-~----------------

Quality Assurance of C. perfringensEpsilon Toxoid Vaccines - ELISAVersus Mouse Neutralisation TestUte Rosskopf-Streicher, Peter Volkers, Kerstin Noeske and Esther WernerPaul-Ehrlich-Institut, D-Langen

SummaryClostridium (C.) perfringens is a Gram-positive anaerobicspore-forming bacterium. Disease caused by C. perfringens in-fection is called enterotoxaemia. C. perfringens strains areclassified on the basis of the lethal exotoxins formed by thebacteria. Epsilon toxin is one of the major lethal toxins andis formed by C. perfringens types Band D.C. perfringens is an ubiquitous bacterium. Infection occurs viafood, water, animal litter or soil. Affected animals includemainly sheep, pigs and cattle. C. perfringens infection mani-fests as pulpy kidney disease and diarrhoea in suckling lambs.Enterotoxaemia development is peracute in most cases. Animalsdie suddenly while grazing on the pasture, without any priorsigns of disease. Therefore, treatment is possible only in veryrare cases.Suitable immunoprophyZactic measures are the treatment ofchoice to combat the disease: Vaccines and immunosera havetherefore been used extensively for a long time.The requirements for quality, efficacy and safety testing of theinactivated vaccines are laid down in the Ph. Eur. in the mono-graph: Clostridium perfringens vaccines for veterinary use. Af-ter a marketing authorisation is attained, the product batchesmust be tested in laboratory animal models for their potencyagainst all vaccine components (Pharmeuropa, 1997).For potency testing (batch control) of C. perfringens types Band D, the induction of specific antibodies against epsilontoxin in rabbits must be verified. For this purpose, 10 rabbitsare immunised twice with the product to be tested. Their bloodis taken 14 days after the last immunisation and the serum ispooled. The pooled serum is then tested for its protective effect.This is done by means of the toxin neutralisation test in mice(optionally also in guinea pigs) in comparison with an interna-tional reference serum. The evaluation criterion is the deathrate of the mice in the test and reference groups after adminis-tration of lethal doses of epsilon toxin. The exact potency of thetest serum is given in International Units (IU). The tested serummust show a minimum content of 5 IV.This in vivo method requires a very high number of experimen-tal animals. Approximately 400 mice (or 50 guinea pigs) areused per vaccine batch.The monograph for C. perfringens vaccines, which has recent-ly been revised, expressly indicates that a validated serologicalmethod may be used for batch testing. In addition, a reference

Zusammenfassung: Wirksamkeitspriifung von C. perfringens Ep-silontoxoid-Impfstoffen - ELISA versus MausneutralisationstestClostridium (C.) perfringens ist ein grampositives Bakterium undanaerober Sporenbildner: Krankheiten, die durch C. perfringensVergiftungen entstehen, werden als Enterotoxdmien bezeichnet.Anhand der letalen Exotoxine, welche die Bakterien bilden, er-folgt die TypendiJferenzierung von C. perfringens. Das Epsilon-toxin ist eines dieser Zetalen Haupttoxine, die durch C. perfrin-gens des Typs B und D gebildet werden.Der Erreger ist ubiquitdr zu finden, eine Aufnahme erfolgt iiberdas Futter, Wasser, Einstreu oder Erdboden. Betroffen sind vorallem Schafe, Schweine und Rinder. Die Erkrankung auj3ert sichbeim Schaf durch die sogenannte Breinierenkrankheit undDurchfiille bei Saugliimmem.Der Verlauf der Enterotoxikdmie ist in den meisten Fallen per-akut, die Tiere verenden ohne vorherige Krankheitserscheinun-gen plotzlick auf der Weide und sind daher therapeutisch nur insehr seltenen Fallen behandelbar.Geeignete immunprophylaktische Maj3nahmen sind das Mittelder Wahl bei der Bekdmpfung der Krankheiten: Impfstoffe undImmunsera werden daher schon seit langem in groj3em Umfangeingesetzi.Die Anforderungen zur Qualitdt, Sicherheit und Wirksamkeit andie inaktivierten Impfstoffe sind im Europdischen Arzneibuch inder Monografie: Clostridium perfringens Impfstoffe fUr Tierefestgeschrieben. Die Produkte miissen nach Erteilung einer Zu-Zassung chargenweise in Labortiermodellen aufihre Wirksamkeitgegeniiber allen Impfstoffkomponenten iiberpriift werden.Fiir die Wirksamkeitspriifung (Chargenpriifung) von C. perfrin-gens Typ B und D ist die Induktion spezifischer Antikorper inKaninchen gegen das Epsilontoxin zu belegen. Hierfiir werdenzundchst 10 Kaninchen mit dem ru priifenden Prdparat zwei-malig immunisiert, das Blut der Tiere 14 Tage nach der letztenImpfung entnommen und das Serum gemischt. DiesesMischserum wird dann auf seine schiitzende Wirkung iiberpriift.Dies erfolgt mittels Toxinneutralisationstest in Mdusen(wahlweise auch Meerschweinchen) im Vergleich zu einem inter-nationalen Referenzserum. Auswertungskriterium ist dieTodesrate der Mouse in den Priif- und Referenzgruppen nachVerabreichung letaler Dosen von Epsilontoxin. Die genaue Wirk-samkeit des Priifimpfstoffes wird in Internationalen Einheiten(IE) angegeben. Das geprufte Serum muj3 einen Mindestgehaltvon 5 IE aufweisen.

