Archives of Virology
Archives of Virology 61, 327--336 (1979)
© by Springer-Verlag 1979
Role of Envelope Proteins of Paramyxoviruses in the Modification of Cell Membrane Antiflens By M. D. EATON Department of Medical Microbiology, Stanford University, Stanford, California, U,S.A. Accepted March 20, 1979
Summary Adsorption of paramyxoviruses to separated membranes of tumor cells produces neoantigens t h a t are immunogenic in syngeneic mice (xenogenization). Thus it became possible to study this process b y using modified virus or virus fractions. Membranes with adsorbed viral derivatives produced an immune response which was measured by cytotoxic and complement fixing antibodies and the appearance of autoimmune disease. The effect of viral preparations with reduced F (fusion) protein activity was compared to treatment of membranes with equivalent amounts of active or inactive virus as measured b y hemagglutination. Viral preparations without hemolytic activity showed diminished adsorption to membranes and the immune response was reduced. Triton X100 and desoxycholate extracted from membranes immunogel~ic material with associated paramyxovirus antigens.
Introduction Modification of t u m o r cell membrane antigens b y myxo- and paramyxoviruses, vesicular stomatitis, vaceinia, type C leukemia virus and others have been described b y several investigators (2, 10, 11). I n these studies homogenates of tumors which were not immunogenic in syngeneic mice showed antigenieity after virus had grown in them. Homogenates of uninfected tumors were not immunogenic. Membrane fractions prepared from an ascites lymphoma infected with parainfluenza virus were shown to be as immunogenic as the cell homogenates whereas membranes from uninfected tumor cells were not (2, 3), and some other fractions from infected cells did not produce immunity to tumor transplant. Syngeneie mice produced antibodies eytotoxic for uninfected tumor cells and cell mediated immunity was demonstrated (1, 3). Also complement fixing antibodies reactive with the membranes and several other t u m o r cell fractions were detected. Antibodies were not stimulated b y similar preparations from uninfected tumor. I n addition to the anti-tumor immunity-, autoantibodies against syngeneic spleen, as well as other tissues, were demonstrated (4). With continued immunization antibodies to deoxynucleoprotein and heterophile reagins also appeared, 22*
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and some of the mice died with a wasting disease characterized b y loss of lymphoid tissue and scarred atrophied kidneys. I t was then found t h a t adsorption of concentrated virus by cell membranes isolated from aseites lymphoma produced immunogenie material t h a t gave results similar to the membranes from infected cells (5). However, autoimmune disease developed more rapidly and tumor transplant resistance, while present in some experiments, could not be demonstrated in others. These observations on the effect of adsorbed virus indicated t h a t modification of cell membrane antigens is a biochemical reaction that can occur in the absence of viral replication causing syngeneic or "self" antigens to appear to the system for cellular and humoral immune response as foreign. Adsorption of vaceinia virus to L cells is said to produce an antigen causing an H-2 dependent response of T lymphocytes as measttred by antiviral immune cytolysis (7). Similar results have been reported for Sendai virus, active or inactivated with UV light or ~-propiolaetone, and Sendal virus envelopes which contain mostly the hemagglutinin-neuraminidase and fusion proteins. Target cells were derived from a mastoeytoma with adsorbed viral preparations (20, 21). Inactivation of the F protein b y trypsin (21) or ethanol (20) resulted in preparations t h a t were ineffective for the formation of target cells lysable b y T lymphocytes although comparable amounts of neuraminidase were adsorbed (20). These studies on H-2 restriction of T cell eytotoxieity with adsorbed virus differ from our experiments to be described, in that our target cells have no infecting or adsorbed virus and lysis is by antibodies. However the several kinds of preparations derived from Sendai virus are very similar to what we used in the preparation of immunogens from isolated ceil membranes. Among the m a n y studies of the interaction of viruses with membranes of intact cells (adsorption and penetration), relatively few have been done with isolated membranes. The envelope proteins of vesicular stomatitis virus have been shown to react with isolated cell membranes in a selective manner, the strongest adsorption being with the glycoprotein for adsorption and penetration (i2). Other investigators have studied the elution and uncoating of enteroviruses by isoIated H e L a eelI plasma membranes (16). The membrane reactive components of paramyxovirus are the H N glycoprotein (hemagglutination and neuraminidasc) and so-called F protein (17) which produces hemolysis and cell fusion. Several procedures have been used to modify the latter protein leaving the H N protein unchanged. We have used these methods to study the effect of the F protein on adsorption of virus to isolated tumor ceil membranes and the resulting antigenic modifications as detected by immunogenieity. Measurements were done on eytotoxie and complement fixing antibodies with syngeneie uninfected lymphoma and spleen and the observation of autoimmune disease in the mice injected with these modified antigens. Materials and Methods Mice
The C 3I-I/Bi/G strain, originally obtained from Dr. Jacob Furth, has been carried by brother X sister mating in our labora%ory, Mice usecl for experiments were either male or female. C 3I-I/HeJ males purchased
from the Jackson Laboratory,
Bar I-Iarbor,
V i r a l M o d i f i c a t i o n of M e m b r a n e A n t i g e n s
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M a i n e , w h i c h differ f r o m t h e C 3 H / B i s t r a i n i n m i n o r t r a n s p l a n t a t i o n a n t i g e n s , were u s e d i n s o m e e x p e r i m e n t s to o b t a i n t u m o r a n t i b o d i e s w i t h o u t a n t i v i r a l a n t i b o d i e s .
Ascites Lymphoma B y i.p. t r a n s p l a n t of l e u k e m i c spleen f r o m C 3 t-I/Bi mice i n f e c t e d w i t h t h e Gross m u r i n e l e u k e m i a v i r u s , a n aseites l y m p h o m a w a s p r o d u c e d . T h e 50 p e r c e n t l e t h a l dose for C 3 t - I / B i m i c e is 5 t o 10 cells a n d i n t h e s y n g e n e i c s t r a i n of mice n o regression of e s t a b l i s h e d t u m o r s h a s e v e r b e e n o b s e r v e d . T u m o r celt e x t r a c t s or m e m b r a n e s fail t o i m m u n i z e e i t h e r C 3 H / B i or C 3 H / I I e , J mice (2). C r u d e t u m o r cell m e m b r a n e p r e p a r a t i o n s from u n i n f e c t e d aseites l y m p h o m a were o b t a i n e d as d e s c r i b e d p r e v i o u s l y (2, 3).
Viruses N e w c a s t l e disease v i r u s (NI)V) s t r a i n Cal a n d p a r a i n f l u e n z a I s t r a i n S e n d a l (SiV) were o b t a i n e d f r o m D r . A. M. l~rinee a n d Dr. J . F. E n d e r s , r e s p e c t i v e l y . A n o t h e r s~rain of SiV o b t a i n e d f r o m Microbiological A s s o c i a t e s w a s u s e d i n s o m e e x p e r i m e n t s . T h e s e s t r a i n s of N D V a n d SiV d i d n o t grow well i n t i s s u e c u l t u r e s of t h e aseites I y m p h o m a b u t d i d a d s o r b to i s o l a t e d t u m o r celt m e m b r a n e s . S t o c k v i r u s e s were p r o p a g a t e d i n t h e a l l a n t o i c sac of 11 -day-old c h i c k e m b r y o s a n d were p a r t i a l l y p u r i f i e d a n d c o n c e n t r a t e d t e n - f o l d b y d i f f e r e n t i a l c e n t r i f u g a t i o n . T h e y were s t o r e d a t - - 6 0 ° C.
Modi/ieation o/ Viral Envelope Proteins SeverM p r o c e d u r e s p u b l i s h e d b y o t h e r s w e r e a p p l i e d i n a t t e m p t s t o r e d u c e or a b o l i s h t h e biological a c t i v i t y of t h e h e m o l y s i s - f u s i o n (F) p r o t e i n l e a v i n g t h e n e u r a m i n i d a s e a n d h e m a g g l u t i n a t i n g a c t i v i t y u n c h a n g e d or o n l y s l i g h t l y d i m i n i s h e d . M e t h o d s u s e d were as follows: 1. T w e e n 8 0 - e t h e r t r e a t m e n t (9, 14) : c o n c e n t r a t e d N D V was s h a k e n for 5 m i n u t e s w i t h T w e e n 80 a t 4 ° C. O n e - t h i r d v o l u m e of e t h e r was a d d e d a n d s h a k i n g c o n t i n u e d for 15 m i n u t e s . A f t e r c e n t r i f u g a t i o n a t 3000 r p m a n d 4 ° C for 20 m i n u t e s , t h e a q u e o u s l a y e r w a s r e m o v e d a n d t h e r e m a i n i n g e t h e r e v a p o r a t e d b y b u b b l i n g t h r o u g h a slow s t r e a m of Ne. 2. F o r m a l d e h y d e a t a c o n c e n t r a t i o n of 0.2 p e r c e n t was i n c u b a t e d w i t h SiV a t 37 ° C for 2 h o u r s . A f t e r s e d i m e n t a t i o n for 90 m i n u t e s a t 18,000 r p m i n a S o r v a l SB 2 c e n t r i f u g e , t h e v i r u s was r e s u s p e n d e d i n t h e original v o l u m e of p h o s p h a t e b u f f e r e d saline (PBS) (14). 3. E g g p a s s a g e SiV was i n o c u l a t e d i n t o a tissue c u l t u r e of b o v i n e k i d n e y cells ( M D B X ) d e r i v e d f r o m a t r a n s p l a n t k i n d l y f u r n i s h e d b y D r . P u r n e l l C h o p p i n (17). Growth under these conditions results in a variant with uncleared F protein. This v i r i o n h a s low i n f e c t i v i t y a n d l a e k s h e m o l y t i c a n d fusion a c t i v i t i e s unless t r e a t e d with certain proteolytie enzymes. 4. E n v e l o p e g l y c o p r o t e i n s of SiV were solubilized w i t h t h e d e t e r g e n t N o n i d e t P 40 a e e o r d i n g to t h e m e t h o d of HOSA~:A a n d S ~ I ~ I z v (6). V i r u s cores c o n t a i n i n g t h e R N A n u e l e o p r o t e i n were r e m o v e d b y e e n t r i f u g a t i o n a t 100,000 × g in a Spinco 40.2 a n g l e c e n t r i f u g e h e a d . E n v e l o p e p a r t i c l e s r e m a i n in t h e s u p e r n a t a n t a n d t h e d e t e r g e n t c a n b e r e m o v e d b y dialysis in S p e c t a p o r t u b i n g w i t h a M.~V. c u t o f f of 12,000 for 72 h o u r s a g a i n s t t h r e e c h a n g e s of P B S a t 4 ° C. S o m e h e m o l y t i c a n d h e m a g g l u t i n a t i n g a c t i v i t y w a s lost i n t h i s p r o c e d u r e . Igernoval of t h e d e t e r g e n t is followed b y r e a s s e m b l y of g l y e o p r o t e i n s 2 a n d 4 i n t o h e m o l y t i c p a r t i c l e s a c c o r d i n g t o /-IosAKA a n d SmMIZV. 5. F o r c o m p a r i s o n w i t h f o r m a l i n i n a c t i v a t e d SiV w h i c h h a s lost h e m o l y t i c a n d fusion a b i l i t y a n o n i n f e c t i o u s p r e p a r a t i o n w h i c h r e t a i n s t h e s e biologieM a c t i v i t i e s w a s o b t a i n e d b y i n a c t i v a t i o n w i t h ~ - p r o p i o l a e t o n e a c c o r d i n g t o t h e m e t h o d of NEFF a n d E~'I)EI~S (13).
