WO2023061993A1 - Polypeptides - Google Patents

Polypeptides Download PDF

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Publication number
WO2023061993A1
WO2023061993A1 PCT/EP2022/078214 EP2022078214W WO2023061993A1 WO 2023061993 A1 WO2023061993 A1 WO 2023061993A1 EP 2022078214 W EP2022078214 W EP 2022078214W WO 2023061993 A1 WO2023061993 A1 WO 2023061993A1
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seq
residue
epitope
scaffold protein
identity
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PCT/EP2022/078214
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Normand Blais
Bruno Correia
Andreas Scheck
Sarah WEHRLE
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Glaxosmithkline Biologicals Sa
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to scaffold proteins presenting an influenza HA stem epitope.
  • influenza vaccines provide only short-term protection via the induction of strain-specific antibodies and require annual reformulation. It is thought that an enhanced vaccine needs to focus the antibody response against conserved neutralizing epitopes. In recent years, several antibodies targeting such a conserved antigenic site in the immunosubdominant stem region of the hemagglutinin (HA) glycoprotein have been isolated.
  • HA hemagglutinin
  • Flu vaccines are most commonly made from an egg-based manufacturing process as either live-attenuated or inactivated-virus formulations.
  • growth in eggs can lead to egg- adapted mutations that decrease immunogenicity (Chen, Zhou, and Jin 2010; Raymond et al. 2016).
  • Influenza immunity is further complicated by immunodominance hierarchies as the main immune response is mounted predominantly against the hemagglutinin (HA) head.
  • HA hemagglutinin
  • An improved vaccine should elicit broadly neutralizing antibodies (bnAbs) against conserved sites that not only protect against drifted strains but also various subtypes.
  • reverse vaccinology proposes the structure-based design of novel vaccine candidates to elicit neutralizing antibodies that are known correlates of protection (Burton 2002; Rappuoli et al. 2016, 201; Plotkin 2010).
  • the reverse vaccinology approach has recently been successfully applied to design novel epitope-focused immunogens for RSV.
  • the present inventors investigated whether mimetics of a conserved HA stem-epitope could be recognized by a broad panel of bnAbs and elicit a potent immune response in mice.
  • the synthetic immunogens were designed to capture the general features of the hydrophobic pocket, only partially relying on structural transplantation of epitope segments while the remaining antigenic surface is mimicked through surface-centric design. It was demonstrated that computationally designed immunogens that mimic a broadly neutralizing stem-epitope bind to a wide panel of broadly neutralizing, stem-specific antibodies and elicit a pan-group antibody response in mice.
  • the results show that epitope mimetics based on heterologous protein scaffolds are able to divert the immune response from the immunodominant head region to a conserved site on the immunosubdominant HA stem.
  • the elicited antibodies are highly specific towards the mimicked site and are cross-reactive to heterologous H1 and H3 strains.
  • the inventors employed a surface-centric design approach together with motif grafting to design novel epitope-focused immunogens. They addressed a conserved site in the hemagglutinin stem that is targeted by multiple broadly neutralizing antibodies.
  • the examples illustrate that the designed immunogens bind site-specific, broadly neutralizing antibodies and elicit strong epitope-focused, cross-reactive immune responses in mice.
  • a scaffold protein comprising at least one region having similarity to an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the scaffold protein has been modified to further resemble the epitope.
  • a scaffold protein wherein the scaffold protein comprises an N- terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4
  • E comprises a sequence sharing at least
  • a method of eliciting an immune response in a subject comprising administering to the subject a scaffold protein wherein the scaffold protein comprises an N-terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a
  • a scaffold protein for use as a medicament wherein the scaffold protein comprises an N-terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4
  • a scaffold protein for use in the treatment or prevention of influenza infection
  • the scaffold protein comprises an N-terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope
  • the polypeptide sequence of the fragment comprises the formula A-B-C- D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4
  • a scaffold protein in the manufacture of a medicament for the treatment or prevention of influenza infection
  • the scaffold protein comprises an N-terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope
  • the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence sharing at least 60% identity with S
  • a scaffold protein comprising an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • a method of eliciting an immune response in a subject comprising administering to the subject a scaffold protein comprising an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C- D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • a scaffold protein for use as a medicament wherein the scaffold protein comprises an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • a scaffold protein for use in the treatment or prevention of influenza infection
  • the scaffold protein comprises an acylhydrolase protein
  • the acylhydrolase protein comprises an influenza hemagglutinin stem epitope
  • the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • a scaffold protein in the manufacture of a medicament for the treatment or prevention of influenza infection
  • the scaffold protein comprises an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope
  • the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • SEQ ID NO: 11 Polypeptide sequence of human ApoE scaffold.
  • SEQ ID NO: 12 Polypeptide sequence of murine ApoE scaffold.
  • SEQ ID NO: 14 Polypeptide sequence of acylhydrolase scaffold region A
  • SEQ ID NO: 22 Polypeptide sequence of a native (or wild type, ‘WT’) acylhydrolase
  • SEQ ID NO: 26 Polypeptide sequence of linker connecting m2e to scaffold protein
  • Fig. 1 Computational design of stem-epitope immunogens.
  • the stem epitope was extracted consisting of a short HSV-loop (1), a VDGW-loop (2), and a regular a-helix (3).
  • the helix and VDGW-loop was queried against the Protein Data Bank (PDB) to retrieve putative templates.
  • the motif was grafted onto a suitable scaffold (PDB ID: 4IYJ) and further improved with directed evolution.
  • SSM site-saturation mutagenesis
  • Fig. 2 Overview of libraries for FI6-focused design affinity maturation.
  • A. The FI6-focused design with targeted structural elements highlighted (grafted epitope helix, hydrophobic pocket, and loop connecting the epitope helix to scaffold). The hydrophobic pocket was targeted with the combinatorial library. The connecting loop was targeted with the SSM library.
  • B. Logo plot of positions targeted in combinatorial library. The library was sorted three times with decreasing concentrations of the FI6 antibody.
  • C Density plot of SSM library. Constructs binding strongly to the FI6 binding antibody and displaying on the yeast surface were sorted as binding population. Constructs without FI6 binding but displaying on yeast were sorted as nonbinding population.
  • D. Heat map of residues enriched in the binding population over the nonbinding population.
  • Fig. 3 Biophysical characterization of relevant designs.
  • Fig. 4 Characterization of designed scaffold proteins.
  • A. Surface plasmon resonance (SPR) measurements of the FI6-focused_04 and stem-epitope_01 design binding to FI6 Fab revealed strong binding of both designs to the antibody with KDs of 6 nM and 44 nM, respectively.
  • C. Negative stain of FI6-focused and stem-epitope designs on ferritin nanoparticles showed correct assembly of the nanoparticle and presentation of the designs.
  • Fig. 5 Schematic overview of nanoparticle construct. Epitope-scaffolds were fused C- terminally to ferritin separated by a GS-linker. They were labelled N-terminally with a His-tag for purification and a TEV cleavage site. The m2e T cell epitope was introduced between epitope-scaffold and His-tag.
  • Fig. 6 Structural characterization of designed immunogens.
  • A. Structural comparison of FI6- focused_03 model to X-ray structure shows overall agreement with a RMSD of 2 A, however, two additional helix-turns were formed on the epitope-helix N-terminal end.
  • Fig.7 Antibody pull down from human PBMCs.
  • PBMCs Peripheral blood mononuclear cells
  • B cells double positive to H1 and FI6-focused_03 design were sorted. Sorted B cells were sequenced and their VH sequences were assigned to their originating germline regions for comparison with known bnAbs.
  • VH3-23 antibody 31-1 B01 shows similar breath and superior affinity compared to FI6.
  • Other sorted antibodies were mainly restricted to group 1 HA binding.
  • D. Stemmimetic positive memory B cells were sorted in a H1 only and a H1/H3 double positive population and both populations were sequenced to assign their corresponding VH genes.
  • Fig.8 Design induced antibody responses.
  • FI6- focused_04 and stem-epitope_01 immunogens were presented on ferritin nanoparticles, H1 HA was given as soluble trimers. All immunogens were adjuvanted with AS03 and injected intramuscularly three times in three week intervals.
  • Fig. 9 Sorting and sequencing of group 1 and group 2 cross- reactive mouse B cells.
  • Fig. 10 Immune protection mediated against lethal challenge with X31 influenza virus in mice (first challenge study).
  • Three weeks after the 3rd injection mice were challenged with 2 LD50 of H3 1968 HK X31 influenza virus and monitored for loss of body weight over 14 days. If an animal lost more than 25% body weight it was sacrificed.
  • Top curves H1_NC99 trimer followed by stem epitope particle
  • middle curves stem epitope only
  • bottom curves H1_NC99 trimer followed by adjuvant only.
  • Fig. 11 Immune protection mediated against lethal challenge with X31 influenza virus in mice (second challenge study).
  • Fig. 12 Protection mediated by stem-mimetic.
  • Influenza hemagglutinin is the major surface antigen of the virion and the primary target of virus neutralizing antibodies.
  • HA is a homotrimeric surface glycoprotein, with each monomer consisting of two disulfide-linked subunits (HA1 , HA2), resulting from the proteolytic cleavage products of a single HA precursor protein.
  • the HA1 chain forms a membrane-distal globular head and a part of the membrane-proximal stem (or ‘stalk’) region.
  • the HA2 chain represents the major component of the stem region.
  • the head of HA mediates receptor binding while the membrane-anchored stem is the main part of membrane fusion machinery.
  • the scaffold proteins are based on either an N-terminal fragment of apolipoprotein E (ApoE) or a putative acylhydrolase protein, in each case incorporating an epitope bound by anti-stem antibodies.
  • ApoE apolipoprotein E
  • a putative acylhydrolase protein in each case incorporating an epitope bound by anti-stem antibodies.
  • the scaffold proteins may themselves be comprised within a construct which comprises further polypeptide sequences.
  • the further polypeptide sequences may include, for example, one or more promoters and/or one or more linkers.
  • the “% sequence identity” between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST v2.0, using standard settings for polypeptide sequences (BLASTP).
  • the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST v2.0, using standard settings for nucleotide sequences (BLASTN).
  • Polypeptide or polynucleotide sequences are said to be the same as or “identical” to other polypeptide or polynucleotide sequences if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C- terminus for polypeptides; from 5’ to 3’ terminus for polynucleotides.
  • a “difference” between polypeptide sequences refers to an insertion, deletion or substitution of a single amino acid residue in a position of the second sequence, compared to the first sequence.
  • Two polypeptide sequences can contain one, two or more such amino acid differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 amino acid residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity).
  • the number of additions, substitutions and/or deletions made to the first sequence to produce the second sequence may be ascertained.
  • An “addition” is the addition of one amino acid residue into the sequence of the first polypeptide (including addition at either terminus of the first polypeptide).
  • a “substitution” is the substitution of one amino acid residue in the sequence of the first polypeptide with one different amino acid residue. Said substitution may be conservative or non-conservative.
  • a “deletion” is the deletion of one amino acid residue from the sequence of the first polypeptide (including deletion at either terminus of the first polypeptide).
  • the naturally occurring amino acids may be referred to as follows: glycine (G or Gly), alanine (A or Ala), valine (V or Vai), leucine (L or Leu), isoleucine (I or lie), proline (P or Pro), phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp), lysine (K or Lys), arginine (R or Arg), histidine (H or His), aspartic acid (D or Asp), glutamic acid (E or Glu), asparagine (N or Asn), glutamine (Q or Gin), cysteine (C or Cys), methionine (M or Met), serine (S or Ser) and Threonine (T or Thr).
  • G or Gly glycine
  • a or Ala valine
  • V or Vai valine
  • leucine L or Leu
  • isoleucine I or lie
  • proline P or Pro
  • a residue may be aspartic acid or asparagine
  • the symbols Asx or B may be used.
  • a residue may be glutamic acid or glutamine
  • the symbols Glx or Z may be used.
  • References to aspartic acid include aspartate, and glutamic acid include glutamate, unless the context specifies otherwise.
  • a “conservative” amino acid substitution is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which is expected to have little influence on the function, activity or other biological properties of the polypeptide.
  • Such conservative substitutions suitably are substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group, as shown in Table 1 below.
  • any residues in a sequence which do not correspond to the residues provided in a reference sequence are conservative substitutions with respect to the residues of the reference sequence.
  • a “corresponding” amino acid residue between a first and second polypeptide sequence is an amino acid residue in a first sequence which shares functionally the same position with an amino acid residue in a second sequence, whilst the amino acid residue in the second sequence may differ in identity from the first.
  • corresponding residues will share the same number if the sequences are the same length. Alignment can be achieved manually or by using, for example, a known computer algorithm for sequence alignment such as NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.
  • references herein to an “epitope” refer to the portion of the target which is bound by the polypeptide, antibody or fragment thereof. Epitopes may also be referred to as “antigenic determinants”.
