Microbes and Infection 15 (2013) 845e848www.elsevier.com/locate/micinf

Highlight

A bacterium’s guide to orienteering in the intestinal lumen*

“BEWELL!Take oral polio vaccine. Tastes good,works fast,and prevents polio” says Wellbee, the Center for Disease Con-trol and Prevention’s national symbol for public health in a 1963poster encouraging the public to receive oral polio vaccine [2].Although those of us who were given polio vaccine without asugar lump may disagree with Wellbee on the taste, we cannotdoubt its effectiveness. Mass immunisation programs as pro-moted by Wellbee in the 1960’s meant that polio virtually dis-appeared in developed countries in the mid-1970’s. In 1988, theWorld Health Organisation (WHO) launched a campaign toeradicate polio from the face of the planet, reducing cases from350,000 reported in that year, to less than 3500 in 2012 [3].

Part of this success can be attributed to the vaccine’s methodof administration. The oral polio vaccine developed by AlbertSabin in the late 1950’s was in fact preceded by an intravenousvaccine developed by John Stalk in 1952 [4]. Both vaccines arehighly effective and are routinely used in the United States andacross Europe [5]. However, it was Sabin’s oral vaccine that waschosen as the saviour in WHO’s campaign. Injections requiresterile needles and trained personal, which makes mass immu-nisation more costly and less safe, particularly in developingcountries that lack the medical infrastructure to administerinjected vaccines. Yet, orally administered vaccines exist foronly a handful of pathogens, which besides polio include rota-virus, Salmonella typhi, and cholera [6].

To understand the challenges faced by oral vaccinationwe justhave to look at the environment into which the vaccine is intro-duced. Assuming that any antigen may survive a bath in gastricacid and proteolytic enzymes, it then has to compete with foodand microbial antigens for absorption by the intestinal tract andthe attention of the mucosal immune system. It’s a crowdedenvironment down there, with ten times more bacteria in theintestinal tract than there are cells in the human body [7].Although on the whole wemay seem blissfully indifferent to thisteeming microbial metropolis, it is clear that host bacteria doinduce immune responses as evidenced by the large number oflymphoid cells present in the intestine and the 3e5 g of IgAproduced per day in a normal human [8]. These factors mean thatcalculating an effective dose is no straightforward affair, andsignificantly larger doses of antigens must be used to achieve alevel of immune response comparable to that of injected vaccines.

* Article highlight of “C5a receptor-targeting ligand-mediated delivery of

dengue virus antigen to M cells evokes antigen-specific systemic and mucosal

immune responses in oral immunization” by S.-H. Kim et al. [1].

1286-4579/$ - see front matter � 2013 Institut Pasteur. Published by Elsevier Ma

http://dx.doi.org/10.1016/j.micinf.2013.10.004

Several strategies have been devised to improve antigenuptake in the intestinal lumen. These include co-administrationof mucosal adjuvants, or coating the antigen with polymer orlipid based particles, or so called ‘mucoadhesives’ such as car-boxymethyl cellulose [6]. Adjuvants often provide danger signalsthat direct and stimulate the immune response to co-administeredantigen. Perhaps some of the most potent mucosal adjuvants arethe bacterially derived enterotoxins, including cholera toxin,(genetically engineered of course so as not to induce a nasty boutof diarrhoea). However in the populous environment of theintestine, such mucosal adjuvants may end up promoting unde-sirable immune responses to food or resident microbes, unlessthey are chemically or genetically linked to the antigen. Alter-natively, antigensmay be delivered by a recombinant bacterial orviral vector. Such microbial vectors however are often stronglyimmunogenic, meaning that the host defence system may mounta response against the vector rather than the desired antigen. Inaddition, bacterial vectors may have to compete with residentbacteria in order to provide doses of antigen required for long-lasting immune responses.

Any potential antigen will undoubtedly be able to navigatebetter the intestinal environment with a set of directions. Indeed,one promising strategy involves telling the antigenwhere to go tohave the best chance of uptake. Microfold (M) cells mediatecontinuous pathogen surveillance by transporting substancesacross epithelial surfaces for subsequent uptake and processingby dendritic cells that are lying in wait [9]. M cells are perfectlyadapted for this function: at their basolateral surface, they possessa unique intraepithelial invagination or ‘pocket’ for cradlinglymphocytes and other antigen-presenting cells, reducing thedistance that antigens have to travel before encountering immunesystem cells. M cells are the main point of access for luminalantigens to underlying mucosal lymphoid tissue, however, theyexist as only 1 out of 10 million epithelial cells in the intestinaltract [10], so finding them can be a challenge. Many pathogenshave developed tricks up their sleeve to do just that, and exploitMcells as a route of entry to underlying host tissues. Understandingthe mechanisms by which some microorganisms selectively useM-cells to cross the intestinal epitheliumwill help us to get usefulmolecules such as vaccine antigens across the intestinal epi-thelium instead of nasty bacteria. Jang and colleagues previouslysuggested that the gram negative bacterium Yersinia enter-ocolitica targetsM cells via the cell surface expression of a ligandtermed OmpH, which interacts with the complement 5a receptoron the surface ofMcells [11,12]. In this issue, they takeadvantage

sson SAS. All rights reserved.