Measurements o/Hemagglutination and Hemolysis Two-fold v i r u s d i l u t i o n s (0.5 ml) in P B S w i t h o u t Ca++ or Mg ++ were m i x e d w i t h a n e q u a l v o l u m e of 0.5 p e r c e n t c h i c k e n e r y t h r o c y t e s in 18 × 100 m m t u b e s a n d allowed to s t a n d a t r o o m t e m p e r a t u r e u n t i l t h e cells h a d s e t t l e d . T h e h i g h e s t d i l a t i o n of v i r u s showing a distinct pattern at the bottom of %he tube was the endpoint.
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F o r m e a s u r e m e n t s of hemolysis t w o - f o l d dilutions of virus set u p as a b o v e were m i x e d w i t h a n equal v o l u m e of 2 p e r c e n t guinea pig e r y t h r o c y t e s a n d i n c u b a t e d for a n h o u r a t 37 ° C. T h e t u b e s w e r e t h e n p l a c e d in t h e r e f r i g e r a t o r u n t i l t h e cells h a d s e t t l e d . The e n d p o i n t was t a k e n as t h e h i g h e s t d i l u t i o n of v i r u s giving a p p r o x i m a t e l y 50 p e r c e n t hemolysis.
Adsorption o] Virus to Crude Membranes M e m b r a n e s u s p e n s i o n s d e r i v e d f r o m a b o u t 10 s cells/m] were m i x e d w i t h a n equal v o l u m e of P B S c o n t a i n i n g i n t a c t v i r u s or m o d i f i e d virus p r e p a r a t i o n s . I n t h e s e experim e n t s t h e I-IA u n i t s / m l of virus p r e p a r a t i o n s v a r i e d f r o m 1000 t o 10,000 H A / m l b u t H A was equalized b y d i l u t i o n of t h e a c t i v e v i r u s p r e p a r a t i o n s . T h e c o n c e n t r a t i o n of a c t i v e virus u s e d for c o m p a r i s o n was t h e r e f o r e a d j u s t e d a c c o r d i n g to I-IA titer. Memb r a n e s a n d v i r u s were t h o r o u g h l y d i s p e r s e d in a D o u n c e h o m o g e n i z e r (Belleo) a n d allowed t o r e m a i n a t 2 5 ° C for 1 hour. M e m b r a n e s w i t h a d s o r b e d v i r u s w e r e t h e n s e d i m e n t e d a t 10a r p m for 20 m i n u t e s a n d w a s h e d t h r e e t i m e s in P B S b y r e s u s p e n d i n g a n d c e n t r i f u g i n g u n t i l less t h a n 10 H A u n i t s / m l r e m a i n e d in t h e s u p e r n a t a n t . This p r o c e d u r e h a s b e e n d e s c r i b e d p r e v i o u s l y in m o r e detail (5). Viral a n t i g e n i r r e v e r s i b l y a t t a c h e d to t h e m e m b r a n e s was m e a s u r e d b y c o m p l e m e n t f i x a t i o n w i t h a n t i v i r a l r a b b i t serum.
A ntisera R a b b i t s w e r e i m m u n i z e d w i t h egg p a s s a g e N D V or SiV b y s u b c u t a n e o u s or i n t r a m u s c u l a r i n j e c t i o n a t m o n t h l y i n t e r v a l s . I n s o m e e x p e r i m e n t s t h e a n t i v i r a l sera were a b s o r b e d w i t h n o r m a l m i n c e d chorioallantoic m e m b r a n e s or o t h e r chick e m b r y o tissue (5). A n t i t u m o r sera w e r e o b t a i n e d b y i m m u n i z i n g C ~ H / H e J mice first w i t h m i n c e d spleen f r o m CaH/Bi mice, t h e n w i t h u n i n f e c t e d ascites l y m p h o m a . B e c a u s e of t h e difference in m i n o r h i s t o e o m p a t i b i l i t y a n t i g e n s b e t w e e n t h e s e t w o s t r a i n s of mice small doses of t h e allogeneie t u m o r were r e j e c t e d b y C~H/I-IeJ mice a f t e r t h e y h a d b e e n i m m u n i z e d w i t h C s H / B i tissue. A n t i s e r a f r o m CaH/Bi mice i m m u n i z e d b y 5 or m o r e i.p. i n j e c t i o n s w i t h t u m o r cell m e m b r a n e s c o n t a i n i n g a d s o r b e d virus f r o m t h e various p r e p a r a t i o n s d e s c r i b e d a b o v e w e r e t e s t e d for a n t i b o d i e s to u n i n f e c t e d t u m o r s a n d n o r m a l s y n g e n e i e spleen b y complement fixation and complement dependent eytotoxieity.
Complement Fixation W e l l - d i s p e r s e d m e m b r a n e s of u n t r e a t e d ascites l y m p h o m a or C3H/Bi spleen ceils o b t a i n e d as d e s c r i b e d p r e v i o u s l y (3) w e r e u s e d as a n t i g e n s to d e t e c t a n t i b o d y r e s p o n s e t o t h e t u m o r or a u t o a n t i b o d i e s as m e a s u r e d w i t h t h e n o r m a l spleen cell m e m b r a n e . The t e s t was p e r f o r m e d w i t h 1.6 to 1.8 u n i t s of guinea pig c o m p l e m e n t , a n t i g e n s 1 : 20, a n d t w o - f o l d d i l u t i o n s of a n t i s e r u m 1:10 to 1:160, all in a m o u n t s of 0.25 ml. A f t e r i n c u b a t i o n a t 36 ° C for 1 h o u r , 0.25 m l of 2 p e r c e n t sensitized s h e e p e r y t h r o c y t e s w e r e a d d e d . S e r u m t i t e r s were s t a t e d as t h e h i g h e s t d i l u t i o n of s e r u m in w h i c h some e r y t h r o e y t e s r e m a i n e d . T h e t i t e r of c o n t r o l s w i t h o u t a n t i g e n , for a n t i c o m p l e m e n t a r y effects of t h e s e r u m , was s u b t r a c t e d f r o m t h e t i t e r w i t h a n t i g e n . T e s t s for a d s o r b e d viral a n t i g e n on t h e m e m b r a n e s were also d o n e b y c o m p l e m e n t f i x a t i o n w i t h a n t i v i r a l r a b b i t sera 1 : 20 or 1 : 40 a n d dilutions of t h e m e m b r a n e p r e p a r a t i o n .
Cytotoxicity Tests Aseites t u m o r cells were a s p i r a t e d f r o m t h e p e r i t o n e u m a n d w a s h e d t h r e e t i m e s in H a n k s ' b a l a n c e d salt solution. N o r m a l spleen cells were o b t a i n e d b y sieving t h e finely m i n c e d C3H/Bi spleen on a 200 m e s h stainless steel screen. E r y t h r o c y t e s w e r e r e m o v e d b y i n c u b a t i o n a t 37 ° C for 20 m i n u t e s in 0.15 ~ NHaCI a n d w a s h i n g . T u m o r or spleen cells w e r e s u s p e n d e d in E a g l e ' s m i n i m u m essential m e d i u m c o n t a i n i n g 17 m ~ h e p e s b u f f e r (N-2 h y d r o x y e t h y l p i p e r a z i n e . N ' 2 e t h a n e sulfonic acid: Calbiochem) a n d 50 ~g/ml of G e n t a m y c i n (Schering Corp.). One p e r c e n t cell s u s p e n s i o n 0.1 ml, was i n c u b a t e d in a w a t e r b a t h a t 36 ° C for 18 h o u r s w i t h 0.1 m l of a n t i s e r u m 1 : 4 a n d 0.1 m l of r a b b i t c o m p l e m e n t 1 : 16 (Grand I s l a n d Biological). T u b e s w e r e s h a k e n periodicMly. C y t o t o x i c i t y w a s d e t e r m i n e d b y t h e d y e exclusion t e s t using 0.1 p e r c e n t t o l u i d i n e blue (3). T h e c y t o t o x i c i n d e x was s t a t e d as t h e p e r c e n t a g e of d e a d cells in t h e t e s t m i n u s
Viral M o d i f i c a t i o n of M e m b r a n e A n t i g e n s
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t h e p e r c e n t a g e of d e a d cells in t h e c o n t r o l c o n t a i n i n g c o m p l e m e n t a n d n o a n t i s e r u m or n o r m a l m o u s e s e r u m . I m m u n e s e r u m a l o n e w a s n o t e y t o t o x i c . S t a i n e d cells i n t h e c o n t r o l s were u s u a l l y 10 t o 20 p e r c e n t of t h e t o t a l a f t e r 18 h o u r s ' i n c u b a t i o n .
