VACCINES BASED ON HEPATITIS B CORE ANTIGENS
Field of the invention
The invention relates to proteins comprising hepatitis B core antigen (HBcAg)
with a sugar attached to an el loop, processes for producing the proteins with the sugar
attached, pharmaceutical compositions comprising 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 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 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 monomelic HBc fails to
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.
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 sugars.
The invention thus provides a protein comprising hepatitis B core antigen
(HBcAg) with a sugar attached to an el loop. The protein may comprise a first and a
second copy of HBcAg in tandem, wherein one or both copies of HBcAg has a sugar
attached to the el 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 el loop;
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
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.
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 filters of 0.8 ,
0.45 and 0.2 (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 (large
peak on left panel of (B)). The numbers above the lanes are fraction numbers collected
from the CL4B column.
Figure 3 : The CL4B void was then passed over an SI000 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 SI000
column.
Figure 4 : (A) 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 baculo-wmon itself.
Figure 5 : Lane A: molecular weight markers, Lane B : unmodified VLP 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 : 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 unloaded VLP has
a value for 30 ug/ml which lies near 0.4 average OD.
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 FIBcAg 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 a sequence which HBcAg may comprise in order to balance
the a-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.
SEQ ID NO: 7 is the sequence of the L0IC-K6 construct.
SEQ ID NO: 8 is the sequence of the LolC-Kl construct.
SEQ ID NO: 9 is a sequence of LolC.
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 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.
Hepatitis B core antigen (HBcAg)
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
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 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 has a
sugar attached to the el loop. Where the protein comprises a first and a second copy of
HBcAg in tandem, one copy of HBcAg has a sugar attached to the el loop. 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 el loop or may comprise a protein
epitope in the el loop. Examples of possible sugars and 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 el loop, the C-terminus and/or the N-terminus. The
el loop of HBcAg can tolerate insertions of e.g. from 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 to
500 aa, from 1 to 400 aa, from 1 to 300 aa, from 1 to 200 aa, from 3 to 100 aa or from 6
to 50 aa. Substitutions may involve a number of amino acids up to, for example, 1, 2, 5,
10, 20 or 50 amino acids over the length of the HBcAg sequence. An extension may be
at the N- or C-terminus of HBcAg. A deletion may be at the N-terminus, C-terminus or
at an internal site of the protein. Substitutions may be made at any position in the
protein sequence. Insertions may also be made at any point in the protein sequence, but
are typically made in surface-exposed regions of the protein such as the el loop. An
inserted sequence may carry a protein epitope. One or more amino acids may be
inserted so that one or more sugars can be subsequently attached. For example, 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acids may be inserted. The amino acids may be
inserted consecutively. Any amino acid inserted for the attachment of a sugar must be
capable of having a sugar attached to it. Examples of such amino acids include lysine,
arginine, asparagine, glutamine, aspartic acid or glutamic acid. For example, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11 or 12 lysines may be inserted. One or more alanines may be inserted
either side of the one or more amino acids which have been inserted for the attachment
of sugar. For 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 FIBcAg 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 FIBcAg sequence in the protein of the invention preferably has
at least 70% sequence identity to the corresponding sequence of a natural HBcAg
protein, such as the protein having the sequence shown in SEQ ID NO: 2 . More
preferably, the identity is at least 80%, at least 90%, at least 97%, at least 98% or at
least 99%. Methods of measuring protein homology are well known in the art and it
will be understood by those of skill in the art that in the present context, homology is
calculated on the basis of amino acid identity (sometimes referred to as "hard
homology").
For example the UWGCG Package (Devereux et al (1984) Nucleic Acids
Research 12: 387-395) provides the BESTFIT program which can be used to calculate
homology (for example used on its default settings). The PILEUP and BLAST
algorithms can be used to calculate homology or line up sequences (typically on their
default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-
300; Altschul, S, F et al (1990) J Mol Biol 215:403-10.
