FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. Title of the Invention
VACCINES BASED ON HEPATITIS B CORE ANTIGENS
2. Applicant(s)
Name Nationality Address
IQUR LIMITED British London Bioscience Innovation Centre 2
Royal College Street London, NW1
0NH, Great Britain
3. Preamble to the description
The following specification particularly describes the invention and the manner in which it is to be performed
2
Field of the invention
The invention relates to proteins comprising hepatitis B core antigen
(HBcAg) with a sugar attached to an e1 loop, processes for producing the proteins
with the sugar attached, pharmaceutical compositions comprising the proteins and
5 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 α-helices which form a characteristic “spike”
10 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 sequence delivers multiple
copies of the antigen. Furthermore, the lack of viral nucleic acid makes them a
particularly safe vector. HBc is particularly interesting as a vaccine carrier since it
15 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
20 dimerise and a VLP does not form. This resulted in a massive loss of
immunogenicity.
Currently there is no licensed vaccine available for the bacterial biothreat
agents Burkholderia pseudomallei and Burkholderia mallei, the causative agents
of melioidosis and glanders respectively.
25
Summary of the invention
The invention is concerned with a vaccine delivery system based on the
hepatitis B (HBV) core protein. A sugar is attached to the HBV core protein
before delivery so that an immune response can be raised against the sugar.
30 The invention thus provides a protein comprising hepatitis B core antigen
(HBcAg) with a sequence of the formula XpZqXr in an e1 loop, wherein X is a
3
negatively charged amino acid residue, Z is a positively charged amino acid
residue, and p, q and r are each independently an integer from 1 to 12, and
wherein a sugar is attached to a Z residue. The protein may comprise a first and a
second copy of HBcAg in tandem, wherein one or both copies of HBcAg has a
5 sugar attached to the e1 loop.
The invention also provides:
- a particle comprising multiple copies of a protein of the invention;
- a process for producing a protein of the invention, which comprises
attaching one or more sugars to the e1 loop;
10 - a pharmaceutical composition comprising a protein of the invention or a
particle of the invention and a pharmaceutically acceptable carrier or diluent;
- a protein of the invention or a particle of the invention for use in a method
of vaccination of the human or animal body;
- use of a protein of the invention or a particle of the invention for the
15 manufacture of a medicament for vaccination of the human or animal body; and
- a method of inducing an immune response in a subject, which method
comprises administering to the subject a protein of the invention or a particle of
the invention.
20 Brief description of the Figures
Figure 1: SDS-PAGE confirmed that tandem cores were found in the
soluble fraction of the yeast lysate. A crude lysate was taken (lane 3), spun at
20,000xg and the supernatant was taken (lane 4). Anything in the pellet (lane 5)
was unusable. The supernatant was diluted (lane 6) and then passed through three
25 filters of 0.8 μm, 0.45 μm and 0.2 μm (lanes 7-9). The material was passed over a
cross-flow filter and the retentate kept (lane 10). This was filtered and then placed
on a CL4B column (lane 11). The void volume was then passed over an S1000
column (lane 12).
Figure 2: VLP were isolated from the void volume of the CL4B column
30 (large peak on left panel of (B)). The numbers above the lanes are fraction
numbers collected from the CL4B column.
4
Figure 3: The CL4B void was then passed over an S1000 column and the
VLP isolated from fractions 12-15. Purity was confirmed by SDS-PAGE and
western blot. The numbers are the tandem core positive fractions collected from
the second S1000 column.
Figure 4: (A) 5 SDS-PAGE with silver staining confirmed that tandem core
was present (marked *), but purity was not as high as in an equivalent yeast
preparation. (B) Electron microscopy identified the major contaminant as baculovirion
itself.
Figure 5: Lane A: molecular weight markers, Lane B: unmodified VLP
10 Lane C: modified VLP.
Figure 6: (A) Schematic representation of sucrose cushion (not to scale),
(B) unbound FITC and (C) FITC-VLP conjugate.
Figure 7: Lane A: molecular weight markers, Lane B: BSA (2mg/ml),
Lane C: BSA (0.5mg/ml), Lane D: Glycoconjugate (2mg/ml) and Lane E:
15 Glycoconjugate (0.5mg/ml).
Figure 8: VLP carrying the LolC insert were tested in an ELISA using
antibodies raised in mice that had been infected with the wild-type Burkholderia
bacterium. The line which corresponds to VLP LolC has a value for 30 ug/ml
which lies between 1.6 and 1.8 average OD. The line which corresponds to
20 unloaded VLP has a value for 30 ug/ml which lies near 0.4 average OD.
Figure 9: Conjugated CPS visualised under electron microscopy.
Immunogold staining Trasmission electron microscopy (TEM). (A) Tris-buffered
saline (TBS) control. (B) anti-CPS mAb.
Figure 10: Efficacy of VLP-CPS conjugation. BALB/c mice were
25 vaccinated 3 times, at two-week intervals and challenged with B. pseudomallei
K96243 by the intraperitoneal route. Significantly greater protection was
achieved with the (B) VLP-CPS conjugate vaccine than with (A) unconjugated
CPS. p=0.0001 Log rank (Mantel-Cox) adjusted for challenge dose. MLD:
minimum lethal dose.
5
Figure 11: CPS specific serum (A) IgG and (B) IgM titres were higher in
VLP-CPS conjugate vaccinated mice than mice vaccinated with unconjugated
CPS.
Brief 5 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.
10 SEQ ID NO: 3 is a sequence which HBcAg may comprise in order to
balance the α-helices.
SEQ ID NO: 4 is the sequence of construct CoHo7e.
SEQ ID NO: 5 is the sequence of construct H3Ho.
SEQ ID NO: 6 is the sequence of the LolC-empty construct.
15 SEQ ID NO: 7 is the sequence of the LolC-K6 construct.
SEQ ID NO: 8 is the sequence of the LolC-K1 construct.
SEQ ID NO: 9 is a sequence of LolC.
SEQ ID NO: 10 is the sequence of a construct comprising the D3-K6-D3
sequence.
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 “a sugar” includes two
25 or more such sugars, or reference to “a protein epitope” includes two or more such
protein epitopes.
All publications, patents and patent applications cited herein, whether
supra or infra, are hereby incorporated by reference in their entirety.
30 Hepatitis B core antigen (HBcAg)
6
HBcAg has 183 or 185 amino acids (aa) depending on the subtype of
HBV. The sequence of the 183 amino acid protein of the ayw subtype plus a 29
amino acid pre-sequence is shown in SEQ ID NO: 2. The mature HBcAg runs
from the Met residue at position 30 to the Cys residue at the extreme C-terminus,
with the sequence 5 from positions 1 to 29 being a pre-sequence.
The protein may comprise two copies of HBcAg forming a dimer. 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 units may be joined directly
10 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 HBcAg in the protein may be native full length HBcAg. The HBcAg
15 has a sugar attached to the e1 loop. A sequence with the formula XpZqXr is
inserted in an e1 loop so that one or more sugars can be subsequently attached to
one or more positively charged residues. X is a negatively charged amino acid
residue, Z is a positively charged amino acid residue and p, q and r are each
independently an integer from 1 to 12. Where the protein comprises a first and a
20 second copy of HBcAg in tandem, one copy of HBcAg has a sugar attached to the
e1 loop via a positively charged residue in a sequence with the formula XpZqXr.
