Archiv für die gesamte Virusforschung
Begründet von R. Doerr
Herausgegeben von S. Gard, Stockholm C. Hallauer, Bern A. Mayr, München W. P. Rowe, Bethesda, Md. J.Vilcek, New York, N.Y.
Vol. 45, 1974
Springer-Verlag Wien New York Archiv für die gesamte Virusforschung
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© 1974 by Springer-Verlag / Wien
Contents/Inhalt: Vol. 45
No. 1—2
Kalinina, Natalya O., Irina V. Scarlat, and V. I. Agol: The Synthesis of Virus-Specific Polypeptides on Polyribosomes Isolated from the Cells In• fected with Encephalomyocarditis Virus (With 3 Figures) 1
Ozaki, Υ., K. Kumagai, M. Kawanishi, and A. Seto: Studies on the Neutra lization of Japanese Encephalitis Virus. III. Analysis of the Neutralization Reaction by Anti-Rabbit-y-Globulin Serum (With 2 Figures) 7
Dmitrieva, Τ. M., and V. I. Agol: Selective Inhibition of the Synthesis of Single-Stranded RNA of Encephalomyocarditis Virus by 2-(a-Hydroxybenzyl) benzimidazole in Cell-Free Systems (With 7 Figures) 17
Aurelian, Laure, and J. D. Strandberg: Biologic and Immunologic Com parison of Two HSV-2 Variants. One an Isolate from Cervical Turnor Cells (With 6 Figures) 27
Berthiaume, I., J. Joncas, and V. Pavilanis: Comparative Structure, Morpho genesis and Biological Characteristics of the Respiratory Syncytial (RS) Virus and the Pneumonia Virus of Mice (PVM) (With 7 Figures) 39 Contents/Inhalt III
Lubiniecki, A. S., J. A. Armstrong, and Mo η to Ho: Eastern Equine Ence phalitis Virus. Quantitative Study of the Effects of Interferon on Virus Re plication (With 4 Figures) 52
Mikhejeva, Angelika, and Yu. Ghendon: The Influence of Supraoptimal Temperature on Poliovirus Type 1 Mahoney Strain Reproduction (With 6 Figures) 65
De Vleesschauwer, L., and S. R. Pattyn: Replication of Arboviruses in Mouse Organ Cultures. I. Results with Middelburg Virus in Organ Cultures and Peritoneal Macrophages (With 5 Figures) 78
Mazur, Natalia, Helena Wrzos, Renata Zahorska, and K. Zakrzewski: Quantitative Haemadsorption Assay of Measles and Parotitis Virus in the Presence of Each Other 86
Sekine, N., and K. Yoshino: Inhibitors against Rabies Virus Present in Normal Rabbit Sera (With 1 Figure) 89
McLaren, C, C. W. Potter, and R. Jennings: Immunity to Influenza in Fer rets. XII. Immunization of Ferrets with TNBP-Split Influenza Virus Vaccine (With 2 Figures) 99
Matthews, T. H. J., Sylvia E. Reed, and D. A. J. Tyrrell: The Effect of Prior Inoculation with an Enterovirus (LEV 4) on Rhinovirus Infection of Volunteers (With 1 Figure) 106
Lemercier, G., D. Schmitt, P. Quenin, A. Jayne et R. Fontanges: La τέ- ponse immunitaire locale au cours de la grippe expe>imentale de la souris Balb/c infectee par myxovirus influenzae A/Hong-Kong/1/68 (H3N2) (Avec 3 Figures) / The Local Immune Response in Experimental Airborne Influenza of Balb/C Mice Infected with Myxovirus Influenza A/Hong-Kong/1/68 (H3N2) 113
Bandlow, G., and U. Koszinowski: Increased Cellular Immunity against Host Cell Antigens Induced by Vacciniavirus (With 2 Figures) 122
Rockborn, G., H. Diderholm, and Z. Dinter: Bovine Viral Diarrhea Virus: Acquired Resistance to Acriflavine as Marker of an Attenuated Strain (With 3 Figures) 128
Lazarus, L. H., and A. Itin: Requirement for Double-Stranded RNA during the in vitro Synthesis of RNA by Foot-and-Mouth Disease Virus Replicase (With 2 Figures) 135
