WO2023061993A1

WO2023061993A1 - Polypeptides

Info

Publication number: WO2023061993A1
Application number: PCT/EP2022/078214
Authority: WO
Inventors: Normand Blais; Bruno Correia; Andreas Scheck; Sarah WEHRLE
Original assignee: Glaxosmithkline Biologicals Sa
Priority date: 2021-10-13
Filing date: 2022-10-11
Publication date: 2023-04-20

Abstract

The present invention relates inter alia to scaffold proteins comprising an influenza hemagglutinin stem epitope.

Description

Polypeptides

TECHNICAL FIELD

The present invention relates to scaffold proteins presenting an influenza HA stem epitope.

BACKGROUND

Every year, infections with influenza lead to 5 million hospitalizations and are lethal for up to 650,000 patients. Additionally, the constant threat of emerging pandemic influenza strains imposes a major risk for global health, emphasizing the need for an effective vaccine. Present 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.

Flu vaccines are most commonly made from an egg-based manufacturing process as either live-attenuated or inactivated-virus formulations. However, 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. However, the head is highly variable as a result of antigenic drift, resulting in strain-specific immune responses. An improved vaccine should elicit broadly neutralizing antibodies (bnAbs) against conserved sites that not only protect against drifted strains but also various subtypes.

In recent years, several attempts have been made to redirect the immune response against the conserved HA stem, following a reverse vaccinology strategy. Starting from structural information of antibody-antigen complexes, 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). With progress in experimental techniques to isolate and analyze B cells as well as the structural characterization of isolated antibodies and their cognate antigens, valuable insights have been gained that facilitate structure-based immunogen design. The reverse vaccinology approach has recently been successfully applied to design novel epitope-focused immunogens for RSV. The obtained results demonstrated that the immune response could be shifted towards subdominant antigenic sites, eliciting neutralizing Abs specific for the targeted epitopes in mice and non-human primates (Correia et al. 2014; Sesterhenn et al. 2019; 2020).

For influenza, several 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. So far, immunogen design approaches for influenza have been mainly focused on removing the immunodominant HA head and stabilizing the stem domain in isolation to overcome the immunodominance of the head. Two promising candidates were reported by Impagliazzo et al. (Impagliazzo et al. 2015) and Yassine et al. (Yassine et al. 2015) demonstrating that antibodies can be elicited against immunosubdominant epitopes on the HA stem. While these headless designs showed great promise in mounting a broader immune response compared to immunizations with full-length HA, the breadth has been limited to subtypes within groups but low cross-group reactivity was observed (Yassine et al. 2015; Impagliazzo et al. 2015; Steel et al. 2010; Wohlbold et al. 2015).

There is a need for further approaches for influenza vaccination.

SUMMARY OF THE INVENTION

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.

Advantages of the embodiments of the invention relative to the prior art may be one or more of the following:

I. Elicitation of bnAbs (e.g. a broad range of bnAbs)

II. Elicitation of bnAbs which are cross- reactive to H1 and H3 influenza strains

III. Elicitation of bnAbs which are cross- reactive to H1, H3 and H5 influenza strains

IV. Diversion of immune response from HA head region to HA stem region

V. Improved immune response

VI. Improved protection against diverse influenza subtypes

VII. Prevention of influenza infection

VIII. Reduction in influenza infection severity

Scaffold proteins

In one aspect there is provided 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.

ApoE-based scaffold proteins

In one aspect there is provided 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. In one aspect there is provided a method of eliciting an immune response in a subject, the method 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.

In one aspect there is provided 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.

In one aspect there is provided a scaffold protein for use in the treatment or prevention of influenza infection 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.

In one aspect there is provided the use of a scaffold protein in the manufacture of a medicament for the treatment or prevention of influenza infection 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.

Acylhydrolase-based scaffold proteins

In one aspect there is provided 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.

In one aspect there is provided a method of eliciting an immune response in a subject, the method 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.

In one aspect there is provided 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.

In one aspect there is provided a scaffold protein for use in the treatment or prevention of influenza infection 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.

In one aspect there is provided the use of a scaffold protein in the manufacture of a medicament for the treatment or prevention of influenza infection 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.

Further aspects of the invention will be evident from the detailed description below.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 Polypeptide sequence of human ApoE scaffold region A

SEQ ID NO: 2 Polypeptide sequence of human ApoE scaffold region B

SEQ ID NO: 3 Polypeptide sequence of human ApoE scaffold region C

SEQ ID NO: 4 Polypeptide sequence of human ApoE scaffold region D

SEQ ID NO: 5 Polypeptide sequence of human ApoE scaffold region E

SEQ ID NO: 6 Polypeptide sequence of murine ApoE scaffold region A

SEQ ID NO: 7 Polypeptide sequence of murine ApoE scaffold region B

SEQ ID NO: 8 Polypeptide sequence of murine ApoE scaffold region C

SEQ ID NO: 9 Polypeptide sequence of murine ApoE scaffold region D

SEQ ID NO: 10 Polypeptide sequence of murine ApoE scaffold region E

SEQ ID NO: 11 Polypeptide sequence of human ApoE scaffold.

SEQ ID NO: 12 Polypeptide sequence of murine ApoE scaffold.

SEQ ID NO: 13 Polypeptide sequence of native (or wild type, ‘WT’) murine ApoE

SEQ ID NO: 14 Polypeptide sequence of acylhydrolase scaffold region A

SEQ ID NO: 15 Polypeptide sequence of acylhydrolase scaffold region B

SEQ ID NO: 16 Polypeptide sequence of acylhydrolase scaffold region C SEQ ID NO: 17 Polypeptide sequence of acylhydrolase scaffold region D

SEQ ID NO: 18 Polypeptide sequence of acylhydrolase scaffold

SEQ ID NO: 19 Polypeptide sequence of human ApoE N-terminal extension

SEQ ID NO: 20 Polypeptide sequence of human ApoE irrelevant region

SEQ ID NO: 21 Polypeptide sequence of human ApoE scaffold comprising extra N- terminal sequences

SEQ ID NO: 22 Polypeptide sequence of a native (or wild type, ‘WT’) acylhydrolase

SEQ ID NO: 23 Polypeptide sequence of murine ApoE irrelevant region

SEQ ID NO: 24 Polypeptide sequence of linker connecting scaffold protein and ferritin

SEQ ID NO: 25 Polypeptide sequence of m2e T cell epitope

SEQ ID NO: 26 Polypeptide sequence of linker connecting m2e to scaffold protein

SEQ ID NO: 27 Polypeptide sequence of a ferritin protein nanoparticle

SEQ ID NO: 28 Polypeptide sequence formula of human ApoE scaffold region B

SEQ ID NO: 29 Polypeptide sequence formula of human ApoE scaffold region D

SEQ ID NO: 30 Polypeptide sequence formula of murine ApoE scaffold region B

SEQ ID NO: 31 Polypeptide sequence formula of murine ApoE scaffold region D

SEQ ID NO: 32 Polypeptide sequence formula of acylhydrolase scaffold region B

SEQ ID NO: 33 Polypeptide sequence formula of acylhydrolase scaffold region C

SEQ ID NO: 34 Polypeptide sequence of FI6-focused design_01

SEQ ID NO: 35 Polypeptide sequence of FI6-focused design_02

SEQ ID NO: 36 Polypeptide sequence of FI6-focused design_03

SEQ ID NO: 37 Polypeptide sequence of FI6-focused design_04

SEQ ID NO: 38 Polypeptide sequence of stem-epitope design_01

SEQ ID NO: 39 Polypeptide sequence of murine ApoE irrelevant region

BRIEF DESCRIPTION OF THE FIGURES

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). For the FI6- focused design (top), 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. Screening of a combinatorial library and site-saturation mutagenesis (SSM) revealed several mutations in the hydrophobic pocket and a loop connecting the epitope helix to the scaffold that improved binding affinity to FI6, resulting in an FI6-focused immunogen. Since structural templates were rare, the epitope was simplified to its core structure, the a-helix. A scaffold was identified in the PDB, and the helix was transplanted onto the scaffold (bottom). The remaining antigenic site was designed in silico to increase the epitope mimicry. Mimicry was evaluated based on the resulting protein surfaces. The stem-epitope mimetic was compared to H1, H3, and H5, demonstrating high epitope mimicry for H1 and H3, and moderate mimicry when compared to H5.

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. A. Circular dichroism (CD) spectra at indicated temperatures. B. Size exclusion chromatography coupled to multi-angle light scattering. C. Thermal denaturation curves. Designed proteins were melted from 20 to 90 °C by CD. The melting temperature (Tm) was determined by the change of ellipticity at the global curve minimum (208 nm).

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. B. Breadth of interaction of both designs displayed on ferritin nanoparticles was assessed, demonstrating the highly focused response of FI6-focused_04 to FI6 Fab, while no interaction was observed with other stem-specific antibodies (CR9114, MEDI-8852). However, stem- epitope_01 exhibited strong binding to all three tested antibodies, suggesting a high mimicry of the designed epitope mimetic and the native antigenic site. 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. B. High epitope mimicry of X-ray structure and H1. C. Structural comparison of the stem-epitope_01 computational model to the solved X-ray structure in complex with CR9114. The computational model showed excellent structural agreement with the solved X-ray structure (RMSD 1.6 A), confirming the recovery of modelled rotamer positions. D. Polar interactions of the stemepitope design in complex with CR9114 were compared to a H5-CR9114 complex, showing full recovery of interactions involving the epitope helix and partial recovery for the remaining epitope. E. The epitope region was well mimicked in the structure when comparing surface shape similarity to the corresponding H5 stem epitope.

