challengens and solutions for improved intestinal vaccination · gut-associated lymphoid tissue...
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Challenges and solutions for improved intestinal vaccination
DEPARTMENT VIROLOGY PARASITOLOGY IMMUNOLOGYLABORATORY OF IMMUNOLOGY
Eric Cox
Laboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Belgium
Eric.Cox@UGent.be
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Enteropathogens of Pigs• Zoonoses
– Salmonella Typhimurium– Salmonella enteritidis (intestinal)– Yersinia enterocolitica– Campylobacter jejuni– Campylobacter coli– Clostridium perfringens type a– Brachyspira pilosicoli– Helicobacter suis (NHPS;
heilmannii)– Enterohemorrhagic E. coli
(subclinical)– Cryptosporidium parvum– Giardia intestinalis
• Similar pathogens as in humans– Enterotoxigenic E. coli– Coronaviruses– Rotaviruses– Ascaris suum
58.6%56.3%
17.2%14.9%17.2%5.7% 6.9%
0.0%10.0%20.0%30.0%40.0%50.0%60.0%70.0%
Coddens et al., 2013. Unpublished results
After weaning
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Protection small intestine
ROUTEparenteral
Too virulent => diseaseToo attenuated => no danger
mucosal
Not mucosa
Can immunomodulation change this?
Oral preferred route
Virulence BarriersTolerance
VACCINEalive dead
AGE passive active
Intestinal Mucosa
Passive immunity
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Challenges of mucosal vaccination
• Escaping maternal (passive ) immunity – Passive milk antibodies (lactogenic immunity)– Passive serum antibodies (colostral immunity)
• Identification of protective antigens• Efficient delivery of protective antigens to mucosa-
associated lymphoid tissue– delivery systems
• Activation of protective immune mechanisms often neutralizing IgA– adjuvants
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Gut-associated lymphoid tissue (GALT)Brandtzaeg and Pabst, 2004
Pig (intestinal length)0 days small intestine ≈ 3.4 m
6- 7 weeks small intestine ≈ 7 m
Adult small intestine ≈ 15 - 20 m
Ileum
Jejunum
Duodenum
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Immunity Oral toleranceT effector cels
DC
DC
T regulator.CD4+ CD4+
PathogensParticulated antigens
Non-replicating solubleantigens
M cell
IgA production
Dendritic cells
DC
Immature antigen -presenting cels
DC
Epithelium
Danger
MUCOSAFOLLICLE-ASSOCIATED EPITHELIUM
Draining lymphnode
DC
DC
DC DCDC
DC
Mucosa-associatedlymphoid tissue
Danger
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Particulatedantigen
M-cel
Gut-associated lymphoid tissue
DCMature DC
Immunity
Follicle-associated epithelium M-cells
Few soluble antigensVirulence factors
Enterocytes
Enterocytes
Lamina propriaMesenteric Lnd
Oral vaccination
Mature DC DC
Danger Danger
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Oral F4 induces mucosal protective IgAresponse
Van den Broeck et al., 1999. Infect Imm
F4 fimbriae oral to weaned pigsPurified by mechanical shearing (±75% pure)1mg F4 at days 0, 1, 2 and 16
D DF4+ ETEC
Control
OrallyImmunized withF4 fimbriae
Chart1
22
33
44
55
66
immunized (n=4)
non-immunized (n=5)
days post infection
F4 + ETEC/ g faeces (x 103)
Fecal excretion after challenge infection with F4+ enterotoxigenic E. coli
0
0
0
12.2
0
1.6
0
0.4
0
0
Sheet1
Days23456
immunized (n=4)00000
non-immunized (n=5)012.21.60.40
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Snoeck et al., 2008. Vet Imm Immunopath.
Ligated loops injected with F4
Binding and uptake of F4 fimbriae
F4 fimbriae in M cell
F4 in enterocytes
Cytokeratin-18
F4 fimbriae
230120100
80
60
50
40
30
Aminopeptidase N (APN)
Blotting of brush border proteins and staining with F4
F4 binds to
Melkebeek et al., 2012. Mucosal Immun.
