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. B U T C H A I A H and B . U . R A O *
Summary: Five different subtypes of foot and mouth disease (FMD) virus
propagated in BHK21 C13 monolayer cell cultures, namely 0 , A ,
A , 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 A ,
A , 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 0 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
A /10, A , 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.
5
5/!0
5/!0
22
22
5
5
22
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 m a x i m u m 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 F M D virus
infectivity in tissue suspension (8, 9, 13, 14, 18) and in cell culture (2, 3, 8) by freezedrying and storage, and these have met with varying degrees of success.
Currently available inactivated F M D 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, H e b b a l , Bangalore 560 024, India.
348
The adjuvant activity of crude saponin (15) or purified saponin, " Q u i l - A " (5,
6,7), was demonstrated with inactivated F M D virus vaccines when incorporated alone
or in combination with aluminium hydroxide gel. However, n o attempt seems to have
been m a d e so far to develop freeze-dried F M D virus vaccine with suitable inactivants
and adjuvants.
T h e present study considers the stability of F M D virus after freeze-drying and
storage and also explores the possibility of developing simple, freeze-dried, binary
ethyleneimine (BEI) inactivated F M D virus vaccines incorporating " Q u i l - A " alone
as adjuvant. The results of assay of live F M D virus and the potency test results of
inactivated F M D virus vaccines before and after freeze-drying and subsequent storage
are reported.
MATERIALS A N D METHODS
Virus strains
Vaccine strains of F M D virus subtypes 0 , A
, A , Q and Asia 1/1 used for
vaccine production at the Indian Veterinary Research Institute were employed in this
study.
5
5 / I 0
2 2
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 a n d / o r 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
A n additive consisting of 50 g lactalbumin hydrolysate and 100 g sucrose in 1 litre
of distilled water was prepared and the p H was adjusted to 7.4 with 1M tris buffer.
It was then sterilised by filtration t h r o u g h 0.45 µm millipore m e m b r a n e and stored
at + 4 ° C .
Adjuvant
Purified saponin " Q u i l - A " (4), supplied by M / s . Superfos, D e n m a r k as a
lyophilised product, was used alone as adjuvant. A 10% stock solution of " Q u i l - A "
was prepared and sterilised by filtration through 0.45 µm millipore m e m b r a n e .
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 " Q u i l - A " (400 µ g / m l ) without aluminium hydroxide. After mixing,
the p H 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 a n d stoppered with vented butyl rubber stoppers. The vaccine
was then rapidly pre-frozen at - 8 0 ° C . T h e partially stoppered vaccine vials in
accessory trays were transferred immediately t o the acrylic chamber of the freezedryer (Model E F 4 Modulyo of Edwards High V a c u u m , West Sussex, England) when
the condenser chamber temperature was - 45°C. The vacuum p u m p was then started,
and drying occurred for a b o u t 19 h o u r s . At the end of the freeze-drying cycle, the
vacuum was 3 X 10- m b . T h e temperature of the product varied from - 8 0 ° 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.
2
The live virus of each subtype was also freeze-dried in a similar manner after mixing
with an equal a m o u n t of the additive.
Vials were stored at - 2 0 ° C and + 4 ° C until testing. T h e 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 a n d / o r
infectivity by plaque titration, as follows:
- Complement fixing antigen titration: The C F viral antigen was titrated in a
micro-test routinely used at this Institute employing homologous virus type specific
guinea pig i m m u n e serum, three units of 5 0 % haemolytic doses of guinea pig
complement and sheep haemolytic system. The titres were expressed as C F U / m l .
- Plaque titration: The virus was assayed for plaque forming units (PFU) in
monolayer cultures of BHK21 C13 cells grown in 25 c m 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 h o u r s . They were then fixed
in 10% formaldehyde solution in normal saline and stained with crystal violet solution
after removal of the fixative and overlay m e d i u m . The plaques were counted and
titres expressed as l o g P F U / m l .
2
10
350
Vaccine potency tests
The monovalent vaccines were tested before (shortly after formulation) and after
freeze-drying and subsequent storage for one year at - 2 0 ° 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 F M D 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 T C I D of homologous F M D virus
subtype and 1B-RS-2 cells in 96-well plastic tissue culture plates. The 5 0 % end-points
were calculated according to the method of Reed and Muench (17). The antibody
titres were expressed as l o g S N values.
