METHOD FOR THE PRODUCTION OF PROTEIN COMPLEXES
AND VACCINE COMPOSITIONS COMPRISING THE SAME
Field of the lnvention
5 The present invention relates to methods for the production of heat shock protein
complexes. The invention further extends to the use of heat shock protein
complexes produced according to the methods of the invention in the preparation
of vaccine compositions for the prevention and treatment of infectious diseases
and cancerous conditions.
10
Background of the lnvention
Heat shock proteins (hsps, HSPs) are a family of highly conserved proteins that
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
15 families: small (hsp20-30kDa); hsp40; hsp60; hsp70; hsp90; and hsp100. Heat
shock 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 heat shock proteins is to chaperone peptides from one cellular
compartment to another and, in the case of diseased cells, heat shock proteins are
20 also known to chaperone viral or tumour-associated peptides to the cell-surface for
presentation to the immune system.
Although heat shock proteins were originally identified in cells subjected to heat
stress, their production can result from a number of other forms of stress, such as
25 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. However, transcriptional
analysis of the genes induced by different stress stimuli show the induction of
distinct sets of genes following exposure of a cell to different stress inducing
30 stimuli (Bacon & Marsh (2007) Curr. Mol. Med. 7:277-86). Moreover, the
individual stress regulons are independently induced (vanBogelen et al. (1 987)
J.Bact. 16926-32 and Wilkes et al. (2009) Applied and Environmental Microbiol.
75981-990) and regulated (Holmes et al. (201 0) Microbiology 156:158-166). In
particular, vanBogelen shows that different stress inducing stimuli cause distinct
heat shock protein genes to be expressed and that the specific expression of
these genes resulted only from one type of stress inducing stimulus when four
5 stress inducing stimuli were tested.
While heat shock proteins can themselves be used as the immunogenic
determinant in vaccine compositions, it has been observed that complexes formed
between heat shock proteins and peptides, in particular antigenic peptide
10 fragments, mediate an enhanced immune response when administered to a
subject. Furthermore, it is known that heat shock proteins can produce complexes
which can be classed as either constitutively produced heat shock protein
complexes or induced heat shock protein complexes. Constitutively produced
heat shock protein complexes are those comprising heat shock proteins which are
15 produced under normal homeostatic conditions. Induced heat shock protein
complexes are produced when a cell is exposed to conditions of stress. When in a
stressed state, the cell upregulates the production of stress proteins, with these
upregulated heat shock proteins being known as induced heat shock proteins.
Furthermore, these induced heat shock proteins form complexes with peptide
20 fragments which are seen to be more immunogenic than complexes formed when
a cell is not under conditions of stress. The enhanced immunogenicity of such
induced heat shock protein complexes over constitutive heat shock protein
complexes has been exemplified in WO 01113943. Without wishing to be bound
by theory, the inventors predict that the enhanced immunogenicity observed with
25 induced heat shock protein complexes is not due to the actual stress protein
component of the complex being different, but due to the stress conditions causing
proteins within the cell to unfold or denature. The heat shock proteins therefore
complex with proteins or protein fragments to prevent them unfolding, or to refold
them, as well as complexing with protein fragments as part of the cell's antigen
30 processing pathway.
Vaccine compositions which comprise heat shock protein complexes (which may
also be referred to as stress protein complexes) as the immunogenic determinant
are widely known. Such vaccines have significant potential as they show the
promise of conferring broad, protective immunity against infection and disease. It
has also been widely documented that heat shock protein complexes are
5 efficacious as vaccines against specific cancers. 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
10 No. WO 0111 3944). Moreover, it has been shown in WO 02120045, WO 0011 0597
and WO 0111 3943 that stress protein complexes isolated from pathogens or
pathogen infected cells are effective as the immunogenic determinant within
vaccines against infectious diseases.
15 There have been various approaches to producing vaccine compositions
comprising stress protein complexes for the treatment and prevention of infectious
diseases. WO 95124923 discloses constitutive heat shock protein complexes
comprising a heat shock protein derived from a host eukaryotic cell and an
antigenic peptide fragment derived from a pathogen. WO 0111 3943 discloses heat
20 shock protein complexes which are induced following the use of a stress inducing
stimulus such as heat or a cytokine such as tumour necrosis factor (TNF). Said
stress proteins may comprise stress proteins derived from a host cell, or,
alternatively, stress proteins derived from an invading pathogen, said stress
proteins being complexed to a peptide derived from the invading pathogen. WO
25 01113944 discloses stress protein complexes which are produced following a
pathogen being subjected to a stress inducing stimulus, wherein the stress protein
and associated peptide fragment are derived directly from the pathogen.
Summary of the invention
30 Following extensive experimentation, the inventors have surprisingly observed that
the production of induced heat shock protein complexes using a plurality of stress
inducing stimuli, typically heat stress and respiratory stress or heat stress and acid
based stress, enhances the quantum of induced stress protein complexes
produced in a cell, when compared to induced stress protein complexes produced
following exposure to a single type of stress inducing stimulus. Specifically, the
inventors have observed that the amount of heat shock protein-peptide complexes
5 (HspCs) which are produced following the exposure of a cell or cells to multiple
stress stimuli can result in a four-fold increase in the number of heat shock proteinpeptide
complexes produced. The production of a higher yield of heat shock
protein-peptide complexes is of significant commercial relevance as large
quantities of heat shock protein complexes would be required for large scale
10 production of a prophylactic or therapeutic vaccine. Moreover, the inventors have
also surprisingly indentified that the induced heat shock protein-peptide complexes
produced using a plurality of stress inducing stimuli, such as heat stress and
respiratory stress or heat stress and acid based stress, are more immunogenic
than induced heat shock protein complexes produced using only a single stress
15 inducing stimulus, such as heat shock alone.
According to a first aspect of the present invention there is provided a method for
the production of stress protein complexes formed between a stress protein and a
peptide, said method comprising, consisting of, or consisting essentially of the
20 steps of:
- culturing cells,
- exposing said cells to a plurality of stress inducing stimuli, and
- purifying the stress protein complexes from the cells.
25 In certain embodiments, the peptide is a peptide fragment. In certain
embodiments, the peptide is an antigenic peptide or an antigenic peptide
fragment. In certain embodiments, the peptide is derived from a pathogenic
organism which typically causes an infectious disease, e.g. a prokaryotic organism
(e.g. gram positive bacteria or gram negative bacteria), protozoa, a virus, a
30 parasite or fungi.
In certain embodiments wherein the peptide is derived from bacteria, the bacteria
are selected from the group consisting of Escherichia, Streptococcus,
Staphylococcus, Bordetella, Corynebacterium, Mycobacterium, Neisseria,
Haemophilus, Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia,
5 Fancisella, Pasturella, 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.
10
In certain embodiments wherein the peptide is derived from a virus, the virus is
selected from the group consisting of human immunodeficiency virus, hepatitis A
virus, hepatitis B, hepatitis C, human papillomavirus, Kaposi's Sarcoma-
Associated Herpesvirus, Herpes Simplex virus, Respiratory Syncytial Virus, Ebola
15 virus, Marburg virus, West Nile virus, St Louis Encephalitis virus, Rift Valley Fever
virus, Influenza viruses, corona virus, 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.
20
In certain embodiments, the peptide is a tumour specific antigen.