  • An antibody binds “essentially the same epitope” as another antibody when they both recognize identical or sterically overlapping epitopes. Commonly used methods to determine whether two antibodies bind to identical or overlapping epitopes are competition assays, which can be configured in a number of different formats (e.g. well plates using radioactive or enzyme labels, or flow cytometry on antigen-expressing cells) using either labelled antigen or labelled antibody.
  • An antibody binds “the same epitope” as another antibody when they both recognize identical epitopes (i.e.
  • an antibody may bind the same epitope as another antibody when all contact points across a specified region of an antigen are identified as the same with the aid of a characterization method such as antibody/antigen cross-linking-coupled MS, HDX, X-ray crystallography, cryo-EM, or mutagenesis.
  • a characterization method such as antibody/antigen cross-linking-coupled MS, HDX, X-ray crystallography, cryo-EM, or mutagenesis.
  • Linear epitopes are formed by a continuous sequence of amino acids in a protein antigen.
  • Conformational epitopes are formed of amino acids that are discontinuous in the protein sequence, but which are brought together upon folding of the protein into its three- dimensional structure.
  • a scaffold protein comprising at least one region having similarity to an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the scaffold protein has been modified to further resemble the epitope.
  • the scaffold protein is not an influenza hemagglutinin stem protein.
  • the scaffold protein is a heterologous scaffold protein with respect to the influenza hemagglutinin stem epitope, i.e. the naturally occurring scaffold protein does not comprise an influenza hemagglutinin stem epitope.
  • the scaffold protein is not derived from influenza hemagglutinin stem protein, more suitably not derived from influenza hemagglutinin protein, more suitably not derived from an influenza protein.
  • influenza hemagglutinin stem epitope is the hydrophobic groove of the influenza hemagglutinin stem protein.
  • influenza hemagglutinin stem epitope is the epitope bound by the FI6 antibody.
  • influenza hemagglutinin stem epitope comprises a part of helix A of hemagglutinin, more suitably all of helix A of hemagglutinin.
  • the scaffold protein does not form part of a nanolipoprotein.
  • the scaffold protein is an immunogen of which the region having similarity to an influenza hemagglutinin stem epitope is an integral part.
  • Apolipoprotein E protein is utilised as a scaffold for an HA stem epitope in the ‘stem epitope’ designs or ‘stem-epitope mimetics’ of the present invention.
  • Apolipoprotein E is a protein which, in its natural context, is involved in the metabolism of fats in the body of mammals.
  • the native polypeptide sequence of an ApoE protein is provided in SEQ ID NO: 13.
  • ApoE is 299 amino acids long and contains multiple amphipathic a-helices.
  • a hinge region connects the N- and C-terminal regions of the protein.
  • the N-terminal region (residues 1-167) forms an anti-parallel four-helix bundle such that the non-polar sides face inside the protein.
  • the C-terminal domain contains three a-helices which form a large exposed hydrophobic surface and interact with those in the N- terminal helix bundle domain through hydrogen bonds and salt-bridges.
  • the C-terminal region also contains a low density lipoprotein receptor (LDLR)-binding site.
  • ApoE is polymorphic with three major alleles (epsilon 2, epsilon 3, and epsilon 4): ApoE-e2 (cys112, cys158), ApoE-e3 (cys112, arg158), and ApoE-e4 (arg112, arg158). Although these allelic forms differ from each other by only one or two amino acids at positions 112 and 158, these differences alter ApoE structure and function.
  • the invention concerns a scaffold protein wherein the scaffold protein comprises an N-terminal fragment of ApoE, wherein the fragment comprises an influenza hemagglutinin stem epitope.
  • the epitope is based on a well-characterized conserved epitope centered around the hydrophobic pocket on the HA stem.
  • the conserved site on the HA stem is a multi-segment epitope, consisting of a 20-residue long a-helix, a four residue VDGW-loop, and a three residue HSV-loop (Fig. 1).
  • solely the 20-residue long a-helix is emulated in the ApoE scaffold according to the invention, omitting the shorter epitope loops.
  • the fragment comprises the formula A-B-C-D-E.
  • A corresponds to the N-terminal region preceding the minor helix epitope region
  • B corresponds to the minor helix epitope region
  • C corresponds to the region between the minor and major helix epitope regions
  • D corresponds to the major helix epitope region
  • E corresponds to the C-terminal region of the fragment.
  • B and D may in some embodiments comprise specific residues which contribute to the epitope.
  • E may in some embodiments comprise specific residues which stabilise the epitope.
  • the fragment comprises a polypeptide sequence of no more than 500 residues, such as no more than 400 residues, such as no more than 300 residues, such as no more than 200 residues, such as no more than 190 residues, such as no more than 185 residues, such as no more than 184 residues, such as no more than 183 residues, such as no more than 182 residues, such as no more than 181 residues.
  • the scaffold protein consists of the N-terminal fragment of apolipoprotein E.
  • the N-terminal fragment of apolipoprotein E consists of the formula A- B-C-D-E.
  • the N-terminal fragment of apolipoprotein E comprises the formula X-Y-A-B-C-D-E wherein only X or Y are present or both X and Y are present and wherein X comprises or consists of SEQ ID NO: 21 and Y comprises or consists of SEQ ID NO: 20.
  • the scaffold protein is an N-terminal fragment derived from human ApoE.
  • A comprises or consists of a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% identity with SEQ ID NO: 1. Most suitably, A comprises or consists of SEQ ID NO: 1.
  • A consists of 40 to 60 residues, such as 45 to 55 residues, such as 50 residues.
  • Certain residues may be present in B to form the epitope.
  • the residue of B corresponding to residue 1 of SEQ ID NO: 2 is L or a conservative substitution thereof, most suitably L; and/or the residue of B corresponding to residue 4 of SEQ ID NO: 2 is A or a conservative substitution thereof, most suitably A; and/or the residue of B corresponding to residue 5 of SEQ ID NO: 2 is I or a conservative substitution thereof, most suitably I; and/or the residue of B corresponding to residue 8 of SEQ ID NO: 2 is M or a conservative substitution thereof, most suitably M; and/or the residue of B corresponding to residue 9 of SEQ ID NO: 2 is K or a conservative substitution thereof, most suitably K.
  • B comprises or consists of a sequence sharing at least 70%, such as at least 80% identity with SEQ ID NO: 2. Most suitably, B comprises or consists of SEQ ID NO: 2.
  • B consists of 5 to 15 residues, such as 7 to 11 residues, such as 9 residues.
  • Suitably B comprises or consists of the sequence LX1X2X3IX4X5MK (SEQ ID NO: 28) wherein Xi is selected from the group consisting of H, K and R; wherein X2 is selected from the group consisting of D and E; wherein X 3 is selected from the group consisting of A, G, I, L, M and V; wherein X4 is selected from the group consisting of A, G, I, L, M and V; and wherein X5 is selected from the group consisting of F, W and Y.
  • Region C LX1X2X3IX4X5MK
  • C comprises or consists of a sequence sharing at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% identity with SEQ ID NO: 3. Most suitably, C comprises or consists of SEQ ID NO: 3.
  • C consists of 10 to 20 residues, such as 13 to 17 residues, such as 14 residues.
  • the residue of D corresponding to residue 1 of SEQ ID NO: 4 is L or a conservative substitution thereof, most suitably L; and/or the residue of D corresponding to residue 2 of SEQ ID NO: 4 is K or a conservative substitution thereof, most suitably K; and/or the residue of D corresponding to residue 4 of SEQ ID NO: 4 is T or a conservative substitution thereof, most suitably T; and/or the residue of D corresponding to residue 5 of SEQ ID NO: 4 is Q or a conservative substitution thereof, most suitably Q; and/or the residue of D corresponding to residue 6 of SEQ ID NO: 4 is N or a conservative substitution thereof, most suitably N; and/or the residue of D corresponding to residue 8 of SEQ ID NO: 4 is I or a conservative substitution thereof, most suitably I; and/or the residue of D corresponding to residue 9 of SEQ ID NO: 4 is D or a conservative substitution thereof, most suitably D; and/or the residue of D corresponding to residue 11 of SEQ ID NO: 4 is I or a
  • D comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 85%, such as at least 90% identity with SEQ ID NO: 4. Most suitably, D comprises or consists of SEQ ID NO: 4.
  • D consists of 15 to 25 residues, such as 18 to 22 residues, such as 19 residues.
  • D comprises or consists of the sequence LKX1TQNX2IDX3ITX4X5VNX6X7A (SEQ ID NO: 29) wherein Xi is selected from the group consisting of A, G, I, L, M and V; wherein X2 is selected from the group consisting of A, G, I, L, M and V; wherein X 3 is selected from the group consisting of A, G, I, L, M and V; wherein X4 is selected from the group consisting of H, K and R; wherein Xs is selected from the group consisting of A, G, I, L, M and V; wherein Xe is selected from the group consisting of D and E; and wherein X? is selected from the group consisting of A, G, I, L, M and V.
  • E comprises or consists of a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% identity with SEQ ID NO: 5. Most suitably, E comprises or consists of SEQ ID NO: 5.
  • E consists of 53 to 65 residues, such as 55 to 59 residues, such as 57 residues.
  • the fragment comprises a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99% identity with SEQ ID NO: 6. More suitably, the fragment consists of a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99% identity with SEQ ID NO: 11. More suitably, the fragment comprises or consists of SEQ I D NO: 11.
  • polypeptide sequence of this exemplary fragment is set out with annotation as follows, wherein underlined residues are mutations introduced to form the epitope.
  • sequence is also provided full length with hyphens separating sequences corresponding to regions A, B, C, D and E:
  • N-terminal extension KVEQAVETEPE, SEQ ID NO: 19
  • PELRQQ following region
  • A consists of a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B consists of a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C consists of a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D consists of a sequence sharing at least 60% identity with SEQ ID NO: 4
  • E consists of a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • the scaffold protein is an N-terminal fragment derived from murine ApoE.
  • A comprises or consists of a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% identity with SEQ ID NO: 6. Most suitably, A comprises or consists of SEQ ID NO: 6. Region B
  • Certain residues may be present in B to form the epitope.
  • the residue of B corresponding to residue 1 of SEQ ID NO: 7 is L or a conservative substitution thereof, most suitably L; and/or the residue of B corresponding to residue 4 of SEQ ID NO: 7 is A or a conservative substitution thereof, most suitably A; and/or the residue of B corresponding to residue 5 of SEQ ID NO: 7 is I or a conservative substitution thereof, most suitably I; and/or the residue of B corresponding to residue 8 of SEQ ID NO: 7 is M or a conservative substitution thereof, most suitably M.
  • B comprises or consists of a sequence sharing at least 70%, such as at least 80% identity with SEQ ID NO: 7. Most suitably, B comprises or consists of SEQ ID NO: 7.
  • B comprises or consists of the sequence LX1X2AIX3X4M (SEQ ID NO: 30) wherein Xi is selected from the group consisting of C, N, P, Q, S and T; wherein X2 is selected from the group consisting of D and E; wherein X 3 is selected from the group consisting of A, G, I, L, M and V; and wherein X4 is selected from the group consisting of F, W and Y.
  • C comprises or consists of a sequence sharing at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% identity with SEQ ID NO: 8. Most suitably, C comprises or consists of SEQ ID NO: 8.
  • the residue of D corresponding to residue 1 of SEQ ID NO: 9 is L or a conservative substitution thereof, most suitably L; and/or the residue of D corresponding to residue 2 of SEQ ID NO: 9 is K or a conservative substitution thereof, most suitably K; and/or the residue of D corresponding to residue 4 of SEQ ID NO: 9 is T or a conservative substitution thereof, most suitably T; and/or the residue of D corresponding to residue 5 of SEQ ID NO: 9 is Q or a conservative substitution thereof, most suitably Q; and/or the residue of D corresponding to residue 6 of SEQ ID NO: 9 is N or a conservative substitution thereof, most suitably N; and/or the residue of D corresponding to residue 8 of SEQ ID NO: 9 is I or a conservative substitution thereof, most suitably I; and/or the residue of D corresponding to residue 9 of SEQ ID NO: 9 is D or a conservative substitution thereof, most suitably D; and/or the residue of D corresponding to residue 11 of SEQ ID NO: 9 is I or a
  • D comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 85%, such as at least 90% identity with SEQ ID NO: 9. Most suitably, D comprises or consists of SEQ ID NO: 9.
  • D comprises or consists of the sequence LKX1TQNX2IDX3ITNX4VNX5X6AE (SEQ ID NO: 31) wherein Xi is selected from the group consisting of A, G, I, L, M and V; wherein X2 is selected from the group consisting of A, G, I, L, M and V; wherein X 3 is selected from the group consisting of A, G, I, L, M and V; wherein X4 is selected from the group consisting of A, G, I, L, M and V; wherein X5 is selected from the group consisting of D and E; and wherein Xe is selected from the group consisting of A, G, I, L, M and V.
  • Xi is selected from the group consisting of A, G, I, L, M and V
  • X2 is selected from the group consisting of A, G, I, L, M and V
  • X 3 is selected from the group consisting of A, G, I, L, M and V
  • E comprises or consists of a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% identity with SEQ ID NO: 10. Most suitably, E comprises or consists of SEQ ID NO: 10.