Fig.7: Antibody pull down from human PBMCs. A. Peripheral blood mononuclear cells (PBMCs) from two recently vaccinated donors were isolated and memory 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. B. Detailed analysis of HVDJ rearrangement in the two donors. Antibodies sorted from donor 63 mainly originated from VH3-30, rearranged with either DH5-12 or DH3-9. Antibodies from donor 31 stemmed from VH3-30 or VH3-23 and showed similar VDJ rearrangements as known neutralizing antibodies. C. Comparing binding affinities to different HAs of sorted antibodies with FI6. The 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. A. Immunization scheme and immunogens used in this study (blue = FI6_focused_04, orange = stem-epitope_01 , red = H1 1999 NC). 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. B. Antibody titers measured against the respective design (blue or orange) and WT scaffold (grey) in sera from final bleeding (day 56 after first injection). Plates were coated with monomeric designs. C. Cross-reactive titers against HA. Sera from D56 tested against H1 1999 NC, H3 1968 HK, H5 2004 VN and H1 1999 NC stem HAs. Plates were coated with trimeric HAs. FI6-focused design is the left bar of each pair and stem-epitope design is the right bar of each pair. D. Antibody titers of designs against H1 and H3 over time. FI6-focused designs are the lower curves and stem-epitope designs are the upper curves. E. Epitope focusing on H1 HA. Binding curves against H1 1999 NC and H1 1999 NC stem KO mutant. Plates were coated with trimeric HAs. Upper curves are H1 1999 NC and lower curves are H1 1999 NC delta stem. F. Virus binding. Binding curves against H3 1968 HK (top curve FI6, next curve down 3x stem-epitope particle, next curve down 3x H1 1999 NC and bottom curve irrelevant sera) and pH1 2009 CA UV-inactivated virus (top curve FI6, next curve down 3x H1 1999 NC, next curve down 3x stem-epitope particle and bottom curve irrelevant sera). G. Cross-reactivity to H1 and H3 of stem-mimetic positive mouse B cells in the draining lymph nodes after 2 and 3 immunizations with stem-mimetic_01 particle.

Fig. 9: Sorting and sequencing of group 1 and group 2 cross- reactive mouse B cells. B cell sorting from draining lymph nodes after three injections with stem-epitope design particle. Balb/c mice (n = 3) were immunized three times at a three-week interval with stem-epitope design particle. Two weeks after the third injection mice were sacrificed and iliac and inguinal lymph nodes were extracted. A. Class switched germinal center and memory B cells were sorted for H1/H3 cross-reactivity. B. Heavy and light chain variable germline gene frequencies in the H1 and H3 double positive population. Total barcode frequencies of all sorted cells are shown.

Fig. 10: Immune protection mediated against lethal challenge with X31 influenza virus in mice (first challenge study). Balb/c mice (n = 5 per group) were vaccinated three times at three- week intervals. Animals were primed with either H1_NC99 trimer or stem-epitope particle and boosted either with stem-epitope particle or adjuvant alone. 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). Balb/c mice (n = 10 per group) were vaccinated three times at three week intervals as described in the Examples. Animals were challenged with 2 LD50 of H3 1968 HK X31 influenza virus three weeks after their third immunization and monitored for loss of body weight over 14 days. If an animal lost more than 25% body weight it was sacrificed.

Fig. 12: Protection mediated by stem-mimetic. A. Antibody-dependent cellular cytotoxicity (ADCC) mediated by design-induced antibodies against three different influenza viruses. Prime immunizations with stem-epitope particle (squares) or H1 (NC99) (triangles, both ways up) were adjuvanted, but H3 (HK68) prime injections (hexagons: top curve in H1PR8 graph, second curve down in H1 CA09 and H3 X31 graphs, and diamonds: bottom curves)) were not. Boost injections with stem-mimetic particle were also adjuvanted in contrast to PBS boost. For the positive control (circles: third curve down in H1 PR8 graph, top curves in H1 CA09 and H3 X31 graphs) a monoclonal antibody was used (for H1 PR8 and CA09 the mouse E7 IgG and for H3 X31 human FI6 IgG). Relative luminescence units (RLU) were determined for all groups and the fold induction over background (negative control) was calculated. The mean values + SEM for all mice in the different groups are shown (n = 5, except 3 x stem-epitope particle (squares) n = 10). B. Percentage of mice per group mediating ADCC activity above background against the different viruses.

DETAILED DESCRIPTION OF THE INVENTION

Influenza hemagglutinin

Influenza hemagglutinin (HA) 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.

Detailed below are the scaffold proteins utilised in the present invention and their amino acid sequences. 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.

In certain embodiments 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.

For the purposes of comparing two closely-related polypeptide sequences, 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). For the purposes of comparing two closely-related polynucleotide sequences, 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).

Alternatively, for the purposes of comparing a first, reference polypeptide sequence to a second, comparison polypeptide sequence, 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).

Using the three letter and one letter codes, 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). Where a residue may be aspartic acid or asparagine, the symbols Asx or B may be used. Where 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.

Table 1

Suitably, 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. Suitably 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. all contact points between the antigen and the antibody are the same). For example, 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.

Further, with the aid of such characterization methods, it is also possible to characterize antibodies which bind essentially the same epitope by recognizing some but not all of the identical contact points. Specifically, such antibodies may share a sufficient number of identical contact points in a specified antigenic region to deliver a broadly equivalent technical effect and/or equivalent antigen interaction selectivity. Additionally, in some instances whereby antibodies recognize essentially the same epitope and confer a broadly equivalent technical effect and/or interaction selectivity, it can also be useful to define the epitope binding footprint by the totality of antigen contacts inclusive of the most N-terminal antigen contact point through to the most C-terminal antigen contact point.

Epitopes found on protein targets may be defined as “linear epitopes” or “conformational epitopes”. 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.

Scaffold proteins

In one embodiment there is provided 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. Suitably 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. In one embodiment, 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.

Suitably the influenza hemagglutinin stem epitope is the hydrophobic groove of the influenza hemagglutinin stem protein. In one embodiment, the influenza hemagglutinin stem epitope is the epitope bound by the FI6 antibody.

Suitably the influenza hemagglutinin stem epitope comprises a part of helix A of hemagglutinin, more suitably all of helix A of hemagglutinin.

In one embodiment, the scaffold protein does not form part of a nanolipoprotein.

In one embodiment, 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

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 (ApoE) 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. Meanwhile, the C-terminal domain (residues 206-299) 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.

In one embodiment 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). However, in one embodiment 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 and 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.

In certain embodiments, 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.

In one embodiment the scaffold protein consists of the N-terminal fragment of apolipoprotein E. In a further embodiment, the N-terminal fragment of apolipoprotein E consists of the formula A- B-C-D-E. In a yet further embodiment, 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. Human ApoE

In one embodiment the scaffold protein is an N-terminal fragment derived from human ApoE.

Region A

Suitably 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.

Suitably A consists of 40 to 60 residues, such as 45 to 55 residues, such as 50 residues.

Region B

Certain residues may be present in B to form the epitope.

Suitably 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.

Suitably 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.

Suitably 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 X₃ 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

Suitably 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.

Suitably C consists of 10 to 20 residues, such as 13 to 17 residues, such as 14 residues.

Region D

Certain residues may be present in D to form the epitope.

Suitably 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 conservative substitution thereof, most suitably I; and/or the residue of D corresponding to residue 12 of SEQ ID NO: 4 is T or a conservative substitution thereof, most suitably T; and/or the residue of D corresponding to residue 15 of SEQ ID NO: 4 is V or a conservative substitution thereof, most suitably V; and/or the residue of D corresponding to residue 16 of SEQ ID NO: 4 is N or a conservative substitution thereof, most suitably N; and/or the residue of D corresponding to residue 19 of SEQ ID NO: 4 is A or a conservative substitution thereof, most suitably A.

Suitably 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.

Suitably D consists of 15 to 25 residues, such as 18 to 22 residues, such as 19 residues. Suitably 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 X₃ 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.

Region E

Suitably 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.

Suitably E consists of 53 to 65 residues, such as 55 to 59 residues, such as 57 residues.

The whole fragment

An exemplary fragment is provided in SEQ ID NO: 11. Suitably, 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.

The polypeptide sequence of this exemplary fragment is set out with annotation as follows, wherein underlined residues are mutations introduced to form the epitope.

Region A

TEWQSGQRWELALGRFWDYLRWVQTLSEQVQ

EELLSSQVTQELRALMDET (SEQ ID NO: 1)

Region B

LKELIAYMK (SEQ ID NO: 2) Region C

ELEEQLTPVAEETR (SEQ ID NO: 3)

Region D

LKLTQNLIDAITRLVNDMA (SEQ ID NO: 4)

Region E DVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGA (SEQ ID NO: 5)

The sequence is also provided full length with hyphens separating sequences corresponding to regions A, B, C, D and E:

TEWQSGQRWELALGRFWDYLRWVQTLSEQVQ

EELLSSQVTQELRALMDET-LKELIAYMK-ELEEQLTPVAEETR-LKLTQNLIDAIT RLVNDMA-DVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDA DDLQKRLAVYQAGA (SEQ ID NO: 21)

An N-terminal extension (KVEQAVETEPE, SEQ ID NO: 19) and following region (PELRQQ, SEQ ID NO: 20) present in native ApoE are considered to be irrelevant to the immunogenicity and stability of the scaffold.

In one embodiment 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 and E consists of a sequence sharing at least 40% identity with SEQ ID NO: 5.

Murine ApoE

In one embodiment the scaffold protein is an N-terminal fragment derived from murine ApoE.

Region A

Suitably 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.

Suitably 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.

Suitably 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.

Suitably 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 X₃ 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.

Region C

Suitably 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.

Region D

Certain residues may be present in D to form the epitope.

Suitably 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 conservative substitution thereof, most suitably I; and/or the residue of D corresponding to residue 12 of SEQ ID NO: 9 is T or a conservative substitution thereof, most suitably T; and/or the residue of D corresponding to residue 13 of SEQ ID NO: 9 is N or a conservative substitution thereof, most suitably N; and/or the residue of D corresponding to residue 15 of SEQ ID NO: 9 is V or a conservative substitution thereof, most suitably V; and/or the residue of D corresponding to residue 16 of SEQ ID NO: 9 is N or a conservative substitution thereof, most suitably N; and/or the residue of D corresponding to residue 19 of SEQ ID NO: 9 is A or a conservative substitution thereof, most suitably A; and/or the residue of D corresponding to residue 20 of SEQ ID NO: 9 is E or a conservative substitution thereof, most suitably E.

Suitably 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.

Suitably 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 X₃ 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.

Region E

Certain residues may be present in E to stabilise the epitope.

Suitably the residue of E corresponding to residue 45 of SEQ ID NO: 10 is V or a conservative substitution thereof, most suitably V; and/or the residue of E corresponding to residue 48 of SEQ ID NO: 10 is A or a conservative substitution thereof, most suitably A. Suitably 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 whole fragment

An exemplary fragment is provided in SEQ ID NO: 12. Suitably, 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.

