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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. REFERENCES [1] [2] [3] [4] [5] [6] [7] Disease Hsp Reference Influenza native extracted and in vitro preparations [185] SV40 native extracted and in vitro preparations [186] HSV-1/2 in vitro preparations [187] [12] Listeria native extracted [188] [13] Tuberculosis native extracted [188] [14] LCMV in vitro preparations [189] [8] [9] [10] [11] The References cited in this table are indicated by the numbers as they appear in the text and bibliography. [15] While the hsps from M. tuberculosis have been shown to be immunodominant antigens recognized by individuals with disease and in contacts (see section 6 above), the development of vaccination approaches using hsps as vaccine candidates has been singularly unsuccessful as the recombinant forms of these proteins (in contrast to DNA vaccines expressing some of the same proteins) and peptides derived from them have been shown to be either poorly immunogenic or to have negligible protective effect in animal models of tuberculosis. However the recognition of the antigen trafficking role of hspCs as distinct from the adjuvant role of hsps has lead to the demonstration that the complexes isolated from current BCG vaccine strains are able to protect against tuberculosis in a mouse aerosol model [191]. This functional and antigenic distinction between hsps and hspCs explains the discrepancies between the results obtained with the use of recombinant hsps and earlier studies utilizing hsps isolated from mycobacteria (i.e. hspCs) and is supported by the lack of immunoreactivity against recombinant hsps observed in animals immunized with BCG hspCs [191]. It should of course be noted, that the use of BCG hspCs as vaccine candidates has the additional advantage of being derived from an organism already licensed for use in humans and with a very long safety record. However the complexity of the HspCs found in these preparations presents a particular problem to the development of manufacturing processes for these vaccines that is currently under investigation. For example production parameters such as growth state of the organisms (log phase or plateau to note just two) remain [17] [16] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] Lindquist, S. (1986) Annu. Rev. Biochem., 55, 1151-1191. Lindquist, S. and Craig, E.A. (1988) Annu. Rev. Genet., 22, 631677. Ellis, R.J. and van der Vies, S.M. (1991) Annu. Rev. Biochem., 60, 321-347. Pelham, H.R.B. (1989) EMBO J., 8, 3171-3176. Colaco, C.A. (1998) Cell Mol. Biol., 44, 883-890. Srivastava, P. (2002) Nat. Rev. Immunoy., 2, 185-194. Wearsch, P.A. and Nicchitta, C.V. (1997) J. Biol. Chem., 272, 5152-5156. Baneyx, F. (2004) Microbial. Cell Factories, 3, 6. Bukau, B. and Horwich, A.L. (1998) Cell, 92, 351-366. Bukau, B., Deuerling, E., Pfund, C. and Craig, E.A. (2000) Cell, 