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Human mammary carcinoma cell line: Infection by an avian myxovirus as a prerequisite for immunopotentiation
Europ. J. Cancer Vol. 11, pp. 59-63. Pergamon Press 1975. Printed in Great Britain
Human Mammary Carcinoma Cell Line: Infection by an Avian Myxovirusas a Prerequisite for Immunopotentiation* CHRISTIAN SAUTER 1, THOMAS B.~CHI 2 and JEAN LINDENMANN 2 aDivision of Oncology, Department of Medicine and 2Division of Experimental Microbiology, Institute of Medical Microbiology, University of Ziirich, 8006 Ziirich, Switzerland Abstract--In view of the proven potentiation of tumor antigen activity produced by infecting tumor cells with myxoviruses, a strain of avian influenza virus (A/Turkey~ England/63, Hay I, Nav 3) previously adapted to grow in HeLa cells was passed serially 8 times in the human mammary carcinoma cell line B T 20. Comparisons of growth curves of the starting v#us with the eighth passage level showed that adaptation had taken place. Immune electron microscopy using ferritin-labelled antiviral antibody revealed viral particles budding at and viral antigens incorporated in cell membranes. The possible use of such a virus for "virus-assisted immunotherapy" is discussed.
INTRODUCTION
primary cultures of human mammary carcinoma.
AUGMENTATION of tumor cell immunogenicity by infection with viruses has been shown in several systems [ 1-5]. Enveloped viruses, mostly influenza viruses, were used for this purpose, since the association of viral and host antigens at membrane sites is apparently important for the augmentation of tumor cell immunogenicity [6]. An essential step for the use of viruses as immunological potentiators in the immunotherapy of human neoplasms is the successful replication of an enveloped virus in the tumor cell in question. Fowl plague virus (FPV), an influenza A virus, has been adapted to several types of human tumor cells [7, 8] and has been shown to replicate regularly in primary cultures of human leukemic cells [9]. In this communication, we report the adaptation of the same virus strain to a human mammary carcinoma cell line as a first step towards obtaining a virus capable of growing in
MATERIAL AND M E T H O D S V/rus The fowl plague virus (A/Turkey/England/ 63, Hav 1, Nav 3, Langham strain) which we used in these experiments had the following passage history: Obtained from H. G. Pereira, World Influenza Center, Mill Hill, London, England, after 4 allantoic passages; the virus was passed 3 times in I C R mouse kidney cell cultures, 18 times in KB ceils, twice in HeLa cells, once in the chick allantois and once in a primary culture of human bronchial carcinoma cells. This virus will be referred to as FPV. (having had no passage in m a m m a r y carcinoma cells). The subscript numbers following FPV will indicate the number of passages in mammary carcinoma cells. Human mammary carcinoma cell line
Accepted 17 June 1974.
The human mammary carcinoma cell line BT 20 [10] was obtained from L. Ozzello, University of Lausanne, Switzerland.
*This investigation was supported by the Julius Mtiller Foundation for Cancer Research, Ziirich, and the Cancer Research Institute, Ziirich Branch.
59
60
Christian Sauter, Thomas B?ichi and Jean Lindenmann
Culture media BT 20 cells were grown in R P M I 1640 (Grand Island Biological Company, Grand Island, N.Y., USA), supplemented with 2 70 fetal calf serum (FCS). Virus adaptation, virus replication, and electron microscopy were done on cells maintained in Eagle's minimum essential medium (MEM) supplemented with 2 % FCS.
Virus adaptation The virus for each passage was taken from the previous passage at the time of first detection ofhemagglutinin. Every 12-24 hr the supernatant was checked for hemagglutinin. For each passage, a 25 cm 2 Falcon plastic bottle (Falcon Plastics, Oxnard, Calif. USA) containing a complete monolayer of BT 20 cells was washed with M E M and infected with 0.33 ml of a virus dilution in M E M containing between 101'° to 103.9 T C I D s o (5070 tissue culture infectious dose) (see Table 1). The infected culture was incubated for 15 min at 37°C with agitation every 5 min for virus adsorption. Four-point-six-six ml of M E M with 2 70 FCS were then added and incubation was continued until detection of hemagglutinin. At this time the supernatant was harvested, and aliquots were sealed in sterile glass ampules and stored at - 8 0 ° C until infectivity titrations or a further adaptation passage could be performed.
Virus replication curves To compare the replication of FPVo and FPV8 in BT 20 cells the following experiment was done: two 75 cm 2 Falcon plastic bottles containing a complete monolayer of BT 20 cells were washed with MEM. To one bottle was added 1 ml of FPVo containing about 102.5 TCIDso, to the other bottle, 1 ml of FPV8 containing also about 102.5 TCIDso. The infected cultures were incubated for 15 rain at 37°C with agitation every 5 min for virus adsorption. Nineteen ml of M E M with 2 7o FCS were then added and incubation was continued. Five ml of supernatant of each bottle were removed (and replaced by 5 ml of fresh medium) at 4, 22, 46, 70, 94, and 176 hr of incubation. The removed samples were checked for hemagglutinin, put into sterile glass ampules and kept at - 8 0 ° C until infectivity titrations could be performed.