Region A

LEWQSNQPWEQALNRFWDYLRWVQTLSDQVQEELQSSQVTQELTALMEDT (SEQ ID NO: 6)

Region B

LTEAIAYM (SEQ ID NO: 7)

Region C

KELEEQLGPVAEETR (SEQ ID NO: 8)

Region D

LKLTQNVIDAITNLVNDMAE (SEQ ID NO: 9)

Region E LRNRLGQYRNEVHTMLGQSTEEIRARLSTHLRKMRKRLMRDAEDVQKALAVYKAGA (SEQ ID NO: 10)

LEWQSNQPWEQALNRFWDYLRWVQTLSDQVQEELQSSQVTQELTALMEDT-LTEAIAYM; KELEEQLGPVAEETR-LKLTQNVIDAITNLVNDMAE- LRNRLGQYRNEVHTMLGQSTEEIRARLSTHLRKMRKRLMRDAEDVQKALAVYKAGA An N-terminal region (PEVTDQ, SEQ ID NO: 39) present in native murine ApoE is considered to be irrelevant to the immunogenicity and stability of the scaffold.

In one embodiment, the ApoE (such as human or mouse ApoE) scaffold does not form part of a nanolipoprotein structure.

Acylhydrolase protein

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. In one embodiment 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). However, in one embodiment 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.

Region A

Certain residues may be present in A to stabilise the scaffold.

Suitably 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 conservative substitution thereof, most suitably E; and/or the residue of A corresponding to residue 73 of SEQ ID NO: 14 is E or a conservative substitution thereof, most suitably E; and/or the residue of A corresponding to residue 80 of SEQ ID NO: 14 is K or a conservative substitution thereof, most suitably K; and/or the residue of A corresponding to residue 83 of SEQ ID NO: 14 is V or a conservative substitution thereof, most suitably V; and/or the residue of A corresponding to residue 87 of SEQ ID NO: 14 is M or a conservative substitution thereof, most suitably M; and/or the residue of A corresponding to residue 99 of SEQ ID NO: 14 is P or a conservative substitution thereof, most suitably P; and/or the residue of A corresponding to residue 104 of SEQ ID NO: 14 is Q or a conservative substitution thereof, most suitably Q; and/or the residue of A corresponding to residue 111 of SEQ ID NO: 14 is K or a conservative substitution thereof, most suitably K; and/or the residue of A corresponding to residue 119 of SEQ ID NO: 14 is A or a conservative substitution thereof, most suitably A.

Suitably 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.

Region B

Certain residues may be present in B to stabilise the scaffold and/or the epitope.

Suitably 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.

Suitably 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.

Suitably 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.

Region C

Certain residues may be present in C to form the epitope.

Suitably 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 conservative substitution thereof, most suitably N; and/or the residue of C corresponding to residue 18 of SEQ ID NO: 16 is I or a conservative substitution thereof, most suitably I; and/or the residue of C corresponding to residue 24 of SEQ ID NO: 16 is F or a conservative substitution thereof, most suitably F; and/or the residue of C corresponding to residue 28 of SEQ ID NO: 16 is F or a conservative substitution thereof, most suitably F; and/or the residue of C corresponding to residue 29 of SEQ ID NO: 16 is V or a conservative substitution thereof, most suitably V; and/or the residue of C corresponding to residue 32 of SEQ ID NO: 16 is A or a conservative substitution thereof, most suitably A; and/or the residue of C corresponding to residue 33 of SEQ ID NO: 16 is Q or a conservative substitution thereof, most suitably Q; and/or the residue of C corresponding to residue 34 of SEQ ID NO: 16 is S or a conservative substitution thereof, most suitably S; and/or the residue of C corresponding to residue 35 of SEQ ID NO: 16 is P or a conservative substitution thereof, most suitably P; and/or the residue of C corresponding to residue 37 of SEQ ID NO: 16 is G or a conservative substitution thereof, most suitably G; and/or the residue of C corresponding to residue 38 of SEQ ID NO: 16 is D or a conservative substitution thereof, most suitably D. Suitably 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. More suitably C comprises or consists of SEQ ID NO: 16.

Suitably 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 X₃ 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 F, W and Y; wherein Xg is selected from the group consisting of A, G, I, L, M and V; wherein X is selected from the group consisting of D and E; wherein Xu is selected from the group consisting of C, N, P, Q, S and T; wherein Xi2 is selected from the group consisting of H, K and R; wherein X13 is selected from the group consisting of A, G, I, L, M and V; wherein Xi₄ is selected from the group consisting of C, N, P, Q, S and T; wherein X is selected from the group consisting of A, G, I, L, M and V; wherein X is selected from the group consisting of D and E; wherein X17 is selected from the group consisting of F, W and Y; wherein X is selected from the group consisting of D and E; wherein X is selected from the group consisting of A, G, I, L, M and V; and wherein X2o is selected from the group consisting of C, N, P, Q, S and T.

Region D

Certain residues may be present in D to stabilise the scaffold.

Suitably the residue of D corresponding to residue 13 of SEQ ID NO: 17 is N or a conservative substitution thereof, most suitably N. Suitably 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.

The whole acylhydrolase protein

An exemplary acylhydrolase protein is provided in SEQ ID NO: 18. Suitably, 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. 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: 18. Suitably the acylhydrolase protein comprises or consists of SEQ ID NO: 18.

The 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.

SDAEKQDPANKKRYAEANKELVRKGKQKNRVVFMGNSITEGWVANDPAFFEDNGYVGRGIS GQTSSHMLERFEEDVIKLKPAWVIMAGTNDIAENAGPYNEEQTFGNIVKMVELARAAKIKVILT SVLP-HAAAPWN ESQK-EATQAIIN LNTRIINYAI EN KI PFVDYFVEMAQSPNGD- LNSSYTRDGVHPNLEGYKVMEALIKKAIDKVL (SEQ ID NO: 18)

In certain embodiments, 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.

In one embodiment 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 and D consists of a sequence sharing at least 50% identity with SEQ ID NO: 17.

In a further embodiment the acylhydrolase protein consists of the formula A-B-C-D. In another embodiment the scaffold protein consists of the acylhydrolase protein.

The scaffold protein may be provided ‘naked’, i.e. not bound to other materials. Alternatively, the scaffold protein may be provided bound to one or more further agents. In a particular embodiment, 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).

Ideally 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.

Suitably the nanoparticle and the scaffold protein are connected by a linker. Suitably the linker consists of 1 to 40 residues, such as 10 to 30 residues. In one embodiment the linker comprises or consists of the polypeptide sequence of SEQ ID NO: 24.

Constructs

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). Such a construct may be structured as set out in Fig. 5.

Suitably in one embodiment the protein comprises an m2e T cell epitope sequence, such as at the N-terminus of the scaffold protein. In one embodiment 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. Consequently, 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.

Influenza virus

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. Finally, the term strain refers to viruses within a subtype that differ from one another in that they have small, genetic variations in their genome.

Antibodies

In one embodiment 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.

Several 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). Suitably the 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.

Upon administration to a subject, the scaffold proteins of the invention elicit antibodies which bind to influenza HA stem. Suitably, the antibodies bind to group 1 and/or group 2 HAs. More suitably the antibodies bind to group 1 and group 2 HAs. For example, 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’).

Suitably 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), 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. Alternatively, 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.

Suitably 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). In a further embodiment, the 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. In a yet further embodiment, 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. 50 nM), less than 4.0 x 10'⁸ M (i.e. 40 nM), less than 3.0 x 10'⁸ M (i.e. 30 nM) or less than 2.0 x 10'⁸ M (i.e. 20 nM). In further embodiments, 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. 2 nM) or less, such as 1.0 x 10'⁹ M (i.e. 1 nM) or less, such as 0.5 x 10'⁹ M (i.e. 0.5 nM) or less, such as 0.3 x 10'⁹ M (i.e. 0.3 nM) or less. 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.

Suitable assays for investigating the efficacy of antibodies in targeting and binding to the stem region are described in the examples, such as ‘ELISA’ and ‘Whole Virus ELISA’ recited under ‘Methods’ below.

Suitably administration of 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.

Adjuvants

In some embodiments the scaffold protein may be administered with an adjuvant. In one embodiment 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 a branched, unsaturated terpenoid ([(CH3)2C[=CHCH2CH2C(CH3)]2=CHCH2-]2; C30H50; 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS Registry Number 7683-64-9). Squalene is readily available from commercial sources or 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).

Any of the a, p, y, 5, E and/or 5, tocopherols can be used, but a-tocopherol (also referred to herein as alpha-tocopherol) is typically used. 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. In some embodiments 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. Suitably 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.

Uniformity of droplet sizes is desirable. 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. Suitably 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, as used herein, 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 Accusizer™ and Nicomp™ series of instruments available from Particle Sizing Systems (Santa Barbara, USA), the Zetasizer™ 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. the intensity-weighted mean hydrodynamic size of the ensemble collection of droplets measured by DLS. The Z-average is derived from cumulants analysis of the measured correlation curve, wherein a single particle size (droplet diameter) is assumed and a single exponential fit is applied to the autocorrelation function. Thus, references herein to average droplet size should be taken as an intensity-weighted average, and ideally the Z-average. Pdl values are easily provided by the same instrumentation which measures average diameter.

In order to maintain a stable submicron emulsion, one or more emulsifying agents (i.e. surfactants) 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. When two or more surfactants are blended, 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. 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 DOWFAX™, Pluronic™ (e.g. F68, F127 or L121 grades) or Synperonic™ 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 (IGEPAL CA-630/NP-40); 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 (Eumulgin™ 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).

Many examples of pharmaceutically acceptable surfactants are known in the art e.g. see Handbook of Pharmaceutical Excipients 6th edition, 2009. Methods for selecting and optimising the choice of surfactant used in a squalene emulsion adjuvant are illustrated in Klucker, 2012. In general, the 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. Especially of interest are 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.

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

If tocopherol is present, the weight ratio of squalene to tocopherol may be 20 or less, such as 10 or less. Suitably the weight ratio of squalene to tocopherol is 0.1 or more. Typically the weight ratio of squalene to tocopherol is 0.1 to 10, especially 0.2 to 5, in particular 0.3 to 3, such as 0.4 to 2. Suitably, 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.

If surfactant is present, typically the weight ratio of squalene to surfactant is 0.73 to 6.6, especially 1 to 5, in particular 1.2 to 4. Suitably, 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. For example, 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.

If tocopherol is present, 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. For example, 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.

If surfactant is present, the amount of surfactant in a single dose, such as a human dose, of squalene emulsion adjuvant is typically at least 0.4 mg. Generally, the amount of surfactant in a single dose, such as a human dose, of 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. For example, 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.

In certain embodiments 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).

High pressure homogenization (HPH or microfluidisation) 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). Briefly, 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. Formulation

In one embodiment, there is provided 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’). Alternatively 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’). When 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. Suitably the scaffold protein and squalene emulsion adjuvant are administered via the same route, in particular intramuscularly.

