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

Editorial Advisory Board / Fachbeirat

V. H. Bon i fas , L a u s a n n e

J . C a s a l s , New Haven , C o n n .

Η. Hana fusa , New York, N .Y .

W . K . J o k l i k , D u r h a m , N . C .

M. Kitaoka, Tokyo

K. M a r a m o r o s c h , Y o n k e r s , N .Y.

M. Matumoto, Tokyo

M. Mussgay , T ü b i n g e n

E. Norrby, S t o c k h o l m

W . P l o w r i g h t , L o n d o n

F. S teck , Bern

J . H. S u b a k - S h a r p e , G l a s g o w

Η. M. T e m i n , Madison, W i s .

D. A . J . Tyrrel l , Harrow

J . D. Ver l inde, Leiden

A . P. W a t e r s o n , London

R. We i l , Geneve

Κ. E. W e i s s , Onderstepoort

H. A . W e n n e r , K a n s a s City, Mo.

V . M. Zhdanov , M o s c o w

Editorial A s s i s t a n t / Redakt ionssekretär: F. Heinz, W i e n

The exclusive copyright for all languages and countries, including the right for photo­mechanical and any other reproductions including microform is transferred to the publisher.

Alle Rechte, einschließlich das der Übersetzung in fremde Sprachen und das der photo­mechanischen Wiedergabe oder einer sonstigen Vervielfältigung, auch in Mikroform, vor­behalten.

© 1974 by Springer-Verlag / Wien

Contents/Inhalt: Vol. 45

No. 1—2

K a l i n i n a , N a t a l y a O . , I r i n a V . S c a r l a t , and V . I. A g o l : T h e S y n t h e s i s of V i r u s - S p e c i f i c Po lypept ides on Po ly r ibosomes Isolated from the C e l l s In­fected with E n c e p h a l o m y o c a r d i t i s V i rus (With 3 Figures) 1

O z a k i , Υ . , K . K u m a g a i , M. K a w a n i s h i , and A . S e t o : Studies on the Neutra­lization of J a p a n e s e Encepha l i t i s V i r u s . III. A n a l y s i s of the Neutralization React ion by A n t i - R a b b i t - y - G l o b u l i n S e r u m (With 2 Figures) 7

D m i t r i e v a , Τ . M., and V . I. A g o l : Select ive Inhibition of the S y n t h e s i s of S i n g l e - S t r a n d e d R N A of Encepha lomyocard i t is V i r u s by 2-(a-Hydroxybenzyl) benzimidazole in C e l l - F r e e S y s t e m s (With 7 F igures ) 17

A u r e l i a n , L a u r e , and J . D . S t r a n d b e r g : Bio logic and Immunologic C o m ­par ison of T w o H S V - 2 Var ian ts . O n e an Isolate from Cervical Turnor C e l l s (With 6 F igures ) 27

B e r t h i a u m e , I., J . J o n c a s , and V . P a v i l a n i s : Comparat ive Structure, Morpho­g e n e s i s and Bio log ica l C h a r a c t e r i s t i c s of the Respiratory Syncytial ( R S ) V i r u s and the P n e u m o n i a V i r u s of Mice (PVM) (With 7 Figures) 39

Contents/ Inhalt III

L u b i n i e c k i , A . S . , J . A . A r m s t r o n g , and Mo η to H o : E a s t e r n Equ ine E n c e ­phalit is V i r u s . Quantitative Study of the Effects of Interferon on V i r u s R e ­plication (With 4 Figures) 52

M i k h e j e v a , A n g e l i k a , and Y u . G h e n d o n : T h e Inf luence of Supraopt ima l T e m p e r a t u r e on Pol iovirus T y p e 1 Mahoney Stra in Reproduct ion (With 6 F igures) 65

D e V l e e s s c h a u w e r , L . , and S . R. P a t t y n : Repl icat ion of A r b o v i r u s e s in M o u s e Organ Cul tures . I. R e s u l t s with Middelburg V i r u s in Organ C u l t u r e s and Per i toneal Macrophages (With 5 F igures ) 78