Extraction o] Viral and Tumor Cell Antigens With Detergent T u m o r cell m e m b r a n e s c a r r y i n g a d s o r b e d v i r a l a n t i g e n s or p r e p a r e d f r o m i n f e c t e d cells were e x t r a c t e d w i t h T r i t o n X 1 0 0 or d e s o x y e h o l a t c i n a n a t t e m p t to o b t a i n soluble i m m u n o g e n i e p r e p a r a t i o n s a n d to t e s t t h e s t a b i l i t y of t h e m o d i f i e d a n t i g e n s . W i t h T r i t o n X 1 0 0 (Calbiochcm) a t 0.5 or 0.25 p e r c e n t m e m b r a n e s c o n t a i n i n g N D V a n t i g e n were e x t r a c t e d i n 0.01 M s o d i u m p h o s p h a t e b u f f e r p H 7.4 for 30 m i n u t e s a t 20 ° C, u s i n g a h o m o g e n i z e r . T h e u n d i s s o l v e d residue was r e m o v e d b y c e n t r i f u g a t i n g a t 104 r p m for 1 h o u r a n d s u s p e n d e d i n P B S e q u a l t o t h e original v o l u m e . T h e d e t e r g e n t i n t h e s u p e r n a t a n t w a s r e m o v e d b y e x t r a c t i n g five t i m e s w i t h 1/3 v o l u m e of e t h e r followed b y b u b b l i n g Ne. D e s o x y c h o l a t e (10 ml) a t a c o n c e n t r a t i o n of 0.5 p e r c e n t i n Tris saline a t p H 7.8 was a d d e d t o a s e d i m e n t of c r u d e m e m b r a n e s f r o m 2 × 10 s cells i n f e c t e d w i t h N D V . A f t e r 30 m i n u t e s a t 37 ° C w i t h m i x i n g , t h e r e s i d u e w a s s e p a r a t e d a t 104 r p m a n d t h e s u p e r n a t a n t d i a l y z e d a g a i n s t 1800 m l of 0.008 M Tris buffer, pI-I 7.4 a t 4 ° C. I n some e x p e r i m e n t s a d d i t i o n of MgC12 0.05 t o 0.2 ~I w a s u s e d to p r e c i p i t a t e t h e a n t i g e n s f r o m the desoxyeholate extract.
Results T a b l e 1 s h o w s t h e h e m a g g l u t i n a t i o n a n d h e m o l y t i c t i t e r of v i r u s p r e p a r a t i o n s t h a t w e r e u s e d f o r a d s o r p t i o n t o i s o l a t e d t u m o r cell m e m b r a n e s . A s d e s c r i b e d b y o t h e r s T w c e n e t h e r t r e a t m e n t , f o r m a l i n (15), a n d g r o w t h i n M D B K t i s s u e c u l t u r e (12) e l i m i n a t e d h e m o l y t i c a c t i v i t y or r e d u c e d t h e t i t e r t o less t h a n 10 b u t t h e hemagg]utination titer was generally unchanged except for SiV grown in MDBK tissue culture and NP40 extract which had lower titers. In this case the compar a b l e a c t i v e v i r u s w a s d i l u t e d t o t h e s a m e H A t i t e r (103) b e f o r e a d s o r p t i o n . T a b l e 1. Measurements o] various viral antigen preparations used /or adsorption to
separated tumor cell membranes T i t e r of v i r u s u s e d for a d s o r p t i o n u n i t s / m l Virus and treatment
ttemagglu tination
Hemolysis
CF t i t e r w i t h a n t i viral serum and virustreated membranes b
NDV active NDV tween, ether c SiV a c t i v e SiV f o r m a l i n d SiV i n a c t . ~ - p r o p i o l a c t o n e SiV a c t i v e (diluted) SiV g r o w n i n M D B K tissue e SiV N P 40 e x t r a c t , dialyzed~
5× 5× 104 6× 2× 103 10 ~ 103
103 < 10 108 < 10 102a 102 < 10 20
80--160 20 160 20--40 80--160 80 20 40
a b c a e f
103 103 103 104
Also m a r k e d c l u m p i n g of t u m o r cell m e m b r a n e s P B S u s e d i n c o m p l e m e n t f i x a t i o n (CF) c o n t a i n e d 50 m ~ MgC13 a n d 12 m • CaC12 E n v e l o p e s d i s r u p t e d , lipids r e m o v e d Inactivated intact virus U n c l e a v e d F0 p r o t e i n p r e c u r s o r V i r u s e n v e l o p e d i s r u p t e d b y d e t e r g e n t r e a s s e m b l e s a f t e r dialysis
332
M.D. EATO:~:
When the amount of viral antigen permanently adsorbed to the membranes was measured by complement fixation with antiviral rabbit serum, titers with membranes treated with non-hemolytic virus preparations were found to be I/4 to 1/s of those with adsorbed active virus. Since the rabbit was immunized by repeated injection of whole active virus, antibodies presumably developed to all envelope proteins and probably also to group specific core proteins. The three nonhemolytic preparations differ with respect to the condition of the F envelope protein. Tween ether disrupts the envelope and removes lipids from the protein so that small fragment hemagglutinins remain (19). Formalin leaves the virus intact but inactivates the F protein which remains antigenieally reactive (15). I n the M D B K preparation an F protein precursor is left uneleaved (17). SiV inactivated with ~-propiolaetone retains its ability to react with cell membranes and produce a permanent attachment of viral antigen since the CF titers of membranes treated with the inactive virus were almost equal to those with active virus. The N P 4 0 viral preparations represent reconstituted viral envelopes with some hemolytie activity but greatly reduced or absent core structure (6). I n addition to the above, envelope glyeoproteins were extracted from partially purified virus b y the method of SCHEID and CHoPP~ (18) with Triton X 100. With these adsorption, to the tumor cell membrane was not significant as measured with antiviral rabbit serum (not shown).
Antibody Response in Syngeneie Mice Mice t h a t had been immunized with the various preparations listed in Table 1 were bled from the heart after five or more i.p. injections and the sere tested for lymphoma antibodies and autoantibodies to norma] syngeneic spleen. Results are shown in Table 2.5~ice receiving membranes treated with NDV Tween-ether or SiV formalinized showed a diminished antibody response as compared to those receiving membranes treated with equal amounts of active virus. Little or no antibodies to splenic tissue were found in mice receiving membranes with adsorbed non-hemolytic virus, whereas the groups receiving the active v i ~ s preparation did develop autoantibodies and several died with the autoimmune disease previously described (4). In the mice receiving antigen derived from SiV inactivated with ~-propiolactone antibodies comparable with those resulting from adsorbed active virus were found after nine injections. Although autoimmune deaths did not occur in this group during immunization, signs of kidney damage were found when the mice were sacrificed at the conclusion of the experiment. Mice receiving membranes t h a t had adsorbed SiV modified by growth in M D B K tissue culture showed diminished antibody response but two mice in this group died of ~utoimmune disease. I t is possible t h a t the F protein precursor in this ease m a y have been split and partially reactivated in rive. The remaining SiV preparation, viral envelope reconstituted from an N P 4 0 extract, produced a moderate but somewhat irregular a,ntibody response and also antoimmune disease. I n some experiments when the numbers of immunizing injections was increased above five, differences between the response to membranes with inactive virus and those with active virus tended to narrow (not shown).
