METHOD FOR THE PURIFICATION OF PROTEIN COMPLEXES
Field of the lnvention
The present invention relates to a novel methodology for the purification of protein
5 complexes. In particular, there is provided a method for the purification of protein
complexes comprising heat shock proteins coupled to peptide fragments. The
invention further extends to the use of the purified protein complexes in the
preparation of vaccine compositions and in the use of the same for the prevention
and treatment of infectious diseases and cancer.
10
Background of the lnvention
Vaccination is widely accepted as the favoured approach to tackle the global
healthcare burden of infectious disease and cancer. However, despite significant
advances in our understanding of the molecular biology relating to infectious
15 disease and cancers, the development of effective vaccines in these areas has
been limited. The most effective vaccines developed use live, attenuated
organisms, however, the safety risk associated with such attenuated pathogens
reverting to virulence has restricted their widespread use. A further major barrier
preventing the wide scale development and use of more effective vaccines is the
20 limited ability to identify candidate pathogen derived proteins that will elicit broad
protective immunity in a specific manner against variant strains of microbial
pathogens.
One particular approach that shows the promise of conferring broad, protective
25 immunity is the use of stress protein complexes as vaccines against infectious
disease and cancer (Colaco et al., (2004) Biochem Soc Trans 32:626-628 and
Zeng et al., (2006) Cancer lmmunol lmmunother 55:329-338). It has also been
widely documented that heat shock proteinlantigenic peptide complexes are
efficacious as vaccines against specific cancers (US Patent No. 5,997,873; US
30 Patent No. 5,935,576, US Patent No. 5,750,119, US Patent No 5,961,979 and US
Patent No. 5,837,251). It has been shown that pathogen derived stress protein
complexes isolated from heat-shocked BCG cells induced T-helper 1 (Thl)
lymphocyte mediated immune responses in a vaccinated host, which conferred
protective immunity against a live challenge in a murine aerosol challenge model
of pulmonary tuberculosis (International PCT Patent Application No. WO
0111 3944). Moreover, it has been shown in WO 02120045, WO 0011 0597 and WO
5 0111 3943 that stress protein complexes isolated from pathogens or pathogen
infected cells are effective as the immunogenic determinant within vaccines
against infectious diseases.
Heat shock proteins (hsps, HSPs) form a family of highly conserved proteins that
10 are widely distributed throughout the plant and animal kingdoms. On the basis of
their molecular weight, the major heat shock proteins are grouped into six different
families: small (hsp20-30kDa); hsp40; hsp60; hsp70; hsp90; and hsp100.
Although heat shock proteins were originally identified in cells subjected to heat
stress, they have been found to be associated with many other forms of stress,
15 such as infection, osmotic stress, cytokine stress and the like. Accordingly, heat
shock proteins are also commonly referred to as stress proteins (SPs) on the basis
that their expression is not solely caused by a heat stress. Members of the hsp60
family include the major chaperone GroEL. These form multimeric complexes with
co-chaperones such as GroES. Many microbial pathogens have additional hsp60
20 families that form distinct complexes from GroEL and some hsp60 family members
may be more immunogenic, such as the hsp65 of mycobacteria. Members of the
hsp70 family include DnaK which can form multimeric complexes with cochaperones
such as DnaJ. Other major hsps include the AAA ATPases, the Clp
proteins, Trigger factor, Hip, HtpG, NAC, Clp, GrpE, SecB and prefoldin.
25
Stress proteins are ubiquitously expressed in both prokaryotic and eukaryotic
cells, where they function as chaperones in the folding and unfolding of
polypeptides. A further role of stress proteins is to chaperone peptides from one
cellular compartment to another and, in the case of diseased cells, stress proteins
30 are also known to chaperone viral or tumour-associated peptides to the cellsurface.
The chaperone function of stress proteins is accomplished through the formation
of complexes between stress proteins and the chaperoned polypeptide.
Chaperoned polypeptides may include peptide fragments, with the formation of
such complexes controlled by an ATP-dependent nucleotide exchange system,
5 which has been most clearly demonstrated for the bacterial Hsp70 homologue,
DnaK (Szabo et a/. PNAS (1 994) Vol 91. 10345-1 0349). Briefly, in its resting
cellular state, DnaK is bound to ATP (adenosine triphosphate) and has a low
affinity for substrate (Palleros et a/. PNAS (1 991 ) Vol. 88. 571 9 -5723). ATP
hydrolysis results in conversion of DnaK to a high-affinity ADP (adenosine
10 diphosphate) state, resulting in the formation of DnaK-ADP-substrate complexes,
where the substrate is typically a polypeptide or protein. Following ADP
dissociation, ATP re-binds to DnaK, resulting in a conformational change that
triggers the release of the correctly folded substrate protein from the complex
(Palleros et a1 J Biol Chem (1 992) Vol 267, No 8, 5279-5285; Palleros et al. Nature
15 (1 993) 365(6447):664-6; Szabo et al. PNAS (1 994) Vol 91. 10345-1 0349). This
final step, resulting in release of substrate, has been shown to require potassium
(K') and magnesium ( ~ g ~in' )a ddition to the binding of ATP (Palleros etal. Nature
(1 993) 365(6447):664-6; Palleros et al. FEBS Letters (1 993) Vol 336, No 1, 124-
128).
20
Heterologous polypeptides or polypeptide fragments complexed with the stress
proteins form stress protein-peptide complexes, which may be referred to as heat
shock protein complexes (HspCs). HspCs are captured by antigen presenting
cells (APCs) to provide a source of antigenic peptides which can be loaded onto
25 major histocompati bility complex (MHC) molecules for cell surface presentation to
the T lymphocytes of the immune system.
Heat shock proteinlantigenic peptide fragment complexes (HspCs) have been
widely studied as cancer vaccines (see, for example US Patent No. 5,997,873 and
30 US Patent No. 5, 935,576) and methods have thus been developed for the
isolation of HspCs from tumour cells for use as effective vaccines against such
tumours. For example, WO 02128407 discloses a method for use in purifying
protein complexes based on the binding affinity of heat shock proteins for heparin.
The two step approach involves heparin affinity chromatography and a subsequent
ion exchange chromatography step which is optional in order to obtain the stress
protein complex preparations. WO 02134205 relates to the purification of HSP70
5 stress protein complexes using Con A Sepharose. These methods however result
in the isolation of individual families of heat shock proteins, and therefore neglect
the use of multiple chaperone proteins as vaccines.
The use of HspCs as cancer vaccines can be significantly improved by the use of
10 multiple chaperone proteins, in particular heat shock proteins (Bleifuss et al 2008)
and thus methods have been developed for the purification of multiple chaperone
proteins and chaperone protein complexes for use in vaccines. For exampleus
Patent No 6,875,849 discloses the use of free-solution isoelectric focusing (FSIEF)
for the purification of HspCs from tumours for use as cancer vaccines. Free
15 flow isoelectric focusing (FF-IEF) can be used to isolate heat shock proteinlpeptide
complexes from pathogens and infected cells for use as the immunogenic
determinant in vaccine compositions for the prevention and treatment of infectious
diseases. However, a key limitation of that technique has been the difficulties
associated with developing a large scale FF-IEF instrument to produce the
20 quantities of heat shock proteinlpeptide complexes (HspCs) which would be
required for large, commercial scale, GMP vaccine manufacture. Moreover, the
use of ampholytes (ampholines) to produce the pH gradient required during the
FF-IEF process results in the introduction of a further contaminant, in addition to
the chaotropes, in the resulting, purified HspC containing preparations. Such
25 contaminants, being unacceptable to Regulatory Authorities, pose a significant
barrier to the use of FF-IEF methodology in the manufacture of HspC containing
vaccine compositions. Interestingly, these inventors report the stability of HspCs
even in the presence use of the chaotropes, such as urea and detergents used
during FF-IEF purification even in the absence of divalent cations and ADP in the
30 process buffers (Bleifuss et al 2008). Additionally, the process of free-flow
isoelectric focussing is slow with a typical run time of 4 hours, during which high
levels of protein degradation result, severely limiting the use of FF-IEF in large
scale production of purified protein complexes.
Summary of the Invention
5 Following extensive experimentation, the present inventors have identified an
improved method for the purification of stress protein-peptide complexes, such as
heat shock proteinlpeptide complexes (HspCs), that can be used to purify multiple
stress protein-peptide complexes which can safely be used for vaccine
manufacture. This methodology separates the protein complexes on the basis of
10 surface charge rather than isoelectric point. In particular, a purification method
has been identified which allows for the rapid purification of a protein complex
comprising a stress protein, such as a heat shock protein, complexed to a peptide
fragment, wherein the yield of purified product is sufficient to allow the preparation
of a commercially acceptable amount of the protein complexes for use in the
15 preparation of a vaccine composition. Advantageously, the purified protein
complexes can be used in the preparation of a vaccine preparation due to the
absence of pharmaceutically unacceptable or undesirable additives or
components introduced during the purification process. Specifically, the inventors
have identified the requirement for specific buffer conditions which prevent the
20 breakdown, or partial dissociation on the stress protein complexes. The
purification method therefore advantageously reduces or ameliorates dissociation
and loss of function of the purified protein complexes when used in a vaccine
composition to elicit an immune response thereagainst, whilst concomitantly
removing the need to use chaotropes or other chemicals such as surfactants to
25 increase the solubility of the protein complexes undergoing purification. Most
surprisingly, the heat shock proteinlantigenic peptide complex (HspC) enriched
preparations (HEPs) purified using the methodology of the invention elicited
significantly enhanced immunity in a vaccinated subject, when compared to the
immunity elicited against similar complexes isolated using standard methodology
30 which did not use the buffer conditions of the present invention. The inventors
have further identified that the utility of the purified protein complexes of the
invention for use in the preparation of a vaccine preparation can be further
enhanced when the cell lysate is buffered in a buffer containing adenosine
diphosphate (ADP) and at least one divalent cation, or in certain embodiments
using only at least one divalent cation. Furthermore, the heat shock
proteinlantigenic peptide complex (HspC) enriched preparations (HEPs) purified
5 using the improved method of this invention in conjunction with a buffer comprising
at least one divalent cation and optionally adenosine diphosphate (ADP) elicited
an even greater protective immune responses in subjects to whom the complexes
were administered.
10 According to a first aspect of the present invention there is provided a method for
the purification from a source mixture of stress protein complexes formed between
a stress protein and a polypeptide, the method comprising the steps of:
(i) providing a source mixture comprising at least one target stress protein
complex comprising a stress protein complexed to a polypeptide,
(ii) determining the isolelectric point (pl) of at least one target stress protein
complex which is to be purified from the source mixture;
(iii) preparing a clarified cell lysate from the source mixture comprising the
identified target stress protein complex;
(iv) subjecting the cell lysate to purification using ion exchange, wherein the
cell lysate is buffered, using a primary buffer comprising at least one
divalent cation, to a pH within 2 units of the pl of the target stress protein
complex, and wherein a secondary buffer provides a salt gradient which is
used to elute a mixture comprising target stress protein complexes.
25 In certain embodiments, the primary buffer further comprises adenosine
diphosphate (ADP) andlor an adenosine diphosphate mimetic. In certain further
embodiments the at least one divalent cation is a magnesium salt, typically
magnesium chloride (MgC12), or a manganese salt. In yet further embodiments the
divalent cation is provided at a concentration of from about 0.1 mM to about 100
30 mM. In one embodiment, the buffer comprises only magnesium salt as the
divalent cation, typically magnesium chloride (MgCI2). In certain embodiments the
adenosine diphosphate is provided at a concentration of from about 0.1 mM to 100
mM.
In certain embodiments, the primary buffer comprises magnesium chloride (MgC12)
5 at a concentration of at least 1 mM and adenosine diphosphate (ADP) at a
concentration of at least I mM. In certain embodiments the buffer further
comprises HEPES (4-(2-hydroxyethy1)-1 -piperazineethanesulfonic acid), which
may be present at a concentration of around 50 mM. Said buffer may have a pH
of around pH 6.8.
10
In certain further embodiments, the primary buffer comprises at least one divalent
cation, but lacks at least one of adenosine diphosphate (ADP), adenosine
triphosphate (ATP), ATPase and/or potassium or a potassium salt. In one
embodiment, the buffer comprises only magnesium salt as the divalent cation,
15 typically magnesium chloride (MgC12).
In certain further embodiments, the primary buffer lacks at least one of: a
chaotrope, a surfactant and/or an ampholyte. Chaotropes (also known as
chaotropic agents, or chaotropic reagents) include urea, guanidine hydrochloride
20 and lithium percolate. Chaotropes are known to act as protein denaturants,
causing a protein to unfold and a resultant change in three dimensional structure.
Ampholytes are molecules which contain both acidic and basic groups. They exist
mostly as zwitterions (a chemical compound which has a net charge of zero) in a
certain range of pH. Surfactants may include anionic surfactants, such as SDS,
25 cationic surfactants, such as CTAC, HTAB and DTAB, non-ionic surfactants, such
as Tween 20, and zwitterionic surfactants, such as DAPS. In certain
embodiments, the primary buffer further lacks at least one of: adenosine
triphosphate (ATP), ATPase, potassium, or a potassium salt.
30 In certain embodiments, the mixture of target stress protein complexes which are
eluted are provided in a preparation which comprises a plurality of stress proteins
of different heat shock protein classes.