Received 18 December 2003; received in final form and accepted for publication 17 February 2004

ALTEX 21, Suppl. Linz 0312004 65

ROSSKOPF-STREICHER ET AL.

serum known as clostridium multicomponent serum has beenavailable since 2000. The objective is to test vaccine batchesagainst this reference and by means of a competitive ELISA de-veloped in the precursor project, using a monoclonal antibodyfor direct determination of specific antitoxins in rabbit sera.This ELISA method was subjected to an international valida-tion to verify whether the protocol and the precision can betransferred within and between the participating laboratories.

Diese in vivo Methode ist mit einem sehr hohen Verbrauchan Versuchstieren verbunden. Pro Impfstoffcharge werden ca.400 Miiuse (oder 50 Meerschweinchen) benotigt.Die zwischenzeitlidi iiberarbeitete Monografie fia C. perfringensImpfstoffe weist ausdriicklich darauf hin, dafJ eine validierteserologische Methode fUr die Chargenpriifung verwendet werdenkann. Seit dem Jahr 2000 steht auch ein Referenzserum; ein so-genanntes Clostridium-Multikomponentenserum, zur Verfiigung.Mit dieser Referen; und mit der im Vorliiuferprojekt entwickeltenTestmethode eines ELISAs unter Verwendung eines mono-klonalen Antikorpers zur direkten Bestimmung spezifischer Anti-toxine in den Kaninchenseren sollen zukunftig Impfstoffchargengepriift werden.Dieser ELISA wurde einer internationalen Validierung unter-zogen, um die Ubertragbarkeit des Protokolls und die Prdzisioninnerhalb und zwischen den teilnehmenden Labors zu iiber-priifen.

Keywords: C. perfringens vaccines, replacement animal test, competitive ELISA, biological reference preparation, validation study

1 Introduction

Diseases of C. perfringens intoxicationare called enterotoxaemias. Five majortoxins are produced by bacteria of theC.perfringens types. Epsilon toxin is onemajor lethal toxin formed by C. perfrin-gens of the B and D types.In sheep, the sickness manifests as

diarrhoea in suckling lambs and so-called pulpy kidney disease. On the basisof the acute progression immunisationis an appropriate means to protect theanimals in addition to avoiding a changeof feeding.Toxoid vaccines are commonly used

for active immunisation. In Germany,four multivalent vaccines containingepsilon toxoid are licensed. For eachbatch of vaccine, quality, safety and effi-cacy must be demonstrated according tothe European Pharmacopoeia (Ph. Eur.)monograph 0363: "Clostridium perfrin-gens vaccine for veterinary use"(Pharmeuropa, 2002).Therefore, 10 rabbits are immunised

twice with the test product, bled twoweeks later. and the sera are pooled.The potency (induction of specific anti-bodies) is measured by comparing thequantity of the serum necessary to pro-tect mice (or other suitable animals)against a fixed dose of toxin with thequantity of reference serum required.

