Note: Descriptions are shown in the official language in which they were submitted.
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MULTIMERIC COMPLEXES OF ANTIGENS AND AN ADJUVANT
This application claims the benefit of priority of
PCT/EP2003/008926, the contents of which are incorporated
herein by reference.
Field of the Invention.
This invention relates to macromolecular assemblies, such as
fusion proteins, comprising an adjuvant and an antigen, which
assemblies provoke an enhanced immune response to the antigen
in comparison to the antigen alone.
Backaround of the Invention.
Adjuvants enhance the immune response to antigens and are
therefore useful in vaccines. However, there are only a
limited number of adjuvants approved for use in humans, and as
stronger adjuvants are known from research on animals, a clear
need exists for stronger immunological adjuvants which are
safe to use in man. For a recent review, see "Advances in
vaccine adjuvants" (Nature Biotechnology, 1999, Volume 17,
pages 1075-1081) and "Recent advances in the discovery and
delivery of vaccine adjuvants" (Nature Reviews in Drug
Discovery, 2003, Volume 2, pages 727-735).
The complement system consists of a set of serum proteins that
25e are important in the response of the immune system to foreign
antigens. The complement system becomes activated when its
primary components are cleaved and the products, alone or with
other proteins, activate additional complement proteins
resulting in a proteolytic cascade. Activation of the
complement system leads to a variety of responses including
increased vascular permeability, chemotaxis of phagocytic
cells, activation of inflammatory cells, opsonisation of
foreign particles, direct killing of cells and tissue damage.
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Activation of the complement system may be triggered by
antigen-antibody complexes (the classical pathway) or a normal
slow activation may be amplified in the presence of cell walls
of invading organisms such as bacteria and viruses (the
alternative pathway). The complement system interacts with
the cellular immune system through a specific pathway
involving C3, a protein central to both classical and
alternative pathways. The proteolytic activation of C3 gives
rise to a large fragment (C3b) and exposes a chemically
reactive internal thiolester linkage which can react
covalently with external nucleophiles such as the cell surface
proteins of invading organisms or foreign cells. As a result,
the potential antigen is "tagged" with C3b and remains
attached to that protein as it undergoes further proteolysis
to iC3b and C3d,g. The latter fragments are, respectively,
ligands for the complement receptors CR3 and CR2; (CR2 is also
referred to as CD21). Thus the labelling of antigen by C3b
can result in a targeting mechanism for cells of the immune
system bearing these receptors.
That such targeting is important for augmentation of the
immune response is first shown by experiments in which mice
were depleted of circulating C3 and then challenged with an
antigen (sheep erythrocytes). Removal of C3 reduced the
antibody response to this antigen (M. B. Pepys, J. Exp. Med.,
140, 126-145, 1974). The role of C3 was confirmed by studies
in animals genetically deficient in either C3 or the upstream
components of the complement cascade which generate C3b, i.e.
C2 and C4 (J.M. Ahearn and D.T. Fearon, Adv. Immunol., 46,
183-219, 1989). More recently, it has been shown that linear
conjugation of a model antigen with more than two copies of
the murine C3d fragment sequence resulted in a very large
(1000-10000-fold) increase in antibody response in mice
compared with unmodified antigen controls (P.W. Dempsey et al,
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Science, 271, 348-350, 1996 W096/17625, PCT/GB95/02851). The
increase could be produced without the use of conventional
adjuvants such as Freund's complete adjuvant, which is too
toxic to be used in humans. The mechanism of this remarkable
effect was demonstrated to be high-affinity binding of the
multivalent C3d construct to CR2 on B-cells, followed by co-
ligation of CR2 with another B-cell membrane protein, CD19 and
with membrane-bound immunoglobulin to generate a signal to the
B-cell nucleus.
However, it has proved difficult to produce large amounts of
homogenous recombinant proteins containing three copies of
C3d. The principal problems have been:
i) the genetic instability of the constructs containing
(three) repeated sequences; and
ii) the folding (or solubilisation and refolding) of the
recombinant protein from inclusion bodies formed in
Escherichia coli.
One approach taken to minimise the genetic instability of
constructs containing repeated copies of the C3d gene is
described in W099/35260 and W001/77324. The technology
described in these applications is to use non-identical
sequences of DNA encoding repeats of C3d.
A multimerisation system using the complement 4 binding
protein (C4bp) is described in W0 91/11461. Human C4b-binding
protein (C4BP) is a plasma glycoprotein of high molecular mass
(570 kDa) which has a spider like structure made of seven
identical alpha-chains and a single beta-chain. The C4bp alpha
chain has a C-terminal core region responsible for assembly of
the molecule into a multimer. According to the standard
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model, the cysteine at position +498 of one C4bp monomer forms
a disulphide bond with the cysteine at position +510 of
another monomer. A minor form comprising only seven alpha-
chains has also been found in human plasma. The natural
function of this plasma glycoprotein is to inhibit the
classical pathway of complement activation.
Most of the alpha-chain of C4bp is composed of eight tandemly
arranged domains of approximately 60 amino acids in length
known as complement control protein (CCP) repeats. W091/11461
proposes that the ability of the C4bp protein to multimerise
can be used to make fusion proteins comprising all or part of
C4bp and a biological protein of interest. Inclusion of one
or more of the CCP repeats (also known as SCRs) were preferred
in the fusion proteins described in WO 91/11461.
W091/11461 suggests that fusion proteins may be used as
vaccines. A number of specific proteins comprising at least
one C4bp SCR region fused to a fragment of hepatitis B a
antigen were made. The a antigen fragments used are core
antigen fragments which are capable of forming multimer
structures.
Libyh M. T. et al., (1997, Blood, 90, 3978-3983) demonstrate
that all CCPs can be deleted (leaving only the C-terminal 57
amino acids) without preventing multimerisation. This
C-terminal region of C4bp is referred to herein as the C4bp
core.
Self-assembling multimeric soluble CD4-C4bp fusion protein
have also been demonstrated in Shinya et al (1999, Biomed &
Pharmacother, Vol. 53: 471) where the fusion proteins were
expressed in the human 293 cell line.
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The use of C4bp is also described in Oudin et a1. (2000,
Journal- of Immunology, Vol. 164:1505).
Christiansen et a1. (2000, Journal of Virology, Vol. 74:4672)
5 discuss the therapeutic use of a CD46-C4bp fusion protein.
W02004/020639 provides a method for obtaining a recombinant
fusion protein in a prokaryotic host, comprising a scaffold of
a C-terminal core protein of C4bp alpha chain optionally fused
to a heterologous polypeptide, said recombinant fusion protein
being capable of forming multimers in soluble form in a
prokaryotic host cell.
Summary of the Invention.
The present invention is based on a novel finding in relation
to a particular class of C4bp fusion proteins. Some of the
prior art discussed above proposes the use of C4bp fusion
proteins to deliver a therapeutic protein of interest, on the
basis that the C4bp moiety is essentially an inert carrier.
W091/11461 discussed above, exemplifies fusion proteins which
comprise a multimer-forming antigen.
In contrast, the present invention is based on the
appreciation that antigens which naturally form multimers are
not desirable for fusions to the C4bp core, as such antigens
may in fact interfere with assembly of the C4bp core into
multimers. In addition, it has been demonstrated that
surprisingly, fusions of a C4bp core to a monomeric antigen
provoke a strong immune response to the antigen. In the
accompanying examples, it is shown that a fusion protein
comprising a monomeric antigen provoked a high titre,
inhibitory antibody response compared to a lower titre, non-
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inhibitory antibody response when antigen was injected with
Freund's adjuvant.
Thus the present invention provides a method for increasing
the immunogenicity of a monomeric antigen by combining it in a
complex with a C4bp core protein. In a preferred method, the
monomeric antigen is covalently bound to a C4bp core protein.
In a highly preferred method, the monomeric antigen is
genetically fused to a C4bp core protein.
The invention also provides a method for inducing high titres
of antibodies against an antigen, and a use for the high titre
antisera produced through the use of the method in the
prevention and/or in the treatment of infectious and malignant
diseases by passive immunisation. In a preferred method, the
high titre antibodies against the antigen are partly purified
by isolating the immunoglobulin fraction of the hyperimmune
plasma or serum, and in a highly preferred method, the
immunoglobulin fraction of the hyperimmune plasma or serum is
isolated from individuals of the same species in which the
antisera will be used to prevent or treat infectious or
malignant diseases.
The present invention thus provides a product which comprises:
a C4bp core protein; and
a monomeric antigen.
The first and second components may be in the form of a fusion
protein. Alternatively, they may be coupled chemically,
through an amino acid side chain either of the first component
or through the side chain of an amino acid which has been
added to the first component specifically to enable the
chemical coupling of the second component.
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The first and second components may be tightly but
noncovalently bound. For example, the side chain of an amino
acid of the first component may be modified to have an
additional biotin group, and this biotin can be used to
combine with streptavidin (where streptavidin is the second
component) or an antigen fused to streptavidin can be combined '
with the first component through this biotin. In another
possibility, biotinylated antigen and biotinylated first
component can be held together firmly but noncovalently by
adding streptavidin and purifying the complexes which result.
For the avoidance of doubt, the designation of "first" and
"second" components does not imply or indicate a specific
linear order in the product of the two components. The two
components may be joined in any order.
Thus where both components are polypeptides and the product is
made as a fusion protein, the N- to C- terminal order of the
two components may be in any permutation.