Brief Reports / Kurze Mitteilungen
Reisenbuk, Valentina, and Silver loks: Australia Antigen and Cell-Mediated Immunity 141
Bykovsky, A. F., I. S. Irlin, and V. M. Zhdanov: A New Group of Oncorna viruses (With 1 Figure) 144
Kascsak, R. J., and M. J. Lyons: Attempts to Demonstrate the Interferon Defense Mechanism in Cultured Mosquito Cells 149
Deig, E. F.: Efficient Production of Large Volumes of Immune Ascitic Fluid from Mice 155
Kurogi, Η., Y. Inaba, E. Takahashi, K. Sato, Y. Goto, T. Omori, and M. Matumoto: New Serotypes of Reoviruses Isolated from Cattle .... 157 IV Contents/Inhalt
Blalock, J. Ε., and G. E. Gifford: Effect of "Aquasol A", Vitamin A, and "Tween 80" on Vesicular Stomatitis Virus Plaque Formation and on Inter feron Action (With 1 Figure) . 161
Plavosin, Livia, and P. Diosi: Non-Productive Infection of Genetically Resist ant Mouse Cells by Murine Cytomegalovirus (With 3 Figures) 165
Diderholm, Η., B. Klingeborn, and Z. Dinter: A Hazy ('Bull's Eye') Plaque Variant of Bovine Viral Diarrhea Virus (With 3 Figures) . 169
Zalan, E., C.Wilson, Dz. Pukitis, and N. A. Labzoffsky: Detection of In fluenza Antibodies by Immunofluorescence Using Antigen-Coated Chicken Erythrocytes (With 1 Figure) 173
Erratum 176 a
No. 3 Gaidamovich, S. Ya, and Sh. A. Kurakhmedova: Hemagglutinating Pro perties of Viruses of the Phlebotomus Fever Group (With 1 Figure) .... 177
Chia, W. K., and M. Savan: Electron Microscopic Observations on Infectious Bovine Rhinotracheitis Virus in Bovine Fetal Tracheal Organ Cultures (With 18 Figures) 185
Klein, P. Α.: Adaptation of Influenza Virus to Growth in Cultured Murine Methylcholanthrene Induced Tumors (With 4 Figures) 199
Dzhivanyan, Τ. I., V. A. Lashkevich, G. G. Bannova, E. S. Sarmanova, Μ. V. Chuprinskaya, J. Vesenjak-Hirjan, and V. Vince: On the Possible Association of the DS Marker of Tick-Borne Encephalitis Virus Strains with Species of Tick Vectors (With 1 Figure) 209
Zhdanov, V. Μ., Ν. N. Bogomolova, V. I. Gavrilov, O. G. Andzhaparidze, P. G. Deryabin, and A. N. Astakhova: Infectious DNA of Tick-Borne Encephalitis Virus (With 5 Figures) 215
Zhdanov, V. M., and Μ. I. Parfanovich: Integration of Measles Virus Nucleic Acid into the Cell Genome (With 5 Figures) 225
Noronha, F., Ε. Dougherty, A. Poco, C. Gries, J. Post, and C. Rickard: Cytological and Serological Studies of a Feline Endogenous C-Type Virus (With 10 Figures) 235
Boyd, Janice E., and R. G. Sommerville: The Antiviral Activity of Some Related Benzo (b) Thiophene Derivatives. I. Initial Screening Tests (With 2 Figures) 249
Boyd, Janice E., and R. G. Sommerville: A Rapid, Simple and Reliable Method for the Screening of Potential Antiviral Compounds in vitro (With 4 Figures) 254
Vande Woude, G. F., and R. Ascione: Translation Products of Foot-and- Mouth Disease Virus-Infected Baby Hamster Kidney Cells (With 5 Figures) . 259
Vigärio, J. D., A. M. Terrinha, and J. F. Moura Nunes: Antigenic Relation• ships among Strains of African Swine Fever Virus (With 2 Figures) . . . 272
Nishibe, Y., and Υ. K. Inoue: Relationship between SMON Virus and Avian Infectious Laryngotracheitis Virus (With 3 Figures) 278 Contents/Inhalt V
McClurkin, A. W., E. C. Pirtle, M. F. Coria, and R. L. Smith: Comparison of Low- and High-Passage Bovine Turbinate Cells for Assay of Bovine Viral Diarrhea Virus (With 2 Figures) 285
