Archives of Virology

Archives of Virology 57, 231--241 (1978)

© by Springer-Verlag 1978

Gel Double Immunodiffusion Studies With Six H u m a n Rhinoviruses By C. B. SMITH

Division of Infectious Diseases, Department of Medicine, University of Utah College of Medicine, Salt Lake City, Utah, U.S.A. With 6 Figures Accepted January 10, 1978

Summary Rabbits were immunized on six occasions during a seven nmnth period with fluorocarbon and sucrose density gradient purified preparations of human rhinovirus types 1 A, 2, 3, 4, 9, or 14. Sera collected 1 week after the final immunization formed 1 or 2 precipitin lines when reacted b y immunodiffusion against fluorocarbon purified preparations of each homologous immunizing virus. Heterologous preeipitin cross reactions were detected between: I ~ V I A antigen and rabbit sera to RV2, l~V9 and t~V14; RV2 antigen and sera to I~V1A; I~V14 antigen and sera to RV3. The heterologous "group" or C-antigenic nature of the cross reactions was suggested by the merging of hetero]ogous precipitin lines formed against rabbit sera with "group" antibody precipitin lines fromed against h u m a n sera and by the location of heterologous reactions close to the antigen well. I n addition, the presence of C-antigenic particles in the fluorocarbon treated RV2 preparation was suggested by the demonstration that a subpopulation of viral particles migrated to a p t { of 4.4 b y isoelectrie focusing.

Introduction

Epidemiologic studies of human rhinovirus infections have been made difficult b y the apparent immunologic heterogeneity of this group of viruses. There are currently over 100 rhinovirus serotypes and diagnosis depends upon virus isolation or the demonstration of a rise in titer of serotype specific neutralizing anti16 Arch. ¥irol. 57/3

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bodies (7). FAULK st al. (5), reported that an antigen common to some of the human rhinoviruses could be demonstrated b y the passive hemagglutination test, and weak cross reactions have been decribed between neutralizing antibodies to some rhinovirus serotypes (3, 6). Unfortunately the low frequency of cross reactions detected by these two tests limits their usefulness as general diagnostic tests for rhinovirus infections. Recently, human rhinoviruses have been shown to resemble other members of the picornavirus group b y existing in two major antigenic froms (1). The native or D-antigenic form of the virus stimulates the formation of neutra]izing antibodies which are generally type specific. Antibodies to the D-antigen of rhinoviruses m a y a]so be detected by the complement fixation (CF), immunodiffusion (ID), and immune electron microscopic (IEM) techniques, and to date these reactions have always been t3~pe-specifie (9, 11, 12, 13). The C-antigenic form of the virus differs in that it is unable to stimulate the formation of neutralizing antibody but instead induces antibodies which cross react with C-antigens of heterologous rhinoviruses and other pieornaviruses by the CF, I D and I E M techniques (9, 13). The C or "group" antigenic particles exist naturally in the less dense natural top component when virions are separated by density gradient, centrifugation, or they m a y be generated from native D-particles by gentle denaturation with heat, acid, or urea (12, 13). The observation that C-antigenic particles of rhinovirus type 2 (RV2) m a y exist as RNA containing full particles or as RNA free e m p t y capsids, and that they m a y also differ in polypeptide composition has led to the conclusion that D- to C-antigenic changes are probably due to eonformational changes in the capsid proteins (1). This hypothesis has been further supported by the observations that for polioviruses and RV2 the two types of antigenic particles m a y be effectively separated by isoeleetric focusing, the D-particles migrating to a p H of 6.2 to 6.5 and the Cparticles migrating to a p H of 4.2 to 4.7 (1, 12). Most adult human sera contain "group" antibodies which react with C-antigens of human picornaviruses, including human rhinoviruses, and human sera have been used to detect rhinovirus C-antigenic reactivity (9). However, sera from animals immunized with a singte rhinovirus serotype are preferable to human sera as reagents for exploring the extent of cross-reactivity between rhinovirus C-antigenie components since most human adults have been exposed to numerous rhinoviruses which can induce various specific and cross-reacting antibodies. DANS st al. (4) used serum from a monkey immunized with rhinovirus type 1A (RV 1 A) and found antibody which cross reacted in the CF test with the C-antigenic natural top component of rhinovirus type 2. Using serum from immunized rabbits, LozcsERe-HoL~4 and YIN (13) described a two-way cross reaction between RV 1A and I~V2 by the CF test and a one-way cross by I D between RV 1A rabbit serum and C-antigenic particles of R V 2 . However, they were unable to demonstrate such cross reactions between these 2 viruses anti RV 14. Other investigators have been unable to demonstrate cross reactions between rhinovirus C-antigens using sera from immunized animals (8, 9). This report describes several new examples of cross reactions using sera from rabbits h~perimmunized with six separate rhinovirus serotypes and C-antigenic components of the six viruses in double immunodiffusion tests.