  • the fragment comprises a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99% identity with SEQ ID NO: 6. More suitably, the fragment consists of a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99% identity with SEQ ID NO: 12. More suitably, the fragment comprises or consists of SEQ ID NO: 12.
  • polypeptide sequence of this exemplary fragment is set out with annotation as follows, wherein underlined residues are mutations introduced to form the epitope.
  • LTEAIAYM (SEQ ID NO: 7)
  • sequence is also provided full length with hyphens separating sequences corresponding to regions A, B, C, D and E:
  • the ApoE (such as human or mouse ApoE) scaffold does not form part of a nanolipoprotein structure.
  • a putative acylhydrolase protein is alternatively used as a scaffold for an HA stem epitope in the ‘FI6-focused’ designs of the present invention.
  • the polypeptide sequence of a native acylhydrolase protein is provided in SEQ ID NO: 22.
  • the invention concerns an acylhydrolase protein, wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope.
  • the epitope is based on a well-characterized conserved epitope centered around the hydrophobic pocket on the HA stem.
  • the conserved site on the HA stem is a multi-segment epitope, consisting of a 20-residue long a-helix, a four residue VDGW-loop, and a three residue HSV-loop (Fig. 1).
  • a multi-segment epitope consisting of a 20-residue long a-helix, a four residue VDGW-loop, and a three residue HSV-loop (Fig. 1).
  • solely the 20-residue long a-helix and a four residue VDGW-loop are emulated in the acylhydrolase protein scaffold according to the invention, omitting the shorter epitope loop.
  • the acylhydrolase protein comprises the formula A-B-C-D wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • Certain residues may be present in A to stabilise the scaffold.
  • the residue of A corresponding to residue 8 of SEQ ID NO: 14 is P or a conservative substitution thereof, most suitably P; and/or the residue of A corresponding to residue 9 of SEQ ID NO: 14 is A or a conservative substitution thereof, most suitably A; and/or the residue of A corresponding to residue 11 of SEQ ID NO: 14 is K or a conservative substitution thereof, most suitably K; and/or the residue of A corresponding to residue 29 of SEQ ID NO: 14 is N or a conservative substitution thereof, most suitably N; and/or the residue of A corresponding to residue 47 of SEQ ID NO: 14 is P or a conservative substitution thereof, most suitably P; and/or the residue of A corresponding to residue 61 of SEQ ID NO: 14 is S or a conservative substitution thereof, most suitably S; and/or the residue of A corresponding to residue 68 of SEQ ID NO: 14 is M or a conservative substitution thereof, most suitably M; and/or the residue of A corresponding to residue 70 of SEQ ID NO: 14 is E or
  • A comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity with SEQ ID NO: 14. More suitably A comprises or consists of SEQ ID NO: 14.
  • Certain residues may be present in B to stabilise the scaffold and/or the epitope.
  • the residue of B corresponding to residue 1 of SEQ ID NO: 15 is H or a conservative substitution thereof, most suitably H; and/or the residue of B corresponding to residue 4 of SEQ ID NO: 15 is A or a conservative substitution thereof, most suitably A; and/or the residue of B corresponding to residue 5 of SEQ ID NO: 15 is P or a conservative substitution thereof, most suitably P; and/or the residue of B corresponding to residue 8 of SEQ ID NO: 15 is E or a conservative substitution thereof, most suitably E; and/or the residue of B corresponding to residue 10 of SEQ ID NO: 15 is Q or a conservative substitution thereof, most suitably Q.
  • B comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 90% identity with SEQ ID NO: 15. More suitably B comprises or consists of SEQ ID NO: 15.
  • B comprises or consists of the sequence HX1X2APX3X4EX5QX6 (SEQ ID NO: 32) wherein Xi is selected from the group consisting of A, G, I, L, M and V; wherein X2 is selected from the group consisting of A, G, I, L, M and V; wherein X3 is selected from the group consisting of F, W and Y; wherein X4 is selected from the group consisting of C, N, P, Q, S and T; wherein Xs is selected from the group consisting of C, N, P, Q, S and T; and wherein Xe is selected from the group consisting of H, K and R.
  • Xi is selected from the group consisting of A, G, I, L, M and V
  • X2 is selected from the group consisting of A, G, I, L, M and V
  • X3 is selected from the group consisting of F, W and Y
  • X4 is selected from the group consisting of C,
  • Certain residues may be present in C to form the epitope.
  • the residue of C corresponding to residue 1 of SEQ ID NO: 16 is E or a conservative substitution thereof, most suitably E; and/or the residue of C corresponding to residue 3 of SEQ ID NO: 16 is T or a conservative substitution thereof, most suitably T; and/or the residue of C corresponding to residue 5 of SEQ ID NO: 16 is A or a conservative substitution thereof, most suitably A; and/or the residue of C corresponding to residue 7 of SEQ ID NO: 16 is I or a conservative substitution thereof, most suitably I; and/or the residue of C corresponding to residue 8 of SEQ ID NO: 16 is N or a conservative substitution thereof, most suitably N; and/or the residue of C corresponding to residue 11 of SEQ ID NO: 16 is T or a conservative substitution thereof, most suitably T; and/or the residue of C corresponding to residue 14 of SEQ ID NO: 16 is I or a conservative substitution thereof, most suitably I; and/or the residue of C corresponding to residue 15 of SEQ ID NO: 16 is N or a
  • C comprises or consists of the sequence EX1TX2AX3INX4X5TX6X7INX8X9IX10X11X12X13 X14FX15X16X17FVX18X19AQSPX20GD (SEQ ID NO: 33) wherein Xi is selected from the group consisting of A, G, I, L, M and V; wherein X2 is selected from the group consisting of C, N, P, Q, S and T; wherein X 3 is selected from the group consisting of A, G, I, L, M and V; wherein X4 is selected from the group consisting of A, G, I, L, M and V; wherein Xs is selected from the group consisting of C, N, P, Q, S and T; wherein Xe is selected from the group consisting of H, K and R; wherein X7 is selected from the group consisting of A, G, I, L, M and V; wherein Xs is selected from the group consisting of the group
  • D comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95% identity with SEQ ID NO: 17. More suitably D comprises or consists of SEQ ID NO: 17.
  • an exemplary acylhydrolase protein is provided in SEQ ID NO: 18.
  • the acylhydrolase protein comprises a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99% identity with SEQ ID NO: 6.
  • the fragment consists of a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 97%, such as at least 98%, such as at least 99% identity with SEQ ID NO: 18.
  • the acylhydrolase protein comprises or consists of SEQ ID NO: 18.
  • polypeptide sequence of this exemplary acylhydrolase protein is set out with annotation as follows wherein underlined residues are mutations introduced to form the epitope, stabilise the epitope, or stabilise the protein generally.
  • Hyphens separate sequences corresponding to regions A, B, C and D.
  • the acylhydrolase protein comprises a polypeptide sequence of no more than 700 residues, such as no more than 500 residues, such as no more than 300 residues, such as no more than 250 residues, such as no more than 230 residues, such as no more than 220 residues.
  • A consists of a sequence sharing at least 50% identity with SEQ ID NO: 14
  • B consists of a sequence sharing at least 70% identity with SEQ ID NO: 15
  • C consists of a sequence sharing at least 80% identity with SEQ ID NO: 16
  • D consists of a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • the acylhydrolase protein consists of the formula A-B-C-D.
  • the scaffold protein consists of the acylhydrolase protein.
  • the scaffold protein may be provided ‘naked’, i.e. not bound to other materials.
  • the scaffold protein may be provided bound to one or more further agents.
  • the scaffold protein is presented on the surface of nanoparticles, such as protein nanoparticles, such as those disclosed in Diaz et al 2018 including ferritin, lumazine and encapsulin. Protein nanoparticles present multiple faces on which antigenic scaffold proteins may be presented.
  • the scaffold protein is most suitably displayed on self-assembling protein nanoparticles, such as most suitably ferritin nanoparticles, such as more suitably insect or bacterial ferritin nanoparticles, such as most suitably H. pylori ferritin nanoparticles (such as those disclosed in Corbett, 2019, WO2013/044203, WO2015/183969 and WO2018/045308; and such as that recited in SEQ ID NO: 27).
  • self-assembling protein nanoparticles such as most suitably ferritin nanoparticles, such as more suitably insect or bacterial ferritin nanoparticles, such as most suitably H. pylori ferritin nanoparticles (such as those disclosed in Corbett, 2019, WO2013/044203, WO2015/183969 and WO2018/045308; and such as that recited in SEQ ID NO: 27).
  • the scaffold protein is displayed on the surface of the nanoparticle, in particular on one or more of the individual faces of the nanoparticle, such as on all faces of the nanoparticle.
  • the nanoparticle and the scaffold protein are connected by a linker.
  • the linker consists of 1 to 40 residues, such as 10 to 30 residues.
  • the linker comprises or consists of the polypeptide sequence of SEQ ID NO: 24.
  • the scaffold protein may be combined in a construct with a nanoparticle as discussed above, and optionally an m2e T cell epitope from the influenza matrix protein, TEV cleavage site and/or 6xHis tag (for purification purposes).
  • a construct may be structured as set out in Fig. 5.
  • the protein comprises an m2e T cell epitope sequence, such as at the N-terminus of the scaffold protein.
  • the m2e T cell epitope sequence comprises a sequence sharing at least 90% identity with SEQ ID NO: 25, such as comprises or consists of SEQ ID NO: 25.
  • the m2e T cell epitope sequence if present, may be connected to the scaffold protein by a linker, such as a linker consisting of 1 to 10 amino acids (e.g. a linker comprising or consisting of the sequence GASG (SEQ ID NO: 26)).
  • the present invention may involve a plurality of antigenic components, for example with the objective to elicit a broad immune response to influenza virus.
  • more than one antigen may be present, more than one polynucleotide encoding an antigen may be present, one polynucleotide encoding more than one antigen may be present or a mixture of antigen(s) and polynucleotide(s) encoding antigen(s) may be present.
  • Polysaccharides such as polysaccharide conjugates may also be present.
  • a ‘Type’ of influenza virus refers to influenza Type A, influenza Type B or influenza type C.
  • the designation of a virus as a specific Type relates to sequence difference in the respective Ml (matrix) protein or NP (nucleoprotein).
  • Type A influenza viruses are further divided into Group 1 and Group 2. These Groups are further divided into subtypes, which refers to classification of a virus based on the sequence of its HA protein. Examples of current commonly recognized subtypes are H1 , H2, H3, H4, H5, H6, H7, H8, H8, H10, H11 , H12, H13, H14, H15 or H16.
  • Group 1 influenza subtypes are H1, H2, H5, H7 and H9.
  • Group 2 influenza subtypes are H4, H6, H8, H10, H11 , H12, H13, H14, H15 and H16.
  • strain refers to viruses within a subtype that differ from one another in that they have small, genetic variations in their genome.
  • the elicited immune response produces anti-Group 1 influenza A stem region antibodies. In a further embodiment, the elicited immune response produces anti- Group 2 influenza A stem region antibodies. Suitably the elicited immune response produces both anti-Group 1 and anti-Group 2 influenza A stem region antibodies, for example anti-H1 and anti-H3 antibodies.
  • bnAbs have been identified that target the HA stem, binding to HAs from group 1 and group 2 (Corti et al. 2011; Dreyfus et al. 2012; Nakamura et al. 2013; Kallewaard et al. 2016; Wu et al. 2015). These antibodies recognize the same conserved site on the HA stem domain around the hydrophobic pocket, engaging the epitope in different orientations. These antibodies include FI6 (Corti et al. 2011), CR9114 (Dreyfus et al. 2012), 39.29 (Nakamura et al. 2013) and MEDI8852 (Kallewaard et al. 2016).
  • influenza hemagglutinin stem epitope is bound by one or more of the FI6 antibody, the CR9114 antibody, the 39.29 antibody or the MEDI-8852. Most suitably, the influenza hemagglutinin stem epitope is bound by the FI6 antibody.
  • the scaffold proteins of the invention elicit antibodies which bind to influenza HA stem.
  • the antibodies bind to group 1 and/or group 2 HAs. More suitably the antibodies bind to group 1 and group 2 HAs.
  • the antibodies bind to H1 (such as 1999 NC) and/or H3 (such as 1968 HK) and/or H5, more suitably the antibodies bind to both H1 and H3 (i.e. they are ‘cross- reactive’).
  • the elicited antibodies originate from the VH3-30 germline region or the VH3-23 germline region.
  • Antibodies comprise stretches of amino acid residues which form an antigen-binding site, capable of binding to an epitope on a target antigen with an affinity (suitably expressed as a Kd value, a Ka value, a kon-rate and/or a koff-rate, as further described herein).
  • affinity represented by the equilibrium constant for the dissociation of an antigen with an antigenbinding polypeptide (KD)
  • KD is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antibody (or fragment thereof): the lesser the value of the KD, the stronger the binding strength between an antigenic determinant and the antigen-binding polypeptide.
  • the affinity can also be expressed as the affinity constant (KA), which is 1/KD.