Virus titrations Virus infectivity assays were performed in 1 6 x l 0 0 m m glass tubes containing chicken
embryo fibroblast monolayer cultures. The monolayers were infected with serial 10-fold dilutions of virus in M E M without serum. Six tubes per dilution were used. After 72 hr of incubation at 37°C, the tubes were screened for cytopathic effects. Tubes with incomplete destruction of the monolayers were checked for hemagglutinin. The TCIDso'S were calculated according to the method of Reed and Muench [11]. The HA titrations were done according to W H O standard procedures.
Antisera Antiserum was prepared to FP virus grown in embryonated hens' eggs. Allantoic fluid with an HA titer of 512 was stored at - 8 0 ° C until used for immunization. Rabbits immunized by 5 weekly intramuscular injections of 2 ml virus with 2 ml complete Freund's adjuvant were bled 14 days after the last injection. The sera, after inactivation (56°C, 30 min) and exhaustive absorption with washed human erythrocytes (0/Rh +), showed a hemagglutination inhibition titer of 1280 against FP virus as tested with fowl red cells. Guinea pig anti-rabbit IgG serum conjugated with ferritin was prepared as described elsewhere [12].
Electron microscopy Cells from infected (16 hr after inoculation with FP virus) or uninfected cultures were washed three times with cold Earle's balanced salt solution (EBSS). The monolayers were incubated with rabbit anti-FP serum for 30 min at 4°C) washed three times with cold EBSS, incubated (30 min, 4°C) with guinea pig antirabbit IgG conjugated with ferritin and washed again three times with cold EBSS. The cells were then fixed (30 min, 4°C), with a mixture of 3 7oo glutaraldehyde and 3 70 acrolein in 0.05 M cacodylate buffer, pH 7.2. All cells were postfixed for 6 0 m i n at 4°C with 27o osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2. The fixed material was left overnight in a 2 7o solution of uranyl acetate before dehydration and embedding in Epon-Araldite. The thin sections were stained with lead citrate.
RESULTS Adaptation of F P V to B T 20 cells FPV was passed serially in BT 20 cells in order to get an optimal adaptation to this cell line. In Table 1, the growth characteristics of the different passages are shown. The virus inputs varied between 101"° to 10 3.9 T C I D s o due to the fact that the viral dilutions had to
Human Mammary Carcinoma Cell Line : Infection by an Avian Myxovirus be chosen arbitrarily, the infectivity titer of the .previous passage being unknown at the time of infection of the following passage. To select for particles capable of replicating in BT 20 cells, the dilutions from one passage to the next were at least 100-fold. Based on our experience with FPV in HeLa cells [7], we expected that a few passages would suffice for optimal adaptation. From the fifth passage on, hemagglutination was regularly detected at 46 hr of incubation. Regardless of the input, the T C I D s o was always around 106/ml at 46 hr of incubation in later passages. We therefore stopped the adaptation experiments at the eigth passage. Comparing the data of the second and sixth passage (same virus inputs, Table 1) the virus
61
log TCID 50/ml
7
3
Virus input 2
Table 1. Serialpassage of FPV in B T 20 monolayer cultures 1,
Virus input* Time to TCIDn0/ml at (Virus from detection of the time of first Passage previous hemagglutinin detection of number passage) (hr) hemagglutinin 1 2 3 4 5 6 7 8
103.9 102.0 101"3 10 x'° 102.5 102.0 103.5 10 3.0
102 84 72 57 46 46 46 46
104.25 104.75 I0 s'5 105.0 105.5 106.0 106.0 10 s's
*TCIDno per monolayer.
replication in the later passage was definitely better with respect to replication speed as well as to infectivity titer. Optimal adaptation may have already been reached by the third or fourth passage, since the delay in hemagglutinin appearance and the somewhat lower infectivity titer could have been due to the very low input in these passages. A good adaptation of FPVo to BT 20 cells therefore seemed to require at least two, but probably four, serial passages.
Comparison of the replicating of FPVo and FPV8 in B T 20 cells Figure 1 shows the growth curves of FPVo (no passage in BT 20 cells) and FPV8 (8 passages in BT 20 cells). The drop of the infectivity titer until four hr of incubation was about the same for both viruses. FPV8 then replicated much faster and to a higher titer than FPVo. For FPV8, hemagglutinin reached a maximal titer of 32 at 176 hr of incubation, whereas for FPVo, no hemagglutinin could be detected until the end of the experiment at 176 hr.
