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Foot-and-mouth disease vaccine potency testing: determination and statistical validation of a model using a serological approach

Paul V. Barnett a,∗ , Robert J. Statham a , Wilna Vosloo b , Daniel T. Haydon c

b a Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK ARC-Onderstepoort Veterinary Institute, Exotic Diseases Division, Private Bag X5, Onderstepoort 0110, South Africa c Department of Zoology, University of Guelph, Guelph, Ont., Canada N1G 2W1

Received 7 October 2002; received in revised form 13 February 2003; accepted 11 March 2003

Abstract European foot-and-mouth disease vaccine manufacturers are required to quantify the efﬁcacy of their product in accordance with the European Pharmacopoeia (EP). The method used most often to establish the potency of foot-and-mouth disease vaccines requires viral challenge of vaccinated cattle. Alternative approaches, such as challenge-free serological assessments have many advantages over existing methods and could be used if robust statistical models could be developed that related antibody titres to protection from challenge. Logistic regression analysis of data from two independent research laboratories, representing six of the seven main serotypes of FMD, permitted the parameterisation of these models and indicated that a signiﬁcant relationship existed between antibody titre and probability of protection. Furthermore, no signiﬁcant differences were observed in the parameters of logistic models ﬁtted to different strains within the serotypes A, O, and SAT-3, or when strains from serotypes A, O, and Asia-1, or SAT-1 and SAT-3, were combined. However, signiﬁcant differences in the model parameters did exist between different laboratories. Using these models a bootstrap analysis suggested that for vaccines that induced consistently high titres, as few as six to eight individual animals could be used to establish with conﬁdence the minimum protective doses that would protect 50% of vaccinated animals. We conclude that a serologically evaluated truncated test that eliminates the need to virus challenge cattle is a credible alternative for quantifying vaccine potency. © 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Foot-and-mouth disease; Vaccine; Potency; Challenge test; Logistic regression; Antibody titre

1. Introduction Inactivated foot-and-mouth disease virus (FMDV) is used in vaccine preparations to control foot-and-mouth disease (FMD), one of the most economically important diseases affecting livestock. These vaccines are used in many parts of the world, particularly where the disease is endemic, including South America, Africa, the Middle East and the Far East. In disease free countries, concentrated inactivated FMD virus antigens are also kept in strategic reserves, so-called antigen banks, which can be rapidly formulated into vaccine during an emergency should an outbreak require additional control measures. In order to assess the quality of the vaccine, all FMD vaccine producers are obliged to instigate a series of tests to establish the safety and efﬁcacy of their product. These tests include a measurement of potency which in accordance

Corresponding author. Tel.: +44-1483-232441; fax: +44-1483-232448. E-mail address: paul.barnett@bbsrc.ac.uk (P.V. Barnett).

∗

with the European Pharmacopoiea (EP) requires that vaccine batches be tested in groups of at least ﬁve cattle inoculated with reduced dose volumes of vaccine so that potency can be expressed in terms of 50% protective doses [1] (PD50 , deﬁned as the factor by which the concentrate may be diluted such that 50% of vaccinated animals are protected). This method has practical and logistical problems, and disadvantages from the perspective of animal welfare. For example, in any extinction point test, approximately 50% of the animals that are not protected will suffer the painful clinical manifestations of the disease and even some protected animals may show primary lesions at the site of challenge. Also, only one valency can be tested in any given trial. Clinically infected animals represent a disease security hazard requiring expensive high security housing. Finally, given the ﬁnancial commitment of purchasing these animals and maintaining them during the trial period the smallest permitted group sizes are often used leading to lack of statistical power and imprecision [2]. However, the European Pharmacopoiea has supported the use of alternative testing methods provided a correlation

