Rev. sci. tech. Off. int. Epiz., 1989, 8 (3), 747-764.
Effect of formaldehyde and binary ethyleneimine (BEI) on the integrity of foot and mouth disease virus capsid M.M. RWEYEMAMU, O. UMEHARA, W. GIORGI, R. MEDEIROS, D. LUCCA NETO and M. BALTAZAR *
Summary: Formaldehyde treatment of foot and mouth disease (FMD) virus caused a statistically significant (p < 0.05) reduction in the concentration of 140S particles (26-72%) for 5 of the 6 South American vaccine virus strains tested. Formaldehyde also reduced the stability of the virus; after storage at 4°C for 6 months, there was a decay of 33-85% in the concentration of 140S particles. In contrast, samples treated with BEI did not undergo a significant reduction in 140S immediately after inactivation or after storage. The efficiency of infectivity inactivation by BEI was influenced by its concentration and temperature. No significant reduction in 140S was observed at 36°C in 24 hours. KEYWORDS: Aphthovirus - Ethyleneimines - Foot and mouth disease Formaldehyde - Inactivation - Vaccines.
INTRODUCTION
It is commonly accepted that the immunogenicity of foot and mouth disease (FMD) vaccines is dependent on the presence of the intact capsid, as either the nucleocapsid with a sedimentation rate of 140S or the natural empty particle which sediments at 75S (10, 15, 20, 37, 40, 44). F o r the majority of strains, the principal immunogenic component is the 140S particle. Several studies have shown a direct relationship between the dose of 140S antigen in vaccine lots a n d the rate, height and persistence of virus neutralising antibody in cattle after b o t h primary and secondary vaccination (8, 9, 29, 30, 34, 39). Hence many modern F M D vaccine manufacturing establishments include the quantification of 140S antigen in the battery of quality control tests during production (5, 2 1 , 22, 26, 3 1 , 34, 38, 42). There are conflicting reports in the literature on the effect of commonly used virus inactivators, formaldehyde and aziridine c o m p o u n d s , on the integrity of F M D virus capsid and thereby o n immunogenicity. Formaldehyde has been largely discredited as an inactivator, mainly because of non-linear inactivation kinetics leading t o residual infective virus which, sometimes, is detectable for several weeks after completion of
* Pfizer International Inc., Laboratorios Pfizer Ltda., Rodovia Presidente Dutra Km 225, CEP 07010 Guarulhos, Sâo Paulo, Brazil.
748 the " i n a c t i v a t i o n " period of 48 hours (12, 19, 41). There have been several instances of F M D outbreaks considered to have been associated with the use of formaldehydeinactivated vaccines (7). In contrast, the aziridine c o m p o u n d s acetylethyleneimine (12), ethyleneimine (17) and binary ethyleneimine (3) have been associated with firstorder virus inactivation kinetics and have, generally, resulted in innocuous vaccines. Such compounds fulfil a recent recommendation by the O I E for inactivated F M D vaccines (33). Nevertheless, some reports in the literature have claimed that both formaldehyde (27) and aziridine compounds (36) are able to destroy the F M D virus capsid and thereby reduce vaccine immunogenicity. A t Pirbright (32, 36), a hybrid inactivation procedure involving sequential treatment of virus with formaldehyde and A E I was devised in order to preserve the 140S particle. As far as we know, the method is not widely employed by F M D vaccine manufacturers. In the present study we investigated the effect of formaldehyde and binary ethyleneimine on South American strains of F M D virus capsid when the c o m p o u n d s were used either singly or in sequence.
MATERIALS A N D METHODS Virus strains The strains of F M D V used in this study were all of South American origin: O Campos/Brasil-58, A Cruzeiro/Brasil-55, A Venceslau/Brasil-76, A Bagé/Brasil-76, A Castellano/Argentina-87 and C Indaial/Brasil-71. They had been adapted to growth in B H K suspension cells (14) for vaccine production. For this study each virus was grown in BHK suspension cells either in a spinner culture of 2 litre volume or in a 3000 litre fermenter. Virus was harvested at m a x i m u m cytopathic effect (c.p.e.), usually after 16 to 24 h. Inactivation with formaldehyde The virus suspension was adjusted to p H 8.0 with glycine buffer and a temperature of 26°C in a water-bath fitted with a magnetic stirrer. Then 1:10 formalin in distilled water was added to the virus suspension to a final concentration of 0.06% v / v formalin. Periodically the virus-containing flask was manually inverted in order to bring virus in the air space above the liquid level (less than 10% of total flask capacity) into contact with inactivator. Inactivation proceeded for 48 h under magnetic agitation. Inactivation with BEI The procedure adopted was similar to that described by Bahnemann (3). BEI was generated by treating 0.1M 2-bromoethylamine hydrobromide with 0.2N N a O H for 1 hour at 37°C. This solution was then added to the virus suspension to give the desired concentration of BEI, i.e. 1% v / v for 1mM BEI; 2 % v / v for 2 m M BEI, etc. Periodically, the virus-containing bottle was inverted in order to inactivate virus in the air space above the liquid level. Temperature and time of inactivation were as specified under " R e s u l t s " . Excess BEI was neutralised at the end of the inactivation period with 0 . 1 % w / v sodium thiosulphate.
