New Generation of Cell Culture Assay for Smallpox Vaccine Potency

The potency of smallpox vaccines produced in the 1970s was testedby titration onto chorioallantoic membranes of fertilized heneggs (CAM assay). The potency specification commonly approvedfor these vaccines was a titer above 10⁸ pock-forming unitsper milliliter. We developed and validated a cell culture titrationassay to have a more reliable potency test. The cell titrationassay and the CAM assay were tested in parallel on 34 first-generationsmallpox vaccine lots. These allowed us to demonstrate thata correlation does exist between the two titration techniquesand to determine a new in-house specification for the cell titrationmethod. This in vitro potency assay will allow us to test first-generationsmallpox vaccines produced on the skin of living animals butwill also give a hint of the potency specification that shouldbe assigned for new generations of cell-derived smallpox vaccines.

INTRODUCTION

Top

Introduction

The human variola virus as well as the vaccinia, cowpox, monkeypox,and camelpox viruses belong to the genus Orthopoxvirus, familyPoxviridae, and are enveloped DNA viruses. An extensive serologicalcross-reactivity exists for all these viruses (6, 7, 8). Variolaand monkeypox viruses are known to produce systemic diseaseswith a generalized rash in humans and primates. The case fatalityrate of variola major was around 30%. The vaccinia virus usuallyproduces a localized lesion at the site of inoculation intothe skin (5, 7).

In 1796, Edward Jenner demonstrated that cowpox infection protectedagainst the variola virus. Then, vaccination against smallpoxstarted with a poxvirus isolated from a cow (named cowpox) followedby the vaccinia virus. Vaccination campaigns beginning in 1958and reactivated in 1967 promoted by the World Health Organization(WHO) allowed the eradication of smallpox in 1977, and WHO recommendedthat vaccination cease in 1980 (1, 3, 4).

In most European countries, the strain of vaccinia virus usedfor vaccination was the Lister strain. In France, smallpox vaccineswere stockpiled in the 1980s (9). The potency of this strategicstockpile was regularly controlled by the French medical agencyby titration on chorioallantoic membranes of chicken embryos(CAM assay). This method was the official method recommendedby WHO and the European Pharmacopoeia for potency testing, andthe titer was to exceed 10⁸ pock-forming unit per milliliter(2, 11).

Because of the possibility that smallpox might be used for bioterrorism,it was decided in 2001 to recheck the potency of the entirestockpile by two different titration techniques. The aim ofour study was to determine if a correlation exists between theCAM assay and the cell titration assay and also to determinea new in-house specification for the cell titration assay. Indeed,the cell titration assay seemed to be a very interesting assaycompared to the CAM assay, because it is more reliable, moreaccurate, technically simpler, and less time-consuming.

MATERIALS AND METHODS

Top

Materials and Methods

Vaccine and standard. Thirty-four lots of freeze-dried smallpox vaccines were producedin the 1980s from the Lister strain. The titer of these vaccinelots was $>=$ 10⁸ pock-forming units per milliliter.The standard RIV 6713/18 (Lister strain produced on calves)donated at the French public health laboratory in the 1980sby WHO and originating from the Rijks Instituut voor Volksgezondheiden Milieu was used as an internal standard in this study.

Vaccine lots and the standard preparation were reconstitutedwith 250 µl and 400 µl, respectively, of Iscove'smodified Dulbecco's medium without fetal bovine serum.

Cells. Vero cells were obtained from the European Directorate for theQuality of Medicines (Council of Europe). Vero cells were maintainedon plastic tissue culture dishes in medium 199 containing 8%fetal bovine serum.

Titration by inoculation onto choriallantoic membrane of fertilized hen eggs. Two adequate dilutions (usually 10^-6 and 10^-7) of each vaccinelot were run in each assay. For each dilution, 10 chicken embryos(12 days old) were inoculated with 100 µl of the dilutiononto the choriallantoic membrane. After 2 days of incubationat 37°C, membranes were collected from the eggs, and thenumber of pocks were counted. The dilution chosen for calculationof the virus concentration (expressed in pock-forming unitsper milliliter) was the dilution which induced a countable numberof lesions exceeding 10 per membrane.