In certain embodiments, exposing the cells to the plurality of stress inducing stimuli
comprises exposing the cells to at least two stress inducing stimuli. Typically, the
25 cells are exposed to two stress inducing stimuli of different types, but may be
exposed to three or more stress inducing stimuli of different types. In certain
embodiments, the stress inducing stimuli can be selected from the group
consisting of, but not limited to, heat stress, respiratory stress (oxygen starvation
or addition), oxidative stress (H202, Fe), acid based stress such as pH change,
30 heavy metal stress, osmotic stress, metabolite restriction and nutrient starvation
such as carbon or iron limitation. In certain preferred embodiments, the stress
inducing stimuli is heat stress and at least one of respiratory stress, oxidative
stress (H202, Fe), acid based stress (pH4), heavy metal stress, osmotic stress,
metabolite restriction or nutrient starvation such as carbon or iron limitation, and in
particular may be heat stress and respiratory stress or heat stress and acid based
stress. Typically the at least two stress inducing stimuli are applied to the cells
5 sequentially. In certain embodiments, the at least two stress inducing stimuli are
applied sequentially.
In certain embodiments, the present invention therefore provides a method for the
production of complexes formed between a stress protein and an antigenic
10 peptide, said method comprising, consisting of, or consisting essentially of the
steps of:
- culturing cells,
- exposing said cells to a heat stress,
- exposing said cells to a respiratory stress, and
- purifying the heat shock protein complexes from the cells.
Typically said cells are exposed to the heat stress and the respiratory stress
simultaneously.
20 In alternative embodiments, the present invention provides a method for the
production of complexes formed between a stress protein and an antigenic
peptide, said method comprising, consisting of, or consisting essentially of the
steps of:
- culturing cells,
25 - exposing said cells to a heat stress,
- exposing said cells to an acid based stress, and
- purifying the heat shock protein complexes from the cells.
Typically said cells are exposed to the heat stress and acid based stress
30 simultaneously, that is, the cells are exposed to the heat stress and the acid based
stress at the same time.
In certain embodiments, the heat stress or heat shock comprises increasing the
heat to which the cultured cells are exposed to a temperature of around 5°C to
10°C above the normal growth temperature of the cells. Accordingly, if the cells
are typically grown at 37"C, then the heat stress may comprise increasing the
5 temperature to which the cells are exposed to about 44°C. In certain
embodiments, said temperature increase is achieved by raising the temperature
within a fermenter, which may be used, for example, to cultivate the cultured
cells to around 42°C to 44°C. In certain embodiments, the temperature
increase is achieved by increasing the temperature from the normal growth
10 temperature at a rate of between or around 0.25"C to 0.5"C per minute (0.25-
0.5"CImin). In certain embodiments, the cells are subjected to a heat stress for
a time period ranging from around 30 minutes to 2.5 hours. In certain
embodiments, the heat stress may typically occur for between 1 to 2 hours.
15 In certain embodiments, the respiratory stress relates to decreasing the amount
of oxygen to which the cultured cells are exposed. Typically, this comprises
restricting the supply of oxygen to the cultured cells from that which causes the
normal physiological growth or homeostasis of the cells. In certain
embodiments, this can be achieved by removing the dissolved oxygen tension
20 (DOT) cascade control as the temperature of the culture increases. In a
preferred embodiment the dissolved oxygen tension can be further limited by
manually reducing the agitation rate, for example, to approximately 320 to 350
rpm. Clark et al., (1 985) Biotechnology and Bioengineering, 27:1507-1511
describes how the dissolved oxygen tension can be controlled in a fermenter
25 via the agitation rate. In a further embodiment, oxygen limitation is achieved by
its partial or complete replacement with carbon dioxide or nitrogen. In certain
preferred embodiments, the respiratory stress is applied to the cells in a
fermenter. In certain embodiments, the respiratory stress relates to increasing
the amount of oxygen to which the cultured cells are exposed.
In certain embodiments, the acid based stress or acid stress comprises
reducing the pH of the cultured cells to a pH below the normal pH at which the
cells are cultured, the normal pH being the pH which causes normal
physiological growth or homeostasis of the cells. In certain embodiments, the
pH is lowered to 5.5, 5, 4.5 or 4. The pH may be lowered by adding an acid,
e.g. hydrochloric acid to the cells. In certain embodiments, the acid based
5 stress is applied to the cells in a fermenter.
The cells may be exposed to the plurality of stress inducing stimuli
simultaneously or sequentially. Accordingly, in certain embodiments, the cells
are subjected to the heat stress prior to being exposed to a second stress, such
10 as the respiratory stress or acid based stress. In a further embodiment, the
cells are subjected to the heat stress and to a second stress, such as the
respiratory stress or acid based stress, concurrently. In a further embodiment,
the cells may be subjected to another stress such as the respiratory stress or
acid based stress first and then subjected to the heat stress.
15
A preferred combination of stresses can be determined by using standard
methods to apply stress inducing stimuli and then quantitating the induction of
stress proteins, such as GroEL and DnaK. Typically the preferred combination of
stresses increases the induction of both GroEL and DnaK. Standard methods
20 which may be used to quantitate the induction of stress proteins include protein gel
analysis, densitometry, immunoblotting and ELISA.
Pathogenic cells
In certain embodiments, the cells are pathogenic cells. In certain embodiments,
25 the pathogenic cells are non-mammalian cells, in particular prokaryotic cells
which may be gram positive or gram negative bacteria. In certain further
embodiments, the pathogenic cells are microbial cells, protozoan cells or
parasitic cells.
30 In certain embodiments, the prokaryotic cells are bacteria selected from the group
consisting of, but not limited to: Escherichia, Streptococcus, Staphylococcus,
Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus,
Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,
Pasturella, Moraxella, Acinetobacter, Erysipelothrix, Branhamella, Actinobacillus,
Streptobacillus, Listeria, Calymmatobacterium, Brucella, Bacillus, Clostridium,
Treponema, Salmonella, Kleibsiella, Vibrio, Proteus, Erwinia, Borrelia, Leptospira,
5 Spirillum, Campylobacter, Shigella, Legionella, Pseudomonas, Aeromonas,
Rickettsia, Chlamydia, Borrelia and Mycoplasma.
In certain embodiments, the bacteria are selected from the group consisting of
Neisseria (e.g . N. meningitidis MC58), Mycobacteria, Clostridium (e.g .
10 Clostridium dificile), Saccharomyces (e.g.S. cerevisiae) and Streptococcus,
e.g. S. pneumoniae. A combination of heat stress and respiratory stress has
been shown to result in higher levels of production of stress protein complexes
in Neisseria, Mycobacteria, Saccharomyces and Clostridium when compared to
stress protein complexes obtained from cells exposed to only one type of stress
15 inducing stimulus. Accordingly, in certain embodiments the cells are Neisseria
(e .g . N. meningitidis MC58), Mycobacteria, Saccharomyces (e .g . S. cerevisiae)
or Clostridium (e.g. Clostridium difficile) and the plurality of stress inducing
stimuli comprises heat stress and respiratory stress. A combination of heat
stress and acid based stress has been shown to result in higher levels of
20 production of stress protein complexes in S. pneumoniae when compared to
stress protein complexes obtained from cells exposed to only one type of stress
inducing stimulus. Accordingly, in certain embodiments the cells are
Streptococcus, e.g, S. pneumoniae, and the plurality of stress inducing stimuli
comprises heat stress and acid based stress. The stress protein complexes
25 obtained from cells exposed to two different types of stress inducing stimuli also
show improved immunogenicity when compared to stress protein complexes
obtained from cells exposed to only one type of stress inducing stimulus.
