How does systemic flagellin immunization induce mucosal IgA?

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

Vaccination has been pivotal to the improved control of infectious diseases seen in the last century. Despite this encouraging progress, infections remain responsible for around 20% of deaths worldwide, primarily in the very young and in the aged, but they also have a considerable impact on our livestock and thus our food chain. This is important, not just for the substantial economic impact it has on the Euro 2 trillion food production business, but because we acquire many deadly infections from our food.

Developing novel vaccines against infectious diseases is highly cost-effective and history demonstrates how effective they can be. Furthermore, as our options to treat bacterial infections using antibiotics diminish, because of increased resistance to these drugs and a paucity of new drugs coming through, vaccination looks increasing attractive as a way to prevent disease developing. Vaccines are so effective at preventing disease because they work by providing protection before the infection is established, i.e. when pathogen numbers are likely to be at their lowest.

In most cases, vaccines provide their protection through the induction of proteins, called antibodies, which are specific for a single part of the bacterial or viral pathogen. These proteins "stick" to the pathogen and prevent it from damaging us or flag up its presence to the cells of the immune system allowing it to be destroyed. Although antibodies are specific, they come in different flavours, so the type of antibody that vaccines induce in the blood that help us kill invading pathogens is called IgG. In contrast, the most common form of antibody at body surfaces, called mucosal sites, is IgA. So both IgG and IgA are highly efficient at protecting us but tend to predominate in different parts of the body. Because vaccines are often given subcutaneously or into the muscle they tend to only induce systemic IgG, but not mucosal IgA. This has prevented us from maximizing the potential for IgA to protect too. This work aims to address this issue.

What we have identified is a way to induce systemic IgG and mucosal IgA at the same time. This is important because it is difficult to achieve. To achieve this we have immunized mice subcutaneously with a key bacterial protein called flagellin. Normally, this protein helps bacteria move and adhere, but here we have used it as a Trojan Horse to induce strong IgG and IgA responses and so potentially exploit the potential of antibody to protect at both mucosal and systemic sites. Because in some parts of the immune system these IgG and IgA responses develop side by side we can examine them for their similarities and differences. This will allow us to generate new technologies to maximize how best to make an efficient vaccine to provide protection at multiple sites throughout the body.

Technical Summary

Vaccination is a key strategy to control infectious disease in humans and economically important animals. Currently, most vaccine strategies rely on the induction of IgG, despite most pathogens entering the host via mucosal routes, where the predominant isotype is IgA. This is because most vaccines are administered systemically and this tends to result in limited IgA induction.

What we have identified is that the flagellin protein, FliC, from Salmonella Typhimurium, when given systemically (e.g. subcutaneously or intraperitoneally), induces IgG in the spleen but IgG and IgA in the mesenteric lymph node (MLN). This co-induction of IgG and IgA in the MLN is surprising since we do not see this in response to other proteins such as ovalbumin when given as a soluble or alum-precipitated protein. When we investigated the mechanism underlying this, we found that it was through the TLR5-mediated recruitment of CD103+CD11b+ dendritic cells (DC) from the lamina propria to the MLN.

Combined with our capacity to identify antigen-specific T and B cells we have a model where we can investigate how IgG and IgA responses can develop concurrently in the same lymph node. This will allow us to better understand the mechanisms through which switching is mediated and controlled and how best to exploit this to develop vaccines that induce systemic and mucosal responses.

One of the key ways this is likely to be regulated is through distinct populations of T follicular helper (Tfh) cells being induced. Using our model we wish to address the hypothesis that the MLN contains multiple populations of Tfh that are induced in parallel yet contribute differently to the regulation of IgA and IgG switching. We will also test the hypothesis that immunizing with sFliC-heterologous antigen conjugates induces IgA responses to the passenger antigen in the MLN and thus sFliC can act as a conjugate carrier for vaccine delivery.

