D8416.PDF

Rev. sci. tech. Off. int. Epiz., 1988, 7 (2), 347-356.
Freeze-drying of foot and mouth disease virus
and its application
in inactivated virus vaccine production
G. BUTCHAIAH and B.U. RAO *
Summary: Five different subtypes of foot and mouth disease (FMD) virus
propagated in BHK21 C13 monolayer cell cultures, namely 05, A5/!0, A22, C¡
and Asia 1/1, were used to study the stability of live virus during freeze-drying
and subsequent storage. Binary ethyleneimine-inactivated FMD virus vaccines
adjuvanted with purified saponin, "Quil-A", without aluminium hydroxide gel,
were examined before and after freeze-drying and storage for potency in guinea
pigs by challenge tests and in cattle by neutralising antibody estimation. The
infectivity titres of virus subtypes A5/!0, A22, C1 and Asia 1/1 were observed
to be retained during the freeze-drying process and with minimal loss when stored
at —20°C for one year. The infectivity of 05 virus was found comparatively
less stable. The complement fixing (CF) antigen titres of all virus subtypes
remained unaffected under these conditions. The test results in guinea pigs and
cattle indicated that the freeze-dried inactivated vaccines of virus subtypes
A5/10, A22, C1 and Asia 1/1 adjuvanted with "Quil-A " alone were potent, and
that they retained their potency after storage at -20°C for at least one year.
KEYWORDS: Aphthovirus - Inactivated vaccines - Lyophilisation - Potency -
Quil-A - Stability - Storage.
INTRODUCTION
It is considered advantageous to freeze-dry viruses and vaccines wherever possible
in order to reduce their volume for storage in cold, to enable easy handling and
transport, and to enhance their keeping quality.
To preserve maximum infectivity for long periods, cell culture-grown foot and
mouth disease (FMD) virus must be stored frozen at very low temperatures. This
makes its handling difficult. Several attempts have been made to preserve FMD virus
infectivity in tissue suspension (8, 9, 13, 14, 18) and in cell culture (2, 3, 8) by freeze-
drying and storage, and these have met with varying degrees of success.
Currently available inactivated FMD virus vaccines adjuvanted with mineral gels
or oils are invariably in liquid form. Although these vaccines are potent, they present
certain disadvantages as to cost, production methods, storage, packing, shipment
and handling under field conditions, particularly in tropical countries. It is not possible
to freeze or freeze-dry these conventional vaccines as the mineral gels or oils lose
their activity in the process (11).
* Southern Regional Station, Indian Veterinary Research Institute, Hebbal, Bangalore 560 024, India.

348
The adjuvant activity of crude saponin (15) or purified saponin, "Quil-A" (5,
6,7), was demonstrated with inactivated FMD virus vaccines when incorporated alone
or in combination with aluminium hydroxide gel. However, no attempt seems to have
been made so far to develop freeze-dried FMD virus vaccine with suitable inactivants
and adjuvants.
The present study considers the stability of FMD virus after freeze-drying and
storage and also explores the possibility of developing simple, freeze-dried, binary
ethyleneimine (BEI) inactivated FMD virus vaccines incorporating "Quil-A" alone
as adjuvant. The results of assay of live FMD virus and the potency test results of
inactivated FMD virus vaccines before and after freeze-drying and subsequent storage
are reported.
MATERIALS AND METHODS
Virus strains
Vaccine strains of FMD virus subtypes 05, A5/I0, A22, Q and Asia 1/1 used for
vaccine production at the Indian Veterinary Research Institute were employed in this
study.
Virus propagation
The virus was used at the sixth passage level in BHK21 C13 monolayer (Glasgow)
cells grown in Eagle's medium in roller bottles. After harvesting from infected cell
cultures the virus was treated with chloroform (1%) and clarified by centrifugation
at 1,000 g for 15 minutes at +4°C and/or filtration through clarifying Seitz filter pads.
Virus inactivation
The virus was inactivated by 0.001 M binary ethyleneimine (BEI) for 20 hours
at 37°C as described by Bahnemann (1). The action of BEI was neutralised by the
addition of cold sodium thiosulphate (2%). The virus inactivation was ascertained
in BHK21 C13 monolayer cells.
Additive
An additive consisting of 50 g lactalbumin hydrolysate and 100 g sucrose in 1 litre
of distilled water was prepared and the pH was adjusted to 7.4 with 1M tris buffer.
It was then sterilised by filtration through 0.45 µm millipore membrane and stored
at +4°C.
Adjuvant
Purified saponin "Quil-A" (4), supplied by M/s. Superfos, Denmark as a
lyophilised product, was used alone as adjuvant. A 10% stock solution of "Quil-A"
was prepared and sterilised by filtration through 0.45 µm millipore membrane.