749 Inactivation kinetics and innocuity testing For measuring inactivation kinetics, samples were collected at hourly intervals up to 4 h after adding inactivator. The inactivator was neutralised and samples titrated for surviving virus infectivity by using the microcolour test (28). The kinetics constant, slope and half-life were calculated. At 24 and 48 hours after commencement of inactivation, samples were collected for innocuity testing. After neutralisation, each sample was inoculated onto duplicate Roux flasks of BHK monolayers maintained on a serum-free medium. The cultures were examined for c.p.e. and complement fixing antigen over three serial passages (2, 16). Quantification of 140S Test virus samples were first concentrated tenfold by using saturated a m m o n i u m sulphate either at 4 ° C overnight or at r o o m temperature (ca. 25°C) for two hours, and resuspended in 0.04M phosphate buffer. Then the concentration of the 140S particles was assayed by sucrose density gradient using the methods of Barteling and Meloen (5) and Doel et al. (21). Briefly, the sample was treated with 0 . 0 0 1 % (w/v) bovine ribonuclease at 36°C for 10 min to degrade any free R N A or ribosome, and then 1 ml of this was layered onto a preformed, linear 10-30% (w/v) sucrose density gradient. The gradients were centrifuged at 40,000 r p m using the Beckmann SW 41 Ti rotor in the Beckmann L8-55M ultracentrifuge for 60 min. Then the gradients were scanned at 259 n m wavelength using the Beckmann DU-7U spectrophotometer fitted with a continuous 0.2 cm pathlength flow-cell. The area under the peak was calculated and used to determine the concentration of 140S particles using the following formula: 140S µ g / m l = where
FR PA FSD S PL E W
= = = = = = =
FRxPAxFSDxlOOO S x P L x E x W x 10
flow rate in m l / m i n area under the peak in c m full scale absorbence optical density unit setting speed of the chart recorder in c m / m i n pathlength of the flow cell in cm extinction point for F M D V , i.e. 78.8 at 259 nm sample volume (ml) applied to gradient 2
(N.B. value divided by 10 to allow for the concentration factor) Under our conditions the method gives a coefficient of variation of 12% for intertest and 5 % for intra-test variation. Statistical analysis The statistical analysis of data was carried out using the R S / 1 programme of BBN Software Products Corporation (Cambridge, USA) operating in the D O S m o d e of IBM on the Microtec X T 2002 microcomputer. The level of rejection of the null hypothesis was set at 0.05.
750 RESULTS Formaldehyde versus BEI on F M D V 140S particle Two series of experiments were carried out. In the first the virus harvest was divided into four lots: one lot was inactivated with formaldehyde (0.06% v / v formalin); the second was inactivated with two doses of 1mM BEI 24 hours apart; the third was treated for the first 24 hours with formaldehyde and then, additionally, with 1mM BEI; the fourth lot was left as a live virus control. The p H of all 4 lots was adjusted to p H 8.0 and they were all incubated under agitation in the same water-bath at 26°C for a total of 48 hours. For each virus strain 4-6 replicate tests were thus carried out. The results of this set of experiments are summarised in Table I a-d and Figure 1, from which it is evident that the reduction in 140S level due to BEI treatment was less than 2 5 % and statistically insignificant for any of the tested strains. In contrast, formaldehyde, either alone or in combination with BEI, reduced 140S by up to 7 2 % . This reduction was evidently primarily due to the action of formaldehyde. By one way analysis of variance the reduction in 140S level caused by formaldehyde was shown to be significant for strains O Campos and A Cruzeiro (p < 0.05). TABLE
la
Effect of inactivators on concentration of 140S antigen (µg/ml) in vaccine virus harvests of strain O Campos Lot no.
Live virus
Formalin xl/26°C/48h
Formalin/BEI 26°C/48h
BEI ImM x2/26°C/48h
1 2 3 4 5
0.900 1.400 0.950 2.280 1.860
0.510 0.830 0.310 1.180 0.880
0.630 0.590 0.860 0.860 1.130
0.860 1.180 0.940 1.860 1.830
Mean STDEV SEM
1.478 0.593 0.265
0.742 0.339 0.152
0.814 0.217 0.970
1.334 0.481 0.215
Anova oneway: p = 0.036 TABLE
lb
Effect of inactivators on concentration of 140S antigen (ng/ml) in vaccine virus harvests of strain A Cruzeiro Lot no.
