fmdv immunology to improve vaccines and vaccination 150509.pdfo1-bfs uv o1-bfs uv + immune igg 0 3 *...
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FMDV immunology to improve
vaccines and vaccination
Early induction of neutralising antibodies- suggesting a T-cell independent antibody response
Depletion of CD4+ T cells
during acute infection with FMDV
VT74-CC30/CC51
VT77-CC30/CC51
VT76-CC30/CC51
VT75-CC30/CC51
CD21 CD21
CD21 CD21
CD4 CD4
CD4CD4
FMDV capsid: T independent and T dependent epitopes
Outcome of this experiment
IgG titre (Day 16pb)
Plasma
cell
number
(Day 3pb)
IgG titre (Day 16pb)
Memory
cell
number
(Day 5pb)y = 0.0002x + 1194.1
R2 = 0.08490
500
1000
1500
2000
0 500000 1000000 1500000
y = -0.0084x + 16423
R2 = 0.379
0
5000
10000
15000
20000
0 500000 1000000 1500000
No correlation between the Ab titres and the peak number of plasma cells or memory
B cells generated.
BoostOva Day 6/7Day 3/4Day 20
Day 5/6
Determination of the kinetics of plasma cells, memory B cells and Ab titre in the
blood following immunisation (and boost immunisation) with ovalbumin:
Plasma cells
Memory B cells
Ab titre
Primary response Secondary response
Calf # 703064 (Immunised with TNP-CGG in QuilA)
0
20,000
40,000
60,000
80,000
100,000
120,000
0 20 40 60 80 100 120
Days
An
ti-T
NP
-ova t
itre
sIgG
IgM
Calf # 503069 (Immunised with TNP-FICOLL in QuilA)
0
1,000
2,000
3,000
4,000
5,000
0 20 40 60 80 100 120
Days
An
ti-T
NP
-ova t
itre
s
IgG
IgM
Immunisation Boost
(day 58)
Immunisation Boost
(day 58) Plasma cells
(day 4pb)
Calf # 703064 (immunised with TNP-CGG)
0
2,000
4,000
6,000
8,000
AS
C n
um
be
r
Calf # 503069 (immunised with TNP-FICOLL)
0
1,000
2,000
3,000
AS
C n
um
be
r
T-dependent vs T-independent
responses
TD
Response
TI
Response
Memory
B cells
(day 6pb)
Immune response to vaccination- rapid induction of antibody
- variable/ short duration of immunity
- variable CD4 T cell response
The way forward
Improve understanding of immune response
Target antigen to antigen presenting cells
Stabilise vaccine antigen
0
50
100
150
200
250
300
Medium O1-BFS O1-BFS + immune
IgG
O1-BFS UV O1-BFS UV +
immune IgG
Co
un
ts p
er
min
ute
x 1
03
*
Dendritic cell targeting of FMDV antigen can be improved
University of
Oxford
Ray Owens
E. coli
mammalian cells
IAH-Epidemiology
Javier Castillo-Olivares/
Satya Parida
MVA
Adenovirus
Sendai virus
University of
Reading
Xiaodong Xu/
Ian Jones
Dr. Saravannan
IVRI India
baculovirus
John Innes Centre
George Lomonossoff
plant virus
yeast
IAH-Microbiology
Alison Burman/
Terry Jackson
Vaccinia virus
National Veterinary Institute
(Denmark)
Graham Belsham
University of Oxford
Elisabeth Fry/
David Stuart
structural biology
ConsortiumIAH-Immunology
Bryan Charleston
co-ordination
Sequences likely to enhance
expression in mammalian cells• At the 5’ end
→ not all the T7 transcripts get capped during co-expression of two recombinant vaccinia viruses
→ 75% initiation downstream of the FMDV IRES occurs at the second AUG
• At the 3’ end
3’UTR
+ poly (A) tail → separately enhance IRES mediated translation
L P2 P3P1VP3 VP1
2A
VP0 3C
3B3
3C
3B3
3'UTR 3'UTR
LbLa
IRESIRES
IRES
+ “La”
→ separately enhance IRES-mediated translation
Proof of principle that an engineered mutation (his to cys) is
consistent with capsid assembly.
Similar approaches can be used for infectious copies.
Structure-based stabilisation of FMDV capsids
Proof of principle that an engineered mutation (his to cys) is
consistent with capsid assembly.
Similar approaches can be used for infectious copies.
Structure-based stabilisation of FMDV capsids
Covalent Cage Particle
Characterisation
CC and WT empties were treated for 2h at 56ºC (or for
30min at pH5), then subjected to sucrose density
gradients.
6 7 8 10 11 12 6 7 8 10 11 12
175
83
62
47.5
32
25
16.5
WT CC
Fraction no. Fraction no.
Assembled empty
particles seen in CC
fractions only.
- Enhanced storage characteristics of formulated products
-Enhanced duration of immunity when combined with depot delivery system
-Improved T cell responses as a consequence of enhanced antigen presentation
Improved stability
Persistence of FMDV antigen- maintaining protective antibody responses
FMDV D46 DAPI
LZ
DZFMDV D46
80 µm
MLN 38DPCI
4 µmDAPI CD21 FMDV
No expression of non-structural
proteins:
-Non-replicating
-Extracellular
HypothesisIn the major wildlife reservoir of FDMV in Sub-Saharan Africa, the buffalo, depots of viable, non-replicating viruses
seed other tissue that results in the production of transmissible virus.
Primary objectives1: Determine whether FMDV capsid and genome are present in buffalo lymphoid tissue.
2: Determine whether secondary sites of FMDV localisation or replication are detectable in buffalo.
3: Determine whether infectious virus can be isolated from buffalo lymphoid tissue or epithelium after the
resolution of clinical signs.
4: Compare the genetic complexity of viral depots in buffalo tissue with virus present in oropharyngeal samples
collected by probang.
5: Develop minimally invasive sampling techniques for lymphoid tissue from buffalo to aid surveillance.
Collaboration with OVI in southern Africa