Gel D o u b l e I m m u n o d i f f u s i o n W i t h R h i n o v i r u s e s

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Materials and Methods Virus Strains R h i n o v i r u s s t r a i n s 1 A (JI-I), 2 ( H G P ) , 3 (Vol), 4 (Vol), 9 (DC) a n d 14 (Bell) were H e L a cell a d a p t e d v i r u s e s o b t a i n e d f r o m Dr. S y l v i a R e e d , C o m m o n Cold R e s e a r c h U n i t , S a l i s b u r y , W i l t s h i r e , E n g l a n d . T h e i d e n t i t y of f i n a l v i r u s h a r v e s t s w a s c o n f i r m e d b y n e u t r a l i z a t i o n w i t h specific a n i m a l a n t i s e r a . Cell Culture l~ethods R h i n o v i r u s s e n s i t i v e I t e L a cells (Ohio I t e L a ) were m a i n t a i n e d a n d p r o p a g a t e d as d e s c r i b e d b y STOTT a n d TY~RELZ (18). V i r u s a n d n e u t r a l i z i n g a n t i b o d y t i t r a t i o n s w e r e p e r f o r m e d i n t r i p l i c a t e roller t u b e s a,t 33 ° C a n d v i r u s t i t e r was d e t e r m i n e d b y c a l c u l a t i n g t h e 50 p e r c e n t tissue c u l t u r e i n f e c t i o u s dose (TCD~0) (15). S e r a were m i x e d w i t h 3 0 - - 3 0 0 TCD~0 of r h i n o v i r u s a t r o o m t e m p e r a t u r e for 1 h o u r before i n o c u l a t i o n i n t o roller t u b e s for m e a s u r e m e n t of n e u t r a l i z i n g a n t i b o d y . V i r u s Growth T w o a n d o n e - h a l f l i t e r W i n c h e s t e r roller b o t t l e s were i n o c u l a t e d w i t h sufficiezlt H e L a cells to p r o d u c e 90 p e r c e n t c o n f l u e n e y a t 24 h o u r s . Ceil g r o w t h m e d i u m was r e m o v e d , t h e cells were w a s h e d t w i c e w i t h P B S a n d 10 m l v i r u s i n o e u l u m was a d d e d t o p r o d u c e a n input, m u l t i p l i c i t y of i n f e c t i o n of 0.5 t o 1 i n f e c t i o u s u n i t s p e r cell. A t t a c h m e n t was a t 37 ° C for 1 h o u r a f t e r w h i c h t h e excess v i r u s i n o c u l u m was r e m o v e d . V i r u s g r o w t h w a s allowed t o p r o c e e d a t 33 ° C i n leucine-free M E M w i t h E a r l e ' s salts (Gibeo) s u p p l e m e n t e d w i t h 1 p e r c e n t h e a t - i n a c t i v a t e d f e t a l b o v i n e s e r u m , penicillin 100 u n i t s / m l a n d g e n t a m i c i n 50 ~g/ml. A f t e r 4 h o u r s of g r o w t h 250 ~Ci of H3 L-leucine ( R a d i o c h e m i e a l Centre, A m e r s h a m , E n g l a n d ) w a s a d d e d to 50 m l of m e d i u m i n e a c h b o t t l e . V i r u s w a s h a r v e s t e d 12 t o 24 h o u r s a f t e r i n o c u l a t i o n w h e n 80 p e r c e n t C P E h a d d e v e l o p e d . T h e ceils w e r e r e m o v e d m e c h a n i c a l l y , p e l l e t e d a t 350 × g for 15 m i n u t e s and resuspended in Eagle's MEM spinner medium supplemented with 5 per cent heati n a c t i v a t e d n e w b o r n calf s e r u m (NBC). T h e cell s u s p e n s i o n w a s r a p i d l y frozen a n d t h a w e d t h r e e t i m e s , t h e cell d e b r i s w a s p e l l e t e d a t 350 × g for 20 m i n u t e s a n d t h e v i r u s h a r v e s t s u p e r n a t e t h e n s t o r e d a t - - 8 0 ° C. V i r u s t i t e r s in logl0 T C D s 0 / m l were 8.5 for I ~ V 1 A , 9.5 for lZV2, 9.2 for R V 3 , 8.2 for 1%V4, 9.5 for R V 9 a n d 9.2 for R V 1 4 . V i r u s Puri]ication A 10 m l v o l u m e of v i r u s h a r v e s t was m i x e d w i t h a 6 m l v o l u m e of t r i c h l o r o t r i f l u o r o ethane (Arklone ]?, ICI) and homogenized for 4 minutes in a MSE homogenizer at maximum speed at 0 ° C. The homogenate was centrifuged for 30 minutes at 350 X g and the aqueous supernate was harvested. This fluorocarbon treated material without f u r t h e r p u r i f i c a t i o n w a s u s e d as t h e a n t i g e n i n d o u b l e i m m u n o d i f f u s i o n tests. M a t e r i a l w h i c h was t o b e a d m i n i s t e r e d to r a b b i t s as t h e i m m u n o g e n was f u r t h e r p u r i f i e d b y p e l l e t i n g a n d suerose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . T h e v i r u s h a r v e s t f r o m 3 roller b o t t l e s ( 15 ml) was c e n t r i f u g e d a t t 75,000 × g for 1.5 h o u r s i n a 8 X 25 a n g l e h e a d (MSE 69590) o n a M S E - 6 5 c e n t r i f u g e , r e s u s p e n d e d i n 0.7 m l of 0.02 ~ Tris b u f f e r ( p H 7.5) b y r e p e a t e d a s p i r a t i o n t h r o u g h a 23 g a u g e needle, a n d s o n i e a t e d for 30 seconds a t low p o w e r i n a 0 ° C w a t e r b a t h . T h e s o n i e a t e d v i r u s was t h e n l a y e r e d o n t h e t o p of a 4.8 m l v o l u m e 1 5 - - 3 3 p e r c e n t c o n t i n u o u s sucrose g r a d i e n t , a n d c e n t r i f u g ed for 1.75 h o u r s a t 1 0 0 , 0 0 0 × g i n a 3 × 5 m l (MSE 99589) s w i n g i n g b u c k e t r o t o r . F r a c t i o n s of 0.15 m l were r e m o v e d f r o m t h e t o p of t h e g r a d i e n t b y d i s p l a c e m e n t f r o m t h e b o t t o m of t h e t u b e w i t h 50 p e r c e n t sucrose. T h e p r e s e n c e of p r o t e i n w a s m o n i t o r e d b y U V a b s o r b a n c e a t 280 n m , 10 V1 s a m p l e s were c o u n t e d o n a P a c k a r d T r i - e a r b l i q u i d s c i n t i l l a t i o n s p e c t r o m e t e r a n d 10 ~1 s a m p l e s were t i t e r e d for virus. I n e a c h i n s t a n c e , f r a c t i o n s t a k e n f r o m t h e m i d d l e of t h e sucrose g r a d i e n t c o n t a i n e d a p e a k of radioaetiv%y which coincided with the peak virus titer. 16"

