Elsevier

Vaccine

Volume 32, Issue 20, 25 April 2014, Pages 2350-2358
Vaccine

Heat shock protein complex vaccination induces protection against Helicobacter pylori without exogenous adjuvant

https://doi.org/10.1016/j.vaccine.2014.02.051Get rights and content

Highlights

  • Hsp complex vaccine induced protection against H. pylori without exogenous adjuvant.

  • First demonstration of protection by a mucosally delivered Hsp complex vaccine.

  • Hsp complex-induced protection was associated with increased gastric cytokines.

  • Hsp complex-induced protection was associated with reduced gastritis severity.

Abstract

Background

The development of a vaccine against the human gastric pathogen Helicobacter pylori, the main causative agent of gastric adenocarcinoma, has been hampered by a number of issues, including the lack of a mucosal adjuvant for use in humans. Heat shock proteins (Hsp), highly conserved molecules expressed by both bacteria and mammalian species, possess a range of functions, including acting as chaperones for cellular proteins and the ability to activate innate immune receptors. Hsp complex (HspC) vaccines, containing Hsp derived from pathogenic bacteria, are immunostimulatory without addition of an exogenous adjuvant and can induce immunity against their chaperoned proteins. In this study we explored in mice the potential utility of a H. pylori HspC vaccine.

Results

Vaccination with H. pylori HspC, by either the subcutaneous or respiratory mucosal route, induced a strong antibody response, elevated gastric cytokine levels and significant protection against subsequent live challenge with this pathogen. The level of protection induced by non-adjuvanted HspC vaccine was equivalent to that which resulted from vaccination with adjuvanted vaccines. While protection induced by immunisation with adjuvanted vaccines was associated with the development of a moderate to severe atrophic gastritis, that induced by H. pylori HspC only resulted in a mild inflammatory response, despite an increase in pro-inflammatory gastric cytokines. This reduced gastritis correlated with an increase in IL-10 and IL-13 levels in the gastric tissues of HspC vaccinated, H. pylori challenged mice.

Conclusions

H. pylori HspC vaccines have the potential to overcome some of the issues preventing the development of a human vaccine against this pathogen: HspC induced protective immunity against H. pylori without addition of an adjuvant and without the induction of a severe inflammatory response. However, complete protection was not obtained so further optimisation of this technology is needed if a human vaccine is to become a reality.

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

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