The invention further provides nucleic acid encoding a fusion
protein of said first and second components. The invention
also provides vectors comprising said nucleic acids and host
cells carrying said vectors.
In another embodiment, the invention provides a method of
making a product comprising:
a C4bp core protein; and
a polypeptide monomeric antigen,
the method comprising expressing nucleic acid encoding the
two components in the form of a fusion protein, and recovering
the product.
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In another embodiment, the invention provides a method of
making a product comprising:
a C4bp core protein;
a non-polypeptide monomeric antigen,
the method comprising expressing nucleic acid encoding the
C4bp core protein, joining said core protein to the antigen,
and recovering the product.
The methods of making the product may be performed in
eukaryotic or prokaryotic cells.
The invention also provides a method of inducing an immune
response to an antigen which method comprises administering to
a subject an effective amount of a product according to the
invention.
The invention also provides the use of a product of the
invention for a method of treatment of the human or animal
body, particularly a method of inducing an immune response.
The invention further provides a pharmaceutical composition
comprising a product of the invention in association with a
pharmaceutically acceptable carrier or diluent.
The invention further provides a method of preparing a
protective immune serum for use in passive immunization
against an infectious agent, said method comprising
vaccinating an animal with a product of the invention,
recovering antiserum from said animal. The antiserum may then
be used in a method of passive immunization of a human
subject. The human subject may be a subject with, or at risk
from, infection with the infectious agent.
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Description of the Drawings.
Figure 1 shows an alignment of C4bp core proteins.
Figure 2 shows the results of expression of proteins in E.
coli.
Figure 3 shows a comparison of a protein of the invention run
on gels under reducing and non-reducing conditions.
Detailed Description of the Invention.
Core protein of C4bp alpha chain.
This is referred to herein as the "C4bp core protein" or "core
protein", or "C4bp scaffold". The terms are used
interchangeably. This protein may be a mammalian C4bp core
protein or a fragment thereof capable of forming multimers and
capable of acting as an adjuvant, or a synthetic or chimeric
variant thereof capable of forming multimers and capable of
acting as an adjuvant.
In this invention, a C4bp core protein or a fragment of the
C4bp alpha chain comprising the core protein, described in
further detail herein, serves as an adjuvant. The human C4bp
core protein of SEQ ID N0: 1 corresponds to amino acids +493
to +549 of full length C4bp protein sequence known in the art
to form multimers.
The invention moreover comprises the use of derivatives of the
C4bp core to increase the immunogenicity of antigens. Such
derivatives comprise mutants thereof, which may contain amino
acid deletions, additions especially the addition of cysteine
residues or substitutions, hybrids or chimeric molecules
formed by fusion of parts of different members of the C4bp
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families and/or circular permutated protein scaffolds, subject
to the maintenance of the adjuvant property described herein.
The invention may also use artificial consensus C4bp sequences
5 based on the alignment of the C4bp core sequences from
multiple species. One example of this class of chimeric
molecule, of the very many possible, is given below (SEQ
ID:20, Figure 1) .
10 Thus the adjuvant may be a C4bp core and optionally one or
more SCRs fused to the core.
In a particularly preferred embodiment, the C4bp component of
the product of the invention is the core protein of C4bp alpha
chain, i.e. the core protein as defined herein not linked to
any C4bp SCR sequences. In such an embodiment, the C4bp core
desirably consists of the residues 1-57 of SEQ ID N0:1 or the
corresponding residues of homologue thereof, or a fragment of
at least 47 amino acids of SEQ ID N0:1 or homologue thereof.
The C4bp core of a product of the invention may additionally
comprise N- or C-terminal extensions such as flexible linkers.
Generally such linkers are a few amino acids in length, such
as from 1 to 20, e.g. from 2 to 10 amino acids in length. One
such linker is a (Glym-Ser)n linker, where m and n are each
independently from 1 to 4. These are used in the art to
attach protein domains to each other. Thus the first
component may be linked to the second by such a linker.
It is preferred that when the first component is the C4bp
core, it is at the C-terminal of the product.
The sequences of a number of mammalian C4bp proteins are
available in the art. These include human C4bp core protein
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(SEQ ID N0: 1). There are a number of homologues of human C4bp
core protein available in the art. There are two types of
homologue: orthologues and paralogues. Orthologues are
defined as homologous genes in different organisms, i.e. the
genes share a common ancestor coincident with the speciation
event that generated them. Paralogues are defined as
homologous genes in the same organism derived from a gene,
chromosome or genome duplication, i.e. the common ancestor of
the genes occurred since the last speciation event.
For example, a search of GenBank and raw genomic trace and EST
(expressed sequence tag) databases indicates mammalian C4bp
core SEQ ID N0:1 homologue proteins in species including
chimpanzees, rhesus monkeys, rabbits, rats, dogs, horses,
mice,. guinea pigs, pigs and cattle. Paralogues and orthologues
of the C4bp of SEQ ID N0:1 have been included in the alignment
in Figure 1.
In total, an alignment of SEQ ID NOs: 1-19 is shown as Figure
1. It can be seen that all nineteen sequences have a high
degree of similarity, though with a greater degree of
variation at the C-terminal end. Further C4bp core proteins
may be identified by searching databases of DNA or protein
sequences, using commonly available search programs such as
BLAST.
Where a C4bp protein from a desired mammalian source is not
available in a database, it may be obtained using routine
cloning methodology well established in the art. In essence,
such techniques comprise using nucleic acid encoding one of
the available C4bp core proteins as a probe to recover and to
determine the sequence of the C4bp core proteins from other
species of interest. A wide variety of techniques are
available for this, for example PCR amplification and cloning
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of the gene using a suitable source of genomic DNA or mRNA
(e.g. from an embryo or an actively dividing differentiated or
tumour cell), or by methods comprising obtaining a cDNA
library from the mammal, e.g. a cDNA library from one of the
above-mentioned sources, probing said library with a known
C4bp nucleic acid under conditions of medium to high
stringency (for example 0.03M sodium chloride and 0.03M sodium
citrate at from about 50°C to about 60°C), and recovering a
cDNA encoding all or part of the C4bp protein of that mammal.
Where a partial cDNA is obtained, the full length coding
sequence may be determined by primer extension techniques.
A fragment of a C4bp core protein capable of forming multimers
may comprise at least 47 amino acids, preferably at least 50
amino acids. The ability of the fragment to form multimers
may be tested by expressing the fragment in a prokaryotic host
cell according to the invention, and recovering the C4bp
fragment under conditions which result in multimerisation of
the full 57 amino acid C4bp core, and determining whether the
fragment also forms multimers. Desirably a fragment of C4bp
core comprises at least residues 6-52 of SEg ID N0: 1 or the
corresponding residues of its homologues.
Variants of C4bp core and fragments capable of forming
multimers, which variants likewise retain the ability to form
multimers (which may be determined as described above for
fragments) may also be used. The variant will preferably have
at least 700, more preferably at least 800, even more
preferably at least 900, for example at least 95% or most
preferably at least 98% sequence identity to a wild type
mammalian C4bp core or a multimer-forming fragment thereof.
In one aspect, the C4bp core will be a core which includes the
glycine appears at position 12, the alanine which appears at
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position 28, the leucines which appear at positions 29, 34,
36, and 41 and the tyrosine which appears at position 32 and
the lysine which appears at position 33 and preferably the two
cysteine residues which appear at positions 6 and 18 of SEQ ID
No: 1. Desirably, the variant will retain the relative spacing
between these residues.
The above-specified degree of identity will be to any one of
SEQ ID NOs: 1-20 or a multimer-forming fragment thereof.
Most preferably the specified degree of identity will be to
SEA ID N0:1 or a multimer-forming fragment thereof.
The degree of sequence identity may be determined by the
algorithm GAP, part of the "Wisconsin package" of algorithms
widely used in the art and available from Accelrys (formerly
Genetics Computer Group, Madison, WI). GAP uses the Needleman
and Wunsch algorithm to align two complete sequences in a way
that maximises the number of matches and minimises the number
of gaps. GAP is useful for alignment of short closely related
sequences of similar length, and thus is suitable for
determining if a sequence meets the identity levels mentioned
above. GAP may be used with default parameters.
Synthetic variants of a mammalian C4bp core protein include
those with one or more amino acid substitutions, deletions or
insertions or additions to the C- or N-termini. Substitutions
are particularly envisaged. Substitutions include
conservative substitutions. Examples of conservative
substitutions include those respecting the groups of similar
amino acids often called the Dayhoff groups. These are as
follows:
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Group 1 D, E, N, Q
Group 2 I, L, V, M
Group 3 F, Y, W
Group 4 K, R, H
Group 5 S, P, T, A, G
_
Group 6 I C
Examples of fragments and variants of the C4bp core protein
which may be made and tested for their ability to form
multimers and to act as an adjuvant thus include SEQ ID NOs:
37 to 44, shown in Table 1 below:
Tabl a 1:
A B C
37 ----GCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQS------ 100
38 ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIEQLELQRDSARQS------ 100
39 ----GSEQVLTGKRLMQSLPNPEDVKMALEVYKLSLEIEQLELQRDSARQSTLDKEL 96
40 ETPEGCEQVLTGKRLMQCLPNPEDVKMALEIYKLTLEIEQLELQRDSARQSTLDKEL 96
41 ETPEGCEQVLTGKRLMQCLPNPEDVKMALEIYKLSLEIKQLELQRDSARQSTLDKEL 96
42 ---EGCEQILTGKRLMQCLPDPEDVKMALEIYKLSLEIKQLELQRDRARQSTL---- 91
43 ETPEGCEQVLTGKRLMQCLPNPEDVKMALEVYKLSLEIKQLELQRDRARQSTLDKEL 96
44 ---EGCEQILTGKRLMQCLPNPEDVKMALEIYKLSLEIEQLELQRDRARQSTLDK-- 95
A=SEQ ID N0; B= sequence, C= o identity, calculated by
reference to a fragment of SEQ ID N0:1 of the same length.
