Heat shock protein complex vaccination induces protection against Helicobacter pylori without exogenous adjuvant
Introduction
The development of an effective vaccine against Helicobacter pylori, the main causative agent of gastric adenocarcinoma [1], gastric mucosal associated lymphoid tissue lymphoma [2] and peptic ulcer disease [3] has been hindered by several key obstacles. These include a poor understanding of the immune mechanisms required for protective immunity, the inability to reliably induce sterilising immunity that completely protects against this infection, and the lack of suitable adjuvants for use in humans [4], [5]. An additional concern has been the development of a post-immunisation gastritis, whereby vaccinated mice subsequently challenged with Helicobacter commonly develop an exacerbated gastritis that is more severe than observed in unvaccinated, infected mice [6], [7]. Hence new approaches are required to overcome these issues.
When released extracellularly, either from host cells or bacteria, heat shock proteins (Hsp) can act as danger-associated molecular pattern molecules (DAMP), potently activating the innate immune system [8]. This has resulted in their evaluation as potential vaccine components, both for the therapeutic treatment of cancers and as prophylactic inducers of protective immunity against pathogenic infections [9], [10]. Hsp possess several key features that promote their potential use in vaccines. Firstly, they are protein chaperones which, when purified from cells, carry bound to them a diverse protein cargo. Secondly, related to their role as a DAMP, they have intrinsic adjuvant activity due to their ability to activate toll-like receptors (TLR) [9], [11], [12]. Purification of Hsp from a bacterial pathogen therefore provides a potential vaccine containing the Hsp itself, as well as its complexed protein cargo, which is immunostimulatory without the requirement of adding an exogenous adjuvant.
The main Hsp possessed by H. pylori are the GroEL/S (58 kDa also called HspB/Hsp60 and 13 kDa also called HspA respectively) and the Dna K/J (also called Hsp70) chaperones [13], [14], and their expression is controlled transcriptionally by the HspR and HrcA repressors [15]. Several studies have demonstrated the potential utility of E. coli expressed recombinant H. pylori GroEL and GroES as protective vaccine antigens when delivered in conjunction with a powerful exogenous adjuvant [16], [17].
Here we present the first examination of a H. pylori Hsp complex vaccine. This study demonstrates that Hsp, derived from H. pylori along with chaperoned cargo, can induce protective immunity. Importantly this is achieved without the addition of an adjuvant, and without inducing a large inflammatory response at the site of infection. We further provide the first demonstration that Hsp complex (HspC) vaccines can be effective when delivered via a mucosal route.
Section snippets
H. pylori culture
H. pylori strain SS1 [18] was cultivated in brain heart infusion broth (BHI; Oxoid) containing 0.02% Amphostat and 5% horse serum (Sigma, St Louis, MO, USA) and grown in microaerophilic conditions for 24 h at 37 °C.
Preparation of H. pylori heat shock protein complex vaccine
Heat shock protein complex vaccines were prepared essentially as described [19]. To prepare the HspC vaccine, H. pylori strain SS1 was lysed using a Triton X-100 solution (40 mM Tris, 1 mM MgCl2, 20 mM NaCl, 0.5% Triton X-100, pH 8.0). The lysate was clarified by centrifugation at 13,000
H. pylori HspC is immunogenic and induces protective immunity when delivered via the subcutaneous route
One-dimensional gel and LC–MSMS analysis of the H. pylori HspC revealed that this complex contained abundant amounts of four different heat shock proteins (Hsp60, DnaK, Hsp90 and grpE) as well as a range of other antigens. Fourteen highly abundant proteins that were identified by LC–MSMS have been marked according to their apparent molecular weights in Fig. 1.
We first examined whether H. pylori HspC was effective when delivered parenterally by injection. Subcutaneous delivery of H. pylori HspC,
Discussion
The development of an effective vaccine against H. pylori has been hindered by several obstacles, including the identification of a suitable mucosal adjuvant for use in a human vaccine. In this study we demonstrate for the first time that a vaccine containing HspC produced from H. pylori, can induce protective immunity without the addition of an exogenous adjuvant. This protective effect could be elicited by H. pylori HspC delivery either by subcutaneous injection or mucosally via the
Acknowledgements
This work was supported by the Victorian Government’s Operational Infrastructure Support Program, ARC Linkage Grant LP120100226 from the Australian Research Council and by ImmunoBiology Limited. PS is supported by a Senior Research Fellowship from the National Health and Medical Research Council of Australia.
Conflict of interest statement: EW, CE, CAC and SM are employees of ImmunoBiology Limited, a company developing vaccines targeted to dendritic cells using Heat shock proteins. The work is
References (44)
- et al.
Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori
Lancet
(1993) - et al.
Oral immunization with urease and Escherichia coli heat-labile enterotoxin is safe and immunogenic in Helicobacter pylori-infected adults
Gastroenterology
(1999) - et al.
Gastritis in urease-immunized mice after Helicobacter felis challenge may be due to residual bacteria
Gastroenterology
(1997) - et al.
Immunological aspects of heat-shock proteins-the optimum stress of life
Molecular Immunology
(2004) - et al.
Caught with their PAMPs down? The extracellular signalling actions of molecular chaperones are not due to microbial contaminants
Cell Stress & Chaperones
(2010) - et al.
A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain
Gastroenterology
(1997) - et al.
Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis
The Journal of Biological Chemistry
(1977) - et al.
Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein
Molecular & Cellular Proteomics: MCP
(2005) - et al.
Muc1 mucin limits both Helicobacter pylori colonization of the murine gastric mucosa and associated gastritis
Gastroenterology
(2007) - et al.
Evaluation of ISCOMATRIX and ISCOM vaccines for immunisation against Helicobacter pylori
Vaccine
(2008)
Tracking the tissue distribution of marker dye following intranasal delivery in mice and chinchillas: a multifactorial analysis of parameters affecting nasal retention
Vaccine
Alkyl hydroperoxide reductase: a candidate Helicobacter pylori vaccine
Vaccine
Vaccination of mice against H pylori induces a strong Th-17 response and immunity that is neutrophil dependent
Gastroenterology
Lipopolysaccharide in bacterial chronic infection: insights from Helicobacter pylori lipopolysaccharide and lipid A
International Journal of Medical Microbiology: IJMM
Helicobacter pylori heat shock protein 60 mediates interleukin-6 production by macrophages via a toll-like receptor (TLR)-2-, TLR-4-, and myeloid differentiation factor 88-independent mechanism
The Journal of Biological Chemistry
Helicobacter pylori infection and the development of gastric cancer
The New England Journal of Medicine
Effect of age on the frequency of active Campylobacter pylori infection diagnosed by the [13C]urea breath test in normal subjects and patients with peptic ulcer disease
The Journal of Infectious Diseases
Why can’t we make an effective vaccine against Helicobacter pylori?
Expert Review of Vaccines
Post-immunisation gastritis and Helicobacter infection in the mouse: a long term study
Gut
Heat-shock proteins as dendritic cell-targeting vaccines--getting warmer
Immunology
Heat shock proteins: conditional mediators of inflammation in tumor immunity
Frontiers in Immunology
Helicobacter pylori heat-shock protein 60 induces interleukin-8 via a Toll-like receptor (TLR)2 and mitogen-activated protein (MAP) kinase pathway in human monocytes
Journal of Medical Microbiology
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