M a z u r , N a t a l i a , H e l e n a W r z o s , R e n a t a Z a h o r s k a , and K . Z a k r z e w s k i : Quantitative Haemadsorpt ion A s s a y of M e a s l e s and Parot i t is V i r u s in the P r e s e n c e of E a c h Other 86

S e k i n e , N. , and K . Y o s h i n o : Inhibitors aga inst R a b i e s V i r u s P r e s e n t in Normal Rabbit S e r a (With 1 Figure) 89

M c L a r e n , C , C . W . Po t te r , and R. J e n n i n g s : Immunity to Inf luenza in Fer ­rets. XI I . Immunization of Ferrets with T N B P - S p l i t Inf luenza V i r u s V a c c i n e (With 2 F igures ) 99

M a t t h e w s , T . H . J . , S y l v i a E . R e e d , and D . A . J . T y r r e l l : T h e Effect of Prior Inoculation with an Enterovirus ( L E V 4) on Rhinov i rus Infection of V o l u n t e e r s (With 1 Figure) 106

L e m e r c i e r , G . , D . S c h m i t t , P . Q u e n i n , A . J a y n e et R . F o n t a n g e s : L a τέ-ponse immunitaire locale au c o u r s de la grippe expe>imentale de la s o u r i s B a l b / c infectee par myxovirus inf luenzae A / H o n g - K o n g / 1 / 6 8 (H3N2) ( A v e c 3 F igures) / T h e Loca l Immune R e s p o n s e in Exper imental A i r b o r n e Inf luenza of B a l b / C Mice Infected with Myxovirus Inf luenza A / H o n g - K o n g / 1 / 6 8 (H3N2) 113

B a n d l o w , G . , and U . K o s z i n o w s k i : I nc reased Ce l lu la r Immunity a g a i n s t Host Cel l A n t i g e n s Induced by V a c c i n i a v i r u s (With 2 F i g u r e s ) 122

R o c k b o r n , G . , H . D i d e r h o l m , and Z . D i n t e r : Bov ine Vi ra l D ia r rhea V i r u s : Acqu i red R e s i s t a n c e to Acr i f lav ine a s Marker of an At tenuated St ra in (With 3 Figures) 128

L a z a r u s , L . H . , and A . I t i n : Requirement for D o u b l e - S t r a n d e d R N A during the in vitro S y n t h e s i s of R N A by Foot -and-Mouth D i s e a s e V i r u s R e p l i c a s e (With 2 F igures) 135

B r i e f R e p o r t s / K u r z e M i t t e i l u n g e n

R e i s e n b u k , V a l e n t i n a , and S i l v e r l o k s : A u s t r a l i a Ant igen and Cel l -Mediated Immunity 141

B y k o v s k y , A . F . , I. S . I r l in , and V . M. Z h d a n o v : A New G r o u p of O n c o r n a ­vi ruses (With 1 Figure) 144

K a s c s a k , R . J . , and M. J . L y o n s : At tempts to D e m o n s t r a t e the Interferon Defense Mechan ism in Cul tured Mosquito C e l l s 149

D e i g , E . F . : Efficient Product ion of Large V o l u m e s of Immune A s c i t i c Fluid from Mice 155

K u r o g i , Η . , Y . I n a b a , E . T a k a h a s h i , K . S a t o , Y . G o t o , T . O m o r i , and M. M a t u m o t o : New Serotypes of R e o v i r u s e s Isolated from Catt le . . . . 157

IV Contents/ Inhalt

B l a l o c k , J . Ε., and G . E . G i f f o r d : Effect of " A q u a s o l A " , Vi tamin A , and " T w e e n 80" on V e s i c u l a r Stomat i t is V i r u s Plaque Format ion and on Inter­feron Act ion (With 1 F igure) . 161

