Foot and mouth disease vaccine potency tests in cattle: the interrelationship of antigen dose, serum neutralizing antibody response and protection from challenge.

The use of tests in cattle remains the basis for evaluating the potency of foot and mouth disease (FMD) vaccines intended for use in cattle. To be able to compare different types of potency test it is essential to have a good understanding of how measurable responses in cattle to vaccination relate to one another. In this paper the interrelationships were examined of log antigen dose (V50), serum neutralizing antibody response (SN50), and protection from challenge (probit), following a single-dose primary vaccination. Estimates of the slopes for each of the three regressions were obtained and these, together with values described by other authors, were used to evaluate how well some of the potency tests for FMD vaccines matched up to Recommendations of the Office International des Epizooties made in 1975.

A potency test method for foot and mouth disease vaccine based on the serum neutralizing antibody response produced in cattle.

A method is described for converting a mean serum neutralizing antibody titre after primary vaccination directly into a percentage protection value using a single regression slope. This has advantages over the indirect method in which a potency value is first estimated on the log antigen dose scale before conversion to a percentage protection value, since to do this requires the use of three separate regression slopes. Test designs for the serological method have been formulated that meet the 1975 Recommendations of the Office International des Epizooties, and which possess minimum potency standards at least as high as those of the European Pharmacopoeia's vaccine dose extinction endpoint method.

A clonal selection based timecourse model for antibody responses to killed vaccine, with applications to foot and mouth disease.

Published models for the timecourse of the immune response are reviewed for their applicability to data from animals that have been injected with killed vaccine. Simple models are required so that statistical fitting procedures become straightforward. A class of models that incorporates the concept of clonal selection has been found useful. Immune memory is described in terms of persistence of mean antibody affinity when the overall concentration of antibody has declined towards background. The model is demonstrated on multipoint affinity distributions and initial negative exponential densities, from which conditions for boundedness can be developed. Predictions are made that are consistent with experiments on the evolution of immunoassay data using foot and mouth disease vaccine.

Sources of variability in foot and mouth disease vaccine potency estimates based on serum neutralizing antibody assay.

Some vaccines can be assayed for potency by measuring the serum antibody response they produce in vaccinated test animals. Using data obtained from potency assays on batches of foot and mouth disease vaccine, the sources of variability in such a method were examined. A linear model is proposed for the analysis of replicate serum neutralizing antibody assays, which represents an improvement on the usual approach of working with only a mean serum assay value for each test animal. Components of variance were calculated, allowing the relative importance of the numbers of test animals, or the numbers of serum assays per test animal, to be estimated in terms of the variability of the overall group mean antibody response. A method is described for calculating fiducial intervals for the serological potency estimates, and it is shown that these intervals are no larger than, and are in fact probably smaller than, those obtained from quantal challenge tests. The results have important implications for the design and analysis of similar biological tests used for other products.

Correlation of 140S antigen dose with the serum neutralizing antibody response and the level of protection induced in cattle by foot-and-mouth disease vaccines.

An analysis was made of data from potency tests on fifteen batches of monovalent foot-and-mouth disease vaccine, comprising five batches each of type O, type A and type C. Regressions were calculated for the relation of percentage protection (probit) versus log 140S antigen dose and for the serum neutralizing antibody titre (log SN50) versus log 140S antigen dose. Type O vaccines required a far higher level (220 ng) of 140S antigen to achieve a 50% protection level (PA50) in cattle than did type A (2.4 ng) and type C (4.36 ng) vaccines. Type O antigen, dose for dose, was as effective at provoking neutralizing antibody as the types A and C antigens. Thus, it would appear that a far higher log SN50 value (2.14) was required for type O vaccines to equate with 50% protection of cattle than was required for type A (1.17) and type C (1.41) vaccines. Prior to 1977, however, the PA50 value for type O vaccine strain was only 1.34 and it was concluded that an antigenic shift was the most likely cause for the large difference between that value and the current PA50 value.