Where deletions of the sequence are made, apart from N- or C-
terminal truncations, these will preferably be limited to no
more than one, two or three deletions which may be contiguous
or non-contiguous.
Where insertions are made, or N- or C-terminal extensions to
the core protein sequence, these will also be desirably
limited in number so that the size of the core protein does
not exceed the length of the wild type sequence by more than
20, preferably by no more than 15, more preferably by no more
than 10, amino acids. Thus in the case of SEQ ID N0: 1, the
core protein, when modified by insertion or elongation, will
desirably be no more than 77 amino acids in length.
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Antigen .
An antigen is any molecule capable of being recognized by an
antibody or T-cell receptor. However, not all antigens are
immunogens. An immunogen is any substance which elicits an
5 immune response. In one aspect, the present invention enables
antigens which are not immunogens to become immunogens, and
those antigens which are weak immunogens to become better
immunogens.
10 An important characteristic of the present invention is that
monomeric antigens are highly preferred when antigens are
produced by being genetically fused to the C4bp core because
they do not impede the assembly of the C4bp core protein into
an oligomeric and therefore functional form.
However in an alternative aspect, the antigens may be non-
monomeric when they are coupled chemically or non-covalently
to the C4bp core protein.
A monomeric antigen may thus fall into two main groups:
1) An antigen which is a fragment or variant of a parent
protein which in its natural state is multimeric (i.e. dimeric
or a higher order multimer), but which antigen itself does not
form multimers under conditions in which the parent protein
does form such multimers; and
2) An antigen which in its natural state is a monomer.
Examples of both types of antigen are discussed further herein
below.
Monomeric antigens have in common that they can be encoded on
a single piece of DNA and when this DNA is fused to DNA
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encoding a C4bp core protein and subsequently translated into
protein, the antigen is linked through a unique point on the
antigen to a single C4bp core protein chain. A simple example
of such an antigen would be lysozyme from hen egg white. The
cDNA encoding the full-length lysozyme open reading frame can
be fused to the C4bp core open reading frame in such a manner
that the assembly of the C4bp part of the resulting fusion
protein is not impeded.
After biosynthesis, a single polypeptide chain fused to a C4bp
core may be processed, for example by proteases, thus
generating new N- and C-termini within the polypeptide chain.
If the two or more chains generated by proteolytic cleavage
remain attached to one another through, for example,
disulphide bonds, the C4bp fusion protein will, at the end of
processing have attached to it a protein which would normally
not be considered monomeric. However, for the purposes of this
invention, proteins of this type are considered monomeric
because they are encoded as a single fusion protein in a
single open reading frame. An example of this type would be
proinsulin, which is processed after biosynthesis to have two
chains, called A and B, which are linked by disulphide bonds.
A fragment of proinsulin, called the C peptide, is removed
following proteolytic processing of the precursor fusion
protein.
The monomeric antigen may be derived from a protein which is
not necessarily monomeric in its natural state. Thus many
antigens found in a polymeric state in Nature can be modified,
for example by protein engineering techniques, so that they
become monomeric. There are three examples. As example of
such an antigen is one derived from the influenza virus
hemagglutinin protein. This is well known to form a complex
trimeric structure in its natural state (Wilson et al. Nature
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289, 366-373, 1981). However, it is possible, by removing the
coiled coil responsible for trimerizing the molecule to obtain
a monomeric fragment. A specific example is provided by the
work of Jeon and Arnon (Viral Immunology 15, 165-176, 2002).
These authors used only residues 96-261 of the hemagglutinin
in order to have a fragment encompassing only the globular
region of the hemagglutinin.
Another example is the Plasmodium merozoite surface protein 1
(MSP1). This large (approximately 200 kDa) protein decorates
the surface of merozoites which are responsible for the blood
stage of malaria infections. It is normally fixed to the
surface of merozoites through a C-terminal GPI anchor (where
GPI is glycosylphosphatidylinisotol). This GPI anchor is
preceded by a hydrophobic stretch of amino acids. As a
consequence of this anchor, neither the full-length MSP1, nor
the C-terminal fragment called MSP1.19 (which remains
membrane-bound even as the merozoite invades erythrocytes) is
ever found in a monomeric state in nature. The same applies to
many membrane proteins which have a single hydrophobic
transmembrane region. The present invention is best practised
by deleting these hydrophobic stretches. See the example
below which describes the fusion of MSP1.19 proteins to C4bp
core proteins.
Thus in one preferred aspect of the invention, the product of
the invention is a fusion of a plasmodium MSP1 monomeric
antigenic fragment fused to a C4bp core protein. The
plasmodium MSP1 antigenic fragment may comprise from about 50
to about 200, preferably from about 50 to about 150, amino
acids. The antigenic fragment may be from any plasmodium
species, such as Plasmodium falciparum or Plasmodium vivax or
Plasmodium ovale or Plasmodium malariae (all of which are
capable of causing illness in humans) or Plasmodium yoelii.
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Although deletions are the easiest method of rendering
monomeric otherwise oligomeric proteins, in some cases,
mutating one or more amino acids may suffice. An example of
this is the CpnlO protein, which in its natural state is a
heptameric protein, like the C4bp core in its principal
isoforms. The mutation of a single amino acid in CpnlO
converts it into a monomeric mutant which makes it suitable
for fusion to a C4bp core protein (Guidry et al. BMC
Biochemistry 4, 14-26, 2003). An alternative approach to
monomerize this protein was to delete N-terminal or C-terminal
amino acids (Llorca et al. Biochem. Biophysica Acta 1337, 47-
56, 1997; Seale and Horowitz, J. Biol. Chem. 270, 30268-30270,
1995) and thereby the regions responsible for inter-subunit
interaction.
In general, for protein that will have a strong propensity to
assemble into oligomeric structures (such as viral capsid
proteins) thus disrupting the assembly of a C4bp core protein
to which it is fused, the principles of deleting the regions
responsible for protein-protein interaction or of mutating
residues at the interface can be applied to obtain monomeric
proteins.
Antigens can be classified into two categories, both of which
are suitable for use with the invention. The first category
is exogenous antigens, and includes all molecules found in
infectious organisms. Bacterial immunogens, parasitic
immunogens and viral immunogens are useful as polypeptide
moieties to create multimeric or hetero-multimeric C4bp fusion
proteins useful as vaccines.
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Bacterial sources of these immunogens include those
responsible for bacterial pneumonia, meningitis, cholera,
diphtheria, pertussis, tetanus, tuberculosis and leprosy.
Parasitic sources include malarial parasites, such as
Plasmodium, as well as trypanosomal and leishmania species.
Viral sources include poxviruses, e.g., smallpox virus, cowpox
virus and orf virus; herpes viruses, e.g., herpes simplex
virus type 1 and 2, B-virus, varicella zoster virus,
cytomegalovirus, and Epstein-Barr virus; adenoviruses, e.g.,
mastadenovirus; papovaviruses, e.g., papillomaviruses such as
HPV16, and polyomaviruses such as BK and JC virus;
parvoviruses, e.g., adeno-associated virus; reoviruses, e.g.,
reoviruses 1, 2 and 3; orbiviruses, e.g., Colorado tick fever;
rotaviruses, e.g., human rotaviruses; alphaviruses, e.g.,
Eastern encephalitis virus and Venezuelan encephalitis virus;
rubiviruses, e.g., rubella; flaviviruses, e.g., yellow fever
virus, Dengue fever viruses, Japanese encephalitis virus,
Tick-borne encephalitis virus and hepatitis C virus;
coronaviruses, e.g., human coronaviruses; paramyxoviruses,
e.g., parainfluenza 1, 2, 3 and 4 and mumps; morbilliviruses,
e.g., measles virus; pneumovirus, e.g., respiratory syncytial
virus; vesiculoviruses, e.g., vesicular stomatitis virus;
lyssaviruses, e.g., rabies virus; orthomyxoviruses, e.g.,
influenza A and B; bunyaviruses e.g., LaCrosse virus;
phleboviruses, e.g., Rift Valley fever virus; nairoviruses,
e.g., Congo hemorrhagic fever virus; hepadnaviridae, e.g.,
hepatitis B; arenaviruses, e.g., 1cm virus, Lasso virus and
Junin virus; retroviruses, e.g., HTLV I, HTLV II, HIV-1 and
HIV-2; enteroviruses, e.g., polio virus 1,- 2 and 3, coxsackie
viruses, echoviruses, human enteroviruses, hepatitis A virus,
hepatitis E virus, and Norwalk-virus; rhinoviruses e.g., human
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rhinovirus; and filoviridae, e.g., Marburg (disease) virus and
Ebola virus.