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the covariance between the estimated slope and intercept.V. it must be standardised and include standard reference reagents and parameterised using substantial data sets from which the models relating titre to protection can be reliably established. • quantify any variation in this relationship between laboratories or as a result of the use of different vaccine strains or serotypes. Pay and Hingley [2] and Ahl et al. Two methods have primarily been used to calculate the degree of protection in a group of vaccinated animals. At the Pirbright Laboratory these tests were performed to either substantiate that a given antigen could be formulated into a ‘high’ potency (≥10 PD50 ) vaccine for acceptance into the International FMD Vaccine Bank [13]. The data is summarised in Table 1 and Fig.P. log antibody titre from the VNT. Serologically-based methods have certain advantages. In this paper. 1. Barnett et al.)). involving just a single vaccine dilution group. and SAT-3). there are strong arguments for replacing the challenge test by in vitro serological assays. the virus neutralising antibody response and the level of induced protection [9]. When no signiﬁcant differences between strains or serotypes were revealed the data were combined and parameters for a single model estimated. or to show stability of the antigen during ultra-low temperature storage. / Vaccine 21 (2003) 3240–3248 3241 can be established between them and the challenge test.000 ID50 into the tongue. by using a truncated version of that speciﬁed in the OIE manual [14]. and between laboratories on the relationship between log antibody titre and protection. Certainly. SAT-1. . The signiﬁcance of all two-way interactions was examined and those revealed to be insignificant at the 5% level were omitted from subsequent analysis. the metabolic inhibition test. normally 8–10 days. Animals were observed closely for the subsequent reading period. Methods 2. serotype. Analysis The data for each vaccinated animal thus constitutes: laboratory. These have included the virus neutralisation test. They allow several FMD vaccines to be tested simultaneously and/or a number of serotypes to be examined in a single polyvalent vaccine and can signiﬁcantly improve the accuracy of the result. The test system must be consistent and reproducible. 2. and the neutralising antibody titre (log 10 SN50 /100 TCID50 ) to the vaccine virus strains estimated from serum samples taken at 21 days post-vaccination using the virus neutralisation test (VNT). Animals were vaccinated (except control individuals) with various dilutions of vaccine of varying valency. strain.2. A correlation was also established between the antigen dose in the vaccine. based at Pirbright in the United Kingdom and the ARC-Onderstepoort Veterinary Institute in South Africa. Data The data comprised the results of vaccine potency trials conducted in the two laboratories (Pirbright and Onderstepoort) on six different serotypes (O. the objective of this analysis was to: • determine the relationship between virus neutralising titre and protection. we have accrued data from two laboratories: The Institute for Animal Health. For this reason many research workers developed and evaluated a variety of serological tests and assessed the correlation of the results with protection. [10] used mean serum antibody titres to calculate a PD50 or a percentage protection. the 95% conﬁdence intervals (95% CI) on T50 as determined using Fiellers theorem [15] and T95 (deﬁned as the titre at which animals are protected with probability 95%). Similarly. Asia-1.1. in which cattle challenge tests and serology have been performed to establish the potency of many different batches of FMD vaccine. Bengelsdorff [11] and van Maanen and Terpstra [12] used a second method in which an individual titre or index was assigned to protection and non-protection and the assessment of a group was calculated from the ‘passed’ and ‘not-passed’ individuals. plaque reduction test. and protected or unprotected status. not least the relatively insigniﬁcant discomfort caused to the animals as a result of vaccination and subsequent blood sampling. Although some disadvantages have been noted with these tests. and characterised by their intercepts and slopes (with accompanying standard errors (S. Stellman [7] developed this approach further by showing how such antibody titres could be used to statistically assess vaccine and Pay and Parker [8] described a method for relating log virus neutralisation titres of cattle sera to the potency of a vaccine in ‘antigen PD50 ’ units. A. and the occurrence of generalised lesions on any one or more feet taken as evidence that the animal was not protected by the vaccination. Encompassing six different serotypes. Logistic regression implemented within MINITAB was used to determine the signiﬁcance of differences between strains within serotypes. • provide a model that could be used to calculate the probability that vaccines achieve potency levels at or in excess of a pre-speciﬁed requirement. it is generally accepted by FMD research workers that there is an excellent correlation between the virus neutralisation antibody titres of primo-vaccinated cattle and their protection from virus challenge at 21 days post-vaccination [3–6]. At this time animals were challenged with homologous virus to the vaccine strain by intradermal inoculation of 10. the laboratory at Onderstepoort performed cattle challenge tests to ensure that their vaccines had potency values of ≥8 PD50 . T50 (deﬁned as the titre at which animals are protected with probability 50%). SAT-2. any form of test would have to meet certain general criteria.E. Appropriate minimal models were determined. mouse protection test and different forms of the enzyme linked immunosorbent assay (ELISA). however. 2.

