Correspondence: Dr S. McNulty, ImmunoBiology Ltd., Babraham Research Campus, Babraham, Cambridge, CB22 3AT, UK. Email: firstname.lastname@example.org
Senior author: Dr S. McNulty
Heat-shock proteins (hsp) provide a natural link between innate and adaptive immune responses by combining the ideal properties of antigen carriage (chaperoning), targeting and activation of antigen-presenting cells (APC), including dendritic cells (DC). Targeting is achieved through binding of hsp to distinct cell surface receptors and is followed by antigen internalization, processing and presentation. An improved understanding of the interaction of hsp with DC has driven the development of numerous hsp-containing vaccines, designed to deliver antigens directly to DC. Studies in mice have shown that for cancers, such vaccines generate impressive immune responses and protection from tumour challenge. However, translation to human use, as for many experimental immunotherapies, has been slow partly because of the need to perform trials in patients with advanced cancers, where demonstration of efficacy is challenging. Recently, the properties of hsp have been used for development of prophylactic vaccines against infectious diseases including tuberculosis and meningitis. These hsp-based vaccines, in the form of pathogen-derived hsp–antigen complexes, or recombinant hsp combined with selected antigens in vitro, offer an innovative approach against challenging diseases where broad antigen coverage is critical.
Seminal studies published by the groups of Steinman and others showed that dendritic cells (DC) function as the key professional antigen-presenting cells (APC), providing a link between the innate and adaptive arms of the immune system. Dendritic cells are central to the generation of adaptive immunity, continuously sampling their vicinity for antigens against which the body might need to react, such as from invading pathogenic microbes. Antigens are taken up by DC in soluble or particulate forms, often facilitated by opsonization by antibody or complement, processed by a series of enzymes and then loaded onto MHC molecules for presentation to T-cells during priming of an immune response. MHC class II usually presents antigenic peptides derived from extracellular organisms to CD4+ T-cells, whereas MHC class I presents peptides derived from intracellular organisms (or cytoplasmic proteins) to CD8+ T-cells. This ensures that the optimum T-cell response is generated: CD4+ T helper cells for antibodies and cell-mediated immunity against extracellular organisms, and CD8+ cytotoxic T-cells against intracellular organisms and cancers. The DC also receive inflammatory signals during infections and cancers; pathogen-associated molecular patterns or danger signals, which are recognized via receptors such as Toll-like receptors and stimulate cytokine secretion and co-stimulatory molecule expression, which further facilitates T-cell responses. Hence, various vaccination strategies aim to target DC because of their pivotal role in adaptive immunity.
Delivering antigens to DC, using strategies that target uptake via surface receptors, including DEC-205, mannose receptor and FcγR1, is an innovative area for developing vaccines and therapeutics. Heat-shock proteins (hsp) carry an antigenic profile or fingerprint of the cells from which they are derived, possess adjuvant activity and bind to receptors on DC to promote uptake. This review highlights the role of hsp in antigen delivery to DC, which forms the basis of a strategy for developing vaccines against cancer and infectious diseases.
Heat-shock protein biology
Within cells, hsp undertake critical and conserved physiological roles. They function as chaperones and co-chaperones binding intracellular polypeptide chains and misfolded proteins, preventing aggregation and supporting folding and transport. Most hsp have at least two functional domains: a polypeptide-binding domain, and an ATPase domain controlling binding and release of polypeptide substrate. Heat-shock proteins are present in organisms as diverse as bacteria and man, protecting proteins from damage during normal physiological activity as well as stressful conditions. As a consequence of their physiological functions, hsp transport multiple proteins as ‘cargo’. Cellular levels of hsp are high, for example in bacteria, hsp70 alone accounts for 1–2% of cellular proteins after heat induction. In eukaryotic cells hsp levels are increased by stressful stimuli including heat, oxidative stress, starvation, hypoxia, irradiation, viral infection and cancerous transformation.[6, 7] The hsp are grouped into several distinct families named according to the molecular weight of family members (Table 1). In man, hsp90, hsp70, hsp60/Chaperonin and hsp40 families have been characterized. In prokaryotes, GroEL (hsp60) and DnaK (hsp70) are the main hsp families.