Antigens from these bacterial, viral and parasitic sources may
5 be used in the production of multimeric proteins useful as
vaccines. The multimers may comprise a mixture of monomers
carrying different antigens.
Antigens from these bacterial, viral and parasitic sources can
10 be considered as exogenous antigens because they are not
normally present in the host and are not encoded in the host
genome. Endogenous antigens, in contrast, are normally
present in the host or are encoded in the host genome, or
both. The ability to generate an immune response to an
15 endogenous antigen is useful in treating tumours that bear
that antigen, or in neutralising growth factors for the
tumour. An example of the first type of endogenous antigen is
HER2, the target for the monoclonal antibody called Herceptin.
An example of the second, growth factor, type of endogenous
20 antigen is gonadotrophin releasing hormone (called GnRH) which
has a trophic effect on some carcinomas of the prostate gland.
Immunogens made using the present invention may be used for
research or therapeutic purposes. For example, research
applications include the generation of antisera to predicted
,gene products in genome sequence data. This requirement
applies to prokaryotic, such as bacterial, and eukaryotic,
including fungal and mammalian, gene products. The antigen may
be any size conventional in the art for vaccines, ranging from
short peptides to very large proteins.
Non-polypeptide immunogens may be, for example, carbohydrates
or nucleic acids. The polysaccharide coats of Neisseria
species or of Streptococcus pneumoniae species are examples of
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21
carbohydrates which may be used for the purposes of the
invention.
Where a non-polypeptide immunogen is part of the product of
the invention, the immunogen may be covalently attached to the
first component of the product using routine synthetic
methods. Generally, the immunogen may be attached to either
the N- or C-terminal of a C4bp core protein comprising the
first component, or to an amino acid side chain group (for
example the epsilon-amino group of lysine or the thiol group
of cysteine), or a combination thereof. More than one
immunogen per fusion protein may be added. To facilitate the
coupling, a cysteine residue may be added to the C4bp core
protein, for example as the N- or C-terminus.
The present invention has many advantages in the generation of
an immune response. For example, the use of multimers can
permit the presentation of a number of antigens,
simultaneously, to the immune system. This allows the
preparation of polyvalent vaccines, capable of raising an
immune response to more than one epitope, which may be present
on a single organism or a number of different organisms.
Accordingly, in a further aspect the monomeric antigen may be
a synthetic antigen comprising two different epitopes, either
from two different organisms or from two different proteins of
the same organism. An example of the latter is a fusion of a
sporozoite antigen sequence, e.g. two or more NANP repeat
sequences from the circumsporozoite protein joined to an MSP1
sequence. A second example of the latter is a fusion of the
M2e sequence described by Neirynck et al. (Nature Medicine 5,
1157- 1163, 1999) fused to a monomeric influenza hemagglutinin
fragment.
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22
Thus, vaccines formed according to the invention may be used
for simultaneous vaccination against more than one disease, or
to target simultaneously a plurality of epitopes on a given
pathogen. The epitopes may be present in single monomer units
or on different monomer units which are combined to provide a
heteromultimer.
C4bp core fusion proteins in particular are useful in the
context of immunisations, because the core protein is normally
present in the serum or plasma of the recipient of the
immunisation, and the core protein does not evoke an immune
response against itself. C4bp proteins are known in a number
of mammalian species, and the appropriate homologues for
mammalian species may be found by those skilled in the art
using standard gene cloning techniques.
Nucleic Acids.
Products of the invention may be produced by expression of a
fusion protein in a prokaryotic or eukaryotic host cell, using
a nucleic acid construct encoding the protein. Where the
antigen is a polypeptide, the expression of the fusion protein
from a nucleic acid sequence can be used to produce a product
of the invention.
Thus the invention provides a nucleic acid construct,
generally DNA or RNA, which encodes a product of the
invention.
The construct will generally be in the form of a replicable
vector, in which sequence encoding the protein is operably
linked to a promoter suitable for expression of the protein in
a desired host cell.
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23
The vectors may be provided with an origin of replication and
optionally a regulator of the promoter. The vectors may
contain one or more selectable marker genes. There are a wide
variety of prokaryotic and eukaryotic expression vectors known
as such in the art, and the present invention may utilise any
vector according to the individual preferences of those of
skill in the art.
A wide variety of prokaryotic host cells can be used in the
method of the present invention. These hosts may include
strains of Escherichia, Pseudomonas, Bacillus, Lactobacillus,
Thermophilus, Salmonella, Enterobacteriacae or Streptomyces.
For example, if E. coli from the genera Escherichia is used in
the method of the invention, preferred strains of this
bacterium to use would include derivatives of BL21(DE3)
including C41(DE3), C43(DE3) or C0214(DE3), as described and
made available in W098/02559.
Even more preferably, derivatives of these strains lacking the
prophage DE3 may be used when the promoter is not the T7
promoter.
Prokaryotic vectors includes vectors bacterial plasmids, e.g.,
plasmids derived from E. coli including ColEI, pCRl, pBR322,
pMB9 and their derivatives, wider host range plasmids, e.g.,
RP4; phage DNAs, e.g., the numerous derivatives of phage
lambda, e.g., NM989, and other DNA phages, e.g., M13 and
filamentous single stranded DNA phages. These and other
vectors may be manipulated using standard recombinant DNA
methodology to introduce a nucleic acid of the invention
operably linked to a promoter.
The promoter may be an inducible promoter. Suitable promoters
include the T7 promoter, the tac promoter, the trp promoter,
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24
the lambda promoters PL or PR and others well known to those
skilled in the art.
A wide variety of eukaryotic host cells may also be used,
including for example yeast, insect and mammalian cells.
Mammalian cells include CHO and mouse cells, African green
monkey cells, such as COS-1, and human cells.
Many eukaryotic vectors suitable for expression of proteins
are known. These vectors may be designed to be chromosomally
incorporated into a eukaryotic cell genome or to be maintained
extrachromosomally, or to be maintained only transiently in
eukaryotic cells. The nucleic acid may be operably linked to
a suitable promoter, such as a strong viral promoter including
a CMV promoter, and SV40 T-antigen promoter or a retroviral
LTR.
To obtain a product of the invention, host cells carrying a
vector of the invention may be cultured under conditions
suitable for expression of the protein, and the protein
recovered from the cells of the culture medium.
Cell culturirig.
Plasmids encoding fusion proteins in accordance with the
invention may be introduced into the host cells using
conventional transformation techniques, and the cells cultured
under conditions to facilitate the production of the fusion
protein. Where an inducible promoter is used, the cells may
initially be cultured in the absence of the inducer, which may
then be added once the cells are growing at a higher density
in order to maximise recovery of protein.
Cell culture conditions are widely known in the art and may be
used in accordance with procedures known as such.
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Although W091/11461 suggests that prokaryotic host cells may
be used in the production of C4bp-based proteins, there was no
experimental demonstration of such production.
5
Recently, it has been found that proteins fused to the C4bp
core produced in the prokaryotic expression systems retain
their functional activity. This is disclosed in
W020041020639, the contents of which are incorporated herein
10 by reference. Such methods may be used in the production of
fusion proteins of the present invention.
Recovery of protein from culture.
Once the cells have been grown to allow for production of the
protein, the protein may be recovered from the cells. Because
15 we have found that surprisingly, the protein remains soluble,
the cells will usually be spun down and lysed by sonication,
for example, which keeps the protein fraction soluble and
allows this fraction to remain in the supernatant following a
further higher speed (e. g. 15,000 rpm for 1 hour)
20 centrifugation.
The fusion protein in the supernatant protein fraction may be
purified further by any suitable combination of standard
protein chromatography techniques. We have used ion-exchange
25 chromatography followed by gel filtration chromatography.
Other chromatographic techniques, such as affinity
chromatography, may also be used.
In one embodiment, we have found that heating the supernatant
sample either after centrifugation of the lysate, or after any
of the other purification steps will assist recovery of the
protein. The sample may be heated to about 70 - 80 °C for a
period of about 10 to 30 minutes.
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26
Depending on the intended uses of the protein, the protein may
be subjected to further purification steps, for example
dialysis, or~to concentration steps, for example freeze
drying.
Compositions and uses thereof.
Products according to the invention may be prepared in the
form of a pharmaceutical composition. The product will be
present with one or more pharmaceutically acceptable carriers
or diluents. The composition will be prepared according to
the intended use and route of administration of the product.
Thus the invention provides a composition comprising a product
of the invention in multimeric form together with one or more
pharmaceutically acceptable carriers or diluents, and the use
of such a composition in methods of immunotherapy for
treatment or prophylaxis of a human or animal subject.
Pharmaceutically acceptable carriers or diluents include those
used in formulations suitable for oral, rectal, nasal, topical
(including buccal and sublingual), vaginal or parenteral
(including subcutaneous, intramuscular, intravenous,
intradermal, intrathecal and epidural) administration. The
formulations may conveniently be presented in unit dosage form
and may be prepared by any of the methods well known in the
art of pharmacy.
Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, etc, a fusion
protein of the invention with optional pharmaceutical
adjuvants in a carrier, such as, for example, water, saline
aqueous dextrose, glycerol, ethanol, and the like, to thereby
form a solution or suspension. If desired, the composition to
be administered may also auxiliary substances such as pH
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27
buffering agents and the like. Actual methods of preparing
such dosage forms are known, or will be apparent, to those
skilled in this art; for example, see Remington's
Pharmaceutical Sciences, Mack Publishing Company, Easton,
Pennsylvania, 19th Edition, 1995.
Compositions according to the invention may additionally
comprise one or more adjuvants, for example mineral salts such
as aluminium hydroxide or calcium phosphate, or cytokines such
as IZ-12 or GM-CSF. A fuller list of suitable adjuvants is
given in Table 1 of Singh and 0'Hagan, Nature Biotechnology,'
17, 1075-1081, 1999, the disclosure of which is incorporated
herein by reference.
Products according to the invention, desirably in the form of
a composition or formulation may be used in methods of
treatment as described herein, by administration of the
product or composition thereof to a human or animal subject.
The amount effective to alleviate the symptoms of the subject
being treated will be determined by the physician taking into
account the patient and the condition to be treated. Dosage
forms or compositions containing active ingredient in the
range of 0.25 to 95% with the balance made up from non-toxic
carrier may be prepared.
Parenteral administration is generally characteri2ed by
injection, either subcutaneously, intramuscularly or
intravenously. Injectables can be prepared in conventional
forms, either as liquid solutions or suspensions, solid forms
suitable for solution or suspension in liquid prior to
injection, or as emulsions. Suitable excipients are, for
example, water, saline, dextrose, glycerol, ethanol or the
like. A more recently devised approach for parenteral
administration employs the implantation of a slow-release or
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28
sustained-release system, such that a constant level of dosage
is maintained. See, e.g., US Patent No. 3,710,795.
Doses of the product will be dependent upon the nature of the
antigen and may be determined according to current practice
a
for administration of that antigen in conventional vaccine
formulations.
Passive Immunisation.
In a further aspect, the invention provides a means for
passive immunisation of a subject with an immune serum
containing antibodies obtained by vaccination of a host
subject with a product of the invention. The host subject may
be a human or non-human mammal. Thus in a further aspect, the
invention provides an immune serum obtained by such a method,
and the use of such an immune serum in a method of treatment
of the human or animal body.
DNA vaccines
In another aspect, the invention provides a eukaryotic
expression vector comprising a nucleic acid sequence encoding
a recombinant fusion protein product of the invention for use
in the treatment of the human or animal body.
Such treatment would achieve its therapeutic effect by
introduction of a nucleic acid sequence encoding an antigen
for the purposes of raising an immune response. Delivery of
nucleic acids can be achieved using a plasmid vector (in
"naked" or formulated form) or a recombinant expression
vector. For a review of DNA vaccination, see Ada G. and
Ramshaw I, in Expert Opinion in Emerging Drugs 8, 27-35,
2003) .
Various viral vectors which can be utilized for gene delivery
include adenovirus, herpes virus, vaccinia or an RNA virus
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29
such as a retrovirus. The retroviral vector may be a
derivative of a murine or avian retrovirus. Examples of
retroviral vectors in which a single foreign gene can be
inserted include, but are not limited to: Moloney murine
leukaemia virus (MoMuLV), Harvey murine sarcoma virus
(HaMuSV), murine mammary tumour virus (MuMTV), and Rous
Sarcoma Virus (RSV). Tnlhen the subject is a human, a vector
such as the gibbon ape leukaemia virus (GaLV) can be utilized.
The vector will include a transcriptional regulatory sequence,
particularly a promoter region sufficient to direct the
initiation of RNA synthesis. Suitable eukaryotic promoters
include the promoter of the mouse metallothionein I gene
(Hamer et al., 1982, J. Moles. Appl. Genet. 1: 273 ); the TK
promoter of Herpes virus (McKnight, 1982, Cell 31: 355 ); the
SV40 early promoter (Benoist et al., 1981, Nature 290: 304 );
the Rous sarcoma virus promoter (Gorman et al., 1982, Proc.
Natl. Acad. Sci. USA 79: 6777); and the cytomegalovirus
promoter (Foecking et al., 1980, Gene 45: 101 ).
Administration of vectors of this aspect of the invention to a
subject, either as a plasmid vector or as part of a viral
vector can be affected by many different routes. Plasmid DNA
can be "naked" or formulated with cationic and neutral lipids
(liposomes) or microencapsulated for either direct or indirect
delivery. The DNA sequences can also be contained within a
viral (e. g., adenoviral, retroviral, herpesvirus, pox virus)
vector, which can be used for either direct or indirect
delivery. Delivery routes include but are not limited to oral,
intramuscular, intradermal (Sato, Y. et al., 1996, Science
273: 352-354), intravenous, intra-arterial, intrathecal,
intrahepatic, inhalation, intravaginal instillation (Bagarazzi
et al., 1997, J Med. Primatol. 26:27), intrarectal,
intratumour or intraperitoneal.
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Thus the invention includes a vector as described herein as a
pharmaceutical composition useful for allowing transfection of
some cells with the DNA vector such that a therapeutic
5 polypeptide will be expressed and have a therapeutic effect,
namely to induce an immune response to an antigen. The
pharmaceutical compositions according to the invention are
prepared by bringing the construct according to the present
invention into a form suitable for administration to a subject
10 using solvents, carriers, delivery systems, excipients, and
additives or auxiliaries. Frequently used solvents include
sterile water and saline (buffered or not). One carrier
includes gold particles, which are delivered biolistically
(i.e., under gas pressure). Other frequently used carriers or
15 delivery systems include cationic liposomes, cochleates and
microcapsules, which may be given as a liquid solution,
enclosed within a delivery capsule or incorporated into food.
An alternative formulation for the administration of gene
20 delivery vectors involves liposomes. Liposome encapsulation
provides an alternative formulation for the administration of
polynucleotides and expression vectors. Liposomes are
microscopic vesicles that consist of one or more lipid
bilayers surrounding aqueous compartments. See, generally,
25 Bakker-Woudenberg et al, 1993, Eur. J. Clin. Microbiol.
Infect. Dis. 12 (Suppl. 1): 561, and Kim, 1993, Drugs 46: 618.
Liposomes are similar in composition to cellular membranes and
as a result, liposomes can be administered safely and are
biodegradable. Depending on the method of preparation,
30 liposomes may be unilamellar or multilamellar, and liposomes
can vary in size with diameters ranging from 0.02 ~aM to
greater than 10 uM. See, for example, Machy et al., 1987,
LIPOSOMES IN CELL BIOLOGY AND PHARMACOLOGY (John Libbey), and
Ostro et al., 1989, American J. Hosp. Phann. 46: 1576.
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31
Expression vectors can be encapsulated within liposomes using
standard techniques. A variety of different liposome
compositions and methods for synthesis are known to those of
skill in the art. See, for example, US-A-4,844,904, US-A-
5,000,959, US-A-4,863,740, US-A-5,589,466, US-A-5,580,859, and
US-A-4,975,282, all of which are hereby incorporated by
reference.
In general, the dosage of administered liposome-encapsulated
vectors will vary depending upon such factors as the patient's
age, weight, height, sex, general medical condition and
previous medical history. Dose ranges for particular
formulations can be determined by using a suitable animal
model.
The invention is illustrated by the following examples.
Example 1 - Plasmodium falciparum MSP1.19-rabbit C4bp fusion
protein.
This example illustrates the fusion of a monomeric antigen
(comprising amino acids 1567-1661 of Plasmodium falciparum
MSP1) to the rabbit core C4bp protein. The fusion protein,
called AVD174, was expressed in, and purified from the
bacterial strain C41(DE3). The fusion protein alone was used
to immunise rabbits without the addition of any adjuvant.
Cloning.
A synthetic 294bp DNA fragment encoding residues 1567-1661 of
the MSP1 protein was digested with NdeI and BamHI and ligated
into pAVD181 previously digested with NdeI and BamHI. This
created an open reading frame encoding the 95 amino acid
MSP1.19 protein fragment fused to the C-terminal 57 residues
of the alpha chain of rabbit C4bp downstream of the T7 late
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32
promoter. The construction, called pAVDl74, was checked by DNA
sequencing.
The nucleotide sequence encoding the AVD174 fusion protein is:
atgttaaacatttcccagcaccagtgcgttaagaaacagtgcccgcagaa
ctctggttgtttccgtcatctggacgagcgtgaagagtgcaaatgtctgc
tgaactacaaacaggaaggtgataaatgtgttgagaacccaaacccgacc
tgtaacgaaaacaacggcggttgtgacgctgatgctaaatgcaccgagga
agacagcggttctaacggtaagaaaatcacctgcgagtgtactaaaccgg
actcctacccgctgttcgacggtatcttttgctccGGATCCGAGGTCCCG
GAAGGCTGTGAGCAGGTGCAAGCGGGTCGCCGTCTCATGCAGTGTCTCGC
AGACCCATACGAAGTGAAAATGGCCCTGGAGGTCTACAAGCTGTCTCTGG
AGATTGAACTCCTGGAACTGCAGCGCGATAAGGCACGTAAAAGCTCTGTG
CTGCGCCAGCTGTAA (SEQ ID N0: 21)
The amino acid sequence of the fusion protein AVD174 encoded
by this construct is as follows:
MLNISQHQCV KKQCPQNSGC FRHLDEREEC KCLLNYKQEG DKCVENPNPT
CNENNGGCDA DAKCTEEDSG SNGKKITCEC TKPDSYPLFD GIFCSGSEVP
EGCEQVQAGR RLMQCLADPY EVKMALEVYK LSLEIELLEL QRDKARKSSV
LRQL (SEQ ID N0: 22)
The residues 1-95 of SEQ ID N0: 22 correspond to residues
1567-1661 of Plasmodium falciparum MSP1 (the monomeric
antigen), and residues 9S-154 of SEQ ID N0: 22 correspond to
the 57 residues of the rabbit C4bp core protein. A GS linker
sequence appears between the two components.