Brief Reports / Kurze Mitteilungen Zakay-Rones, Ζ., G. Spira, and R. Levy: Local Antibody Formation in the Brain of Chickens (With 3 Figures) 290 Sergeant, Α., J. C. D'Halluin, A. P. Verbert, and V. Krsmanovic: Effect of Ionic Strength on Adenovirus 2 DNA Transcription by KB Cell DNA- Dependent RNA Polymerases I, II, and III (With 1 Figure) 294
Wigand, R-, and W. Klein: Properties of Adenovirus Substituted with lodo- deoxyuridine (With 1 Figure) 298
No. 4 Seemayer, Ν. H., and V. Defendi: Analysis of Minimal Functions of Simian Virus 40 (With 5 Figures) 301 Peleg, J., and V. Stollar: Homologous Interference in Aedes aegypti Cell Cultures Infected with Sindbis Virus (With 4 Figures) 309 Lecatsas, G., O. W. Prozesky, and F. Scheepers: The Cytopathology and Development of a Human Polyomavirus (B.K.) (With 13 Figures) 319 Rossi, C. R., and G. K. Kiesel: Complement-Requiring Neutralizing Anti bodies in Cattle to Infectious Bovine Rhinotracheitis Virus (With 1 Figure) 328 Paul, N. R., A. J. Rhodes, J. B. Campbell, and N. A. Labzoffsky: Rubella Precipitin Response in Natural Infection and in Vaccination (With 3 Figures) 335 Iwakata, S., L. P. Morrissey, A. J. Rhodes, and N. A. Labzoffsky: The Use of Trypsin-Treated Human Group 0 Erythrocytes in Rubella Hemagglutina- tion-lnhibition Test 352
Brief Reports / Kurze Mitteilungen Barteling, S. J., and R. H. Meloen: A Simple Method for the Quantification of 140 S Particles of Foot-and-Mouth Disease Virus (FMDV) (With 1 Figure) 362 Harter, D. H., Eugenia T. Gamboa, and J. E. Coward: Replication of Measles Virus in Dolphin Kidney Cells (With 5 Figures) 365 Bessho, Hiroko, Yoko Nishibe, and Υ. K. Inoue: Morphology of SMON Virus (With 2 Figures) 370 Lomniczi, B.: Multiplication of Different Newcastle Disease Virus Strains in Chicken Brain (With 2 Figures) 373 Zylber-Katz, Ester, A. Lazar, and Pnina Weisman: Electron Microscopic Studies on Frog Virus 3 Infection in HeLa Cells at Permissive and Non- Permissive Temperatures (With 6 Figures) 376 Shibuta, Η., T. Ishikawa, R. Hondo, Y. Aoyama, K. Kurata, and M. Matu- moto: Varicella Virus Isolation from Spinal Ganglion (With 1 Figure) ... 382 Van der Veen, J., and A. Mes: Serological Classification of Two Mouse Adenoviruses 386
Listed in Current Contents Archiv für die gesamte Virusforschung 45, 122—127 (1974) © by Springer-Verlag 1974
Increased Cellular Immunity against Host Cell Antigens Induced bv Vaccinia virus1
By
G. BANDLOW and XL KOSZINOWSKI Hygiene Institute of the University of Göttingen, Federal Republic of Germany
With 2 Figures Received March 4, 1974
Summary Guinea pigs show an increased cell-mediated immunity against host cell anti• gens after active immunization with vaccinia virus infected tissue culture cells. This immunologic adjuvant effect was observed in a heterologous system com• prising primary rabbit kidney cells, primary fibroblasts from the mouse, permanent monkey kidney cells and in vivo infected mouse brain cells. The quantitative determination of the cellular immune response was carried out both in macrophage migration inhibition and in lymphocyte transformation tests. Cell-mediated immunity appears on the third day after immunization. A prerequisite for the adjuvant effect is the reproduction of the virus in the cell against which the increas• ed cell-mediated immune response is directed.
