CENTRM.ELANDBOUWCATALOGU S Promotor: dr.A .va nKauune n hoogleraar ind emoleculair ebiologi e Co-promotor: dr.A.C .Minso n hoogleraar ind evirologi e aand eUniversit y of Cambridge,Cambridge , Engeland C.S. McLean-Pieper MONOCLONAL ANTIBODIEST O HERPES SIMPLEX VIRUS TYPE 2 Proefschrift terverkrijgin gva nd egraa dva n doctor ind elandbouwwetenschappen , opgeza gva nd erecto rmagnificus , dr.C.C .Oosterlee , hoogleraar ind e veeteeltwetenschap inhe topenbaa r teverdedige n opvrijda g3 Septembe r 1982 desnamiddag st evie ruu r ind eaul a vand e landbouwhogeschoolt eWageningen . 'Ut.i&fW'Oi ACKNOWLEDGEMENTS. I would like to thank the following people for their help during the various stages of the work described in this thesis. Without them it would never have been written. First of all, Tony Minson, whose support and encouragement as my supervisor have been invaluable. He was always available to give advice and practical help when problems.arose. I have learned much form his critical supervision, both in the practical work and in the writing of this thesis. Ab van Kammen, especially for his help and comments during the writing. TonyNash ,fo rhi sinvaluabl ehel pwit h theanima l experiments, andfo rhi sdiscussio nan dcomment so nman y aspectso fth ework . David Hancock,fo rhi sexcellen t technicalassistanc e duringth e laterstage so fth eproject . AnneBuckmaster ,wh oprovide dth edat a involving theantibodie s AP7an dAP12 ,an dwa salway savailabl efo rfriendl ydiscussion . ProfessorP .Wildy ,wh omad e itpossibl e form et owor k inth e departmento fpathology . DrS .Buchan ,wh oprovide d someo fth edat ao nantibod yLP1 . Andal lth emember so fth edivisio n of virology, forprovidin g sucha pleasan tatmospher et owor kin . I also want tothan km yhusband , Robert,whos e help withth e processing of this thesis.Hi scontinuou s support hasbee n invaluable. Asa Ph. Di sals o thecompletio no fa scientifi c education,I would liket othan km yparent sfo rgivin gm eth eopportunit yt o got ouniversity ,an dfo rprovidin g sucha goo d homebackground . fijtuoiio)) ^02 ,STEIXINGEN I. Theobservatio nb ySteven san dCook ,tha tanti-Herpe sIg Gi s importantfo rmaintainin glaten tinfection s isinvalidate d in theligh to fSekizaw ae tal' sfinding stha ti nth eabsenc eo f herpesspecifi c antibodies,viru slatenc ywa sstil lmaintained . Sekizawa,T. ,Openshaw ,H. ,Wohlenberg ,C .an dNotkins ,A.L . (1980). Science 210:1026-1028 . Stevens,Y.G .an dCook ,M.L . (1974). J. Immunol.113 :1685-1693 . II. Congenital humancytomegaloviru s infectionsar ea majo rcaus e ofabnormalitie si nth enewborn ,bu ti nvie wo fth epossibl e oncogenicpotentia l of thevirus ,th evaccinatio no fyoun gwome n withliv eattenuate dviru si sunjustified . Elek,S.P . andStern ,H . (1974). Lancet1 :15 . Osborne,J.E . (1981). J. Inf.Dis .143 :618-630 . III. RNAtumo rviruse sar eno timportan tagent so fhuma ncancer ,bu t thestud yo f theseviruse sha sle d toth e identificationo fgene s andprotein s involved inth e inductiono fa wid erang eo fneoplasti c diseases. Parada,L.F. ,Tabin ,C.Y. ,Shih ,C .an dWeinberg ,R.A . (1982. Nature 297:474-478 . Kaplan,H.S . in "MolecularBiolog yo fRNA-Tumo rViruses" ,Ed . J.R. Stephenson. Acad.Press ,Ne wYor k (1980). IV. Thedevelopmen to f single-cellprotoplast s forth estud yo fplan t virusreplicatio n inth eearl y1970' sheralde d ane wer ai nplan t virology. However,th eoutcom eha sbee ndisappointin g intha t theprogres smad e inplan tviru sreplicatio n inth epas tte nyear s doesno tcompar ewit hth esucces si n-th estud yo fanima lviru s replication following theintroductio no fanima l tissueculture . Tabeke,I. ,Otsuka ,Y .an dAoki ,S . (1968). Plantan dCel lPhysiol . 