The protein has an estimated molecular weight of 17,319
Daltons, and a theoretical pI of 5.05.
Expression.
The plasmid pAVD174 encoding the Plasmodium falciparum-rabbit
C4bp core protein was expressed in the E. coli strain
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C41(DE3). The transformed cells were grown in LB medium at
37°C to an OD600 of approximately 0.6, then expression was
induced with IPTG to a final concentration of 0.5mM, and the
culture was grown for a further three hours at 37°C at which
point the cells were harvested by centrifugation.
Purification of AVD174 fusion protein
The protein AVD174 was purified from 1 litre of C41(DE3)
cells. All of the fusion protein was found in the soluble
fraction after the cells were lysed by sonication in a buffer
containing 20mM MES pH6.5 and 5mM EDTA. The supernatant after
centrifugation was loaded on a HitrapS column.
Cationic column (Hi Trap S)
The column was equilibrated in 20 mM MES pH 6.5, 20mM EDTA
buffer (buffer A). The protein was eluted with a gradient of
10 column volumes from Buffer A to Buffer B (buffer A plus
0.5M NaCl). AVD174 eluted at a concentration of approximately
200mM NaCl.
The HiTrapS fractions containing AVD174 were concentrated
using a Millipore concentrator (cut-off 30 K) and then loaded
on a Gel Filtration column.
Gel filtration column (Superdex 200 26/60 prep grade)
A Superdex 200 26/60 column was equilibrated with 20mM Tris
buffer pH8, 150mM NaCl, and the concentrated AVD174 protein
from the HiTrapS fractions was loaded.
The protein eluted in two peaks. The correctly folded and
assembled protein eluted in 156 mls, whereas an earlier, minor
peak eluting at 120 mls is not correctly assembled or folded.
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Biophysical characterisation
The oligomeric state of C4bp fusion proteins containing
disulphide bonds (present in all except the murine core
protein: see Figure 1) can be checked easily by comparing the
behaviour of the protein on an SDS-PAGE gel in the presence
and absence of the reducing agent beta-mercaptoethanol (BME).
The AVD177 protein has an apparent size of approximately
140kDa in the absence of BME, whereas in the presence of BME,
it is reduced and runs with an apparent size of just over
20kDa.
Mass spectrometry
The.AVD174 protein was examined by electrospray mass
spectrometry after reduction by BME and alkylatiori by N-ethyl
maleimide (NEM). Results showed the addition of 14 NEM
molecules (each of 125 Da) to the protein of which the
molecular weight was determined to be 19,072 Da.
Endotoxin levels
The level of endotoxin in the purified protein was determined
using the LAL (limulus amoebocyte lysate) test kit form
Biowhittaker to be 21 EU per milligram of protein.
Example 2 - Plasmodium falciparum MSP1.19-human C4bp fusion
protein.
This example illustrates the fusion of a monomeric antigen
(comprising amino acids 1567-1661 of Plasmodium falciparum
MSP1) to the human C4bp core protein. The fusion protein was
expressed in, and purified from, the bacterial strain
C41(DE3). The fusion protein alone can be used to immunise
humans without the addition of any adjuvant.
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Cloning.
A synthetic 294bp DNA fragment encoding residues 1567-1661 of
the MSP1 protein was digested with NdeI and BamHI and ligated
into pAVD181 preciously digested with NdeI and BamHI. This
5 created an open reading frame encoding the 95 amino acid
MSP1.19 protein fragment fused to the C-terminal 57 residues
of the alpha chain of human C4bp downstream of the T7 late
promoter. The construction, called pAVD177, was checked by DNA
sequencing.
The nucleotide sequence encoding the AVD177 fusion protein is:
atgttaaacatttcccagcaccagtgcgttaagaaacagtgcccgcagaa
ctctggttgtttccgtcatctggacgagcgtgaagagtgcaaatgtctgc
tgaactacaaacaggaaggtgataaatgtgttgagaacccaaacccgacc
tgtaacgaaaacaacggcggttgtgacgctgatgctaaatgcaccgagga
agacagcggttctaacggtaagaaaatcacctgcgagtgtactaaaccgg
actcctacccgctgttcgacggtatcttttgctccGGATCCgagaccccc
gaaggctgtgaacaagtgctcacaggcaaaagactcatgcagtgtctccc
aaacccagaggatgtgaaaatggccctggaggtatataagctgtctctgg
aaattgaacaactggaactacagagagacagcgcaagacaatccactttg
gataaagaactataa (SEQ ID N0: 23)
The amino acid sequence of the fusion protein AVD177 encoded
by this construct is as follows:
MLNISQHQCV KKQCPQNSGC FRHLDEREEC KCLLNYKQEG DKCVENPNPT
CNENNGGCDA DAKCTEEDSG SNGKKITCEC TKPDSYPLFD GIFCSGSETP
EGCEQVLTGK RLMQCLPNPE DVKMALEVYK LSLEIEQLEL QRDSARQSTL
DKEL (SEQ ID N0: 24)
The residues 1-95 of SEQ ID N0: 24 correspond to residues
1567-1661 of Plasmodium falciparum MSP1 (the monomeric
antigen), and residues 98-154 of SEQ ID N0: 24 correspond to
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36
the 57 residues of the human C4bp core protein. A GS linker
sequence appears between the two components.
The protein has an estimated molecular weight of 17,261
Daltons, and a theoretical pI of 4.72.
Expression.
Expression of protein is shown in Figure 2. An SDS-PAGE gel
showing the expression of AVD177 protein in three different
strains, C41(DE3), BL21(DE3) and C43(DE3),either uninduced (U)
or after induction at 37°C or 30°C is presented. The lanes on
the gel are as follows:
Lane 1: molecular weight markers (in descending order: 66, 60,
46, 36, 28, 20, 14, 12, 6 kDa)
Lane 2: C41(DE3) before induction
Lane 3: C41(DE3) three hours after induction at 37°C;
Lane 4: C41(DE3) three hours after induction at 30°C;
Lane 5: BL21(DE3) before induction
Lane 6: BL21(DE3) three hours after induction at 37°C;
Lane 7: BL21(DE3) three hours after induction at 30°C;
Lane 8: C43(DE3) before induction
Lane 9: C43(DE3) three hours after induction at 37°C;
Lane 10: C43(DE3) three hours after induction at 30°C.
As can be seen, good expression was obtained in C41(DE3) and
in the strain C43(DE3). In contrast, no expression was found,
under the conditions tested, in the strain BL21(DE3): see
Figure 2. Cultures were grown in LB medium at 30°C and at
37°C
to an optical density (0D600) of approximately 0.6 and then
expression was induced by the addition of IPTG to a final
concentration of 0.5mM.
Purification of AVDZ77 fusion protein
The protein AVD177 was purified from 1 litre of C41(DE3) cells
grown for three hours after induction at 37°C. All of the
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fusion protein was found in the soluble fraction after lysis
of the bacterial pellet. Cells were lysed by sonication in a
buffer containing 20mM MES pH6.5 and 5mM EDTA. The supernatant
after centrifugation was loaded on a MonoS column.
Cationic column (Mono S HR 10/10)
The column was equilibrated in 20 mM MES pH 6.5, 20mM EDTA
buffer (buffer A). The protein was eluted with a gradient of
column volumes from Buffer A to Buffer B which was buffer A
10 plus 0.5M NaCl. AVD177 eluted at a concentration of
approximately 200mM NaCl.
The MonoS fractions containing AVD177 were concentrated using
a Millipore concentrator (cut-off 30 K) and then loaded on a
Gel Filtration column.
Gel filtration column (Superdex 200 26/60 prep grade)
A Superdex 200 26/60 column was equilibrated with 20mM Tris
buffer pH8, 150mM NaCl, and the concentrated AVD177 protein
from the MonoS fractions was loaded.
The protein eluted in two peaks. The correctly folded and
assembled protein eluted in 150 mls, whereas an earlier, minor
peak eluting at 115 mls is not correctly assembled or folded.
Biophysical characterisation
The oligomeric state of C4bp fusion proteins containing
disulphide bonds (present in all except the murine core
protein: see Figure 1) can be checked easily by comparing the
behaviour of the protein on an SDS-PAGE gel in the presence
and absence of the reducing agent beta-mercaptoethanol (BME).
The results are shown in Figure 3, which shows an SDS-PAGE gel
showing the AVD177 protein run under reducing conditions (left
+BME) and under non-reducing conditions (right -BME) separated
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by molecular weight markers (in descending order: 66, 60, 46,
36, 28, 20, 14, 12, 6 kDa). As can be seen from Figure 3, the
AVD177 protein has an apparent size of approximately 140kDa in
the absence of BME, whereas in the presence of BME, it is
reduced and runs with an apparent size of just over 20kDa.
Mass spectrometry
The AVD177 protein was examined by electrospray mass
spectrometry after reduction by BME and alkylation by N-ethyl
maleimide (NEM). Results showed the addition of 14 NEM
molecules (each of 125 Da) to the protein of which the
molecular weight was determined to be 19,015 Da.