  • Affinity can be determined by known methods, depending on the specific antigen of interest. For example KD may be determined by the method recited in the Examples section under method 1.14. Any KD value less than 10' 6 is considered to indicate binding.
  • the antibody binds to the influenza hemagglutinin stem epitope with a binding affinity (KD) of less than 3.0 x 10' 7 M (i.e. 300 nM) or less than 1.5 x 10' 7 M (i.e. 150 nM).
  • KD is 1.3 x 10' 7 M (i.e. 130 nM) or less, such as 1.0 x 10' 7 M (i.e. 100 nM) or less.
  • the KD is less than 6.0 x 10' 8 M (i.e. 60 nM), such as less than 5.0 x 10' 8 M (i.e.
  • the KD may be 1.0 x 10' 8 M (i.e. 10 nM) or less, such as 7.0 x 10' 9 M (i.e. 7 nM) or less, such as 6.0 x 10' 9 M (i.e. 6 nM) or less, such as 5.0 x 10 -9 M (i.e. 5 nM) or less, such as 2.0 x 10 -9 M (i.e.
  • the KD of the antibody may be established by the method titled ‘Surface plasmon resonance to measure binding affinities’, as detailed under the Examples section below.
  • the scaffold protein induces an immune response that is at least 2- fold, such as at least 5-fold, such as at least 10- fold, such as at least 100-fold greater than that of influenza HA stem.
  • the scaffold protein may be administered with an adjuvant.
  • the adjuvant may be a squalene emulsion adjuvant.
  • the term ‘squalene emulsion adjuvant’ as used herein refers to a squalene-containing oil-in-water emulsion adjuvant.
  • Squalene is readily available from commercial sources or may be obtained by methods known in the art. Squalene shows good biocompatibility and is readily metabolised.
  • the squalene emulsion adjuvant may comprise one or more tocopherols, suitably wherein the weight ratio of squalene to tocopherol is 20 or less (i.e. 20 weight units of squalene or less per weight unit of tocopherol or, alternatively phrased, at least 1 weight unit of tocopherol per 20 weight units of squalene).
  • a-tocopherol also referred to herein as alpha-tocopherol
  • D-alpha-tocopherol and D/L-alpha-tocopherol can both be used.
  • Tocopherols are readily available from commercial sources or may be obtained by methods known in the art.
  • the squalene emulsion adjuvant contains alpha-tocopherol, especially D/L-alpha-tocopherol.
  • Squalene emulsion adjuvants will typically have a submicron droplet size. Droplet sizes below 200 nm are beneficial in that they can facilitate sterilisation by filtration. There is evidence that droplet sizes in the 80 to 200 nm range are of particular interest for potency, manufacturing consistency and stability reasons (Klucker, 2012; Shah, 2014; Shah, 2015; Shah, 2019). Suitably the squalene emulsion adjuvant has an average droplet size of less than 1 um, especially less than 500 nm and in particular less than 200 nm.
  • the squalene emulsion adjuvant has an average droplet size of at least 50 nm, especially at least 80 nm, in particular at least 100 nm, such as at least 120 nm.
  • the squalene emulsion adjuvant may have an average droplet size of 50 to 200 nm, such as 80 to 200 nm, especially 120 to 180 nm, in particular 140 to 180 nm, such as about 160 nm.
  • a polydispersity index (Pdl) of greater than 0.7 indicates that the sample has a very broad size distribution and a reported value of 0 means that size variation is absent, although values smaller than 0.05 are rarely seen.
  • the squalene emulsion adjuvant has a polydispersity of 0.5 or less, especially 0.3 or less, such as 0.2 or less.
  • the droplet size means the average diameter of oil droplets in an emulsion and can be determined in various ways e.g. using the techniques of dynamic light scattering and/or single-particle optical sensing, using an apparatus such as the AccusizerTM and NicompTM series of instruments available from Particle Sizing Systems (Santa Barbara, USA), the ZetasizerTM instruments from Malvern Instruments (UK), or the Particle Size Distribution Analyzer instruments from Horiba (Kyoto, Japan). See Light Scattering from Polymer Solutions and Nanoparticle Dispersions Schartl, 2007. Dynamic light scattering (DLS) is the preferred method by which droplet size is determined. The preferred method for defining the average droplet diameter is a Z-average i.e.
  • one or more emulsifying agents are generally required.
  • Surfactants can be classified by their ‘HLB’ (Griffin’s hydrophile/lipophile balance), where a HLB in the range 1-10 generally means that the surfactant is more soluble in oil than in water, whereas a HLB in the range 10-20 means that the surfactant is more soluble in water than in oil.
  • HLB values are readily available for many surfactants of interest or can be determined experimentally, e.g. polysorbate 80 has a HLB of 15.0 and TPGS has a HLB of 13 to 13.2. Sorbitan trioleate has a HLB of 1.8.
  • the resulting HLB of the blend is typically calculated by the weighted average e.g. a 70/30 wt% mixture of polysorbate 80 and TPGS has a HLB of (15.0 x 0.70) + (13 x 0.30) i.e. 14.4.
  • a 70/30 wt% mixture of polysorbate 80 and sorbitan trioleate has a HLB of (15.0 x 0.70) + (1.8 x 0.30) i.e. 11.04.
  • Surfactant(s) will typically be metabolisable (biodegradable) and biocompatible, being suitable for use as a pharmaceutical.
  • the surfactant can include ionic (cationic, anionic or zwitterionic) and/or non-ionic surfactants.
  • ionic cationic, anionic or zwitterionic
  • non-ionic surfactants The use of only non-ionic surfactants is often desirable, for example due to their pH independence.
  • the invention can thus use surfactants including, but not limited to: the polyoxyethylene sorbitan ester surfactants (commonly referred to as the Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, especially polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM, PluronicTM (e.g., the polyoxyethylene sorbitan ester surfactants (commonly referred to as the Tweens or polysorbates), such as polysorbate 20 and polysorbate 80, especially polysorbate 80; copolymers of ethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO), sold under the DOWFAXTM, PluronicTM (e.g.
  • Tweens or polysorbates such as polysorbate 20 and polysorbate 80, especially polysorbate 80
  • SynperonicTM tradenames such as linear EO/PO block copolymers, for example poloxamer 407, poloxamer 401 and poloxamer 188; octoxynols, which can vary in the number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol) being of particular interest;
  • octylphenoxy polyethoxyethanol
  • phospholipids such as phosphatidylcholine (lecithin); polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such as polyoxyethylene 4 lauryl ether (Brij 30, Emulgen 104P), polyoxyethylene-9-lauryl ether and polyoxyethylene 12 cetyl/stearyl ether (EumulginTM B1, cetereth-12 or polyoxyethylene cetostearyl ether); sorbitan esters (commonly known as the Spans), such as sorbitan trioleate (Span 85), sorbitan monooleate (Span 80) and sorbitan monolaurate (Span 20); or tocopherol derivative surfactants, such as alpha-tocopherol-polyethylene glycol succinate (TPGS).
  • TPGS alpha-tocopherol-polyethylene glycol succinate
  • surfactant component has a HLB between 10 and 18, such as between 12 and 17, in particular 13 to 16. This can be typically achieved using a single surfactant or, in some embodiments, using a mixture of surfactants.
  • Surfactants of particular interest include: poloxamer 401, poloxamer 188, polysorbate 80, sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, in combination with each other or in combination with other surfactants.
  • polysorbate 80 sorbitan trioleate, sorbitan monooleate and polyoxyethylene 12 cetyl/stearyl ether either alone, or in combination with each other.
  • a particular surfactant of interest is polysorbate 80.
  • a particular combination of surfactants of interest is polysorbate 80 and sorbitan trioleate.
  • a further combination of surfactants of interest is sorbitan monooleate and polyoxyethylene cetostearyl ether.
  • the squalene emulsion adjuvant comprises one surfactant, such as polysorbate 80. In some embodiments the squalene emulsion adjuvant comprises two surfactants, such as polysorbate 80 and sorbitan trioleate or sorbitan monooleate and polyoxyethylene cetostearyl ether. In other embodiments the squalene emulsion adjuvant comprises three or more surfactants, such as three surfactants.
  • the weight ratio of squalene to tocopherol may be 20 or less, such as 10 or less.
  • the weight ratio of squalene to tocopherol is 0.1 or more.
  • the weight ratio of squalene to tocopherol is 0.1 to 10, especially 0.2 to 5, in particular 0.3 to 3, such as 0.4 to 2.
  • the weight ratio of squalene to tocopherol is 0.72 to 1.136, especially 0.8 to 1, in particular 0.85 to 0.95, such as 0.9.
  • the weight ratio of squalene to surfactant is 0.73 to 6.6, especially 1 to 5, in particular 1.2 to 4.
  • the weight ratio of squalene to surfactant is 1.71 to 2.8, especially 2 to 2.4, in particular 2.1 to 2.3, such as 2.2.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant is typically at least 1.2 mg. Generally, the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant is 50 mg or less. The amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.2 to 20 mg, in particular 1.2 to 15 mg.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.2 to 2 mg, 2 to 4 mg, 4 to 8 mg or 8 to 12.1 mg.
  • the amount of squalene in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.21 to 1.52 mg, 2.43 to 3.03 mg, 4.87 to 6.05 mg or 9.75 to 12.1 mg.
  • the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant is typically at least 1.3 mg. Generally, the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant is 55 mg or less. The amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.3 to 22 mg, in particular 1.3 to 16.6 mg.
  • the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.3 to 2 mg, 2 to 4 mg, 4 to 8 mg or 8 to 13.6 mg.
  • the amount of tocopherol in a single dose, such as a human dose, of squalene emulsion adjuvant may be 1.33 to 1.69 mg, 2.66 to 3.39 mg, 5.32 to 6.77 mg or 10.65 to 13.53 mg.
  • the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant is typically at least 0.4 mg.
  • the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant is 18 mg or less.
  • the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.4 to 9.5 mg, in particular 0.4 to 7 mg.
  • the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.4 to 1 mg, 1 to 2 mg, 2 to 4 mg or 4 to 7 mg.
  • the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant may be 0.54 to 0.71 mg, 1.08 to 1.42 mg, 2.16 to 2.84 mg or 4.32 to 5.68 mg.
  • the squalene emulsion adjuvant may consist essentially of squalene, surfactant and water. In certain other embodiments the squalene emulsion adjuvant may consist essentially of squalene, tocopherol, surfactant and water. Squalene emulsion adjuvants may contain additional components as desired or required depending upon the intended final presentation and vaccination strategy, such as buffers and/or tonicity modifying agents, for example modified phosphate buffered saline (disodium phosphate, potassium biphosphate, sodium chloride and potassium chloride).
  • buffers and/or tonicity modifying agents for example modified phosphate buffered saline (disodium phosphate, potassium biphosphate, sodium chloride and potassium chloride).
  • High pressure homogenization may be applied to yield squalene emulsion adjuvants comprising tocopherol which demonstrate uniformly small droplet sizes and long-term stability (see EP0868918 and W02006/100109).
  • oil phase composed of squalene and tocopherol may be formulated under a nitrogen atmosphere.
  • Aqueous phase is prepared separately, typically composed of water for injection or phosphate buffered saline, and polysorbate 80.
  • Oil and aqueous phases are combined, such as at a ratio of 1:9 (volume of oil phase to volume of aqueous phase) before homogenisation and microfluidisation, such as by a single pass through an in-line homogeniser and three passes through a microfluidiser (at around 15000 psi).
  • the resulting emulsion may then be sterile filtered, for example through two trains of two 0.5/0.2 urn filters in series (i.e. 0.5/0.2/0.5/0.2), see WO2011/154444.
  • Operation is desirably undertaken under an inert atmosphere, e.g. nitrogen. Positive pressure may be applied, see WO2011/154443.
  • an immunogenic composition comprising the scaffold protein of the invention and a pharmaceutically acceptable diluent or carrier.
  • the scaffold protein (and optionally a squalene emulsion adjuvant) may be administered as a formulation containing the scaffold protein and squalene emulsion adjuvant (‘co-formulation’ or ‘co-formulated’).
  • the scaffold protein and squalene emulsion adjuvant may be administered as a first formulation containing the scaffold protein and a second formulation containing the squalene emulsion adjuvant (‘separate formulation’ or ‘separately formulated’).
  • the scaffold protein and squalene emulsion adjuvant may be administered through the same or different routes, to the same or different locations, and at the same or different times.
  • the scaffold protein and squalene emulsion adjuvant may be administered via various suitable routes, including parenteral, such as intramuscular or subcutaneous administration.
  • the scaffold protein and squalene emulsion adjuvant may be administered via different routes.
  • the scaffold protein and squalene emulsion adjuvant are administered via the same route, in particular intramuscularly.
  • the scaffold protein and squalene emulsion adjuvant are desirably administered to locations with sufficient spatial proximity such that the adjuvant effect is adequately maintained.
  • spatial proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at to the same location.
  • the adjuvant effect seen with administration to the same location is defined as the level of increase observed as a result of administration of the scaffold protein and squalene emulsion adjuvant to the same location compared with administration of the scaffold protein alone.