Table 1. Heat-shock protein (hsp) families
Heat-shock proteins expressed in mammals are shown in bold.
Stress proteins are ubiquitous and can be detected readily in normal human plasma samples. Absolute levels of extracellular hsp vary markedly between individuals. For example, reported levels for human plasma hsp60 range between < 1 ng/ml and 1 mg/ml and between 100 pg/ml and 160 ng/ml for serum hsp72. Levels of hsp are dynamic during normal physiological activities; exercise increases hsp72 levels in serum by fourfold to eightfold. Therefore, extracellular hsp are continuously present in the circulation of normal individuals and can be increased transiently by several fold without apparent pathology.
In addition to functioning as intracellular protein chaperones, hsp modulate the immune system by stimulating both innate and adaptive responses. The term ‘chaperokine’ has been used to describe the dual activity of hsp functioning as both chaperone and cytokine. Once released from a host or pathogen cell, hsp bind to cellular receptors to trigger an innate immune response, including maturation of DC and secretion of pro-inflammatory cytokines and chemokines, for example RANTES (Regulated on Activation Normal T-cell Expressed and Secreted), through Toll-like receptor activation. Processing of cargo proteins carried by hsp occurs, leading to antigen presentation on MHC. Hence hsp link the innate and acquired immune responses to pathogens and have the potential to function as vaccine adjuvants in infections and cancer. For example, hsp70 is an effective and safe adjuvant in neonatal mice and functions effectively via mucosae to generate protective cell-mediated immune responses against herpes simplex virus type-1. Moreover, modified hsp are also capable of inducing cytokine responses. For example, a fusion protein containing Bacillus Calmette–Guérin (BCG)-derived hsp70 and Mycobacterium leprae-derived major membrane protein binds to human DC stimulating production of interleukin-12 p70 through Toll-like receptor 2.
Cellular receptors for hsp
Dendritic cells and other cell types possess multiple receptors that bind hsp but the identities and functions of those proposed to modulate the immune system in vivo are not fully understood. The expression profile of these receptors is broad, including, but not limited to, multiple immune, epithelial, endothelial and fibroblast cells and multiple cell types of the central nervous system. Receptors for which evidence supports a role in hsp binding and their distribution on immune cells are shown (Table 2).
Table 2. Heat shock protein (hsp) receptors on cells of the immune system
The relative contribution made by each receptor type to the binding and internalization of hsp by DC is poorly understood. For example, with the exception of Scavenger Receptor type A and CD91, the hsp binding functionality of each receptor has not been assessed in vivo. Furthermore, investigations show that for gp96, non-specific endocytosis/pinocytosis mechanisms account for a fraction of internalization.
Heat-shock proteins deliver antigen peptides to DC for T-cell priming
Heat-shock proteins deliver peptides as cargo to DC (Fig. 1) leading to MHC presentation for priming of adaptive immunity. Increased levels of pathogen-derived hsp caused by inflammatory stimuli such as fever, result in a concomitant increase in pathogen-specific antigens carried as hsp complexes. The uptake of hsp complexes by DC enables efficient capture and presentation of pathogen-specific antigens and the mounting of a specific immune response against the infectious agent through the generation of CD4+ T-cell responses. The capture of pathogen-specific antigens ‘chaperoned’ in hsp complexes also results in their uptake and MHC class I restricted presentation to specific T-cells, so eliciting CD8+ cytotoxic T-cell responses. It has been shown through the use of inhibitors, that hsp90 plays a significant natural role in chaperoning antigenic peptides in presentation.
Human DC pulsed with peptide-loaded mycobacterial hsp70 generate potent antigen-specific cytotoxic T-cell responses, dependent on an hsp70-stimulated calcium signalling cascade. Delivery of peptides is achieved significantly through extracellular hsp binding to cellular receptors, followed by internalization. Antigens need to be bound or linked to hsp to facilitate uptake, simple mixing is not adequate. The hsp70–peptide complexes reach endosomal compartments that fuse with vesicles containing recycling MHC class I–peptide complexes. Protein fragments chaperoned by hsp and not intact proteins are sufficient for priming CD8+ T-cell responses.
