IAEA/FAO IAEA/FAO Antibody titres in FMD type A strains: comparison of methodologies to predict cross-protection Tesfaalem T.Sebhatu, Klaas Weerdmeester, Froukje van Hemert-Kluitenberg, Aldo Dekker 17 – 19 April 2012, Hazyview, South Africa
IAEA/FAOIAEA/FAO
Antibody titres in FMD type A strains:
comparison of methodologies to predict
cross-protection
Tesfaalem T.Sebhatu, Klaas Weerdmeester, Froukje van Hemert-Kluitenberg, Aldo Dekker
17 – 19 April 2012, Hazyview, South Africa
IAEA/FAO
Outline
� VP1 gene sequence of all 10 FMDV type A strains
� Difference between serological tests
� Virus neutralisation test (VNT)
� Neutralisation index test (NIT)
� Liquid Phase Blocking ELISA (LPBE)
� Statistical analysis
� Mean titres
� r1 values
� Scaling responses
� Cluster analysis
� Principal Component analysis (PCA)
IAEA/FAO
Research goals:
� Comparison of test methodologies
�VNT, NIT, and LPBE
�Which serological methodology gives the highest
confidence?
�Does difference between test methods predict significant
variation?
IAEA/FAO
Source of the 10 FMDV Serotype A strains
used in the study
WRLFMD, Pirbright, U.K.
� A/ERI/2/98
� A/SUD/2/84
� A/KEN/12/2005
� A/ETH/13/2005
� A/MAU/1/2006
CVI, Lelystad, Netherlands
� A22/IRQ/24/64
� A/TUR/20/2006
� A/TUR/14/98
� A/IRN/02/97
� A10/Holland/42
IAEA/FAO
Animal experiment
� 50 cattle plus 5 controls carried out in Eritrea
� 10 different FMDV type A strains
� Cultured on BHK-21 cells
� Inactivated with binary ethylenimine (BEI)
� One cycle of polyethylene glycol (PEG) concentration
� Quantitative sucrose density gradient analysis was used to determine the 146S antigen concentration
� Vaccination with 10 µg antigen in 2 ml Aluminium hydroxide saponin adjuvant (formulation on site)
� Sera 21 day post-vaccination
IAEA/FAO
Serological test methods
� Virus neutralisation
� Varying serum dilution- versus fixed virus concentration
� Neutralisation index
� Fixed serum dilution - versus variable virus concentration
� Liquid Phase Blocking ELISA
� Using subtype specific Guinea-pig and Rabbit semi-purified Ig fractions (saturated Ammonium Sulphate)
IAEA/FAO
Statistical analysis
� Mean titres
� r1-values
� Mean scaled titre (titre – mean)/sd
� Cluster analysis using Euclidian distance
� Principal component analysis (PCA)
IAEA/FAO
VP1 gene sequence of the 10 FMDV type A strains
� A dendrogram
constructed from the VP1
gene of the 10 FMDV
serotype A strains using
BEAST displaying the
posterior value (0 – 1)
indicating the confidence
of the split. FMDV
serotype O1 manisa/1969
was used as an outgroup
to construct the rooted
tree.
IAEA/FAO
Results of the 3 serological
methods
� Huge dataset, difficult to interpret
� On the X-axis the different strains used in the test
� On the Y-axis the 10log
mean antibody titre
� For each vaccine barplots of VNT (black), NIT (grey) & LPBE (Light grey) results
� Very similar titres in VNT and NIT
� Mostly homologous titres are the highest
1 2 3 4 5 6 7 8 9 10
1 - A10 Holland
01
23
4
1 2 3 4 5 6 7 8 9 10
2 - A22 Iraq
01
23
4
1 2 3 4 5 6 7 8 9 10
3 - A SUD/2/84
01
23
4
1 2 3 4 5 6 7 8 9 10
4 - A IRN/2/97
01
23
4
1 2 3 4 5 6 7 8 9 10
5 - A TUR/14/98
01
23
4
1 2 3 4 5 6 7 8 9 10
6 - A ERI/2/98
01
23
4
1 2 3 4 5 6 7 8 9 10
7 - A ETH/13/2005
01
23
4
1 2 3 4 5 6 7 8 9 10
8 - A KEN/12/2005
01
23
4
1 2 3 4 5 6 7 8 9 10
9 - A MAU/1/2006
01
23
4
1 2 3 4 5 6 7 8 9 10
10 - A TUR/20/2006
01
23
4
IAEA/FAO
Comparison of vaccine response (African strains)
- Mostly homologous titres are the highest
- LPBE has a lower discriminatory capacity for differences between strains, but
gives a more consistent result when compared with VN & NI
A/ERI/2/98 Vaccine A/MAU/1/2006 Vaccine
IAEA/FAO
Comparison of vaccine response (Middle East strains)
A/TUR/20/2006 Vaccine A22/IRQ/24/64 Vaccine
IAEA/FAO
VNT Results
� Difference between antibody response induced by the vaccine compared to the response of the virus to the sera made by vaccination against the other strains
� Low response of vaccine against heterologous strains
� Relatively good response by sera from heterologous strains
IAEA/FAO
r1 Values
� Comparison r1-values VNT (black), NIT (dark grey) and LPBE (light grey)
� Huge difference between neutralising and ELISA antibodies
� A/SUD/2/84, A/IRN/2/97 and A/TUR/20/2006 showed high VNT & NIT r1 values (>1.0)
� Because responses in different tests are not on the same scale
1 2 3 4 5 6 7 8 9 10
1 - A10 Holland