Viral M o d i f i c a t i o n of M e m b r a n e A n t i g e n s
333
Extraction o] Viral and T u m o r Antigen F r o m Membranes I n a t t e m p t s to solubilize i m m u n o g e n i c m a t e r i a l f r o m m e m b r a n e s w i t h associated viral antigen, we used Triton X t00 which dissolves viral glycoprotein (18) a n d i n a f e w e x p e r i m e n t s d e s o x y c h o l a t e (see M a t e r i a l s a n d M e t h o d s ) . T h e r e s u l t s s h o w n i n T a b l e 3 i n d i c a t e t h a t b o t h v i r a l a n d t u m o r cell a n t i g e n s w e r e Table 2. I m m u n e response o] mice receiving membranes with adsorbed intact virus or
modi]ied viral preparations Virus
Complement fixation titer ~
Cytotoxieity index a
preparation
L y m p h o m a N spleen
L y m p h o m a N spleen
disease b
40 10
20 10=~
24 10
37 21
4/6 0/6
A c t i v e 104 H A c
30
10
35
37
2/6
Formalin c -propiolaetone a A c t i v e 10~ I-IA M D B K culture c N P 40 e x t r a c t c
10 ~7 50 30 10 ± 30
0 20 30 15 10~
12
0
0/6
9 25 12 0
44 40 0 31
0/6 3/6 2/6 3/6
Deaths with autoimmune
ND V Active c T w e e n 80-ether c
Sendai
A v e r a g e of 2 d e t e r m i n a t i o n s each. P o o l e d sera f r o m 3 - - 6 s u r v i v i n g m i c e t a k e n a t about. 3 m o n t h s a f t e r s t a r t of i m m u n i z a t i o n b N u m b e r of d e a t h s / n u m b e r i n j e c t e d . Mice s h o w e d w a s t i n g a n d s e v e r l y s c a r r e d kidn e y s w i t h i n t e r s t i t i a l i n f i l t r a t i o n (see r e f e r e n c e 4). D e a t h s a t 3 t o 6 m o n t h s c Five injections a Nine injections T a b l e 3. Properties o] antigens extracted with Triton X I O 0 or desoxycholate ]rom the membranes o] tumor cells in which N D V had grown or membranes with adsorbed N D V Antigen CF with
Extract
of membranes
I-IA titer
Hemolysin titer
Antiviral serum
Antitumor serum b
T u m o r CF antibody response in mice c
160 -0 10
40 20 0 --
80 80 0 t0
40 -40 10
80 40 0a 20
80 20
0 --
40 20
20 --
40 40
Triton NDV grown a NDV adsorbed Uninfected membrane Residue from Triton extraction
DOG N D V growna R e s i d u e f r o m DOC e x t r a c t i o n s
a Culture of 1 p e r c e n t t u m o r cells in E a g l e ' s M i n i m a l E s s e n t i a l M e d i u m w i t h 5 p e r c e n t calf s e r u m in slowly rolled b o t t l e s . V i r u s i n o c u l u m 1 : 200 d i l u t i o n of allantoie fluid b F r o m C 3 H / I - I e J m i c e : see Materials a n d M e t h o d s Six or m o r e m i c e p e r g r o u p a Mice receiving w h o l e m e m b r a n e s w i t h o u t v i r u s s h o w e d CF t i t e r s of less t h a n 10 (refs. 4, 5)
334
M.D. EATOn:
extracted. Hemagglutination and hemolysis was found in the Triton extracts and these were inhibited by antiviral serum. Finely dispersed membrane residues when mixed with chicken erythrocytes formed at low dilutions hemadsorption complexes that settled to the bottom of the tubes in a pattern like hemagglutination. Complement fixation tests were done with antiviral rabbit serum and with antiserum against uninfected whole tumor cells from C3H/HeJ mice. Both viral and tumor cell antigen were found in the extracts, but these were absent or present in only low titers in the residues. Material reactive with anti-tumor serum only was extracted by Triton from uninfected membranes. Mice were injected with extracts and residues to test for immune response. Antibodies giving complement fixation ~Jth tumor celt antigen (last column of Table 3) were found in all of the sera except those from mice receiving extracts of uninfected membranes. However, the immunized mJee showed little or no resistance to tumor Challenge, indicating t h a t the extraction procedure was destructive of the tumor specific transplant antigen. Fatal autoimmune disease was seen in some of the animals receiving Triton extracts and residues indicating t h a t the observed complement fixing antibody response was mostly against "self" antigens. Diseussion
By electron microscopy it appears that SiV envelopes can fuse with the cell membrane and remain there after cell disruption (8) thus giving permanent attachment of some viral antigens. As shown in Table 1 modification of the F protein activity reduces this irreversible adsorption to cell membranes. I t is not clear what role the H N protein has in this process. Possibly with intact viral envelopes, the p r i m a r y adsorption is effeeted b y H ~~ protein followed b y fusion of the F prorein and elution of the neuraminidase protein. Ten-fold dilution of active SiV used for adsorption did not reduce proportionately the ~mount of virus antigen on membranes. F r o m our data the neuraminidase b y itself does not bring about the observed immunogenic changes in cell membrane antigen. I n each experiment membranes were treated with approximately equivalent amounts of H N protein but variable F protein activity. The results indicate that the F protein is a major factor in permanent adsorption and in causing cell membrane antigens to become immunogenic in syngeneic hosts. The hemolytic activity of extracted SiV proteins is dependent on reassembly of the envelopes (19) after removal of detergent and on the participation of certain phospholipids. Removal of these lipids b y ether reduces or abolishes hemolytic activity but Tween-ether preparations injected into rabbits produce antibodies to all viral proteins including the F protein (15). Virus inactivated with formalin has similar antigenicity but lacks hemolytic activity. On the other hand ~-propiolaetone inactivates virus without dsetruction of the abihty of the F protein to modify cell membranes antigens. Also non-infectious virus produced by lysis with N P 4 0 followed b y removal of the RNA protein cores and reassembly of the envelopes (6) has hemolytic and celt antigen modifying activity. The identity of the membrane antigens modified b y virus is unknown but an association with H-2 antigens has been suggested (20). We have shown t h a t immunogenic material containing both viral and cell antigens can be extracted
Viral Modification of Membrane Antigens
335
b y T r i t o n X 1 0 0 from m e m b r a n e s of infected cells or m e m b r a n e s t h a t have adsorbed virus. Solubilization is a step t o w a r d f u r t h e r analysis of cell antigens a n d associated viral envelope proteins.
Aeknowledi]ments The author thanks Brian and David Kelsall, Michael Faria, Jearmine Boyer and Susan Almquist for their very helpful assistance in the laboratory. This research was supported b y a grant (IM-71 B) from the American Cancer Society.
Referenees 1. AL~QUIST, S. J. P., EATON, M. D. : Cell mediated i m m u n i t y in mice immunized with modified syngeneic Gross lymphoma antigen. Cell. Immuno]. 37, 383--389 (1978). 2. EATON, M. D., tlELLE~, J. A., SCALA, A. I¢. : E n h a n c e m e n t of lymphoma celi immunogenieity by infection with nononcogenic virus. Cancer 1%es. 33, 3293--3298 (1973). 3. EATON, M. D., A~MQVlST, S. J. P. : Antibody response of syngeneic mice to membrane antigens from NDV-infected lymphoma. Proc. Soc. exp. Biol. Med. 148, 1090--1094 (1975). 4. EATON, M. D., ALMQIIIST, S. J. t ). : A u t o i m m u n i t y induced by injection of virusmodified cell membrane antigens in syngeneic mice. Infect. I m m u n . 15, 322--328 (1977). 5. EATON, M. D., ALMQIYIST,S. J. P. : Tumor i m m u n i t y and a u t o i m m u n i t y produced by injection of antigens modified by adsorbed virus. (In preparation.) 6. HOSAKA, Y., SmMIZU, Y. K. : Artificial assembly of envelope particles of H V J (Sendal virus). 1. Assembly of hemolytic and fusion factors from envelopes solubilized by Nonidet P40. Virology 49, 627- -640 (1972). 7. HOPEL, A. J., BABLANIAN,1%., COLE, G. A. : Inductive requirements for the generation of virus-specific T lymphoeytes. 1. Nature of the host cell-virus interaction that triggers the secondary poxvirus-specific cytotoxic T lymphocyte induction. J. Immunol. 121, 736--743 (1978). 8. HOWE, C., MORGAN,C. : Interaction between Sendal virus and h u m a n erythrocytcs. J. Virol. 3, 70--81 (1969). 9. JoI~N, T. J., FULGII~ITI, V. : Parainfluenza 2 virus : increase in hemagglutinin titcr on treatment with Tween-80 and ether. Proc. Soc. exp. Biol. Med. 121, 109--112 (1966). 10. KOBAYASHI, I-I., KODAN_A, T., GOTOHDA, E.: Xenogenization of tumor cells. t{okkaido University Medical Library Series 9 (1977). 11. L I ~ I ) E ~ N N , J., KLEIN, P. A. : Viral oncolysis. Increased immunogenicity of host ceil antigens associated with influenza virus. J. exp. Med. 126, 93--108 (1967). 12. MomclsoN, T. G., MoQuAIN, C. : Assembly of viral membranes. 1. Association of vesicular stomatitis virus membrane proteins and membranes in a cell free system. J. ViroI. 21, 4 5 t - - 4 5 8 (1977). 13. NnFE, J. M., E~-D~s, J. : Poliovirus replication and eytopathogenicity in monolayer hamster cell cultures fused with ~-propiolactone inactivated Sendai virus. Proe. Soe. exp. Biol. Med. 127, 269--267 (1968). 14. NOIgRBY, E. : HemaggIutination by measles virus. A simple procedure for production of high potency antigen for hemagglutination-inhibition(HI) tests. Proe. Soe. exp. Biol. Med. 111, 814~818 (1962). 15. OaVELL, C., NOR~BY, E. : Immunologic properties of purified Sendal virus glyeoproteins. J. Immunol. 119, 1882-1887 (1977). 16. I~OESlNG, T. G., TOSELLI, P. A., C~OW~LL, 1%. L. : Elution and uneoating of Coxsaekievirus B 3 by isolated tIeLa cell plasma membranes. J. Virol. 15, 654--667 (1975).
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17. S0ttEID, A., Ct{OPt'IN, P.. Identification of biologic activities of paramyxovirus glyeoprotein. Activation of cell fusion, hemolysis and infectivity b y proteolytic cleavage of an inaetive precursor. Virology 57, 475--490 (1974). 18. SCI~EID, A., Cl~oPPi~r, P.: Isolation and purifieation of the envelope proteins of Newcastle disease virus. J. Virol. 11, 263--271 (1973). 19. SHi~iiZl:, K., HOSAKA, Y., SI~I~IZU, Y. K. : Solubilization of envelopes of H V J (Sendai virus) with alkali-emasol treatment and reassembly of envelope particles with removal of detergent. J. Virol. 9, 842--850 (1972). 20. SUGAMURA,K., SHI~IZr~, K., BAC~, F. I-I. : I n v o l v e m e n t of fusion activity of ultraviolet light-inactivated Sendal virus in formation of target antigens recognized by eytotoxie T cells. J. exp. Med. 148, 276--287 (1978). 21. KOZlSrOWSKI, U., GE~ING, M. J., WATERFIELD, M. : T-cell cytotoxicity in the absence of viral protein synthesis. Nature 267, 160--163 (1977). Authors' address : Dr. M. D. EATosr, D e p a r t m e n t of Medical Microbiology, Stanford University, Stanford, CA 94305, U.S.A.