The other copy of HBcAg may be native HBcAg, may be a modified version of
HBcAg as described herein, may have a sugar attached to the e1 loop or may
comprise a protein epitope in the e1 loop. Examples of possible sugars and
25 protein epitopes are 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 e1 loop, the C-terminus
30 and/or the N-terminus. The e1 loop of HBcAg can tolerate insertions of e.g. from
7
1 to 500 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 t 5 o 500 aa, from 1 to 400 aa, from 1 to 300 aa, from 1 to 200 aa, from 3 to
100 aa or from 6 to 50 aa. Substitutions may involve a number of amino acids up
to, for example, 1, 2, 5, 10, 20 or 50 amino acids over the length of the HBcAg
sequence. An extension may be at the N- or C-terminus of HBcAg. A deletion
may be at the N-terminus, C-terminus or at an internal site of the protein.
10 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 e1 loop. An inserted sequence
may carry a protein epitope. A sequence with the formula XpZqXr is inserted in an
e1 loop so that one or more sugars can be subsequently attached. Where the
15 protein comprises a first and a second copy of HBcAg in tandem, only one copy
may have a sugar attached to the e1 loop via a positively charged residue in a
sequence with the formula XpZqXr or both copies may have a sugar attached to the
e1 loop via a positively charged residue in a sequence with the formula XpZqXr.
Where both copies have a sugar attached to the e1 loop via a positively charged
20 residue in a sequence with the formula XpZqXr, the sequence of formula XpZqXr
may be the same for both copies or may be different. X is a negatively charged
amino acid residue, Z is a positively charged amino acid residue and p, q and r are
each independently an integer from 1 to 12. Any amino acid inserted for the
attachment of a sugar must be capable of having a sugar attached to it. Z may be
25 lysine or arginine or a combination of these two amino acids. Z is preferably
lysine. The value of q may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. For example,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 lysines may be inserted. X is a negatively
charged amino acid and therefore may be aspartic acid or glutamic acid or a
combination of these two amino acids. X is preferably aspartic acid. The value of
30 p may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The value of r may be 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11 or 12. The value of p may be equal to r. Preferably the total
8
value of p and r is equal to the value of q. For example, a possible sequence could
have p as 3, q as 6 and r as 3. A preferred sequence has Z as lysine, X as aspartic
acid, p as 3, q as 6 and r as 3. One or more alanines may be inserted either side of
the amino acids which have been inserted for the attachment of sugar. For
5 example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20
alanines may be inserted. The alanines may be inserted consecutively. 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 e1
10 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
15 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
20 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.
25 Software for performing BLAST analyses is publicly available through the
National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
This algorithm involves first identifying high scoring sequence pair (HSPs) by
identifying short words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a word of the
30 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
9
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 va 5 lue; 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
10 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.
15 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
20 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 e1 loop of HBcAg is at positions 68 to 90 of the mature sequence, and
a protein epitope may be inserted anywhere between these positions. Amino acids
for the attachment of a sugar as discussed above may be inserted anywhere
25 between these positions. Preferably, the epitope or amino acids for the attachment
of sugar is inserted in the region from positions 69 to 90, 71 to 90 or 75 to 85.
Most preferred is to insert the epitope or the amino acids for the attachment of
sugar between amino acid residues 79 and 80 or between residues 80 and 81.
When a protein epitope or amino acids for the attachment of sugar is inserted, the
30 entire sequence of HBcAg may be maintained, or alternatively the whole or a part
of the e1 loop sequence may be deleted and replaced by the protein sequence.
10
Thus, amino acid residues 69 to 90, 71 to 90 or 75 to 85 may be replaced by a
protein epitope or amino acids for the attachment of sugar. Where a protein
epitope or amino acids for the attachment of sugar replaces e1 loop sequence, the
replacement sequence is generally not shorter than the sequence that it replaces.
A C-terminal truncation 5 of HBcAg will generally not go beyond aa 144
because if any further truncation is made particles may not form. Thus, the
deleted amino acids may, for example, comprise aa 144 to the C-terminal aa (aa
183 or 185), aa 150 to the C-terminal aa, aa 164 to the C-terminal aa or aa 172 to
the C-terminal aa. The C-terminus of HBcAg binds DNA, and truncation of the
10 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 the protein of the invention. The particle can be in the form of
15 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
20 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.
A linker may join adjacent HBcAg copies. A linker may be present on
one or both sides of the sequence of the formula XpZqXr in the e1 loop. The linker
25 between adjacent HBcAg copies is generally a chain of amino acids at least 1.5
nm (15 Å) in length, for example from 1.5 to 10 nm, from 1.5 to 5 nm or from 1.5
to 3 nm. This linker may, for example, comprise 4 to 40 aa or 10 to 30 aa,
preferably 15 to 25 aa. The linker present on one or both sides of the sequence of
the formula XpZqXr is typically shorter, for example from 3 to 20 aa such as from
30 4 to 12 aa. A linker is generally flexible. The amino acids in a linker may, for
example, include or be entirely composed of glycine, serine and/or proline. For
11
example, a linker may comprise one, two or more repeats of the sequence GlynSer
(GnS) where n is 1, 2, 3, 4, 5, 6, 7 or 8. Alternatively, a linker may comprise one
or more GlyPro (GP) dipeptide repeats. The number of repeats may, for example,
be from 1 to 18, preferably from 1 to 12. A linker may comprise repeats of the
GnS sequence with different value 5 s for n. In the case of G2S repeats, the use of 5,
6 or 7 repeats in a linker joining adjacent HBcAg copies has been found to allow
the formation of particles. 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.
10 The two α-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
15 may comprise an inserted sequence which acts to “balance” the α-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 protein
epitope 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
20 amino acids are preferably uncharged amino acids such as alanine, leucine, serine
and threonine. The inserted sequence is preferably AAALAAA (SEQ ID NO: 3).
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.
25 Sugar
The term “sugar” refers to polysaccharides, oligosaccharides and
monosaccharides. The protein comprises HBcAg with a sugar attached to an e1
loop. A sequence with the formula XpZqXr is inserted in an e1 loop so that one or
more sugars can be subsequently attached to one or more positively charged
30 residues. The protein may comprise a first and a second copy of HBcAg in
12
tandem. Where there are two copies of HBcAg in tandem, one or both copies of
HBcAg has a sugar attached to the e1 loop.
There may be more than one sugar attached to the e1 loop. The e1 loop
may have more than one type of sugar attached. The e1 loop may have different
sugars attached. Where there are two copies of 5 HBcAg in tandem, there may be a
different sugar or different sugars attached to the e1 loop in each HBcAg. It may
be useful for simultaneously inducing an immune response to more than one
pathogen or allergen if the sugars are derived from more than one pathogen or
allergen. The sugar may be part of a glycoprotein so that the glycoprotein is
10 attached to the e1 loop.
The sugar may be derived from any pathogen or allergen. The sugar may
comprise a T-cell or a B-cell epitope. If it is a T-cell epitope, it may be a cytotoxic
T-lymphocyte (CTL) epitope or a T-helper (Th) cell epitope (e.g. a Th1 or Th2
epitope). There may be more than one epitope present. If there is more than one
15 epitope present, one of the epitopes may be a T-helper cell epitope and another
may be 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 sugar depends on the disease that it is wished to raise an
immune response or vaccinate against. The sugar may, for example, be from a
20 pathogenic organism, a cancer-associated antigen or an allergen. The pathogenic
organism may, for example, be a virus, a bacterium or a protozoan. The sugar
may be from any of the sources described herein from which a protein epitope
may be derived, such as pathogenic organisms and cancers, and which comprise a
sugar.
