Overview of the Immune Response and it’s Relevance to Vaccine

Efficacy

James A. Roth, DVM, PhDInstitute for International Cooperation in

Animal BiologicsIowa State University

What is the basis for protective immunity for the disease in question

• Circulating antibody?– IgM, IgG?

• Mucosal antibody?– IgA, IgE?

• Cell-mediated immunity?- Cytokine secretion (TH1 vs TH2)?- Cytotoxic T cells?- Gamma Delta T cells?

• What are the important antigens?

Pathogenic Mechanisms

• Adherence to Mucosa• Invasive parasite• Exotoxin/Endotoxin• Viremia• Septicemia• Intracytoplasmic Growth• Growth in phagosome• Infect epithelial cells

Defense Mechanisms

• Mucosal Antibody (IgA)• IgE• Neutralizing Antibody• Neutralizing Antibody• Opsonizing Antibody• Cytotoxic T cells• TH1 Cytokines• Gamma Delta T cells

RoittRoitt, I., , I., BrostoffBrostoff, J., Male, D. 1998. , J., Male, D. 1998. ImmunologyImmunology. 5th Ed. . 5th Ed. MosbyMosby. Philadelphia. pg17. Philadelphia. pg17

Functional T-cell subsets

Lymphocyte Type Antigen Recognition

B cells Intact, unprocessed molecules

TH1 and TH2 cells Exogenous peptides processed and presented on MHC II

Tc cells Endogenous peptides processed and presented on MHC I

Gamma Delta T cells Intact, unprocessed molecules

Lymphocyte Type Response to Antigen

B cells Produce AntibodiesTH1 cells Secrete cytokines which

enhance phagocyte killingTH2 cells Secrete cytokines which

help antibody production (IgA and IgE)

Tc cells Cytotoxic for cells synthesizing foreign proteins

Gamma Delta T cells Secrete cytokines and/or cytotoxic for cells with abnormal surface antigens

Parham, P., The Immune System. 2000, Garland

Publishing, Fig. 6.32

Selection of effector mechanisms by TH1 and TH2 cells

T Helper Cell T Cytotoxic Cell

Exogenous Pathway of Antigen

PresentationAbbas, et al. Cellular and

Molecular Immunology, 1997

Abbas, et al. Cellular and Molecular Immunology, 1997

Endogenous Pathway of Antigen

Presentation

Parham, P., The Immune System. 2000, Garland Publishing, Fig 12.5

ISCOMs Carry Peptides into the Cytoplasm

Roitt, I. et al. Immunology,

1996

Allele-specific motifs in peptides eluted from MHC class II molecules

Roitt, I. et al. Immunology,

1996

Binding peptides from class I molecules

Abbas, A.K., Lichtman, A.H., Pober, J.S. 1994. Cellular and Molecular Immunology. 2nd Ed. W.B. Saunders Co., Philadelphia.

Bacterial Antigens

ExotoxinEndotoxin

Tizard, Ian R. Veterinary Immunology: An Introduction, 6th ed. ,2000

Viral Antigens

Roitt, I., Brostoff, J., Male, D. Immunology. 1985

Roitt, I. et al, Immunology,

1996

Critical molecules involved in antigen presentation

Detection of Antigen Specific T cell subset Activation

(CD25)

Flow Cytometer

Flow Cytometer

(% T cells stimulated)(MFI)

(% T cells unstimulated)(MFI)Expression Index =

UnstimulatedUnstimulated

T Cell

CD4

StimulatedStimulated

T Cell

CD4CD25

CD25

CD25

CD

25

CD25

Methods

Modified live Modified live and inactivated and inactivated vaccinesvaccines

ImmunizeImmunize

Isolate PBMC from Isolate PBMC from immune & naïve calvesimmune & naïve calves

Stimulate with Ag: Stimulate with Ag: live, inactivated, live, inactivated,

recombinantrecombinant

Two-color flow cytometry

Monoclonal Ab Monoclonal Ab stainingstaining

IL2R IL2R (CD25)(CD25)

CD4CD4 CD8CD8 γδγδ TCRTCR

1) Anti 1) Anti -- IL2RIL2R

2) Anti 2) Anti -- CD4, CD4, CD8 or CD8 or γδγδ TCRTCR

Flow cytometry output

CD 4 CD 4

CD

25

CD

25

CD 4 CD 4 Negative ResponseNegative Response Positive ResponsePositive Response

Induction of T Lymphocytes Specific for BVD Virus in Calves With

Maternal Antibody

Janice Endsley1, Julia Ridpath2, John Neill2, James Roth1

1Iowa State University2USDA ARS National Animal Disease Center

Endsley et. al., Viral Immunol, 17:13-23, 2004

Objective

To determine if infection with virulent BVDV in the presence of maternal antibody will induce antigen specific T cells and protective immunity without inducing antibody

Experimental Design

• Pooled colostrum from BVDV hyper-immunized cows fed to 12 calves.

• Six calves inoculated with BVDV 1373 at 6 to 20 days of age.

• All calves challenged with BVDV 1373 at 8 months of age.

