of helix CD, as indicated in In certain embodiments, the HA1 N- terminal stem segment comprises the amino acids 1-x of HA1, and the HA1 C- terminal stem segment comprises the amino acids y-end (i.e. the C-terminal amino acid of HA1) of HA1. Thus, in certain embodiments, the deletion in the HA1 segment comprises the amino acid sequence from the amino acid at position x+1 up to and including the amino acid at position y-1.In certain embodiments, the polypeptides do not comprise the signal sequence. In certain embodiments, the HA1 N- terminal segment thus comprises the amino acid p-x of HA1, wherein p is the first amino acid of the mature HA molecule (e.g. p=18 in case of SEQ ID NO: 1). The skilled person will be able to prepare the polypeptides described herein without the signal peptides (e.g. amino acids 1-17 of SEQ ID NO: 1).

In certain embodiments, the polypeptides described herein contain the intracellular sequences of HA and the transmembrane domain. In other embodiments, the polypeptides described herein do not comprise the intracellular sequences of HA and the transmembrane domain. In certain embodiments, the intracellular and transmembrane sequence, e.g. the amino acid sequence from position (or the equivalent of) 519, 520, 521, 522, 523, 524, 525, 526, 527, 526, 528, 529, or 530 of the HA2 domain to the C-terminus of the HA2 domain has been removed.

The polypeptides described herein do not comprise the full length HA1.

In certain embodiments, the polypeptides are glycosylated.

In certain embodiments, the immunogenic polypeptides are substantially smaller than HA0, preferably lacking all or substantially all of the globular head of HA. Preferably, the immunogenic polypeptides are no more than 360, preferably no more than 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280, 275, or 270 amino acids in length. In certain embodiments, the immunogenic polypeptides are 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 polypeptides further comprise 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 HA 1 and HA2 domains are based.

The polypeptides described herein comprise the conserved stem domain epitopes of the group 1 cross-neutralizing antibody CR6261 (as disclosed in WO2008/028946) and/or of the antibody CR9114 (as described in WO2013/007770), an antibody capable of binding to and neutralizing both group 1 and group 2 influenza A viruses, as well as influenza B viruses. It also described are HA stem domain polypeptides, wherein said polypeptides stably present the epitopes of the antibody CR6261 and/or CR9114, as indicated by binding of said antibody or antibodies to said polypeptides. In an embodiment, the polypeptides do not bind to CR8020 and CR8057 (described in ), which are monoclonal antibodies that binds to H3 influenza viruses only. The influenza hemagglutinin stem domain polypeptides described herein are suitable for use in immunogenic compositions (e.g. vaccines) capable of generating immune responses against a plurality of influenza virus A and/or B strains. In an embodiment, the influenza hemagglutinin stem domain polypeptides are capable of generating immune responses against influenza A virus strains of phylogenetic group 1 and/or group 2, in particular against influenza virus strains of both phylogenetic group 1 and group 2. In an embodiment, the polypeptides are capable of generating an immune response against homologous influenza virus strains. In an embodiment, the polypeptides are capable of generating an immune response against heterologous influenza virus strains of the same and/or different subtypes. In a further embodiment, the polypeptides are capable of generating an immune response to influenza virus strains of both phylogenetic group 1 and group 2 and influenza B virus strains.

The polypeptides described herein may be used e.g. in stand alone therapy and/or prophylaxis and/or diagnosis of a disease or condition caused by an influenza virus, in particular a phylogenetic group 1 or 2 influenza A virus and/or an influenza B virus, or in combination with other prophylactic and/or therapeutic treatments, such as (existing or future) vaccines, antiviral agents and/or monoclonal antibodies.

In a further aspect, described are nucleic acid molecules encoding the influenza HA stem domain polypeptides. In yet another aspect, described are vectors comprising the nucleic acids encoding the immunogenic polypeptides.

In a further aspect, described are methods for inducing an immune response in a subject, the method comprising administering to the subject a polypeptide and/or nucleic acid molecule described herein.

In another aspect, described are immunogenic compositions comprising a polypeptide and/or a nucleic acid molecule described herein. The immunogenic compositions described herein can be in any form that allows for the compositions to be administered to a subject, e.g. mice, ferrets or humans. In a specific embodiment, the immunogenic compositions are suitable for human administration. The polypeptides, nucleic acid molecules and compositions may be used in methods of preventing and/or treating an influenza virus disease and/or for diagnostic purposes. The compositions may further comprise a pharmaceutically acceptable carrier or excipient. In certain embodiments, the compositions described herein comprise, or are administered in combination with, an adjuvant.

In another aspect, described are polypeptides, nucleic acids and/or immunogenic compositions for use as a vaccine. Also described are immunogenic polypeptides, nucleic acids, and/or immunogenic compositions for use as a vaccine in the prevention and/or treatment of a disease or condition caused by an influenza virus A subtype of phylogenetic group 1 and/or 2 and/or influenza B virus.

The various embodiments and uses of the polypeptides according to the invention will become clear from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE FIGURES shows a model of the HA monomer in the pre-fusion state as present in the native trimer. HA 1 is shown in light grey, HA2 is shown in dark grey. Helix A (an important part of the epitope of CR6261) and helix CD (part of the trimer interface) are indicated, as is the loop connecting these secondary structure elements. The C-terminus of HA1 and the N-terminus of HA2 are also indicated. The fusion peptide is located at the N-terminus of HA2.

Binding of soluble polypeptides of the invention to monoclonal antibodies CR6261 and CR9114 using biolayer interferometry. Top panels show individual binding curves for immobilized monoclonal antibodies exposed to varying concentrations of soluble polypeptides of the invention, bottom panels show the steady state analysis used to estimate K . A: s127H1 SEQ ID NO: 66. B: s86B4 SEQ ID NO: 67. C: s74H9 SEQ ID NO: 65. D: s6E12 SEQ ID NO: 69. E: s55G7 SEQ ID NO: 68.

Size exclusion chromatograms of soluble polypeptides of the invention in the absence and presence of Fab Fragments of CR8020, CR6261 and CR9114. For all soluble polypeptides of the invention complex formation is observed for CR6261 and CR9114 Fab fragments, but not for CR8020 Fab fragments. A: s127H1 SEQ ID NO: 66. B: s86B4 SEQ ID NO: 67. C: s74H9 SEQ ID NO: 65. D: s6E12 SEQ ID NO: 69. E: s55G7 SEQ ID NO: 68.

Evaluation of protective efficacy of polypeptide of the invention s74H9 SEQ ID NO: 65 and s86B4 SEQ ID NO: 67 in a lethal influenza H1N1 A/NL/602/09 challenge model. A: Top row: Survival, mean body weight change and median clinical score for the negative (PBS) and positive control (CR6261) groups. Bottom row: Survival, mean body weight change and median clinical score for the experimental groups immunized with s74H9 or s86B4. For reasons of comparison the negative control PBS group is also shown. B: Immunogenicity of s74H9 (SEQ ID NO: 65) and s86B4 (SEQ ID NO: 67).

Top row: left and middle panel: immunization induces antibodies capable of recognizing the cognate antigen (s74H9 left, s86B4 middle panel) as well as full length HA from H1N1 A/Brisbane/59/07 (right panel) as determined by ELISA. Bottom row: The induced antibodies are capable of competing with CR9114 for binding to full length HA from H1N1 A/Brisbane/59/07 in a competition ELISA (left panel). For reasons of comparison competition levels by unlabeled CR9114 (i.e. self-competition) and the non-binding monoclonal antibody CR8020, both serially diluted from 5 μg/ml starting concentration, are indicated in a separate graph.

Evaluation of protective efficacy of polypeptide of the invention s127H1 (SEQ ID NO: 66) in a lethal influenza H1N1 A/Puerto Rico/8/1934 challenge model. A: Top row: Survival, mean body weight change and median clinical score for the negative (PBS) and positive control (CR6261) groups. Bottom row: Survival, mean body weight change and median clinical score for the experimental group immunized with s127H1 (SEQ ID NO: 35). For reasons of comparison the negative control PBS group is also shown.

B: Immunogenicity of s127H1 (SEQ ID NO: 35). Top row:: immunization induces antibodies capable of recognizing the cognate antigen s127H1 (SEQ ID NO: 35) (left panel) as well as full length HA from H1N1 A/Brisbane/59/07 (right panel) as determined by ELISA. Bottom row: The induced antibodies are capable of competing with CR9114 for binding to full length HA from H1N1 A/Brisbane/59/07 in a competition ELISA (left panel). For reasons of comparison competition levels by unlabeled CR9114 (i.e. self- competition) and the non-binding monoclonal antibody CR8020, both serially diluted from 5 μg/ml starting concentration, are indicated in a separate graph.

Observed and expected frequency of occurrence of an amino acid at the indicated position in Set 1 sequences that show improved binding to CR6261. Values are expressed as percentage of the total number of Set 1 sequences that show improved CR6261 binding. Expected values are calculated as 100% divided by the number of variable amino acids at each position included in the set.

Frequency of occurrence of combinations of amino acids in improved CR6261 binders from Set1. Sequences with improved binding to CR6261 were grouped according to the presence of amino acids at the positions indicated on the left, and the frequency of each combination was calculated as a percentage of the total number of Set 1 sequences that show improved CR6261 binding. Combinations that are more prevalent have been boxed. A. Analysis of combination sequences in the fusion peptide area. B. Analysis of the B-loop area.

Observed and expected frequency of occurrence of an amino acid at the indicated position in Set 2 sequences that show improved binding to CR6261 Values are expressed as percentage of the total number of Set 2 sequences that show improved CR6261 binding. Expected values are calculated as 100% divided by the number of variable amino acids at each position included in the set.

Frequency of occurrence of combinations of amino acids in improved CR6261 binders from Set 2. Sequences with improved binding to CR6261 were grouped according to the presence of amino acids at the positions indicated on the left, and the frequency of each combination was calculated as a percentage of the total number of Set 2 sequences that show improved CR6261 binding. Combinations that are more prevalent have been boxed. Variation at position 402 was not included in the analysis as all improved binding sequences contain Met at this position , whereas for position 352 only sequences containing either Phe or Tyr were taken into account. A. Analysis of combination sequences in the fusion peptide area. B. Analysis of the B-loop area.

DEFINITIONS Definitions of terms as used in the present disclosure are given below.

An amino acid described herein can be any of the twenty naturally occurring (or ‘standard’ amino acids) or variants thereof, such as e.g. D-proline (the D-enantiomer of proline), or any variants that are not naturally found in proteins, such as e.g. norleucine. The standard amino acids can be divided into several groups based on their properties.

Important factors are charge, hydrophilicity or hydrophobicity, size and functional groups.

These properties are important for protein structure and protein–protein interactions. Some amino acids have special properties such as cysteine, that can form covalent disulfide bonds (or disulfide bridges) to other cysteine residues, proline that forms a cycle to the polypeptide backbone, and glycine that is more flexible than other amino acids. Table 2 shows the abbreviations and properties of the 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. Percent identity is the percentage of amino acid residues in a candidate sequence that are identical (i.e., the amino acid residues at a given position in the alignment are the same residue) or similar (i.e., the amino acid substitution at a given position in the alignment is a conservative substitution, as discussed below), to the corresponding amino acid residue in the peptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence homology. Sequence homology, including percentages of sequence identity and similarity, are determined using sequence alignment techniques well- known in the art, such as by visual inspection and mathematical calculation, or more preferably, the comparison is done by comparing sequence information using a computer 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)).

"Conservative substitution" refers to replacement of an amino acid of one class is with another amino acid of the same class. In particular embodiments, a conservative substitution does not alter the structure or function, or both, of a polypeptide. Classes of amino acids for the purposes of conservative substitution include hydrophobic (e.g. Met, Ala, Val, Leu), neutral hydrophilic (e.g. Cys, Ser, Thr), acidic (e.g. Asp, Glu), basic (e.g.

Asn, Gln, His, Lys, Arg), conformation disrupters (e.g. Gly, Pro) and aromatic (e.g. 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, in particular an influenza virus infection. In specific embodiments, a term "disease" refers to the pathological state resulting from the presence of the virus in a cell or a subject, or by the invasion of a cell or subject by the virus. In certain embodiments, the condition is a disease in a subject, the severity of which is decreased by inducing an immune response in the subject through the administration of an immunogenic composition.

As used herein, the term "effective amount" in the context of administering a therapy to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s). In certain embodiments, an "effective amount" in the context of administration of a therapy to a subject refers to the amount of a therapy which is sufficient to achieve a reduction or amelioration of the severity of an influenza virus infection, disease or symptom associated therewith, such as, but not limited to a reduction in the duration of an influenza virus infection, disease or symptom associated therewith, the prevention of the progression of an influenza virus infection, disease or symptom associated therewith, the prevention of the development or onset or recurrence of an influenza virus infection, disease or symptom associated therewith, the prevention or reduction of the spread of an influenza virus from one subject to another subject , the reduction of hospitalization of a subject and/or hospitalization length, an increase of the survival of a subject with an influenza virus infection or disease associated therewith, elimination of an influenza virus infection or disease associated therewith, inhibition or reduction of influenza virus replication, reduction of influenza virus titer; and/or enhancement and/or improvement of the prophylactic or therapeutic effect(s) of another therapy. In certain embodiments, the effective amount does not result in complete protection from an influenza virus disease, but results in a lower titer or reduced number of influenza viruses compared to an untreated subject. Benefits of a reduction in the titer, number or total burden of influenza virus include, but are not limited to, less severe symptoms of the infection, fewer symptoms of the infection and a reduction in the length of the disease associated with the infection.

The term "host", as used herein, is intended to refer to an organism or a cell into which a vector such as a cloning vector or an expression vector has been introduced. The organism or cell can be prokaryotic or eukaryotic. Preferably, the host comprises isolated host cells, e.g. host cells in culture. The term "host cells" merely signifies that the cells are modified for the (over)-expression of the polypeptides described herein. It should be understood that the term host is intended to refer not only to the particular subject organism or cell but to the progeny of such an organism or cell as well. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent organism or cell, but are still included within the scope of the term "host" as used herein.

The term "included" or "including" as used herein is deemed to be followed by the words "without limitation".

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

Influenza viruses are classified into influenza virus types: genus A, B and C. The term "influenza virus subtype" as used herein refers to influenza A virus variants that are characterized by combinations of the hemagglutinin (H) and neuramidase (N) viral surface proteins. According to the present disclosure influenza virus subtypes may be referred to by their H number, such as for example "influenza virus comprising HA of the H3 subtype", "influenza virus of the H3 subtype" or "H3 influenza", or by a combination of a H number and an N number, such as for example "influenza virus subtype H3N2" or "H3N2". The term "subtype" specifically includes all individual "strains", within each subtype, which usually result from mutations and show different pathogenic profiles, including natural isolates as well as man-made mutants or reassortants and the like. Such strains may also be referred to as various "isolates" of a viral subtype. Accordingly, as used herein, the terms "strains" and "isolates" may be used interchangeably. The current nomenclature for human influenza virus strains or isolates includes the type (genus) of virus, i.e. A, B or C, the geographical location of the first isolation, strain number and year of isolation, usually with the antigenic description of HA and NA given in brackets, e.g. A/Moscow/10/00 (H3N2).

Non-human strains also include the host of origin in the nomenclature. The influenza A virus subtypes can further be classified by reference to their phylogenetic group.

Phylogenetic analysis has demonstrated a subdivision of hemagglutinins into two main groups: inter alia the H1, H2, H5 and H9 subtypes in phylogenetic group 1 ("group 1" influenza viruses) and inter alia the H3, H4, H7 and H10 subtypes in phylogenetic group 2 ("group 2" influenza viruses).

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

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

As used herein, in certain embodiments the numbering of the amino acids in HA is based on the numbering of amino acids in HA0 of a wild type influenza virus, e.g. the numbering of the amino acids of the H1N1 influenza strain A/Brisbane/59/2007 (SEQ ID NO: 1). As used herein, the wording "the amino acid at position "x" in HA" thus means the amino acid corresponding to the amino acid at position x in HA0 of the particular wild type influenza virus, e.g. A/Brisbane/59/2007 (SEQ ID NO: 1; wherein the amino acids of the HA2 domain have been indicated in italics). It will be understood by the skilled person that equivalent amino acids in other influenza virus strains and/or subtypes can be determined by multiple sequence alignment. Note that, in the numbering system used throughout this application 1 refers to the N-terminal amino acid of an immature HA0 protein (SEQ ID NO: 1). The mature sequence starts e.g. on position 18 of SEQ ID NO: 1. It will be understood by the skilled person that the leader sequence (or signal sequence) that directs transport of a protein during production (e.g. corresponding to amino acids 1-17 of SEQ ID NO: 1), generally is not present in the final polypeptide, that is e.g. used in a vaccine. In certain embodiments, the polypeptides described herein thus comprise an amino acid sequence without the leader sequence, i.e. the amino acid sequence is based on the amino acid sequence of HA0 without the signal sequence.

"Polypeptide" refers to a polymer of amino acids linked by amide bonds as is known to those of skill in the art. As used herein, the term can refer to a single polypeptide chain linked by covalent amide bonds. The term can also refer to multiple polypeptide chains associated by non-covalent interactions such as ionic contacts, hydrogen bonds, Van der Waals contacts and hydrophobic contacts. Those of skill in the art will recognize that the term includes polypeptides that have been modified, for example by post-translational processing such as signal peptide cleavage, disulfide bond formation, glycosylation (e.g., N-linked and O-linked glycosylation), protease cleavage and lipid modification (e.g. S-palmitoylation).

"Stem domain polypeptide" refers to a polypeptide that comprises one or more polypeptide chains that make up a stem domain of a naturally-occurring (or wild-type) hemagglutinin (HA). Typically, a stem domain polypeptide is a single polypeptide chain (i.e. corresponding to the stem domain of a hemagglutinin HA0 polypeptide) or two polypeptide chains (i.e. corresponding to the stem domain of a hemagglutinin HAl polypeptide in association with a hemagglutinin HA2 polypeptide). According to the present disclosure, a stem domain polypeptide comprises one or more mutations as compared to the wild-type HA molecule, in particular one or more amino acid residues of the wild-type HA may have been substituted by other amino acids, not naturally occurring on the corresponding position in a particular wild-type HA. Stem domain polypeptides according to the present disclosure can furthermore comprise one or more linking sequences, as described below.