Accordingly, advantageously the methodology of the present invention provides a
mixture of purified stress protein complexes wherein this mixture of stress proteins
comprises different stress protein complexes. That is, the stress protein
components of the stress protein complex are a mixture derived from a plurality
5 (i.e. more than one) heat shock protein class or family, for example, there may be
a mixture of heat shock proteins derived from the classes HSPGO, HSP70 and/or
HSPSO, or from any other heat shock protein class which is present in a eukaryotic
cell, or a pathogenic cell. Specifically any stress protein complex which is present
in the cell lysate of a pathogen cell, or a cell infected with a pathogen can be
10 purified by the method of the present invention and therefore present in the final
purified product. This mixture of different heat shock proteins is significant as the
subsequent administration of the purified complexes as the immunogenic
determinant in a vaccine composition results in an enhanced immune response
being elicited against the vaccine composition by the immunised host. This
15 enhanced immune response confers improved long term protective immunity
against the pathogen against which the subject is being immunised.
Accordingly, in certain aspects, the invention extends to a method of purifying
and/or isolating multiple stress protein complexes from a cell lysate obtained from
20 a pathogenic cell, a cell infected with a pathogen, or a tumour cell. Typically the
stress protein complexes comprise stress proteins of different stress protein
classes. Said purified and/or isolated stress protein complexes, or a preparation
or mixture comprising the same, can then typically be used as the immunogenic
determinant in a vaccine composition to elicit an immune response and associated
25 protective immunity against the pathogen or tumour cell from which the lysate is
derived, or against a pathogen which is infecting a cell from which the lysate is
derived. Accordingly, such method would comprise the steps of:
(i) providing a clarified cell lysate from the source mixture comprising the
identified target stress protein complex;
30 (ii) subjecting the cell lysate to purification using ion exchange, wherein the
cell lysate is buffered, using a buffer comprising at least one divalent cation,
to a pH within 2 units of the pl of the target stress protein complex, and
wherein a salt gradient is used to elute the target stress protein complexes,
and
(iii) obtaining an enriched preparation comprising a plurality of stress protein
complexes.
In certain embodiments, the purifying and/or isolating multiple stress protein
complexes or a preparation or mixture comprising the same, comprise stress
proteins derived from different families of stress proteins, thus the purified product
contains a mixture of stress protein complexes wherein the stress proteins can be
10 derived from different stress protein families, for example, the purified mixture may
comprise a plurality of different heat shock protein types which form complexes
with peptides.
In certain further aspects, the invention extends to the use of a purified fraction or
15 mixture obtained from a purification methodology, typically ion exchange
purification, in particular ion exchange chromatography which is performed
according to a method of the present invention, or which purification methodology
uses a buffer solution according to the present invention, as the immunogenic
determinant in a vaccine composition to prepare a vaccine composition.
20
Buffer solutions
In certain further aspects, the invention extends to a buffer solution for use in
performing a protein purification method, in particular ion exchange, such as ion
exchange chromatography, wherein the purification methodology is based upon
25 the purification, isolation and/or separation of a proteinaceous mixture derived
from a cell or cell culture in order to isolate and/or purify a plurality of stress protein
complexes therefrom, where the buffer solution comprises at least one divalent
cation. In certain embodiments the at least one divalent cation is a magnesium
salt, typically magnesium chloride. Further suitable divalent cations are disclosed
30 herein. In certain embodiments, the buffer further comprises adenosine
diphosphate (ADP). In certain embodiments, the buffer lacks at least one of, or all
of: adenosine triphosphate (ATP), ATPase, potassium, or a potassium salt,
chaotropes, ampholytes and surfactants.
Vaccine compositions
5 In various further aspects, the invention extends to vaccine compositions, or to
compositions which mediate or elicit and immune response, which comprise the
purified HspC-enriched lysates or purified and/or isolated multiple stress protein
complexes or a preparation or mixture comprising the same, which are obtained
by the methods of the invention. Said vaccine compositions are typically
10 administered to mammals, in particular humans in order to confer protective
immunity against a pathogen. However, due to the acknowledged high level of
homology between stress proteins from different species, vaccine compositions
may be used to vaccinate a wide variety of animals.
15 As such, a further aspect of the invention provides a vaccine composition
comprising, as the immunogenic determinant, a purified HspC-enriched eluate
fraction or lysate obtained by the present invention. In a preferred embodiment,
the invention provides a vaccine composition comprising a purified stress proteinpolypeptide
complex (HspC)-enriched eluate fraction or lysate, which is obtained
20 by the purification method of the present invention, wherein the lysate is buffered
in a buffer comprising at least one divalent cation. The buffer may further
comprise adenosine diphosphate. In certain embodiments, the buffer lacks ATP
and/or potassium.
25 A yet further aspect provides a vaccine composition for use in elicting an immune
response in a subject, wherein the immunogenic determinant is purified and/or
isolated multiple stress protein complexes or a preparation or mixture comprising
the same, obtained using the purification method of the present invention.
Specifically, the purification method of the invention comprises isoelectric
30 chromatography which uses the buffer solution described herein to improve the
stability and therefore the antigenicity of the stress protein complexes purified from
a source proteinaceous mixture.
In certain embodiments, the purified multiple stress protein complexes may be
isolated, such that the vaccine composition comprises, as the immunogenic
determinant, purified and isolated complexes as the immunogenic determinant of
5 the vaccine composition.
In certain further aspects the present invention provides the use of a vaccine
composition according to the invention, or of a purified andlor isolated mixture of
stress proteinlpeptide complexes obtained using the methodology of the invention,
10 for use in medicine.
In certain further aspects the present invention provides for the use of the purified
mixture of stress protein-peptide complexes or of a preparation comprising the
same, in the preparation of a medicament for the treatment of an infectious
15 disease or a cancerous or a malignant condition.
In certain further aspects, the present invention provides a plurality of stress
protein-peptide complexes or of a preparation or mixture comprising the same,
which is purified by the method of the first aspect of the present invention for use
20 in a vaccine composition for the treatment or prevention of an infectious disease or
a cancerous or a malignant condition.
In certain embodiments, the purified and isolated stress protein complexes or the
vaccine compositions containing the same are administered as a prophylactic
25 vaccine. In certain further embodiments, the purified stress protein complexes,
preparations comprising the same, or the vaccine compositions containing the
same are administered as a therapeutic vaccine.
In various further aspects, the present invention extends to the use of the purified
30 and isolated stress protein complexes, or to preparations or mixtures comprising
the same, or to vaccine compositions containing the same, as a booster vaccine to
enhance the immune response generated in a host against a pathogen or cancer
antigen to which the subject has previously been exposed to, typically by way of
infection or due to the previous administration of a primary vaccine.
Compositions of the invention may be lyophilised or in aqueous form, i.e. solutions
5 or suspensions. Liquid formulations of this type allow the compositions to be
administered direct from their packaged form, without the need for reconstitution in
an aqueous medium, and are thus ideal for injection. Compositions may be
presented in vials, or they may be presented in ready filled syringes. The syringes
may be supplied with or without needles. A syringe will include a single dose of the
10 composition, whereas a vial may include a single dose or multiple doses (e.g. 2
doses).
In certain embodiments, a vaccine composition according to the invention is
formulated for in vivo administration to a subject, such that they confer an antibody
15 titre superior to the criterion for seroprotection for each antigenic component for an
acceptable percentage of human subjects. This is an important test in the
assessment of a vaccine's efficacy throughout the population. Antigens with an
associated antibody titre above which a host is considered to be seroconverted
against the antigen are well known, and such titres are published by organisations
20 such as WHO. In one embodiment, more than 80% of a statistically significant
sample of subjects is seroconverted, in another embodiment more than 90% of a
statistically significant sample of subjects is seroconverted, in a further
embodiment more than 93% of a statistically significant sample of subjects is
seroconverted and in yet another embodiment 96-1 00% of a statistically significant
25 sample of subjects is seroconverted. The amount of antigen in each vaccine dose
is selected as an amount which induces an immunoprotective response without
significant, adverse side effects in typical vaccines. Such amount will vary
depending on which specific immunogens are employed. In certain embodiments,
the vaccine composition may also elicit a Thl lymphocyte cell mediated immune
30 response. Such an immune response is desirable when protecting a subject
against an intracellular pathogen. In certain embodiments, vaccine compositions
comprising the purified stress protein complexes of the invention elicit an immune
response in a host which comprises both a cell mediated and humoral (antibody
mediated) immune response.
In various further aspects, the present invention provides a method for producing a
5 vaccine composition comprising the step of mixing the purified stress protein
complexes or a preparation or mixture comprising the same, of the invention
together with at least one pharmaceutically acceptable excipient, carrier or diluent.
In one embodiment of the present invention there is provided a vaccine
composition for use in a medicament for the treatment or prevention of a
10 pathogenic disease, such as that caused by infection by a pathogenic bacteria
selected from the group comprising, but not limited to: Bordetella pertussis,
Clostridium tetani, Clostridium difficile, Corynebacterium diphtheriae,
Haemophilus influenzae b, Mycobacterium tuberculosis and leprae, Salmonella
typhi, Streptococcus pneumonia Vibrio Cholerae and Neisseria meningitides. In a
1 5 further embodiment of the present invention there is provided a vaccine
composition for use in a medicament for the treatment or prevention of a
pathogenic disease, such as that caused by infection by a pathogenic or
oncogenic virus selected from the group comprising, but not limited to: Influenza,
Hepatitis, Herpes, HIV, HPV, RSV, Polyoma, CMV, EBV, Rotovirus, Norovirus
20 and SARS. In a yet further embodiment of the present invention there is provided
a vaccine composition for use in a medicament for the treatment or prevention of
cancer and neoplastic disease.
Additionally, a method of immunising a subject, typically a human, against disease
25 caused Bordetella pertussis, Clostridium tetani, Clostridium difficile,
Corynebacterium diphtheriae, Haemophilus influenzae type b, Mycobacterium
tuberculosis and leprae, Salmonella typhi, Vibrio Cholerae, Streptococcus
pneumonia, Neisseria meningitidis and pathogenic and oncogenic viruses, which
method comprises administering to the host an immunoprotective dose of the
30 vaccine of the invention is further provided.
The amount of antigen (i.e. the immunogenic determinant) in each vaccine dose is
selected as an amount which induces an immunoprotective response in the
vaccinated subject without significant adverse side effects. The amount of antigen
will vary depending upon which specific immunogen is employed and how it is
5 presented, however, it will be understood that the enhanced immune response
mediated against the purified complexes of the invention will mean that an
enhanced immune response will be mediated against an amount of complexes
purified using the present methodology, when compared to a vaccine composition
comprising a similar amount of protein complexes obtained using the purification
10 methods known in the art.
The invention further provides for the use of the purified HspC-enriched lysates, or
isolated stress proteins derived therefrom in a method of vaccinating a subject to
induce immunity against a pathogen derived infectious disease or cancerous or
15 malignant condition.
Accordingly a yet further aspect of the invention provides for a method of
vaccinating a subject against a pathogen derived infectious disease or a
cancerous condition, said method comprising the steps of:
20 - providing a vaccine composition comprising, as the immunogenic
determinant, a purified stress protein complex enriched preparation
obtained according to the method of the present invention, said
purified stress protein-enriched preparation being derived from a
cancerous cell, a pathogen, or a cell infected with a pathogen
against which protective immunity is desired, and comprising
different stress protein types as a mixture within the purified
preparation, and
- administering a vaccine composition comprising the stress protein
complex-enriched preparation to a subject in a therapeutically
effective or prophylactically effective amount sufficient to elicit an
immune response in the subject against the stress protein complexenriched
preparation.
As used herein, the term "vaccine composition" means any composition containing
an immunogenic determinant which stimulates the immune system in a manner
5 such that it can better respond to subsequent challenges, pathogenic infections or
oncogenesis. It will be appreciated that a vaccine usually contains an
immunogenic determinant and optionally an adjuvant, the adjuvant serving to nonspecifically
enhance the immune response to the immunogenic determinant.
10 In certain embodiments, the subject is an animal, typically a human. The methods
of the invention can also be used to purify stress protein complexes for use in a
vaccine composition for the treatment of other animals such as horses, cattle,
goats, sheep, swine and birds.
15 In certain embodiments, the microbial pathogen from which the purified stress
protein complexes (HspC-enriched preparations) of the invention are derived, may
be selected on the grounds that it causes disease or infection. The vaccine
compositions provided by the invention may be used either prophylactically or
therapeutically. The inventors however recognise that the compositions may be
20 particularly useful as prophylactic vaccines due to their economy of production and
their ability to elicit a protective immune response against the pathogen from which
the peptide, or the peptide and stress protein is derived.
The inventors have further surprisingly identified that stress protein-peptide
25 complexes which are obtained using the methods of the invention can be used as
"booster" vaccinations, said booster vaccinations enhancing the immunity provided
in a subject against a pathogen or a cancerous condition, wherein the initial
immunity was conferred by vaccination with a live or attenuated vaccine, or by a
vaccine composition wherein the immunogenic determinant was a stress protein-
30 peptide complex.
Accordingly, a yet further aspect of the invention provides for a method of boosting
a protective immune response in a subject against a pathogen derived infectious
disease or a cancerous condition, wherein said protective immune response has
been elicited by the previous administration of a live or attenuated vaccine or of a
5 stress protein-peptide complex comprising a peptide derived from the pathogen
against which immunity is desired, said method comprising the steps of:
- providing a composition comprising a stress proteinlpeptide
complex-enriched preparation obtained according to the method of
the present invention, said purified stress proteinlpeptide complexenriched
preparation being derived from a cancerous cell, a
pathogen infected cell or a pathogen against which protective
immunity is desired and comprising different stress protein types as
a mixture within the purified preparation, and
administering a composition comprising the stress proteinlpeptide complex-
15 enriched preparation to a subject in an amount sufficient to elicit an immune
response in the subject against the stress proteinlpeptide complex-enriched
preparation.
