Supervisor: Dr Phil Sutton
Helicobacter pylori is estimated to infect approximately half the global human population. Infection leads to a range of serious disease pathologies, the most important of these being gastric cancer. Triple drug-therapy is currently used to treat patients suffering from H. pylori infection, however drug resistant strains are emerging. Current vaccines do not fully protect the host from H. pylori infection, which is critical to avert the development of associated disease. Thus, new ways of improving the current vaccination strategies, by optimising vaccination regimes and identifying the immune parameters involved in protection would greatly assist in the prevention of disease associated with H. pylori infection.
Research Questions
Can the host immune response to Helicobacter pylori infection be improved through an optimised vaccination regime?
What immune parameters can be identified in response to Helicobacter pylori which correlate with improved protection following vaccination with the optimised regime?
Do novel adjuvants, which can be used in humans, protect against Helicobacter
pylori infection?
Project
The aims of this project are to improve the efficacy of H. pylori vaccines and test novel adjuvants that could be useful in humans. The goals of this project are as follows.
1. Test the efficacy of adjuvants that are potentially useful in a human H.
pylori vaccine.
2. Test the efficacy of an optimised H. pylori vaccination regime and identify
specific immune correlates of protection, through analysis of the cellular
and humoral immune responses.
3. Assess the applicability of an optimised H. pylori vaccine against different
genetic backgrounds by testing the optimised regime in different mouse strains.
4. Test the efficacy of therapeutic vaccination, using an optimised vaccination
regime.
Proposed Research Plan, Procedures and Current Status
Model development
In order to achieve the goals listed above, it was first necessary to establish
the mouse model of H. pylori infection at the Centre for Animal Biotechnology
(CAB).
Specific tasks and techniques successfully achieved for model development
include:
- culture and maintenance of H. pylori (strain - SS1) in vitro and in vivo
- preservation of glycerol bacterial stocks for long term storage
- production of H. pylori lysate for use as vaccine
- development of other techniques such as colony forming assay (CFA) for estimations
of bacterial numbers; splenocytes proliferation assays for assessment of specific
antigen responses and cytokine production.
- orogastric gavage technique for infecting mice and delivering vaccines
Problems that were overcome
During the course of specific antibody ELISA development, it was noticed that
there were unusually high background specific IgG levels in sera from naïve
animals that had not been vaccinated with or exposed to H. pylori. After
extensive testing by PCR, which involved isolation of DNA from mouse faecal
and tissue samples, it was suspected that animals in the animal experiment
room were infected with another helicobacter species. Faecal samples were
sent to a diagnostic laboratory for verification of infection and were found
to be positive for H. hepaticus, a common, intestinal helicobacter, easily
transmitted and often found in rodent animal houses.
Although the implications of infection with another helicobacter species are unclear, it may be important to my results if other immune parameters I am measuring are affected. The problem of infection by H. hepaticus has been addressed by altering the mouse husbandry conditions. A new room with restricted access has been set up in a separate part of the animal house. All husbandry is carried out by myself or other trained members of the helicobacter research team at CAB. All new animals brought in are certified helicobacter-free and faecal samples have been collected for future verification of this status. If this level of husbandry improvement is still not sufficient to prevent infection by Helicobacter spp. filter-top cages will be purchased, which are designed to prevent the spread of organisms transmitted by contaminated bedding and faeces from adjacent cages.
Optimised Regime
The optimised vaccination regime was repeated to verify the results obtained
by Dr Phil Sutton at the UNSW (experiments numbered CDS1 and CDS2). Measurements
of splenocyte proliferation, cytokine secretion and serum and mucosal antibodies
were taken to attempt to identify specific immune parameters involved in
protection. Tests for the applicability of the optimised H. pylori vaccine
against different genetic backgrounds was commence another different mouse
strain (BALB/c).
Summary of research progress
(i) Timeline
First Year
• Establish mouse model of infection, commence animal trials for the optimised
regime and adjuvant studies
• Optimise assays and techniques and commence analysis of samples collected
from animal trials
• Commence preparation of methods for thesis and write-up of completed experiments
Second Year
Animal trials
• repeat the optimised regime trial in C57BL/6 mice
• test the optimised regime in other mouse strains to test the applicability
across different genetic backgrounds (BALB/c, QS outbred, CBA, C3H/He) and in
native-Helicobacter-free mice
• test a higher dose of antigen in the optimised regime; test other adjuvants;
test another vaccine formulation
Lab work
• Optimise assays; IgA ELISA; IFN-γ ELISA; Western blots/2D gels;
antibody agglutination
Thesis writing
• Continue with methods and mannan-adjuvant trial
Third Year; planned timeline of work to be completed
Animal trials
• Complete animal trials for optimised regime
• Repeat adjuvant trials and other trials (potentially 4 in total), pending
results of previous trials
Lab work
• Immunohistochemistry; 2D gels; develop and apply method for eluting specific
antibodies from agglutinated bacteria and use in various analyses
Thesis writing
• Complete writing up thesis
Publications and Presentations
Publications
C. Skene and P. Sutton. "Saponin-adjuvanted particulate vaccines for clinical
use". Methods. 2006 in press.
Conference Presentations
C. Skene, P. Sutton. "An evaluation of agglutinating antibodies against
Helicobacter pylori induced by infection and vaccination". 35th Annual
Scientific Meeting of the Australasian Society for Immunology, Melbourne, Australia,
4-8 December 2005. Poster. (Abstract published in; Tissue Antigens, 66(5):545,
November 2005).
Oral presentations
Centre for Animal Biotechnology, Parkville (February 2005). Confirmation seminar.
Helicobacter pylori vaccines.
Centre for Animal Biotechnology, Parkville (September 2005). Seminar. Helicobacter pylori; a novel vaccine target.
Centre for Animal Biotechnology, Parkville (May 2004). Lab talk. Progress in the development of a mouse model of H. pylori infection at CAB.
Centre for Animal Biotechnology, Parkville (October 2004). Lab talk. Detection of Helicobacter spp. in mice, by PCR.
Centre for Animal Biotechnology, Parkville (June 2005). Lab talk. Optimised Vaccination Regime for H. pylori.