Field of the invention

The invention relates to proteins comprising hepatitis B core antigen (HBcAg) and influenza virus A surface polypeptide M2 or an immunogenic fragment thereof. The invention also relates to particles formed from the proteins, nucleic acid molecules encoding the proteins, processes for producing the proteins, pharmaceutical compositions containing the proteins and use of the proteins to induce an immune response in a subject.

Background of the invention

The Hepatitis B virus core (HBc) protein has a somewhat unique structure comprised of two anti-parallel a-helices which form a characteristic "spike" structure. Two HBc molecules then spontaneously dimerise to form a twin spike bundle. This bundle is the building block of a virus like particle (VLP). VLPs are attractive vaccine systems since their highly repetitious nature delivers multiple copies of the antigen. Furthermore, the lack of viral nucleic acid makes them a safe vector. HBc is particularly interesting as a vaccine carrier since it has several sites into which antigenic sequences may be inserted. The extreme immunogenicity of HBc is then also imparted to the inserted sequence, thus making that too immunogenic. The optimal insertion site is the Major Insertion Region (MIR). However, it was shown previously that when a large or hydrophobic sequence is inserted into the MIR, then monomeric HBc fails to dimerise and a VLP does not form (Pumpen & Grens 2001), thus making the vaccine ineffective.

Influenza virus is a member of the Orthomyxoviridae family. There are three subtypes of influenza viruses designated A, B, and C that infect humans. The influenza virion contains a segmented negative-sense RNA genome. The enveloped influenza A virions have three membrane proteins, hemagglutinin (HA), neuraminidase (NA) and proton ion-channel protein (M2); a matrix protein (Ml) just below the lipid bilayer; a ribonucleoprotein core consisting of 8 viral RNA segments and three proteins

(polymerase acidic protein (PA), polymerase basic protein 1 (PBl) and polymerase basic protein 2 (PB2)); and nonstructural protein 2 (NS2). Influenza B virions have four proteins in the envelope: HA, NA, NB, and BM2. Like the M2 protein of influenza A virus, the BM2 protein is a proton channel that is essential for the uncoating process. The NB protein is believed to be an ion channel, but it is not required for viral replication in cell culture.

Influenza C viruses are somewhat different. Like the influenza A and B viruses, the core of influenza C viruses consists of a ribonucleoprotein made up of viral RNA and four proteins. The Ml protein lies just below the membrane, as in influenza A and B virions. A minor viral envelope protein is CM2, which functions as an ion channel. The major influenza C virus envelope glycoprotein is called HEF (hemagglutinin-esterase- fusion) because it has the functions of both the HA and the NA.

The HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the cell, and are immunodominant epitopes for virus neutralization and protective immunity. However, these proteins can, and often do, change from strain to strain. Due to the variability of these two proteins, a broad spectrum, long lasting influenza vaccine has so far not been developed. The influenza vaccine commonly used has to be adapted almost every year to follow the antigenic drift of the virus. When more drastic changes occur in the virus, known as an antigenic shift, the vaccine is no longer protective.

Summary of the invention

The invention is concerned with a vaccine delivery system based on the hepatitis B (HBV) core protein. Current vaccines to influenza virus require them to be redesigned each year due to the rapid mutation rate of the virus which causes the emergence of escape variants not contained in the previous year vaccines. They rely on predicting the predominant circulating influenza strain but they are rendered suboptimal when there is a mismatch between vaccine and circulating strains. In addition they cannot protect against new, previously unseen, viral strains. One solution is to design vaccines based on the conserved protein domains of influenza, which remain largely unchanged from year to year and are conserved in new emergent variants. Many previous attempts to target conserved domains have failed because these regions have low immunogenicity by themselves or cannot be displayed in their natural conformation outside the wild type virus. The inventors have managed to insert into a tandem construct a conserved region from influenza virus A surface polypeptide M2 (influenza matrix protein 2). The conserved region was influenza virus A surface polypeptide M2 ectodomain (M2e). The resulting VLP is able to generate seroconversion to the relevant M2 peptide and confer protection from a lethal H1N1 influenza infection (see Example 1). The inventors have also managed to insert into a tandem construct a conserved region from influenza hemagglutinin (HA). The resulting VLP is able to generate seroconversion to HA protein and confer protection from a lethal homologous H1N1 influenza infection (see Example 2). Further, the inventors have also managed to simultaneously insert into a tandem construct conserved regions from both influenza hemagglutinin (HA) and matrix 2 protein ectodomain (M2e). The resulting VLP is able to generate seroconversion to HA protein and M2e peptide and confer protection from a lethal homologous H1N1 influenza infection (Example 3). The inventors also produced a vaccine comprising two different VLPs: the tandem construct for the first VLP comprising conserved regions from both influenza HA and M2e; and the tandem construct for the second VLP comprising a conserved region from influenza HA from a different subtype of influenza. Together, the two VLPs comprise 5 different conserved antigens from influenza and can be delivered simultaneously as a single vaccine. The combination vaccine is able to generate seroconversion to group- 1 and group-2 HA protein, as well as M2e peptide and confer protection from lethal H1N1 and H3N2 influenza infections (Example 4).

The inventors found that inserting native M2e into the el loop resulted in the VLPs not forming properly because there was protein mis-folding. The inventors surprisingly found that this problem could be overcome by adding a linker sequence which flanked the M2e insert, and/or by substituting one or both of the two cysteine residues at positions 17 and 19 of M2e with an alternative amino acid. The linker allows the formation of VLPs which are able to induce seroconversion and provide protection from pathogenic challenge. For example, rows 1 and 4 of Table 2 in Example 1 demonstrate that introducing a linker between the M2e and the el loop results in VLPs which can induce seroconversion. Thus, the linker provides a means of inserting M2e into the tandem construct which would, without the linker, lead to mis-folding and therefore would not be able to produce VLP that are effective at inducing immune responses.

Discussed further below are examples of immunogenic polypeptides which may be incorporated into the tandem construct. In particular, the current inventors have found that tandem constructs comprising the influenza virus A surface polypeptide M2 ectodomain and/or the stalk domain of hemagglutinin (HA) produce VLPs which are particularly effective at inducing seroconversion and providing protection from challenge with influenza (see the Examples).

The substitution of cysteine with serine at position 17 and/or 19 of M2e allows the formation of VLPs which are able to induce seroconversion and provide protection from pathogenic challenge. Without the substitution of one or both cysteines, the VLPs do not form properly, unless a linker is inserted between the M2e insert and el loop as described above. For example, rows 1 to 3 of Table 2 in Example 1 demonstrate that substituting the cysteine with serine at position 17 and/or 19 can result in VLPs which can induce seroconversion where seroconversion is absent using VLPs with the native cysteine at position 17 and 19. It is thought that the presence of the substitution of the cysteine residues prevents the formation of disulphide bonds which may disrupt the formation of the VLPs.

Thus, the invention provides a protein comprising a first and a second copy of

HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop. The influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is flanked by a linker on one or both sides that joins the polypeptide or fragment to the

HBcAg sequence.

The invention also provides a protein comprising a first and a second copy of HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop and the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid.

The protein of the invention may also comprise another immunogenic

polypeptide in the other copy of HBcAg. This immunogenic polypeptide may be derived from influenza. However, it may also be derived from a different pathogen or allergen. Therefore the protein of the invention is useful for inducing an immune response to influenza virus and, depending on what other immunogenic polypeptides are present, it may also be useful for simultaneously inducing an immune response to a different pathogen or allergen.

The inventors also found that the potential problem of mis-folding of inserted antigenic peptides could be resolved by presenting the antigenic peptide in the el loop of one of the copies of HBcAg and having a "null" insert in the el loop of the second copy of HBcAg. In particular, the inventors developed a further type of VLP (VLP2) by inserting into one copy of HBcAg in the tandem construct a conserved region from influenza H3N2 virus hemagluttinin HA2 protein domain (LAH3). They found that inserting only a short sequence, of less than 20 amino acids, into the second copy of HBcAg allowed the first insert (LAH3) to configure properly and conferred greater solubility to the whole VLP (Example 4). Specifically, they inserted a single Lysine (K) residue flanked by a short flexible linker region made up of Glycine and Serine residues, which is effectively a "null" insert.

Thus, the invention also provides a protein comprising a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids. The fragment of HA is optionally from influenza virus hemagglutinin HA2 protein domain, and further optionally from influenza virus subtype H3N2. The second copy of HBcAg may comprise, in the el loop, a Lysine (K) residue flanked on each side by a linker sequence comprising Glycine and Serine residues.

The invention further provides a protein comprising a first and a second copy of HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop according to the invention, wherein the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids. The second copy of HBcAg may comprise, in the el loop, a Lysine (K) residue flanked on each side by a linker sequence comprising Glycine and Serine residues.

The invention also provides:

a particle comprising multiple copies of one or more proteins of the invention; a nucleic acid molecule encoding a protein of the invention;

a host cell comprising one or more nucleic acid molecules of the invention;

a process for producing a protein of the invention, which process comprises culturing a host cell containing a nucleic acid molecule which encodes the protein under conditions in which the protein is expressed, and recovering the protein; a pharmaceutical composition comprising a protein of the invention, a particle of the invention or a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier or diluent;

a vaccine comprising a protein of the invention, a particle of the invention or a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier or diluent;

a method of inducing an immune response against influenza in a subject, which method comprises administering to the subject a protein of the invention, a particle of the invention or a nucleic acid molecule of the invention;

- a protein of the invention, a particle of the invention or a nucleic acid molecule of the invention for use in a method of vaccination of the human or animal body against influenza;

use of a protein of the invention, a particle of the invention or a nucleic acid molecule of the invention for the manufacture of a medicament for vaccination of the human or animal body against influenza.

The proteins, particles, nucleic acids, pharmaceutical compositions and vaccines of the invention may be used on their own to protect against influenza or they may be used in combination. The use of different VLPs comprising different influenza antigens in combination enables a broader level of protection, for example from different subtypes of influenza simultaneously. With this in mind, the inventors developed a combination vaccine comprising a mixture of two VLPs, which together contain 5 conserved antigens from influenza HA and M2e which can be delivered simultaneously as a single vaccine (Example 4).

Thus, the invention also provides:

- a pharmaceutical composition comprising: (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which one or both of the copies of HBcAg comprises, in the el loop, influenza virus A surface polypeptide M2 or an immunogenic fragment thereof flanked on one or both sides by a linker that joins the polypeptide or fragment to HBcAg sequence; and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; and a pharmaceutically acceptable carrier or diluent;

a pharmaceutical composition comprising (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop and the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid; and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; and a pharmaceutically acceptable carrier or diluent;

a vaccine comprising: (i) a first protein, a particle comprising

multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which one or both of the copies of HBcAg comprises, in the el loop, influenza virus A surface polypeptide M2 or an immunogenic fragment thereof flanked on one or both sides by a linker that joins the polypeptide or fragment to HBcAg sequence; and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; and a pharmaceutically acceptable carrier or diluent;

a vaccine comprising (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop and the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid; and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; and a pharmaceutically acceptable carrier or diluent;

a method of inducing an immune response against influenza in a subject, which method comprises administering to the subject: (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which one or both of the copies of HBcAg comprises, in the el loop, influenza virus A surface polypeptide M2 or an immunogenic fragment thereof flanked on one or both sides by a linker that joins the polypeptide or fragment to HBcAg sequence; and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids;

a method of inducing an immune response against influenza in a subject, which method comprises administering to the subject: (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop and the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid; and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids;

(i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first

and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which one or both of the copies of HBcAg comprises, in the el loop, influenza virus A surface polypeptide M2 or an immunogenic fragment thereof flanked on one or both sides by a linker that joins the polypeptide or fragment to HBcAg sequence; in combination with (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; for use in a method of vaccination of the human or animal body against influenza;

(i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop and the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid; in combination with (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; for use in a method of vaccination of the human or animal body against influenza;

- use of (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which one or both of the copies of HBcAg comprises, in the el loop, influenza virus A surface polypeptide M2 or an immunogenic fragment thereof flanked on one or both sides by a linker that joins the polypeptide or fragment to HBcAg sequence; in combination with (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; for the manufacture of a medicament for vaccination of the human or animal body against influenza; and

- use of (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the protein comprises a first and a second copy of HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop and the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid; in combination with (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; for the manufacture of a medicament for vaccination of the human or animal body against influenza. Brief description of the Figures

Figure 1 : Immunisation with Tandem Core containing Influenza 3 xM2e sequence generated antibody which recognises recombinant M2e peptide. Serum was collected 3 weeks after primary immunisation from 5 mice immunised with the Tandem Core +Influenza 3xM2e VLP (circle), Adjuvant only (triangle) or 14C2 monoclonal Ab to M2e peptide (square). Serum pools from 5 mice were tested in duplicate by ELISA to M2e, at 3 different serum dilutions.

Figure 2: Immunisation with Tandem Core VLP containing 3xM2e insert mitigates weight loss during a lethal flu challenge. 4 weeks after immunisation with Tandem Core containing 3xM2e insert (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Percentage weight was calculated as:

% weight = 100- (100 x (weight at day 0 -weight at day n /weight at day 0).

5 mice per group, representative of 2 individual experiments. Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 3 : Immunisation with Tandem Core VLP containing 3xM2e insert reduces clinical illness during a lethal flu challenge. 4 weeks after immunisation with Tandem Core containing HA 3xM2e (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Group clinical score was calculated as the addition of the individual clinical scores for each mouse in the group, 5 mice per group. Individual clinical scores were determined by the scale shown in Table 3. Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 4: Immunisation with Tandem Core VLP containing 3xM2e abrogates mortality following a lethal PR8 influenza challenge. 4 weeks after immunisation with Tandem Core containing 3xM2e insert (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Percent survival was determined as:

% survival =100 - (100* ( no. mice day 0- no. of surviving mice day(n)/ no. mice at day 0)

Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 5: Schematic depicting surface interface of influenza virus. Modified from

Park et al, J. Virol. March 1998 vol. 72 no. 3 2449-2455.

Figure 6: Immunisation with Tandem Core containing Influenza stalk sequence generated antibody which recognises recombinant hemagglutinin protein. Serum was collected 3 weeks after primary immunisation from 5 mice immunised with the Tandem Core +Influenza stalk VLP (circle), Adjuvant only (triangle) or PR8 infected mice (square). Serum pools were tested in duplicate by ELISA to rHA from A/PR8 HlNl, at 3 different serum dilutions.

Figure 7: Immunisation with Tandem Core VLP containing HA stalk insert mitigates weight loss during a lethal flu challenge. 4 weeks after immunisation with Tandem Core containing HA stalk insert (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Percentage weight was calculated as:

% weight = 100- (100 x (weight at day 0 -weight at day n /weight at day 0).