In certain further embodiments, the stress proteinlpeptide complex containing
20 vaccines of the present invention may be used for the boosting of immune
responses in animals that have been previously immunised with other subunit,
multi-subunit, carbohydrate or conjugate vaccines. In yet further embodiments,
the stress proteinlpeptide complex vaccines of the present invention can be used
to boost the immune responses against a target antigen in animals that have been
25 previously immunised with nucleic acid or live vaccines. In yet further
embodiments, the stress proteinlpeptide complex containing vaccine compositions
of the present invention provide for the boosting of immune responses mediated in
subjects that have been previously immunised against a pathogen or cancer
specific antigen.
30
In certain further aspects, the present invention extends to vaccine compositions
comprising the stress protein-peptide complexes purified by the invention for use
in the boosting of immune responses in animals, wherein the animal has
previously been vaccinated with a vaccine composition comprising at least one
pathogen derived antigen, a pathogen, in particular an attenuated pathogen, or a
cancer specific antigen. Typically the peptide component is derived from the same
5 pathogen or cancerous cell, as that which provided the immunogenic determinant
for the initial vaccination.
In certain yet further aspects, the present invention extends to vaccine
compositions comprising the stress protein-peptide complexes purified by the
10 invention for use in the boosting of immune responses in animals, wherein the
animal has previously been exposed to a pathogen or cancer expressing antigens
that are present in the stress protein complexes.
In certain further embodiments, the present invention provides compositions for
15 the preparation of cellular vaccines such as dendritic cells (DCs) which have been
pulsed with the purified stress proteinlpeptide complex-enriched preparations of
the invention. Administration of such pulsed dendritic cells to subject will result in
a T-cell mediated response being directed against the stress proteinlantigenic cell
complex. Such a therapy can be particularly effective when treating a subject with
20 a cancerous or malignant condition. In such embodiments, typically the stress
proteinlpeptide complex is derived from a cancerous cell.
In certain embodiments, the vaccine composition of the invention may be replaced
with a composition for inducing an immune response, or by a composition for
25 eliciting an immune response, said compositions typically comprising the same
immunogenic determinants as those provide in the vaccine compositions
described herein.
Brief Description of the Figures
Figure 1 shows the SDS-PAGE analysis of the purification of protein
complexes from BCG bacterial cell lysates (A) and the analysis of these
samples for the Hsp71 and Hsp65 proteins by Western blotting (B). V1 is
the parent high speed spin lysate from BCG which was processed further to
yield V2, HspCs purified using conventional isoelectric focussing methods,
and V3, HEPs purified using the first method of this invention.
Figure 2 shows the immunogenicity of the HEPs isolated from BCG (IEX)
using the first method of this invention, compared to the parent lysate (LSS)
and HspCs isolated using conventional isoelectric focussing methods (IEF).
Figure 3 shows SDS-PAGE analysis of the purification of protein complexes
from Neisseria meningitidis, eluted by a step salt gradient (A) and the
analysis of these samples for the Hsp70, Hsp65 and PorA proteins by
Western blotting (B).
Figure 4 shows the immunogenicity of the HEPs isolated from Neisseria
meningitidis using the isoelectric chromatography based purification method
of the present invention (IEC HspC) compared to the HEPs isolated from
Neisseria meningitidis using conventional isoelectric focussing methods
(IEF HspC). Though both HspCs showed significantly better opsonisation
activity against heterologous strains than the current outer membrane
vesicle vaccine (H44176 OMV), the IEC HspCs showed better cross-strain
immunogenicity than the IEF HspCs.
Figure 5A shows Western blot analysis of HEPs from BCG bacterial cell
lysates purified in the presence of ADP + a divalent cation. Lane 1 shows
Coomasie blue stained SDS-PAGE, while lanes 2 to 4 show recognition of
Hsp65, Hsp71 and Ag85, respectively, by Western blot. Figure 5B shows
Coomaise blue stained SDS-PAGE of HEPs from BCG bacterial cell lysates
purified in the presence of divalent cation (BCG 001/09), plus ADP (HspC
Vac) or ATP (BCG 002109).
Figure 6 shows the immunogenicity of the HEPs isolated from BCG. Figure
6A shows the cell mediated immunity induced by HEP vaccines isolated
from BCG bacterial cell lysates either in the absence (IEC (2 vaccs) or
presence of ADP + a divalent cation (IEC (2vaccs) +ADP/M~~c')o mpared
to vaccination with live BCG (BCG Wk 8) and BCG primed with a HspC
boost (BCGIIEC). Figure 6B shows the Thl biased humoral immunity
induced by HEP vaccines isolated from BCG bacterial cell lysates either in
the presence of ADP and a divalent cation (VI), ATP and a divalent cation
(V2) or divalent cation alone (V3) and significant enhancement of the
antibody response to the current live BCG vaccine (BCG) by boosting with
the HspC vaccine (BCG and VI).
Figure 7A shows the reduction of lung colony counts in HEP- immunised
animals challenged with live TB. Animals were immunised with saline as a
negative control, live BCG bacteria (BCG), HEPs isolated from BCG
bacterial cell lysates either in the absence (IEC) or presence of ADP + a
divalent cation (IEC), or primed with live BGC and boosted with HEPs
isolated by the improved method of the invention (BCG +IEC ).
Figure 7B shows the reduction of lung colony counts in animals immunised
or boosted with HEPs and challenged with live TB. Animals were
immunised with saline as a negative control, live BCG vaccine (BCG),
HEPs isolated from BCG bacterial cell lysates in presence of ADP plus
divalent cation (VI), divalent cation alone (V3) or divalent cation plus ATP
to disrupt the HspC complex (V2), or primed with BCG and boosted with the
HEP vaccine (BCG + VI).
Figure 8 shows Coomassie Blue stained SDS-PAGE (lanes 1,4) and
Western blot analysis of Hsp6O and Hsp70 in HEPs purified from Neisseria
meningitidis either in the absence (lanes 1 to 3) or presence (lanes 4 to 6)
of ADP and a divalent cation.
Figure 9 shows Coomasie Blue stained SDS-PAGE analysis of the
purification of HEPs from CHO cells, eluted by a step salt gradient (A) and
the analysis of these samples for the Hsp70 (B) and Hsp6O (C) proteins by
Western blotting. Lane 1 : Mw markers, lane 2: flow through, lane 3: wash,
lane 4: 150mM elution, lane 5:150 mM elution, lane 6: 250mM elution, lane
7: 250 mM elution, lane 8: 350 mM elution, lane 9: 350 mM, lane 10: 500
mM elution, lane 11 : 500 mM elution, lane 12: 1 M elution.
Detailed Description of the Invention
The present invention provides an improved method for the purification of a
mixture of complexes comprising a stress protein complexed to a peptide or
peptide fragment from a source mixture, typically a cell lysate. The improved
10 method of the invention provides for protein complexes to be purified using ion
exchange based methods, without the need to use chemicals such as chaotropes,
surfactants and ampholytes (ampholines) in the purification methodology. Such a
method is advantageous as the presence of extraneous ingredients in
pharmaceutical preparations is generally undesirable because it causes instability
15 and dissociation of the stress proteinlpeptide complexes which can result in the
stress protein complexes being less immunogenic. Hence, the purified complexes
obtained using the method of the invention can be used to produce improved
vaccine preparations which elicit enhanced immune responses in the subjects to
whom the vaccine compositions are administered. Furthermore, ATP is known to
20 cause the dissociation of stress proteinlpeptide complexes. However, in the
absence of ATP, HspCs are generally regarded to be very stable even when
chaotropic agents such as detergents and 7M Urea are used in their purifications.
The inventors have surprisingly identified the requirement for stabilisation of the
stress protein complexes during purification to yield HspCs that are more
25 immunogenic than similar complexes obtained using purification techniques such
as traditional isoelectric focusing (IEF). The inventors have provided an improved
ion exchange chromatography methodology which not only prevents the
dissociation of the stress protein complexes, due to the presence of at least one
divalent cation in the buffer, such as a magnesium salt, but also optionally
30 adenosine diphosphate, which is present in the buffer at a concentration which
minimises the dissociation of stress protein complexes. The presence of stable
stress protein complexes, as well as the provision of a mixture of stress proteins of
different types provides a mixture which can be used as the immunogenic
determinant in a vaccine composition and which will elicit an enhanced immune
response, over that elicited by a stress protein complex purified using methods
which are known in the art.
5
The method of the invention is further advantageous in that it provides a
purification method which has a greater capacity than previous used protein
purification techniques, such as isoelectric focusing, for the purification of stress
protein complexes, such that the large scale, commercially viable, cost effective
10 production of vaccines containing such complexes can be achieved.
Furthermore, the inventors have surprisingly identified that stress protein
complexes which are purified using the methods of the invention are more
immunogenic than similar complexes obtained using purification techniques such
15 as traditional isoelectric focusing (IEF). The improved method of the invention
provides for protein complexes to be purified using specific buffer compositions
and conditions that yield HEPs that show enhanced immunity. Hence, the HspC
complexes obtained by the methods of the invention are more immunogenic than
those purified using standard purification methods known in the art and can be
20 used to produce improved vaccine preparations.
Source mixture
Typically the stress protein complexes of the invention are purified or isolated from
a source mixture. In certain embodiments, the source mixture is a mixture which
25 comprises at least one stress proteinlpeptide fragment complex (for which
purification is desired) and one or more contaminants. Non-limiting examples of
contaminants present in the source mixture may include: host cell proteins other
than stress proteins or stress protein complexes, host cell metabolites, host cell
constitutive proteins, nucleic acids, endotoxins, chemical product related
30 contaminants, lipids, media additives and media derivatives.
In certain embodiments, the source mixture is a proteinaceous mixture, or is
derived from a cell lysate, or a cell homogenate. In certain embodiments, the cell
lysate or homogenate is derived from a prokaryotic cell, typically a pathogenic
prokaryotic cell, wherein said prokaryotic cell may be an intracellular or
5 extracellular pathogenic bacteria. In certain further embodiments, the cell lysate
may be derived from a cell infected with a prokaryotic cell. In further
embodiments, the cell lysate or homogenate is derived from a eukaryotic cell, such
as a eukaryotic cell infected with a pathogen, for example a prokaryotic pathogen.
In certain embodiments, the cell lysate or homogenate is derived from a tumour
10 cell, a cancerous cell mass or tissue, or a cell derived from a biopsy. In certain
embodiments, the cells are cells derived from cell culture where the cells are
transformed or transfected. In certain further embodiments, the cell lysate or
homogenate can be obtained directly from a host cell or pathogenic organism, or
from a cell infected by a pathogenic organism. The pathogenic organism may be
15 selected from the group consisting of, but not limited to: (i) a virus, for example,
influenza, papilloma virus, herpes simplex virus, hepatitis virus (type A, B or C),
HIV, measles and the like, (ii) an intracellular protozoa, such as a trypanosome; or
(iii) a cell infected with a prokaryote, in particular an intracellular bacteria, such as
a bacteria of the species Mycobacteria or Neisseria. Further examples of source
20 mixtures that can be purified using the method of the present invention include
harvested cell culture fluid, cell culture supernatant and conditioned cell culture
supernatant. Furthermore, the cell lysate can be derived from a tumour cell
In embodiments where the source mixture is derived from, or comprised of a cell
25 lysate, the lysate may be obtained by any suitable means known to the person
skilled in the art, including, but not limited to: (i) mechanical means, such as
sonication, cavitation, freeze-thaw cycles, the use of a cell homogeniser such as a
French press, Dounce homogeniser or motor driven glass/TEFLON homogenizer;
(ii) cell lysis using a detergent; or (iii) osmotic lysis by bringing the cells into
30 contact with a hypotonic buffer or hypertonic buffer as required. In certain
embodiments where cell lysis is used to produce the source mixture, proteinase
inhibitors may further be added to the source mixture.
In certain embodiments, where the source mixture is derived from a homogenised
cell preparation, such as a cell lysate or a tissue sample, the homogenate may be
centrifuged, at least once, for example at 10,000g for 30 minutes. The
5 supernatant can then be collected and subjected to further centrifugation, or be
prepared for purification using the ion exchange based methodology described
herein. In certain further embodiments the centrifugation step may be replaced or
complimented by a filtration step.
10 In certain embodiments, the source mixture is a proteinaceous mixture of proteins,
typically a solution comprised of a plurality of proteins. In certain further
embodiments, the source mixture is a cell lysate derived from cancerous cells,
pathogenic organisms, cells infected with pathogenic organisms, or cell cultures
comprising pathogenic organisms, or cells infected therewith.
15
In certain embodiments, the method of the invention may be used to extract, purify
andlor obtain a protein complex from a natural or biosynthetic source. In certain
further embodiments, the method may be used to purify a synthetic or recombinant
stress protein complex from a cell culture or other protein mixture.
20
In certain embodiments, the purified complex is present within at least one
fraction, such as an eluate fraction. Typically the at least one fraction comprises
one or more stress proteinlpeptide complex. Said fraction may be referred to as a
purified product or purification product, and may further be called a heat shock
25 proteinlantigenic peptide complex (HspC) enriched preparation (HEP).
