Download Mycobacterial Heat Shock Proteins as Vaccines - A Model

Current Molecular Medicine 2007, 7, 339-350
339
Mycobacterial Heat Shock Proteins as Vaccines - A Model of
Facilitated Antigen Presentation
K. Barry Walker*,1 , James Keeble1 and Camilo Colaco2
1Biotherapeutics
Group, NIBSC, Blanche Lane, South Mimms, Potters Bar EN6 3QG, UK
2ImmunoBiology
Ltd., Babraham Bioincubator, Babraham, Cambridge CB2 4AT, UK
Abstract: Heat shock proteins (hsps) are a highly conserved family of proteins, first recognized by
their upregulated expression in response to host exposure to raised temperatures. Further study has
revealed that they have numerous functions in the cell, primarily as chaperones mediating both the
correct folding of nascent polypeptide chains and the dissolution of aggregated protein complexes. The
energy requirement for this chaperone activity is provided by the ATPase activity found in most
families of hsps and thus the peptide binding capacity is controlled by ATP hydrolysis. The structural
consequence of this is that hsps isolated in situ are found complexed to chaperoned peptides (hspCs).
Much previous work has implicated hsps in the immune response to pathogens and recent studies
have shown that the interaction of hsps with antigen presenting cells, such as dendritic cells (DCs),
mediates the integration of the innate and acquired immune responses. This central role for hspCs in
immunity is facilitated by their dual function in both innate immunity, with the induction of cytokines
and the maturation of DCs mediated by the hsp component, and acquired immunity, with the trafficking
of antigens chaperoned in hspCs for antigen presentation by the mature DCs.
Keywords: Heat shock proteins, antigen presentation, vaccines, tuberculosis, chaperone.
GENERAL BACKGROUND TO HEAT SHOCK
PROTEINS
for cell survival and knock out cells/organisms of
these cannot be produced as deletions are fatal.
Heat shock proteins (hsps) are a family of highly
conserved molecules that are found in both
prokaryotic
and
eukaryotic
organisms.
First
discovered as a group of proteins that are upregulated by stress, they are thus also referred to as
stress induced proteins. The hsps are highly
inducible upon environmental stress (heat oxygen;
pH; nutrient deprivation) and their dramatic upregulation provides some level of protection against
the external stress by preventing protein aggregation
and denaturing [1]. This led to the initial hypothesis
that their major function was in stress management,
reviewed in [1]. However it was soon recognised that
these proteins (or highly homologous polypeptides)
were some of the most abundant constitutively
expressed proteins found in cells, comprising up to
5-10% total cellular protein levels. Moreover, they
were often found bound to nascent polypeptide
chains and thus a more fundamental protein
housekeeping function was proposed. Chaperones
are involved in variety of functions including protein
folding and unfolding, degradation, the prevention of
aggregation and the assembly of multisubunit
complexes, reviewed in [2]. Consistent with this
fundamental role for hsps, many of the hsp genes
(hsp60, hsp70 and hsp90) are known to be essential
Much initial work on hsps focused on the
elucidation of their protein chaperone function and
their role in thermotolerance
[3].
However,
subsequent studies on the molecular biology of hsps
and their induction, led to the elucidation not only of
the mechanism of membrane protein synthesis but
also that of transcriptional control of genes [4]. More
recently, attention has focused on the role of these
proteins in immunity and, most importantly, on the
integration of innate and acquired immunity [5]. This
central role of hsps in immunity reflects their
utilisation by the immune system and in particular by
the key antigen presenting cell, the dendritic cell
(DC), as both maturation-inducing signal molecules
and antigen-trafficking vehicles [5-7]. In this article
we will review the current literature on mycobacterial
hsps and discuss their potential as vaccines.
*Address correspondence to this author at the Biotherapeutics Group,
NIBSC, Blanche Lane, South Mimms, Potters Bar EN6 3QG, UK; Tel:
+44 (0) 1707 641473; Fax: +44 (0) 1707 641057; E-mail:
[email protected]
1566-5240/07 $50.00+.00
1. Hsp Functions
Protein House Keeping Functions
Hsps are primarily classified not only by their
molecular size, but also by their function (See Table
1). The main polypeptide families are the hsp60
(GroEL), hsp70 (DnaK), and hsp100 (ClpB) proteins.
The former two families function primarily as
chaperones in the folding of nascent polypeptide
chains, whereas the latter functions primarily in
refolding of mis-folded and aggregated proteins [812]. The functions of the other hsp families, including
the small hsps, CCT and hsp90 proteins, are less
well characterized but they appear to also act as
© 2007 Bentham Science Publishers Ltd.
340 Current Molecular Medicine, 2007, Vol. 7, No. 4
Table 1.
Walker et al.
