1. BVDV and Vaccination of the Young Animal James A. Roth, DVM, PhD, DACVM Center for Food Security and Public Health College of Veterinary Medicine Iowa State University

2. 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

3. 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

4. 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

5. Roitt, I., Brostoff, J., Male, D. Immunology. 1985 Internal and external antigens surrounding the viral genetic material external antigensinternal antigens non-protective protective non-protective

6. Exogenous (MHCII) and Endogenous (MHCI) Pathways of Antigen Presentation

7. 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

8. T Cell Activation Antigen stimulated UnstimulatedStimulated TT CD25 (IL-2R) Cell surface marker Cytokine

9. 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 T IFN  IL-4 CD 8  CD 25

10. Immunize calves Collect blood samples from vaccinated and control calves Monitoring T Cell Responses by CD25 and Cytokine  Expression Analysis

11. 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 IFN  Expression Analysis

12. Induction of Antigen Specific T Cell Subset Activation to Bovine Respiratory Disease Viruses by MLV Vaccine Platt, 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

13. 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 10 8 TCID 50 /ml) Nasal secretion collection for virus titration –Days 0-14 post-challenge Experimental Design

14. 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

15. BHV-1 Results Pre- vaccination Week 0 Post- vaccination Weeks 4, 5, 6, 8 CD25 Expression Index 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

16. BVDV Type 1 Results CD25 Expression Index Pre- vaccination Week 0 Post- vaccination Weeks 4, 5, 6, 8 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

17. BVDV Type 2 Results CD25 Expression Index Pre- vaccination Week 0 Post- vaccination Weeks 4, 5, 6, 8 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

18. BHV-1 Results  %IFN  + Pre- vaccination Week 0 Post- vaccination Weeks 4, 5, 6, 8 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

19. BVDV Type 1 Results  %IFN  + Pre- vaccination Week 0 Post- vaccination Weeks 4, 5, 6, 8 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

20. 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 Statistically significant (p<0.01) (p<0.05)

21. 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

22. 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

23. 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.

24. Net increase of %CD25+ area under the curve analysis results ( p<0.05)

25. Net increase of IFN  area under the curve analysis results ( p<0.05)

26. Induction of T Lymphocytes Specific for Bovine Viral Diarrhea Virus in Calves with Maternal Antibody Janice Endsley 1, Julia Ridpath 2, John Neill 2, Matt Sandbulte 1, James Roth 1 1 Iowa State University, 2 USDA ARS National Animal Disease Center Viral Immunology 17:13-23, 2004

27. 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

28. 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

29. 100 101 102 103 104 105 106 107 024681012 14161820 Day Post Inoculation No Colostrum First inoculation Colostrum Temp (F) Temperature (After 1 st Inoculation)

30. 0 0.5 1 1.5 2 2.5 3 3.5 4 12345678910 Month after first BVDV inoculation SVN Titer (log10) ColostrumColostrum, BVDV Neutralizing Antibody Responses to BVDV Type 2

31. 0 5 10 15 20 25 30 0-10 wks11-20 wks21-32 wks Colostrum Colostrum, BVDV Weeks after first BVDV inoculation Expression Index P = 0.07 P = 0.04 Activation of CD4 T Cells by BVDV Type 2

32. 0 5 10 15 20 25 30 0-10 wks11-20 wks21-32 wks P = 0.008 P = 0.10 Colostrum Colostrum, BVDV Weeks after first BVDV inoculation Expression Index Activation of CD8 T Cells by BVDV Type 2

33. 0 5 10 15 20 25 30 0-10 wks11-20 wks21-32 wks P = 0.04 P = 0.006 P = 0.04 Colostrum Colostrum, BVDV Weeks after first BVDV inoculation Expression Index Activation of  T Cells by BVDV Type 2

34. IFN  ng/ml IFN  Production (ELISA)

35. 100 101 102 103 104 105 106 107 02 468101214161820 Days post challenge Challenge Colostrum + Virus Colostrum No Colostrum Temp (F) Temperature (After Challenge)

36. Virus Isolation from Buffy Coats (After Challenge) Days post challenge

37. Mean SN Titers to BVDV 1373 After Challenge Colostrum + Virus Colostrum No Colostrum

38. Effect of maternal antibody on T cell and antibody responses to BVDV type 2 modified live virus vaccine and virulent challenge Ratree Platt 1, Philip W. Widel 2, Lyle D. Kesl 3, James A. Roth 1 1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011, USA 2 Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO 64507, USA 3 Veterinary Resources, Inc., Ames, IA 50010, USA Supported by Boehringer Ingelheim Vetmedica, Inc

39. 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

40. Experimental design wk0 wk3 wk6 wk9 wk11 wk12 wk13 wk14 VaccinationChallenge Clinical signs, rectal temperature, and body weight gain observation SVN test CMI assay

41. 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

42. 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

43. Mean log 2 BVDV type 2 SVN titer 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

44. 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

45.  % IFN  + * Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age

46.  % IL-4+ * Statistically different (p<0.05) * Statistically different (p<0.01) from control group of the same age

47. 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

48. 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

49. Mean log 2 BVDV type 2 SVN titer 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

50. 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

51. Immunosuppression by BVDV and MLV BVDV Vaccine

52. Suppression of Neutrophil Iodination by Virulent BVDV Roth et al, AJVR 42:244-250, 1981 Controls NADL BVDV IN 1015-74 BVDV IM

53. Suppression of Neutrophil Iodination by MLV BVDV and ACTH Roth and Kaeberle, AJVR 44:2366-2372, 1983

54. 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

55. 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?