Without wishing to be bound by theory, the inventors have identified that an
enhanced immune response can be elicited in a subject who is administered a
vaccine composition which comprises, as the immunogenic determinant, stress
30 proteinlantigenic peptide fragment complex(es) which are derived from a
cancerous cell, a pathogenic cell, a cell infected by a pathogenic organism, or a
prokaryotic or eukaryotic cell which has been genetically modified such that it
expresses a heterologous protein which is derived from a cancerous cell or a
pathogen which causes an infectious disease in a host, wherein the heterologous
protein causes an immune response to be mounted there-against when
administered to a subject. As such, in certain embodiments, the purified product
5 typically comprises a mixture of antigenic peptidelheat shock protein complexes.
Stress protein
In certain embodiments, the stress protein complex can be a heat shock protein
complex (HspC) comprising a heat shock protein which is complexed to a peptide
10 fragment.
In certain embodiments, the heat shock protein can be any suitable heat shock
protein which is derived from the cell lysate which is to be purified. In certain
embodiments, the heat shock protein may be selected from any one of the families
15 of the group comprising, but not limited to: hsp20-30kD; hsp40; hsp60; hsp70;
hsp90; and hsp100. In certain further embodiments, the stress protein may be a
protein which is classed as a chaperone protein. Such a protein may include, but
is not limited to proteins selected from the group consisting of: DnaK, DnaJ,
GroEL, GroES, hspX, acr2, AAA+, clpAIB, HtpG, TRIC, CCT, IbpA, IbpB,
20 calrecticulin, hsp40, hsp70, hsp72, hsp90, grp94, grp75, BiPlgrp78, grp75/mt,
gp96 and small hsps.
In certain embodiments, the target stress protein complex comprises a heat shock
proteinlantigenic peptide fragment complex derived from a host cell which has
25 been genetically modified to constitutively express stress protein genes, andlor
express a heterologous protein, such as an antigenic peptide or peptide fragment.
In certain further embodiments, the cell may be a host cell expressing a
heterologous gene, for example an insect cell infected with a baculovirus vector
construct comprising an antigenic gene of interest. In yet further embodiments,
30 the cell may be a cancerous cell derived from a human or animal subject.
In certain embodiments, where a mixture of complexes is provided, this may
comprise heat shock proteins of one particular family, for example, the hsp70 or
hsp60 families, although it is preferred that the mixture comprises different heat
shock protein complexes derived from different families. The method of the
5 present invention would provide a method for the purification of all complexes
comprising a heat shock protein complexed to an (antigenic) peptide fragment,
irrespective of the identity, molecular weight or size of the antigenic peptide or
peptide fragment.
10 In certain further embodiments, the heat shock proteinlantigenic peptide complex
(HspC) enriched preparations (HEP) comprise heat shock proteins from different
stress protein families or classes, such as hsp60, hsp65, hsp70 and hsp90, said
families being co-purified as a mixture using the methods of the invention.
15 In certain further embodiments, the heat shock proteinlantigenic peptide complex
(HspC) enriched preparations (HEP) may be heat shock proteinlpeptide fragment
complexes of a particular molecular weight. In certain embodiments, the stress
protein complexes have a molecular weight in the range of 50KDa to SOOKDa.
20 Antigenic peptide fragment
In certain embodiments, the polypeptide which is conjoined to the stress protein to
for the stress protein complex (HspC) is a peptide fragment, that is, the peptide
fragment is a fragment of a larger polypeptide or protein. Typically the peptide is
an antigenic peptide, that is, a proinflammatory response would be mediated
25 against the polypeptide in a host to whom the peptide is administered. The
peptide or antigenic peptide should be suitable to allow a T cell (cell mediated) or
antibody mediated (humoral) immune response to be raised against it in the host
to whom the stress protein complex is administered. Typically, the polypeptide is
derived from a pathogen, or a cell infected with a pathogenic cell, against which an
30 immune response is desired. In certain further embodiments, the polypeptide is
derived from a malignant or cancerous cell, or a cell lysate containing the same,
wherein the polypeptide or peptide fragment is a tumour specific antigen.
In certain embodiments, the peptide is complexed to the stress protein in a noncovalent
manner. In certain further embodiments, the peptide is complexed to the
stress protein by means of a covalent bond.
5
In certain embodiments, the peptide fragment is an antigenic peptide fragment
derived from a pathogenic organism wherein the pathogenic organism typically
causes an infectious disease in a host. In certain embodiments, the pathogenic
cell may be a prokaryotic cell, such as a gram positive or gram negative bacteria,
10 or an intracellular or extracellular bacterial pathogen. In certain further
embodiments, the pathogen is a viral pathogen, or a peptide fragment derived
therefrom. In certain further embodiments, the pathogen may be a protozoa, a
parasite or a fungi, such as a yeast.
15 In certain embodiments, the pathogenic cell from which the antigenic peptide is
derived may be prokaryote selected from the group consisting of, but not limited to
members of the genus Escherichia, Streptococcus, Staphylococcus, Bordetella,
Corynebacterium, Mycobacterium, Neisseria, Haemophilus, Actinomycetes,
Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella, Pasturella,
20 Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus,
Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium,
Treponema, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira,
Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas,
Rickettsia, Chlamydia, Borrelia and Mycoplasma.
25
In certain embodiments, the antigenic peptide fragment may be a viral peptide.
The virus from which the peptide can be derived may be selected from the group
consisting of, but not limited to: human immunodeficiency virus (HIV), hepatitis A
virus (HAV), hepatitis B (HBV), hepatitis C (HCV), any other hepatitis-associated
30 virus, human papillomavirus (HPV) especially high-risk oncogenic human
papillomavirus types, Kaposi's Sarcoma-Associated Herpesvirus (KSHV) (also
known as Human Herpesvirus-8 (HHV-8)), Herpes Simplex virus (HSV) (any
subtype), Respiratory Syncytial Virus (RSV) and associated respiratory viruses,
Influenza viruses including avian influenza and swine influenza, particularly if
these are transmissible to humans, coronaviruses including SARS-associated
Coronavirus (SARS-CoV), rhinovirus, adenovirus, SIV, rotavirus, human papilloma
5 virus, arbovirus, measles virus, polio virus, rubella virus, mumps virus, papova
virus, cytomegalovirus, varicella-zoster virus, varicella virus, huntavirus and any
emergent virus, in particular Ebola virus, Marburg virus, West Nile virus (WNV), St
Louis Encephalitis virus (SLEV), Rift Valley Fever virus (RVFV) and other
members of the Bunyaviridae.
10
In certain embodiments where the antigenic peptide fragment is derived from a
protozoan pathogen, the protozoa may typically be an intracellular protozoan, such
as leishmania or trypanosoma.
15 In embodiments where the antigenic peptide fragment is derived from a yeast or
fungi, said fungi may be derived from a genus selected from the group comprising:
Acremonium, Alternaria, Amylomyces, Arthoderma, Aspergillus, Aureobasidium,
Blastochizomyces, Botrytis, Candida, Cladosporium, Crytococcus, Dictyostelium,
Emmonsia, Fusarium, Geomyces, Geotrichum, Microsporum, Neurospora,
20 Paecilomyces, Penicillium, Pilaira, Pityrosporum, Rhizopus, Rhodotorula,
Saccharomyces, Stachybotrys, Trichophyton, Trichoporon, or Yarrowia.
In certain embodiments, the antigenic peptide fragment may be derived from a
tumour cell. In such embodiments, typically the antigenic peptide fragment is, or is
25 a fragment of, a tumour specific antigen. In certain embodiments the tumour cell
may be derived from a cancerous or malignant condition selected from the group
including, but not limited to, Acute and Chronic Myelogenous Leukemia (AML,
CML), Follicular Non-Hodgkins lymphoma, malignant melanoma, Hairy Cell
leu kaemia, multiple myeloma, carcinoid tumours with carcinoid syndrome and liver
30 and lymph node metastases, AIDS related Kaposi's sarcoma, renal cell
carcinoma, adenocarcinoma of the large bowel, squamous cell carcinoma of the
head and neck. Furthermore, it would be well known to the person skilled in the
art that some infectious diseases can cause cancer in subjects whom they infect.
Accordingly, administration of the complexes purified according to the
methodology of this invention wherein the polypeptide is derived from an infectious
disease can be used to treat or prevent cancer. For example, a complex
5 comprising a polypeptide derived from human papillomavirus can be used to treat
or prevent cervical cancer in a suitable subject.
In certain further embodiments, the antigenic peptide fragment which is complexed
to the stress protein is a heterologous protein or peptide fragment which is
10 expressed in a host cell by recombinant means, for example, by introduction into
the host cell of a vector or similar construct. In certain embodiments, the
heterologous antigen may be derived from a bacterial pathogen, a viral pathogen
or is a tumour specific antigen.
15 In certain further embodiments, the host cell may be a eukaryotic cell which is
infected by an intracellular pathogen. In such embodiments, the stress protein
complex may comprise a heat shock protein derived from the host cell complexed
to an antigenic peptide fragment derived from the intracellular pathogen, or a
stress protein and peptide fragment which are both derived from the intracellular
20 pathogen.
In certain embodiments of the invention, the source mixture is a cell lysate which is
produced from a cell population which has been exposed to a stress inducing
stimuli which is suitable to cause the induced (as opposed to the constitutive)
25 expression of stress proteins, typically heat shock proteins. In certain
embodiments, the stress inducing stimuli is selected from the group comprising,
but not limited to: heat shock, osmotic shock, pressure and nutrient deprivation. In
certain other embodiments, the stress induction is achieved by the genetic
modification of a cell to cause the constitutive expression of a heat shock protein
30 gene. In one such embodiment the genetic modification is the inactivation of
repressor genes that suppress the expression of stress proteins such as the hspR
and HrcA repressor genes in microbial pathogens. Other such genetic
modifications are described in WO 20021020045 and citations referred to therein.
For the non-genetic induction of stress proteins, the optimum conditions for
5 inducing the stress proteins can readily be determined by simple trial and error
with the effect of a change of stress stimuli being assessed with regard to levels of
stress protein production using conventional techniques, such as those described
in Current Protocols in Immunology, Wiley Interscience, 1997. Other such
conditions are described in WO 20011013944 and the citations referred to therein.
10
In one embodiment, at least one heat shock proteinlantigenic peptide complex
(HspC) enriched preparations (HEP) which is purified using the methods of the
present invention comprise heat shock proteinlantigenic peptide fragment
complexes (HspCs), which include, but which are not limited to hspC65, hspC70,
15 hspC9O and hspC100.
Ion Exchange conditions
The method of the invention is based on separating proteins using a methodology
based on ion exchange chromatography. However, the presently described
20 method has been improved over standard ion exchange chromatography protocols
and procedures currently used in the art, through the removal of chemicals, which
are typically present in the buffer solution, which may adversely affect protein
structure and integrity, which may cause the dissociation or partial dissociation of
protein complexes, or which may result in contaminants being present in the
25 purified fractions. The method therefore employs a modified ion exchange
methodology to produce an enriched or purified preparation comprising stress
protein complexes, which may also be referred to as an HEP (HspC enriched
preparation).
30 Ion exchange chromatography (IEC) relies on charge-charge interactions between
proteins present in the sample mixture (cell lysate) and the charges immobilized
on the resin or matrix used. Ion exchange chromatography may take the form of
cation exchange chromatography, in which positively charged ions bind to a
negatively charged resin, or anion exchange chromatography, in which the protein
binding ions have a negative charge, and the immobilized functional matrix or
resin has a positive charge. Once the protein present in the sample mixture is
5 bound to the resin, the column is washed to equilibrate this with a starting buffer.
Typically this buffer is of low ionic strength. In the methods of the present
invention, the inventors employ the use of a buffer which comprises at least one
divalent cation, such as magnesium or manganese, which is typically provided in a
salt form, such as magnesium chloride. The buffer may further comprise
10 adenosine diphosphate, which is present in the buffer at a level which will
significantly inhibit the dissociation of the stress proteinlpeptide complexes. Such
a buffer has been identified by the inventors as protecting the structural stability of
the stress protein complexes, as the buffer prevents the dissociation of the stress
protein-peptide complexes during the purification process. The method of the
15 present invention provides that the bound stress protein complexes are then
eluted off in fractions by changing the salt gradient present in the column, typically
through the use of a second buffer solution, such as sodium chloride (NaCI) or a
sodium chloride based solution. The eluted fractions are collected, with the
fractions eluted at different pls (isoelectric points) containing different stress
20 protein complexes. As the pl of the desired protein complexes has been identified,
the eluted fraction containing the complexes of interest will be readily identifiable.
Typically the methodology of the present invention will result in a purified (or
isolated) mixture of stress protein complexes. Unlike traditional ion exchange
chromatography based purification methods which result in the recovery of stress
25 protein complexes of one family (such as HSP70), the methodology of the present
invention provides a purified product (which may also be referred to as an isolated
product wherein a mixture of different stress proteins is present. For example, if
the cell lysate is derived from a eukaryotic cell infected with a pathogen, then the
resulting purified product may comprise stress protein complexes, wherein the
30 stress protein is at least 2 of HSPGO or HSP70. Any other heat shock protein
found within the cell lysate may also be present in the purified product.
Furthermore, if during the preparation of the cell lysate, the pathogen which is
infecting the eukaryotic cell is also lysed, then the methods of the present
invention further allow for pathogen derived stress proteinlpeptide complexes to be
present in the purified product. For example, the purified product may comprise a
mixture of stress protein complexes where the stress proteins may be selected
5 from the group comprising: HSP40, HSPGO, HSP70, HSP84, HSPSO, Dna-K and
Dna-J.