Major Families of hsps and their Characteristics
Hsp Family
Structural Features
Members
Intracellular
Location
Reference
Small Hsps
Varied, often large oligomeric
structures
hsp10, GroES. hsp16, α-crystalin, hsp20,
hsp25, hsp26, hsp27
Cytosol
[24, 31-38]
Hsp40
Dimeric
hsp40, DnaJ, Sis1
Cytosol
[52]
Hsp47
Monomer
Trimer
hsp47
Endoplasmic
reticulum
[13]
CCT
hetero-oligomeric complex
TRiC (60 Kd family)
cytosol
[14]
Calreticulin
Monomeric
Calreticulin, Calnexin
Endoplasmic
reticulum
[15]
Hsp60
2 stacked heptameric rings
hsp60, hsp65, GroEL
Cytosol
Mitocondria
[8-12]
Hsp70
Monomeric
hsp71, hsc70(hsp73) hsp110/SSE, DnaK,
SSC1, SSQ1, ECM10, Grp78(BIP),
Grp170
Cytosol
Mitocondria
Endoplasmic
reticulum
[8-12]
Hsp90
Non covalent homodimers
hsc84, hsp86, HTPG, Gp96 (Grp94,
endoplasmin)
Cytosol
Endoplasmic
reticulum
[77-79]
Hsp100
Multimeric complexes with hsp70
and hsp25
hsp104, Hsp110, Clp proteins, Hsp78
Cytosol
Mitocondria
[8, 10, 79, 91,92]
chaperones for specialized or essential polypeptides,
directing the folding, re-folding and stabilization of
polypeptide domains, thereby preventing these
proteins from taking non-functional conformations [815].
gene family is also thought to function in the
degradation of denatured proteins and it is thought
that the interaction of hsp100 with the small hsps, as
opposed to Dna K/J, determines if the final outcome
of dis-aggregation is unfolding or degradation [21].
The classic protein folding pathway of nascent
polypeptides involves the most ubiquitous hsp family,
the hsp60/65 gene family, which is found in both
eukaryotes and prokaryotes. In
prokaryotes,
hsp60/65 is called GroEL and the gene for this
protein is found in an operon that also contains the
chaperone GroES (or hsp10) with which it cooperates to mediate the correct folding of the
majority of cellular proteins. In addition to the
GroEL/ES complex, the classic pathway also
requires
the
function
of
the
hsp40/hsp70
chaperones which stabilise nascent polypeptides in
a linear, unfolded state and either mediate their
folding directly or deliver the partially folded
polypeptide to the GroEL/GroES complex. In
prokaryotes, this gene family is called DnaJ/DnaK
and these genes are again usually found in the
same operon that is located distinct from, and
controlled independently of, the GroEL/GroES
operon [16-19].
Finally, it is thought that there may be additional
small families of specific hsps that chaperone
abundant cellular proteins that are involved in
specialized and essential functions within the cells.
These include the hsp90 gene family that are
chaperones for steroid hormone receptors and
nuclear proteins and the TRiC/CCT gene families
that chaperone the folding of the cytoskeletal
proteins tubulin and actin [12,14,22].
In addition to the classic protein folding of
nascent polypeptides, hsps also function in the
recovery of misfolded and denatured proteins, by
binding to protein aggregates and the unfolding and
refolding of the polypeptide chains contained
therein. In prokaryotes, this disaggregation function
is primarily performed by the Clp gene family the
main members of which are the ClpA/B genes in
procaryotes and hsp100/104 polypeptides found in
the cytosol of eukaryotic cells. These hsps function
in a complex with the DnaK/J hsps in the refolding of
the disaggregated polypeptide chains [20]. The Clp
2. ATP/ADP Involvement
All the major Hsp families are associated with
ATPase activity that is integral to their function as
molecular chaperones. Thus the binding of ATP
results in conformational changes of the hsp that
expose the peptide binding sites, while the
hydrolysis of ATP is thought to provide the energy
required to mediate the folding of the nascent
polypeptide chains. However, while this model is
supported by structural studies on the hsp60 and
hsp70 systems, the Hsp100 protein family probably
utilizes the hydrolysis of ATP to release unfolded
polypeptide chains from their protein aggregates. In
the hsp60 system, ATP binding brings about a
conformational change in the hsp that exposes its
peptide binding core, allowing peptides to enter the
peptide binding chamber. This is then followed by
the binding of the hsp10 co-chaperone which closes
the chamber and ATP hydrolysis to ADP then
energises the folding of nascent polypeptide chain in
a hydrophobic environment as shown in Fig. (1). In
the hsp70 system, ATP binding brings about a
Mycobacterial Heat Shock Proteins as Vaccines
Current Molecular Medicine, 2007, Vol. 7, No. 4
conformational change in the hsp that exposes its
peptide binding site, allowing peptides to enter the
binding cleft. ATP hydrolysis to ADP then closes this
cleft [11,23-25] and the nascent protein can
undergo folding without interference from other
constituents of the intracellular environment as
shown in Fig. (2). Hsp90 also requires ATP/ADP
binding to bring about a conformational change for
polypeptide binding and folding [26].
