Preparation of specific antisera against adenoviruses by affinity bead immunization (ABI)

Journal of Immunological Methods, 26 (1979) 141--149 Q Elsevier/North-Holland Biomedical Press

141

PREPARATION OF SPECIFIC ANTISERA AGAINST ADENOVIRUSES BY A F F I N I T Y B E A D I M M U N I Z A T I O N ( A B I )

MATS E. JOHANSSON, GORAN WADELL *, P. AKE JACOBSSON and LENNART SVENSSON Department of Virology, National Bacteriological Laboratory, S-105 21 Stockholm, and * Department of Virology, Karolinska Institute, S-105 21 Stockholm, Sweden

(Received 18 July 1978, accepted 16 September 1978)

Certain adenovirus types can be replicated only to low titer in tissue cultures. Other, such as adenovirus strains associated with infantile gastroenteritis, cannot be replicated in vitro. A method which allows preparation of specific antisera has therefore been evaluated. The procedure involves coupling of group-specific antibodies against adenovirus capsid subunits to CNBr-activated Sepharose 4B; reaction of crude virus suspensions with immobilized adenovirus-specific IgG; elimination of contaminating material by extensive washing using a wide pH range; and immunization with adenovirus immunogens immobilized on the beads. Efficient immunization was obtained with immunogen doses of both 50 ng and 50 pg. The immunization procedure which has been designated affinity bead immunization (ABI) could therefore have a wide applicability in cases where the relevant immunogen constitutes a minor fraction of a crude preparation.

INTRODUCTION We h a v e r e c e n t l y d e s c r i b e d an a p p l i c a t i o n o f a f f i n i t y c h r o m a t o g r a p h y f o r p r e p a r a t i o n o f t y p e - s p e c i f i c a n t i s e r a against a d e n o v i r u s { J o h a n s s o n a n d Wadell, 1 9 7 8 ) . Certain a d e n o v i r u s t y p e s o f clinical relevance g r o w p o o r l y or n o t a t all in tissue c u l t u r e s . I t is p e r t i n e n t t o p r e p a r e diagnostic r e a g e n t s specific f o r a d e n o v i r u s t y p e (Ad) 8, w h i c h causes e p i d e m i c k e r a t o - c o n j u n c tivitis, a n d e n t e r i c a d e n o v i r u s e s w h i c h are r e s p o n s i b l e f o r infantile gastroenteritis. Purified i m m u n o g e n s o f t h e s e a d e n o v i r u s e s are d i f f i c u l t to o b t a i n by conventional purification methods. All m a m m a l i a n a d e n o v i r u s e s share group-specific antigens localized on the p a r t o f t h e ' h e x o n s w h i c h f o r m the i n t e r i o r o f the virus capsid. T h e t y p e specific d e t e r m i n a n t s are e x p o s e d on t h e e x t e r i o r o f t h e capsid ( N o r r b y , 1 9 6 9 ) . S t r u c t u r a l viral s u b u n i t s , i.e. soluble a d e n o v i r u s c o m p o n e n t s such as h e x o n s , p e n t o n s a n d fibers are p r o d u c e d in vast excess d u r i n g a d e n o v i r u s i n f e c t i o n s (White et al., 1 9 6 9 ) . We t h e r e f o r e e v a l u a t e d the possible use o f affinity chromatography techniques for preparation of purified type-specific i m m u n o g e n s f r o m c r u d e virus s u s p e n s i o n s o f Ad 8 a n d e n t e r i c adenoviruses.

142 The present paper presents results of evaluation of two alternative methods. The first procedure comprised adsorption of the antigen to the immunoadsorbent followed by elution of the purified immunogen with glycineHC1 buffer at pH 2.8. In an alternative m e t h o d antigen adsorbed on the immunoadsorbent was used as immunogen. This procedure was designated affinity bead immunization (ABI) and was definitely the most efficient technique. The efficiency of ABI was therefore compared with other immunization schedules. MATERIAL AND METHODS A d 8 is a causative agent of epidemic kerato-conjunctivitis. Soluble viral components were obtained by 3 cycles of freeze-thawing at --70°C and room temperature, respectively, of an unfraetionated suspension of Ad 8 infected cells followed by centrifugation at 100,000 × g for 60 min. A d 31 is frequently isolated from patients with gastroenteritis (Belian et al., 1968; Moritsugu, 1969). Soluble viral antigens were extracted as described above. A d 33 was used as a model system. Virions were purified as described by Winberg and Wadell (1977). Intact Ad 33 virions were dissociated into subunits by 20 cycles of freeze-thawing. The viral subunits were labeled with l~sI by lactoperoxidase according to Thorell and Johansson (1971) and the protein c o n t e n t was determined according to Lowry et al. (1951). The specific activity expressed as eounts/min/ttg protein was used to determine the a m o u n t of viral antigen bound to the immunoadsorbent. Enteric adenovirus type 1 was extracted from the feces of a 5-year-old diabetic boy with severe diarrhea. This isolate could n o t be replicated in tissue culture. The polypeptide composition of purified virions has been analyzed by SDS-polyacrylamide gel eleetrophoresis according to the procedure used for classification of h u m a n adenoviruses as described (Wadell, 1979). The polypeptide composition of this enteric isolate differed distinctly from the polypeptide pattern of the established h u m a n adenovirus types (Wadell et al., in preparation). The soluble viral components, which were used in the preparation of immunogens, were obtained by sonication (20 kHz, 4 × 15 see) followed by ultraeentrifugation (100,000 × g , 60 min) of a 10% feces suspension in PBS. The pellet was discarded. Immobilization o f viral antigens on the immunoadsorbent. The IgG fractions, twice precipitated with 50% saturated (NH4)2SO4 of a rabbit-antiadeno-2 virion serum, prepared as described for anti-virion serum by Johansson and Wadell (1978) was coupled to CNBr-activated Sepharose 4B ® (Pharmacia, 1974) at a concentration of 10 mg IgG/ml gel. The anti-adeno serum contained mainly high-avidity antibodies (Wadell and Norrby, 1969; Werblin et al., 1973) largely directed against adeno group-antigen, as confirmed by tests against heterologous adenovirus types. The gel was saturated with blue dextran (Heinzel et al., 1976) after

143 coupling with IgG to avoid non-specific binding of proteins. The a m o u n t of antigen reacting with the immunoadsorbent was chosen so that the antibody c o m p l e m e n t fixing units (CFU) exceeded the antigen CFU at least 4-fold. The antigen was allowed to react with the immunoadsorbent overnight at room temperature. