Journal of Virological Methods, Elsevier
A simplified plaque assay for varicella vaccine J. Husson-van
‘lnstitut d’ Hygiene et d’ Epidemiologic, Departement Microbiologic, Brussels, Belgium; ‘Smifh Kline - RIT, Division Bioiogique, Rixensart, Belgium (Accepted
Summary A simple and accurate plaque assay is described for potency testing of attenuated varicella vaccine. Assays were performed on the African green monkey kidney continuous cell line CV-1, in multidish-plates, under a semi-solid carboxymethylcellulose overlay. The test is economical and yields accurate individual titre estimates, the reliability of which may be assessed by parallel titration of reference preparations. Varicella
Introduction A live varicella vaccine was recently developed (Takahashi et al., 1975, 1985; Hayakawa et al., 1984). A convenient, economical and reliable method for routine quantitation of successive batches is thus a necessity. Varicella-zoster (v-z) virus is notoriously heat labile and difficult to grow. Although numerous human and primate cell cultures have been reported as susceptible to v-z virus infection, few cell systems are convenient for practical use in the control laboratory (Weller et al., 1958; Taylor-Robinson, 1959; Caunt et al., 1964, 1969; Sefcovicova, 1971; Harbour et al., 1975; Gilden et al., 1978; Darai et al., 1978; Grose et al., 1978). Factors affecting quantitative isolation of v-z virus have been investigated and various plaque assays have been described (Rapp et al., 1963; Rager-Zisman et al., 1973; Gerna et al., 1976; Hondo et al., 1976; Asano et al., 1978; Levin et al., 1984). It was stated that potency testing of varicella vaccine reCorrespondence to: J. Husson-van Vliet, Institut d’ Hygiene biologie, rue J. Wytsman, 1050, Brussels, Belgium. 0166-0934/87/$03.50
1987 El sevier Science
et d’ Epidemologie,
quires strictly standardized methods and that. even when retaining optimal conditions, important variations may occur from one test to another (D’Hondt et al., 1985). Results obtained when titrating varicella vaccine by using a cytopathic microassay in a continuous line of monkey kidney cells were recently assessed (Husson-van Vliet, 1987). The purpose of the present paper is to describe a simple and reliable plaque assay using the W-1 continuous ceil line in plastic multidish-plates, under a semisolid overlay.
Cell cultures African green monkey kidney cells CV-1 were used between passage levels 40 and 50. Cells were grown in equal parts of Basal Medium Eagle (BME) and Eagle’s Minimum Essential Medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 50 pgiml gentamycin. The same medium with only 5% FCS was used for maintenance. For the assays, cells from 3-7 day confluent cultures were resuspended in growth medium (100000 cells/ml) and a single homogeneous pool of cells was distributed in the plates to be used within 3 days. Vaccine preparations A single routine batch of freeze-dried vaccine (OKA-RIT strain) was used throughout the tests. Monodose vials were rehydrated with and diluted in chilled dilution medium (phosphate buffered saline containing 5% sucrose. 0.1% Na-glutamate and 10% FCS) (Asano et al., 1978). Serial dilution steps were made by hand, using predistributed volumes of diluent (Cornwall syringe) and fixed-volume micropipettes with disposable tips. All vials were kept on crushed ice during assays and the dilutions were distributed immediately after preparation. Plaque assay The tests were performed in &well (35 mm diameter) plastic plates with covers (Nunc). As a rule. 3-6 replicate wells were used for each dilution of a four-fold series issuing from each sample. Aliquots (100 ~1) of the dilutions were adsorbed for 1 h on drained, preformed monolayers with frequent rocking in a humidified incubator at 36°C with 5% COZ. After adsorption, the monolayers were overlayed with 5 ml maintenance medium containing 2% w/v of sodium carboxymethylcellulose (CMC, Koch Light, U.K.). Cultures were incubated for 7 days at 36°C in a humidified 5% COZ incubator. The dose-response curve was analysed from the counts obtained in an assay designed to assess the suitability of the linear relationship: m, = f x,
115 _* __._._/ “,.__
\ __\. */_*““. / .““X
Fig. 1. Plaques
on different occasions with the same batch of attenuated CV-1 cells after 7 days at 36°C.
where mi is the expected mean density at dilution X, of a suspension assumed to have a true mean density of I’ infective particles per unit volume. For this particular assay, multiple aliquots of a single series of 5 two-fold (0.3 log) dilutions were distributed in parallel in identical plates.
Fixing, staining, counting and computing The plates were fixed on the seventh day, with 10% form01 saline, and stained with carbol fuchsin solution. They were examined with the naked eye on an illuminated ground. The lesions were scored; the titers, expressed as plaque forming units (pfu)/O.l ml, were evaluated by the method of maximum likelihood (Roberts et al., 1965). programmed on a HP41C pocket calculator (unpublished data). The estimation procedure assumes Poissonian variation and proportionality of counts to dilution factor. With respect to these assumptions, the program determines the range of dilutions suitable for estimation of the density and yields an estimate with a smaller true standard error than the more usual one, i.e. the arithmetic mean of the estimates from the individual dilutions.
Plaques morphology Lesions were generally well-rounded, with sharp boundaries and a clear center. They reached the threshold of visibility around the fourth day after inoculation and then increased in size to a diameter of 1 - 2 mm (presumably depending on the cell sensitivity since the same vaccine batch was used throughout the tests) (Fig. 1).
TABLE 1 Analysis of counts for parallel plates for each of a series of p dilutions of varicella vaccine by the method of maximum likelihood. Dilution Factor X,
Parallel counts r,,
88, 84, 60, 60, 62, 63, 70, 91, 71. 63, 76, 72
39, 42, 38, 44, 31, 30, 45, 30, 301 37, 35, 29, 45, 30, 44
22, 19, 14, 23, 15, 15, 19, 22, 14, 14, 23, 17, 19, 15, 20, 20
11, 9. 7. 16, 7, 8, 15, 10, 4, 8, 9, 12, 5, 9, 9
3, 7, 6, 2, 4, 7, 4, 6, 10, 3, 4, 2
1 = lln,m, = l/n, Tx, 2 = m, = f’x, with f = X, X, r, I Qr$, = 580.9 Where i = index of dilution (i = l,...,p) and
j = index of counts within dilution (j = 1,. ,a,)
Total R, Weight for total (1)
Expected mean count (2)
TABLE 2 Analysis and significance tests according to Roberts’ method Source of variation Regression Total deviation from regression Deviations of dilution means from regression Between parallel piates within dilutions Total
69 4 65 70
P = 0.98 P = 0.22
The present sum of squares were I= times those in the usual regression analysis. 0”
For the particular assessment of dose-response curve, the p sets on ni observed parallel counts (i= 1,. . . ,p) and their analysis, according to above method, are indicated in Tables 1 and 2.
Fig. 2. Dose-response curve obtained with attenuated variceha vaccine in CV-1 cells: the coarse straight fine was traced through the mean log counts; the fine lines join the 95% confidence limits of Poissonian variation around the mean log counts.
As shown in Table 2. there is no evidence of heterogeneity or departure from equation (I ). Log-decimal counts were plotted against log-decimal dilutions (Fig. 2). A straight iine was traced through the mean log counts. The expected Poissonian variation between counts within dilutions was estimated by the root mean count and the corresponding approximated 95% confidence limits were traced (log units). We determined, according to Cooper (1969), that the theoretical maximum number of plaques allowing individual counting on the well surface without overcrowding was 70 for a 35 mm diameter well and a plaque diameter of 4 mm (to ensure that each actual lesion is surrounded by an intact zone for convenience and reliability of plaque scoring).
The reproducibility of the assay for routine control purposes was estimated by repeatedly testing several samples of the same vaccine batch on various occasions for more than 2 mth using different CV-I cell passages, the other conditions remaining identical: this conforms more to the usual situation in the vaccine control laboratory than a restricted assay design. The titers observed through these tests ranged from 217 - 740 pfuiO.1 ml with a 33% global variation around the general mean titer equal to SO7 pfuiO.1 ml (n = 15). Combining the standard errors of titer estimates (ranging from 4 - 8%:) with a probable error of 7G/c per step dilution. precision varied from 7 - 10% of the titer estimate, resulting in a 8% precision for the general mean titer. No correlation was found between titer and passage fevel. However, for a single sample assayed in paralfel on a Iow and a high celt passage, the titers varied from 3 to 1. but on other occasions, the Iatter high ceil passage yielded much higher titer estimates. When assaying five samples in parallel and under identical conditions, test-to-test variation was 18% of the mean.
Discussion From the above results, it is obvious that the present plaque counting system meets the requirements of the vaccine controller from many points of view. Analysis of dose-response relationship is consistent with the single-particle concept and proves the adequacy of the plaque assay. For routine application of the assay, two-fold dilution steps are clearly less adequate than four-fold ones, especially if few rephcate wells are used, due to excessive overlapping of counts variation ranges, as shown in Fig. 2. A similar observation was made previously in the case of end-point titrations with other viruscell systems (Allan et al., 1968; Husson-van Vliet., 1986). The use of a monkey kidney continuous cell line is advantageous; this usual substrate is less difficult to grow and also less sensitive to adverse conditions than most others reported as highly susceptible to v-z virus. The African green monkey kidney cell line Vero was previously used successfuHy for growing v-z virus (Caunt et
al., 1969; Harhour et al., 1975). but in our case, the plaques developed much more slowly in Vera than in W-l celts, thereby proionging the assay, Among the cell substrates usually cited for v-z quant~~at~on, human embryonic Iung fibroblasts (HELF) were mainly used under agar ur methylcellulose, or even liquid medium overlay. However, the risk of clumping is higher in this substrate than in CV-1 cells due to the different plaque morphology; the lesions are elongated along the lines of the fibroblastic cell making it necessary to use larger culture dishes in order to maintain convenience and reliability of plaque scoring. In addition, we found no need to use DEAE-dextran in the overlay (as reported in the case of HELF cells) since the lesions in CV-1 cells were clear-cut and reached a convenient size within a suitable time. This was already observed for the plaque assay of Simian virus 40 on CV-1 cells (Fendrick et al., 1983). Hence, we found the CV-1 continuous ceII line more suitable for the standardj~ation of a plaque counting assay. Due to the inherent fragility of v-z virus, we prefer to perform the tests using preformed monolayers instead of using the alternative cell-suspension method of inoculation: the former procedure allows the quality of the monolayer-s to be examined before inoculating them while ensuring better adsorption conditions. Despite accuracy of the present estimates, it is obvious that variations still occur between replicate samples of the same preparation, as it was already observed for other virus-cell systems (Hebert et al.. 198C)), making it necessary to titrate several samples in the same test in parallel, Although reproducibility of the plaque assay has been seldom characterized statistically. a survey of the literature showed that the present titer variation was consistent with that observed eisewhere in case of other virus-ceit systems (French et al., 1959; Gomwalk., 1980; Mann et al.. 1980). In conclusion, the plaque assay performed in plastic muttidish-plates. using the continuaus cell line CV-1 under a CMC overlay is suitable for routine potency testing of varicella vaccine, in so far as on each particular occasion, the response level of the cell system can be assessed. The test is easy to perform and economical in materials, It yields accurate individual estimates, the consistency of which is improved by using standard cell passage levels. The parallel titration of reference preparations (Thornton et ai., 1984) allows gauging of uncontrollable susceptibility variations of the cell cultures.
Grateful acknowledgement is made to Smith Kline-RIT (Rixensart) for kindly providing vaccine preparation and cells. Acknowledgement is also made to Mr. J. Bouquiaux and M. Cosse for the photographs and to Dr. J.-J. Claustriaux (Faculte SC. Agr, Gembloux) for valuable statistical advice.
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