Live virus
Formalin x l/26°C/48h
Formalin/BEI 26°C/48h
BEI ImM x 2/26°C/48h
1 2 3 4 5 6
0.330 0.400 0.610 0.570 0.570 0.760
0.010 0.070 0.210 0.010 0.010 0.250
0.010 0.010 0.050 0.070 0.070 0.500
0.430 0.330 0.720 0.520 0.480 0.410
Mean STDEV SEM
0.540 0.154 0.063
0.093 0.109 0.045
0.118 0.189 0.077
0.482 0.133 0.054
Anova oneway: p = 0.001
751 TABLE
le
Effect of inactivators on concentration of 140S antigen (¡xg/ml) in vaccine virus harvests of strain A Venceslau Live virus
Formalin x l/26°C/48h
Formalin/BEI 26°C/48h
BEI ImM X 2/26°C/48h
1 2 3 4
0.240 0.700 0.300 0.990
0.110 0.170 0.010 0.200
0.070 0.140 0.140 0.500
0.210 0.400 0.260 0.860
Mean STDEV SEM
0.558 0.530 0.177
0.122 0.084 0.042
0.212 0.194 0.097
0.432 0.296 0.148
Lot no.
Anova oneway: p = 0.112
TABLE
Id
Effect of inactivators on concentration of 140S antigen (¡jLg/ml) in vaccine virus harvests of strain C Indaial Live virus
Formalin x l/26°C/48h
Formalin/BEI 26°C/48h
BEI ImM x 2/26°C/48h
1 2 3 4
1.330 1.500 0.750 1.400
0.950 0.470 0.710 1.200
0.710 0.720 0.650 1.170
1.000 1.030 0.710 1.380
Mean STDEV SEM
1.245 0.337 0.169
0.832 0.314 0.157
0.812 0.240 0.120
1.030 0.274 0.137
Lot no.
Anova oneway: p = 0.185
In the second set of experiments a direct comparison was m a d e of formaldehyde plus BEI treatment versus BEI alone. For each strain 8-10 replicate sets of inactivations were carried out. The results are summarised in Table II and Figure 2. The statistical analysis hypothesis investigated was whether the residual 140S concentration was significantly lower after formaldehyde than after BEI treatment, as the first set of experiments had already indicated a destructive effect for formaldehyde. Therefore, a one-tailed t-test was applied to the data. It was demonstrated for five of the six South American virus strains tested that formaldehyde significantly reduced the concentration of 140S particles. Effect of formaldehyde on stability of stored virus Pairs of eight lots of harvested virus inactivated either with BEI alone ( I m M x 2 for 48 hours) or formaldehyde plus BEI (as described above) were stored at 3-6°C for six m o n t h s . They were assayed for 140S content at the beginning and end of the storage period.
1
Effect of inactivators on concentration of 140S antigen (%) in FMD vaccine virus harvests
FIG.
752
NO 00 OO
o
NO NO
ON
as
BEI A-Bagé
un o
NO
un
ON
CN CN
un O
O
en en un un oo en CN en
o
r-
o
o
o
NO NO ON
o o
O
O o r— o
O
o o un
en vo
010'
o o
O
o o
O
o
o
o
O
o
o
en en CN CN oo en r-; o O o "°. O Ö O 010'
o o un t O o
o ro
o o Ö o o
1.120
o
NO
en en
p-
eN en NO ON CN o
d d d CN
en oo en r» en CN o NO
ON
o d d
d d d
rNO o
m CN ten un O
o o o
d d d
010
010'
o
o o o ND NO oo ON CN OO o en
en
oo
d S*
1.587 oo
oo
ON
m
CN o NO o en O 'O
O
O
CN
o
BEI A-Venc
o
r-
CN
CN
Formal. A-Venc
CN un r- NO O o
O
1.508
1.380
0.710
1.000
1.030
~
m
oo
1.359
o
NO OO
^ d d
0.388
Formal. A-Bagé
o r - m oo un un oo un r— en ON o
ON
d d
O NO O NO r— O
00 C*N
o O O CN
BEI A-Cruz
ON
rt
Un CN o
o
un (--* un
NO
o ^
o
Formal. A-Cruz
en un S en CN d
NO
•sl- en r— en en oo NO
NO ON
oo
as
as
BEI C-Inda rmal. Inda
OO
o o o
010'
en
un un CN un un O
O
d d d
oo ON
o
o o o un o O o
ON
ON
CN
CN ©
CN r-
NO
o
d d d
O
oo
Os
o NO OO
O
o
o
o
oo
NO
en
O NO
ON ON
CN O
^
un
un
ON
CN
o
o
OO 1
" —'
—
oo oo en r-
r--
CN o
o
o
o
O
o
ON
o
SEM
o
Mean
o NO 00
STDEV
BEI O-Cam
a
• a
Formal O-Cam
Effect of formaldehyde and BEI on FMDV 140S concentration
oo
Os
O)
o
en e
o
en
O
o
ON
o
NO OO O
CN
C*N
NO
un
O
o NO oo
O CN en un un o
o
un
OO
NO
r-
oo
o
o
NO
r— oo
o
r-
NO
en
NO
One-tailed t-test. p values: O-Camp = 0.0005; A-Cruz = 0.0011; A-Venc = 0.0275; C-Inda = 0.0334; A-Bagé = 0.3410; A-Cast = 0.018
H
^
z<
TABLE II
oo
O t - en en o en ON r- o so CN ON o rCN CN CN CN ^
0.148
o r-
0.417
•- t— Un
rmal. •Cast
BEI A-Cast
753
(O/u)
UOIJBJJIISOUCO S u r i 2
Effect of formaldehyde and BEI on FMDV 140S concentration
FIG.