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

As used herein, the term "wild-type" in the context of a virus refers to influenza viruses that are prevalent, circulating naturally and producing typical outbreaks of disease.

DETAILED DESCRIPTION Influenza viruses have a significant impact on global public health, causing millions of cases of severe illness each year, thousands of deaths, and considerable economic losses.

Current trivalent influenza vaccines elicit a potent neutralizing antibody response to the vaccine strains and closely related isolates, but rarely extend to more diverged strains within a subtype or to other subtypes. In addition, selection of the appropriate vaccine strains presents many challenges and frequently results in sub-optimal protection.

Furthermore, predicting the subtype of the next pandemic virus, including when and where it will arise, is currently impossible.

Hemagglutinin (HA) is the major envelope glycoprotein from influenza A viruses which is the major target of neutralizing antibodies. Hemagglutinin has two main functions during the entry process. First, hemagglutinin mediates attachment of the virus to the surface of target cells through interactions with sialic acid receptors. Second, after endocytosis of the virus, hemagglutinin subsequently triggers the fusion of the viral and endosomal membranes to release its genome into the cytoplasm of the target cell. HA comprises a large ectodomain of ~500 amino acids that is cleaved by host- derived enzymes to generate 2 polypeptides that remain linked by a disulfide bond. The majority of the N- terminal fragment (HA1, 320-330 amino acids) forms a membrane-distal globular domain that contains the receptor-binding site and most determinants recognized by virus- neutralizing antibodies. The smaller C-terminal portion (HA2, ~180 amino acids) forms a stem-like structure that anchors the globular domain to the cellular or viral membrane. The degree of sequence homology between subtypes is smaller in the HA1 polypeptides (34% - 59% homology between subtypes) than in the HA2 polypeptide (51%- 80% homology).

The most conserved region is the sequence around the cleavage site, particularly the HA2 N- terminal 23 amino acids, which is conserved among all influenza A virus subtypes (Lorieau et al., 2010). Part of this region is exposed as a surface loop in the HA precursor molecule (HA0), but becomes inaccessible when HA0 is cleaved into HA1 and HA2.

Most neutralizing antibodies bind to the loops that surround the receptor binding site and interfere with receptor binding and attachment. Since these loops are highly variable, most antibodies targeting these regions are strain-specific, explaining why current vaccines elicit such limited, strain-specific immunity. Recently, however, fully human monoclonal antibodies against influenza virus hemagglutinin with broad cross-neutralizing potency were generated. Functional and structural analysis have 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, , WO2010/130636, ).

Stem domain polypeptides stably presenting the epitopes of these antibodies are described in the co-pending patent application . At least some of the stem domain polypeptides described herein stably present the epitope of CR6261 and/or CR9114 and are immunogenic in mice. At least some of the stem domain polypeptides described herein stably present the epitope of CR8020 and are immunogenic in mice.

According to the present disclosure new HA stem domain polypeptides have been designed presenting these epitopes. These polypeptides can be used to create a universal epitope-based vaccine inducing protection against a broad range of influenza strains.

Like in the previously described stem domain polypeptides, the highly variable and immunodominant part, i.e. the head domain, is first removed from the full length HA molecule to create a stem domain polypeptide, also called mini-HA, in order to redirect the immune response towards the stem domain where the epitopes for the broadly neutralizing antibodies are located. The broadly neutralizing antibodies mentioned above were used to probe the correct folding of the newly created molecules, and to confirm the presence of the neutralizing epitopes.

The new stem domain polypeptides described herein show increased binding of the antibodies, in particular CR6261 and/or CR9114, as compared to binding of those antibodies to the stem polypeptides described earlier ().

The stem domain polypeptides described herein are capable of presenting the conserved epitopes of the membrane proximal stem domain HA molecule to the immune system in the absence of dominant epitopes that are present in the membrane distal head domain. To this end, part of the primary sequence of the HA0 protein making up the head domain is removed and reconnected, either directly or, in some embodiments, by introducing a short flexible linking sequence (‘linker’) to restore the continuity of the polypeptide chain. The resulting polypeptide sequence is further modified by introducing specific mutations that stabilize the native 3-dimensional structure of the remaining part of the HA0 molecule.

The present disclosure thus describes polypeptides comprising (a) an influenza hemagglutinin HA1 domain that comprises an HA1 N- terminal stem segment, covalently linked by a linking sequence of 0-50 amino acid residues to an HA1 C- terminal stem segment, and (b) an influenza hemagglutinin HA2 domain, wherein the hemagglutinin stem domain polypeptide is resistant to protease cleavage at the junction between HA1 and HA, and wherein on or more amino acids in the HA1 and HA2 domain have been mutated. In the polypeptides described herein, the HA1 and HA2 domain thus comprise one or more mutations as compared to the HA1 and HA2 domain of a wild-type influenza hemagglutinin on which the HA stem domain polypeptide is based.

According to the present disclosure, the stem domain polypeptides are based on HA of an influenza virus comprising HA of the H1 subtype.

In certain embodiments, the polypeptides described herein comprise one or more mutations on position 337, 340, 352 or 353 of SEQ ID NO: 1, or equivalent positions in other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 352 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 353 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 337 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 340 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

In certain embodiments, the polypeptides described herein comprise one or more mutations in the HA2 amino acid sequence connecting the C-terminal residue of helix A to the N-terminal residue of helix CD, as indicated in In certain embodiments, one or more of the amino acids on position 402, 406, 409, 413 and 416 (numbering refers to SEQ ID NO: 1), or equivalent positions in other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 402 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 406 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 409 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 413 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

In certain embodiments, the polypeptides described herein comprise at least one mutation on position 416 of SEQ ID NO: 1, or on an equivalent position of other influenza viruses.

The polypeptides described herein do not comprise the full length HA1.

In certain embodiments, the polypeptides further comprise one or more additional mutations in the HA1 and/or HA2 domain, as compared to the amino acid sequence of the HA of which the HA 1 and HA2 domains are derived.Thus, the stability of the stem polypeptides is further increased.

According to the present disclosure, the "HA1 N-terminal segment" refers to a polypeptide segment that corresponds to the amino-terminal portion of the HA1 domain of an influenza hemagglutinin (HA) molecule. In certain embodiments, the HA1 N-terminal polypeptide segment comprises the amino acids from position 1 to position x of the HA1 domain, wherein amino acid on position x is an amino acid residue within HA1. The term "HA1 C-terminal segment" refers to a polypeptide segment that corresponds to the carboxy-terminal portion of an influenza hemagglutinin HA1 domain. In certain embodiments, the HA1 C-terminal polypeptide segment comprises the amino acids from position y to and including the C-terminal amino acid of the HA1 domain, wherein the amino acid on position y is an amino acid residue within HA1. According to the present disclosure y is greater than x, thus a segment of the HA1 domain between the HA1 N- terminal segment and the HA1 C-terminal segment, i.e. between the amino acid on position x and the amino acid on position y of HA1, has been deleted, and in some embodiments, replaced by a linking sequence.

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

In certain embodiments, the polypeptides do not comprise the signal sequence. Thus in certain embodiments, the HA1 N-terminal segment comprises the amino acid p-x of HA1, wherein p is the first amino acid of the mature HA molecule (e.g. p=18 in case of SEQ ID NO: 1). The skilled person will be able to prepare the polypeptides described herein without the signal peptides (e.g. amino acids 1-17 of SEQ ID NO: 1).

According to the present disclosure, the hemagglutinin stem domain polypeptides are resistant to protease cleavage at the junction between HA1 and HA2. It is known to those of skill in the art that the Arg (R) - Gly (G) sequence spanning HA1 and HA2 is a recognition site for trypsin and trypsin-like proteases and is typically cleaved for hemagglutinin activation. Since the HA stem domain polypeptides described herein should not be activated, the influenza hemagglutinin stem domain polypeptides described herein are resistant to protease cleavage. According to the present disclosure, thus 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 HA1 C-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 polypeptides are glycosylated.

According to the present disclosure, the influenza hemagglutinin stem domain polypeptides are based on HA of influenza viruses of the H1 subtype. With "based on" it is meant that the N-terminal segments, and/or C-terminal segments of the HA1 domain and/or the HA2 domains have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the corresponding N-terminal and/or C-terminal segments of HA1 and/or the HA2 domains of any naturally occurring influenza hemagglutinin of a H1, H3 and/or H5 subtype known to those of skill in the art or later discovered. In certain embodiments, the influenza hemagglutinin stem domain polypeptides are based on an influenza hemagglutinin of a group 1 influenza A virus.

According to the present disclosure, the polypeptides are based on H1 HA, i.e. HA comprising an amino acid sequence from an influenza virus of the H1 subtype. In a particular embodiment, the polypeptides comprise hemagglutinin stem domains from or based on HA of an influenza A virus comprising HA of the H1 subtype, such as from the influenza virus A/Brisbane/59/2007 (H1N1) (SEQ ID NO:1), as described below. It will be understood by the skilled person that also other influenza A viruses comprising HA of the H1 subtype may be used according to the present disclosure. In certain embodiments, the polypeptides comprise hemagglutinin stem domains based on HA of an influenza A H1 virus selected from Table 3.

In certain embodiments, the polypeptides comprise a HA1 N-terminal polypeptide segment comprising the amino acids from position 1 to position x of the H1 HA1 domain, wherein x is any amino acid between the amino acid on position 46 and the amino acid on position 60, such as the amino acid on position 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59, preferably wherein x is 52, 53, 55 or 59. Preferably, the polypeptides comprise a HA1 N-terminal segment without the signal sequence, i.e. a HA1 N-terminal segment comprising the amino acids from position 18 (e.g. for H1 HA, such as SEQ ID NO: 1), or an equivalent position in other H1 influenza virus strains (see e.g. Table 3), to position x of the HA1 domain. In certain embodiments, the HA1 N-terminal segment thus comprises the amino acids from position p (wherein p=18 for H1 HA in SEQ ID NO: 1 or an equivalent position on other H1 HAs), to position x of the HA1 domain.

In certain embodiments, the HA1 C-terminal polypeptide segment comprises the amino acids from position y to and including the C-terminal amino acid of the H1 HA1 domain, wherein y is any amino acid between the amino acid on positions 290 and the amino acid on position 325 of H1 HA1, preferably wherein y is 291, 303, 318, or 321.

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

According to the present disclosure, the stem polypeptides comprise one or more mutations, i.e. amino acid substitutions, in the HA1 domain and/or the HA2 domain, as compared to the amino acid sequence of corresponding wild-type influenza virus HA1 and/or HA2 domains, i.e. the influenza virus on which the stem polypeptides are based.

In certain embodiments, one or more amino acid residues close to the HA0 cleavage site (residue 343 in SEQ ID NO: 1) have been mutated. In certain embodiments, one or more of the amino acid residues on position 337, 340, 352, or 353 of SEQ ID NO: 1, or equivalent positions in other influenza viruses, have been mutated, i.e. are substituted by an amino acid that is not occurring at the corresponding position in the amino acid sequence of the HA of the wild-type influenza virus on which the stem polypeptide is based. Table 7 shows the the naturally occurring amino acid variation.

In certain embodiments the polypeptides comprise one or more of the mutations as indicated in Table 1.

In certain embodiments, the mutated amino acid residue on position 337 (HA1 domain) is selected from the group consisting of I, E, K, V, A, and T.

In certain embodiments, the mutated amino acid residue on position 340 (HA1 domain) is selected from the group consisting of I, K, R, T, F, N, S and Y.

In certain embodiments, the mutated amino acid residue on position 352 (HA2 domain) is selected from the group consisting of D, V, Y, A, I, N, S, and T.

In certain embodiments, the mutated amino acid residue on position 353 (HA2 domain) is selected from the group consisting of K, R, T, E, G, and V.

In certain embodiments the mutated amino acid introduces a consensus N- glycoslation e.g. N-X-T/S (where X is any naturally ccuring amino acid except P) in the sequence as is for example the case for I340N in SEQ ID NO: 6 In certain embodiments, the mutated amino acid is an amino acid that does not naturally occur in sequences of the same subtype.

It is again noted that the numbering of the amino acids is based on the numbering of amino acids in H1 HA0, in particular the numbering of the amino acids of the H1N1 influenza strain A/Brisbane/59/2007 (SEQ ID NO: 1). The skilled person will be able to determine the equivalent amino acids in HA of other influenza virusesand thus will be able to determine equivalent mutations, see e.g. Table 3 for the sequence alignment of different H1 influenza viruses.

In certain embodiments, the HA2 domain comprises one or more mutations in the HA2 amino acid sequence connecting the C-terminal residue of helix A to the N-terminal residue of helix CD (. The H1 HA2 amino acid sequence connecting the C- terminal residue of helix A and the N-terminal residue of helix CD comprises the amino acid sequence comprising residues 402-418 of influenza HA (numbering according to SEQ ID NO: 1), comprising the amino acid sequence MNTQFTAVGKEFN(H/K)LE(K/R) (SEQ ID NO: 8).

In certain embodiments, the amino acid sequence connecting the C-terminal residue of helix A to the N-terminal residue of helix CD, i.e. the region comprising the amino acid residues 402-418 of influenza HA of serotype H1 (numbering according to SEQ ID NO: 1) comprises the amino acid sequence X NTQX TAX GKEX N(H/K)X E(K/R) (SEQ ID NO: 52). 1 2 3 4 5 In certain embodiments the polypeptides comprise one or more of the mutations in the H1 HA2 domain as indicated in Table 1.

In certain embodiments, one or more of the amino acids on position 402, 406, 409, 413 and 416 (numbering refers to SEQ ID NO: 1), i.e one or more of the amino acids X , X , X X and X have been mutated, i.e. comprise an amino acid that is not 1 2 3, 4 5 occurring at those positions in a wild-type influenza virus on which the stem polypeptide is based.

In certain embodiments, the mutated amino acid on position 402, i.e. X is an amino acid selected from the group consisting of M, E, K, V, R and T.

In certain embodiments, the mutated amino acid on position 406, i.e. X is an amino acid selected from the group consisting of F, I, N, T, H, L and Y, preferably I, L or is an In certain embodiments, the mutated amino acid on position 409, i.e. X3, amino acid selected from the group consisting of V, A, G, I, R, F and S, preferably A, I or F.

In certain embodiments, the mutated amino acid on position 413, i.e. X , is an amino acid selected from the group consisting of F, I, N, S, T, Y, E, K, M, and V, preferably I, Y, M or V.

In certain embodiments, the mutated amino acid on position 416, i.e. X is an amino acid selected from the group consisting of L, H, I, N, R, preferably I.

Combinations of these mutations are also possible.

In certain embodiments, the HA1 N- terminal stem segment comprises the amino acid residues 1-52 of HA1, preferably the amino acid residues 18-52 of HA1, and the HA1 C- terminal stem segment comprises the amino acid residues 321-343 of HA1, wherein the amino acid on position 343, i.e. R343, has been mutated and is an amino acid other than R, preferably glutamine (Q). In certain embodiments, the HA1 N- terminal stem segment consists of the amino acid residues 1-52 of HA1, preferably the amino acid residues 18-52 of HA1, and the HA1 C- terminal stem segment consists of the amino acid residues 321-343 of HA1.

In certain embodiments, the polypeptides selectively bind to the antibodies CR6261 and/or CR9114. In an embodiment, the polypeptide does not bind to the antibody CR8057. In an embodiment, CR6261 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; CR9114 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In an embodiment, CR8057 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

As described above, the polypeptides comprise an influenza hemagglutinin HA1 domain that comprises an HA1 N- terminal stem segment that is covalently linked by a linking sequence of 0- 50 amino acid residues to the HA1 C- terminal stem segment. The linking sequence does not occur in naturally occurring, or wild-type, HA. In certain embodiments, the linker is a peptide that comprises one amino acid residue, two or less amino acid residues, three or less amino acid residues, four or less amino acid residues, five or less amino acid residues, ten or less amino acid residues, 15 or less amino acid residues, or 20 or less amino acid residues or 30 or less amino acid residues or 40 or less amino acid residues or 50 or less amino acid residues. In a specific embodiment, the linking sequence is a sequence selected from the group consisting of G, GS, GGG, GSG, GSA, GSGS, GSAG, GGGG, GSAGS, GSGSG, GSAGSA, GSAGSAG, and GSGSGSG.

In certain embodiments, the HA1 N-terminal segment is directly linked to the HA1 C-terminal segment, i.e. the polypeptides do not comprise a linking sequence.

According to the present disclosure, removal of the cleavage site between HA1 and HA2 can be achieved by mutation of R (in a small number of cases K) to Q at the P1 position (see e.g. Sun et al, 2010 for an explanation of the nomenclature of the cleavage site (position 343 in SEQ ID NO: 1). A mutation to Q is preferred but S, T, N, D or E are alternatives.

According to the present disclosure, one or more disulfide bridges are introduced in the stem domain polypeptides, preferably between amino acids of (or the equivalent of) position 324 and 436 in H1 A/Brisbane/59/2007 (SEQ ID NO: 1). In certain embodiments, the polypeptides thus further comprise the mutation R324C in the HA1 domain and T436C in the HA2 domain. Equivalent positions can be easily determined by those skilled in the art by aligning the sequences using a suitable algorithm such as Clustal, Muscle etc. Engineered disulfide bridges are created by mutating at least one (if the other is already a cysteine), but usually two residues that are spatially close into cysteine, that will spontaneously or by active oxidation form a covalent bond between the sulfur atoms of these residues.

Influenza HA in its native form exists as a trimer on the cell or virus membrane.

In certain embodiments the intracellular and transmembrane sequence is removed so that a secreted (soluble) polypeptide is produced following expression in cells. Methods to express and purify secreted ectodomains of HA have been described (see e.g. Dopheide et al 2009; Ekiert et al 2009, 2011; Stevens et al 2004, 2006; Wilson et al 1981). A person skilled in the art will understand that these methods can also be applied directly to stem domain polypeptides described herein in order to achieve expression of secreted (soluble) polypeptide. Therefore these polypeptides are also encompassed in the present disclosure.

In certain embodiments, the polypeptides described herein contain the intracellular sequences of HA and the transmembrane domain. In other embodiments, the intracellular and transmembrane sequences, e.g. the amino acid sequence from position (or the equivalent of) 519, 520, 521, 522, 523, 524, 525, 526, 527, 526, 528, 529, or 530 of the HA2 domain to the C-terminus of the HA2 domain (numbering according to SEQ ID NO: 1) have been removed to produce a soluble polypeptide following expression in cells.