MYCOBACTERIAL HSP CHARACTERISTICS
Small Hsps - Hsp10 (cpn10, GroES)
The Mycobacterium tuberculosis chaperonin10
antigen (M. tuberculosis Cpn10), also known as
hsp10, or MPB57, or the 10-14 kDa antigen, is a
secreted protein with homology with the GroES
family [27]. It shows about 44% homology with the E.
coli form of GroES and 90% sequence homology to
the M. leprae form [28-30].
Hsp10 interacts with hsp60 (GroEL), in
conjunction with ATP to bring about conformational
change of GroEL to bring about protein folding [3133]. The GroEL/GroES complex assembles as a
barrel structure which provides a hydrophobic
environment for polypeptide chains emerging from
ATP
341
the ribosome. The two heptameric rings of GroEL
form the walls of a double-barreled structure with a
hydrophobic central chamber and the GroES forms
the lids at either end as shown in Fig. (1). The
eukaryotic hsp10 family is also involved in the correct
folding of new proteins in the mitochondria where it
interacts with the mitochondrial hsp60 and mediates
the folding and assembly of proteins and is involved
in the sorting of certain proteins, such as the Rieske
Fe/S protein.
Hsp16.3 Hsp14/16 ( -Crystallin)
The mycobacterium hsp16.3 (also referred to as
14 or 16 kDa antigen) was initially identified as an
immunodominant antigen and is homologous to the
major vertebrate eye lens structural protein
α-crystallin [34,35]. Hsp 16.3 has a nonameric
structure in vitro [36], assembled and re-assembled
via trimer and hexamer intermediates [37, 38].
Homology between species ranges from 27 to 31%
[39] and there are functional similarities between the
M. tuberculosis hsp16.3 and human αΒ-crystallin
even though homology between them is only 18%
[40]. The 18Kd immuno-dominant protein of M.
leprae is also related to eukaryotic small hsp [41].
This α-crystallin homolog is distinct from the M.
A
B
ATP
ATP
C
D
ADP
ATP
ADP
Hsp10 (GroES)
Peptide (Unfolded)
Hsp60 (GroEL)
Peptide (Folded)
Fig. (1). The GroEL-GroES cycle.
The GroEL structure is a double ring each comprising of 7 subunits each with an ATP binding site. Each ring has a peptide
binding apical domain. Nascent or unfolded peptide binds to this domain (A) ATP binding brings about a conformational
change of each subunit which shifts the peptide into the interior cavity of the ring, which is then topped with GroES (This is
done by alternating ATP cycles in each ring), (B) The peptide cannot escape and can undergo folding in an isolated
environment (C) ADP removal and the binding of ATP brings about GroES unbinding and the folded peptide can escape (D).
342 Current Molecular Medicine, 2007, Vol. 7, No. 4
tuberculosis protein [35] and the difference in
sequence provides the basis of immuno-diagnostic
assays that distinguish between these organisms
[34,35].
The α-crystallin functions as a molecular
chaperone [42-44] and confers cellular thermoresistance by preventing protein aggregation [45].
Hsp16.3 of M. tuberculosis is also capable suppressing thermal aggregation of proteins [36,46].
Hsp16.3 is a membrane associated protein [47] that
is minimally expressed in logarithmically growing
cultures of M. tuberculosis. Hsp16.3 is not normally
observed but is rapidly induced when growth enters
stationary phase, when cell wall thickening and
localisation of Hsp16.3 is observed [39,48]. Hsp16.3
expression has been linked to oxygen depleted nonreplicating persistence [49] and stationary phase
growth [50]. In addition Hsp16.3 has also been
implicated with the scavenging of reactive oxygen
intermediates
and
thus
aiding
survival
in
macrophages [51]. This hypothesis is supported, in
part, by the observations that survival of M.
tuberculosis H37Rv-hsp16.3 gene knockouts was
reduced in macrophages over 6 days in culture,
whereas the uptake was comparable to the wild type
organism. Interestingly, this reduced survival in the
hsp16.3 knockouts was lost if the organsisms were
grown to log phase in long term macrophage
cultures [52]. In vivo studies have shown that such
knockouts exhibit increased bacterial growth [53]
and pathology in a murine tuberculosis model [54],
thus implicating the product as an important immune
target for the modulation of the hosts immune
response [54].
Hsp40 (DnaJ)
Hsp 40 is homologous to the DnaJ protein, a
chaperone protein that is found in the cytosol and
highly conserved between bacteria species [55]. It is
also found in the endoplasmic reticulum in yeast and
mammalian cells [56,57].