66

A content of 5 IU epsilon antitoxin permillilitre of C. perfringens vaccine oftypes B and D is prescribed. The revisionof the monograph explicitly favours vali-dated serological methods for controllingvaccine batches above the in vivo test(see Tab. 1).With the production of a specific

monoclonal antibody against epsilontoxin and the development of an ELISAfor the quantification of antibody induc-tion in vaccinated rabbits by Elvira Ebert(Ebert et al., 1998), a first important stepwas taken towards replacing the mouseneutralisation test. The next step was thesupply of the "Clostridia rabbit (multi-component) antiserum BRP batch 1" bythe European Directorate for the Qualityof Medicines (EDQM), allowing theevaluation of test sera by means of thisreference, which has a defined content on

epsilon antitoxin of 11 IV (Lucken et aI.,2001).

2 Animals, material andmethods

2.1 Laboratory animalsRabbits of the breed "White NewZealand" were purchased from CharlesRiver, Kisslegg, Germany. Body weightwas in the range of 1,800 g, and age wasbetween 3 and 6 months at the time ofimmunisation. Animals were kept infloor husbandry and were fed with rawvegetables and commercial pellets; waterwas available ad libitum.

2.2 VaccinesFour C. perfringens epsilon toxoid vac-cines of different compositions were in-

Tab. 1: Potency test of C. perfringens toxoid vaccines

up to now in the future

2-fold immunisation of 2-fold immunisation of10 rabbits 10 rabbitstest category: low exposure test category persisting

I-mouse neutralisation test (fn~vFvio)~.

serOlogy (in vitro)test category: severe suffering test category replaced

Amount of animals per batch: Amount of animals per batch:• 10 rabbits • 10 rabbits• approx. 400 mice or 50 guinea pigs • no more mice or guineapigs

ALTEX 21, Suppl. Linz 0312004

m..... ROSSKOPF-STREICHER ET AL.---~--------------------------------------

2.3 ImmunisationprocedureEach vaccine was administered to 10rabbits as required in the monograph"Clostridium perfringens vaccines for Tab. 2: Vaccine constituentsveterinary use". The animals receivedone dose as stated on the label and asecond dose after four weeks. Bloodwas taken two weeks after the secondimmunisation by cardiac puncture underanaesthesia. The samples were cen-trifuged (6,000 g for 15 min), and equalvolumes of individual sera of one vac-cine group were combined to produce aserum pool. Vials with 1 rnl serum eachwere freeze-dried and stored at 2°_8°C.

eluded (Tab. 2). All vaccines are combi-nation products and are licensed for theGerman and European markets.

2.4 ReferenceserumThe Clostridia rabbit (multi-component)antiserum BRP Batch 1 was used as ref-erence with an assigned activity of 11 IUC. perfringens epsilon antitoxin per vial.

2.5 Negative serumThe negative control serum was preparedby taking blood from unvaccinated rab-bits. The status of the animals was speci-fied pathogen free (spf). The serumpreparation was performed as describedin 2.3.

2.6 Monoclonal antibodyThe monoclonal antibody (mAb) 5B7against epsilon toxin was generated inhybridoma cells at the Paul-Ehrlich-In-stitut cultured in vitro (miniPERM). In aserum neutralisation test (SNT) usingMDCK cells, no cytopathogenic effect(CPE) was observed after addition oftrypsin-activated epsilon toxin at a dilu-tion of 1:6,400 of the hybridoma super-natant. Protection was also shown in amouse challenge test: The supernatant,diluted 1:10, was mixed separately with10 different toxin concentrations each.The mice were protected even against thehighest amount of toxin (270-fold LD50)(Ebert et aI., 1999).

ples are titrated and pre-incubated. With-out washing the plate, the purified mAb5B7 against epsilon toxin is added ascompetition against the polyclonal serabeing tested. In a further step, peroxidase

labelled goat anti-mouse IgG binds to themAbs. The reaction is visualised by ad-dition of tetramethyl-benzidine/hydrogenperoxide as substrate. Controls includedon each plate are a negative serum and

