Virus Inoculation in Mice Bearing Ehrlich Ascitic Tumors: Antigen Production and Tumor Regression

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1 INFTION AND IMMUNITY, JUIY 1982, p /82/723-5$2./ Vl. 37, N. 1 Virus Inculatin in Mice Bearing hrlich Ascitic Tumrs: Antigen Prductin and Tumr Regressin NORMA. MTTLR,* DLPHIN H. LARK, AND JORDI ASALS Tandil Virus Research Labratry, Faculty f Veterinary Sciences, Natinal University f entral Buens Aires Prvince, Pint 399, Tandil 7, Buens Aires, Argentina Received 25 Nvember 1981/Accepted 1 March 1982 hrlich ascitic carcinma, as develped in albin mice, has been used as a surce f hemagglutinating and cmplement-fixing antigens, and it prved t be suitable fr ne type f antigen, r bth, fr at least 12 viruses f 16 tested. Hemagglutinins were btained with members f arbvirus grups A, B, and ; cmplement-fixing antigens were btained fr at least ne member f each antigenic grup tested. hrlich ascitic tumr was cmpared with sarcma 18 as a surce f antigens; althugh sarcma 18 shwed many advantages ver hrlich tumr, the latter gave, in general, better results fr cmplement-fixing antigens. Onclytic effect with cmplete recvery f the mice was bserved in sme instances. The highest recvery rate resulted with ng and UNA viruses (4%), and the secnd highest rate resulted with dengue 2, St. Luis, Hazara, and Uukuniemi viruses (2%). The best survival was bserved, in decreasing rder, with ng, St. Luis, dengue 2, Tacaribe, Sindbis, Junin, and Amapari viruses. Previus studies (7) have shwn that sarcma 18, when prpagated intraperitneally (i.p.) in intact mice, prvides an excellent system fr the multiplicatin f sme arbviruses, with the assciated appearance f hemagglutinating (HA) and cmplement-fixing (F) antigens in the ascitic fluid. This reprt describes similar studies with hrlich ascitic tumr, cmpares the tw sets f results, and prvides data n tumr regressin assciated with sme f the viral infectins. 23 MATRIALS AND MTHODS Mice. Adult mice f the harles River D(R)-1 strain were used thrughut. hrlich ascitic tumr. A muse bearing an hrlich ascitic tumr was btained frm A.. Sartrelli, Department f Pharmaclgy, Yale University Schl f Medicine, in January The ascitic tumr had undergne an undetermined number f passages in albin mice. In the preliminary tests t determine the dse level f ascitic fluid that wuld mst rapidly prduce ascitic tumrs and yet permit the mice t survive lng enugh fr antigen develpment, grups f 3, 44, and 5 mice were inculated i.p. with.1 ml f undiluted fluid.2 ml f fluid diluted 1:1, and.2 ml f undiluted fluid, respectively. The inculum f.2 ml f undiluted fluid was chsen fr the experiments, since it gave a gd yield f ascitic fluid by day 8 pst-inculatin in cmbinatin with an average survival time f better than 14 days (14.4 ± 4.7) (Table 1). Viruses. The fllwing 16 viruses (1, 6) were used: astern equine encephalitis () (TenBreck), Sindbis (gar339), and Una (BeArl3136) viruses f antigenic grup A; dengue 2 (TR1751) and St. Luis encephalitis (SL) (Partn) viruses f grup B; Apeu (BeAn8) and Marituba (BeAnl5) viruses f grup ; Orpuche virus (TR976) f the Simbu grup; Uukuniemi virus (S-23) f the Uukuniemi grup; rimean hemrrhagic fever virus, ng (IbAnlO2) and Hazara (JD28), f the ng grup; Amapari (BeAn7563), Junin (XJ and Nguera), Tacaribe (TR11573), and Tamiami (D W 1777) viruses f the Tacaribe grup; and lrad tick fever virus (ndn). Virus stcks cnsisting f a 1 r 2 suspensin f infected newbrn muse brain tissue were used fr inculatin; all were lyphilized preparatins held at 4, except fr the virus stck, which was kept wet frzen at -7. Dilutins were made in.75% bvine albumin in phsphate-buffered saline (ph 7.2). The lyphilized virus stcks were knwn t cntain at least 14 suckling muse intracerebral 5 lethal dses in the.1-ml vlume inculated; titers were determined fr the virus stck when used. xperimental design. Mice in grups f 5 were inculated i.p. with hrlich ascitic tumr. After 8 t 1 days, when marked abdminal swelling signalled the develpment f fluid, they were inculated i.p. with virus. Thereafter, fr as lng as any mice survived, r TABL 1. Survival f mice after i.p. inculatin f different dilutins f hrlich ascitic tumr N. f Dse (ml) AST' SD mice 3.1, undiluted 15.5 ± , diluted 1: t , undiluted "AST, Average survival time in days = X. Dwnladed frm n Nvember 15, 218 by guest