Hsp40 is a co-chaperone that regulates the
hsp70 (DnaK) protein folding system [58,59]. The
interactions between the two hsps are complex and
much has been discovered through the study of the
E.coli DnaK/J system that is involved in the classical
folding of nascent polypeptide chains [60] as shown
in Fig. (2). There are two major functions associated
with hsp40; it assists in the delivery of peptide
substrate to hsp70 [61-63] and it controls the
ATPase activity that mediates the conformational
changes in hsp70 that allow the exposure of its
peptide binding cleft [59,60,64-66]. In prokaryotes,
the hsp40 gene (DnaJ) is located in the same
operon as its co-chaperone hsp70 (DnaK). In M.
tuberculosis there is also a second DnaJ homolog
which is unlinked to the DnaK/J operon but may also
be associated with a homologue of DnaK [67].
Mutations that affect hsp40-peptide and ATP
binding to hsp70 result in aberrant function [18].
However these functional defects are not restricted
Walker et al.
to the mis-folding of nascent polypeptides but also
inhibit the dissolution of protein aggregates and it is
now recognized that the hsp70/40 chaperones also
function in the disaggregation and refolding of misfolded protein aggregates, consistent with their
proposed role in the repair of heat-induced protein
damage and thermotolerance [62].
Hsp60/65 (cpn60-1, cpn60-2 or GroEL1/2)
Hsp60 is homologous to
GroEL
[29,68].
Homology to GroEL of different species is typically
50-60%, with the M. tuberculosis, M. leprae and BCG
proteins sharing greater than 95% sequence
homology [69] raising the potential for cross reactivity
between species [70]. It is also a common protein
that is shared by many prokaryotes and eukaryotes
and the mycobacterial hsp60 shares 41% homology
with the human protein [71]. As in other bacteria, the
M. tuberculosis hsp60 gene (GroEL1/cpn60-1) is
found in a single operon together with its cochaperone GroES [72]. However M. tuberculosis,
along with other members of the actinomyces, has a
second hsp60 chaperone (hsp65) encoded by a
separate gene (GroEL2/cpn60-2) that is not linked to
the GroEL1 operon [71]. Hsp65 (cpn60-2) shows an
amino acid sequence homology of 61% to hsp60
(Cpn60-1) and 53% to the GroEL of E. coil [71].
Hsp60 functions as a complex with hsp10 in the
classical GroES-GroEL protein folding system. This
complex has been extensively reviewed [8-12], thus
only a brief overview will be given as shown in Fig.
(1). GroEL assembles as a functional double ring
structure each of which is comprised of 7 hsp60
subunits and contains an ATP binding site [23,32].
Each ring has a peptide binding apical domain that
binds nascent or unfolded peptides and the binding
of ATP to this domain brings about a conformational
change of each subunit that shifts the peptide into
the interior cavity of the ring. The subsequent
binding of GroES closes off the ring cavity and
provides an isolated hydrophobic environment for
the correct folding of the nascent polypeptide away
from the hydrophilic environment of the cytosol as
shown in Fig. (1). The hydrolysis of the bound ATP
provides the energy requirement for the folding and
ADP removal is mediated by the binding of new ATP
that also brings about GroES unbinding and the
folded peptide is released from the complex as
shown in Fig. (1).
The crystal structure of GroEL2 of M. tuberculosis
has been elucidated and appears distinct from
GroEL1 in that it forms dimers rather than hexamers
and it is thus thought that GroEL2/hsp65 may play a
function distinct from GroEL1/hsp60 [73]. This
hypothesis appears to be supported by the lack of a
GroES analogue in the operon containing GroEL2.
However, surprisingly, the hsp60/GroEL1 gene is not
essential for survival whereas deletion of the
hsp65/GroEL2 gene is lethal [74]. The significance
of having two close homologs of the same protein is
currently under investigation [73,74].
Mycobacterial Heat Shock Proteins as Vaccines
Current Molecular Medicine, 2007, Vol. 7, No. 4
Both hsp60 and hsp65 are highly immunogenic
and their expression is up regulated by stress
[17,21,29,68,71,75].
Interestingly,
the
immunogenicity of the hsp60/65 proteins in subjects with
tuberculosis appears to be associated predominantly
with hsp65 [28,76].
Hsp70 (DnaK)
Like the hsp10-hsp60 system this too has been
extensively reviewed elsewhere [8-12]; again only a
brief overview will be given and a schematic of the
chaperone cycle of the DnaK/J complex is shown in
Fig. (2).
The hsp70 gene family, typified by the DnaK
protein of E. coli, is highly conserved in all
prokaryotes and shares a 40% sequence homology
with the eukaryotic chaperone [1,16]. This protein is
highly stress inducible and functions in the classical
protein folding pathway with co-chaperone hsp40
(DnaJ) [18,77,78]. The hsp40 (DnaJ) is thought to
stabilise peptides in a linear, unfolded state and
deliver them to the hsp70 (Dnak) chaperone, where
the ATPase activity of the latter provides the
required energy for the folding of the peptide to its
final native conformation [60-62] as shown in Fig. (2).
The hsp40 has also been shown to modulate the
343
ATPase activity of hsp70 in the complex [79].
Although the functional mechanism of hsp70/40
complex functions is distinct from the hsp60/65 family
of proteins described earlier, the two chaperone
complexes show functional complimentarity in the
classical pathway for the folding of nascent
polypeptide chains [3,9-12]. However, in addition to
their role in the folding of nascent proteins both
hsp40 and 70 are involved in the disaggregation of
protein complexes mediated by the hsp100 (ClpB)
chaperone family of proteins [20,21] and determine
the ultimate fate of misfolded polypeptides between
refolding or degradation [59-66].
Hsp90 (gp96, Grp94)
Members of the hsp90 family are primarily
associated with core functions in eukaryotes, where
they are found predominantly in the ER (gp96) but
also in the cytosol (hsp90) [80,81]. In eukaryotes the
hsp90 gene family is thought to mediate the folding
of specialized groups of proteins such as steroid
receptors [82]. The hsp90 gene family is also
ubiquitous in fungi where they have been proposed
to play a variety of roles [83]. Homologues of the
cytosolic hsp90 are also found in bacteria and show
up to 40% homology to mammalian proteins [2,84].
C
D
A
ADP
ATP
ADP
B
ATP
E
F
ATP
ATP
Hsp70(Open)
Hsp40
GrpE
Peptide (Unfolded)
ADP
Hsp70(Closed)
Peptide (folded)
Fig. (2). Hsp40/70 (DnaJ/K) cycle.
Hsp40 binds peptides independently of hsp70 (A). Hsp40-peptide complex interacts with hsp70-ATP complex (open state)
(B). Hsp40 delivers peptide into the binding cleft of hsp70 (C). ATP to ADP hydrolysis brings dissociation of hsp40, hsp70
changes into closed form (D). GrpE bind to hsp70 and trigger release of ADP (E). ATP binds and releases GrpE and hsp70
returns to an open state (F).
344 Current Molecular Medicine, 2007, Vol. 7, No. 4
Structural studies of hsp90 have revealed that
the molecule functions as a homodimer with the two
monomers associating in a tail to tail orientation [8588]. Each subunit of the homodimer has ATP
binding domain and, like hsp70/hsp40, hsp90
requires ATP/ADP binding for peptide interaction
[89,90]. ATP binding has been observed to bring
about a conformational change in hsp90, and is
believed
to
bring
about
dimerisation
and
encapsulation around peptides [26,91]. However,
the exact nature and relevance to peptide binding
and an ability to help protein folding is still unclear
[89]. In contrast to the cytosolic form hsp90, gp96
shows weak ATP binding [7,92,93].
Hsp100 (Clp)
Members of the hsp100 family were originally
described as factors for thermotolerance and
constitute proteins homologous to the ClpA/B
chaperones of E. coli [94]. These hsps are members
of the AAA+ protein family [95] and are involved in a
wide range of functions primarily associated with their
distinct role in the disassembly of protein aggregates
and the refolding or degradation of denatured
polypeptide chains [96,97]. Hsp100 appears to be
similar to the classical GroEL in both structure and
function, as it assembles to form a hexadimer
providing a hydrophobic cavity for protein folding
[8,10,82]. Both families utilise ATP hydrolysis for their
function, though the hsp100 chaperones ultilise the
energy for the disaggregation of polypeptide chains
from denatured protein complexes rather than their
folding [21,82]. In the refolding function, members of
this family operate as co-chaperones with DnaK/J
[21,82], whereas it is thought likely that by
association with the small hsp families, Hsp100
functions in the degradation of the misfolded
polypeptide chains instead [21,82].
IMMUNODOMINANCE AND IMMUNOGENICITY
Both humans and rodents that have been
infected with M. tuberculosis, or those that have
been vaccinated with BCG, produce antibodies and
T and B cells that are reactive to a variety of
antigens and a number of hsps are represented
among the immuno-dominant molecules [98,99].
Classical murine vaccination studies in which mice
were vaccinated with M. tuberculosis or M. leprae
preparations revealed antibody responses against 6
major hsp antigens [98,99]. Of these the small hsps
(hsp10/ GroES and
hsp16.3/α-crystallin) and
members of the hsp60 and hsp70 family have been
identified as being dominant immune recognition
targets [75,100-102]. In addition, many hsps are
found to be abundant in culture filtrates, which have
been shown to elicit good protective immunity in
animal models [75,101]. These have thus attracted
much interest as antigens for serological diagnostic
assays and as potential vaccine candidates
[69,103,104].
Walker et al.