754
755 The results obtained are summarised in Table III. For BEI inactivated samples, there was no significant d r o p in the 140S level over the study period. However, the formaldehyde-treated samples had shown a 33-85% reduction in 140S level; this decay was shown to be statistically significant by the one-tailed t-test (p < 0.05).
TABLE
Effect of BEI or formalin Virus lot no. O-504 O-505 O-506 A-V-260 A-V-263 C-IN-291 C-IN-293 A-Cr-146
III
on stability
of FMDV
140S
BEI-start µg/ml)
BEI 6 months (µg/ml)
Percent residua]
Formalinstart (µg/ml)
Formalin 6 months (µg/ml)
Percent residual
1.670 2.119 0.941 0.394 0.192 1.508 1.587 0.348
1.839 1.925 1.150 0.449 0.417 1.744 1.593 0.362
100.00 90.84 100.00 100.00 100.00 100.00 100.00 100.00
0.705 0.852 0.461 0.163 0.067 0.966 1.008 0.249
0.286 0.406 0.308 0.070 0.010 0.354 0.659 0.100
40.57 47.65 66.81 42.94 14.93 36.65 65.38 40.16
Values > 100% recorded as 100% One-tailed t-test: stored BEI-treated samples n.s. loss (p = 0.599); formald. samples decay significant (p = 0.0410)
Effect of temperature of BEI inactivation on 140S particles FMDV inactivation with BEI is usually carried out at 35-37°C with a concentration of 1.0-1.5mM applied either once for 24 hours or repeated for an additional 24 hour period (i.e. total 48 h). A recent report by Bahnemann et al. (4) implied that the virus breaks down under these conditions, and these authors adopted a higher dose of BEI (3mM) at 26°C for only 24 h. Two sets of experiments were carried out to investigate this. In the first set, for each virus strain, 5-15 lots of harvest were inactivated in parallel either by one dose of 3mM BEI at 26°C for 48 hours or by 2 doses each of 1mM BEI at 36°C with the second dose after the initial 24 hours. Results of 140S assay are shown in Table IV and Figure 3 from which it can be deduced that no significant difference was detected between the lower BEI dose at 36°C and the higher BEI dose at 26°C (p > 0.3). In the second set of experiments comparison was made of 3mM BEI at 26°C with 3mM at 36°C for a total period of 48 h. In each case a single dose was applied. Each virus strain was thus tested in ten replicates with different virus harvests. Results are summarised in Table V. There was no significant difference in the level of 140S detected after inactivation at either temperature (p > 0.7), although in general there was a tendency for slightly lower values at 36°C after 48 hours than at 2 6 ° C . Efficiency of inactivation of virus infectivity by different BEI procedures BEI was tested in concentrations of l-3mM at 26°C or 36°C over 48-hour periods. Samples for inactivation kinetics titration were collected at times 0, 1, 2, 3, 4 hours
One tailed t-test p values for O-Camp = 0.3441; A-Cruz = 0.3302; A-Venc = 0.6817; C-Inda = 0.3800
Effect of BEI dose and temperature on concentration of FMDV 140S
TABLE IV
(µg/ml)
756
757
FIG.
3
Effect of temperature and BEI dose on F M D V 140S concentration
c/3
n
m
NO
OO
ON
0.209
SEM
1.351
1.297
0.395
0.071
0.225
0.391
0.064
0.203 0.069
0.218
0.372
0.746
0.463 0.146
0.147
0.718 0.465
0.760
1.370
0.134
0.422
0.685
1.221
0.098
0.044
0.193
0.610
1.195
1.007
2.113
0.180
0.570
1.131
1.120
0.188
1.412
1.552
1.147
0.919
0.261
1.319
1.799
1.821
1.562
0.159
>o
0.180
0.569
0.684
0.226
1.182
0.992 0.071
1.351
0.558
1.149
1.344
1.904
1.836
1.282 0.706
o
0.175
0.554
0.723
0.346
0.664
0.061
1.648 0.745
1.977
0.673
0.589 0.552
0.781 0.681
1.296 0.615
0.833
0.965
o
Anova oneway p values for O-Camp = 0.728; A-Cruz = 0.966; A-Vences = 0.932; C-Inda = 0.966
1.503
2
0.660
1.934
0.325
0.424 0.288
1.412 0.792
1.127
1.218
0.775 0.613
0.777 0.645
0.016
0.034
0.010 0.944
0.311
0.475
0.778
0.655
m
STDEV
2.010
0.427
0.635
0.056
0.253
0.468
0.317
0.512
0.450
0.537
0.645 0.704
0.304 0.099
Vences. Vences. Indaial Indaial Indaial 36°C/24h 36°C/48h 26°C/48h 36°C/24h 36°C/48h
OO OO
Mean
2.000
2.316
0.690
0.098
0.795
1.644
0.265
0.328
0.413
0.506
0.499 0.164
q
2.263
0.469
0.034
0.638
1.458
0.820
0.835
1.578
0.954
Vences. 26°C/48h
oo
2.694
1.755
0.834
1.701
1.107
1.590
2.226
0.592
0.968
1.055
0.582
0.903
0.840
1.102
1.495
1.299
Campos Campos Cruzeiro Cruzeiro Cruzeiro 36°C/24h 36°C/48h 26°C/48h 36°C/24h 36°C/48h
1.528
Campos 26°C/48h
Effect of temperature of FMD V inactivation with 3mM BEI on 140S (µg/ml)
TABLE V
758
759 and for innocuity testing in B H K monolayer cells at 24 and 48 h o u r s . The results are summarised in Tables VI and VII.
