KATIE BOLAND DVM, PHD JUNE 15, 2013 VACCINATION OF CATTLE WITH ESCHERICHIA COLI O157:H7-DERIVED PROTEINS RESULTS IN HUMORAL AND CELLULAR IMMUNE RESPONSES.

KAT I E BO L A N D DV M , P H DJ U N E 1 5 , 2 0 1 3

VACCINATION OF CATTLE WITH ESCHERICHIA COLI O157:H7-DERIVED PROTEINS RESULTS

IN HUMORAL AND CELLULAR IMMUNE RESPONSES BUT DOES NOT CONFER PROTECTION AGAINST SUBSEQUENT

CHALLENGE

1

E. COLI O157:H7

• Causes disease in humans• Diarrhea (MMRW, 1985)

• Hemolytic-uremic syndrome (HUS) (Riley LW, Remis RS, Helgerson SD, et al, 1983)

• Renal failure, hemolytic anemia and thrombocytopenia

• Pathogenesis• Attaching and effacing (A/E) lesions

• Pedestal formation• Dissolution of brush border

• Shiga toxins

• Prevention is key

2

RESERVOIR

• Cattle are major source of contamination (Armstrong et al., 1996)

• Transient colonization• Variable shedding

• Meat• Produce• Water

• Recto-anal junction• Lymphoid rich tissue

3

PRE-PROCESSING CONTROL

• Decontamination• Probiotics• Diet• Antimicrobials

http://www.chadcompany.com/MVC-006F.JPG

4

A/E LESIONS5

(Gauthier, Finlay 2002)

6

1: Mucosal immunization of naïve cattle with recombinant intimin induces a more robust adaptive

immune response at the recto-anal junction than subcutaneous immunization

2: An induced adaptive immune response to recombinant E. coli O157:H7 proteins is associated with

decreased colonization

HYPOTHESES

TRIAL SETUP

• Trials 1, 2 and 3• 3 animals per group

• Trial 1: Cholera toxin B subunits (rectal) and Freund’s (SQ)• Trial 2: TLR 7/8 agonist (rectal and SQ)• Trial 3: TLR 4 agonist (rectal and SQ)

• Immunized weeks 0, 3, 6

7

CELLULAR RESPONSES

      INT OVA

Trial Treatment Adjvant Week 0 Week 9 Week 0 Week 9

1 rectal CTB 0 2 0 2  SQ CFA 0 3 0 22 rectal TLR7/8 0 3 0 1  SQ TLR7/8 0 3 0 13 rectal TLR4 2 2 0 2  SQ TLR4 3 3 0 1

      INT OVA

Trial Treatment Adjvant Week 9 Week 9

1 rectal CTB 2 2  SQ CFA 2 02 rectal TLR7/8 3 2  SQ TLR7/8 2 33 rectal TLR4 0 0  SQ TLR4 0 0

Systemic PBMC Responses

Local MRLN Responses

8

TRIAL 4

• Is the MRNL response targeted?• TLR 7/8 agonist• All get SQ immunization week 0• Immunized again weeks 8 and 11• All SQ immunizations on same side

• Evaluate MRLNs as well and prescapular LNs

9

10

34545 34549 34551 34553 34590 32888 32896 34548 34570 34577

1

10

100

1000

Con A INT OVA

Stim

ulat

ion

Inde

x

Animal NumbersRectal Subcutaneous

*

*

*

*

* *

*

* **

*

**

*

*

Trial 4 PBMC Responses Week 11

11

34545 34549 34551 34553 34590 32888 32896 34548 34570 34577

1

10

100

1000

Con A INT OVA

Stim

ulat

ion

Inde

x

Rectal Subcutaneous

*

*

*

*

**

*

* *

*

** *

*

*

**

*

*

* *

**

*

***

Trial 4 MRLN Responses

Animal Numbers

12

Draining Non-draining Mesorectal0

10

20

30

40

50

60

70

80

90

100

110

120

130S

timu

latio

n In

de

x

Lymph Nodes

Trial 4 LN Stimulation Summary INT 10g

SERUM ANTIBODY RESPONSES

• All 4 trials:• Significant increases in titers to INT• Significant increases in titers to OVA in SQ groups