Antigen Adjuvant Preservative(No.of vaccines) (No.of vaccines) (No.of vaccines)

C. petiringens type B (4) Aluminiumhydroxide (2) Thiomersal (3) ~C. petiringens type C (4) Alaun (1) - Formaldehyde (2)C. petiringens type D (4) EmulsifiedOil(1)

-tr:chauvoei (3)

C. septicum (3)

I~VYi type B (3)C. novyi type D (1)C. tetani (3)E. coli (1)

Mannheimia haem%Ytica: (1}Pasteurella treha/osi (1)

The numbers in brackets are representing the amount of vaccines inwhich the constituentis to find e.g.: C. petiringens type B (4) indicates, it is included in all used vaccines.

r~I:::::.-.:.. ../ .: SUDstrate: .. .

: .Te~r~ nie~hyt ben~dilie :.. hyarogeFl' peroxide .r·-:-· tI·.. ·~

PO

Peroxidase labelled

anti-rnouse antibody

Antigen specific

Antigen-specificpolyclonal antibody

2.7 ELISAThe applied method is an indirect com-petitive ELISA. First, the epsilon toxin isused as antigen to coat the multi-wellplates. In a second step, the serum sam- Fig. 1: Principle of the indirect competitive ELISA.

ALTEX 21, SuppL Linz 03/2004 67

ROSSKOPF-STREICHER ET AL. m....-------------------------~--

"':t:: 40,0c;:)'ie0:;::;co

30,0E~g>-Q 20,0c~Q,CIl>~ 10,0a;a:

TS 1

mean = arithmetic mean

TS 2 TS 3 TS4

Fig. 2: Relative potency values calculated for each test serum, representing the mean value of four measurements in eachlaboratory. Required content per millilitre vaccine: 5 IU.

the conjugate control. The tested sera arecompared with the reference serum(clostridia rabbit antiserum, BRP Batch1) also titrated on each plate. Figure 1shows the principle of the test.

2.8 Study designThe study was performed in sevenlaboratories of different internationalauthorities. In order to assure anonymity,each laboratory was coded with a capitalletter: Laboratory A to Laboratory G.The test method was developed by

Elvira Ebert and had already been pre-validated in four laboratories (Ebert etal., 1999). For the validation study - incontrast to the pre-validation study -some modifications of the test protocolwere undertaken, including the use of the"Clostridia rabbit (multi-component) an-tiserum BRP Batch 1" as a reference tocompare the potency of the tested sera.The aim of the validation study was toprove reproducibility within different in-ternationallaboratories.Therefore, the participants were sup-

plied with the ELISA kit, including thereference serum, the monoclonal antibody,the test sera, the control sera, the conju-gate, skim milk powder as well as the mul-ti-well plates and mylar sealing tapes.

68

Test sera 1-4

I DA DB DO DE []F IIG []mean I

The participants were asked to testeach of the four test serum samples fourtimes on four different days (in order toevaluate the inter-day precision).

2.9 Statistical methodsThe potency of the test preparations wascalculated by analysing individual assaysas parallel line assays (Finney, 1978),comparing the response with log concen-tration. If necessary, a square-root trans-formation was applied to the responsein order to obtain better linearity. Forassessment of the intra- and inter-labora-tory variation, the geometric coefficientof variation (GCV) (Kirkwood, 1979)was provided for each sample.All analyses were performed using

SAS, Version 8.2 (SAS, 1999-2001).

3 Results

All seven participating laboratories pro-vided their results. The data of one labo-ratory had to be excluded from the anal-ysis owing to technical problems.Linearity was given at dilutions of 1:16to 1:128.The intra-assay precision coefficients

of variation were found to be in the rangeof 10% up to 20%, depending on the

laboratory. The test demonstrated goodreproducibility between the different par-ticipants. Figure 2 shows the calculatedpotency values for the four test sera andtheir mean value. All sera indicated apotency above the required 5 ID. The re-sults were very close. A greater variationwas found only within the results for theproduct with the highest potency.