5 mice per group, representative of 2 individual experiments. Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 8: Immunisation with Tandem Core VLP containing HA stalk insert reduces clinical illness during a lethal flu challenge. 4 weeks after immunisation with Tandem Core containing HA stalk insert (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Group clinical score was calculated as the addition of the individual clinical scores for each mouse in the group, 5 mice per group.

Individual clinical scores were determined by the scale shown in Table 3. Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 9: Immunisation with Tandem Core VLP containing HA stalk insert abrogates mortality following a lethal PR8 influenza challenge. 4 weeks after immunisation with Tandem Core containing HA stalk insert (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Percent survival was determined as:

% survival =100 - (100* ( no. mice day 0- no. of surviving mice day(n)/ no. mice at day 0)

Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 10: Schematic showing HA protein stalk region. Modified from Kaminski and Lee, Front Immunol. 2011; 2: 76. Published online Dec 16, 2011. Prepublished online Sep 12, 2011.doi: 10.3389/fimmu.2011.00076.

Figure 11 : Immunisation with Tandem Core containing Influenza HA stalk and 3x M2e sequences generated antibody which recognises recombinant hemagglutinin protein and M2e peptide. Serum was collected 3 weeks after primary immunisation from 5 mice immunised with the Tandem Core VLP (circle), Adjuvant only (triangle) or PR8 infected mice (square). A monoclonal antibody to M2e (CI 4) was used as a positive control in the M2e ELISA (cross). Serum pools were tested in duplicate by ELISA to rHA from A/PR8 H1N1 or M2e peptide, at 3 different serum dilutions.

Figure 12: Immunisation with Tandem Core VLP containing Influenza stalk and 3x M2e sequences mitigates weight loss during an influenza challenge. 4 weeks after immunisation with Tandem Core VLP (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Percentage weight was calculated as:

% weight = 100- (100 x (weight at day 0 -weight at day n /weight at day 0).

5 mice per group, representative of 2 individual experiments. Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 13 : Immunisation with Tandem Core VLP containing Influenza stalk and 3x M2e sequences reduces clinical illness during a flu challenge. 4 weeks after immunisation with Tandem Core VLP (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Group clinical score was calculated as the addition of the individual clinical scores for each mouse in the group, 5 mice per group.

Individual clinical scores were determined by the scale shown in Table 3. Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 14: Immunisation with Tandem Core VLP containing Influenza stalk and 3x M2e sequences abrogates mortality following a lethal PR8 influenza challenge. 4 weeks after immunisation with Tandem Core VLP (circle) or Adjuvant only (triangle), mice were infected with 5xmLD50 of PR8 virus. Percent survival was determined as: % survival =100 - (100* (no. mice day 0- no. of surviving mice day (n)/ no. mice at day 0).

Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 15: Schematic of Tandem Core VLP containing Influenza derived inserts in both MIRs. Core 1 is shown in cross-hatch containing the HA stalk insert at the MIR. Core 2 is shown in black containing the triple M2e insert. The dimer shown (A) assembled into a virus like particle (VLP) which displays both Core 1 and 2 together with their inserts on the outside (B). Figure 16: Schematic of Tandem Core VLPl and VLP2 containing Influenza derived inserts in one or both MIRs. Core 1 shown in cross-hatch, containing the HA stalk insert at the MIR. Core 2 is shown in black containing the triple M2e insert or the "null", Lysine residue-containing, insert. The dimer shown (A) assembled into a virus like particle (VLPl), similarly the building block for VLP2 is shown (B). The assembled VLPs display both Core 1 and 2 together with their inserts on the outside (C).

Figure 17: Model depicting secondary structure of influenza virus HA stalk inserts inside VLPl (HA2.3) and VLP2 (LAID).

Figure 18: Immunisation with Tandem Core VLPs containing Influenza stalk and M2e sequences generated antibody which recognises recombinant hemagglutinin protein and M2e peptide. Points on the graph represent the average of 2 ELISA absorbance values from pooled serum of 5 mice per group. Seroconversion to hemagglutinin from H3N2 is shown triangles, to H1N1 as squares and M2 ectodomain as diamond shapes. The negative control is representative of reactivity to all 3 proteins from the adjuvant- only pooled serum (circles). This experiment was repeated 3 separate times.

Figure 19: Immunisation with Tandem Core VLP containing influenza derived inserts mitigates weight loss during an influenza challenge. 4 weeks after immunisation with Tandem Core VLPs (squares) or Adjuvant only (circles), mice were infected with 5xmLD50 of PR8 virus (2a) or X31 H3N2 (2b). Percentage weight was calculated as: % weight = 100- (100 x (weight at day 0 -weight at day n /weight at day 0). 5 mice per group, representative of 2 individual experiments. Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Figure 20: Immunisation with Tandem Core VLP containing influenza derived inserts reduces clinical illness during a flu challenge. 4 weeks after immunisation with Tandem Core VLPs (squares) or Adjuvant only (circles), mice were infected with

5xmLD50 of PR8 virus (3a) or X31 H3N2 (3b). Group clinical score was calculated as the addition of the individual clinical scores for each mouse in the group, 5 mice per group. Individual clinical scores were determined by the scale shown in Table 3. Mice that reached 20% weight loss, or an individual score of 15, either perished or were culled according to UK Home Office guidelines.

Figure 21 : Immunisation with Tandem Core VLPs containing influenza derived inserts abrogates mortality following a lethal influenza challenge. 4 weeks after immunisation with Tandem Core VLP (squares) or Adjuvant only (circles), mice were infected with 5x mLD50 of PR8 H1N1 (4a) or 3x mLD50 X31 H3N2 (4b) virus.

Percent survival was determined as: % survival =100 - (100* (no. mice day 0- no. of surviving mice day (n)/ no. mice at day 0). Mice that reached 20% weight loss either perished or were culled according to UK Home Office guidelines.

Brief description of the sequences

SEQ ID NO: 1 is the 183 amino acid protein of the ayw subtype plus a 29 amino acid pre-sequence of HBcAg and the corresponding nucleotide sequence.

SEQ ID NO: 2 is the 183 amino acid protein of the ayw subtype plus a 29 amino acid pre-sequence of HBcAg.

SEQ ID NO: 3 is the M2 amino acid sequence from influenza virus A strain A/34/PR8.

SEQ ID NO: 4 is a possible linker sequence for linking adjacent HBcAg units, for flanking the immunogenic polypeptide insert in the el loop or for joining together multiple immunogenic polypeptides in the el loop.

SEQ ID NO: 5 is a possible linker sequence for linking adjacent HBcAg units, for flanking the immunogenic polypeptide insert in the el loop or for joining together multiple immunogenic polypeptides in the el loop. SEQ ID NO: 5 is three repeats of the sequence of SEQ ID NO: 4.

SEQ ID NO: 6 is a wild-type M2e sequence.

SEQ ID NO: 7 is the same as the sequence of SEQ ID NO: 6 except that the cysteine at position 17 has been substituted with a serine.

SEQ ID NO: 8 is the same as the sequence of SEQ ID NO: 6 except that the cysteine at positions 17 and 19 have both been substituted with a serine.

SEQ ID NO: 9 is the universal M2e consensus sequence except that each cysteine at positions 17 and 19 have been substituted with a serine.

SEQ ID NO: 10 is a variant M2e sequence.

SEQ ID NO: 11 is the amino acid sequence of a tandem core with three M2e sequences inserted in one copy of HBcAg. The three M2e sequences inserted are flanked on both sides by a linker sequence. One linker spans amino acids 80 to 94 of SEQ ID NO: 11 and the other spans amino acids 167 to 181 of SEQ ID NO: 11 (amino acids shown in italics in Example 1). The linker sequences are identical to the sequence of SEQ ID NO: 5. The three M2e sequences span amino acids 95 to 166 of SEQ ID NO: 11 (amino acids shown underlined in Example 1). The M2e sequence spanning amino acids 95 to 118 of SEQ ID NO: 11 is identical to the sequence of SEQ ID NO: 9. The M2e sequence spanning amino acids 119 to 142 of SEQ ID NO: 11 is identical to the sequence of SEQ ID NO: 8. The M2e sequence spanning amino acids 143 to 166 of SEQ ID NO: 11 is identical to the sequence of SEQ ID NO: 10.

SEQ ID NO: 12 is the amino acid sequence of a tandem core with a HA stalk insert in one copy of HBcAg. The HA stalk insert spans amino acids 80 to 151 of SEQ ID NO: 12 (amino acids shown underlined in Example 2). The HA stalk insert spans amino acids 403-474 of the HA protein isolated from influenza A virus HlNl/Lux/09.

SEQ ID NO: 13 is the amino acid sequence of a tandem core with a HA stalk insert in one copy of HBcAg and three M2e sequences inserted in the other copy of HBcAg. The HA stalk insert spans amino acids 80 to 151 of SEQ ID NO: 13 (amino acids shown double underlined in Example 3). The HA stalk insert spans amino acids 403-474 of the HA protein isolated from influenza A virus HlNl/Lux/09. The three

M2e sequences inserted are flanked on both sides by a linker sequence. One linker spans amino acids 328 to 342 of SEQ ID NO: 13 and the other spans amino acids 415 to 429 of SEQ ID NO: 13 (amino acids shown in italics in Example 3). The linker sequences are identical to the sequence of SEQ ID NO: 5. The three M2e sequences span amino acids 343 to 414 of SEQ ID NO: 13 (amino acids shown underlined in Example 3). The M2e sequence spanning amino acids 343 to 366 of SEQ ID NO: 13 is identical to the sequence of SEQ ID NO: 9. The M2e sequence spanning amino acids 367 to 390 of SEQ ID NO: 13 is identical to the sequence of SEQ ID NO: 8. The M2e sequence spanning amino acids 391to 414 of SEQ ID NO: 13 is identical to the sequence of SEQ ID NO: 10.

SEQ ID NO: 14 is the same sequence as SEQ ID NO: 6 with an addition of an -

OH group at the C-terminus.

SEQ ID NO: 15 is a sequence which HBcAg may comprise in order to balance the a-helices.

SEQ ID NO: 16 is a possible linker sequence for linking adjacent HBcAg units, for flanking the immunogenic polypeptide insert in the el loop or for joining together multiple immunogenic polypeptides in the el loop. It was used as a linker adjacent to the M2e inserts in VLP1 in Example 4. SEQ ID NO: 17 is the DNA sequence of a tandem core with a HA stalk insert in one copy of HBcAg and three M2e sequences inserted in the other copy of HBcAg (VLPl in Example 4).

SEQ ID NO: 18 is the amino acid sequence of a tandem core with a HA stalk insert in one copy of HBcAg and three M2e sequences inserted in the other copy of

HBcAg (VLPl in Example 4). The HA stalk insert spans amino acids 80 to 151 of SEQ ID NO: 18 (amino acids shown underlined in Example 4). The HA stalk insert spans amino acids 403-474 of the HA protein isolated from influenza A virus HlNl/Lux/09. The three M2e sequences inserted are flanked on both sides by a linker sequence. One linker spans amino acids 328 to 342 of SEQ ID NO: 18 and the other spans amino acids 415 to 428 of SEQ ID NO: 18 (amino acids shown in italics in Example 4). The linker sequences are the sequences of SEQ ID NO: 5 and SEQ ID NO: 16 respectively. The three M2e sequences span amino acids 343 to 414 of SEQ ID NO: 18 (amino acids shown underlined in Example 4). The M2e sequence spanning amino acids 343 to 366 of SEQ ID NO: 18 is identical to the sequence of SEQ ID NO: 9. The M2e sequence spanning amino acids 367 to 390 of SEQ ID NO: 18 is identical to the sequence of SEQ ID NO: 8. The M2e sequence spanning amino acids 391 to 414 of SEQ ID NO: 18 is identical to the sequence of SEQ ID NO: 10.

SEQ ID NO: 19 is the DNA sequence of a tandem core with a HA stalk insert in one copy of HBcAg and a "null" insert in the other copy of HBcAg (VLP2 in Example 4).

SEQ ID NO: 20 is the amino acid sequence of a tandem core with a HA stalk insert in one copy of HBcAg and a "null" insert in the other copy of HBcAg (VLP2 in Example 4). The HA stalk insert spans amino acids 80 to 134 of SEQ ID NO: 20 (amino acids shown underlined in Example 4). The HA stalk insert spans amino acids 421-475 of the HA protein isolated from influenza A virus H3N2/HK/68. The "null" insert spans amino acids 311 to 324 of SEQ ID NO: 20 and corresponds to SEQ ID NO: 21.

SEQ ID NO: 21 is the amino acid sequence of the "null" insert that was inserted into the second copy of HBcAg in VLP2 in Example 4.

SEQ ID NO: 22 is the amino acid sequence of the HA stalk spanning amino acids

403-474 of the HA protein isolated from influenza A virus HlNl/Lux/09. This corresponds to amino acids 80 to 151 of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 18. SEQ ID NO: 23 is the amino acid sequence of the HA long alpha helix spanning amino acids 420-474 of the HA protein isolated from influenza A virus HlNl/Lux/09. This corresponds to amino acids 97 to 151 of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 18.

SEQ ID NO: 24 is the amino acid sequence of the HA stalk region spanning amino acids 421-475 of the HA protein isolated from influenza A virus H3N2/HK/68. This corresponds to amino acids 80 to 134 of SEQ ID NO: 20.

Detailed description of the invention

In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an immunogenic polypeptide" includes two or more such polypeptides.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

The current inventors have developed tandem core constructs which have overcome difficulties associated with introducing immunogenic polypeptides, including influenza virus A surface polypeptide M2, into the HBcAg cores.

The tandem constructs are a genetic fusion of two HBcAg genes such that the resulting recombinant protein forms two parallel "spikes" which are indistinguishable from wild type core proteins which naturally dimerise. The tandem core proteins form VLPs in a manner similar to monomeric core proteins. "Tandem core", "tandem construct" and "tandem core construct" are used interchangeably herein. The terms may be used to describe tandem HBcAg cores which do or do not have an immunogenic polypeptide in the el loop of one or both copies of HBcAg.

Provided are tandem core constructs comprising a linker on one or both sides of influenza virus A surface polypeptide M2 or an immunogenic fragment thereof which is inserted in the el loop of one or both copies of HBcAg. Also provided are tandem core constructs which comprise influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop of one or both copies of HBcAg and in which the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid. Also provided are tandem core constructs comprising hemagglutinin (HA) or an immunogenic fragment thereof inserted in the el loop of one copy of HBcAg and a sequence of less than 20 amino acids, optionally comprising a Lysine (K) residue flanked by Glycine and Serine residues, inserted into the other copy of HBcAg. The features described herein may apply to any of the tandem constructs described above. For example, the influenza virus A surface polypeptide M2 polypeptide or immunogenic fragment thereof in the tandem core construct described above which comprises the linker on one or both sides of the influenza virus A surface polypeptide M2 polypeptide or immunogenic fragment thereof may have substituted cysteines at position 17 and/or position 19.