P l a v o s i n , L i v i a , and P . D i o s i : Non-Product ive Infection of Genet ica l ly R e s i s t ­ant Mouse C e l l s by Murine Cytomegalov i rus (With 3 F igures ) 165

D i d e r h o l m , Η . , B . K l i n g e b o r n , and Z . D i n t e r : A Hazy ( 'Bu l l ' s Eye ' ) P laque Variant of Bovine Viral D ia r rhea V i r u s (With 3 F igures) . 169

Z a l a n , E . , C . W i l s o n , D z . P u k i t i s , and N . A . L a b z o f f s k y : Detect ion of In­f luenza Ant ibod ies by I m m u n o f l u o r e s c e n c e Using A n t i g e n - C o a t e d C h i c k e n Erythrocytes (With 1 F igure) 173

E r r a t u m 176 a

No. 3 G a i d a m o v i c h , S . Y a , and S h . A . K u r a k h m e d o v a : Hemagglutinating P r o ­

perties of V i r u s e s of the P h l e b o t o m u s Fever Group (With 1 Figure) . . . . 177

C h i a , W . K . , and M. S a v a n : Electron Microscop ic Observa t ions on Infect ious Bovine Rhinotrachei t is V i r u s in Bovine Fetal T r a c h e a l Organ Cu l tu res (With 18 F igures) 185

K l e i n , P . Α . : Adaptat ion of Inf luenza V i r u s to Growth in Cul tured Murine Methylcholanthrene Induced T u m o r s (With 4 F igures) 199

D z h i v a n y a n , Τ . I., V . A . L a s h k e v i c h , G . G . B a n n o v a , E . S . S a r m a n o v a , Μ . V . C h u p r i n s k a y a , J . V e s e n j a k - H i r j a n , and V . V i n c e : On the P o s s i b l e A s s o c i a t i o n of the D S Marker of T i c k - B o r n e Encephal i t is V i r u s S t ra ins with S p e c i e s of T i c k V e c t o r s (With 1 Figure) 209

Z h d a n o v , V . Μ . , Ν . N . B o g o m o l o v a , V . I. G a v r i l o v , O . G . A n d z h a p a r i d z e , P . G . D e r y a b i n , and A . N . A s t a k h o v a : Infectious D N A of T i c k - B o r n e Encephal i t is V i r u s (With 5 F i g u r e s ) 215

Z h d a n o v , V . M., and Μ . I. P a r f a n o v i c h : Integration of Meas les V i r u s Nucle ic A c i d into the Ce l l G e n o m e (With 5 F igures) 225

N o r o n h a , F . , Ε. D o u g h e r t y , A . P o c o , C . G r i e s , J . P o s t , and C . R i c k a r d : Cytological and S e r o l o g i c a l S t u d i e s of a Fel ine Endogenous C - T y p e V i r u s (With 10 F igures) 235

B o y d , J a n i c e E . , and R . G . S o m m e r v i l l e : T h e Antiviral Activity of S o m e Related Benzo (b) T h i o p h e n e Derivat ives. I. Initial S c r e e n i n g T e s t s (With 2 F igures) 249

B o y d , J a n i c e E . , and R . G . S o m m e r v i l l e : A Rapid, S imple and Rel iable Method for the S c r e e n i n g of Potential Antiviral C o m p o u n d s in vitro (With 4 F igures) 254

V a n d e W o u d e , G . F . , and R . A s c i o n e : Transla t ion Products of Foot -and-Mouth D i s e a s e V i rus - In fec ted Baby Hamster Kidney Ce l ls (With 5 F igures ) . 259

V i g ä r i o , J . D. , A . M. T e r r i n h a , and J . F . M o u r a N u n e s : Ant igen ic Relat ion­s h i p s among S t r a i n s of A f r i c a n S w i n e Fever V i rus (With 2 F igures) . . . 272

N i s h i b e , Y . , and Υ . K . I n o u e : Relat ionship between S M O N V i r u s and A v i a n Infectious Laryngotrachei t is V i r u s (With 3 F igures) 278