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Se~ I m m u n e sera to e a c h of t h e r h i n o v i r u s e s were p r e p a r e d in y o u n g a d u l t r a b b i t s . F o r t h e p r i m a r y i m m u n i z a t i o n , f r a c t i o n s f r o m t h e m i d d l e t h i r d of t h e sucrose g r a d i e n t s were p o o l e d to e n s u r e t h e p r e s e n c e of b o t h C- a n d ] ) - a n t i g e n i c particles. Pools were d i l u t e d w i t h a n e q u a l v o l u m e of P B S a n d p a s s e d t h r o u g h a 0.45 m i c r o n millipore filter. E q u a l v o l u m e s of i m m u n o g e n a n d c o m p l e t e F r e u n d ' s a d j u v a n t (CFA) were emulsified a n d a d m i n i s t e r e d t o r a b b i t s b y single 0.5 m l i n j e c t i o n s in a f o o t p a d a n d i n t r a m u s c u l a r l y (i.m.). T h e first b o o s t e r i m m u n i z a t i o n w a s g i v e n a t 5 weeks in t h e f o r m of 0.3 m l of filtered a n t i g e n emulsified w i t h a n equM v o l u m e of i n c o m p l e t e F r e u n d ' s a d j u v a n t i . m . Sera collected 1 w e e k a f t e r t h i s b o o s t e r i m m u n i z a t i o n c o n t a i n e d low levels of n e u t r a l i z i n g a n t i b o d y , h o w e v e r , p r e c i p i t a t i n g a n t i b o d y was n o t d e t e c t a b l e . A t t h i s t i m e t i t r a t i o n of t h e filtered v i r u s p r e p a r a t i o n i n d i c a t e d t h a t f i l t r a t i o n was a s s o c i a t e d w i t h a 2~-3 Iogl0 fall i n v i r u s t i t e r , t h e r e f o r e , u n f i l t e r e d sucrose g r a d i e n t f r a c t i o n s were u s e d i n all s u b s e q u e n t i m m u n i z a t i o n s . T h e a n i m a l s were i n o c u l a t e d for t h e t h i r d t i m e 10 weeks a f t e r i n i t i a l i m m u n i z a t i o n w i t h 1.2 m t of a n e q u a l v o l u m e of u n f i l t e r e d v i r u s emulsified w i t h C F A w h i c h was s u p p l e m e n t e d w i t h 1.5 m g of g r o u n d h e a t - k i l l e d t u b e r c l e bacilli ( h u m a n t y p e s C, DT, P N , o b t a i n e d from Dr. T e n Feizi). T h i s inoculat i o n was a d m i n i s t e r e d i n 15 20 s e p a r a t e i n t r a d e r m a l i n j e c t i o n s a c c o r d i n g to t h e m e t h o d of VIATUKAI$IS et al. (20). A d d i t i o n a l b o o s t e r doses of a n t i g e n were g i v e n i . v . a t 4 m o n t h s a n d i . m . a t 5 a n d 7 m o n t h s a n d t h e a n i m a l s were e x s a n g u i n a t e d t w e e k after the final immunization at which time maximum precipitating antibody activity w a s p r e s e n t . T h e s e sera h a d reciprocal n e u t r a l i z i n g a n t i b o d y t i t e r s a g a i n s t h o m o l o g o u s v i r u s of 80 for R V 1 A a n d b e t w e e n 1280 a n d 4000 for r e m a i n i n g a n t i g e n s . t I u m a n sera collected before a n d 3 weeks a f t e r e x p e r i m e n t a l i n f e c t i o n w i t h R V 2 were s u p p l i e d b y Dr. S y l v i a R e e d , C o m m o n Cold U n i t , Salisbury, E n g l a n d .