Aerobic cells
30 In certain embodiments, the cells are aerobic cells, for example pathogenic
aerobic cells. During the normal growth of cells in culture, the cells will have an
optimum requirement for oxygen. For example, an aerobic cell will require
oxygen for growth and survival. The depletion of the oxygen level will limit the
ability of an aerobic cell to grow. The absence of oxygen in the growth medium
will most likely result in the death of the cell. Any particular cell will have a
preferred dissolved oxygen tension (DOT), this being the oxygen supply
5 provided to the growing and stationary cells in culture. For example, a cell
culture may be grown with a dissolved oxygen tension (DOT) at a range of
between >I 0% to 50%. In certain preferred embodiments, the dissolved
oxygen tension (DOT) may be provided at a level of, or around >20% or of
about >30%.
10
Facultative anaerobic cells
In certain embodiments, the cells are facultative anaerobic cells, for example
pathogenic anaerobic cells. A facultative anaerobic organism is an organism
which usually makes ATP by aerobic respiration. However, when oxygen is not
15 present, the facultative anaerobe can switch to fermentation. Facultative
anaerobes therefore survive in the presence of oxygen, while obligate anaerobes
die. Further, the concentration of oxygen and fermentable material in the growth
environment will influence whether the facultative anaerobe uses aerobic
respiration or fermentation to derive energy. The depletion of the oxygen level will
20 therefore limit the ability of a facultative anaerobe to grow. However, the absence
of oxygen in the growth medium or environment may result in a switch to
anaerobic growth. Accordingly, exposing a facultative anaerobic cell to respiratory
stress will involve carefully managing the dissolved oxygen tension (DOT) to which
the cells are exposed in order to induce respiratory stress. The application of
25 respiratory stress may also involve the removal of fermentable material from the
growth environment.
Anaerobic cells
In certain embodiments, the cells are anaerobic cells, for example pathogenic
30 anaerobic cells. In a certain embodiment of the invention, stress protein
complexes may be derived from anaerobic pathogens (obligate anaerobes), in
particular anaerobic bacteria, which cannot grow in the presence of oxygen.
Accordingly, the application of respiratory stress to obligate anaerobes requires
oxygen to be added to the culture medium, rather than removed, in order to
confer an oxidative stress linked stress inducing stimulus. Accordingly, an
anaerobic cell culture may be exposed to respiratory stress by growing it in the
5 presence of a dissolved oxygen tension (DOT) at a range of between >I 0% to
50%. In certain preferred embodiments, the dissolved oxygen tension (DOT)
may be provided at a level of, or around >20% or of about >30%.
Accordingly, in certain embodiments the method comprises a method for the
10 production of complexes formed between a stress protein and an antigenic
peptide or antigenic peptide fragment which is derived from an anaerobic
pathogen, said method comprising, consisting of, or consisting essentially of the
steps of:
- culturing anaerobic pathogenic cells in an environment in which oxygen
is not present,
- exposing said cells to a heat stress,
- further exposing said cells to a respiratory stress, said stress comprising
increasing the amount of oxygen to which the cultured cells are
exposed, and
- purifying the stress protein complexes from the anaerobic pathogenic
cells.
In certain embodiments, said cells are exposed to the heat stress and respiratory
stress simultaneously, that is, the cells are exposed to the heat stress and the acid
25 based stress at the same time.
In certain embodiments, nitrogen may be present in the culture medium to
compensate for the lack of oxygen.
30 Cancerous cells
In certain embodiments, the cells are cancerous cells. Accordingly in certain
embodiments the present invention provides a method for the production of
complexes formed between a stress protein and an antigenic peptide, said method
comprising, consisting of, or consisting essentially of the steps of:
- culturing cancerous cells,
- exposing said cells to a heat stress,
- exposing said cells to a respiratory stress or an acid based stress, and
- purifying the heat shock protein complexes from the cancerous cells.
Typically said cells are exposed to the heat stress and the acid based stress or
respiratory stress simultaneously, that is, the cells are exposed to the heat stress
10 and the acid based stress at the same time.
Genetically modified cells
In certain embodiments, the invention extends to a cell or cells which have
been genetically modified to constitutively express heat shock proteins, e.g. by
15 deleting hspR and/or hrcA. Said cells can be further subjected to one or more
additional stress inducing stimuli in accordance with the present invention.
Typical additional stress inducing stimuli include respiratory stress such as
oxygen limitation, pH stress (for example acid stress at pH4) and metabolite
restriction such as carbon or iron limitation.
20
The prokaryotic heat shock protein families DnaJ, DnaK, GroEL and GroES are
encoded in operons, with the initial gene in the operon being a control gene which
suppresses the expression of the heat shock protein genes contained within the
operon. In Streptomyces and Helicobacterfor example, expression of the hspR
25 gene suppresses the expression of DnaJ and DnaK. Deletion of the hspR gene
therefore results in a genetically modified microbe that constitutively expresses
heat shock proteins (see Bucca et al. (2003) Mol. Microbiol50(1)153-166).
However, it should be noted that the two major heat shock protein families DnaK
and GroEL are regulated by two different regulons hspR and hrcA. Of these heat
30 shock regulons, the hsp70 / DnaK regulon is under the control of hspR, while the
hsp60 /groEL regulon is under the control of hrcA. The deletion of both these
genes would be required to maximally upregulate both Dnak and GroEL heat
shock protein expression (Holmes et al. (201 0) Microbiology 156:158-166 amd
Aravindhan V. et al. (2009) FEMS Microbial Lett. 292 42-49). Homologous
operons have been identified in a number of recently sequenced microbes,
including other strains of Streptomyces and Mycobacterium tuberculosis and the
5 commonly used related vaccine strain BCG.
Other repressor genes may also control the expression of other stress proteins
and these may also be genetically modified to provide modified microbes that
constitutively express stress proteins. These include, but are not limited to, the
10 transcriptional control genes sigma and rho and the stress-gene regulatory protein
genes hrcA, MerR and HmrR.
Accordingly, the present invention may further extend to the use of a genetically
modified pathogen which has been genetically modified to knock out or disable at
15 least one repressor gene which controls the expression of a heat shock protein,
wherein said genetically modified pathogen is subjected to respiratory stress or
acid stress prior to the isolation and/or purification of the induced heat shock
protein complexes for use as the immunogenic determinants in vaccine
compositions.
20
In certain embodiments, the cells are cells which have been genetically modified
such that they express a heterologous protein which is derived from a cancerous
cell. In alternative embodiments, the cells are cells which has been genetically
modified such that they express a heterologous protein derived from a pathogen
25 which causes an infectious disease in a host.
Infected Cells
In certain embodiments, the cells are host cells which are infected with a
pathogenic organism. The complexes may be formed from a heat shock
30 protein derived from the host cells complexed to a peptide fragment derived
from the invading pathogen, or from a heat shock protein and peptide fragment
which are both derived from the invading pathogen.
Accordingly, in certain embodiments the invention provides a method for the
production of complexes formed between a heat shock protein and a peptide
fragment, said method comprising, consisting of, or consisting essentially of the
5 steps of:
- culturing cells which are infected with a pathogen,
- exposing said cells to multiple stress inducing stimuli, and
- purifying the heat shock protein complexes from the cultured cells.