TABLE V I
Inactivation kinetics of FMD V by BEI Mean half-life in minutes Virus strain 0 Campos A Venceslau A Cruzeiro C Indaial Overall mean STDEV N
lmM/26°C
2mM/26°C
3mM/26°C
lmM/36°C
2mM/36°C
70.59 71.79 83.24 185.94 107.24 72.60 13.00
39.87 30.20 77.59 53.39 44.90 22.41 17.00
33.79 34.27 119.00 34.93 51.09 43.61 15.00
36.55 35.28 75.62 28.18 41.17 17.36 13.00
16.33 23.60 24.61 13.30 18.31 4.98 10.00
Overall mean and standard deviation refer to total n observations
TABLE V I I
Innocuity Virus strain O A A C
Campos Venceslau Cruzeiro Indaial
Total Percent
lmMx2 26°C/48h
results of FMD V inactivation (Positive/total)
2mM/26°C 3mM/26°C 3mM/26°C 48h 24h 48h
by BEI lmMx2 36°C/48h
2mM/36°C 24h
1/6 1/13 0/2 1/4
2/6 1/6 0/2 0/4
3/11 3/7 1/6 0/4
0/34 2/23 0/17 0/12
3/19 0/2 0/9 0/7
0/25 0/19 0/13 0/10
3/23 13
3/18 16
7/28 25
2/86 2
3/37 8
0/67 0
Positive = virus detected
When BEI inactivation was performed at 26°C, regardless of concentration, or at a concentration of 1mM regardless of temperature, there-was a risk of incomplete inactivation of virus infectivity. Procedures at 36°C with BEI doses of 2 m M or above were found to be safe in terms of F M D V innocuity.
DISCUSSION Aziridine c o m p o u n d s are efficient inactivators of F M D virus for vaccine manufacture (24, 35). However, workers at Pirbright demonstrated that certain strains of serotype SAT 2 were particularly labile to acetylethyleneimine, and that this destructive effect of A E I could be overcome by pre-fixing the virus with formaldehyde
760 (32, 36). It seemed possible, therefore, that the stability of F M D V vaccines would be improved by always treating virus harvests with formaldehyde before complete inactivation with an aziridine c o m p o u n d , such as BEI. However, several studies had indicated that formaldehyde itself might be deleterious to some strains of F M D V . For example, Wild and Brown (44) observed that although formaldehyde-inactivated F M D virus sedimented at 140S, its immunising activity for guinea pigs was much lower t h a n the A E I inactivated preparation. Girard et al. (27) found that by using BEI, instead of formalin, to inactivate a strain of type O virus from Turkey, there was a marked improvement in the potency for cattle of the corresponding vaccine. Adamowicz et al. (1) demonstrated that formaldehyde degraded the 140S particles of types O and C virus but not type A . In a more recent study, Ferris et al. (25) found that formaldehyde had a variable effect on 140S particles, depending on virus strain: it stabilised a strain of serotype SAT 2, had little influence on the 140S of some strains, produced a reduction of 140S for some and totally degraded other strains (e.g. A France 1/68). In several cases these authors also observed that pretreatment of virus with formaldehyde resulted in antigens of reduced immunogenicity for guinea pigs. Most of these studies, however, were based on a limited number of observations and often there was no statistical analysis of the observed effect. In the present study, an attempt was m a d e to obtain data from statistically analysable sets of replicates. By so doing, we have shown that 5 of 6 South American strains of FMDV, of serotypes O , A , C, suffered a significant reduction in 140S compared with intact virus or virus inactivated with BEI. The exception was strain A Bagé, which was not significantly susceptible to the action of formaldehyde. Further, we have shown that upon storage at 4 ° C , formaldehyde-treated 140S particles degrade significantly over six m o n t h s in contrast to BEI-treated virus. Ferris et al. (25) found that only strain C Resende decayed upon storage at 4 ° C . But in agreement with our results is the work of Czelleng et al. (18) who found that of the virus strains studied, there was only 3-30% residual complement fixing activity after a storage period of five weeks. Therefore, it can be concluded that far from being a general stabilising agent, formaldehyde can often degrade the immunising antigen (140S particles) of F M D V . These observations would seem t o be at variance with the general experience by users of formalin who claim high potency for formaldehyde-inactivated antigen. The apparent anomaly is probably explicable on the following grounds. Firstly, such claims m a y express sentiment more t h a n experimental evidence. Secondly, because of the curvilinear inactivation kinetics by formaldehyde, it