101, 119-122. Netzer, W.J. and Hartl, F.U. (1998) Trends Biochem. Sci., 23, 6873. Young, J.C., Agashe, V.R., Siegers, K. and Hartl, F.U. (2004) Nat. Rev. Mol. Cell Biol., 5, 781-791. Dafforn, T.R., Della, M. and Miller, A.D. (2001) J. Biol. Chem., 276, 49310-49319. Llorca, O., Smyth, M.G., Carrascosa, J.L., Willison, K.R., Radermacher, M., Steinbacher, S. and Valpuesta, J.M. (1999) Nat. Struct. Biol., 6, 639-642. Michalak, M., Milner, R.E., Burns, K. and Opas, M. (1992) Biochem. J., 285 (Pt 3), 681-692. Bardwell, J.C. and Craig, E.A. (1984) Proc. Natl. Acad. Sci. USA, 81, 848-852. Barreiro, C., lez-Lavado, E., tek, M. and Martin, J.-F. (2004) J. Bact., 186, 4813-4817. Gassler, C.S., Buchberger, A., Laufen, T., Mayer, M.P., Schroder, H., Valencia, A. and Bukau, B. (1998) Proc. Natl. Acad. Sci. USA, 95, 15229-15234. Nicchitta, C.V. (2003) Nat. Rev. Immunol., 3, 427-432. Watanabe, Y.H. and Yoshida, M. (2004) J. Biol. Chem., 279, 15723-15727. Stirling, P.C., Lundin, V.F. and Leroux, M.R. (2003) EMBO Rep., 4, 565-570. Leroux, M.R., Melki, R., Gordon, B., Batelier, G. and Candido, E.P. (1997) J. Biol. Chem., 272, 24646-24656. Ranson, N.A., Farr, G.W., Roseman, A.M., Gowen, B., Fenton, W.A., Horwich, A.L. and Saibil, H.R. (2001) Cell, 107, 869-879. Saibil, H. (2000) Curr. Opin. Struct. Biol., 10, 251-258. Saibil, H.R. and Ranson, N.A. (2002) Trends Biochem. Sci., 27, 627-632. Csermely, P., Kajtar, J., Hollosi, M., Jalsovszky, G., Holly, S., Kahn, C.R., Gergely, P., Jr., Soti, C., Mihaly, K. and Somogyi, J. (1993) J. Biol. Chem., 268, 1901-1907. Baird, P.N., Hall, L.M. and Coates, A.R. (1988) Nucleic Acids Res., 16, 9047. Chua-Intra, B., Ivanyi, J., Hills, A., Thole, J., Moreno, C. and Vordermeier, H.M. (1998) Immunology, 93, 64-72. Shinnick, T.M., Vodkin, M.H. and Williams, J.C. (1988) Infect. Immun., 56, 446-451. Shinnick, T.M., Plikaytis, B.B., Hyche, A.D., Van Landingham, R.M. and Walker, L.L. (1989) Nucleic Acids Res., 17, 1254. Cobb, A.J. and Frothingham, R. (1999) Vet. Microbiol., 67, 31-35. Grallert, H. and Buchner, J. (2001) J. Struct. Biol., 135, 95-103. Ma, J., Sigler, P.B., Xu, Z. and Karplus, M. (2000) J. Mol. Biol., 302, 303-313. de Jong, W.W., Leunissen, J.A. and Voorter, C.E. (1993) Mol. Biol. Evol., 10, 103-126. Verbon, A., Hartskeerl, R.A., Schuitema, A., Kolk, A.H., Young, D.B. and Lathigra, R. (1992) J. Bacteriol., 174, 1352-1359. Chang, Z., Primm, T.P., Jakana, J., Lee, I.H., Serysheva, I., Chiu, W., Gilbert, H.F. and Quiocho, F.A. (1996) J. Biol. Chem., 271, 7218-7223. Abulimiti, A., Fu, X., Gu, L., Feng, X. and Chang, Z. (2003) J. Mol. Biol., 326, 1013-1023. Abulimiti, A. and Chang, Z. (2003) Biochem. (Mosc.), 68, 269-274. Yuan, Y., Crane, D.D. and Barry, C.E., III. (1996) J. Bacteriol., 178, 4484-4492. Valdez, M.M., Clark, J.I., Wu, G.J.S. and Muchowski, P.J. (2002) Eur. J. Biochem., 269, 