Immunodi//usion Tests T h e m i c r o m e t h o d of TYI~IaELL (19) was u s e d for t h e d o u b l e i m m u n o d i f f u s i o n assay. Slides were i n c u b a t e d for 3 or 4 d a y s a t r o o m t e m p e r a t u r e a n d r i n s e d w i t h pI-I 7.2 P B S for 24 h o u r s before s t a i n i n g w i t h 0.1 p e r c e n t Coomassie blue. Gels were p h o t o g r a p h e d before d r y i n g .

Isoelectric Focusing T h e m e t h o d of KORA~-T a n d LONBERG-HoLsI (I0) was u s e d for isoelectric focusing of r h i n o v i r u s part, ieles. L i n e a r g r a d i e n t s of 3 6 - - 1 0 p e r c e n t sucrose were g e n e r a t e d i n 1 p e r c e n t (w/v) a m p h o l y t e s ( L K B P r o d u c t s , B r o m m a , Sweden) p H r a n g e 3 . 5 - - 1 0 . T h e v i r u s w a s a d d e d in a 200 ~xl v o l u m e of 18 p e r c e n t sucrose i n 1 p e r c e n t a m p h o l y t e s o l u t i o n to t h e a p p r o x i m a t e m i d d l e of t h e g r a d i e n t . E l e e t r o p h o r e s i s was a t 4 ° C w i t h 2 - - 0 . 5 m a m p / g r a d i e n t for 5 hours. F r a c t i o n s were r e m o v e d f r o m t h e t o p of t h e g r a d i e n t b y a s p i r a t i o n , 20 ~1 s a m p l e s of e a c h f r a c t i o n were c o u n t e d i n a s e i n t i l l a t i o n s p e c t r o m e t e r a n d t h e p H w a s d e t e r m i n e d a t r o o m t e m p e r a t u r e a f t e r d i l u t i o n of s a m p l e s w i t h 6 p a r t s d e i o n i z e d distilled w a t e r .

Results Rabbit sera collected prior to immunization did not form preeipitin bands when reacted in the double immunodiffusion test against viral antigens. Sera

collected one week after the final (7 months) booster immunization with RV2 produced several precipitin bands when reacted against fluorocarbon treated homologous viral antigen (Fig. l, reaction with serum 2 b). Control studies indicated that this serum and sera from rabbits immunized with the other 5 rhinoviruses also formed precipitin bands when reacted with NBC serum and the IIeLa cell material. Sera were therefore, treated with NBC serum and HeLa cell lysate (0.I ml of 107 lleLa cells/ml lysate, 0.2 ml NBC and 0.8 ml rabbit immune serum) and incubated for 2 hours at 4 ° C prior to testing. Treated sera g a v e no reactions

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with t I e L a cell a n d NBC serum control antigens. E a c h serum gave either a single precipitin b a n d (Fig. 1) or 2 precipitin b a n d s (Fig. 2, 3, & 4) when reacted with homologous antigen. W h e n 2 b a n d s were visible the "specific" precipitin b a n d (D-antigen reaction) was located n e a r the serum well while t h e heterologous or " g r o u p " precipitin b a n d (C-antigen reaction) was located near the center a n t i g e n well (17).

Fig. 1. Immunodiffusion of I~V2 (center well) against untreated rabbit antisera to RV2 collected 1 week after the finM (7 month) immunization (2b); tIeLa cell and NBC serum treated antisera to RV2 collected 1 week after the 5th booster (5 months) (2a) and the final booster (7 months) immunization (2e); HeLa cell and NBC treated serum collected 1 week after the final (7 months) booster immunization with RV 1A ( 1), RV 3 (3) and RV 9 (9). The precipit,in line formed between RV 2 antigen and treated antiserum to RV 2 (2e) merges in a line of identity with the precipitin line formed with treated antiserum to RV 1 A (1) Fig. 2. Immunodiffusion of tl.V2 (center well) against treated rabbit antisera to RV 1A (1), RV 2 (2 e), and sera eolIeeted before and after experimental infections with RV2 from h u m a n volunteer No. 1 (Ha and Hb) and volunteer No. 2 (He and tId). The group or heterologous line of precipitate formed with rabbit antisera to RV 1 A and RV 2 merges with the group line of precipitate formed with each of the h u m a n sera. The arrow points to the second "specific" line of precipitate formed between RV 2 antigen and rabbit antiserum to tCV 2

A cross reaction was seen b e t w e e n R V 2 a n t i g e n a n d t r e a t e d r a b b i t sera to R V 2 a n d R V 1 A (Fig. i). No heterologous group reactions were seen b e t w e e n t~V2 a n t i g e n a n d t r e a t e d r a b b i t antisera to R V 3 a n d R V 9 (Fig. 1), or to I~V4 a n d 1~V14 (not shown). I n Figure 2, a f a i n t specific (D-antigen) precipitin line is visible between R V 2 a n t i g e n a n d t r e a t e d r a b b i t sera to R V 2 (indicated b y arrow). A l t h o u g h the m u c h denser group antibody- p r e c i p i t a t i o n b a n d which formed b e t w e e n R V 2 Ag a n d R V 2 antisera does n o t have as acute a n angle of deviation at it passes b e t w e e n the 1%V2 a n t i g e n a n d the I~V 1 A r a b b i t s e r u m as was shown i n Figure 1, the extension of the line p a s t the R V 1 A s e r u m a n d 1~V2 a n t i g e n zone a n d the t e n d e n c y for this line to b e n d a w a y from the t~V 1 A serum

236

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well is consistent with a cross reaction between the R V 2 antigen and the R V 2 and R V t A rabbit sera. Figure 2 shows t h a t the group a n t i b o d y line which formed between R V 2 antigen and R V 2 a n d R V 1 A rabbit antisera merged in a line of identity with the group a n t i b o d y line formed between R V 2 antigen and postinfection sera collected from two volunteers who had been experimentally infected with R V 2 . The we-infection serum (Ha) from volunteer ~ 1 m a y have contained some precipitating antibodies to R V 2 since the group a n t i b o d y line bends a w a y from this serum, however a clear acute deviation in this line is not visible. The post-infection serum from volunteer @l does form a more prominent line of precipitation with the R V 2 antigen t h a n the preinfection serum indicating t h a t an increase in C-reactive antibodies was associated with t~V2 infection. There was no difference in the prominence of the pre- and post-infection serum group lines from volunteer @2. Although these volunteers exhibited 64-fold rises in titer of neutralizing a n t i b o d y to R V 2 , we were unable to detect, a second :'specific" preeipitin line with the homologous rhinoviral antigen which had been described in post-rhinovirus infection sera b y H u G ~ s st al. (9).