is quite probable that some residual live virus in the vaccine could contribute to the potency of antigens inactivated only with formaldehyde. Thirdly, it can be argued that adsorption of virus onto aluminium hydroxide before inactivation might preserve the virus capsid from the destructive effects of formaldehyde. However, in their re-evaluation of F M D inactivation by formaldehyde, Barteling and Woortmeyer (6) did not confirm this preserving effect of aluminium hydroxide. It should be noted that with adsorbed antigen, it is difficult to quantify effects on 140S owing to the inefficiency of quantitative elution of virus from aluminium hydroxide (23, 4 1 , 43). A further explanation of why the deleterious effects of formaldehyde on virus capsid may not become readily apparent in routine potency tests, lies in the fact that the relationship between antigen dose and antibody response for F M D vaccines is sigmoid rather than linear (9, 39). The effect of changes in antigen content can be masked by responses being in the upper plateau of the sigmoid curve. Finally our studies with BEI have shown that concentration (l-3mM) and temperature of virus inactivation (26°C or 36°C) are important for complete
761 inactivation of infectivity, but probably not as critical as indicated by the observation of Bahnemann (4) in terms of 140S particle integrity. We have not been able to confirm his observation of thermal lability of South American F M D V strains at 36°C within 24 h o u r s . Our results agree with those of Brown and others (11, 13) who showed that at 37°C F M D virus loses infectivity on account of in situ R N A degeneration, but without breakdown of the capsid into sub-units. It is probable that culture constituents or environmental conditions, other t h a n temperature, or the quality of the primary chemical, bromoethylamine, or the efficiency of its conversion to BEI may be more critical in the effect of inactivation on the 140S particle t h a n simply temperature (26°C or 36°C) per se. It is important, therefore, that these parameters be standardised within the production environment of a vaccine manufacturer.
* * *
EFFET DU FORMOL ET DE L'ÉTHYLÈNE-IMINE BINAIRE (EIB) SUR L'INTÉGRITÉ DE LA CAPSIDE DU VIRUS APHTEUX. - M.M. Rweyemamu, O. Umehara, W. Giorgi, R. Medeiros, D. Lucca Neto et M. Baltazar. Résumé : Le traitement du virus aphteux par le formol a provoqué une réduction statistiquement significative (p < 0,05) de la concentration des particules 140S (26 à 72%) chez cinq des six souches sud-américaines de virus vaccinal testées. Le formol a également réduit la stabilité du virus ; après stockage à 4°C pendant six mois, la concentration des particules 140S a baissé de 33 à 85%. En revanche, pour les échantillons traités par l'éthylène-imine binaire (EIB), il n'y a pas eu de réduction significative des particules 140S, ni immédiatement après l'inactivation ni après stockage. L'efficacité de l'inactivation du pouvoir infectant par l'EIB a été influencée par la concentration du produit et par la température. Aucune réduction significative des 140S n'a été observée en 24 heures à 36°C. MOTS-CLÉS : Aphthovirus - Ethylène-imines - Fièvre aphteuse - Formol Inactivation - Vaccins. * * *
EFECTO DEL FORMOL Y DEL ETILENOIMINO BINARIO (EIB) EN LA INTEGRIDAD DE LA CAPSIDA DEL VIRUS AFTOSO. - M.M. Rweyemamu, O. Umehara, W. Giorgi, R. Medeiros, D. Lucca Neto y M. Baltazar. Resumen: El tratamiento del virus aftoso con formol ha provocado una reducción estadísticamente significativa (p < 0,05) de la concentración de partículas 140S (del 26 al 72%) en cinco de las seis cepas sudamericanas de virus vacunal probadas. El formol también redujo la estabilidad del virus. Así, al cabo de 6 meses de almacenamiento a 4 °C, la concentración de las partículas 140S había disminuido entre un 33 y un 85%. En cambio, para las muestras tratadas con etilenoimino binario (EIB), no hubo disminución significativa de las partículas 140S ni inmediatamente después de la inactivación ni después del almacenamiento. La eficacia de la inactivación del poder infectante por el EIB fue influenciada por la concentración del producto y por la temperatura. No se observó ninguna reducción significativa de las 140S en 24 horas a 36°C.
762 PALABRAS CLAVE: Aftovirus - Etilenoiminos - Fiebre aftosa - Formol Inactivación - Vacunas.
* * * REFERENCES 1.
2. 3. 4.
5.
6.
7. 8. 9.
10. 11.
12.
13. 14.
15. 16.
