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Assessment of an end-point microassay for yellow fever vaccine
Biologic&
(1990) 18, 25-27
Assessment
* lnstitut d’Hygi&ne
of an End-point Microassay Fever Vaccine Jocelyne Husson van Vliet* et d-Epidkmiologie, Departement Microbiologic, 74-7 050 Brussels, Belgium
for Yellow
rue J. Wytsman,
Abstract. A statistical analysis was performed on the results obtained in titrations of yellow fever vaccine using a cytopathic end-point microassay in the Vero cell line. The present test system appeared less sensitive than the conventional plaque assay in PS cells but the consistency in titres was satisfactory since both test-to-test and within-test variations were small. In addition, the test is easy to perform, economical and fast.
Introduction The methods for determining the infectious titre of yellow fever vaccines are either the LDSo mouse assay or the plaque counting assay in cell cultures, as recommended in the current WHO requirements.’ The latter technique is adapted from the microplaque procedure in porcine kidney cells PS described for arboviruses by De Madrid and Porterfleld.2 The purpose of this paper is to evaluate the reliability of the cytopathic effect (cpe) end-point microtechnique in order to ascertain if the procedure is suitable for routine control purposes. From the two recommended cell-substrates (either Vero or PS cells), we choose the Vero line, which is already used in the control laboratory for many other routine tests, is easy to grow and has low sensitivity to adverse environmental conditions. Material
and methods
Cell cultures African green monkey kidney cells Vero3 were used at passage levels 165-175. The cells were grown in Eagle’s Minimum Essential Medium (MEM) supplemented with 5% heat-inactivated fetal calf serum (FCS) and 50 pg/ml gentamycin. The same medium with only 2% FCS was used for maintenance. Vaccine preparation A single batch of freeze-dried commercial yellow fever vaccine (attenuated 17D strain of yellow fever virus) was used in all the tests. This preparation (stored at -70°C) has been used for more than 10 1045-1056/90/010025+03
$03.00/O
years in our laboratory as an in-house reference, the titre of which has been estimated throughout 60 routine tests in PS cells by the usual plaque assay: 4.55 log plaque-forming units (pfu) per dose as general mean (N = 60) with a variation coefficient of 6.3%. The virus preparation, in vials containing five doses, was rehydrated and diluted with chilled dilution medium (MEM containing 1% FCS and 50 pg/ml gentamycin). The dilutions were distributed immediately after preparation. The cpe microassay In brief, the tests were performed in plastic microtitre plates (Nunc, 96 flat-bottomed wells) sealed with pressure-sensitive film (Falcon). The vaccine inoculum (200 ,ol of serial lo-fold dilutions of vaccine) and Vero cells (75 ~1 of a 200 000 cells/ml suspension) were added consecutively using 10 wells per dilution. The microplates were incubated at 36°C and stained on the seventh day (10% form01 saline as fixative and carbol fuchsin as staining solution). They were examined macroscopically on an illuminated viewing box for recording infection as an all-or-none effect. The titres-expressed in log CCID50/dosP and their standard error were estimated using Karber’s formulae.4
Results Assays were performed on 25 occasions. Five parallel titrations were done on each occasion, using replicate @ 1990 The International
Association of Biological Standardization
26
J. Husson van Vliet
series of dilutions starting from one the vaccine preparation and using a preparation. The titres obtained are Table 1. Variance analysis of titres obtained for preparation is summarized in Table 2.
sample of single cell shown in
Test 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Mean
4.02 4.10 4.38 4.68 4.12 4.52 4.74 4.66 5.16 4.20 4.32 4.34 4.18 4.46 4.70 4.68 4.76 4.86 4.36 4.42 4.38 4.20 4.18 4.30 4.92 4.47
(3.90420) (3*90430) (4.00-4.50) (4.50-4.80) (3.90440) (4.40-4.70) (4.60490) (4.40-4.90) (4.90-5-40) (4.00-4.40) (4-20-4.40) (4*10-4*50) (4.00-4.40) (4.10-4.60) (4-50-4.90) (4.60-4.80) (4-40-5.10) (4-60-5.10) (4.20-4*60) (4.30-4-50) (4.20-4.50) (4.00-4.40) (4-00-4-30) (4-00-4-60) (4-80-5.00) (3*90-5.40)
the vaccine
SD
%CV
0.11 o-19 0.22 0.11 0.19 0.13 o-11 0.21 0.24 o-14 0.08 o-17 o-15 0.21 0.16 0.08 0.30 0.21 0.15 o-11 0.11 O-16 o-13 0.24 0.08 O-16
2.7 4.6 5.0 2.4 4.6 2.9 2.3 4.5 4.7 3.3 1.9 3.9 3.6 4.7 3.4 1.7 6.3 4.3 3.4 2.5 2.5 3.8 3.1 5.6 1.6 3.6
SD: standard deviation: % CV: coefficient of variation (o/o). Table 2. Variance analysis of titres obtained for a yellow fever vaccine preparation by cpe microassay in Vero cells Variation source Between-test Within-test Total *
Degrees of freedom
SS
MS
F*
24 100 124
10.20 2.86 13.06
O-425 0.029
14.86
1oo = 2.59 at the confidence level e = 0.001.