Contents/ Inhalt V

M c C l u r k i n , A . W . , E . C . P i r t l e , M. F . C o r i a , and R . L . S m i t h : C o m p a r i s o n of L o w - and H i g h - P a s s a g e Bovine Turb inate C e l l s for A s s a y of Bovine Viral D ia r rhea V i r u s (With 2 F igures) 285

B r i e f R e p o r t s / K u r z e M i t t e i l u n g e n

Z a k a y - R o n e s , Ζ . , G . S p i r a , and R. L e v y : Loca l Ant ibody Formation in the Brain of C h i c k e n s (With 3 F igures) 290

S e r g e a n t , Α . , J . C . D ' H a l l u i n , A . P . V e r b e r t , and V . K r s m a n o v i c : Effect of Ionic Strength on A d e n o v i r u s 2 D N A Transcr ip t ion by K B Cel l D N A -Dependent R N A P o l y m e r a s e s I, II, and III (With 1 F igure) 294

W i g a n d , R- , and W . K l e i n : Propert ies of A d e n o v i r u s Subst i tuted with lodo-deoxyuridine (With 1 Figure) 298

No. 4 S e e m a y e r , Ν . H . , and V . D e f e n d i : A n a l y s i s of Minimal Funct ions of S imian

V i r u s 40 (With 5 F igures ) 301

P e l e g , J . , and V . S t o l l a r : Homologous Interference in Aedes aegypti Cel l Cu l tu res Infected with S indb is V i r u s (With 4 F i g u r e s ) 309

L e c a t s a s , G . , O . W . P r o z e s k y , and F . S c h e e p e r s : T h e Cytopathology and Development of a Human P o l y o m a v i r u s ( B . K . ) (With 13 F igures) 319

R o s s i , C . R . , and G . K . K i e s e l : Complement -Requ i r ing Neutralizing Ant i ­bodies in Catt le to Infectious Bovine Rhinotrachei t is V i r u s (With 1 Figure) 328

P a u l , N . R . , A . J . R h o d e s , J . B . C a m p b e l l , and N . A . L a b z o f f s k y : Rubel la Precipit in R e s p o n s e in Natural Infection and in V a c c i n a t i o n (With 3 F igures) 335

I w a k a t a , S . , L . P . M o r r i s s e y , A . J . R h o d e s , and N . A . L a b z o f f s k y : T h e U s e of T ryps in -T rea ted Human Group 0 Erythrocytes in Rube l la Hemagglut ina-tion-lnhibition T e s t 352

B r i e f R e p o r t s / K u r z e M i t t e i l u n g e n

B a r t e l i n g , S . J . , and R. H . M e l o e n : A S i m p l e Method for the Quantif ication of 140 S Par t ic les of Foot-and-Mouth D i s e a s e V i r u s ( F M D V ) (With 1 Figure) 362

Har ter , D. H . , E u g e n i a T . G a m b o a , and J . E . C o w a r d : Repl icat ion of Meas les V i rus in Dolphin Kidney Ce l ls (With 5 F igures ) 365

B e s s h o , H i r o k o , Y o k o N i s h i b e , and Υ . K . I n o u e : Morphology of S M O N V i rus (With 2 F igures) 370

L o m n i c z i , B . : Multiplication of Different Newcas t le D i s e a s e V i rus St ra ins in Chicken Brain (With 2 Figures) 373

Z y l b e r - K a t z , E s t e r , A . L a z a r , and P n i n a W e i s m a n : Electron Microscopic S tud ies on Frog V i r u s 3 Infection in H e L a C e l l s at P e r m i s s i v e and Non-Permiss ive Tempera tu res (With 6 F igures) 376

S h i b u t a , Η . , T . I s h i k a w a , R. H o n d o , Y . A o y a m a , K . K u r a t a , and M. M a t u -m o t o : Var ice l la V i rus Isolation from Sp ina l Gangl ion (With 1 Figure) . . . 382