Highly purified human recombinant hsp70 enhances cross-presentation of exogenous antigens on MHC class I resulting in better antigen-specific T-cell stimulation. Here T-cell stimulation was a function of the degree of complex formation between hsp70 and peptides and correlated with improved antigen delivery to endosomal compartments. hsp70 enhanced cross-presentation by different APC including DC and B cells and antigen-specific T-cell activation occurred in the absence of innate signals transmitted by hsp70. Heat shock protein 90-mediated cross-presentation of ovalbumin-derived antigens involves binding of hsp90–ovalbumin complexes to Scavenger Receptor expressed by Endothelial Cells-I on the surface of APC. Internalization is driven through a regulated, endocytic pathway. Peptides are loaded either directly onto MHC class I in endosomes, or undergo cytosomal processing by aminopeptidases and proteases. Extracellular hsp90 can therefore convey antigenic peptides through an efficient endocytosis pathway in APC and facilitate presentation in a regulated manner. Heat-shock proteins can also mediate by the same mechanism cross-presentation of exogenous HIV antigens.
Distinct hsp possess differing abilities to induce cross-presentation of antigens. For example, an extract prepared from human melanoma lines contained the four major chaperone proteins hsp/HSC 70, hsp90, Grp94/gp96 and calreticulin. These hsp were functional, enhancing presentation of exogenous peptides, but superior activity was observed for the hsp70-rich preparation. Small hsp fragments are sufficient to link peptides and to be taken up by receptors on APC including CD91 and Scavenger Receptor type A, and can be used in immunotherapy of tumours and vaccine development.
To replicate a physiological response to natural infections, so as to maximize immune protection, it is necessary for a vaccine to contain multiple hsp families and associated antigens, hence delivering a broad range of antigens thereby maximizing antigen coverage and protection. The identity and range of cargo carried are dependent upon the types and diversity of hsp present within a vaccine. Gp96, hsp70 and hsp90 each bind distinct antigen peptide precursors. For Escherichia coli, GroEL binds to approximately 250 of the 2400 cytosolic proteins and a recent study demonstrated that for folding in vivo, 57 proteins are bona fide obligate GroEL substrates.
Deletion of GroEL is lethal in E. coli, as is the deletion of the two chaperones Trigger Factor and DnaK (hsp70) that chaperone a significant subset of GroEL target proteins. For cancer, a chaperone-rich cell lysate is more effective than purified hsp alone in generating tumour-specific responses in multiple murine models.[55, 56] The chaperone-rich cell lysate vaccine has a more pronounced immunological effect per unit material of protein than any one of the individual chaperone proteins that it contains used independently as vaccines.
Heat-shock proteins as cancer therapeutics
Immune responses can be generated by hsp against tumour antigens, despite immune evasion processes mediated for example by regulatory T-cells. The potential role for hsp in the immune response to cancer was recognized by Srivastava and colleagues, who proposed that hsp complexed with antigenic peptides, released from tumour cells (or virus-infected cells) in vivo during lysis, are taken up by APC, and the potential use of hsp in cancer immunotherapy has been demonstrated extensively. Of interest, immunization of mice with gp96 can induce a regulated immune response resulting either in tumour immunity or down-regulation, depending on the immunization dose used. Heat-shock protein-based vaccines have been shown to activate tumour-specific immunity, triggering the proliferation and cytotoxic capabilities of cancer-specific CD8+ T-cells, inhibiting tumour growth. The hsp also activate natural killer cells to impart anti-tumour responses. Exogenous antigens chaperoned by hsp are presented by MHC class I molecules and recognized by CD8+ T lymphocytes offering one mechanism for the classical phenomenon of cross-presentation as well as offering a role within the immune danger theory.[62, 63] Lysates from heat-shocked tumour cells provide an optimal source of tumour antigens, generating DC with improved cross-presentation capacity.
