Transcript Document

Development and assessment of
multivalent recombinant vaccines for
bovine respiration disease
Dr Tim Mahony
Queensland Alliance Agriculture & Food Innovation
The University of Queensland
[email protected]
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 0
Day 50
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
$60-100m
1m head
North America
$1-5b US
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
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–
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Immunocompetence
Balancing act
Recombination
Latent infections
BRD vaccines
•
Bovine herpesvirus 1 (BoHV-1)
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Live attenuated vaccine
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Delivered intranasally
One shot at induction
Replicates
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DNA virus
Typically very stable
Known virulence factors
Ideal vaccine vector
Rapid onset of innate immune response
Interferon based protection
Protection during key period of adaption
Recombinant vector
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–
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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
KanR
gE
KanR
CapR
PCR,
Transposition
CapR
KanR
KanR
Cells
rBoHV-1, gE-
KanR
UL52
Circ
UL54
UL50
UL53
UL51
UL49.5
UL49
UL48
UL47
UL46
UL44
UL43
20 kbp
Tn5-22
UL42
UL41
Tn5-56
Tn5-5
UL40
UL39
Tn5-20
Tn5-84
UL38
Tn5-48
Tn5-53
Tn5-67
Tn5-80
Tn5-55
Tn5-74
Tn5-49
Tn5-12
UL37
Tn5-87
UL36
40 kbp
Tn5-45
Tn5-47
Tn5-54
UL35
Tn5-46
UL33
UL34
Tn5-15
Tn5-24
Tn5-65
Tn5-40
UL31
Tn5-41
Tn5-76
Tn5-51
UL28
UL32
UL30
UL26.5
UL29
UL27
UL26
60 kbp
Tn5-25
Tn5-37
Tn5-44
Tn5-38
UL24
UL25
Tn5-16
Tn5-68
Tn5-86
Tn5-13
Tn5-36
Tn5-100
UL19.5
UL23
UL22
UL21
UL20
UL17
UL19
UL18
UL16
UL15
80 kbp
Tn5-28
Tn5-50
Tn5-73
NsiI
UL13
Tn5-85
Tn5-89
Tn5-77
Tn5-99
Tn5-81
Tn5-91
Tn5-64
Tn5-31
UL3.5
UL11
UL14
UL12
UL9
UL10
UL8
Tn5-34
Tn5-30
UL7
UL6
UL5
UL3
UL4
UL0.7
UL2 UL1
100 kbp
Tn5-66
Tn5-95
Tn5-62
UL0.5
IRS
bICP0
Tn5-63
Tn5-98
Tn5-2
Tn5-35
Tn5-90
Tn5-93
US8
Tn5-83
Tn5-88
US9 bICP22
In-4
Ori S
Tn5-7
In-1 Ori S
In-2
Tn5-82
Tn5-72
US1.67
bICP4
Tn5-71
Tn5-69
Tn5-6
Tn5-92
In-1
Tn5-39
bICP22
In-2
Tn5-9
US2
US3
US4
US6
120 kbp
Tn5-60
Tn5-42
Tn5-32
Tn5-79
Tn5-78
Tn5-70
TRS
In-10
US7
Tn5-75
In-3
LRORF2
Tn5-26
Tn5-27
bICP4
In-9
135.3 kbp
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
– HIV gp120
– HIV gag
x1,000
x40
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
8
6
4
2
0
recV FD
recV
RG
BoHV-1
UV
recV FD
UV
BVDV
• Lowest clinical scores in vaccinates
• Comparable to Rhinogard
• Shed less BoHV-1, for less time
BVDV-1 challenge
Virus detection
dpi
4
6
8
11
Vaccine
Control
Treatment 1
Treatment 2
BC
-
-
-
+
+
+
-
-
-
-
+
-
NS
-
-
-
-
-
-
-
-
-
-
-
-
BC
-
-
-
+
-
+
-
-
-
-
-
-
NS
-
-
-
-
-
-
-
-
-
-
-
-
BC
-
-
-
-
-
-
-
-
-
-
-
-
NS
-
-
-
+
-
+
-
-
-
-
-
-
BC
-
-
-
-
-
-
-
-
-
-
-
-
NS
-
+
-
-
-
-
-
-
-
-
-
-
Aguirreburualde et al. (2013) Vet Immunol Immunopathol 151:315– 324
Days post infection
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
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
BVDV Groups - Clincal Scores
BHV1 Groups - Clincal Scores
15
15
5
0
0
C
on
tr
ol
s
5
in
at
ed
10
Va
cc
10
Score
20
Score
20
Lower clinical scores in BHV vaccinated / Challenge group - protection
Equivalent low clinical scores in BVDV-1 challenge groups – no disease
Bacterial challenge
Mh Colonies (Log10)
10
BoHV-1 Controls
BoHV-1 Vaccinated
8
6
4
2
0
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
D3
D7
P2
D3
P3
D7
D3
VP
D7
P4
D3 D7
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
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–
–
–
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
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•
•
•
•
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Jenny Gravel
Fiona McCarthy
Rebecca Kann
Trish Eats
Peter Young
NBRDI Team
Queensland Animal Science Precinct
Department of Primary Industries & Fisheries
Meat & Livestock Australia