V a n d e r V e e n , J . , and A . M e s : Sero log ica l C l a s s i f i c a t i o n of T w o Mouse A d e n o v i r u s e s 386

L i s t e d in C u r r e n t C o n t e n t s

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. B A N D L O W and X L K O S Z I N O W S K I

Hygiene Inst i tute of the University of Göttingen, Federal Republic of Germany

W i t h 2 Figures

Received March 4, 1974

Summary

Guinea pigs show an increased cell-mediated i m m u n i t y against host cell a n t i ­gens after active immunizat ion w i t h vaccinia v irus infected tissue culture cells. This immunologic ad juvant effect was observed i n a heterologous system com­prising p r i m a r y rabb i t k idney cells, p r i m a r y fibroblasts f r om the mouse, permanent monkey k idney cells and in vivo infected mouse bra in cells. The quant i ta t ive determination of the cellular immune response was carried out b o t h i n macrophage migrat ion i n h i b i t i o n and i n lymphocyte transformation tests. Cell-mediated i m m u n i t y appears on the t h i r d day after immunizat ion . A prerequisite for the ad juvant effect is the reproduction of the virus i n the cell against which the increas­ed cell-mediated immune response is directed.

1. Introduction

Ever since the publ icat ion of R A M O N i n 1925 ( l ) o n the production of d iphther ia and tetanus ant i tox ins , an increase i n the immunogenic i ty of dif ferent substances has been aimed at the use of adjuvants. N o w more t h a n 70 dif ferent adjuvants are known , and i n the meantime several immunological adjuvants have atta ined great importance i n medicine, especially i n the product ion of vaccines. Essentially three substances have won great importance: a l u m i n i u m compounds, substances which are k n o w n as Freund's adjuvants and t h i r d l y endotoxins of gramnegative bact­eria. A l l these adjuvants are substances, which increase the immunogenic i ty of other substances i n a completely unspecific way.

I n preceding papers we have reported t h a t viruses act as immunological adjuvants (2). Guinea pigs being act ively immunized against heterologous virus-infected cells showed an increased product ion 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 " L a n d Niedersachsen".

Β A N D L O W et CiL: Cellular I m m u n i t y against Host Cell Antigens by Vacciniavirus 123

i i n wh i ch the v irus had been mul t ip l i ed , thus indicat ing t h a t the virus acts as a very j-specific a d j u v a n t . I n this connection we wanted to f i n d out whether viruses, 1 besides increasing the product ion of cytotoxic antibodies, can also improve the * cell-mediated immune response. For the following experiments we chose the vaccinia v i rus , as i n our previous studies on humoral antibodies i t has proved to be

fa ve ry good immunological ad juvant (3).

2 . Mater ia ls a n d Methods

2.1. Virus and Cell Culture Vaccinia virus strain W R was grown in cell cultures of BHK-cells (106·5 TCIDso/ml),

Vero-cells (10 δ TCIDso), RK-cells (105 TCID 5 o/ml) , embryonic guinea pig fibroblasts ((10*TCID 5o/ml) as well as in vivo i n the brain of adult N M R I mice (10 8 TCID 5 o) . "Vaccinia virus strain Elstree was cultivated in human embryonic cells (106 TCIDso/ml).

Herpes simplex virus strain " H o f " was grown in BHK-cells (105·3 TCIDso)-A l l cell cultures were grown in Eagle's Minimum Essential Medium w i t h Earle's

1BSS. Infect iv i ty t itrations were performed in A G M K cells.