Fig. 3. Immunodiffusion of I/~VtA (center welt) against treated rabbit, antisera to ~ V 1 A (1), RV2 (2), and RV9 (9). A line of identity is formed nearest the antigen welt for M1 reactions tested. The '%peeifie" preeipitin line formed betweer~ R,V i A antigen and antiserum to RV 1A can be seen lying nearer to the antiserum well Fig. 4. Immunodiffusion of 1RV1A (center well) against treated rabbit antisera to t ~ V I A (t), R V 4 (4), 1KV9 (9), and ~ V 1 4 (t4). A group line of identity is formed between the RV 1A antigen and antisera to I~V 1 A, RV 9, and t~,V 14. No preeipitin line formed between RV i A antigen and antiserum to I~V 4 I%V 1 A viral antigen formed heterologous group preeipitin lines with treated rabbit antisera to t~V2 and R V 9 (Fig. 3) and 1%V14 (Fig. 4). The junctions between the homologous R V t A antigen-antibody precipitin line and lines formed between P~V 1A antigen and antisera to 1%V9 and 1%V2 (Fig. 3) and R V 14 (Fig. 4) show weak single spurs which suggest t h a t these antisera also contain small a m o u n t s of specific and non-cross reacting antibodies. The group a n t i b o d y

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precipitate which formed between RV 1A and these rabbit immune sera merged in a line of identity with group precipitates formed between RV 1A antigen and the same human sera tested in Figure 2. No group line of precipitate was detectable between R V 1 A antigen and antisera to R V 4 (Fig. 4) or to l~V3 (not shown). The "specific" precipitin line between RV 1 A antigen and t~V I A serum is detectable near the serum well in both Figure 3 and 4. The faint precipitin line in the "specific" area between RV 1 A antigen and serum to R V 9 in Figure 4 is probably an artifact since it is not seen in Figure 3. Heating R V 1 A and RV2 antigens to 56 ° C for 5 minutes in an a t t e m p t to change D- to C-antigenic particles (13) caused specific precipitin lines to disappear but did not enhance the group precipitin lines. t~V14 viral antigen formed a heterologous group precipitin line only with rabbit antisera to RV3 (Fig. 5). In separate tests, this precipitin line merged with the group line which formed between t~V 14 antigens and human sera. Single precipitin lines were formed when l~V3, RV4, and I~V9 viral antigens were reacted with homologous rabbit antisera, however, these antigens failed to form heterologous group precipitin lines.

i

Fig. 5. Immunodiffusion of I~V 14 (center well) against treated antisera to /~V 14 (14) and RV3 (3). A group line of identity is formed between RV14 antigen and both antisera To demonstrate the presence of C-antigenic particles in our antigenic preparations, we separated fluorocarbon treated l~V2 virions by isoelectric focusing (Fig. 6), The major component consisted of particles migrating to a pI-I of 6.8 (D-antigenic) and the minor component migrated to a p H of 4.4 [C-antigenic according to the criteria of 14ORA~T et al. (12)]. The isoelectrie focusing technique did not prove to be useful for preparing purified C- and D-antigenic materials since samples taken from both sub-population peaks failed to react with homologous rabbit antisera in the immunodiffusion assay.

238

C.B. SS~tT~: IO°oo~il,

9

"

pH

8

-400 Qo

7 6-

-300

pH 5 -200

CPM H3

o

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5

I

10 15 20 FRACTION NUMBER

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25

Fig. 6, Isoeleetrie focusing of fluorocarbon purified sH L-leueine labeled tgV-2 virions in a linear gradient of 36--10 per cent sucrose. Virus was added in 18 per cent sucrose and 1 per cent ampholyte to the middle of the gradient and focusing was carried out for 5 hours at 4 ° C. Radioactivity was determined in 20 ~1 samples of each fraction and pH was determined after dilution of samples with 6 parts of deionized distilled water. Radioactivity ( ), p H (. • .) 1)iseussion A fluorocarbon purified preparation of t~V2 virions was shown to cross react b y immunodiffusion with rabbit antibodies to R V 1 A , confirming the initial report of LONI~ERG-HOLM and YIN (13). New cross reactions detected b y immunediffusion in the present study were between R V 1 A virions and rabbit antisera to R V 2 , I{V9 and t~V14 and between R V t 4 virions and rabbit antisera to 1~V3. C~'oss reactivity between R V t A C-antigenic particles and rabbit antisera to l~V2 has been previously demonstrated b y the CF test (13). Some cross reactivity between R V t 4 and R V 3 has been demonstrated b y the neutralization test after cross immunization of rabbits (3), however, it is difficult to associate these observations with our findings since neutralizing antibodies are associated with I)antigenic reactivity. The heterologous " g r o u p " or C-antigenic nature of the cross reactions detected in the present s t u d y is indicated b y the location of the heterologous precipitin line nearer to the antigen well t h a n the homologous "specific" preeipitin line (9, 17), and b y the merging (line of identity) between heterologous preeipitin reactions with rabbit antisera and the group a n t i b o d y line formed with h u m a n sera. The presence of C-antigenic partieles in the fluorocarbon treated R V 2 preparation is also suggested b y the demonstration t h a t a subpopulation of viral particles migrated to a p H of 4.4 b y isoelectric focusing. KORA~T et al. (12) have demonstrated t h a t naturally occurring and artifically produced C-antigenic particles of rhinovirnses can be easily separated from native D-antigenic particles b y this technique. Due to the instability of C-antigen particles at low p H (8, 12), we were unable to use the isoelectrie focusing method to prepare C-antigenic material.