Examples of hsp-based therapeutics in cancer trials are detailed in Table 3. To date, one hsp vaccine, Vitespen, is licensed and marketed. The hsp gp96, the master chaperone for Toll-like receptors, is the major component of Vitespen. Chaperoning by gp96 increases uptake over unchaperoned peptides in vitro by two orders of magnitude and immunization of mice with 5 ng gp96–peptide complexes, results in generation of a peptide-specific CD4+ T-cell response. In April 2008, Vitespen was approved in Russia as a patient-specific adjuvant treatment of kidney cancer for individuals at intermediate-risk for disease recurrence. Outside Russia, Vitespen is an investigational vaccine designed to treat cancer with the intent of minimizing side-effects. It has been studied extensively in clinical trials in Phase I and II settings, demonstrating efficacy in some but not all trials. Phase III studies have been completed in which over 1300 patients with renal cell carcinoma or malignant melanoma have been treated. Essentially neither toxicity nor autoimmunity induced by Vitespen was observed.
Table 3. Examples of heat-shock protein (hsp) -based cancer therapeutics in clinical trials
Vitespen Gp96 based vaccine derived from patient tumour
Various cancers including, but not limited to, kidney, liver, ovarian, colorectal, glioma and melanoma
Although pre-clinical studies with Vitespen were promising, clinical studies show limited efficacy. This outcome may be a consequence of differences in the hsp content of Vitespen used for initial in vivo models compared with the vaccine used for clinical trials. Pre-clinical studies reported utilised vaccines containing gp96 or hsp70, while clinical studies utilised vaccine containing only gp96. Critically, gp96 and hsp70 have distinct functions as endoplasmic reticulum (ER) luminal and cytoplasmic chaperones, respectively, and thus bind distinct client proteins. Heat-shock protein 70 binds a variety of cytoplasmic proteins and isolation of this hsp from tumour cells will result in the purification of intact hsp–client protein complexes. In contrast, gp96 binds membrane proteins such as integrins and Toll-like receptors and is essential for chaperoning peptides in the ER. As the clinical production processes used do not contain detergents, peptides bound to gp96 in Vitespen are unlikely to result from tumour client proteins. Hence differences between the bound peptides in gp96 isolated from the homogeneous tumour tissue in the animal models and the heterogeneous tumour tissue from patients in the clinical trials may also account for the limited efficacy reported for Vitespen. Other key issues concerning the future development of such a vaccine are the correct and effective dose of hsp, and which patients to target.
Other hsp provide alternatives to gp96 for cancer vaccine development. Vaccination with hsp70 derived from the Meth A sarcoma, established dose-dependent immunity to challenge with Meth A sarcoma in mice. A suggestion to use cell lines or allogeneic tumours as a more generic source of hsp complex carrying common cancer antigens, has not been taken further. High molecular weight chaperone complexes, hsp110- or grp170-tyrosinase-related protein 2 peptide (TRP2175–192), were superior to conventional chaperones as a vaccine platform to deliver tumour-derived antigens. In addition, the immunization with chaperones combined to two different melanoma antigens (gp100, TRP2) significantly improved anti-tumour efficacy compared with either of the single antigen vaccines, demonstrating that hsp combination vaccines can offer increased efficacy. In a Phase II clinical trial, vaccination with autologous tumour-derived gp96–peptide complex vaccine (hsp complex-96) together with granulocyte–macrophage colony-stimulating factor and interferon-α was associated with mild local and systemic toxicity. Vaccination was proven to instigate both tumour-specific T-cell-mediated and natural killer cell responses in some patients. However, neither immunological nor clinical responses were improved compared with those recorded in a previous study investigating hsp complex-96 monotherapy. A recent study has provided the first evidence in man of patient-specific immune responses against autologous tumour-derived peptides bound to gp96.
