Defining the molecular basis of H7 flagellin as an adhesin and mucosal adjuvant for vaccine development

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 are bacteria that cause serious gastrointestinal disease in humans. Toxins released by the bacteria cause life-threatening damage to the kidneys and brain. Cattle are the main reservoir host for the bacteria and humans become infected initially by ingesting food or drink that has been contaminated, directly or indirectly, from animal faeces. One way to guard against human infection with EHEC O157 is to block or limit shedding of the organism from cattle. This can be done via vaccination of cattle. Our previous research has demonstrated that the whip-like 'flagella' of EHEC O157 that are usually used for bacterial movement, can bind to the intestinal lining of cattle to promote colonisation. If we vaccinate cattle with purified flagella, specific antibodies are generated in the gut that block this binding and limit bacterial colonisation. However, a subset of these antibodies have a negative impact in that they bind to a region of the flagella that interferes with how the host recognizes and responds to the real infection when it occurs. There are two main aims of the research. The first is to define the specific region within the flagellin molecule required for flagella binding and to use this in a vaccine preparation to study how it limits colonisation of cattle by EHEC O157. It is anticipated that by using only the region of flagellin required for binding in the gastrointestinal tract of cattle or by altering recognition of the region that stimulates inflammatory responses in mammals, we should be able to produce an effective vaccine containing flagellin that significantly reduces EHEC O157 shedding from cattle. The second part of the research is to understand how the vaccination of cattle with whole flagellin stimulates such a good antibody response on the surface of the animals gastrointestinal tract and then exploit this understanding to generate responses to other molecules linked to flagellin. Finding ways to stimulate this 'mucosal' immunity is important for the development of vaccines to many infectious diseases.

Technical Summary

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is an important food-borne pathogen that causes serious human infections ranging in symptoms from self-limiting diarrhoea or haemorrhagic colitis to life-threatening haemolytic-uremic syndrome. Cattle are the primary asymptomatic reservoir for EHEC O157 and human infections can occur by consumption of contaminated food or drink, direct contact with animals or person-to-person spread. A key objective is to reduce the carriage or prevalence of EHEC O157:H7 in cattle to limit human exposure. Development of effective interventions in cattle will rely on understanding the bacterial factors involved in colonization of the gastrointestinal tract. We have demonstrated that EHEC O157 predominately colonises the terminal rectum of cattle and that H7 flagella functions as an adhesin for EHEC O157 when binding to epithelial cells cultured from this rectal site. Comparative binding studies with other flagella indicate that distinct motifs in the central (D2 and D3) domains are likely to confer the binding specificity. Systemic immunization of cattle with purified H7 flagellin reduces the proportion of animals that become colonised. However, in calves that do become colonised, bacterial shedding levels are at least as high as non-vaccinated controls. Both IgG and IgA are induced following H7 flagellin immunization and inhibit EHEC O157:H7 adherence in vitro. However, IgG blocks TLR5 signalling and this may impact on shedding levels following immunization. The aims are: (1) to define the region of FliC(H7) required for binding specificity and to generate an H7 flagellin fragment or H7 variant that can be used to induce antibodies that block binding but that do not inhibit TLR5 signalling by wild type H7 flagellin; (2) to investigate how systemic vaccination with H7 flagellin results in specific IgA responses and to use this information to generate a mucosal response to a heterologous antigen.

Planned Impact

This research, as reflected by the investment form Novartis Animal Health (NAH), has two areas that could be translated for commercial benefit in the medium term. The first is the use of H7 or modified H7 flagella in a cattle/ruminant vaccine to prevent or limit EHEC O157 shedding and therefore could have a direct impact on food safety, human health and the sustainability of UK farming. In addition to NAH, we are also in discussion with Bioniche who already have a provisional licence for an EHEC O157 cattle vaccine in Canada and would like to pursue its further development and use in Europe potentially through our grouping. They may consider licensing an H7 derivative to add to their current vaccine base as we have data that this improves the efficacy of the vaccine. The research proposed also aims to develop a mucosal adjuvant technology based on flagellin hybrids that could be applicable to other antigens, initially targeting ruminant animal diseases. We consider that the research will add to the knowledge economy, hopefully providing opportunities to develop other vaccines based on the information we generate and protect. We already have a patent pending on the applications of H7 described above, currently being filed in Europe and multiple countries worldwide. The research fits directly under the BBSRC priorities of 'Animal Health', 'Food Security' and 'Global Security' in aiming to carry out research into host-pathogen interactions that can be applied to reduce the threat from infectious diseases to the UK, thereby improving the sustainability of UK agriculture and reducing our dependence on imported food. In addition, a functional understanding of this TLR5 agonist should allow its exploitation for therapeutic intervention, in line with the BBSRC drive to more 'synthetic biology' in which key molecules are used directly or modified for therapeutic or diagnostic purposes. This research funding would continue to support a multi-institute grouping that was established as a result of DEFRA Fellowship funding in 1999 and provides a world-leading centre for expertise on enterohaemorrhagic E. coli; that is also currently expanding into other significant zoonotic diseases. The grouping has introduced many veterinary scientists into research that have gone on to contribute in companies, further research and to agencies, such as EFSA. This type of research support underpins the capacity to train undergraduate and post-graduate researchers in this important area. Undergraduate and postgraduate students that train in the laboratory experience a wide-range of skills and build their value for future contribution to scientific advancement and wealth creation. The grouping has not only disseminated information in journals and at conferences, but has provided reports for government agencies including DEFRA, FSA and HPS that can impact on advice and policy. Examples of this include the information on EHEC O157 rectal carriage and recent input into the Godstone petting farm enquiry in Southern England. Both SAC and Moredun have extensive connections into the agricultural industry and prepare articles and hold events for knowledge exchange with farmers and producers. Significant efforts have been made by the grouping in Scotland to raise awareness of O157 and to provide information for risk management by rural communities and countryside users. An additional impact at the educational level comes from articles for the press and and the radio. We have also produced an animation of EHEC colonisation of the gastro-intestinal tract a few years ago that the SGM has now posted on You Tube and this has a already been viewed 10,000 times (http://www.youtube.com/watch?v=EKLAZfCSf3g). We aim to continue to produce this type of information that engages the public and can be used in schools. Students from my laboratory (DG) have participated in the Edinburgh Science Festival and in directly promoting this subject in Schools.