2 4.. V) - -D u u n ~ H I en ' D u - ' c. I p LI.. c - t = '- O 1-: n r- I WV) 1-* _s 4L) >.> 1 u > l Z r. D < ' " O ;z ",en f es xx es ~ It I cn.u,.. * - ul-ilii m m mmm. L-S 4- c ;7.. L. i-.22. id:i cn Z Z~ z> 3 '6. zus u; until regressin f the tumr, 2 t 5 ml f ascitic fluid was withdrawn daily frm the muse in each grup that shwed the greatest degree f abdminal swelling; much larger quantities f fluid culd have been harvested if desired. The ascitic fluids were immediately frzen and kept at -2 until prepared as antigens fr use in HA and F tests (2, 3). In the case f the muse grups inculated with Apeu and Marituba viruses, hwever, ascitic fluids harvested n days 3 t 6 after virus inculatin were pled by virus and inculated int additinal grups f tumr-bearing mice. The average survival times fr mice were determined frm the day they received hrlich tumr fluid t the day they died and are reprted as means with standard deviatins (5); when a given virus was inculated int mre than ne muse grup, the results are thse determined fr all f the mice s used. Median survival times were als calculated as affrding mre accurate measurement fr grups in which sme mice survived lnger than 2.5 r 3 mnths (at which time they were apparently healthy and were withdrawn frm the experiment). Fr statistical analysis, the significance levels fr survival were determined by the rati f the differences between the means t their standard errrs (5). HA antigens. Fr preparatin f HA antigens ascitic fluids were thawed and separated frm cellular debris. The fluids were extracted twice with 2 vlumes f acetne, dried n a vacuum pump fr 1 h, and then rehydrated in 5 vlumes f brate-saline (ph 9.) as described previusly fr sarcma 18 fluids (7). Hydratin tk place quickly, and the antigens culd be used n the same day withut being centrifuged. HA and hemagglutinatin inhibitin assays were thse f larke and asals (3) adapted t a micrtechnique (9). Serial twfld dilutins f antigens, starting at 1:4, were tested at phs ranging frm 5.8 t 7.2 with intervals f.2 ph units. Results are given as the ptimum titer at any ph; if mre than 1 grup f mice were inculated with the same virus suspensin, the results shwn in the tables are als the ptimum btained in each day. ach HA antigen was tested fr specificity in the hemagglutinatin inhibitin test with its hmlgus and least tw unrelated immune sera r ascitic fluids. Fr this purpse immune fluids were extracted twice with acetne, and gse cells were adsrbed. F antigens. The F antigens used were the fluids freed f cellular debris and heated fr 2 min at 6 in a water bath. Serial twfld dilutins starting at 1:2 were made in Vernal buffer and tested against hmlgus and unrelated immune fluids (immune fluids were sera r ascitic fluids). The F tests were carried ut in grid tritatins as described by asals (2). Fr each immune fluid, a cntrl experiment was perfrmed with a crrespnding reference sucrse-acetne-extracted muse brain antigen (muse liver antigen in the case f Marituba virus) (3). RSULTS Specific HA antigens were btained with three arbviruses f grup A (, Sindbis, and Una viruses), SL virus f grup B, and Apeu and Marituba viruses f grup ; Apeu and Dwnladed frm n Nvember 15, 218 by guest 24 MTTLR, LARK, AND ASALS INFT. IMMUN.