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
2 - A22 Iraq
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
3 - A SUD/2/84
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
4 - A IRN/2/97
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
5 - A TUR/14/98
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
6 - A ERI/2/98
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
7 - A ETH/13/2005
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
8 - A KEN/12/2005
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
9 - A MAU/1/2006
0.0
0.4
0.8
1 2 3 4 5 6 7 8 9 10
10 - A TUR/20/2006
0.0
0.4
0.8
IAEA/FAO
Scaled titres
� A clear difference in the scale of the response for each test strain
� For each test strain (titre-mean titre)/sd
� Comparison scaled titres VNT (black), NIT (dark grey) and LPBE (light grey)
� In all tests all homologous scaled titres are the highest
Scaled titre
10log
1 2 3 4 5 6 7 8 9 10
1 - A10 Holland
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
2 - A22 Iraq
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
3 - A SUD/2/84
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
4 - A IRN/2/97
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
5 - A TUR/14/98
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
6 - A ERI/2/98
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
7 - A ETH/13/2005
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
8 - A KEN/12/2005
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
9 - A MAU/1/2006
-2-1
01
2
1 2 3 4 5 6 7 8 9 10
10 - A TUR/20/2006
-2-1
01
2
IAEA/F
AO
A MAU/1/2006
A TUR/20/2006
A22 Iraq
A TUR/14/98
A ETH/13/2005
A SUD/2/84
A ERI/2/98
A KEN/12/2005
A10 Holland
A IRN/2/97
5 6 7 8 9 10
Height
A10 Holland
A IRN/2/97
A SUD/2/84
A ETH/13/2005
A KEN/12/2005
A MAU/1/2006
A ERI/2/98
A TUR/20/2006
A22 Iraq
A TUR/14/98
5 6 7 8 9 10
Height
Cluste
rs b
ased o
n E
uclid
ean d
ista
nce
Based o
n scaled
VNT titre
s
Based o
n scaled
NIT
titres
IAEA/FAO
A T
UR
/20/2
006
A S
UD
/2/8
4
A M
AU
/1/2
006
A22 I
raq
A10 H
olla
nd
A E
TH
/13/2
005
A K
EN
/12/2
005
A E
RI/
2/9
8
A I
RN
/2/9
7
A T
UR
/14/9
8
23
45
67
He
igh
t
Clusters based on Euclidean distance
� Scaled LPBE titre
� African strains (red
circle) cluster more
closely than the Asian
strains (blue circle)
� Different serological techniques produce different clusters
� Different statistical techniques produce different clusters (not shown)
IAEA/FAO
A22 I
raq
A10 H
olla
nd
A K
EN
/12/2
005
A E
RI/
2/9
8
A E
TH
/13/2
005
A I
RN
/2/9
7
A T
UR
/14/9
8
A T
UR
/20/2
006
A S
UD
/2/8
4
A M
AU
/1/2
006
34
56
78
He
igh
t
Clusters based on Euclidean distance
� LPBE scaled r1 cluster
� Clustering of the African and Middle East strains in multi groups indicating no antigenic similarity between the strains
� African strains clustered slightly better than the Asian strains
IAEA/FAO
VNT principal component analysis on strains
-0.35 -0.30 -0.25
-0.2
0.0
0.2
0.4
PC1
PC
2
IAEA/FAO
VNT principal component analysis on sera
-0.2 -0.1 0.0 0.1 0.2
-0.2
-0.1
0.0
0.1
0.2
PC1
PC
2
IAEA/FAO
NIT principal component analysis on strains
NIT
-0.38 -0.36 -0.34 -0.32 -0.30 -0.28 -0.26
-0.4
-0.2
0.0
0.2
0.4
PC1
PC
2
IAEA/FAO
-0.2 -0.1 0.0 0.1 0.2
-0.2
-0.1
0.0
0.1
0.2
PC1
PC
2NIT principal component analysis on sera
IAEA/FAO
LPBE principal component analysis on strains
LPBE
-0.36 -0.34 -0.32 -0.30 -0.28 -0.26 -0.24
-0.4
-0.2
0.0
0.2
0.4
PC1
PC
2
IAEA/FAO
-0.3 -0.2 -0.1 0.0 0.1 0.2
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
PC1
PC
2LPBE principal component analysis on sera
IAEA/FAO
Conclusion
� Neutralisation tests, VNT and NIT, have a higher specificity than LPBE
� Therefore neutralisation tests have a better discrimination capacity of strains
� r1 values are difficult to interpret as values greater than 1 are observed
� Scaled titres can reduce variation between tests
� Cluster analysis does not produce consistent results and not comparable with genetic information
� PCA showed high variations on sera and strains
� Antigenic evolution was found to be more punctuated than genetic evolution, as the change of virion proteins in response to antibody selection is more frequent.
IAEA/FAO
Acknowledgement
� This study was sponsored by the International Atomic Energy Agency (IAEA), Vienna, Austria
� Ministry of Agriculture, Eritrea
� National Veterinary Laboratory staff, Asmara, Eritrea
� FAO Representative Office, Eritrea
IAEA/FAO
THANK YOU