Production of Influenza Virus Complement-Fixing Antibody

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 1975, p Copyright C) 1975 Americn Society for Microbiology Vol. 2, No. 4 Printed in U.S.A. Production of Influenz Virus Complement-Fixing Antibody in Mouse Ascitic Fluid LENDELL A. WHITE,* WILLIAM C. GAMBLE, AND W. ADRIAN CHAPPELL Bureu of Lbortories, Center for Disese Control, Atlnt, Georgi Received for publiction 17 June 1975 Suspension cultures of BHK-21/13s cells infected with either A/Hong Kong/8/68 or B/Msschusetts/3/66 strins of influenz were used to prepre type-specific influenz ribonucleoprotein-immunizing ntigens. These ntigens were used in compring four immuniztion schedules in the production of immune mouse scitic fluids. Lrge volumes of scitic fluids were obtined by these schedules. These fluids contined type-specific complement-fixing ntibody nd were devoid of nonspecific host tissue or heterologous cross-rections. Guine pig ser contining ntibodies to the soluble or ribonucleoprotein (RNP) ntigen of the influenz virus re used in identifying influenz types A nd B by the complement fixtion (CF) test. These type-specific ntiser, prepred following procedures reported by Chppell et l. (5) nd Lennette nd Schmidt (12), serve s qulity controls for the CF test in the serodignosis of influenz infections. Problems in obtining guine pigs without prmyxovirus CF or hemgglutintion-inhibition (HAI) ntibodies nd in keeping these nimls free of infection with other respirtory gents stimulted serch for more suitble method for producing influenz type-specific immune regents. Immune mouse scitic fluid (IAF) hs been shown to be comprble to immune serum in potency nd specificity of rections in CF, HAI, nd neutrliztion tests (15). Severl investigtors hve described methods for producing lrge mounts of IAF-contining ntibodies in high titers. Herrmnn nd Engle (8) demonstrted HAI ntibody levels for influenz nd Newcstle disese virus in srcom 180-induced scitic fluid. Ksel et l. (11) produced orthomyxovirus nd prmyxovirus HAI ntibodies nd coxsckievirus-neutrlizing ntibodies in mouse scitic fluids by immunzing with virl ntigen plus bcteril-djuvnt mixture. Berkovich (1) nd Fbiyi nd Wenner (7) described CF nd neutrlizing ntibodies to picornviruses in mouse scites formed with Erlich tumor cells. In recent yers, srcom 180/TG cells in combintion with Freund djuvnt hve been used extensively in the preprtion of rbovirus IAF contining CF, HAI, nd neutrlizing ntibodies (2, 15, 16). We hve investigted the preprtion nd use of IAF prepred ginst type-specific influenz RNP ntigens s replcement for immune guine pig serum. MATERIALS AND METHODS Mice. Rndom-bred, 8-week-old, ICR, white, femle mice were used to propgte srcom 180/TG cells nd to produce scitic fluid. Srcom 180/TG. Srcom 180/TG cells were received from Aln C. Srtorelli. They were mintined s 10% cell suspension in 10% glycerol sline nd stored t -70 C. Immunized mice were inoculted in the peritonel cvity with 0.2 ml of 10% suspension of recent pssge of srcom 180/TG cells to stimulte scites formtion. Adjuvnt. Freund complete djuvnt (FCA) consisting of 85% Drkeol 6-VR (Pennsylvni Refining Co., Butler, P.), 15% Arlcel A (Hill Top Reserch, Inc., Mimiville, Ohio), nd 50 mg of desiccted Mycobcterium butyricum (Difco Lbortories, Detroit, Mich.) ws utoclved t 15 lb/in2 t 121 C for 45 min nd stored t 4 C until used. For immuniztion, ech ntigen ws mixed with n equl volume of FCA. A stble emulsion ws prepred by vigorously shking the mixture in n Interntionl Centrifuge (model PR-2) equipped with shker hed no nd operted t 1,b00 rpm for 30 min t 4 C (9). Influenz viruses. Influenz strins A/Hong Kong/8/68 (H3N2) nd B/Msschusetts/3/66 were used in prepring virl nd RNP-immunizing ntigens. The llntoic cvity of 10-dy-old embryonted chicken eggs ws inoculted with 0.2 ml of 10-3 virus dilution in phosphte-buffered sline, ph 7.2. After incubtion t 33 C for 3 dys, the eggs were chilled t 4 C, nd the llntoic fluids were pooled nd clrified by centrifugtion t 500 x g for 15 min. These llntoic fluids were used in the intrnsl (i.n.) inocultion of mice s well s in the inoculum for cell cultures used in prepring RNPimmunizing ntigens. The hemgglutintion (HA) titers of the llntoic fluids rnged from 1:320 to 1:1280. The infectivity titers of the inocul of the BHK-21/13s suspension cultures were determined by hemdsorption in primry rhesus kidney cell cultures (12). These titers were 1058 men infective 287

2 288 WHITE, GAMBLE, AND CHAPPELL doses per ml for A/Hong Kong/8/68 nd men infective doses for B/Msschusetts/3/66. Cell cultures. The BHK-21/13s suspension cultures were grown in Wistr growth medium (5) in 1- liter, screw-cp, Erlenmeyer flsks on mgnetic stirrer. The Wistr growth medium ws Egle miniml essentil medium prepred in Erle blnced slt solutions modified to contin double concentrtion of vitmins nd mino cids (except 1 x glutmine), supplemented with 0.1 mg of ferric nitrte per liter, 4.5 g of glucose per liter, 10% tryptose phosphte broth (Difco Lbortories), nd 10% hetinctivted fetl clf serum. Cultures were incubted t 34 C nd were subdivided every 7 dys by diluting 50-ml liquot of cell suspension to strting cell concentrtion of 5 x 105 cells per ml. Every 48 h n dditionl 200 ml of growth medium ws dded until the finl volume reched 800 ml. Before inocultion, spinner cultures were centrifuged t 500 x g for 15 min, nd the cells were resuspended in 200 ml of mintennce medium consisting of Egle miniml essentil medium without serum. An llntoic fluid inoculum ws dded to the cell suspension nd llowed to dsorb with continuous gittion for 1 h t 34 C. After dsorption, the volume of medium ws incresed to 800 ml, giving n verge cell count of 1.5 x 106 cells per ml. The suspension cultures were monitored dily for HA titer nd djusted to mintin ph of 7.2 to 7.4. Cells were counted dily, nd the vibility ws determined by trypn blue permebility. The cultures were hrvested when the suspension contined 85 to 95% nonvible cells nd were stored t -70 C until processed. Preprtion of RNP ntigens. The cell culture mteril ws processed ccording to modifiction of the technique of Rott et l. (14). The culture ws concentrted to one-eighth of the originl volume by using dilysis membrnes nd crystlline Crbowx (PEG-20M) t 4 C (3). Virl hemgglutinins were removed by exhustive dsorption with humn "O" erythrocytes. The RNP ntigen ws precipitted by cidifiction with 1 N HCl to ph 4.5 t 4 C. The precipitte ws pcked by centrifugtion t 600 x g for 30 min nd resuspended in 0.01 M tris(hydroxymethyl)minomethne buffer, ph 8.5, t concentrtion 'of one-twentieth of the originl volume. Immuniztion of mice. Mice were immunized in groups of 25 ccording to the immuniztion schedules presented in Tble 1. Influenz-infected llntoic fluids were given by the i.n. route in 0.25-ml mounts s the first inoculum of schedules 1 nd 4. Emulsions of equl volumes of RNP ntigen nd FCA were dministered intrperitonelly i.p. in 0.5- ml mounts in ll of the schedules. Ascitic fluid nd serum. Ascitic fluids were withdrwn by prcentesis with n 18-guge needle ttched to 30-ml syringe. Mice were initilly tpped 7 to 10 dys fter they were inoculted with srcom cells, nd the timing of lter tppings depended upon the ccumultion of fluid. The fluids from ech tp were pooled nd llowed to stnd t room temperture for pproximtely 1 h. The fibrinous clot ws seprted from the fluid by filtrtion through double lyer of sterile guze. The fluids were centrifuged t 16,000 x g for 30 min nd stored t -70 C. Bcteril contmintion ws eliminted by filtrtion through 0.22-,um membrne filter (Millipore Corp., Bedford, Mss.). All surviving mice were bled t the end of the experiment, nd the ser were pooled. Serology. The