22
t,6
70
94
l'lrhm
Fig. 1. Replication of FPVo (4~-------O, no passages in B T 20 cells) and FPVs ( 0 O, eight passages in B T 20 cells) in B T 20 cell monolayers.
Cytopathogenic effect of F P V on B T 20 cells A slight cytopathogenic effect (CPE) of FPV on BT 20 cells was seen at the time of the first detection of hemagglutinin. At this stage, cytoplasmic vacuolization and a few rounded cells were observed. Twenty-four hours later, an almost complete CPE was seen (Fig. 2B). Most of the ceils had detached from the bottom of the plastic bottle, were round, and took up trypan blue stain. Electron microscopy In cultures fixed 16 hr after inoculation with FP virus, virions were frequently found to be attached to cell surfaces (Fig. 3). Occasionally, budding of virus was observed at bulging segments of the cytomembrane (Fig. 4). In addition to the morphological features characterizing these structures as viruses, immunological tagging by ferritin labelled antibody revealed viral antigenicity (Figs. 3-6). Attached or budding virus particles invariably were tagged heavily with labelled antibodies visualized by the electron dense core of the ferritin molecule. The specificity of the reaction was established by treatment of uninfected cultures with the same reactants; here, all ferritin molecules were washed out because the antiviral antibody was not bound to the cells (Fig. 7). Reaction of antibodies with membranes of infected cultures not revealing any characteristic viral differentiation demonstrated the presence of viral antigens also in segments of
Fig. 2.
Typical lymphoblast : the dense chromatin is disposed in a thin peripheral rim; the nucleolus (arrow) is compact. / x 8250).
Fig. 3. Ring-shaped nucleolu~ cell: the nucleolar (arrow)components are disposed in a characteristical ring-like disposition(inset). The dense chromatin is s~milar to that observed in typical lymphoblasls. ( x 9500; inset : x 20700). Fig. 4. X cell : the chromatin is condensed in an irregular peripheral strand and in blocks dispersed in the nucleoplasm and around the large nucleolus (arrow). ( x 10750).
Fig. 2.
Phase-contrast photomicrographs of B T 20 cell monolayers, x 320. A. Uninfected control culture. B. 94 hr after infection with FPVs.
(to face p. 62)
62
Christian Sauter, Thomas Btichi and Jean Lindenmann
cellular surfaces where neither budding nor adsorbed virus particles were seen (Figs. 3 and 6). In infected cultures, a few ceils were seen which completely failed to react with the antisera and which showed no structural signs of virus replication (Fig. 5). At the stages of infection studied in the electron microscope, the cytopathic effects (16 hr) were inconspicuous. The aspect and structural integrity of nucleus and cytoplasmic organelles in uninfected cells treated under the same conditions of incubation (Fig. 7) were essentially the same as in infected cultures (Fig. 6).
DISCUSSION In the present report we have shown that an avian influenza A virus (fowl plague virus), after several adaptation passages, replicated well in a human m a m m a r y carcinoma cell line (BT 20). Virological as well as immunotherapeutical aspects of this finding wilt now be discussed.
Virological aspects In contrast to human influenza A strains and to the Brescia [13, 14] and Dutch strains of avian influenza A [15, 16], the avian influenza A we used in these experiments proved to be easily adaptable to human tumor cells. For adaptation to BT 20 cells we chose FPV already adapted to HeLa cells, since both cell lines have an epithelial origin. After serial passage of the HeLa adapted FPV in human leukemic cells (which have a mesothelial origin), adaptation to HeLa cells was partially lost [7], indicating that there might be such a property as "epithelial adaptation". T h a t we were still dealing with Fowl Plague virus and not with some contaminant picked up during the long passage history of our strain was indicated by the electron microscopical findings: the budding particles had the typical morphology of myxoviruses. Their reactivity with an antiserum prepared against the egg grown virus not passaged in BT 20 cells showed their antigenic similarity to the starting virus. The same antiserum, incidentally, reacted to high titer with a subline of the original strain passed only a few times in eggs.
Immunological aspects In order for immunological potentation of cellular antigens by viruses to occur, it seems essential that a full cycle of viral replication in the relevant cells be obtained [17]. This condition appears to have been met in the present experiments. The budding of virus particles was strikingly similar to that seen in Ehrlich ascites tumor cells infected with the WSA strain of influenza virus [18], a system where immunological potentiation has been studied extensively [1, 2]. The use of ferritin labelled antiviral antibody also revealed incorporation of viral antigens at membrane sites showing no other evidence of viral activity (Fig. 3 and 6). This indicated intimate associations of viral and cellular antigens at various stages of the infectious process, thus creating numerous opportunities for those hapten-carrier relationships to become established which are thought to form the basis of immunological potentiation [17]. With the virus in its present state of adaptation to a stable cell line of human m a m m a r y tumor origin, it should be easier to grow it in newly established tissue cultures from fresh m a m m a r y tumors. This, of course, would be a prerequisite should "virus-assisted immunotherapy" of cancer patients, using their own virus-infected autologous tumor as a vaccine, be contemplated. If one were to become convinced that certain established tumor cell lines, such as the BT 20 line used in the present experiments, contained antigens shared by spontaneously occurring tumors and amenable to immunological potentiation by viruses, then a much easier technical approach would become feasible: BT 20 cells could be grown in vast quantities and infected with virus by procedures which have become standard practice in the manufacture of viral vaccines. To prove, however, that BT 20 cells, or any other established tumor cell line carries relevant antigens will require some sort of immunological test showing clearly a correlation between a patient's capacity to react against these cells and his clinical course.
Acknowledgements--The authors wish to acknowledge the excellent assistance of Mrs. M. Badoux, Mrs. S. Ekenbark, Miss R. Keller, and Miss R. Leemann. We thank Prof. L. Ozzello for supplying the BT 20 cell line.
REFERENCES 1.
J. LINDENMANNand P. A. KLEIN, Viral oncolysis: increased immunogenicity
of host cell antigen associated with influenza virus. 3". exp. Med. 126, 93 (1967).
Human Mammary Carcinoma Cell Line: l ~ a i o n by an Avian Myxovirus 2. 3. 4. 5 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
J. LINDENMANNand P. A. KLEIN, Immunological aspects of viral oncolysis. Rec. Results Cancer Res. 9, 1 (1967). C. BOONE, K. Br~eKMAN and P. BR~'DCH.~T, Tumor immunity induced in mice with cell-free homogenates of influenza virus-infected tumor cells. Nature (Lond.) 231, 265 (1971). C . W . BOONE, M. PARANJPE, T. O ~ and R. GILLETTE, Virus-augmented tumor transplantation antigens: evidence for a helper antigen mechanism. Int. J. Cancer 13, 543 (1974). M . D . EATON, J. A. HELLER and A. R. SCALA, Enhancement of lymphoma cell immunogenicity by infection with non-oncogenic virus. Cancer Res. 33, 3293 (1973). J. LINDZNMANN, The use of viruses as immunological potentiators. Ciba Foundation Symposium 18, (new series) 197 (1973). A. GENDER, CaR. SAtrrER and J. LI~EN~a~N, Fowl plague virus adapted to human epithelial tumor cells and human myeloblasts in vitro--I. Characteristics and replication in monolayer cultures. Arch. ges. Virusforsch.40, 137 (1973). A. GEm3ER, CHR. SAUTERand J. LINDENMANN,Fowl plague virus adapted to human epithelial tumor cells and human myeloblasts in vitro---II. Replication in human leukemic myeloblast cultures. Arch. ges. Virusforsch. 40, 255 (1973). CHR.SAUTER,U. BAUMBERGER,S. EKENBARKand J. LINDENMANN,Replication of an avian myxovirus in primary cultures of human leukemic ceils. Cancer Res. 33, 3002 (1973). E.Y. LASFARGUESand L. OZZELLO, Cultivation of human breast carcinomas. J. nat. Cancer Inst. 21, 1131 (1958). L . J . REED and H. MUENCrI, A simple method of estimating fifty per cent endpoints. Amer. J. Hyg. 27, 493 (1938). M. AGUET and TH. BXCHI, Comparison of the direct and indirect immunoferritin tagging of Sendai virus antigen. Ann. Immunol. (Inst. Pasteur) 124 C, 407 (1973). C. HALLAUER,Studien tiber die Variabilit~it des Htihnerpestvirus im Gewebeexplantat. Arch. ges. Virusforsch. 1, 70 (1939). C. HALLAUER and G. KRONAUER, Weitere Versuche zur Ztichtung und Abeandlung von klassischem Gefltigelpestvirus in menschlichen Gewebeexplantaten. Arch. ges. Virusforsch. 9, 232 (1959). H . E . PEREIRA,Multiplication of fowl plague virus in chicken embryo cell suspensions. J. Path. Bact. 65, 259 (1953). D . M . CHAPRONIEREand H. G. PEREIRA, Propagation of fowl plague and of Newcastle disease viruses in cultures of embryonic human lung. Brit. J. exp. Path. 36, 607 (1955). J. LINDENMANN,Viruses as immunological adjuvants in cancer. Biochim. Biophys. Acta (Review) 290, 555 (1974). Ta. BXcHI, W. GERHARD,J. LINDEN~CrANNand K. MOHLETHALER,Morphogenesis of influenza A virus in Ehrlich ascites tumor cells as revealed by thinsectioning and freeze-etching. J. Virol. 4, 769 (1969).