V.065 4. The test is conservative because the variance of a mixed binomial process (in which each ‘trial’ succeeds or fails with a different probability) is n i=1 νi (1 − νi ). If n cattle in a potency trial are vaccinated with vaccine diluted X-fold and the hypothesis that ν ¯ ≤ 0.44 (40) 2.68 (55) 1. .200 −4.34 (32) 2.067 2.760 −5. We can.008 0.714 1. t3 .150 1. Barnett et al.141 1.988 1.326 1.123 −6. ν ¯ .469 1.79 (10) 1. of animals protected (%) 37 (80) 31 (86) 19 (73) 53 (52) 32 (88) 22 (85) 49 (64) 7 (58) 44 (57) 1 (14) 2 (14) 22 (67) Average neutralising Ab titre of vaccinated animals 2.29 (24) 1. The 95% conﬁdence regions (95% CR) for adopted models were also determined using methods described in Sokal and Rolf [16].780 4.18 12.581 −0. a1 Covariance (a0 .116 0.020 −0.726 9.450 1.96 1.540 1.79 (55) 1.673 5.482 4. O: Onderstepoort) Serotype Asia-1 O A Challenge strains P: India P: Manisa P: Lausanne P: Cruzeiro P: Thailand P: Iraq O: Sar/9/81 O: Bot/1/77 O: Zim/7/83 O: KNP/19/89 O: Bec/1/65 O: KNP/10/90 No.252 2. is given by: ν ¯ = 1/n n i=1 νi .409 1.920 −8.72 (5) 0.5.5 then it can be concluded that the PD50 of the vaccine is greater than X.560 1.594 1. The expected proportion of protected cattle. The expected probability of protection (νi ) for an individual animal with titre ti is given by: νi = exp(a0 + a1 ti ) 1 + exp(a0 + a1 ti ) (1) where a0 and a1 are the intercept and slope of the associated logistic regression model relating protection to titre. . tn .965 1.967 2. with log titres t1 .258 Insufﬁcient data 0.307 1. / Vaccine 21 (2003) 3240–3248 Table 1 Summary of the data (P: Pirbright.728 1.843 2. of animals challenged 46 36 26 102 36 26 77 12 77 7 14 33 No.860 −0.517 1.30 1.160 1. An estimate of whether or not this differs signiﬁcantly from 50% of the group can be approximated n n/2 by inspecting the quantity p = i=0 ν ¯ i (1 − ν ¯ )(n−i) i which constitutes a (one-tailed) test of the hypothesis that ν ¯ ≤ 0.18 1.773 5.310 4.02 1. in a group comprising n individuals.243 0.820 −1. which is always greater than the variance for a homogenous process with the same mean (in which each ‘trial’ succeeds or fails with the same probability)—which is nν ¯ (1 − ν ¯ ) [17].67 (92) 2.996 2.855 1.3242 P.36 (24) 1.805 −0. Thus.417 1.14 3.567 1.5 is rejected in favour of the hypothesis that ν ¯ > 0. use a bootstrap analysis to investigate the relationship between the number of animals in such challenge-free trials.002 −5. Virus/lab Vaccine/challenge strain n a0 S.164 1.E.091 1.977 1.889 −10.780 −6.581 1.840 0.66 (10) 1.086 2. and use the model combined over all strains for that serotype reported in Table 2 to calculate the mean and variance of the expected number of animals Table 2 Parameters corresponding to logistic regression models ﬁtted to subsets of the data in which no signiﬁcant heterogeneity could be detected at the 5% level Model no.157 4.803 4.339 1. in principle. and the probability that signiﬁcantly more than half the animals in such trials would be protected.43 5.554 1.287 −1.118 .668 −13. assuming the process to be homogenous when it is in fact mixed should lead to an overestimate of p.912 1.957 4.000 −6. .932 1. t2 .162 Insufﬁcient data 0.70 (25) SAT-1 SAT-2 SAT-3 The number of animals challenged includes control unvaccinated animals.054 8.377 1.486 0.31 0.25 (32) 2. a0 a1 S.923 1.807 −6.934 0.41 1. a1 ) T50 T50 (95% CI) T95 Onderstepoort 1 SAT-1 2 SAT-1 3 SAT-1 4 SAT-2 5 SAT-2 6 SAT-2 7 SAT-3 Pirbright 8 9 10 11 A O Asia-1 All 3 combined Sar/9/81 Bot/1/77 Sar/9/81 + Bot/1/77 Zim/7/83 KNP/19/89 Zim/7/83 + KNP/19/89 Bec/1/65 + KNP/10/90 Cruzeiro + Thailand + Iraq Manisa + Lausanne India Cruzeiro + Thailand + Iraq + Manisa + Lausanne + India 77 12 89 77 7 84 47 164 62 46 272 −5.740 1.634 0.858 1.749 1. We can randomly assign all vaccinated individuals in a trial of n animals a titre from the set of observed titres for that serotype.658 −1.155 −15.190 −102.744 1.E.