In certain embodiments, a soluble polypeptide described herein can be created by deletion of the polypeptide sequence from residue (the equivalent of) 514 to the C- terminus (numbering according to SEQ ID NO: 1), Alternatively, additional residues can be included in the polypeptide described herein, e.g. by deleting the sequence from residue 515, 516, 517, 518, 519, 520, 521, 522, 523. Optionally, a his-tag sequence (HHHHHH (SEQ ID NO: 20) or HHHHHHH (SEQ ID NO: 21)) may be added, for purification purposes, optionally connected through a linker. Optionally the linker may contain a proteolytic cleavage site to enzymatically remove the his-tag after purification.

In certain embodiments, a soluble polypeptide described herein can be created by deletion of the polypeptide sequence from residue (the equivalent of) 524, 525, 526, 527, 528, 529, 530, 531 or 532 (numbering according to SEQ ID NO: 1). Optionally, a his-tag sequence (HHHHHH (SEQ ID NO: 20) or HHHHHHH (SEQ ID NO: 21)) may be added, for purification purposes, optionally connected through a linker. Optionally the linker may contain a proteolytic cleavage site to remove the his-tag after purification.

The soluble polypeptides can be further stabilized by introducing a sequence known to form trimeric structures, such as the foldon sequence (e.g. as described herein).

Polypeptides obtained as described above are also encompassed in the present disclosure The native HA exists as a trimer on the cell surface. Most of the interactions between the individual monomers that keep the trimer together are located in the head domain while in the stem domain trimerization is mediated by the formation of a trimeric coiled coil motif. After removal of the head the tertiary structure is destabilized and therefore modifications are needed in order to increase protein stability. By strengthening the helical propensity of the helix CD a more stable protein can be created. In certain embodiments, the sequence MKQIEDKIEEIESKQ (SEQ ID NO: 5), derived from GCN4 and also known to trimerize is introduced at (the equivalent of) position 419-433.

In certain embodiments, the polypeptides are further stabilized by introducing a sequence known to form trimeric structures, i.e.

GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 3) at the C-terminus of HA2, optionally connected through a linker. The linker may optionally contain a cleavage site for processing afterwards according to protocols well known to those skilled in the art.

To facilitate purification of the soluble form a tag sequence may be added, e.g. a his tag (HHHHHHH (SEQ ID NO: 20) or HHHHHH (SEQ ID NO: 21)) or FLAG tag (DYKDDDDK) (SEQ ID NO: 22) or a combination of these, optionally connected via short linkers. The linker may optionally contain (part of) a proteolytic cleavage site, e.g.

RSLVPR (SEQ ID NO: 23) (thrombin) or IEGR (SEQ ID NO: 24) (Factor X) for processing afterwards according to protocols well known to those skilled in the art. The processed proteins are also encompassed in the present disclosure.

In certain embodiments, the C-terminal part of the HA2 domain from position 520-565 has been deleted (numbering according to SEQ ID NO: 1) and replaced by SGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHH H (SEQ ID NO: 4).

Applicants have previously identified broadly neutralizing antibodies isolated from primary human B-cells from vaccinated individuals some of which were specific for group 1 (e.g. CR6261, as described in ) and some of which were specific for group 2 influenza viruses (e.g. CR8020 as described in ).

Detailed analysis of the epitopes of these monoclonal antibodies has 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 partly explained the fact that the antibodies are group-specific. With the identification of CR9114-like antibodies that cross-react with many group 1 and 2 HA molecules, as described below, it has become clear that it is possible for the human immune system to elicit very broad neutralizing antibodies against influenza viruses. However, given the need for a yearly vaccination scheme these antibodies are apparently not, or only to a very low extent elicited following infection or vaccination with (seasonal) influenza viruses of subtypes H1 and/or H3.

According to the present disclosure polypeptides are described that mimic the specific epitopes of CR6261 and/or CR9114, and that can be used as immunogenic polypeptides, e.g. to elicit cross-neutralizing antibodies when administered in vivo, either alone, or in combination with other prophylactic and/or therapeutic treatments. With "cross-neutralizing antibodies", antibodies are meant that are capable of neutralizing at least two, preferably at least three, four, or five different subtypes of influenza A viruses of phylogenetic group1, and/or at least two, preferably at least three, four, or five different subtypes of influenza A viruses of phylogenetic group 2, and/or at least two, different subtypes of influenza B viruses, in particular at least all virus strains that are neutralized by CR6261 and CR9114.

The polypeptides described herein do not comprise the full length HA1. In certain embodiments, the immunogenic polypeptides are substantially smaller than HA0, preferably lacking all or substantially all of the globular head of HA. Preferably, the immunogenic polypeptides are no more than 360, preferably no more than 350, 340, 330, 320, 310, 305, 300, 295, 290, 285, 280, 275, or 270 amino acids in length. In an embodiment, 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 polypeptides selectively bind to the antibodies CR6261 and/or CR9114. In certain embodiments, the polypeptide does not bind to the antibody CR8057. CR6261 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; CR9114 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12; CR8020 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18.

CR8057 comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14.

As described above, the polypeptides comprise an influenza hemagglutinin HA1 domain that comprises an HA1 N- terminal stem segment that is covalently linked by a linking sequence of 0- 50 amino acid residues to the HA1 C- terminal stem segment. The linking sequence, if present, does not occur in naturally occurring, or wild-type, HA. In certain embodiments, the linker is a peptide that comprises one amino acid residue, two or less amino acid residues, three or less amino acid residues, four or less amino acid residues, five or less amino acid residues, ten or less amino acid residues, 15 or less amino acid residues, or 20 or less amino acid residues or 30 or less amino acid residues or 40 or less amino acid residues or 50 or less amino acid residues. In a specific embodiment, the linking sequence is a sequence selected from the group consisting of G, GS, GGG, GSG, GSA, GSGS, GSAG, GGGG, GSAGS, GSGSG, GSAGSA, GSAGSAG, and GSGSGSG.

In certain embodiments, the polypeptides comprise the amino acid sequence: DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGL RNX1PSX2QSQGLFGAIAGX3X4EGGWTGMVDGWYGYHHQNEQGSGYAADQKS TQNAINGITNKVNSVIEKX5NTQX6TAX7GKEX8NKX9ERMKQIEDKIEEIESKQIWC YNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDE CMESVKNGTYDYPKYSEESKLNREKIDGVSGRDYKDDDDKLVPRGSPGSGYIPE APRDGQAYVRKDGEWVLLSTFLGHHHHHH (SEQ ID NO: 53), wherein X is an amino acid selected from the group consisting of E, I, K, V, A, and T; X is an amino acid selected from the group consisting of I, K, R, T, F, N, S and Y; X is an amino acid selected from the group consisting of D, F, V, Y, A, I, N, S, and T; X is an amino acid selected from the group consisting of I, K, R, T, E, G and V; X is an amino acid selected from the group consisting of E, K, M, V, R, and T; X is an amino acid selected from the group consisting of F, I, N, S, T, Y, H, and L; X is an amino acid selected from the group consisting of A, G, I, R, T, V, F, and S; X is an amino acid selected from the group consisting of F, I, N, S, T, Y, G, E, K, M, and V; and X is an amino acid selected from the group consisting of H, I, L, N, R, and S.

In certain embodiments, the polypeptides comprise the amino acid sequence: DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGL RNX1PSX2QSQGLFGAIAGX3X4EGGWTGMVDGWYGYHHQNEQGSGYAADQKS TQNAINGITNKVNSVIEKX NTQX TAX GKEX NKX ERMKQIEDKIEEIESKQIWC 6 7 8 9 YNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDE CMESVKNGTYDYPKYSEESKLNREKIDG (SEQ ID NO: 54), wherein X is an amino acid selected from the group consisting of E, I, K, V, A, and T; X is an amino acid selected from the group consisting of I, K, R, T, F, N, S and Y; X is an amino acid selected from the group consisting of D, F, V, Y, A, I, N, S, and T; X is an amino acid selected from the group consisting of I, K, R, T, E, G and V; X is an amino acid selected from the group consisting of E, K, M, V, R, and T; X is an amino acid selected from the group consisting of F, I, N, S, T, Y, H, and L; X is an amino acid selected from the group consisting of A, G, I, R, T, V, F, and S; X is an amino acid selected from the group consisting of F, I, N, S, T, Y, G, E, K, M, and V; and X is an amino acid selected from the group consisting of H, I, L, N, R, and S.

In certain embodiments, the polypeptides comprise the amino acid sequence: DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGL RNX PSX QSQGLFGAIAGX X EGGWTGMVDGWYGYHHQNEQGSGYAADQKS 1 2 3 4 TQNAINGITNKVNSVIEKX NTQX TAX GKEX NKX ERMKQIEDKIEEIESKQIWC 6 7 8 9 YNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDE CMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEG (SEQ ID NO: 70), wherein X is an amino acid selected from the group consisting of E, I, K, V, A, and T; X is an amino acid selected from the group consisting of I, K, R, T, F, N, S and Y; X is an amino acid selected from the group consisting of D, F, V, Y, A, I, N, S, and T; X is an amino acid selected from the group consisting of I, K, R, T, E, G and V; X is an amino acid selected from the group consisting of E, K, M, V, R, and T; X is an amino acid selected from the group consisting of F, I, N, S, T, Y, H, and L; X is an amino acid selected from the group consisting of A, G, I, R, T, V, F, and S; X is an amino acid selected from the group consisting of F, I, N, S, T, Y, G, E, K, M and V; and X is an amino acid selected from the group consisting of H, I, L, N, R, and S.

In certain embodiments, the polypeptides comprise the amino acid sequence: DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGL PSX QSQGLFGAIAGX X EGGWTGMVDGWYGYHHQNEQGSGYAADQKS RNX1 2 3 4 TQNAINGITNKVNSVIEKX NTQX TAX GKEX NKX ERMKQIEDKIEEIESKQIWC 6 7 8 9 YNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCNDE CMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQILAIYSTVASSLVLLVS LGAISFWMCSNGSLQCRICI (SEQ ID NO: 71), wherein X is an amino acid selected from the group consisting of E, I, K, V, A, and T; X is an amino acid selected from the group consisting of I, K, R, T, F, N, S and Y; X is an amino acid selected from the group consisting of D, F, V, Y, A, I, N, S, and T; X is an amino acid selected from the group consisting of I, K, R, T, E, G and V; X is an amino acid selected from the group consisting of E, K, M, V, R, and T; X is an amino acid selected from the group consisting of F, I, N, S, T, Y, H, and L; X is an amino acid selected from the group consisting of A, G, I, R, T, V, F, and S; X is an amino acid selected from the group consisting of F, I, N, S, T, Y, G, E, K, M and V; and X is an amino acid selected from the group consisting of H, I, L, N, R, and S.

The influenza hemagglutinin stem domain polypeptides can be prepared according to any technique deemed suitable to one of skill, including techniques described below.

Thus, the immunogenic polypeptides described herein may be synthesized as DNA sequences by standard methods known in the art and cloned and subsequently expressed, in vitro or in vivo, using suitable restriction enzymes and methods known in the art. The present disclosure thus also relates to nucleic acid molecules encoding the above described polypeptides. The present disclosure further relates to vectors comprising the nucleic acids encoding the polypeptides described herein. In certain embodiments, a nucleic acid molecule according to the present disclosure is part of a vector, e.g. a plasmid. Such vectors can easily be manipulated by methods well known to the person skilled in the art, and can for instance be designed for being capable of replication in prokaryotic and/or eukaryotic cells. In addition, many vectors can directly or in the form of an isolated desired fragment there from be used for transformation of eukaryotic cells and will integrate in whole or in part into the genome of such cells, resulting in stable host cells comprising the desired nucleic acid in their genome. The vector used can be any vector that is suitable for cloning DNA and that can be used for transcription of a nucleic acid of interest. When host cells are used it is preferred that the vector is an integrating vector. Alternatively, the vector may be an episomally replicating vector.

The person skilled in the art is capable of choosing suitable expression vectors, and inserting the nucleic acid sequences described herein in a functional manner. To obtain expression of nucleic acid sequences encoding polypeptides, it is well known to those skilled in the art that sequences capable of driving expression can be functionally linked to the nucleic acid sequences encoding the polypeptide, resulting in recombinant nucleic acid molecules encoding a protein or polypeptide in expressible format. In general, the promoter sequence is placed upstream of the sequences that should be expressed. Many expression vectors are available in the art, e.g. the pcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BD Sciences, pCMV-Script from Stratagene, etc, which can be used to obtain suitable promoters and/or transcription terminator sequences, polyA sequences, and the like. Where the sequence encoding the polypeptide of interest is properly inserted with reference to sequences governing the transcription and translation of the encoded polypeptide, the resulting expression cassette is useful to produce the polypeptide of interest, referred to as expression. Sequences driving expression may include promoters, enhancers and the like, and combinations thereof. These should be capable of functioning in the host cell, thereby driving expression of the nucleic acid sequences that are functionally linked to them. The person skilled in the art is aware that various promoters can be used to obtain expression of a gene in host cells. Promoters can be constitutive or regulated, and can be obtained from various sources, including viruses, prokaryotic, or eukaryotic sources, or artificially designed. Expression of nucleic acids of interest may be from the natural promoter or derivative thereof or from an entirely heterologous promoter (Kaufman, 2000). Some well-known and much used promoters for expression in eukaryotic cells comprise promoters derived from viruses, such as adenovirus, e.g. the E1A promoter, promoters derived from cytomegalovirus (CMV), such as the CMV immediate early (IE) promoter (referred to herein as the CMV promoter) (obtainable for instance from pcDNA, Invitrogen), promoters derived from Simian Virus 40 (SV40) (Das et al, 1985), and the like.

Suitable promoters can also be derived from eukaryotic cells, such as methallothionein (MT) promoters, elongation factor 1 α (EF-1 α) promoter (Gill et al., 2001), ubiquitin C or UB6 promoter (Gill et al., 2001), actin promoter, an immunoglobulin promoter, heat shock promoters, and the like. Testing for promoter function and strength of a promoter is a matter of routine for a person skilled in the art, and in general may for instance encompass cloning a test gene such as lacZ, luciferase, GFP, etc. behind the promoter sequence, and test for expression of the test gene. Of course, promoters may be altered by deletion, addition, mutation of sequences therein, and tested for functionality, to find new, attenuated, or improved promoter sequences. According to the present disclosure, strong promoters that give high transcription levels in the eukaryotic cells of choice are preferred.

The constructs may be transfected into eukaryotic cells (e.g. plant, fungal, yeast or animal cells) or suitable prokaryotic expression systems like E. coli using methods that are well known to persons skilled in the art. In some cases a suitable ‘tag’ sequence (such as for example, but not limited to, a his-, myc-, strep-, or flag-tag) or complete protein (such as for example, but not limited to, maltose binding protein or glutathione S transferase) may be added to the sequences described herein to allow for purification and/or identification of the polypeptides from the cells or supernatant. Optionally a sequence containing a specific proteolytic site can be included to afterwards remove the tag by proteolytic digestion.

Purified polypeptides can be analyzed by spectroscopic methods known in the art (e.g. circular dichroism spectroscopy, Fourier Transform Infrared spectroscopy and NMR spectroscopy or X-ray crystallography) to investigate the presence of desired structures like helices and beta sheets. ELISA, Octet and FACS and the like can be used to investigate binding of the polypeptides described herein to the broadly neutralizing antibodies described before (CR6261, CR9114, CR8057). Thus, polypeptides according to the present disclosure having the correct conformation can be selected.

The present disclosure further relates to immunogenic compositions comprising a therapeutically effective amount of at least one of the polypeptides and/or nucleic acids described herein. In certain embodiments, the compositions comprise polypeptides comprising hemagglutinin stem domains from (or based on) HA of one influenza subtype, e.g. based on HA of an influenza virus comprising HA of e.g. a H1 or H3 subtype. In certain embodiments, the compositions comprise polypeptides comprising hemagglutinin stem domains based on HA of two or more different influenza subtypes, e.g. compositions comprising both polypeptides comprising hemagglutinin stem domains based on HA of the H1 subtype and polypeptides comprising hemagglutinin stem domains based on HA of the H3 subtype.

The immunogenic compositions preferably further comprise a pharmaceutically acceptable carrier. In the present context, the term "pharmaceutically acceptable" means that the carrier, at the dosages and concentrations employed, will not cause unwanted or harmful effects in the subjects to which they are administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company ; Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press [2000]). The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can e.g. be employed as liquid carriers, particularly for injectable solutions. The exact formulation should suit the mode of administration. The polypeptides and/or nucleic acid molecules preferably are 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 solutions can then be lyophilized or filled into pharmaceutical dosage containers. The pH of the solution generally is in the range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5.

The present disclosure also relates to influenza HA stem domain polypeptides, a nucleic acid molecules and/or vectors as described above for use in inducing an immune response against influenza HA protein. The present disclosure also relates to methods for inducing an immune response in a subject, the method comprising administering to a subject, a polypeptide, nucleic acid molecule and/or immunogenic composition as described above. A subject according to the present disclosure preferably is a mammal that is capable of being infected with an infectious disease-causing agent, in particular an influenza virus, or otherwise can benefit from the induction of an immune response, such subject for instance being a rodent, e.g. a mouse, a ferret, or a domestic or farm animal, or a non-human-primate, or a human. Preferably, the subject is a human subject. The present disclosure thus describes methods for inducing an immune response to an influenza virus hemagglutinin (HA), in particular of a group 1 and/or group 2 influenza A virus, such as an influenza virus comprising HA of the H1, H2, H3, H4, H5, H7 and/or H10 subtype, and/or of an influenza B virus, in a subject utilizing the polypeptides, nucleic acids and/or immunogenic compositions described herein. In some embodiments, the immune response induced is effective to prevent and/or treat an influenza virus infection caused group 1 and/or group 2 influenza A virus subtypes and/or influenza B viruses. In some embodiments, the immune response induced by the polypeptides, nucleic acids and/or immunogenic compositions described herein is effective to prevent and/or treat an influenza A and/or B virus infection caused by two, three, four, five or six subtypes of influenza A and/or B viruses.

Since it is well known that small proteins and/or nucleic acid molecules do not always efficiently induce a potent immune response it may be necessary to increase the immunogenicity of the polypeptides and/or nucleic acid molecules by adding an adjuvant.