• Monitor antibody and T cell subset responses

0

0.5

1

1.5

2

2.5

3

3.5

4

1 2 3 4 5 6 7 8 9 10

Colostrum Colostrum, BVDV

Serum Neutralizing Antibody to Type 2 BVDV

Months after first BVDV challenge

SN T

iter (

log

10)

Activation of CD4+ T cells by BVDV2 890

0

5

10

15

20

25

30

0-10 wks 11-20 wks 21-32 wks

Colostrum Colostrum, BVDV

Weeks after first BVDV challenge

Exp

ress

ion

Inde

x P = 0.07

P <0.05

Activation of CD8+ T cells by BVDV2 890

0

5

10

15

20

25

30

0-10 wks 11-20 wks 21-32 wks

Colostrum Colostrum, BVDV

Weeks after first BVDV challenge

Exp

ress

ion

Inde

x

P < 0.01

P = 0.10

Activation of γδ T cells by BVDV2 890

0

5

10

15

20

25

30

0-10 wks 11-20 wks 21-32 wks

Colostrum Colostrum, BVDV

Exp

ress

ion

Inde

x

Weeks after first BVDV challenge

P < 0.05 P < 0.01P < 0.05

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

C o lo s t ru m , n o B V D V C o lo s t ru m , B V D V W e e k s a f te r f i r s t B V D V c h a l le n g e

P < 0 .0 1

P < 0 .0 1

P < 0 .0 1

Interferon Gamma in Supernatants From Mononuclear Cells Stimulated with BVDV 890

IFNγ

ng/m

l

0-10 wks 11-20 wks 21-32 wks

Rectal Temperature After BVDV 1373 Challenge at 8 Months of Age

99.0100.0101.0102.0103.0104.0105.0106.0

0 2 4 6 8 10 12 14Days After Infection

Deg

rees

Fah

renh

eit

PreviouslyChallenged

Not PreviouslyChallenged

Total Leukocyte Counts After BVDV 1373 Challenge at 8 Months of Age

0

2,000

4,000

6,000

8,000

10,000

12,000

14,000

0 2 4 6 9 11 13Days After Infection

Plat

elet

s

PrevioslyChallenged

Not PreviouslyChallenged

DAYS POST CHALLENGE

Previously Challenged

Not Previously Challenged

2 4 6 8 10 12

0/5 0/5 0/5 0/5 0/5 0/5

3/6 4/6 6/6 6/6 2/6 2/6

Isolation of BVD Virus from Buffy Coat After Second Challenge

ConclusionsWhen challenged with virulent BVDV, calves with high maternal antibody titers to BVDV:

•Did not produce an antibody response to BVDV

•Did develop memory CD4+, CD8+, and γδ T cells

•Did produce an anamnestic antibody response upon subsequent challenge

•Are protected from BVDV challenge in the absence of detectable serum neutralizing antibody

T Cell-Populations Responsive to Bovine Respiratory Syncytial Virus in

Seronegative Calves

Matt Sandbulte and James RothVet Immunol Immunopathol, 84:111-123, 2002

Two-Fold Testing for BRSV Immunity in Beef Calves

Calves tested for virus-specific neutralizing antibody and T cells

16 identified as seronegative

Among these, T cell responses were detected in several.

#94

#95

#96

#97

#99

#100

#101

#132

#134

#135

#136

#138

#139

#141

0

5

10

15

20

25

30

35

CD

25 E

xpre

ssio

n In

dex

T Cell Responses to BRSV in Seronegative Calves

CD4CD8GD

Why T Cells Without Antibody?

Hypothesis:Those calves were previously infected with BRSV in the presence of maternal antibody. The humoral response was blocked, but T cells were primed.

Test:Vaccinate naïve calves and T cell-positive calves with BRSV. Does the latter group have immunologic memory, i.e. develop superior antibody and T cell responses?

CD4+ T Cell Responses

02468

101214161820

Week -4 Week -2 Week 0 Week 2 Week 4 Week 6

CD

25 E

xpre

ssio

n In

dex Group 1

Group 2Group 3 †‡

†‡

‡

†‡ †‡

*

*

†‡

CD8+ T Cell Responses

05

101520253035404550

Week -4 Week -2 Week 0 Week 2 Week 4 Week 6

CD

25 E

xpre

ssio

n In

dex

Group 1Group 2Group 3

†‡

‡

† *

†‡

*

†‡ ‡

Gamma delta T Cell Responses

0

20

40

60

80

100

120

Week -4 Week -2 Week 0 Week 2 Week 4 Week 6

CD

25 E

xpre

ssio

n In

dex Group 1

Group 2Group 3

†‡ †‡

†‡*

†‡

†‡

*

Neutralizing Antibody Responses

* Group 2 > Group 1 † Group 3 > Group 1 ‡ Group 3 > Group 2

0

1

2

3

4

5

6

7

Week -8 Week -4 Week 0 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6

Log(

2) S

VN T

iter Naïve / No treatment

Naïve / Vaccine

CMI(+) / Vaccine† ‡

† ‡

† ‡ † ‡

**

†

†

Limitations to Technology for DetectingLimitations to Technology for DetectingT Cell Subset ActivationT Cell Subset Activation

•• Day to day and animal to animal variability require Day to day and animal to animal variability require that large numbers of animals be used and that they that large numbers of animals be used and that they be sampled repeatedlybe sampled repeatedly

•• Expensive; labor and equipment intensiveExpensive; labor and equipment intensive

•• Lymphocytes should be set up the same day as Lymphocytes should be set up the same day as collected collected

•• Valid comparisons can only be made on lymphocytes Valid comparisons can only be made on lymphocytes assayed in parallelassayed in parallel