1806-1813. Nerland, A.H., Mustafa, A.S., Sweetser, D., Godal, T. and Young, R.A. (1988) J. Bacteriol., 170, 5919-5921. Horwitz, J. (1992) Proc. Natl. Acad. Sci. USA, 89, 10449-10453. Jakob, U., Gaestel, M., Engel, K. and Buchner, J. (1993). J. Biol. Chem., 268, 1517-1520. Mycobacterial Heat Shock Proteins as Vaccines [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] Merck, K.B., Groenen, P.J., Voorter, C.E., Haard-Hoekman, W.A., Horwitz, J., Bloemendal, H. and de Jong, W.W. (1993) J. Biol. Chem., 268, 1046-1052. van den IJssel, P.R., Overkamp, P., Knauf, U., Gaestel, M. and de Jong, W.W. (1994) FEBS Lett., 355, 54-56. Yang, H., Huang, S., Dai, H., Gong, Y., Zheng, C. and Chang, Z. (1999) Protein Sci., 8, 174-179. Lee, B.Y., Hefta, S.A. and Brennan, P.J. (1992) Infect. Immun., 60, 2066-2074. Cunningham, A.F. and Spreadbury, C.L. (1998) J. Bacteriol., 180, 801-808. Desjardin, L.E., Hayes, L.G., Sohaskey, C.D., Wayne, L.G. and Eisenach, K.D. (2001) J. Bacteriol., 183, 5311-5316. Hu, Y. and Coates, A.R. (1999) J. Bacteriol., 181, 1380-1387. Abulimiti, A., Qiu, X., Chen, J., Liu, Y. and Chang, Z. (2003) Biochem. Biophys. Res. Commun., 305, 87-93. Yuan, Y., Crane, D.D., Simpson, R.M., Zhu, Y.Q., Hickey, M.J., Sherman, D.R. and Barry, C.E., III. (1998) Proc. Natl. Acad. Sci. USA, 95, 9578-9583. Hu, Y., Movahedzadeh, F., Stoker, N.G. and Coates, A.R. (2006) Infect. Immun., 74, 861-868. Stewart, J.N., Rivera, H.N., Karls, R., Quinn, F.D., Roman, J. and Rivera-Marrero, C.A. (2006) Microbiology, 152, 233-244. Bardwell, J.C., Tilly, K., Craig, E., King, J., Zylicz, M. and Georgopoulos, C. (1986) . J. Biol. Chem., 261, 1782-1785. Cheetham, M.E., Brion, J.P. and Anderton, B.H. (1992) Biochem. J., 284 (Pt 2), 469-476. Feldheim, D., Rothblatt, J. and Schekman, R. (1992) Mol. Cell Biol., 12, 3288-3296. Gething, M.J. and Sambrook, J. (1992) Nature, 355, 33-45. Laufen, T., Mayer, M.P., Beisel, C., Klostermeier, D., Mogk, A., Reinstein, J. and Bukau, B. (1999) Proc. Natl. Acad. Sci. USA, 96, 5452-5457. Karzai, A.W. and McMacken, R. (1996) J. Biol. Chem., 271, 1123611246. Gamer, J., Multhaup, G., Tomoyasu, T., McCarty, J.S., Rudiger, S., Schonfeld, H.J., Schirra, C., Bujard, H. and Bukau, B. (1996) EMBO J., 15, 607-617. Schroder, H., Langer, T., Hartl, F.U. and Bukau, B. (1993) EMBO J., 12, 4137-4144. Szabo, A., Langer, T., Schroder, H., Flanagan, J., Bukau, B. and Hartl, F.U. (1994) Proc. Natl. Acad. Sci. USA, 91, 10345-10349. Liberek, K., Skowyra, D., Zylicz, M., Johnson, C. and Georgopoulos, C. (1991) J. Biol. Chem., 266, 14491-14496. Wawrzynow, A. and Zylicz, M. (1995) J. Biol. Chem., 270, 1930019306. Wawrzynow, A., Banecki, B., Wall, D., Liberek, K., Georgopoulos, C. and Zylicz, M. (1995) J. Biol. Chem., 270, 19307-19311. Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry III, C.E., Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, M.A., Rajandream, M.A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J.E., Taylor, K., Whitehead, S. and Barrell, B.G. (1998) Nature, 393, 537-544. Shinnick, T.M. (1987) J. Bacteriol., 169, 1080-1088. Shinnick, T.M., Sweetser, D., Thole, J., van Embden, J. and Young, R.A. (1987) Infect. Immun., 55, 1932-1935. Young, D.B., Ivanyi, J., Cox, J.H. and Lamb, J.R. (1987) Immunol. Today, 8, 215-219. Kong, T.H., Coates, A.R.M., Butcher, P.D., Hickman, C.J. and Shinnick, T.M. (1993) Proc. Natl. Acad. Sci. USA, 90, 2608-2612. Stewart, G.R., Wernisch, L., Stabler, R., Mangan, J.A., Hinds, J., Laing, K.G., Young, D.B. and Butcher, P.D. (2002) Microbiology, 148, 3129-3138. Qamra, R. and Mande, S.C. (2004) J. Bacteriol., 186, 8105-8113. Qamra, R., Srinivas, V. and Mande, S.C. (2004) J. Mol. Biol., 342, 605-617. Young, D., Lathigra, R., Hendrix, R., Sweetser, D. and Young, R.A. (1988) Proc. Natl. Acad. Sci. USA, 85, 4267-4270. Ahmad, S., Amoudy, H.A., Thole, J.E., Young, D.B. and Mustafa, A.S. (1999) Scand. J. Immunol., 49, 515-522. Cogelja, C.G., Horne, B.E., Kelley, W.L., Schwager, F., Georgopoulos, C. and Genevaux, P. (2006) J. Biol. Chem., 281, 12436 - 12444 . Garsia, R.J., Hellqvist, L., Booth, R.J., Radford, A.J., Britton, W.J., Astbury, L., Trent, R.J. and Basten, A. (1989) Infect. Immun., 57, 204-212. Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C. and Zylicz, M. (1991) Proc. Natl. Acad. Sci. USA, 88, 2874-2878. Argon, Y. and Simen, B.B. (1999) Semin. Cell Dev. Biol., 10, 495505. Mazzarella, R.A. and Green, M. (1987) J. Biol. Chem., 262, 88758883. Jakob, U., Meyer, I., Bugl, H., Andre, S., Bardwell, J.C. and Buchner, J. (1995) J. Biol. Chem., 270, 14412-14419. Burnie, J.P., Carter, T.L., Hodgetts, S.J. and Matthews, R.C. (2006) FEMS Microbiol. Rev., 30, 53-88. Bardwell, J.C. and Craig, E.A. (1987) Proc. Natl. Acad. Sci. USA, 84, 5177-5181. Bracher, A. and Hartl, F.U. (2005) Structure (Camb.), 13, 501-502. Facciponte, J.G., Wang, X.Y., MacDonald, I.J., Park, J.E., Arnouk, H., Grimm, M.J., Li, Y., Kim, H., Manjili, M.H., Easton, D.P. and Subjeck, J.R. (2006) Cancer Immunol. Immunother., 55, 339-346. Current Molecular Medicine, 2007, Vol. 7, No. 4 [87] [88] [89] [90] [91] [92] [93] [94] [95] [96] [97] [98] [99] [100] [101] [102] [103] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] [125] [126] 349 Immormino, R.M., Dollins, D.E., Shaffer, P.L., Soldano, K.L., Walker, M.A. and Gewirth, D.T. (2004) J. Biol. Chem., 279, 4616246171. Sastry, S. and Linderoth, N. (1999) J. Biol. Chem., 274, 1202312035. Prodromou, C., Roe, S.M., O'Brien, R., Ladbury, J.E., Piper, P.W. and Pearl, L.H. (1997) Cell, 90, 65-75. Rosser, M.F.N. and Nicchitta, C.V. (2000) J. Biol. Chem., 275, 22798-22805. Richter, K. and Buchner, J. (2001) J. Cell Physiol., 188, 281-290. Rosser, M.F., Trotta, B.M., Marshall, M.R., Berwin, B. and Nicchitta, C.V. (2004) Biochemistry., 43, 8835-8845. Soldano, K.L., Jivan, A., Nicchitta, C.V. and