5 0
10
5 0
RESULTS A N D DISCUSSION
Stability of FMD virus during freeze-drying and storage
Initially, the effect of freeze-drying and subsequent storage on live F M D 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 4 6 % to 6 2 % , with higher retention of infectivity when
stored for over one year at - 2 0 ° C rather t h a n + 4 ° C . The stability of O virus
infectivity during freeze-drying and storage was comparatively low. In contrast, the
C F 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.
5
Earlier investigators have recorded their experiences and varying degrees of success
in freeze-drying and storing crude F M D 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).
Virus
subtype
d
vo
CN 1 — 1
o
VO
Os
?
O
TH
en
CM
en
# en
<N CN
m
en
CN
PFU
*—i
O
CFU
vo
6.50
'—1
4.79
5.05
CN
<
r~
00
en
CN
en
i—i
CN
o
O
6.37
CFU
PFU
5.30
93
5.19
to
5.74
en
5.87
en
Os
5.85
O
5.89
ro
?o
r-
en
Os
10
Figures in parentheses indicate virus survival rates.
PFU = Log plaque forming units/ml.
CFU = Complement fixing units/ml.
CFU
en
<N
80
4.06
en
CN
r--
4.65
oo
oo
6.14
3.94
3.65
4.60
<N
5.40
>—<
5.86
o
CN
en
CN
5.95
en
CN
r-
6.04
en
es
en
<N
6.40
00
6.19
(59%)
6.22
r-
00
6.42
5.46
en
CN
6.43
r00
Asia 1/1
O
vo
6.43
6.80
o
VO
TH
6.77
6.89
o
vo
6.87
6.49
O
SO
vo
7.14
5.87
6.26
O
PFU
oo
Os
CFU
6.83
7.00
O
en
en
7.20
en
O
7.10
en
80
so
en
en
73
73
3.65
4.08
fn
4.35
so
4.76
U
OS
VO
80
4.33
4.46
o
oo
4.73
OS
5.05
rH
7.31
CFU
Storage tennperature and period in months
-20°C
o
Tt
M
TH
PFU
5.98
PFU
After
freezedrying
+
5.60
(46%)
93
Before
freezedrying
en
Virus
assay
Virus titres
Stability of live foot and mouth disease virus during freeze-drying and subsequent storage
TABLE I
351
en
Os
.—<
Os
o
352
By the methods of assay employed in this study, for virus titres to be significantly
different from each other, two end-points would have to differ by more than
0.5 l o g P F U / m l in the case of infectivity titres, and by more than twice the values
of C F U / m l in the case of C F antigen titres. This difference was not apparent with
most of the freeze-dried preparations of A
, A , C1 and Asia 1/1 virus subtypes
stored at - 2 0 ° C .
10
5 / 1 0
2 2
In conclusion, the cell culture-grown F M D virus could withstand the freeze-drying
process, and it should be stored at - 2 0 ° C rather t h a n + 4 ° C to retain maximum
infectivity. By contrast, C F antigen titres remained more or less constant irrespective
of the storage temperature.
The loss in virus infectivity titre rather than immunogenicity might occur during
freeze-drying and subsequent storage. Since the infectivity of F M D virus was retained
with minimal loss in the present study, the immunogenicity would not be adversely
affected. This was verified by testing the potency of freeze-dried inactivated F M D
virus vaccines in animals.
Potency of freeze-dried inactivated FMD virus vaccines
L a b o r a t o r y assay showed that the virus subtypes A
, A , C1 and Asia 1/1
incorporated into the vaccines had good titres. Their infectivity titres (log P F U / m l )
were 7.45, 7.32, 6.45 and 6.50, and C F titres ( C F U / m l ) were 160, 160, 320 and 160
respectively. O virus titres were relatively low with an infectivity titre of 6.20 and
C F titre of 80.
5 / 1 0
2 2
10
5
In the present study, binary ethyleneimine was used instead of formaldehyde in
F M D virus inactivation for preparation of vaccines without aluminium hydroxide gel.
Table II summarises the results of potency tests in guinea pigs of F M D vaccines
before and after freeze-drying and storage. Monovalent vaccines of virus subtypes
A
, A , C1 and Asia 1/1 adjuvanted with " Q u i l - A " alone, which were in aqueous
form before freeze-drying, protected guinea pigs with high C-index values. These
results agree with those of other workers who reported that crude saponin (15) or
purified saponin, " Q u i l - A " (5, 6, 7), when incorporated into inactivated F M D virus
vaccines of types O, A and C as adjuvant without aluminium hydroxide gel, was able
to stimulate antibody production in cattle at levels comparable to mineral oil or geladjuvanted vaccines that could confer protection against the disease.