In certain embodiments, the immunogenic compositions described herein comprise, or are administered in combination with, an adjuvant. The adjuvant for administration in combination with a composition described herein may be administered before, concomitantly with, or after administration of said composition. Examples of suitable adjuvants include aluminium salts such as aluminium hydroxide and/or aluminium phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene- water emulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see e.g. US 5,057,540; WO 90/03184, WO 96/11711, , ); bacterial or microbial derivatives, examples of which are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating 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, known immunopotentiating technologies may be used, such as fusing the polypeptides described herein to proteins known in the art to enhance immune response (e.g. tetanus toxoid, CRM197, rCTB, bacterial flagellins or others) or including the polypeptides in virosomes, or combinations thereof. Other non-limiting examples that can be used are e.g. disclosed by Coffman et al. (2010).

In an embodiment, the influenza hemagglutinin stem domain polypeptides described herein are incorporated into viral-like particle (VLP) vectors. VLPs generally comprise a viral polypeptide(s) typically derived from a structural protein(s) of a virus. Preferably, the VLPs are not capable of replicating. In certain embodiments, the VLPs may lack the complete genome of a virus or comprise a portion of the genome of a virus. In some embodiments, the VLPs are not capable of infecting a cell. In some embodiments, the VLPs express on their surface one or more of viral (e.g., virus surface glycoprotein) or non-viral (e.g., antibody or protein) targeting moieties known to one skilled in the art.

In a specific embodiment, the polypeptide described herein is incorporated into a virosome. A virosome containing a polypeptide according to the present disclosure may be produced using techniques known to those skilled in the art. For example, a virosome may be produced by disrupting a purified virus, extracting the genome, and reassembling particles with the viral proteins (e.g., an influenza hemagglutinin stem domain polypeptide) and lipids to form lipid particles containing viral proteins.

The present disclosure also relates to the above-described polypeptides, nucleic acids and/or immunogenic compositions for inducing an immune response in a subject against influenza HA, in particular for use as a vaccine. The influenza hemagglutinin stem domain polypeptides, nucleic acids encoding such polypeptides, or vectors comprising such nucleic acids or polypeptides described herein thus may be used to elicit neutralizing antibodies against influenza viruses, for example, against the stem region of influenza virus hemagglutinin. The present disclosure in particular relates to polypeptides, nucleic acids, and/or imunogenic compositions as described above for use as a vaccine in the prevention and/or treatment of a disease or condition caused by an influenza A virus of phylogenetic group 1 and/or phylogenetic group 2 and/or an influenza B virus. In an embodiment, the vaccine may be used in the prevention and/or treatment of diseases caused by two, three, four, five, six or more different subtypes of phylogenetic group 1 and/or 2 and/or influenza B viruses. The polypeptides described herein may be used after synthesis in vitro or in a suitable cellular expression system, including bacterial and eukaryotic cells, or alternatively, may be expressed in vivo in a subject in need thereof, by expressing a nucleic acid coding for the immunogenic polypeptide. Such nucleic acid vaccines may take any form, including naked DNA, plasmids, or viral vectors including adenoviral vectors.

Administration of the polypeptides, nucleic acid molecules, and/or immunogenic compositions according to the present disclosure can be performed using standard routes of administration. Non-limiting examples include parenteral administration, such as intravenous, intradermal, transdermal, intramuscular, subcutaneous, etc, or mucosal administration, e.g. intranasal, oral, and the like. The skilled person will be capable to determine the various possibilities to administer the polypeptides, nucleic acid molecules, and/or immunogenic compositions according to the present disclosure, in order to induce an immune response. In certain embodiments, the polypeptide, nucleic acid molecule, and/or immunogenic composition (or vaccine) is administered more than one time, i.e. in a so- called homologous prime-boost regimen. In certain embodiments where the polypeptide, nucleic acid molecule, and/or immunogenic composition is administered more than once, the administration of the second dose can be performed after a time interval of, for example, one week or more after the administration of the first dose, two weeks or more after the administration of the first dose , three weeks or more after the administration of the first dose, one month or more after the administration of the first dose, six weeks or more after the administration of the first dose, two months or more after the administration of the first dose, 3 months or more after the administration of the first dose, 4 months or more after the administration of the first dose, etc, up to several years after the administration of the first dose of the polypeptide, nucleic acid molecule, and/or immunogenic composition.

It is also possible to administer the vaccine more than twice, e.g. three times, four times, etc, so that the first priming administration is followed by more than one boosting administration. In other embodiments, the polypeptide, nucleic acid molecule, and/or immunogenic composition according to the present disclosure is administered only once.

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

The present disclosure further describes methods for preventing and/or treating an influenza virus disease in a subject utilizing the polypeptides, nucleic acids and/or compositions described herein. In a specific embodiment, a method for preventing and/or treating an influenza virus disease in a subject comprises administering to a subject in need thereof an effective amount of a polypeptide, nucleic acid and/or immunogenic composition, as described above. A therapeutically effective amount refers to an amount of the polypeptide, nucleic acid, and/or composition as defined herein, that is effective for preventing, ameliorating and/or treating a disease or condition resulting from infection by a group 1 or 2 influenza A virus, and/or an influenza B virus. Prevention encompasses inhibiting or reducing the spread of influenza virus or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection by an influenza virus. Ameloriation as used in herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of influenza infection.

Those in need of treatment include those already inflicted with a condition resulting from infection with a group 1 or a group 2 influenza A virus, or an influenza B virus, as well as those in which infection with influenza virus is to be prevented. The polypeptides, nucleic acids and/or compositions described herein thus may be administered to a naive subject, i.e., a subject that does not have a disease caused by influenza virus infection or has not been and is not currently infected with an influenza virus infection, or to subjects that already are and/or have been infected with an influenza virus.

In an embodiment, prevention and/or treatment may be targeted at patient groups that are susceptible to influenza virus infection. Such patient groups include, but are not limited to e.g., the elderly (e.g. ≥ 50 years old, ≥ 60 years old, and preferably ≥ 65 years old), the young (e.g. ≤ 5 years old, ≤ 1 year old), hospitalized patients and patients who have been treated with an antiviral compound but have shown an inadequate antiviral response.

In another embodiment, the polypeptides, nucleic acids and/or immunogenic compositions may be administered to a subject in combination with one or more other active agents, such as existing, or future influenza vaccines, monoclonal antibodies and/or antiviral agents, and/or antibacterial, and/or immunomodulatory agents. The one or more other active agents may be beneficial in the treatment and/or prevention of an influenza virus disease or may ameliorate a symptom or condition associated with an influenza virus disease. In some embodiments, the one or more other active agents are pain relievers, anti- fever medications, or therapies that alleviate or assist with breathing.

Dosage regimens of the polypeptides and/or nucleic acid molecules described herein can be adjusted to provide the optimum desired response (e.g., a therapeutic response). A suitable dosage range may for instance be 0.1-100 mg/kg body weight, preferably 1-50 mg/kg body weight, preferably 0.5-15 mg/kg body weight. The precise dosage of the polypeptides and/or nucleic acid molecules to be employed will e.g. depend on the route of administration, and the seriousness of the infection or disease caused by it, and should be decided according to the judgment of the practitioner and each subject's circumstances. For example, effective doses vary depending target site, physiological state of the patient (including age, body weight, health), and whether treatment is prophylactic or therapeutic.

Usually, the patient is a human but non-human mammals including transgenic mammals can also be treated. Treatment dosages are optimally titrated to optimize safety and efficacy.

The polypeptides described herein may also be used to verify binding of monoclonal antibodies identified as potential therapeutic candidates. In addition, the polypeptides described herein may be used as diagnostic tool, for example to test the immune status of an individual by establishing whether there are antibodies in the serum of such individual capable of binding to the polypeptide described herein. The present disclosure thus also relates to an in vitro diagnostic method for detecting the presence of an influenza infection in a patient said method comprising the steps of a) contacting a biological sample obtained from said patient with a polypeptide according to the present disclosure; and b) detecting the presence of antibody-antigen complexes.

The polypeptides described herein may also be used to identify new binding molecules or improve existing binding molecules, such as monoclonal antibodies and antiviral agents.

The invention is further illustrated in the following examples and figures. The examples are not intended to limit the scope of the invention in any way.

EXAMPLES Example 1: Preparation of novel stem based polypeptides discloses influenza hemagglutinin stem domain polypeptides, compositions and vaccines and methods of their use in the field of prevention and/or treatment of influenza. Here we describe additional sequences of stem domain polypeptides derived from the full length HA of H1N1 A/Brisbane/59/2007 (SEQ ID NO: 1). The novel stem domain polypeptides are obtained by site-directed mutation of H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) and present the broadly influenza neutralizing epitope of CR6261 (Throsby et al, 2009; Ekiert et al 2010) and/or CR9114.

H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) was derived from the full length HA of H1N1 A/Brisbane/59/2007 (SEQ ID NO: 1) by taking the following steps: 1. Removal of the cleavage site in HA0. Cleavage of wild type HA at this site results in HA1 and HA2. The removal can be achieved by mutation of R to Q at the P1 position (see e.g. Sun et al, 2010 for an explanation of the nomenclature of the cleavage site (position 343 in SEQ ID NO: 1). 2. Removal of the head domain by deleting amino acids 53 to 320 from SEQ ID NO; 1.

The remaining N- and C-terminal parts of the sequence were joined by a four residue flexible linker, GGGG. 3. Increasing the solubility of the loop (between the A-helix and the CD helix) formed by (the equivalent of) residues 402 to 418 in H1 A/Brisbane/59/2007 (SEQ ID NO: 1) in order to both increase the stability of the pre-fusion conformation and to destabilize the post-fusion conformation of the modified HA. In H1-mini2- cluster1+5+6-GCN4 (SEQ ID NO: 2) mutations F406S, V409T, F413G and L416S (numbering refers to SEQ ID NO: 1) were introduced 4. Introducing a disulfide bridge between amino acids at position 324 and 436 in H1 A/Brisbane/59/2007; this is achieved by introducing mutations R324C and Y436C. (numbering refers to SEQ ID NO: 1) 5. Introducing the GCN4 derived sequence MKQIEDKIEEIESKQ (SEQ ID NO: 5), that is known to trimerize, at position 419-433 (numbering refers to SEQ ID NO: 1).

In certain embodiments, the polypeptides described herein contain the intracellular sequences of HA and the transmembrane domain. In other embodiments, the sequence of the transmembrane and intracellular domain have been deleted from position (or the equivalent thereof, as determined from sequence alignment) 519, 520, 521, 522, 523, 524, 525, 526, 526, 527, 528, 529, or 530 of HA2 to the C-terminus of HA2 (numbering according to SEQ ID NO: 1) so that a secreted (soluble) polypeptide is produced following expression in cells. The soluble polypeptide can be further stabilized by introducing a sequence known to form trimeric structures, i.e. the foldon sequence AYVRKDGEWVLL (SEQ ID NO: 3), optionally connected through a short linker, as described above. The linker may optionally contain a cleavage site for processing afterwards according to protocols well known to those skilled in the art. To facilitate purification and detection of the soluble form a tag sequence may be optionally added, e.g. a histidine tag (HHHHHHH (SEQ ID NO: 20) or HHHHHH (SEQ ID NO: 21) or a FLAG tag (DYKDDDDK; SEQ ID NO: 22) or combination of these, optionally connected via short linkers. The linker may optionally contain (part of) a proteolytic cleavage site, e.g. LVPRGS (SEQ ID NO: 23) (thrombin) or IEGR (SEQ ID NO: 24) (Factor X) for processing afterwards according to protocols well known to those skilled in the art. The processed proteins are also encompassed in the present disclosure.

An example of such a C-teminal sequence combining FLAG-tag, thrombin cleavage site, foldon, and His sequences is SEQ ID NO: 4 FLAG- thrombin-foldon-His.

This sequence was combined with a soluble form of H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) sequence to create the parental sequence (SEQ ID NO: 6) that was used to create novel polypeptides described herein by mutagenesis. This sequence does not contain the leader sequence corresponding to amino acids 1-17 of SEQ ID NO: 1 and 2.

The stem domain polypeptides are created by deleting the part of the hemagglutinin sequence that encodes the head domain of the molecule and reconnecting the N- and C-terminal parts of the sequence on either side of the deletion through a linker as described in PCT/2012/073706 and above. The removal of the head domain leaves part of the molecule that was previously shielded from the aqueous solvent exposed, potentially destabilizing the structure of the polypeptides described herein. For this reason residues in the B-loop (in particular amino acid residue 406 (F and S in SEQ ID NO: 1 and 2, respectively), 409 (V and T) 413 (F and G) and 416 (L and S) were mutated in various combinations using parental sequence SEQ ID NO: 6 as the starting point.

SEQ ID NO: 6 was created from H1-mini2-cluster1+5+6-GCN4 (SEQ ID NO: 2) by removing the leader sequence, and replacing residues 520-565 with a Flag-thrombin- foldon--his sequence (SEQ ID NO: 4).

Similarly, in the area around the fusion peptide a number of hydrophobic residues are exposed to the solvent, caused by the fact that, unlike the native full length HA, the polypeptides described herein cannot be cleaved and undergo the associated conformational change that buries the hydrophobic fusion peptide in the interior of the protein. To address this issue some or all of the residues I337, I340, F352 and I353 in SEQ ID NO: 2 were also mutated.

The helix A of HA is an important part of the epitopes of broadly neutralizing epitopes of CR6261, CR9114 and FI6.v3. The amino acid residue M402 (numbering refers to SEQ ID NO: 1) sits at the C-terminal end of this helix, and to further stabilize the helix structure this residue was also targeted to create novel polypeptides described herein. Two different sets of mutant polypeptides are disclosed in Table 1.

Table 1: Mutations created in SEQ ID NO: 6. Corresponding amino acids in SEQ ID NO: 1 (full length, wt HA) and SEQ ID NO: 6 are also indicated.

Set 1 Position residue amino acids introduced ID SEQ ID NO: 1 NO: 6 337 I I E, I, K, V 340 I I I, K, R, T 352 F F D, F, V, Y 353 I I I, K, R, T 402 M M E, K, M, V 406 F S F, I, N, T, Y, S 409 V T A, G, I, R, T, V 413 F G F, I, N, S, T, Y, G 416 L S H, I, L, N, R, S Set 2 Position residue amino acids introduced ID SEQ ID NO: 1 NO: 6 337 I I A, E, I, K, T, V 340 I I F, I, N, S, T, Y 352 F F A, D, F, I, N, S, T, V, Y 353 I I E, G, I, K, R, V 402 M M M, R, T 406 F S F, H, L, Y, 409 V T F, I, S, T 413 F G E, K, M, V 416 L S I, L, R, S Example 2. Detection of polypeptide expression and binding to broadly neutralizing antibodies DNA sequences encoding the polypeptides described herein were transformed into Pichia pastoris or transfected into HEK293F cells using protocols well known to persons skilled in the art. Constructs used for expression in mammalian cells contained the HA leader sequence (residue 1-17 in SEQ ID NO: 1 and 2), whereas in constructs used for expression in P. pastoris the HA leader sequence was replaced with the yeast alpha factor leader sequence (SEQ ID NO: 7). In this way expressed protein are directed towards the cell culture medium thus allowing binding and expression to be determined without further purification of the polypeptides described herein. All sequences contained the FLAG-foldon-HIS C-terminal sequence (SEQ ID NO: 4).

Monoclonal antibody binding (CR6261, CR9114, CR8020) to polypeptides described herein was determined by ELISA. To this end ELISA plates were treated overnight with a 2 μg/ml monoclonal antibody solution (20 μl/well) at 4 C. After removal of the antibody solution the remaining surface was blocked with 4% solution of non-fat dry milk powder in PBS for a minimum of 1 h at room temperature. After washing of the plates, 20 μl of cell culture medium (neat or diluted) was added to each well and incubated for at least 1 h at room temperature. ELISA plates were then washed and 20 μl of anti-FLAG-HRP antibody solution (Sigma A8952, 2000 times diluted in 4% non-fat dry milk in PBS-Tween) was added. After incubation (1h at room temperature) plates were washed once more, and 20 μl luminescent substrate (Thermoscientific C#34078) was added to develop the signal. Alternatively, a colorimetric detection method can be used to develop the signal.

Expression of polypeptides described herein was determined from a homogeneous time-resolved fluorescence assay (for a general description see e.g. Degorce et al., Curr.

Chem. Genomics 2009 3: 22-32). To this end a mixture of Terbium (Tb) labeled anti- FLAG monoclonal antibody (donor) and Alexa488 labeled anti-His monoclonal antibody (acceptor) (HTRF solution) was prepared by adding 210.5 μl Anti-FLAG-TB (stock solution 26 μg/ml) and 1.68 ml of anti-HIS-488 (stock solution 50 μg/ml) to 80 ml of a 1 to 1 mixture of culture medium and 50 mM HEPES + 0.1% BSA. 19 μl of HTRF solution was added to each well of a ELISA plate and 1 μl of culture medium was added. Upon excitation and after a delay to allow interfering short-lived background signals arising from other compounds (proteins, media components etc) to decay, the ratio of fluorescence emission at 520 and 665 nm was determined. This is a measure of total protein content in the sample and is used to normalize the mAb binding signals between different experiments.

The polypeptides described herein listed in Table 4 and 5 were expressed in P.Pastoris following protocols well known to those skilled in the art. Culture medium was collected and binding to CR6261 binding of and expression of the stem domain polypeptides was determined as described above. Since the response in the binding assay scales with the concentration of expresses protein, ELISA binding signal was normalized for protein expression by comparing the ratio of binding signal over the signal in the HTRF assay for each expressed sequence. All expressed protein exhibit higher ratio’s of CR626 binding to HTRF signal compared to the parental sequence of SEQ ID NO: 6.

The polypeptides described herein listed in Table 6 were expressed in HEK293F cells following protocols well known to those skilled in the art. Culture medium was collected and binding to CR6261 binding of and expression of the stem domain polypeptides was determined as described above. The ratio of CR6261 binding to HTRF signals was calculated and compared to the ratio calculated for the parental sequence SEQ ID NO: 6. The results are listed in Table 6; all expressed proteins exhibit higher ratios, indicating that the stem polypeptides of the present disclosure show increased binding of CR6261.