2.2. Animals and Immunization Pirbright guinea pigs, weighing 250 g, were used. Experiments were performed i n

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 / m l Thrombo-

vetren, Promonta, Hamburg) blood was sedimented wi th a 10 per cent added volume of 5 per cent dextran in saline (MG 250,000, Pharmacia, Uppsala, Sweden) for 45 min­utes 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 w i t h 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 w i t h 30 per cent homologous heat-inactivated guinea pig serum, and left standing for 1 hour at ~J-4° C. The cells in the supernatant f luid were then washed twice w i t h Hanks' BSS, and f inally 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 m l . Cell cultures were set up consisting of 1 χ 106

mononuclear cells in a total volume of 1 ml in disposable culture tubes (Greiner, Nürtin­gen, Nr. 160S) at +37° C in a 5 per cent COa-air atmosphere. Twelve hours before processing the cultures, 1.5 μ-Ci of tr i t iated thymidine (spec, act iv i ty 23,000 mCi/mMol thymidine, Radiochemical Centre Amersham), was added to each vial . Uptake of the isotope into D N A 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 m l of sodiumthioglycollate (Difco Laboratories, Detroit) . The exudate cells were washed three times and incubated at 21° C for 60 min­utes in TCI99 w i t h 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 w i t h these cells adjusted to 107 cells/ml, then sealed w i t h "Seal-Ease" (Adams, Parsippang, N.J.) at one end, and centrifuged at 1000 r . p . m . for

124 G . B A N D L O W and U . K O S Z I N O W S K I :

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 w i t h 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 w i t h the simultaneously pre­pared controls without antigen-containing medium and calculated as follows:

average migration w i t h antigen Migration (%) = 5. x 1 0 0 average migration without antigen

3 . Results

3.1. Increased Cellular Immunity against Cell Antigens Induced by Vaccinia Virus

P i r b r i g h t guinea pigs were immunized against dif ferent cells infected in vitro w i t h vaccinia v i rus . Controls were set up w i t h non-infected cells. One week after the antigen-injection, cellular i m m u n i t y against the respective host cell antigens was tested both b y the macrophage migrat ion i n h i b i t i o n test and the lymphocyte transformation test. The results thus obtained u n i f o r m l y show t h a t virus-infected heterologous cells induce a far better immune response t h a n non-infected cells (Fig. 1). B o t h tests yielded closely correlated results (Fig. 2). The blast cell t rans­format ion was done w i t h peripheral blood lymphocytes as well as spleen l ympho ­cytes; yet the former ones provided much better results. This cellular immune response is demonstrable already three days after the antigen in ject ion . I n the autologous system, however, we d i d not detect any cellular i m m u n i t y against p r i m a r y embryonic guinea pig fibroblasts.

3.2. Intracellular Virus Replication as a Prerequisite for the Immunological Adjuvant Effect

I n order to induce an increased cellular immune response against heterologous host cell antigens, the virus must replicate i n the cell. A lready 3 hours after i n f e c t i o n — i . e . before any mature v irus particles have been formed—increased immunogenic i ty can be demonstrated. S imply m i x i n g the v irus and non-infected cells immediate ly before inject ion does not lead to any ad juvant effect. S imi lar ly , no ad juvant effect was observed, when v irus and non-infected cells were inoculated separately in to the two h i n d legs.

I n contrast, we found an increase of the cellular i m m u n i t y directed against those cel ls—Vero cells i n the present case—in which the v irus had been grown. L i v e vaccinia v irus does not appear necessary for the enhancing effect in vivo. since t reatment of virus-infected cell homogenates w i t h 0.075 per cent (v /v) of formaldehyde may inact ivate v irus completely, yet w i t h o u t reducing the a c t i v i t y of the cell antigen.

3.3. Specificity of the Increased Immune Response

I n d u c t i o n of lymphocyte mediated i m m u n i t y b y dif ferent heterologous cells proved to be h igh ly specific. Guinea p ig lymphocytes sensitized against one part i c ­ular cell species d i d not cross react w i t h other heterologous cells. The induced

Cellular I m m u n i t y against Host Cell Antigens by Vaeciniavirus 125

esffect proved to be s t r i c t l y virus-dependent. Guinea pigs act ively preimmunized aigainst vaccinia v irus showed no ad juvant effect. Likewise, i f the virus-infected ccell homogenate is incubated w i t h anti-vaccinia virus ant ibody (Vacuman Berna) boefore being inoculated i n guinea pigs, an increased cellular immune response can m o t be observed. This means, t h a t virus-specific antigens are a prerequisite for