Over-expression of hsp70 increases significantly the immunogenicity of cancer cell extracts; with the mechanism of cell death influencing both hsp70 expression levels and the immunogenicity of cell extracts. In addition to hsp complex from hsp70 (hsp70C), synthetic peptide-mimics of hsp70C can modulate positively the immune response against tumours and therefore provide an additional approach for therapeutic intervention. Heat shock protein 70 derived from tumours of characterized antigenic makeup could be used as a generic subunit tumour vaccine. Vaccines derived from tumours or cell lines that have undergone heating to increase the abundance of hsp may provide an innovative approach. For example, vaccination with heated autologous prostate cancer cells elicits protection against tumour challenge in 60% of vaccinated rats, compared with 0% protection in control rats receiving vaccines from non-shocked cells, together with an increase in the T helper type 1 (interferon-γ) response.
Heat shock protein 70 extracted from DC fused to patient-derived ovarian cancer or breast cancer cells (hsp70.PC-F) were tested as tumour vaccines. The hsp70.PC-F induced T-cells expressing higher levels of interferon-γ and with increased killing capacity for tumour cells, compared with those induced by hsp derived from tumour cells, although these were characterized by a higher content of tumour antigens and the detection of hsp such as hsp90 and hsp110. In a mouse tumour model, vaccination with hsp60, hsp70, gp96 and hsp110/peptides, combined with treatment with a low-dose of cyclophosphamide plus interleukin-12, induced a marked immunological response directed against autologous tumours.
Heat-shock protein vaccines: an innovative solution for infectious diseases
Heat-shock proteins possess broad utility as vaccine components. For example, marketed adjuvants often possess side-effects (e.g. ulceration); hsp adjuvants avoid such effects. The abilities of hsp to drive innate stimulation and deliver antigens are now being exploited in prophylactic vaccines against infectious diseases. In one approach, hsp-based vaccines have been produced by over-expressing the influenza virus nucleoprotein in cultured cells before purification of gp96. The gp96 preparation was well tolerated in mice; with preliminary results suggesting that a cellular immune response was induced, providing a novel strategy to develop vaccines against virus targets.
There are several published approaches to prepare hsp complexes, including ion exchange and hydroxyapatite column chromatography and immunoprecipitation with antibodies coupled to magnetic beads. In an innovative approach, hsp70C have been extracted from plant cells expressing viral antigens[86, 87] using the same ADP-chromatography purification protocol described for animal hsp70, a method able to prevent the release of the naturally chaperoned peptides. Plant-derived hsp70C were shown to activate the immune system inducing both activation of MHC class I-restricted polyclonal T-cell responses and antibody production in mice of different haplotypes without the need of adjuvant co-delivery. These results indicate that hsp70C derived from plants producing recombinant antigens may be used to formulate multi-epitope vaccines.
Several investigational prophylactic vaccines containing hsp and hsp complex are in development. For example, a tuberculosis vaccine based on hsp complex from BCG (T-BioVax) has demonstrated good efficacy in the mouse Mycobacterium tuberculosis aerosol challenge model.[89, 90] ImmunoBiology Ltd is also developing a vaccine against meningitis (MenBioVax) derived from heat-shocked Neisseria meningitidis. Both T-BioVax and MenBioVax contain multiple hsp families derived from the stressed bacterium of interest to maximize efficacy. MenBioVax provides protection against lethal challenge in a mouse model of meningococcal septicaemia. Sera obtained from mice immunized with this vaccine show promising bactericidal and opsonophagocytic responses against a panel of N. meningitidis strains.
HerpV, a vaccine consisting of 32 synthetic 35mer HSV-2 peptides representative of all phases of viral replication, non-covalently complexed with recombinant human hsp70 protein, is well tolerated and safe. This was the first hsp-based vaccine to show immune responses against viral antigens in humans. Vaccinated subjects demonstrated a statistically significant CD4+ T-cell response to HSV-2 antigens, with the majority of subjects also having a significant CD8+ T-cell response.