DuodenumJejunumIleum
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Oral targeting with APN-specific rabbit antibodies
3
4
5
6
7
8
9
10
11
0 7 13 20 27
Log
2 (ti
ter)
Time (d)
IgG
IgG + CT
anti-APN
anti-APN + CT
N=4
N=6
N=4
15 min after injection in small intestinal loop
IgA titer
Oral 1 mg antibodies
Antibodies binding to APN are endocytosedby enterocytes
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Particulatedantigen
M-cel
Gut-associated lymphoid tissue
DCMature DC
Immunity
Follicle-associated epithelium M-cells
Few soluble antigensVirulence factors
Enterocytes
Enterocytes
Lamina propriaMesenteric Lnd
Oral vaccination
PathogenLive attenuated vaccine
Mature DC DC
Soluble antigen neutralized by milk antibodies during suckling period
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Yeast particles for delivering antigen
Targeting particles to intestinal receptor (APN) for more efficient uptake in the gut
Targeting yeast particles
Baert et al., 2015. Journal of Controled Release
Baert et al., 2016. International Journal of Nanomedicine
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FedF
Saccharomyces cerevisiae
Acid/basictreatment
antigen complexed with tRNA
Baert et al., 2015. J Contr Rel
Yeast particles as antigen delivery vehicle
F18 fimbriae
E.coli
Load with antigen
Baert et al., 2016. Int J Nanomedicine
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Conjugation of APN-specific monoclonal to particles
BSA*FITC or FedF
Yeastmicroparticle
anti-APN monoclonal
Stained with IgG-AlexaFluor 647
Confocal microscopy
UncoatedYeast particle
Anti-APN coatedYeast particle
Baert et al., 2015. J Contr Rel
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Lumen
Lumen
Lumen
LumenLumen
Lumen
Lamina Propria Jejunal PP Ileal PP
Control
aAPNYeast
particle
APN-targeting results in a higher transcytosis of GPs through intestinal epithelial cells ex vivo (explants)
Green = particles or APNBlue = nucleusRed = actin filament
1 hour incubation
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Different groups
2. FedF
D0 D1 D13 D14 D28
Stomach acid blocker
Stomach acid blocker Booster
Primer dose End
Ig IgA IgG IgA
1. Control 3. FedF GP4. anti-APN FedF GP Serum ELISA
PBMCsfrom blood
ELISPot
ELISPotELISA
APN-targeting results in a higher production of FedF-specific antibodies in vivo
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Particulatedantigen
M-cel
Gut-associated lymphoid tissue
DCMature DC
Immunity
Follicle-associated epithelium M-cells
Few soluble antigensVirulence factors
Enterocytes
Enterocytes
Lamina propriaMesenteric Lnd
Oral vaccination
PathogenLive attenuated vaccine
Mature DC DC
Soluble antigen neutralized by milk antibodies during suckling period
Bacterial vectors might interfere with antibiotic treatment
Viral vectors sufficient local replication
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Particulatedantigen
M-cel
Gut-associated lymphoid tissue
DCMature DC
Immunity
Follicle-associated epithelium M-cells
Few soluble antigensVirulence factors
Enterocytes
Enterocytes
Lamina propriaMesenteric Lnd
Oral vaccination
Live Vector -no neutralization by milk antibodies
-not sensitive to antimicrobials-adjuvant (danger)
Mature DC DC
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APN targeting seems a promising strategy to induce protective immunity upon oral vaccination Piglets & other species (human, sheep, …) Functionalised nanoparticles (targeting, TLR ligands) Targeting soluble antigens
Anti-APN linked to antigenAnti-APN linked to particle
DC
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AcknowledgementsFormer PhD students )Prof. dr. Wim Van den Broeckdr. Frank Verdonck (Ablynx)dr. Petra Tiels (VIB-UGent)dr. Veerle Snoeck (Ablynx)dr. Kristien Rasschaert (B&D)dr. Vesna Melkebeekdr. Philippe Bellot (USG Professionals)dr. Kim Baert (Anacura)
Lab of Immunology (UGent)Prof dr. Bruno Goddeeris (KULeuven)Prof. dr. H. Favoreel dr. Bert Devriendt (post-doc)
Lab of Pharmaceutical Technology (UGent)Prof. dr. Jean-Paul RemonProf. dr. Bruno De Geest
Vrije Universiteit Brussel (VUB)Prof. dr. Henri Degreve and Han Remaut
Financial support
Lab of Pharmaceutical Biotechnology (Ugent)Prof dr. Dieter Deforcedr. Kelly Tilleman
Faculty of VeterinaryMedicine
Challenges and solutions for improved intestinal vaccinationEnteropathogens of PigsProtection small intestineChallenges of mucosal vaccination�Slide Number 5Slide Number 6Slide Number 7Oral F4 induces mucosal protective IgA response Slide Number 9Slide Number 10Slide Number 11Targeting yeast particlesSlide Number 13Conjugation of APN-specific monoclonal to particlesSlide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20
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