Gewirth, D.T. (2003) J. Biol. Chem., 278, 48330-48338. Zavilgelsky, G.B., Kotova, V.Y., Mazhul', M.M. and Manukhov, I.V. (2002) Biokhimiya, 67, 1191-1198. Weibezahn, J., Bukau, B. and Mogk, A. (2004) Microb. Cell Fact., 3, 1. Katiyar-Agarwal, S., Agarwal, M., Gallie, D.R. and Grover, A. (2001) Crit. Rev. Plant Sci., 20, 277-295. Weber-Ban, E.U., Reid, B.G., Miranker, A.D. and Horwich, A.L. (1999) Nature, 401, 90-93. Results of a World Health Organization-sponsored workshop on monoclonal antibodies to Mycobacterium leprae. (1985) Infect. Immun., 48, 603-605. Results of a World Health Organization-sponsored workshop to characterize antigens recognized by mycobacterium-specific monoclonal antibodies. (1986) Infect. Immun., 51, 718-720. Chandramuki, A., Bothamley, G.H., Brennan, P.J. and Ivanyi, J. (1989) J. Clin. Microbiol., 27, 821-825. Jackett, P.S., Bothamley, G.H., Batra, H.V., Mistry, A., Young, D.B. and Ivanyi, J. (1988) J. Clin. Microbiol., 26, 2313-2318. Young, R.A. and Elliott, T.J. (1989) Cell, 59, 5-8. Lim, J.H., Kim, H.J., Lee, K.S., Jo, E.K., Song, C.H., Jung, S.B., Kim, S.Y., Lee, J.S., Paik, T.H. and Park, J.K. (2004) FEMS Microbiol. Lett., 232, 51-59. Wedlock, D.N., Skinner, M.A., De Lisle, G.W., Vordermeier, H.M., Hewinson, R.G., Hecker, R., Van Drunen Littel-Van Den Hurk, Babiuk, L.A. and Buddle, B.M. (2005) Vet. Immunol. Immunopath., 106, 53-63. Barnes, P.F., Mehra, V., Rivoire, B., Fong, S.J., Brennan, P.J., Voegtline, M.S., Minden, P., Houghten, R.A., Bloom, B.R. and Modlin, R.L. (1992) J. Immunol., 148, 1835-1840. Launois, P., N'Diaye, M.N., Cartel, J.L., Mane, I., Drowart, A., Van Vooren, J.P., Sarthou, J.L. and Huygen, K. (1995) Infect. Immun., 63, 88-93. Mehra, V., Bloom, B.R., Bajardi, A.C., Grisso, C.L., Sieling, P.A., Alland, D., Convit, J., Fan, X.D., Hunter, S.W., Brennan, P.J. and Modlin, R.L. (1992) J. Exp. Med., 175, 275-284. Minden, P., Kelleher, P.J., Freed, J.H., Nielsen, L.D., Brennan, P.J., McPheron, L. and McClatchy, J.K. (1984) Infect. Immun., 46, 519-525. Rojas, R.E. and Segal-Eiras, A. (1996) FEMS Immunol. Med. Microbiol., 15, 189-198. Uyemura, K., Ohmen, J.D., Grisso, C.L., Sieling, P.A., Wyzykowski, R., Reisinger, D.M., Rea, T.H. and Modlin, R.L. (1992) Infect. Immun., 60, 4542-4548. Kim, J., Sette, A., Rodda, S., Southwood, S., Sieling, P.A., Mehra, V., Ohmen, J.D., Oliveros, J., Appella, E., Higashimoto, Y., Rea, T.H., Bloom, B.R. and Modlin, R.L. (1997) J. Immunol., 159, 335343. Chua-Intra, B., Wilkinson, R.J. and Ivanyi, J. (2002) Infect. Immun., 70, 1645-1647. Fattorini, L., Creti, R., Nisini, R., Pietrobono, R., Fan, Y., Stringaro, A., Arancia, G., Serlupi-Crescenzi, O., Iona, E. and Orefici, G. (2002) J. Med. Microbiol., 51, 1071-1079. Lalvani, A., Nagvenkar, P., Udwadia, Z., Pathan, A.A., Wilkinson, K.A., Shastri, J.S., Ewer, K., Hill, A.V., Mehta, A. and Rodrigues, C. (2001) J. Infect. Dis., 