Cell titration. The cells were plated on 96-well plates (7,000 cells/well) inIscove's modified Dulbecco's medium with 5% fetal bovine serumthe day before titration. Three independent dilution seriesof the vaccine samples and of the internal standard were preparedin Iscove's modified Dulbecco's medium without fetal bovineserum. Cells were inoculated with 100 µl of a 10^-5 to10^-9 dilution, and for each dilution, eight wells were infected.The 96-well plates were incubated at 37°C, and cytopathiceffect was read 9 to 10 days after inoculation.

Precision of cell titration method. The precision of the cell culture method was assessed with thereference vaccine. In order to obtain a normal distributionof the data, a logarithmic transformation was applied to thetiters. The experimental design consisted of six groups of assayscomplying with conditions of intermediate precision (assaysperformed independently with the same method in the same laboratoryon different days). Within each group, four assays were carriedout under conditions ensuring repeatability (assays performedindependently with the same method, in the same laboratory,with the same equipment, on the same day). The homogeneity ofwithin-test variances was verified by a Cochran's test. To evaluatethe repeatability and intermediate precision variances, a variancecomponents analysis was performed.

Normality and consistency of cell titration assay. A test was run to determine whether the distribution could beadequately modeled by a log-normal distribution. The chi-squaretest divides the range of the distribution into 20 equally probableclasses and compares the number of observations in each classto the number expected.

A control chart was set up to analyze the consistency of thefirst-generation vaccine stockpile. In order to obtain a normaldistribution of the data, a logarithmic transformation was appliedto the titers. The control limits were calculated as m₀ ±3s₀, where m₀ is the mean and s₀ is the estimation of the truestandard deviation of the titer distribution.

Correlation between in vivo (CAM assay) and in vitro (cell titration) methods. The relationship between each pair of variables was performedby fitting a logarithmic model: log₁₀(in vivo titer) = a + bx log₁₀(in vitro titer). This statistical study required a regressionanalysis, including an analysis of variance. The Pearson productmoment correlation between each pair of variables was calculated.This correlation coefficient ranges between -1 and +1 and measuresthe strength of the linear relationship between the variables.The P value tests the statistical significance of the estimatedcorrelations. A P value below 0.05 indicates statistically significantnonzero correlation at the 95% confidence level.

RESULTS

Top

Results

Precision of cell titration method. An evaluation of the precision of the cell titration methodwas performed with the reference vaccine. Cochran's test showedthat the variances of the six groups (assays) were homogeneous(P = 0.392). The precision results (Table 1) showed acceptablerepeatability (within-test variance) and intermediate precision(between-test variance). The precision of the method was calculatedas ±0.27 log₁₀.

View this table:
[in this window]
[in a new window]
TABLE 1. Precision of the cell titration method, assessed by using the reference vaccine: within-test and between-test variance component analysis

Normality and consistency of cell titration assay. Because the P value for the chi-square test performed equaled0.06, we could not reject the assumption that the distribution(logarithmic transformation of the data) came from a normaldistribution.

A control chart of vaccine production (34 results) was set upto analyze the consistency of the cell titration method (Fig.1). The mean titer was around 8.02 log₁₀ tissue culture infectiousdoses (TCID₅₀)/ml. The upper and lower control limits were 9.47log₁₀ TCID₅₀/ml and 6.57 log₁₀ TCID₅₀/ml, respectively.

View larger version (13K):
[in this window]
[in a new window]
FIG. 1. Control chart of 34 smallpox vaccine lots for the cell culture method, expressed as log₁₀(TCID₅₀/ml).

Correlation between in vivo (CAM assay) and in vitro (cell titration) methods. A statistical description of the relationship between each pairof variables was performed (Table 2 and Fig. 2).

View this table:
[in this window]
[in a new window]
TABLE 2. Correlations between in vivo (CAM assay) and in vitro (cell culture titration) methods

View larger version (23K):
[in this window]
[in a new window]
FIG. 2. Correlation between in vivo (CAM assay) and in vitro (cell culture titration) methods. The inner bounds show 95.0% confidence limits for the regression line. The outer bounds show 95.0% prediction limits for future observations. The arrow represents the in vitro titer corresponding to a predicted in vivo titer of 8.0 log₁₀.