J., G A L A M P O I X J. & P R U N E T P. (1977). - Contrôles physicochimiques de routine d'antigènes aphteux concentrés virulents et inactivés. Develop. biol. Standard., 35, 301-310. A N D E R S O N E.C., C A P S T I C K P.B., M O W A T G . N . & L E E C H F.B. (1970). - In vitro method for safety testing of foot and mouth disease vaccines. J. Hyg., Camb., 68, 159-172. B A H N E M A N N H.G. (1975). - Binary ethylenimine as an inactivant for foot-and-mouth disease virus and its application for vaccine production. Arch. Virol., 47, 47-56. B A H N E M A N N H.G., M E S Q U I T A J., A S T U D I L L O V. & D O R A F. (1987). - The production and application of an oil adjuvant vaccine against foot and mouth disease in cattle. In Modern approaches to animal cell technology (R.E. Spier & J.B. Griffiths, eds.). Butterworths, Sevenoaks, England, 628-640. B A R T E L I N G S.J. & M E L O E N R.H. (1974). - A simple method for the quantification of 140S particles of foot and mouth disease virus (FMDV). Arch. ges. Virusforsch., 45, 362-364. B A R T E L I N G S.J. & W O O R T M E Y E R R. (1984). - Formaldehyde inactivation of foot-andmouth disease virus. Conditions for the preparation of safe vaccine. Arch. Virol., 80, 103-117. B E C K E. & S T R O H M A I E R K. (1987). - Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. J. Virol., 61, 1621-1629. B L A C K L . , R W E Y E M A M U M.M. & B O G E A. (1984). - Revaccination response of cattle as a function of the 140S FMD antigen dose. J. comp. Path., 94, 417-424. B L A C K L . , N I C H O L L S M.J., R W E Y E M A M U M.M., F E R R A R I R. & Z U N I N O M.A. (1986). Foot-and-mouth disease vaccination: a multifactorial study of the influence of antigen dose and potentially competitive immunogens on the response of cattle of different ages. Res. vet. Sci., 40, 303-307. B R O W N F. (1972). — Structure-function relationships in foot-and-mouth disease virus. In Immunity in viral and rickettsial diseases (A. Kohn & M.A. Klingberg, eds.), 19-28. B R O W N F., C A R T W R I G H T B. & S T E W A R T D . L . (1963). - The effect of various inactivating agents on the viral and ribonucleic acid infectivities of foot-and-mouth disease virus and on its attachment to susceptible cells. J. gen. Microbiol., 31, 179-186. B R O W N F., H Y S L O P N . S T . G . , C R I C K J. & M O R R O W A.W. (1963). - The use of acetylethyleneimine in the production of inactivated foot-and-mouth disease vaccines. J. Hyg., Camb., 61, 337-344. B R O W N F. & W I L D T.F. (1966). - The effect of heat on the structure of foot-and-mouth disease virus and the viral ribonucleic acid. Biochim. Biophys. Acta, 119, 301-308. C A P S T I C K P.B., G A R L A N D A.J.M., C H A P M A N W . G . & M A S T E R S R.C. (1967). - Factors affecting the production of foot-and-mouth disease virus in deep suspension cultures of BHK 21 clone 13 cells. J. Hyg., Camb., 65, 273-280. C O W A N K.M. (1973). - Antibody response to viral antigens. Adv. Immunol., 17, 195-253. CPFA (1980). - Manual de procedimientos para el control de vacuna antiaftosa. Serie de manuales técnicos N ° 2, Centro Panamericano de Fiebre Aftosa (CPFA), Rio de Janeiro, Brazil. ADAMOWICZ P H . , GUERCHE
763 17.
CuNLiFFE H.R. (1973). - Inactivation of foot-and-mouth disease virus with ethylenimine. Appl. Microbiol., 26, 747-750.
18. C Z E L L E N G F . , F A R K A S L., P E R E N Y I T T., F A Z E K A S A . , S Z A L A I F . , Z S I T V A I K . & E R D E L Y I
K. (1978). - The effect of ethyleneimine and formaldehyde inactivation on the immunogenicity of foot-and-mouth disease virus. XVth Conf. of the OIE Commission on Foot and Mouth Disease, Report No. 503, OIE, Paris. 19.
D O E L T.R. (1985). - Inactivation of viruses produced in animal cell cultures. In Animal cell biotechnology, vol. 2 (R.E. Spier & J . B . Griffiths, eds.). Academic Press, London, 129-149.
20.
DOEL
T.R. & C H O N G W.K.T. (1982). - Comparative immunogenicity of 146S, 75S and 12S particles of foot-and-mouth disease virus. Arch. Virol., 73, 185-191.
21.
D O E L T.R., F L E T T O N B.W. & S T A P L E R . F . (1981). - Further developments in the quantification of small RNA viruses by U.V. photometry of sucrose density gradients. Develop, biol. Standard., 50, 209-219. 22. D O E L T.R. & M O W A T G . N . (1985). - An international collaborative study on foot-andmouth disease virus assay methods. 2. Quantification of 146S particles. J. biol. Standard., 13, 335-344.
23.
D O E L T.R. & S T A P L E R . F . (1981). - Elution of foot-and-mouth disease virus from aluminium hydroxide vaccines. Rep. Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease. FAO, Rome, 34-42.
24. F E R N A N D E Z A.A., C A S A S O L A S C O A G A R., BAHNEMANN H.G., ASTUDILLO S O N D A H L M . S . , G O M E S I., B A L T A R J . & F E R N A N D E Z G . (1985). - Production and
V.M.,
quality control of foot and mouth disease vaccines in South America. Rep. Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease. FAO, Rome, 50-55.