Example 3: Purification and characterization of polypeptides of the invention To further characterize polypeptides described herein, the immunogens 127H1 (SEQ ID NO: 55), 86B4 (SEQ ID NO: 56), 74H9 (SEQ ID NO: 57), 6E12 (SEQ ID NO: 58) and 55G7 (SEQ ID NO: 59) were cultured and purified to homogeneity. In order to obtain a highly pure preparation of a polypeptide described herein, HEK293F cells were transfected with expression vector pcDNA2004 containing the genes encoding soluble forms of 127H1 (SEQ ID NO: 55), 86B4 (SEQ ID NO: 56), 74H9 (SEQ ID NO: 57), 6E12 (SEQ ID NO: 58) and 55G7 (SEQ ID NO: 59). Soluble forms were created in this case by replacement of residue 519-565 (numbering refers to SEQ ID NO: 1) with sequence RSLVPRGSPGHHHHHH (SEQ ID NO: 69). It will be understood by the skilled person that the leader sequence (or signal sequence) that directs transport of a protein during production (corresponding to amino acids 1-17 of SEQ ID NO: 1) will not be present in the secreted final polypeptide. The amino acid sequences of the soluble secreted proteins are given as SEQ ID NO: 65 to 69.

To produce the polypeptides described herein 1.0* 10 vc/mL were seeded by spinning down HEK293F cells (Invitrogen) at 300 g for 5 min and resuspending in 300 mL pre-warmed Freestyle™ medium per SF1000 flask. This culture was incubated for 1 hour at 37 °C, 10% CO2 at 110 rpm in a multitron incubator. After 1 hour the plasmid DNA was pipetted in 9.9 mL Optimem medium to a concentration of 1.0 μg/mL in the 300 mL culture volume. In parallel 440 μL 293fectin® was pipetted in 9.9 mL Optimem medium and incubated for 5 minutes at room temperature. After 5 minutes the plasmid DNA/Optimem mix was added to the 293fectin®/Optimem mix and incubated at room temperature for 20 minutes. After the incubation the plasmid DNA/293fectin® mix was added drop wise to the cell suspension. The transfected cultured was incubated at 37 °C, 10% CO2 and 110 rpm in a multitron incubator. At day 7 cells were separated from the culture medium by centrifugation (30 minutes at 3000 g), while the supernatant containing the soluble polypeptides described herein was filtrated over a 0.2 μm bottle top filter for further processing.

For purification purposes ca 1400 ml culture supernatant from cells transfected with genes encoding the soluble forms (as described above) of polypeptides described herein 127H1 (SEQ ID NO: 55 ), 86B4 (SEQ ID NO: 56 ), 74H9 (SEQ ID NO: 57), 6E12 (SEQ ID NO: 58) or 55G7 (SEQ ID NO: 59) was applied to a 24 ml Ni Sepharose HP column, pre- equilibrated in wash buffer (20 mM TRIS, 500 mM NaCl, pH 7.8). Following a washing step with 10 mM Imidaze in wash buffer the bound polypeptides described herein were eluted with a step-wise gradient of 300 mM imidazole in wash buffer. The elution peaks were collected, buffer exchanged, concentrated, and applied to a size exclusion column for further purification (Superdex 200). Fractions were collected during elution and analyzed for protein content by SDS-PAGE and Western Blot (using a monoclonal antibody specific for histidine-tags). Fractions containing the purified polypeptides described herein were collected and used for further analysis. Purity was determined by Size exclusion chromatography and was above 90% in all cases. Characteristics of the purification procedures are listed in table 8.

To analyze the binding reaction between polypeptides described herein and to confirm the presence of the conformational epitopes of CR6261 and CR9114, the complexation of these antibodies with the purified protein was studied by biolayer interferometry (Octet Red , Forte Bio). To this end, biotinylated CR6261, CR9114 were immobilized on streptavidin coated sensors, which subsequently were exposed first to a solution of the purified polypeptide described herein to measure the rate of association and then to a wash solution to measure the rate of dissociation. The immobilized CR6261and CR9114 both recognize the polypeptides described herein as evidenced by the clear responses after exposure to the soluble form of polypeptides described herein. To estimate the dissociation constant for the binding interaction a titration was performed using a 2-fold dilution series. Sensors containing immobilized CR6261 were exposed to solutions containing the soluble polypeptides described herein at concentrations of 750, 375, 163, 81, 40, 20 and 10 nM, respectively, except in the case of the purified s55G7 (SEQ ID NO: 68) where a concentration range of 2000, 1000, 500, 250, 125, 63 and 31 nM was used. Similarly, sensors containing immobilized CR9114 were exposed to solutions containing the soluble polypeptides described herein at concentrations of 300, 150, 75, 38, 19, 9 and 5 nM, respectively. In all cases, the final response after 4000 seconds was recorded, plotted as a function of the polypeptide concentration, and a fit to a steady state 1:1 binding model was performed to calculate an apparent dissociation constant K (see figure 2A to E). Values determined in this way are listed in Table 8.

The binding between polypeptides described herein s127H1 (SEQ ID NO: 66), s86B4 (SEQ ID NO: 67), s74H9 (SEQ ID NO: 65), s6E12 (SEQ ID NO: 69) and s55G7 (SEQ ID NO: 68) and Fab fragments of CR6261, CR9114 and as control CR8020 was studied by analytical size exclusion chromatography combined with multi-angle light scattering (SEC- MALS). This technology allows the simultaneous separation and estimation of the molecular size for different molecular species and/or complexes. The results are shown in figure 3A to E, and summarized in Table 9. The data indicate that the polypeptides described herein are monomeric and form a 1:1 complex with Fab fragments of CR6261 and CR9114, but not CR8020. This is in line with the specificity of the binding reactions of the Fab fragments, since CR6261 and CR9114 bind to HA’s derived from group 1, whereas CR8020 does not.

In conclusion we have shown that soluble forms of polypeptides described herein 127H1 (SEQ ID NO: 55), 86B4 (SEQ ID NO: 56), 74H9 (SEQ ID NO: 57), 6E12 (SEQ ID NO: 581) and 55G7 (SEQ ID NO: 59) can be produced and purified to homogeneity. The purified soluble polypeptides described herein are capable of binding broadly neutralizing monoclonal antibodies CR6261 and CR9114 with high affinity, confirming the presence of the corresponding neutralizing epitopes in these stem domain polypeptides.

Example 4: Evaluation of protective efficacy of polypeptides of the invention in a lethal influenza challenge model In order to evaluate the protective efficacy of polypeptides described herein in a lethal influenza challenge model, groups of 10 female BALB/c mice (age 6-8 weeks) were immunized 3 times at 3 week intervals with 30 µg of purified s86B4 (SEQ ID NO: 67) and s74H9 (SEQ ID NO: 65) adjuvated with 10 µg Matrix-M. As a positive control for the challenge model CR6261 (15 mg/kg) was administered i.m. 1 day prior to challenge, while immunization with PBS served as a negative control. Four weeks after the last immunization mice were challenged with 25xLD50 heterologous challenge virus (H1N1 A/NL/602/09) and monitored daily (survival, weight, clinical scores) for 3 weeks.

Pre-challenge serum is tested in ELISA assays for binding to the polypeptide described herein used for immunization (to verify correct immunization), binding to soluble full length HA form H1N1 A/Brisbane/59/07 and competition for binding to full length HA with the broadly neutralizing antibody monoclonal antibody CR9114 (to determine whether induced antibodies bind at close proximity to the broadly neutralizing CR9114 epitope). The results are shown in Figure 4A and B.

The results show that the experiment is valid since all mice in the PBS control group succumb to infection at or before day 8 post challenge, whereas the positive control group (15mg/kg CR6261, 1 day before challenge) is fully protected. In contrast to the PBS treated mice, 4 out of 10 of the mice immunized with polypeptide described herein s86B4 (SEQ ID NO: 67) or s74H9 (SEQ ID NO: 65) survive the lethal challenge. This results in an increased survival time and reduced clinical score for groups immunized with polypeptide described herein compared to the PBS control group.

The ELISA data (see Figure 4B) using either the cognate antigen (i.e. either s86B4 or s74H9) or the full length HA as the antigen indicate that both polypeptides described herein s74H9 and s86B4 are immunogenic and induce antibodies that are capable of recognizing full length HA.

To further understand the immunological response to the immunization a competition binding ELISA was performed. To this end plate bound full length HA is incubated with serial diluted serum samples, after which CR9114-biotin at a predetermined concentration is added. After further incubation, the amount of CR9114- biotin bound is quantified. Data are analysed using linear regression of OD versus log dilution, expressed as ‘slope OD’ (∆OD/10 fold dilution). The data show that the antibodies that are capable of competing for binding with the broadly neutralizing antibody CR9114 are induced by immunization with polypeptides described herein. As a comparison, levels induced by unlabeled CR9114 (i.e. self-competition) and the non- binding monoclonal antibody CR8020, both serially diluted from 5 μg/ml starting concentration are indicated in a separate graph.

In conclusion, these data show that immunization with polypeptides described herein s86B4 (SEQ ID NO: 67) or s74H9 (SEQ ID NO: 65) can protect mice against lethal infection with influenza. Both polypeptides are immunogenic, induce antibodies that can bind to full length HA and at least part of these antibodies bind at, or close to, the epitope of the broadly neutralizing epitope of monoclonal antibody CR9114.

Example 5: Evaluation of protective efficacy of another polypeptide of the invention in a lethal influenza challenge model In order to evaluate the protective efficacy of another polypeptide described herein s127H1 (SEQ ID NO: 66) in a lethal influenza challenge model, groups of 10 female BALB/c mice (age 6-8 weeks) were immunized 3 times at 3 week intervals with µg of purified s127H1 adjuvated with 10 µg Matrix-M. As a positive control for the challenge model, broadly neutralizing antibody monoclonal antibody CR6261 (15 mg/kg) was administered i.m. 1 day prior to challenge, while immunization with PBS served as a negative control. Four weeks after the last immunization mice were challenged with 25xLD50 heterologous challenge virus (H1N1 A/Puerto Rico/8/34) and monitored daily (survival, weight, clinical scores) for 3 weeks. Pre-challenge serum is tested in ELISA assays for binding to polypeptide described herein s127H1 that was used for immunization (to verify correct immunization), binding to soluble H1N1 A/Brisbane/59/07 full length HA and competition for binding to full length HA with the broadly neutralizing antibody monoclonal antibody CR9114 (to determine whether induced antibodies bind at close proximity to the broadly neutralizing CR9114 epitope).

The results are shown in Figure 5A and B.

The results show that the experiment is valid since all mice in the PBS control group succumb to infection at day 7 post challenge, whereas the positive control group (15mg/kg CR6261, 1 day before challenge) is fully protected. In contrast to the PBS treated mice, 7 out of 10 of the mice immunized with polypeptide described herein s127H1 (SEQ ID NO: 66) survive the lethal challenge. This resulted in an increased survival proportion, increased survival time and reduced clinical score for groups immunized with polypeptide described herein s127H1 compared to the PBS control group.

The ELISA data using s127H1 or the soluble full length HA as the antigen indicate that the polypeptide described herein s127H1 is immunogenic and induces antibodies that are capable of recognizing full length HA (see figure 5B). To further understand the immunological response to the immunization a competition binding ELISA was performed. To this end plate bound full length HA is incubated with serial diluted serum samples, after which CR9114-biotin at a predetermined titrated concentration is added. After further incubation, the amount of CR9114-biotin bound is quantified using streptavin -conjugated horse radish peroxidase following protocols well known in the art. Data are analysed using linear regression of OD versus log dilution, expressed as ‘slope OD’ (∆OD/10 fold dilution). The data show that the antibodies that are capable of competing for binding with the broadly neutralizing antibody CR9114 are induced by immunization with polypeptides described herein, as indicated by the elevated levels of competition observed in figure 5B. As a comparison, levels induced by unlabeled CR9114 (i.e. self-competition) and the non-binding monoclonal antibody CR8020, both serially diluted from 5 μg/ml starting concentration are indicated in a separate graph.

In conclusion we have shown that immunization with polypeptides described herein s127H1 (SEQ ID NO: 66) can protect mice against lethal infection with influenza.

The polypeptide is immunogenic, induces antibodies that can bind to full length HA and at least part of these antibodies bind at, or close to, the epitope of the broadly neutralizing epitope of monoclonal antibody CR9114.

Example 6: Analysis of improved CR6261 binders from set 1 and set 2.

To further understand which sequences can lead to increased binding of CR6261 compared to the parent sequence SEQ ID NO: 6, the sequences listed in Table 4 and 5 were further analyzed. To this end, for each targeted position the relative frequency of each of the variable amino acids present in the set of improved binders was calculated and compared to the expected frequency if there would be no preference for any of the variable amino acids (see figure 6 and 8 for sequences of set 1 and 2, respectively). The data show that some amino acids are more prevalent at certain positions in sequences that show improved CR6261 binding.

Both in set 1 and 2, high prevalence is observed for Met at position 402 (numbering refers to SEQ ID NO: 1) in the sequences with improved binding, although in set 1 Val is also observed in some cases. Similarly, at position 352 high prevalence of the aromatic amino acids Phe and Tyr, at position 416 increased prevalence of Ser (although not very strong in set 1), and for position 337 increased prevalence of Lys is observed in both sets. It should be noted that Ser at position 416 introduces a putative N-glycosylation site at Asn414. Furthermore, at position 340 Asn is highly prevalent in improved CR6261 binders in set 2, whereas in set 1 Thr is more prevalent; both mutations introduce a putative glycosylation site at position 340: Asn leads to a putative N-glycosylation site, whereas Thr leads to a putative O-glycosylation site. At position 353 Thr is highly prevalent in improved binders from set 1, whereas in improved binders from set 2 Val is the most prevalent amino acid. At positions 406 and 409 the most prevalent amino acids in improved CR6261 binders are hydrophobic: at position 406 Ile and Phe (set 1) and Phe and Leu (set 2) are the most prevalent, at position 409 Ile and Val ( set 1) and Phe and Ile (set 2) are the most prevalent. Finally, at position 413 Phe is the most prevalent amino acid among improved CR6261 binders from set 1, whereas Glu is the most prevalent among improved binders from set 2.

The sequences of the improved CR6261 binders were analyzed further to determine whether some combinations of amino acids were more prevalent among improved CR6261 binders than others. To this end the number of improved binder sequences containing a particular combination of amino acids was counted and plotted as shown in Figure 7A, B (improved binders from set 1) and Figure 9A,B (set 2). The data were organized according to the different areas addressed in the mutant sets of polypeptides, i.e. the area of the fusion peptide (residue no 337, 340, 352 and 353; Figure 7A and 9A for improved binders from set 1 and 2, respectively) and the B-loop (residue no 406, 409, 413 and 416; figure 7B and 9B). In view of the very high prevalence of Met at position 402, this position was not included in the analysis to detect frequently occurring combinations as nearly all combinations of other positions with position 402 contain Met.

Among improved CR6261 binders from set 1, combinations of either Phe or Tyr at position 352 with either Ile or Thr at position 353 are most prevalent (boxed areas in Figure 7A). With respect to combinations of amino acids in the B-loop area, Ile at position 406 combined with either Phe or Tyr (i.e. an aromatic amino acid) at position 413 and Tyr at position 406 combined with N at position 413 show increased prevalence among improved CR6261 binders (boxed areas in Figure 7B).

For the analysis of sequences of improved CR6261 binders among the polypeptides from set 2 focusing on the fusion peptide area, only sequences containing either Phe or Tyr at position 352 were taken into account. Most prevalent among improved CR6261 binders with Phe or Tyr at position 352 are sequences with Asn at position 340 (see boxed area in Figure 9A). For the B-loop area sequences with either Phe, Leu or Tyr at position 406, in combination with either Glu, Met or Val at position 413 (boxed areas in Figure 9B) are most prevalent among improved CR6261 binders.

In conclusion, the analysis shows that in the fusion peptide area the introduction of a Thr at position 353, in particular in combination with an aromatic residue (Phe or Tyr) at position 352 can improve the binding of CR6261 to soluble polypeptides described herein. Furthermore, Lys at position 337 or introduction of a putative N-glycosylation site by introducing Asn at position 340 can also contribute to improved binding of CR6261.

In the B-loop area a large hydrophobic residue (Phe, Tyr, Leu or Ile) can contribute to improved binding to CR6261. In particular, Phe, Leu, or Tyr at position 406 in combination with either Glu, Met, or Val at position 413 can improve binding of CR6261. Polypeptides described herein comprising these residues at the described positions are preferred embodiments of the present disclosure.