The isoelectric point (pl) is the pH at which a particular molecule, such as the
protein complexes of the invention, has no net electrical charge. The pl value of a
10 protein or protein complex can be determined from its primary sequence, or
empirically using conventional isoelectric focussing techniques and commercially
available equipment. The pl value of the protein complex can be used to affect the
solubility of the protein at a particular pH. Protein molecules contain both acidic
and basic functional groups. Further, amino acids may be positive, negative or
15 neutral in charge. These factors give a protein its overall charge. At a pH lower
than their pl, proteins carry a net positive charge. At a pH above their pl, proteins
carry a net negative charge. Proteins have minimum solubility in salt solutions at
the pH which corresponds to their pl. This can lead to the protein complex
precipitating out of solution. It is therefore desirable that when varying the salt
20 gradient in accordance with the method of the invention, that the pH is not lowered
from the initial pH as this may result in the protein complexes precipitating out of
solution. Typically therefore, once the pH is set, it is not increased. In certain
embodiments, the increase of the salt gradient results in an increase in pH.
25 As such, in certain embodiments, the method comprises varying the salt
concentration of a buffer solution used in the column matrix, for example by using
a buffer solution comprising sodium chloride. Typically this variation in the salt
gradient of the buffer causes the stress protein complexes to be eluted, typically in
fractions consistent with changes in the salt concentration. Accordingly, in certain
30 embodiments, the progressive addition of the elution buffer provides a salt
gradient. Typically, the elution buffer contains sodium chloride (NaCI), and the salt
gradient can be varied by varying the presence of sodium chloride (NaCI) in the
elution buffer to which the matrix is exposed. In certain embodiments, sodium
chloride may be present in the buffer at a concentration of 150mM, 250mM,
350mM, or 500mM. In certain embodiments, the elution buffer provides a pH
gradient which can be varied as the constituents of the buffer are varied. In certain
5 embodiments the elution buffer comprises at least one divalent cation, and
optionally adenosine diphosphate.
In certain embodiments, the stress protein complexes are present in fractions
collected which are eluted and which have a pl in the range of pl 4 to pl 8. In
10 certain embodiments, the pl of the stress proteins which are to be purified may be
firstly determined by isoelectric focussing.
In certain embodiments, the purification methodology is not performed in the
presence of (i.e. it is performed in the absence of) urea or a similar compound or
15 solution. In certain other embodiments, ampholytes, chaotropes and/or surfactants
are not used in the purification methodology. In certain preferred embodiments the
buffer contains at least one divalent cation and may further contain adenosine
diphosphate.
20 Typically the method comprises the steps of (i) applying the source mixture to an
ion exchange matrix, (ii) adjusting the pH, varying the salt gradient across the ion
exchange matrix, and (iii) collecting the eluted fractions, wherein said fractions
comprise purified or enriched stress protein complexes, the elution of which is
caused by the changing of the salt gradient under specific conditions. In certain
25 embodiments, the matrix is a resin. In further embodiments, the matrix is a
membrane. Typically the matrix is comprised of charged particles.
In certain embodiments, the ion exchange is performed using an ion exchange
membrane absorber which serves to separate complex protein mixtures into basic
30 and acidic fractions. The inventors have identified that that form of ion exchange
results in a method which is convenient, fast and reproducible and which therefore
can produce a consistently high yield of stable stress protein complexes, which
have their integrity maintained, as would be required for the production of
commercial quantities of a vaccine preparation which comprised a protein
component, such as a stress protein-peptide complex as the immunogenic
determinant. Furthermore, in certain embodiments where stress protein
5 complexes are for use as the immunogenic determinant in the preparation of
vaccine compositions and wherein the stress protein complexes are purified in the
presence of a buffer comprising at least one divalent cation, and in certain
preferred embodiments adenosine diphosphate, the inventors have identified that
such stress protein complexes have superior utility in mediating or enhancing
10 immune responses elicited by the use of such vaccines as the complexes remain
in a more stable state, that is, the complexes do not dissociate to become a
separate stress protein and peptide fragment. In certain embodiments, the buffer
lacks at least one of adenosine triphosphate (ATP), ATPase, potassium, or a
potassium salt, chaotropes, ampholytes and surfactants.
15
In one embodiment, the heat shock proteinlantigenic peptide complex (HspC)
enriched preparations (HEP) can be eluted from the ion exchange
chromatography medium using any suitable elution buffer known to the skilled
person in order to maintain protein integrity. Typically the elution buffer comprises
20 a salt, such as sodium chloride, as described hereinbefore. The elution buffer may
further comprise a phosphate or TRlS based buffer, acetate, citrate or a hydrogen
ion buffer.
The terms "ion-exchange" and "ion-exchange chromatography" as used herein
25 refer to a chromatographic process in which an ionizable solute of interest (e.g., a
protein complex of interest provided in a cell lysate) interacts with an oppositely
charged ligand linked to a solid phase ion exchange material under appropriate
conditions of pH and conductivity, such that the solute of interest interacts nonspecifically
with the charged compound more or less than the solute impurities or
30 contaminants in the mixture. The contaminating solutes in the mixture can be
washed from a column of the ion exchange material or are bound to or excluded
from the resin, faster or slower than the solute of interest. "Ion-exchange
chromatography" specifically includes cation exchange, anion exchange, and
mixed mode chromatographies.
In certain embodiments, the ion exchange chromatography medium includes an
5 ion exchange column. Typically, the ion exchange column includes a high flow
base, such as an agarose or sepharose high flow base. Optionally the high flow
base includes a surface extender, such as animal free dextran. Suitable ion
exchange media include both cation and anion exchange resins and columns
including those derivatized with quaternary ammonium salts and sulphonic
10 moieties, for examples the CAPTOQ~co~lu mn and CAPTOS~re~si ns (GE
Healthcare Limited). In further embodiments the ion exchange chromatography
medium includes a mixed multimodal ion exchange resins, for example the
CaptoTMMMCa nd CAPTOTMAdherec olumns (GE Healthcare).
15 In certain embodiments, the cell lysates are buffer exchanged into 50mM
phosphate buffer pH6.8. In certain embodiments, the ion exchange column
includes a high flow base, preferably an agarose high flow base. In certain
embodiments, the high flow base includes a surface extender, e.g. animal free
dextran, and a Q ligand, e.g. a quaternary ammonium salt.
20
The phrase "ion exchange material" refers to a solid phase that is negatively
charged (i.e. a cation exchange resin) or positively charged (i.e. an anion
exchange resin). In one embodiment, the charge can be provided by attaching one
or more charged ligands (or adsorbents) to the solid phase, e.g. by covalent
25 linking. Alternatively, or in addition, the charge can be an inherent property of the
solid phase (e.g. as is the case for silica, which has an overall negative charge).
In certain embodiments, where the ion exchange chromatography is cation
exchange chromatography, the cation exchange chromatography step employs a
30 ligand selected from the group comprising, but not limited to: sulfonate, carboxylic,
carboxymethyl sulfonic acid, sulfoisobutyl, sulfoethyl, carboxyl, sulphopropyl,
sulphonyl, sulphoxyethyl and orthophosphate.
A "cation exchange resin" refers to a solid phase which is negatively charged, and
which has free cations for exchange with cations in an aqueous solution passed
over or through the solid phase. Any negatively charged ligand attached to the
solid phase suitable to form the cation exchange resin can be used, e.g., a
5 carboxylate, sulfonate and others as described below. Commercially available
cation exchange resins include, but are not limited to, for example, those having a
sulfonate based group (e.g., Monos, Minis, Source 15s and 30S, SP Sepharose
FAST FLOWTMS, P Sepharose High Performance from GE Healthcare, Toyopearl
SP-650s and SP-650M from Tosoh, Macro-Prep High S from BioRad, Ceramic
10 HyperD S, Trisacryl M and LS SP and Spherodex LS SP from Pall Technologies);
a sulfoethyl based group (e.g., Fractogel SE from EMD, or Poros S-10 and S-20
from Applied Biosystems); a sulphopropyl based group (e.g., TSK Gel SP 5PW
and SP-5PW-HR from Tosoh, Poros HS-20 and HS 50 from Applied Biosystems);
a sulfoisobutyl based group (e.g., Fractogel EMD SO3 from EMD); a sulfoxyethyl
15 based group (e.g., SE52, SE53 and Express-Ion S from Whatman), a
carboxymethyl based group (e.g., CM Sepharose Fast Flow from GE Healthcare,
Hydrocell CM from Biochrom Labs Inc., Macro-Prep CM from BioRad, Ceramic
HyperD CM, Trisacryl M CM, Trisacryl LS CM, from Pall Technologies, Matrex
Cellufine C500 and C200 from Millipore, CM52, CM32, CM23 and Express - Ion C
20 from Whatman, Toyopearl CM-650S, CM-650M and CM-650C from Tosoh);
sulfonic and carboxylic acid based groups (e.g. BAKER BOND^^ Carboxy-Sulfon
from J.T. Baker); a carboxylic acid based group (e.g., WP CBX from J.T Baker,
DOWEX MAC-3 from Dow Liquid Separations, Amberlite Weak Cation
Exchangers, DOWEX~W~ea k Cation Exchanger, and Diaion Weak Cation
25 Exchangers from Sigma- Aldrich and Fractogel EMD COO- from EMD); a sulfonic
acid based group (e. g., Hydrocell SP from Biochrom Labs Inc., DOWEX~Fi~ne
Mesh Strong Acid Cation Resin from Dow Liquid Separations, UNOsphere S, WP
Sulfonic from J. T. Baker, Sartobind S membrane from Sartorius, Amberlite Strong
Cation Exchangers, DOWEX~St~ro ng Cation and Diaion Strong Cation
30 Exchanger from Sigma-Aldrich); and a orthophosphate based group (e.g., pl 1
from Whatman).
If desirable, a cation exchange membrane can be used instead of a cation
exchange resin, for example, Sartobind S (Sartorius; Edgewood, NY).
In certain embodiments, where the ion exchange chromatography is anion
exchange chromatography, the anion exchange chromatography step may employ
5 a ligand selected from the group consisting of: quaternary ammonium or amine,
dethylamine, diethylaminopropyl, amino, timethylammoniumethyl, trimethylbenzyl
ammonium, dimethylethanolbenzyl ammonium, polyamine.
An "anion exchange resin" refers to a solid phase which is positively charged, thus
10 having one or more positively charged ligands attached thereto. Any positively
charged ligand attached to a solid phase suitable to form the anionic exchange
resin can be used, such as quaternary amino groups. For example, a ligand used
in AEC can be a quaternary ammonium, such as quaternary alkylamine and
quaternary al ky la1 kanol amine, or amine, diethylamine, diethylaminopropyl, amino,
15 timethylammoniumethyl, trimethylbenzyl ammonium, dimethylethanolbenzyl
ammonium, and polyamine. Alternatively, for AEC, a membrane having a
positively charged ligand, such as a ligand described above, can be used instead
of an anion exchange resin.
Commercially available anion exchange resins include, but are not limited to,
20 DEAE cellulose, Poros PI 20, PI 50, HQ 10, HQ 20, HQ 50, D 50 from Applied
Biosystems, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX Sepharose
Fast Flow, Q Sepharose high Performance, QAE SEPHADEXTM and FAST Q
SEPHAROSETM from GE Healthcare, WP PEI, WP DEAM, WP QUAT from J.T.
Baker, Hydrocell DEAE and Hydrocell QA from Biochrom Labs Inc., UNOsphere
25 Q, Macro-Prep DEAE and Macro-Prep High Q from Biorad, Ceramic HyperD Q,
ceramic HyperD DEAE, Q HyperZ, Trisacryl M and LS DEAE, Spherodex LS
DEAE, QMA Spherosil LS, QMA Spherosil M from Pall Technologies, DOWEX
Fine Mesh Strong Base Type I and Type II Anion Resins and DOWEX
MONOSPHER E 77, weak base anion from Dow Liquid Separations, Matrex
30 Cellufine A200, A500, Q500, and Q800 from Millipore, Fractogel EMD TMAE3
Fractogel EMD DEAE and Fractogel EMD DMAE from EMD, Amberlite weak and
strong anion exchangers type I and II, DOWEX weak and strong anion exchangers
type I and II, Diaion weak and strong anion exchangers type I and II, Duolite from
Sigma- Aldrich, TSK gel Q and DEAE 5PW and 5PW-HR, Toyopearl SuperQ-
5 650S, 650M and 650C3 QAE-550C and 650S, DEAE- 650M and 650C from
Tosoh, and QA52, DE23, DE32, DE51, DE52, DE53, Express-Ion D and Q from
Whatman.
If desirable, an anion exchange membrane can be used instead of an anion
exchange resin. Commercially available anion exchange membranes include, but
10 are not limited to, SARTOBIND Q~~ from Sartorius, MUSTANG Q~~ from Pall
Technologies and INTERCEPT Q~~ membrane from Millipore.
In certain embodiments, the anion exchange chromatography is performed at a pH
of from about pH 5.0 to about pH 9.0 and at a conductivity of from about 0.5 to
about 5 mS/cm. In certain embodiments, the cation exchange chromatography is
15 performed at a pH of from about pH 4.0 to about pH 9.0 and at a conductivity of
from about 0.5 to about 15 mS/cm. In certain embodiments, the mixed mode
chromatography is performed at a pH of from about pH 4.0 to about pH 9.0 and at
a conductivity of from about 0.5 to about 15 mS/cm.
20 The "pl" or "isoelectric point" of a polypeptide refers to the pH at which the
polypeptide's positive charge balances its negative charge. The pl can be
calculated according to various conventional methodologies, e.g., from the net
charge of the amino acid and/or sialic acid residues on the polypeptide or
determined empirically using isoelectric focusing techniques.
