VACCINES BASED ON HEPATITIS B CORE ANTIGENS
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-esterasefusion)
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. 201 1; 2 : 76. Published online Dec 16, 201 1. Prepublished
online Sep 12, 201 1.doi: 10.3389/fimmu.201 1.00076.
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 adjuvantonly
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 2 1: 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: 2 1.
SEQ ID NO: 2 1 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 FIBV. The
sequence of the 183 amino acid protein of the ayw subtype plus a 29 amino acid presequence
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 Nterminus,
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 positivevalued
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, 7 1 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, 7 1 to 90 or 75 to 85 may be
replaced by an immunogenic polypeptide. Where an immunogenic polypeptide replaces
e l 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 Cterminal
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 2 1 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 Ser (G 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 G2S 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 G2S. 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 Cterminal
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 Cterminus
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, PLOSONE, 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.
Immunogenicpolypeptide
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, Treponemapallidua, Pseudomonas, Bordetella pertussis, Brucella,
Franciscella tulorensis, Helicobacter pylori, Leptospria interrogans, Legionella
pnumophila, Yersiniapestis, 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 genito-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, PB1, 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 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.
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 asM2 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 asM2 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 (RSV) 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,
HI4, HI5 or HI6 and/or from any of the NA subtypes such as Nl, N2, N3, N4, N5, N6,
N7, N8 orN9. 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 90/1 1092,
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 g 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] (oilin-
water emulsion + MPL + QS-21), Montanide ISA-51 and ISA-720 (stabilised waterin-
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; GGGGSGGGGSGGGGS (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
seq uence
Influenza MSLLTEVETPTRNGWGSKSNGSSD (GGGGS) (SEQ Yes
M 2 (SEQ ID NO: 9) ID NO: 4) x3
MSLLTEVETPIRNEWGSRSNGSSD
(SEQ ID NO: 8)
MSLLTEVETPTRN EWESRSSGSSD
(SEQ ID 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 15 g VLP material, 2C^g SAS (sigma adjuvant system- Sigma
Aldrich), 20 g Pierce Imject Alum (Thermo Scientific), and sterile saline solution to a
total volume of . For booster immunisations, administered 7 and 14 days after
primary, individual mice received a subcutaneous (s.c.) injection containing 5ug VLP
material, 20 g MLPA (Sigma), 20g muramyl dipeptide (Sigma), 20g Pierce Imject
Alum (Thermo Scientific) and made up to 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 6.25 g/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 wereprotected 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:
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHT
ALRQAIL CWGELMTLATWVGNNLEGS
MNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYH
DSNVK LYEKVRSQLKNNA
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 15 g VLP material, g SAS (sigma adjuvant system- Sigma
Aldrich), g Pierce Imject Alum (Thermo Scientific), and sterile saline solution to a
total volume of . For booster immunisations, administered 7 and 14 days after
primary, individual mice received a subcutaneous (s.c.) injection containing 5ug VLP
material, 0 g MLPA (Sigma), 20 g muramyl dipeptide (Sigma), 20g Pierce Imject
Alum (Thermo Scientific) and made up ΐ ΐ ΐ 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 PBSTween20
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 wereprotected 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
DSNVKNLYEKVRSOLKNNA
SGRDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVW
IRTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPY
KEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI
LCWGELMTLATWVGNNLEF
GGGGSGGGGSGGGGS
MSLLTEVETPTRNGWGSKSNGSSD
MSLLTEVETPIRNEWGSRSNGS 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 15 g VLP material, 20 g SAS (sigma adjuvant system- Sigma
Aldrich), 20 g Pierce Imject Alum (Thermo Scientific), and sterile saline solution to a
total volume of . For booster immunisations, administered 7 and 14 days after
primary, individual mice received a subcutaneous (s.c.) injection containing 5ug VLP
material, 20g MLPA (Sigma), 20g muramyl dipeptide (Sigma), 20g Pierce Imject
Alum (Thermo Scientific) and made up ΐ ΐ ΐ 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 6.25 g/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 wereprotected 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 nonimmunised
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 VLP1 is termed M2e x 3 henceforth.
The M2e sequences are flanked by flexible spacers: GGGGSGGGGSGGGGS (SEQ ID
NO: 5) and GGGGSGGGGSGGGG (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
MNTQFTAVGKEFNHLEKRIENLNKK VDDGFLDIWTYNAELLVLLENERTLDYH
DSNVKNLYEKVRSQLKNNA
SGRDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVW
IRTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPY
KEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAI
LCWGELMTLATWVGNNLEF
GGGGSGGGGSGGGGS
MSLLTEVETPTRNGWGSKSNGSSD
MSLLTEVETPIRNEWGSRSNGS SD
MSLLTEVETPTRNEWESRS SGSSD
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, g SAS (sigma adjuvant system- Sigma Aldrich),
g Pierce Imject Alum (Thermo Scientific), and sterile saline solution to a total
volume of . For booster immunisations, administered 7 and 14 days after primary,
individual mice received a s.c. injection containing 15ug VLP material, g MLPA
(Sigma), g muramyl dipeptide (Sigma), 2C^g Pierce Imject Alum (Thermo
Scientific) and made up to 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 6.25 g/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 X3 1 (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 X3 1 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 X3 1 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 nonneutralising
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.