Software for performing BLAST analyses is publicly available through the
National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This
algorithm involves first identifying high scoring sequence pair (HSPs) by identifying
short words of length W in the query sequence that either match or satisfy some
positive-valued threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighbourhood word score threshold
(Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating
searches to find HSPs containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score can be increased.
Extensions for the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence is reached. The
BLAST algorithm parameters W, T and X determine the sensitivity and speed of the
alignment. The BLAST program uses as defaults a word length (W) of 11, the
BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci.
USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a
comparison of both strands.
The BLAST algorithm performs a statistical analysis of the similarity between
two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:
5873-5787. One measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of the probability by
which a match between two nucleotide or amino acid sequences would occur by chance.
For example, a sequence is considered similar to another sequence if the smallest sum
probability in comparison of the first sequence to the second sequence is less than about
1, preferably less than about 0.1, more preferably less than about 0.01, and most
preferably less than about 0.001.
The el loop of HBcAg is at positions 68 to 90 of the mature sequence, and a
protein epitope may be inserted anywhere between these positions. Amino acid for the
attachment of a sugar as discussed above may be inserted anywhere between these
positions. Preferably, the epitope or amino acid for the attachment of sugar is inserted
in the region from positions 69 to 90, 7 1 to 90 or 75 to 85. Most preferred is to insert
the epitope or the amino acid for the attachment of sugar between amino acid residues
79 and 80 or between residues 80 and 81. When a protein epitope or an amino acid for
the attachment of sugar 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 a protein epitope or an amino acid for the attachment of sugar. Where a
protein epitope or an amino acid for the attachment of sugar replaces el loop sequence,
the replacement sequence is generally not shorter than the sequence that it replaces.
A C-terminal truncation of HBcAg will generally not go beyond aa 144 because
if any further truncation is made particles may not form. Thus, the deleted amino acids
may, for example, comprise aa 144 to the C-terminal aa (aa 183 or 185), aa 150 to the
C-terminal aa, aa 164 to the C-terminal aa or aa 172 to the C-terminal aa. The Cterminus
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 the protein 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 linker between adjacent HBcAg copies 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. A preferred
linker comprises one or more repeats of the sequence Gly Ser (G S) where n is 2, 3, 4,
5, 6, 7 or 8 . 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. 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.
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 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 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.
Sugar
The term "sugar" refers to polysaccharides, oligosaccharides and
monosaccharides. The protein comprises HBcAg with a sugar attached to an el loop.
The protein may comprise a first and a second copy of HBcAg in tandem. Where there
are two copies of HBcAg in tandem, one or both copies of HBcAg has a sugar attached
to the el loop.
There may be more than one sugar attached to the el loop. The el loop may
have more than one type of sugar attached. The el loop may have different sugars
attached. Where there are two copies of HBcAg in tandem, there may be a different
sugar or different sugars attached to the el 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 attached to the el loop.
The sugar is attached to one or more amino acids in the el loop. The one or
more amino acids for attachment of the sugar may be inserted into the el loop as
described herein. The one or more amino acids for attachment of the sugar may be
amino acids which occur naturally in HBcAg. Examples of such amino acids include
lysine, arginine, asparagine, glutamine, aspartic acid or glutamic acid. The sugar may
be attached to more than one naturally occurring amino acid. The sugar may be
attached to a naturally occurring amino acid and an inserted amino acid.
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 Tlymphocyte
(CTL) epitope or a T-helper (Th) cell epitope (e.g. a Thl or Th2 epitope).
There may be more than one epitope present. If there is more than one 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 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.
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 Burkholderia, for example, Burkholderia
pseudomallei or Burkholderia mallei. CPS comprises an unbranched homopolymer of
1-3 linked 2-0 acetyl-6-deoxy -P-D-manno-heptopyranose. Therefore the sugar may
comprise an unbranched homopolymer of 1-3 linked 2-0 acetyl-6-deoxy -P-D-mannoheptopyranose.
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 than 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.
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 e l loop and the second
copy comprises a protein epitope in the e l loop. The "first copy" may b e either the Nterminal
or C-terminal copy.