349
Vaccine preparation
The inactivated viral antigen of each subtype was separately mixed with an equal
amount of the additive. Simple monovalent vaccines were prepared by the addition
of the adjuvant "Quil-A" (400 µg/ml) without aluminium hydroxide. After mixing,
the pH of the vaccine was adjusted to 7.4 with tris buffer if required.
Freeze-drying procedure
The monovalent vaccine of each virus subtype was dispensed in 1.0 ml quantities
into 6.5 ml glass vials and stoppered with vented butyl rubber stoppers. The vaccine
was then rapidly pre-frozen at -80°C. The partially stoppered vaccine vials in
accessory trays were transferred immediately to the acrylic chamber of the freeze-
dryer (Model EF4 Modulyo of Edwards High Vacuum, West Sussex, England) when
the condenser chamber temperature was - 45°C. The vacuum pump was then started,
and drying occurred for about 19 hours. At the end of the freeze-drying cycle, the
vacuum was 3 X 10-2 mb. The temperature of the product varied from -80°C to
25°C (ambient) during the period of freeze-drying. The vials were closed under vacuum
with the rubber stoppers and sealed with aluminium caps.
The live virus of each subtype was also freeze-dried in a similar manner after mixing
with an equal amount of the additive.
Vials were stored at -20°C and +4°C until testing. The freeze-dried product
was reconstituted by adding 1 ml of sterile distilled water to each vial just before assay.
Estimation of residual moisture content
The residual moisture content of the final freeze-dried product was determined
by placing at least three samples from each batch in a vacuum desiccator over
phosphorous pentoxide at ambient temperature until constant weight was achieved.
The loss in weight of the sample was then calculated.
Virus assay
The freeze-dried product was assayed for complement fixing (CF) antigen and/or
infectivity by plaque titration, as follows:
- Complement fixing antigen titration: The CF viral antigen was titrated in a
micro-test routinely used at this Institute employing homologous virus type specific
guinea pig immune serum, three units of 50% haemolytic doses of guinea pig
complement and sheep haemolytic system. The titres were expressed as CFU/ml.
- Plaque titration: The virus was assayed for plaque forming units (PFU) in
monolayer cultures of BHK21 C13 cells grown in 25 cm2 plastic tissue culture flasks.
Serial 10-fold dilutions of the virus were inoculated onto the cultures using at least
three cultures for each dilution. After one hour virus absorption at 37°C, 0.8% agarose
overlay medium was placed on the cell sheet and allowed to set. The cultures were
incubated at 37°C in an inverted position for about 66 hours. They were then fixed
in 10% formaldehyde solution in normal saline and stained with crystal violet solution
after removal of the fixative and overlay medium. The plaques were counted and
titres expressed as log10 PFU/ml.