CLAIMS
1. A protein comprising 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.
2 . The protein according to claim 1, wherein 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.
3 . 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.
4 . The protein according to any one of the preceding claims, wherein one
copy of HBcAg comprises the influenza virus A surface polypeptide M2 or immunogenic
fragment thereof in the el loop and the other copy of HBcAg comprises another
immunogenic polypeptide.
5 . The protein according to any one of the preceding claims, wherein the
protein comprises the following components:
[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] -
[the other immunogenic polypeptide] - [the part of the second copy of HBcAg that is Cterminal
to the el loop].
6 . The protein according to any one of the preceding claims, wherein the
protein comprises the following components:
[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] - [the other immunogenic polypeptide] - [fifth linker] - [the part of the
second copy of HBcAg that is C-terminal to the el loop]
7 . The protein according to any one of claims 4 to 6, wherein the other
immunogenic polypeptide is an influenza virus polypeptide or immunogenic fragment
thereof.
8 . The protein according to claim 7, wherein the influenza virus polypeptide
or immunogenic fragment thereof is hemagglutinin (HA) or an immunogenic fragment
thereof, wherein the fragment of HA is optionally the HA stalk region.
9 . The protein according to any one of claims 1 to 3, wherein one copy of
HBcAg comprises the influenza virus A surface polypeptide M2 or immunogenic
fragment thereof in the el loop and the other copy of HBcAg comprises, in the el loop, a
sequence of less than 20 amino acids.
10. The protein according to claim 9, wherein the second copy of HBcAg
comprises, in the el loop, a Lysine (K) residue flanked on each side by a linker sequence
comprising Glycine and Serine residues.
11. The protein according to claim 9 or 10, wherein the protein comprises the
following components:
[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].
12. The protein according to any of claims 9 to 11, wherein the second copy
of HBcAg comprises the sequence GSGSGGGKGGGSGS (SEQ ID NO: 21) in the el
loop.
13. The protein according to any one of claims 2 to 12, wherein the alternative
amino acid at position 17 and/or 19 of the influenza virus A surface polypeptide M2 or
the immunogenic fragment thereof is serine.
14. The protein according to any one of the preceding claims, wherein there is
more than one copy of the influenza virus A surface polypeptide M2 or immunogenic
fragment thereof in the or each el loop.
15. The protein according to claim 14, wherein there are from 2 to 5 copies of
the influenza virus A surface polypeptide M2 or immunogenic fragment thereof in the or
each el loop.
16. The protein according to claim 14, wherein there are 3 copies of the
influenza virus A surface polypeptide M2 or immunogenic fragment thereof in the or
each el loop.
17. The protein according to any one of claims 14 to 16, wherein there is a
linker between each copy of influenza virus A surface polypeptide M2 or immunogenic
fragment thereof.
18. The protein according to any one of the preceding claims, wherein the
immunogenic fragment of influenza virus A surface polypeptide M2 is the influenza
virus A surface polypeptide M2 ectodomain (M2e).
19. The protein according to claim 18, wherein the or each copy of M2e in the
or each el loop is the universal M2e consensus sequence or the universal M2e consensus
sequence with up to 6 amino acid substitutions, additions or deletions.
20. The protein according to claim 19, wherein there are three copies of M2e
in the or each el loop and the first copy is the universal M2e consensus sequence, the
second copy is a common variant found in H3N2 or H7H7 and the third copy is a
common variant found in H5N1 .
2 1. The protein according to any one of the preceding claims, wherein the
tandem copies of HBcAg are joined by a linker.
22. The protein according to any one of the preceding claims, wherein:
(a) the or each linker is at least 1.5 nm in length; and/or
(b) the or each linker comprises one or multiple copies of the sequence
GlynSer (GnS) wherein n is from 2 to 8 .
23. The protein according to claim 22, wherein the or each linker is
GGGGSGGGGSGGGGS (SEQ ID NO: 5).
24. 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,
influenza virus 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.
25. The protein according to claim 24, wherein:
(a) the fragment of HA is from influenza H3N2 virus hemagglutinin HA2 protein
domain; and/or
(b) the second copy of HBcAg comprises, in the el loop, a Lysine (K) residue
flanked on each side by a linker sequence comprising Glycine and Serine residues.