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
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.
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 different proteins.
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 Thl
or Th2 epitope). In a preferred embodiment of the invention, one of the epitopes is a Thelper
cell epitope and another is a B-cell or a CTL epitope. The presence of the Thelper
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 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 (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, 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 epitope 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,
Yersinia pestis, Streptococcus (types A and B), Pneumococcus, Meningococcus,
Hemophilus influenza (type b), Complybacteriosis, Moraxella catarrhalis,
Donovanosis, and Actinomycosis, fungal pathogens including Candidiasis and
Aspergillosis, and parasitic pathogens including Toxoplasma gondii, Taenia, Flukes,
Roundworms, Flatworms, Amebiasis, Giardiasis, Cryptosporidium, Schitosoma,
Pneumocystis carinii, Trichomoniasis and Trichinosis.
The epitope 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 epitope 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 Bcell
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, differentation 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 FIPV types. Further examples of
particular tumour antigens include MART-1, Melan-A, p97, beta-HCG, GalNAc,
MAGE-1, MAGE-2, MAGE-4, MAGE- 12, MUCl, MUC2, MUC3, MUC4, MUCl 8,
CEA, DDC, P IA, 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 epitopes for use in the invention include epitopes
from 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 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 may be a fragment
of any of the proteins listed in Table 1.
Table 1 - proteins
microarray analysis." J Infect Dis. 201 1 Apr
1;203(7):1002-1 1.
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 (Rpl) Human sero-positive, healthy.
Tippayawat etal, "Burkholderia pseudomallei
proteins presented by monocyte-derived dendritic
cells stimulate human memory T cells in vitro." Infect
Immun. 201 1 Jan;79(l):305-13.
Tandem repeat sequence (Rp2) Human sero-positive, healthy.
Tippayawat etal., "Burkholderia pseudomallei
proteins presented by monocyte-derived dendritic
cells stimulate human memory T cells in vitro." Infect
Immun. 201 1 Jan;79(l):305-13.
Omp85 (Outer membrane Efficacy in animal model.
protein) Su et al. , "Immunization with the recombinant
Burkholderia pseudomallei outer membrane protein
Omp85 induces protective immunity in mice."
Vaccine. 2010 Jul 12;28(31):5005-1 1.
Hcp2 (type VI secretion Efficacy in animal model.
protein) Burtnick et al., "The cluster 1 type VI secretion
system is a major virulence determinant in
Burkholderia pseudomallei." Infect Immun. 201 1
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 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 el loop.
A LolC protein sequence described herein is:
ALGVAALIVVLSVMNGFQKEVRDRMLSVLAHVEIFSPTGSMPDWQLTAKEARL
RSVIGAAPYVDAQALLTRQDAVSGVMLRGVEPSLEPQVSDIGKDMKAGALT
ALAPGQFGIVLGNALAGNLGVGVGDKVTLVAPEGTITPAGMMPRLKQFTVVGI
FESGHYEYDSTLAMIDIQDAQALFRLPAPTGVRLRLTDMQKAPQVARELAHTL
SGDLYIRDWTQQNKTWF SAVQIEKRMMFIILTLIIAVAAFNLVS SLVMTVTNKQ
ADIAILRTLGAQPGSFMKIFVVQGVTIGFVGTATGVALGCLIAWSIPWLIPMIEHA
FGVQFLPP SVYFISELP SELVAGDVIKIGVIAGS (SEQ ID NO: 9)
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 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 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 third column
may be substituted for each other.
Table 2
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 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.
Processfor 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 el loop. As described herein,
sugar is attached to one or more amino acids in the el loop. The sugar may be attached
by reductive amination of the oxidized sugar to an amino acid in the el 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 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.
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 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 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 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. /zHBlOl). 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 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.
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 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 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 FIBcAg and
the sugar). Where the protein comprises two copies of FIBcAg, 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 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 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 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 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.
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 any of the pathogens described herein, for example
Burkholderia. The protein could simultaneously vaccinate 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 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 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 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 or particle 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 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, 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 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 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.