Gel Double Immunodiffusion With Rhinoviruses

239

The C-antigenieity of our fluorocarbon purified virus antigen preparations could not be increased by alteration of the D-antigenic component b y mild heat treatment as has been described b y LO~BEI~G-HozM and YI¢~ (13). This difference m a y be due to the fact that their starting material consisted of sucrose and CsC1 gradient purified D-antigenic material while our virus preparation was relatively anpurified and therefore already contained C-antigenic material. Several investigators have been unable to detect antibodies to picornavirus C-antigenic materials in experimental animals when testing sera after the usual 4 - - 8 week period of immunization (9, 17). Others have noted that repeated infection of experimental animals with different virus serotypes from the same picornavirus group was necessary to develop antibody to C-antigenic components (16). Using a relatively vigorous immunization schedule we did not detect cross reacting antibodies b y the immunodiffusion test until 5 months after primary immunization, and we found the most reactive antisera in the final bleed at 7 months. However, because our first two immunizations contained reduced amounts of infectious virus (and presmnably viral antigen), due to filtration effects, it is possible that other investigators m a y find t h a t C-reactive antibodies will appear before 7 months. Although we have described several new cross reactions between hyperimmune experimental animal sera and C-antigens of rhinoviruses, our observations indicate that such cross reactivity m a y not extend completely throughout the human rhinovirus group. The RV 1A antigenic preparation proved to be the strongest and most broadly reacting source of C-antigenic material, yet it failed to detect C-reactive antibodies in rabbit sera to R V 3 and RV4. On the other hand, RV14, a relatively weak source of C-antigenic material did show cross reactivity with rabbit antiserum to RV3. In his review of cross reactions between neutralizing antibodies to rhinoviruses, F o x (6) described several virus groups which appeared to share antigenicity. Perhaps extended studies of C-antigenicity of rhinoviruses using hyperimmmle animal sera will permit definition of a small number of C-antigenic groups. H u m a n sera eolleeted before and after experimental infection with RV2 contained group antibodies which precipitated with C-antigenic components of I~V 1A, RV2, and RV14. An increased prominence of these group precipitin lines was detectable in the post-infection serum from one of the two individuals tested. However, the immunodiffusion test is not very sensitive for assessing changes in titer of antibodies, and it is possible that more sensitive tests such as the OF test or radioimrnunoassay m a y ultimately be useful as a general rhinovirus serological test. Several years ago, MOGABGAB (14) reported antibody rises to a crude R V 1 A viral OF antigen in adults following presumably viral acute respiratory illnesses. Several examples of heterologous rises were detected in the same paired human sera when they were reacted with OF antigens to other picornaviruses, suggesting that increases in titer of group or "C" reactive antibodies followed acute respiratory viral illness. However, interest in the OF test for diagnosis of rhinovirus infections subsequently declined when CHAPPLE st al. (2) using a more purified antigenic preparation of RV2, failed to detect antibody rises in 13 volunteers following experimental I~V2 virus infection. Our demonstration t h a t R V 1 A was a much better source of cross reacting C-antigenic material

240

C . B . SMITH:

t h a n was R V 2 m a y explain the discrepancies b e t w e e n these two reports. B a s e d on the conditions used ill this study, I~V 1 A would appear to be a good source of C-antigenic c o m p o n e n t s in f u t u r e a t t e m p t s to develop a CF or r a d i o i m m u n o a s s a y for r h i n o v i r u s " g r o u p " or C-antigen r e a c t i v e antibodies in h u m a n sere following rhinovirus infections.

Acknowledgments This work was conducted while the author was a visiting investigator at the Medical Research Council Clinical Research Centre, Harrow, England and was supported by a Senior International Fellowship of the Fogarty International Center. The author is indebted to Drs. D. A. J. TyrrelI, S. E. Reed, M. R. Maenaughton and C. Golden for helpful advice.