10 In certain embodiments, the multiple stress inducing stimuli comprises at least 2
stress inducing stimuli which are applied to the cells simultaneously and which
can include heat stress and respiratory stress or heat stress and acid based
stress.
15 The methods of the present invention advantageously provide a mixture of purified
heat shock protein complexes wherein this mixture of heat shock protein
complexes comprises different subtypes of heat shock proteins. That is, the heat
shock protein components of the heat shock protein complexes may be heat
shock proteins of different families of heat shock subtypes. For example, there
20 may be a mixture of heat shock proteins derived from classes selected from, but
not limited to, HSPGO, HSP70 and/or HSPSO, or from any other heat shock protein
class which is present in a eukaryotic cell or a pathogenic cell. In certain
embodiments, it is preferred that the heat shock protein complexes comprise or
consist of heat shock proteins of the GroEL (the prokaryotic heat shock protein
25 which is equivalent to HSPGO in mammalian cells) and/or DnaK (the prokaryotic
heat shock protein which is equivalent to HSP70 in mammalian cells) heat shock
protein families. Accordingly, in certain embodiments of the invention, the
purification methods used herein are used to purify protein complexes wherein the
heat shock protein component comprises DnaK and GroEL.
Inducing and purifying heat shock protein complexes
In certain embodiments, the heat shock protein complexes are purified or isolated
from a cell lysate obtained from the cell, e.g. the pathogenic cell, cancerous cell or
the cell infected with a pathogen, which has been subjected to the stress-inducing
stimuli.
5
Typically the stress protein complexes comprise heat shock proteins of different
heat shock protein classes. In certain embodiments, the heat shock proteins are
of the subtypes DnaK and/or GroEL. Said purified and/or isolated heat shock
protein complexes, or a preparation or mixture comprising the same, can then
10 typically be used as the immunogenic determinant in a vaccine composition to
elicit an immune response and associated protective immunity against the
pathogen or cancerous cell from which the stress protein complexes are derived,
or against a pathogen which is infecting a cell from which the stress protein
complexes are derived.
15
In certain embodiments, purifying the heat shock protein complexes comprises:
(i) providing a clarified cell lysate from the culture cells, wherein the cell
lysate comprises the stress protein complexes,
(ii) subjecting the cell lysate to purification using ion exchange, wherein
20 the cell lysate is buffered to a pH within 2 units of the pl of the target
heat shock protein complexes and wherein a salt gradient is used to
elute the heat shock protein complexes, and
(iii) obtaining an enriched preparation comprising the heat shock protein
complexes.
25
In certain embodiments, the buffer comprises divalent cations which may be
provided at a concentration of from about 0.1 mM to 100mM. In certain
embodiments, the divalent cation is a magnesium salt and/or a manganese salt.
In certain embodiments, the buffer further comprises ADP (adenosine
30 diphosphate) which may be provided at a concentration of from about O.lmM to
1OOmM.
In certain embodiments the heat shock protein complexes comprise heat shock
proteins of the classes DnaK and/or GroEL.
Vaccine compositions
5 Typically, said purified heat shock protein complexes obtained by the foregoing
aspect of the invention can be used as the immunogenic determinant in a vaccine
composition for use in mediating an immune response against the pathogen from
which the stress protein complexes were derived.
10 In various further aspects, the invention therefore extends to vaccine
compositions, or to compositions which mediate or elicit an immune response,
which comprise the heat shock protein complexes which are obtained by the
methods of the invention. Said vaccine compositions are typically administered to
mammals, in particular humans, in order to confer protective immunity against a
15 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.
As such, a further aspect of the invention provides a vaccine composition
20 comprising, as the immunogenic determinant, purified heat shock protein
complexes obtained by the method of the present invention.
In certain further aspects, the present invention provides a vaccine composition
according to the invention, or a purified and/or isolated mixture of heat shock
25 protein complexes obtained using the methods of the invention, for use in
medicine.
In certain further aspects the present invention provides the use of heat shock
protein complexes produced in accordance with any of the foregoing aspects of
30 the invention in the preparation of a medicament for the prevention or treatment of
an infectious disease or a cancerous or malignant condition.
In certain further aspects, the present invention provides heat shock protein
complexes for use in a vaccine composition for the treatment or prevention of an
infectious disease or a cancerous or a malignant condition.
5 In certain embodiments, the heat shock protein complexes or the vaccine
compositions containing the same are administered as a prophylactic vaccine. In
certain further embodiments, the purified stress protein complexes or the vaccine
compositions comprising the same are administered as therapeutic vaccines.
10 In various further aspects, the present invention extends to the heat shock protein
complexes or to preparations or mixtures comprising the same, or to vaccine
compositions containing the same, for use 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
15 due to the previous administration of a primary vaccine.
Compositions of the invention may be lyophilised or in aqueous form, i.e. solutions
or suspensions. Liquid formulations of this type allow the compositions to be
administered direct from their packaged form, without the need for reconstitution in
20 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
composition, whereas a vial may include a single dose or multiple doses (e.g. 2
doses).
25
In various further aspects, the present invention provides a method for producing a
vaccine composition comprising the step of mixing the purified heat shock protein
complexes of the invention, or a preparation or mixture comprising the same,
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
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 Mycobacterium
5 leprae, Salmonella typhi, Streptococcus pneumonia, Vibrio Cholerae and
Neisseria meningitides. In a 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
10 to, influenza, hepatitis, herpes, human immunodeficiency virus (HIV), human
papaloma virus (HPV), respiratory synctial virus (RSV), Polyoma, cytomegalovirus
(CMV), Epstein-Bar virus (EBV), Rotovirus, Norovirus, coronavirus, hepatitis A
virus (HAV), hepatitis B (HBV), hepatitis C (HCV), human papillomavirus (HPV),
Kaposi's Sarcoma-Associated Herpesvirus (KSHV), Herpes Simplex virus (HSV),
15 Respiratory Syncytial Virus, Ebola virus, Marburg virus, West Nile virus (WNV), St
Louis Encephalitis virus (SLEV), Rift Valley Fever virus (RVFV), Influenza viruses,
coronaviruses, rhinovirus, adenovirus, SIV, rotavirus, arbovirus, measles virus,
polio virus, rubella virus, mumps virus, papova virus, varicella-zoster virus,
varicella virus, huntavirus and cytomegalovirus.
20
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.
25 Additionally, a yet further aspect of the invention extends to a method of
immunising a subject, typically a human, against disease caused by Bordetella
pertussis, Clostridium tetani, Clostridium difficile, Corynebacterium diphtheriae,
Haemophilus influenzae type b, Mycobacterium tuberculosis and leprae,
Salmonella typhi, Vibrio Cholerae, Streptococcus pneumonia, Neisseria
30 meningitidis and pathogenic and oncogenic viruses, which method comprises
administering to the host an immunoprotective dose of the vaccine of the
invention.
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
5 will vary depending upon which specific immunogen is employed and how it is
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 complexes produced using
the present methodology, when compared to a vaccine composition comprising a
10 similar amount of protein complexes obtained using the production methods
known in the art.
The invention further provides for the use of the heat shock protein complexes of
the invention in a method of vaccinating a subject to induce immunity against a
15 pathogen derived infectious disease or cancerous or 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 heat shock protein complex provided according to
any one of the methods of the present invention, said heat shock
protein complex being derived from a cancerous cell, a pathogenic
cell or a cell infected with a pathogen or expressing a heterologous
antigen which has been subjected to heat shock and respiratory or
acid based stress inducing stimuli against which protective
immunity is desired, and comprising different heat shock protein
types as a mixture, and
- administering the vaccine composition comprising the heat shock
protein complex to the subject in a therapeutically effective or
prophylactically effective amount sufficient to elicit an immune
response in the subject against the heat shock protein complex.