Hepatitis B core antigen (HBcAg)

Tandem Core is a virus like particle (VLP) based on FIBcore protein which is known to be highly immunogenic and has the ability to confer immunogenic properties to protein inserts within its structure. Furthermore the virus-like properties of Tandem Core can display foreign antigens while maintaining structural epitopes on a multimeric display platform. Additionally Tandem Core is able to carry multiple inserts due to its double insertion site.

FIBcAg has 183 or 185 amino acids (aa) depending on the subtype of FIB V. The sequence of the 183 amino acid protein of the ayw subtype plus a 29 amino acid pre- sequence is shown in SEQ ID NO: 2. The mature HBcAg runs from the Met residue at position 30 to the Cys residue at the extreme C-terminus, with the sequence from positions 1 to 29 being a pre-sequence.

The protein comprises two copies of HBcAg (the "first copy" and the "second copy") forming a dimer. "Copies" and "units" of HBcAg are used interchangeably herein. Dimers of HBcAg form the structural building blocks of VLPs. The HBcAg units are generally joined together in a head-to-toe fashion, i.e. the C-terminus of one unit is joined to the N-terminus of the adjacent unit. The "first copy" may be either the N-terminal or C-terminal copy. The units may be joined directly by a covalent bond (e.g. a peptide bond), but preferably they are joined by a linker which spaces the adjacent units apart and thereby prevents any problem with disruption of the packing of adjacent units. The nature of the linker is discussed below. The joined dimer forms the "tandem construct", "tandem core" or "tandem core construct" which can have immunogenic polypeptides inserted into one or both el loops.

The HBcAg in the protein may be native full length HBcAg. However, in accordance with one aspect of the invention, at least one of the units is a modified form of HBcAg having the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof in the el loop. The other copy of HBcAg may be native HBcAg or may be a modified version of HBcAg as described herein. The modified version of HBcAg may have another immunogenic polypeptide in the el loop. The tandem construct may have the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof inserted in the el loop of both HBcAg copies. There may be more than one type of immunogenic polypeptide in one HBcAg copy. Examples of possible immunogenic polypeptides are discussed herein.

In accordance with another aspect of the invention, one of the units is a modified form of HBcAg having the influenza virus hemagglutinin (HA) polypeptide or an immunogenic fragment thereof in the el loop. The other copy of HBcAg may be native HBcAg or may be a modified version of HBcAg as described further herein.

The immunogenic polypeptide may be flanked on one or both sides by a linker. Therefore a tandem construct may contain one or more linkers flanking the immunogenic polypeptide in one copy of the HBcAg or in both copies of the HBcAg. If there is more than one immunogenic polypeptide in one or each of the el loop or if there is more than one copy of the same immunogenic polypeptide in one or each el loop then there can be linkers flanking one or each of the immunogenic polypeptides on one or both sides of the immunogenic polypeptides. Each of the immunogenic polypeptides may or may not be flanked on one of both sides by a linker. There can be flanking linkers which join adjacent immunogenic polypeptides and/or flanking linkers which join the immunogenic polypeptide to the el loop. The tandem construct may also have a linker joining the HBcAg units. The nature of the linkers is discussed below.

As a general rule, any modifications are chosen so as not to interfere with the conformation of HBcAg and its ability to assemble into particles. Such modifications are made at sites in the protein which are not important for maintenance of its conformation, for example in the el loop, the C-terminus and/or the N-terminus. The el loop of HBcAg can tolerate insertions of e.g. from 1 to 600 amino acids without destroying the particle-forming ability of the protein. The HBcAg sequence may be modified by substitution, insertion, deletion or extension. The size of insertion, deletion or extension may, for example, be from 1 to 600 aa, from 1 to 500 aa, from 1 to 400 aa, from 1 to 300 aa, from 1 to 200 aa, from 3 to 100 aa or from 6 to 50 aa. Substitutions may involve a number of amino acids up to, for example, 1, 2, 5, 10, 20 or 50 amino acids over the length of the HBcAg sequence. An extension may be at the N- or C-terminus of HBcAg. A deletion may be at the N- terminus, C-terminus or at an internal site of the protein. Substitutions may be made at any position in the protein sequence. Insertions may also be made at any point in the protein sequence, but are typically made in surface-exposed regions of the protein such as the el loop. An inserted sequence may carry an immunogenic polypeptide. More than one modification may be made to each HBcAg unit. Thus, it is possible to make a terminal extension or deletion and also an internal insertion. For example, a truncation may be made at the C-terminus and an insertion may be made in the el loop.

Each part of the HBcAg sequence in the protein of the invention preferably has at least 70% sequence identity to the corresponding sequence of a natural HBcAg protein, such as the protein having the sequence shown in SEQ ID NO: 2. More preferably, the identity is at least 80%, at least 90%, at least 97%, at least 98% or at least 99%. Methods of measuring protein homology are well known in the art and it will be understood by those of skill in the art that in the present context, homology is calculated on the basis of amino acid identity (sometimes referred to as "hard homology").

For example the UWGCG Package (Devereux et al (1984) Nucleic Acids Research 12: 387-395) provides the BESTFIT program which can be used to calculate homology (for example used on its default settings). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290- 300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:

5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The el loop of HBcAg is at positions 68 to 90 of the mature sequence, and an immunogenic polypeptide may be inserted anywhere between these positions.

Preferably, the immunogenic polypeptide is inserted in the region from positions 69 to 90, 71 to 90 or 75 to 85. Most preferred is to insert the immunogenic polypeptide between amino acid residues 79 and 80 or between residues 80 and 81. When an immunogenic polypeptide is inserted, the entire sequence of HBcAg may be maintained, or alternatively the whole or a part of the el loop sequence may be deleted and replaced by the protein sequence. Thus, amino acid residues 69 to 90, 71 to 90 or 75 to 85 may be replaced by an immunogenic polypeptide. Where an immunogenic polypeptide replaces el loop sequence, the replacement sequence is generally not shorter than the sequence that it replaces.

A C-terminal truncation of HBcAg will generally not go beyond aa 144 because if any further truncation is made particles may not form. Thus, the deleted amino acids may, for example, comprise aa 144 to the C-terminal aa (aa 183 or 185), aa 150 to the C- terminal aa, aa 164 to the C-terminal aa or aa 172 to the C-terminal aa. The C-terminus of HBcAg binds DNA, and truncation of the C-terminus therefore reduces or completely removes DNA from preparations of HBcAg and HBcAg hybrid proteins.

The protein of the invention forms particles which preferably resemble the particles formed by native HBcAg. The particle of the invention comprises multiple copies of one or more proteins of the invention. The particle can be in the form of a VLP. The particles of the invention are typically at least 10 nm in diameter, for example from 10 to 50 nm or from 20 to 40 nm in diameter, but preferably they are about 27 nm in diameter (which is the size of native HBcAg particles). They comprise multiple HBcAg units, for example from 150 to 300 units, but generally they are fixed to about 180 or about 240 units (which are the numbers of units in native HBcAg particles). As the protein of the invention can be a dimer, this means that the number of protein monomers in the particles may be from 75 to 150 but is generally about 90 or about 120.

The two a-helices that comprise the HBc spike region are not symmetrical and so the resulting MIR does not point completely vertically from the VLP, but is slightly offset. Molecular modelling thus suggests that any antigen that was inserted may lie parallel to the VLP, rather than at right angles. This could possibly lead to steric hindrance and a decrease in immunogenicity. The HBcAg may comprise an inserted sequence which acts to "balance" the a-helices by adding an extra turn or turns to the first helix (which lies at positions 50 to 73 of the mature sequence). This results in the presentation of an inserted immunogenic polypeptide in a perpendicular orientation to the VLP. This may be achieved by inserting from 3 to 12 amino acids (e.g. 3, 5 or 7 amino acids) into HBcAg. These amino acids are preferably uncharged amino acids such as alanine, leucine, serine and threonine. The inserted sequence is preferably AAALAAA (SEQ ID NO: 15). The insertion may be at a site between amino acids 50 and 75 of the mature sequence, for example at a site between residues 60 and 75 or residues 70 and 73.

The particle of the invention may comprise more than one protein of the invention, i.e. a mixed particle. The inventors found that inserting a "null" insert of less than 20 amino acids in length, and as further described herein, into one copy of HBcAg in the tandem construct allows the inserted antigen(s) in the other copy of HBcAg to fold and/or be presented correctly. A protein comprising one type of inserted antigen(s) in one copy of HBcAg and a "null" insert in the other copy of HBcAg can be combined, into the same particle, with a protein comprising a different type of inserted antigen(s) in one copy of HBcAg and a "null" insert in the other copy of HBcAg. This allows the particle to comprise multiple copies of both types of inserted antigen(s), well spaced apart and in a stable form. The inserted antigens are essentially placed with a "spacer" between each other, thus providing room to correctly fold. Monomeric HBcAg would not be able to achieve this.

Linkers

The linker between adjacent HBcAg units, flanking the inserts in the el loop and/or joining adjacent inserts in the el loop is generally a chain of amino acids at least 1.5 nm (15 A) in length, for example from 1.5 to 10 nm, from 1.5 to 5 nm or from 1.5 to 3 nm. It may, for example, comprise 4 to 40 aa or 10 to 30 aa, preferably 15 to 21 aa. The linker is generally flexible. The amino acids in the linker may, for example, include or be entirely composed of glycine, serine and/or proline. For example, the linker may comprise one or more repeats of the sequence Gly _n Ser (G _n S) where n is 2, 3, 4, 5, 6, 7 or 8. A preferred linker comprises one, two or more repeats of the sequence

GlyGlyGlyGlySer (GGGGS) (SEQ ID NO: 4). For example, GGGGSGGGGSGGGGS (SEQ ID NO: 5). Alternatively, the linker may comprise one or more GlyPro (GP) dipeptide repeats. The number of repeats may, for example, be from 1 to 18, preferably from 3 to 12. In the case of G ₂ S repeats, the use of 5, 6 or 7 repeats has been found to allow the formation of particles. A preferred linker between adjacent HBcAg units is 7 repeats of G ₂ S. The linker may correspond to the hinge region of an antibody; this hinge region is thought to provide a flexible joint between the antigen-binding and tail domains of antibodies.

An example of the structure of a tandem construct which contains linkers comprises the following:

[the part of the "first copy" of HBcAg that is N-terminal to the el loop] - [first linker] - [influenza virus A surface polypeptide M2 or immunogenic fragment thereof] - [second linker] - [the part of the "first copy" of HBcAg that is C-terminal to the el loop] - [third linker] - [the part of the "second copy" of HBcAg that is N-terminal to the el loop] - [optional immunogenic polypeptide such as HA stalk] - [the part of the "second copy" of HBcAg that is C-terminal to the el loop]

If there is more than one linker in the tandem construct then they may be the same or different from one another. For example, they may be all the same, they may all be different from one another, two or more linkers may be the same but different from one or more other linkers, and so on. Where there is an immunogenic polypeptide in the "second copy", then the inserted sequence in each of the el loops may have a linker on one or both sides. If there are linkers on both sides of both inserts then an example of the structure of a tandem construct comprises the following:

[the part of the "first copy" of HBcAg that is N-terminal to the el loop] - [first linker] - [influenza virus A surface polypeptide M2 or immunogenic fragment thereof] - [second linker] - [the part of the "first copy" of HBcAg that is C-terminal to the el loop] - [third linker] - [the part of the "second copy" of HBcAg that is N-terminal to the el loop] - [fourth linker] - [optional immunogenic polypeptide such as HA stalk] - [fifth linker] - [the part of the "second copy" of HBcAg that is C-terminal to the el loop]

The tandem core constructs of one aspect of the invention comprise an influenza virus A surface polypeptide M2 or immunogenic fragment thereof in the el loop of one copy of HBcAg and optionally another immunogenic polypeptide in the el loop of the other copy of HBcAg. The immunogenic polypeptide in the other copy of HBcAg

(described as the "optional immunogenic polypeptide" in the arrangements above) may be any immunogenic polypeptide as described herein. The immunogenic polypeptide therefore may be another influenza polypeptide or an immunogenic fragment thereof. It may be the influenza virus A surface polypeptide M2 or an immunogenic fragment thereof. Therefore there may be influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop of both copies of HBcAg. The influenza virus A surface polypeptide M2 or the immunogenic fragment thereof in each el loop may be the same or different. The immunogenic polypeptide may be derived from HA. For example, the immunogenic polypeptide may be HA stalk or an immunogenic fragment thereof. Therefore there may be influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop of one copy of HBcAg and HA stalk or an immunogenic fragment thereof in the el loop of the other copy of HBcAg.

The tandem core constructs of another aspect of the invention comprise the influenza virus hemagglutinin (HA) polypeptide or an immunogenic fragment thereof in the el loop of one copy of HBcAg. The el loop of the other copy of HBcAg comprises a sequence of less than 20 amino acids.

As described herein there may be one or more copies of the immunogenic polypeptide in the el loop of one copy of HBcAg. For example, there may be one or more copies of influenza virus A surface polypeptide M2 or the immunogenic fragment thereof in one el loop. There may be one or more copies of HA stalk or the

immunogenic fragment thereof in one el loop. There may be up to 2, 3, 4, 6 or 8 copies of an immunogenic polypeptide in one el loop. There may be multiple copies of an immunogenic polypeptide in each el loop. For example, the tandem construct may comprise one, two, three, four or five copies of influenza virus A surface polypeptide M2 or the immunogenic fragment thereof in the el loop of one HBcAg and one, two or three copies of HA stalk or the immunogenic fragment thereof in the el loop of the other HBcAg. The tandem construct may therefore comprise three copies of influenza virus A surface polypeptide M2 or the immunogenic fragment thereof in the el loop of one

HBcAg and one copy of HA stalk or the immunogenic fragment thereof in the el loop of the other HBcAg. Further, the tandem construct may comprise three copies of influenza virus A surface polypeptide M2 or the immunogenic fragment thereof in the el loop of one HBcAg and two or three copies of HA stalk or the immunogenic fragment thereof in the el loop of the other HBcAg. Each copy in one el loop may be the same or different. For example, there may be two, three, four or five (preferably three) different sequences of influenza virus A surface polypeptide or immunogenic fragment thereof in one el loop. There may be linkers between copies of the immunogenic polypeptide inserted into one el loop. Therefore there may be linkers which join one or more immunogenic polypeptides to the el loop as well as linkers which join together multiple immunogenic polypeptides inserted into one el loop. For example the arrangement of the tandem construct may be as follows:

[the part of the "first copy" of HBcAg that is N-terminal to the el loop] - [first linker] - [influenza virus A surface polypeptide M2 or immunogenic fragment thereof] - [second linker] - [influenza virus A surface polypeptide M2 or immunogenic fragment thereof] [third linker] - [influenza virus A surface polypeptide M2 or immunogenic fragment thereof] - [fourth linker] - [the part of the "first copy" of HBcAg that is C- terminal to the el loop] - [fifth linker] - [the part of the "second copy" of HBcAg that is N-terminal to the el loop] - [optional immunogenic polypeptide such as HA stalk] - [the part of the "second copy" of HBcAg that is C-terminal to the el loop] Influenza virus A surface polypeptide M2 (M2)

The purpose of the protein of the invention is that it can be used to induce an immune response to influenza, particularly influenza virus A, and can therefore be used as an influenza vaccine. The protein of one aspect of the invention has the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof inserted into the el loop of one or both copies of HBcAg. A protein of the invention may have M2 or the immunogenic fragment thereof inserted into the el loop of both copies of HBcAg. The influenza virus A surface polypeptide M2 is a proton-selective ion channel protein, integral in the viral envelope of the influenza virus. The channel itself is a homotetramer (consists of four identical M2 units), where the units are helices stabilized by two disulfide bonds. The influenza virus A surface polypeptide M2 unit consists of three protein domains: the 24 amino acids on the N-terminal end, exposed to the outside environment, the 19 hydrophobic amino acids on the transmembrane region, and the 54 amino acids on the C-terminal end, oriented towards the inside of the viral particle. The full length sequence of M2 protein from influenza A virus strain A/34/PR8 is shown in SEQ ID NO: 3.