1. Introduction Ever since the publication of RAMON in 1925 (l)on the production of diphtheria and tetanus antitoxins, an increase in the immunogenicity of different substances has been aimed at the use of adjuvants. Now more than 70 different adjuvants are known, and in the meantime several immunological adjuvants have attained great importance in medicine, especially in the production of vaccines. Essentially three substances have won great importance: aluminium compounds, substances which are known as Freund's adjuvants and thirdly endotoxins of gramnegative bact• eria. All these adjuvants are substances, which increase the immunogenicity of other substances in a completely unspecific way. In preceding papers we have reported that viruses act as immunological adjuvants (2). Guinea pigs being actively immunized against heterologous virus- infected cells showed an increased production of humoral antibodies against the host cells used. These cytotoxic antibodies were directed only against the cell type
1 Supported by a grant from the "Land Niedersachsen". Β AND LOW et CiL: Cellular Immunity against Host Cell Antigens by Vacciniavirus 123 iin which the virus had been multiplied, thus indicating that the virus acts as a very j-specific adjuvant. In this connection we wanted to find out whether viruses, 1 besides increasing the production of cytotoxic antibodies, can also improve the * cell-mediated immune response. For the following experiments we chose the vaccinia virus, as in our previous studies on humoral antibodies it has proved to be fa very good immunological adjuvant (3).
2. Materials and Methods
2.1. Virus and Cell Culture Vaccinia virus strain WR was grown in cell cultures of BHK-cells (106·5 TCIDso/ml),
δ 5 Vero-cells (10 TCIDso), RK-cells (10 TCID5o/ml), embryonic guinea pig fibroblasts
8 ((10*TCID5o/ml) as well as in vivo in the brain of adult NMRI mice (10 TCID5o). "Vaccinia virus strain Elstree was cultivated in human embryonic cells (106 TCIDso/ml). Herpes simplex virus strain "Hof" was grown in BHK-cells (105·3 TCIDso)- All cell cultures were grown in Eagle's Minimum Essential Medium with Earle's 1BSS. Infectivity titrations were performed in AGMK cells.
2.2. Animals and Immunization Pirbright guinea pigs, weighing 250 g, were used. Experiments were performed in igroupes of 5 animals each. The virus infected and non infected cell suspensions were adjusted to 800 μg• protein/ml. Inoculation of the suspensions took place as described before (2). 2.3. Assay for Blast Cell Transformation Guinea pigs were bled out by cardiac puncture. The heparinized (25 U/ml Thrombo- vetren, Promonta, Hamburg) blood was sedimented with a 10 per cent added volume of 5 per cent dextran in saline (MG 250,000, Pharmacia, Uppsala, Sweden) for 45 min utes at 37° C to obtain a plasma rich in peripheral white blood cells. The spleens of the guinea pigs were removed aseptically, cut in fragments and homogenized by hand in a glass grinder with the addition of Eagle's Medium. The suspension was centrifuged at 11500 rev/min for 5 minutes, the sediment re-suspended in medium TC 199 with 30 per cent homologous heat-inactivated guinea pig serum, and left standing for 1 hour at ~J-4° C. The cells in the supernatant fluid were then washed twice with Hanks' BSS, and finally resuspended in culture medium TC 199 with 15 per cent guinea pig serum. The suspension of the washed peripheral white blood cells as well as the spleen cells was adjusted to 106 viable lymphocytes per ml. Cell cultures were set up consisting of 1 χ 106 mononuclear cells in a total volume of 1 ml in disposable culture tubes (Greiner, Nürtin• gen, Nr. 160S) at +37° C in a 5 per cent COa-air atmosphere. Twelve hours before processing the cultures, 1.5 μ-Ci of tritiated thymidine (spec, activity 23,000 mCi/mMol thymidine, Radiochemical Centre Amersham), was added to each vial. Uptake of the isotope into DNA was assayed at the end of cultivation. The proliferative response was expressed as an isotope incorporation index, representing the ratio of the mean counts/minute of replicate culture vials containing antigen divided by mean counts/ minute of replicate control culture vials in the same experiment.