846 Highlight / Microbes and Infection 15 (2013) 845e848

ofYersinia’s ingenuity tomaximise the introduction of an entirelydifferent beast to the mucosal immune system; the EDIII antigenof the Dengue 2 (DENV2) virus [1].

Dengue virus is carried by the tropical dwelling Aedesmosquito. Although ingestion may not be the typical point ofentry, (studies on the transmission of Dengue virus by swal-lowing carrier mosquitoes are unsurprisingly lacking), immunecells induced by antigen in the gut canmigrate to the circulation,allowing systemic humoral and cellular immune responses [13].Jang and colleagues show that EDIII genetically conjugated tothe OmpH targeting ligand was taken up by M cells in orallyvaccinated mice and induced both mucosal immune responsesand systemic immune responses. Although an initial inductionof systemic responses following oral vaccination is a step in theright direction, one of the major challenges in developingmucosal vaccines is the possible induction of ‘oral tolerance’;the idea that extended and repeated exposure to mucosal anti-gens may reduce systemic immune responses against that anti-gen. Immune responses in both systemic and mucosalcompartments were heightened in the EDIII-OmpH orallyvaccinated group following systemic challenge with EDIII,suggesting the absence of oral tolerance in this case.

More specific challenges remain regarding Dengue fever. TheDengue virus exists as four different serotypes (DENV1-4). Pri-mary infection with any of the four serotypes results in a lifelongimmunity to that serotype and a temporary immunity to others.However, any individual previously infected with DENV andunfortunate enough to be the chosenmeal for amosquito carryinga different DENV serotype is likely to experience a more severerange of symptoms the second time round [14]. This immuno-logical priming amongst serotypes means that any potentialvaccine needs to induce strong neutralising antibodies against allfour serotypes simultaneously. Nonetheless, the results reportedhere provide a basis for the development of a much needed oralDengue vaccine. Just don’t forget the spoonful of sugar.

Interview with Yong-Suk Jang

Yong-Suk Jang is a group leader at the Department ofMolecular Biology, Chonbuk National University, Korea.

What triggered your interest in oral vaccination as animmunisation strategy for DENV?

In addition to immunological priming of virus infectionamong different serotypes of DENV, one of the great tasksin DENV vaccine development for developing and under-developed countries is lowering the cost of vaccinationand securing the ease of vaccine delivery. We believe thatoral mucosal vaccine is an ideal strategy against DENVinfection in that sense. In addition, recent studies showingthat DENV is predominantly localized in lymphoid and gut-associated tissues including Peyer’s patches supported therationale for developing oral mucosal vaccine against DENVinfection. However, there are obstacles in oral mucosalvaccine development such as low efficiency in antigendelivery to immune inductive sites and possible oral toler-ance induction. That is why many efforts have been con-centrated to develop effective mucosal vaccine adjuvant. Wealso have been interested in developing antigen targetingstrategy into mucosal immune inductive site and identifiedseveral ligands including OmpH which is reported in thismanuscript.

What was your first reaction when you faced the results?Did you expect them?

Yes, we were kind of expecting the results. Actually, weidentified a ligand which is capable of targeting the conjugatedantigen into M cells in mucosal immune inductive site. Whilewe were unravelling action mechanism of the ligand, we foundthat the amino acid sequence of the ligand showed highhomology with that of OmpH expressed in Yersinia enter-ocolitica which exploits M cells for its infection. We tried tomake longer ligand than the previous one to establish a stabledelivery system andmade the OmpH ligand. Because the ligandwas designed based on the previous one which was efficient inantigen delivery, we expected the positive results although it isalways exciting to get positive and conclusive results.

How will the project go on?

The basic goal of our team in developing oral mucosalvaccine is to develop the vaccine by using plant-basedexpression system because the antigen-expressing plantmaterials do not need cold-storage for preservation and pro-duction cost is quite low. Therefore, we are collaborating withthe plant molecular biologists for this project and trying toproduce various forms of DENV antigens in transgenic plantmaterials.

What is the take-home message of the article?

If you use efficient targeting and delivery strategy, oralmucosal compartment can be used as a good delivery target forvaccine delivery.

Do you have a personal motto, quote or leading sentence?

Don’t be stressed and try to be happy while you are doingyour job!

In a nutshell

� Coupling of the Yersinia enterocolitica OmpH ligandto the EDIII antigen of Dengue virus resulted in thetargeting of EDIII to antigen uptake M cells of theintestinal epithelium in mice.