Received January
17, 1979
Archives of Virology 61, 327--336 (1979)
© by Springer-Verlag 1979
Role of Envelope Proteins of Paramyxoviruses in the Modification of Cell Membrane Antiflens By M. D. EATON Department of Medical Microbiology, Stanford University, Stanford, California, U,S.A. Accepted March 20, 1979
Summary Adsorption of paramyxoviruses to separated membranes of tumor cells produces neoantigens t h a t are immunogenic in syngeneic mice (xenogenization). Thus it became possible to study this process b y using modified virus or virus fractions. Membranes with adsorbed viral derivatives produced an immune response which was measured by cytotoxic and complement fixing antibodies and the appearance of autoimmune disease. The effect of viral preparations with reduced F (fusion) protein activity was compared to treatment of membranes with equivalent amounts of active or inactive virus as measured b y hemagglutination. Viral preparations without hemolytic activity showed diminished adsorption to membranes and the immune response was reduced. Triton X100 and desoxycholate extracted from membranes immunogel~ic material with associated paramyxovirus antigens.
Introduction Modification of t u m o r cell membrane antigens b y myxo- and paramyxoviruses, vesicular stomatitis, vaceinia, type C leukemia virus and others have been described b y several investigators (2, 10, 11). I n these studies homogenates of tumors which were not immunogenic in syngeneic mice showed antigenieity after virus had grown in them. Homogenates of uninfected tumors were not immunogenic. Membrane fractions prepared from an ascites lymphoma infected with parainfluenza virus were shown to be as immunogenic as the cell homogenates whereas membranes from uninfected tumor cells were not (2, 3), and some other fractions from infected cells did not produce immunity to tumor transplant. Syngeneie mice produced antibodies eytotoxic for uninfected tumor cells and cell mediated immunity was demonstrated (1, 3). Also complement fixing antibodies reactive with the membranes and several other t u m o r cell fractions were detected. Antibodies were not stimulated b y similar preparations from uninfected tumor. I n addition to the anti-tumor immunity-, autoantibodies against syngeneic spleen, as well as other tissues, were demonstrated (4). With continued immunization antibodies to deoxynucleoprotein and heterophile reagins also appeared, 22*
0304-8608/79/0061/0327/$ 02.00
328
M.D. EA'ro~:
and some of the mice died with a wasting disease characterized b y loss of lymphoid tissue and scarred atrophied kidneys. I t was then found t h a t adsorption of concentrated virus by cell membranes isolated from aseites lymphoma produced immunogenie material t h a t gave results similar to the membranes from infected cells (5). However, autoimmune disease developed more rapidly and tumor transplant resistance, while present in some experiments, could not be demonstrated in others. These observations on the effect of adsorbed virus indicated t h a t modification of cell membrane antigens is a biochemical reaction that can occur in the absence of viral replication causing syngeneic or "self" antigens to appear to the system for cellular and humoral immune response as foreign. Adsorption of vaceinia virus to L cells is said to produce an antigen causing an H-2 dependent response of T lymphocytes as measttred by antiviral immune cytolysis (7). Similar results have been reported for Sendai virus, active or inactivated with UV light or ~-propiolaetone, and Sendal virus envelopes which contain mostly the hemagglutinin-neuraminidase and fusion proteins. Target cells were derived from a mastoeytoma with adsorbed viral preparations (20, 21). Inactivation of the F protein b y trypsin (21) or ethanol (20) resulted in preparations t h a t were ineffective for the formation of target cells lysable b y T lymphocytes although comparable amounts of neuraminidase were adsorbed (20). These studies on H-2 restriction of T cell eytotoxieity with adsorbed virus differ from our experiments to be described, in that our target cells have no infecting or adsorbed virus and lysis is by antibodies. However the several kinds of preparations derived from Sendai virus are very similar to what we used in the preparation of immunogens from isolated ceil membranes. Among the m a n y studies of the interaction of viruses with membranes of intact cells (adsorption and penetration), relatively few have been done with isolated membranes. The envelope proteins of vesicular stomatitis virus have been shown to react with isolated cell membranes in a selective manner, the strongest adsorption being with the glycoprotein for adsorption and penetration (i2). Other investigators have studied the elution and uncoating of enteroviruses by isoIated H e L a eelI plasma membranes (16). The membrane reactive components of paramyxovirus are the H N glycoprotein (hemagglutination and neuraminidasc) and so-called F protein (17) which produces hemolysis and cell fusion. Several procedures have been used to modify the latter protein leaving the H N protein unchanged. We have used these methods to study the effect of the F protein on adsorption of virus to isolated tumor ceil membranes and the resulting antigenic modifications as detected by immunogenieity. Measurements were done on eytotoxie and complement fixing antibodies with syngeneie uninfected lymphoma and spleen and the observation of autoimmune disease in the mice injected with these modified antigens. Materials and Methods Mice
The C 3I-I/Bi/G strain, originally obtained from Dr. Jacob Furth, has been carried by brother X sister mating in our labora%ory, Mice usecl for experiments were either male or female. C 3I-I/HeJ males purchased
from the Jackson Laboratory,
Bar I-Iarbor,
V i r a l M o d i f i c a t i o n of M e m b r a n e A n t i g e n s
329
M a i n e , w h i c h differ f r o m t h e C 3 H / B i s t r a i n i n m i n o r t r a n s p l a n t a t i o n a n t i g e n s , were u s e d i n s o m e e x p e r i m e n t s to o b t a i n t u m o r a n t i b o d i e s w i t h o u t a n t i v i r a l a n t i b o d i e s .
Ascites Lymphoma B y i.p. t r a n s p l a n t of l e u k e m i c spleen f r o m C 3 t-I/Bi mice i n f e c t e d w i t h t h e Gross m u r i n e l e u k e m i a v i r u s , a n aseites l y m p h o m a w a s p r o d u c e d . T h e 50 p e r c e n t l e t h a l dose for C 3 t - I / B i m i c e is 5 t o 10 cells a n d i n t h e s y n g e n e i c s t r a i n of mice n o regression of e s t a b l i s h e d t u m o r s h a s e v e r b e e n o b s e r v e d . T u m o r celt e x t r a c t s or m e m b r a n e s fail t o i m m u n i z e e i t h e r C 3 H / B i or C 3 H / I I e , J mice (2). C r u d e t u m o r cell m e m b r a n e p r e p a r a t i o n s from u n i n f e c t e d aseites l y m p h o m a were o b t a i n e d as d e s c r i b e d p r e v i o u s l y (2, 3).
Viruses N e w c a s t l e disease v i r u s (NI)V) s t r a i n Cal a n d p a r a i n f l u e n z a I s t r a i n S e n d a l (SiV) were o b t a i n e d f r o m D r . A. M. l~rinee a n d Dr. J . F. E n d e r s , r e s p e c t i v e l y . A n o t h e r s~rain of SiV o b t a i n e d f r o m Microbiological A s s o c i a t e s w a s u s e d i n s o m e e x p e r i m e n t s . T h e s e s t r a i n s of N D V a n d SiV d i d n o t grow well i n t i s s u e c u l t u r e s of t h e aseites I y m p h o m a b u t d i d a d s o r b to i s o l a t e d t u m o r celt m e m b r a n e s . S t o c k v i r u s e s were p r o p a g a t e d i n t h e a l l a n t o i c sac of 11 -day-old c h i c k e m b r y o s a n d were p a r t i a l l y p u r i f i e d a n d c o n c e n t r a t e d t e n - f o l d b y d i f f e r e n t i a l c e n t r i f u g a t i o n . T h e y were s t o r e d a t - - 6 0 ° C.
Modi/ieation o/ Viral Envelope Proteins SeverM p r o c e d u r e s p u b l i s h e d b y o t h e r s w e r e a p p l i e d i n a t t e m p t s t o r e d u c e or a b o l i s h t h e biological a c t i v i t y of t h e h e m o l y s i s - f u s i o n (F) p r o t e i n l e a v i n g t h e n e u r a m i n i d a s e a n d h e m a g g l u t i n a t i n g a c t i v i t y u n c h a n g e d or o n l y s l i g h t l y d i m i n i s h e d . M e t h o d s u s e d were as follows: 1. T w e e n 8 0 - e t h e r t r e a t m e n t (9, 14) : c o n c e n t r a t e d N D V was s h a k e n for 5 m i n u t e s w i t h T w e e n 80 a t 4 ° C. O n e - t h i r d v o l u m e of e t h e r was a d d e d a n d s h a k i n g c o n t i n u e d for 15 m i n u t e s . A f t e r c e n t r i f u g a t i o n a t 3000 r p m a n d 4 ° C for 20 m i n u t e s , t h e a q u e o u s l a y e r w a s r e m o v e d a n d t h e r e m a i n i n g e t h e r e v a p o r a t e d b y b u b b l i n g t h r o u g h a slow s t r e a m of Ne. 2. F o r m a l d e h y d e a t a c o n c e n t r a t i o n of 0.2 p e r c e n t was i n c u b a t e d w i t h SiV a t 37 ° C for 2 h o u r s . A f t e r s e d i m e n t a t i o n for 90 m i n u t e s a t 18,000 r p m i n a S o r v a l SB 2 c e n t r i f u g e , t h e v i r u s was r e s u s p e n d e d i n t h e original v o l u m e of p h o s p h a t e b u f f e r e d saline (PBS) (14). 3. E g g p a s s a g e SiV was i n o c u l a t e d i n t o a tissue c u l t u r e of b o v i n e k i d n e y cells ( M D B X ) d e r i v e d f r o m a t r a n s p l a n t k i n d l y f u r n i s h e d b y D r . P u r n e l l C h o p p i n (17). Growth under these conditions results in a variant with uncleared F protein. This v i r i o n h a s low i n f e c t i v i t y a n d l a e k s h e m o l y t i c a n d fusion a c t i v i t i e s unless t r e a t e d with certain proteolytie enzymes. 4. E n v e l o p e g l y c o p r o t e i n s of SiV were solubilized w i t h t h e d e t e r g e n t N o n i d e t P 40 a e e o r d i n g to t h e m e t h o d of HOSA~:A a n d S ~ I ~ I z v (6). V i r u s cores c o n t a i n i n g t h e R N A n u e l e o p r o t e i n were r e m o v e d b y e e n t r i f u g a t i o n a t 100,000 × g in a Spinco 40.2 a n g l e c e n t r i f u g e h e a d . E n v e l o p e p a r t i c l e s r e m a i n in t h e s u p e r n a t a n t a n d t h e d e t e r g e n t c a n b e r e m o v e d b y dialysis in S p e c t a p o r t u b i n g w i t h a M.~V. c u t o f f of 12,000 for 72 h o u r s a g a i n s t t h r e e c h a n g e s of P B S a t 4 ° C. S o m e h e m o l y t i c a n d h e m a g g l u t i n a t i n g a c t i v i t y w a s lost i n t h i s p r o c e d u r e . Igernoval of t h e d e t e r g e n t is followed b y r e a s s e m b l y of g l y e o p r o t e i n s 2 a n d 4 i n t o h e m o l y t i c p a r t i c l e s a c c o r d i n g t o /-IosAKA a n d SmMIZV. 5. F o r c o m p a r i s o n w i t h f o r m a l i n i n a c t i v a t e d SiV w h i c h h a s lost h e m o l y t i c a n d fusion a b i l i t y a n o n i n f e c t i o u s p r e p a r a t i o n w h i c h r e t a i n s t h e s e biologieM a c t i v i t i e s w a s o b t a i n e d b y i n a c t i v a t i o n w i t h ~ - p r o p i o l a e t o n e a c c o r d i n g t o t h e m e t h o d of NEFF a n d E~'I)EI~S (13).