  • the scaffold protein and squalene emulsion adjuvant are desirably administered to a location draining to the same lymph node, such as to the same limb, in particular to the same muscle.
  • the scaffold protein and squalene emulsion adjuvant are administered intramuscularly to the same muscle.
  • the scaffold protein and squalene emulsion adjuvant are administered to the same location.
  • the spatial separation of administration locations may be at least 5 mm, such as at least 1 cm.
  • the spatial separation of administration locations may be less than 10 cm, such as less than 5 cm apart.
  • the scaffold protein and squalene emulsion adjuvant are desirably administered with sufficient temporal proximity such that the adjuvant effect is adequately maintained.
  • temporal proximity is sufficient to maintain at least 50%, especially at least 75% and in particular at least 90% of the adjuvant effect seen with administration at the same time.
  • the adjuvant effect seen with administration at the same time is defined as the level of increase observed as a result of administration at (essentially) the same time compared with administration of the scaffold protein without squalene emulsion adjuvant.
  • scaffold protein and squalene emulsion adjuvant When administered as separate formulations, scaffold protein and squalene emulsion adjuvant may be administered within 12 hours.
  • the scaffold protein and squalene emulsion adjuvant are administered within 6 hours, especially within 2 hours, in particular within 1 hour, such as within 30 minutes and especially within 15 minutes (e.g. within 5 minutes).
  • the delay between administration of the scaffold protein and squalene emulsion adjuvant may be at least 5 seconds, such as 10 seconds, and in particular at least 30 seconds.
  • the scaffold protein When administered as separate formulations, if the scaffold protein and squalene emulsion adjuvant are administered with a delay, the scaffold protein may be administered first and the squalene emulsion adjuvant administered second. Alternatively, the squalene emulsion adjuvant is administered first and the scaffold protein administered second. Appropriate temporal proximity may depend on the order or administration.
  • the scaffold protein and squalene emulsion adjuvant are administered without intentional delay (accounting for the practicalities of multiple administrations).
  • the scaffold protein and squalene emulsion adjuvant may initially be provided in various forms which facilitate manufacture, storage and distribution.
  • certain components may have limited stability in liquid form, certain components may not be amendable to drying, certain components may be incompatible when mixed (either on a short- or long-term basis).
  • certain components may be provided in separate containers the contents of which are subsequently combined.
  • the skilled person will appreciate that many possibilities exist, although it is generally desirable to have a limited number of containers and limited number of required steps to prepare the final coformulation or separate formulations for administration.
  • the scaffold protein may be provided in liquid or dry (e.g. lyophilised) form.
  • the preferred form will depend on factors such as the precise nature of the scaffold protein, e.g. if the scaffold protein is amenable to drying, or other components which may be present.
  • the squalene emulsion adjuvant is provided in liquid form.
  • the invention provides a composition comprising a scaffold protein and a squalene emulsion adjuvant.
  • a composition comprising a scaffold protein and a squalene emulsion adjuvant.
  • the scaffold protein and squalene emulsion adjuvant are provided as a liquid co-formulation.
  • a liquid co-formulation enables convenient administration at the point of use.
  • a composition (such as those containing scaffold protein or squalene emulsion adjuvant) intended for combination with other compositions prior to administration need not itself have a physiologically acceptable pH or a physiologically acceptable tonicity; a formulation intended for administration should have a physiologically acceptable pH and should have a physiologically acceptable osmolality.
  • the pH of a liquid preparation is adjusted in view of the components of the composition and necessary suitability for administration to the subject.
  • the pH of a formulation is generally at least 4, especially at least 5, in particular at least 5.5 such as at least 6.
  • the pH of a formulation is generally 9 or less, especially 8.5 or less, in particular 8 or less, such as 7.5 or less.
  • the pH of a formulation may be 4 to 9, especially 5 to 8.5, in particular 5.5 to 8, such as 6.5 to 7.4 (e.g. 6.5 to 7.1).
  • solutions should have a physiologically acceptable osmolality to avoid excessive cell distortion or lysis.
  • a physiologically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic.
  • the formulations for administration will have an osmolality of 250 to 750 mOsm/kg, especially 250 to 550 mOsm/kg, in particular 270 to 500 mOsm/kg, such as 270 to 400 mOsm/kg.
  • Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced® Model 2020 available from Advanced Instruments Inc. (USA).
  • Liquids used for reconstitution will be substantially aqueous, such as water for injection, phosphate buffered saline and the like.
  • Buffers may be selected from acetate, citrate, histidine, maleate, phosphate, succinate, tartrate and TRIS.
  • the buffer may be a phosphate buffer such as Na/Na2PO4, Na/K2PO4 or K/K2PO4.
  • the formulations used in the present invention have a dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.6 ml, in particular a dose volume of 0.45 to 0.55 ml, such as 0.5 ml.
  • the volumes of the compositions used may depend on the subject, delivery route and location, with smaller doses being given by the intradermal route or if both the scaffold protein and squalene emulsion adjuvant are delivered to the same location.
  • a typical human dose for administration through routes such as intramuscular is in the region of 200 ul to 750 ml, such as 400 to 600 ul, in particular about 500 ul, such as 500 ul.
  • Stabilisers may be present. Stabilisers may be of particular relevance where multidose containers are provided as doses of the final formulation(s) may be administered to subjects over a period of time.
  • Approaches for establishing strong and lasting immunity often include repeated immunisation, i.e. boosting an immune response by administration of one or more further doses. Such further administrations may be performed with the same immunogenic compositions (homologous boosting) or with different immunogenic compositions (heterologous boosting).
  • the present invention may be applied as part of a homologous or heterologous prime/boost regimen, as either the priming or a/the boosting immunisation.
  • the scaffold protein and squalene emulsion adjuvant may therefore be part of a multi-dose administration regime.
  • the scaffold protein and squalene emulsion adjuvant may be provided as a priming dose in a multidose regime, especially a two- or three- dose regime, in particular a two-dose regime.
  • the scaffold protein and squalene emulsion adjuvant may be provided as a boosting dose in a multidose regime, especially a two- or three- dose regime, such as a two-dose regime.
  • Priming and boosting doses may be homologous or heterologous.
  • the scaffold protein and squalene emulsion adjuvant may be provided as a priming dose and boosting dose(s) in a homologous multidose regime, especially a two- or three-dose regime, in particular a two-dose regime.
  • the scaffold protein and squalene emulsion adjuvant may be provided as a priming dose or boosting dose in a heterologous multidose regime, especially a two- or three-dose regime, in particular a two-dose regime, and the boosting dose(s) may be different (e.g. scaffold protein; or an alternative antigen presentation such as protein or virally vectored antigen - with or without adjuvant, such as squalene emulsion adjuvant).
  • the protein is administered as part of a homologous prime-boost regime (such as three administrations of the protein).
  • the protein is administered as part of a heterologous prime-boost regime (such as a prime administration of an HA stem protein (e.g. H1 NC99) followed by one or more administrations of the protein.
  • the time between doses may be two weeks to six months, such as three weeks to three months. Periodic longer-term booster doses may be also be provided, such as every 2 to 10 years.
  • the squalene emulsion adjuvant may be administered to a subject separately from the scaffold protein, or the adjuvant may be combined, either during manufacturing or extemporaneously, with the scaffold protein to provide an immunogenic composition for combined administration.
  • administration of scaffold proteins of the invention is intended for prophylaxis, i.e. for administration to a subject which is not infected with influenza virus. In one embodiment, administration of scaffold proteins of the invention is intended for treatment, i.e. for administration to a subject which is infected with influenza virus.
  • a single dose of scaffold protein is 0.001 to 1000 ug, especially 0.01 to 100 ug, in particular 0.1 to 50 ug.
  • a suitable single dose of scaffold protein is 10 to 30 ug, especially 15 to 25 ug, in particular about 20 ug.
  • a single dose of scaffold protein is suitably 1 to 3 ug, especially 1.5 to 2.5 ug, in particular about 2 ug.
  • the scaffold protein is for use as a medicament, such as for use in the prevention of, or vaccination against, influenza e.g. administered to a person (e.g. subject) at risk of influenza infection.
  • a method of prevention and/or treatment of influenza disease comprising the administration of a scaffold protein as described herein to a person in need thereof, e.g. to a person (e.g. subject) at risk of influenza infection, e.g. an elderly person (age 50 or over, particularly age 65 or over).
  • the proteins of the invention are generally intended for administration to mammalian subjects, in particular human subjects.
  • the subject may be a wild or domesticated animal.
  • Mammalian subjects include for example cats, dogs, pigs, sheep, horses or cattle.
  • the subject is human.
  • the subject to be treated may be of any age.
  • the subject is a human infant (up to 12 months of age).
  • the subject is a human child (less than 18 years of age).
  • the subject is an adult human (aged 18-59).
  • the subject is an older human (aged 60 or greater).
  • Doses administered to younger children, such as less than 12 years of age, may be reduced relative to an equivalent adult dose, such as by 50%.
  • Nucleotide sequences encoding the scaffold proteins of the invention may be synthesized, and/or cloned and expressed according to techniques well known to those in the art. See for example, Sambrook, et al. Molecular Cloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989).
  • the polynucleotide sequences will be codon optimised for a particular recipient host cell using standard methodologies.
  • a DNA construct encoding a scaffold protein sequence can be codon optimised for expression in other hosts e.g. bacteria, mammalian or insect cells. Suitable host cells may include bacterial cells such as E. Coli, fungal cells such as yeast, insect cells such as Drosophila S2, Spodoptera Sf9, SfOO+ or Hi-5 and animal cells such as CHO. Miscellaneous
  • composition “comprising” and variants thereof such as “comprises” are to be interpreted as including the stated element (e.g., integer) or elements (e.g., integers) without necessarily excluding any other elements (e.g., integers).
  • a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
  • x in or “approximately” in relation to a numerical value x is optional and means, for example, x+10% of the given figure, such as x+5% of the given figure.
  • a process comprising a step of mixing two or more components does not require any specific order of mixing.
  • components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.
  • a scaffold protein comprising at least one region having similarity to an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the scaffold protein has been modified to further resemble the epitope.
  • a method of eliciting an immune response in a subject comprising administering to the subject a scaffold protein comprising at least one region having similarity to an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the scaffold protein has been modified to further resemble the epitope.
  • a scaffold protein for use as a medicament wherein the scaffold protein comprises at least one region having similarity to an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the scaffold protein has been modified to further resemble the epitope.
  • a scaffold protein for use in the treatment or prevention of influenza infection wherein the scaffold protein comprises at least one region having similarity to an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the scaffold protein has been modified to further resemble the epitope.
  • Use of a scaffold protein in the manufacture of a medicament for the treatment or prevention of influenza infection wherein the scaffold protein comprises at least one region having similarity to an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the scaffold protein has been modified to further resemble the epitope.
  • the scaffold protein comprises an N- terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4
  • E comprises a sequence sharing at least 40% identity with SEQ ID
  • the method of eliciting an immune response in a subject comprising administering to the subject a scaffold protein wherein the scaffold protein comprises an N-terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence
  • the scaffold protein for use as a medicament according to clause 3, wherein the scaffold protein comprises an N-terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4
  • the scaffold protein for use in the treatment or prevention of influenza infection according to clause 4 wherein the scaffold protein comprises an N-terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence sharing at least 60% identity with SEQ
  • a scaffold protein in the manufacture of a medicament for the treatment or prevention of influenza infection according to clause 5 wherein the scaffold protein comprises an N-terminal fragment of apolipoprotein E protein (ApoE) wherein the fragment comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the fragment comprises the formula A-B-C-D-E, wherein A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1, wherein B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D comprises a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E comprises a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B comprises a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C comprises a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D comprises a sequence sharing at
  • A consists of a sequence sharing at least 40% identity with SEQ ID NO: 1
  • B consists of a sequence sharing at least 60% identity with SEQ ID NO: 2
  • C consists of a sequence sharing at least 40% identity with SEQ ID NO: 3
  • D consists of a sequence sharing at least 60% identity with SEQ ID NO: 4
  • E consists of a sequence sharing at least 40% identity with SEQ ID NO: 5.
  • A comprises or consists of a sequence sharing at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% identity with SEQ ID NO: 1.
  • B comprises or consists of a sequence sharing at least 70%, such as at least 80% identity with SEQ ID NO: 2.
  • C comprises or consists of a sequence sharing at least 50%, such as at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90% identity with SEQ ID NO: 3.
  • D comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 85%, such as at least 90% identity with SEQ ID NO: 4.
  • D comprises or consists of the sequence LKX1TQNX2IDX3ITX4X5VNX6X7A (SEQ ID NO: 29) wherein Xi is selected from the group consisting of A, G, I, L, M and V; wherein X2 is selected from the group consisting of A, G, I, L, M and V; wherein X 3 is selected from the group consisting of A, G, I, L, M and V; wherein X4 is selected from the group consisting of H, K and R; wherein Xs is selected from the group consisting of A, G, I, L, M and V; wherein Xe is selected from the group consisting of D and E; and wherein X?