microtiter Lbortory Brnch Complement Fixtion technique (13) ws used in performing the CF test. Block titrtions were used to determine the optiml IAF, ser, nd ntigen dilutions. The HA titers were determined by mcrotechnique, nd HAI tests were performed by using the microtiter procedures described by Hierholzer et l. (10). The serologic ntigens were prepred ccording to previously described techniques (5). RESULTS Infected BHK-21/13s suspension cultures were monitored dily for the presence of hemgglutinins nd for the percentge of vible cells remining in the culture. The results of the culture monitorings nd the CF titers of the immunizing ntigens re presented in Tble 2. The cultures were hrvested for the preprtion of RNP-immunizing ntigens when more thn 85% of the cells were no longer vible or on dy 4 fter inocultion. Dily observtions of TABLE 1. Dy Immuniztion schedules in producing immune mouse scitic fluid Schedule no. J. CLIN. MICROBIOL [ 4 0 IN IP IP IN 7 Ipb IP IP 14 IP IP IP 21 IP Srcomc IP 23 IP 28 Srcom IP 30 IP 33 Prcentesis 35 Srcom IP 37 IP 40 Prcentesis 42 IP 47 Prcentesis 49 IP 57 Srcom 59 IP 69 Prcentesis IN, Intrnsl instilltion of 0.25 ml of infectious llntoic fluids. b IP, Intrperitonel injection of 0.5 ml of n equl volume emulsion of RNP ntigen nd FCA. e IP injection of 0.2 ml of 10% suspension of srcom 180/TG cells.

3 VOL. 2, 1975 PRODUCTION OF CF ANTIBODY IN ASCITIC FLUID 289 TABLE 2. Results ofdily monitoring ofbhk-21113s suspension cultures forpreprtion of RNP-immunizing ntigens Vible cell HA titerb count (x 104 no. VuID0 inocu- of RNP nlo. ID50 titer lum (ml) Zero At ntigenc time hr vest Expt Virus nd Vol of cells/ml) Dys fter inocultion CF titer I AIHK/8/ < ID5dml < II < < I B/mss/3/ < ID5dml II < < I Uninoculted cell <2 <2 <2 <2 <2 <4 II cultures <2 <2 <2 <2 <2 <4 ID50, Men infective dose. b HA titer expressed s the highest dilution of ntigen producing complete hemgglutintion with chicken erythrocytes. c CF titer expressed s dilution fctor of the optiml ntigen dilution s determined in block titrtions with specific ntiser. the infected suspension cultures showed n increse in hemgglutinins from dy 0 to the time of hrvest nd corresponding decrese in cell vibility. When 3 volumes of inoculum were compred, greter increse in HA titer ws observed with the smller inoculum, but t the time of hrvest, comprble HA titers were observed with ll cultures. Similr results were observed with the type A nd B influenz cultures. No correltion ws demonstrted between the rte of decrese of cell vibility nd the size of the inoculum. After processing, the CF titers of the type A/RNP ntigens rnged from 1:128 to 51:512, wheres those of the type B/RNP ntigens rnged from 1:16 to 1:128. No pprent ssocition ws observed between the size of inoculum nd either the CF titer or the rte of decrese in cell vibility in the suspension cultures. Neither HA nor CF rections could be demonstrted with smples collected from uninoculted control cultures. The vibility of the cells in these cultures declined t slower rte thn the inoculted cultures, nd the cell mortlity did not exceed 50% t the time of hrvest on dy 4. Crude nd RNP ntigens of influenz types A TABLE 3. Effect of immuniking schedule with influenz AlHong Kongl8/68 (H3N2) on type-specific CF ntibody titer in mouse scitic fluid Ascitic fluid CF ntibody titerb Immunizing tp (dy fter schedule immuniztion Type Type strted) AIRNPc B/RNPc 1. Whole virus plus A (37) 64 <8 A/RNP B (42) 128 <8 C (47) 64 <8 2. A/RNP A (30) 128 <8 B (35) 128 <8 C (40) 128 <8 3. A/RNP A (47) 256 <8 B (51) 256 <8 C (58) 128 <8 4. Whole virus plus A (64) 256 <8 A/RNP B (68) 128 <8 C (75) 128 <8 Refer to Tble 1 for description of immunizing schedule. b CF titer expressed s dilution fctor of the optiml ntigen dilution s determined in block titrtions with specific ntiser. c CF ntigen used in serologicl test.