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, 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.
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.
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 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 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 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 or particle of the invention and an adjuvant may be administered
sequentially or simultaneously, preferably simultaneously. The two entities may be
administered in the same or different compositions, preferably the same composition.
An adjuvant is delivered so that an adjuvant effect is seen, that is the immune response
seen will differ from that if the adjuvant had not been administered with the antigen.
The two entities may be administered at the same or different sites, preferably the same
sites. Preferably, the two entities are administered in the same composition at the same
site at the same time preferably via injection.
Any suitable adjuvant may be used. Currently used vaccine adjuvants include:
Inorganic compounds, such as aluminium salts (e.g. aluminium hydroxide and
aluminium phosphate) or calcium phosphate. Aluminium salts are otherwise known as
alum.
Oil emulsions and surfactant based formulations, e.g. MF59 (microfluidised
detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2]
(oil-in-water emulsion + MPL + QS-21), Montanide ISA-51 and ISA-720 (stabilised
water-in-oil emulsion).
- Particulate adjuvants, e.g. virosomes (unilamellar liposomal vehicles
incorporating e.g. influenza haemagglutinin), AS04 ([SBAS4] Al salt with MPL),
ISCOMS (structured complex of saponins and lipids), and polylactide co-glycolide
(PLC).
Microbial derivatives (natural and synthetic), e.g. monophosphoryl lipid A
(MPL), Detox (MPL +M. Phlei cell wall skeleton), AGP [RC-529] (synthetic acylated
monosaccharide), DC Chol (lipoidal immunostimulators able to self organise into
liposomes), OM-174 (lipid A derivative), CpG motifs (synthetic oligonucleotides
containing immunostimulatory CpG motifs), and modified LT and CT (genetically
modified bacterial toxins to provide non-toxic adjuvant effects).
Endogenous human immunomodulators, e.g. hGM-CSF or hIL-12 (cytokines
that can be administered either as protein or plasmid encoded), and Immudaptin (C3d
tandem array).
Inert vehicles, such as gold particles.
Preferably the adjuvant used is alum. Most preferably the adjuvant is a mixture
of aluminium hydroxide and magnesium hydroxide, for example Inject alum (Pierce
Laboratories).
The invention is illustrated by the following Example:
Example
Materials & Methods
Design of constructs
All tandem core clones are derived from the parental construct CoHo7e. In this
version of tandem core, a-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 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:
MDIDPYKEFGATVELL SFLPSDFFPSVRDLLDTASALYREALESPEHC SPHHTAL
RQAILCWGELMTLATWVGNNLEGSAGGGRDPASRDLVVNYVNTNMGLKIRQ
LLWFHI SCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVGGSSGG
SGGSGGSGGSGGSGGSTMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALY
REALESPEHCSPHHTALRQAILCWGELMTLATWVGNNLEFAGASDPASRDLVV
NYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILS
TLPETTVVL (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 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 a-helices as described above. The synthetic inserts were ligated to
produce H3Ho. The final sequences was verified and was as follows:
MSDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTA
LRQAILCWGELMTLATWVAAALAAAEGSDPASRDLVVNYVNTNMGLKIRQLL
WFHI SCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVGGS SGGS
GGSGGSGGSTMDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESP
EHCSPHHTALRQAILCWGELMTLATWVAAALAAAESGDPASRDLVVNYVNTN
MGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTV
VLE
(SEQ ID NO: 5)
An alternative 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 a-helical folding were found at both the N and C termini.
Thus, insertion of the antigen would be from an a-helical secondary structure into the
a-helix of the FIBc 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 successful. LolC-empty
sequence:
MDIDPYKEFGATVELL SFLPSDFFPSVRDLLDTASALYREALESPEHC SPFIHTAL
RQAILCWGELMTLATWVAAALAAAEGSALGVAALIVVLSVMNGFQKEVRDR
MLSVLAHVEIFSPTGSMPDWQLTAKEARLNRSVIGAAPYVDAQALLTRQDAVS
GVMLRGVEPSLEPQVSDIGKDMKAGALTALAPGQFGIVLGNALAGNLGVGVG
DKVTLVAPEGTITPAGMMPRLKQF TVVGIFESGHYEYDSTLAMIDIQDAQALFR
LPAPTGVRLRLTDMQKAPQ VARELAHTLSGDL YIRDWTQQNKTWF SAVQIEKR
MMFIILTLIIAVAAFNLVSSLVMTVTNKQADIAILRTLGAQPGSIMKIFVVQGVTI
GFVGTATGVALGCLIAWSIPWLIPMIEHAFGVQFLPPSVYFISELPSELVAGDVIK
IGVIAGSDPASRDLVVNYVNTNMGLKIRQLLWFHI SCLTFGRETVLEYLVSFGV
WIRTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSTMDIDPYKEFGATV
ELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTL
ATWVAAALAAAE SGDPASRX)LVVNYVNTNMGLKIRQLLWFHI SCLTFGRETV
LEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVLE (SEQ ID NO: 6)
Two variations of the LolC insertion were then made by cleaving core 2 of the
H3Ho LolC-empty construct using Pstl and Xhol. Synthetic inserts containing either
hexa-lysine or a single lysine flanked by repeating alanine residues were then ligated in.
Again, sequencing confirmed their identity. L0IC-K6 sequence:
MDIDPYKEFGATVELL SFLPSDFFPSVRDLLDT ASALYREALESPEHC SPFIHTAL
RQAILCWGELMTLATWVAAALAAAEGSALGVAALIVVLSVMNGFQKEVRDR
MLSVLAHVEIFSPTGSMPDWQLTAKEARLNRSVIGAAPYVDAQALLTRQDAVS
GVMLRGVEPSLEPQVSDIGKDMKAGALTALAPGQFGIVLGNALAGNLGVGVG
DKVTLVAPEGTITP AGMMPRLKQF TVVGIFE SGHYEYDSTLAMIDIQDAQALFR
LPAPTGVRLRLTDMQKAPQ VARELAHTLSGDL YIRDWTQQNKTWF SAVQIEKR
MMFIILTLIIAVAAFNLVSSLVMTVTNKQADIAILRTLGAQPGSIMKIFVVQGVTI
GFVGTATGVALGCLIAWSIPWLIPMIEHAFGVQFLPPSVYFISELPSELVAGDVIK
IGVIAGSDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGV
WIRTPPAYRPPNAPIL STLPETTVVGGS SGGSGGSGGSGGSTMDIDPYKEFGATV
ELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTL
ATWVAAALAAAESGGSGSKKKKKKGSGSSGDPASRDLVVNYVNTNMGLKIR
QLLWFHI SCLTFGRETVLEYLVSFGVWIRTPP AYRPPNAPIL STLPETTVVLE
(SEQ ID NO: 7)
LolC-Kl sequence:
MDIDPYKEFGATVELL SFLPSDFFPSVRDLLDT ASALYREALESPEHC SPHHTAL
RQAILCWGELMTLATWVAAALAAAEGSALGVAALIVVLSVMNGFQKEVRDR
MLSVLAHVEIFSPTGSMPDWQLTAKEARLNRSVIGAAPYVDAQALLTRQDAVS
GVMLRGVEPSLEPQVSDIGKDMKAGALTALAPGQFGIVLGNALAGNLGVGVG
DKVTLVAPEGTITP AGMMPRLKQF TVVGIFE SGHYEYD STLAMIDIQDAQALFR
LPAPTGVRLRLTDMQKAPQVARELAHTLSGDLYIRDWTQQNKTWFSAVQIEKR
MMFIILTLIIAVAAFNLVSSLVMTVTNKQADIAILRTLGAQPGSIMKIFVVQGVTI
GFVGTATGVALGCLIAWSIPWLIPMIEHAFGVQFLPPSVYFISELPSELVAGDVIK
IGVIAGSDPASRDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGV
WIRTPPAYRPPNAPILSTLPETTVVGGSSGGSGGSGGSGGSTMDIDPYKEFGATV
ELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTL
ATWVAAALAAAESGGSGSGGGKGGGSGSSGDPASRDLVVNYVNTNMGLKIR
QLLWFHI SCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVLE
(SEQ ID NO: 8)
Design of plasmids
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 pOETl (Oxford Expression Technologies) for
baculovirus. The H3Ho sequence was inserted into the multi-cloning site of pPICz
using Mfel and pspOMI, whereas pOETl was inserted using pspOMI and Bell.