2.4. Assay for Macrophage Migration Inhibition Macrophages were obtained from the peritoneal cavity of Pirbright strain guinea pigs 72 hours after injection of 20 ml of sodiumthioglycollate (Difco Laboratories, Detroit). The exudate cells were washed three times and incubated at 21° C for 60 min utes in TCI99 with 15 per cent homologous heat-inactivated guinea pig serum and 20 per cent antigen (virus cellsuspension or cell suspension alone, 1000γ protein/ml). Glass capillaries were filled with these cells adjusted to 107 cells/ml, then sealed with "Seal-Ease" (Adams, Parsippang, N.J.) at one end, and centrifuged at 1000 r.p.m. for 124 G. BANDLOW and U. KOSZINOWSKI :
5 minutes. Each capillary was then cut at the border of the packed cells and placed in plastic culture chambers (Greiner, Nürtingen), where they were held in place by a small amount of sterile silicone. The chambers were filled with medium containing 10 per cent antigen (1000 γ/ml) or medium without antigen as control and incubated at 37° C. Migration areas were determined after 16 and 24 hours by tracing their outline on a microscope projection screen and measured by planimetry. The effect of antigens on macrophage migration was determined by comparison with the simultaneously pre pared controls without antigen-containing medium and calculated as follows: average migration with antigen Migration (%) = 5. x 100 average migration without antigen
3. Results
3.1. Increased Cellular Immunity against Cell Antigens Induced by Vaccinia Virus
Pirbright guinea pigs were immunized against different cells infected in vitro with vaccinia virus. Controls were set up with non-infected cells. One week after the antigen-injection, cellular immunity against the respective host cell antigens was tested both by the macrophage migration inhibition test and the lymphocyte transformation test. The results thus obtained uniformly show that virus-infected heterologous cells induce a far better immune response than non-infected cells (Fig. 1). Both tests yielded closely correlated results (Fig. 2). The blast cell trans formation was done with peripheral blood lymphocytes as well as spleen lympho cytes; yet the former ones provided much better results. This cellular immune response is demonstrable already three days after the antigen injection. In the autologous system, however, we did not detect any cellular immunity against primary embryonic guinea pig fibroblasts.
3.2. Intracellular Virus Replication as a Prerequisite for the Immunological Adjuvant Effect
In order to induce an increased cellular immune response against heterologous host cell antigens, the virus must replicate in the cell. Already 3 hours after infection—i.e. before any mature virus particles have been formed—increased immunogenicity can be demonstrated. Simply mixing the virus and non-infected cells immediately before injection does not lead to any adjuvant effect. Similarly, no adjuvant effect was observed, when virus and non-infected cells were inoculated separately into the two hind legs. In contrast, we found an increase of the cellular immunity directed against those cells—Vero cells in the present case—in which the virus had been grown. Live vaccinia virus does not appear necessary for the enhancing effect in vivo. since treatment of virus-infected cell homogenates with 0.075 per cent (v/v) of formaldehyde may inactivate virus completely, yet without reducing the activity of the cell antigen.