25
The pH and conductivity of the chromatography buffer are selected such that the
HspCs of interest are bound to the IEC resin used. Examples of buffers suitable
for use as an elution buffer may include a phosphate or TRlS based buffer,
acetate, citrate and the hydrogen ion buffers (Good et al., 1966 Biochemistry 5:
30 467-477).
The term "elution buffer", as used herein, refers to a buffer used to elute the
protein complexes of interest from the resin. The pH and conductivity of the
elution buffer are selected such that the protein complexes of interest are eluted
5 from the CEC resin used in the process. Examples of buffers suitable for use as
an elution buffer may include a phosphate or TRlS based buffer, acetate, citrate,
glycine, histine and the Good buffers.
In certain embodiments, the elution buffer is sodium chloride (NaCI) which may be
10 used at a concentration of from about 50mM to about 1500mM. In certain further
embodiments, the elution buffer comprises at least one divalent cation, and in
certain embodiments adenosine diphosphate. In certain embodiments, the buffer
lacks at least one of adenosine triphosphate (ATP), ATPase, potassium, or a
potassium salt, chaotropes, ampholytes and surfactants.
15
The pH of the elution buffer can be from about pH 3 to about pH 10, more
preferably pH from about pH 4 to about pH 9. In certain embodiments, the pH of
the buffer is about pH 6.8.
20 The present invention further extends to HspC-enriched lysates (HEL) which are
purified according to the methods of the present invention. Said HspC-enriched
lysates may also be known as an HspC-enriched fraction (HEF) or as HspC
enriched compositions (HEC). Accordingly, a further aspect of the present
invention provides at least one HspC-enriched lysate purified by the method of
25 invention for use in the preparation of a vaccine composition. Typically the HspCenriched
lysate is derived from at least one eluted fraction obtained from the ion
exchange method of the present invention.
In certain embodiments, the HspC-enriched lysate comprises a complex formed
30 between a stress protein (heat shock protein) and a polypeptide or peptide
fragment, in particular an antigenic peptide fragment. In certain embodiments, the
purified HspC-enriched lysate is derived from a microbial host, prokaryotic, viral or
protozoal pathogen, a eukaryotic host cell infected with a pathogen, or from a
malignant or cancerous cell. In a preferred embodiment, the purified complex is a
stress proteinlpeptide complex comprises a mixture of stress proteins wherein the
stress protein can be selected from the group consisting of small hsps, hsp65,
5 hsp70, hsp90 and hsp100.
Ion Exchange Chromatography (IEC) relies on charge-charge interactions
between the proteins in the sample and the charges immobilized on the resin of
choice. In general IEC can be subdivided into cation exchange chromatography or
10 anion exchange chromatography. For example ~ a p t(ao po~sit~ivel~y ch arged
anion exchanger) may be utilised when the target protein complex is predicted to
be negatively charged at pH6.8 (see table I ) and captosTM (a negatively charged
anion exchanger) may be utilised when the target protein complex is predicted to
be positively charged at pH6.8.
15
Table 1 Predicted binding of Hsps to CaptoQ column
Protein MW (kDa) pl Predicted binding to
CaptoQ*
Hsp9O 75 4.7 strong
Hsp71 67 4.9 strong
Hsp6O (Cpn60.11 56 4.7 strong
groEL1)
Hsp65 (Cpn60.21 57 4.6 strong
groEL2)
HspX 16 5 strong
BCG-A (GroES) 11 4.6 strong
"phosphate buffer pH6.8 used for column equilibration
Moreover, the distribution of the isoelectric point (pl) of proteins in a proteome is
20 universal for all prokaryotes and can be represented as a bimodal distribution or
"butterfly effect" such that approximately 60% of the proteins have a pl 17 and
40% of the proteome has a pl 28 (see Table 1). For example, where stress protein
complexes from mycobacterial cultures are used to generate a cell lysate, it would
be expected that at pH 6.8 at least 40% of the mycobacterial contaminating
proteome would be readily removed from the purified complex and hence also
5 from vaccine product using captoQTM IEC. The skilled man will readily understand
that the methods of the present invention may be performed using any suitable ion
exchange medium which is a high performance medium.
Important technical issues to consider when developing a robust protein
10 purification strategy include a short process run time and careful consideration of
the buffer composition. The present inventors have provided a method for the
purification of protein complexes and in doing have overcome issues associated
with protein degradation, modification or disruption of the proteins complexes
during the purification process. Although immunogenic HspC enriched vaccines
15 have been prepared with free flow isoelectric focusing (FF-IEF), typical run times
were 4 hours and it was also necessary to maintain protein solubility with
chaotropes such as urea. This chaotrope has been shown to have destabilizing
effects on both macromolecular structure and protein function. Encouragingly,
captoQTM due to the chemical stability of the high flow 'protein friendly' matrix
20 results in a shorter run time and offers a greater flexibility of buffer choice. The
present invention has surprisingly reduced the time taken to prepare 3mg of HspC
(stress protein complex) enriched preparation from 10mg of starting lysate to
approximately 2 hours. Moreover protein solubility was maintained without the
need for chaotropes or surfactants. Additionally, protein degradation levels were
25 reduced. The buffer compositions of the present invention minimised the disruption
of the protein complexes during the purification process and the HEPs produced
by the methods of the present invention showed enhanced immunogenicity and
protection against infection. Preferred buffer comprise at least one divalent cation
and optionally adenosine diphosphate
The methods of the present invention have been used to prepare potential
vaccines against tuberculosis (TB), meningitis and influenza and are simple,
predictable, and straightforward, with the process performance defined almost
exclusively by the isoelectric point of the target proteins and the buffer pH. The
vaccine composition may comprise at least 2 two of the major heat shock proteins
thought to be important in eliciting protective immunity in a host, specifically Hsp6O
5 or GroEL, and Hsp70 or DnaK families and homologues.
The methods of the present invention have the advantage of being scalable and
rapid with the possibility of processing litres of lysate to generate Kg amounts of
purified protein complex and vaccine composition.
10
Administration of vaccine compositions
In certain embodiments, the vaccine compositions of the invention may further
comprise at least one adjuvant. In certain embodiments, the adjuvant is selected
from the group consisting of, but not limited to; Freund's complete adjuvant,
15 Freund's incomplete adjuvant, Quil A, Detox, ISCOMs and squalene. Further
suitable adjuvants include mineral gels or an aluminium salt such as aluminium
hydroxide or aluminium phosphate, but may also be a salt of calcium, iron or zinc,
or may be an insoluble suspension of acylated tyrosine, or acylated sugars, or
may be cationically or anionically derivatised saccharides, polyphosphazenes,
20 biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives
(e.g. of reduced toxicity), 3-0-deacylated MPL, quil A, Saponin, QS21, Freund's
Incomplete Adjuvant (Difco Laboratories, Detroit, MI), Merck Adjuvant 65 (Merck
and Company, Inc., USA), AS-2, AS01 , AS03, AS04, AS1 5 (GSK, USA), MF59
(Chiron, Sienna, Italy), CpG oligonucleotides, bioadhesives and mucoadhesives,
25 microparticles, liposomes, outer membrane vesicles, polyoxyethylene ether
formulations, polyoxyethylene ester formulations, muramyl peptides or
imidazoquinolone compounds.
The vaccine compositions or purified andlor isolated stress protein complexes of
30 the present invention may be administered to a subject in need of treatment via
any suitable route. Typically the composition is administered parenterally.
Examples of other possible routes for parenteral administration include, but are not
limited to; intravenous, intracardial, intraarterial, intraperitoneal, intramuscular,
intracavity, subcutaneous, transmucosal, inhalation or transdermal. Routes of
administration may further include topical and enteral, for example, mucosal
(including pulmonary), oral, nasal, rectal. The formulation may be a liquid, for
5 example, a physiologic salt solution containing non-phosphate buffer at pH 6.8-
7.6, or a lyophilised or freeze dried powder.
In certain embodiments, the composition is deliverable as an injectable
composition. For intravenous injection, the stress protein complexes will be in the
10 form of a parenterally acceptable aqueous solution which is pyrogen-free and has
suitable pH, isotonicity and stability. Those of relevant skill in the art are well able
to prepare suitable solutions using, for example, isotonic vehicles such as sodium
chloride injection, Ringer's injection or, Lactated Ringer's injection. Preservatives,
stabilisers, buffers, antioxidants and/or other additives may be included, as
15 required.
In certain embodiments, the injection method can be needless or may use a
needle which penetrates the dermis. In certain further embodiments the vaccine is
suitable for oral administration, or can be administered transdermally, or by
20 pulmonary delivery. In certain embodiments, the vaccine composition is
administered as a prophylactic vaccine. In certain embodiments, the vaccine
composition is administered as a therapeutic vaccine. In yet further embodiments
the vaccine composition is administered as a booster vaccine to any previously
administered vaccine mediated by a primary immunisation schedule.
25
Pharmaceutical compositions for oral administration may be in tablet, capsule,
powder or liquid form. A tablet may comprise a solid carrier such as gelatine or an
adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier
such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
30 Physiological saline solution, dextrose or other saccharide solution or glycols such
as ethylene glycol, propylene glycol or polyethylene glycol may be included.
Examples of the techniques and protocols mentioned above and other techniques
and protocols which may be used in accordance with the invention can be found in
Remington's Pharmaceutical Sciences, 18th edition, Gennaro, A.R., Lippincott
Williams & Wilkins; 20th edition ISBN 0-91 2734-04-3 and Pharmaceutical Dosage
5 Forms and Drug Delivery Systems; Ansel, H.C. et al. 7th Edition ISBN 0-683305-
72-7, the entire disclosures of which is herein incorporated by reference.
The vaccine compositions or purified and/or isolated stress protein complexes of
the present invention may also be administered via microspheres, liposomes,
10 other microparticulate delivery systems or sustained release formulations placed in
certain tissues including blood.
Dosage regimens can include a single administration of the composition of the
invention, or multiple administrative doses of the composition. The compositions
15 can further be administered sequentially or separately with other therapeutics and
medicaments which are used for the treatment of the condition for which the
composition of the present invention is being administered to treat.
The actual amount administered, and rate and time-course of administration, will
20 depend on the nature and severity of what is being treated. Prescription of
treatment, e.g. decisions on dosage etc, is ultimately within the responsibility and
at the discretion of general practitioners and other medical doctors, and typically
takes account of the disorder to be treated, the condition of the individual patient,
the site of delivery, the method of administration and other factors known to
25 practitioners.
Definitions
Unless otherwise defined, all technical and scientific terms used herein have the
meaning commonly understood by a person who is skilled in the art in the field of
30 the present invention.
Throughout the specification, unless the context demands otherwise, the terms
'comprise' or 'include', or variations such as 'comprises' or 'comprising', 'includes'
or 'including' will be understood to imply the inclusion of a stated integer or group
of integers, but not the exclusion of any other integer or group of integers.
5
As used herein, terms such as "a", "an" and "the" include singular and plural
referents unless the context clearly demands otherwise. Thus, for example,
reference to "an active agent" or "a pharmacologically active agent" includes a
single active agent as well as two or more different active agents in combination,
10 while references to "a carrier" includes mixtures of two or more carriers as well as
a single carrier, and the like.
The terms "peptide", "polypeptide" and "protein" are used herein interchangeably
to describe a series of at least two amino acids covalently linked by peptide bonds
15 or modified peptide bonds such as isosteres. No limitation is placed on the
maximum number of amino acids which may comprise a peptide or protein.
Furthermore, the term polypeptide extends to fragments, analogues and
derivatives of a peptide, wherein said fragment, analogue or derivative retains the
same biological functional activity as the peptide from which the fragment,
20 derivative or analogue is derived.
As used herein, the term "treatment" and associated terms such as "treat" and
"treating" mean the eliciting of protective immune response against an
immunogenic determinant in order to confer long term protective immunity against
25 the pathogen or cancer cell from which the immunogenic determinant of the
vaccine composition is derived. The term 'treatment' therefore refers to any
regimen that can benefit a subject. The treatment may be in respect of an existing
condition or may be prophylactic (preventative treatment). Treatment may include
curative, alleviative or prophylactic effects.
As used herein, the term "therapeutically effective amount" means the amount of a
stress protein complex or vaccine composition of the invention which is required to
induce a protective immune response against an infectious disease or cancerous
condition. As used herein, the term "prophylactically effective amount" relates to
the amount of a multiple stress protein complex or vaccine composition which is
required to prevent the initial onset, progression or recurrence of an infectious
5 disease or cancerous condition. The term "therapeutic" does not necessarily imply
that a subject is treated until total recovery. Similarly, "prophylactic" does not
necessarily mean that the subject will not eventually contract a disease condition.
A "subject" in the context of the present invention includes and encompasses
10 mammals such as humans, primates and livestock animals (e. g. sheep, pigs,
cattle, horses, donkeys); laboratory test animals such as mice, rabbits, rats and
guinea pigs; and companion animals such as dogs and cats. It is preferred for the
purposes of the present invention that the mammal is a human. The term
"subject" is interchangeable with the term "patient" as used herein.
15
As used herein, the terms "mount", "mounted", "elicit" or "elicited" when used in
relation to an immune response mean an immune response which is raised
against the immunogenic determinant of a vaccine composition which is
administered to a subject. Typically the immunogenic determinant of the vaccine
20 composition comprises the isolated and/or purified stress protein complexes
obtained using the methods of the present invention.
As used herein, the term "immune response" includes T cell mediated and/or B
cell mediated immune responses that are influenced by modulation of T cell co-
25 stimulation. The term immune response further includes immune responses that
are indirectly effected by T cell activation such as antibody production (humoral
responses) and the activation of cytokine responsive cells such as macrophages.