Table 2. Standard amino acids, abbreviations and properties Amino Acid 3-Letter 1-Letter Side chain Side chain charge (pH 7.4) polarity alanine Ala A nonpolar Neutral arginine Arg R polar Positive asparagine Asn N polar Neutral aspartic acid Asp D polar Negative cysteine Cys C nonpolar Neutral glutamic acid Glu E polar Negative glutamine Gln Q polar Neutral glycine Gly G nonpolar Neutral histidine His H polar positive (10%) neutral(90%) isoleucine Ile I nonpolar Neutral leucine Leu L nonpolar Neutral lysine Lys K polar Positive methionine Met M nonpolar Neutral phenylalanine Phe F nonpolar Neutral proline Pro P nonpolar Neutral serine Ser S polar Neutral threonine Thr T polar Neutral tryptophan Trp W nonpolar Neutral tyrosine Tyr Y polar Neutral valine Val V nonpolar Neutral Table 3. Sequence alignment of H1 sequences according to particular embodiments of the invention 1. A/Solomon Islands/6/2003 (H1N1) (SEQ ID NO: 25) 2. A/Brisbane/59/2007 (H1N1) (SEQ ID NO: 1) 3. A/New Caledonia/20/1999(H1N1) (SEQ ID NO: 26) 4. A/California/07/2009 (H1N1) (SEQ ID NO: 27) . A/swine/Hubei/S1/2009(H1N1) (SEQ ID NO: 28) 6. A/swine/Haseluenne/IDT2617/2003(H1N1) (SEQ ID NO: 29) 7. A/NewYork/8/2006(H1N1) (SEQ ID NO: 30) 8. A/SolomonIslands/3/2006(H1N1) (SEQ ID NO: 31) 9. A/NewYork/146/2000(H1N1) (SEQ ID NO: 32) . A/NewYork/653/1996(H1N1) (SEQ ID NO: 33) 11. A/Beijing/262/1995(H1N1) (SEQ ID NO: 34) 12. A/Texas/36/1991(H1N1) (SEQ ID NO: 35) 13. A/Singapore/6/1986(H1N1) (SEQ ID NO: 36) 14. A/Chile/1/1983(H1N1) (SEQ ID NO: 37) . A/Baylor/11515/1982(H1N1) (SEQ ID NO: 38) 16. A/Brazil/11/1978(H1N1) (SEQ ID NO: 39) 17. A/USSR/90/1977(H1N1) (SEQ ID NO: 40) 18. A/NewJersey/8/1976(H1N1) (SEQ ID NO: 41) 19. A/Denver/1957(H1N1) (SEQ ID NO: 42) . A/Albany/4835/1948(H1N1) (SEQ ID NO: 43) 21. A/FortMonmouth/1/1947(H1N1) (SEQ ID NO: 44) 22. A/Cameron/1946(H1N1) (SEQ ID NO: 45) 23. A/Weiss/1943(H1N1) (SEQ ID NO: 46) 24. A/Iowa/1943(H1N1) (SEQ ID NO: 47) . A/Bellamy/1942(H1N1) (SEQ ID NO: 48) 26. A/PuertoRico/8/1934(H1N1) (SEQ ID NO: 49) 27. A/WSN/1933(H1N1) (SEQ ID NO: 50) 28. A/SouthCarolina/1/1918(H1N1) (SEQ ID NO: 51) 1. MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60 2. MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL ENSHNGKLCL 60 3. MKAKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60 4. MKAILVVLLY TFATANADTL CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDKHNGKLCK 60 . MEAKLFVLFC AFTALKADTF CVGYHANYST HTVDTILEKN VTVTHSVNLL ENSHNGKLCS 60 6. MEAKLFVLFC AFTALKADTI CVGYHANNST DTVDTILEKN VTVTHSINLL ENNHNGKLCS 60 7. MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60 40 8. MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60 9. MKAKLLVLLC AFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 . MKAKLLVLLC AFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 11. MKAKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCL 60 12. MKAKLLVLLC AFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 45 13. MKAKLLVLLC AFTATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 14. MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDNHNGKLCK 60 . MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 16. MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 17. MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 50 18. MKAKLLVLLC AFTATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 19. MKAKLLILLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 . MKAKLLVLLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 21. MKAKLLILLC ALTATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 22. MKAKLLILLC ALSATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 55 23. MKARLLVLLC ALAATDADTI CIGYHANNST DTVDTILEKN VTVTHSVNLL EDSHNGKLCR 60 24. MKARLLVLLC ALAATDADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 . MKARLLVLLC AIAATDADTI CIGYHANNST DTVDTILEKN VTVTHSVNLL EDSHNGKLCR 60 26. MKANLLVLLC ALAAADADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCR 60 27. MKAKLLVLLY AFVATDADTI CIGYHANNST DTVDTIFEKN VAVTHSVNLL EDRHNGKLCK 60 28. MEARLLVLLC AFAATNADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL EDSHNGKLCK 60 *:. *::** :: :: ***: ********** *****::*** *:******** *: ****** 1. LKGIAPLQLG NCSVAGWILG NPECELLISR ESWSYIVEKP NPENGTCYPG HFADYEELRE 120 2. LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVEKP NPENGTCYPG HFADYEELRE 120 3. LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVETP NPENGTCYPG YFADYEELRE 120 4. LRGVAPLHLG KCNIAGWILG NPECESLSTA SSWSYIVETP SSDNGTCYPG DFIDYEELRE 120 . LNGKIPLQLG NCNVAGWILG NPKCDLLLTA NSSSYIIETS KSKNGACYPG EFADYEELKE 120 6. LNGKAPLQLG NCNVAGWILG NPECDLLLTV DSWSYIIETS NSKNGACYPG EFADYEELRE 120 7. LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVETP NPENGTCYPG YFADYEELRE 120 8. LKGIAPLQLG NCSVAGWILG NPECELLISR ESWSYIVEKP NPENGTCYPG HFADYEELRE 120 9. LKGTAPLQLG NCSIAGWILG NPECESLFSK ESWSYIAETP NPKNGTCYPG YFADYEELRE 120 . LKGTAPLQLG NCSVAGWILG NPECESLFSK ESWSYIAETP NPENGTCYPG YFADYEELRE 120 11. LKGIAPLQLG NCSVAGWILG NPECESLISK ESWSYIVETP NPENGTCYPG YFADYEELRE 120 12. LKGIAPLQLG NCSVAGWILG NPKCESLFSK ESWSYIAETP NPENGTCYPG YFADYEELRE 120 13. LKGIAPLQLG NCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 120 14. LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 120 . LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 120 16. LKGIAPLQLG KCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 120 17. LKGIAPLQLG KCNIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 120 18. LKGIAPLQLG NCSIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 120 19. LKGKAPLQLG NCNIAGWVLG NPECESLLSN RSWSYIAETP NSENGTCYPG DFADYEELRE 120 . LKGIAPLQLG KCNIAGWILG NPECESLFSK KSWSYIAETP NSENGTCYPG YFADYEELRE 120 21. LKGIAPLQLG KCNIAGWILG NPECESLLSK RSWSYIAETP NSENGACYPG DFADYEELRE 120 22. LKGIAPLQLG KCNIAGWILG NPECESLLSK RSWSYIAETP NSENGACYPG DFADYEELRE 120 23. LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVEIP NSENGTCYPG DFTDYEELRE 120 24. LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVETP NSENGTCYPG DFIDYEELRE 120 . LKGIAPLQLG KCNIAGWILG NPECESLLSE RSWSYIVETP NSENGTCYPG DFIDYEELRE 120 26. LKGIAPLQLG KCNIAGWLLG NPECDPLLPV RSWSYIVETP NSENGICYPG DFIDYEELRE 120 27. LKGIAPLQLG KCNITGWLLG NPECDSLLPA RSWSYIVETP NSENGACYPG DFIDYEELRE 120 28. LKGIAPLQLG KCNIAGWLLG NPECDLLLTA SSWSYIVETS NSENGTCYPG DFIDYEELRE 120 *:* ***:** :*.::**:** **:*: * . *****.* . ...** **** * ******* 1. QLSSVSSFER FEIFPKESSW PNHTTT-GVS ASCSHNGESS FYKNLLWLTG KNGLYPNLSK 179 2. QLSSVSSFER FEIFPKESSW PNHTVT-GVS ASCSHNGESS FYRNLLWLTG KNGLYPNLSK 179 40 3. QLSSVSSFER FEIFPKESSW PNHTVT-GVS ASCSHNGKSS FYRNLLWLTG KNGLYPNLSK 179 4. QLSSVSSFER FEIFPKTSSW PNHDSNKGVT AACPHAGAKS FYKNLIWLVK KGNSYPKLSK 180 . QLSTVSSFER FEIFPKAISW PDHDATRGTT VACSHSGVNS FYRNLLSTVK KGNSYPKLSK 180 6. QLSTVSSFER FEIFPKATSW PNHDTTRGTT ISCSHSGANS FYRNLLWIVK KGNSYPKLSK 180 7. QLSSVSSFER FEIFPKESSW PNHTVT-GVS ASCSHNGKSS FYRNLLWLTG KNGLYPNLSK 179 45 8. QLSSVSSFER FEIFPKESSW PNHTTT-GVS ASCSHNGESS FYKNLLWLTG KNGLYPNLSK 179 9. QLSSVSSFER FEIFPKDSSW PNHTVTKGVT ASCSHNGKSS FYKNLLWLTE KNGLYPNLSK 180 . QLSSVSSFER FEIFPKESSW PNHTVTKGVT ASCSHNGKSS FYKNLLWLTE KNGLYPNLSK 180 11. QLSSVSSFER FEIFPKESSW PNHTVT-GVT ASCSHNGKSS FYRNLLWLTE KNGLYPNLSN 179 12. QLSSVSSFER FEIFPKESSW PNHTVTKGVT TSCSHNGKSS FYRNLLWLTK KNGLYPNVSK 180 50 13. QLSSVSSFER FEIFPKESSW PNHTVTKGVT ASCSHKGRSS FYRNLLWLTK KNGSYPNLSK 180 14. QLSSVSSFER FEIFPKESSW PKHNVTKGVT AACSHKGKSS FYRNLLWLTE KNGSYPNLSK 180 . QLSSVSSFER FEIFPKESSW PKHSVTRGVT ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK 180 16. QLSSVSSFER FEIFPKERSW PKHNITRGVT ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK 180 17. QLSSVSSFER FEIFPKERSW PKHNVTRGVT ASCSHKGKSS FYRNLLWLTE KNGSYPNLSK 180 55 18. QLSSVSSFER FEIFPKESSW PNHTVTKGVT ASCSHKGRSS FYRNLLWLTK KNGSYPNLSK 180 19. QLSSVSSFER FEIFPKERSW PNHTTR-GVT AACPHARKSS FYKNLVWLTE ANGSYPNLSR 179 . QLSSVSSFER FEIFPKERSW PKHNITRGVT AACSHKGKSS FYRNLLWLTE KNGSYPNLNK 180 21. QLSSVSSFER FEIFPKERSW PKHNITRGVT AACSHAGKSS FYKNLLWLTE TDGSYPKLSK 180 22. QLSSVSSFER FEIFPKGRSW PEHNIDIGVT AACSHAGKSS FYKNLLWLTE KDGSYPNLNK 180 60 23. QLSSVSSFER FEIFPKESSW PKHNTARGVT AACSHAGKSS FYRNLLWLTE KDGSYPNLKN 180 24. QLSSVSSFER FEIFSKESSW PKHTTG-GVT AACSHAGKSS FYRNLLWLTE KDGSYPNLNN 179 . QLSSVTSFER FEIFPKETSW PKHNTTKGVT AACSHAGKCS FYRNLLWLTE KDGSYPNLNN 180 26. QLSSVSSFER FEIFPKESSW PNHNTN-GVT AACSHEGKSS FYRNLLWLTE KEGSYPKLKN 179 27. QLSSVSSLER FEIFPKESSW PNHTFN-GVT VSCSHRGKSS FYRNLLWLTK KGDSYPKLTN 179 28. QLSSVSSFEK FEIFPKTSSW PNHETTKGVT AACSYAGASS FYRNLLWLTK KGSSYPKLSK 180 *****:*:*: ****.* ** *:* **: .:*.: * **:**:**. . **::.. 1. SYANNKEKEV LVLWGVHHPP NIGDQRALYH KENAYVSVVS SHYSRKFTPE IAKRPKVRDQ 239 2. SYANNKEKEV LVLWGVHHPP NIGNQKALYH TENAYVSVVS SHYSRKFTPE IAKRPKVRDQ 239 3. SYVNNKEKEV LVLWGVHHPP NIGNQRALYH TENAYVSVVS SHYSRRFTPE IAKRPKVRDQ 239 4. SYINDKGKEV LVLWGIHHPS TSADQQSLYQ NADAYVFVGS SRYSKKFKPE IAIRPKVRXX 240 5. SYTNNKGKEV LVIWGVHHPP TDSVQQTLYQ NKHTYVSVGS SKYYKRFTPE IVARPKVRGQ 240 6. SYTNNKGKEV LVIWGVHHPP TDSDQQTLYQ NNHTYVSVGS SKYYQRFTPE IVTRPKVRGQ 240 7. SYANNKEKEV LVLWGVHHPP NIGDQRALYH TENAYVSVVS SHYSRRFTPE IAKRPKVRDQ 239 8. SYANNKEKEV LVLWGVHHPP NIGDQRALYH KENAYVSVVS SHYSRKFTPE IAKRPKVRDQ 239 9. SYVNKKGKEV LVLWGVHHPS NMGDQRAIYH KENAYVSVLS SHYSRRFTPE IAKRPKVRDQ 240 10. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSVVS SHYSRRFTPE ITKRPKVRDQ 240 11. SYVNNKEKEV LVLWGVHHPS NIRDQRAIYH TENAYVSVVS SHYSRRFTPE IAKRPKVRGQ 239 12. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSVVS SHYSRRFTPE IAKRPKVRDQ 240 13. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSVVS SHYNRRFTPE IAKRPKVRDQ 240 14. SYVNNKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSVVS SHYNRRFTPE IAKRPKVRNQ 240 15. SYVNDKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSVVS SHYNRRFTPE IAKRPKVRDQ 240 16. SYVNNKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSVVS SNYNRRFTPE IAKRPKVRGQ 240 17. SYVNNKEKEV LVLWGVHHPS NIEDQKTIYR KENAYVSVVS SNYNRRFTPE IAERPKVRGQ 240 18. SYVNNKEKEV LVLWGVHHPS NIGDQRAIYH TENAYVSVVS SHYNRRFTPE IAKRPKVRDQ 240 19. SYVNNQEKEV LVLWGVHHPS NIEEQRALYR KDNAYVSVVS SNYNRRFTPE IAKRPKVRDQ 239 20. SYVNNKEKEV LVLWGVHHPS NIEDQKTLYR KENAYVSVVS SNYNRRFTPE IAERPKVRGQ 240 21. SYVNNKEKEV LVLWGVHHPS NIEDQKTLYR KENAYVSVVS SNYNRRFTPE IAERPKVRGQ 240 22. SYVNKKEKEV LILWGVHHPP NIENQKTLYR KENAYVSVVS SNYNRRFTPE IAERPKVRGQ 240 23. SYVNKKGKEV LVLWGVHHPS SIKEQQTLYQ KENAYVSVVS SNYNRRFTPE IAERPKVRDQ 240 24. SYVNKKGKEV LVLWGVHHPS NIKDQQTLYQ KENAYVSVVS SNYNRRFTPE IAERPKVRGQ 239 25. SYVNKKGKEV LVLWGVHHPS NIKDQQTLYQ KENAYVSVVS SNYNRRFTPE IAERPKVRGQ 240 26. SYVNKKGKEV LVLWGIHHPP NSKEQQNLYQ NENAYVSVVT SNYNRRFTPE IAERPKVRDQ 239 27. SYVNNKGKEV LVLWGVHHPS SSDEQQSLYS NGNAYVSVAS SNYNRRFTPE IAARPKVKDQ 239 28. SYVNNKGKEV LVLWGVHHPP TGTDQQSLYQ NADAYVSVGS SKYNRRFTPE IAARPKVRDQ 240 ** *.: *** *:***:***. . :*: :* . :*** * : *.*.::*.** *: ****: 1. EGRINYYWTL LEPGDTIIFE ANGNLIAPRY AFALSRGFGS GIINSNAPMD ECDAKCQTPQ 299 2. EGRINYYWTL LEPGDTIIFE ANGNLIAPRY AFALSRGFGS GIINSNAPMD KCDAKCQTPQ 299 3. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNAPMD ECDAKCQTPQ 299 40 4. EGRMNYYWTL VEPGDKITFE ATGNLVVPRY AFAMERNAGS GIIISDTPVH DCNTTCQTPK 300 . AGRMNYYWTL FDQGDTITFE ATGNLIAPWH AFALKKGSSS GIMLSDAQVH NCTTKCQTPH 300 6. AGRMNYYWTL LDQGDTITFE ATGNLIAPWH AFALNKGPSS GIMISDAHVH NCTTKCQTPH 300 7. EGRINYYWTL LEPGDTIIFE ANGNLIAPRF AFALSRGFGS GIITSNAPMD ECDAKCQTPQ 299 8. EGRINYYWTL LEPGDTIIFE ANGNLIAPRY AFALSRGFGS GIINSNAPMD ECDAKCQTPQ 299 45 9. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIIISNASMG ECDAKCQTPQ 300 . EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMG ECDAKCQTPQ 300 11. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNAPMN ECDAKCQTPQ 299 12. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDAKCQTPQ 300 13. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDAKCQTPQ 300 50 14. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDAKCQTPQ 300 . EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNVSMD ECDAKCQTPQ 300 16. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDTKCQTPQ 300 17. AGRINYYWTL LEPGDTIIFE ANGNLIAPWH AFALNRGFGS GIITSNASMD ECDTKCQTPQ 300 18. EGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMD ECDAKCQTPQ 300 55 19. SGRMNYYWTL LEPGDTIIFE ATGNLIAPWY AFALSRGPGS GIITSNAPLD ECDTKCQTPQ 299 . AGRINYYWTL LEPGDTIIFE ANGNLIAPWH AFALSRGFGS GIITSNASMD ECDTKCQTPQ 300 21. AGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRDFGS GIITSNASMD ECDTKCQTPQ 300 22. AGRINYYWTL LEPGDTIIFE ANGNLIAPWY AFALNRGIGS GIITSNASMD ECDTKCQTPQ 300 23. AGRMNYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMH ECDTKCQTPQ 300 60 24. AGRINYYWTL LKPGDTIMFE ANGNLIAPWY AFALSRGFGS GIITSNASMH ECDTKCQTPQ 299 . AGRMNYYWTL LEPGDTIIFE ANGNLIAPWY AFALSRGFGS GIITSNASMH ECNTKCQTPQ 300 26. AGRMNYYWTL LKPGDTIIFE ANGNLIAPMY AFALRRGFGS GIITSNASMH ECNTKCQTPL 299 27. HGRMNYYWTL LEPGDTIIFE ATGNLIAPWY AFALSRGFES GIITSNASMH ECNTKCQTPQ 299 28. AGRMNYYWTL LEPGDTITFE ATGNLIAPWY AFALNRGSGS GIITSDAPVH DCNTKCQTPH 300 **:****** ::***.* ** *.***:.* . ***: *. * *** *:..: .*::.**** 1. GAINSSLPFQ NVHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 2. GAINSSLPFQ NVHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 3. GAINSSLPFQ NVHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 4. GAINTSLPFQ NIHPITIGKC PKYVKSTKLR LATGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 . GALKNNLPLQ NVHLFTIGEC PKYVKSTQLR MATGLRNIPS IQSRGLFGAI AGFIEGGRTG 360 6. GALKSNLPFQ NVHPSTIGEC PKYVKSTQLR MATGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 7. GAINSSLPFQ NVHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 8. GAINSSLPFQ NVHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 9. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNVPS IQSRGLFGAI AGFIEGGWTG 360 . GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 11. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 12. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 13. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 14. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 . GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 16. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 17. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 18. GAINSSLPFQ NVHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 19. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS VQSRGLFGAI AGFIEGGWTG 359 . GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 21. GAINSSLPFQ NIHPVTIGEC PKYVKSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 22. GAINSSLPFQ NIHPFTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWDG 360 23. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 24. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 . GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 26. GAINSSLPYQ NIHPVTIGEC PKYVRSAKLR MVTGLRNIPS IQSRGLFGAI AGFIEGGWTG 359 27. GSINSNLPFQ NIHPVTIGEC PKYVRSTKLR MVTGLRNIPS IQYRGLFGAI AGFIEGGWTG 359 28. GAINSSLPFQ NIHPVTIGEC PKYVRSTKLR MATGLRNIPS IQSRGLFGAI AGFIEGGWTG 360 *:**:.**:* *:**.***:* ****:*:*** :.*****:** :* ******* ******** * 1. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419 40 2. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419 3. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419 4. MVDGWYGYHH QNEQGSGYAA DLKSTQNAID EITNKVNSVI EKMNTQFTAV GKEFNHLEKR 420 . MIDGWYGYHH QNEQGSGYAA DQKSTQIAID GINNKANSVI GKMNIQLTSV GKEFNSLEKR 420 6. MIDGWYGYHH QNEQGSGYAA DQKSTQIAID GINNKVNSII EKMNTQFTSV GKEFNDLEKR 420 45 7. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419 8. MVDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419 9. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSII EKMNTQFTAV GKEFNKLEKR 420 . MIDGWYGYHH QNEQGSGYAA DQKSTQNAID GITNKVNSVI EKMNTQFTAV GKEFNKLERR 420 11. MMDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 419 50 12. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 420 13. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 420 14. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSII EKMNTQFTAV GKEFNKLEKR 420 . MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 420 16. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 420 55 17. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 420 18. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLERR 420 19. MMDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 419 . MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 420 21. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN WITNKVNSVI EKMNTQFTAV GKEFNKLEKR 420 60 22. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNKLEKR 420 23. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNNLEKR 420 24. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNNLEKR 419 . MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNNLEKR 420 26. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNIQFTAV GKEFNKLEKR 419 27. MIDGWYGYHH QNEQGSGYAA DQKSTQNAIN GITNKVNSVI EKMNTQFTAV GKEFNNLEKR 419 28. MIDGWYGYHH QNEQGSGYAA DQKSTQNAID GITNKVNSVI EKMNTQFTAV GKEFNNLERR 420 *:******** ********** * *******: *******:* **** ***** *****:**:* 1. MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 2. MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 3. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 4. IENLNKKVDD GFLDIWTYNA ELLVLLENER TLDYHDSNVK NLYEKVRSQL KNNAKEIGNG 480 5. KENLNKTVDD RFLDVWTFNA ELLVLLENQR TLEFHDLNIK SLYEKVKSHL RNNDKEIGNG 480 6. IENLNKKVDD GFLDVWTYNA ELLILLENER TLDFHDFNVK NLYEKVKSQL RNNAKEIGNG 480 7. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 8. MENLNKKVDD GFIDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 9. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDLNVK NLYEKVKNQL KNNAKEIGNG 480 10. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKTQL KNNAKEIGNG 480 11. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 12. MENLNKKVDD GFLDIWTYNA ELLVLLENGR TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 480 13. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 480 14. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 480 15. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 480 16. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 480 17. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 480 18. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 480 19. MENLNKKVDD GFMDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKNQL RNNAKELGNG 479 20. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 480 21. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKNQL RNNAKEIGNG 480 22. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKNQL RNNAKEIGNG 480 23. MENLNKKVDD GFLDIWTYNA ELLILLENER TLDFHDSNVK NLYEKVKSQL RNNAKEIGNG 480 24. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKNQL RNNAKEIGNG 479 25. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL RNNAKEIGNG 480 26. MENLNNKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVK NLYEKVKSQL KNNAKEIGNG 479 27. MENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDLNVK NLYEKVKSQL KNNAKEIGNG 479 28. IENLNKKVDD GFLDIWTYNA ELLVLLENER TLDFHDSNVR NLYEKVKSQL KNNAKEIGNG 480 :****:**** **:******* ***:**** * ***:** **: ******:.** :*****:*** 1. CFEFYHKCND ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539 2. CFEFYHKCND ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539 3. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539 4. CFEFYHKCDN TCMESVKNGT YDYPKYSEEA KLNREEIDGV KLESTRIYQI LAIYSTVASS 540 40 5. CFEFYHKRDN ECLECVKNGT YNYPKYSEES KFNREEIVGV KLESMGIHQI LAIYSTVASS 540 6. CFEFYHKCDN ECMESVKNGT YNYPKYSEES KLNREKIDGV KLESMGVHQI LAIYSTVASS 540 7. CFEFYHKCND ECMESVKNGT YDYPKYSEES KLNRERIDGV KLESMGVYQI LAIYSTVASS 539 8. CFEFYHKCND ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539 9. CFEFYHKCNN ECMESVKNGT YDYPKYSKES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 45 10. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 11. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539 12. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNRGKIDGV KLESMGVYQI LAIYSTVASS 540 13. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 14. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 50 15. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 16. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 17. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 18. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 19. CFEFYHKCDN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYRI LAIYSTVASS 539 55 20. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 21. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 22. CFEFYHKCNN ECMESVKNGT YDYPKFSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 23. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 24. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTAASS 539 60 25. CFEFYHKCNN ECMESVKNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 540 26. CFEFYHKCDN ECMESVRNGT YDYPKYSEES KLNREKVDGV KLESMGIYQI LAIYSTVASS 539 27. CFEFYHKCDN ECMESVRNGT YDYPKYSEES KLNREKIDGV KLESMGVYQI LAIYSTVASS 539 28. CFEFYHKCDD ACMESVRNGT YDYPKYSEES KLNREEIDGV KLESMGVYQI LAIYSTVASS 540 ********:: *****:*** *****:*:*: **** .:*** **** :*:* ******.*** 1. LVLLVSLGAI SFWMCSNGSL QCRICI 565 2. LVLLVSLGAI SFWMCSNGSL QCRICI 565 3. LVLLVSLGAI SFWMCSNGSL QCRICI 565 4. LVLVVSLGAI SFWMCSNGSL QCRICI 566 5. LVLLVSLGAI SFWMCSNGSL QCRVCI 566 6. LVLLVSLGAI SFWMCSNGSL QCRICI 566 7. LVLLVSLGAI SFWMCSNGSL QCRICI 565 8. LVLLVSLGAI SFWMCSNGSL QCRICI 565 9. LVLLVSLGAI SFWMCSNGSL QCRICI 566 10. LVLLVSLGAI SFWMCSNGSL QCRICI 566 11. LVLLVSLGAI SFWMCSNGSL QCRICI 565 12. LVLLVSLGAI SFWMCSNGSL QCRICI 566 13. LVLLVSLGAI SFWMCSNGSL QCRICI 566 14. LVLLVSLGAI SFWMCSNGSL QCRICI 566 15. LVLLVSLGAI SFWMCSNGSL QCRICI 566 16. LVLLVSLGAI SFWMCSNGSL QCRICI 566 17. LVLLVSLGAI SFWMCSNGSL QCRICI 566 18. LVLLVSLGAI SFWMCSNGSL QCRICI 566 19. LVLLVSLGAI SFWMCSNGSL QCRICI 565 20. LVLLVSLGAI SFWMCSNGSL QCRICI 566 21. LVLLVSLGAI SFWMCSNGSL QCRICI 566 22. LVLLVSLGAI SFWMCSNGSL QCRICI 566 23. LVLLVSLGAI SFWMCSNGSL QCRICI 566 24. LVLLVSLGAI SFWMCSNGSL QCRICI 565 25. LVLLVSLGAI SFWMCSNGSL QCRICI 566 26. LVLLVSLGAI SFWMCSNGSL QCRICI 565 27. LVLLVSLGAI SFWMCSNGSL QCRICI 565 28. LVLLVSLGAI SFWMCSNGSL QCRICI 566 ***:****** ********** ****** Table 4. Polypeptides expressed in P. pastoris.

