dr.jamesrothvaccinationinneonates.ppt
TRANSCRIPT
BVDV and Vaccination of the Young Animal
James A. Roth, DVM, PhD, DACVM
Center for Food Security and Public HealthCollege of Veterinary Medicine
Iowa State University
Issues Regarding BVDV Vaccination in the Young Calf
• Protection by maternal antibody• Immunocompetence of the young calf• Induction of protective immune mechanisms
by MLV vs. killed vaccines• Cross protection of antigenic variants• Interference by maternal antibody• Immunosuppression by MLV vaccine
Pathogenic Mechanisms
Adherence to mucosa Mucosal antibody (IgA)
Parasites IgE
Exotoxin/Endotoxin Neutralizing antibody
Viremia Neutralizing antibody
Septicemia Opsonizing antibody
Intracytoplasmic growth
Virus replication
Cytotoxic T cells
Types 1 and 2 Interferons
Intracellular growth Th1 cytokines
Infect epithelial cells Gamma delta T cells
Defense Mechanisms
Pathogenic Mechanisms
Adherence to mucosa Mucosal antibody (IgA)
Parasites IgE
Exotoxin/Endotoxin Neutralizing antibody
Viremia Neutralizing antibody
Septicemia Opsonizing antibody
Intracytoplasmic growth
Virus replication
Cytotoxic T cells
Types 1 and 2 Interferons
Intracellular growth Th1 cytokines
Infect epithelial cells Gamma delta T cells
Defense Mechanisms
Respiratory Virus Infection
Roitt, I., Brostoff, J., Male, D. Immunology. 1985
Internal and external antigens surrounding the viral genetic material
external antigens internal antigens
non-protective protective non-protective
Exogenous (MHCII) and Endogenous (MHCI) Pathways of Antigen Presentation
Detection of Antigen Specific T Cell-
Mediated Immunity to Bovine Respiratory
Disease Viruses by Flow Cytometry
T Helper cells (CD4)
Cytotoxic T cells (CD8)
Gamma Delta T cells
T Cell Activation
Antigen stimulated
Unstimulated Stimulated
T T
CD25 (IL-2R)
Cell surface marker
Cytokine
Multi-parameter Flow Cytometry
• PBMC are incubated with antigens for 4-5 days.
• Cell surface markers and intracellular cytokines are stained with different fluorochromes and analyzed by flow cytometer.
Detects expression of CD25, intracellular IFN, and IL-4 in T cell subsets.
CD 4
T
T
TIFN
IL-4
CD 8
CD 25
• Immunize calves
• Collect blood samples from vaccinated and control calves
Monitoring T Cell Responses by CD25 and CytokineExpression Analysis
• Isolate peripheral blood mononuclear cells (PBMC)
• Incubate PBMC in vitro with antigens in microtiter plates
• Stain cell phenotype markers and activation markers
Monitoring T Cell Responses by CD25 and IFNExpression Analysis
Induction of Antigen Specific T Cell Subset Activation to Bovine Respiratory Disease
Viruses by MLV VaccinePlatt, R., W. Burdett, and J. A. Roth. 2006. Induction
of antigen specific T cell subset activation to bovine respiratory disease viruses by a modified-live virus vaccine. Am J Vet Res, 67:1179-1184, 2006
Supported by Intervet
• Vaccination with modified-live virus vaccine (BHV-1, BRSV, BVDV types 1 and 2, and PI3) (Vista vaccine – Intervet)
– Week 0
• Blood collection for CMI assay
– Weeks 0, 4, 5, 6, 8, 24, 25, 26, 27
• Challenge
– BHV-1 Cooper strain on week 25 (2 ml of 108 TCID50/ml)
• Nasal secretion collection for virus titration
– Days 0-14 post-challenge
Experimental Design
In vitro stimulation of PBMC• Unstimulated
• Mitogen stimulated
• Live virus stimulated
– BHV-1 (Bovishield)
– BRSV (Bovishield)
– BVDV type 1 (TGAN-NADC)
– BVDV type 2 (890-NADC)
CMI assay
BHV-1 Results
0
10
20
30
40
50
60
70
80
90
Mean
(Week
0)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
70
80
90
Mean
(Week
45
68
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
70
80
90
Mean
(Week
24
25
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
70
80
90
Mean
(Week
26
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
70
80
90
Mean
(Week
27
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
Pre-vaccination
Week 0
Post-vaccination
Weeks 4, 5, 6, 8
CD25 Expression Index
Subset All PBMC CD4 CD8
gd Non T cells Statistically significant (p<0.01) (p<0.05)
Pre- challenge
Weeks 24, 25
Week 1 post-challenge
Week 26
Week 2 post-challenge