The use of serum neutralizing antibody assay for the determination of the potency of foot and mouth disease (FMD) vaccines in cattle.

The Permanent Commission of O.I.E. on foot and mouth disease has proposed a series of potency standards for foot and mouth disease vaccines. No totally in vitro assay has yet been developed to satisfy these requirements, and most Control Authorities require a potency assay to be carried out in cattle using challenge with virulent virus. There are various reasons why challenge tests should be replaced by serological tests, but a stumbling block to this is the requirement that the 50% protective antibody level (PA50) is valid only for a specific combination of vaccine virus seed lot with cattle virus seed lot in a laboratory. The results of challenge and antibody tests for 38 U.K. Control Authority vaccine batch tests were analysed to examine the correlation between potencies measured by the two methods. The correlation between the methods was high and the proportion of misclassification of batches was low. It is suggested that the results provide a good basis for future use of the serological method, but that it is still sometimes necessary to carry out challenge tests.

The use of a logistic model for the quantitative interpretation of indirect sandwich enzyme labelled immunosorbent assays (ELISA) for antibodies and antigens in foot and mouth disease.

A three parameter logistic model is described for the analysis of profiles of optical density vs log dose for indirect sandwich ELISA tests in foot and mouth disease. The model describes the observed phenomenon of saturability with increasing dose, and its parameters can be interpreted in terms of molecular binding events. A computer program to fit the model is described. An approximate statistical test is developed which can be used to test for departures from equivalence for replicate profiles. It is found that correlation of the optical density values to a standard reference reaction considerably improves reproducibility.

An indirect sandwich enzyme labelled immunosorbent assay for the detection of foot and mouth disease virus immunizing antigen in tissue culture harvests.

The optimum conditions for an indirect sandwich enzyme-linked immunosorbent assay for foot and mouth disease virus 140S antigen assay are described. Factors which could contribute to the variation in the test were investigated and a calibration coefficient for the conversion of ELISA values to antigen concentration in micrograms of 140S antigen per millilitre was calculated. Antigen mass in nine tissue culture harvests was estimated and these correlated well with estimates made by sucrose density gradients (r = 0.95).

The differentiation of foot and mouth disease virus strains using an indirect sandwich enzyme-linked immunosorbent assay saturation model.

Sigmoid saturation curves were fitted to the results of titrations of antiserum to foot and mouth disease virus against homologous and heterologous virus strains. Differentiation of strains was readily evident from the different levels of the homologous and heterologous curves. These differences could be quantified by comparison of the saturation curve parameters K and PRmax. Factors which affect variations in K and PRmax and their biological significance were investigated by varying the first phase antibody and the antigen used in the test. PRmax was found to represent an overall combining potential of the antigen with both sera used in the sandwich test. K, which was theoretically a measure of affinity, also reflected antibody titre. Relationships measured using this model were found to correlate with the reference test system--two-dimensional microneutralization.

Graphical method for comparative statistical study of vaccine potency tests.

Producers and consumers are interested in some of the intrinsic characteristics of vaccine potency assays for the comparative evaluation of suitable experimental design. A graphical method is developed which represents the precision of test results, the sensitivity of such results to changes in dosage, and the relevance of the results in the way they reflect the protection afforded in the host species. The graphs can be constructed from Producer's scores and Consumer's scores on each of the scales of test score, antigen dose and probability of protection against disease. A method for calculating these scores is suggested and illustrated for single and multiple component vaccines, for tests which do or do not employ a standard reference preparation, and for tests which employ quantitative or quantal systems of scoring.

Some applications of a graphical method for the comparative statistical study of vaccine potency assays.

Using examples drawn from some commonly-used methods for vaccine potency assay, we show how a graphical method of representing three key characteristics, precision, sensitivity and relevance, can be used to compare basic methods and variations in test design. We suggest that this method can be of considerable value to Control Authorities in establishing test designs and potency standards, and to vaccine manufacturers in formulating vaccines which will have a high probability of meeting these potency standards.