The influenza virus A surface polypeptide M2 to be inserted into the el loop of HBcAg is derived from influenza virus A. It can be derived from the sequence in SEQ ID NO: 3. A full length influenza virus A surface polypeptide M2, i.e. the full 97 amino acid sequence, may be inserted into the el loop of HBcAg. For example, the full length sequence of SEQ ID NO: 3 may be inserted. The influenza virus A surface polypeptide M2 may be a naturally occurring M2 protein or may be a variant of a naturally occurring influenza virus A surface polypeptide M2.

More than one copy of influenza virus A surface polypeptide M2 or an immunogenic fragment thereof may be inserted into the el loop of one or both copies of HBcAg. For example, 2, 3, 4, 5, 6, 7 or 8 copies of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof may be inserted in the el loop of one or both copies of HBcAg. For example, 1, 2 or 3 copies may be inserted in the el loop of one or both copies of HBcAg. Therefore 1, 2 or 3 copies of influenza virus A surface polypeptide M2 or the immunogenic fragment thereof may be inserted in both el loops or in one of the el loops. Where there is more than one copy of influenza virus A surface polypeptide M2 or the immunogenic fragment thereof in the el loop, the sequences for each copy may be identical or may be different. If the sequences are different, they can be inserted in any order. For example, the "first copy", "second copy", "third copy" and so on may be in any order from the N-terminus to the C- terminus in the el loop. For example, the "third copy" may be N-terminus to the "first copy".

An influenza virus A surface polypeptide M2 sequence is set out in SEQ ID NO:

3. The sequence of the influenza virus A surface polypeptide M2 may have homology with SEQ ID NO: 3 or any naturally occurring influenza virus A surface polypeptide M2, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity, for example over the full sequence or over a region of at least 20, for example at least 30, at least 40, at least 50, at least 60, at least 80 or more contiguous amino acids. Methods of measuring protein homology are well known in the art and are discussed above in relation to the HBV core protein.

The homologous protein typically differs from the naturally occurring influenza virus A surface polypeptide M2 sequence by substitution, insertion or deletion, for example from 1, 2, 3, 4, 5 to 8 or more substitutions, deletions or insertions. The substitutions are preferably 'conservative' and may be made, for example, according to Table 1. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

Table 1

The sequence of the influenza virus A surface polypeptide M2 or immunogenic fragment thereof insert may be derived from any subtype of influenza type A (see e.g. Sharp 2002 Cell Vol. 108, 305-312, "Origins of Human Virus Diversity" and Shi et al 2010, PLOSO E, 5(12) "A Complete Analysis of HA and NA Genes of Influenza A Viruses"). For example, from any of the HA subtypes such as HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15 or H16 and/or from any of the NA subtypes such as Nl, N2, N3, N4, N5, N6, N7, N8 or N9. Preferably the influenza virus A surface polypeptide M2 or immunogenic fragment thereof insert may be derived from H1N1, H5N1, H3N2, H7N7, H1N2, H2N2, H7N3, H5N2, H1N7, H9N2, H7N2 or H10N7. Even more preferably the influenza virus A surface polypeptide M2 or immunogenic fragment thereof insert may be derived from H3N2, H5N1, H1N1 or H7N7.

An immunogenic fragment of influenza virus A surface polypeptide M2 to be used as an insert is a shortened version of a full length influenza virus A surface polypeptide M2 that retains the ability of inducing an immune response. In some instances, a fragment may be at least 10%, such as at least 20%, at least 30%, at least 40% or at least 50%, preferably at least 60%, more preferably at least 70%, still more preferably at least 80%, even more preferably at least 90% and still more preferably at least 95% of the length of a naturally occurring influenza virus A surface polypeptide M2 sequence or the sequence of SEQ ID NO: 3. For example a fragment may be from 6 to 96 aa, from 6 to 50 aa or from 6 to 25 aa in length.

Preferably the immunogenic fragment is from a region of influenza virus A surface polypeptide M2 that is exposed on the surface of the virion. It is preferred that the immunogenic fragment is influenza virus A surface polypeptide M2 ectodomain (M2e), which is the external domain of the influenza virus A surface polypeptide M2 protein. The sequence of M2e may be a universal M2e consensus sequence. SEQ ID NO: 9 shows a universal sequence which has had the cysteines at positions 17 and 19 substituted with serines. The sequence of M2e may be a variant of a universal M2e consensus sequence. The sequence of the M2e may have homology with a universal M2e consensus sequence or any naturally occurring M2e, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity, for example over the full sequence or over a region of at least 8, for example at least 10, at least 15 or at least 20 or more contiguous amino acids. Methods of measuring protein homology are well known in the art and are discussed above in relation to the HBV core protein. The sequence may vary by substitution, addition and/or deletion of one or more amino acid. For example, there may be up to 18, up to 15, up to 12, up to 10 or up to 5 substitutions, deletions or additions. The sequence may vary by only deletions, only additions or only substitutions. The sequence may vary by a combination of deletion and addition, deletion and substitution, addition and substitution, or deletion, addition and substitution. Preferably there are one, two, three, four, five or six deletions, additions and substitutions. The substitutions are preferably 'conservative' and may be made, for example, according to Table 1 above. Examples of substituted forms of the M2e sequence are used in Examples 1 and 3. The sequence of M2e may be derived from any subtype of the influenza virus A, such as those listed above in relation to influenza virus A surface polypeptide M2. It is preferred that the most common variants of M2e are used. For example, see the sequences for M2e used in Examples 1 and 3. The immunogenic fragment of influenza virus A surface polypeptide M2 can be any of these sequences. Some of these sequences have had the cysteines at positions 17 and/or 19 substituted with serines. Any of the sequences of influenza virus A surface polypeptide M2 or the immunogenic fragment thereof such as M2e can be modified in this way as discussed further below. The M2e sequence from influenza A virus strain A/34/PR8 is the first 24 amino acids of SEQ ID NO: 3. A fragment of influenza virus A surface polypeptide M2 can comprise or be amino acids 1 to 24 of SEQ ID NO: 3.

Substitution or deletion of cysteines in influenza virus A surface polypeptide M2

The cysteine amino acid at position 17, the cysteine amino acid at position 19 or the cysteine amino acid at both positions 17 and 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof can be deleted or substituted with an alternative amino acid. There can be a combination of deletion and substitution. For example, the cysteine at position 17 is deleted and the cysteine at position 19 is substituted or the cysteine at position 17 is substituted and the cysteine at position 19 is deleted. Positions 17 and 19 of influenza virus A surface polypeptide M2 or the immunogenic fragment thereof are positions 17 and 19 from the N-terminus of the mature influenza virus A surface polypeptide M2. For example, positions 17 and 19 of SEQ ID NO: 3. As described above, the fragment of influenza virus A surface polypeptide M2 can be M2e. Therefore the cysteine at position 17, the cysteine at position 19 or both of the cysteines at positions 17 and 19 of M2e can be deleted or substituted with an alternative amino acid.

The "alternative amino acid" can be any amino acid which is not cysteine and which enables the formation of VLPs which in turn can induce immune responses in a subject. The substitutions are preferably 'conservative' and may be made, for example, according to Table 1 above. The cysteine is preferably substituted with serine, threonine or methionine. Serine is most preferred. Example M2e sequences which have one or both cysteines substituted are shown in Table 2 of Example 1.

Immunogenic polypeptide

The flexibility of the tandem core system means that the protein of the invention, in addition to comprising M2 or an immunogenic fragment thereof, may comprise one or more further immunogenic polypeptides. For example, the protein of the invention may comprise one or more further influenza virus derived immunogenic polypeptides in order to induce an excellent immune response to influenza virus. Alternatively, the protein of the invention may comprise one or more immunogenic polypeptides derived from a different source such as a different pathogen or allergen, in order to simultaneously induce immune responses to influenza virus and to a different pathogen or allergen. Therefore, although the protein of the invention must have the influenza virus A surface polypeptide M2 or an immunogenic fragment thereof inserted into the el loop of at least one copy of HBcAg, the el loop of the other copy of HBcAg in the protein may comprise any other type of immunogenic polypeptide(s).

The immunogenic polypeptide comprises a sequence of amino acids which is capable of inducing an immune response. The immunogenic polypeptide may be conformational or linear. It may be, for example, a sequence of from 6 to 600 aa, 6 to 300 aa, 6 to 200 aa, 50 to 200 aa, 100 to 200 aa, 6 to 120 aa, 20 to 90 aa, 40 to 90 aa or 60 to 90 aa.

Large and/or hydrophobic insertions can be accommodated without VLP disruption. The immunogenic polypeptide to be used as an insert may be of any suitable size that does not disrupt VLP formation. It is preferably less than 100 kDa, for example less than 80 kDa, less than 60 kDa, less than 40 kDa, less than 20 kDa, less than 10 kDa or less than 5 kDa. It may be more than 5 kDa, 10 kDa, 20 kDa, or 30 kDa.

The protein of the invention may contain more than one immunogenic polypeptide, for example up to 2, 3, 4, 6 or 8 immunogenic polypeptides. More than one copy of an immunogenic polypeptide may be inserted in one or both copies of HBcAg; for example, from 2 to 8 copies may be inserted, e.g. 2, 3, 4, 5, 6, 7 or 8 copies may be inserted. Where there are two or more immunogenic polypeptides in the protein of the invention, they may be from the same or different organisms and from the same or different proteins.

The immunogenic polypeptide may comprise one or more T-cell or B-cell epitopes. If it comprises a T-cell epitope, it may be a cytotoxic T-lymphocyte (CTL) epitope or a T-helper (Th) cell epitope (e.g. a Thl or Th2 epitope). In a preferred embodiment of the invention, the immunogenic polypeptide comprises a T-helper cell epitope and a B-cell or a CTL epitope. The presence of the T-helper cell epitope enhances the immune response against the B-cell or CTL epitope.

The choice of immunogenic polypeptide depends on the disease that it is wished to vaccinate against or treat. The immunogenic polypeptide may, for example, be from a pathogenic organism, a cancer-associated antigen or an allergen. The pathogenic organism may, for example, be a virus, a bacterium or a protozoan.

The immunogenic polypeptide may be derived from any pathogen, such as but not limited to, a virus, including a member of the orthomyxoviridae (including for instance influenza A, B and C viruses), adenoviridae (including for instance a human adenovirus), Caliciviridae (such as Norwalk virus group), herpesviridae (including for instance HSV-1, HSV-2, EBV, CMV and VZV), papovaviridae (including for instance Human Papilloma Virus - HPV), poxviridae (including for instance smallpox and vaccinia), parvoviridae (including for instance parvovirus B19), reoviridae (including for instance a rotavirus), coronaviridae (including for instance SARS),flaviviridae

(including for instance yellow fever, West Nile virus, dengue, hepatitis C and tick-borne encephalitis), picornaviridae (including enteroviruses, polio, rhinovirus, and hepatitis A), togaviridae (including for instance rubella virus), filoviridae (including for instance Marburg and Ebola), paramyxoviridae (including, a parainfluenza virus, respiratory syncitial virus (RSV), mumps and measles), rhabdoviridae (including for instance rabies virus), bunyaviridae (including for instance Hanta virus), retroviridae (including for instance HIV and HTLV - Human T-cell Lymphoma virus) and hepadnaviridae

(including for instance hepatitis B).

The immunogenic polypeptide may be derived from bacteria, including

Burkholderia, M. tuberculosis, Chlamydia, N. gonorrhoeae, Shigella, Salmonella, Vibrio Cholera, Treponema pallidua, Pseudomonas, Bordetella pertussis, Brucella,

Franciscella tulorensis, Helicobacter pylori, Leptospria interrogans, Legionella pnumophila, Yersinia pestis, Streptococcus (types A and B), Pneumococcus, Meningococcus, Hemophilus influenza (type b), Complybacteriosis, Moraxella catarrhalis, Donovanosis, and Actinomycosis, fungal pathogens including Candidiasis and Aspergillosis, and parasitic pathogens including Toxoplasma gondii, Taenia, Flukes, Roundworms, Flatworms, Amebiasis, Giardiasis, Cryptosporidium, Schitosoma, Pneumocystis carinii, Trichomoniasis and Trichinosis.

The immunogenic polypeptide may be derived from a pathogen that infects through a) the respiratory tract, b) the geni to-urinary system or c) the gastrointestinal tract. Examples of such pathogens include a) members of the adenoviridae,

paramyxoviridae and poxviridae, rhinovirus, influenza, and Hanta virus, b) Ureaplasma urealyticum, Neisseria gonorrhoeae, Gardnerella vaginalis, Trichomonas vaginalis, Treponema pallidum, Chlamydia trachomatis, Haemophilus ducreyi, herpes simplex virus, HPV, HIV, Candida albicans, Treponema pallidum, and Calmatobacterium granulomatis, and c) Shigella, Salmonella, Vibrio Cholera, E.coli, Entamoeba histolytica, Campylobacter, Clostridium, Yersinia, rotavirus, norovirus, adenovirus, astrovirus, Roundworms, Flatworms, Giardiasis, and Cryptosporidium.