Development of hsp vaccines is based on the need to emulate safely, the mechanism by which protection is established during a normal infection. Extracellular, pathogen-derived hsp are routinely present during infection and transport pathogen proteins as cargo. During infection, because of bacterial lysis, multiple pathogen hsp will be visible to the host in parallel. The identity of cargo proteins will depend upon the family and type of hsp chaperone. The meningococcal stress protein MSP63, a member of the hsp60 family, has been shown in man to be immunogenic during natural meningococcal infection. Genes encoding hsp, including DnaK, GroEL, GroES, DnaJ, GrpE and ClpB, were shown by transcriptional profiling to be up-regulated several fold in N. meningitidis in human blood during bacteraemia.
The similarity of pathogen-derived hsp to human hsp raises the hypothetical possibility of enhanced self recognition induced by vaccines enriched for pathogen hsp. Theoretically, this could occur as a consequence of the presentation of host proteins to DC by vaccine-derived hsp and the induction of autoimmune responses induced by the vaccine hsp. The potential for antibodies produced in mice against plant hsp70 to cross-react, either with murine hsp70 or human hsp70, has been investigated and found to be absent despite the significant structural similarities between the three isoforms.
Significantly, as a consequence of the manufacturing process, hsp are present in many marketed vaccines against infectious diseases, notably in whole cell vaccines and vaccines derived from cell extracts. The extensive, safe use of vaccines containing hsp therefore provides compelling evidence against safety concerns. For example, whole cell vaccines are used widely and possess acceptable safety profiles. Antibodies to hsp65 were found in sera from children vaccinated with DTP (diphtheria, tetanus, pertussis) vaccine administered extensively in Europe and the USA. Antibodies against BCG hsp develop naturally in infants in 6–12 months, even without BCG vaccination. The safety of human exposure to N. meningitidis hsp was obtained from administration of marketed vaccines that contain hsp. Such vaccines have been used since the 1980s and the safety records are excellent.
Perspective: new vaccine approaches are needed urgently
From the pioneering work of Benjamin Jesty and subsequent developments from Edward Jenner to the present day, vaccines have delivered and continue to deliver significant improvements to global health. Smallpox is eradicated, polio has been controlled and the frequency of childhood diseases such as measles has reduced. However, the most successful vaccines have been against diseases where the causal pathogen does not have major anti-immune defence mechanisms. Many pathogens, including hepatitis C and human immunodeficiency viruses, M. tuberculosis, Helicobacter pylori and Plasmodium falciparum have evolved complex immune evasion strategies and probably require high level effector T-cell activation for their eradication. So far, these pathogens have proved intractable to existing vaccination strategies. Bioterrorism, emerging and re-emerging infectious diseases and changes in population demographics and target groups will shape the need for new vaccines. These challenges drive the requirement for new efficacious vaccines produced at low cost and therefore innovative technologies are urgently required. Several such approaches involve the targeting of vaccine antigens to DC, the key controllers of the immune response.
Heat-shock proteins possess significant properties that support their inclusion in the next generation of vaccines to target DC: first, hsp are natural adjuvants; second, hsp deliver multiple antigens that can induce adaptive immune responses to provide broad coverage against pathogens and effective cancer therapy; and third, data show that they are safe constituents of existing vaccines. Most marketed vaccines generate antibody responses but hsp vaccines can also generate cellular immunity, a tightly regulated process varying between individuals in part because of MHC differences. Heat-shock protein complexes derived from cells carry a broad antigenic peptide fingerprint, which helps to avoid both pathogen and immune escape mechanisms. Critically, manufacturing approaches for hsp-containing vaccines against infectious diseases provide low cost production. Although hsp vaccines provide an exciting and innovative strategy for the development of much needed new vaccines, data from clinical trials are now needed to confirm that they provide an effective new approach in man.
We wish to acknowledge TSB grant number 1204_BCF_CDS_R1 21601-155139 awarded from the UK innovation agency, the Technology Strategy Board, as part of the UK government-backed Biomedical Catalyst. Dr McNulty is a project manager for ImmunoBiology Ltd, a vaccine development company based at the Babraham Research Campus, Cambridge.
ImmunoBiology Ltd develops innovative anti-infective vaccines based on hsp and has a number of patents in this field.