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Frenkel Lecture
Utilising scientific advances to improve FMDV vaccines
Löffler and Frosch (1897) demonstrated the aetiological agent was a filterable
particle. FMD was the first animal disease to be attributed to a virus.
Demonstration that cattle could be protected by passive immuisation using
sera from convalescent animals
Diverse antigenic nature of the virus was recognised and led to the description
of the seven serotypes
The first practical inactivated vaccine was developed by Waldmann et al. (1937)
using virus from infected cattle. Formaldehyde inactivation in the presence of
aluminium hydroxide gel.
Waldmann type vaccine production was further improved by the work of
Frenkel (1947). Suspensions of the epithelial cells were prepared from the
tongues of healthy cattle, maintained and infected in vitro. The Frenkel
procedure was used for vaccine production for many years.
BHK monolayers could be used for the growth and titration of FMDV, Mowat
and Chapman (1962).
BHK cells grown in suspension, Capstick et al. (1962)
Suspension cells produced in large scale fermenters, Telling and Elsworth
(1965)
Binary ethyleneimine (BEI) adopted as a safe inactivation method
(Bahnemann, 1973).
High quality vaccines based on concentrated and purified inactivated FMD
antigens.
Storage of highly concentrated materials at very low temperature for long
periods of time, the basis of emergency antigen banks (Doel and Pullen,
1990).
Development of improved adjuvants
The Lymph Node
Capsule
B cell
zone
Afferent
Lymphatic Vessel
Marginal
Sinus
Post-capillary
venulles
Primary follicle
Secondary follicle
Cortex
Medullary cords
macrophages
and plasma cells
T cell zone
Paracortical
area
Abbas et al., 1991
Vein
Artery
Induction of a primary immune response
Innate Immunity
rder
o
b
l
a
eli
Epith
DC
precursor
Cytokines
Eosinophil
NK cell
B
cells
Macrophage
B
cells
CTLs
Helper T cells
GC
CTLs
Helper T cells
Acute infection with FMDV serotype O in cattle
Early induction of neutralising antibodies
- suggesting a T-cell independent antibody response
Neutralising antibody to FMDV is produced despite the absence
of a pronounced specific proliferative response
Neutralising Antibody in serum
200
Neutralising titre
150
VN89
100
VN90
pos
50
inconclusive
neg
0
0
1
2
3
4
5
6
7
8
10
12
14
Da ys post infe ction
Clinical score VS study day
12
Clinical score
10
8
6
VN89
VN90
4
2
0
D-2 D-1 D0
D1
D2
D3
D4
D5
D6
D7
D8
D9 D10 D11 D12 D13 D14 D15 D16 D17 D18 D19 D20
Study day
Infection with FMDV does not appear to induce a
detectable proliferative response against FMDV antigen
Proliferation data for donor VN90
Proliferation data for donor VN89
700000
700000
600000
600000
500000
500000
Day-9
400000
Day 9
300000
Day 12
mean cpm
Day 5
Day 5
400000
Day 9
300000
Day 12
Day 19
Day 19
200000
200000
100000
100000
0
0
Media
PW M
vaccine antigen
Media
PW M
Antigen
vaccine antigen
Antige n
Clinical score VS study day
12
10
C lin ic a l s c o r e
mean cpm
Day-9
8
6
VN89
VN90
4
2
0
D-2 D-1 D0 D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11 D12 D13 D14 D15 D16 D17 D18 D19 D20
Study day
Miriam Windsor
poster
Depletion of CD4+ T cells
during acute infection with FMDV
VT74-CC30/CC51
CD21
CD4
VT76-CC30/CC51
CD21
VT75-CC30/CC51
CD21
CD4
CD4
VT77-CC30/CC51
CD21
CD4
VT74 MM1A/CC51
CD21
CD3
VT76 MM1A/CC51
CD21
VT75 MM1A/CC51
CD21
CD3
CD3
VT77 MM1A/CC51
CD21
CD3
CD4 T cell depletion studies
3ABC ELISA
G-H loop peptide
ELISA
FMDV capsid: T independent and T dependent epitopes
Interaction of FMDV with dendritic cells
Antibody from immune cattle can form complexes with FMDV
which allow the virus to enter, replicate in, and kill moDC
O1K-Cad2 with
immune2C2
serum (1/100)
O1K-Cad2 with non2C2serum (1/100)
immune
O1K-Cad2
alone
2C2
500
500
400
400
400
300
4.88%
200
300
Count
500
C ount
C ount
Stained
for viral
nonstructural
proteins
indicating
replication
at 6 hours
postinfection
4.18%
200
200
100
100
100
0
0
0
10
0
10
1
2
10
10
FL2-H
3
10
4
10
0
10
1
2
10
10
FL2-H
3
10
4
49.40%
300
10
0
10
1
2
10
10
FL2-H
3
10
4
Targeting dendritic cells with live FMDV: reduced T cell
responses
800
*
700
Counts per minute x 10
2
600
500