Expression and CR6261 binding were determined as described and the ratio of binding and expression signals calculated.

Fusion peptide area B-loop SET1 337 340 352 353 402 406 409 413 416 fold increase CR6261 HTRF of ratio over F, I, N, S, A, G, I, R, F, I, N, S, H, I, L, N, clone binding ratio E, I, K, V I, K, R, T D, F, V, Y I, K, R, T E, K, M, V parental H1 T, Y T, V T, Y R, S signal signal mini-HA KI Y T M F I N R 239E11 1076944 1492 721,81 121,52 127H1 800024 6572 121,73 20,49 KK F T M Y I Y S 171E5 879704 11508 76,44 12,87 KT F T M I A F S KK F T M I V F N 239D2 570424 9279 61,47 10,35 KI Y T V Y I F S 247B2 414984 7583 54,73 9,21 KT F T M Y A Y H 253D4 395824 7546 52,45 8,83 252F5 421824 8621 48,93 8,24 VK Y T M Y V Y N 220C9 1086064 22606 48,04 8,09 KT F T M F T Y L 125D3 139824 2937 47,61 8,02 KK F T M Y G T H VK F T M Y I N H 137C11 416504 9167 45,44 7,65 KT F T M I V Y H 131B5 844344 20419 41,35 6,96 233F11 583024 14389 40,52 6,82 KK Y T M T I G S 234C5 377864 9465 39,92 6,72 II Y T M F T N L 115A1 1176904 30389 38,73 6,52 KK Y T M I V Y I KK Y T M I V I S 185G7 505864 13560 37,31 6,28 KK Y T M T T S S 275D4 327344 9030 36,25 6,10 244B8 273744 7757 35,29 5,94 IT Y T M Y A I S 252B8 284984 8252 34,54 5,81 KI Y T M S I N L VK Y T M I V F H 213C11 667024 20624 32,34 5,44 KT Y K V S G Y L 174G3 491184 15320 32,06 5,40 KI Y T M Y V N R 125D10 133904 4241 31,57 5,31 127A7 233064 7498 31,08 5,23 ET Y T M I I I L 304G11 110504 3588 30,8 5,19 KK Y K M F T F S 162A11 364024 11939 30,49 5,13 VK Y T M F A F I IK Y T M I A I L 271F10 315304 10348 30,47 5,13 IT Y I M I I I N 218G11 958504 33710 28,43 4,79 251C8 269544 9634 27,98 4,71 KT Y K M Y I N L 258A6 165624 6004 27,59 4,64 IT Y T M Y T F H 134A4 456304 17366 26,28 4,42 KI Y I M I A Y N EI Y T M Y V S S 214C11 317904 12120 26,23 4,42 KK Y T M T V I I 182G8 399864 15262 26,2 4,41 113E7 966064 38018 25,41 4,28 KK F T M Y T I H 230G9 854584 34093 25,07 4,22 KK Y T M Y T F R KT F I V I I Y L 222G4 419064 16996 24,66 4,15 IT Y T M I I F N 182D7 418944 17096 24,51 4,13 KT Y T M S A N H 272H2 263264 10844 24,28 4,09 191C8 309064 12753 24,23 4,08 IT Y T V I A F I 123C10 237824 9843 24,16 4,07 KI Y K M F A T L 284B9 1663504 70812 23,49 3,95 KT Y R M I R T L KK F I M I I N S 134A3 531784 23414 22,71 3,82 KK Y T M S V T H 188F4 287384 12888 22,3 3,75 189B7 336344 15207 22,12 3,72 ET F T M Y V F N 148D5 329144 14994 21,95 3,70 ET Y I M F G S H 194C8 242304 11113 21,8 3,67 IT F T M F V F I KT Y K M F V S I 188A8 279144 13001 21,47 3,61 VT Y T M Y T N N 162B3 279584 13159 21,25 3,58 204C5 832784 39330 21,17 3,56 VK F T V I I Y L 216E5 334904 15873 21,1 3,55 VT F T M F R Y R VR Y I M I I Y S 129C2 199464 9486 21,03 3,54 EI F T M F I Y S 286E8 158704 7662 20,71 3,49 KR Y T V I V F S 264G4 180504 8751 20,63 3,47 214C4 302264 14709 20,55 3,46 II F T V F A S S 125A8 212224 10327 20,55 3,46 KI F T V I V Y I SET1 Fusion peptide area B-loop 337 340 352 353 402 406 409 413 416 fold increase CR6261 HTRF of ratio over F, I, N, S, A, G, I, R, F, I, N, S, H, I, L, N, clone binding ratio E, I, K, V I, K, R, T D, F, V, Y I, K, R, T E, K, M, V signal parental H1 T, Y T, V T, Y R, S signal mini-HA IT Y I M Y T F L 123G2 498584 24442 20,4 3,43 EK Y K M F I I H 187C6 345464 16932 20,4 3,43 134H10 591704 29253 20,23 3,41 KT Y T V I T F I 187H10 299224 15289 19,57 3,29 KT Y I M I G F L 101D4 336584 17243 19,52 3,29 IK Y I M I IS N KK Y R M F I S N 193B6 206904 10650 19,43 3,27 IR F T V I I N N 137C5 295944 15406 19,21 3,23 112F3 449824 24169 18,61 3,13 VR F I M I I Y S 176A5 193104 10476 18,43 3,10 IT F T V F IF I 213B2 131704 7178 18,35 3,09 KK Y T M T V F L IK F T M Y G Y H 307A10 114984 6348 18,11 3,05 ET F I M F G T I 126C3 219944 12413 17,72 2,98 263B6 151184 8800 17,18 2,89 IT Y I M S T Y I 138F11 147864 8788 16,83 2,83 ER Y R M F V F L 134D3 303504 18129 16,74 2,82 ER F I M Y T F S VT Y I V I A F S 131D5 344504 20857 16,52 2,78 KT Y I M Y A F H 138F8 347704 21081 16,49 2,78 301F11 116904 7108 16,45 2,77 VT F T VY I S H 112G6 543944 33149 16,41 2,76 VR Y I M F I S I 245C9 180024 10980 16,4 2,76 VR F T VF V T L VT Y T VF V F S 123E2 477064 29184 16,35 2,75 VT Y T M Y I T R 266A11 90584 5696 15,9 2,68 104C4 521224 34458 15,13 2,55 VK Y I M F G F N 194E4 408584 27424 14,9 2,51 EK F T M I T F I 206B11 358744 24697 14,53 2,45 VR Y T M F T I L KT Y K M I V T N 192C4 343184 23932 14,34 2,41 125H3 317384 22785 13,93 2,35 IT F T M I A Y R 145C9 182344 13108 13,91 2,34 IT F I V Y IS N 243D6 132144 9596 13,77 2,32 IR F T M N V Y R 182D3 142664 10487 13,6 2,29 IT Y R M F A G S VK F I M F V F N 181H9 310504 23153 13,41 2,26 163E3 183544 14033 13,08 2,20 EK Y K M I V I L 145E7 132224 10312 12,82 2,16 IT F K V I IF S 275G3 115104 9180 12,54 2,11 VT Y I M T A S S IR F T M T G F S 191D5 123824 10048 12,32 2,07 VT Y I V I A F S 188G10 142504 11593 12,29 2,07 171F6 140464 11555 12,16 2,05 KT Y T M S T Y L 125C2 83624 7009 11,93 2,01 II F T V I T S S 206B8 285824 24166 11,83 1,99 VI Y T M I T F H IK F T M F R F S 145F2 498504 42457 11,74 1,98 KT Y T M N G S S 199F3 328504 29850 11,01 1,85 181H11 186664 17205 10,85 1,83 VT Y T M I I N R 188C8 113344 10520 10,77 1,81 IK Y T M S T Y L 189E10 188864 18252 10,35 1,74 KT Y T M S G S S VT Y I M Y T T I 146G7 533864 52422 10,18 1,71 182H2 109624 10976 9,99 1,68 KI F T V I I T L 262B9 94744 9584 9,89 1,66 IK Y T M F R F R 145E8 211504 21732 9,73 1,64 EK F K V I V F I 249B11 145184 14995 9,68 1,63 KK F T M S T G H KR D I M F I N N 182C6 92944 9939 9,35 1,57 SEQ ID NO: 6 AV + 2SD 9,28 1,56 SEQ ID NO: 6 AV 238077 40100 5,94 1,00 Table 5. Polypeptides expressed in P. pastoris.

