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

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 Unpublished results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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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