When administered as separate formulations, the scaffold protein and squalene emulsion adjuvant are desirably administered to locations with sufficient spatial proximity such that the adjuvant effect is adequately maintained. For example, 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.

Suitably the scaffold protein and squalene emulsion adjuvant are administered intramuscularly to the same muscle. In certain embodiments, 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.

When administered as separate formulations, the scaffold protein and squalene emulsion adjuvant are desirably administered with sufficient temporal proximity such that the adjuvant effect is adequately maintained. For example, 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.

When administered as separate formulations, scaffold protein and squalene emulsion adjuvant may be administered within 12 hours. Suitably 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.

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.

Desirably, the scaffold protein and squalene emulsion adjuvant are administered without intentional delay (accounting for the practicalities of multiple administrations).

In addition to co-formulated or separately formulated presentations of the scaffold protein and squalene emulsion adjuvant for direct administration, the scaffold protein and squalene emulsion adjuvant may initially be provided in various forms which facilitate manufacture, storage and distribution. For example, 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). Independent of whether the scaffold protein and squalene emulsion are co-formulated at administration, they 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. Typically 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).

For parenteral administration, 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. Suitably 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. As mentioned above, the requirement for buffer and/or tonicity modifying agents will depend on the on both the contents of the container being reconstituted and the subsequent use of the reconstituted contents. 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.

Suitably, 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.

Administration

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.

Administration of the scaffold protein and squalene emulsion adjuvant may therefore be part of a multi-dose administration regime. For example, 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. Consequently, 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. Alternatively, 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).

In one embodiment the protein is administered as part of a homologous prime-boost regime (such as three administrations of the protein). In an alternative embodiment, 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.

In one embodiment, 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.

Suitably 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. Alternatively a suitable single dose of scaffold protein is 10 to 30 ug, especially 15 to 25 ug, in particular about 20 ug. Alternatively, a single dose of scaffold protein is suitably 1 to 3 ug, especially 1.5 to 2.5 ug, in particular about 2 ug. Treatment

In one embodiment, 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.

In a further aspect, there is provided 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. In one embodiment the invention, the subject is human.

The subject to be treated may be of any age. In one embodiment the subject is a human infant (up to 12 months of age). In one embodiment the subject is a human child (less than 18 years of age). In one embodiment the subject is an adult human (aged 18-59). In one embodiment 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). In some embodiments, the polynucleotide sequences will be codon optimised for a particular recipient host cell using standard methodologies. For example, 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

Throughout the specification, including the claims, where the context permits, the term “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). Thus a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

The term “about” 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.

As used herein, the singular forms “a,” “an” and “the” include plural references unless the content clearly dictates otherwise.

Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus 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.

Clauses

Clauses further illustrating the invention are as follows:

1. 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.

2. A method of eliciting an immune response in a subject, the method 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 according to clause 1 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. The method of eliciting an immune response in a subject according to clause 2, the method 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. 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. 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. The use of 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. The scaffold protein, method or use according to any one of clauses 1 to 10, wherein A consists of a sequence sharing at least 40% identity with SEQ ID NO: 1 , wherein B consists of a sequence sharing at least 60% identity with SEQ ID NO: 2, wherein C consists of a sequence sharing at least 40% identity with SEQ ID NO: 3, wherein D consists of a sequence sharing at least 60% identity with SEQ ID NO: 4 and wherein E consists of a sequence sharing at least 40% identity with SEQ ID NO: 5. The scaffold protein, method or use according to any one of clauses 1 to 11, wherein 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. The scaffold protein, method or use according to any one of clauses 1 to 12, wherein B comprises or consists of a sequence sharing at least 70%, such as at least 80% identity with SEQ ID NO: 2. The scaffold protein, method or use according to any one of clauses 1 to 13, wherein 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. The scaffold protein, method or use according to any one of clauses 1 to 14, wherein 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. The scaffold protein, method or use according to any one of clauses 1 to 15, wherein 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. The scaffold protein, method or use according to any one of clauses 1 to 16, wherein A comprises or consists of SEQ ID NO: 1. The scaffold protein, method or use according to any one of clauses 1 to 17, wherein 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 X₃ 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. The scaffold protein, method or use according to any one of clauses 1 to 18, wherein B comprises or consists of SEQ ID NO: 2. The scaffold protein, method or use according to any one of clauses 1 to 19, wherein C comprises or consists of SEQ ID NO: 3. The scaffold protein, method or use according to any one of clauses 1 to 20, wherein 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 X₃ 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. The scaffold protein, method or use according to any one of clauses 1 to 21 , wherein D comprises or consists of SEQ ID NO: 4. The scaffold protein, method or use according to any one of clauses 1 to 22, wherein E comprises or consists of SEQ ID NO: 5. The scaffold protein, method or use according to any one of clauses 1 to 23, wherein A comprises consists of SEQ ID NO: 1 or a sequence comprising conservative substitutions to SEQ ID NO 1 , wherein B comprises or consists of SEQ ID NO: 28, wherein C comprises or consists of SEQ ID NO: 3 or a sequence comprising conservative substitutions to SEQ ID NO: 3, wherein D comprises of consists of SEQ ID NO: 29 and wherein E comprises or consists of SEQ ID NO: 5 or a sequence comprising conservative substitutions to SEQ ID NO: 5. The scaffold protein, method or use according to any one of clauses 1 to 24, wherein A consists of SEQ ID NO: 1 , wherein B consists of SEQ ID NO: 2, wherein C consists of SEQ ID NO: 3, wherein D consists of SEQ ID NO: 4 and wherein E consists of SEQ ID NO: 5. The scaffold protein, method or use according to any one of clauses 1 to 25, wherein the scaffold protein comprises 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: 11. The scaffold protein, method or use according to clause 26, wherein the scaffold protein comprises or consists of SEQ ID NO: 11 . The scaffold protein, method or use according to any one of clauses 1 to 27, wherein the residue of B corresponding to residue 1 of SEQ ID NO: 2 is L or a conservative substitution thereof; and/or the residue of B corresponding to residue 4 of SEQ ID NO: 2 is A or a conservative substitution thereof; and/or the residue of B corresponding to residue 5 of SEQ ID NO: 2 is I or a conservative substitution thereof; and/or the residue of B corresponding to residue 8 of SEQ ID NO: 2 is M or a conservative substitution thereof; and/or the residue of B corresponding to residue 9 of SEQ ID NO: 2 is K or a conservative substitution thereof. The scaffold protein, method or use according to clause 28, wherein the residue of B corresponding to residue 1 of SEQ ID NO: 2 is L or a conservative substitution thereof; and the residue of B corresponding to residue 4 of SEQ ID NO: 2 is A or a conservative substitution thereof; and the residue of B corresponding to residue 5 of SEQ ID NO: 2 is I or a conservative substitution thereof; and the residue of B corresponding to residue 8 of SEQ ID NO: 2 is M or a conservative substitution thereof; and the residue of B corresponding to residue 9 of SEQ ID NO: 2 is K or a conservative substitution thereof. The scaffold protein, method or use according to clause 28, wherein the residue of B corresponding to residue 1 of SEQ ID NO: 2 is L; and/or the residue of B corresponding to residue 4 of SEQ ID NO: 2 is A; and/or the residue of B corresponding to residue 5 of SEQ ID NO: 2 is I; and/or the residue of B corresponding to residue 8 of SEQ ID NO: 2 is M; and/or the residue of B corresponding to residue 9 of SEQ ID NO: 2 is K. The scaffold protein, method or use according to clause 30, wherein the residue of B corresponding to residue 1 of SEQ ID NO: 2 is L; and the residue of B corresponding to residue 4 of SEQ ID NO: 2 is A; and the residue of B corresponding to residue 5 of SEQ ID NO: 2 is I; and the residue of B corresponding to residue 8 of SEQ ID NO: 2 is M; and the residue of B corresponding to residue 9 of SEQ ID NO: 2 is K. The scaffold protein, method of use according to any one of clauses 1 to 31 , wherein the residue of D corresponding to residue 1 of SEQ ID NO: 4 is L or a conservative substitution thereof; and/or the residue of D corresponding to residue 2 of SEQ ID NO: 4 is K or a conservative substitution thereof; and/or the residue of D corresponding to residue 4 of SEQ ID NO: 4 is T or a conservative substitution thereof; and/or the residue of D corresponding to residue 5 of SEQ ID NO: 4 is Q or a conservative substitution thereof; and/or the residue of D corresponding to residue 6 of SEQ ID NO: 4 is N or a conservative substitution thereof; and/or the residue of D corresponding to residue 8 of SEQ ID NO: 4 is I or a conservative substitution thereof; and/or the residue of D corresponding to residue 9 of SEQ ID NO: 4 is D or a conservative substitution thereof,; and/or the residue of D corresponding to residue 11 of SEQ ID NO: 4 is I or a conservative substitution thereof; and/or the residue of D corresponding to residue 12 of SEQ ID NO: 4 is T or a conservative substitution thereof; and/or the residue of D corresponding to residue 15 of SEQ ID NO: 4 is V or a conservative substitution thereof; and/or the residue of D corresponding to residue 16 of SEQ ID NO: 4 is N or a conservative substitution thereof; and/or the residue of D corresponding to residue 19 of SEQ ID NO: 4 is A or a conservative substitution thereof. 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 or a conservative substitution thereof; and the residue of D corresponding to residue 2 of SEQ ID NO: 4 is K or a conservative substitution thereof; and the residue of D corresponding to residue 4 of SEQ ID NO: 4 is T or a conservative substitution thereof; and the residue of D corresponding to residue 5 of SEQ ID NO: 4 is Q or a conservative substitution thereof; and the residue of D corresponding to residue 6 of SEQ ID NO: 4 is N or a conservative substitution thereof; and the residue of D corresponding to residue 8 of SEQ ID NO: 4 is I or a conservative substitution thereof; and the residue of D corresponding to residue 9 of SEQ ID NO: 4 is D or a conservative substitution thereof; and the residue of D corresponding to residue 11 of SEQ ID NO: 4 is I or a conservative substitution thereof; and the residue of D corresponding to residue 12 of SEQ ID NO: 4 is T or a conservative substitution thereof; and the residue of D corresponding to residue 15 of SEQ ID NO: 4 is V or a conservative substitution thereof; and the residue of D corresponding to residue 16 of SEQ ID NO: 4 is N or a conservative substitution thereof; and the residue of D corresponding to residue 19 of SEQ ID NO: 4 is A or a conservative substitution thereof. 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/or the residue of D corresponding to residue 19 of SEQ ID NO: 4 is A. 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. The scaffold protein, method or use according to any one of clauses 1 to 35 wherein 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, method or use according to any one of clauses 1 to 36, wherein the scaffold protein consists of the N-terminal fragment of apolipoprotein E. The scaffold protein, method or use according to any one of clauses 1 to 37, wherein the N-terminal fragment of apolipoprotein E consists of the formula A-B-C-D-E. The scaffold protein, method or use according to any one of clauses 1 to 37, wherein the N-terminal fragment of apolipoprotein E comprises or consists of 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 a sequence sharing at least 60% identity with SEQ ID NO: 21 and Y comprises or consists of a sequence sharing at least 60% identity with SEQ ID NO: 20. The scaffold protein, method or use according to clause 39, wherein 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 according to clause 1 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. The method of eliciting an immune response in a subject according to clause 2, the method 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.