F24
3. Variations
Cpe microassay
Table 1. Titres (log CCID 50/dose) obtained by the cpe microassay in Vero cells, when testing in 125 identical assays the same yellow fever vaccine preparation (25 tests X five parallel titrations) Mean titre (range) in log CCID50/dose (five titrations per test)
Table
in titres (% of the general mean) Plaque assay in PS cells
in Vero cells
Test-to-test variation Within-test variation Overall variation (N = 125)
6.3 3.8
6.3* -
7.3
-
*N=60. Titre variations were expressed as percentages of the general mean titre of the vaccine (Table 3). The test-to-test and within-test variations shown in Table 3 were computed from Table 2 by the method of Dagnelie.5 To allow comparison, the value previously obtained by means of the plaque assay in PS cells was also indicated. The precision of the titre estimate was computed and expressed as a percentage of the general mean titre of the vaccine: the standard error mean value was O-19 log-units (ranging from 0.10 to O-28) and represented 4.3 % of the general mean titre of the vaccine. Discussion From the above results and analysis, the cpe microassay described here could be a interesting alternative for the routine potency test of yellow fever vaccine. As expected, because of the uniformity of the experimental conditions, the ,within-test results show a good homogeneity, with a small within-test variation. Though the above CCIDsO mean titre was comparable to the mean pfu titre previously obtained by the usual plaque assay in PS cells, the present test system is less sensitive. Indeed, it has been established that in systems of equal sensitivity, CCIDso titres will be 1.44 times higher than pfu titres.6 The present feature was presumably due to differences in the susceptibility of the cells since it was found that in similar assay conditions, higher numbers of pfu can be obtained in PS than in Vero cells.7 On the other hand, the test-to-test variation (6.3%) was identical in both test systems as indicated in Table 3. Moreover in the cpe microsystem, the actual within-test variation was small (3.8%) and consistent with the 4.3% mean standard error of the estimates of the titres.
Microasssy
for yellow fever vaccine
In critical assays, where precise quantitation is desired, the pIaque technique is often preferred over the cpe assay. However, the present test-to-test and within-test variations are lower than most values obtained for other virus-cell systems.‘-” These results indicate that the present test, if rigorously standardized, could retain the accuracy of the plaque assay. Moreover, the cpe microassay may be advantageous for routine use since it reduces the time and the manipulations required, especially since staining of the microplates makes the all-or-none responses as easy to read as plaque counting. The test is also very economical in reagents and plastic ware. In conclusion, the present microassay could be a reliable method for the routine potency test of yellow fever vaccine. The test is rapid and easy to perform; it yields accurate individual estimates, the reliability of which may be assessed by parallel titration of reference preparations, gauging of possible cell susceptibility variations. Hence, the present cpe microsystem could meet the requirements of the vaccine controller on many counts, provided that further standardized assays establish the relation with the conventional plaque assay and the LDso mouse assay recommended in the current WHO requirements.l
27
Acknowiedgements
I am grateful to Dr J.-J. Glaustriaux (Chaire de Statistiques de la Faculte des SC. Agr. Gembloux) for statistical advice, Mr M Putseys for competent cell culture assistance, Mrs R De Brandt for previously performing the plaque tests, and Mr PE Lemoine for his support. References 1. WHO Requirements for yellow fever vaccine. WHO Expert Committee on Biological Standardization. WHO Tech Rep Ser 1976; 594: 23. 2. De Madrid AT, Porterfield JS. Bull WHO 1969; 40:113. 3. Yasumura Y, Kawakita Y. Nippon Rinsho 1963; 21: 1201. 4. RhodesAJ, van Rooyen C. Textbook of Virology. Baltimore: Williams & Williams, 1968. 5. Dagnelie P. Thkories et M&hodes Statistiques, Vol. 2. PressesAgronomiques de Gembloux (Belgium), 1975. 6. Cooper PD. In: Smith KM, Lauffer ML, eds. Advances in Virus Research. New York: Academic Press, 1961: 319. 7. Seagroatt V, Magrath DI. J Biol Stand 1983; 11: 47. 8. Kenny MT, Schell K. J Biol Stand 1975; 3: 291. 9. Mann GF, Allison LMC, CopelandJA, Agostini CFM, Zukerman AJ. J Biol Stand 1980; 8: 219. 10. Husson-van Vliet J. J Biol Stand 1987; 15: 385. Received for publication 30 March 1989; accepted 15 June 1989.