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Inhibit/on of Macrophage Migration

D Blood,

Spleen

\ Blast fis/i Transformation /{Η3Τ Incorporation)

Vacc. RK

RfC Vacc. Zero

Vero Vacc. M.Brain Vacc. ΰ.Ρ/'g M.Brain u.Pig

IFig. 1. Delayed hypersensitivity in guinea pigs which have been immunized w i t h differ­ent 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. B A N D L O W and U . K O S Z I N O W S K I :

the ad juvant effect. On the other hand, the a d j u v a n t effect is no t o n l y inducible b y vaccinia v irus . Indeed, Ave were able to show the same, though less effective enhanced immunogenic i ty of cells b y using vesicular s tomati t is v i rus as well as herpes simplex v irus .

4. Discussion

Vaccinia v irus acts as an immunological ad juvant , t h a t is, i t increases cellular immune reaction against heterologous cell antigens i n guinea pigs. The same effect could also be demonstrated b y using herpes simplex v irus and vesicular stomatit is v irus . Parameters for the delayed type of hypersensit iv i ty were incorporation of t r i t i a t e d thymid ine in to transformed guinea p ig tymphocytes as wel l as the inh ib i ­t i on of macrophage migra t i on . B o t h tests, which have been used successfully i n the guinea pig system by several authors (4, 5, 6), correlated closely. Our experi­ments show t h a t virus-enhanced immunogenic i ty can be demonstrated w i t h differ­ent heterologous cells, infected in vitro and in vivo b y vaccinia v i rus . The enhance­ment of the heterologous cell antigen a c t i v i t y produced b y vaccinia-virus is h igh ly specific for the cell species t h a t has been infected b y the v i r u s : immunizat i on of guinea pigs w i t h homogenates of dif ferent virus-infected, heterologous cells d id not confer any cross i m m u n i t y . Against syngeneic normal host cells we f ound no such virus-enhanced immunogenic i ty .

The mode of action of the ad juvant effect induced b y vaccinia v i rus differs i n several respects f r om t h a t of commonly used adjuvants such as a lumin ium hydroxyde or Freund's adjuvants . One prerequisite for the immunological ad juvant effect is the replication of the v irus i n the cell, against whi ch the cellular i m m u n i t y w i l l be directed. Non-infected cells mixed w i t h v i rus before inoculat ion failed to immunize against the cells. Likewise ineffective is the separate in jec t ion of vaccinia virus and non-infected cell homogenates. This provides fur ther evidence that the cell antigen enhancing effect of the v irus is no t s imply clue to a nonspecific s t imulat ion of the immune system.

L ive vaccinia v irus does not seem necessary for the observed ad juvant effect. Virus-infected cell homogenates t h a t had been completely inact ivated w i t h f o r m ­aldehyde retained the increased immunogenic i ty . These experiments confirm the f indings of L I N D E N M A N N ( 7 ) , H Ä K K I N E N (8) and. B O O N E (9) who showed t h a t t reatment of virus-infected t u m o r cells w i t h formaldehyde d i d no t reduce their virus-induced immunogenic i ty . I m m u n i z a t i o n of mice w i t h homogenates of influenza virus-infected t u m o r cells conferred i m m u n i t y to challenge w i t h tumor cells, whereas immunizat ion w i t h the same dose of homogenate of non-infected t u m o r cells d i d not .