44. The 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.

45. The use of 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.

46. The scaffold protein, method or use according to any one of clauses 41 to 45, wherein 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.

47. The scaffold protein, method or use according to any one of clauses 41 to 46, wherein 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. 48. The scaffold protein, method or use according to any one of clauses 41 to 47, wherein 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.

49. The scaffold protein, method or use according to any one of clauses 41 to 48, wherein 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.

50. The scaffold protein, method or use according to any one of clauses 41 to 49, wherein 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.

51 . The scaffold protein, method or use according to any one of clauses 41 to 50, wherein the scaffold protein comprises 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.

52. The scaffold protein, method or use according to clause 51 , wherein the scaffold protein comprises or consists of SEQ ID NO: 18.

53. The scaffold protein, method or use according to any one of clauses 41 to 52, wherein the residue of A corresponding to residue 8 of SEQ ID NO: 14 is P or a conservative substitution thereof; and/or the residue of A corresponding to residue 9 of SEQ ID NO: 14 is A or a conservative substitution thereof; and/or the residue of A corresponding to residue 11 of SEQ ID NO: 14 is K or a conservative substitution thereof; and/or the residue of A corresponding to residue 29 of SEQ ID NO: 14 is N or a conservative substitution thereof; and/or the residue of A corresponding to residue 47 of SEQ ID NO: 14 is P or a conservative substitution thereof; and/or the residue of A corresponding to residue 61 of SEQ ID NO: 14 is S or a conservative substitution thereof; and/or the residue of A corresponding to residue 68 of SEQ ID NO: 14 is M or a conservative substitution thereof; and/or the residue of A corresponding to residue 70 of SEQ ID NO: 14 is E or a conservative substitution thereof; and/or the residue of A corresponding to residue 73 of SEQ ID NO: 14 is E or a conservative substitution thereof; and/or the residue of A corresponding to residue 80 of SEQ ID NO: 14 is K or a conservative substitution thereof; and/or the residue of A corresponding to residue 83 of SEQ ID NO: 14 is V or a conservative substitution thereof; and/or the residue of A corresponding to residue 87 of SEQ ID NO: 14 is M or a conservative substitution thereof; and/or the residue of A corresponding to residue 99 of SEQ ID NO: 14 is P or a conservative substitution thereof; and/or the residue of A corresponding to residue 104 of SEQ ID NO: 14 is Q or a conservative substitution thereof; and/or the residue of A corresponding to residue 111 of SEQ ID NO: 14 is K or a conservative substitution thereof; and/or the residue of A corresponding to residue 119 of SEQ ID NO: 14 is A or a conservative substitution thereof. The scaffold protein, method or use according to clause 53, wherein the residue of A corresponding to residue 8 of SEQ ID NO: 14 is P or a conservative substitution thereof; and the residue of A corresponding to residue 9 of SEQ ID NO: 14 is A or a conservative substitution thereof; and the residue of A corresponding to residue 11 of SEQ ID NO: 14 is K or a conservative substitution thereof; and the residue of A corresponding to residue 29 of SEQ ID NO: 14 is N or a conservative substitution thereof; and the residue of A corresponding to residue 47 of SEQ ID NO: 14 is P or a conservative substitution thereof; and the residue of A corresponding to residue 61 of SEQ ID NO: 14 is S or a conservative substitution thereof; and the residue of A corresponding to residue 68 of SEQ ID NO: 14 is M or a conservative substitution thereof; and the residue of A corresponding to residue 70 of SEQ ID NO: 14 is E or a conservative substitution thereof; and the residue of A corresponding to residue 73 of SEQ ID NO: 14 is E or a conservative substitution thereof; and the residue of A corresponding to residue 80 of SEQ ID NO: 14 is K or a conservative substitution thereof; and the residue of A corresponding to residue 83 of SEQ ID NO: 14 is V or a conservative substitution thereof; and the residue of A corresponding to residue 87 of SEQ ID NO: 14 is M or a conservative substitution thereof; and the residue of A corresponding to residue 99 of SEQ ID NO: 14 is P or a conservative substitution thereof; and the residue of A corresponding to residue 104 of SEQ ID NO: 14 is Q or a conservative substitution thereof; and the residue of A corresponding to residue 111 of SEQ ID NO: 14 is K or a conservative substitution thereof; and the residue of A corresponding to residue 119 of SEQ ID NO: 14 is A or a conservative substitution thereof. The scaffold protein, method or use according to clause 53, wherein the residue of A corresponding to residue 8 of SEQ ID NO: 14 is P and/or the residue of A corresponding to residue 9 of SEQ ID NO: 14 is A; and/or the residue of A corresponding to residue 11 of SEQ ID NO: 14 is K; and/or the residue of A corresponding to residue 29 of SEQ ID NO: 14 is N; and/or the residue of A corresponding to residue 47 of SEQ I D NO: 14 is P; and/or the residue of A corresponding to residue 61 of SEQ ID NO: 14 is S; and/or the residue of A corresponding to residue 68 of SEQ ID NO: 14 is M; and/or the residue of A corresponding to residue 70 of SEQ ID NO: 14 is E; and/or the residue of A corresponding to residue 73 of SEQ ID NO: 14 is E; and/or the residue of A corresponding to residue 80 of SEQ ID NO: 14 is K; and/or the residue of A corresponding to residue 83 of SEQ ID NO: 14 is V; and/or the residue of A corresponding to residue 87 of SEQ ID NO: 14 is M; and/or the residue of A corresponding to residue 99 of SEQ ID NO: 14 is P; and/or the residue of A corresponding to residue 104 of SEQ ID NO: 14 is Q; and/or the residue of A corresponding to residue 111 of SEQ ID NO: 14 is K; and/or the residue of A corresponding to residue 119 of SEQ ID NO: 14 is A. The scaffold protein, method or use according to clause 55, wherein the residue of A corresponding to residue 8 of SEQ ID NO: 14 is P and the residue of A corresponding to residue 9 of SEQ ID NO: 14 is A; and the residue of A corresponding to residue 11 of SEQ ID NO: 14 is K; and the residue of A corresponding to residue 29 of SEQ ID NO:

14 is N; and the residue of A corresponding to residue 47 of SEQ ID NO: 14 is P; and the residue of A corresponding to residue 61 of SEQ ID NO: 14 is S; and the residue of A corresponding to residue 68 of SEQ ID NO: 14 is M; and the residue of A corresponding to residue 70 of SEQ ID NO: 14 is E; and the residue of A corresponding to residue 73 of SEQ ID NO: 14 is E; and the residue of A corresponding to residue 80 of SEQ ID NO: 14 is K; and the residue of A corresponding to residue 83 of SEQ ID NO: 14 is V; and the residue of A corresponding to residue 87 of SEQ ID NO: 14 is M; and the residue of A corresponding to residue 99 of SEQ ID NO: 14 is P; and the residue of A corresponding to residue 104 of SEQ ID NO: 14 is Q; and the residue of A corresponding to residue 111 of SEQ ID NO: 14 is K; and the residue of A corresponding to residue 119 of SEQ ID NO: 14 is A. The scaffold protein, method or use according to any one of clauses 41 to 56, wherein the residue of B corresponding to residue 1 of SEQ ID NO: 15 is H or a conservative substitution thereof; and/or the residue of B corresponding to residue 4 of SEQ ID NO:

15 is A or a conservative substitution thereof; and/or the residue of B corresponding to residue 5 of SEQ ID NO: 15 is P or a conservative substitution thereof; and/or the residue of B corresponding to residue 8 of SEQ ID NO: 15 is E or a conservative substitution thereof; and/or 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 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 any one of clauses 41 to 60, wherein the residue of C corresponding to residue 1 of SEQ ID NO: 16 is E or a conservative substitution thereof; and/or the residue of C corresponding to residue 3 of SEQ ID NO: 16 is T or a conservative substitution thereof; and/or the residue of C corresponding to residue 5 of SEQ ID NO: 16 is A or a conservative substitution thereof; and/or the residue of C corresponding to residue 7 of SEQ ID NO: 16 is I or a conservative substitution thereof; and/or the residue of C corresponding to residue 8 of SEQ ID NO: 16 is N or a conservative substitution thereof; and/or the residue of C corresponding to residue 11 of SEQ ID NO: 16 is T or a conservative substitution thereof; and/or the residue of C corresponding to residue 14 of SEQ ID NO: 16 is I or a conservative substitution thereof; and/or the residue of C corresponding to residue 15 of SEQ ID NO: 16 is N or a conservative substitution thereof; and/or the residue of C corresponding to residue 18 of SEQ ID NO: 16 is I or a conservative substitution thereof; and/or the residue of C corresponding to residue 24 of SEQ ID NO: 16 is F or a conservative substitution thereof; and/or the residue of C corresponding to residue 28 of SEQ ID NO: 16 is F or a conservative substitution thereof; and/or the residue of C corresponding to residue 29 of SEQ ID NO: 16 is V or a conservative substitution thereof; and/or the residue of C corresponding to residue 32 of SEQ ID NO: 16 is A or a conservative substitution thereof; and/or the residue of C corresponding to residue 33 of SEQ ID NO: 16 is Q or a conservative substitution thereof; and/or the residue of C corresponding to residue 34 of SEQ ID NO: 16 is S or a conservative substitution thereof; and/or the residue of C corresponding to residue 35 of SEQ ID NO: 16 is P or a conservative substitution thereof; and/or the residue of C corresponding to residue 37 of SEQ ID NO: 16 is G or a conservative substitution thereof; and/or the residue of C corresponding to residue 38 of SEQ ID NO: 16 is D or a conservative substitution thereof. 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 to residue 24 of SEQ ID NO: 16 is F or a conservative substitution thereof; and the residue of C corresponding to residue 28 of SEQ ID NO: 16 is F or a conservative substitution thereof; and the residue of C corresponding to residue 29 of SEQ ID NO: 16 is V or a conservative substitution thereof; and the residue of C corresponding to residue 32 of SEQ ID NO: 16 is A or a conservative substitution thereof; and the residue of C corresponding to residue 33 of SEQ ID NO: 16 is Q or a conservative substitution thereof; and the residue of C corresponding to residue 34 of SEQ ID NO: 16 is S or a conservative substitution thereof; and the residue of C corresponding to residue 35 of SEQ ID NO: 16 is P or a conservative substitution thereof; and the residue of C corresponding to residue 37 of SEQ ID NO: 16 is G or a conservative substitution thereof; and the residue of C corresponding to residue 38 of SEQ ID NO: 16 is D or a conservative substitution thereof. 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/or the residue of C corresponding to residue 29 of SEQ ID NO: 16 is V; and/or the residue of C corresponding to residue 32 of SEQ ID NO: 16 is A; and/or the residue of C corresponding to residue 33 of SEQ ID NO: 16 is Q; and/or the residue of C corresponding to residue 34 of SEQ ID NO: 16 is S; and/or the residue of C corresponding to residue 35 of SEQ ID NO: 16 is P; and/or the residue of C corresponding to residue 37 of SEQ ID NO: 16 is G; and/or the residue of C corresponding to residue 38 of SEQ ID NO: 16 is D. 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 the residue of C corresponding to residue 32 of SEQ ID NO: 16 is A; and the residue of C corresponding to residue 33 of SEQ ID NO: 16 is Q; and the residue of C corresponding to residue 34 of SEQ ID NO: 16 is S; and the residue of C corresponding to residue 35 of SEQ ID NO: 16 is P; and the residue of C corresponding to residue 37 of SEQ ID NO: 16 is G; and the residue of C corresponding to residue 38 of SEQ ID NO: 16 is D. The scaffold protein, method or use according to any one of clauses 41 to 64, wherein the residue of D corresponding to residue 13 of SEQ ID NO: 17 is N or a conservative substitution thereof. The scaffold protein, method or use according to clause 65, wherein the residue of D corresponding to residue 13 of SEQ ID NO: 17 is N. The scaffold protein, method or use according to any one of clauses 41 to 66, wherein A comprises or consists of SEQ ID NO: 14. The scaffold protein, method or use according to any one of clauses 41 to 67, wherein 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. The scaffold protein, method or use according to any one of clauses 42 to 68, wherein B comprises or consists of SEQ ID NO: 15. The scaffold protein, method or use according to any one of clauses 41 to 69, wherein 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 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 F, W and Y; wherein Xg is selected from the group consisting of A, G, I, L, M and V; wherein X is selected from the group consisting of D and E; wherein Xu is selected from the group consisting of C, N, P, Q, S and T; wherein Xi2 is selected from the group consisting of H, K and R; wherein X is selected from the group consisting of A, G, I, L, M and V; wherein Xi₄ is selected from the group consisting of C, N, P, Q, S and T; wherein Xis is selected from the group consisting of A, G, I, L, M and V; wherein X is selected from the group consisting of D and E; wherein X17 is selected from the group consisting of F, W and Y; wherein Xis is selected from the group consisting of D and E; wherein X is selected from the group consisting of A, G, I, L, M and V; and wherein X2o is selected from the group consisting of C, N, P, Q, S and T.

71. The scaffold protein, method or use according to any one of clauses 41 to 70, wherein C comprises or consists of SEQ ID NO: 16.

72. The scaffold protein, method or use according to any one of clauses 41 to 71 , wherein D comprises or consists of SEQ ID NO: 17.

73. The scaffold protein, method or use according to any one of clauses 41 to 72, wherein 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.

74. The scaffold protein, method or use according to any one of clauses 41 to 73, wherein A consists of SEQ ID NO: 14, B consists of SEQ ID NO: 15, C consists of SEQ ID NO: 16 and D consists of SEQ ID NO: 17.

75. The scaffold protein, method or use according to any one of clauses 41 to 74, wherein 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.

76. The scaffold protein, method or use according to any one of clauses 41 to 75, wherein the acylhydrolase protein consists of the formula A-B-C-D. 77. The scaffold protein, method or use according to any one of clauses 41 to 76, wherein the scaffold protein consists of the acylhydrolase protein.

78. The scaffold protein, method or use according to any one of clauses 1 to 77, wherein the protein is presented on a nanoparticle.

79. The scaffold protein, method or use according to clause 78, wherein the nanoparticle is a protein nanoparticle.

80. The scaffold protein, method or use according to clause 79, wherein the protein nanoparticle is ferritin.

81. The scaffold protein, method or use according to clause 80, wherein the ferritin is selected from bacterial or insect ferritin.

82. The scaffold protein, method or use according to clause 81 , wherein the ferritin is bacterial ferritin.

83. The scaffold protein, method or use according to clause 82, wherein the ferritin is H. pylori ferritin.

84. The scaffold protein, method or use according to clause 83, wherein the H. pylori ferritin comprises or consists of SEQ ID NO: 27.

85. The scaffold protein, method or use according to any one of clauses 78 to 84, wherein the nanoparticle and the scaffold protein are connected by a linker.

86. The scaffold protein, method or use according to clause 85, wherein the linker consists of 1 to 40 residues.

87. The scaffold protein, method or use according to clause 86, wherein the linker consists of 10 to 30 residues.

88. The scaffold protein, method or use according to any one of clauses 85 to 87, wherein the linker comprises the polypeptide sequence of SEQ ID NO: 24. 89. The scaffold protein, method or use according to clause 85, wherein the linker consists of the polypeptide sequence of SEQ ID NO: 24.

90. The scaffold protein, method or use according to any one of clauses 1 to 89, wherein the scaffold protein is linked to a T cell epitope sequence.

92. The scaffold protein, method or use according to clause 91 , wherein the T cell epitope sequence is an m2e T cell epitope sequence.

93. The scaffold protein, method or use according to clause 82, wherein the m2e T cell epitope sequence comprises a sequence sharing at least 90% identity with SEQ ID NO: 25.

94. The scaffold protein, method or use according to clause 93, wherein the m2e T cell epitope sequence comprises or consists of SEQ ID NO: 25.

95. The scaffold protein, method or use according to any one of clauses 90 to 94, wherein the T cell epitope sequence is at the N-terminus of the scaffold protein.

96. The scaffold protein, method or use according to any one of clauses 90 to 95, wherein the T cell epitope sequence is connected to the scaffold protein by a linker.

97. The scaffold protein, method or use according to clause 96, wherein the linker consists of 1 to 10 amino acids.

98. The scaffold protein, method or use according to clause 97, wherein the linker comprises or consists of the sequence GASG (SEQ ID NO: 26).

99. The scaffold protein, method or use according to any one of clauses 1 to 98 wherein the influenza hemagglutinin stem epitope is bound by the FI6 antibody.

100. The scaffold protein, method or use according to any one of clauses 1 to 99 wherein the influenza hemagglutinin stem epitope is bound by the CR9114 antibody.

101. The scaffold protein, method or use according to any one of clauses 1 to 100 wherein the influenza hemagglutinin stem epitope is bound by the 39.29 antibody. 102. The scaffold protein, method or use according to any one of clauses 1 to 101 wherein the influenza hemagglutinin stem epitope is bound by the MEDI-8852 antibody.

103. The scaffold protein, method or use according to any one of clauses 1 to 102 wherein the stem epitope is bound by antibodies which bind to H1 influenza strains.

104. The scaffold protein, method or use according to any one of clauses 1 to 103 wherein the stem epitope is bound by antibodies which bind to H3 influenza strains.

105. The scaffold protein, method or use according to any one of clauses 1 to 104 wherein the stem epitope is bound by antibodies which bind to H5 influenza strains.

106. The scaffold protein, method or use according to any one of clauses 99 to 105 wherein the antibody binds to the influenza hemagglutinin stem epitope with a binding affinity (KD) of less than 3.0 x 10'⁷ M (/.e. 300 nM) or less than 1.5 x 10'⁷ M (/.e. 150 nM). In a further embodiment, 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. In a yet further embodiment, the KD is less than 6.0 x 10^-8 M (/.e. 60 nM), such as less than 5.0 x 10'⁸ M (/.e. 50 nM), less than 4.0 x 10'⁸ M (/.e. 40 nM), less than 3.0 x 10^-8 M (/.e. 30 nM) or less than 2.0 x 10^-8 M (/.e. 20 nM). In further embodiments, 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.

107. The scaffold protein, method or use according to any one of clauses 1 to 106, wherein 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.

108. The scaffold protein, method or use according to any one of clauses 1 to 106, wherein a single dose of scaffold protein is 10 to 30 ug, especially 15 to 25 ug, in particular about 20 ug.

109. The scaffold protein, method or use according to any one of clauses 1 to 106, wherein a single dose of protein is 1 to 3 ug, especially 1.5 to 2.5 ug, in particular about 2 ug.

110. The scaffold protein, method or use according to any one of clauses 1 to 109, wherein the scaffold protein is administered with a squalene emulsion adjuvant. The scaffold protein, method or use according to clause 110, wherein the scaffold protein and squalene emulsion adjuvant are administered as a co-formulation. The scaffold protein according to any one of clauses 1 to 111 , for intramuscular administration. 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. The scaffold protein, method or use according to any one of clauses 110 to 114, wherein the protein and squalene emulsion adjuvant are 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, method or use according to any one of clauses 110 to 115, wherein the protein and squalene emulsion adjuvant are administered to the same location. The scaffold protein, method or use according to any one of clauses 110 to 116, wherein the protein and squalene emulsion adjuvant are administered concurrently. The scaffold protein, method or use according to any one of clauses 1 to 117, wherein administration of 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 according to any one of clauses 1 to 118, for administration to a subject which is not infected with influenza virus. The scaffold protein, method or use according to any one of clauses 1 to 119, wherein the elicited immune response reduces partially or completely the severity of one or more symptoms and/or time over which one or more symptoms of influenza virus infection are experienced by the subject. 121. The scaffold protein, method or use according to any one of clauses 1 to 120, wherein the elicited immune response reduces the likelihood of developing an established influenza virus infection after challenge.

122. The scaffold protein, method or use according to any one of clauses 1 to 121 , wherein the elicited immune response slows progression of influenza.