25 Preferably, the pathogenic organism is derived from a bacterium. The
bacterium may be Burkholderia, for example, Burkholderia pseudomallei or
Burkholderia mallei. The pathogenic organism may comprise common capsule
polysaccharide (CPS). The sugar may comprise antigen from CPS. The sugar
may comprise one or more epitopes from CPS. CPS may be derived from
30 Burkholderia, for example, Burkholderia pseudomallei or Burkholderia mallei.
CPS comprises an unbranched homopolymer of 1-3 linked 2-O acetyl-6-deoxy-β-
13
D-manno-heptopyranose. Therefore the sugar may comprise an unbranched
homopolymer of 1-3 linked 2-O acetyl-6-deoxy-β-D-manno-heptopyranose. CPS
has been identified as a major virulence determinant in both B. pseudomallei and
B. mallei where loss of CPS expression in B. pseudomallei increases the MLD in
mice from 70 cfu to greater tha 5 n 106 cfu. The CPS of B. pseudomallei has been
demonstrated to provide partial protection against subsequent challenge in the
mouse model, whilst passive transfer of antibodies raised against CPS can also
provide protection.
10 Protein epitope
The protein of the invention may comprise a first and a second copy of
HBcAg in tandem, wherein the first copy has a sugar attached to the e1 loop and
the second copy comprises a protein epitope in the e1 loop. The “first copy” may
be either the N-terminal or C-terminal copy.
15 The protein epitope comprises a sequence of amino acids which raises an
immune response. The epitope may be conformational or linear. It may be, for
example, in a sequence of from 6 to 500 aa, 20 to 500 aa, 50 to 500 aa, 100 to 500
aa, 200 to 500 aa, 300 to 500 aa or 300 to 400 aa.
Large and/or hydrophobic insertions can be accommodated without VLP
20 disruption. The protein epitope 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.
25 The protein of the invention may contain more than one protein epitope,
for example up to 2, 3, 5 or 8 protein epitopes. More than one copy of an epitope
may be inserted in the copy of HBcAg; for example, from 2 to 8 copies may be
inserted. Where there are two or more protein epitopes in the protein of the
invention, they may be from the same or different organisms and from the same or
30 different proteins.
14
The epitope may be a T-cell or a B-cell epitope. If it is a T-cell epitope, it
may be a cytotoxic T-lymphocyte (CTL) epitope or a T-helper (Th) cell epitope
(e.g. a Th1 or Th2 epitope). In a preferred embodiment of the invention, one of
the epitopes is a T-helper cell epitope and another is a B-cell or a CTL epitope.
The presence of the T-helper 5 cell epitope enhances the immune response against
the B-cell or CTL epitope.
The choice of epitope depends on the disease that it is wished to vaccinate
against. The epitope may, for example, be from a pathogenic organism, a cancerassociated
antigen or an allergen. The pathogenic organism may, for example, be
10 a virus, a bacterium or a protozoan.
The epitope 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
15 (including for instance HSV-1, HSV-2, EBV, CMV and VZV), papovaviridae
(including for instance Human Papilloma Viruse - 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,
20 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
25 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 epitope may be derived from bacteria, including Burkholderia,
M.tuberculosis, Chlamydia, N.gonorrhoeae, Shigella, Salmonella, Vibrio
30 Cholera, Treponema pallidua, Pseudomonas, Bordetella pertussis, Brucella,
Franciscella tulorensis, Helicobacter pylori, Leptospria interrogaus, Legionella
15
pnumophila, Yersinia pestis, Streptococcus (types A and B), Pneumococcus,
Meningococcus, Hemophilus influenza (type b), Complybacteriosis, Moraxella
catarrhalis, Donovanosis, and Actinomycosis, fungal pathogens including
Candidiasis and Aspergillosis, and parasitic pathogens including Toxoplasma
5 gondii, Taenia, Flukes, Roundworms, Flatworms, Amebiasis, Giardiasis,
Cryptosporidium, Schitosoma, Pneumocystis carinii, Trichomoniasis and
Trichinosis.
The epitope may be derived from a pathogen that infects through a) the
respiratory tract, b) the genito-urinary system or c) the gastrointestinal tract.
10 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,
15 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 epitope to be used in the invention may be derived from a cancer such
20 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,
25 differentation antigens such as tyrosinase, gp100, 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, GaINAc, MAGE-1, MAGE-2, MAGE-4, MAGE-12, MUC1,
MUC2, MUC3, MUC4, MUC18, CEA, DDC, P1A, EpCam, melanoma antigen
30 gp75, Hker 8, high molecular weight melanoma antigen, K19, Tyrl, Tyr2,
members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen),
16
PSMA (prostate specific membrane antigen), prostate secretary protein, alphafetoprotein,
CA125, CA19.9, TAG-72, BRCA-1 and BRCA-2 antigen.
Examples of other candidate epitopes for use in the invention include
epitopes from the following antigens: the influenza antigens HA (hemagglutinin),
NA 5 (neuraminidase), NP (nucleoprotein/nucleocapsid protein), M1, 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;
10 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,
15 and myc; and house dust mite allergen.
Preferably, the protein epitope is derived from Burkholderia, for example
Burkholderia pseudomallei or Burkholderia mallei. The protein epitope may be
derived from any of the proteins listed in Table 1. The protein epitope may
comprise or consist of any of the proteins listed in Table 1. The protein epitope
20 may be a fragment of any of the proteins listed in Table 1.
Table 1 - proteins
Protein Comment
LolC (ABC transporter) Efficacy in animal model.
Harland et al., “Identification of a LolC homologue in
Burkholderia pseudomallei, a novel protective antigen
for melioidosis.” Infect Immun. 2007
Aug;75(8):4173-80.
PotF(ABC transporter) Efficacy in animal model.
17
Harland et al., “Identification of a LolC homologue in
Burkholderia pseudomallei, a novel protective antigen
for melioidosis.” Infect Immun. 2007
Aug;75(8):4173-80.
OppA(ABC transporter) Human convalescent sera.
Suwannasaen et al., “Human immune responses to
Burkholderia pseudomallei characterized by protein
microarray analysis.” J Infect Dis. 2011 Apr
1;203(7):1002-11.
Efficacy in animal model.
Harland et al., “Identification of a LolC homologue in
Burkholderia pseudomallei, a novel protective antigen
for melioidosis.” Infect Immun. 2007
Aug;75(8):4173-80.
Tandem repeat sequence (Rp1) Human sero-positive, healthy.
Tippayawat et al., “Burkholderia pseudomallei
proteins presented by monocyte-derived dendritic
cells stimulate human memory T cells in vitro.” Infect
Immun. 2011 Jan;79(1):305-13.
Tandem repeat sequence (Rp2) Human sero-positive, healthy.
Tippayawat et al., “Burkholderia pseudomallei
proteins presented by monocyte-derived dendritic
cells stimulate human memory T cells in vitro.” Infect
Immun. 2011 Jan;79(1):305-13.
Omp85 (Outer membrane
protein)
Efficacy in animal model.
