Download Katie Boland, DVM

1
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
KATIE BOLAND DVM, PHD
JUNE 15, 2013
2
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
3
RESERVOIR
• Cattle are major source of contamination (Armstrong
et al., 1996)
• Transient colonization
• Variable shedding
• Meat
• Produce
• Water
• Recto-anal junction
• Lymphoid rich tissue
4
PRE-PROCESSING CONTROL
•
•
•
•
Decontamination
Probiotics
Diet
Antimicrobials
http://www.chadcompany.com/MVC-006F.JPG
5
A/E LESIONS
(Gauthier, Finlay 2002)
6
HYPOTHESES
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
7
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
8
CELLULAR RESPONSES
Systemic PBMC Responses
INT
OVA
Trial
Treatment
Adjvant
Week 0
Week 9
Week 0
Week 9
1
rectal
SQ
rectal
SQ
rectal
SQ
CTB
CFA
TLR7/8
TLR7/8
TLR4
TLR4
0
0
0
0
2
3
2
3
3
3
2
3
0
0
0
0
0
0
2
2
1
1
2
1
2
3
Local MRLN Responses
INT
OVA
Trial
Treatment
Adjvant
Week 9
Week 9
1
rectal
SQ
rectal
SQ
rectal
SQ
CTB
CFA
TLR7/8
TLR7/8
TLR4
TLR4
2
2
3
2
0
0
2
0
2
3
0
0
2
3
9
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
10
Trial 4 PBMC Responses Week 11
1000
Con A
INT
OVA
Stimulation Index
100
*
*
*
10
*
*
*
*
**
*
*
*
*
*
*
1
34545 34549 34551 34553 34590 32888 32896 34548 34570 34577
Rectal
Subcutaneous
Animal Numbers
11
Trial 4 MRLN Responses
1000
*
*
*
*
*
**
*
*
*
*
*
*
*
**
*
*
*
*
100
Stimulation Index
Con A
INT
OVA
*
*
10
*
**
*
*
1
34545 34549 34551 34553 34590 32888 32896 34548 34570 34577
Rectal
Subcutaneous
Animal Numbers
12
Trial 4 LN Stimulation Summary INT 10g
130
120
110
100
Stimulation Index
90
80
70
60
50
40
30
20
10
0
Draining
Non-draining
Lymph Nodes
Mesorectal
13
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
14
HYPOTHESES
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
15
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 weeks
Evaluated PBMC and serum antibody responses
16
Challenge 1 Colonization Levels
Log10(CFU E. coli O157:H7/Swab)
7
6
Immunized Animals
Sham-Immunized Animals
5
4
3
2
1
0
0
1
3
8
14
21
28
Days Post-Challenge
35
42
49
17
Challenge 1 Colonization Levels by PBMC Responses
Log10(CFU E. coli O157:H7/Swab)
7
6
High Responders
Low Responders
5
4
3
2
1
0
0
1
3
8
14
21
28
Days Post-Challenge
35
42
49
18
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
19
CHALLENGE SUMMARY
Trial
Trial 1
Trial 2
Trial 3
Trial 4
Trial
Groups
Animals
Immunization
Post
Challenge
Challenge
Treatment
Adjuvant
Route
Total
doses
Bacteria
Total CFUs
Route
Culture
Time
Immunized
5
INT, OVA,
EspB, TIR
R837
Rectal
4
A
1010
Rectal
49
Sham
5
1x PBS
none
Rectal
4
A
1010
Rectal
49
Immunized
6
INT, OVA,
Esp A,
EspB, TIR
R848
SQ
3
A
1010
Oral
28
Sham
6
1x PBS
none
SQ
3
A
1010
Oral
28
Oral
10
109 CFU
total B
none
Oral
1
B
109
Oral
56
Rectal
10
109 CFU
total B
none
Rectal
1
B
109
Rectal
56
Oral
10
109 CFU
total B
none
Oral
2
B
107
Rectal
35
20
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
21
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
22
THANK YOU!
Kevin Lahmers
Tim Baszler
Tom Besser
Wendy Brown
Doug Call
Esther Trueblood
Tovah Kerr
Susan Smart
Emma Karel
Fred Loaiza
Lonnie Austin
Alex Beck
Andrea Hayles
Carolyn Bohach
Eric Sutten
Haiqing Sheng
Claudia Deobald Kathleen Sutten
Allison Vilander
Claire Miller
WADDL Pathologists and residents
VMP Faculty and staff
Graduate Students
Officemates
Friends
Family
ELISA
Samples
Negative control
1:10
1:20
Positive if >
average + 2 SD
1:1280
Titer is last positive dilution