123. The scaffold protein, method or use according to any one of clauses 119 to 122, wherein the influenza virus is H3 1968 HK X31 influenza virus.

124. The scaffold protein, method or use according to any one of clauses 119 to 123, wherein the scaffold protein (such as ApoE) does not form part of a nanolipoprotein.

125. 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.

126. The method according to any one of clauses 2, 7, 42 or 78 to 125, wherein the subject is a human.

127. A nucleic acid encoding the scaffold protein according to any one of clauses 1 to 125.

EXAMPLES Methods

FI6-focused designs

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).

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. Residues contributing to the dimerization (ddG < -0.8) and exposed hydrophobic residues in the interface were selected and submitted to sequence design. We generated 50 models and selected the best design based on overall Rosetta energy units (REU). Eight mutations were selected that drastically decreased the predicted binding energy, suggesting disruptive interactions, while maintaining an overall energy for the model on par with the native monomeric subunit. To improve stability of the resulting monomeric design variant, 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.

The polypeptide sequences of the designs were as shown in Table 2 below: Table 2

The 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.

Stem-epitope mimetics

Based on previous results that indicated a lack of suitable protein scaffolds for the complex epitope, it was decided to focus on identifying scaffolds based on the regular a-helix of the epitope. A scaffold search was performed against a subset of the PDB containing monomeric proteins with helical secondary structural elements and a length of 50 to 250 amino acids with a RMSD cutoff at 1 A, similar as described above but using Rosetta MotifGraft protocol (Silva, Correia, and Procko 2016) to align the structures, resulting in 7,655 matches. To narrow the set of potential scaffolds the RMSD was lowered to a threshold of 0.3 A and allowing no steric clashes, resulting in 1 ,525 matches. 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. Four interface positions were identified that demonstrated low resemblance of the antigenic site and these positions were subjected to surface-centric design. RosettaSurf-site was performed on these positions, sampling all 20 amino acids at each position and evaluating their impact on overall epitope mimicry. Individual mutations were evaluated based on their shape similarity to the antigenic site as observed in the H1-FI6 complex (PDB ID: 3ZTN), their propensity to form a-helices (Chou and Fasman 1978), and their biochemical properties, preferring hydrophobic residues to mimic the hydrophobic pocket of the epitope. Designed variants were evaluated based on predicted binding energy to the FI6 antibody, followed by manual inspection of the best ranking designs. A rational mutation I98A was introduced, as the designed amino acids did not match the native epitope residues, resulting in the stem-epitope_01. An additional variant (stem-epitope_02) introducing two potential disulfide bonds connecting positions 53-97 and 96-137, for improved stability was also produced. 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).

The polypeptide sequence of the design, based on murine ApoE, is shown in Table 3 below:

Table 3

Although included in the design above, the initial N-terminal amino acids GEPEVTDQ (SEQ ID NO: 23) were considered irrelevant for the immunogenicity and stability of the scaffold.

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.

Yeast surface display of single designs

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. Cells were washed once and incubated for additional 30 min with FITC-conjugated anti-cMyc antibody (sigma, #SAB4700448) and PE-conjugated anti-human Fc (invitrogen, #12-4998-82). Cells were washed and analyzed using a Gallios flow cytometer (Beckman Coulter).

Combinatorial libraries

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.

Yeast surface display of libraries

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

Design scaffolds

DNA sequences were produced for bacterial expression with a c-terminal 6x His Tag. 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.

Cells were grown at 37°C till they reached an OD600 of 0.7 and induced with 1mM IPTG. 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. 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 AKTA™ pure system (Cytivia).

Antibodies

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 Expi293™ 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 AKTA™ 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.

Recombinant Hemagglutinin

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 Expi293™ expression system from Thermo Fisher Scientific. Supernatant was collected 6 days post transfection, filtered and purified via Ni-NTA affinity or StrepTrap™ HP, for GCN4 HA versions, followed by size exclusion on a HiLoad® 16/600 Superdex® 200pg column on an AKTA™ pure system (cytivia).

Design nanoparticles

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 Expi293™ expression system from Thermo Fisher Scientific. Supernatant was collected 6 days post transfection, filtered and purified via Ni-NTA affinity followed by size exclusion on a HiLoad® 16/600 Superose 6 pg column on an AKTA™ pure system (cytivia).

Surface plasmon resonance to measure binding affinities

SPR measurements were performed on a Biacore 8K (cytivia) with HBS-EP+ as running buffer (10 mM HEPES pH 7.4, 150 mM NaCI, 3 mM EDTA, 0.005% v/v Surfactant P20, GE Healthcare). Ligands were immobilized on a CM5 chip (cytivia #29104988) via amine coupling. Approximately 1000 response units (RU) of IgG were immobilized, and designed proteins were injected as analyte in two-fold serial dilutions. The flow rate was 30 pl/min with a contact time of 120 s followed by 800 s dissociation time. After each injection, the surface was regenerated using 0.1 M glycine at pH 2.5. Data were fitted using 1:1 Langmuir binding model within the Biacore 8K analysis software (cytivia #29310604).

Size exclusion chromatography multi-angle light scattering (SEC-MALS)

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 Far-LIV circular dichroism spectra were measured using a Chirascan™ 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).

Next-generation sequencing

After sorting, 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. For bioinformatic analysis, sequences were translated in the correct reading frame, and enrichment values were computed for each sequence.

Cell line and viruses

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.

Serum binding to virus infected cells

The assay was performed as previously described (Angeletti et al., 2019). Briefly, MDCK cells were infected using A/Puerto Rico/8/34 (H1N1), A/California/07/2009 (H1 N1), A/HKx31 (H3N2) viruses, at MOI = 3 for 5h at 37°C in DM EM medium with gentamycin (Thermo Fisher, Waltham, MA) and TPCK-Trypsin (BioNordika, Soina, Sweden). Viruses were propagated in 10 days old embryonated chicken eggs (VALO BioMedia, Osterholz-Scharmbeck, Germany) or MDCK SIAT 1 cells. After incubation, cells were incubated with a serial dilution of sera for 60 min at room temperature. After washes with DPBS + 5% FBS + 0.5mM EDTA (Thermo Fisher, Waltham, MA), cells were stained with BV421 anti-mouse kappa light chain (clone 187.1) (BD biosciences, San Jose, CA) for 30 min at 4°C. Cells were washed again and permeabilized using a fixation/permeabilization buffer from the Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher, Waltham, MA) for either 30min or overnight at 4°C. Then, cells were stained with an AF488 coupled (AF488 Protein Labelling Kit, Thermo Fisher, Waltham, MA) anti-influenza A virus NP (clone H16-L10-4R5 (HB65)) antibody (Bio X Cell, Lebanon, NH) in permeabilization buffer. The samples were acquired on a CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, IN) and analyzed with the GraphPad Prism9 software.

ADCC Assay

ADCC assay was performed as previously described (Kosik et al. 2019). Briefly, 10,000 MDCK cells were seeded into 96-well white flat bottom plates (Thermo Fisher, Waltham, MA) and left to settle overnight. The following day, cells were washed twice with DPBS (Thermo Fisher, Waltham, MA) and infected with following viruses at MOI=3 in RPMI 1640 medium with 4% ultralow-IgG serum (Thermo Fisher, Waltham, MA): A/Puerto Rico/8/34 (H1 N1), A/California/07/2009 (H1N1) and A/HKx31 (H3N2). After 6 h, 25 pl of an 1 :40 dilution of mouse sera were combined with 50,000 in 25 pl of effector cells (Jurkat cell line expressing luciferase gene under control of the NFAT response element and stably expressing FcyRIV; Promega) in RPMI 1640 medium with 4% ultralow-IgG serum (Thermo Fisher, Waltham, MA). The serumeffector cell mixture was added onto the MDCK cells (effector cells:target cells = 5:1). After overnight incubation at 37°C in 5% CO2, 50 pl of Bright-Glo Luciferase Assay lysis/substrate buffer (Promega) was added and luminescence was measured within 5 min using a SpectraMax® i3x plate reader (Molecular Devices, San Jose, CA). Fold induction was calculated over a no sera containing control using GraphPad Prism9 software.

Mouse immunizations

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.

Challenge For the challenge, 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.

ELISA

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. 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.

Whole Virus ELISA

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. After another wash, goat anti mouse H+L chain IgG coupled to peroxidase (Vector Laboratories, Burlingame, CA) was used for staining. Plates were washed again and then developed with TMB (Thermo Fisher, Waltham, MA) and H2SO4. Plates were read at 405nm on a plate reader (Tecan, Mannedorf, Switzerland).

B cell sorting and sequencing

Animals 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. Cells were stained in DPBS + 5% FBS + 0.5mM EDTA with anti-lgD BV786 (BD Biosciences, San Jose, CA), anti- CD3e BV510 (BD Biosciences, San Jose, CA), anti-NK1.1 BV510 (Biolegend, San Diego, CA), anti-B220 APC-Cy7 (BD Biosciences, San Jose, CA), anti- GL7 BV421 (eBiosciences, San Diego, CA), anti- CD38 FITC (BD Biosciences, San Jose, CA), recombinant HAH1 California (Mammalian Protein Expression Core Facility, University of Gothenburg) and recombinant HAH3 Brisbane (National Institutes of Health, Bethesda, MD) conjugated to APC and PE (Thermo Fisher, Waltham, MA) respectively. The labelled cells were run and data acquired using BD LSR Fortessa X-20 or BD FACSAria Fusion (BD Biosciences), data was analyzed using Flow-Jo software (TreeStar).

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. In short, GEMs (gelbeads in emulsion) were generated using the Chromium Next GEM Chip K (10x Genomics, Pleasanton, CA) and cells were barcoded while a reverse transcription reaction was performed. Next, cDNA was amplified and samples were split. From this step on, either V(D)J or 5’ Gene Expression Dual Index Libraries were generated using the Dual Index Kit TT Set A. For the 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

To design immunogens eliciting a protective immune response, 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). 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. Based on subsequent searches, 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). Initial binding affinity of the computationally designed protein to FI6 was low, however, specific to the mimicked site when compared to WT protein. To improve binding affinity of the FI6-focused design, a computationally derived combinatorial library was screened including residues adjacent to the grafted epitope helix for tighter binding to the FI6 antibody and could improve binding to a KD of 320 nM (FI6-focused_02). In the next step, a further increase in binding affinity was sought using a site-saturation mutagenesis (SSM) library by sampling positions that could be relevant for the accurate presentation of the stem site and surrounding residues (aa 93-106 and aa 123-189) (Fig. 2).