Su et al., “Immunization with the recombinant
Burkholderia pseudomallei outer membrane protein
18
Omp85 induces protective immunity in mice.”
Vaccine. 2010 Jul 12;28(31):5005-11.
Hcp2 (type VI secretion
protein)
Efficacy in animal model.
Burtnick et al., “The cluster 1 type VI secretion
system is a major virulence determinant in
Burkholderia pseudomallei.” Infect Immun. 2011
Apr;79(4):1512-25.
Preferably, the protein epitope is derived from LolC protein. The
following paragraphs discuss LolC protein but the discussion applies equally to
any of the proteins listed 5 in Table 1. The LolC protein may be a naturally
occurring LolC protein or may be a variant of a naturally occurring LolC protein.
The protein epitope may comprise or consist of LolC protein. Therefore full
length LolC or a fragment thereof may be inserted into the e1 loop.
A LolC protein sequence described herein is:
10
ALGVAALIVVLSVMNGFQKEVRDRMLSVLAHVEIFSPTGSMPDWQLTAK
EARLNRSVIGAAPYVDAQALLTRQDAVSGVMLRGVEPSLEPQVSDIGKD
MKAGALTALAPGQFGIVLGNALAGNLGVGVGDKVTLVAPEGTITPAGM
MPRLKQFTVVGIFESGHYEYDSTLAMIDIQDAQALFRLPAPTGVRLRLTD
15 MQKAPQVARELAHTLSGDLYIRDWTQQNKTWFSAVQIEKRMMFIILTLII
AVAAFNLVSSLVMTVTNKQADIAILRTLGAQPGSIMKIFVVQGVTIGFVGT
ATGVALGCLIAWSIPWLIPMIEHAFGVQFLPPSVYFISELPSELVAGDVIKIG
VIAGS (SEQ ID NO: 9)
20 The sequence of the LolC protein may have homology with SEQ ID NO: 9 or any
naturally occurring LolC protein, 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
19
example over the full sequence or over a region of at least 20, for example at least
50, at least 100, at least 150, at least 200, at least 250, at least 300, or at least 350
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 5 differs from the naturally occurring
LolC 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 2. Amino acids
in the same block in the second column and preferably in the same line in the
10 third column may be substituted for each other.
Table 2
ALIPHATIC Non-Polar G A P
I L V
Polar-uncharged C S T M
N Q
Polar-charged D E
K R
AROMATIC H F W Y
15
A fragment of LolC protein to be used as an insert is a shortened version
of a full length LolC protein 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
20 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 LolC sequence or the sequence of SEQ ID NO: 9. For example a
fragment may be from 6 to 354 aa, from 6 to 300 aa, from 6 to 200 aa, from 6 to
100 aa, from 6 to 50 aa or from 6 to 25 aa in length.
20
Process for attaching the sugar to the protein
The invention provides a process for producing a protein of the invention.
The process comprises attaching one or more sugars to the e1 loop. As described
herein, sugar is attached t 5 o one or more amino acids in the e1 loop. The sugar
may be attached by reductive amination of the oxidized sugar to an amino acid in
the e1 loop. The amino acid may be lysine. Sodium periodate may be used to
oxidise the sugar. Oxidising the sugar generates terminal aldehyde residues.
Terminal aldehyde residues of the sugar can be reductively aminated to primary
10 amines using sodium cyanoborohydride in PBS at pH 7.5. Before attaching the
sugar, the protein can be purified.
The process may further comprise making the protein prior to the
attachment of the sugar. The generation of the protein prior to the attachment of
the sugar is described in more detail below.
15
Making the protein prior to sugar attachment
Prior to attaching the sugar, the protein may be made by recombinant
DNA technology. The nucleic acid molecules may be made using known
techniques for manipulating nucleic acids. Where the protein comprises two
20 copies of HBcAg, typically, two separate DNA constructs encoding the two
HBcAg copies are made and then joined together by overlapping PCR.
Prior to attaching the sugar, the protein 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
25 include bacteria such as E. coli, yeast, mammalian cells and other eukaryotic cells,
for example insect Sf9 cells.
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
30 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
21
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, f 5 or 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
10 suitable for use in E. coli strains (such as E. coli HB101). 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
15 protein is carried out in mammalian cells, either in vitro or in vivo, mammalian
promoters may be used. Tissue-specific promoters, for example hepatocyte cellspecific
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
20 human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters and
adenovirus promoters. All these promoters are readily available in the art.
The protein 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
25 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.
30 Inducing an immune response
22
A protein or particle of the invention can be used to induce an immune
response. The protein or particle may be used as a vaccine. The protein or
particle may be used to raise multiple simultaneous immune responses to all the
components (the HBcAg and the sugar). Where the protein comprises two copies
of HBcAg, 5 the multiple immune responses may also include further immune
responses against additional sugars and/or immune responses against the protein
epitope. If all the sugars, and optional protein epitopes, are derived from the same
source then this can induce an enhanced immune response against that source, for
example a pathogen. If all the sugars, and optional protein epitopes, are derived
10 from more than one source then this can induce simultaneous immune responses
against the different sources, for example more than one pathogen.
The protein or particle 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
15 provides a pharmaceutical composition (e.g. a vaccine composition) comprising a
protein of the invention or a particle comprising multiple copies of the protein of
the invention and a pharmaceutically acceptable carrier or diluent. The
composition may further comprise an adjuvant. The composition can be used for
vaccination of the human or animal body. The composition may be used to
20 vaccinate against any of the pathogens described herein. In particular, the
composition may be used to vaccinate against Burkholderia, for example,
Burkholderia pseudomallei or Burkholderia mallei.
A protein of the invention or a particle of the invention can be used in a
method of vaccination of the human or animal body. The invention provides use
25 of a protein of the invention or a particle of the invention for the manufacture of a
medicament for vaccination of the human or animal body. The protein or particle
may be used to vaccinate against any of the pathogens described herein. In
particular, the composition may be used to vaccinate against Burkholderia, for
example, Burkholderia pseudomallei or Burkholderia mallei.
30 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,
23
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 any of the
pathogens described herein, for example Burkholderia. The protein could
simultaneously vaccinate 5 an individual to any of a wide range of diseases and
conditions depending on the sugar and optional protein epitope which the protein
comprises. Such diseases and conditions include any of those described herein
and HBV, HAV, HCV, foot-and-mouth disease, polio, herpes, rabies, AIDS,
dengue fever, yellow fever, malaria, tuberculosis, whooping cough, typhoid, food
10 poisoning, diarrhoea, meningitis and gonorrhoea. The sugars and protein epitopes
are chosen so as to be appropriate for the disease against which the vaccine is
intended to provide protection.
The invention provides a method of inducing an immune response in a
subject comprising administering to the subject the protein or particle of the
15 invention. Preferably the immune response is against Burkholderia, for example,
Burkholderia pseudomallei or Burkholderia mallei.
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
20 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
25 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
30 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
24
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 or particle of the invention is for administration to a subject.
It may be 5 administered simultaneously or sequentially with an adjuvant.
Therefore the composition of the invention comprising the protein or particle 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,
10 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
15 example, compositions containing the protein or particle 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 or particle together with a
pharmaceutically acceptable carrier or diluent. The composition optionally
20 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
25 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,
30 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
25
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, sucr 5 ose, 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
10 substances is available in REMINGTON‟S PHARMACEUTICAL SCIENCES
(Mack Pub. Co., N.J. 1991), incorporated herein by reference.