(1990) 18, 25-27
Assessment
* lnstitut d’Hygi&ne
of an End-point Microassay Fever Vaccine Jocelyne Husson van Vliet* et d-Epidkmiologie, Departement Microbiologic, 74-7 050 Brussels, Belgium
for Yellow
rue J. Wytsman,
Abstract. A statistical analysis was performed on the results obtained in titrations of yellow fever vaccine using a cytopathic end-point microassay in the Vero cell line. The present test system appeared less sensitive than the conventional plaque assay in PS cells but the consistency in titres was satisfactory since both test-to-test and within-test variations were small. In addition, the test is easy to perform, economical and fast.
Introduction The methods for determining the infectious titre of yellow fever vaccines are either the LDSo mouse assay or the plaque counting assay in cell cultures, as recommended in the current WHO requirements.’ The latter technique is adapted from the microplaque procedure in porcine kidney cells PS described for arboviruses by De Madrid and Porterfleld.2 The purpose of this paper is to evaluate the reliability of the cytopathic effect (cpe) end-point microtechnique in order to ascertain if the procedure is suitable for routine control purposes. From the two recommended cell-substrates (either Vero or PS cells), we choose the Vero line, which is already used in the control laboratory for many other routine tests, is easy to grow and has low sensitivity to adverse environmental conditions. Material
and methods
Cell cultures African green monkey kidney cells Vero3 were used at passage levels 165-175. The cells were grown in Eagle’s Minimum Essential Medium (MEM) supplemented with 5% heat-inactivated fetal calf serum (FCS) and 50 pg/ml gentamycin. The same medium with only 2% FCS was used for maintenance. Vaccine preparation A single batch of freeze-dried commercial yellow fever vaccine (attenuated 17D strain of yellow fever virus) was used in all the tests. This preparation (stored at -70°C) has been used for more than 10 1045-1056/90/010025+03
$03.00/O
years in our laboratory as an in-house reference, the titre of which has been estimated throughout 60 routine tests in PS cells by the usual plaque assay: 4.55 log plaque-forming units (pfu) per dose as general mean (N = 60) with a variation coefficient of 6.3%. The virus preparation, in vials containing five doses, was rehydrated and diluted with chilled dilution medium (MEM containing 1% FCS and 50 pg/ml gentamycin). The dilutions were distributed immediately after preparation. The cpe microassay In brief, the tests were performed in plastic microtitre plates (Nunc, 96 flat-bottomed wells) sealed with pressure-sensitive film (Falcon). The vaccine inoculum (200 ,ol of serial lo-fold dilutions of vaccine) and Vero cells (75 ~1 of a 200 000 cells/ml suspension) were added consecutively using 10 wells per dilution. The microplates were incubated at 36°C and stained on the seventh day (10% form01 saline as fixative and carbol fuchsin as staining solution). They were examined macroscopically on an illuminated viewing box for recording infection as an all-or-none effect. The titres-expressed in log CCID50/dosP and their standard error were estimated using Karber’s formulae.4
Results Assays were performed on 25 occasions. Five parallel titrations were done on each occasion, using replicate @ 1990 The International
Association of Biological Standardization
26
J. Husson van Vliet
series of dilutions starting from one the vaccine preparation and using a preparation. The titres obtained are Table 1. Variance analysis of titres obtained for preparation is summarized in Table 2.
sample of single cell shown in
Test 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Mean
4.02 4.10 4.38 4.68 4.12 4.52 4.74 4.66 5.16 4.20 4.32 4.34 4.18 4.46 4.70 4.68 4.76 4.86 4.36 4.42 4.38 4.20 4.18 4.30 4.92 4.47
(3.90420) (3*90430) (4.00-4.50) (4.50-4.80) (3.90440) (4.40-4.70) (4.60490) (4.40-4.90) (4.90-5-40) (4.00-4.40) (4-20-4.40) (4*10-4*50) (4.00-4.40) (4.10-4.60) (4-50-4.90) (4.60-4.80) (4-40-5.10) (4-60-5.10) (4.20-4*60) (4.30-4-50) (4.20-4.50) (4.00-4.40) (4-00-4-30) (4-00-4-60) (4-80-5.00) (3*90-5.40)
the vaccine
SD
%CV
0.11 o-19 0.22 0.11 0.19 0.13 o-11 0.21 0.24 o-14 0.08 o-17 o-15 0.21 0.16 0.08 0.30 0.21 0.15 o-11 0.11 O-16 o-13 0.24 0.08 O-16
2.7 4.6 5.0 2.4 4.6 2.9 2.3 4.5 4.7 3.3 1.9 3.9 3.6 4.7 3.4 1.7 6.3 4.3 3.4 2.5 2.5 3.8 3.1 5.6 1.6 3.6
SD: standard deviation: % CV: coefficient of variation (o/o). Table 2. Variance analysis of titres obtained for a yellow fever vaccine preparation by cpe microassay in Vero cells Variation source Between-test Within-test Total *
Degrees of freedom
SS
MS
F*
24 100 124
10.20 2.86 13.06
O-425 0.029
14.86
1oo = 2.59 at the confidence level e = 0.001.