Endotoxin levels
The level of endotoxin in the purified protein was determined
using the LAL (limulus amoebocyte lysate) test kit form
Biowhittaker to be 38 EU per milligram of protein.
Example 3 - Mutant Plasmodium falci arum MSP1.19-rabbit C4bp
fusion protein.
By way of example, a second Plasmodium falciparum MSP1.19-
rabbit C4bp protein is described here. This differs
principally in having a distinct codon usage to pAVD174 and
pAVD177 for the monomeric antigen gene and also contains three
amino acid changes (described in Uthaipibull et al., J Mol
Biol. 307, 1381-1394, 2001). This fusion protein was called
AVD178.
The nucleotide sequence encoding the AVD178 fusion protein is:
atgctgaatatttcccagcaccagtgcgtaaagaaacagtgtcctcagaa
ctctggttgcttccgccatctggacgaacgcgaatattgcaaatgccgtc
tgaactacaaacaggaaggtgacaagtgcgttctgaacccgaacccaact
tgtaacgagaacaacggtggctgcgatgctgatgctaaatgcactgaaga
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agacagcggttctaacggcaaaaaaatcacctgcgagtgcaccaaaccgg
acagctatccgctgttcgacggcattttttgttctggatccGAGGTCCCG
GAAGGCTGTGAGCAGGTGCAAGCGGGTCGCCGTCTCATGCAGTGTCTCGC
AGACCCATACGAAGTGAAAATGGCCCTGGAGGTCTACAAGCTGTCTCTGG
AGATTGAACTCCTGGAACTGCAGCGCGATAAGGCACGTAAAAGCTCTGTG
CTGCGCCAGCTGTAA (SEQ ID N0: 25)
The amino acid sequence of the fusion protein AVD178 encoded
by this construct is as follows:
MLNISQHQCVKKQCPQNSGCFRHLDEREYCKCRLNYKQEGDKCVhNPNPTCNENNGGCDADA
KCTEEDSGSNGKKITCECTKPDSYPLFDGIFCSGSEVPEGCEQVQAGRRLMQCLADPYEVKM
ALEVYKLSLEIELLELQRDKARKSSVLRQL (SEQ ID N0: 26)
The three mutant amino acids are in bold and underlined.
The residues 1-95 of SEQ ID N0: 25 correspond to residues
1567-1661 of Plasmodium falciparum MSP1 (the monomeric
antigen) with three mutations, and residues 98-154 of SEQ ID
N0: 24 correspond to the 57 residues of the rabbit C4bp core
protein. A GS linker sequence appears between the two
components.
Expression, purification and characterisation of AVDZ7~
This was carried out essentially as described for the AVD174
and AVD177 proteins. Elution from the.HiTrapS column was in
approximately 200mM. On gel filtration, the oligomeric protein
eluted in a volume of 159m1s. On mass spectrometry, with 14
NEM residues per monomer, the molecular mass was 19, 133 Da.
The endotoxin level was measured at 58EU per milligram of
protein.
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Example 4 - Plasmodium yoelii MSP1.19-murine C4bp fusion
protein.
C1 oning
The AVD108 protein was prepared as follows: a synthetic DNA
5 fragment encoding MSP1.19 and a part of MSP1.33 from
Plasmodium yoelii was cloned as an Ndel-BamHI fragment
unpstream from the C-terminal 54 amino acids of the murine
C4bp alpha chain.
10 The nucleotide sequence encoding the fusion protein AVD108 was
as follows:
ATGAGATCTCACATTGCCTCTATTGCTTTGAACAACTTGAACAAGTCTGG
TTTGGTAGGAGAAGGTGAGTCTAAGAAGATTTTGGCTAAGATGCTGAACA
TGGACGGTATGGACTTGTTGGGTGTTGACCCTAAGCATGTTTGTGTTGAC
15 ACTAGAGACATTCCTAAGAACGCTGGATGTTTCAGAGACGACAACGGTAC
TGAAGAGTGGAGATGTTTGTTGGGTTACAAGAAGGGTGAGGGTAACACCT
GCGTTGAGAACAACAACCCTACTTGCGACATCAACAACGGTGGATGTGAC
CCAACCGCCTCTTGTCAAAACGCTGAATCTACCGAAAACTCCAAGAAGAT
TATTTGCACCTGTAAGGAACCAACCCCTAACGCCTACTACGAGGGTGTTT
20 TCTGTTCTTCTTCCGGATCCGAGGCCTCTGAAGACCTTAAGCCTGCGCTT
ACAGGCAACAAGACCATGCAGTATGTGCCAAATTCACACGATGTGAAAAT
GGCTCTGGAGATCTACAAGCTGACTCTGGAGGTTGAACTACTACAGCTCC
AGATACAAAAGGAGAAACACACTGAAGCACACTAA (SEQ ID N0: 27)
25 The amino acid sequence of the protein AVD108 was as follows:
MRSHIASIAL NNLNKSGLVG EGESKKILAK MLNMDGMDLL GVDPKHVCVD
TRDIPKNAGC FRDDNGTEEW RCLLGYKKGE GNTCVENNNP TCDINNGGCD
PTASCQNAES TENSKKIICT CKEPTPNAYY EGVFCSSSGS EASEDLKPAL
TGNKTMQYVP NSHDVKMALE IYKLTLEVEL LQLQIQKEKH TEAH (SEQ ID N0:
30 28)
The residues 3-138 of SEQ ID N0: 28 correspond to residues
1619-1753 of Plasmodium yoelii MSP1 (the monomeric antigen),
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and residues 141-194 of SEQ ID N0: 28 correspond to the 54
residues of the rabbit C4bp core protein. A GS linker
sequence appears between the two components, and a short
restriction site encoded sequence precedes the first
component.
Expression of AVD108
The protein AVD108 was expressed in the E. coli strain
C41(DE3). A three litre culture was grown in LB medium at 37°C
to an optical density (0D600) of approximately 0.6 and then
expression was induced by the addition of IPTG to a final
concentration of 0.7mM. Four hours after induction, the cells
were harvested by centrifugation. The cells were lysed in
buffer A (50mM Tris pH9, 5mM EDTA) and debris removed by
centrifugation.
Purification and characterisation of AVD108
The protein AVD108 was purified using four column
chromatography steps. In the first anion-exchange
chromatographic step, a DEAF HR16/10 column was used. The
protein was loaded in buffer A and eluted in a gradient with
buffer B " which was buffer A plus 1M NaCI. AVD108 eluted in a
broad peak between 180-300mM NaCl.
In the second hydrophobic interaction chromatographic step,
the pooled fractions containing AVD108 from the DEAF column
were loaded on a Macro-Prep Phenyl Sepharose column and eluted
in a gradient of decreasing salt from 1M to OM NaCl. In the
final two chromatographic steps, the AVD108 protein was
purified by gel filtration on a Superdex 5200 26/60 column.
The first time, the protein was denatured by adding urea to
final concentration of 8M and incubating overnight at 4°C. The
monomer eluted from this column in a volume of 203m1s. After
renaturation by dialysis against Buffer H (20mM Tris pH7.5,
150mM NaCl) overnight at 4°C, before being loaded on the same
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column now equilibrated in Buffer H. The oligomers now eluted
in a volume of 164m1s. By mass spectrometry, the mass was
determined as 21,257 Da. Endotoxin levels by the LAL test kit
from Biowhittaker was 4 EU per milligram of protein.
Example 5 - Immunisation using Plasmodium falciparum MSP1.19-
rabbit C4bp fusion protein.
Innnunisation
The AVD174 protein prepared as described above in Example 1
was used to immunise three New Zealand White (NZW) rabbits.
The immunisation schedule was as follows: each rabbit received
four injections at two-weekly intervals (in other words, on
days 0, 14, 28 and 42). Each injection was subcutaneous and
contained 345 micrograms (ar 20 nanomoles) of protein in a
buffered isotonic saline solution without the addition of any
known adjuvant.
In parallel, three NZW rabbits were immunised with 212
micrograms (or 20 nanomoles) of AVD172 protein in Freund's
adjuvant. The AVD172 is the same as AVD174 but lacks the C-
terminal 57 amino acids from rabbit C4bp. Tt has the following
amino acid sequence:
MLNZSQHQCV KKQCPQNSGC FRHLDEREEC KCLLNYKQEG DKCVENPNPT
CNENNGGCDA DAKCTEEDSG SNGKKITCEC TKPDSYPLFD GIFCS (SEQ ID N0:
29) .
The immunisation schedule was as follows: each rabbit received
four injections at two-weekly intervals (in other words, on
days 0, 14, 28 and 42). The first injection for each rabbit
was in Complete Freund's Adjuvant and was administered
intradermally, whereas the three following injections were
given in Incomplete Freund's Adjuvant, and were administered
subcutaneously.
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Antibody titres
Antibody titres one week after the last injection (i.e; on day
63) against MSP1 on the surface of Plasmodium falciparum
merozoites were measured by indirect immunofluorescence (as
described in Zing et al. Vaccine 15, 1562-1567, 1997 for F.
yoelii) on acetone-fixed smears of P, falciparum infected
erythrocytes.
The two highest titres in the rabbits immunised with the
AVD174 protein were 1/81,920. In the rabbits immunised with
the AVD172 protein in Freund's adjuvant, the two highest
titres were 1/20,480.