Alternatively, the protein or particle and/or the adjuvant may be
encapsulated, adsorbed to, or associated with, particulate carriers. Suitable
particulate carriers include those derived from polymethyl methacrylate polymers,
15 as well as PLG microparticles derived from poly(lactides) and poly(lactide-coglycolides).
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.
20 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
25 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.
30 Typically, the protein or particle of the invention is administered to a
subject in an amount that will be effective in modulating an immune response. An
26
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. A
prophylactically 5 effective amount is an amount which prevents the onset of one or
more symptoms of the disease or disorder.
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 vaccine may be given in a single dose schedule or a multiple dose
10 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
15 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 or particle of the invention and an adjuvant may be
20 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
25 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:
27
- 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
5 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified
saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + QS-21), Montanide
ISA-51 and ISA-720 (stabilised water-in-oil emulsion).
- Particulate adjuvants, e.g. virosomes (unilamellar liposomal vehicles
incorporating e.g. influenza haemagglutinin), AS04 ([SBAS4] Al salt with MPL),
10 ISCOMS (structured complex of saponins and lipids), and polylactide coglycolide
(PLG).
- 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
15 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
20 (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
25 alum (Pierce Laboratories).
The invention is illustrated by the following Examples:
Example 1
30
Materials & Methods
28
Design of constructs
All tandem core clones are derived from the parental construct CoHo7e. In
this version of tandem core, α-helices are “balanced” as described above, and both
copies of HBc have the nucleic acid binding region removed. Thus, the construct
is designated a homo-tandem since 5 both versions of core are essentially identical,
the only differences being silent mutations to allow for altered restriction sites.
The sequence of tandem core CoHo7e used was:
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPH
10 HTALRQAILCWGELMTLATWVGNNLEGSAGGGRDPASRDLVVNYVNTN
MGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLP
ETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPYKEFGATVELLSFLPSDF
FPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVG
NNLEFAGASDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEY
15 LVSFGVWIRTPPAYRPPNAPILSTLPETTVVL (SEQ ID NO: 4)
The H3Ho construct was based on the original CoHo version of tandem
core, as outlined in international application WO 2001/077158. A hexa-lysine
insert was designed which would produce "hotspots" of reactivity onto which CPS
20 could be bound using standard amine chemistry. These were designed
synthetically and included a redesigned MIR which included the sequence
AAALAAA (SEQ ID NO: 3) to “balance” the -helices as described above. The
synthetic inserts were ligated to produce H3Ho. The final sequences was verified
and was as follows:
25
MSDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPH
HTALRQAILCWGELMTLATWVAAALAAAEGSDPASRDLVVNYVNTNM
GLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPE
TTVVGGSSGGSGGSGGSGGSTMDIDPYKEFGATVELLSFLPSDFFPSVRDL
30 LDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVAAALAAA
29
ESGDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGV
WIRTPPAYRPPNAPILSTLPETTVVLE
(SEQ ID NO: 5)
An alternative 5 approach was to insert a protein antigen isolated from
Burkholderia. The LolC protein has been shown previously to be immunogenic
and so was chosen as a potential insert. However, in order to ensure that assembly
of the VLP was not impeded, regions of -helical folding were found at both the
N and C termini. Thus, insertion of the antigen would be from an -helical
10 secondary structure into the -helix of the HBc spike. Currently, there is no
crystallographic data for LolC so the structure was predicted using the PSIPRED
algorithm (http://bioinf.cs.ucl.ac.uk/psipred/). The predicted insertion and
flanking regions were synthesised chemically and inserted into core 1 of H3Ho
using standard ligation techniques. Final sequencing confirmed that this had been
15 successful. LolC-empty sequence:
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPH
HTALRQAILCWGELMTLATWVAAALAAAEGSALGVAALIVVLSVMNGF
QKEVRDRMLSVLAHVEIFSPTGSMPDWQLTAKEARLNRSVIGAAPYVDA
20 QALLTRQDAVSGVMLRGVEPSLEPQVSDIGKDMKAGALTALAPGQFGIV
LGNALAGNLGVGVGDKVTLVAPEGTITPAGMMPRLKQFTVVGIFESGHY
EYDSTLAMIDIQDAQALFRLPAPTGVRLRLTDMQKAPQVARELAHTLSGD
LYIRDWTQQNKTWFSAVQIEKRMMFIILTLIIAVAAFNLVSSLVMTVTNK
QADIAILRTLGAQPGSIMKIFVVQGVTIGFVGTATGVALGCLIAWSIPWLIP
25 MIEHAFGVQFLPPSVYFISELPSELVAGDVIKIGVIAGSDPASRDLVVNYVN
TNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILST
LPETTVVGGSSGGSGGSGGSGGSTMDIDPYKEFGATVELLSFLPSDFFPSV
RDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVAAAL
AAAESGDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVS
30 FGVWIRTPPAYRPPNAPILSTLPETTVVLE (SEQ ID NO: 6)
30
Two variations of the LolC insertion were then made by cleaving core 2 of
the H3Ho LolC-empty construct using PstI and XhoI. Synthetic inserts containing
either hexa-lysine or a single lysine flanked by repeating alanine residues were
then ligated in. Again, sequencing confirmed their identity. LolC-K6 sequence:
5
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPH
HTALRQAILCWGELMTLATWVAAALAAAEGSALGVAALIVVLSVMNGF
QKEVRDRMLSVLAHVEIFSPTGSMPDWQLTAKEARLNRSVIGAAPYVDA
QALLTRQDAVSGVMLRGVEPSLEPQVSDIGKDMKAGALTALAPGQFGIV
10 LGNALAGNLGVGVGDKVTLVAPEGTITPAGMMPRLKQFTVVGIFESGHY
EYDSTLAMIDIQDAQALFRLPAPTGVRLRLTDMQKAPQVARELAHTLSGD
LYIRDWTQQNKTWFSAVQIEKRMMFIILTLIIAVAAFNLVSSLVMTVTNK
QADIAILRTLGAQPGSIMKIFVVQGVTIGFVGTATGVALGCLIAWSIPWLIP
MIEHAFGVQFLPPSVYFISELPSELVAGDVIKIGVIAGSDPASRDLVVNYVN
15 TNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILST
LPETTVVGGSSGGSGGSGGSGGSTMDIDPYKEFGATVELLSFLPSDFFPSV
RDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVAAAL
AAAESGGSGSKKKKKKGSGSSGDPASRDLVVNYVNTNMGLKIRQLLWF
HISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVLE (SEQ
20 ID NO: 7)
LolC-K1 sequence:
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPH
25 HTALRQAILCWGELMTLATWVAAALAAAEGSALGVAALIVVLSVMNGF
QKEVRDRMLSVLAHVEIFSPTGSMPDWQLTAKEARLNRSVIGAAPYVDA
QALLTRQDAVSGVMLRGVEPSLEPQVSDIGKDMKAGALTALAPGQFGIV
LGNALAGNLGVGVGDKVTLVAPEGTITPAGMMPRLKQFTVVGIFESGHY
EYDSTLAMIDIQDAQALFRLPAPTGVRLRLTDMQKAPQVARELAHTLSGD
30 LYIRDWTQQNKTWFSAVQIEKRMMFIILTLIIAVAAFNLVSSLVMTVTNK
QADIAILRTLGAQPGSIMKIFVVQGVTIGFVGTATGVALGCLIAWSIPWLIP
31
MIEHAFGVQFLPPSVYFISELPSELVAGDVIKIGVIAGSDPASRDLVVNYVN
TNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILST
LPETTVVGGSSGGSGGSGGSGGSTMDIDPYKEFGATVELLSFLPSDFFPSV
RDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVAAAL
AAAESGGSGSGGGKGGGSGSSGDPASRDLVVNYVNTNMGLKIRQLLW5 F
HISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVLE (SEQ
ID NO: 8)
Design of plasmids
10 Protein expression was carried out using two systems; the yeast Pichia
pastoris and a baculovirus vector. This required the use of two different plasmids,
specifically, pPICz (Invitrogen) for yeast and pOET1 (Oxford Expression
Technologies) for baculovirus. The H3Ho sequence was inserted into the multicloning
site of pPICz using MfeI and pspOMI, whereas pOET1 was inserted using
15 pspOMI and BclI.
