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
Outline

• What is bovine respiratory disease?

• The role of pathogens in BRD

• Vaccines for BRD

• Testing of BRD vaccines

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
The pathway to BRD
BRD in the field

Induction
Day 50
Day 0

Pre Feedlot
Peak BRD
Day 21

Cattle are most at risk in the first three weeks on feed

BRD in the field

Population of 35,000; 18% treated for BRD

True cost of BRD?

Australia North America

$60-100m $1-5b US
1m head 10m head

vetmed.wsu.edu
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
 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)
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
Live viral vectors

• Advantage
– Single dose
– Follows similar course of infection
– Rapid non-specific protection

• Disadvantage
– Immunocompetence
– Balancing act
– Recombination
– Latent infections
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
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
BRD vaccines

gE
KanR

KanR

CapR CapR
PCR,
Transposition KanR

KanR
Cells KanR

rBoHV-1, gE-
 UL52 UL50

Circ UL54 UL53 UL51 UL49.5 UL49 UL48 UL47 UL46 UL44 UL43
20 kbp
Tn5-22 Tn5-5 Tn5-56 Tn5-20 Tn5-80 Tn5-48 Tn5-74 Tn5-55 Tn5-87
Tn5-84 Tn5-53 Tn5-49
Tn5-67 Tn5-12

UL42 UL41 UL40 UL39 UL38 UL37 UL36

40 kbp

Tn5-45 Tn5-54 Tn5-46 Tn5-15 Tn5-24 Tn5-65 Tn5-40 Tn5-41 Tn5-76 Tn5-51
Tn5-47

UL35 UL33 UL31 UL28 UL26.5

UL34 UL32 UL30 UL29 UL27 UL26

60 kbp
Tn5-25 Tn5-37 Tn5-44 Tn5-38 Tn5-86 Tn5-16 Tn5-36 Tn5-100
Tn5-13 Tn5-68

UL24 UL19.5 UL17 UL16

UL25 UL23 UL22 UL21 UL20 UL19 UL18 UL15

80 kbp
Tn5-28 Tn5-50 Tn5-73 Tn5-85 Tn5-77 Tn5-99 Tn5-91 Tn5-64 Tn5-31 Tn5-34
NsiI Tn5-89 Tn5-81 Tn5-30

UL13 UL11 UL3.5 UL3 UL0.7

UL14 UL12 UL10 UL9 UL8 UL7 UL6 UL5 UL4 UL2 UL1
100 kbp
Tn5-66 Tn5-95 Tn5-27 Tn5-63 Tn5-98 Tn5-35 Tn5-90 Tn5-75 Tn5-6 Tn5-72
Tn5-62 Tn5-2 Tn5-92

IRS
UL0.5 bICP0 In-3 US1.67

LRORF2 bICP4 In-4 Ori S In-1 bICP22 US2 US3 US4 US6
120 kbp
In-2
Tn5-26 Tn5-71 Tn5-83 Tn5-7 Tn5-39 Tn5-60 Tn5-32 Tn5-78
Tn5-69 Tn5-88 Tn5-9 Tn5-42 Tn5-79 Tn5-70

TRS
In-10
In-9
US7 US8 US9 bICP22 In-1 Ori S bICP4
135.3 kbp
In-2
Tn5-93 Tn5-82
Recombinant vaccines: BVDV-1

Use major immunological determinant

Virus Taxonomy 8th ICTV Ed. Fauquet et al.

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
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
RNA processing

• Cryptic Splice Sites (CSS)

– By definition difficult to identify

• Use consensus donor sites / acceptor

• Sites detected are method dependent

RNA processing
• Flavivirus
– HCV

• First demonstration of cryptic splicing

– Classical Swine Fever
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

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
Codon counting
• Codon bias

• Many dramatic examples

– Bovine papillomavirus 1 x1,000
– HIV gp120 x40
– HIV gag x300

• Consistent, bacterial, yeast, plant &

mammalian systems
Gustafsson et al. 2004 TIB 22:346

Andre et al. 1998 J Virol 72:1497

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
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
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
Vaccine trials
• BRDC is a multifactorial disease

• How to measure efficacy?

• Virus shedding
– Duration
– Quantity

• Clinical parameters
– Temperature
– Respiration
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

Dual challenge model
20

18

16

14

12

10

recV FD recV RG UV recV FD UV

BoHV-1 BVDV

• Lowest clinical scores in vaccinates

• Comparable to Rhinogard
• Shed less BoHV-1, for less time
BVDV-1 challenge
Virus detection

dpi Vaccine Control Treatment 1 Treatment 2

4 BC - - - + + + - - - - + -
NS - - - - - - - - - - - -
6 BC - - - + - + - - - - - -
NS - - - - - - - - - - - -
8 BC - - - - - - - - - - - -
NS - - - + - + - - - - - -
11 BC - - - - - - - - - - - -
NS - + - - - - - - - - - -

Aguirreburualde et al. (2013) Vet Immunol Immunopathol

151:315– 324
BVDV-1 challenge

Strain 1 Strain 2 Strain 3 Strain 4 Strain 5 Control

N=3 N=4 N=4 N=4 N=6 N=2
1 0 0 0 0 0 0
2 0 1 2 0 1 0
Days post infection

3 1 0 2 0 3 0
4 1 0 2 0 1 0
5 0 0 1 0 2 0
6 0 0 4 1 2 0
7 0 0 4* 0* 2* 0
12 1 0 1 0 1 0
14 0 0 0 0 1 0
21 0 0 0 0 0 0
BoHV-1 challenge

BHV1 Groups - Clincal Scores BVDV Groups - Clincal Scores

20 20

15 15
Score

Score
10 10

5 5

0 0
s d
ol te
s

ed

r
t na
ol

on
at

i
tr

c
in
on

C c
cc

Va
C

Va

Lower clinical scores in BHV vaccinated / Challenge group - protection

Equivalent low clinical scores in BVDV-1 challenge groups – no disease

Bacterial challenge

10
BoHV-1 Controls
Mh Colonies (Log10)

8 BoHV-1 Vaccinated

0
20

21

22

24

25

26
ay

ay

ay

ay

ay

ay
D

Days Post-Vaccination
BVDV-1 challenge

• No overt clinical distinction

• Some animals virus positive

• All animals seroconverted

• Analyses of blood cellular composition

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
Vaccine stability

P1 P2 P3 P4
D3 D7 D3 D7 D3 D7 D3 D7

VP VP

Recovered BAC clones show highly similar genomes

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
 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

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
Next big challenge

• Registration & commercialisation of GMO

• Industry perception

• Public & consumer perceptions

• Regulator acceptance
– DIR50
– Research requirements
Summary

• BRD remains a big issue

• Vaccines provide possible solution

• Recombinant vaccines

• Need to better understand pathogens

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