9:115-124 . V. Therecen tproposa lb yPrusine re tal . fora "prion"a sth e infectiousscrapi eagen t ishighl yunorthodox . AsKimberli n pointsout ,a muc h simplerworkin ghypothesis ,involvin g infectious nucleic acid,whic h fitsal lth eestablishe d factsca nb efound . Kimberlin,R.H . (1982). Nature 297:107-108 . Prusiner,S.B . (1982). Science 216.-136 . VI. Thewidesprea d useo f liveattenuate d poliovirusvaccine sha s ledt oth evirtua leliminatio no fpoliomyelitis ,bu toffer sn o i prospectso feliminatin g thevirus . Countriesusin g theliv e attenuatedvaccin e should adopt thepolic yalread yuse d inSwede n and theNetherland san dbegi nusin gkille dvaccine s inorde r to achieveeliminatio no fth evirus . Horstmann,D.M. ,Niederman ,J.C .an dPaul ,J.R . (1959).J.Amer . Med.Ass .170 :1-8 . Salk,J .an d Salk,D . (1977). Science195 :834-847 . VII. Theus eo fPMS Fa sa proteas e inhibitordurin g theisolatio no f plantprotein si so flittl evalue . Borserse tal . (1982). Virology116 :236-249 . 7111. The studyo f temperature-sensitivemutant so fanima lviruse sca n yieldusefu linformation ,bu tth eoccurrenc eo f silent,doubl ean d suppressormutation smak e interpretationo fresult sfro mth estud y of singlevira lmutant shazardous . Ahmed,R. ,Chakraborty ,P.R. , Graham,A.F. ,Raraig ,R.F .an d Fields,B.N . (1980). J.Virol .34 :383-389 . Cox,N.J. ,Kendal ,A.P. ,Maassab ,H.F. ,Scholtissek ,C .an d Spring,S.B . (1981). In "Thereplicatio no fNegativ eStran d Viruses".D.H.L .Bisho pan dR.W .Compans ,eds .p.405 . Elsevier, NorthHolland . IX. TheEnglis h systemo fcompetitiv eUniversit yentranc e examination ispreferabl et oth eDutc h systemo fa lottery . Proefschriftva nCorneli aS .McLean-Piepe r 3Septembe r 1982 TABLE OF CONTENTS. Scopeo f the Investigations 1 Chapter 1:Introductio n 3 Significanceo fmonoclona l antibodies invira lresearc h 4 HerpesSimple xViru sprotein s 5 Controlo fvira lprotei nsynthesi s 6 Polypeptideso f thevirio n 7 Glycoproteins :structur ean d function 7 Glycoproteins astarget so f theimmun esyste m 9 Glycoproteins astarget so f neutralising antibodies 10 Roleo fglycoprotein s inADC Can dAbC ' 11 Glycoproteins astarget so fcytotoxi cT-cell s 11 Cross-reactive antigens inth eHerpe sViruse s 12 Clinicalapplication so fmonoclona lantibodie s toHerpe s SimplexViru s 13 Chapter 2;Fusio nexperiment s 15 Introduction 15 Materialsan dMethod s Virus 15 Cells 16 Mice 16 ReversePassiv eHaemagglutinatio n 16 Couplingo f antibody toplasti c surfaces 16 Labellingo fprotein susin g 125I 17 Radioimmunoassay 17 Immunisationschedul e 17 Fusion 18 Cloning 18 Labellingo f secreted imunoglobulins 18 Ouchterlony test 19 Results A.Preliminar y Experiments 19 Immunisationschedul e 19 Assaysystem s 19 Discussion 21 Immunisation 21 Assaymethod s 22 B.Fusio nexperiment s 22 Raisingo f serumo rasiti c fluid 26 Immunoglobulins secretedb y thehybridoma' s 26 Discussion 27 Chapter 3;Antibodie s LP1an d LP4. 