Planned Impact

Society
The importance of this work extends significantly beyond supporting academic research. By understanding how antibody responses develop we are helping understand how to improve vaccine development. This is vital. In our highly mobile society the risks from infectious diseases are increasing due to increased travel and altered global migration and extend also into the food-chain. This is because many of our infections are actively acquired from food (e.g. Salmonella) or livestock rearing acts as an incubator and enables pathogen diversity (e.g. influenza virus). In parallel, we have a decreasing efficacy of anti-microbial treatments due to resistance and only a modest number of anti-virals available. Vaccination is a cost-effective approach that can protect against infection at the extremes of age in those groups that are most susceptible. Furthermore, vaccination is an acceptable intervention to society at large, and as the media response to the recent measles outbreak demonstrates, is one that is diminishing in controversy. Indeed, the measles outbreak demonstrates the importance of vaccination programmes to protecting society and the consequences when there is insufficient vaccine coverage.

Industry

This work will have a marked impact upon industry. The vaccine market is estimated to be worth $52 billion by 2016 and is likely to increase as more vaccines are developed and are required due to the increasing challenge of anti-microbial resistance. Flagellin is a molecule of interest both as a vaccine component and as a carrier, as demonstrated in the "Benefits" section above. This is aided by its safe use in man its ability to induce immunity in the absence of exogenous adjuvant. Therefore, the benefits to industry from this work are two-fold. First, there is understanding the nature of the immune response to flagellin itself and second, there is the impact improved understanding of fate decisions in immunoglobulin switching will deliver.

Publications

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Hitchcock JR (2015) Inflammation drives thrombosis after Salmonella infection via CLEC-2 on platelets. in The Journal of clinical investigation

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Bravo-Blas A (2019) Serovar Typhimurium Travels to Mesenteric Lymph Nodes Both with Host Cells and Autonomously. in Journal of immunology (Baltimore, Md. : 1950)

 
Description This work has generated multiple exciting avenues of research, which evolved from BBSRC grant BB/F022778/1.

Using the bacterial motility protein flagellin, that triggers an immune response at least in part through a molecule called TLR5, we have shown that the presence of flagellin can moderate the extent of a Th1 response to Salmonella infection. Th1 responses are needed for clearance of active infections, but what is not clear is how much of a Th1 response is needed. Where this research becomes highly significant for translation is that it helps identify a way to limit the size of a Th1 response. This is important because a lot of the illness we suffer from infection is not caused by damage driven from the bacterium but rather it is due to our immune response to the organism. This is called immunopathology and is responsible for many of the consequences of inflammatory diseases. Therefore, we may be able to use flagellin to help moderate the worst excesses of this condition.

Secondly, we have identified a single cell type that is important for controlling antibody responses to flagellin in the gut. This is important since it helps understand how to enhance vaccine responses at the gut, which could help prevent infections from developing before they spread into the body. A really exciting finding that came from this is that being able to mount an immune response in the gut lead to a better immune response throughout the body. Therefore, using flagellin to help drive immune responses to vaccines may improve the protection they offer.
Exploitation Route This has been detailed above but in summary:

1. We can help moderate immunpathology caused by our immune response becoming overactive

2. We can target a single cellular subset to improve vaccine responses at the gut and throughout the body.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Australian National University 
Organisation Australian National University (ANU)
Department College of Medicine, Biology & Environment (CMBE)
Country Australia 
Sector Academic/University 
PI Contribution We worked together on different aspects of TfH development
Collaborator Contribution Publication in JEM
Impact PMID: 21708925
Start Year 2009
 