This demonstrated that fusing a monomeric antigen to a C4bp
core can induce higher antibody titres than administering the
same monomeric antigen in Freund's adjuvant.
Characterisation of antibodies produced
High titres of antibodies may not suffice to prevent or treat
an infection, and it is known that the specificity of the
antibodies produced on immunisation with an antigen can be of
critical importance. Guevara Patino et al. (J.Exp.Med. 186,
1689-1699, 1997) have described in detail methods for assaying
blocking and inhibitory antibodies against MSP1.19. These
methods were used to see if there were inhibitory antibodies
present (which are useful because they block the processing of
MSP1.42 into MSP1.33 and MSP1.19 and thus prevent erythrocyte
invasion).
Inhibitory antibodies were only found among the antibodies
induced by the AVD174 protein. None were found in the
antisera of rabbits immunised with AVD172 protein. In
contrast, AVD172 in Freund's adjuvant can induce blocking
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antibodies, as can natural Plasmodium infections in man and
these are deleterious (see Guevara Patino et al., op. cit.).
Example 6 - Immunisation using Plasmodium yoelii MSP1.19-
murine C4bp fusion protein.
I~nunisation of mice
The AVD108 protein prepared as described in Example 5 was used
to immunise six BALB/c mice. No adjuvant was used, and the
protein was in a buffered isotonic saline solution. Forty
micrograms (1.9 nanomoles) of protein was used per injection.
Each mouse was injected three times, subcutaneously, at four-
weekly intervals (in other words, on days 0, 28 and 56).
In parallel, six BALB/c mice were immunised with twenty-three
micrograms (also 1.9 nanomoles) of the AVD183 protein, which
is the same as AVD108 but lacking the murine C4bp C-terminal
54 amino acids (i.e. it is the P.yoelii MSP1.19 protein
alone). Twenty-three micrograms of this protein (in the same
buffered isotonic saline solution used for AVD108) was used
per injection. Each mouse was injected three times,
subcutaneously, on days 0, 28 and 56.
Antilaody titres
Antibody titres two weeks after the last injection (i.e; on
day 70) against MSP1 on the surface of Plasmodium yoelii
merozoites were measured by indirect immunofluorescence (as
described in Ling et al. Vaccine 15, 1562-1567, 1997) on
acetone-fixed smears of P.yoelii infected erythrocytes.
The results showed that five of six mice immunised with the
AVD108 protein had titres of 1/40,960, while the sixth mouse
had a titre of 1/10,240. In contrast, no antibodies against
MSP1 could be detected in any of the mice immunised with the
AVD183 protein at a dilution of 1/80. This demonstrated that
fusing the monomeric MSP1.19 antigen to a C4bp core could
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increase the titre of antibodies obtained up to five-hundred-
fold.
Parasite challenge
Both groups of six mice immunised as described above were
5 challenged with a lethal dose of 5,000 P.yoelii infected
erythrocytes. Again, this assay has been described by Zing et
al. (op. cit.). The six mice immunised with the AVD183
protein all died within seven days of the parasite challenge.
On the other hand, five of the six mice immunised with AVD108
10 were alive and free of parasites in their blood (as assessed
by Giemsa staining of thin blood smears examined
microscopically). The sixth mouse, which had a day 70 titre of
1/10,240 died nineteen days after the challenge; over 70% of
this mouse's erythrocytes were seen to be infected by Giemsa
15 staining on day 19.
In conclusion, this challenge experiment demonstrated that
immunisation with a monomeric antigen fused to a C4bp core
protein alone in the absence of any known adjuvants could
20 protect against an otherwise lethal Plasmodium infection. It
is believed at present that this represents the first instance
of successful vaccination against Plasmodium infection using
just a single protein unaccompanied by any known adjuvant.
25 Example 7 - Influenza hemagglutinin-C4bp fusion proteins.
This example illustrates the fusion of a monomeric antigen
(comprising residues 91-261 of the HA1 hemagglutinin protein
of influenza A virus) to the human, rabbit and murine core
C4bp proteins. Normally, the full-length HAl is assembled into
30 a trimer on the surface of virions cells, so that using only
this peptide fragment effectively converts it into a monomeric
antigen. The fusion proteins, called AVD272 to AVD274, are
expressed in, and purified from the bacterial strain C41(DE3).
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These fusion proteins alone are used to immunise mice and
rabbits without the addition of any adjuvant.
In AVD272, the HA1 fragment (described in Jeon and Arnon,
Viral Immunology, 15, 165-176, 2002) is fused to the marine
C4bp scaffold.
The amino acid sequence of AVD272 is as follows:
kafsncypyd vpdyaslrsl vassgtlefi tegftwtgvt qnggsnackr
gpgsgffsrl nwltksgsty pvlnvtmpnn dnfdklyiwg ihhpstnqeq
tslyvqasgr vtvstrrsqq tiipnigsrp wvrglssris iywtivkpgd
vlvinsngnl iaprgyfkmr GSEASEDLKP ALTGNKTMQY VPNSHDVKMA
LEIYKLTLEV ELLQLQIQKE KHTEAH (SEQ ID N0: 30)
In AVD273, the HA1 fragment is fused to the rabbit C4bp
scaffold.
The amino acid sequence of AVD273 is as follows:
kafsncypyd vpdyaslrsl vassgtlefi tegftwtgvt qnggsnackr
gpgsgffsrl nwltksgsty pvlnvtmpnn dnfdklyiwg ihhpstnqeq
tslyvqasgr vtvstrrsqq tiipnigsrp wvrglssris iywtivkpgd
vlvinsngnl iaprgyfkmr GSEVPEGCEQ VQAGRRLMQC LADPYEVKMA
LEVYKLSLEI ELLELQRDKA RKSSVLRQL (SEQ ID NO: 31)
In AVD274, the HA1 fragment was fused to the human C4bp
scaffold.
The amino acid sequence of AVD274 is as follows:
kafsncypyd vpdyaslrsl vassgtlefi tegftwtgvt qnggsnackr
gpgsgffsrl nwltksgsty pvlnvtmpnn dnfdklyiwg ihhpstnqeq
tslyvqasgr vtvstrrsqq tiipnigsrp wvrglssris iywtivkpgd
vlvinsngnl iaprgyfkmr GSETPEGCEQ VLTGKRLMQC LPNPEDVKMA
LEVYKLSLEI EQLELQRDSA RQSTLDKEL (SEQ ID N0: 32)
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Mice are immunised three times subcutaneously with the AVD272
protein (2 nanomoles) without the addition of any adjuvant at
two-weekly intervals (i.e. on days 0, 14, and 28) and show
appreciable antibody titres (as determined essentially as
described by Jeon et al., op.cit.) and significant protection
against a lethal challenge with 5 LD50 doses of the mouse
adapted A/PR/8/34 strain.
Example 8 - Influenza M2 peptide-C4bp fusion proteins
This example illustrates the fusion of a monomeric antigen
(comprising residues 2-24, the extracellular part, of the M2
protein of influenza A virus) to the human, rabbit and marine
core C4bp proteins. Normally, the full-length M2 is assembled
into a tetramer in virions and infected cells, so that using
only this peptide fragment effectively converts it into a
monomeric antigen. The fusion proteins, called AVD275 to
AVD278, were expressed in, and purified from the bacterial
strain C41(DE3). These fusion proteins alone were used to
immunise mice~and rabbits without the addition of any
adjuvant.
In AVD275, the extracellular M2 peptide (described in Neirynck
et al. Nature Medicine 5, 1157- 1163, 1999) was fused to the
marine C4bp scaffold.
The amino acid sequence of AVD275 was as follows:
SLLTEVETPI RNEfnlGCRCND SSDGSEASED LKPALTGNKT MQYVPNSHDV
KMALEIYKLT LEVELLQLQI QKEKHTEAH (SEQ TD N0: 33)
In AVD276, the extracellular M2 peptide was fused to the
rabbit C4bp scaffold.
The amino acid sequence of AVD276 was as follows:
SLLTEVETPI RNEWGCRCND SSDGSEVPEG CEQVQAGRRL MQCLADPYEV
KMALEVYKLS LEIELLELQR DKARKSSVLR QL (SEQ ID N0: 34)
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In AVD277, the extracellular M2 peptide was fused to the human
C4bp scaffold.
The amino acid sequence of AVD277 was as follows:
SLLTEVETPT RNEWGCRCND SSDGSETPEG CEQVLTGKRL MQCLPNPEDV
KMALEVYKLS LEIEQLELQR DSARQSTLDK EL (SEQ ID NO: 35)
In AVD278, a variant of the extracellular M2 peptide in which
both cysteines were replaced by serine residues was fused to
the human C4bp scaffold.
The amino acid sequence of AVD278 was as follows:
SLLTEVETPI RNEWGSRSND SSDGSETPEG CEQVLTGKRL MQCLPNPEDV
KMALEVYKLS LEIEQLELQR DSARQSTLDK EL (SEQ ID N0: 36)
Mice immunised three times subcutaneously with the AVD275
protein (2 nanomoles) without the addition of any adjuvant at
two-weekly intervals (i.e. on days 0, 14, and 28) show
appreciable antibody titres (as determined essentially as
described by Neirynck et al., op.cit.) and significant
protection against a lethal challenge with 5 LD50 doses of the
mouse adapted A/PR/8/34 strain.