400
300
200
100
0
Medium
Vaccine antigen
O1K-Cad2
O1K-Cad2 +
im m une IgG
Persistence of FMDV antigen
- maintaining protective antibody responses
MLN 38DPCI
LZ
FMDV D46
DZ
FMDV D46 DAPI
80 µm
No expression of non-structural
proteins:
-Non-replicating
-Extracellular
DAPI CD21 FMDV
Nick Juleff
presentation
4 µm
Immune response to FMDV infection
Innate Immunity
rder
o
b
l
a
eli
Epith
DC
precursor
Cytokines
Eosinophil
NK cell
B
cells
Macrophage
B
cells
CTLs
Helper T cells
GC
CTLs
Helper T cells
Immune response to vaccination
- rapid induction of antibody
- variable/ short duration of immunity
- variable CD4 T cell response
Assessment of neutralising antibody titres and specific T cell proliferative
response post FMDV vaccination
a
c
Antibody response
10000
1 yr
1
1 yr
27
/0
4/
11 06
/0
5/
25 06
/0
5/
08 06
/0
6/
22 06
/0
6/
06 06
/0
7/
20 06
/0
7/
03 06
/0
8/
17 06
/0
8/
31 06
/0
8/
14 06
/0
9/
28 06
/0
9/
12 06
/1
0/
26 06
/1
0/
09 06
/1
1/
23 06
/1
1/
07 06
/1
2/
21 06
/1
2/
04 06
/0
1/
18 07
/0
1/
01 07
/0
2/
07
07/05/06
07/04/06
07/03/06
07/02/06
07/01/06
07/12/05
07/11/05
07/10/05
600000
07/09/05
10
07/08/05
10
07/07/05
100
07/06/05
100
07/05/05
1000
07/04/05
1000
1
Antibody response
10000
300000
500000
250000
b
d
400000
200000
300000
150000
200000
100000
50000
100000
0
0
1
2
3
4
5
6
7
T cell response: open bars FMDV
8
9
10
Veronica Carr
poster
FMDV capids: poor stability/ variable stability
Stability of A and SAT2 capsids (146S) at 49oC
Tim Doel (1981)
Dendritic cell targeting of FMDV antigen can be improved
300
*
Co u n ts p er m in u te x 10
3
250
200
150
100
50
0
Medium
O1-BFS
O1-BFS + immune
IgG
O1-BFS UV
O1-BFS UV +
im mune IgG
Immune response to FMDV vaccine
Innate Immunity
rder
o
b
l
a
eli
Epith
DC
precursor
Cytokines
Eosinophil
NK cell
B
cells
Macrophage
B
cells
CTLs
Helper T cells
GC
CTLs
Helper T cells
The way forward
Improve understanding of immune response
Stabilise vaccine antigen
Target antigen to antigen presenting cells
B cell responses
Which B cells can be detected in the
blood?
Short-lived plasmacytes:
Not clear
Memory B cells:
Need to be stimulated prior to
the ELISPOT assay in order to
induce their differentiation into
plasmocytes
McHeyzer-Williams et al.; Annu. Rev. Immunol. 2005
Long-lived plasmacytes:
No stimulation required but only
detectable when transiting from
the lymphoid organs towards the
bone marrow
Assessment of OVA-specific plasma and
memory B cells (IgG) following
immunisation
Ab titres
Anti-ovalbumin IgG titre
A:
107
106
1000000
105
100000
4
10
10000
3
10
1000
101002
10101
1001
107
106
1000000
105
100000
4
10
10000
3
10
1000
101002
10101
1001
10000000
10000000
0
10
20
30
40
50
0
5
10
15
20
25
30
5
10
15
20
25
30
20
25
30
Induced-ASC number
per 106 cultured PBMC
C: Memory B cells
ASC number per 106 PBMC
ASC number per 106 PBMC
B: Plasma cells
250
2000
200
1500
150
1000
100
500
50
0
0
0
10
20
30
40
50
600
0
20000
15000
400
10000
200
5000
0
0
0
10
20
30
40
Days post-immunisation
(primary response)
50
0
5
10
15
Days post-boost immunisation
(secondary response)
Eric Lefevre
poster
Plasma cell/ Memory B cell frequency in blood of infants
after immunisation with Men C vaccines (Kelly et al., Blood 2006)
Plasma cells
Memory cells
Antigen stability
Structural analysis at the Diamond light source
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
Proof of principle that an engineered mutation (his to cys) is
consistent with capsid assembly.
Similar approaches can be used for infectious copies.
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.
CC
WT
175
83
Assembled empty
particles seen in CC
fractions only.
62
47.5
32
25
16.5
6
7
8
10
Fraction no.
11
12
6
7
8
Fraction no.
10
11
12
Improved stability
- 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
Establishing a cycle of rational vaccine design
Development of a suite of in vitro and in vivo assays to relate the consequences
of improved stability and antigen presenting cell targeting on immunogenicity.
Application of CD4 and CD8 T cell and B cell assays to predict enhanced duration
of immunity by new vaccine candidates.
Implement a rapid iterative cycle of developing and testing new vaccine candidates
DEFRA
BBSRC