A l ready 2 — 3 hours after vaccinia v irus infection the immunogenic i ty of the host cell is increased, t h a t is before any mature particles of vaccinia viruses are produced. This coincides w i t h a change of the host cell surface which also appears 2 — 3 hours after v irus infection ( 1 0 ) . Therefore the ad juvant effect produced b y vaccinia virus seems t o be due to a close association between host cell antigens and antigens produced b y the v irus . These complex antigens apparent ly occur i n the plasma membrane of the host cell, as we were able to observe the same enhancing effect b y using plasma membranes isolated f r om infected B H K - c e l l s ( 1 0 ) . B O O N E ( 1 1 ) recently demonstrated t h a t membrane fractions prepared f rom tumor

Cellular I m m u n i t y against Host Cell Antigens by Vacciniavirus 127

cells infected w i t h influenza virus showed an enhanced tumor - t ransp lantat i on -a n t i g e n - a c t i v i t y . This and our own observations lead us t o believe t h a t the potent ia l i m m u n o g e n is a host-cell-virus antigen complex formed, i n the plasma membrane of t h e host cell. The virus-specific par t of this supposed complex antigen can be abolished b y t reat ing the virus-cell homogenates w i t h v i rus specific ant ibody and t h u s , the ad juvant effect does not work a,ny more.

One m a y suppose t h a t the protective immune reaction against t u m o r specific antigens is p r i m a r i l y due to cellular i m m u n i t y . Our experiments presented here m a y give evidence t h a t viruses can take over the role of immunological adjuvants in a t u m o r system and possibly increase a specific cellular immune response directed against tumor cell antigens. Fur ther experiments about v irus enhanced t u m o r cell antigens are i n progress.

Acknowledgement We thank Mrs. E. Heyner and Mrs. G. Kühn for skil l ful technical assistance.

References 1. R A M O N , G.: Sur la production des antitoxines. C R . Acad. Sei. (Paris) 181, 157

(1925). 2. B A N D L O W , G., F. K I E L I N G und R. T H O M S S E N : Viren als immunologische A d j u -

vantien für die Produktion von Antikörpern gegen Wirtszellantigene. Arch. ges. Virusforsch. 34, 287 (1971).

3. B A N D L O W , G., W. F I S C H E R und R. T H O M S S E N : Untersuchungen zum Mechanismus der immunologischen A d j u vans Wirkung des Vacciniavirus. Arch. ges. Virusforsch. 38 , 192 (1972)!

4. B L O O M , Β. R., and B. B E N N E T T : Mechanism of a reaction in vitro associated w i t h delayed-type hypersensitivity. Science 153, 80 (1966).

5. D A V I D , J . R., S. A L - A S K A R I , H . S. L A W R E N C E , and L . T H O M A S : Delayed hyper­sensitivity in vitro. I . The specificity of inhibition of cell migration antigens. J . Immunol . 93 , 264 (1964).

ί. W O L S T E N C R O F T , R. Α . , and D. C. D U M O N D E : 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 w i th specific antigen. Immunology 18, 599 (1970).

1. L I N D E N M A N N , J . , and P. A. K L E I N : Immunological aspects of viral oncolysis. Ree. Res. Cancer Res. 2, 1 (1967).

6. H Ä K K I N E N , I . , and P. H A L O N E N : Induction of tumour immuni ty 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. B O O N E , C, K . B L A C K M A N , and P. B R A U D S C H A F T : Tumour immunity induced in mice with cell-free homogenates of influenza virus-infected tumour cells. Nature (Lond.) 231, 265 (1971).

K . B A N D L O W , G., U . K O S Z I N O W S K I und R. T H O M S S E N : Gesteigerte Immunogenität von Plasmamembranen vacciniavirusinfizierter BHK-Zel len . Arch. ges. Virusforsch. 40, 63 (1973).

11. B O O N E , C , T. O R M E , K . B L A C K M A N , and R. G I L L E T T E : Preparation of membrane fractions with enhanced tumor-transplantation antigen act iv i ty from tumor cells infected wi th influenza virus. J . nat. Cancer Inst . 51 , 1141 (1973).

Authors' address: P. D. Dr. G. B A N D L O W , Hygiene-Inst i tut der Universität, K'euzbergring 57, D-3400 Göttingen, Federal Republic of Germany.