Protein expression of tandem core in yeast
Yeast were transformed by electroporation with 300ng of linearised
plasmid DNA. The yeast were then streaked onto YPD plates containing
20 100μg/ml Zeocin and the largest clones selected. These clones were then
individually challenged with increasing concentrations of Zeocin and the most
resistant clone selected to be transformed a second time by electroporation. The
process was repeated using selection plates with higher levels of Zeocin for clone
selection. In this way, high copy-number clones were developed which had vastly
25 improved VLP expression levels. Large scale yeast cultures were set up in
200mls of YPD. After approximately 4 days, the media was replaced with
induction media and the yeast induced using methanol (0.8% for 72 hrs). After
this time period, the yeast cells were harvested by centrifugation at 1500g and the
pellets stored at -80oC before purification.
32
Protein expression of tandem core in baculovirus
Recombinant virus was produced by co-transfection in Sf9 insect cells.
Duplicate reaction mixtures, each containing flashBACPRIME viral DNA
(100ng) and transfer vector 5 DNA (500ng; pOET1-K6-lolc) together with
Lipofectin liposome forming reagent (baculoFECTIN), were added to 35mm2
dishes with Sf9 insect cells seeded at a density of 1×106 cells/dish. The dishes
were then incubated at 28oC for 5 days, following which the medium containing
each virus was harvested into sterile tubes. A stock of virus was created from 50
ml of Sf9 cells, at a density of 2×106 10 cells/ml, infected with 0.5 ml of the cotransfection
mix. The infected shake culture was incubated for 5 days at 28oC and
then harvested by centrifugation at 500xg for 20 min at 4oC. For expression of
protein, Sf9 cells were seeded in 35mm2 dishes at 1×106 cells/dish, whereas Tni
cells were seeded at 0.5×106 cells/dish. Each dish was infected with the virus at
moi 5. Following 72 hrs incubation at 28o15 C the cell pellets and supernatants were
harvested from each dish.
Purification of protein
Regardless of the expression vector used, or the nature of the insert,
20 purification was carried out in a similar manner. Induced cells were harvested and
spun down (300xg) before being resuspended in lysis buffer (20mM Tris pH 8.4,
5mM EDTA, 5mM DTT, 2mM AEBSF) at a ratio of 2.5g wet weight/10mls lysis
buffer. The resulting solution was then passed through a microniser
(AVP Gaulin LAB 40) set at 500psi three times. Detergent (Triton X100) was
25 added to make a 0.5% solution and the lysate spun for 30mins (25,000xg) before
harvesting the supernatant. The clarified supernatant was passed through a 0.8um
dead end filter (Nalgene), followed by a 0.45um and finally a 0.2um filtration.
This material was diluted ten-fold (20mM Tris pH 8.4, 5mM EDTA) before being
passed over a tangential flow device with a 1MDa molecular weight cut-off
30 (Pellicon). This step both removed low molecular weight contaminants and
reduced volume down to 25mls.
33
The concentrated lysate was then applied to an XK26/92 column packed
with Sepharose CL4B resin driven by an Akta Pure FPLC system. The buffer was
20mM Tris pH 8.4, 5mM EDTA. The fractions were monitored at 260, 280 and
350nm. The void volume was collected since this contains large proteins
including VLP. The 5 remainder of protein isolated over the column was discarded.
The void volume fractions were pooled and concentrated using tangential flow
(Pellicon) back down to 10mls. This material was then passed over an XK26/55
column packed with Sephacryl S1000 resin. This resin has a much larger pore size
and is capable of resolving VLP from other large proteins. Previously, we had
10 calibrated the column using recombinant monomeric HBc (Biospacific Inc) and
therefore knew when assembled VLP would elute. These fractions were collected
and concentrated over tangential flow a final time.
Identity and qualification of protein
15 Samples were routinely stored at each step of the purification process, thus
allowing in-process monitoring. It was known that whilst each expression system
undoubtedly made VLP, not all tandem core proteins achieved this final
conformation. Therefore, the most important contaminant that needed to be
removed was actually tandem core itself in either the monomeric or misfolded
20 state. However, SDS-PAGE and western blotting are, by definition, denaturing
techniques so these had to be coupled with knowledge of protein size which could
be estimated using the retention time on the FPLC columns.
Samples containing tandem core were characterised by electrophoresis at
all stages of the process on 12.5% SDS-polyacrylamide gels (Laemmli, 1970)
25 followed by Coomassie blue staining. Western Blot analyses were performed as
described (Konig, et al., 1998) using a monoclonal primary antibody against HBc
protein (10E11[Abcam]) followed by a mouse secondary antibody conjugated to
horseradish peroxidase and the chemiluminescent substrate ECLplus (Amersham
Pharmacia). Protein concentrations were measured by the Bradford method
30 (BioRad).
34
Electron microscopy
All samples were diluted to 0.1 mg / ml in 20 mM Tris HCl pH 8 and
sonicated in a water bath sonicator for 45 seconds immediately prior to adsorption
to grids. Formvar / carbon coated copper grids (400 mesh) were placed carbon
side down on dr 5 oplets of the diluted samples on parafilm. Material was allowed to
adsorb for 10 min. Grids were then washed in 4 changes of 1 % uranyl acetate.
The grid was incubated for 20 seconds with the final change of uranyl acetate
prior to blotting and air drying. Grids were viewed and digitally imaged on a FEI
Tecnai G2 TEM. Images were taken at a magnification of 87,000x, 43,000x and
10 26,000x.
ELISA for antigenicity of LolC
A 96-well microtiter plate was coated for 24 hours at 4oC with 100μL/well
of purified LolC (standard, 2-0.03 μg/mL), VLPs (negative control, 30-0.2μg/mL)
15 and VLP-LolC (test sample, 30-0.2μg/mL). All samples were serially diluted twofold
in phosphate-buffered saline (1x Dulbecco‟s PBS, Invitrogen). Each well was
washed three times with PBS-0.05% Tween 20, and blocked with 200μL of PBS
containing 5% (w/v) skimmed milk powder for 1 hour at 37oC. Each well was
then washed three times with PBS-0.05% Tween 20 and a 1:1000 dilution of sera
20 from mice vaccinated with endotoxin-free LolC protein added (100μL/well) and
incubated for 1 hour at 37oC. Following three washes in PBS-Tween 20, a 1:2000
dilution of IgG goat anti-mouse horseradish peroxidase conjugate was added to
each well (100μL) and incubated for 1 hour at 37oC. Each well was washed a
further six times in PBS-Tween 20 and bound conjugate detected with
25 ABTS/hydrogen peroxide substrate (100μL/well) with incubation at room
temperature for 20 minutes prior to optical density measurement at 414nm.