4 Discussion

The vaccines used for the study all clear-ly passed the potency requirements. Thisresult was in agreement with the infor-mation given by the manufacturers. Theinduced potencies were in the range of10 international units per rn1 for thevaccines 1, 3 and 4 (TS 1, TS 3 andTS 4). The Vaccine 2 induced muchhigher antibody titres as can be seen inthe corresponding test serum 2. Like theother products as well, it is a multivalentvaccine containing different antigens.The details given on the package insertindicate a very high content of bindingunits of Clostridium perfringens epsilontoxoid. Therefore it could be concludedthat the high content directly correlateswith the increased antibody induction inthe rabbit sera.

ALTEX 21, Suppl. Linz 03/2004

ROSSKOPF-STREICHER ET AL.-~----------------

The validation study confirmed thetransferability and reproducibility of thecompetitive ELISA and, for this reason,the applicability of testing vaccine batch-es for their content of epsilon toxin.A high specificity is given on the basisof the monoclonal antibody: This mono-clonal demonstrated protection againstepsilon toxin both in the cell test and in amouse protection test. In the ELISA, thepolyclonal rabbit sera must competeagainst the monoclonal, and the amountof displacement correlates with the effi-cacy of the test product. The second tool,the clostridia rabbit reference preparationwith a defined content allows the calcula-tion of the potency using a parallel linemodel.

For testing of one vaccine batch, themonograph prescribes two immunisa-tions of ten rabbits four weeks apart.Fourteen days after the second immuni-sation, the animals are bled and equalblood volumes of each rabbit are blendedin a serum pool. Investigations on the re-duction of this number of animals unfor-tunately confirmed the necessity of theten animals. However, sufficient bloodfor the ELISA can be obtained bypuncture of the ear artery, so that killingof the animals is not necessary.

With the availability of this serologicalassay the use of the mouse neutralisationtest will no longer be justified. Manufac-turers as well as control authorities willbe able to test the content of epsilon

ALTEX 21. Suppl. Linz 0312004

toxin in vaccines by this alternativemethod. The advantages are summarisedas follows:• Complete abolition of the mouse neu-tralisation test,• reduction of time of exposure,• no exposure with infectious material.

ReferencesEbert, E., Oppling, V, Werner, E. und

Cussler, K. (1998). Entwicklung undPravalidierung von Alternativme-thoden zur Wirksamkeitspriifung vonClostridium perfringens- Impfstoffen.ALTEX 15, Supplement, 59-61.

Ebert, E., Oeppling V, Werner E. andCussler K. (1999). Development andprevalidation of two different ELISAsystems for the potency testing ofClostridium perfringens b- and e-tox-oid containing veterinary vaccines.FEMS Immunology and MedicalMicrobiology 24, 299-311.

Finney, D. J. (1978). Statistical methodsin biological assay (3rd Edition). Lon-don: Charles Griffin.

Kirkwood T. B. L. (1979). Geometricmeans and measures of dispersion.Biometrics 35, 908-909.

Lucken, R., Daas, A. and Esposito-Farese, M.-E. (2001). Collaborativestudy for the establishment of a Euro-pean Pharmacopoeia Biological Refer-ence Preparation for Clostridia anti-serum for serological potency testing

of clostridial vaccines for veterinaryuse. Pharmeuropa Spec. Issue Biol.2001 Feb; 2000 (2), 65-87.

Pharmeuropa (1997). Clostridium per-fringens vaccine for veterinary use.Pharmeuropa vol. 9, No 4, 648-651.

Pharmeuropa (2002). Clostridium per-fringens vaccine for veterinary use.monograph 0363. Pharmeuropa 2002Jul 14.3, 2250-2251.

SAS 1999 - 2001. SAS Institute Inc.,Cary NC.

AcknowledgementsThe study was supported by the GermanMinistry of Education and Research(project 0312636). We would like tothank EDQM for contributing theClostridia rabbit antiserum Ph. Eur. BRP1, and we are very grateful to the partici-pants for their participation and co-oper-ation in this study. We also thankMelanie Schindler for performing thetests and preparing the study.

Correspondence toUte Rosskopf-StreicherPaul-Ehrlich-InstitutPaul-Ehrlich-Str. 51-59D-63225 LangenGermanyphone: +49-6103-77 7415fax: +49-6103-77 1254e-mail: rosut@pei.de

69