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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
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
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