5 / 1 0
2 2
However, our earlier studies (unpublished data) showed that such aqueous vaccines
without aluminium hydroxide gel rapidly deteriorated, with substantial loss in potency
when stored at usual refrigeration temperatures for more than a few days. Storing
these vaccines by freezing at low temperatures makes their handling very difficult.
In the current study, we therefore tried to improve their keeping quality by freezedrying.
The potency of the vaccines of virus subtypes A
, A , Q and Asia 1/1 seems
to be retained without loss during the freeze-drying process. In fact, these freezedried vaccines retained their potency even after storage for at least one year at - 20°C.
Thus, they met the requirements of potency in guinea pigs when tested by the method
of Lucam et al. (12). Since their C-index values were within the 9 5 % confidence limits
of the original values for the same vaccines before freeze-drying, their original potency
should be considered as retained without significant loss after freeze-drying and storage
(Table II).
5 / 1 0
2 2
353
TABLE
Results
II
of potency tests in guinea pigs of inactivated FMD
vaccines before and after freeze-drying
and storage
virus
Potency in guinea pigs (C-index)
Vaccine
virus
subtype
O5
Before
freeze-drying
After
freeze-drying
Freeze-dried and
stored at - 20°C
for 12 months
1.78
(1.48-2.14)
1.23
(1.03-1.48)
not done
A
5/10
4.10
(3.42-4.92)
4.00
(3.33-4.80)
3.83
(3.19-4.60)
A
22
3.65
(3.04-4.38)
3.59
(2.99-4.31)
3.50
(2.92-4.20)
4.10
(3.42-4.92)
4.25
(3.54-5.10)
3.88
(3.23-4.66)
2.30
(1.92-2.76)
2.40
(2.0-2.88)
2.65
(2.21-3.18)
c1
Asia
1/1
Figures in parentheses indicate 95% confidence limits.
The apparent
to 4.25 and that
freeze-dried Asia
variation (Table
increase in the potency of C1 vaccine from C-index value of 4.10
of Asia 1/1 vaccine from 2.30 to 2.40 after freeze-drying and of
1/1 vaccine from 2.40 to 2.65 after storage could be due to biological
II).
None of the vaccinated cattle developed any adverse reaction. Table III summarises
the neutralising antibody levels in cattle over an observation period of 3 months after
vaccination with a single dose of the freeze-dried monovalent F M D vaccine. There
was good antibody response in cattle vaccinated with A
, A , Q and Asia 1/1
vaccines with titres ranging from 1.97 to 2.82 at 30 days post-vaccination.
Furthermore, no substantial loss in antibody titres was observed at the end of 3 months
with the titres ranging from 1.71 to 2.46. The antibody response in cattle at 30 days
post-vaccination appears to be uniform since the values for twice the standard
deviation lie in the range of 0.42-0.74. The antibody response to O vaccine was
comparatively low with a maximum titre of only 1.38 at 15 days post-vaccination.
5 / 1 0
2 2
5
The poor potency of O vaccine could be due to inadequate viral antigen
incorporation into the vaccine, degradation of antigen during freeze-drying or t o the
possibility that " Q u i l - A " alone was not an effective adjuvant for this virus antigen.
5
In conclusion, the freeze-dried and stored F M D vaccines incorporating BEIinactivated virus and " Q u i l - A " as adjuvant had a potency well above the minimum
acceptable level. These vaccines are simple, cheap, easy to c o m p o u n d , store, pack
and transport. It is also possible to reduce the dose of the vaccine by reconstitution
of the freeze-dried product with an appropriately reduced quantity of the diluent.
Such freeze-dried vaccines are easy to handle and would be more acceptable in the
field provided their efficacy was not markedly inferior to the conventional liquid
vaccines adjuvanted with mineral gel or oil.