� Oral immunisation with OmpH-EDIII induced bothmucosal and systemic immune responses as assessed bythemeasurement of serum IgG and fecal IgA fourweeksafter immunisation. An immune response upon the oraladministration of EDIII alone could not be detected.

� Immune responses to the OmpH-EDIII were heavilyskewed towards a Th2 response.

� Oral vaccination did not induce systemic tolerance.Instead, higher numbers of EDIII-specific IgG andIgA secreting cells were detected following systemicchallenge at four weeks after oral immunisation withOmpH-EDIII compared to EDIII alone.

847Highlight / Microbes and Infection 15 (2013) 845e848

What advice would you give to the young next-generationscientists?

I believe that good outcomes will follow when you enjoydoing research and concentrate on it.

What is your favourite hang-out method after a tough dayat the lab?

It is really relieving for me when I have time to talk withmy family at the end of a stressful day. Since my children arealready grown-up and have moved to another city and abroad,taking a walk with my wife along the small river near myhome gives me a good rest.

If you could travel back in time e what historicalpersonality would you like to meet and what scientific discoveryto assist to?

I wonder people know about Dr. H.W. Lee, a Koreanvirologist who found and characterized Hantaan virus. Hewas a really great and respectable scientist. His efforts toidentify unknown virus without commercial experimentalmaterials including good antibodies were absolutely amaz-ing. I want to learn his persistency and great insight on theresearch.

If you could travel forward in time e what eventualinvention would you like to check out?

As an immunologist, I am interested in vaccines whichare not available yet. Especially, I want to check outwhether effective vaccine against HIV infection would bedeveloped.

Background

Oral vaccination is a safe and cost efficientmethodfor preventing infectious diseases. However, thisimmunisation strategy is currently employedagainst only a handful of pathogens, since anypotential vaccinemustfirst negotiate theharshandpopulated environment of the gastrointestinaltract. Numerous mucosal adjuvants have beendevised to improveantigenuptakeby the intestinallumen. One promising strategy involves the tar-geting of specialised epithelial cells called M cells,which ensure the surveillance of ingested antigensby transporting substances across the epithelialsurfaces to underlying dendritic cells. Manypathogens seem to do this quite well already.Understanding how such micro-organisms use Mcells to cross epithelial membranes will lead to thedevelopment of improved strategies for mucosalvaccine delivery.

References

[1] S.-H. Kim, et al., C5a receptor-targeting ligand-mediated delivery of

dengue virus antigen to M cells evokes antigen-specific systemic and

mucosal immune responses in oral immunization, Microbes Infect. 15

(2013) 895e902.

848 Highlight / Microbes and Infection 15 (2013) 845e848

[2] http://phil.cdc.gov/phil/details.asp?pid¼7224.

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game, J. Gen. Virol., 93, 457e474.

[5] http://en.wikipedia.org/wiki/Polio_vaccine.

[6] J. Mestecky, H. Nguyen, C. Czerkinsky, H. Kiyono, Oral immunization:

an update, Curr. Opin. Gastroenterol. 24 (2008) 713e719.

[7] R.E. Ley, D.A. Peterson, J.I. Gordon, Ecological and evolutionary forces

shaping microbial diversity in the human intestine, Cell 124 (2006)

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[8] J. Mestecky, M.W. Russell, C.O. Elson, Perspectives on mucosal vac-

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[9] S.C. Corr, C.C. Gahan, C. Hill, M-cells: origin, morphology and role in

mucosal immunity and microbial pathogenesis, FEMS Immunol. Med.

Microbiol. 52 (2008) 2e12.[10] R. Kuolee, W. Chen, M cell-targeted delivery of vaccines and ther-

apeutics, Expert Opin. Drug Delivery 5 (2008) 693e702.

[11] S.H. Kim, K.W. Seo, J. Kim, K.Y. Lee, Y.S. Jang, The M cell-targeting

ligand promotes antigen delivery and induces antigen-specific immune

responses in mucosal vaccination, J. Immunol. 185 (2010) 5787e5795.

[12] S.H. Kim, et al., M cells expressing the complement C5a receptor are

efficient targets for mucosal vaccine delivery, Eur. J. Immunol. 41 (2011)

3219e3229.

[13] L. Wang, R.L. Coppel, Oral vaccine delivery: can it protect against

non-mucosal pathogens? Expert Rev. Vaccines 7 (2008) 729e738.[14] S. Murrell, S.C. Wu, M. Butler, Review of dengue virus and the

development of a vaccine, Biotechnol. Adv. 29 (2011) 239e247.

Emma Louise WaltonUniversite Paris 7, UMR7216 Epigenetics and Cell Fate,

35 rue Helene Brion, Paris 75013, FranceE-mail address: ewalton86@gmail.com

4 October 2013