Measurements o/Hemagglutination and Hemolysis Two-fold v i r u s d i l u t i o n s (0.5 ml) in P B S w i t h o u t Ca++ or Mg ++ were m i x e d w i t h a n e q u a l v o l u m e of 0.5 p e r c e n t c h i c k e n e r y t h r o c y t e s in 18 × 100 m m t u b e s a n d allowed to s t a n d a t r o o m t e m p e r a t u r e u n t i l t h e cells h a d s e t t l e d . T h e h i g h e s t d i l a t i o n of v i r u s showing a distinct pattern at the bottom of %he tube was the endpoint.
330
M . D . EATON:
F o r m e a s u r e m e n t s of hemolysis t w o - f o l d dilutions of virus set u p as a b o v e were m i x e d w i t h a n equal v o l u m e of 2 p e r c e n t guinea pig e r y t h r o c y t e s a n d i n c u b a t e d for a n h o u r a t 37 ° C. T h e t u b e s w e r e t h e n p l a c e d in t h e r e f r i g e r a t o r u n t i l t h e cells h a d s e t t l e d . The e n d p o i n t was t a k e n as t h e h i g h e s t d i l u t i o n of v i r u s giving a p p r o x i m a t e l y 50 p e r c e n t hemolysis.
Adsorption o] Virus to Crude Membranes M e m b r a n e s u s p e n s i o n s d e r i v e d f r o m a b o u t 10 s cells/m] were m i x e d w i t h a n equal v o l u m e of P B S c o n t a i n i n g i n t a c t v i r u s or m o d i f i e d virus p r e p a r a t i o n s . I n t h e s e experim e n t s t h e I-IA u n i t s / m l of virus p r e p a r a t i o n s v a r i e d f r o m 1000 t o 10,000 H A / m l b u t H A was equalized b y d i l u t i o n of t h e a c t i v e v i r u s p r e p a r a t i o n s . T h e c o n c e n t r a t i o n of a c t i v e virus u s e d for c o m p a r i s o n was t h e r e f o r e a d j u s t e d a c c o r d i n g to I-IA titer. Memb r a n e s a n d v i r u s were t h o r o u g h l y d i s p e r s e d in a D o u n c e h o m o g e n i z e r (Belleo) a n d allowed t o r e m a i n a t 2 5 ° C for 1 hour. M e m b r a n e s w i t h a d s o r b e d v i r u s w e r e t h e n s e d i m e n t e d a t 10a r p m for 20 m i n u t e s a n d w a s h e d t h r e e t i m e s in P B S b y r e s u s p e n d i n g a n d c e n t r i f u g i n g u n t i l less t h a n 10 H A u n i t s / m l r e m a i n e d in t h e s u p e r n a t a n t . This p r o c e d u r e h a s b e e n d e s c r i b e d p r e v i o u s l y in m o r e detail (5). Viral a n t i g e n i r r e v e r s i b l y a t t a c h e d to t h e m e m b r a n e s was m e a s u r e d b y c o m p l e m e n t f i x a t i o n w i t h a n t i v i r a l r a b b i t serum.
A ntisera R a b b i t s w e r e i m m u n i z e d w i t h egg p a s s a g e N D V or SiV b y s u b c u t a n e o u s or i n t r a m u s c u l a r i n j e c t i o n a t m o n t h l y i n t e r v a l s . I n s o m e e x p e r i m e n t s t h e a n t i v i r a l sera were a b s o r b e d w i t h n o r m a l m i n c e d chorioallantoic m e m b r a n e s or o t h e r chick e m b r y o tissue (5). A n t i t u m o r sera w e r e o b t a i n e d b y i m m u n i z i n g C ~ H / H e J mice first w i t h m i n c e d spleen f r o m CaH/Bi mice, t h e n w i t h u n i n f e c t e d ascites l y m p h o m a . B e c a u s e of t h e difference in m i n o r h i s t o e o m p a t i b i l i t y a n t i g e n s b e t w e e n t h e s e t w o s t r a i n s of mice small doses of t h e allogeneie t u m o r were r e j e c t e d b y C~H/I-IeJ mice a f t e r t h e y h a d b e e n i m m u n i z e d w i t h C s H / B i tissue. A n t i s e r a f r o m CaH/Bi mice i m m u n i z e d b y 5 or m o r e i.p. i n j e c t i o n s w i t h t u m o r cell m e m b r a n e s c o n t a i n i n g a d s o r b e d virus f r o m t h e various p r e p a r a t i o n s d e s c r i b e d a b o v e w e r e t e s t e d for a n t i b o d i e s to u n i n f e c t e d t u m o r s a n d n o r m a l s y n g e n e i e spleen b y complement fixation and complement dependent eytotoxieity.
Complement Fixation W e l l - d i s p e r s e d m e m b r a n e s of u n t r e a t e d ascites l y m p h o m a or C3H/Bi spleen ceils o b t a i n e d as d e s c r i b e d p r e v i o u s l y (3) w e r e u s e d as a n t i g e n s to d e t e c t a n t i b o d y r e s p o n s e t o t h e t u m o r or a u t o a n t i b o d i e s as m e a s u r e d w i t h t h e n o r m a l spleen cell m e m b r a n e . The t e s t was p e r f o r m e d w i t h 1.6 to 1.8 u n i t s of guinea pig c o m p l e m e n t , a n t i g e n s 1 : 20, a n d t w o - f o l d d i l u t i o n s of a n t i s e r u m 1:10 to 1:160, all in a m o u n t s of 0.25 ml. A f t e r i n c u b a t i o n a t 36 ° C for 1 h o u r , 0.25 m l of 2 p e r c e n t sensitized s h e e p e r y t h r o c y t e s w e r e a d d e d . S e r u m t i t e r s were s t a t e d as t h e h i g h e s t d i l u t i o n of s e r u m in w h i c h some e r y t h r o e y t e s r e m a i n e d . T h e t i t e r of c o n t r o l s w i t h o u t a n t i g e n , for a n t i c o m p l e m e n t a r y effects of t h e s e r u m , was s u b t r a c t e d f r o m t h e t i t e r w i t h a n t i g e n . T e s t s for a d s o r b e d viral a n t i g e n on t h e m e m b r a n e s were also d o n e b y c o m p l e m e n t f i x a t i o n w i t h a n t i v i r a l r a b b i t sera 1 : 20 or 1 : 40 a n d dilutions of t h e m e m b r a n e p r e p a r a t i o n .
Cytotoxicity Tests Aseites t u m o r cells were a s p i r a t e d f r o m t h e p e r i t o n e u m a n d w a s h e d t h r e e t i m e s in H a n k s ' b a l a n c e d salt solution. N o r m a l spleen cells were o b t a i n e d b y sieving t h e finely m i n c e d C3H/Bi spleen on a 200 m e s h stainless steel screen. E r y t h r o c y t e s w e r e r e m o v e d b y i n c u b a t i o n a t 37 ° C for 20 m i n u t e s in 0.15 ~ NHaCI a n d w a s h i n g . T u m o r or spleen cells w e r e s u s p e n d e d in E a g l e ' s m i n i m u m essential m e d i u m c o n t a i n i n g 17 m ~ h e p e s b u f f e r (N-2 h y d r o x y e t h y l p i p e r a z i n e . N ' 2 e t h a n e sulfonic acid: Calbiochem) a n d 50 ~g/ml of G e n t a m y c i n (Schering Corp.). One p e r c e n t cell s u s p e n s i o n 0.1 ml, was i n c u b a t e d in a w a t e r b a t h a t 36 ° C for 18 h o u r s w i t h 0.1 m l of a n t i s e r u m 1 : 4 a n d 0.1 m l of r a b b i t c o m p l e m e n t 1 : 16 (Grand I s l a n d Biological). T u b e s w e r e s h a k e n periodicMly. C y t o t o x i c i t y w a s d e t e r m i n e d b y t h e d y e exclusion t e s t using 0.1 p e r c e n t t o l u i d i n e blue (3). T h e c y t o t o x i c i n d e x was s t a t e d as t h e p e r c e n t a g e of d e a d cells in t h e t e s t m i n u s
Viral M o d i f i c a t i o n of M e m b r a n e A n t i g e n s
331
t h e p e r c e n t a g e of d e a d cells in t h e c o n t r o l c o n t a i n i n g c o m p l e m e n t a n d n o a n t i s e r u m or n o r m a l m o u s e s e r u m . I m m u n e s e r u m a l o n e w a s n o t e y t o t o x i c . S t a i n e d cells i n t h e c o n t r o l s were u s u a l l y 10 t o 20 p e r c e n t of t h e t o t a l a f t e r 18 h o u r s ' i n c u b a t i o n .