• Variable responses to OVA in rectal groups

• In trial 1 and 4:• Titers in SQ groups significantly increased compared to

rectal groups

13

14

1: Mucosal immunization of naïve cattle with recombinant intimin induces a more robust adaptive

immune response at the recto-anal junction than subcutaneous immunization

(reject)

2: An induced adaptive immune response to recombinant E. coli O157:H7 proteins is associated with

decreased colonization

HYPOTHESES

CHALLENGE TRIALS

Trials 1 and 2

• 6 animals per group• Immunized or sham• 4(trial 1) or 3 (trial 2)

doses• Challenged

• Rectal (trial 1)• Oral (trial 2)

Trials 3 and 4

• Trial 3• 10 animals per group• Previous oral or rectal

exposure• Oral or rectal challenge

• Trial 4• Oral group from trial 3• Rectal challenge

Colonization levels followed for 4-7 weeksEvaluated PBMC and serum antibody responses

15

16

0 1 3 8 14 21 28 35 42 490

1

2

3

4

5

6

7Lo

g 10(C

FU

E.

coli

O15

7:H

7/S

wab

)

Days Post-Challenge

Immunized Animals Sham-Immunized Animals

Challenge 1 Colonization Levels

17

0 1 3 8 14 21 28 35 42 490

1

2

3

4

5

6

7

High Responders Low Responders

Log 10

(CF

U E

. co

li O

157:

H7/

Sw

ab)

Days Post-Challenge

Challenge 1 Colonization Levels by PBMC Responses

SERUM ANTIBODY RESPONSES

• In trials 1 and 2:• All animals had significant increases in titers to all

proteins• No change 2 weeks post-challenge

• In trials 3 and 4• Variable, low responses to all proteins• Tended to be higher in trial 4

• In all trials:• Did not correlate with colonization duration

18

Trial Trial

GroupsAnimals Immunization Challenge

Post Challenge

Treatment Adjuvant RouteTotal doses

Bacteria Total CFUs RouteCulture Time

Trial 1 Immunized 5INT, OVA, EspB, TIR

R837 Rectal 4 A 1010 Rectal 49

Sham 5 1x PBS none Rectal 4 A 1010 Rectal 49

Trial 2 Immunized 6INT, OVA,

Esp A, EspB, TIR

R848 SQ 3 A 1010 Oral 28

Sham 6 1x PBS none SQ 3 A 1010 Oral 28

Trial 3 Oral 10109 CFU total B

none Oral 1 B 109 Oral 56

Rectal 10109 CFU total B

none Rectal 1 B 109 Rectal 56

Trial 4 Oral 10109 CFU total B

none Oral 2 B 107 Rectal 35

CHALLENGE SUMMARY

19

CHALLENGE CONCLUSIONS

• Immunization and experimental exposure induced lymphoproliferative responses

• Immunization induced significant antibody responses

• No significant increase in responses after challenge

• Neither lymphoproliferative nor serum antibody responses conferred protection against colonization

20

OVERALL SUMMARY

• Mucosal immunization does not induce a more robust regional immune response compared to SQ immunization • Regional responses overall more robust than systemic

and are targeted in SQ immunization

• Induced immune responses are not protective against challenge

21

22

Tovah KerrSusan SmartEmma KarelFred LoaizaLonnie Austin

Alex BeckAndrea HaylesEric SuttenKathleen SuttenAllison VilanderClaire Miller

Carolyn BohachHaiqing ShengClaudia Deobald

THANK YOU!

Kevin LahmersTim BaszlerTom BesserWendy BrownDoug CallEsther Trueblood

WADDL Pathologists and residentsVMP Faculty and staffGraduate StudentsOfficematesFriendsFamily

ELISA

1:10

1:20

1:1280

Negative controlSamples

Positive if > average + 2 SD

Titer is last positive dilution