Referenees 1. BU~r~E~WOICT~r, B. E., G~NE~T, t~. 1~., I~ORAN% B. D., LON~E~G-HoL~, K., YIN, 1~. H. : Replication of rhinoviruses. Arch. Virol. 51, 169--189 (1976). 2. CI~APPLE, P. J., HEAD, B., TYRRELL, D. A. J. : A complement fixing antigen from an M. l~hinovirus. Arch. ges. Virusforsch. 21, 123--126 (1967). 3. COONEY, M. K., WISE, J. A., I~2ENNY, G. E., F o x , J. P. : Broad antigenic relationships among rhinovirus serotypes revealed by cross-immunization of rabbits with different serotypes. J. Immunol. 114, 635--639 (1975). 4. D±Ns, P. E., ~o~s¥~H, B. 1~., CI~ANoeK, R. M.: Density of infectious virus and complement.fixing antigens of two rhinovirus strains. J. Bacteriol. 91, 1605--1611 (1966). 5. FAULK, W. P., VYAS, G. N., PalLLIPS, C. A., FUDENBERG, H. H., CHISM,I~. : Passive hemagglutination test. for anti-rhinovirus antibodies. Nature (New Biol.) 231, t01 to 104 (197t). 6. F o x , J. P. : Is a rhinovirus vaccine possible ? Amer. J. Epidemiol. 103, 345 354 (1976). 7. GWALTNEY, J. M., JI¢. : P~hinoviruses. Yale J. Biol. Med. 48, 1 % 4 5 (1975). 8. HUGI4ES, J. H., CHEMA, S., LtN, N., CO~AN~, 1~,. M., HA~PA~IAN, V. V. : Acid lability of rbinoviruses: loss of C and D antigenicity after treatment af p H 3.0. J. Immunol. 112, 9t9--925 (I974).

9. HUGHES, J. H., GN~av, J. M., HILTY, M. D., CHE~,

S., 0~TOLE~CGHI, A. C., H A M PARIAN, V. V. : Pieornaviruses : rapid differentiation and identification b y i m m u n e

electronmicroseopy and immunodiffusion. J. med. Microbiol. 10, 203--212 (1977). 10. KogAN~, B. D., LONBE~G-HozM, K. : Zonal electrophoresis and isoeleetric focusing of protein and virus particles in density gradients of small volume. Anal. Biochem. 59, 75--82 (1972). 11. KO~ANT, B. D., LON~E~G-IIOL~, K., NOBLE, J., STASI~¥, J. T. : Naturally occurring and artifieally produced components of rhinoviruses. Virology 48, 71--86 (1972). 12. KOR~NT, t3. D., LONBEgG-HOL~I, K., YIN, F. I-I., NOBLE-I][AI~VE¥,J. : Fractionation of biologically active and inactive populations of human rhinovirus Type 2. Virology 63, 384--394 (1975). 13. LONBE~G-HOL3f, K., YI~', F. H.: Antigenic determinents of infective and inactivated human rhi~mvirus Type 2. J. Virol. 12, 114--123 (t973). t4. MOGABG.4.~, W. J. : 2060 Virus (ECHO 28) in K B cell cultures. Characteristics, complement-fixation and antigenic relationships ~o some other respiroviruses. Amer. J. Hyg. 76, 15--26 (1962). 15. REED, L. J., MIUENCH,H. : A simple method of estimating fifty percent, endpoints. Amer. J. Hyg. 27, 493--497 (1938). 16. SOItNIDT, N. J., DENNIS, T., LENNETTE, E. H., He, H. H., StJ:IlgOTO, T. T.: Antibody responses of rhesus (maeaea mulatta) monkeys experimentally infected with coxsaekie viruses of group B and group A, Type 9. I. Antibody responses within the coxsackie virus group. J. Immunol. 95, 54-~69 (1965).

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17. ScH~II)% N. J., LE~I~ETTE, E. H. : Gel double diffusion studies with group B and group A, Type 9 coxsackie viruses. I. The technique and reactions obtained with hyperimmune animal sera and human sera. J. Immunol. 89, 85--95 (1961). 18. STOTT, E. J., TYI~I~LI~, D. A. J. : Some improved techniques for the study of rhinoviruses using I-IeLa cells. Arch. ges. Virusforsch. 23, 236--244 (1968). 19. T¥~I~]~I~I~, D. A. J. : Immunodiffusion with IZhinoviruses. In: WEII~, D. i . (ed.), Handbook of Experimental Immunology, 2nd ed., 37.16. Oxford: Blackwell Scientific Publications 1973. 20. VIATUKAITIS, J., ROBBINS, J. B., NIESC~I,AG, E., l~oss, G. T.: A method for producing specific antisera with small doses of immunogen. J. clin. Endocr. and Metab. 33, 988--991 (1971). Author's address: C. B. S)~IT~I, M.D., Division of Infectious Diseases, University of U t a h Medical Center, 50 North Medical Drive, Salt Lake City, UT84132, U.S.A. Received September 16, 1977