Fusion peptide area B-loop Set 2 337340 352353 402406 409413 416 fold increase CR6261 of ratio HTRF A, E, I, K, T, F, I, N, S, T, A, D, F, I, N, E, G, I, K, R, M, R, T F, H, L, Y F, I, S, T E, K, M, V I. L. R. S clone binding ratio over V Y S, T, V, Y V signal signal parental SEQ ID NO: 6 KN Y K M F I M I 86B4 1077144 13862 77,7 13,08 TN YV M Y F E R 7A7 987824 13452 73,43 12,36 55G7 616184 8767 70,28 11,83 KN Y V M Y I M L 71H2 1109984 16750 66,27 11,16 KN F K M L I V S 86B3 900904 14448 62,35 10,50 KN Y K M L I V R 71A4 1064144 17597 60,47 10,18 TN YV M Y F E R 51G3 460304 7773 59,22 9,97 TI F V M L F E S 84B8 582144 10091 57,69 9,71 KN Y I M F F M S 79C2 364184 7116 51,18 8,62 TN Y R M F T V S 69G8 481344 9479 50,78 8,55 I N F R M L I V L 79D5 702584 13981 50,25 8,46 AN F K M L F V L 54H4 291744 5857 49,81 8,39 KI Y K M L I E L 11H6 427384 9146 46,73 7,87 K N YE M F TE S 90A9 413664 9025 45,84 7,72 KS Y V M Y T V S 75G5 1011384 26695 37,89 6,38 ES Y V M L F E R 8A10 360104 9630 37,39 6,29 KN Y V M L I V R 72D4 329944 8881 37,15 6,25 VN F R M F S M S 74H9 1283144 35494 36,15 6,09 KN F K M Y F M S 88C5 471424 13355 35,3 5,94 KN Y R M L I V R 61A9 383064 10864 35,26 5,94 T N FR M FFE L 86H9 457344 13340 34,28 5,77 KN F G M F T V S 71D3 1573024 46711 33,68 5,67 IS Y V M F I V L 9C6 270984 8235 32,91 5,54 K T Y V M YTK I 81F11 317824 9964 31,9 5,37 KI F V M F F V S 84E10 255064 7996 31,9 5,37 IN F R M F S V S 71C4 1350144 44339 30,45 5,13 KN F G M F I V S 84D3 84424 2920 28,91 4,87 EN F K M L I E S 96H8 205904 7224 28,5 4,80 K YYK M F I M S 85A7 235704 8416 28,01 4,72 KN Y E M L F V R 50G10 264144 9470 27,89 4,70 TN F E M F F V S 6A1 299824 10912 27,48 4,63 AN F R M F F M S 91C4 1157424 44837 25,81 4,35 KN F G M L I M R 2C4 258264 10139 25,47 4,29 IN F V M F IV L 63C3 188184 7625 24,68 4,15 E TYK M L F V L 850 196024 8115 24,16 4,07 KN V G M F F V I 67C10 306104 12907 23,72 3,99 ET F V M F F M L 10F9 165984 7113 23,34 3,93 II Y V M Y F E R 4C1 385504 16548 23,3 3,92 KN S V M F I E I 86G3 183944 7995 23,01 3,87 TS Y V M F T V L 51G10 215264 9727 22,13 3,73 AN Y R M F I K S 58A5 90744 4142 21,91 3,69 VT F R M L I M S 56F8 235344 10823 21,74 3,66 IN F E M F T E L 67C11 209184 9856 21,22 3,57 K Y Y I M FFE I 91C8 333584 16012 20,83 3,51 KN F G M L I K S 48B11 302864 14946 20,26 3,41 IN A G M L I E S 78F11 84104 4155 20,24 3,41 I I FR M Y FE I Fusion peptide area B-loop Set 2 337340 352353 402406 409413 416 fold increase CR6261 of ratio HTRF A, E, I, K, T, F, I, N, S, T, A, D, F, I, N, E, G, I, K, R, M, R, T F, H, L, Y F, I, S, T E, K, M, V I. L. R. S clone binding ratio over V Y S, T, V, Y V signal signal parental SEQ ID NO: 6 IY F V M Y F E I 76A10 136984 6841 20,02 3,37 II Y V M F F V S 55H2 58104 2984 19,47 3,28 KN A G M F I M S 74D7 358784 18453 19,44 3,27 TS F V M Y T V S 11B4 166464 8679 19,18 3,23 TTF E M F S M S 56F4 185984 9740 19,09 3,21 KN S R M Y I E S 71E7 202704 10688 18,97 3,19 48B10 102904 5480 18,78 3,16 I FFK M L F M S 48D11 120584 6807 17,71 2,98 EY Y V M F T V S 35H3 106224 6092 17,44 2,94 VS F V M L S M R 53G10 107784 6188 17,42 2,93 TN F V M L T V S 86F1 158624 9145 17,35 2,92 II F V M Y I V I 9C10 114144 6595 17,31 2,91 II Y V M H S V S 6E12 372504 22044 16,9 2,85 EN F I M L F V L 2D9 316024 19245 16,42 2,76 KN N I M Y F E L 27B10 187344 11465 16,34 2,75 K NNV M L F E S 79F8 185264 11801 15,7 2,64 IN V I M F T E S 11F4 150824 9996 15,09 2,54 IY F V M Y F V L 60A2 92664 6166 15,03 2,53 E N Y V M FSE L 58C8 277144 18603 14,9 2,51 AS Y I M L S E L 12C6 289184 20023 14,44 2,43 IN S V M L I E L 89F11 84824 5908 14,36 2,42 TI YI M L S V S 96G5 108264 7589 14,27 2,40 VN F I M Y F M S 29C2 177904 12921 13,77 2,32 KN F G M Y F M R 56D2 145624 10658 13,66 2,30 E T F I M FFK S 66C8 184544 13591 13,58 2,29 KN V I M L F V L 69D2 445704 34266 13,01 2,19 V F F V M YTE S 75E9 134504 10422 12,91 2,17 I I F G M FSE I 97G10 253104 20061 12,62 2,12 E S F I M FFE I 36E4 196104 15917 12,32 2,07 IN N K M F F V L 7D9 77824 6320 12,31 2,07 KN F V M F F M L 1F2 148544 12244 12,13 2,04 KN Y V M F F M I 76D10 113664 9729 11,68 1,97 TN A K M L T E S 36H2 171144 14761 11,59 1,95 TN Y K M H F M R 86G2 69704 6069 11,49 1,93 EN F V M L I E R 63D3 145784 13100 11,13 1,87 KN I G M F T E L 96A7 83304 7575 11 1,85 VI F V M F S V S 36D6 71304 6569 10,85 1,83 IN A G M F T E I 91F10 14784 1394 10,6 1,78 TN YG M F I E R 80F10 90864 8609 10,55 1,78 IS V V M L I E S 75H8 103304 10074 10,25 1,73 A NNV M F F M S 57B8 58384 5800 10,07 1,70 KI Y I M F F V I 8D7 73424 7324 10,03 1,69 KN F V M L F E L 58A11 53264 5363 9,93 1,67 VT Y I M F T V S 7B6 60384 6137 9,84 1,66 KI S E M F I M S 87H5 78104 7994 9,77 1,64 EI F I M F F V S 70F6 418624 43334 9,66 1,63 KN I G M L T E R 26H1 79744 8268 9,64 1,62 EN F I M L S V I 78G2 56704 6055 9,36 1,58 VI Y G M L F E S SEQ ID NO: 6 AV + 2SD 9,28 1,56 SEQ ID NO 238077 40100 5,94 1,00 Table 6. Polypeptides expressed in HEK293F.

Expression and CR6261 binding were determined as described and the ratio of binding and expression signals calculated. The mutations included in each clone are indicated in Table 4 and 5. fold increase of CR6261 ratio over binding HTRF parental SEQ ID signal signal NO: 6 Clone ratio 127H1 24150000 327363 73,77 4,25 86B4 19970680 334887 59,63 3,44 171E5 6625080 235511 28,13 1,62 7A7 6191080 242461 25,53 1,47 71H2 21080360 336346 62,67 3,61 220C9 8493560 162872 52,15 3,00 131B5 5725640 139561 41,03 2,36 115A1 9557640 175377 54,50 3,14 74H9 26144240 344988 75,78 4,37 71C4 6413600 214495 29,90 1,72 91C4 8442400 245138 34,44 1,98 113E7 13005960 260748 49,88 2,87 6E12 15326000 309443 49,53 2,85 181H9 11892520 324690 36,63 2,11 SEQ ID NO: 6 AV 5661550 326077 17,36 1,00 Table 7. Naturally occuring sequence variation at the indicated positions in % of total number of sequences for each subtype position amino acid H1 H3 H5 H7 337 V 67 99 19 100 I 32 1 2 T 0,8 3 S 73 Y 0,1 N 0,5 A 2 G 0,1 340 I 99 21 98 V 0,43 T 0,03 0,5 K 97 R 2 47 G 29 E 0,3 S 2 352 F 100 100 100 100 353 I 99,9 100 100 100 L 0,1 402 M 100 100 T 99,8 100 S 0,02 Table 8. Purification and strength of mAb binding of polypeptides of the invention app app SEQ ID Volume Yield Purity K K NO: supernatant (mg/l of from HP- CR6261 CR9114 (ml) culture) SEC (%) (nM) (nM) s127H1 35 1376 9.0 100.0 130 10 s86B4 36 1380 9.0 96.0 150 13 s55G7 37 1460 18.1 100.0 150 9 s74H9 34 1335 11.3 99.7 130 10 s6E12 38 1479 13.1 90.8 390 34 Table 9. Molecular weights as determined by SEC-MALS for polypeptides of the invention and their complexes with Fab fragments of CR6261 and CR9114. Theoretical (theor) values are estimated on the basis of the sequence of the polypeptide of the invention (assuming a monomer) and an additional contribution of approximately 10 kDa from attached glycans. The molecular weights of the Fab fragments of CR6261, CR9114 and CR8020 were also determined by SEC-MALS, and were 48, 49 and 47 kDa, respectively.

SEQ ID MW MW complex with MW complex with NO: (kDa) CR6261 (kDa) CR9114 (kDa) Theor Observed Theor Observed Theor Observed s127H1 35 40 39 87 74 86 83 s86B4 36 40 40 88 75 87 83 s55G7 37 40 40 90 66 87 80 s74H9 34 40 41 89 72 88 83 s6E12 38 40 40 88 67 87 80 REFERENCES Bommakanti et al. (2010), PNAS 107(31): 13701-13706.

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Wilson et al (1981) Nature 289: 366 The term "comprising" as used in this specification and claims means "consisting at least in part of". When interpreting statements in this specification, and claims which include the term "comprising", it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

SEQUENCES SEQ ID NO 1: H1 Full length (A/Brisbane/59/2007) MKVKLLVLLC TFTATYADTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL 50 ENSHNGKLCL LKGIAPLQLG NCSVAGWILG NPECELLISK ESWSYIVEKP 100 NPENGTCYPG HFADYEELRE QLSSVSSFER FEIFPKESSW PNHTVTGVSA 150 SCSHNGESSF YRNLLWLTGK NGLYPNLSKS YANNKEKEVL VLWGVHHPPN 200 IGDQKALYHT ENAYVSVVSS HYSRKFTPEI AKRPKVRDQE GRINYYWTLL 250 EPGDTIIFEA NGNLIAPRYA FALSRGFGSG IINSNAPMDK CDAKCQTPQG 300 AINSSLPFQN VHPVTIGECP KYVRSAKLRM VTGLRNIPSI QSRGLFGAIA 350 GFIEGGWTGM VDGWYGYHHQ NEQGSGYAAD QKSTQNAING ITNKVNSVIE 400 KMNTQFTAVG KEFNKLERRM ENLNKKVDDG FIDIWTYNAE LLVLLENERT 450 LDFHDSNVKN LYEKVKSQLK NNAKEIGNGC FEFYHKCNDE CMESVKNGTY 500 DYPKYSEESK LNREKIDGVK LESMGVYQIL AIYSTVASSL VLLVSLGAIS 550 FWMCSNGSLQ CRICI 565 SEQ ID NO: 2: H1-mini2-cluster1+5+6-GCN4 MKVKLLVLLC TFTATYA DTI CIGYHANNST DTVDTVLEKN VTVTHSVNLL 50 ENGGGGKYVC SAKLRMVTGL RNIPSIQSQG LFGAIAGFIE GGWTGMVDGW 100 YGYHHQNEQG SGYAADQKST QNAINGITNK VNSVIEKMNT QSTATGKEGN 150 KSERMKQIED KIEEIESKQI WCYNAELLVL LENERTLDFH DSNVKNLYEK 200 VKSQLKNNAK EIGNGCFEFY HKCNDECMES VKNGTYDYPK YSEESKLNRE 250 KIDGVKLESM GVYQILAIYS TVASSLVLLV SLGAISFWMC SNGSLQCRIC 300 I 301 SEQ ID NO: 3: foldon GYIPEAPRDGQAYVRKDGEWVLLSTFL SEQ ID NO: 4: FLAG-thrombin-foldon-HIS SGRDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH SEQ ID NO: 5: MKQIEDKIEEIESKQ SEQ ID NO: 6: H1-mini2-cluster1+5+6-GCN4 without leader sequence and with FLAG-thrombin-foldon-HIS DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNIPSIQ SQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEK MNTQSTATGKEGNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNL 40 YEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVSG RDYKDDDDKLVPRGSPGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGHHHHHH SEQ ID NO: 7: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTN NGLLFINTTIASIAAKEEGVSLEKREAEA SEQ ID NO 8: H1 consensus sequence residue 402-418 (numbering according to SEQ ID NO:1) 402 MNTQFTAVG KEFN(H/K)LE(K/R) 418 >SC09-114 VH PROTEIN (SEQ ID NO: 11) QVQLVQSGAEVKKPGSSVKVSCKSSGGTSNNYAISWVRQAPGQGLDWMGGISPIFGSTAY AQKFQGRVTISADIFSNTAYMELNSLTSEDTAVYFCARHGNYYYYSGMDVWGQGTTVTVS >SC09-114 VL PROTEIN (SEQ ID NO: 12) SYVLTQPPAVSGTPGQRVTISCSGSDSNIGRRSVNWYQQFPGTAPKLLIYSNDQRPSVVP DRFSGSKSGTSASLAISGLQSEDEAEYYCAAWDDSLKGAVFGGGTQLTVL >CR6261 VH PROTEIN (SEQ ID NO: 9) E V Q L V E S G A E V K K P G S S V K V S C K A S G G P F R S Y A I S W V R Q A P G Q G P E W M G G I I P I F G T T K Y A P K F Q G R V T I T A D D F A G T V Y M E L S S L R S E D T A M Y Y C A K H M G Y Q V R E T M D V W G K G T T V T V S S >CR6261 VL PROTEIN (SEQ ID NO: 10) Q S V L T Q P P S V S A A P G Q K V T I S C S G S S S N I G N D Y V S W Y Q Q L P G T A P K L L I Y D N N K R P S G I P D R F S G S K S G T S A T L G I T G L Q T G D E A N Y Y C A T W D R R P T A Y V V F G G G T K L T V L G >SC08-057 VH PROTEIN (SEQ ID NO: 13) EVQLVESGGGLVQPGGSLRLSCAASGFTDSVIFMSWVRQAPGKGLECVSIIYIDDSTYYA DSVKGRFTISRHNSMGTVFLEMNSLRPDDTAVYYCATESGDFGDQTGPYHYYAMDV >SC08-057 VL PROTEIN (SEQ ID NO: 14) 40 QSALTQPASVSGSPGQSITISCTGSSGDIGGYNAVSWYQHHPGKAPKLMIYEVTSRPSGV SDRFSASRSGDTASLTVSGLQAEDEAHYYCCSFADSNILI >SC08-020 VH PROTEIN (SEQ ID NO: 17) QVQLQQSGAEVKTPGASVKVSCKASGYTFTRFGVSWIRQAPGQGLEWIGWISAYNGDTYYAQKFQ ARVTMTTDTSTTTAYMEMRSLRSDDTAVYYCAREPPLFYSSWSLDN >SC08-020 VL PROTEIN (SEQ ID NO: 18) EIVXTQSPGTLSLSPGERATLSCRASQSVSMNYLAWFQQKPGQAPRLLIYGASRRATGIPDRISG SGSGTDFTLTISRLEPADFAVYYCQQYGTSPRT SEQ ID NO: 55: 127H1 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQYTAIGKEYNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 56 : 86B4 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNKPSNQSQGLFGAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQFTAIGKEMNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 59 : 55G7 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNKPSNQSQGLFGAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQYTAIGKEMNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 57 : 74H9 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR 40 MVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQYTAFGKEMNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI 45 SEQ ID NO: 60 : 115A1 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNKPSKQSQGLFGAIAGYTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQITAVGKEYNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDG 50 VKLESMGVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 61 : 71H2 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNKPSNQSQGLFGAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQLTAIGKEVNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 62 : 181H9 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNVPSKQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQFTAVGKEFNKNERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 58 : 6E12 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNEPSNQSQGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQLTAFGKEVNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 63 :220C9 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNKPSTQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQFTATGKEYNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 64 : 113E7 MKVKLLVLLCTFTATYADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLR MVTGLRNKPSKQSQGLFGAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNK VNSVIEKMNTQYTATGKEINKHERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVK NLYEKVKSQLKNNAKEIGNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESM GVYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI SEQ ID NO: 65: s74H9 DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLF GAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGK EMNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI 40 GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHH SEQ ID NO: 66: s127H1 DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLF GAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGK EYNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI 45 GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHH SEQ ID NO: 67: s86B4 DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLF GAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGK EMNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHH SEQ ID NO: 68: s55G7 DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLF GAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGK EMNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHH SEQ ID NO: 69: s6E12 DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLF GAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGK EVNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGRSLVPRGSPGHHHHHH SEQ ID NO: 72: s74H9-long DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLF GAIAGFKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAFGK EMNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEG SEQ ID NO: 73: s127H1-long DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSKQSQGLF GAIAGFTEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGK EYNKSERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEG SEQ ID NO: 74: s86B4-long DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLF GAIAGYKEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQFTAIGK EMNKIERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNRE KIDGVKLESMGVYQIEG SEQ ID NO: 75: s55G7-long DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNKPSNQSQGLF 40 GAIAGYVEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQYTAIGK EMNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEG SEQ ID NO: 76: s6E12-long DTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLENGGGGKYVCSAKLRMVTGLRNEPSNQSQGLF GAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNSVIEKMNTQLTAFGK EVNKLERMKQIEDKIEEIESKQIWCYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEI GNGCFEFYHKCNDECMESVKNGTYDYPKYSEESKLNREKIDGVKLESMGVYQIEG

Claims

1. An influenza hemagglutinin (HA) stem domain polypeptide comprising an amino acid 5 sequence selected from the group consisting of SEQ ID NO: 55, SEQ ID NO: 66 and SEQ ID NO: 73.

2. A nucleic acid molecule encoding a polypeptide according to claim 1. 10

3. A vector comprising the nucleic acid molecule of claim 2.

4. A composition comprising the polypeptide of claim 1, and/or a nucleic acid molecule according to claim 2 and/or a vector according to claim 3. 15

5. A use of an influenza HA stem domain polypeptide according to claim 1, a nucleic acid according to claim 2, and/or a composition according to claim 4 in the manufacture of a medicament.

6. A use as claimed in claim 5, wherein the medicament is a vaccine.

7. A use of an influenza HA stem domain polypeptide according to claim 1, a nucleic acid molecule according to claim 2 and/or a composition according to claim 4 in the manufacture of a medicament for inducing an immune response against influenza HA protein in a patient in need thereof.

8. A use of an influenza HA stem domain polypeptide according to claim 1, a nucleic acid molecule according to claim 2 and/or a composition according to claim 4 in the manufacture of a medicament for the prophylaxis and/or treatment of influenza infection in a patient in need thereof.

9. An influenza hemagglutinin (HA) stem domain polypeptide as claimed in claim 1 substantially as herein described with reference to any example thereof.

10. A nucleic acid mdolecule as claimed in claim 2, substantially as herein described with 5 reference to any example thereof.

11. A vector as claimed in claim 3, substantially as herein described with reference to any example thereof. 10

12. A compostion as claimed in claim 4, substantially as herein described with reference to any example thereof.

13. A use as claimed in any one of claims 5-8, substantially as herein described with reference to any example thereof.