Extraction o] Viral and Tumor Cell Antigens With Detergent T u m o r cell m e m b r a n e s c a r r y i n g a d s o r b e d v i r a l a n t i g e n s or p r e p a r e d f r o m i n f e c t e d cells were e x t r a c t e d w i t h T r i t o n X 1 0 0 or d e s o x y e h o l a t c i n a n a t t e m p t to o b t a i n soluble i m m u n o g e n i e p r e p a r a t i o n s a n d to t e s t t h e s t a b i l i t y of t h e m o d i f i e d a n t i g e n s . W i t h T r i t o n X 1 0 0 (Calbiochcm) a t 0.5 or 0.25 p e r c e n t m e m b r a n e s c o n t a i n i n g N D V a n t i g e n were e x t r a c t e d i n 0.01 M s o d i u m p h o s p h a t e b u f f e r p H 7.4 for 30 m i n u t e s a t 20 ° C, u s i n g a h o m o g e n i z e r . T h e u n d i s s o l v e d residue was r e m o v e d b y c e n t r i f u g a t i n g a t 104 r p m for 1 h o u r a n d s u s p e n d e d i n P B S e q u a l t o t h e original v o l u m e . T h e d e t e r g e n t i n t h e s u p e r n a t a n t w a s r e m o v e d b y e x t r a c t i n g five t i m e s w i t h 1/3 v o l u m e of e t h e r followed b y b u b b l i n g Ne. D e s o x y c h o l a t e (10 ml) a t a c o n c e n t r a t i o n of 0.5 p e r c e n t i n Tris saline a t p H 7.8 was a d d e d t o a s e d i m e n t of c r u d e m e m b r a n e s f r o m 2 × 10 s cells i n f e c t e d w i t h N D V . A f t e r 30 m i n u t e s a t 37 ° C w i t h m i x i n g , t h e r e s i d u e w a s s e p a r a t e d a t 104 r p m a n d t h e s u p e r n a t a n t d i a l y z e d a g a i n s t 1800 m l of 0.008 M Tris buffer, pI-I 7.4 a t 4 ° C. I n some e x p e r i m e n t s a d d i t i o n of MgC12 0.05 t o 0.2 ~I w a s u s e d to p r e c i p i t a t e t h e a n t i g e n s f r o m the desoxyeholate extract.
Results T a b l e 1 s h o w s t h e h e m a g g l u t i n a t i o n a n d h e m o l y t i c t i t e r of v i r u s p r e p a r a t i o n s t h a t w e r e u s e d f o r a d s o r p t i o n t o i s o l a t e d t u m o r cell m e m b r a n e s . A s d e s c r i b e d b y o t h e r s T w c e n e t h e r t r e a t m e n t , f o r m a l i n (15), a n d g r o w t h i n M D B K t i s s u e c u l t u r e (12) e l i m i n a t e d h e m o l y t i c a c t i v i t y or r e d u c e d t h e t i t e r t o less t h a n 10 b u t t h e hemagg]utination titer was generally unchanged except for SiV grown in MDBK tissue culture and NP40 extract which had lower titers. In this case the compar a b l e a c t i v e v i r u s w a s d i l u t e d t o t h e s a m e H A t i t e r (103) b e f o r e a d s o r p t i o n . T a b l e 1. Measurements o] various viral antigen preparations used /or adsorption to
separated tumor cell membranes T i t e r of v i r u s u s e d for a d s o r p t i o n u n i t s / m l Virus and treatment
ttemagglu tination
Hemolysis
CF t i t e r w i t h a n t i viral serum and virustreated membranes b
NDV active NDV tween, ether c SiV a c t i v e SiV f o r m a l i n d SiV i n a c t . ~ - p r o p i o l a c t o n e SiV a c t i v e (diluted) SiV g r o w n i n M D B K tissue e SiV N P 40 e x t r a c t , dialyzed~
5× 5× 104 6× 2× 103 10 ~ 103
103 < 10 108 < 10 102a 102 < 10 20
80--160 20 160 20--40 80--160 80 20 40
a b c a e f
103 103 103 104
Also m a r k e d c l u m p i n g of t u m o r cell m e m b r a n e s P B S u s e d i n c o m p l e m e n t f i x a t i o n (CF) c o n t a i n e d 50 m ~ MgC13 a n d 12 m • CaC12 E n v e l o p e s d i s r u p t e d , lipids r e m o v e d Inactivated intact virus U n c l e a v e d F0 p r o t e i n p r e c u r s o r V i r u s e n v e l o p e d i s r u p t e d b y d e t e r g e n t r e a s s e m b l e s a f t e r dialysis
332
M.D. EATO:~:
When the amount of viral antigen permanently adsorbed to the membranes was measured by complement fixation with antiviral rabbit serum, titers with membranes treated with non-hemolytic virus preparations were found to be I/4 to 1/s of those with adsorbed active virus. Since the rabbit was immunized by repeated injection of whole active virus, antibodies presumably developed to all envelope proteins and probably also to group specific core proteins. The three nonhemolytic preparations differ with respect to the condition of the F envelope protein. Tween ether disrupts the envelope and removes lipids from the protein so that small fragment hemagglutinins remain (19). Formalin leaves the virus intact but inactivates the F protein which remains antigenieally reactive (15). I n the M D B K preparation an F protein precursor is left uneleaved (17). SiV inactivated with ~-propiolaetone retains its ability to react with cell membranes and produce a permanent attachment of viral antigen since the CF titers of membranes treated with the inactive virus were almost equal to those with active virus. The N P 4 0 viral preparations represent reconstituted viral envelopes with some hemolytie activity but greatly reduced or absent core structure (6). I n addition to the above, envelope glyeoproteins were extracted from partially purified virus b y the method of SCHEID and CHoPP~ (18) with Triton X 100. With these adsorption, to the tumor cell membrane was not significant as measured with antiviral rabbit serum (not shown).
Antibody Response in Syngeneie Mice Mice t h a t had been immunized with the various preparations listed in Table 1 were bled from the heart after five or more i.p. injections and the sere tested for lymphoma antibodies and autoantibodies to norma] syngeneic spleen. Results are shown in Table 2.5~ice receiving membranes treated with NDV Tween-ether or SiV formalinized showed a diminished antibody response as compared to those receiving membranes treated with equal amounts of active virus. Little or no antibodies to splenic tissue were found in mice receiving membranes with adsorbed non-hemolytic virus, whereas the groups receiving the active v i ~ s preparation did develop autoantibodies and several died with the autoimmune disease previously described (4). In the mice receiving antigen derived from SiV inactivated with ~-propiolactone antibodies comparable with those resulting from adsorbed active virus were found after nine injections. Although autoimmune deaths did not occur in this group during immunization, signs of kidney damage were found when the mice were sacrificed at the conclusion of the experiment. Mice receiving membranes t h a t had adsorbed SiV modified by growth in M D B K tissue culture showed diminished antibody response but two mice in this group died of ~utoimmune disease. I t is possible t h a t the F protein precursor in this ease m a y have been split and partially reactivated in rive. The remaining SiV preparation, viral envelope reconstituted from an N P 4 0 extract, produced a moderate but somewhat irregular a,ntibody response and also antoimmune disease. I n some experiments when the numbers of immunizing injections was increased above five, differences between the response to membranes with inactive virus and those with active virus tended to narrow (not shown).