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 induced heat
shock protein complexes of the invention are derived can 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 particularly useful as
20 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 heat shock protein is derived.
The inventors have further surprisingly identified that heat shock protein
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 complex comprising a peptide derived from the pathogen against
which immunity is desired, said method comprising the steps of:
- providing a composition comprising a heat shock protein complex
or complexes prepared according to any foregoing method of the
present invention, said purified heat shock protein complex or
complexes 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,
and
- administering the composition to the subject in an amount sufficient
to elicit an immune response in the subject against the stress
protein complex or complexes.
In certain further embodiments, the heat shock protein complex containing
vaccines of the present invention may be used for boosting immune responses in
20 animals that have been previously immunised with other subunit, multi-subunit,
carbohydrate or conjugate vaccines. In yet further embodiments, the heat shock
protein complex vaccines of the present invention can be used to boost the
immune responses against a target antigen in animals that have been previously
immunised with nucleic acid or live vaccines. In yet further embodiments, the heat
25 shock protein complex containing vaccine compositions of the present invention
provide for boosting immune responses mediated in subjects that have been
previously immunised against a pathogen or cancer specific antigen.
In certain further aspects, the present invention extends to vaccine compositions
30 comprising the heat shock protein complexes induced by the methods of the
present invention for use in boosting 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.
In certain further aspects, the present invention extends to vaccine compositions
5 comprising the heat shock protein complexes produced by the invention for use in
boosting immune responses in animals, wherein the animal has previously been
exposed to a pathogen or cancer antigens derived from the same or related cells
as those from which the heat shock protein complexes have been derived.
10 In certain further embodiments, the present invention provides compositions for
the preparation of cellular vaccines such as dendritic cells (DCs) which have been
pulsed with the purified stress protein complexes of the invention. Administration
of such pulsed dendritic cells to a subject will result in a T-cell mediated response
being directed against the heat shock protein complexes. Such a therapy can be
15 particularly effective when treating a subject with a cancerous or malignant
condition. In such embodiments, typically the heat shock protein complexes are
derived from a cancerous cell.
In certain embodiments, the vaccine composition of the invention may be replaced
20 with a composition for inducing an immune response, or by a composition for
eliciting an immune response, said compositions typically comprising the same
immunogenic determinants as those provided in the vaccine compositions
described herein.
25 Brief Description of the Figures
Figure 1A shows the time course of heat shock protein induction from cells
which are stressed with both heat shock and oxygen limitation (respiratory
stress). Lane 2 shows pre-induction protein levels and lanes 3-7 show 30
minute intervals of a 0-2 hour stress induction. Increasing amounts of
protein (GroEL and DnaK) can be seen in the bands at 60 and 70KD in
lanes 3 to 7, these representing increased amounts of GroEL (70kDa) and
DnaK (6OkDa). Lanes 8 and 9 show increasing amounts of hsp60 and
hsp70 standards, with these being the major heat shock proteins induced.
Lanes 1 and 11 contain molecular weight (MW) markers.
Figure 1 B shows a comparison of heat shock protein production following
heat shock alone (lane I ) and heat shock and respiratory stress (oxygen
limitation) (lane 2). Lane MW shows molecular weight markers and the
lanes marked "Hsp stds" shows purified hsp60 and hsp70 proteins. A
comparison of lanes 1 and 2 shows that there is a significantly higher level
of heat shock protein induction in lane 2, this being depicted by the
presence of significantly darker bands, corresponding to the bands seen in
the hsp60 and hsp70 marker lanes (Hsp stds),
Figure 2 (A-F) shows the immune response to heat shock protein
complexes made from cells subjected to heat shock only (groups 1 and 2)
and to a combination of heat shock and respiratory stress (group 3) as
assayed by antibody-dependent opsonophagocytosis (OPA) of
fluorescently labelled clinically relevant Neisserial strains (panels A-F). The
positive control (column I ) shows results using sera from animals
vaccinated with an outer membrane vesicle (OMV) preparation from the
homologous strain. Group 1 shows results using sera from animals
vaccinated with complexes produced using heat stress inducing stimulus
only, purified by Ion-exchange using a HEPES based buffer. Group 2
shows results using sera from animals vaccinated with complexes produced
using heat stress inducing stimulus only, purified by Ion-exchange using a
Tris based buffer. Group 3 shows results using sera from animals
vaccinated with complexes produced using a combination of heat shock
and respiratory stress stimulii, using the same Tris buffer purification as
used in Group 2.
Figure 3 shows the time course of stress protein induction from
S.pneumoniae cells which are stressed with a number of distinct stress
stimuli, namely oxidative, osmotic, heavy metal and acid stress. Increasing
amounts of heat shock proteins (hsp60 and hsp70) can be seen in the
Western blots using antibodies against GroEL and DnaK to analyse stress
inducing stimuli for use in combination with heat shock or a combination of
heat and respiratory stress.
Figure 4 shows the induction of stress proteins from S.pneumoniae cells
which are stressed for either 5 or 15 minutes with a combination of heat and
acid stress. Heat shock was constant at 42OC and acid shock was at pH
4.5, 5 and 5.5. The optimal combination of heat shock and pH 5 can be
determined from a comparison of the Western blots using antibodies
against hsp60 and hsp70 in lanes 7 and 8, which show a clear improved
induction of both GroEL and DnaK as compared to lanes 1 to 4 which show
cells subjected to only a single stress inducing stimulus.
Figure 5 shows the immune response to stress protein complexes made
from cells subject to either heat stress alone (HS vaccine) or a double
stress combination of heat and acid stress (DS vaccine), as assayed by
antibody-dependent opsonophagocytosis assay (OPA) of fluorescently
labelled S.pneumoniae strain Rxl. The positive control is sera from mice
immunised with whole killed cells from the homologous strain (Rxl) and the
isolated stress protein complexes from the single stress (heat shock only)
were additionally tested at a higher dose (68yg) than the double stress
vaccine (50yg).
Figure 6 shows the breadth of the immune response to stress protein
complexes made from cells subject to either heat stress alone (HS vaccine)
or a double stress combination of heat and acid (DS vaccine), as assayed
by enzyme-linked immunoassay (ELISA) against clinically relevant
S.pneumoniae strains of different, heterlogous, serotypes. The positive
control is sera from mice immunised with whole killed cells from the strain
Rxl and the stress protein complexes from the single stress (heat shock
only) were additionally tested at a higher dose (68yg) than the double
stress vaccine (50yg).
Detailed Description of the Invention
5 The present invention provides stress protein-peptide complexes, wherein the
production of the stress protein is induced following the cell being subjected to a
plurality of stress inducing stimuli, typically a heat shock and a respiratory stress or
a heat shock and an acid based stress.
10 The inventors have identified that stressing a cell with at least two stress inducing
stimuli results in a significantly higher level of heat shock proteins being induced,
with this increase in production being assessed by the inventors as comprising at
least a two-fold increase, preferably three- or four-fold, over protein levels
produced following exposure to a single stress inducing stimulus. This is
15 particularly surprising for disparate stress inducing stimuli such as heat and
respiratory stress as these are generally thought to be subject to control by
different genetic and transcriptional elements. Accordingly, it is entirely
unexpected that following the exposure of a prokaryotic cell to heat stress, the
amount of heat shock protein produced in response to that stress inducing
20 stimulus can be further (and significantly) enhanced by exposing the cell to a
secondary stress inducing stimulus.