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 10C^g/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 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 -80°C before purification.
Protein expression of tandem core in baculovirus
Recombinant virus was produced by co-transfection in Sf9 insect cells.
Duplicate reaction mixtures, each containing flashBACPREVIE viral DNA (lOOng) and
transfer vector DNA (500ng; pOETl-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 28°C for 5
days, following which the medium containing each virus was harvested into sterile
tubes. A stock of vims was created from 50 ml of Sf9 cells, at a density of 2 106
cells/ml, infected with 0.5 ml of the co-transfection mix. The infected shake culture was
incubated for 5 days at 28°C and then harvested by centrifugation at 500xg for 20 min at
4°C. 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 28°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, 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/lOmls lysis buffer. The
resulting solution was then passed through a microniser (AVP Gaulin LAB 40) set at
500psi three times. Detergent (Triton XI00) was 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 lMDa
molecular weight cut-off (Pellicon). This step both removed low molecular weight
contaminants and reduced volume down to 25mls.
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
remainder of protein isolated over the column was discarded. The void volume fractions
were pooled and concentrated using tangential flow (Pellicon) back down to lOmls.
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 calibrated the column using recombinant monomeric
FIBc (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
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 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) followed by
Coomassie blue staining. Western Blot analyses were performed as described (Konig, et
al, 1998) using a monoclonal primary antibody against HBc protein (10E1 1[Abeam])
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 (BioRad).
Electron microscopy
All samples were diluted to 0 .1mg / ml in 20 mM Tris HC1 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
droplets 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 26,000x.
ELISAfor antigenicity ofLolC
A 96-well microtiter plate was coated for 24 hours at 4°C with of
purified LolC (standard, 2-0.03 g/mL), VLPs (negative control, 30-0^g/mL) and
VLP-LolC (test sample, 30-0^g/mL). All samples were serially diluted two-fold in
phosphate-buffered saline (lx Dulbecco's PBS, Invitrogen). Each well was washed
three times with PBS-0.05% Tween 20, and blocked with 2 of PBS containing 5%
(w/v) skimmed milk powder for 1 hour at 37°C. Each well was then washed three times
with PBS-0.05% Tween 20 and a 1:1000 dilution of sera from mice vaccinated with
endotoxin-free LolC protein added (10C^L/well) and incubated for 1 hour at 37°C.
Following three washes in PBS-Tween 20, a 1:2000 dilution of IgG goat anti-mouse
horseradish peroxidase conjugate was added to each well () and incubated for 1
hour at 37°C. Each well was washed a further six times in PBS-Tween 20 and bound
conjugate detected with ABTS/hydrogen peroxide substrate () 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 in
PBS ( 1 ml) and the reaction mixture was left at room temperature for 1 hour. Excess
sodium weto-periodate was removed using a PD-10 desalting column (GE Healthcare)
equilibrated with PBS. The oxidized CPS was added to a solution of protein at 5 mg/ml
( 1 ml) in PBS. 20 ΐ 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 ΐ of NaB¾ [1M
NaB¾ in 10 mMNaOH] was added, after agitation the reaction was 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 warehousing speed-vac (Thermo
Scientific).
The concentrated protein sample was purified on an Xpress FPLC purification
system. The conjugate solution was injected onto an S500 sepharose 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.
Results
Protein isolationfrom 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 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
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 IMDa molecular weight cut-off. This served to retain
the large VLPs but removed some of the low molecular weight contaminants.
