dr.jamesrothvaccinationinneonates.ppt

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?