Week 27
BVDV Type 1 Results
CD25 Expression Index
Pre-vaccination
Week 0
Post-vaccination
Weeks 4, 5, 6, 8
0
10
20
30
40
50
60
Mean
(Week
0)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
Mean
(Week
45
68
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
Mean
(Week
24
25
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
Mean
(Week
26
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
Mean
(Week
27
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
Subset All PBMC CD4 CD8
gd Non T cells Statistically significant (p<0.01) (p<0.05)
Pre- challenge
Weeks 24, 25
Week 1 post-challenge
Week 26
Week 2 post-challenge
Week 27
BVDV Type 2 Results
CD25 Expression Index
Pre-vaccination
Week 0
Post-vaccination
Weeks 4, 5, 6, 8
0
10
20
30
40
50
60
Mean
(Week
0)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
Mean
(Week
45
68
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
Mean
(Week
24
25
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
Mean
(Week
26
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
0
10
20
30
40
50
60
Mean
(Week
27
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
Subset All PBMC CD4 CD8
gd Non T cells Statistically significant (p<0.01) (p<0.05)
Pre- challenge
Weeks 24, 25
Week 1 post-challenge
Week 26
Week 2 post-challenge
Week 27
BHV-1 Results
%IFN +
Pre-vaccination
Week 0
Post-vaccination
Weeks 4, 5, 6, 8
-2
-1
0
1
2
3
4
Mean
(Week
0)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
45
68
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
24
25
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
26
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
27
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
Subset All PBMC CD4 CD8
gd Non T cells Statistically significant (p<0.01) (p<0.05)
Pre- challenge
Weeks 24, 25
Week 1 post-challenge
Week 26
Week 2 post-challenge
Week 27
BVDV Type 1 Results
%IFN +
Pre-vaccination
Week 0
Post-vaccination
Weeks 4, 5, 6, 8
-2
-1
0
1
2
3
4
Mean
(Week
0)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
45
68
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
24
25
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
26
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
27
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
Subset All PBMC CD4 CD8
gd Non T cells Statistically significant (p<0.01) (p<0.05)
Pre- challenge
Weeks 24, 25
Week 1 post-challenge
Week 26
Week 2 post-challenge
Week 27
BVDV Type 2 Results
%IFN +
Pre-vaccination
Week 0
Post-vaccination
Weeks 4, 5, 6, 8
Pre- challenge
Weeks 24, 25
Week 1 post-challenge
Week 26
Week 2 post-challenge
Week 27
-2
-1
0
1
2
3
4
Mean
(Week
0)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
45
68
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
24
25
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
26
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
-2
-1
0
1
2
3
4
Mean
(Week
27
)
All
PB
MC
CD
4
CD
8
gd
Non T
cells
All
PB
MC
CD
4
CD
8
gd
Non T
cells
Control Vaccinated
Subset within Group
Subset All PBMC CD4 CD8
gd Non T cells Statistically significant (p<0.01) (p<0.05)
Efficacy of Killed BVDV Vaccines for Induction of T Cell Mediated Immunity?
Humoral and T cell-mediated immune responses to bivalent killed bovine viral diarrhea virus vaccine in beef cattle. Ratree Platt, Christopher Coutu, Todd Meinert, James A. Roth.
Vet Immunol Immunopathol. 122:8-15, 2008.
Supported by Pfizer Animal Health
Materials and Methods
Animals and vaccines
• Two groups of 10, 9-12 month old, BVDV seronegative, castrated male crossbred beef cattle were used.
• Gr. 1 served as negative mock-vaccinated control.
• Group 2 was vaccinated subcutaneously twice, 3 weeks apart, with 2 ml of modified live BHV-1, PI3, and BRSV diluted in diluent containing killed BVDV type 1 (strain 5960) and type 2 (strain 53637) in an adjuvant containing Quil A, Amphigen, and cholesterol
Materials and Methods
Cell-mediated immunity responses
• Anticoagulated blood samples were collected on vaccination day and days 28, 35, 56 and 70 post-vaccination.