3 VOL. 37, 1982 Marituba viruses were inculated as virus-infected hrlich ascitic tumr fluid rather than as muse brain (Table 2). N hemagglutinins were detected with the ther nine viruses. Of the viruses used, dengue 2, Hazara, Uukuniemi, and Orpuche viruses are knwn t yield hemagglutinins by ther techniques. The rest f the viruses used in the test never shwed hemagglutinins with any system, and therefre we d nt knw whether they really have any. T determine the relatinship between the amunt f inculated virus and the time and amunt f antigen prductin, five grups f mice were inculated with different dses f virus (Table 2). The highest titer was btained with the highest inculum in the shrtest time, reaching a titer f 1:4,96 n day 2 pst-inculatin. The time f appearance f hemagglutinins was frm 1 t 5 days depending n the virus and the dses inculated. They were nt detected later than day 1 after virus inculatin. Specific F antigens, hwever, were btained with at least ne virus f each antigenic grup tested (Table 3). Psitive results were btained with, Sindbis, Una, dengue 2, SL, Apeu, Marituba, Hazara, Orpuche, Uukuniemi, lrad tick fever, and Amapari viruses (Table 3). Mst f the viruses gave the best F titers between days 5 and 7 except Uukuniemi and Amapari viruses, which had ptima n days 11 and 19, respectively. mpletely negative results were shwn by the rest f the members f the Tacaribe grup and by ng virus, and it was nt because f lack f samples since tapping was perfrmed up t 24 days pst-inculatin fr mst f them. By cmparisn, althugh sarcma 18 shwed many advantages ver hrlich ascitic tumr as a surce f antigens, the latter gave, in general, better results fr F antigens. Tumr regressin ccurred with ng and Una viruses (4% recvery rate) and t a lesser degree (2%) with dengue 2, SL, Hazara, and Uukuniemi viruses (Table. Mice lked healthy and were kept fr 3 mnths, after which they were discarded. Survival time f carcinmatus mice were prlnged significantly (P <.1) with the fllwing viruses, given in decreasing rder f prbability values: ng, SL, dengue 2, Tacaribe, Sindis, Junin, and Amapari viruses. Other viruses had less effect (P <.5) like Hazara, lrad tick fever, and Orpuche viruses, and the ther viruses diminished r had n effect n time f survival (Table 5). DISUSSION Our first apprach fr antigens in an ascitic tumr, sarcma 18, prduced gd results with ^3>I -x VIRUS INOULATION IN TUMOR-BARING MI 25 3XS. ) *O)-- w _. Pz<, 'Q S.9., 5 i ~p < *-. t'o tuj ( 3 < -~.9. *s. z. p) -.1 n=; ev r -a.9)i (r r"=b c ) gl ( t -q~~~~~~~~~.k ~ ~ -' - QO. -i w as ax & I-A )-. wi - -L "-AtJ t%j t- %1 I7~ I - >t JI -&A % O% %%s 4 tj ( % SS I P Z., P, > ti ta r) ON ( D I\J >!. > r) > r) lw Q ,, cs r II III4< I%<I ( ( < I1 : D (A In, 91 ( t. ip a ( r _.).-. l _.9 _.) _ 3.9 ( Dwnladed frm n Nvember 15, 218 by guest

4 26 MTTLR, LARK, AND ASALS TABL 4. Survival f hrlich ascitic tumr-bearing mice inculated with arbviruses Virus Median Mean" SD" Mrtality rati' Una ±3.23 3/5 Dengue ±7.84 4/5 SL ±1.11 4/5 ng ±3.5 3/5 Hazara ± /5 Uukuniemi ±5.78 4/5 a Means and standard deviatins are based n deaths. b The mrtality rati is the number f deaths/ttal number f mice. Fr tumr-bearing mice receiving n virus, the mrtality rati was 124/124. many arbviruses (7). Hwever, different types f tumral cells may lead t different results. The waiting times fr btainable sarcma 18 r hrlich tumrs in mice are practically the same when the same inculum f.2 ml f fresh, undiluted ascitic fluid is used; the average survival times fr mice after inculatin f tumr cells withut further inculatin f viruses were 13.3 days fr sarcma 18 and 14.4 fr hrlich tumr; hwever, the standard deviatin fr sarcma 18 was ±2.6, whereas that fr hrlich tumr was ±4.7. hrlich tumrs seem t have disadvantages as cmpared with sarcma 18 in the sense that the yield f ascitic fluid frm sarcmatus mice is larger (mre prminent abdmens) plus the fluid recvered frm the ascites is abut 9% f the ttal fr sarcma 18 and nly 8% fr hrlich tumr. HA antigen titers are higher fr sarcma 18, except fr grup where hrlich tumr had higher titers; hwever, in the case f grup viruses infected ascitic fluid instead f muse brain was inculated, and this step was nt attempted with sarcma 18. Owing t the fact that individual mice were used fr antigen titers and sme ascites taps were better than thers, individual variatin cannt be ignred. Hwever, a similar phenmenn is