29 Introduction 29 Materialsan dmethod s 29 Cellsan dviru s 29 Radioimmunoassay 29 Fluorescence 29 Preparationo f isotope-labelled antigen 29 Immunoprecipitation 30 Summary 63 Samenvatting 64 Tableo fantibodie s 66 References 67 Abbreviations 79 Identificationo f targetantigen s 30 Neutralisation 31 Results 31 Identificationo f targetantigen s 31 LP1 31 LP4 32 Cross-reactivity withothe rHerpe sSimple xViru sstrain s 32 Immunofluorescence 32 Discussion 35 Chapter 4;Antibodie s toglycosylate d proteins 37 Introduction 37 Materialsan dmethod s 37 Virusstrains ,cell san dmediu m 37 R.I.A. 37 Neutralisation 37 Immunoprecipitation 38 Fluorescence Rosetting 39 Couplingo f IgGt osepharos ebead s 39 Purificationo f IgG 39 Results 39 I.Antibodie sdirecte d againstg D 39 Immunoprecipitations 40 Neutralisation 40 Cross-reactivity withHSV- 1 41 Competitionbindin g 43 Fluorescence 44 Rosetting 44 Identificationo fgD-precurso r 47 II.Antibodie sdirecte d againstg Can dg E 47 Immunoprecipitations 47 Cross-reactivityo f LP5an d LP6wit hHSV- 1 48 Neutralisation 48 Fluorescence 48 III.Antibodie sLP7 ,LP 8an d LP9 48 Discussion 50 Antibodies tog D 50 Antibody LP5 50 Antibody LP6 AntibodiesLP7 ,LP 8an dLP 9 52 Chapter 5;Antivira lrol eo f LP2an dLP 3i nviv o 53 Introduction 53 Materials andmethod s 54 Virus,cell s 54 Mice 54 Inoculationo fmic e 54 Infectivityassay s 54 Seraan dasciti c fluids 54 Results 54 Effecto f LP2an d LP3o n infectionwit hHSV- 1 55 Effecto fLP 2an dLP 4o n infectionwit hHSV- 2 55 Effecto f LP2an d LP3o nth eestablishmen to f latency 58 Discussion 59 Discussion 61 SCOPE OF THE INVESTIGATIONS. In 1975, a method was developed by Kolher and Milstein for the production of large quantities of monospecific antibodies.They isolated a HAT-sensitive mouse myeloma cell line which, after fusion with spleen cells from an immunised mouse, produced hybrid cells of predifined specificity. Large quantities of monoclonal antibodies against any given antigen can be obtained in this way. The technique has been used successfully for the production of monoclonal antibodies against many different antigens, such as tumor antigens, lymphocyte markers, human leucocyte interferon and many viral antigens. (Koprowski et al, 1978; McMichael at al,1979; Secher and Burke,1980; Wiktor and Koprowski,1978; Gerhard et al,1977.) These antibodies have proved useful tools to probe and characterise many biological systems. Their application to clinical diagnosis and therapy and to large scale purification of certain antigens will lead to a further understanding of the antigenic determinants of the virus, the mechanism by which they infect cells, the importance of neutralising antibodies and the interrelationships of different viral proteins. They will provide a source of specific reagents that can be used to clearly define antigenic variationss in a given virus type. In recent years,there have been many reports on identification and characterisation of Herpes Simplex Virus proteins, (for a review: see Spear and Roi zman ,1 980 ). The research in this field has been greatly hampered by the complexity of the virus, the great number of virus-specific proteins and the lack of knowledge regarding their interrelationships. Monoclonal antibodies will be useful tools in providing more information about the unique identity and relationships between the proteins coded for by this virus.