Immune Responses to Individual Hsps
Hsp10 (cpn10, GroES)
M. tuberculosis hsp10 is a secreted protein that is
highly immunogenic and a major target for both
antibodies [101] and reactive T-cell clones [105110]. The epitopes recognized by reactive T-cells
have been determined [111] and in the case of M.
tuberculosis, epitope recognition is directed against
the N-terminal peptides [112]. However, even though
it is a highly immunogenic protein, its ability to elicit
protective immunity is limited and requires additional
use of adjuvant to enhance its efficacy [113].
Nonetheless, its immunodominance has proved
useful in the development of specific and sensitive
immunodiagnostic assays that can distinguish
between individuals who are BCG vaccinated and
those who are infected with M. tuberculosis [114,
115]. Even though the M. tuberculosis protein
contains more than 90% sequence homology to
other bacterial GroES polypeptides, species specific
antibodies are mounted towards it [28,31,110,116]
and the antigenic specificity makes it likely that
assays for antibody against this protein will replace
the classic PPD skin test for diagnosing tuberculosis
infection.
Hsp16.3 ( -Crystallin)
Hsp16.3 has been characterised as a major
membrane protein and is linked with virulence
[47,48,117]. Levels of expression of hsp16.3 are
modified during different growth phases. During invitro culture levels are increased as growth enter
stationary phase [39,50,118]. It is produced in BCG
under conditions of oxygen deficiency [119], and
localization of the 16 kDa alpha-crystallin homologue
to regions of cell wall thickening has been observed
[48]. As previously mentioned hsp16.3 has been
implicated as an important immune target for the
modulation of the hosts immune response [53,54].
For example DNA vaccines coding for ESAT-6 and
α-crystallin have caused reduced bacillary growth in
animal models [120,121].
T cell clones from naturally-converted PPDpositive healthy subjects react strongly to this
antigen [122,123]. Like hsp10, hsp16.3 does not
show a high level of homology from species to
species and T-cell clones for M. leprae and the M.
tuberculosis gene products do not cross react [124].
Hsp60/65 (cpn60-1, cpn60-2 GroEL)
Highly immunoreactive, hsp60/65, along with
hsp10, are perhaps the most immuno-dominant antigens of M. tuberculosis. Multiple immuno-dominant
epitopes of hsp60/65 have been characterised using
monoclonal antibodies [125]. It is estimated that 1020 % of all mycobacterial reactive T-cells are specific
for hsp65 in mice immunised with M. tuberculosis or
BCG [126]. High levels of anti hsp60/65 T cell
reactivity is also seen in human TB contacts and
those infected with M. tuberculosis [127,128] and
the T cell epitopes have been characterised and
mapped [129].
Mycobacterial Heat Shock Proteins as Vaccines
This immunodominance has resulted in much
interest in hsp60/65 as potential vaccine candidates
(See section 6). However, as an abundant,
ubiquitous protein the potential for self cross
reactivity needs to be considered [70]. It has been
reported that T cells reactive to M. tuberculosis
hsp65 have the potential to react to self proteins
[130] and immune responses to bacterial hsp60/65
have been postulated to be involved in the
development of atherosclerosis, and possibly arthritis
[131-133]. Paradoxically anti-hsp60/65 responses
have also been postulated in the development of
tolerance and therefore may provide an avenue for
the treatment of autoimmune diseases [134-137].
In addition to its specific use as a vaccine against
tuberculosis, hsp60/65 has also been shown to have
potent innate immune effects with both the induction
of chemokines and maturation of dendritic cells
[5,138-140]. This has led to the development of a
number of hsp65-antigen fusion proteins for the
treatment of both infectious diseases and cancer
[102,137]. The hsp65 used in these vaccine
adjuvant applications is the product of the GroEL2
gene and this is the basis of the DNA vaccines being
developed for prevention and treatment of
tuberculosis [141-149]. The value of GroEL1 as a
vaccine candidate is largely unknown.
Hsp70 (DnaK)
Despite the high degree of homology between
the hsp70 proteins from mycobacteria and
eucaryotes [78], hsp 70 is also a dominant immune
target in microbial infections with both T and B cells
responses against this antigen [150]. As for the
hsp60/65 family, these hsps also have potent
stimulation potential for the innate immune system.
They have been shown to both induce betachemokines and stimulate dendritic cell maturation,
thus also enhancing adaptive immunity [139,151].
Moreover, the complex of hsp70 with chaperoned
peptide has been shown to act as a delivery system
for chaperoned peptides [139,152]. This antigen
trafficking function required the intact protein and is
distinct from the domains being required for its
adjuvant activity [153-155]. Thus much interest has
recently been focused on hspCs comprising hsp70
as vaccines in cancer and infectious diseases
[140,156,157].
HSP’S AS VACCINE CANDIDATES
Hsp65 DNA Vaccines
As immunodominant antigens in M. tuberculosis
infection, hsps have attracted much interest as
possible vaccine candidates against TB. In this
context, hsp65 has been the most intensely studied
as it is perhaps the most immunodominant antigen,
with high antibody titers mounted towards it
[75,98,99] and an estimated 10-20 % of all T-cells
being specific for hsp65 in infected mice [126].