Furthermore, the inventors have surprisingly identified that heat shock protein
complexes which are produced using the methods of the invention are more
25 immunogenic than similar complexes obtained following a single stress inducing
stimulus, or when compared to heat shock protein complexes which are
constitutively produced. Hence, the heat shock protein complexes produced by
the methods of the invention are more immunogenic than those produced using
standard production methods known in the art and can be used to produce
30 improved vaccine preparations.
Heat shock protein complexes
In certain embodiments, the heat shock protein complex can be a heat shock
protein complex (HspC) comprising a heat shock protein which is complexed to a
peptide or peptide fragment. In certain embodiments, the heat shock protein can
5 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 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
10 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, calrecticulin, hsp40, hsp70, hsp72, hsp90, grp94, grp75,
BiPlgrp78, grp75/mt, gp96 and small heat shock proteins (hsps). In certain
embodiments, it is preferred that the heat shock protein is GroEL and / or DnaK.
15
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
20 present invention provides a method for the purification of all complexes
comprising a heat shock protein complexed to a (antigenic) peptide, irrespective of
the identity, molecular weight or size of the peptide.
In certain embodiments, the target heat shock protein complex comprises a heat
25 shock protein complex derived from a host cell which has been genetically
modified to constitutively express stress protein genes, and/or 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 a yeast cell carrying an expression vector construct comprising an
30 antigenic gene of interest. In yet further embodiments, the cell may be a
cancerous cell derived from a human or animal subject.
In certain further embodiments, heat shock protein complex (HspC) enriched
preparations (HEPs) comprise heat shock proteins from different stress protein
families or classes, such as DnaK, GroEL, hsp60, hsp65, hsp70 and hsp90, said
families being co-purified as a mixture using the methods of the invention.
5
In certain further embodiments, the heat shock protein complex (HspC) enriched
preparations (HEPs) may be heat shock protein complexes of a particular
molecular weight. In certain embodiments, the stress protein complexes have a
molecular weight in the range of 50KDa to 9OOKDa.
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, ASOI, 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 stress protein complexes of the present invention
30 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 or rectal. The formulation may be a liquid, for example, a physiologic salt
5 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 needleless 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
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
30 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 heat shock protein complexes of the present
invention may also be administered via microspheres, liposomes, other
microparticulate delivery systems or sustained release formulations placed in
certain tissues including blood.
5
Dosage regimens can include a single administration of the composition of the
invention, or multiple administrative doses of the composition. The compositions
can further be administered sequentially or separately with other therapeutics and
medicaments which are used for the treatment of the condition for which the
10 composition of the present invention is being administered to treat.
The actual amount administered, and rate and time-course of administration, will
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
15 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
practitioners.
20 Definitions
As herein defined, the term "stress inducing stimulus" means a stimulus which
is capable of inducing a stress response within a cell or cells subjected to the
stimulus. As herein defined, the term "plurality of stress inducing stimuli" or
"multiple stress inducing stimuli" refers to at least two stress inducing stimuli
25 and means two, three or more stress inducing stimuli. The stress inducing
stimulus may include, but is not limited to, respiratory stress, cultivation under
limited nutrient levels, exposure to a cytokine (such as tumour necrosis factor
(TNF) or interferon gamma (IFN-gamma)), osmotic shock of a pathogen (in
particular, once it has been cultivated to statutory growth phase by the addition
30 of high concentrations of an electrolyte, such as sodium chloride, to the
cultivation medium), acid based stress, pH variation, metabolite restriction or
nutrient starvation, such as iron or carbon limitation, cultivation under high
pressure, exposure to heavy metals and exposure to oxidising agents.
Unless otherwise defined, all technical and scientific terms used herein have the
5 meaning commonly understood by a person who is skilled in the art in the field of
the present invention.
Throughout the specification, unless the context demands otherwise, the terms
"comprise" or "include", or variations such as "comprises" or "comprising",
10 "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.
As used herein, terms such as "a", "an" and "the" include singular and plural
referents unless the context clearly demands otherwise. Thus, for example,
15 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,
while references to "a carrier" includes mixtures of two or more carriers as well as
a single carrier, and the like.
20 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
the pathogen or cancer cell from which the immunogenic determinant of the
vaccine composition is derived. The term 'treatment' therefore refers to any
25 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
30 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
disease or cancerous condition. The term "therapeutic" does not necessarily imply
that a subject is treated until total recovery. Similarly, "prophylactic" does not
5 necessarily mean that the subject will not eventually contract a disease condition.
A "subject" in the context of the present invention includes and encompasses
mammals such as humans, primates and livestock animals (e. g. sheep, pigs,
cattle, horses, donkeys); laboratory test animals such as mice, rabbits, rats and
10 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.
As used herein, the terms "mount", "mounted", "elicit" or "elicited" when used in
15 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
composition comprises the isolated and/or purified stress protein complexes
obtained using the methods of the present invention.
20
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 costimulation.
The term immune response further includes immune responses that
are indirectly effected by T cell activation such as antibody production (humoral
25 responses) and the activation of cytokine responsive cells such as macrophages.
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
30 scope of the invention. Although the invention has been described in connection
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
taken as, an acknowledgment or any form of suggestion that this prior art forms
5 part of the common general knowledge in any country.
EXAMPLES
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
10 be construed as being limiting on the present invention.
Example 1 - Improved induction and immunogenicity of stress proteins
in gram negative organisms by multiple stress stimuli
Neisserial strains (N. lactamica and MC58) were initially grown in 500mL non-
15 baffled Erlenmeyer flasks containing 100mL Frantz medium at 37°C with
shaking at 180rpm for 12 hours and then inoculated into a 60L fermenter
containing 54L Frantz medium supplemented with essential amino acids.The
culture was grown at 37°C with dissolved oxygen tension (DOT) maintained at
>30% by agitation cascade to a maximum of 500rpm. DOT was measured
20 using a galvanic dissolved oxygen probe (New Brunswick Scientific) or a redox
sensor (Mettler Toledo). Final fermentation cultures were heat stressed by
raising the temperature of the fermenter to 44°C at a rate of 0.25 - 0.5"CImin.
In some cultures, an additional stress to heat shock was applied by oxygen
limitation (respiratory stress). This was achieved by removing the dissolved
25 oxygen tension (DOT) cascade control as the temperature of the culture rose
towards 44°C and manually reducing the agitation rate to approximately 320 -
350rpm. Samples for product analysis were removed at pre, 0, 1 and 2 hours
post stressing and induction of heat shock proteins analysed by SDS-PAGE
analysis and western blotting using standard equipment and protocols
30 (Invitrogen).
Typical results obtained are shown in Figure I, which clearly shows the time
dependent induction of GroEL and DnaK by the use of respiratory stress as a
supplement to heat stress (Figure IA, lanes 2-7) as can be identified by comigration
with recombinant standards (Fig IA, lanes 8-1 0) and confirmed by
5 western blotting. The unexpectedly additive effects of disparate stress stimuli is
clearly demonstrated by a direct comparison (Fig 1 B) of cultures subjected to
only heat shock (lane 1) and a combination of heat and respiratory stress (lane
2) which shows a clear enhancement of the major heat shock protein families
hsp60 and hsp70.