63
Human Mammary Carcinoma Cell Line: Infection by an Avian Myxovirusas a Prerequisite for Immunopotentiation* CHRISTIAN SAUTER 1, THOMAS B.~CHI 2 and JEAN LINDENMANN 2 aDivision of Oncology, Department of Medicine and 2Division of Experimental Microbiology, Institute of Medical Microbiology, University of Ziirich, 8006 Ziirich, Switzerland Abstract--In view of the proven potentiation of tumor antigen activity produced by infecting tumor cells with myxoviruses, a strain of avian influenza virus (A/Turkey~ England/63, Hay I, Nav 3) previously adapted to grow in HeLa cells was passed serially 8 times in the human mammary carcinoma cell line B T 20. Comparisons of growth curves of the starting v#us with the eighth passage level showed that adaptation had taken place. Immune electron microscopy using ferritin-labelled antiviral antibody revealed viral particles budding at and viral antigens incorporated in cell membranes. The possible use of such a virus for "virus-assisted immunotherapy" is discussed.
INTRODUCTION
primary cultures of human mammary carcinoma.
AUGMENTATION of tumor cell immunogenicity by infection with viruses has been shown in several systems [ 1-5]. Enveloped viruses, mostly influenza viruses, were used for this purpose, since the association of viral and host antigens at membrane sites is apparently important for the augmentation of tumor cell immunogenicity [6]. An essential step for the use of viruses as immunological potentiators in the immunotherapy of human neoplasms is the successful replication of an enveloped virus in the tumor cell in question. Fowl plague virus (FPV), an influenza A virus, has been adapted to several types of human tumor cells [7, 8] and has been shown to replicate regularly in primary cultures of human leukemic cells [9]. In this communication, we report the adaptation of the same virus strain to a human mammary carcinoma cell line as a first step towards obtaining a virus capable of growing in
MATERIAL AND M E T H O D S V/rus The fowl plague virus (A/Turkey/England/ 63, Hav 1, Nav 3, Langham strain) which we used in these experiments had the following passage history: Obtained from H. G. Pereira, World Influenza Center, Mill Hill, London, England, after 4 allantoic passages; the virus was passed 3 times in I C R mouse kidney cell cultures, 18 times in KB ceils, twice in HeLa cells, once in the chick allantois and once in a primary culture of human bronchial carcinoma cells. This virus will be referred to as FPV. (having had no passage in m a m m a r y carcinoma cells). The subscript numbers following FPV will indicate the number of passages in mammary carcinoma cells. Human mammary carcinoma cell line
Accepted 17 June 1974.
The human mammary carcinoma cell line BT 20 [10] was obtained from L. Ozzello, University of Lausanne, Switzerland.
*This investigation was supported by the Julius Mtiller Foundation for Cancer Research, Ziirich, and the Cancer Research Institute, Ziirich Branch.
59
60
Christian Sauter, Thomas B?ichi and Jean Lindenmann
Culture media BT 20 cells were grown in R P M I 1640 (Grand Island Biological Company, Grand Island, N.Y., USA), supplemented with 2 70 fetal calf serum (FCS). Virus adaptation, virus replication, and electron microscopy were done on cells maintained in Eagle's minimum essential medium (MEM) supplemented with 2 % FCS.
Virus adaptation The virus for each passage was taken from the previous passage at the time of first detection ofhemagglutinin. Every 12-24 hr the supernatant was checked for hemagglutinin. For each passage, a 25 cm 2 Falcon plastic bottle (Falcon Plastics, Oxnard, Calif. USA) containing a complete monolayer of BT 20 cells was washed with M E M and infected with 0.33 ml of a virus dilution in M E M containing between 101'° to 103.9 T C I D s o (5070 tissue culture infectious dose) (see Table 1). The infected culture was incubated for 15 min at 37°C with agitation every 5 min for virus adsorption. Four-point-six-six ml of M E M with 2 70 FCS were then added and incubation was continued until detection of hemagglutinin. At this time the supernatant was harvested, and aliquots were sealed in sterile glass ampules and stored at - 8 0 ° C until infectivity titrations or a further adaptation passage could be performed.
Virus replication curves To compare the replication of FPVo and FPV8 in BT 20 cells the following experiment was done: two 75 cm 2 Falcon plastic bottles containing a complete monolayer of BT 20 cells were washed with MEM. To one bottle was added 1 ml of FPVo containing about 102.5 TCIDso, to the other bottle, 1 ml of FPV8 containing also about 102.5 TCIDso. The infected cultures were incubated for 15 rain at 37°C with agitation every 5 min for virus adsorption. Nineteen ml of M E M with 2 7o FCS were then added and incubation was continued. Five ml of supernatant of each bottle were removed (and replaced by 5 ml of fresh medium) at 4, 22, 46, 70, 94, and 176 hr of incubation. The removed samples were checked for hemagglutinin, put into sterile glass ampules and kept at - 8 0 ° C until infectivity titrations could be performed.