The immunogenic polypeptide to be used in the invention may be derived from a cancer such as, but not limited to, cancer of the lung, pancreas, bowel, colon, breast, uterus, cervix, ovary, testes, prostate, melanoma, Kaposi's sarcoma, a lymphoma (e.g. EBV-induced B-cell lymphoma) and a leukaemia. Specific examples of tumour associated antigens include, but are not limited to, cancer-testes antigens such as members of the MAGE family (MAGE 1, 2, 3 etc), NY-ESO-1 and SSX-2,

differentiation antigens such as tyrosinase, gplOO, PSA, Her-2 and CEA, mutated self antigens and viral tumour antigens such as E6 and/or E7 from oncogenic HPV types. Further examples of particular tumour antigens include MART-1, Melan-A, p97, beta- HCG, GalNAc, MAGE- 1 , MAGE-2, MAGE-4, MAGE- 12, MUC 1 , MUC2, MUC3 , MUC4, MUC 18, CEA, DDC, P1A, EpCam, melanoma antigen gp75, Hker 8, high molecular weight melanoma antigen, K19, Tyrl, Tyr2, members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen), PSMA (prostate specific membrane antigen), prostate secretary protein, alpha-fetoprotein, CA125, CA19.9, TAG-72, BRCA- 1 and BRCA-2 antigen.

Examples of other candidate immunogenic polypeptide for use in the invention include the following antigens: the influenza antigens HA (hemagglutinin), NA

(neuraminidase), P (nucleoprotein/nucleocapsid protein), Ml, M2, PB1, PB2, PA, NS1 and NS2; the HIV antigens gp 120, gp 160, gag, pol, Nef, Tat and Ref; the malaria antigens CS protein and Sporozoite surface protein 2; the herpes virus antigens EBV gp340, EBV gp85, HSV gB, HSV gD, HSV gH, HSV early protein product,

cytomegalovirus gB, cytomegalovirus gH, and IE protein gP72; the human papilloma virus antigens E4, E6 and E7; the respiratory syncytial virus antigens F protein, G protein, and N protein; the pertactin antigen of B. pertussis; the tumor antigens carcinoma CEA, carcinoma associated mucin, carcinoma P53, melanoma MPG, melanoma P97, MAGE antigen, carcinoma Neu oncogene product, prostate specific antigen (PSA), prostate associated antigen, ras protein, and myc; and house dust mite allergen.

Preferably, the immunogenic polypeptide is derived from influenza virus. The immunogenic polypeptide may be derived from influenza virus A, B or C. Preferably it is derived from influenza virus A. The immunogenic polypeptide may be derived from any influenza antigens such as the HA (hemagglutinin), NA (neuraminidase), P (nucleoprotein/nucleocapsid protein), Ml, M2, PB 1, PB2, PA, NS1 and NS2 antigens and in particular the M2, HA and NA and antigens. The immunogenic polypeptide is preferably the influenza virus A surface polypeptide M2 or immunogenic fragment thereof as described herein.

The immunogenic polypeptide is preferably hemagglutinin (HA) or an immunogenic fragment thereof. The above discussion in relation to influenza virus A surface polypeptide M2 and the immunogenic fragment thereof also relates to HA. The sequence of HA may have homology with any naturally occurring HA, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity, for example over the full sequence or over a region of at least 20, for example at least 30, at least 50, at least 70, at least 100, at least 150, at least 200 or more contiguous amino acids. Methods of measuring protein homology are well known in the art and are discussed above in relation to the HBV core protein.

The immunogenic polypeptide is preferably HA or an immunogenic fragment thereof. A fragment of HA may be derived from HAl or HA2. A fragment may be from 6 to 565 aa, from 6 to 300 aa, from 6 to 200, or from 6 to 100 aa in length. The fragment of HA may be HA2, which is also known as the stalk region of HA. Figure 10 shows the structure of HA stalk region. The fragment may comprise the known domains Loop B, Helix C, Helix CD and Helix D of the HA2 monomer (see Figure 10). Examples of fragments of HA for insertion in the el loop are shown in Table 4. The inserted HA stalk sequence can be the amino acids 80 to 151 of SEQ ID NO: 12. The sequence of HA stalk may have homology with any naturally occurring HA stalk, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity, for example over the full sequence or over a region of at least 8, for example at least 10, at least 20, at least 30, at least 50, at least 60, at least 70 or more contiguous amino acids. Methods of measuring protein homology are well known in the art and are discussed above in relation to the HB V core protein. The sequence may vary by substitution, addition and/or deletion of one or more amino acid. For example, there may be up to 18, up to 15, up to 12, up to 10 or up to 5 substitutions, deletions or additions. The sequence may vary by only deletions, only additions or only substitutions. The sequence may vary by a combination of deletion and addition, deletion and substitution, addition and substitution, or deletion, addition and substitution. Preferably there are one, two, three, four, five or six deletions, additions and substitutions. The substitutions are preferably 'conservative' and may be made, for example, according to Table 1 above.

More than one copy of HA or an immunogenic fragment thereof may be inserted into the el loop. For example, 2, 3, 4, 5, 6, 7 or 8 copies of HA or the immunogenic fragment thereof may be inserted in the el loop. For example, 1, 2 or 3 copies may be inserted in the el loop. Therefore 1, 2 or 3 copies of HA or the immunogenic fragment thereof may be inserted the el loop. Where there is more than one copy of HA or the immunogenic fragment thereof in the el loop, the sequences for each copy may be identical or may be different. If the sequences are different, they can be inserted in any order. For example, the "first copy", "second copy", "third copy" and so on may be in any order from the N-terminus to the C-terminus in the el loop. For example, the "third copy" may be N-terminus to the "first copy".

In accordance with one aspect of the invention, the tandem construct comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the "first copy" of HBcAg and HA or an immunogenic fragment thereof in the "second copy" of HBcAg. As described herein, the "first copy" may be either the N-terminal or C-terminal copy. "Null" insert in one copy of HBcAg

In accordance with another aspect of the invention, the tandem construct comprises a "null" insert in one copy of HBcAg. The inventors found that this allows the antigen in the other copy of HBcAg to fold and/or be presented correctly. A "null" insert is a short sequence, typically of less than 20 amino acids in length, that allows the antigen in the other copy of HBcAg to fold and/or be presented correctly.

In more detail, the inventors inserted a conserved region from influenza H3N2 virus HA2 protein domain (LAH3) into one copy of HBcAg in the tandem construct. They found that inserting a short sequence, of less than 20 amino acids, into the second copy of HBcAg allowed the first insert (LAH3) to configure properly and conferred greater solubility to the whole VLP compared with expressing LAH3 in a monomeric core (Example 4). Specifically, they inserted into the second copy of HBcAg a sequence comprising single Lysine (K) residue flanked by a short flexible linker region made up of Glycine and Serine residues (a "null" insert). Such an insert could be used in one copy of HBcAg in a tandem construct with any antigen in the second copy of HBcAg to help the antigen to fold and/or be presented correctly.

Thus, the invention provides a protein comprising a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which one copy of HBcAg comprises, in the el loop, influenza virus A surface polypeptide M2 or an immunogenic fragment thereof flanked on one or both sides by a linker that joins the polypeptide or fragment to HBcAg sequence and the other copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids.

The invention also provides a protein comprising a first and a second copy of HBcAg in tandem, in which one copy of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop and the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid and the other copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids.

The M2 or fragment thereof, in accordance with either of these aspects of the invention, can be an M2 or fragment thereof as described above.

The invention also provides a protein comprising a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, HA or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids.

The HA or fragment thereof, in accordance with this aspect of the invention, can be an HA or fragment thereof as described above. The fragment of HA could, for example, be from HA2 protein domain, such as from influenza H3N2 virus, such that the protein can be used to immunise against influenza H3N2 virus infection. For example, the fragment may comprise amino acids 421 to 475 of the HA protein isolated from influenza A virus H3N2. The fragment may comprise SEQ ID NO: 24 or a sequence having homology with the sequence. A sequence having homology with a naturally occurring HA sequence is described above. There may be more than one copy of the HA or fragment thereof, also as decribed above.

The second copy of HBcAg comprises a short sequence in the el loop, i.e. a "null" insert. The sequence allows the insert in the first copy HBcAg to configure properly and/or confers greater solubility to the whole VLP compared with expressing the insert in the first copy of HBcAg in a monomeric HBcAg core. The sequence is less than 20 amino acids in length, for example less than or equal to 18, less than or equal to 15, less than or equal to 12, less than or equal to 10, less than or equal to 5, or less than or equal to 3 amino acids in length. For example, the sequence may be 18, 16, 14, 12, 10, 8, 6, 4, 2, 1 or 0 amino acids in length. The sequence may be 14 amino acids in length, as in SEQ ID NO: 21. The sequence may comprise a Lysine (K) residue flanked on each side by a linker sequence (the "second" and "third" linkers in the structure below). The linker is generally flexible. The amino acids in the linker may, for example, include or be entirely composed of Glycine and Serine residues. The Lysine residue may, for example, be flanked by a linker sequence comprising 1 to 10 Glycine or Serine residues, such as 2 to 8, or 3 to 7 Glycine or Serine residues.

An example of the structure of a tandem construct comprising M2 or an immunogenic fragment thereof comprises the following:

[the part of the first copy of HBcAg that is N-terminal to the el loop] - [first linker] - [influenza virus A surface polypeptide M2 or immunogenic fragment thereof] - [second linker] - [the part of the first copy of HBcAg that is C-terminal to the el loop] - [third linker] - [the part of the second copy of HBcAg that is N-terminal to the el loop] - [fourth linker] - [Lysine (K) residue] - [fifth linker] - [the part of the second copy of HBcAg that is C-terminal to the el loop].

An example of the structure of a tandem construct comprising HA or an immunogenic fragment thereof comprises the following:

[the part of the first copy of HBcAg that is N-terminal to the el loop] -

[hemagglutinin (HA) or an immunogenic fragment thereof] - [the part of the first copy of HBcAg that is C-terminal to the el loop] - [first linker] - [the part of the second copy of HBcAg that is N-terminal to the el loop] - [second linker] - [Lysine (K) residue] - [third linker] - [the part of the second copy of HBcAg that is C-terminal to the el loop].

The second copy of HBcAg may comprise the sequence of SEQ ID NO: 21, or a homologous sequence, in the el loop. A sequence may have homology with SEQ ID NO: 21, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%), at least 98%> or at least 99% identity, for example over the full sequence or over a region of at least 6, for example at least 8, at least 10, at least 12 or more contiguous amino acids. Methods of measuring protein homology are well known in the art and are discussed above in relation to the HBV core protein. The sequence may vary by substitution, addition and/or deletion of one or more amino acid. For example, there may be up to 8, up to 6, up to 4, or up to 2 substitutions, deletions or additions. The sequence may vary by only deletions, only additions or only substitutions. The sequence may vary by a combination of deletion and addition, deletion and substitution, addition and substitution, or deletion, addition and substitution. Preferably there are one, two, three, four, five or six deletions, additions and substitutions. The substitutions are preferably 'conservative' and may be made, for example, according to Table 1 above.

A protein comprising antigen(s) in one copy of HBcAg and a null insert in the second copy of HBcAg can be expressed in a particle comprising multiple copies of the protein. Alternatively, a protein comprising an antigen (such as M2 or an immunogenic fragment thereof) in one copy of HBcAg and a null insert in the second copy of HBcAg can be used in combination with a protein comprising a second antigen (such as HA or an immunogenic fragment thereof) in one copy of HBcAg and a null insert in the second copy of HBcAg to create a mixed particle. The null inserts allow the two antigens to be presented well spaced apart, resulting in a stable particle. Making the proteins of the invention

The proteins of the invention are generally made by recombinant DNA

technology. The invention includes a nucleic acid molecule (e.g. DNA or RNA) encoding a protein of the invention, such as an expression vector. The nucleic acid molecules may be made using known techniques for manipulating nucleic acids.

Typically, two separate DNA constructs encoding two HBcAg units are made and then joined together by overlapping PCR.

A protein of the invention may be produced by culturing a host cell containing a nucleic molecule encoding the protein under conditions in which the protein is expressed, and recovering the protein. Suitable host cells include bacteria such as E. coli, yeast, mammalian cells and other eukaryotic cells, for example insect Sf9 cells.

More than one protein of the invention may be produced simultaneously by transforming a host cell with more than one nucleic acid molecule encoding a protein of the invention. For example, a host cell may be transformed with a nucleic acid molecule encoding a protein comprising an antigen (such as M2 or an immunogenic fragment thereof) in one copy of FIBcAg and a null insert in the second copy of FIBcAg and a nucleic acid molecule encoding a protein comprising a second antigen (such as HA or an immunogenic fragment thereof) in one copy of FIBcAg and a null insert in the second copy of FIBcAg. The two proteins may be encoded on the same or separate nucleic acid molecules.

The vectors constituting nucleic acid molecules according to the invention may be, for example, plasmid or virus vectors. They may contain an origin of replication, a promoter for the expression of the sequence encoding the protein, a regulator of the promoter such as an enhancer, a transcription stop signal, a translation start signal and/or a translation stop signal. The vectors may also contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene in the case of a mammalian vector. Vectors may be used in vitro, for example for the production of RNA or used to transform or transfect a host cell. The vector may also be adapted to be used in vivo, for example in a method of gene therapy or DNA vaccination.

Promoters, enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. For example, prokaryotic promoters may be used, in particular those suitable for use in E. coli strains (such as E. coli HBlOl). A promoter whose activity is induced in response to a change in the surrounding environment, such as anaerobic conditions, may be used. Preferably an htrA or nirB promoter may be used. These promoters may be used in particular to express the protein in an attenuated bacterium, for example for use as a vaccine. When expression of the protein of the invention is carried out in mammalian cells, either in vitro or in vivo, mammalian promoters may be used. Tissue-specific promoters, for example hepatocyte cell-specific promoters, may also be used. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RS V) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters and adenovirus promoters. All these promoters are readily available in the art.

A protein according to the invention may be purified using conventional techniques for purifying proteins. The protein may, for example, be provided in purified, pure or isolated form. For use in a vaccine, the protein must generally be provided at a high level of purity, for example at a level at which it constitutes more than 80%, more than 90%, more than 95% or more than 98% of the protein in the preparation. However, it may be desirable to mix the protein with other proteins in the final vaccine formulation.

Inducing an immune response

A protein, particle or nucleic acid of the invention can be used to induce an immune response, particularly against influenza such as influenza virus A. The protein, particle or nucleic acid may be used as a vaccine. Provided is a method of inducing an immune response in a subject, comprising administering to the subject a protein, particle or nucleic acid of the invention. An adjuvant may be administered in combination with the protein, particle or nucleic acid. The protein, particle or nucleic acid may be used to raise multiple simultaneous immune responses to all the components (the HBcAg, influenza virus A surface polypeptide M2, or HA and possibly one or more other immunogenic polypeptides). If the immunogenic polypeptides are also derived from influenza then this can induce an enhanced immune response against influenza. If the immunogenic polypeptide is not derived from influenza then this can induce

simultaneous immune responses against the source of the immunogenic polypeptide and influenza. If there is more than one immunogenic polypeptide as well as influenza virus A surface polypeptide M2, then the more than one immunogenic polypeptide may be from one source, such as a pathogen or allergen, or from different sources, such as more than one pathogen or allergen. If all the immunogenic polypeptides are derived from more than one source then this can induce simultaneous immune responses against the different sources, for example more than one pathogen or allergen. One of the advantages of the invention is that it allows precise control over the ratio of different immunogenic polypeptides to be delivered in a vaccine. For example, the ratio of the influenza virus A surface polypeptide M2 in a first copy of HBcAg to immunogenic polypeptide in a second copy of HBcAg can be precisely 1 : 1.