Publications

10 25 50

publication icon
Fernandez-Brando RJ (2016) Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides. in Antimicrobial agents and chemotherapy

publication icon
Matthews L (2013) Predicting the public health benefit of vaccinating cattle against Escherichia coli O157. in Proceedings of the National Academy of Sciences of the United States of America

 
Description Main findings of the research are:
1. We have demonstrated that bovine TLR5 is active (there was published debate about this), and that bovine TLR5 and human TLR5 show functional variation in terms of the types of flagellin they respond to. This is important as it means that if using flagellin as an agonist in an adjuvant then there are specific flagellins that may be more effective to use in each host species.
2 The bovine and human TLR5 also demonstrate differences in the signalling pathways they use. This is exemplified by our finding that each TLR really only functions optimally in its cognate host cell background, i.e. bovine TLR in bovine cells and human TLR5 in human cells. When switched they lose significant function in terms of activation of inflammatory signalling. The reasons why these differences have evolved between the species isnot currently understood.
3. Cloning of the Flu M2 ectodomain into H7 flagellin was achieved leading to expression on the flagella. These purified flagella were used to vaccinate cattle and while the constructs generated excellent IgG responses to M2, IgA responses were negligible. We consider this is due to the IgA response being mainly confirmational and T-cell independent and so a specific response to the purified M2 was not detectable. Conjugation methods and perhaps larger co-antigens need to be tested for further analysis of H7 as an adjuvant.
4. We have demonstrated that a vaccine containing a combination of H7 flagellin, intimin and EspA antigens is effective at reducing colonisation of E. coli O157 in cattle. Moreover, we have now demonstrated that the vaccine is able to block colonisation of Shiga toxin positive (Stx+) E. coli O157 as much of our previous work was tested using Stx- isolates due to cost issues. We are currently seeking commercial partners for wider testing of the vaccine in field trials towards licencing. We also intend to test flagellin adjuvancy in other livestock species including pigs and chickens.
5. Modelling based on our vaccine efficacy data indicates it should prevent animal to animal transmission and 'super-shedding' in production systems.
6. We have evidence that the flagella of E. coli and Salmonella can penetrate into eukaryotic cell membranes and that has consequences for adherence and subsequent pathology. This work is still to be published but direct interactions with Salmonella flagella and the actin cytoskeleton have been demonstrated.
Exploitation Route We still hope to obtain further funding to explore the adjuvant activity of flagella, especially in cattle and swine. We also still aim to have the IP licenced and field trials of the vaccine to limit E. coli O157 excretion from cattle to protect public health.
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description We have continued to pursue intellectual property (patents) on the use of H7 flagellin both as an adjuvant as as a component of a three component vaccine to prevent EHEC O157 excretion from cattle. We are still in negotiation with commercial partners to test the vaccine in a commercial feedlot system and we now hope this will happen in 2019. Positive results from such a trial would be anticipated to lead to support to licence the vaccine for market.
First Year Of Impact 2013
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description Food Standard Agency mitigation of risk from EHEC O157
Amount £2,063,845 (GBP)
Funding ID FS101055 
Organisation Food Standards Agency (FSA) 
Sector Public
Country United Kingdom
Start 01/2014 
End 08/2017
 
Description Impact Acceleration Account
Amount £19,259 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 07/2016
 
Description The capacity and pathogenic potential of bacteria that internalise into plant tissue
Amount £37,462 (GBP)
Organisation Food Standards Agency (FSA) 
Sector Public
Country United Kingdom
Start 04/2014 
End 03/2016
 
Description Collaboration with USDA Nebraska 
Organisation U.S. Department of Agriculture USDA
Country United States 
Sector Public 
PI Contribution Exchange of STEC sequence information. Analysis of PacBio long read sequencing of human and cattle isolates
Collaborator Contribution Exchange of STEC sequence information. Carrying out of PacBio sequencing. Discussion of feedlot trials for E. coli O157 vaccine
Impact Publications are provided in main list
Start Year 2014
 
Title IMMUNOGENIC OMPOSITIONS CONTAINING ESCHERICHIA COLI H7 FLAGELLA AND METHODS OF USE THEREOF 
Description Immunogenic compositions containing Escherichia coli O157:H7 flagella including fusion proteins and methods using the immunogenic compositions are disclosed. Inducing an immune response in an animal to Escherichia coli O157:H7 flagella will result in prevention of colonization by Escherichia coli O157:H7 in the animal or a reduction in the amount of Escherichia coli O157:H7 infecting the animal. The immune composition will prevent or reduce the attachment of Escherichia coli O157:H7 to cells within the animal. 
IP Reference WO2009050474 
Protection Patent granted
Year Protection Granted 2015
Licensed No
Impact A commercial partner has supported further research due to the IP and still working to achieve wider scale commercialisation
 
Description Press release and TV news item 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Press releases and one TV news report about our vaccine research and machine learning prediction of the human health threat of specific E. coli strains
Year(s) Of Engagement Activity 2013,2016
 
Description Prison visit (Shotts) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact Presented a workshop at Shotts prison to a small group working on a prison magazine.
Year(s) Of Engagement Activity 2014