354
T A B L E III
Neutralising antibody titres in cattle after
with freeze-dried inactivated FMD virus
Vaccine
virus
subtype
A
5/10
A
22
C
,
Asia 1/1
Postvaccination
period
(days)
vaccination
vaccines
Neutralising antibody titres (log10 SN )
50
Group
mean
Standard
deviation
0
15
30
60
90
<0.90
1.38
1.32
1.26
1.14
0.20
0.16
0.18
0.13
0
15
30
60
90
<0.90
2.70
2.78
2.61
2.53
0.21
0.21
0.17
0.24
0
15
30
60
90
<0.90
2.67
2.70
2.63
2.46
0.24
0.21
0.21
0.20
0
15
30
60
90
<0.90
2.82
2.82
2.70
. 2.52
0.22
0.22
0.18
0.22
0
15
30
60
90
<0.90
2.13
1.97
1.74
1.71
0.20
0.37
0.36
0.41
Number of
cattle tested
_
5
_
6
_
6
_
5
_
5
ACKNOWLEDGEMENTS
The authors wish to thank the Director of the Institute for encouragement and
providing facilities to carry out this work.
*
355
LYOPHILISATION DU VIRUS APHTEUX : APPLICATION A LA PRODUCTION DE
VIRUS-VACCINS INACTIVÉS. - G. Butchaiah et B.U. Rao.
Résumé : Les auteurs ont utilisé cinq sous-types différents du virus aphteux
(0¡, A / ,
A22, C1 et Asia 1/1), multipliés en culture de cellules BHK21 C13
en monocouche, pour étudier l'effet de la lyophilisation et du stockage des virus
vivants sur leur stabilité. Des virus-vaccins anti-aphteux, inactivés par l'éthylèneimine binaire et adjuvés avec la saponinepurifiée «Quil-A», sans gel d'hydroxyde
d'aluminium, ont été examinés du point de vue de leur activité, avant et après
lyophilisation et stockage, par épreuve virulente sur cobayes et évaluation des
anticorps neutralisants chez les bovins. Il a été observé que les sous-types du
virus A /10, A , C1 et Asia 1/1 conservaient leurs titres infectants au cours
de la lyophilisation, et que ces titres baissaient de façon minimale après un an
de stockage à -20°C. Comparativement, l'infectivité du virus O s'est montrée
moins stable. Pour tous les sous-types du virus, les titres antigéniques fixant
le complément sont restés inchangés dans ces conditions. Les résultats des
épreuves sur cobayes et sur bovins ont montré que les vaccins inactivés lyophilisés
des sous-types A
, A , C1 et Asia 1/1, ayant «Quil-A» pour seul adjuvant,
étaient actifs et le restaient après stockage à —20°Cpendant au moins un an.
5 10
5
22
5
5 / 1 0
22
MOTS-CLÉS : Activité - Aphthovirus - Lyophilisation - Quil-A - Stabilité Stockage - Vaccins inactivés.
*
* *
LIOFILIZACIÓN DEL VIRUS AFTOSO: APLICACIÓN EN LA PRODUCCIÓN DE
VIRUS-VACUNAS INACTIVADAS. - G. Butchaiah y B.U. Rao.
Resumen: Los autores utilizaron cinco subtipos diferentes de virus aftosos (O5,
A ,
A , C1 y Asia 1/1), multiplicados en cultivos de células BHK21 C13
en monocapa, para estudiar el efecto producido por la liofilización y el
almacenamiento de los virus vivos en su estabilidad. Se examinaron virus-vacunas
antiaftosas, inactivados por etileno-imina binario, con saponina purificada
«Quil-A» como adyuvante y sin hidróxido de aluminio, desde el punto de vista
de su actividad, antes y después de la liofilización y almacenamiento, mediante
prueba virulenta en cobayos y evaluación de anticuerpos neutralizantes en los
bovinos. Se observó que los subtipos del virus A ,
A , C1 y Asia 1/1
conservaban sus títulos infectantes durante la liofilización y que estos títulos
bajaban en grado mínimo al cabo de un año de almacenamiento a —20°C.
Comparativamente, la infecciosidad del virus O se reveló menos estable. En
todos los subtipos del virus, los títulos antigénicos que fijaban el complemento
conservaron estas condiciones intactas. Los resultados de las pruebas en cobayos
y bovinos demostraron que las vacunas inactivadas liofilizadas de los subtipos
A ,
A , C1 y Asia 1/1, con «Quil-A» como único adyuvante, eran activas
y seguían siéndolo después de un almacenamiento a -20°C de por lo menos
un año.
5/l0
22
5/10
22
s
5/10
22
PALABRAS CLAVE: Actividad - Aftovirus - Almacenamiento - Estabilidad Liofilización - Quil-A - Vacunas inactivadas.
*
* *
356
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