26. The protein according to claim 24 or 25, wherein the protein comprises
the following components:
[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].
27. The protein according to any one of claims 24 to 26, wherein the second
copy of HBcAg comprises the sequence GSGSGGGKGGGSGS (SEQ ID NO: 21) in the
el loop.
28. A particle comprising multiple copies of one or more proteins as claimed
in any one of the preceding claims.
29. The particle of claim 28 comprising multiple copies of a protein as
claimed in any one of claims 9 to 23 and of a protein as claimed in any one of claims 24
to 27.
30. A nucleic acid molecule encoding a protein as claimed in any one of
claims 1 to 27.
31. The nucleic acid molecule according to claim 30, which is an expression
vector.
32. A host cell comprising one or more nucleic acid molecules as claimed in
claim 30 or 31.
33. A process for producing a protein as claimed in any one of claims 1 to 27,
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.
34. A pharmaceutical composition comprising a protein as claimed in any one
of claims 1 to 27, a particle as claimed in claim 28 or 29 or a nucleic acid molecule as
claimed in claim 30 or 31, and a pharmaceutically acceptable carrier or diluent.
35. A pharmaceutical composition comprising a protein as claimed in any one
of claims 1 to 23 and a protein as claimed in any one of claims 24 to 27; a particle
comprising a protein as claimed in any one of claims 1 to 23 and a particle comprising a
protein as claimed in any one of claims 24 to 27; or a nucleic acid molecule encoding a
protein as claimed in any one of claims 1 to 23 and a nucleic acid molecule encoding a
protein as claimed in any one of claims 24 to 27; and a pharmaceutically acceptable
carrier or diluent.
36. A vaccine comprising a protein as claimed in any one of claims 1 to 27, a
particle as claimed in claim 28 or 29 or a nucleic acid molecule as claimed in claim 30 or
31, and a pharmaceutically acceptable carrier or diluent.
37. A vaccine comprising a protein as claimed in any one of claims 1 to 23
and a protein as claimed in any one of claims 24 to 27; a particle comprising a protein as
claimed in any one of claims 1 to 23 and a particle comprising a protein as claimed in
any one of claims 24 to 27; or a nucleic acid molecule encoding a protein as claimed in
any one of claims 1 to 23 and a nucleic acid molecule encoding a protein as claimed in
any one of claims 24 to 27; and a pharmaceutically acceptable carrier or diluent.
38. The pharmaceutical composition according to claim 34 or 35 or the
vaccine according to claim 36 or 37, further comprising an adjuvant.
39. A method of inducing an immune response against influenza in a subject,
which method comprises administering to the subject a protein as claimed in any one of
claims 1 to 23, a particle as claimed in claim 28 or 29 or a nucleic acid molecule as
claimed in claim 30 or 31.
40. A method of inducing an immune response against influenza in a subject,
which method comprises administering to the subject a protein as claimed in any one of
claims 1 to 23 and a protein as claimed in any one of claims 24 to 27; a particle
comprising a protein as claimed in any one of claims 1 to 23 and a particle comprising a
protein as claimed in any one of claims 24 to 27; or a nucleic acid molecule encoding a
protein as claimed in any one of claims 1 to 23 and a nucleic acid molecule encoding a
protein as claimed in any one of claims 24 to 27.
41. The method according to claim 39 or 40, wherein administration is in
combination with an adjuvant.
42. A protein as claimed in any one of claims 1 to 23, a particle as claimed in
claim 28 or 29 or a nucleic acid molecule as claimed in claim 30 or 31, for use in a
method of vaccination of the human or animal body against influenza.
43. A protein as claimed in any one of claims 1 to 23 and a protein as claimed
in any one of claims 24 to 27; a particle comprising a protein as claimed in any one of
claims 1 to 23 and a particle comprising a protein as claimed in any one of claims 24 to
27; or a nucleic acid molecule encoding a protein as claimed in any one of claims 1 to 23
and a nucleic acid molecule encoding a protein as claimed in any one of claims 24 to 27,
for use in a method of vaccination of the human or animal body against influenza.
44. Use of a protein as claimed in any one of claims 1 to 23, a particle as
claimed in claim 28 or 29 or a nucleic acid molecule as claimed in claim 30 or 31, for the
manufacture of a medicament for vaccination of the human or animal body against
influenza.
45. Use of a protein as claimed in any one of claims 1 to 23 and a protein as
claimed in any one of claims 24 to 27; a particle comprising a protein as claimed in any
one of claims 1 to 23 and a particle comprising a protein as claimed in any one of claims
24 to 27; or a nucleic acid molecule encoding a protein as claimed in any one of claims 1
to 23 and a nucleic acid molecule encoding a protein as claimed in any one of claims 24
to 27, for the manufacture of a medicament for vaccination of the human or animal body
against influenza.