3.3. Specificity of the Increased Immune Response
Induction of lymphocyte mediated immunity by different heterologous cells proved to be highly specific. Guinea pig lymphocytes sensitized against one partic ular cell species did not cross react with other heterologous cells. The induced Cellular Immunity against Host Cell Antigens by Vaeciniavirus 125 esffect proved to be strictly virus-dependent. Guinea pigs actively preimmunized aigainst vaccinia virus showed no adjuvant effect. Likewise, if the virus-infected ccell homogenate is incubated with anti-vaccinia virus antibody (Vacuman Berna) boefore being inoculated in guinea pigs, an increased cellular immune response can mot be observed. This means, that virus-specific antigens are a prerequisite for
Inhibit/on of Macrophage Migration
Blood, D \ Blast fis/i Transformation /{Η3Τ Incorporation) I Spleen I t ι t ψο
Vacc. RfC Vacc. Vero Vacc. M.Brain Vacc. ΰ.Ρ/'g RK Zero M.Brain u.Pig IFig. 1. Delayed hypersensitivity in guinea pigs which have been immunized with differ ent cells infected by vaccinia virus 20 30 W SO 60 Inhibition of Migration (%) Fig. 2. Correlation of blast cell transformation and inhibition of macrophage migration 126 G. BANDLOW and U. KOSZINOWSKI : the adjuvant effect. On the other hand, the adjuvant effect is not only inducible by vaccinia virus. Indeed, Ave were able to show the same, though less effective enhanced immunogenicity of cells by using vesicular stomatitis virus as well as herpes simplex virus. 4. Discussion Vaccinia virus acts as an immunological adjuvant, that is, it increases cellular immune reaction against heterologous cell antigens in guinea pigs. The same effect could also be demonstrated by using herpes simplex virus and vesicular stomatitis virus. Parameters for the delayed type of hypersensitivity were incorporation of tritiated thymidine into transformed guinea pig tymphocytes as well as the inhibi• tion of macrophage migration. Both tests, which have been used successfully in the guinea pig system by several authors (4, 5, 6), correlated closely. Our experi• ments show that virus-enhanced immunogenicity can be demonstrated with differ• ent heterologous cells, infected in vitro and in vivo by vaccinia virus. The enhance• ment of the heterologous cell antigen activity produced by vaccinia-virus is highly specific for the cell species that has been infected by the virus: immunization of guinea pigs with homogenates of different virus-infected, heterologous cells did not confer any cross immunity. Against syngeneic normal host cells we found no such virus-enhanced immunogenicity. The mode of action of the adjuvant effect induced by vaccinia virus differs in several respects from that of commonly used adjuvants such as aluminium hydroxyde or Freund's adjuvants. One prerequisite for the immunological adjuvant effect is the replication of the virus in the cell, against which the cellular immunity will be directed. Non-infected cells mixed with virus before inoculation failed to immunize against the cells. Likewise ineffective is the separate injection of vaccinia virus and non-infected cell homogenates. This provides further evidence that the cell antigen enhancing effect of the virus is not simply clue to a nonspecific stimulation of the immune system. Live vaccinia virus does not seem necessary for the observed adjuvant effect. Virus-infected cell homogenates that had been completely inactivated with form• aldehyde retained the increased immunogenicity. These experiments confirm the findings of LINDENMANN (7), HÄKKINEN (8) and. BOONE (9) who showed that treatment of virus-infected tumor cells with formaldehyde did not reduce their virus-induced immunogenicity. Immunization of mice with homogenates of influenza virus-infected tumor cells conferred immunity to challenge with tumor cells, whereas immunization with the same dose of homogenate of