1.1. and the P-value indicative of whether signiﬁcantly more than half the animals were protected. allows us to compute the probability that signiﬁcantly more than half the animals in a trial would be protected as a function of the number of animals in the trial. Histograms showing the frequency distribution of titres induced by vaccines of different FMD serotypes.P. protected. The relationship between virus neutralising titre and protection No signiﬁcant interactions between neutralising antibody titre and serotype. Repeating this process 500 times each for a range of different possible sizes of trials.V. Results 3. / Vaccine 21 (2003) 3240–3248 3243 Fig. 3. Barnett et al. or antibody titre and strain were .

and 1. 2. A.05): a signiﬁcant relationship existed between titre and probability of protection against Sar/9/81 (P < 0. Determining the probability that a particular test vaccine has a speciﬁed/required PD50 Suppose that a vaccine concentrate of serotype Asia-1 from Pirbright was diluted X-fold and used to vaccinate eight test animals. Analysis of all three serotypes combined also revealed no signiﬁcant differences in intercepts between serotypes. O. 3. Therefore.843. SAT-2.252. In addition. 2.067. 3a and b). / Vaccine 21 (2003) 3240–3248 detected in any of the analyses described below. 3. and (KNP/10/90 + Bec/1/65) representing the three SAT strains are compared. Barnett et al. Institute for Animal Health. Analysis indicated a highly signiﬁcant effect of laboratory on the relationship between titre and protection. Pirbright Signiﬁcant relationships existed between antibody titre and probability of protection for all strains for which data were available for serotypes Asia-1. 3. When strains Sar/9/81.3244 P. ARC-Onderstepoort Veterinary Institute Of the two strains of SAT-1 examined.001) but insufﬁcient data existed to establish such a relationship for the other SAT-2 strain. the conﬁdence regions in parameter space which encapsulate the ‘true’ parameters governing the relationship between titre and protection with 95% probability. respectively (for models with strains combined within serotypes). The parameter estimates. . Zim/7/83. are angled ellipses (see Fig. An observation previously reported using independent data from tests involving other O. namely.001) but not against Bot/1/77 (presumably a consequence of the small number of potency results available for this strain). Manisa and Lausanne. signiﬁcant differences in model intercept were established between Sar/9/81 and Bot/1/77 (P < 0. no signiﬁcant differences were revealed in the intercepts of logistic models ﬁtted to the three strains of the A serotype. O. At 21 days post-vaccination. T95 values for serotypes A. A22 Iraq and A24 Cruzeiro. This means that uncertainty in the estimated value of the intercept is negatively correlated with uncertainty in the estimate of the slope. KNP/19/89. The estimates of slope and intercept for the logistic regressions exhibit negative covariance (see Table 2).3. a signiﬁcant relationship between titre and protection existed for the SAT-2 strain Zim/7/83 (P < 0.001). 