4 290 WHITE, GAMBLE, AND CHAPPELL nd B were dministered ccording to the four schedules presented in Tble 1. The mice in schedules 1 nd 4 were inoculted by the i.n. route with infected llntoic fluid, nd subsequent inocultions were mde with RNP ntigens. The mice in schedules 2 nd 3 received only the RNP ntigen for different periods of time. The CF ntibody titers of the IAF obtined from tppings t 4- to 5-dy intervls re compred in Tbles 3 nd 4. Homologous ntibody titers of type A influenz scitic fluids rnged from 1:64 to 1:256 (Tble 3). Homologous ntibody titers of type B influenz scitic fluids rnged from 1:16 to 1:128 (Tble 4). Schedule 4 ppered to be best for type B immuniztions, wheres schedules 2, 3, or 4 produced comprble results for type A immuniztion. Comprble volumes of scitic fluids were obtined with ll of the immuniztion schedules. IAF yields of processed fluids verged pproximtely 10 ml per mouse. All scitic fluids were tested for heterologous s well s homologous CF titers. No heterologous CF titers for orthomyxoviruses or prmyxoviruses were observed. Control fluids collected from mice immunized with norml ntigens were negtive in the CF test. Antiser obtined from the mice immeditely fter the third tpping hd CF titer comprble to tht of the IAF. DISCUSSION Guine pig ntiser hve been used for number of yers in the typing of orthomyxoviruses. The immuniztion schedule routinely used to produce type-specific, CF ntiser requires bout 56 dys (5). In the volume production of these ntiser s reference regents, we hve hd difficulty in obtining guine pigs free of prmyxovirus ntibodies. If these regents re to comply with the Center for Disese Control specifictions (4), they must not contin ny heterologous orthomyxovirus or prmyxovirus ntibodies. To meet these specifictions, we hve hd to pretest lrge numbers of guine pigs to obtin nimls suitble for immuniztion. Mny lbortories, including our own, produce immune mouse scitic fluids for vriety of viruses, nd we believed tht scitic fluids might be stisfctory replcement for influenz RNP guine pig ntiser. We determined erly in these investigtions tht ser from norml mice nd scitic fluids obtined fter immuniztion with control ntigens were devoid of CF ntibodies for orthomyxoviruses nd prmyxoviruses. This indicted tht the vilbility of stisfctory mice would reduce the problems ssocited with guine pigs. J. CLIN. MICROBIOL. TABLE 4. Effect of immunizing schedule with influenz BlMsschusettsl3/66 on type-specific CF ntibody titer in mouse scitic fluid Ascitic fluid CF ntibody titerb Immunizing tp (dy fter schedule immuniztion Type Type strted) B/RNPc A/RNPc 1. Whole virus plus A (37) 32 <8 B/RNP B (42) 32 <8 C (47) 32 <8 2. B/RNP A (30) 16 <8 B (35) 16 <8 C (40) 16 <8 3. B/RNP A (47) 64 <8 B (51) 32 <8 C (58) 128 <8 4. Whole virus plus A (64) 128 <8 B/RNP B (68) 128 <8 C (75) 128 <8 Refer to Tble 1 for description of immunizing schedule. b CF titer expressed s dilution fctor of the optiml ntigen dilution s determined in block titrtions with specific ntiser. ' CF ntigen used in serologicl test. Four immuniztion schedules were compred by using homotypic RNP ntigens prepred from type A nd B influenz viruses. In schedules 1 nd 4, mice were initilly given infective llntoic fluids by intrnsl instilltion. This ws followed by 4 intrperitonel injections of prtilly purified RNP t different time intervls. Schedules 2 nd 3 consisted of RNP injections without the initil dose of infective virus. There ppered to be no substntil difference in ntibody response to influenz type A regrdless of the schedule. Antibody response in schedule 2 ws simlr to tht in the other schedules, which lsted longer. There ws no dvntge in using the longer schedules or in giving the initil dose of infectious llntoic fluid by the i.n. route. Schedules 3 nd 4 ppered to produce better ntibody response thn schedules 1 nd 2 with influenz type B. As with influenz type A, there ws no definite dvntge in giving the initil i.n. dose of infectious llntoic fluid. A high nd specific CF ntibody titer my be produced for either influenz type A or B with RNP ntigen with schedule 3. In ll groups of mice immunized with both types of influenz, ntibody titers persisted for t lest 10 dys fter the first prcentesis. Thus, scitic fluid cn be collected severl times from the sme mice. Other investigtors (8, 11) hve used immune