183, 469-477. Shams, H., Klucar, P., Weis, S.E., Lalvani, A., Moonan, P.K., Safi, H., Wizel, B., Ewer, K., Nepom, G.T., Lewinsohn, D.M., Andersen, P. and Barnes, P.F. (2004) J. Immunol., 173, 1966-1977. Chua-Intra, B., Peerapakorn, S., Davey, N., Jurcevic, S., Busson, M., Vordermeier, H.M., Pirayavaraporn, C. and Ivanyi, J. (1998) Infect. Immun., 66, 4903-4909. Zhang, H., Fu, X., Jiao, W., Zhang, X., Liu, C. and Chang, Z. (2005) Biochem. Biophys. Res. Commun., 330, 1055-1061. Lim, A., Eleuterio, M., Hutter, B., Murugasu-Oei, B. and Dick, T. (1999) J. Bacteriol., 181, 2252-2256. Tabira, Y., Ohara, N., Ohara, N., Kitaura, H., Matsumoto, S., Naito, M. and Yamada, T. (1998) Res. Microbiol., 149, 255-264. Khera, A., Singh, R., Shakila, H., Rao, V., Dhar, N., Narayanan, P.R., Parmasivan, C.N., Ramanathan, V.D. and Tyagi, A.K. (2005) Vaccine, 23, 5655-5665. Lakey, E.K., Margoliash, E. and Pierce, S.K. (1987) Proc. Natl. Acad. Sci. USA, 84, 1659-1663. Oftung, F., Borka, E. and Mustafa, A.S. (1998) FEMS Immunol. Med. Microbiol., 20, 319-325. Wilkinson, R.J., Wilkinson, K.A., De Smet, K.A., Haslov, K., Pasvol, G., Singh, M., Svarcova, I. and Ivanyi, J. (1998) Scand. J. Immunol., 48, 403-409. Mehra, V., Bloom, B.R., Torigian, V.K., Mandich, D., Reichel, M., Young, S.M., Salgame, P., Convit, J., Hunter, S.W., McNeil, M. (1989) J. Immunol., 142, 2873-2878. Buchanan, T.M., Nomaguchi, H., Anderson, D.C., Young, R.A., Gillis, T.P., Britton, W.J., Ivanyi, J., Kolk, A.H., Closs, O., Bloom, B.R. (1987) Infect. Immun., 55, 1000-1003. Kaufmann, S.H., Vath, U., Thole, J.E., Van Embden, J.D. and Emmrich, F. (1987) Eur. J. Immunol., 17, 351-357. 350 Current Molecular Medicine, 2007, Vol. 7, No. 4 [127] [128] [129] [130] [131] [132] [133] [134] [135] [136] [137] [138] [139] [140] [141] [142] [143] [144] [145] [146] [147] [148] [149] [150] [151] [152] [153] [154] [155] [156] [157] [158] [159] Oftung, F., Mustafa, A.S., Husson, R., Young, R.A. and Godal, T. (1987) J. Immunol., 138, 927-931. Ottenhoff, T.H., Ab, B.K., Van Embden, J.D., Thole, J.E. and Kiessling, R. (1988) J. Exp. Med., 168, 1947-1952. Lamb, J.R., Ivanyi, J., Rees, A.D., Rothbard, J.B., Howland, K., Young, R.A. and Young, D.B. (1987) EMBO J., 6, 1245-1249. Munk, M.E., Schoel, B., Modrow, S., Karr, R.W., Young, R.A. and Kaufmann, S.H. (1989) J. Immunol., 143, 2844-2849. Anderton, S.M., van der, Z.R., Noordzij, A. and Van Eden, W. (1994) J. Immunol., 152, 3656-3664. Ford, P., Gemmell, E., Walker, P., West, M., Cullinan, M. and Seymour, G. (2005) Clin. Diagn. Lab. Immunol., 12, 259-267. Prakken, B.J., Roord, S., Ronaghy, A., Wauben, M., Albani, S. and Van Eden, W. (2003) Springer Sem. Immunopathol., 25, 47-63. Ben Nun, A., Mendel, I., Sappler, G. and Kerlero, d.R. (1995) J. Immunol., 154, 2939-2948. Chandawarkar, R.Y., Wagh, M.S., Kovalchin, J.T. and Srivastava, P. (2004) Int. Immunol., 16, 615-624. Cohen, I.R. and