2.2.086. A15 Thailand. the Zim/7/83 was found to be modelled by a lower intercept to the SAT-1 and SAT-3 strains (which were not distinguishable) indicating that this SAT-2 strain requires a higher titre to achieve the same level of protection compared to Sar/9/81. and 2. these animals were Fig. No signiﬁcant differences between titre and protection could be established for the SAT-3 strains Bec/1/65 and KNP/10/90 so these were combined and a signiﬁcant relationship between titre and protection was apparent (P < 0. KNP/10/90 and Bec/1/65. and SAT-3 vaccines which were 1.4.141. Similarly.858. The best ﬁtting models to four different subsets of the data. we therefore proceeded to analyse results from the different laboratories separately.V. The probabilities of protection with different titres of antibody for different subsets of the data sets that are statistically indistinguishable are indicated in Fig. of the O strain. and C vaccine strains [18]. or the two strains. and Asia-1 vaccines were 2. This is reﬂected in the T95 values for SAT-1. respectively (once again for models with strains combined within serotypes). covariances and T50 and T95 values are summarised in Table 2. with standard errors. 2. and A.

65. diluted Y-fold.0046. Using these titres and the estimated model (number 3) for the combined SAT-1 results from this laboratory from Table 2. and 10 (see Table 2). were: 2. / Vaccine 21 (2003) 3240–3248 3245 Fig. 2. 2. ti . 1. 3. 2.25. . Barnett et al. we would estimate that the expected proportion of animals protected would be ν ¯ = (1/10) 10 i=1 (exp(a0 + a1 ti ))/(1 + exp(a0 + a1 ti )) = 0. 1.10.30.15. 2. 2. The conﬁdence regions are for models 3. but from these results we anticipate that over 90% would be protected.15. As a second example suppose that 10 test animals had been vaccinated with a SAT-1 vaccine from Onderstepoort. and log antibody titre determined. n n/2 The quantity i=0 ν ¯ i (1 − ν ¯ )(n−i) yields a P-value of i 0.60. 2.P.5%) of these animals with these titres were protected from challenge). 1. The angle in the orientation of these ellipses arises from the negative covariance between estimates of slope and intercepts reported in Table 2. 6. Suppose the 21-day log titres were: 1.90260 (in reality seven of eight (87. 1.70. 9.80.50.40. 2. and 7 and 8.V. we would estimate that the expected proportion of animals protected would be ν ¯ = (1/8) 8 i=1 (exp(a0 + a1 ti ))/(1 + exp(a0 + a1 ti )) = 0. Using these titres and the estimated model (a0 and a1 — model number 10) this laboratory from Table 2. 1. and 1. The conﬁdence regions for the models ﬁtted to the data from vaccine potency trials for serotypes from the two laboratories. bled. so the probability that the PD50 really is X or less is in this case very small and we could be 99.70.54% conﬁdent that the PD50 of the stored vaccine concentrate was greater than X.40.90.85. Suppose the eight log titres. If the PD50 of the test vaccine was X then we would expect 50% of the animals to be protected.85.10. and 1.882 (in reality 7 of the 10 (70%) animals with these titres were protected from challenge). 3.15. 3.