Virus titrations Virus infectivity assays were performed in 1 6 x l 0 0 m m glass tubes containing chicken
embryo fibroblast monolayer cultures. The monolayers were infected with serial 10-fold dilutions of virus in M E M without serum. Six tubes per dilution were used. After 72 hr of incubation at 37°C, the tubes were screened for cytopathic effects. Tubes with incomplete destruction of the monolayers were checked for hemagglutinin. The TCIDso'S were calculated according to the method of Reed and Muench [11]. The HA titrations were done according to W H O standard procedures.
Antisera Antiserum was prepared to FP virus grown in embryonated hens' eggs. Allantoic fluid with an HA titer of 512 was stored at - 8 0 ° C until used for immunization. Rabbits immunized by 5 weekly intramuscular injections of 2 ml virus with 2 ml complete Freund's adjuvant were bled 14 days after the last injection. The sera, after inactivation (56°C, 30 min) and exhaustive absorption with washed human erythrocytes (0/Rh +), showed a hemagglutination inhibition titer of 1280 against FP virus as tested with fowl red cells. Guinea pig anti-rabbit IgG serum conjugated with ferritin was prepared as described elsewhere [12].
Electron microscopy Cells from infected (16 hr after inoculation with FP virus) or uninfected cultures were washed three times with cold Earle's balanced salt solution (EBSS). The monolayers were incubated with rabbit anti-FP serum for 30 min at 4°C) washed three times with cold EBSS, incubated (30 min, 4°C) with guinea pig antirabbit IgG conjugated with ferritin and washed again three times with cold EBSS. The cells were then fixed (30 min, 4°C), with a mixture of 3 7oo glutaraldehyde and 3 70 acrolein in 0.05 M cacodylate buffer, pH 7.2. All cells were postfixed for 6 0 m i n at 4°C with 27o osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2. The fixed material was left overnight in a 2 7o solution of uranyl acetate before dehydration and embedding in Epon-Araldite. The thin sections were stained with lead citrate.
RESULTS Adaptation of F P V to B T 20 cells FPV was passed serially in BT 20 cells in order to get an optimal adaptation to this cell line. In Table 1, the growth characteristics of the different passages are shown. The virus inputs varied between 101"° to 10 3.9 T C I D s o due to the fact that the viral dilutions had to
Human Mammary Carcinoma Cell Line : Infection by an Avian Myxovirus be chosen arbitrarily, the infectivity titer of the .previous passage being unknown at the time of infection of the following passage. To select for particles capable of replicating in BT 20 cells, the dilutions from one passage to the next were at least 100-fold. Based on our experience with FPV in HeLa cells [7], we expected that a few passages would suffice for optimal adaptation. From the fifth passage on, hemagglutination was regularly detected at 46 hr of incubation. Regardless of the input, the T C I D s o was always around 106/ml at 46 hr of incubation in later passages. We therefore stopped the adaptation experiments at the eigth passage. Comparing the data of the second and sixth passage (same virus inputs, Table 1) the virus
61
log TCID 50/ml
7
3
Virus input 2
Table 1. Serialpassage of FPV in B T 20 monolayer cultures 1,
Virus input* Time to TCIDn0/ml at (Virus from detection of the time of first Passage previous hemagglutinin detection of number passage) (hr) hemagglutinin 1 2 3 4 5 6 7 8
103.9 102.0 101"3 10 x'° 102.5 102.0 103.5 10 3.0
102 84 72 57 46 46 46 46
104.25 104.75 I0 s'5 105.0 105.5 106.0 106.0 10 s's
*TCIDno per monolayer.
replication in the later passage was definitely better with respect to replication speed as well as to infectivity titer. Optimal adaptation may have already been reached by the third or fourth passage, since the delay in hemagglutinin appearance and the somewhat lower infectivity titer could have been due to the very low input in these passages. A good adaptation of FPVo to BT 20 cells therefore seemed to require at least two, but probably four, serial passages.
Comparison of the replicating of FPVo and FPV8 in B T 20 cells Figure 1 shows the growth curves of FPVo (no passage in BT 20 cells) and FPV8 (8 passages in BT 20 cells). The drop of the infectivity titer until four hr of incubation was about the same for both viruses. FPV8 then replicated much faster and to a higher titer than FPVo. For FPV8, hemagglutinin reached a maximal titer of 32 at 176 hr of incubation, whereas for FPVo, no hemagglutinin could be detected until the end of the experiment at 176 hr.