• Specific T cell responses assays for BVDV types 1 and 2 were tested for – The up-regulation of CD25 by multi-parameter flow
cytometry – The expression of IFN in cultured cells supernatants by
indirect ELISA.
Net increase of %CD25+
-400.0
-200.0
0.0200.0
400.0
600.0
800.01000.0
1200.0
1400.0
BVDV1
BVDV2
BVDV1
BVDV2
BVDV1
BVDV2
BVDV1
BVDV2
BVDV1
BVDV2
BVDV1
BVDV2
All PBMC CD4 CD8 CD8+gd gd Non T cells
Control Vaccinated
Net increase of %CD25+ area under the curve analysis results ( p<0.05)
Net IFN gamma expression
0.0
50000.0
100000.0
150000.0
200000.0
250000.0
BVDV 1 BVDV 2
Control Vaccinated
Net increase of IFN area under the curve analysis results ( p<0.05)
Induction of T Lymphocytes Specific for Bovine Viral Diarrhea Virus in Calves with
Maternal Antibody
Janice Endsley1, Julia Ridpath2,
John Neill2, Matt Sandbulte1, James Roth1
1Iowa State University, 2USDA ARS National Animal Disease Center
Viral Immunology 17:13-23, 2004
Calves infected with Bovine Viral
Diarrhea virus in the presence of passive
antibody will develop CD4, CD8, and T
cells specific for BVDV, without a detectable
antibody response and will be protected from
subsequent challenge.
Hypothesis
• Pooled colostrum from BVDV hyper-immunized cows was fed to 12 calves.
• Six of 12 calves were inoculated with BVDV type 2 (strain 1373) at 2 to 5 weeks of age.
• Three calves received no colostrum and no BVDV inoculation.
• Three calves received no colostrum and were challenged at 2 to 5 weeks of age (all died).
• All surviving calves were challenged with BVDV type 2 (strain 1373) at 8 to 9 months of age.
Experimental Design
100
101
102
103
104
105
106
107
0 2 4 6 8 10 12 14 16 18 20
Day Post Inoculation
No ColostrumFirst inoculation Colostrum
Tem
p (F
)
Temperature (After 1st Inoculation)
0
0.5
1
1.5
2
2.5
3
3.5
4
1 2 3 4 5 6 7 8 9 10
Month after first BVDV inoculation
SV
N T
iter
(log1
0)
Colostrum Colostrum, BVDV
Neutralizing Antibody Responses to BVDV Type 2
0
5
10
15
20
25
30
0-10 wks 11-20 wks 21-32 wks
Colostrum Colostrum, BVDV
Weeks after first BVDV inoculation
Exp
ress
ion
Inde
x
P = 0.07
P = 0.04
Activation of CD4 T Cells by BVDV Type 2
0
5
10
15
20
25
30
0-10 wks 11-20 wks 21-32 wks
P = 0.008
P = 0.10
Colostrum Colostrum, BVDV
Weeks after first BVDV inoculation
Exp
ress
ion
Inde
x
Activation of CD8 T Cells by BVDV Type 2
0
5
10
15
20
25
30
0-10 wks 11-20 wks 21-32 wks
P = 0.04 P = 0.006P = 0.04
Colostrum Colostrum, BVDV
Weeks after first BVDV inoculation
Exp
ress
ion
Inde
x
Activation of T Cells by BVDV Type 2
0
20
40
60
80
100
120
140
0 - 10 Weeks 11 - 20 Weeks 21 - 32 Weeks
Colostrum, No BVDV Colostrum, BVDV
IFN
ng/m
lIFN Production (ELISA)
100
101
102
103
104
105
106
107
0 2 4 6 8 10 12 14 16 18 20
Days post challenge
Challenge
Colostrum + VirusColostrum + Virus
ColostrumColostrum
No ColostrumNo Colostrum
Tem
p (
F)
Temperature (After Challenge)
Virus Isolation from Buffy Coats (After Challenge)
Days post challenge
Group 2 4 6 8 10 12
Colostrum + BVDV 0/5 0/5 0/5 0/5 0/5 0/5
Colostrum 3/6 4/6 6/6 6/6 2/6 2/6
No colostrum 2/3 3/3 3/3 3/3 0/3 0/3
Mean SN Titers to BVDV 1373 After Challenge
Colostrum + VirusColostrumNo Colostrum
Effect of maternal antibody on T cell and antibody responses to BVDV type 2 modified live virus
vaccine and virulent challenge
Ratree Platt1, Philip W. Widel2, Lyle D. Kesl3, James A. Roth1
1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011, USA2 Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO 64507, USA
3 Veterinary Resources, Inc., Ames, IA 50010, USA
Supported by Boehringer Ingelheim Vetmedica, Inc
Materials and Methods
• Animals– Three age groups of bottle-fed Holstein
calves, (1-2 week, 4-5 week, and 7-8 week old), 12 calves in each group