Chemical conjugation of CPS to VLP
Sodium meta-periodate (6 mg, 0.3 mmol) and CPS (5 mg) were dissolved
30 in PBS (1 ml) and the reaction mixture was left at room temperature for 1 hour.
Excess sodium meta-periodate was removed using a PD-10 desalting column (GE
35
Healthcare) equilibrated with PBS. The oxidized CPS was added to a solution of
protein at 5 mg/ml (1 ml) in PBS. 20 μl of NaBH3CN [1 M in 10 mMNaOH] was
added to the solution and it was left for four days at room temperature in the dark.
20 μl of NaBH4 [1M NaBH4 in 10 mMNaOH] was added, after agitation the
reaction w 5 as left for 40 min. The solution was diluted in MQ-H2O and
extensively dialyzed against ammonium bicarbonate buffer [20 mM, pH 7.8] and
concentrated in vacuousing speed-vac (Thermo Scientific).
The concentrated protein sample was purified on an ÄKTA Xpress FPLC
purification system. The conjugate solution was injected onto an S500 sepharose
10 SEC column XK 26/60 (GE Healthcare) and eluted using ammonium bicarbonate
buffer (20 mM, pH 7.8) at 1 ml/min. All fractions (2.5 ml) were collected and
analysed for carbohydrate using the phenol:sulphuric acid assay and by TEM and
dot-blot analysis. The pooled fractions were concentrated in vacuo (speed-vac,
Thermo Scientific) and dialyzed into PBS.
15
Results
Protein isolation from yeast samples
Samples were taken throughout the process and thus it was possible to
track the purity enrichment as the process proceeded. Most importantly, regardless
20 of the insert present, the majority of tandem core was found in the soluble fraction
after the initial centrifugation post-lysis. Only a minority of core protein was
found in the pellet from this spin which suggests that chimeric VLP remain
soluble despite their complex composition.
The isolation of VLP from either yeast or Baculovirus lysates is somewhat
25 unusual since it has been found that affinity chromatography is not readily
compatible with tandem core. Hence, the method that evolved was based on
gradual refinements of size class within the sample. Initially, very large debris
was removed by filtration, leaving particles less than 200nm and below present.
The samples were then passed over a tangential flow filter with 1MDa molecular
30 weight cut-off. This served to retain the large VLPs but removed some of the low
molecular weight contaminants.
36
The samples were then separated using CL4B size exclusion
chromatography (SEC). This matrix has a relatively small pore size and very large
material, including VLPs, will not enter the resin. Thus, large material passes
directly through the column and is found in the void volume. However, a
considerable 5 amount of small proteins do enter the resin and are retarded.
Therefore, by retaining only the void volume the samples are effectively enriched.
SDS-PAGE and western blot once again shows that the majority of tandem core
is, indeed, found in the CL4B void volume.
The CL4B void sample was then passed over an S1000 SEC column. This
10 is conventionally used to isolate large molecules such as nucleic acids, but also is
capable of resolving VLP from other large debris. The column had previously
been calibrated using recombinant HBc which were known to be VLP of a similar
size to tandem core VLP (34.6nm). Thus, it was possible to determine that tandem
core VLP should be found in the final 1/3 of the column elution. This was, indeed,
15 the case and pure VLP were isolated from these fractions. This was confirmed by
electron microscopy.
Figures 1 to 3 contain data for protein isolation from yeast samples. SDSPAGE
confirmed that tandem cores were found in the soluble fraction of the yeast
lysate (Figure 1). Figure 2 shows VLP were isolated from the void volume of the
20 CL4B column (large peak on left panel of Figure 2B). The CL4B void was then
passed over an S1000 column and the VLP isolated from fractions 12-15. Purity
was confirmed by SDS-PAGE and western blot (Figure 3).
Protein isolation from baculovirus samples
25 Tandem core proteins were also found in the supernatant from the initial
25,000xg spin, once again suggesting that the VLP were soluble. SDS-PAGE and
western blot confirmed that very little protein was lost to the insoluble pellet. In
most respects, the isolation of VLP from baculovirus was very similar to that
from yeast. However, there was one major difference since it was common to find
30 that baculo-virion co-purified with tandem core. This was detectable both in SDS37
PAGE as a 50KDa band (Figure 4A) and also was clearly visible as long tubules
in electron micrographs (Figure 4B).
Despite several purification iterations, it was not possible to purify the
VLP made in Baculovirus to homogeneity. Hence, the preferred expression
5 system for VLP is the yeast system.
Conjugation to CPS
In order to demonstrate the feasibility of the conjugation approach,
Fluorescein isothiocyanate (FITC) was conjugated to VLP using the techniques
10 outlined previously.
It should be noted that since SDS-PAGE is, by definition, a denaturing
technique, these data show that the tandem core building block has been
effectively modified since its molecular weight has increased (Figure 5).
However, when these conjugated particles were run on a sucrose cushion, a
15 fluorescent band was seen in the VLP region thus supporting the fact that
conjugation had been achieved without destruction of the VLP (Figure 6).
Similarly, we further demonstrate that conjugation of CPS itself is also
possible by conjugating this to bovine serum albumin (Figure 7). Thus, we have
shown that conjugation of CPS is possible using our defined chemical method and
20 that conjugation to VLP is also present. Therefore, the ligation of CPS directly to
VLP should also be feasible.
Antigenicity and immunogenicity
The conjugation of CPS to VLPs should not radically alter the
25 glycoprotein‟s tertiary structure. In vivo testing of the conjugate is underway.
However, an alternative approach is to insert an antigen directly into the
tandem core molecule. Whilst this is likely to lead to VLP which are highly
decorated with the inserted antigen, it does impose potentially severe steric
restrictions on the folding of the insert since it is tethered at both ends. To
30 examine this, VLP carrying the LolC insert were tested in an ELISA using
antibodies raised in mice that had been infected with the wild-type Burkholderia
38
bacterium. Remarkably, the LolC carrying VLP were recognised with almost as
high an affinity as wild-type Lolc protein itself. There was a small response to
unloaded VLP, but the response was clearly predominantly to the insert (Figure
8). For Figure 8, the line which corresponds to VLP LolC has a value for 30 ug/ml
which lies between 1.6 and 1.8 5 average OD. The line which corresponds to
unloaded VLP has a value for 30 ug/ml which lies near 0.4 average OD.
Discussion
The immunogenicity of monomeric core protein is well established, as is
10 its ability to accept antigenic inserts into its MIR. However, it is equally well
documented that the technology has a major weakness because the core dimers no
longer form when large or hydrophobic inserts are added, leading to a failure of
VLP formation. The development of tandem core constructs overcomes this major
limitation.