Young, D.B. (1991) Immunol. Today, 12, 105-110. Young, R.A. (1990) Annu. Rev. Immunol., 8, 401-420. Basu, S. and Srivastava, P.K. (2000) Cell Stress Chaperones, 5, 443-451. Lehner, T., Bergmeier, L.A., Wang, Y., Tao, L., Sing, M., Spallek, R. and van der, Z.R. (2000) Eur. J. Immunol., 30, 594-603. Lehner, T., Wang, Y., Whittall, T., McGowan, E., Kelly, C.G. and Singh, M. (2004) Biochem. Soc. Trans., 32, 629-632. Silva, C.L., Silva, M.F., Pietro, R.C. and Lowrie, D.B. (1996) Infect. Immun., 64, 2400-2407. Bonato, V.L., Lima, V.M., Tascon, R.E., Lowrie, D.B. and Silva, C.L. (1998) Infect. Immun., 66, 169-175. Bonato, V.L.D., Goncalves, E.D.C., Soares, E.G., Junior, R.R.S., Sartori, A., Coelho-Castelo, A.A.M. and Silva, C.L. (2004) Immunol., 113, 130-138. Silva, C.L., Bonato, V.L., Coelho-Castelo, A.A., De Souza, A.O., Santos, S.A., Lima, K.M., Faccioli, L.H. and Rodrigues, J.M. (2005) Gene Ther., 12, 281-287. Lukacs, K.V., Lowrie, D.B., Stokes, R.W. and Colston, M.J. (1993) J. Exp. Med., 178, 343-348. Tascon, R.E., Colston, M.J., Ragno, S., Stavropoulos, E., Gregory, D. and Lowrie, D.B. (1996) Nat. Med., 2, 888-892. Lowrie, D.B., Silva, C.L., Colston, M.J., Ragno, S. and Tascon, R.E. (1997) Vaccine, 15, 834-838. Lowrie, D.B., Tascon, R.E., Bonato, V.L.D., Lima, V.M.F., Faccioli, L.H., Stavropoulos, E., Colston, M.J., Hewinson, R.G., Moelling, K. and Silva, C.L. (1999) Nature, 400, 269-271. Lowrie, D.B. and Silva, C.L. (2000) Vaccine, 18, 1712-1716. McKenzie, K.R., Adams, E., Britton, W.J., Garsia, R.J. and Basten, A. (1991) J. Immunol., 147, 312-319. Asea, A., Kraeft, S.K., Kurt-Jones, E.A., Stevenson, M.A., Chen, L.B., Finberg, R.W., Koo, G.C. and Calderwood, S.K. (2000) Nat. Med., 6, 435-442. Srivastava, P. (2002) Ann. Rev. Immunol., 20, 395-425. Rudiger, S., Mayer, M.P., Schneider-Mergener, J. and Bukau, B. (2000) J. Mol. Biol., 304, 245-251. Wendling, U., Paul, L., van der Zee, R., Prakken, B., Singh, M. and van Eden, W. (2000) J. Immunol., 164, 2711-2717. Zhang, J. and Walker, G.C. (1996) J. Biol. Chem., 271, 1966819674. Udono, H. and Srivastava, P.K. (1994) J. Immunol., 152, 53985403. Udono, H. and Srivastava, P.K. (1993) J. Exp. Med., 178, 13911396. Husson, R.N. and Young, R.A. (1987) Proc. Natl. Acad. Sci. USA, 84, 1679-1683. Mehra, V., Sweetser, D. and Young, R.A. (1986) Proc. Natl. Acad. Sci. USA, 83, 7013-7017. Received: July 07, 2006 Revised: August 08, 2006 Accepted: August 08, 2006 Walker et al. [160] [161] [162] [163] [164] [165] [166] [167] [168] [169] [170] [171] [172] [173] [174] [175] [176] [177] [178] [179] [180] [181] [182] [183] [184] [185] [186] [187] [188] [189] [190] [191] Young, R.A., Mehra, V., Sweetser, D., Buchanan, T., Clark-Curtiss, J., Davis, R.W. and Bloom, B.R. (1985) Nature, 316, 450-452. Lowrie, D.B., Tascon, R.E., Colston, M.J. and Silva, C.L. (1994) Vaccine, 12, 1537-1540. Silva, C.L. and