The SSM library approach allows a thorough sampling of a large number of relevant positions. The best individual mutations from the library were combined and 16 variants were screened for improved binding to FI6. All tested variants boosted affinity and the best binding design (KD = 0.26 nM), FI6-focused_03, which was comparable to H1 HA affinity, was selected (Fig. 1). 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. To counter the destabilizing mutations, the design was computationally assessed for stabilizing positions, identifying twelve residue positions that could be improved by point mutations. Three candidates were tested for thermostability and a set of mutations were identified that improved stability which were combined in the FI6-focused_04 design. The final design, 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).

Following the results of the FI6-focused_04 immunogen design, it was concluded that while protein scaffolds closely matching the epitope’s structure were rare, immunogens could be engineered by grafting core epitope elements and improving overall mimicry of the antigenic site through structure-based design. Accordingly, the regular epitope a-helix was focused on as anchor for new designs, performing scaffold searches solely with the helical segment. As expected, the number of putative scaffolds increased. To identify optimal candidate scaffolds that could maximize epitope mimicry, several filters were applied including predicted binding energy, number of atomic clashes, and number of potential side chain interactions between the scaffold and the FI6 antibody. Mouse apolipoprotein E (ApoE) was identified (PDB ID: 1YA9 (Hatters, Peters-Libeu, and Weisgraber 2005)), a regular four-helix bundle that closely mimicked the epitope helix (backbone RMSD 0.5 A) and offered sufficient surface area to mimic the complete stem epitope (Fig. 1). Since ApoE is involved in lipid transport and highly abundant in serum (Lusis et al. 1987), it was hypothesized that the immune response towards the scaffold itself would be low in mice. After grafting the epitope helix side chains onto the scaffold, 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).

To increase immunogenicity and B cell cross-linking of the FI6-focused_04 and the stem- epitope_01 design, each of them were fused to a ferritin nanoparticle (Helicobacter pylori), respectively (Fig. 5).

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.

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).

In parallel, the crystal structure of the epitope-stem mimetic was solved in complex with CR9114 to a resolution of 2.7 A (Fig. 6C). Comparison of structure and model revealed close structural agreement with a RMSD of 1.6 A. Close inspection of the side chain placement revealed that rotamers generally adapted the designed conformations and many of the polar and hydrophobic interactions were accurately retained. The structure of this design was compared to H5 in complex with CR9114 (PDB ID: 4FQI (Dreyfus et al. 2012)). Polar interactions of the residues surrounding the epitope helix were partially recovered, while polar interactions formed by the epitope helix were presented completely (Fig. 6D). Additionally, the epitope surface of the stem-epitope mimetic’s crystal structure resembled the surface of H5 HA complex with CR9114 (Fig. 6E), demonstrating successful mimicry of the epitope.

Binding properties

To assess the capability of the FI6-focused design to engage known anti-HA antibody lineages, its ability to bind memory B cells in human peripheral blood mononuclear cells (PBMCs) was investigated. To ensure specificity to HA, cells double positive to H1 stem and FI6-focused_03 were sorted from two recently vaccinated donors (Figure 7A). For the stemmimetic, design positive memory B cells were sorted in a H1 only and a H1/H3 double positive population and sequenced (Figure 7D.

A major population of pulled down antibodies from both designs were derived from the VH3- 23, a germline gene that is known to give rise to potent bnAbs (Joyce et al. 2016).

Sequencing of these antibodies confirmed the strong focusing of the FI6-focused_03 design towards FI6-like antibodies as they were almost exclusively originating from VH3-30, the same germline region as FI6 (Fig. 7B). While FI6 binds HAs from group 1 and group 2 as the result of affinity maturation caused by somatic mutations, germline FI6 (FI6-GL) is restricted to group 1 (Corti et al. 2011), in line with observations that all sorted antibodies from VH3-30 were binding predominantly to group 1 HAs (Fig. 7C). While antibodies with similar VDJ rearrangement and HCDR3 lengths as FI6 were found in both donors, one of the donors also produced VH3-30 antibodies with a unique VDJ rearrangement not previously discovered (63- 1A12).

One of the isolated antibodies (31-1 B01) 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).

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.

Together these results show that the designs can engage relevant human antibody lineages that can provide broad influenza neutralization.

Induction of antibodies in mice

To investigate the ability of these designs to elicit antibodies in vivo, 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. Testing antibody titers against the WT scaffold of the stem-epitope_01 immunogen confirmed strong epitope-focusing (Fig. 8B). As the scaffold protein of stem- epitope_01 is an endogenous mouse protein it was hypothesized that elicited antibodies were epitope-specific and did not target the scaffold due to B cell tolerance. The immuno-silent scaffold protein renders this design an ideal immunogen, as it promotes a commonly subdominant epitope to become immunodominant. In the next step, the elicited antibodies from both designs were tested for cross-reactivity to group 1 and 2 HAs. Both designs showed comparable titers to H1 , however, the stem-epitope mimetic showed significantly higher titers to H3 (Fig. 8C, D). Nevertheless, in the same animals the titers against H3 are considerably lower than against the stem-epitope design, even though the majority of antibodies were specific for the grafted epitope (Figure 8B). This discrepancy is most likely resulting from antibodies which approach the epitope in an angle that is not accessible on full-length HA, similar to results from previous studies (Schneemann et al. 2012).

To determine the epitope focusing on HA, the sera were tested against a KO version of H1 containing a glycan in the stem epitope that has been reported to disrupt binding to stemspecific Abs (Darricarrere et al. 2021) (Fig. 8E). For both designs the glycan knocked out binding almost completely, confirming the specificity of the elicited antibodies to the selected stem epitope. In contrast, sera from mice immunized three times with full-length HA did not show decreased binding to the KO version, demonstrating the poor response against this subdominant site. The results are supported by earlier studies reporting that even after immunization with headless HAs this epitope is targeted poorly in mice (Sangesland et al. 2019). Due to the distinct epitope focusing and the close mimicking of H3 it was decided to concentrate further experiments on the stem-epitope_01 mimetic.

To examine if the elicited antibodies not only bind to recombinant antigens but also to natural antigens on viruses, 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.

To obtain information about the antibodies induced by the stem-epitope_01 mimetic at the monoclonal level, 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. Together with the previous observations in mouse sera, this result suggests that this design is a closer mimic of the H3 stem rather than the H1 stem. Despite this imbalance, there was still a proportion of antibodies with the ability to engage both, H3 and H1 HAs. Induction of such bnAbs has remained difficult and could e.g. not be achieved with H1 and H3 mosaic nanoparticles in mice (Cohen et al. 2021).

To provide further insight, spleens and draining lymph nodes were examined two weeks after the 2^nd and the 3^rd injection and class switched B cells were analyzed for design and HA binding (Figure 8G). While the proportion of design positive B cells remained stable between the 2^nd and 3^rd injection, a clear increase of the H1/H3 cross-reactive antibodies after the 3^rd injection could be observed. These results furthermore point out that the induced HA positive antibodies are almost entirely cross-reactive to H1 and H3.

To allow for a more detailed analysis of the elicited cross- reactive antibodies, a paired heavy and light chain sequence library was prepared from single B cells. The generated cDNA library was sequenced via next-generation sequencing, resulting in the recovery of approximately 1000 antibody sequences with variable heavy (VH) and light chains (VL) originating from various germlines. Two VH chains (VH3-1 and VH9-3) and one light chain (VL3-12) showed an increased enrichment (Fig. 9B).

Protection Studies

Since stem antibodies are known to protect mainly via antibody-dependent cellular cytotoxicity and not by direct viral neutralization, their functionality can be best assessed in vivo (DiLillo et al. 2016). Therefore, 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). Three weeks after the last injection, 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. Here, 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).

Antibody-Dependent Cellular Cytotoxicity

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. In contrast, the heterologous prime boost injections, especially with H3 prime, showed more consistent ADCC activation. Moreover, prime injections with an H3 HA trimer showed the strongest and broadest ADCC signals, reinforcing our previous hypothesis that the surface mimicry is biased towards H3 HAs. REFERENCES

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WG2006/100109

WO20 11/154444

WO20 11/154443

WO20 13/044203

WO2015/183969

WO20 18/045308

EP0868918

Claims

Claims 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 according to claim 1 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. The scaffold protein according to claim 2, wherein A comprises a sequence sharing at least 70% identity with SEQ ID NO: 1; B comprises a sequence sharing at least 80% identity with SEQ ID NO: 2; C comprises a sequence sharing at least 70% identity with SEQ ID NO: 3; D comprises a sequence sharing at least 80% identity with SEQ ID NO: 4; and E comprises a sequence sharing at least 70% identity with SEQ ID NO: 5. The scaffold protein according to claim 3, wherein A comprises SEQ ID NO: 1 ; B comprises SEQ ID NO: 2; C comprises SEQ ID NO: 3; D comprises SEQ ID NO: 4; and E comprises SEQ ID NO: 5. The scaffold protein according to any one of claims 1 to 4, wherein the scaffold protein comprises a sequence sharing at least 70% identity with SEQ ID NO: 11. The scaffold protein according to claim 5, wherein the scaffold protein comprises SEQ ID NO: 11. The scaffold protein according to claim 1 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.

8. The scaffold protein according to claim 7, wherein A comprises a sequence sharing at least 70% identity with SEQ ID NO: 14; B comprises a sequence sharing at least 90% identity with SEQ ID NO: 15; C comprises a sequence sharing at least 90% identity with SEQ ID NO: 16; and D comprises a sequence sharing at least 70% identity with SEQ ID NO: 17.

9. The scaffold protein according to either claim 7 or 8, wherein the scaffold protein comprises a sequence sharing at least 70% identity with SEQ ID NO: 18.

10. The scaffold protein according to claim 9, wherein the scaffold protein comprises SEQ

ID NO: 18.

11. The scaffold protein according to any one of claims 1 to 10, wherein the scaffold protein is presented on a ferritin nanoparticle.

12. The scaffold protein according to any one of claims 1 to 11, wherein the scaffold protein is linked to a T cell epitope sequence.

13. The scaffold protein according to any one of claims 1 to 12 wherein the influenza hemagglutinin stem epitope is bound by the FI6 antibody.

14. The scaffold protein according to any one of claims 1 to 13 wherein the influenza hemagglutinin stem epitope is bound by antibodies which bind to H1 and H3 influenza strains.

15. The scaffold protein according to either claim 13 or 14 wherein the antibody binds to the influenza hemagglutinin stem epitope with a binding affinity (KD) of less than 100 nM.