22
t,6
70
94
l'lrhm
Fig. 1. Replication of FPVo (4~-------O, no passages in B T 20 cells) and FPVs ( 0 O, eight passages in B T 20 cells) in B T 20 cell monolayers.
Cytopathogenic effect of F P V on B T 20 cells A slight cytopathogenic effect (CPE) of FPV on BT 20 cells was seen at the time of the first detection of hemagglutinin. At this stage, cytoplasmic vacuolization and a few rounded cells were observed. Twenty-four hours later, an almost complete CPE was seen (Fig. 2B). Most of the ceils had detached from the bottom of the plastic bottle, were round, and took up trypan blue stain. Electron microscopy In cultures fixed 16 hr after inoculation with FP virus, virions were frequently found to be attached to cell surfaces (Fig. 3). Occasionally, budding of virus was observed at bulging segments of the cytomembrane (Fig. 4). In addition to the morphological features characterizing these structures as viruses, immunological tagging by ferritin labelled antibody revealed viral antigenicity (Figs. 3-6). Attached or budding virus particles invariably were tagged heavily with labelled antibodies visualized by the electron dense core of the ferritin molecule. The specificity of the reaction was established by treatment of uninfected cultures with the same reactants; here, all ferritin molecules were washed out because the antiviral antibody was not bound to the cells (Fig. 7). Reaction of antibodies with membranes of infected cultures not revealing any characteristic viral differentiation demonstrated the presence of viral antigens also in segments of
Fig. 2.
Typical lymphoblast : the dense chromatin is disposed in a thin peripheral rim; the nucleolus (arrow) is compact. / x 8250).
Fig. 3. Ring-shaped nucleolu~ cell: the nucleolar (arrow)components are disposed in a characteristical ring-like disposition(inset). The dense chromatin is s~milar to that observed in typical lymphoblasls. ( x 9500; inset : x 20700). Fig. 4. X cell : the chromatin is condensed in an irregular peripheral strand and in blocks dispersed in the nucleoplasm and around the large nucleolus (arrow). ( x 10750).
Fig. 2.
Phase-contrast photomicrographs of B T 20 cell monolayers, x 320. A. Uninfected control culture. B. 94 hr after infection with FPVs.
(to face p. 62)
62
Christian Sauter, Thomas Btichi and Jean Lindenmann
cellular surfaces where neither budding nor adsorbed virus particles were seen (Figs. 3 and 6). In infected cultures, a few ceils were seen which completely failed to react with the antisera and which showed no structural signs of virus replication (Fig. 5). At the stages of infection studied in the electron microscope, the cytopathic effects (16 hr) were inconspicuous. The aspect and structural integrity of nucleus and cytoplasmic organelles in uninfected cells treated under the same conditions of incubation (Fig. 7) were essentially the same as in infected cultures (Fig. 6).
DISCUSSION In the present report we have shown that an avian influenza A virus (fowl plague virus), after several adaptation passages, replicated well in a human m a m m a r y carcinoma cell line (BT 20). Virological as well as immunotherapeutical aspects of this finding wilt now be discussed.
Virological aspects In contrast to human influenza A strains and to the Brescia [13, 14] and Dutch strains of avian influenza A [15, 16], the avian influenza A we used in these experiments proved to be easily adaptable to human tumor cells. For adaptation to BT 20 cells we chose FPV already adapted to HeLa cells, since both cell lines have an epithelial origin. After serial passage of the HeLa adapted FPV in human leukemic cells (which have a mesothelial origin), adaptation to HeLa cells was partially lost [7], indicating that there might be such a property as "epithelial adaptation". T h a t we were still dealing with Fowl Plague virus and not with some contaminant picked up during the long passage history of our strain was indicated by the electron microscopical findings: the budding particles had the typical morphology of myxoviruses. Their reactivity with an antiserum prepared against the egg grown virus not passaged in BT 20 cells showed their antigenic similarity to the starting virus. The same antiserum, incidentally, reacted to high titer with a subline of the original strain passed only a few times in eggs.
