OS18 - Open Session 2018 Borgo Egnazia (Puglia), Italy 29 - 31 /10/2018 EuFMD Sessions\Open Session\Archive-2018\Open 2018 - Italy\6. Online & Streaming\PPTs

1. The use of reverse genetics to
facilitate the growth of FMDV for
the production of vaccines.
Dr Stephen Berryman1
Femke Feenstra2, Jose Coco-Martin2 and Toby Tuthill1
1Pirbright Institute 2Boehringer Ingelheim Animal Health

2. • Current FMDV vaccines are chemically inactivated virus preparations produced in large-
scale mammalian cell culture (usually suspension BHK cells)
• There is a periodic need to produce new vaccines against emerging field strains
• Production of new vaccine strains involves adaptation of field viruses to cell culture in
order to improve growth characteristics
• Measures that improve the speed by which new vaccine strains can be adapted to cell
culture could have a large impact by increasing the amount of vaccine available globally
for FMD control.
FMDV Vaccine Development

3. • Tissue culture adaption is often associated with acquisition of the ability to use
receptors not normally used by FMDV:
- Field Strains: Use RGD-dependent αv integrins (αvβ1, αvβ3, αvβ6, αvβ8)
- Tissue Culture Adapted Viruses: Can use alternative receptors including Heparan
Sulphate, and non-HS receptors e.g. JMJD6
• The changes associated with the ability to use non-integrin receptors often involve the
acquisition of positively charged residues around the HS binding site or the 5-fold
symmetry axis (Maree et al. 2010, Berryman et al. 2013, Dill et al. 2017.).
• Aim – Introduce previously described cell culture adaptation mutations by design
using reverse genetics, and assess if this leads to immediate and improved growth in
cell culture
FMDV Receptors and Cell Culture Adaptation
O1BFS 1860 Pentamer
HS binding
site
5-fold
symmetry axis

4. Infectious Clone based on Tissue culture Adapted
type O FMDV (pO1K)
Field Strain VP2-2A (Synthetic DNA)
Chimeric Infectious Clone
- VP2, VP3, VP1, 2A from field strain
- Backbone based on tissue culture adapted type O FMDV
Chimeric Infectious Clones – Capsid Switching

5. • First stage – Make chimeric infectious copies carrying wild type capsids from one
example each of the four most prevalent serotypes of FMDV (A, Asia, O, SAT-2)
• Second stage – introduce targeted mutations into the wild type field capsid coding
region of the infectious copy plasmids, in order to enable the chimeric viruses to grow
better in cell culture when rescued
• 2 categories of mutation: 1) HS binding site and 2) 5-fold symmetry axis
2) Example 5 fold symmetry changes type A
(Berryman et al. 2013)
1) Example HS binding site changes type A
Chimeric Infectious Clones – Engineering Changes at the HS
binding site and 5-fold symmetry axis.

6. From Plasmid to Virus
Linear cDNA clone (e.g. pO1K/A)
Synthetic RNA
Transfect RNA
in to BHK
Passage 1 Passage 2
Recover virus by
passage in BTY
(WT capsids) or
BHK (mutant
capsids)

7. Most (But Not all) Variants Produce Viable Virus
Virus could be
recovered on
BTY but no
CPE on BHK
Capsid
(VP2-2A)
Back
bone
Recovered?
A WT Type O 
A KK Type O 
A RK Type O 
A HS Type O 
Asia-1 WT Type O 
Asia-1 KK Type O 
Asia-1 RK Type O 
Asia-1 HS Type O 
Capsid
(VP2-2A)
Back
bone
Recovered?
O WT Type O 
O KK Type O 
O RK Type O 
O HS Type O 
SAT2 WT Type O 
SAT2 KGR Type O ?
SAT2 KKR Type O 
SAT2 KHR Type O 
SAT2 HS Type O 

8. Sequencing of Recovered Viruses
• Viral RNA was extracted from recovered viruses, converted to cDNA via a non-PCR
based protocol, and sequenced on the Illumina Miseq
• WT viruses – No Capsid protein amino acid changes
– Should still have WT phenotype.
• In some adapted viruses extra positively charged residues could be seen at the
consensus level
– not unexpected and indicates extra adaptation during recovery process.

9. No
Virus
WT KK HS WT KK RK
AsiaI capsid A capsid
No
Virus WT KK RK HS WT KKR
O capsid SAT2 capsid
BTY
αvβ6 +
HS+          
CHO
Integrin –
HS+          
BHK
αvβ3+
HS+
         
Viruses have expected properties –
BTY – All cause CPE
CHO – Only mutants cause CPE (Indicates non-integrin receptor usage)
BHK – All mutants caused CPE, WT less CPE
CPE?
CPE?
CPE?
WT KK HS WT KK RK WT KK RK HS WT KKR
WT KK HS WT KK RK WT KK RK HS WT KKR
Phenotype of Recovered Viruses – Which cell lines can they infect?