The mycobacterial hsp65 genes have been
cloned and sequenced and show a high level of
Current Molecular Medicine, 2007, Vol. 7, No. 4
345
homology between mycobacterium species [68,158160]. The development of DNA vaccine approaches
lead to recognition of the potential of mycobacterium
hsp65 DNA expression vectors as potential vaccine
candidates, in particular by Lowrie et al. [146-149].
Initial work showed that macrophage derived cells
(J774) transformed with an M. leprae hsp65 DNA
plasmid produced hsp65 and conferred protection in
mice challenged intravenously with a high dose of M.
tuberculosis [145,161]. However it is interesting that
vaccination with hsp65 protein alone did not confer
significant protection. Adoptive transfer showed that
CD4 and CD8 cells specific to hsp65 were elicited
and conferred protection [161]. These cell types and
frequency have been characterised [141,162].
Further work showed that vaccination with DNA
plasmids coding the M. leprae hsp65 could confer
protection via specific cellular responses in mice
challenged intravenously [146], and this was
extended to other mycobacterium antigens, including
hsp70 [147]. Particularly interesting was work which
showed that hsp65 DNA plasmids could be used as
a therapeutic agent in mice [142-144,147,148].
However these studies have recently become
controversial, as hsp65 DNA vaccines did not elicit a
therapeutic action when used in a more
physiologically relevant model, a low dose murine
aerosol challenge system [163]. Moreover, an
exacerbation of pathology similar to the Koch
phenomenon was observed in the lungs, bringing
into question safety issues of using such agents in a
population already infected with M. tuberculosis
[164,165]. In this respect, recent studies have
suggested that the risk of exacerbated pathology
may not be specifically associated with the use of
the hsp65 gene itself but may be a feature of DNA
vaccination per se [165-167]. If the latter
observations are confirmed, they would sound a
cautionary note for the development of DNA
vaccines
for
TB.
This
issue
has
been
comprehensively reviewed recently [168].
Hsp as Antigen Carriers and Adjuvants
Immunogenic Potential of HspCs
The protein binding and chaperonin functions of
hsps have lead to the proposal that members of this
family of proteins may act to deliver specific bound
peptides in a targeted way to the immune system
[5,6]. Peptide binding to hsps has been recognized
since the 1980’s when studies of protein extracts led
to the identification of complexes of hsps and
chaperoned peptides (hspCs) as antigenic moieties
that
induced
specific
anti-tumour
immunity
[1,2,156,157]. Initial studies focused on the stressinduced gp96 proteins but later studies showed that
hspCs comprising other hsp families also formed
complexes capable of trafficking bound peptides for
the loading of MHC molecules and antigen
presentation in DCs [1,2,5,138,169]. The nature of
the bound peptides has been of great interest and
successful efforts have been made by a number of
346 Current Molecular Medicine, 2007, Vol. 7, No. 4
Walker et al.
HspC
CD14
CD36
CD91
CD40
TLR2
TLR4
hsp
IL-12, TNF-a
IL-1b, GM-CSF
NO,RANTES
MIP-1a,MCP-1
Receptors-
Peptide
+CD91
+Scavenger
Receptor
CD40
MHC Class
Endocytosis
Crosspresentation
CD8+
T Cell
CD80/86
Nucleus
TCR
Increased MHC class ll
expression via upregulation
of vesicle maturation
APC (DC)
CD4+
T Cell
MHC Class ll
Fig. (3). Interaction of chaperoned proteins (hspCs) and antigen-presenting cells (DC): maturation of DC and presentation
on both Class I and II MHC.
groups to isolate specific hspCs and characterise the
bound peptides and these have been shown to be
the basis of the antigenic specificity elicited by these
complexes
[121,152,157,170-172].
In
most
instances, the peptides isolated from these hspCs
have consisted of well recognized immunogenic
epitopes for both CD4 and CD8 cells (MHC class II
and MHC class I presentation pathways), firmly
establishing the role of hsps in facilitating and
presenting antigenic peptides to the immune system
in a manner consistent with the eliciting of an
effective and specific immune response [5,6].
Hsp Receptors
The details of the pathways from cytosolic hspCs
to antigen presentation to an appropriate cell have
been the focus of much detailed study and it has
become evident that there are a number of potential
pathways involved [57,173-175]. Hsps have been
shown to bind to a number of cell surface molecules,
some directly involved in immune responses, others
less clearly so [176-180] (See Table 2). The
interaction of hsp/hsp peptide complexes and
antigen presenting cells (APC) in particular is central
to the understanding of the elicitation of antigen
specific immune responses. It is particularly important
to distinguish between hsp receptors that mediate
the upregulation of innate immunity and those
involved in the antigen trafficking necessary for
antigen presentation as these appear to differentially
regulate the adjuvant and specific antigenic
functions of hspCs [5-7]. For example, the receptors
CD91 and Lox-1 are distinct receptor families
involved in endocytosis and antigen trafficking and,
as such, elicit acquired immune responses, whereas
TLR-2/4 receptors mediate activation of intracellular
signaling and upregulation of innate immunity
[176,178,181,182] as shown in Fig. (3) and Table 2.