which makes the test very costly and involves the examination of virus challenged animals to establish the protective ability of the vaccines. even using as many as 18 animals would not necessarily indicate 50% protection with any degree of conﬁdence. so the probability that the PD50 really is Y or less is in this case n n/2 ν ¯ i (1 − ν ¯ )(n−i) = 0. or because different relationships hold for the different serotypes studied in each of the different laboratories cannot be determined from the data available. previous studies have also documented differences in the test results between different laboratories [19]. However. The results are then . O. This takes into account not only the active substance. six to eight animals might provide sufﬁcient statistical power to conﬁdently establish required PD50 values. This is fewer animals than that currently stipulated by the European Pharmacopoiea. 4. In general. The number of animals required in a trial to establish with 95% conﬁdence that the vaccine protected 50% of vaccinated animals obviously depends on the data used but some examples using data analysed in this study are shown in Fig.V. We developed a simple statistical framework for analysis of challenge-free trials for which existing data suggests that for some serotypes. results from different strains of the same serotype usually conformed to a single logistic model (this was the case for three serotypes represented by more than one strain: A. 4. / Vaccine 21 (2003) 3240–3248 Fig. If the titres from each serotype analysed here were representative generally of those obtainable from vaccines prepared from these serotypes then only modest numbers of animals (as few as six) are required to establish that the most effective and consistent vaccines (those of serotype O and Asia-1) protect half the animals when diluted as they were in these trials. Barnett et al. The parameterisation of these models for each serotype enables the use of challenge-free trials to estimate the effectiveness of these vaccines.2% would be protected. 6–10). If the PD50 of the test vaccine was Y then we would expect 50% of the animals to be protected. Whether this is because of methodological differences in the adopted procedures. For the more variable titres obtained from SAT-1 and SAT-3 vaccines. we i=0 i could be 99. 4. This is true for foot-and-mouth disease vaccine as this vaccine always incorporates either aluminium hydroxide/saponin or mineral oil-based adjuvants. This in vivo potency assessment usually involves cattle. The probability of being able to show that the PD50 is signiﬁcantly greater than that used in these vaccine trials as a function of the number of individuals used in the trial. and SAT-3). Discussion This analysis reveals that the relationships documented between vaccinally induced antibody titre and protection against clinical foot-and-mouth disease differs between two different laboratories. i. which further limits accessibility and adds to the overall cost. Usually potency testing of inactivated vaccines is performed on the target species. but other components such as the adjuvants which are incorporated to further enhance the immunity of the vaccine. The number of individuals (the number of re-sampled titres) in each trial is indicated on the x-axis.3246 P. but from these results we anticipate that 88.66% conﬁdent that the PD50 of the vaccine tested was greater than Y. Parameter values are those for the combined strains model for each serotype (numbers 3. The extremely infectious nature of FMDV means that high level containment facilities are required to perform such tests. antigen.e. it appears that so long as sample sizes were not too small. Each point is the proportion of 500 re-sampled sets of titre values (from the indicated serotype) in which signiﬁcantly more than 50% of individuals in the set would be protected.0034 and in this case.

