1. Development and assessment of multivalent recombinant vaccines for bovine respiration disease Dr Tim Mahony Queensland Alliance Agriculture & Food Innovation The University of Queensland t.mahony@uq.edu.au

2. Outline What is bovine respiratory disease? The role of pathogens in BRD Vaccines for BRD Testing of BRD vaccines

3. Bovine Respiratory Disease (BRD) Respiratory Disease in feedlot cattle –Multi-factorial disease Environmental factors –Stress/climate/transport/food/density/social Pathogen exposure –>4 Viruses –>3 Bacteria Most susceptible 2 to 3 weeks –Peak about 21 days

4. The pathway to BRD

5. BRD in the field Cattle are most at risk in the first three weeks on feed Pre Feedlot Induction Day 0 Peak BRD Day 21 Day 50

6. BRD in the field Population of 35,000; 18% treated for BRD

7. True cost of BRD? Australia$60-100m 1m head North America $1-5b US 10m head vetmed.wsu.edu

8. Bovine respiratory disease Four viruses are associated with BRD: Bovine herpesvirus (BoHV-1) Bovine viral diarrhoea virus 1 (BVDV-1) Bovine parainfluenza 3 virus Bovine respiratory syncytial virus Bovine coronavirus Plus others Bacteria are also associated with BRD: Mannheimia haemolytica Pasteurella multocida Mycoplasma bovis Plus others Most significant disease in feedlot cattle –$1 Billion in North America

9. BRD vaccines “Four” main viral agents Bovine herpesvirus 1 (BoHV-1) –Large DNA genome (ds, 135 kbp) Bovine viral diarrhea virus (BVDV) –Medium RNA genome (ss, +ve, 12.5 kb) Bovine respiratory syncytial virus –Medium RNA genome (ss, -ve, 14.5 kb) Bovine coronavirus –Large RNA genome (ss, +ve, 31 kb) Bovine parainfluenza virus 3 –Medium RNA genome (ss, -ve, 14.5 kb)

10. Current vaccines Rhinogard –Developed by DAF (Zoetis) –Specifically for feedlot sector Pestigard –Developed by NSW Dept Ag (Zoetis) Bovilis-MH® vaccine –Developed by Beef CRC (MSD) Bovilis-MH +IBR® vaccine –MSD North America

11. Live viral vectors Advantage –Single dose –Follows similar course of infection –Rapid non-specific protection Disadvantage –Immunocompetence –Balancing act –Recombination –Latent infections

12. BRD vaccines Bovine herpesvirus 1 (BoHV-1) –DNA virus –Typically very stable –Known virulence factors –Ideal vaccine vector Live attenuated vaccine –Delivered intranasally –One shot at induction Replicates –Rapid onset of innate immune response –Interferon based protection –Protection during key period of adaption Recombinant vector –Large genome –Able to accommodate insertion/deletions –Stable replication –Easily manipulated –Host specific –Marker vaccines, DIVA

13. BRD vaccines Develop BoHV-1 as a multivalent vector –Insertion of antigens Need technology to rapidly generate new vaccines First generation recombinant vaccine –BoHV-1 carrying BVDV antigens Developed a BoHV-1 infectious clone system –Mutant libraries –Insertion libraries

14. BRD vaccines Cap R Kan R rBoHV-1, gE - Kan R Cells Cap R gE Kan R PCR, Transposition

16. Recombinant vaccines: BVDV-1 Virus Taxonomy 8 th ICTV Ed. Fauquet et al. Use major immunological determinant

17. Recombinant vaccines BoHV-1 replicates in the nucleus BVDV replicates in the cytoplasm –Is not subject to RNA processing Splicing? microRNA? Unknowns? –Is not subject to transport (mRNA) –Translation inhibition

18. Recombinant vaccines BoHV-1 –High G/C –Common viral mechanism BVDV –High A/T –Different codon usages Infect same host –Replicate in different cells/tissues

19. RNA processing Cryptic Splice Sites (CSS) –By definition difficult to identify Use consensus donor sites / acceptor Sites detected are method dependent

20. RNA processing Flavivirus –HCV First demonstration of cryptic splicing –Classical Swine Fever

21. Cryptic splicing May occur Equine infectious anemia virus –Lentivirus (?) Zhou et al. 2002 Vet Micro 88:127 Unpublished observations –BVDV –BRSV BVDV Case Study Strain C86

22. BVDV E2 Schmitt et al. 1999 J Gen Virol 80:2839 BVDV E2 –Removed CSS & polyA; changed codons Able to demonstrate expression –C86 Genomic 6 Donor / 3 Acceptor –C86 Synthetic 3 Donor / 2 Acceptor