Viral M o d i f i c a t i o n of M e m b r a n e A n t i g e n s
333
Extraction o] Viral and T u m o r Antigen F r o m Membranes I n a t t e m p t s to solubilize i m m u n o g e n i c m a t e r i a l f r o m m e m b r a n e s w i t h associated viral antigen, we used Triton X t00 which dissolves viral glycoprotein (18) a n d i n a f e w e x p e r i m e n t s d e s o x y c h o l a t e (see M a t e r i a l s a n d M e t h o d s ) . T h e r e s u l t s s h o w n i n T a b l e 3 i n d i c a t e t h a t b o t h v i r a l a n d t u m o r cell a n t i g e n s w e r e Table 2. I m m u n e response o] mice receiving membranes with adsorbed intact virus or
modi]ied viral preparations Virus
Complement fixation titer ~
Cytotoxieity index a
preparation
L y m p h o m a N spleen
L y m p h o m a N spleen
disease b
40 10
20 10=~
24 10
37 21
4/6 0/6
A c t i v e 104 H A c
30
10
35
37
2/6
Formalin c -propiolaetone a A c t i v e 10~ I-IA M D B K culture c N P 40 e x t r a c t c
10 ~7 50 30 10 ± 30
0 20 30 15 10~
12
0
0/6
9 25 12 0
44 40 0 31
0/6 3/6 2/6 3/6
Deaths with autoimmune
ND V Active c T w e e n 80-ether c
Sendai
A v e r a g e of 2 d e t e r m i n a t i o n s each. P o o l e d sera f r o m 3 - - 6 s u r v i v i n g m i c e t a k e n a t about. 3 m o n t h s a f t e r s t a r t of i m m u n i z a t i o n b N u m b e r of d e a t h s / n u m b e r i n j e c t e d . Mice s h o w e d w a s t i n g a n d s e v e r l y s c a r r e d kidn e y s w i t h i n t e r s t i t i a l i n f i l t r a t i o n (see r e f e r e n c e 4). D e a t h s a t 3 t o 6 m o n t h s c Five injections a Nine injections T a b l e 3. Properties o] antigens extracted with Triton X I O 0 or desoxycholate ]rom the membranes o] tumor cells in which N D V had grown or membranes with adsorbed N D V Antigen CF with
Extract
of membranes
I-IA titer
Hemolysin titer
Antiviral serum
Antitumor serum b
T u m o r CF antibody response in mice c
160 -0 10
40 20 0 --
80 80 0 t0
40 -40 10
80 40 0a 20
80 20
0 --
40 20
20 --
40 40
Triton NDV grown a NDV adsorbed Uninfected membrane Residue from Triton extraction
DOG N D V growna R e s i d u e f r o m DOC e x t r a c t i o n s
a Culture of 1 p e r c e n t t u m o r cells in E a g l e ' s M i n i m a l E s s e n t i a l M e d i u m w i t h 5 p e r c e n t calf s e r u m in slowly rolled b o t t l e s . V i r u s i n o c u l u m 1 : 200 d i l u t i o n of allantoie fluid b F r o m C 3 H / I - I e J m i c e : see Materials a n d M e t h o d s Six or m o r e m i c e p e r g r o u p a Mice receiving w h o l e m e m b r a n e s w i t h o u t v i r u s s h o w e d CF t i t e r s of less t h a n 10 (refs. 4, 5)
334
M.D. EATOn:
extracted. Hemagglutination and hemolysis was found in the Triton extracts and these were inhibited by antiviral serum. Finely dispersed membrane residues when mixed with chicken erythrocytes formed at low dilutions hemadsorption complexes that settled to the bottom of the tubes in a pattern like hemagglutination. Complement fixation tests were done with antiviral rabbit serum and with antiserum against uninfected whole tumor cells from C3H/HeJ mice. Both viral and tumor cell antigen were found in the extracts, but these were absent or present in only low titers in the residues. Material reactive with anti-tumor serum only was extracted by Triton from uninfected membranes. Mice were injected with extracts and residues to test for immune response. Antibodies giving complement fixation ~Jth tumor celt antigen (last column of Table 3) were found in all of the sera except those from mice receiving extracts of uninfected membranes. However, the immunized mJee showed little or no resistance to tumor Challenge, indicating t h a t the extraction procedure was destructive of the tumor specific transplant antigen. Fatal autoimmune disease was seen in some of the animals receiving Triton extracts and residues indicating t h a t the observed complement fixing antibody response was mostly against "self" antigens. Diseussion
By electron microscopy it appears that SiV envelopes can fuse with the cell membrane and remain there after cell disruption (8) thus giving permanent attachment of some viral antigens. As shown in Table 1 modification of the F protein activity reduces this irreversible adsorption to cell membranes. I t is not clear what role the H N protein has in this process. Possibly with intact viral envelopes, the p r i m a r y adsorption is effeeted b y H ~~ protein followed b y fusion of the F prorein and elution of the neuraminidase protein. Ten-fold dilution of active SiV used for adsorption did not reduce proportionately the ~mount of virus antigen on membranes. F r o m our data the neuraminidase b y itself does not bring about the observed immunogenic changes in cell membrane antigen. I n each experiment membranes were treated with approximately equivalent amounts of H N protein but variable F protein activity. The results indicate that the F protein is a major factor in permanent adsorption and in causing cell membrane antigens to become immunogenic in syngeneic hosts. The hemolytic activity of extracted SiV proteins is dependent on reassembly of the envelopes (19) after removal of detergent and on the participation of certain phospholipids. Removal of these lipids b y ether reduces or abolishes hemolytic activity but Tween-ether preparations injected into rabbits produce antibodies to all viral proteins including the F protein (15). Virus inactivated with formalin has similar antigenicity but lacks hemolytic activity. On the other hand ~-propiolaetone inactivates virus without dsetruction of the abihty of the F protein to modify cell membranes antigens. Also non-infectious virus produced by lysis with N P 4 0 followed b y removal of the RNA protein cores and reassembly of the envelopes (6) has hemolytic and celt antigen modifying activity. The identity of the membrane antigens modified b y virus is unknown but an association with H-2 antigens has been suggested (20). We have shown t h a t immunogenic material containing both viral and cell antigens can be extracted
Viral Modification of Membrane Antigens
335
b y T r i t o n X 1 0 0 from m e m b r a n e s of infected cells or m e m b r a n e s t h a t have adsorbed virus. Solubilization is a step t o w a r d f u r t h e r analysis of cell antigens a n d associated viral envelope proteins.
Aeknowledi]ments The author thanks Brian and David Kelsall, Michael Faria, Jearmine Boyer and Susan Almquist for their very helpful assistance in the laboratory. This research was supported b y a grant (IM-71 B) from the American Cancer Society.
Referenees 1. AL~QUIST, S. J. P., EATON, M. D. : Cell mediated i m m u n i t y in mice immunized with modified syngeneic Gross lymphoma antigen. Cell. Immuno]. 37, 383--389 (1978). 2. EATON, M. D., tlELLE~, J. A., SCALA, A. I¢. : E n h a n c e m e n t of lymphoma celi immunogenieity by infection with nononcogenic virus. Cancer 1%es. 33, 3293--3298 (1973). 3. EATON, M. D., A~MQVlST, S. J. P. : Antibody response of syngeneic mice to membrane antigens from NDV-infected lymphoma. Proc. Soc. exp. Biol. Med. 148, 1090--1094 (1975). 4. EATON, M. D., ALMQIIIST, S. J. t ). : A u t o i m m u n i t y induced by injection of virusmodified cell membrane antigens in syngeneic mice. Infect. I m m u n . 15, 322--328 (1977). 5. EATON, M. D., ALMQIYIST,S. J. P. : Tumor i m m u n i t y and a u t o i m m u n i t y produced by injection of antigens modified by adsorbed virus. (In preparation.) 6. HOSAKA, Y., SmMIZU, Y. K. : Artificial assembly of envelope particles of H V J (Sendal virus). 1. Assembly of hemolytic and fusion factors from envelopes solubilized by Nonidet P40. Virology 49, 627- -640 (1972). 7. HOPEL, A. J., BABLANIAN,1%., COLE, G. A. : Inductive requirements for the generation of virus-specific T lymphoeytes. 1. Nature of the host cell-virus interaction that triggers the secondary poxvirus-specific cytotoxic T lymphocyte induction. J. Immunol. 121, 736--743 (1978). 8. HOWE, C., MORGAN,C. : Interaction between Sendal virus and h u m a n erythrocytcs. J. Virol. 3, 70--81 (1969). 9. JoI~N, T. J., FULGII~ITI, V. : Parainfluenza 2 virus : increase in hemagglutinin titcr on treatment with Tween-80 and ether. Proc. Soc. exp. Biol. Med. 121, 109--112 (1966). 10. KOBAYASHI, I-I., KODAN_A, T., GOTOHDA, E.: Xenogenization of tumor cells. t{okkaido University Medical Library Series 9 (1977). 11. L I ~ I ) E ~ N N , J., KLEIN, P. A. : Viral oncolysis. Increased immunogenicity of host ceil antigens associated with influenza virus. J. exp. Med. 126, 93--108 (1967). 12. MomclsoN, T. G., MoQuAIN, C. : Assembly of viral membranes. 1. Association of vesicular stomatitis virus membrane proteins and membranes in a cell free system. J. ViroI. 21, 4 5 t - - 4 5 8 (1977). 13. NnFE, J. M., E~-D~s, J. : Poliovirus replication and eytopathogenicity in monolayer hamster cell cultures fused with ~-propiolactone inactivated Sendai virus. Proe. Soe. exp. Biol. Med. 127, 269--267 (1968). 14. NOIgRBY, E. : HemaggIutination by measles virus. A simple procedure for production of high potency antigen for hemagglutination-inhibition(HI) tests. Proe. Soe. exp. Biol. Med. 111, 814~818 (1962). 15. OaVELL, C., NOR~BY, E. : Immunologic properties of purified Sendal virus glyeoproteins. J. Immunol. 119, 1882-1887 (1977). 16. I~OESlNG, T. G., TOSELLI, P. A., C~OW~LL, 1%. L. : Elution and uneoating of Coxsaekievirus B 3 by isolated tIeLa cell plasma membranes. J. Virol. 15, 654--667 (1975).
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17. S0ttEID, A., Ct{OPt'IN, P.. Identification of biologic activities of paramyxovirus glyeoprotein. Activation of cell fusion, hemolysis and infectivity b y proteolytic cleavage of an inaetive precursor. Virology 57, 475--490 (1974). 18. SCI~EID, A., Cl~oPPi~r, P.: Isolation and purifieation of the envelope proteins of Newcastle disease virus. J. Virol. 11, 263--271 (1973). 19. SHi~iiZl:, K., HOSAKA, Y., SI~I~IZU, Y. K. : Solubilization of envelopes of H V J (Sendai virus) with alkali-emasol treatment and reassembly of envelope particles with removal of detergent. J. Virol. 9, 842--850 (1972). 20. SUGAMURA,K., SHI~IZr~, K., BAC~, F. I-I. : I n v o l v e m e n t of fusion activity of ultraviolet light-inactivated Sendal virus in formation of target antigens recognized by eytotoxie T cells. J. exp. Med. 148, 276--287 (1978). 21. KOZlSrOWSKI, U., GE~ING, M. J., WATERFIELD, M. : T-cell cytotoxicity in the absence of viral protein synthesis. Nature 267, 160--163 (1977). Authors' address : Dr. M. D. EATosr, D e p a r t m e n t of Medical Microbiology, Stanford University, Stanford, CA 94305, U.S.A.
Received January
17, 1979