Since the P value in the analysis of variance table was lessthan 0.01, there was a statistically significant relationshipbetween the in vivo and in vitro results at the 99% or higherconfidence level. The R² statistic indicates that the modelas fitted explains 30% of the variability in the in vivo results.The correlation coefficient equals 0.55, indicating a moderatelystrong relationship between the in vivo (CAM assay) and in vitro(cell titration) results.

Moreover, the regression analysis indicates that a titer ofgreater than 7.6 log₁₀ TCID₅₀/ml with the in vitro method wouldhave been found above the in vivo specification of 8.0 log₁₀pock-forming units per milliliter at the 95% confidence level.

DISCUSSION

Top

Discussion

The idea to propose another technique to control the potencyof smallpox vaccines is not new and was developed in the 1970s(10). The eradication of smallpox and the discontinuance ofvaccination in 1980 were probably the reasons why this methodwas no longer pursued. Because of the new possibility that thevariola virus may be used as a biological weapon and that somecountries have a smallpox vaccine stockpile, it is again opportuneto develop and standardize a method of titration on cell culturefor smallpox vaccines. Because the reference method of titrationin all regulatory documents was the CAM assay (titration inchorioallantoic membranes of chicken embryos), we decided tocompare the cell titration assay to the reference method.

If we consider the results, as expected, a significant relationshipwas observed between the in vivo (CAM assay) and the in vitro(cell titration) methods. The correlation coefficient of r =0.552 is not particularly high (R² = 30%), as can be seen fromFig. 2. This is not surprising, given that all the vaccine lotstested were of similar potency. A large component of the variationis therefore the independent assay-to-assay variation for eachmethod, which is not expected to be correlated. A more realisticassessment of the agreement between methods could only be obtainedwith data from vaccines of a wider range of potencies, includingsubpotent vaccines. Moreover, as shown with the control chart,the lack of trends, the low precisions, and the fact that nopoint is out of the control limits allowed us to consider thatour in vitro control method is suitable to monitor the consistencyof smallpox vaccine production.

Indeed, the consistency and the correlation studies led to definea lower in-house specification for our in vitro method, 7.6log₁₀ TCID₅₀/ml.

Concerning the cell titration method, as expected, because ofthe uniformity of the experimental conditions, the precisionwas acceptable and usual for this type of technique (±0.27log₁₀). The control chart implemented with our internal referenceconfirmed this (±0.31 log₁₀) (data not shown). The controlchart became a criterion which must be verified to validateor invalidate the assay in routine testing.

Both potency methods used to assay the French stockpile of freeze-driedsmallpox vaccines produced in the 1970s confirmed the very goodstability of this vaccine stored at -20°C. This informationis very important in view of the production of a new generationof cell-derived vaccines that could be stable as freeze-driedlots for at least 25 years for an improbable act of bioterrorism.

This new validated method of cell titration will allow regulatoryagencies and also manufacturers to link the potency of first-generationsmallpox vaccines to that of second-generation vaccines. Thepotency specification of the cell-derived vaccines could notbe a transposition of the potency specification of the CAM assaybut will depend on the potency control method and on its correlationwith the reference method (CAM assay).

Potency assay by cell titration could be included in all internationalrecommendations such as the European guidelines, European Pharmacopoeiamonographs, and WHO recommendations. This reliable and simplemethod should allow performance of the potency assay throughoutdevelopment and during the process of quality control of smallpoxvaccines.

ACKNOWLEDGMENTS

We are very grateful to Suzanne Sierzputowski for helpful suggestionsand technical assistance.

FOOTNOTES

* Corresponding author. Mailing address: Agence Française de Sécurité Sanitaire des Produits de Santé, 321 avenue Jean Jaurès, 69007 Lyon, France. Phone: (33) 4 72 76 06 10. Fax: (33) 4 72 76 06 15. E-mail: Isabelle.Leparc-Goffart{at}afssaps.sante.fr.

REFERENCES

Top