bserved with HA antigens in pled infected muse brains r sera frm many newbrn mice; HA antigens reach a peak that is nt lng lasting, and 24 h later there may be n HA activity at all, as seems t be the case with Sindbis virus (Table 2), where the HA antigen titer appeared n day 5 (1:256) and was negative frm day 6 n. Rutine wrk indicates that there is a mment in the grwing curve f a virus in a hst, tissue, r cell system where the prductin f HA antigen is best, with a range f psitivity arund it in time, and the same is true fr F antigens. Pssible explanatins fr this phenmenn are many: ne is the frmatin f hemagglutinatin inhibitin antibdies bserved in the TABL 5. ffect f virus inculatin n average survival time f hrlich tumr-bearing mice' Virus Median Mean SD N. micef mice Nne Sindbis Apeu Marituba Orpuche lrad tick fever Junin (XJ) Junin (Prtill/Nguera) Amapari Tacaribe Tamiami ' All mice in these experiments died. INFT. IMMUN. same ascitic fluids, at least in sarcmatus mice (7); anther is the presence f lipid r lipprtein substances, since acetne remves lack f HA activity in treated sera. The treatment we used here is like that described fr muse serum (3), and n further investigatins were dne t determine whether all f the lipids were remved in all instances; as a rutine technique applied t the ascitic fluids it seems t be gd enugh. Perhaps due t its high cellular cntent, hrlich tumr yielded better F antigens fr mst f the viruses tested. Many f them prduced a better yield f antigens in 5 t 6 days; these viruses generally kill newbrn mice in 5 r 6 days r less. Viruses with lnger times fr yielding antigens, like Uukuniemi and Amapari viruses, are knwn fr having lnger average survival time in newbrn mice; frm 6 t 12 days and 13.7 t 18 days, respectively (1). Multiplicatin f viruses in tumr cells has been knwn fr many years frm studies f virus-induced nclysis f slid tumrs (8), f ascitic tumrs (, and f tumr cells in vitr (1). Hwever, at the time these experiments n tumrs in mice were perfrmed many f the viruses yielding the best results were nt available since they were islated later (1) like ng, Hazara, and Uukuniemi viruses frm the family Bunyaviridae and Una virus, Alphavirus (arbvirus A) frm the Tgaviridae family islated in The same is true fr Tacaribe, Junin, and Amapari viruses, members f the family Arenaviridae islated in 1956, 1958, and 1964 respectively, which prduced a lnger survival time in carcinmatus mice. Prlngatin f carcinmatus muse survival and knwn ability f the viruses t grw in mice seem t crrelate with viruses that nrmally are nt pathgenic fr adult mice inculated i.p. (1). Dwnladed frm n Nvember 15, 218 by guest

5 VOL. 37, 1982 This bservatin seems t indicate that the viruses may have the ability t multiply and destry the carcinmatus cells withut killing the animal. AKNOWLDGMNTS We are very grateful t Rbert Shpe and lin White fr their advice and help in the preparatin f this manuscript. N..M. is investigatr f the mmissin f Scientific Investigatins frm Buens Aires Prvince, Argentina. VIRUS INOULATION IN TUMOR-BARING MI 27 LITRATUR ITD 1. Berge, T Internatinal catalgue f arbviruses including certain ther viruses f vertebrates, 2nd ed. Department f Health, ducatin and Welfare publicatin n. (D Department f Health, ducatin and Welfare, Washingtn, D.. 2. asals, J Methds Virl. 3: larke, D. H., and J. asals Techniques fr hemagglutinatin and hemagglutinatin-inhibitin with arthrpd-brne viruses. Am. J. Trp. Med. Hyg. 7: Kprwski, H Ascites tumrs as culture media in quantitative grwth studies f viral agents. Ann. N.Y. Acad. Sci. 63: Li, J.. R In. Brthers (ed.), Statistical inference, vl. 1, p Ann Arbr, Mich. 6. Mettler, N Islatin f a micrtatbite frm patients with hemlytic uremic syndrme and thrmbticthrmpcytpenic purpura and frm mites in the United States. New ngl. J. Med. 281: Mettler, N.., J. asals, D. larke, W. Dwns, and R. Shpe Use f sarcma 18 t prepare hemagglutinating and cmplement-fixing antigens fr viruses in adult mice. Prc. Sc. xp. Bil. Med. 136: Mre, A The destructive effects f the virus f Russian Far astern encephalitis n the transplantable muse sarcma 18. ancer 2: Sever, J. L Applicatin f a micrtechnique t viral serlgical investigatins. J. Immunl. 88: Stim, T. B., G. A. Fisher, W. G. Dwns, and M. Y. W. hu ytlytic activity f arbviruses in murine leukemic lymphblasts. ancer hemther. Rep. 2: Dwnladed frm n Nvember 15, 218 by guest