  • A comprises consists of SEQ ID NO: 1 or a sequence comprising conservative substitutions to SEQ ID NO 1
  • B comprises or consists of SEQ ID NO: 28
  • C comprises or consists of SEQ ID NO: 3 or a sequence comprising conservative substitutions to SEQ ID NO: 3
  • D comprises of consists of SEQ ID NO: 29
  • E comprises or consists of SEQ ID NO: 5 or a sequence comprising conservative substitutions to SEQ ID NO: 5.
  • the scaffold protein method of use according to clause 32, wherein the residue of D corresponding to residue 1 of SEQ ID NO: 4 is L; and/or the residue of D corresponding to residue 2 of SEQ ID NO: 4 is K; and/or the residue of D corresponding to residue 4 of SEQ ID NO: 4 is T; and/or the residue of D corresponding to residue 5 of SEQ ID NO: 4 is Q; and/or the residue of D corresponding to residue 6 of SEQ ID NO: 4 is N; and/or the residue of D corresponding to residue 8 of SEQ ID NO: 4 is I; and/or the residue of D corresponding to residue 9 of SEQ ID NO: 4 is D; and/or the residue of D corresponding to residue 11 of SEQ ID NO: 4 is I; and/or the residue of D corresponding to residue 12 of SEQ ID NO: 4 is T; and/or the residue of D corresponding to residue 15 of SEQ ID NO: 4 is V; and/or the residue of D corresponding to residue 16 of SEQ ID NO: 4 is N; and/
  • the scaffold protein method of use according to clause 34, wherein the residue of D corresponding to residue 1 of SEQ ID NO: 4 is L; and the residue of D corresponding to residue 2 of SEQ ID NO: 4 is K; and the residue of D corresponding to residue 4 of SEQ ID NO: 4 is T; and the residue of D corresponding to residue 5 of SEQ ID NO: 4 is Q; and the residue of D corresponding to residue 6 of SEQ ID NO: 4 is N; and the residue of D corresponding to residue 8 of SEQ ID NO: 4 is I; and the residue of D corresponding to residue 9 of SEQ ID NO: 4 is D; and the residue of D corresponding to residue 11 of SEQ ID NO: 4 is I; and the residue of D corresponding to residue 12 of SEQ ID NO: 4 is T; and the residue of D corresponding to residue 15 of SEQ ID NO: 4 is V; and the residue of D corresponding to residue 16 of SEQ ID NO: 4 is N; and the residue of D corresponding to residue 19 of SEQ ID NO: 4 is A.
  • N-terminal fragment of apolipoprotein E comprises the formula X-Y-A-B-C-D-E wherein only X or Y are present or both X and Y are present and wherein X comprises or consists of SEQ ID NO: 21 and Y comprises or consists of SEQ ID NO: 20.
  • the scaffold protein comprises an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • the method of eliciting an immune response in a subject comprising administering to the subject a scaffold protein comprising an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • the scaffold protein for use as a medicament according to clause 3, wherein the scaffold protein comprises an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • scaffold protein for use in the treatment or prevention of influenza infection according to clause 4, wherein the scaffold protein comprises an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • a scaffold protein in the manufacture of a medicament for the treatment or prevention of influenza infection according to clause 5, wherein the scaffold protein comprises an acylhydrolase protein wherein the acylhydrolase protein comprises an influenza hemagglutinin stem epitope, wherein the polypeptide sequence of the acylhydrolase protein comprises the formula A-B-C-D, wherein A comprises a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B comprises a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C comprises a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D comprises a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • A consists of a sequence sharing at least 50% identity with SEQ ID NO: 14, wherein B consists of a sequence sharing at least 70% identity with SEQ ID NO: 15, wherein C consists of a sequence sharing at least 80% identity with SEQ ID NO: 16 and wherein D consists of a sequence sharing at least 50% identity with SEQ ID NO: 17.
  • A comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98%, such as at least 99% identity with SEQ ID NO: 14.
  • B comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 90% identity with SEQ ID NO: 15.
  • C comprises or consists of a sequence sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95% identity with SEQ ID NO: 16.
  • scaffold protein comprising or consists of a sequence sharing at least 50%, such as at least 70%, such as at least 90%, such as at least 95%, such as at least 98% identity with SEQ ID NO: 18.
  • scaffold protein comprising or consists of SEQ ID NO: 18.
  • the scaffold protein, method or use according to clause 57 wherein the residue of B corresponding to residue 1 of SEQ ID NO: 15 is H or a conservative substitution thereof; and the residue of B corresponding to residue 4 of SEQ ID NO: 15 is A or a conservative substitution thereof; and the residue of B corresponding to residue 5 of SEQ ID NO: 15 is P or a conservative substitution thereof; and the residue of B corresponding to residue 8 of SEQ ID NO: 15 is E or a conservative substitution thereof; and the residue of B corresponding to residue 10 of SEQ ID NO: 15 is Q or a conservative substitution thereof.
  • the scaffold protein, method or use according to clause 57 wherein the residue of B corresponding to residue 1 of SEQ ID NO: 15 is H; and/or the residue of B corresponding to residue 4 of SEQ ID NO: 15 is A; and/or the residue of B corresponding to residue 5 of SEQ ID NO: 15 is P; and/or the residue of B corresponding to residue 8 of SEQ ID NO: 15 is E; and/or the residue of B corresponding to residue 10 of SEQ ID NO: 15 is Q.
  • the scaffold protein, method or use according to clause 59 wherein the residue of B corresponding to residue 1 of SEQ ID NO: 15 is H; and the residue of B corresponding to residue 4 of SEQ ID NO: 15 is A; and the residue of B corresponding to residue 5 of SEQ ID NO: 15 is P; and the residue of B corresponding to residue 8 of SEQ ID NO: 15 is E; and the residue of B corresponding to residue 10 of SEQ ID NO: 15 is Q.
  • the scaffold protein, method or use according to clause 61 wherein the residue of C corresponding to residue 1 of SEQ ID NO: 16 is E or a conservative substitution thereof; and the residue of C corresponding to residue 3 of SEQ ID NO: 16 is T or a conservative substitution thereof; and the residue of C corresponding to residue 5 of SEQ ID NO: 16 is A or a conservative substitution thereof; and the residue of C corresponding to residue 7 of SEQ ID NO: 16 is I or a conservative substitution thereof; and the residue of C corresponding to residue 8 of SEQ ID NO: 16 is N or a conservative substitution thereof; and the residue of C corresponding to residue 11 of SEQ ID NO: 16 is T or a conservative substitution thereof; and the residue of C corresponding to residue 14 of SEQ ID NO: 16 is I or a conservative substitution thereof; and the residue of C corresponding to residue 15 of SEQ ID NO: 16 is N or a conservative substitution thereof; and the residue of C corresponding to residue 18 of SEQ ID NO: 16 is I or a conservative substitution thereof; and the residue of C corresponding
  • the scaffold protein, method or use according to clause 61 wherein the residue of C corresponding to residue 1 of SEQ ID NO: 16 is E; and/or the residue of C corresponding to residue 3 of SEQ ID NO: 16 is T; and/or the residue of C corresponding to residue 5 of SEQ ID NO: 16 is A; and/or the residue of C corresponding to residue 7 of SEQ ID NO: 16 is I; and/or the residue of C corresponding to residue 8 of SEQ ID NO: 16 is N; and/or the residue of C corresponding to residue 11 of SEQ ID NO: 16 is T; and/or the residue of C corresponding to residue 14 of SEQ ID NO: 16 is I; and/or the residue of C corresponding to residue 15 of SEQ ID NO: 16 is N; and/or the residue of C corresponding to residue 18 of SEQ ID NO: 16 is I; and/or the residue of C corresponding to residue 24 of SEQ ID NO: 16 is F; and/or the residue of C corresponding to residue 28 of SEQ ID NO: 16 is F; and
  • the scaffold protein, method or use according to clause 63 wherein the residue of C corresponding to residue 1 of SEQ ID NO: 16 is E; and the residue of C corresponding to residue 3 of SEQ ID NO: 16 is T; and the residue of C corresponding to residue 5 of SEQ ID NO: 16 is A; and the residue of C corresponding to residue 7 of SEQ ID NO: 16 is I; and the residue of C corresponding to residue 8 of SEQ ID NO: 16 is N; and the residue of C corresponding to residue 11 of SEQ ID NO: 16 is T; and the residue of C corresponding to residue 14 of SEQ ID NO: 16 is I; and the residue of C corresponding to residue 15 of SEQ ID NO: 16 is N; and the residue of C corresponding to residue 18 of SEQ ID NO: 16 is I; and the residue of C corresponding to residue 24 of SEQ ID NO: 16 is F; and the residue of C corresponding to residue 28 of SEQ ID NO: 16 is F; and the residue of C corresponding to residue 29 of SEQ ID NO: 16 is V; and
  • B comprises or consists of the sequence HX1X2APX3X4EX5QX6 (SEQ ID NO: 32) wherein Xi is selected from the group consisting of A, G, I, L, M and V; wherein X2 is selected from the group consisting of A, G, I, L, M and V; wherein X3 is selected from the group consisting of F, W and Y; wherein X4 is selected from the group consisting of C, N, P, Q, S and T; wherein Xs is selected from the group consisting of C, N, P, Q, S and T; and wherein Xe is selected from the group consisting of H, K and R.
  • Xi is selected from the group consisting of A, G, I, L, M and V
  • X2 is selected from the group consisting of A, G, I, L, M and V
  • X3 is selected from the group consisting of F, W and Y
  • X4 is selected from the group consisting of C,
  • C comprises or consists of the sequence EX1TX2AX3INX4X5TX6X7INX8X9IX10X11X12X13X14FX15X16X17FVX18X19AQSPX20GD (SEQ ID NO: 33) wherein Xi is selected from the group consisting of A, G, I, L, M and V; wherein X2 is selected from the group consisting of C, N, P, Q, S and T; wherein Xs is selected from the group consisting of A, G, I, L, M and V; wherein X4 is selected from the group consisting of A, G, I, L, M and V; wherein Xs is selected from the group consisting of C, N, P, Q, S and T; wherein Xe is selected
  • A comprises or consists of SEQ ID NO: 14 or a sequence comprising conservative substitutions to SEQ ID NO: 14;
  • B comprises or consists of SEQ ID NO: 32;
  • C comprises or consists of SEQ ID NO: 33;
  • D comprises or consists of SEQ ID NO: 17 or a sequence comprising conservative substitutions to SEQ ID NO: 17.
  • acylhydrolase protein comprises a polypeptide sequence of no more than 700 residues, such as no more than 500 residues, such as no more than 300 residues, such as no more than 250 residues, such as no more than 230 residues, such as no more than 220 residues.
  • KD binding affinity
  • the KD is 1.3 x 10' 7 M (/.e. 130 nM) or less, such as 1.0 x 10' 7 M (/.e. 100 nM) or less.
  • the KD is less than 6.0 x 10 -8 M (/.e.
  • the KD may be 1.0 x 10' 8 M (/.e. 10 nM) or less, such as 7.0 x 10 -9 M (/.e 7 nM) or less, such as 6.0 x 10' 9 M (/.e. 6 nM) or less, such as 5.0 x 10' 9 M (/.e. 5 nM) or less.
  • the scaffold protein, method or use according to any one of clauses 1 to 111 wherein the protein is administered intramuscularly.
  • the scaffold protein, method or use according to any one of clauses 110, 112 or 113, wherein the scaffold protein and squalene emulsion adjuvant are administered as separate formulations.
  • scaffold protein The scaffold protein, method or use according to any one of claims 119 to 124, wherein the scaffold protein is an immunogen of which the region having similarity to an influenza hemagglutinin stem epitope is an integral part.
  • the structural segments comprising the conserved HA stem epitope around the hydrophobic pocket were extracted from the H1 crystal structure in complex with the FI6 antibody (PDB ID: 3ZTN).
  • the epitope consists of three segments, a three residue HSV-loop (residues 28-30, chain A), a four residue VDGW-loop (residues 18-21, chain B), and an a-helix (residues 38-57, chain B).
  • a structural search was performed of the epitope against the Protein Data Bank (Version August 2018) containing 141 ,920 protein structures to identify putative scaffold candidates based on the local similarity.
  • the search was performed using the MASTER software (Zhou and Grigoryan 2015) with a backbone RMSD threshold below 2 A, however, no suitable scaffolds were detected according to both, local structural features or overall topology.
  • a second search was performed, omitting the HSV-loop of the epitope to increase chances of local structural matches, resulting in 45,616 matches with backbone RMSD below 2 A.
  • the potential scaffold set was narrowed down by restricting the protein length to 50-250 residues and evaluating the accessibility of the epitope to the FI6 antibody in terms of predicted binding energy and atomic clashes. The remaining candidates were inspected manually to select scaffolds which present the epitope in its native conformation and provide additional surface area to mimic the entire antigenic site.
  • a putative acylhydrolase was selected (PDB ID: 4IYJ) that matches to the trimmed epitope with a RMSD of 1.44 A and the epitope side chains of the a-helix and VDGW-loop were transplanted onto the scaffold (FI6-focused_01).
  • 45 design variants were expressed in yeast and screened for binding to the FI6 antibody. Based on the screening on the yeast surface, one design was identified that showed specific interaction with the antibody. Since initial binding of the design protein to the FI6 antibody was relatively low, binding was improved through a combinatorial library by sampling positions adjacent to the epitope helix, resulting in a 320 nM KD binder (FI6-focused_02) i.e. having binding improved to a dissociation constant (KD) of 320 nM, as measured by Surface Plasmon Resonance (SPR).
  • SPR Surface Plasmon Resonance
  • the binding affinity of the FI6-focused design was further improved by a subsequent single site mutation (site-saturation mutagenesis or SSM) library, sampling epitope positions and surrounding residues around the grafted site (aa 93-106 and aa 123-189). See Figures 1 and 2. Best individual mutations were combined to improve binding affinity with the least amount of additional mutations. In total 16 variants were screened for improved binding to FI6 and all design variants boosted affinity, with the best binding design, FI6-focused_03, resulting in a KD of 0.26 nM. Since the native protein scaffold forms a homodimer, several mutations were introduced to disrupt dimer-formation.
  • SSM site-saturation mutagenesis
  • Residues contributing to the dimerization (ddG ⁇ -0.8) and exposed hydrophobic residues in the interface were selected and submitted to sequence design.
  • a BLAST search NCBI Resource Coordinators 2018 was performed of the WT protein scaffold sequence and used to construct a position-specific scoring matrix (PSSM) used during subsequent sequence design. Mutations were selected that improved local residue REU and were not part of the epitope region, resulting in twelve mutations in total to increase thermostability of the protein, resulting in the final design, named FI6-focused_04.
  • amino acid residues which were introduced into the putative acylhydrolase sequence to produce the final design, FI6-focused design_04, are those recited under regions A to D of ‘Acylhydrolase protein’ in the description above and which are underlined in the sequence provided under ‘The whole acylhydrolase protein’ in the description above.
  • Potential scaffolds were further filtered by assessing accessibility of the FI6 antibody and computing the number of putative contacts between the scaffold and antibody in the epitope region to evaluate the potential to improve overall epitope mimicry. Based on these selection criteria the top 50 matches were manually evaluated and mouse apolipoprotein E (ApoE, PDB ID: 1YA9) was selected as design candidate. The sidechains of the epitope helix were transplanted onto the scaffold using Rosetta MotifGraft and three mutations on the scaffold, not part of the interface, were introduced to resolve steric hindrance with epitope residues. Next, the overall epitope mimicry was evaluated based on surface similarity using RosettaSurf to identify positions with low epitope mimicry.
  • stem-epitope_02 A rational mutation I98A was introduced, as the designed amino acids did not match the native epitope residues, resulting in the stem-epitope_01.
  • the proteins were recombinantly expressed in E. coli and purified. Both proteins were monomeric, correctly folded, and bound to antibody FI6 with KDs of 44 nM and 48 nM, for the stem-epitope_01 and stem-epitope_02, respectively.
  • the introduced disulfide bonds in stem-epitope_02 did not increase stability as evaluated by its melting temperature and thus stem-epitope_01 was used for further analysis. Data analysis was performed with the help of the rstoolbox Python library (Bonet et al. 2019) and protein structures were visualized using PyMOL (Schrodinger, LLC 2015).
  • polypeptide sequence of the design based on murine ApoE, is shown in Table 3 below:
  • the amino acid residues which were introduced into the N-terminal ApoE fragment to produce the final design, Stem-epitope design_01 are those recited under regions A to E of ‘Murine ApoE’ (and ‘Human ApoE’) in the description above and which are underlined in the sequences provided under ‘The whole fragment’ in the description above.
  • DNA sequences of all designs were produced with homology overhangs for cloning.
  • DNA was transformed with linearized pCTcon2 vector (Addgene #41843) into EBY-100 yeast using the Frozen-EZ Yeast Transformation II Kit (Zymo Research).
  • Transformed yeast were passaged once in minimal glucose medium (SDCAA) before induction of surface display in minimal galactose medium (SGCAA) overnight at 30°C.
  • Transformed cells were washed with PBS + 0.05% BSA and incubated with different concentrations of FI6 antibody for 2h at 4°C.
  • Combinatorial sequence libraries were constructed by assembling multiple overlapping primers containing degenerate codons at selected positions for combinatorial sampling of the epitope. Primers were mixed (10 pM each), and assembled in a PCR reaction (55°C annealing for 30 sec, 72°C extension time for 1 min, 25 cycles). To amplify full-length assembled products, a second PCR reaction was performed, with forward and reverse primers specific for the full- length product. The PCR product was desalted and used for transformation.
  • Combinatorial libraries and SSM libraries were transformed as linear DNA fragments in a 5:1 ratio with linearized pCTcon2 vector as described previously (Chao et al. 2006) into EBY-100 yeast. Transformation efficiency generally yielded around 107 transformants.
  • Library cultures were prepared for sorting similar to single designs. Labelled cells were sorted on a Sony SH800 cell sorter. For combinatorial libraries, sorted cells were grown in SDCAA and prepared similarly for two additional rounds of sorting. After the 3rd sort cells were plated on SDCAA plates and single colonies were sequenced. SSM libraries were only sorted once and grown in liquid culture for plasmid prep. Protein expression and purification
  • Plasmids were transformed in E. coli BL21 (DE3) and grown overnight in LB medium supplemented with Ampicillin. Overnight cultures were used to inoculate the main culture at an OD600 of 0.1.
  • FI6- focused design versions were incubated for 4-5h at 37°C.
  • Stem-epitope design was incubated overnight at 22°C.
  • Cultures were harvested by centrifugation. Pellets were resuspended in lysis buffer (50 mM Tris, pH 7.5, 500 mM NaCI, 5% Glycerol, 1 mg/ml lysozyme, 1 mM PMSF, and 1 pg/ml DNase) and sonicated on ice for a total of 12 minutes, in intervals of 15 s sonication followed by 45 s pause.
  • lysis buffer 50 mM Tris, pH 7.5, 500 mM NaCI, 5% Glycerol, 1 mg/ml lysozyme, 1 mM PMSF, and 1 pg/ml DNase
  • the lysates were clarified by centrifugation (48,000 g, 20 min) and purified via Ni-NTA affinity chromatography followed by size exclusion on a HiLoad® 16/600 Superdex® 200pg column on an AKTATM pure system (Cytivia).
  • the DNA sequence of all used human antibodies were ordered from Twist Bioscience and cloned into a pHLsec vector for mammalian expression containing a C-terminal human Fc fragment for heavy chain cloning and no Tag for light chain cloning.
  • Antibodies were produced using the Expi293TM expression system from Thermo Fisher Scientific. Supernatant was collected 6 days post transfection and purified via protein A affinity chromatography and subsequent size exclusion on a HiLoad® 16/600 Superdex® 200pg column on an AKTATM pure system (Cytivia).
  • Plasmids encoding the CR9114 Fab heavy and light chains for X-ray crystallography were dually transfected into Expi293 cells with the Fab heavy chain also encoding a Strep Tag II at the C terminus.
  • Cell supernatant was harvested at day 5 when cells reached -80% viability, diafiltered to remove destinbiotin from the supernatant, then CR9114 Fab was purified using a StrepTrap HP column (GE Healthcare).
  • the Strep Tag II was proteolytically cleaved using TEV protease (AcTEV protease, Thermo Fisher Scientific) prior to size exclusion chromatography in buffer containing 10 mM Tris pH 7.5, 150 mM NaCI.
  • Plasmids encoding for H1_NC99, H1_stem_NC99, H3_HK68, H5_VN05, V7_ShO7 were kindly provided by the NIH. All HAs contained a C-terminal T4 trimerization site, Avi-Tag and 6x His Tag. Modified versions as stem-epitope KO mutants, GCN4 trimerization sites and stem constructs were ordered as linear dsDNA inserts from Twist Bioscience and cloned into the VRC vector from NIH. All recombinant HAs carry the Y98F mutation in the receptor-binding domain. HAs were produced using the Expi293TM expression system from Thermo Fisher Scientific.
  • the sequences of the FI6-focused design_04 and stem-epitope design were cloned into a pHLsec vector with an N-terminal 6x His Tag and a C-terminal ferritin from Helicobacter pylori (GenBank ID: QAB33511.1). Designs and ferritin were connected by a GS linker containing one glycosylation site (GGSGGSGGSGGSNGTGGSGGS, SEQ ID NO: 24). Ferritin-design nanoparticles were produced using the Expi293TM expression system from Thermo Fisher Scientific.
  • Size exclusion chromatography with an online multi-angle light scattering device (miniDAWN TREOS, Wyatt) was used to determine the oligomeric state and molecular weight for the protein in solution.
  • Purified proteins were concentrated to 1 mg/ml in PBS (pH 7.4), and injected into a Superdex 75 300/10 GL column (cytivia) with a flow rate of 0.5 ml/min, and UV280 and light scattering signals were recorded.
  • Molecular weight was determined using the ASTRA software (version 6.1, Wyatt).
  • Circular Dichroism spectra were measured using a ChirascanTM spectrometer (AppliedPhotophysics) in a 1-mm path-length cuvette.
  • the protein samples were prepared in a 10 mM sodium phosphate buffer at a protein concentration between 20 and 50 pM. Wavelengths between 200 nm and 250 nm were recorded with a scanning speed of 20 nm min-1 and a response time of 0.125 secs. All spectra were averaged two times and corrected for buffer absorption. Temperature ramping melts were performed from 20 to 90°C with an increment of 2 °C/min. Thermal denaturation curves were plotted by the change of ellipticity at the global curve minimum to calculate the melting temperature (Tm).
  • yeast cells were grown in SDCAA medium, pelleted and plasmid DNA was extracted using Zymoprep Yeast Plasmid Miniprep II (Zymo Research) following the manufacturer’s instructions.
  • the coding sequence of the designed variants was amplified using vector-specific primer pairs, Illumina sequencing adapters were attached using an additional overhang PCR, and PCR products were desalted on PCR purification columns (Qiaquick PCR purification kit, Qiagen).
  • Next generation sequencing was performed using an Illumina MiSeq 2 x 150 bp paired end sequencing (300 cycles), yielding between 0.45-0.58 million reads/sample.
  • sequences were translated in the correct reading frame, and enrichment values were computed for each sequence.
  • MDCK-cells were cultured in DM EM (ThermoFisher Scientific) supplemented with 10% heat inactivated FBS (ThermoFisher Scientific) under 5% CO2 atmosphere at 37°C.
  • MDCK-SIAT1 Were cultured as MDCK but with the addition of 500 pg/ml geneticin (Gibco).
  • the following viruses were employed in this study: A/Puerto Rico/8/34 (H1N1), A/California/07/2009 (H1 N1) and A/HKx31 (H3N2). Viruses were propagated in 10 days old embryonated chicken eggs (VALO BioMedia) or MDCK SIATI cells.
  • AF488 coupled AF488 Protein Labelling Kit, Thermo Fisher, Waltham, MA
  • anti-influenza A virus NP clone H16-L10-4R5 (HB65)
  • Bio X Cell Riverside, NH
  • the samples were acquired on a CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, IN) and analyzed with the GraphPad Prism9 software.
  • mice Female six-week-old Balb/cjRJ mice were acclimatized for one week. Hemagglutinins were used at a concentration of 20 pg/mL and design particles at 40 pg/mL. Immunogens were diluted with PBS (pH 7.4) to the intended concentration and mixed 1 :1 with AS03 adjuvant right before the injection. Each mouse was injected intramuscularly in the hind leg with 50 pL, corresponding to 1 pg of HA and 2 pg of design particles. Immunizations were performed on day 0, 21 and 42. Tail bleedings (-100 pL) were performed on day 0, 14 and 35. On day 56 mice were sacrificed, mice were anaesthetized with isoflurane and blood was drawn by cardiac puncture.
  • mice were assigned to groups of five or ten animals. Injections were given three weeks apart intramuscular into the left hind. Either 2pg of the stem design particle, 1 pg of H1 m2e, only adjuvant or only PBS were administered. Three weeks after the final dose, animals were challenged intranasally with a lethal dose of 1x106 TCID50 X-31 (H3N2) (National Institutes of Health, Bethesda, MD) virus. The following two weeks, mice were weighed every day and sacrificed if their weight dropped below 75% of the initial weight.
  • H3N2 TCID50 X-31
  • Nunc MediSorp plates were coated with antigen (recombinant HA, design scaffolds or wildtype scaffolds) overnight at 4°C in PBS (pH 7.4). Plates were blocked with blocking buffer (PBS + 0.05% Tween + 5% skimmed milk powder (sigma, #70166) for 2h at room temperature (RT). Plates were washed 4 times with PBST (PBS + 0.05% Tween). Mouse sera was serially diluted in dilution buffer (PBS + 1% BSA) and incubated for 2h at RT. Plates were washed again 4 times with PBST.
  • blocking buffer PBS + 0.05% Tween + 5% skimmed milk powder (sigma, #70166)
  • Anti-mouse-Fc HRP-conjugated antibody was diluted 1 :5000 in dilution buffer and incubated 1h at RT. Plates were washed again 4 times and developed by adding 100 pL of TMB solution per well. The reaction was stopped after 5 minutes 100 pl with 0.5M HCI. The absorbance at 450 nm was measured on a Tecan Safire 2 plate reader, and the antigen specific titers were determined as the reciprocal of the serum dilution yielding a signal two-fold above the background.
  • X31(H3N2) and California 0709 (H1 N1) virus (National Institutes of Health, Bethesda, MD) were UV-inactivated on ice for 30 min.
  • 96-well plates (Greiner Bio-One GmbH, Kremsmunster, Austria) were then coated with either whole UV-inactivated virus or HAH1 purified from PR8 virus as previously described (Angeletti et al. 2019). After at least an overnight incubation at 4°C, plates were blocked with 2% BSA. Subsequently, after washing three times, sera to be tested were diluted in serial 2-fold dilutions down the plate. Plates were then incubated at 37°C for 1.5h.
  • mice were immunized three times with the stem design particle intra muscularly into the left hind leg, three weeks apart between immunizations and one week after the last injection, mice were sacrificed. Iliac and Inguinal lymph nodes were pooled and analysed.
  • IgD negative, H1 H3 double-positive B cells were sorted with the Fusion Cell Sorter (BD Biosciences, San Jose, CA) with a 100 pm nozzle into BSA-coated tubes supplied with 5% FBS buffer. Cells were further processed with Chromium Next GEM Single Cell 5’ Reagent Kits v2 for Dual Index (10x Genomics, Pleasanton, CA) following the given instructions.
  • GEMs gelbeads in emulsion
  • cDNA was amplified and samples were split.
  • V(D)J or 5’ Gene Expression Dual Index Libraries were generated using the Dual Index Kit TT Set A.
  • V(D)J library the V(D)J cDNA was first amplified. After every step, a quality control was performed using a Qubit Fluorometer (Thermo Fisher, Waltham, MA) and after the last step, samples were run on a TapeStation (Agilent Technologies, Santa Clara, CA) to determine the average fragment size. Samples were then sequenced with the Illumina NovaSeq sequencing system (Illumina, San Diego, CA) and processed according to the guidelines given by 10xGenomics. Obtained data were QC-checked and analysed and antibodies were selected based on mutation rate and expansion of the clones.
  • Example 1 Design of immunogens mimicking a conserved epitope in the hemagglutinin stem
  • a well-characterized conserved epitope centered around the hydrophobic pocket on the HA stem was selected.
  • This stem epitope is commonly targeted by broadly neutralizing antibodies (bnAbs) such as FI6 (Corti et al. 2011), CR9114 (Dreyfus et al. 2012), 39.29 (Nakamura et al. 2013), or MEDI- 8852 (Kallewaard et al. 2016) (Fig. 1).
  • bnAbs broadly neutralizing antibodies
  • the conserved site on the HA stem is a multi-segment epitope, consisting of a 20-residue long a-helix, a four residue VDGW-loop, and a three residue HSV-loop (Fig. 1).
  • the stem-epitope was extracted from a crystal structure of H1 hemagglutinin in complex with the FI6 antibody (PDB ID: 3ZTN (Corti et al. 2011)) and potential protein scaffolds were identified by querying the Protein Data Bank (PDB) (Berman et al. 2000) for structurally similar proteins. However, due to the irregular and discontinuous nature of the epitope, close matches were absent.
  • a scaffold was selected (PDB ID: 4IYJ) that closely mimicked the a-helix and the 4-residue loop (backbone RMSD 1.44 A), omitting the shorter epitope loop. It was hypothesized that the design could be able to mimic core features of the epitope even though close structural matches were absent. Suitability of the scaffold was confirmed by evaluating the predicted binding energy and atomic clashes of the scaffold and FI6 antibody. After transplantation of the epitope helix and 4-residue loop onto the selected protein scaffold, computational sequence design was performed to optimize binding towards the FI6 antibody, resulting in the FI6- focused_01 design (Fig. 1).
  • the SSM library approach allows a thorough sampling of a large number of relevant positions.
  • a second round of computational sequence design was performed, introducing point mutations to monomerize the scaffold which forms a homodimer in its native state. Monomerization was necessary to enable efficient display on protein nanoparticles.
  • the native homodimerization interface of the protein scaffold is located opposite to the transplanted epitope and was disrupted with eight mutations, but entailed overall decreased protein stability.
  • FI6-focused_04 was well-folded and monomeric as confirmed by CD and SEC-MALS (Fig. 3) and bound FI6 with 6 nM as measured by surface plasmon resonance (SPR) (Fig. 4A).
  • epitope mimicry was evaluated based on the overall surface mimicry between the protein scaffold and HA with RosettaSurf, and confirmed high similarity to the native epitope (Fig. 1). Based on the observed similarities, computational sequence design was performed to further improve epitope mimicry by 130% over the WT scaffold based on epitope surface shape similarity, resulting in the stem-epitope_01 design. In addition, epitope mimicry was evaluated by predicting binding energies to CR9114 and MEDI-8852, showing similar values to those of FI6, indicating that the stem-epitope mimetic could engage a range of bnAbs. The stem-epitope_01 mimetic was confirmed to adopt an a-helical fold by CD, was monomeric, and bound with a KD of 44 nM to FI6 (Fig. 4A, Fig. 3).
  • Ferritin assembles from 24 subunits, allowing the multivalent display of proteins resulting in enhanced binding kinetics through avidity (Kanekiyo et al. 2013). For both designs well-formed particles could be observed (Fig. 4C). While the FI6-focused_04 particle only bound with high affinity to FI6, the stem-epitope_01 particle showed strong binding to FI6, MEDI-8852 and CR9114, demonstrating its improved binding breadth (Fig. 4B). To further enhance the immune response, a known T cell epitope from the influenza matrix protein (m2e) was fused via a linker to the N-terminus of the scaffold proteins (Eliasson et al. 2018) (Fig. 5). The m2e sequence is provided in SEQ ID NO: 25 and the linker sequence (GASG) is provided in SEQ ID NO: 26.
  • m2e sequence is provided in SEQ ID NO: 25 and the linker sequence (GASG) is provided in SEQ ID NO: 26.
  • Example 2 Structural characterization of designed stem-epitope mimetics
  • the crystal structure of the FI6-focused_03 design was solved in complex with FI6 Fab at 1.95 A resolution (Fig. 6A). Comparing the solved structure and designed model demonstrated close structural similarity with a RMSD of 2 A. It was observed that the grafted epitope helix was elongated by two additional turns at the N-terminal end. This was likely attributed to a mutation introduced as part of the epitope (P144T), forming the transition of helix to loop in the native scaffold. However, the introduced mutation benefits the interaction with FI6 and mimicry of the stem epitope was still high when compared to H1 HA (Fig. 6B).
  • PBMCs peripheral blood mononuclear cells
  • One of the isolated antibodies originated from VH3-23, a germline that is known to give rise to potent bnAbs (Joyce et al. 2016) and was highly cross-reactive towards group 1 and group 2 HAs (Fig. 7C).
  • Known antibodies from the same germline did not cross-react with the design, most likely as the result of higher mutational load of the selected antibodies.
  • Previously described antibodies as well as 31-1 B01 isolated in this study were able to neutralize viruses from group 1 and 2 with similar potencies (31-1C12, 63-1A12, 63-1C07 not tested).
  • H1 While all of the stem-mimetic positive B cells that were cross-reactive to H1 and H3 harbored the same antibody (31-1 B01), the H1 only population mainly contained antibodies from VH1-69 ( Figure 7D), which represents the major human VH region giving rise to group 1 specific HA stem antibodies. Its ability to engage this class of antibodies could be beneficial for the robust induction of pan-group 1 bnAbs.
  • naive Balb/c mice were injected three times with nanoparticles displaying either FI6-focused_04 or stem-epitope_01 immunogen, adjuvanted with AS03 (Fig. 8A). Both designs were immunogenic as seen by the high, design-induced antibody titers. Since these designs were based on natural, heterologous proteins, the respective WT scaffolds were used to determine the proportion of antibodies targeting the epitope. The antibody response against the WT scaffolds reflects the proportion of non-epitope-specific antibodies. Thus, a lower response to the WT scaffold correlates with high epitope-focusing.
  • a viral ELISA was performed. ELISA plates were coated with UV- inactivated virus and binding titers were determined. Serum antibodies elicited by the stemepitope design showed virus binding on par with three times H1 full-length trimer immunization against a heterologous pH1 Ca09 virus. Notably, design-induced antibodies exhibited superior binding to an H3 HK68 virus (Fig. 8F). Similar results were obtained using a flow cytometric assay to detect binding to nascent virions on infected MDCK cells. Considering that the stemepitope mimetic carries a single antigenic site presenting ⁇ 6% of the HA surface area, the enhanced binding breadth of the elicited antibodies is remarkable.
  • B cells were isolated from mice immunized with stem-epitope_01 particles and evaluated their cross-reactivity to group 1 and group 2 HAs. Spleens and draining lymph nodes were examined two weeks after the 2 nd and 3rd injection and H1/H3 cross-reactive cells in memory and germinal-center B cell population were considered. The B cell analysis confirmed previous observations with sera that the majority of the HA positive antibodies bound to H3 only. However, approximately 2% of the isolated antibodies were cross-reactive for H1 and H3 (Fig. 9A). No antibodies specific to H1 only were detected.
  • VH variable heavy
  • VL variable heavy chain
  • mice were immunized three times with different combinations of an H1 NC99 trimer and the stem-epitope particle, either in a homologous prime-boost (3x stemepitope design), heterologous prime-boost (H1 NC99 prime and 2x stem-epitope design), or prime only (H1 NC99) regime (Fig. 10).
  • mice were challenged with a lethal dose of an H3 X31 virus.
  • mice that received at least two injections of the stem-epitope design particle had a survival rate of 80%. In contrast, mice that were only injected with recombinant H1 NC99 HA trimer had a survival rate of only 20% (Fig. 10).
  • the viral challenge study clearly demonstrates the superiority of the design induced antibodies. All groups experienced significant weight loss, however, the groups that received the stemepitope design particle started recovering four to five days after infection while mice receiving only H1 NC99 continued to lose weight.
  • a second challenge study confirmed protection mediated by the stem-epitope design particle, with larger groups of animals.
  • 8 out of 10 mice were protected from a lethal H3 X31 virus challenge after receiving three immunisations with the stem-epitope design particle (Figure 11).
  • This stem-epitope design represents the first HA immunogen based on a heterologous protein that confers protection to an influenza virus challenge.
  • Other existing HA immunogens are built upon modified or trimmed HA proteins. Most of these immunogens show better protection against related strains but struggle to protect against diverse subtypes (Krammer et al. 2013; Schotsaert et al. 2016; Sutton et al. 2017).
  • Stem-specific antibodies are known to be weakly neutralizing and mainly protect via Fc- receptor mediated cellular pathways (DiLillo et al. 2016). Therefore, the activation of antibodydependent cellular cytotoxicity (ADCC) was evaluated through the elicited antibodies in mouse sera. Immunizations with three times stem-epitope design were compared with heterologous prime boost immunizations of H1 or H3 trimers as prime injections and stem-mimetic particle or PBS boost injections ( Figure 12A).
  • HA prime only injections did not show ADCC activity against any of the three tested viruses, H1 N1 PR8, H1N1 CA07/09 and H3N2 X31, while all groups boosted with the stem-mimetic particle showed ADCC activity against at least one virus (Figure 12A).
  • the results show that the stem-mimetic is able to induce functional cross- reactive antibodies against H1 and H3 viruses and boost pre-existing HA antibodies in a heterologous prime boost scheme. Nevertheless, it has to be noted that not all mice showed ADCC activation (Figure 12B), particularly the homologous immunization with three times stem-mimetic particle was limited to activation in a minority of mice.
  • Impagliazzo A., F. Milder, H. Kuipers, M. V. Wagner, X. Zhu, R. M. B. Hoffman, R. van Meersbergen, et al. 2015. “A Stable Trimeric Influenza Hemagglutinin Stem as a Broadly Protective Immunogen.” Science 349 (6254): 1301-6. https://doi.Org/10.1126/science.aac7263. luliano, A Danielle, Katherine M Roguski, Howard H Chang, David J Muscatello, Rakhee Palekar, Stefano Tempia, Cheryl Cohen, et al. 2018. “Estimates of Global Seasonal Influenza- Associated Respiratory Mortality: A Modelling Study.” The Lancet 391 (10127): 1285-1300. https://doi.Org/10.1016/S0140-6736(17)33293-2.

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Abstract

La présente invention concerne, entre autres, des protéines d'échafaudage comprenant un épitope à tige d'hémagglutinine de la grippe.
PCT/EP2022/078214 2021-10-13 2022-10-11 Polypeptides WO2023061993A1 (fr)

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EP0868918A2 (fr) 1993-12-23 1998-10-07 SMITHKLINE BEECHAM BIOLOGICALS s.a. Vaccins
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