The protein, particle or nucleic acid may be employed alone or as part of a composition including, but not limited to, a pharmaceutical composition, a vaccine composition or an immunotherapeutic composition. The invention therefore provides a pharmaceutical composition (e.g. a vaccine composition) comprising a protein of the invention, a particle comprising multiple copies of the protein of the invention or a nucleic acid molecule encoding the protein of the invention and a pharmaceutically acceptable carrier or diluent. Also, as explained above, the invention provides "mixed" particles comprising more than one type of protein of the invention. Thus, the invention also provides a pharmaceutical composition, a vaccine composition or an

immunotherapeutic composition comprising such mixed particles. The composition may further comprise an adjuvant. The composition can be used for the treatment of the human or animal body. The composition can be used for vaccination of the human or animal body. Provided is a method of treatment of a human or animal subject comprising administering the composition to the subject. The composition may be used to vaccinate against any of the pathogens described herein. In particular, the composition may be used to vaccinate against influenza, such as influenza virus A.

A protein of the invention, a particle of the invention or a nucleic acid of the invention can be used in a method of treatment of the human or animal body. Provided is a method of treatment of a human or animal subject comprising administering to the subject a protein of the invention, a particle of the invention or a nucleic acid of the invention. An adjuvant may be administered in combination with the protein, particle or nucleic acid. Also provided is use of a protein of the invention, a particle of the invention or a nucleic acid of the invention for the manufacture of a medicament for treatment of the human or animal body. The protein, particle or nucleic acid may be used to treat the pathogens described herein. In particular, the protein, particle or nucleic acid may be used for the treatment of influenza, such as influenza virus type A.

A protein of the invention, a particle of the invention or a nucleic acid of the invention can be used in a method of vaccination of the human or animal body. Provided is a method of vaccination of the human or animal subject comprising administering to the subject a protein of the invention, a particle of the invention or a nucleic acid of the invention. The invention provides use of a protein of the invention, a particle of the invention or a nucleic acid of the invention for the manufacture of a medicament for vaccination of the human or animal body. The protein, particle or nucleic acid may be used to vaccinate against any of the pathogens described herein. In particular, the protein, particle or nucleic acid may be used to vaccinate against influenza virus, such as influenza virus A.

The principle behind vaccination is to induce an immune response in a host so as to generate an immunological memory in the host. This means that, when the host is exposed to the virulent pathogen, it mounts an effective (protective) immune response, i.e. an immune response which inactivates and/or kills the pathogen. The invention forms the basis of a vaccine against influenza virus and depending on what other immunogenic polypeptides are included in the protein, could simultaneously vaccinate an individual to any of a wide range of other diseases and conditions, such as HBV, HAV, HCV, foot-and-mouth disease, polio, herpes, rabies, AIDS, dengue fever, yellow fever, malaria, tuberculosis, whooping cough, typhoid, food poisoning, diarrhoea, meningitis and gonorrhoea. The immunogenic polypeptides in the protein of the invention are chosen so as to be appropriate for the disease against which the vaccine is intended to provide protection.

The protein, particle or nucleic acid of the invention has the capability of inducing immune responses against any or all subtypes of influenza virus A. The protein, particle or nucleic acid of the invention therefore has the capability of vaccinating against any or all subtypes of influenza virus A (see e.g, Sharp 2002 Cell Vol. 108, 305-312, "Origins of Human Virus Diversity" and Shi et al 2010, PLOSONE, 5(12) "A Complete Analysis of HA and NA Genes of Influenza A Viruses"). For example, any of the HA subtypes such as HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, H14, H15 or H16 and/or from any of the NA subtypes such as Nl, N2, N3, N4, N5, N6, N7, N8 or N9. Preferably subtypes HlNl, H5N1, H3N2, H7N7, H1N2, H2N2, H7N3, H5N2, H1N7, H9N2, H7N2 and/or H10N7. Even more preferably subtypes H3N2, H5N1, H1N1 and/or H7N7. For example, the tandem construct can comprise influenza virus A surface polypeptide M2 or an immunogenic fragment thereof from multiple subtypes of influenza virus A and may therefore be used as a universal vaccine inducing immune responses, and thus providing protection, against a subset or all subtypes of influenza virus A. For example, a tandem construct comprising universal M2e sequence and one, two or more variants of the universal M2e sequence (as described herein) may induce immune responses against a subset or all subtypes of influenza virus A. For example, see the triple Me2 containing tandem constructs in Examples 1 and 3. The one, two or more variants of universal M2e sequence may be one or more (e.g. one, two or three) of the most common variants found in the HA subtypes such as HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, Hl l, H12, H13, H14, H15 or H16 and/or from any of the NA subtypes such as Nl, N2, N3, N4, N5, N6, N7, N8 or N9. Preferably subtypes H1N1, H5N1, H3N2, H7N7, H1N2, H2N2, H7N3, H5N2, H1N7, H9N2, H7N2 and/or H10N7. Even more preferably subtypes H3N2, H5N1, H1N1 and/or H7N7.

Also, as an example, the tandem construct can comprise influenza virus A surface polypeptide M2 or an immunogenic fragment thereof and one or more further

immunogenic polypeptides derived from one or more subtypes of influenza virus A and may therefore be used as a universal vaccine inducing immune responses, and thus providing protection, against a subset or all subtypes of influenza virus A. For example, a tandem construct comprising universal M2e sequence and one, two or more variants of the universal M2e sequence (as described herein) and one or more (e.g. one, two or three) copies of HA or an immunogenic fragment thereof (e.g. HA stalk) may induce immune responses against a subset or all subtypes of influenza virus A. For example, see the tandem constructs in Example 3. The one, two or more variants of universal M2e sequence may be one or more (e.g. one, two or three) of the most common variants found in the HA subtypes such as HI, H2, H3, H4, H5, H6, H7, H8, H9, H10, HI 1, H12, H13, HI 4, HI 5 or HI 6 and/or from any of the NA subtypes such as Nl, N2, N3, N4, N5, N6, N7, N8 or N9. Preferably subtypes H1N1, H5N1, H3N2, H7N7, H1N2, H2N2, H7N3, H5N2, H1N7, H9N2, H7N2 and/or H10N7. Even more preferably subtypes H3N2, H5N1, H1N1 and/or H7N7. The immunogenic polypeptide (e.g. HA or immunogenic fragment thereof, such as HA stalk) may also be derived from any influenza virus A subtype such as any of those listed above. Also, as an example, the tandem construct can comprise influenza virus HA or an immunogenic fragment thereof (e.g. HA stalk) region which may also be derived from any influenza virus A subtype such as any of those listed above.

Multiple different tandem constructs, each containing different influenza virus antigens from the same or different subtypes of influenza can be prepared for

simultaneous use in a method of vaccination of the human or animal body against influenza. For example, a tandem construct comprising influenza virus A surface polypeptide M2 or an immunogenic fragment thereof of the invention can be used in combination with a tandem construct comprising HA or an immunogenic fragment thereof of the invention. In particular, a tandem construct comprising influenza virus A surface polypeptide M2 or an immunogenic fragment thereof of the invention providing protection from H1N1, H1N7 and/or H5N1 influenza infection could be used in combination with a tandem construct comprising HA or an immunogenic construct of the invention providing protection from H3N2 influenza protection. For example, see the tandem constructs in Example 4.

Thus, the invention also provides pharmaceutical compositions and vaccines comprising more than one different protein, particle or nucleic acid of the invention. For example, provided are pharmaceutical compositions and vaccines comprising (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, wherein the first protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which one or both of the copies of HBcAg comprises, in the el loop, influenza virus A surface polypeptide M2 or an immunogenic fragment thereof flanked on one or both sides by a linker that joins the polypeptide or fragment to HBcAg sequence, and optionally the second copy of HBcAg comprises, in the el loop, another immunogenic polypeptide or a sequence of less than 20 amino acids; and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; and a pharmaceutically acceptable carrier or diluent. Also provided are pharmaceutical compositions and vaccines comprising (i) a first protein, a particle comprising multiple copies of the first protein, or a nucleic acid encoding a protein, wherein the protein comprises a first and a second copy of HBcAg in tandem, in which one or both of the copies of HBcAg comprises influenza virus A surface polypeptide M2 or an immunogenic fragment thereof in the el loop and the cysteine amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or the immunogenic fragment thereof is deleted or substituted with an alternative amino acid, and optionally the second copy of HBcAg comprises, in the el loop, another immunogenic polypeptide or a sequence of less than 20 amino acids; and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, wherein the protein comprises a first and a second copy of hepatitis B core antigen (HBcAg) in tandem, in which the first copy of HBcAg comprises, in the el loop, hemagglutinin (HA) or an immunogenic fragment thereof, wherein the fragment of HA is optionally the HA stalk region, and the second copy of HBcAg comprises, in the el loop, a sequence of less than 20 amino acids; and a pharmaceutically acceptable carrier or diluent.

The first and second proteins in the pharmaceutical composition or vaccine may provide protection against any of the subtypes of influenza described above, but preferably provide protection against different subtypes. For example, the first protein may provide protection against H1N1, H1N7 and/or H5N1 and the second protein may provide protection against H3N2 influenza infection (as for the example tandem constructs in Example 4).

Therefore, the first and second proteins of the invention may be used together in a method of inducing an immune response against influenza in a subject. The method may comprise administering to the subject (i) the first protein of the invention, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, and (ii) a second protein of the invention, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein. The two entities may be administered in the same or different compositions, preferably the same composition.

Also provided is (i) a first protein of the invention, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, and (ii) a second protein of the invention, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, for use in a method of vaccination of the human or animal body against influenza. Also provided is use of (i) a first protein of the invention, a particle comprising multiple copies of the first protein, or a nucleic acid encoding the first protein, and (ii) a second protein, a particle comprising multiple copies of the second protein, or a nucleic acid encoding the second protein, for the manufacture of a medicament for vaccination of the human or animal body against influenza. The terms "individual" and "subject" are used interchangeably herein to refer to any member of the subphylum cordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs as well as pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like. The terms do not denote a particular age. Thus, both adult and newborn individuals are intended to be covered. The methods described herein are intended for use in any of the above vertebrate species, since the immune systems of all of these vertebrates operate similarly.

In some instances, the invention may be administered to any suitable subject and in particular any suitable subject of a given species, preferably a suitable human subject. Thus, as many subjects as possible may, for instance, be subject to administration without emphasis on any particular group of subjects. For instance, a population of subjects as a whole, or as many as possible, may be subject to administration.

The protein, particle or nucleic acid of the invention is for administration to a subject. It may be administered simultaneously or sequentially with an adjuvant.

Therefore the composition of the invention comprising the protein, particle or nucleic acid may also comprise an adjuvant. The composition of the invention may be one which is to be delivered by injection (such as intradermal, subcutaneous, intramuscular, intravenous, intraosseous, and intraperitoneal), transdermal particle delivery, inhalation, topically, orally or transmucosally (such as nasal, sublingual, vaginal or rectal).

The compositions may be formulated as conventional pharmaceutical

preparations. This can be done using standard pharmaceutical formulation chemistries and methodologies, which are available to those skilled in the art. For example, compositions containing the protein, particle or nucleic acid with or without an adjuvant can be combined with one or more pharmaceutically acceptable excipients or vehicles to provide a liquid preparation. Thus also provided is a pharmaceutical composition comprising the protein, particle or nucleic acid together with a pharmaceutically acceptable carrier or diluent. The composition optionally comprises an adjuvant.

Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present. These carriers, diluents and auxiliary substances are generally pharmaceutical agents which may be administered without undue toxicity and which, in the case of antigenic compositions will not in themselves induce an immune response in the individual receiving the composition. Pharmaceutically acceptable carriers include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. It is also preferred, although not required, that the preparation will contain a pharmaceutically acceptable carrier that serves as a stabilizer, particularly for peptide, protein or other like molecules if they are to be included in the composition. Examples of suitable carriers that also act as stabilizers for peptides include, without limitation, pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, and the like. Other suitable carriers include, again without limitation, starch, cellulose, sodium or calcium phosphates, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEGs), and combination thereof. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in REMINGTON' S

PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991), incorporated herein by reference.

Certain facilitators of nucleic acid uptake and/or expression ("transfection facilitating agents") can also be included in the compositions, for example, facilitators such as bupivacaine, cardiotoxin and sucrose, and transfection facilitating vehicles such as liposomal or lipid preparations that are routinely used to deliver nucleic acid molecules. Anionic and neutral liposomes are widely available and well known for delivering nucleic acid molecules (see, e.g., Liposomes: A Practical Approach, (1990) RPC New Ed., IRL Press). Cationic lipid preparations are also well known vehicles for use in delivery of nucleic acid molecules. Suitable lipid preparations include DOTMA (N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), available under the tradename Lipofectin™ , and DOTAP (l,2-bis(oleyloxy)-3-

(trimethylammonio)propane), see, e.g., Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416; Malone et al. (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081; US Patent Nos 5,283, 185 and 5,527,928, and International Publication Nos WO 90/11092, WO 91/15501 and WO 95/26356. These cationic lipids may preferably be used in association with a neutral lipid, for example DOPE (dioleyl phosphatidylethanolamine). Still further transfection-facilitating compositions that can be added to the above lipid or liposome preparations include spermine derivatives (see, e.g., International Publication No. WO 93/18759) and membrane-permeabilizing compounds such as GALA,

Gramicidine S and cationic bile salts (see, e.g., International Publication No. WO 93/19768).

Alternatively, the protein, particle or nucleic acid and/or the adjuvant may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules. For example, polynucleotides can be precipitated onto carriers in the presence of a polynucleotide condensing agent and a metal ion chelating agent. Preferred condensing agents include cationic polymers, in particular polyamines, and in particular a polyargine or a polylysine. In a preferred instance the polyamine is (Arg)4 or (Arg)6. Reference may be made to the techniques discussed in WO2004/208560 which may be employed.