non-infected tumor cells did not. Already 2—3 hours after vaccinia virus infection the immunogenicity of the host cell is increased, that is before any mature particles of vaccinia viruses are produced. This coincides with a change of the host cell surface which also appears 2—3 hours after virus infection (10). Therefore the adjuvant effect produced by vaccinia virus seems to be due to a close association between host cell antigens and antigens produced by the virus. These complex antigens apparently occur in the plasma membrane of the host cell, as we were able to observe the same enhancing effect by using plasma membranes isolated from infected BHK-cells (10). BOONE (11) recently demonstrated that membrane fractions prepared from tumor Cellular Immunity against Host Cell Antigens by Vacciniavirus 127 cells infected with influenza virus showed an enhanced tumor-transplantation- antigen-activity. This and our own observations lead us to believe that the potential immunogen is a host-cell-virus antigen complex formed, in the plasma membrane of the host cell. The virus-specific part of this supposed complex antigen can be abolished by treating the virus-cell homogenates with virus specific antibody and thus, the adjuvant effect does not work a,ny more. One may suppose that the protective immune reaction against tumor specific antigens is primarily due to cellular immunity. Our experiments presented here may give evidence that viruses can take over the role of immunological adjuvants in a tumor system and possibly increase a specific cellular immune response directed against tumor cell antigens. Further experiments about virus enhanced tumor cell antigens are in progress. Acknowledgement We thank Mrs. E. Heyner and Mrs. G. Kühn for skillful technical assistance. References 1. RAMON, G.: Sur la production des antitoxines. CR. Acad. Sei. (Paris) 181, 157 (1925). 2. BANDLOW, G., F. KIELING und R. THOMSSEN: Viren als immunologische Adju- vantien für die Produktion von Antikörpern gegen Wirtszellantigene. Arch. ges. Virusforsch. 34, 287 (1971). 3. BANDLOW, G., W. FISCHER und R. THOMSSEN: Untersuchungen zum Mechanismus der immunologischen Ad ju vans Wirkung des Vacciniavirus. Arch. ges. Virusforsch. 38, 192 (1972)! 4. BLOOM, Β. R., and B. BENNETT: Mechanism of a reaction in vitro associated with delayed-type hypersensitivity. Science 153, 80 (1966). 5. DAVID, J. R., S. AL-ASKARI, H. S. LAWRENCE, and L. THOMAS: Delayed hyper sensitivity in vitro. I. The specificity of inhibition of cell migration antigens. J. Immunol. 93, 264 (1964). ί. WOLSTENCROFT, R. Α., and D. C. DUMONDE: In vitro studies of cell-mediated immunity. I. Induction of lymphocyte transformation by a soluble 'mitogenic* factor derived from interaction of sensitized guinea-pig lymphoid cells with specific antigen. Immunology 18, 599 (1970). 1. LINDENMANN, J., and P. A. KLEIN: Immunological aspects of viral oncolysis. Ree. Res. Cancer Res. 2, 1 (1967). 6. HÄKKINEN, I., and P. HALONEN: Induction of tumour immunity in mice with antigens prepared from influenza and vesicular stomatitis virus grown in suspension culture of Ehrlich ascites cells. J. nat. Cancer Inst. 46, 1161 (1971). S. BOONE, C, K. BLACKMAN, and P. BRAUDSCHAFT: Tumour immunity induced in mice with cell-free homogenates of influenza virus-infected tumour cells. Nature (Lond.) 231, 265 (1971). K. BANDLOW, G., U. KOSZINOWSKI und R. THOMSSEN: Gesteigerte Immunogenität von Plasmamembranen vacciniavirusinfizierter BHK-Zellen. Arch. ges. Virusforsch. 40, 63 (1973). 11. BOONE, C, T. ORME, K. BLACKMAN, and R. GILLETTE: Preparation of membrane fractions with enhanced tumor-transplantation antigen activity from tumor cells infected with influenza virus. J. nat. Cancer Inst. 51, 1141 (1973). Authors' address: P. D. Dr. G. BANDLOW, Hygiene-Institut der Universität, K'euzbergring 57, D-3400 Göttingen, Federal Republic of Germany.