25.
F E R R I S N . P . , D O N A L D S O N A.I., B A R N E T T I.T.R. & OSBORNE R.W. (1984). - Inactivation, purification and stability of 146S antigens of foot and mouth disease virus for use as reagents in the complement fixation test. Rev. sci. tech. Off. int. Epiz., 3 (2), 339-350.
26.
G A R L A N D A . J . M . , M O W A T G.N. & F L E T T O N B . (1977). - An evaluation of some methods of assay of foot-and-mouth disease antigen for vaccines. Develop. biol. Standard., 35, 323-332.
27.
GIRARD H.C., BAYRAMOGLU
29.
MORGAN
30.
MOWAT
32.
MOWAT
O., E R O L N. & B U R G U T A. (1977). - Inactivation of O1 FMD virus by the binary ethylene imine (BEI). Bull. Off int. Epiz., 87, 201-217. 28. M A R T I N W . B . & C H A P M A N W.G. (1961). - The tissue culture colour test for assaying the virus and neutralizing antibody of foot-and-mouth disease and its application to the measurement of immunity in cattle. Res. vet. Sci., 2, 53-61. D . O . , M C K E R C H E R P . D . & B A C H R A C H H.L. (1970). - Quantification of the antigenicity and immunogenicity of purified foot-and-mouth disease virus vaccine for swine and steers. Appl. Microbiol., 20, 770-774.
G.N. (1972). - Quantities of purified antigen required to immunize swine against foot-and-mouth disease. Bull. Off. int. Epiz., 77, 887-897. 31. M O W A T G.N. & D O E L T.R. (1982). - Improvements in methods for the assessment of antigens for FMD vaccines. Proc. 16th Conf. of the OIE Foot and Mouth Disease Commission. OIE, Paris, Vol. I, 127-138. G.N., M A S T E R S R.C. & P R I N C E M . J . (1973). - Enhancement of immunising potency of a foot-and-mouth disease vaccine for cattle by treatment of the antigen with formaldehyde. Arch. ges. Virusforsch., 41, 365-370.
33. OIE (1986). - Final Report and Recommendation No. 18. Proc. 17th Conf. of the OIE Foot and Mouth Disease Commission. OIE, Paris, 400.
764 34.
P A Y T . W . F . & H I N G L E Y P . (1987). - Correlation of 140S antigen dose with the serum neutralizing antibody response and the level of protection induced in cattle by foot-andmouth disease vaccines. Vaccine, 5, 60-64.
35.
T . W . F . , T E L L I N G R . C . & T H O R N E A.C. (1981). - Inactivation of FMD virus with aziridines. Rep. Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease. FAO, Rome, 55-57.
36.
R O W L A N D S D.J., S A N G A R D.V. & B R O W N F. (1972). - Stabilizing the immunizing antigen of foot-and-mouth disease virus by fixation with formaldehyde. Arch. ges. Virusforsch., 39, 274-283.
PAY
37.
R O W L A N D S D.J., S A N G A R D . V . & B R O W N F. (1975). - A comparative chemical and serological study of the full and empty particles of foot-and-mouth disease virus. J. gen. Virol., 26, 227-238. 38. R W E Y E M A M U M . M . (1982). - Developments in the biochemical and immunoassay assessment of foot-and-mouth disease vaccine antigens. First Int. Conf. on the Impact of Viral Diseases on the Development of Latin American Countries and the Caribbean Region. Rio de Janeiro, Brazil, Vol. 1, 437-450.
39.
R W E Y E M A M U M . M . , B L A C K L., B O G E A., T H O R N E A.C. & T E R R Y G . M . (1984). - The relationship between 140S antigen dose in aqueous foot-and-mouth disease vaccines and the serum antibody response of cattle. J. biol. Standard., 12, 111-120.
40.
RWEYEMAMU M . M . , TERRY G.M. & P A Y
42.
T E L L I N G R.C. (1975). - Industrial production of FMD vaccine using BHK 21 suspension cells. Some comparative results relating in vitro assays and cattle potency. Rep. Meeting of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease. FAO, Rome, 95-100.
43.
VISSER N. & W O O R T M E Y E R R. (1982). - Quantification, characterization and safety testing of foot-and-mouth disease virus antigens eluted from alhydrogel vaccines. Develop. biol. Standard., 50, 277-283.
44.
W I L D T.F. & B R O W N F. (1968). - A study of the physical properties of the immunizing antigen of foot-and-mouth disease virus and the effect of various inactivating agents on its structure. Arch. ges. Virusforsch., 24, 86-103.
T . W . F . (1979). - Stability and immunogenicity of empty particles of foot-and-mouth disease virus. Arch. Virol., 59, 69-79. 41. S I M O N E F. D E , B U G N E T T I M., P A N I N A G.F., B A R E I S. & M E L L A N O D. (1981). Quantification and safety of FMD virus antigen eluted from vaccine. Rep. Session of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease. FAO, Rome, 30-33.