EXAMPLES
30 The present invention will now be described with reference to the following
examples which are provided for the purpose of illustration and are not intended to
be construed as being limiting on the present invention.
Example 1 : Preparation of HspC-enriched preparations from BCG cellular
lvsates
BCG cell pellets from mid-log heat shocked cultures were lysed and clarified by
5 centrifugation. Cell pellets were resuspended in sterile PBS containing an EDTAfree
protease inhibitor cocktail. Resuspended cells were lysed using sonication, a
Beadbeater or passed through an Emulsiflex C5 high pressure homogeniser and
collected in a sterile bag. Benzonase (250 UImL) was added to the lysate. The
samples were then homogenised a further two times, the cellular lysate transferred
10 to centrifuge tubes and cell debris removed by centrifugation for 20 minutes at
6000 g. The clarified lysate was collected and centrifuged for a further 60 minutes
at 14000 g and the supernatant was removed and referenced as high speed
clarified lysates. 10ml of clarified lysates were desalted and buffer exchanged into
50mM phosphate buffer pH 6.8. The protein concentration of the sample was
15 determined and an IEC (ion exchange chromatography) HspC enriched
preparation was prepared by column chromatography as follows. 10mg of protein
was loaded on to a ~ a p tcolum~n ~at a~ flo w rate of 0.5mllminute. After
extensive washing of the column with 50mM phosphate buffer, pH 6.8 proteins
were batch eluted using increasing concentrations of NaCl (1 50mM, 300mM,
20 500mM and 1 M). Eluted fractions containing Hsp70 and Hsp65 were analysed
using SDS-PAGE and Western blotting using commercial antisera against DnaK
and GroEL. Examples of the HEPs prepared are shown in Figures 1A (SDSPAGE)
and 1 B (Western blotting). In some preparations, a mixed modal MMC
column was used with loading in 50mM Acetate, 1 M NaCI, ImM MgCI2 at pH5 and
25 elution in the same buffer at pH 8.0.
Example 2: Enhancing complex stability in the preparation of HspC-enriched
preparations from BCG cellular lvsates
BCG cell pellets from mid-log heat shocked cultures were lysed and clarified by
30 centrifugation. Cell pellets were resuspended in sterile PBS containing an EDTAfree
protease inhibitor cocktail. Resuspended cells were lysed using sonication, a
Beadbeater or passed through a Emulsiflex C5 high pressure homogeniser and
collected in a sterile bag. Benzonase (250 UImL) was added to the lysate. The
samples were then homogenised a further two times, the cellular lysate transferred
to centrifuge tubes and cell debris removed by centrifugation for 20 minutes at
6000 g. The clarified lysate was collected and centrifuged for a further 60 minutes
5 at 14000 g and the supernatant was removed and referenced as high speed
clarified lysates. 10ml of clarified lysates were desalted and buffer exchanged into
50 mM HEPES, 1 mM MgCI2, pH 6.8 with or without 1 mM ADP. The HspC
enriched preparation was isolated using column chromatography on a ~ a p t o ~ ~ ~
column. After extensive washing of the column with 50 mM HEPES, 1 mM MgCI2,
10 pH 6.8, with or without 1 mM ADP, proteins were batch eluted using increasing
concentrations of NaCl (1 50mM, 350mM, 500mM and 1 M). Eluted fractions
containing Hsp70 and Hsp65 were analysed using SDS-PAGE and Western
blotting using commercial antisera against DnaK (Hsp70), GroEL (Hsp65) and
Ag85. Examples of the HEPs prepared are shown in Figure 5A. Control HEPs
15 containing disrupted HspC complexes were prepared using buffer compositions
with ATP replacing ADP (Fig5B). Although the HEPs prepared in the various
buffers did not show significant differences in their Coomasie Blue stained protein
profiles (Fig 5B), they differed greatly in their immunogenicity as shown in
Example 3 below.
20
Example 3: Immunogenicity of BCG derived HspC-enriched preparations
BCG HEPs were used to immunise BalbC mice and spleens harvested from the
immunised animals 28 days after immunisation. Spleens were collected into
RPMI-1640 and single cell suspensions were made by pressing the spleens
25 through 70 pm cell strainers using a 5 mL syringe plunger into a 50 mL Falcon
tube. Cells were counted using trypan blue exclusion on a KOVA glasstic slide
haemocytometer and the production of interferon gamma (IFN-y) assayed in a
recall response to TB antigens.
30 2x1 o6 splenocytes were added to each well in a 24 well tissue culture plate (Nunc)
in 1 mL culture medium and to each well, one of the following antigens was added:
BSA (1 0 pglml), Con A (1 0 pglml), TB whole cell lysate (WCL at 50 to 1.56
pgImL), HEPs, IEF HspCs or Ag85 (1 0 pg1mL).
Culture supernatants from the re-stimulated wells were tested for IFN-y IL-2, IL-4
5 and IL-5 using a murine ELlSA kit according to the manufacturer's protocol (R&D
Systems). Figure 2 shows typical results obtained with spleen cells from
immunised mice re-stimulated with WCL in vitro. The results show that HEPs
isolated from BCG (IEX) induced a strong IFN-y response in the immunised
animals, stronger than the parent BCG lysates (LSS) and much stronger than
10 HspCs isolated using conventional free-flow isoelectric focussing method
previously described (IEF) for the isolation of multiple HspC families. Recall
responses to WCL were comparable to those seen with Con A and significant but
smaller responses were seen against Ag85. The in vitro IFN-y responses also
translated into in vivo protection against live TB challenge in the mouse aerosol
15 challenge model. Figure 6 shows the detailed immunogenicity of the HEPs
isolated from BCG. Figure 6A shows typical results for cell mediated immunity
obtained assayed using spleen cells from immunised mice re-stimulated with WCL
in vitro. The results show that HEPs isolated from BCG as in Example 2, in the
presence of ADP and divalent cations (IEC (2 vaccs) and ADPIMg), induced a
20 strong IFN-y response in the immunised animals, stronger than the HEPs isolated
from BCG as in Example 1 (IEC (2 vaccs), and similar to the response seen in
animals immunised with live BCG and boosted with these HEPs (BCGIIEC (2
vaccs)) .
25 Figure 6B shows the Thl-bias of the humoral immunity induced by HEP vaccines
isolated from BCG bacterial cell lysates either in the presence of ADP + a divalent
cation (VI), ATP + a divalent cation (V2) or divalent cation alone (V3) and
demonstrate the enhanced immunogenicity of HEPs isolated using the improved
buffer compositions of the present invention. Fig.6B also shows the significant
30 enhancement of the Thl induced IgG2a antibody response to the current live BCG
vaccine (BCG) by boosting with the HspC vaccine (BCG + V1) prepared by the
improved methods of the present invention.
Example 4: Protective Immunity induced by BCG HspC vaccines
5 HEPs isolated from BCG as in Examples 1 (IEC) and 2 (IEC mod) were used to
immunise groups of 8-1 0 na'ive or live BCG-primed BalbC mice. Control animals
were immunised with either saline or live BCG vaccine (Statens Serum Institute).
HspC vaccines were dosed at 75pg with an interval of 4 weeks between prime and
booster vaccinations and 4 weeks after the final immunisations the animals were
10 challenged with 50-100 CFU of live TB strain H37Rv. Lungs were harvested from
the immunised animals 28 days after challenge and lung homogenates plated in
triplicates to quantitate lung colonisation by TB. Figure 7 shows typical CFU
recovered from the lungs 4 weeks post-challenge. Animals immunised with HEPs
purified using the improved buffer compositions of the present invention show
15 significantly enhanced protection as assessed by reduced lung Cfu on live
challenge (Fig 7A. IEC mod versus IEC) and also boosted the protection induced
by the current live BCG vaccine (Fig 7A. BCG versus BCG and IEC). HEP
vaccines prepared using the HspCs isolated as in Example 2 in the presence of
ADP and a divalent cation (IEC mod) showed a significant protection as assayed
20 by a reduction in lung colony counts, even better than the protection afforded by
the live BCG vaccine (BCG). Boosting of animals immunised with live BCG with
HspC vaccines (BCG and IEC) showed a significantly improved protection
compared to those receiving BCG alone (BCG) as assessed by a further reduction
in lung colony counts after H37Rv challenge.
25
To demonstrate the absolute requirement for the stabilisation of the HspC
complexes for the enhanced protective immunogenicity seen, HEPs isolated using
the the improved buffer compositions of the present invention were compared to
HEPs isolated in the presence of ATP to disrupt the HspC complexes. The results
30 obtained are shown in Fig 7B and show significant protection in animals
immunised with HEPs isolated in the presence of a divalent cation (V3) and ADP
(VI) compared to those purified in the presence of ATP to disrupt the HspC
complexes (V2). The results also show the boosting of animals primed with the
current BCG live vaccine (BCG) immunised with HEP vaccines (BCG and VI).
Example 5: Preparation of HEPs from Neisseria
5 Cultures of an acapsulate variant of Neisseria meningitidis strain MC58 (Mol
Microbiol. 1995, Nov; 18(4):741-54) were heat shocked at 44°C and killed by
treatment with the antibiotic gentamicin. Cells were processed to produce HEPs
as described in Examples 1 and 2. In brief, cells were lysed by cycles of freezing
and thawing or sonication and clarified by centrifugation for 20 minutes at 6,000g.
10 Clarified extract was loaded onto a column packed with CaptoQ ion exchange
resin. After extensive washing of the column with 50mM phosphate buffer, pH 6.8
proteins were batch eluted using increasing concentrations of NaCl (1 50mM,
350mM, 500mM). Eluted fractions containing Hsp70 and Hsp65 were analysed
using SDS-PAGE (Figure 3A). Fractions eluted by 150 mM and 350 mM NaCl
15 were combined and dialysed into PBS. Vaccine was assessed by gel
electrophoresis and Western blotting for the presence of the major hsp families
and the outer membrane porin, PorA. Results are shown in Figure 3B.
Figure 8 shows HEPs from Neisseria meningitidis strain MC58 purified in the
20 absence of ADP and a divalent cation (A) according to the method in Example 1,
and in the presence of ADP and a divalent cation (B) according to the method in
Example 2, and western blotted for the presence of GroEL (hsp60) and DnaK
(hsp70).
25 Example 6: Immunogenicity of Neisserial HEPs
The HEPs prepared from Neisseria meningitidis strain MC58 or Neisseria
lactamica according to the method of Example 3 were used to immunise mice in
order to generate sera for assessment of cross strain responses. Sera from
immunised animals were pooled and assessed for their ability to elicit cross-strain
30 antibody-mediated opsonophagocytosis using the following clinical Neisserial
strains; MC58, H44176-SL, M01-240101, M01-240013, M01-240149, M01-240185
and M01-240355. For assay, serum samples were incubated with killedfluoresence-
labelled bacteria and Ig G-depleted baby rabbit complement for 7.5
min at 37°C. HL60 cells differentiated with 0.8% DMF, were added and samples
incubated for 7.5 min before the addition of ice cold DPBS to stop the reaction.
Samples were analysed by flow cytometry and data expressed as a fluorescence
5 index value (FI-C'). For all strains, serum obtained from mice vaccinated with the
MC58-derived HEPs induced opsonisation responses significantly greater than
those obtained with serum from non-vaccinated controls and animals immunised
with hspCs purified using conventional isoelectric focussing methods or with a
commercial outer membrane vesicle (H44176 OMV) vaccine candidate. The
10 results obtained with the heterologous strain M01-240101 are shown in Figure 4
and show the cross-strain protection obtained with the HEPs (IEC HspC),
conventionally purified hspCs (IEF HspC) and the OMV vaccine (H44176 OMV).
The HEPs vaccine also generated higher opsonisation values than the IEF HspC
vaccine against both MC58 and the H44176 strain from which the OMV vaccine
15 was derived. HEPs isolated from a commensal Neisseria, N.lactamica using the
methods in Examples 1 and 2 showed significant cross-serotype protection of
mice in a lethal challenge study, with complete survival of all mice against
peritoneal injection of live MC58.
20 Example 7: Purification and immunoaenicity of HEPs from Streptococcus
pneumoniae
Streptococcus pneumonia strain D39 was grown in Hoeprich's medium, heat
shocked at 42°C for 45 minutes and heat-killed at 56°C for 15 minutes. Cells were
harvested by centrifugation, resuspended in 50 mM HEPES, 1 mM MgC12, 1 mM
25 ADP pH 6.8 and disrupted in an Emulsiflex C5. The lysate was clarified by
centrifugation and loaded on a CaptoQ column and eluted in lysis buffer containing
300mM NaCI. The purified HEPs were used to immunize BalbC mice and the sera
analysed for antibodies against the various S.pneumoniae strains by ELISA. The
HEPs vaccines induced good antibodies against the immunization strain D39 and
30 cross reaction with strains 23F and TIGR4. The humoral response induced further
showed a Thl lymphocyte cell bias with the production of IgG2a subtype
antibodies being greater than the production of lgGl subtype antibodies.