10. Adaptive Changes Increase Infectious Titres in BHK Cells
1
10
100
1000
10000
100000
1000000
10000000
100000000
1E+09
Titre(PFU/ml)
BHK
BTY
A and Asia Viruses- Mutated Viruses have slightly higher BHK titres than WT
O and SAT Viruses- WT has very poor BHK titre so mutated viruses have greatly enhanced
BHK titres (although BTY titres similar to WT)

11. A WT
1/16
1/32
1/64
1/128
1/256
1/512
Final
Serum
Dilution
1/1024
None
A(1) Serum
A KK
A(1) Serum
A RK
ABS
Sera
Only
Antisera
Only
controls
A(1)
A(1) Serum
A(1)
A WT
ABS 1/16
A KK
ABS 1/16
A RK
ABS 1/16
• VNT on BTY cells using existing cattle sera generated using viruses closely related to our strains
• Antigenic properties of WT and mutant viruses are compared
• (can’t use BHK/IBRS-2 due to poor growth O WT and SAT WT)
Antibody titre is
the dilution of
antibody which
gives 50%
protection
R1 Value is the
ratio of antibody
titre for mutant
divided by that
for WT
Comparing Antigenic properties of Viruses by VNT on BTY
- Example – Type A

12. Most Variants Show Similar Neutralisation Titres to WT in VNT
0.50 =
R1 Value vs WT
For Vaccine
matching R1
>0.3 considered
a match
R1 >0.3 for all
except SAT-2
KKR
0.50 = R1 Value vs WT
0.83 1.17 1.14 1.14
0.57 0.57 0.50
0.15
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
LogAntibodyTitre
Log Antibody Titre for WT and Mutant Viruses

13. “Adapted” Viruses Grow Faster than WT in Adherent BHK – CPE Assay
Type A Viruses (+SAT Mutant) Asia Viruses
SAT KKR
A Turk KK
A Turk RK
A Turk WT
No Virus
Asia KK
Asia HS
Low MOI
No Virus
Asia WT
Low MOI
Note for Type O and SAT Viruses
-No WT as not high enough BHK titre
for even for the low MOI used here
OUKG KK
No Virus
OUKG HS
OUKG RK
Type O Viruses
For All Serotypes tested – Mutant
Viruses show faster growth than WT
Low MOI

14. • All mutants show quicker CPE compared to the WT variant
1 0 2 0 3 0 4 0
-5 0
0
5 0
1 0 0
1 5 0
T im e p o s t in fe c tio n (h )
CPE(%)
O 1 K -A w t
O 1 K -A K K
O 1 K -A R K
1 0 2 0 3 0 4 0
-5 0
0
5 0
1 0 0
1 5 0
T im e p o s t in fe c tio n (h )
CPE(%)
O 1 K -A s ia I w t
O 1 K -A s ia I K K
O 1 K -A s ia I H S
1 0 2 0 3 0 4 0
-5 0
0
5 0
1 0 0
1 5 0
T im e p o s t in fe c tio n (h )
CPE(%)
O 1 K -S A T 2 w t
O 1 K -S A T 2 K K R
1 0 2 0 3 0 4 0
-5 0
0
5 0
1 0 0
1 5 0
T im e p o s t in fe c tio n (h )
CPE(%)
O 1 K -O K K
O 1 K -O R K
O 1 K -O H S
“Adapted” Viruses Grow Faster than WT in Suspension BHK-21 Cells
Data – Femke Feenstra
(Boehringer Ingelheim)

15. ELISA
• O - WT does not grow, so the
mutations improve the yield
• KK/RK significantly higher
yields compared to HS
ELISA
• A - No significant difference
between WT and mutants
0 1 0 2 0 3 0 4 0
0
1
2
3
T im e p o s t in fe c tio n (h )
146Syield(ELISAunits)
O 1 K -A w t
O 1 K -A K K
O 1 K -A R K
0 1 0 2 0 3 0 4 0
0
2
4
6
8
T im e p o s t in fe c tio n (h )
146Syield(ELISAunits)
O 1 K -O K K
O 1 K -O R K
O 1 K -O H S*
*
**
Type O capsid
Type A capsid
Introduced Capsid Changes Can Enhance 146S Yield in Suspension BHK
Data – Femke Feenstra
(Boehringer Ingelheim)

16. UV peak
• WT yield higher that
mutants.
• Yield HS significantly lower
than KK
UV Peak
• Yield mutant higher than wt,
since WT does not grow
0 1 0 2 0 3 0 4 0
0
5 0
1 0 0
T im e p o s t in fe c tio n (h )
146Syield(%ofhighestyieldinthisstudy)
O 1 K -A s ia I w t
O 1 K -A s ia I K K
O 1 K -A s ia I H S
* *
*
0 1 0 2 0 3 0 4 0
0
5 0
1 0 0
T im e p o s t in fe c tio n (h )
146Syield(%ofhighestyieldinthisstudy)
O 1 K -S A T 2 w t
O 1 K -S A T 2 K K R
*
**
*
Data – Femke Feenstra
(Boehringer Ingelheim)
Asia-I capsid
SAT-2 capsid
Introduced Capsid Changes Can Enhance 146S Yield in Suspension BHK
Introduced capsid changes greatly
enhance 146S yield versus WT for the SAT-
2 and type O viruses (but not A and Asia)

17. • We were able to rescue viruses carrying capsid proteins from Type A, AsiaI, O and SAT-
2 field strains in a type O backbone.
• Viruses carrying mutations at the HS binding site and/or 5-fold axis designed to
enhance growth in BHK cells were also rescued, and gained ability to infect CHO cells
indicative of alternative receptor usage.
• Mutants (except SAT-2 KKR) showed response comparable to WT in neutralisation
assays in BTY – Need animal experiments to determine if SAT-2 KKR is really a problem.
• Mutants show faster CPE than WT in adherent BHK.
• Mutants show faster CPE than WT in suspension BHK cells.
• SAT-2 and type O mutants (i.e. those where WT equivalents grew poorly in BHK)
showed enhanced 146S yield over WT in suspension BHK cells.
• These recombinant techniques could prove highly valuable to speed up new
vaccine strain development by reducing the need for time consuming tissue
culture adaptation and increasing the success rate of growing field strains in
tissue culture.
Summary

18. Acknowledgements
Pirbright Institute
Toby Tuthill
Terry Jackson
Veronica Fowler
Anna Ludi
Ginette Wilsden
Boehringer Ingelheim Animal Health
Jose Coco-Martin
Femke Feenstra
Hélène Gaude