Once formulated the compositions can be delivered to a subject in vivo using a variety of known routes and techniques. For example, the liquid preparations can be provided as an injectable solution, suspension or emulsion and administered via parenteral, subcutaneous, intradermal, intramuscular, intravenous intraosseous and intraperitoneal injection using a conventional needle and syringe, or using a liquid jet injection system. Liquid preparations can also be administered topically to skin or mucosal tissue (e.g. nasal, sublingual, vaginal or rectal), or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of

administration include oral administration, suppositories, and active or passive transdermal delivery techniques. The protein, particle or nucleic acid of the invention is administered to a subject in an amount that will be effective in modulating an immune response. An appropriate effective amount will fall in a relatively broad range but can be readily determined by one of skill in the art by routine trials. The "Physicians Desk Reference" and "Goodman and Gilman's The Pharmacological Basis of Therapeutics" are useful for the purpose of determining the amount needed. Typically, the protein or particles are administered in a dose of from 0.1 to 200 mg, preferably from 1 to 100 mg, more preferably from 10 to 50 mg body weight. The nucleic acid of the invention may be administered directly as a naked nucleic acid construct using techniques known in the art or using vectors known in the art. The amount of nucleic acid administered is typically in the range of from 1 μg to 10 mg, preferably from 100 mg to 1 mg. The vaccine may be given in a single dose schedule or a multiple dose schedule, for example in from 2 to 32 or from 4 to 16 doses. The routes of administration and doses given above are intended only as a guide, and the route and dose may ultimately be at the discretion of the physician.

In some cases after an initial administration a subsequent administration of the composition of the invention may be performed. In particular, following an initial administration a subject may be given a "booster". The booster may be, for instance, a dose chosen from any of those mentioned herein. The booster administration may, for instance, be at least a week, two weeks, four weeks, six weeks, a month, two months or six months after the initial administration.

The protein, particle or nucleic acid of the invention and an adjuvant may be administered sequentially or simultaneously, preferably simultaneously. The two entities may be administered in the same or different compositions, preferably the same composition. An adjuvant is delivered so that an adjuvant effect is seen, that is the immune response seen will differ from that if the adjuvant had not been administered with the antigen. The two entities may be administered at the same or different sites, preferably the same sites. Preferably, the two entities are administered in the same composition at the same site at the same time preferably via injection.

Any suitable adjuvant may be used. Currently used vaccine adjuvants include: - Inorganic compounds, such as aluminium salts (e.g. aluminium hydroxide and aluminium phosphate) or calcium phosphate. Aluminium salts are otherwise known as alum. Oil emulsions and surfactant based formulations, e.g. MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil- in-water emulsion + MPL + QS-21), Montanide ISA-51 and ISA-720 (stabilised water- in-oil emulsion).

- Particulate adjuvants, e.g. virosomes (unilamellar liposomal vehicles

incorporating e.g. influenza haemagglutinin), AS04 ([SBAS4] Al salt with MPL), ISCOMS (structured complex of saponins and lipids), and polylactide co-glycolide (PLC).

Microbial derivatives (natural and synthetic), e.g. monophosphoryl lipid A (MPL), Detox (MPL + M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated monosaccharide), DC Chol (lipoidal immunostimulators able to self organise into liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), and modified LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant effects).

- Endogenous human immunomodulators, e.g. hGM-CSF or hIL-12 (cytokines that can be administered either as protein or plasmid encoded), and Immudaptin (C3d tandem array).

Inert vehicles, such as gold particles.

Preferably the adjuvant used is alum. Most preferably the adjuvant is a mixture of aluminium hydroxide and magnesium hydroxide, for example Inject alum (Pierce Laboratories) which is not suitable for use in humans.

The invention is illustrated by the following Examples: Example 1

Materials and Methods:

VLP sequence:

Tandem Core containing inserts from influenza virus conserved proteins in the

MIR was produced in BL21 E.coli. The sequence corresponding to the insert belongs to the region of influenza virus matrix protein 2 ectodomain (M2e). It spans 24 amino acids encoding the known N-terminal external sequence of M2 protein (Figure 5); MSLLTEVETPIRNEWGCRCNGSSD (SEQ ID NO: 6). This wild type sequence was modified to replace the Cysteine residues (which affect VLP formation) at position 17 and 19 (underlined above), for Serine residues. The final insert contains 3 variations of this sequence. The first version is the universal M2e consensus sequence except that the Cysteine residues at positions 17 and 19 have been substituted with Serines (SEQ ID NO: 9). The second (SEQ ID NO: 8) and third (SEQ ID NO: 10) are mutated versions of the universal sequence which correspond to the most common variants found in H3N2 and H5N1 influenza viruses except that the Cysteine residues at positions 17 and 19 have been substituted with Serines. The M2e sequences are flanked by a flexible spacer made up of 15 amino acids; GGGGS GGGGS GGGGS (SEQ ID NO: 5). Other constructs containing variations of M2e sequence identity, copy number and flanking sequences were also produced; these are described in Table 2 below.

Table 2. Table of alternative VLP generated with variations on insert sequence.

Between

each M2e

sequence

I nfluenza MSLLTEVETPTRNGWGSKSNGSSD (GGGGS) (SEQ Yes

M2 (SEQ I D NO: 9) I D NO: 4) x3

MSLLTEVETPI RNEWGSRSNGSSD

(SEQ I D NO: 8)

MSLLTEVETPTRNEWESRSSGSSD

(SEQ I D NO: 10)

* Flanking sequences are upstream and downstream of M2e sequence

$ This insert has the flanking sequence indicated between M2e sequences. The amino acid sequence of Tandem Core with 3x M2e insert (single letter aa code) is shown below. Amino acids from Tandem Core are in bold and those from Influenza M2 are underlined, the flexible linking region is in italics:

MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHT ALRQAILCWGELMTLATWVGNNLEGS GGGGSGGGGSGGGGS MSLLTEVETPTRNGWGSKSNGSSD MSLLTEVETPIRNEWGSRSNGS SD MSLLTEVETPTRNEWESRSSGSSD GGGGSGGGGSGGGGS

GRDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWI RTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPY KEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMTLATWVGNNLEFAGASDPASRDLVVNYVNTNMGLKIRQLLWF HISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGR SPRRRTPSPRRRRSQSPRRRRSQSRESQCLEHHHHHH- (SEQ ID NO: 11)

Theoretical pI/Mw: 6.29 / 51438.34 Animals:

6-8 week old Balb/C female mice were purchased from Harlan (Wyton, UK) and housed in IVC category 2 containment facilities. All animal care and procedures were performed in accordance with Home Office UK regulations on animal use for experimental purposes.

Immunisation:

For primary immunisation, individual mice were given an intraperitoneal (i.p.) injection containing 15μg VLP material, 2C^g SAS (sigma adjuvant system- Sigma Aldrich), 20μg Pierce Imject Alum (Thermo Scientific), and sterile saline solution to a total volume of ΙΟΟμΙ. For booster immunisations, administered 7 and 14 days after primary, individual mice received a subcutaneous (s.c.) injection containing 5ug VLP material, 20μg MLPA (Sigma), 20μg muramyl dipeptide (Sigma), 20μg Pierce Imject Alum (Thermo Scientific) and made up to ΙΟΟμΙ final volume with sterile saline solution. One day after the final booster, mice were bled by facial vein extraction to confirm seroconversion. Control groups were immunised with adjuvants only, not containing VLP material.

Seroconversion:

Detection of anti-M2e antibodies was performed by ELISA. 96-well Nunc

Maxisorp plates were coated with 6.25μg/ml M2e peptide sequence

MSLLTEVETPIRNEWGCRCNGS SD-OH (SEQ ID NO: 14) (Activotec) in 1M NaCl buffer. Coated plates were washed 3x with PBS-Tween20 0.05% and blocked with 10%> milk solution for IHr at 37°C. Diluted serum in 2.5% milk was added and incubated at 37°C for IHr then washed 3x as before. TMB substrate (Sigma) was added for 20 minutes and reaction was stopped with 1M H2SO4. Absorbance at 450nm with a 630nm correction was read using the Tecan Sunrise plate reader and analysed by Magellan™ software. All wells were run in duplicate with at least 3 dilution repeats.

Challenge:

4 weeks after primary immunisation, mice were infected with a 5x mLD50 dose of A/PR8 H1N1 influenza virus. Virus was administered intranasally (i.n.) after mice were anaesthetised by intraperitoneal (i.p.) administration of a ketamine/xylasine 2: 1 mixture in saline. Mice were weighed at time of infection (day 0) and semi-daily thereafter until full recovery was made. Mice were also scored using the sickness scale found in Table 3. Results:

Immunised mice seroconverted to M2e peptide from influenza virus.

3 weeks following primary immunisation, anti-serum from mice was collected, pooled and tested for reactivity against synthetic M2e peptides from influenza virus by ELISA. Mice immunised with the Tandem Core VLP containing the 3x M2e insert generated antibody which could bind M2e peptide from influenza virus, whereas mice immunised with adjuvant only did not (Figure 1).

Immunised mice were protected from lethal A/PR8 H1N1 influenza virus infection.

4 weeks following primary immunisation, mice were challenged with 5x mLD50 of PR8 influenza virus. Mice in the Adjuvant only group lost weight rapidly and presented with high clinical scores (Figures 2 and 3), reaching 100% mortality by day 8 post-infection (Figure 4). Conversely mice immunised with Tandem Core with influenza 3xM2e insert reached a peak weight loss of 8% associated with a much milder clinical score, and began recovery by day 7, making a full recovery by day 12 post infection

(Figures 2 and 3). Survival in the immunised group was 100% (Figure 4). Taken together these results show that mice which received Tandem Core containing the influenza 3xM2e insert showed less weight loss, lower morbidity and no mortality following a lethal challenge of PR8 influenza virus. Protection conferred by Tandem Core was not sterile, either because the anti- M2e antibody titre was not high enough to protect fully or because the anti-M2e protects via a non-neutralising mechanism. Alternatively protection may be mediated via a non-antibody dependent mechanism but it correlates well with anti-M2e production levels. Regardless of the specific mechanism protection is conferred by vaccination with Tandem Core VLP as non-immunised mice show much more severe symptoms and high mortality. Table 3. Severity scale for influenza symptoms. Individual clinical scores were determined using the scale below.

Example 2

Materials and Methods: VLP sequence:

Tandem Core containing inserts from influenza virus conserved proteins in the MIR was produced in BL21 E.coli. The sequence corresponding to the insert belongs to the stalk region of influenza virus hemagglutinin HA2 protein domain. It spans amino acids encoding the known domains; Loop B, Helix C, Helix CD and Helix D of the HA2 monomer (Figure 10). This sequence spans amino acids (aa) 403-474 of the HA protein isolated from influenza A virus HlNl/Lux/09. There were other constructs containing variations of the HA stalk insert sequence, these are described in Table 4 below.

Table 4. Table of alternative VLP generated with variations on insert sequence.

The amino acid sequence of Tandem Core with HA stalk influenza insert (single letter aa code) is shown below. Amino acids from Tandem Core are in bold and those from Influenza HA are underlined: MDIDPYKEFGAT VELLSFLPSDFFPS VRDLLDTASALYREALESPEHCSPHHT ALRQAIL CWGELMTLATWVGNNLEGS

MNTQFTAVGKEF HLEKRIE L KKVDDGFLDIWTYNAELLVLLE ERTLDYH D SNVK L YEK VRS QLKNN A

SGRDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVW IRTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPY KEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMTLATWVGNNLEFAGASDPASRDLVVNYVNTNMGLKIRQLLWF HISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRDRGR SPRRRTPSPRRRRSQSPRRRRSQSRESQCLEHHHHHH- (SEQ ID NO: 12)

Theoretical pI/Mw: 6.98 / 50293.85

Animals:

6-8 week old Balb/C female mice were purchased from Harlan (Wyton, UK) and housed in IVC category 2 containment facilities. All animal care and procedures were performed in accordance with Home Office UK regulations on animal use for

experimental purposes.

Immunisation:

For primary immunisation, individual mice were given an intraperitoneal (i.p.) injection containing 15μg VLP material, 2C^g SAS (sigma adjuvant system- Sigma Aldrich), 2C^g Pierce Imject Alum (Thermo Scientific), and sterile saline solution to a total volume of ΙΟΟμΙ. For booster immunisations, administered 7 and 14 days after primary, individual mice received a subcutaneous (s.c.) injection containing 5ug VLP material, 20μg MLPA (Sigma), 20μg muramyl dipeptide (Sigma), 20μg Pierce Imject Alum (Thermo Scientific) and made up ΐοΐθθμΐ final volume with sterile saline solution. One day after the final booster, mice were bled by facial vein extraction to confirm seroconversion. Control groups were immunised with adjuvants only, not containing VLP material.

Seroconversion:

Detection of anti-HA antibodies was performed by ELISA. 96-well Nunc

Maxisorp plates were coated with ^g/ml rHA from A/PR8 influenza virus (Life

Technologies) in carbonate bi-carbonate buffer. Coated plates were washed 3x with PBS- Tween20 0.05% and blocked with 10% milk solution for IHr at 37°C. Diluted serum in 2.5%) milk was added and incubated at 37°C for IHr then washed 3x as before. TMB substrate (Sigma) was added for 20 minutes and reaction was stopped with 1M H2SO4. Absorbance at 450nm with a 630nm correction was read using the Tecan Sunrise plate reader and analysed by Magellan™ software. All wells were run in duplicate with at least 3 dilution repeats.

Challenge:

4 weeks after primary immunisation, mice were infected with a 5x mLD50 dose of A/PR8 H1N1 influenza virus. Virus was administered intranasally (i.n.) after mice were anaesthetised by intraperitoneal (i.p.) administration of a ketamine/xylasine 2: 1 mixture in saline. Mice were weighed at time of infection (day 0) and semi-daily thereafter until full recovery was made. Mice were also scored using the sickness scale found in Table 3 above. Results:

Immunised mice seroconverted to rHA protein from A/PR8 H1N1 influenza virus.

3 weeks following primary immunisation, anti-serum from mice was collected, pooled and tested for reactivity against recombinant hemagglutinin protein from PR8 influenza virus by ELISA. Mice immunised with the Tandem Core VLP containing the HA stalk insert generated antibody which could bind rHA protein from influenza virus, whereas mice immunised with adjuvant only did not (Figure 6).

Immunised mice were protected from lethal A/PR8 H1N1 influenza virus infection.