15 Whilst the utility of tandem core as a delivery system for inserted protein
antigens has been demonstrated elsewhere, it is also possible to use the system in
a chemical conjugation mode. In this case, non-specific linker amino acids are
inserted into the MIR and disease specificity comes from the chemical
conjugation of antigens to these aforementioned target amino acids. This
20 technique further expands the antigens that tandem core can carry since
glycoproteins can be conjugated which would not be possible to add using
conventional cloning means. The multimeric nature of VLPs means that multiple
copies of the target conjugate are added per VLP. Given that 90-120 HBc dimers
are present in every VLP, very high antigen delivery densities can be reached. It
25 is, of course, possible to combine chemical conjugation with specific antigen
insertion, thus making a chimeric molecule with both a specific protein and
specific glycoprotein simultaneously.
The preferred expression system for VLP is the yeast Pichia pastoris.
However, multiple systems can be used including bacteria, Baculovirus and even
30 plant based expression. These data prove that the specificity of the system comes
entirely from the primary protein sequence and is not related to post-translational
39
modifications which may be present in a particular expression system.
Furthermore, we have demonstrated that the purification strategy used is
applicable to any expression system and is thus likely to be scaleable to an
industrial process.
5
Example 2
CPS was attached to an e1 loop of a tandem core via a lysine in a sequence
with the formula XpZqXr, where X is aspartic acid, Z is lysine, p is 3, q is 6 and r
10 is 3. The tandem core construct had the following sequence:
MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPH
HTALRQAILCWGELMTLATWVGNNLEGSGGSGGGSGSGRDPASRDLVV
NYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPN
15 APILSTLPETTVVGGSSGGSGGSGGSGGSGGSGGSTMDIDPYKEFGATVEL
LSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELM
TLATWVGNNLEFGSGSGGDDDKKKKKKDDDGGSGSASDPASRDLVVNY
VNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPI
LSTLPETTVVRRRDRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC
20 (SEQ ID NO: 10)
The sequence of 3 aspartic acid residues, 6 lysine residues and 3 aspartic acid
residues is underlined in the above sequence of the construct, This construct was
tested for its ability to induce immune responses in mice. The results are set out
25 below.
Results
Conjugation of CPS to VLPs
CPS was oxidised with sodium periodate and conjugated to the VLP with
30 sodium cyanoborohydride. Conjugated CPS was visualised under electron
microscopy (Figure 9).
40
Efficacy of the CPS-VLP conjugate
BALB/c mice were vaccinated 3 times, at two-week intervals and
challenged with B. pseudomallei K96243 by the intraperitoneal route.
Significantly greater protection was achieved with the VLP-CPS conjugate
5 vaccine than with unconjugated CPS (Figure 10).
CPS specific serum IgG and IgM titres were higher in VLP-CPS conjugate
vaccinated mice than mice vaccinated with unconjugated CPS (Figure 11).
Discussion
10 A VLP-CPS conjugate gives significantly greater protection than
unconjugated CPS against an intra-peritoneal B. pseudomallei challenge.
34
WE CLAIM:
1. A protein comprising hepatitis B core antigen (HBcAg) with a sequence
of the formula XpZqXr in an e1 loop, wherein X is a 5 negatively charged amino acid
residue, Z is a positively charged amino acid residue, and p, q and r are each
independently an integer from 1 to 12, and wherein a sugar is attached to a Z residue.
2. A protein comprising a first and a second copy of HBcAg in tandem,
10 wherein one or both copies of HBcAg has a sequence of the formula XpZqXr as defined
in claim 1 in an e1 loop and a sugar attached to a Z residue.
3. The protein according to claim 2, wherein the first copy of HBcAg has a
sequence of the formula XpZqXr as defined in claim 1 in an e1 loop and a sugar attached
15 to a Z residue and the second copy comprises a protein epitope in the e1 loop.
4. The protein according to any one of the preceding claims, wherein Z is
lysine or arginine.
20 5. The protein according to any one of the preceding claims, wherein X is
aspartic acid or glutamic acid.
6. The protein according to any one of the preceding claims, wherein Z is
lysine and X is aspartic acid.
25
7. The protein according to any one of the preceding claims, wherein p, q
and r are each independently an integer from 1 to 6.
8. The protein according to any one of the preceding claims, wherein the
30 total of p plus r is equal to q.
9. The protein according to any one of the preceding claims, wherein p is 3,
q is 6 and r is 3.
35
10. The protein according to any one of the preceding claims, wherein X is
aspartic acid, Z is lysine, p is 3, q is 6 and r is 3.
11. The protein according to any one of the preceding claims, wherein the
sequence of formula 5 XpZqXr is flanked by multiple alanines.
12. The protein according to any one of the preceding claims, wherein the
sugar or sugars are derived from a bacterium.
10 13. The protein according to claim 12, wherein the bacterium is
Burkholderia.
14. The protein according to claim 13, wherein the bacterium is
Burkholderia pseudomallei or Burkholderia mallei.
15
15. The protein according to any one of the preceding claims, wherein the
sugar or sugars comprise common capsule polysaccharide (CPS).
16. The protein according to any one of the preceding claims, wherein the
20 sugar or sugars comprise an unbranched homopolymer of 1-3 linked 2-O acetyl-6-
deoxy-β-D-manno-heptopyranose.
17. The protein according to any one of claims 3 to 16, wherein the protein
epitope is from Burkholderia.
25
18. The protein according to any one of claims 3 to 17, wherein the protein
epitope is from Burkholderia pseudomallei or Burkholderia mallei.
19. The protein according to any one of claims 3 to 18, wherein the protein
30 epitope is from LolC, PotF, OppA, Rp1, Rp2, Omp85 or Hcp2.
20. The protein according to any one of claims 2 to 19, wherein the tandem
copies of HBcAg are joined by a linker.
36
21. The protein according to claim 20, wherein the linker is at least 1.5 nm in
length.
22. The protein according to claim 20 or 21, wherein the linker comprises
multiple copies of 5 the sequence GlynSer (GnS) where n is from 2 to 8.
23. The protein according to any one of the preceding claims, wherein the
HBcAg comprises the sequence AlaAlaAlaLeuAlaAlaAla (AAALAAA; SEQ ID NO:
3).
10
24. A particle comprising multiple copies of a protein as claimed in any one
of the preceding claims.
25. A process for producing a protein as claimed in any one of claims 1 to
15 23, which process comprises attaching sugar to the e1 loop.
26. The process according to claim 25, wherein the sugar is attached to the
e1 loop by reductive amination.
20 27. The process according to claims 26, wherein the sugar is oxidised to
generate a terminal aldehyde residue which is reductively aminated to primary amine in
the e1 loop.
28. A pharmaceutical composition comprising a protein as claimed in any
25 one of claims 1 to 23 or a particle as claimed in claim 24, and a pharmaceutically
acceptable carrier or diluent.
29. A protein according to any one of claims 1 to 23 or a particle according
to claim 24, for use in a method of vaccination of the human or animal body.
30
30. The protein or particle according to claim 29 for use in a method of
vaccination of the human or animal body against Burkholderia.
53
31. Use of a protein according to any one of claims 1 to 23 or a particle
according to claim 24, for the manufacture of a medicament for vaccination of the
human or animal body.
32. The use according 5 to claim 31, for vaccination against
Burkholderia.
33. A method of inducing an immune response in a subject, which
method comprises administering to the subject a protein as claimed in any one of
10 claims 1 to 23 or a particle as claimed in claim 24.
34. The method according to claim 33, for inducing an immune
response against Burkholderia.