23. Codon counting Codon bias Many dramatic examples –Bovine papillomavirus 1x1,000 –HIV gp120x40 –HIV gagx300 Consistent, bacterial, yeast, plant & mammalian systems Gustafsson et al. 2004 TIB 22:346 Andre et al. 1998 J Virol 72:1497

24. The BVDV E2 “Solution” Make some changes Site directed mutagenesis –Effective –Efficient depending on number –Expensive Synthetic genes –Effective/Efficient –One stop shop –Cost effective

25. The “solution” Remove “cryptic” splice sites Change codon usage to high G/C –90% identity @ nucleotide level –100% Identity @ amino acid level Add on signal sequence –Erns BVDV –gD BoHV-1

26. Prototype vaccine Demonstrated expression in transfected cells Insert E2 cassette into BoHV-1 genome Rescue virus Cross neutralisation studies Recombinant BoHV-1 neutralised by BVDV +ve sera

27. Vaccine trials BRDC is a multifactorial disease How to measure efficacy? Virus shedding –Duration –Quantity Clinical parameters –Temperature –Respiration

28. Dual Challenge Model Vaccinate - Day 0 –Temps & swabs for 7 - 14 days Viral Challenge - Day 14 –BoHV-1 – Strain 3932 –Temps, swabs & clinical signs Bacterial Challenge - Day 19 –Mannheimia haemolyticia –Temps, swabs & clinical signs Trial ends Day 35

30. Dual challenge model 0 2 4 6 8 10 12 14 16 18 20 Lowest clinical scores in vaccinates Comparable to Rhinogard Shed less BoHV-1, for less time recV FDrecVRGUV recV FD BoHV-1BVDV

31. BVDV-1 challenge dpiVaccineControlTreatment 1Treatment 2 4BC---+++----+- NS------------ 6BC---+-+------ NS------------ 8BC------------ NS---+-+------ 11BC------------ NS-+---------- Aguirreburualde et al. (2013) Vet Immunol Immunopathol 151:315– 324 Virus detection

32. BVDV-1 challenge Strain 1Strain 2Strain 3Strain 4Strain 5Control N=3N=4 N=6N=2 1000000 2012010 3102030 4102010 5001020 6004120 7004*0*2*0 12101010 14000010 21000000 Days post infection

33. Lower clinical scores in BHV vaccinated / Challenge group - protection Equivalent low clinical scores in BVDV-1 challenge groups – no disease BHV1 Groups - Clincal Scores Score Controls Vaccinated 0 5 10 15 20 BoHV-1 challenge

34. Bacterial challenge Days Post-Vaccination Mh Colonies (Log10) Day 20Day 21Day 22Day 24Day 25Day 26 0 2 4 6 8 10 BoHV-1 Controls BoHV-1 Vaccinated

35. BVDV-1 challenge No overt clinical distinction Some animals virus positive All animals seroconverted Analyses of blood cellular composition

36. Vaccine stability Rescue of BAC after virus isolation Provides mechanism accurately assess in vivo properties of viral population Isolate virus from nasal swabs –Reclone into bacteria –Restriction enzyme profile Sequencing

37. Vaccine stability P1P2P3P4 D3D3D7D3D3D7D7 V P VP D7 Recovered BAC clones show highly similar genomes

38. Future studies Understanding viral virulence BoHV-1 –Still see BRD in vaccinated cattle –Latest trial results BVDV-1 –Distinction of challenge subtle –Vaccination suggests benefit –Variable, larger numbers –High and low virulence

39. BRD and BoHV-1: Future Constructed a V155 infectious clone (1961) –Developing as multivalent vector –Sequencing is complete –Establish a “molecular baseline” Completely sequence additional genomes –Using new generation sequencing technologies –Provide fine genetic resolution –Antigenic drift / antigenic shift / host Genomic comparisons –Molecular basis for variation Direct vaccine/field strain challenge trials

40. Assessment Prototype Vaccine –Provided strong protection in BoHV-1 model –BVDV Challenge Vaccine is stable –Co-infection? No evidence of instability –BoHV-1 in general –Transgene

41. Next big challenge Registration & commercialisation of GMO Industry perception Public & consumer perceptions Regulator acceptance –DIR50 –Research requirements

42. Summary BRD remains a big issue Vaccines provide possible solution Recombinant vaccines Need to better understand pathogens

43. Acknowledgements Jenny Gravel Fiona McCarthy Rebecca Kann Trish Eats Peter Young NBRDI Team Queensland Animal Science Precinct Department of Primary Industries & Fisheries Meat & Livestock Australia