Thus the interaction between hspCs and antigen
presenting cells such as the DC involves two linked
and
interacting
consequences.
The
primary
interaction is the potent adjuvant like capacity of the
hsp’s to induce a range of pro-inflammatory
molecules – including cytokines and chemokines
[151,178,181]. Closely associated with this process
is the subsequent uptake of the hsp/peptide
complex (hspC) and the processing and loading of
antigenic peptide on the MHC molecules, resulting in
antigen presentation and specific MHC-restricted
immune recognition [5,6]. In addition the interaction
of APC and hsps has been shown to result in a wide
range of intracellular signaling activity culminating in
APC maturation and modulation of expression of a
wide range of surface molecules. These changes in
turn potentially modify the responsiveness of the cell,
and its migratory-homing patterns [169,178,179,
183]. Antigen responsiveness is facilitated through
this combination of antigen internalisation and
Mycobacterial Heat Shock Proteins as Vaccines
Current Molecular Medicine, 2007, Vol. 7, No. 4
Protein
347
hs
p7
0
Grp170
TAP
Tapasin
MHC class I
Calnexin
hs
p9
0
10
p1
hs
Proteasome
Calreticulin
gp96
Endoplasmic
Reticulum
Cytosol
Fig. (4). Intracellular Processing of Proteins and hsp-chaperoned antigen loading onto MHC Class I.
processing within cells that are being stimulated
through activation of cytokine and chemokine
receptors [5] and shown in Figs. (3) and (4).
Table 2. Hsp Receptors and their Ligands
Receptor
Ligand/s
Reference
CD91
gp96
hsp70
CRT
hsp90
[173]
Lox-1
hsp70
[182]
TLR-2/4
hsp70
hsp60
gp96
[179]
[178]
[180]
SR-A
gp96
[180]
CD40
hsp70
[177]
CD14
hsp70
[151]
CD36
gp96
[184]
The References cited in this table are indicated by the numbers as they
appear in the text and bibliography.
The subsequent molecular and cellular events
downstream of hsp binding to its cognate receptor
remain to be elucidated. Fundamental questions
remain about the pathways from receptor binding to
antigen presentation and the degree to which these
pathways may themselves be modulated by, for
example, cytokine activation of the cell or co-receptor
ligation. The role of non-APC’s that may express hsp
receptors remains unknown, although it has become
evident that a number of non-APC cell populations
do express appropriate receptors – for example
platelets and thrombocytes can bind gp96
[176,177,179,183,184] as shown in Figs. (3) and (4).
There undoubtedly remains a significant body of
work to be done in clarifying the roles and
interactions of the receptors for hsp’s and their
functions. It should be remembered that these
processes are dynamic and therefore there is ample
scope for modulation of responses at many levels. It
is not at all clear if APC’s at different stages of
activation express differing repertoires of relevant
receptors for hsp’s, or even if differing populations,
possibly derived from differing anatomical locations,
have similar degrees of receptor heterogeneity.
CONCLUSIONS
Infectious disease can be controlled by the host’s
immune responses, particularly if these responses
have been boosted and focused by antigen specific
vaccination. For the reasons already outlined, hsppeptide complexes could have an ability to enhance
induction of antigen specific responsiveness to
protective antigens derived from infectious agents.
This approach has been explored over the last
decade using a wide range of diseases and disease
models [185-189] (See Table 3).
The most cost effective approach in combating
infectious disease is through effective vaccination.
348 Current Molecular Medicine, 2007, Vol. 7, No. 4
Walker et al.
Mass vaccination campaigns have been remarkably
effective in reducing the burden of disease in society
as a whole, however there remain a number of
infectious diseases where the development of
improved vaccines is a priority, one of these being
tuberculosis. The experience with BCG over the last
80 years has emphasized the need for vaccines that
are not limited by host exposure to environmental
mycobacteria. This has driven the development of
subunit vaccines [164,166,167,190] and, in this
respect, it should be noted that as potential “multisubunit vaccines”, the mycobacterial hspCs present
attractive candidates for this approach.
Table 3. Clinical
Indications
Interventions
for
HspC
Based
unclear and much work remains to be done to clarify
their effects on the composition of the hspCs
produced. Nonetheless the broad immunity elicited
by hspCs and their “multi subunit” nature justifies
inclusion as major vaccine candidates.
These
characteristics also suggest that BCG hspCs could
be used to boost existing immunity elicited by BCG
vaccination, tuberculosis infection or environmental
mycobacteria.
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While the hsps from M. tuberculosis have been
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