10
The stressed cell pellets were also resuspended in PBS, lysed using an
Emulsiflex C5 homogeniser and used to prepare heat-shock protein complex
(HspC) enriched vaccine compositions by Ion exchange chromatography as
described in PCT Patent Application No. WO 20101026432. The vaccines were
15 used to immunise groups of 8 mice and the antibody responses quantified for
functionality using complement binding and opsonophagocytosis assays as
described in WO 201 01026432. Typical results obtained are shown in Figure 2
which clearly show improved immune responses elicited by vaccine
compositions comprising, as an immunogenic determinant, heat shock protein
20 complexes derived from cultures that had been subjected to both heat and
respiratory stress (group 3), as compared to vaccine compositions comprising,
as an immunogenic determinant, heat shock protein complexes derived from
cultures that had been subjected to heat shock alone (group 2), as assayed by
opsonophagocytosis (Figure 2). The cross-reactive immunogenicity induced
25 was assayed against a number of fluorescently labelled heterologous Neisserial
strains and normalised to a homologous OMV vaccine as the positive control.
The improved cross-reactive immunogenicity elicited against a number of
clinically relevant Neisserial strains, M01-240013, M01-240101, M01-240149,
M01-240185 and M01-240355 and H44176-SL, covering a broad spectrum of
30 heterlogous circulating serotypes, is illustrated in Fig.2A-F.
Example 2 - lmproved induction and immunoaenicity of stress proteins
in gram positive organisms by multiple stress stimuli
The Mycobacterial vaccine strain, BCG Danish (Statens Serum Institute) was
grown in a Sauton media supplanted with 0.1% Tween 80 and antifoam
5 emulsion C (Sigma). Fermentation was carried out in a 3L bioreactor (Braun)
using 21 cultures grown at 37°C with shaking at 360rpm with dissolved oxygen
tension (DOT) maintained at >20% by agitation cascade to a maximum of
500rpm. Final fermentation cultures were heat stressed (heat shocked) by
raising the temperature of the fermenter to 44°C at a rate of 0.25 - 0.5"CImin
10 for 1 hr. In some cultures oxygen limitation (oxidative stress) was achieved by
removing the dissolved oxygen tension (DOT) cascade control as the
temperature of the culture rose towards 44°C and manually reducing the
agitation rate to approximately 320 - 350rpm.
15 The results obtained showed a clear additive induction of the major heat shock
proteins GroEL and DnaK by the use of respiratory stress in addition to heat
stress. Heat-shock protein enriched vaccine compositions were prepared using
ion exchange chromatography as described in WO 20011026432 and to
immunise groups of mice and rabbits. The antibody responses again showed
20 improved immune responses elicited by vaccine compositions wherein the
immunogenic determinant comprises heat shock protein complexes derived
from cultures that had been subjected to both heat and respiratory stress, as
assayed by western blotting and ELlSA using serum from the immunised
animals. The improved immunogenicity resulted in an increased protection
25 against aerosol challenge with live H37Rv, with a further 0.8 log reduction in
lung colony forming units in mice immunised with vaccine compositions from
the double stressed compared to single stressed BCG.
Example 3 - lmproved induction and immunoaenicity of stress proteins
30 in facultative anaerobes by multiple stress stimuli
Laboratory strain Rxl of Streptococcus pneumoniae was grown in Hoeprich
Medium, pH 7.5 at 37°C in a shaking incubator in an atmosphere of 5% C02.
Cultures were seeded using 0.5ml of a master stock (OD 0.3) into 40ml media
and grown at 50rpm for 5-6 hours (OD 0.2). Cultures were then subjected to
multiple stress inducing stimuli, including heat shock at 40°C for 30-60 minutes,
respiratory shock by removal of the C02 source, pH stress by addition of HCI to
5 adjust culture to pH 5 and iron restriction. Samples of the cultures were
analysed for the induction of heat shock proteins by SDS-PAGE analysis and
Western blotting using standard equipment and protocols (Invitrogen).
Comparisons of the induction of GroEL (hsp60) and DnaK (hsp70) by the
various stress stimuli (Fig.3) were used to select the most promising
10 combinations. Fig.4 shows the results obtained using the selected combination
of heat shock and acid stress (lanes 8,9 and 12,13) which showed a clear
improvement in the induction of both GroEL and DnaK over the induction by
either heat shock (lane 3) or acid stress (lanes 4-6) alone.
15 Heat-shock protein enriched vaccine compositions (HEPs) were prepared from
the Rxl cultures subjected to heat shock or a double stress combination of heat
and acid stress using ion exchange chromatography as described in WO
20011026432 and used to immunise groups of mice. The antibody responses
elicited showed clearly improved immune responses mediated by vaccine
20 compositions from multiple stimuli as assayed by western blotting, ELlSA
(Fig.5) and OPA (Fig.6) using serum from the immunised animals.
HEPs were isolated from S.pneumoniae subjected to either just heat shock at
42°C or a double stress combination of heat and acid shock (pH5) and used to
25 immunise mice. The HEP vaccine from the single stress was also used at a
higher dose (68yg and 50yg) than the vaccine from the double stress (50yg)
and compared to a vaccine of whole killed pathogen as a positive control. The
HEP vaccines from the S.pneumoniae cells induced by the double stress (DS
vaccine) show clearly improved immunogenicity over the single stress vaccine
30 (HS vaccine) even when the latter is used at a significantly higher dosage (Figs.
5 & 6). This improved immunogenicity is seen not just in the affinity and avidity
of the antibodies elicited as assessed by the OPA assay (Fig. 5), but also in the
cross-reactive breadth of the antibodies produced as assessed by ELlSA
against clinically relevant S.pneumoniae strains of different serotypes (Fig. 6).
The heterologous stains used to demonstrate the improved breadth of immunity
induced by the HEP vaccines isolated from the cells subjected to the double
5 stress, covered both serotypes present in the current commercial 13-valent but
not the 7-valent vaccines (serotype 19A) as well serotypes of escape variants
not covered by these vaccines and of emerging clinical importance (serotypes 8
and 22F) in pneumococcal disease. The breadth of protective immunity
induced by the double stress HEP vaccines was also significantly better than
10 that observed with the use of a whole cell killed vaccine.
Example 4 - Improved induction and immunogenicity of stress proteins
in obligate anaerobes by multiple stress stimuli
Laboratory strains of double toxin mutants of Clostridium difficile were grown in
15 TY Medium, pH6.8 at 37°C in a shaking incubator in an atmosphere of
H2:C02:N2(r atio 10:10:80) at 50rpm to achieve an OD of 0.5-0.7 and the
cultures were then subjected to a combination of heat and respiratory stress by
incubation at 44°C for 2 hours in an ambient atmosphere. Samples of the
cultures were analysed for the induction of heat shock proteins by SDS-PAGE
20 analysis and Western blotting using standard equipment and protocols
(Invitrogen). Heat-shock protein enriched vaccine compositions were prepared
using ion exchange chromatography as described in WO 20011026432 and to
immunise groups of mice. The antibody responses again showed improved
immune responses mediated by vaccine compositions wherein the
25 immunogenic determinant comprises heat shock protein complexes derived
from cultures that had been subjected to both heat and respiratory stress, as
assayed by western blotting and titration of the blocking of bacterial adhesion to
epithelial cell cultures.