p. [6] Bercan A. [9] Pay TWF. A further important consideration is that often FMD vaccines can be multivalent containing three or four different strains of virus and it is not possible to undertake a challenge test with more than a single strain of virus at any one time. The use of serum neutralising antibody assay for the determination of the potency of foot-and-mouth disease vaccines in cattle. 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. Lorenz R. However.22–24]. Controle quantitatif du vaccin antiaphtheux. Thesis. These results are then extrapolated into percentages of protected animals within the vaccine dose groups and converted to a PD50 value by the Kärber method [20] or some similar procedure. Fontaine J. Vaccine 1987. Haydon was supported by the Wellcome Trust. individual analysis of the data from each laboratory showed that. ﬁrst. Report of the Meeting of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease.63:875–6. Czechoslovakia. In summary. Speciﬁcally. 99–111. Sur la corrélation entre le test de séro-neutralization sur sourceux et immunisation anti-aphtheuse cro´ ısee chez les bovins. it does show. Acknowledgements Daniel T. and thus induce a wider range of titres than would be expected from non-experimental use. Beziehung zwischen serumneutralisation und immunitat. References [1] European Pharmacopoeia 1997.7.V. FAO.12:1–11. Analyse statistique de la relation liant les titres d’anticorps neutralisant au pourcentage de protection bovine. Rec Med Vet (Alfort) 1968.5:60–4. either black or white. Such tests are often based on relatively small numbers of animals. Mh Tierheildk 1960. This test has been viewed as both statistically weak and inaccurate with a 90% conﬁdence interval for the PD50 value lying anywhere between 45 and 220% of the potency [21]. we do not claim that the titres included in the analysis here are representative of those that would be expected from ﬁeld use of market ready product. Data for serotype C from Pirbright could not be used in this analysis because no records of vaccinated but unprotected animals existed—the vaccine always protected vaccinates. Bericht uber die ergebnisse der grossversuchs 1957–1958 zur wirkaamkeitsprufung der Maulund klavenseuche-koncentratvakzine nach pyl unf der originalvakzine nach waldmann und kobe. [8] Pay TWF. O. The bootstrap analysis of the number of animals required to conclude with reasonable conﬁdence that signiﬁcantly more than half the animals in a group are protected (Fig. providing an alternative method for evaluating the vaccine potency required. Wittman G. 4) is only intended for illustratory purposes. Bull Off Int Epizoot 1969. therefore. to establish the lowest dilution of vaccine that would protect 50% of the animals. [2] Pay TWF. and second.64:153–61. Dohotaru U. and thus a logistic model could not be ﬁtted. [7] Stellman C.35:369–83. a signiﬁcant relationship can be established between antibody titre and probability of protection. that if a vaccine regularly induces high levels of protection then this number could be quite modest—possibly as low as 6–8. . Haas B. [3] van Bekkum JG. with no intermediary level in which an animal could be perceived as being partially protected. For example. that its very straightforward to use this kind of data to suggest the number of animals required in such trials. Results are.144:325–51.103:252–61. Develop Biol Stand 1986. [10] Ahl R. Rome. Bornarel P. Develop Biol Stand 1977. Relation entre titre d’anticorps neutralisants et protection des animaux après vaccination antiaphteuse. Hingley PJ. Barnett et al. and Asia-1 or SAT-1 and SAT-3 are combined. Lang R. The data of this sort collected at Onderstepoort and Pirbright permits a feasibility study of this approach. Some statistical and experimental design problems in the assessment of FMDV vaccine potency. Parker NJ. that avoids the use of virus challenge and requires only a single group of cattle administered with a vaccine diluted to a level equal to or greater than the required potency value (PD50 ) potency. O. This of course means that animals not protected will suffer the painful clinical manifestations of FMD and even some protected animals may undergo some pain due to the development of primary lesions at the site of challenge. In order to develop logistic models it is important that the data span a wide range of titres. 20–23 September 1988. Lorenz RJ. Hingley PJ. Uhlmann W. Tomescu A. Ann Ins Pasteur 1962. Lang R. We thank Victoria Edge for helpful discussions. Terre J. the European Pharmacopoeia monograph for foot-and-mouth disease vaccines requires three vaccine dilution groups of ﬁve animals per group.71:381–92. Foot-and-Mouth Disease (Ruminants) Vaccine 1997. statistical analysis of serological and protection data accrued from challenge/potency tests performed at two independent research laboratories and encompassing vaccine strains representing six of the seven serotypes of FMD indicates a signiﬁcant effect of laboratory on the rela- tionship between neutralising antibody titre and protection. A test approach that relies on large numbers of deﬁned sera that have an established correlation with potency and protection in the target species therefore seems an attractive alternative. [5] Mackowiak C. Assessment of potency of foot-and-mouth disease vaccines by means of antibody assays with sera from vaccinated cattle. no signiﬁcant differences were observed in the parameters of logistic models ﬁtted to combined strains within serotypes A. Prague. IV. By far the most explored area of investigation has been the correlation between protection and serological parameters such as neutralising antibody [2. and SAT-3. Petermann MB. Camand R. provided reasonable sample sizes are available. Data for some strains and serotypes (very usefully) includes vaccine used at higher dilutions. Neutraliserende antistoffen in sera van regen mond and Klauwzeer geente runderen. [4] Hecke F. Appendix 17. 1959. Furthermore. / Vaccine 21 (2003) 3240–3248 3247 based on whether generalisation of disease has occurred at sites other than that of challenge. Muntiu N. Utrecht. However. University of Utrecht.P. or when the strains from serotypes A. These models permit the development of a truncated test.