4 weeks following primary immunisation, mice were challenged with 5x mLD50 of PR8 influenza virus. Mice in the Adjuvant only group lost weight rapidly and presented with high clinical scores (Figures 7 and 8), reaching 100% mortality by day 8 post-infection (Figure 9). Conversely mice immunised with Tandem Core with influenza stalk insert reached a peak weight loss of 15% associated with a much milder clinical score, and began recovery by day 8, making a full recovery by day 16 post infection (Figures 7 and 8). Survival in the immunised group was 100%) (Figure 9). Taken together these results show that mice which received Tandem Core containing the influenza stalk insert showed less weight loss, lower morbidity and no mortality following a lethal challenge of PR8 influenza virus. Protection conferred by Tandem Core was not sterile either because the anti-HA antibody titre was not high enough to protect fully or because the anti-HA protects via a non-neutralising mechanism. Alternatively protection may be mediated via a non-antibody dependent mechanism but it correlates well with anti-HA production levels. Regardless of the specific mechanism protection is conferred by vaccination with Tandem Core VLP as non-immunised mice show much more severe symptoms and high mortality. Example 3

Materials and Methods: VLP sequence:

Tandem Core containing inserts from influenza virus conserved proteins within the two major insertion regions (MIR) was produced in BL21 E.coli (Figure 15). The sequence corresponding to the first insert belongs to the stalk region of influenza virus hemagglutinin HA2 protein domain. It spans amino acids encoding the known domains; Loop B, Helix C, Helix CD and Helix D of the HA2 monomer (Figure 10). This sequence spans amino acids (aa) 403-474 of the HA protein isolated from influenza A virus HlNl/Lux/09. The sequence corresponding to the second insert belongs to the region of influenza virus matrix protein 2 ectodomain (M2e). It spans 24 amino acids encoding the known N-terminal external sequence of M2 protein;

MSLLTEVETPIRNEWGCRCNGSSD (SEQ ID NO: 6) (Figure 5). This wild type sequence was modified to replace the Cysteine residues (which affect VLP formation) at position 17 and 19 (underlined above), for Serine residues. The final insert contains 3 variations of this sequence. The first version is the universal M2e consensus sequence except that the Cysteine residues at positions 17 and 19 have been substituted with Serines (SEQ ID NO: 9). The second (SEQ ID NO: 8) and third (SEQ ID NO: 10) are mutated versions of the universal sequence which correspond to the most common variants found in H7N7 and H5N1 influenza viruses except that the Cysteine residues at positions 17 and 19 have been substituted with Serines. The amino acid sequence of Tandem Core with HA stalk influenza insert and M2e x 3 is shown below (single letter aa code). Amino acids from Tandem Core are in bold and those from Influenza are underlined:

MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHT ALRQAIL CWGELMTLATWVGNNLEGS MNTOFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYH D SNVKNL YEK VRS OLKNN A

SGRDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVW IRTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPY KEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMTLATWVGNNLEF

GGGGSGGGGSGGGGS MSLLTEVETPTRNGWGSKSNGSSD MSLLTEVETPIR EWGSRSNGS SD MSLLTEVETPTRNEWESRSSGSSD

GGGGSGGGGSGGGGS

ASDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIR TPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQS RESQCLEHHHHHH- (SEQ ID NO: 13)

Theoretical pI/Mw: 6.17 / 59834.78

Animals:

6-8 week old Balb/C female mice were purchased from Harlan (Wyton, UK) and housed in IVC category 2 containment facilities. All animal care and procedures were performed in accordance with Home Office UK regulations on animal use for

experimental purposes. Immunisation:

For primary immunisation, individual mice were given an intraperitoneal (i.p.) injection containing 15μg VLP material, 20μg SAS (sigma adjuvant system- Sigma Aldrich), 20μg Pierce Imject Alum (Thermo Scientific), and sterile saline solution to a total volume of ΙΟΟμΙ. For booster immunisations, administered 7 and 14 days after primary, individual mice received a subcutaneous (s.c.) injection containing 5ug VLP material, 20μg MLPA (Sigma), 20μg muramyl dipeptide (Sigma), 20μg Pierce Imject Alum (Thermo Scientific) and made up ΐοΐθθμΐ final volume with sterile saline solution. One day after the final booster, mice were bled by facial vein extraction to confirm seroconversion. Control groups were immunised with adjuvants only, not containing VLP material. Seroconversion:

Detection of anti-HA or anti-M2e antibodies was performed by ELISA. 96-well Nunc Maxisorp plates were coated with ^g/ml rHA from A/PR8 influenza virus (Life Technologies) in carbonate bi-carbonate buffer, or 6.25μg/ml M2e peptide sequence MSLLTEVETPIRNEWGCRCNGS SD-OH (SEQ ID NO: 14) (Activotec) in 1M NaCl buffer. Coated plates were washed 3x with PBS-Tween20 0.05% and blocked with 10% milk solution for IHr at 37°C. Diluted serum in 2.5% milk was added and incubated at 37°C for IHr then washed 3x as before. Goat-anti-Mouse IgG -Peroxidase secondary Ab was added at 1/2500 dilution (Sigma) and incubated at 37°C for IHr then washed 3x as before. TMB substrate (Sigma) was added for 20 minutes and reaction was stopped with 1M H2SO4. Absorbance at 450nm with a 630nm correction was read using the Tecan Sunrise plate reader and analysed by Magellan™ software. All wells were run in duplicate with at least 3 dilution repeats. Challenge:

4 weeks after primary immunisation, mice were infected with a 5x mLD50 dose of A/PR8 H1N1 influenza virus. Virus was administered intranasally (i.n.) after mice were anaesthetised by intraperitoneal (i.p.) administration of a ketamine/xylasine 2: 1 mixture in saline. Mice were weighed at time of infection (day 0) and semi-daily thereafter until full recovery was made. Mice were also scored using the sickness scale found in Table 3.

Results: Immunised mice seroconverted to rHA protein from A/PR8 H1N1 influenza virus and M2e peptide.

3 weeks following primary immunisation, anti-serum from mice was collected, pooled and tested for reactivity against recombinant hemagglutinin protein from PR8 influenza virus and M2e peptide by ELISA. Mice immunised with the Tandem Core VLP containing the HA stalk and 3x M2e inserts generated antibody which could bind rHA protein from influenza virus as well as M2e peptide, whereas mice immunised with adjuvant only did not (Figure 11). Immunised mice were protected from A/PR8 H1N1 influenza virus infection.

4 weeks following primary immunisation, mice were challenged with 5x mLD50 of PR8 influenza virus. Mice in the Adjuvant only group lost weight rapidly and presented with high clinical scores (Figures 12 and 13), reaching 50% mortality by day 10 post-infection (Figure 14). Conversely mice immunised with Tandem Core VLP reached a peak weight loss of 5% associated with a much milder clinical score, and began recovery by day 6, making a full recovery by day 10 post infection (Figures 12 and 13). Survival in the immunised group was 100% (Figure 14). Taken together these results show that mice which received Tandem Core containing the influenza derived insert showed less weight loss, lower morbidity and no mortality following a lethal challenge of PR8 influenza virus. Protection conferred by Tandem Core was not sterile either because the anti-influenza antibody titre was not high enough to protect fully or because VLP vaccination protects via a non-neutralising mechanism. Regardless of the specific mechanism, protection is conferred by vaccination with Tandem Core VLP as non- immunised mice show much more severe symptoms and high mortality.

Example 4

Materials and Methods:

VLP sequence:

Tandem Core VLPs containing inserts from influenza virus conserved protein domains within the two major insertion regions (MIR) (Figure 16) were produced in Pichia Pastoris yeast. The first VLP (VLPl) contains two influenza inserts; one in each MIR. The first insert is the sequence corresponding the stalk region of influenza H1N1 virus hemagglutinin HA2 protein domain. It spans amino acids encoding the known domains; Loop B, Helix C, Helix CD and Helix D of the HA2 monomer (Figure 10). This sequence comprises amino acids (aa) 403-474 of the HA protein isolated from influenza A virus HlNl/Lux/09. This insert of VLPl is referred to as HA2.3 henceforth (Figure 17).

The sequence corresponding to the second insert on VLPl belongs to the region of influenza virus matrix protein 2 ectodomain (M2e). It spans 24 amino acids encoding the known N-terminal external sequence of M2 protein (Figure 5); MSLLTEVETPIRNEWGCRCNGSSD (SEQ ID NO: 6). This wild type sequence was modified to replace the Cysteine residues (which affect VLP formation) at position 17 and 19 (underlined above), for Serine residues. The final insert contains 3 variations of this sequence. The first version is the universal M2e consensus sequence except that the Cysteine residues at positions 17 and 19 have been substituted with Serines (SEQ ID NO: 9). The second (SEQ ID NO: 8) and third (SEQ ID NO: 10) are mutated versions of the universal sequence which correspond to the most common variants found in H7N7 and H5N1 influenza viruses except that the Cysteine residues at positions 17 and 19 have been substituted with Serines. This triple insert of VLP 1 is termed M2e x 3 henceforth. The M2e sequences are flanked by flexible spacers: GGGGSGGGGSGGGGS (SEQ ID NO: 5) and GGGGS GGGGS GGGG (SEQ ID NO: 16).

The full DNA sequence for VLPl is SEQ ID NO: 17 and below the amino acid sequence of VLPl is shown (single letter aa code). Amino acids from Tandem Core are in bold, those from Influenza are underlined and flexible linking regions are in italics:

MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHT ALRQAILCWGELMTLATWVGNNLEGS

MNTQF T A VGKEFNHLEKRIENLNKK VDD GFLDIWT YN AELL VLLENERTLD YH

D SNVKNL YEK VRS QLKNN A SGRDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVW

IRTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPY

KEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI

LCWGELMTLATWVGNNLEF

GGGGSGGGGSGGGGS MSLLTEVETPTRNGWGSKSNGSSD

MSLLTEVETPIRNEWGSRSNGS SD

MSLLTEVETPTRNEWESRS SGS SD

GGGGSGGGGSGGGG

ASDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIR TPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQS RESQC

(SEQ ID NO: 18) Theoretical pI/Mw: 6.01 / 58769.66

The second VLP (VLP2) contains a sequence in its first MIR corresponding the stalk region of influenza H3N2 virus hemagglutinin HA2 protein domain. It spans amino acids encoding the known domains Helix C, Helix CD and Helix D which are sometimes collectively termed "long alpha-helix" or "LAH" (Figure 10). This sequence comprises amino acids (aa) 421-475 of the HA protein isolated from influenza A virus

H3N2/HK/68. This insert will be referred to as LAH3 henceforth (Figure 17).

The second insert of VLP2 is a single Lysine (K) residue flanked by a flexible linker region made up of Glycine and Serine residues. The sequence of the insert used is GSGSGGGKGGGSGS (SEQ ID NO: 21). This is effectively a null insert which allows the first insert (LAH3) to configure properly and confers greater solubility to the whole VLP2.

The full DNA sequence for VLP2 is SEQ ID NO: 19 and below the amino acid sequence of VLP2 is shown (single letter aa code). Amino acids from Tandem Core are in bold, those from Influenza are underlined and linker sequences are in italics:

MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHT ALRQAILCWGELMTLATWVGNNLEGS RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTRRQLREN A

SGRDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVW IRTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPY KEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI LCWGELMTLATWVGNNLEF

GSGSGGGKGGGSGS

ASDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIR

TPPAYRPPNAPILSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQS

RESQC

(SEQ ID NO: 20)

Theoretical pI/Mw: 6.77 / 48186.40

Animals: 6-8 week old BALB/c female mice were purchased from Harlan (Wyton, UK) and housed in IVC category II containment facilities. All animal care and procedures were performed in accordance with Home Office UK regulations on animal use for experimental purposes.

Immunisation:

For primary immunisation, individual mice were given an i.p. injection containing 3C^g VLP material, 2C^g SAS (sigma adjuvant system- Sigma Aldrich), 2C^g Pierce Imject Alum (Thermo Scientific), and sterile saline solution to a total volume of ΙΟΟμΙ. For booster immunisations, administered 7 and 14 days after primary, individual mice received a s.c. injection containing 15ug VLP material, 2C^g MLPA (Sigma), 2C^g muramyl dipeptide (Sigma), 2C^g Pierce Imject Alum (Thermo

Scientific) and made up to ΙΟΟμΙ final volume with sterile saline solution. One day after the final booster, mice were bled to confirm seroconversion. Control groups were immunised with adjuvants only, not containing VLP material.

Seroconversion:

Detection of anti-HA or anti-M2e antibodies was performed by ELISA. 96-well Nunc Maxisorp plates were coated with ^g/ml rHA (Life Technologies) from A/PR8 or X31 influenza virus in carbonate bi-carbonate buffer, or 6.25μg/ml M2e peptide sequence MSLLTEVETPIRNEWGCRCNGSSD-OH (SEQ ID NO: 14) (Activotec) in 1M NaCl buffer. Coated plates were washed 3x with PBS-Tween20 0.05% and blocked with 10% milk solution for lHr at 37C. Diluted serum in 2.5% milk was added and incubated at 37C for lHr then washed 3x as before. Goat-anti-Mouse IgG -Peroxidase secondary Ab was added at 1/2500 dilution (Sigma) and incubated at 37C for lHr then washed 3x as before. TMB substrate (Sigma) was added for 20 minutes and reaction was stopped with 1M H2SO4. Absorbance at 450nm with a 630nm correction was read using the Tecan Sunrise plate reader and analysed by Magellan™ software. All wells were run in duplicate with at least 3 dilution repeats.

Challenge:

4 weeks after primary immunisation, mice were infected with a 5x mLD50 dose of A/PR8 H1N1 or 3x mLD50 X31 H3N2 influenza virus. Virus was administered i.n. after mice were anaesthetised with isofluorane delivered via an oxygen diffusion chamber. Mice were weighed at time of infection (day 0) and daily thereafter until full recovery was made. Mice were also scored using the sickness scale found in Table 3. Results:

Immunised mice seroconverted to rHA protein from H1N1 (A/PR8), H3N2 (X31) influenza virus and M2e peptide.

3 weeks following primary immunisation, anti-serum from 5 mice was collected, pooled and tested for reactivity against recombinant hemagglutinin protein from PR8

(HI) or X31 (H3) influenza virus and M2e peptide by ELISA. Mice immunised with the Tandem Core VLP1 and VLP2 containing the HA stalk and M2e inserts generated antibody which could bind rHA protein from influenza virus as well as M2e peptide. Mice immunised with adjuvant only (neg-) did not test positive to any influenza antigens (Figure 18).

Immunised mice with Tandem Core VLP were protected from H1N1 and H3N2 influenza virus infection.

4 weeks following primary immunisation, mice were challenged with 5x mLD50 of PR8 H1N1 (a) or 3x mLD50 X31 H3N2 (b) influenza virus. Mice in the adjuvant-only group presented with rapid weight loss (Figures 19a and 19b) and high clinical scores (Figures 20a and 20b), with a high degree of mortality by day 8 post-infection (Figures 21a and 21b). Conversely mice immunised with Tandem Core VLP reached a peak weight loss of 10% associated with a much milder clinical score, and began recovery by days 5-7, making a full recovery by day 15 post infection (Figures 19 and 20). Survival in the immunised groups was 100% (Figure 21). Taken together these results show that mice which received Tandem Core VLP1 and VLP2 containing the influenza derived inserts showed less weight loss, lower morbidity and no mortality following a lethal challenge of either PR8 H1N1 or X31 H3N2 influenza virus. Protection conferred by Tandem Core was permissive to infection either because the anti-influenza antibody titre was not high enough for sterile immunity or because VLP vaccination protects via a non- neutralising mechanism. Regardless of the specific mechanism, a clear benefit is conferred by vaccination with Tandem Core VLPs as mice in the negative control group show much more severe symptoms and high mortality.