F24
3. Variations
Cpe microassay
Table 1. Titres (log CCID 50/dose) obtained by the cpe microassay in Vero cells, when testing in 125 identical assays the same yellow fever vaccine preparation (25 tests X five parallel titrations) Mean titre (range) in log CCID50/dose (five titrations per test)
Table
in titres (% of the general mean) Plaque assay in PS cells
in Vero cells
Test-to-test variation Within-test variation Overall variation (N = 125)
6.3 3.8
6.3* -
7.3
-
*N=60. Titre variations were expressed as percentages of the general mean titre of the vaccine (Table 3). The test-to-test and within-test variations shown in Table 3 were computed from Table 2 by the method of Dagnelie.5 To allow comparison, the value previously obtained by means of the plaque assay in PS cells was also indicated. The precision of the titre estimate was computed and expressed as a percentage of the general mean titre of the vaccine: the standard error mean value was O-19 log-units (ranging from 0.10 to O-28) and represented 4.3 % of the general mean titre of the vaccine. Discussion From the above results and analysis, the cpe microassay described here could be a interesting alternative for the routine potency test of yellow fever vaccine. As expected, because of the uniformity of the experimental conditions, the ,within-test results show a good homogeneity, with a small within-test variation. Though the above CCIDsO mean titre was comparable to the mean pfu titre previously obtained by the usual plaque assay in PS cells, the present test system is less sensitive. Indeed, it has been established that in systems of equal sensitivity, CCIDso titres will be 1.44 times higher than pfu titres.6 The present feature was presumably due to differences in the susceptibility of the cells since it was found that in similar assay conditions, higher numbers of pfu can be obtained in PS than in Vero cells.7 On the other hand, the test-to-test variation (6.3%) was identical in both test systems as indicated in Table 3. Moreover in the cpe microsystem, the actual within-test variation was small (3.8%) and consistent with the 4.3% mean standard error of the estimates of the titres.
Microasssy
for yellow fever vaccine
In critical assays, where precise quantitation is desired, the pIaque technique is often preferred over the cpe assay. However, the present test-to-test and within-test variations are lower than most values obtained for other virus-cell systems.‘-” These results indicate that the present test, if rigorously standardized, could retain the accuracy of the plaque assay. Moreover, the cpe microassay may be advantageous for routine use since it reduces the time and the manipulations required, especially since staining of the microplates makes the all-or-none responses as easy to read as plaque counting. The test is also very economical in reagents and plastic ware. In conclusion, the present microassay could be a reliable method for the routine potency test of yellow fever vaccine. The test is rapid and easy to perform; it yields accurate individual estimates, the reliability of which may be assessed by parallel titration of reference preparations, gauging of possible cell susceptibility variations. Hence, the present cpe microsystem could meet the requirements of the vaccine controller on many counts, provided that further standardized assays establish the relation with the conventional plaque assay and the LDso mouse assay recommended in the current WHO requirements.l
27
Acknowiedgements
I am grateful to Dr J.-J. Glaustriaux (Chaire de Statistiques de la Faculte des SC. Agr. Gembloux) for statistical advice, Mr M Putseys for competent cell culture assistance, Mrs R De Brandt for previously performing the plaque tests, and Mr PE Lemoine for his support. References 1. WHO Requirements for yellow fever vaccine. WHO Expert Committee on Biological Standardization. WHO Tech Rep Ser 1976; 594: 23. 2. De Madrid AT, Porterfield JS. Bull WHO 1969; 40:113. 3. Yasumura Y, Kawakita Y. Nippon Rinsho 1963; 21: 1201. 4. RhodesAJ, van Rooyen C. Textbook of Virology. Baltimore: Williams & Williams, 1968. 5. Dagnelie P. Thkories et M&hodes Statistiques, Vol. 2. PressesAgronomiques de Gembloux (Belgium), 1975. 6. Cooper PD. In: Smith KM, Lauffer ML, eds. Advances in Virus Research. New York: Academic Press, 1961: 319. 7. Seagroatt V, Magrath DI. J Biol Stand 1983; 11: 47. 8. Kenny MT, Schell K. J Biol Stand 1975; 3: 291. 9. Mann GF, Allison LMC, CopelandJA, Agostini CFM, Zukerman AJ. J Biol Stand 1980; 8: 219. 10. Husson-van Vliet J. J Biol Stand 1987; 15: 385. Received for publication 30 March 1989; accepted 15 June 1989.