Example 8: Purification and immunoaenicity of HEPs from baculovirus
infected insect cells
Recombinant baculovirus expressing influenza H3 Panama haemaggluttinin and
5 hepatitis C virus El and E2 polypeptides as fusion proteins with a human IgG Fc
fragment were used to infected Sf9 insect cells. Infected cells were grown for 72
hours in Insect-Xpress protein-free media and cells pelleted at 4,500rpm for 10
minutes in a Jouan GR4 22 centrifuge. Cell pellets were resuspended and lysed
on ice in 10mM Tris-HCI, pH 6.8 containing 0.2% NP40, I mglml pepstatin and
10 0.2mM PMSF using a dounce homogenizer. The lysate was centrifuged at
12,000g for 15 minutes and the supernatant centrifuged at 100,000g for 30
minutes to yield a clarified lysate which was then loaded onto a ~ a p tcolum~n.~ ~
The column was washed in 10mM Tris-HCI, pH 6.8 containing 100mM NaCl and
the HEPs eluted with a 150-350mM NaCl salt gradient. The purified HEPs were
15 used to immunise mice and rabbits and sera from the immunized animals assayed
by Western blotting and inhibition of hemaggluttination.
Example 9: Purification and immunoaenicity of HEPs from tumour cells
EL4 and A20 cells were grown in RPMl media, lysed in buffers containing non-
20 ionic using a Potter homogeniser and tumour cell HEPs purified and tested for
immunogenicity by Western blotting as in Example 6.
Example 10: Purification and immunoaenicity of HEPs from CHO cells
expressing heterloaous antigens
25 CHO cells and CHO cells expressing Fc-fusion proteins were grown in CHO CD
media (Gibco). CHO cells were harvested, washed in PBS, resuspended in 50 mM
HEPES, 150 mM NaCl pH6.8 either with or without 1 mM ADP and I mM MgCl and
lysed by sonication. The lysate was clarified by centrifugation followed by filtration
through 0.8 yM and then 0.2 yM filters, diluted lox in 50mM HEPES pH6.8 and
30 the HEPs purified using an AKTA chromatography system on a 1 ml CaptoQ
column. The column was washed with 20 ml buffer containing 50 mM HEPES
pH6.8 and B, 50 mM HEPES, 20 mM NaCl pH6.8 either with or without added
1 mM ADP and I mM MgCl and the HEPs eluted in wash buffer containing
increasing salt concentration of 150 mM NaCI, 250 mM NaCI, 350 mM and 500
mM NaCI. HEPs were run on SDS-PAGE gels and either stained for protein with
Coomassie (Figure 9A) or Western blotted for either hsp60 (antibody SPA-875,
5 Stressgene) or Hsp70 (antibody SPA-81 1, Stressgene) (Figure 9B and 9C
respectively). The Coomassie staining demonstrates the captured proteins by
CaptoQ resin from the CHO lysate and good separation of these proteins by step
gradient elution. The Western blots demonstrate the elution of the HEPs from the
CaptoQ column with 150 mM NaCI.
10
All documents referred to in this specification are herein incorporated by reference.
Various modifications and variations to the described embodiments of the
inventions will be apparent to those skilled in the art without departing from the
scope of the invention. Although the invention has been described in connection
15 with specific preferred embodiments, it should be understood that the invention as
claimed should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes of carrying out the invention which
are obvious to those skilled in the art are intended to be covered by the present
invention. Reference to any prior art in this specification is not, and should not be
20 taken as, an acknowledgment or any form of suggestion that this prior art forms
part of the common general knowledge in any country.

Claims
1. A method for the purification of complexes formed between a stress protein
and a polypeptide, the method comprising the steps of:
5 (i) providing a source mixture comprising at least one target stress protein
complex formed from a stress protein complexed to a polypeptide,
(ii) determining the isoelectric point (pl) of at least one target stress protein
complex which is to be purified from the source mixture;
(iii) preparing a clarified cell lysate from the source mixture comprising the
identified stress protein complex;
(iv) subjecting the cell lysate to purification using ion exchange, wherein the
cell lysate is buffered with a primary buffer comprising at least one divalent
cation to a pH within 2 units of the pl of the target stress protein complex,
and wherein a secondary buffer providing a salt gradient is used to elute a
mixture of target stress protein complexes.
2. The method as claimed in claim 1 wherein the target stress protein
complexes which are eluted are provided in a preparation comprise a
plurality of stress proteins of different heat shock protein classes.
3. The method as claimed in claim 1 or claim 2 wherein the buffer of step (iv)
further comprises adenosine diphosphate.
4. The method as claimed in claim 3 wherein the adenosine diphosphate is
25 provided at a concentration of from about 0.1 mM to 1 OOmM, and wherein
the divalent cation is provided at a concentration of about 0.1 mM to
1 OOmM.
5. The method as claimed in any one of claims 1 to 4, wherein the at least one
divalent cation is a magnesium salt and/or a manganese salt.
6. A method as claimed in any one of claims 1 to 5 wherein the primary buffer
lacks at least one of adenosine triphosphate (ATP), ATPase andlor
potassium or a potassium salt.
5 7. The method as claimed in any preceding claim wherein the primary buffer
does not include chaotropes, surfactants, uear or ampholytes.
8. The method as claimed in any preceding claim wherein the secondary
buffer is an elution buffer to provide the salt gradient.
10
9. The method as claimed in claim 8 wherein elution buffer comprises sodium
chloride.
10. The method as claimed in claim 8 or 9 wherein the sodium chloride is
15 provided in the elution buffer at a concentration of about 50mM to 500mM
11. The method as claimed in claim 8 or 9 wherein the pH of the elution buffer
is from about pH3 to about pH10.
20 12. The method as claimed in claim 8 or 9 wherein the pH of the elution buffer
is from about pH4 to about pH9.
13. The method as claimed in claim 8 or 9 wherein the pH of the elution buffer
is about pH6.8.
25
14. The method as claimed in any preceding claim wherein the ion exchange is
ion exchange chromatography.
15. The method as claimed in any preceding claim wherein the ion exchange is
30 cation exchange chromatography.
16. The method as claimed in any one of claims 1 to 14 wherein the ion
exchange is anion exchange chromatography.
17. The method as claimed in any one of claims 1 to 14 wherein the ion
5 exchange is mixed mode chromatography.
18. The method as claimed in any preceding claim wherein the ion exchange is
performed using an ion exchange membrane absorber which serves to separate
complex protein mixtures into basic and acidic fractions.
10
19. The method as claimed in any preceding claim wherein the solid phase of
the ion exchange is a resin or a membrane.
20. A method as claimed in any preceding claim wherein the stress protein
15 complexes are eluted in fraction which comprise complexes with a pl of 4.5 to 6.5.
21. The method as claimed in claim 16 wherein the anion exchange
chromatography is performed at a pH of from about 5.0 to about 9.0 and at a
conductivity of from about 0.5 to about 5 mSIcm.
20
22. The method as claimed in claim 15 wherein the cation exchange
chromatography is performed at a pH of from about 4.0 to about 9.0 and at a
conductivity of from about 0.5 to about 1 5 mSIcm.
25 23. The method as claimed in any preceding claim wherein the stress protein is
a heat shock protein.
24. The method as claimed in claim 18 wherein the complex protein mixture
comprises one or more heat shock proteins selected from the group consisting of:
30 hsp20-30kD, hsp40, hsp60, hsp70, hsp90, hsp100, calrecticulin, hsp72, grp94,
grp75 BiPlgrp78, grp75lmt and gp96.
25. The method as claimed in any preceding claim wherein the stress proteinpeptide
fragment complex is derived from the group consisting of: a cancerous
cell, a pathogenic cell, a cell infected by a pathogenic organism, a cell which has
been genetically modified such that it expresses a heterologous protein which is
5 derived from a cancerous cell, a cell which has been genetically modified such that
it expresses a heterologous protein derived from a pathogen which causes an
infectious disease in a host.
26. The method as claimed in any preceding claim wherein the stress protein
10 complexes have a molecular weight in the range of 50KDa to 9OOKDa.
27. The method as claimed in any preceding claim wherein the peptide of the
stress protein peptide fragment is derived from a pathogenic organism which
typically causes an infectious disease.
15
28. The method as claimed in claim 27 wherein the pathogenic organism is
selected from the group consisting of a prokaryotic cell, a protozoa, a virus, a
parasite or a fungi.
20 29. The method as claimed in claim 27 wherein the prokaryotic organism is a
gram positive bacteria or a gram negative bacteria.
30. The method as claimed in claim 29 wherein the bacteria is selected from
the species selected from the group consisting of: Escherichia, Streptococcus,
25 Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria,
Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia,
Fancisella, Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Bran hamel la,
Actinobacillus, Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus,
Clostridium, Treponema, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia,
30 Leptospira, Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas,
Aeromonas, Rickettsia, Chlamydia, Borrelia and Mycoplasma.
31. The method as claimed in claim 27 wherein the peptide fragment is derived
from a viral peptide.
33. The method as claimed in claim 26 wherein the virus may be selected from
5 the group comprising: human immunodeficiency virus (HIV), hepatitis A virus
(HAV), hepatitis B (HBV), hepatitis C (HCV), human papillomavirus (HPV),
Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Herpes Simplex virus (HSV),
Respiratory Syncytial Virus, Ebola virus, Marburg virus, West Nile virus (WNV), St
Louis Encephalitis virus (SLEV), Rift Valley Fever virus (RVFV), Influenza viruses,
10 coronaviruses, rhinovirus, adenovirus, SIV, rotavirus, human papilloma virus,
arbovirus, measles virus, polio virus, rubella virus, mumps virus, papova virus,
varicella-zoster virus, varicella virus, huntavirus and cytomegalovirus.
33. The method as claimed in claim 27 wherein the peptide fragment is derived
15 from a protozoan pathogen.
34. The method as claimed in claim 27 wherein the peptide fragment is a
tumour specific antigen.
20 35. A vaccine composition comprising a purified stress protein-peptide complex
eluate fraction or lysate obtained by the method of any one of claims 1 to 34.
36. Use of a purified stress protein-peptide complex obtained by the method of
any one of claims 1 to 34 in the preparation of a medicament for the treatment of
25 an infectious disease.
37. Use of a purified stress protein-peptide complex obtained by the method of
any one of claims 1 to 34 in the preparation of a medicament for the treatment of a
cancerous or a malignant condition.
38. A stress protein-peptide complexed which is purified by the method of any
one of claims 1 to 34 for use in a vaccine composition for treating an infectious
disease.
5 39. A stress protein-peptide complexed which is purified by the method of any
one of claims 1 to 34 for use in a vaccine composition for treating a cancerous or a
malignant condition.
40. A method of vaccinating a subject against a pathogen derived infectious
10 disease or a cancerous condition, said method comprising the steps of:
- providing a vaccine composition comprising, as the immunogenic
determinant,a purified stress proteinlpeptide complex-enriched
preparation obtained according to the method of any one of claims
1 to 34, and
- administering a therapeutically effective or phrophylactically
effective amount of the vaccine to a subject in an amount sufficient
to elicit an immune response in the subject against the stress
proteinlpeptide complex enriched preparation.
20 41. A method of boosting a protective immune response mediated by a primary
immunisation schedule in a subject against a pathogen derived infectious disease
or a cancerous condition, wherein said protective immune response has been
elicited by the previous administration of a live or attenuated vaccine or a vaccine
composition as claimed in claim 35, said method comprising the steps of:
25 - providing a composition comprising, as the immunogenic
determinant, a purified stress protein complex-enriched preparation
obtained according to the method of any one of claims 1 to 34, ,
and
- administering a therapeutically or prophylactically effective amount
of the composition to a subject in an amount sufficient to enhance
the immune response in the subject against the stress protein
complexes.
42. A method of boosting a protective immune response mediated by a primary
immunisation schedule in a subject against a pathogen derived infectious disease
or a cancerous condition, wherein said protective immune response has been
5 elicited by the previous administration of a nucleic acid or protein vaccine, said
method comprising the steps of:
- providing a composition comprising, as the immunogenic
determinant, a purified stress protein complex obtained according
to the method of any one of claims 1 to 34, and
- administering a therapeutically effective or prophylactically effective
amount of a composition comprising the stress protein complexes
to a subject in an amount sufficient to elicit an immune response in
the subject against the stress protein complexes.
15 43. A method of boosting a protective immune response mediated by a primary
immunisation schedule in a subject against a pathogen derived infectious disease
or a cancerous condition, wherein said protective immune response has been
elicited by the previous exposure to the pathogen or cancer, said method
comprising the steps of:
20 - providing a composition comprising, as the immunogenic
determinant, a purified stress protein complex obtained according
to the method of any one of claims 1 to 34, and
- administering a therapeutically effective or prophylactically effective
amount of a composition comprising the stress protein complexes
to a subject in an amount sufficient to elicit an immune response in
the subject against the stress protein complexes.
44. A buffer solution for use in the purification of protein complexes using ion
exchange, wherein the buffer comprises at least one divalent cation, and wherein
30 the buffer lacks at least one of adenosine triphosphate (ATP), ATPase, potassium,
or a potassium salt, chaotropes, ampholytes and surfactants.
45. The buffer solution as claimed in claim 44 wherein the buffer further comprises
adenosine diphosphate.
46. The buffer solution as claimed in claim 44 or 45 wherein the at least one divalent
cation is a magnesium salt.
47. The buffer solution as claimed in claim 46 wherein the magnesium salt is magnesium
chloride (MgCl2).
48. The buffer solution as claimed in any one of claims 44 to 47 wherein the divalent
cation is a manganese salt.
49. The buffer solution as claimed in any one of claims 44 to 48 wherein the divalent
cation is provided at a concentration of from about 0.1 mM to about 100 mM.
50. The buffer solution as claimed in any one of claims 44 to 49 wherein the buffer further
comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).
51. The buffer solution as claimed in any one of claims 45 to 50 wherein the adenosine
diphosphate is provided at a concentration of from about 0.1 mM to 100 mM.
Dated this 9th day of January 2012