Description Babraham 
Organisation Babraham Institute
Country United Kingdom 
Sector Private 
PI Contribution We contribute to the collaboration through our immunohistology skills
Collaborator Contribution They provide unique mouse models and reagents
Impact The RNA-binding protein HuR is essential for the B cell antibody response. Diaz-Muñoz MD, Bell SE, Fairfax K, Monzon-Casanova E, Cunningham AF, Gonzalez-Porta M, Andrews SR, Bunik VI, Zarnack K, Curk T, Heggermont WA, Heymans S, Gibson GE, Kontoyiannis DL, Ule J, Turner M. Nat Immunol. 2015 Apr;16(4):415-25. doi: 10.1038/ni.3115. Epub 2015 Feb 23. PMID: 25706746 Free PMC Article
Start Year 2011
 
Description Development of low cost passive vaccination tools based on IgY 
Organisation Sanovo Technology Group
Country Denmark 
Sector Private 
PI Contribution As a result of funding from CiC I have met with representatives of Sanovo. We have signed a MTA and are currently identifying how to perform proof-in-principle experiments on how to manufacture cheap, passive vaccines consumed by travellers as they go from high income, low infection risk, to low income, high infection risk environments. The company will generate the IgY based on our immunogens. This is still ata very early stage.
Collaborator Contribution Please see above
Impact Immunology, vaccinology, protein production,
Start Year 2016
 
Description Flagellin as a vaccine carrier 
Organisation Moredun Research Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We have established links to generate new vaccines against veterinary associated infections
Collaborator Contribution Expertise in the veterinary field
Impact Only recently established. No outputs yet
Start Year 2019
 
Description MRC Centre for Immune Regulation 
Organisation University of Birmingham
Department MRC Centre for Immune Regulation
Country United Kingdom 
Sector Public 
PI Contribution Contribution to a theme in the Centre: Induction and regulation of immunity in secondary lymphoid tissues. My work examining how immune responses develop to pathogens and their components offers broad cross-theme collaborations.
Collaborator Contribution Access to material and equipment as well as MRC students
Impact From this collaboration nearly all of my publications and patent are due, in part, to this collaboration.
 
Description UCT 
Organisation University of Cape Town
Country South Africa 
Sector Academic/University 
PI Contribution We have published a number of papers together examining how antibody responses and vaccines work. We have conducted research visits to UCT to perform the work there and also collaborated by providing advice and reagents
Collaborator Contribution They have provided unique reagents and resources enabling us to conduct experiments and studies not possible in their absence.
Impact doi: 10.1007/978-1-4939-1489-0_4 doi: 10.1371/journal.ppat.1003662 doi: 10.1371/journal.pntd.0003341
Start Year 2009
 
Description University of Lund 
Organisation Lund University
Country Sweden 
Sector Academic/University 
PI Contribution The post-doctoral fellow Dr Adriana Flores-Langarica made two visits to Lund to work collaboratively on the project. The resources used (animal models, consumables etc) were covered by Lund. This is a significant cost and research benefit to us as it made available unique resources.
Collaborator Contribution They provided unique models for our studies
Impact The first manuscript from this work is in preparation
Start Year 2013
 
Description Vaccine development with GSK Vaccines for Global Health 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We are developing a novel generation of vaccines. We have the expertise to assess how they function
Collaborator Contribution Our partners generate the vaccine candidates
Impact This has just started
Start Year 2017
 
Description Secondary school student work experience 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Type Of Presentation Workshop Facilitator
Geographic Reach Local
Primary Audience Schools
Results and Impact 1 student for work experience.

This has developed into a longer-term relationship. For instance, I will host another student next year and have arranged to talk at the school next year.
Year(s) Of Engagement Activity 2012
 
Description Sun interview 
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 This was an interview in The Sun regarding the MMR controversy and the benefits of vaccination.

Feedback from the public was positive
Year(s) Of Engagement Activity 2014
 
Description cafe scientifique 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Lots of discussions

Email exchanges setting up links with the public
Year(s) Of Engagement Activity 2014
 
Description school visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talks at local schools about animal experimentation and immunology, vaccinology and infectious disease. During this it became clear it is important alos to offer career advice. This is something also pursued by members of my group

Increased awareness and involvement with other schools
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014