Lowrie, D.B. (2000) Infect. Immun., 68, 3269-3274. Taylor, J.L., Ordway, D.J., Troudt, J., Gonzalez-Juarrero, M., Basaraba, R.J. and Orme, I.M. (2005) Infect. Immun., 73, 51895193. Basaraba, R.J., Izzo, A.A., Brandt, L. and Orme, I.M. (2006) Vaccine, 24, 280-286. Orme, I.M. (2006) Tuberculosis (Edinb.), 86, 68-73. Orme, I.M. (2006) Vaccine, 24, 2-19. Orme, I.M. (2005) Vaccine, 23, 2105-2108. Lowrie, D.B. (2006) Vaccine, 24, 1983-1989. Heath, W.R. and Carbone, F.R. (2001) Annu. Rev. Immunol., 19, 4764. Barrios, C., Georgopoulos, C., Lambert, P.H. and Del Giudice, G. (1994) Clin. Exp. Immunol., 98, 229-233. Nieland, T.J., Tan, M.C., Monne-van Muijen, M., Koning, F., Kruisbeek, A.M. and van Bleek, G.M. (1996) Proc. Natl. Acad. Sci. USA, 93, 6135-6139. Zhu, X., Zhao, X., Burkholder, W.F., Gragerov, A., Ogata, C.M., Gottesman, M.E. and Hendrickson, W.A. (1996) Science, 272, 1606-1614. Binder, R.J., Blachere, N.E. and Srivastava, P.K. (2001) J. Biol. Chem., 276, 17163-17171. Freeman, B.C. and Morimoto, R.I. (1996) EMBO J., 15, 2969-2979. Tobian, A.A.R., Canaday, D.H., Boom, W.H. and Harding, C.V. (2004) J. Immunol., 172, 5277-5286. Basu, S., Binder, R.J., Ramalingam, T. and Srivastava, P.K. (2001) Immunity, 14, 303-313. Becker, T., Hartl, F.U. and Wieland, F. (2002) J. Cell Biol., 158, 1277-1285. Vabulas, R.M., Ahmad-Nejad, P., da Costa, C., Miethke, T., Kirschning, C.J., Hacker, H. and Wagner, H. (2001) J. Biol. Chem., 276, 31332-31339. Vabulas, R.M., Ahmad-Nejad, P., Ghose, S., Kirschning, C.J., Issels, R.D. and Wagner, H. (2002) J. Biol. Chem., 277, 1510715112. Berwin, B., Hart, J.P., Rice, S., Gass, C., Pizzo, S.V., Post, S.R. and Nicchitta, C.V. (2003) EMBO J., 22, 6127-6136. Wang, Y., Kelly, C.G., Singh, M., McGowan, E.G., Carrara, A.S., Bergmeier, L.A. and Lehner, T. (2002) J. Immunol., 169, 2422-2429. Delneste, Y., Magistrelli, G., Gauchat, J., Haeuw, J., Aubry, J., Nakamura, K., Kawakami-Honda, N., Goetsch, L., Sawamura, T., Bonnefoy, J. and Jeannin, P. (2002) Immunity, 17, 353-362. Theriault, J.R., Mambula, S.S., Sawamura, T., Stevenson, M.A. and Calderwood, S.K. (2005) FEBS Lett., 579, 1951-1960. Panjwani, N., Popova, L., Febbraio, M. and Srivastava, P.K. (2000) Cell Stress Chaperones, 5, 391. Heikema, A., Agsteribbe, E., Wilschut, J. and Huckriede, A. (1997) Immunol. Lett., 57, 69-74. Srivastava, P.K. (2000) Nat. Immunol., 1, 363-366. Navaratnam, M., Deshpande, M.S., Hariharan, M.J., Zatechka, J. and Srikumaran, S. (2001) Vaccine, 19, 1425-1434. Zugel, U., Sponaas, A.M., Neckermann, J., Schoel, B. and Kaufmann, S.H.E. (2001) Infect. Immun., 69, 4164-4167. Ciupitu, A.M., Petersson, M., O'Donnell, C.L., Williams, K., Jindal, S., Kiessling, R. and Welsh, R.M. (1998) J. Exp. Med., 187, 685691. Sable, S.B., Verma, I. and Khuller, G.K. (2005) Vaccine, 23, 41754184. Colaco, C.A., Bailey, C.R., Keeble, J. and Walker, K.B. (2004) Biochem. Soc. Trans., 32, 626-628.