30 Example 5 - Improved induction of stress proteins by multiple stress in
fungal microorganisms
S.cerevisiae strain ATCC 20602 was grown in Difco YM growth medium, pH5 at
30°C in a 51 benchtop BioFlo 31 0 fermenter (New Brunswick Scientific) at a
dissolved oxygen concentration of 30% achieved by an agitiation speed of 2-
800rpm. After 24 hours the culture was subjected to a combination of heat and
5 respiratory stress by stopping the oxygen feed and raising the temperature to 40°C
for 1 hour. Samples of the cultures were analysed for the induction of heat shock
proteins by SDS-PAGE analysis and Western blotting using standard equipment
and protocols (Invitrogen).
Claims
1. A method for the production of stress protein complexes formed between a
stress protein and a peptide, said method comprising the steps of:
5 (i) culturing cells,
(ii) exposing said cells to a plurality of stress inducing stimuli, and
(iii) purifying the stress protein complexes from the cells.
2. The method as claimed in claim 1 wherein the plurality of stress inducing
10 stimuli comprises at least two stress inducing stimuli selected from the group
consisting of heat stress, respiratory stress, oxidative stress, acid based stress,
heavy metal stress, osmotic stress, metabolite restriction and nutrient starvation
3. The method as claimed in claim 1 or 2 wherein the plurality of stress
15 inducing stimuli includes heat stress.
4. The method as claimed in claim 3 wherein the heat stress comprises
increasing the heat to which the cultured cells are exposed to a temperature of
around 5 - 10°C greater than the normal growth temperature of the cells.
20
5. The method as claimed in any one of claims 1 to 4 wherein the plurality of
stress inducing stimuli includes respiratory stress.
6. The method as claimed in claim 5 wherein the respiratory stress
25 comprises decreasing the amount of oxygen to which the cultured cells are
exposed from that which causes normal physiological growth or homeostasis of
the cells.
7. The method as claimed in claim 5 or 6 wherein the cells are exposed to
30 the heat stress prior to being exposed to the respiratory stress.
8. The method as claimed in claim 5 or 6 wherein the cells are exposed to
the heat stress and to the respiratory stress concurrently.
9. The method as claimed in claim 5 or 6 wherein the cells are exposed to
5 the respiratory stress prior to being exposed to the heat stress.
10. The method as claimed in any one of claims 1 to 4 wherein the plurality of
stress inducing stimuli includes acid based stress.
10 1 1. The method as claimed in claim 10 wherein the acid based stress
comprises decreasing the pH at which the cultured cells are exposed from that
which causes normal physiological growth or homeostasis of the cells.
12. The method as claimed in claim 10 or 11 wherein the cells are exposed
15 to the heat stress prior to being exposed to the acid based stress.
13. The method as claimed in claim 10 or 11 wherein the cells are exposed
to the heat stress and to the acid based stress concurrently.
20 14. The method as claimed in claim 10 or 11 wherein the cells are exposed
to the acid based stress prior to being exposed to the heat stress.
15. The method as claimed in any one of claims 2 to 14 wherein the cells are
subjected to the heat stress for a time period ranging from 1 to 2 hours.
25
16. The method as claimed in any preceding claim wherein the cells are
selected from the group consisting of pathogenic cells, cancerous cells, cells
infected by a pathogenic organism, cells which have been genetically modified to
constitutively express heat shock proteins, cells which have been genetically
30 modified such that they express a heterologous protein which is derived from a
cancerous cell and cells which has been genetically modified such that they
express a heterologous protein derived from a pathogen which causes an
infectious disease in a host.
17. The method as claimed in claim 16 wherein the cells are pathogenic cells.
5
18. The method as claimed in claim 17 wherein the pathogenic cells are
selected from the group consisting of gram positive prokaryotic cells, gram
negative prokaryotic cells, microbial cells, protozoan cells, viruses, fungi and
parasitic cells.
10
19. The method as claimed in claim 18 wherein the prokaryotic cells are
selected from the group consisting of Escherichia, Streptococcus, Staphylococcus,
Bordetella, Corynebacterium, Mycobacterium, Neisseria, Haemophilus,
Actinomycetes, Streptomycetes, Nocardia, Enterobacter, Yersinia, Fancisella,
15 Pasturella, 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.
20
20. The method as claimed in any one of claims 1 to 9 wherein the cells are
selected from the group consisting of Neisseria, Mycobacteria, Saccharomyces
and Clostridium.
25 21. The method as claimed in any one of claims 10 to 15 wherein the cells are
Streptococcus.
22. The method as claimed in any preceding claim wherein the peptide is a
tumour specific antigen.
23. The method as claimed in claim 3 or 4 wherein the cells are anaerobic
pathogenic cells and the plurality of stress inducing stimuli includes exposing said
cells to oxidative stress, said oxidative stress comprising increasing the amount of
oxygen to which the cultured cells are exposed.
24. The method as claimed in any preceding claim wherein the step of purifying
5 the stress protein complexes comprises the steps of:
- providing a clarified cell lysate which comprises the stress protein
complexes,
- subjecting the cell lysate to purification using ion exchange, wherein the
cell lysate is buffered to a pH within 2 units of the pl of a target stress
protein complex, and wherein a salt gradient is used to elute the stress
protein complexes, and
- obtaining an enriched preparation comprising the heat shock protein
complexes.
15 25. The method as claimed in any preceding claim wherein the heat shock
protein complexes comprise one or more heat shock proteins selected from the
group consisting of hsp20-30kD, hsp40, hsp60, hsp70, hsp90, hspl00,
calrecticulin, hsp72, grp94, grp75 BiPlgrp78, grp75lmt and gp96.
20 26. The method as claimed in any preceding claim wherein the heat shock
protein complexes comprise GroEl and DnaK.
27. The method as claimed in any preceding claim wherein the stress protein
complexes have a molecular weight in the range of 50KDa to 9OOKDa.
25
28. A vaccine composition comprising a stress protein complex obtained by the
method of any one of claims 1 to 27.
29. Use of a purified stress protein complex produced by the method of any one
30 of claims 1 to 27 in the preparation of a medicament for the treatment of an
infectious disease or a cancerous or malignant condition.
30. A stress potein complex produced by the method of any one of claims 1 to 27 for use in treating an infections diseases or a cancerous or malignant condition.
31. A method of vaccinating a subject against a pathogen derived infections diseases or a cancerous condition, said method comprising the steps of:
-provided a vaccine composition comprising, as the immunogenic determinant, purified stress protein complexes produced according to the method of any one of claims 1 to 27, and
-administering a therapeutically effective or prophylactically effective amount of the vaccine composition to the subject in an amount sufficient to elicit an immune response in the subject against the stress protein complex.
32. A method of boosting a protect immune response mediated by a primary immunisation schedule in a subject against a pathogen derived infections disease or a canserous conditions, wherein said protective immune response has been elicite by previous exposure to the pathogen or cancerous condition or by previous administreation of a vaccine selected from the group consisting of a live vaccine, an attenuated vaccine, a vaccine compositions as claimed in claim 28, a nucleic acid vaccine and a protein vaccine, saim method comprising the steps of:
-provided a composition comprising, as the immunogenic determinant, purified stress protein complexes produced according to the method of any one of claims 1 to 27, and
-administering a therapeutically or prophylactically effective amount of the composition to the subjecct in an amount sufficient to enhance the immune response in the subject against the stress protein complexes.