Immunological aspects In order for immunological potentation of cellular antigens by viruses to occur, it seems essential that a full cycle of viral replication in the relevant cells be obtained [17]. This condition appears to have been met in the present experiments. The budding of virus particles was strikingly similar to that seen in Ehrlich ascites tumor cells infected with the WSA strain of influenza virus [18], a system where immunological potentiation has been studied extensively [1, 2]. The use of ferritin labelled antiviral antibody also revealed incorporation of viral antigens at membrane sites showing no other evidence of viral activity (Fig. 3 and 6). This indicated intimate associations of viral and cellular antigens at various stages of the infectious process, thus creating numerous opportunities for those hapten-carrier relationships to become established which are thought to form the basis of immunological potentiation [17]. With the virus in its present state of adaptation to a stable cell line of human m a m m a r y tumor origin, it should be easier to grow it in newly established tissue cultures from fresh m a m m a r y tumors. This, of course, would be a prerequisite should "virus-assisted immunotherapy" of cancer patients, using their own virus-infected autologous tumor as a vaccine, be contemplated. If one were to become convinced that certain established tumor cell lines, such as the BT 20 line used in the present experiments, contained antigens shared by spontaneously occurring tumors and amenable to immunological potentiation by viruses, then a much easier technical approach would become feasible: BT 20 cells could be grown in vast quantities and infected with virus by procedures which have become standard practice in the manufacture of viral vaccines. To prove, however, that BT 20 cells, or any other established tumor cell line carries relevant antigens will require some sort of immunological test showing clearly a correlation between a patient's capacity to react against these cells and his clinical course.
Acknowledgements--The authors wish to acknowledge the excellent assistance of Mrs. M. Badoux, Mrs. S. Ekenbark, Miss R. Keller, and Miss R. Leemann. We thank Prof. L. Ozzello for supplying the BT 20 cell line.
REFERENCES 1.
J. LINDENMANNand P. A. KLEIN, Viral oncolysis: increased immunogenicity
of host cell antigen associated with influenza virus. 3". exp. Med. 126, 93 (1967).
Human Mammary Carcinoma Cell Line: l ~ a i o n by an Avian Myxovirus 2. 3. 4. 5 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
J. LINDENMANNand P. A. KLEIN, Immunological aspects of viral oncolysis. Rec. Results Cancer Res. 9, 1 (1967). C. BOONE, K. Br~eKMAN and P. BR~'DCH.~T, Tumor immunity induced in mice with cell-free homogenates of influenza virus-infected tumor cells. Nature (Lond.) 231, 265 (1971). C . W . BOONE, M. PARANJPE, T. O ~ and R. GILLETTE, Virus-augmented tumor transplantation antigens: evidence for a helper antigen mechanism. Int. J. Cancer 13, 543 (1974). M . D . EATON, J. A. HELLER and A. R. SCALA, Enhancement of lymphoma cell immunogenicity by infection with non-oncogenic virus. Cancer Res. 33, 3293 (1973). J. LINDZNMANN, The use of viruses as immunological potentiators. Ciba Foundation Symposium 18, (new series) 197 (1973). A. GENDER, CaR. SAtrrER and J. LI~EN~a~N, Fowl plague virus adapted to human epithelial tumor cells and human myeloblasts in vitro--I. Characteristics and replication in monolayer cultures. Arch. ges. Virusforsch.40, 137 (1973). A. GEm3ER, CHR. SAUTERand J. LINDENMANN,Fowl plague virus adapted to human epithelial tumor cells and human myeloblasts in vitro---II. Replication in human leukemic myeloblast cultures. Arch. ges. Virusforsch. 40, 255 (1973). CHR.SAUTER,U. BAUMBERGER,S. EKENBARKand J. LINDENMANN,Replication of an avian myxovirus in primary cultures of human leukemic ceils. Cancer Res. 33, 3002 (1973). E.Y. LASFARGUESand L. OZZELLO, Cultivation of human breast carcinomas. J. nat. Cancer Inst. 21, 1131 (1958). L . J . REED and H. MUENCrI, A simple method of estimating fifty per cent endpoints. Amer. J. Hyg. 27, 493 (1938). M. AGUET and TH. BXCHI, Comparison of the direct and indirect immunoferritin tagging of Sendai virus antigen. Ann. Immunol. (Inst. Pasteur) 124 C, 407 (1973). C. HALLAUER,Studien tiber die Variabilit~it des Htihnerpestvirus im Gewebeexplantat. Arch. ges. Virusforsch. 1, 70 (1939). C. HALLAUER and G. KRONAUER, Weitere Versuche zur Ztichtung und Abeandlung von klassischem Gefltigelpestvirus in menschlichen Gewebeexplantaten. Arch. ges. Virusforsch. 9, 232 (1959). H . E . PEREIRA,Multiplication of fowl plague virus in chicken embryo cell suspensions. J. Path. Bact. 65, 259 (1953). D . M . CHAPRONIEREand H. G. PEREIRA, Propagation of fowl plague and of Newcastle disease viruses in cultures of embryonic human lung. Brit. J. exp. Path. 36, 607 (1955). J. LINDENMANN,Viruses as immunological adjuvants in cancer. Biochim. Biophys. Acta (Review) 290, 555 (1974). Ta. BXcHI, W. GERHARD,J. LINDEN~CrANNand K. MOHLETHALER,Morphogenesis of influenza A virus in Ehrlich ascites tumor cells as revealed by thinsectioning and freeze-etching. J. Virol. 4, 769 (1969).
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