Sutmöller P. FAO. Appendix 6.102:193–8. [15] Finney DJ. Hingley PJ. Report of the Session of the Research Group of [19] [20] [21] [22] [23] [24] . p. Further studies on the assessment of potency of FMD vaccines by means of antibody assays with sera from vaccinated cattle. Czechoslovakia.10:699–706. / Vaccine 21 (2003) 3240–3248 the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease. Session of the Research Group of the European Commission for the Control of Foot-and-Mouth Disease. OIE Listed Diseases. Vieira A. Potency estimation of FMD vaccines according to antibody assay results. Rio de Janeiro. p. Gomes I. 1971. 3rd ed.1. Pay TWF. [14] Anon. Barnett et al. The relationship of neutralizing antibody titres for FMDV and the protection of cattle. Probit analysis. Denmark. 4th ed.1. Manual of Standards for Diagnostic Tests and Vaccines. Arche Exp Pathol Pharmakol 1931. Carabin H. [12] van Maanen C. Sutmoller P.V. [18] Ahl R.20:1505–14.1. Haas B. 45–9. [16] Sokal RR. An introduction to probability theory and its applications. 1968. Ahl R. [13] Barnett PV. Testing the efﬁcacy of FMD vaccines: relationships between test infection results and corresponding neutralization titres of vaccinated cattle. 15–18 October 1985. Brazil. Rome. Alternative potency test of FMD vaccines and results of comparative antibody assays in different cell systems and ELISA. Terpstra C. Boletin del Centro Panamericano Fiebre Aftosa 1984.162:480. Report of the Meeting of the Research Group of the Standing Technical Committee of the European Commission for the Control of Foot-and-Mouth Disease. Astudillo VM.39–40:57–62. FAO. Biometry. June 1990. Ofﬁce International des Epizooties. p. Vaccine 2002. Berliner und Munchener Tierarztliche Wochenschrift 1989. Vaccine 1992. Karber G. p. New York: Freeman. Part 2. Rome. Rolf FJ. [17] Feller W. Chapter 2. 20–23 September 1989. Prague. 77– 92. Rome. serum neutralizing antibody response and protection from challenge. 1988. Beitrag zur kollekiven Behandlung pharmakologischer Reihenversuche. Foot-and-Mouth Disease.3248 P. Lorenz RJ. Section 2. 1995. reduction and reﬁnement. [11] Bengelsdorff HJ. Lindholm. A review of emergency foot-and-mouth disease (FMD) vaccines. Hendriksen CFM. 51–60. Wittman G. Comparison of the liquid phase blocking sandwich ELISA and the serum neutralisation test to evaluate immunity in potency tests for foot-and-mouth disease vaccines. Foot-and-mouth disease vaccine potency tests in cattle: the interrelationship of antigen dose. Boletin del Centro Panamericano de Fiebre Aftosa 1980.45–50:31–4. London: Cambridge University Press. 2000. 91–8. Wittmann G. Laboratory animals in vaccine production and control: replacement. New York: Wiley. Dordretch: Kluwer Academic Publishers. FAO. 3rd ed.

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