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 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 SI000 SEC column. This 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, 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. SDS-PAGE
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 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 isolationfrom baculovirus samples
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 that baculovirion
co-purified with tandem core. This was detectable both in SDS-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 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 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 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 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 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 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 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 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 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.
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 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 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 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 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.
CLAIMS
1. A protein comprising hepatitis B core antigen (HBcAg) with a sugar
attached to an el loop.
2 . The protein according to claim 1, comprising a first and a second copy of
HBcAg in tandem, wherein one or both copies of HBcAg has a sugar attached to the el
loop.
3 . The protein according to claim 2, wherein the first copy has a sugar
attached to the el loop and the second copy comprises a protein epitope in the el loop.
4 . The protein according to any one of the preceding claims, wherein the
sugar or sugars are attached to lysine, arginine, asparagine, glutamine, aspartic acid or
glutamic acid in the el loop.
5 . The protein according to claim 4, wherein the el loop with the sugar or
sugars attached comprises 1 to 12 consecutive lysines.
6 . The protein according to claim 5, wherein the el loop with the sugar or
sugars attached comprises 1 or 6 consecutive lysines.
7 . The protein according to any one of claims 4 to 6, wherein the lysine or
lysines are flanked by multiple alanines.
8 . The protein according to any one of the preceding claims, wherein the
sugar or sugars are derived from a bacterium.
9 . The protein according to claim 8, wherein the bacterium is Burkholderia.
10. The protein according to claim 9, wherein the bacterium is Burkholderia
pseudomallei or Burkholderia mallei.
11. The protein according to any one of the preceding claims, wherein the
sugar or sugars comprise common capsule polysaccharide (CPS).
12. The protein according to any one of the preceding claims, wherein the
sugar or sugars comprise an unbranched homopolymer of 1-3 linked 2-0 acetyl-6-
deoxy -P-D-manno-heptopyranose.
13. The protein according to any one of claims 3 to 12, wherein the protein
epitope is from Burkholderia.
14. The protein according to any one of claims 3 to 13, wherein the protein
epitope is from Burkholderia pseudomallei or Burkholderia mallei.
15. The protein according to any one of claims 3 to 14, wherein the protein
epitope is from LolC, PotF, OppA, Rpl, Rp2, Omp85 or Hcp2.
16. The protein according to any one of claims 2 to 15, wherein the tandem
copies of HBcAg are joined by a linker.
17. The protein according to claim 16, wherein the linker is at least 1.5 nm in
length.
18. The protein according to claim 16 or 17, wherein the linker comprises
multiple copies of the sequence Gly Ser (G S) where n is from 2 to 8 .
19. The protein according to any one of the preceding claims, wherein the
HBcAg comprises the sequence AlaAlaAlaLeuAlaAlaAla (AAALAAA; SEQ ID NO:
3).
20. A particle comprising multiple copies of a protein as claimed in any one
of the preceding claims.
21. A process for producing a protein as claimed in any one of claims 1 to
19, which process comprises attaching sugar to the e l loop.
22. The process according to claim 21, wherein the sugar is attached to the
el loop by reductive amination.
23. The process according to claims 22, wherein the sugar is oxidised to
generate a terminal aldehyde residue which is reductively aminated to primary amine in
the el loop.
24. A pharmaceutical composition comprising a protein as claimed in any
one of claims 1 to 19 or a particle as claimed in claim 20, and a pharmaceutically
acceptable carrier or diluent.
25. A protein according to any one of claims 1 to 19 or a particle according
to claim 20, for use in a method of vaccination of the human or animal body.
26. The protein or particle according to claim 25 for use in a method of
vaccination of the human or animal body against Burkholderia.
27. Use of a protein according to any one of claims 1 to 19 or a particle
according to claim 20, for the manufacture of a medicament for vaccination of the
human or animal body.
28. The use according to claim 27, for vaccination against Burkholderia.
29. 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 claims 1 to 19
or a particle as claimed in claim 20.
30. The method according to claim 29, for inducing an immune response
against Burkholderia.