350
Vaccine potency tests
The monovalent vaccines were tested before (shortly after formulation) and after
freeze-drying and subsequent storage for one year at -20°C. The vaccine potency
was assayed in guinea pigs and cattle.
Healthy adult guinea pigs of required body weight were vaccinated with a dose
of 0.25 ml subcutaneously and challenged with guinea pig adapted virulent virus at
21 days post-vaccination following the C-index method of Lucam et al. (12).
As for cattle, groups free from FMD virus neutralising antibodies in their sera
were vaccinated subcutaneously with a 2.5 ml dose of monovalent vaccine. The animals
were bled at different periods up to 3 months after vaccination for collection of serum.
They were also examined for any possible local or systemic reaction after vaccination.
Serum samples were assayed for neutralising antibody levels in a micro-test
employing two-fold serial serum dilutions, 100 TCID50 of homologous FMD virus
subtype and 1B-RS-2 cells in 96-well plastic tissue culture plates. The 50% end-points
were calculated according to the method of Reed and Muench (17). The antibody
titres were expressed as log10 SN50 values.
RESULTS AND DISCUSSION
Stability of FMD virus during freeze-drying and storage
Initially, the effect of freeze-drying and subsequent storage on live FMD virus
was studied. At least three separate experiments for each virus subtype were conducted.
A minimum of three samples at a time from each batch of virus were used for assay.
The residual moisture content in each batch of freeze-dried product was found to
be less than 2%.
The results presented in Table I are the mean values of assays from all experiments
carried out with a single virus subtype. The survival rates after freeze-drying for
different subtypes varied from 46% to 62%, with higher retention of infectivity when
stored for over one year at -20°C rather than +4°C. The stability of O5 virus
infectivity during freeze-drying and storage was comparatively low. In contrast, the
CF antigen titres of all virus subtypes remained more or less unaffected both during
the freeze-drying process and subsequent storage, irrespective of the storage
temperature.
Earlier investigators have recorded their experiences and varying degrees of success
in freeze-drying and storing crude FMD virus in the form of infected mouse muscle
(18), cattle tongue epithelium (13, 14), tissue culture and tissue suspension virus (3,
8). Purified virus was found to degrade rapidly during freeze-drying and storage (10).
Occasional values of virus assay found out of line in the present study (such as
increased virus titres after freeze-drying or subsequent storage; Table I) could be due
to test variation. Such deviations from the straight line could also be due to uneven
drying of the product if the virus assay titres at the time interval before and after
were in line (16).

351
TABLE I
Stability of live
foot
and
mouth
disease virus during freeze-drying and
subsequent
storage
Virus titres
M
TH
3.65
VO
5.87
en
en
4.79
r-
00
3.94
00
4.06
en
Os
months
U
o
Tt
+
OS
so
4.08 4.35
73 80
6.49 6.26
O
vo
O
SO
5.46 5.05
r-
00
en
CN
4.60 3.65
en
<N
r-
oo
5.30 4.65
93 80
nperature
and period
in
fn
rH
4.33 4.76
73 80
6.80 6.89
o
vo
TH
o
VO
6.43 6.22
en
es
en
CN
5.86 5.40
en
CN
r--
oo
5.74 5.19
t-
o
.—<
en
Os
Storage ten
-20°C
OS
4.46
o
oo
6.83
O
vo
'—1
6.50
O
o
CN
5.95
en
CN
5.87
en
Os
so
4.73
en
Os
7.00
en
en
6.43
en
CM
6.04
en
<N
5.85
O
o
en
5.05
O
oo
7.20
O
vo
*—i
6.77
en
>—<
<N 6.40
r~
00
5.89
r-
o
After
freeze-
drying
5.60
(46%)
93
7.10
?
CN
O
vo
1—1
6.87
#
<N
m
en
CN
6.19
(59%)
en
CN
6.14
? r-
o
Before freeze-
drying
5.98
en
Os
7.31
o
VO
TH
7.14
en
CN
6.42
en
i—i
CN
6.37
en
Os
Virus
assay
PFU CFU PFU
CFU PFU CFU PFU
CFU PFU CFU
Virus subtype
d
o
CN
< O
Asia
1/1
Figures in parentheses indicate
virus
survival rates.
PFU =
Log10
plaque
forming
units/ml.
CFU = Complement
fixing
units/ml.