– Eight calves were vaccinated and 4 calves served as non-vaccinated controls
• Vaccine– MLV vaccine (Express™5)
• BVDV type 2 challenge – Virulent strain 1373
Experimental design
wk0 wk3 wk6 wk9 wk11 wk12 wk13 wk14
Vaccination Challenge
Clinical signs, rectal temperature, and body weight gain observation
SVN test
CMI assay
Cell-mediated immunity assay
• Multi-parameter flow cytometry (6 fluorochromes) using BVDV type 2 (strain 890) as recall antigen
• Detected IL-2 receptor (CD25) and intracellular IFN and IL-4 expressions in major T cell subsets (CD4+, CD8+, TCR+)
CD25 expression index = (%CD25+)(MFI) of stimulated cells (%CD25+)(MFI) of unstimulated cells
%IFN+ = %IFN+ of stimulated cells - %IFN+ of unstimulated cells
%IL-4+ = % IL-4+ of stimulated cells - % IL-4+ of unstimulated cells
Protection from virulent challenge
• Clinical signs included depression, anorexia, nasal and ocular discharges, oral lesion, respiratory signs, cough, and diarrhea
• Rectal temperature
• Body weight gain
Mean log2 BVDV type 2 SVN titer
0
2
4
6
8
10
12
Wk 0 Wk 3 Wk 6 Wk 9 Wk 12 Wk 13 Wk 14
Weeks post-vaccination
Mean
log2
BV
DV
type 2
SV
N t
iter
wk Control 1-2
wk Control 4-5
wk Control 7-8
wk Vaccinated 1-2
wk Vaccinated 4-5
wk Vaccinated 7-8
Challenge
Levels not connected by same letter are significantly different within same group. * Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age
CD25 Expression Index
Levels not connected by same letter are significantly different within same group. * Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age
% IFN+
* Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age
% IL-4+
* Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age
Mean cumulative clinical score
Levels not connected by same letter are significantly different within same group. * Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age
Challenge
Mean rectal temperature
Levels not connected by same letter are significantly different within same group. * Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age
Challenge
Mean log2 BVDV type 2 SVN titer
0
2
4
6
8
10
12
Wk 0 Wk 3 Wk 6 Wk 9 Wk 12 Wk 13 Wk 14
Weeks post-vaccination
Mean
log2
BV
DV
type 2
SV
N t
iter
wk Control 1-2
wk Control 4-5
wk Control 7-8
wk Vaccinated 1-2
wk Vaccinated 4-5
wk Vaccinated 7-8
Challenge
Levels not connected by same letter are significantly different within same group. * Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age
Response to BHV1, BRSV, and PI3
• No antibody response in any age group of calves
• In contrast to BVDV1 and BVDV2 there was no evidence of T cell responsiveness to BHV1, BRSV, or PI3 in any age group of calves
• Since the calves were not challenged with BHV1, BRSV, or PI3, the influence of vaccination on resistance to challenge is not known
Immunosuppression by BVDV and MLV BVDV Vaccine
Suppression of Neutrophil Iodination by Virulent BVDV
Roth et al, AJVR 42:244-250, 1981
ControlsNADL BVDV IN1015-74 BVDV IM
Suppression of Neutrophil Iodination by MLV BVDV and ACTH
Roth and Kaeberle, AJVR 44:2366-2372, 1983
Issues Regarding BVDV Vaccination in the Young Calf
• Protection by maternal antibody• Immunocompetence of the young calf• Induction of protective immune mechanisms
by MLV vs. killed vaccines• Cross protection of antigenic variants• Interference by maternal antibody• Immunosuppression by MLV vaccine
Important Open Questions Regarding BVDV
Vaccination in Young Calves
• Will Killed vaccines induce T cell mediated immunity when given in the presence of maternal antibody?
• Careful characterization of immunosuppression by MLV vaccines
• Are MLV vaccines immunosuppressive when given to a calf with maternal antibody?
• Safety of MLV vaccines given to young calf still with the cow?