5 VOL. 2, 1975 PRODUCTION OF CF ANTIBODY IN ASCITIC FLUID 291 mouse scitic fluids s substitute for ntiser for orthomyxoviruses; however, they were interested primrily in HAI or neutrlizing ntibodies. The immunizing ntigens used to stimulte these ntibodies were crude infectious llntoic fluids. Since we were interested in producing type-specific CF ntibodies, prtilly purified, homotypic influenz RNP ws used s the ntigenic mteril. The scitic fluids collected in these investigtions were not only free of specific HAI ntibodies to influenz virus, they were lso devoid of nonspecific inhibitors for influenz A/Hong Kong/8/68, which hve been reported in ser of norml guine pigs nd severl other species of nimls (6). ACKNOWLEDGMENTS We thnk Mrion T. Colemn (now decesed) nd Wlter R. Dowdle, Virology Division, Center for Disese Control, for their dvice nd comments during these experiments, nd the Virl nd Rickettsil Products Brnch stff for its technicl ssistnce. LITERATURE CITED 1. Berkovich, S A simple rpid method for production of virl ntibody in mice. Proc. Soc. Exp. Biol. Med. 111: Brndt, W. E., E. L. Buescher, nd F. M. Hetrick Production nd chrcteriztion of rbovirus ntibody in mouse scitic fluid. Am. J. Trop. Med. Hyg. 16: Bucc, M. A., H. L. Csey, nd J. F. Winn Method of concentrtion of virl dignostic regents using hydrophilic gents. Proc. Soc. Exp. Biol. Med. 104: Center for Disese Control Chlmydil, mycoplsml, rickettsil nd virl, p. A1-18. Specifictions nd evlution methods for immunologicl nd microbiologicl regents, vol. 2. Center for Disese Control, Atlnt, G. 5. Chppell, W. A., M. A. Bucc, L. A. White, nd W. C. Gmble Production mnul-virl, rickettsil, chlmydil, mycoplsml regents, 5th ed. p. 29. Center for Disese Control, Atlnt, G. 6. Colemn, M. T.,nd W. R. Dowdle Properties of the Hong Kong influenz virus. 1. Generl chrcteristics of the Hong Kong virus. Bull. W. H : Fbiyi, A., nd H. A. Wenner Enterovirus ntibodies in mouse scitic fluids fter immuniztion. J. Immunol. 94: Herrmnn, E. C., Jr., nd C. Engle Tumor cellinduced mouse scites fluid s source of virl ntibodies. Proc. Soc. Exp. Biol. Med. 98: Hierholzer, J. C., W. C. Gmble, K. D. Quist, nd W. A. Chppell Comprison ofimmuniztion methods for producing reference denovirus ntiser in horses. Appl. Microbiol. 24: Hierholzer, J. C., M. T. Suggs, nd E. C. Hll Stndrdized virl hemgglutintion-inhibition test. II. Description nd sttisticl evlution. Appl. Microbiol. 18: Ksel, J. A., R. Liebermn, nd H. A. Smith Production of virl ntibodies in scitic fluid of mice. Virology 9: Lennette, E. H., nd N. J. Schmidt (ed.) Dignostic procedures for virl nd rickettsil infections, 4th ed., p Americn Public Helth Assocition, Inc., New York. 13. Ntionl Communicble Disese Center Stndrdized dignostic complement fixtion method nd dpttion to micro test, 1st ed. Ntionl Communicble Disese Center, Atlnt, G. 14. Rott, R., A. P. Wterson, nd I. M. Red Chrcteriztion of "soluble" ntigens derived from cells infected with Sendi nd Newcstle disese viruses. Virology 21: Srtorelli, A. C., D. S. Fischer, nd W. G. Downs Use of Srcom 180/TG to prepre hyperimmune scitic fluid in the mouse. J. Immunol. 96: Tiksingh, E. S., I. Spence, nd W. G. Downs The use of djuvnt nd srcom 180 cells in the production of mouse hyperimmune scitic fluids to rboviruses. Am. J. Trop. Med. Hyg. 15: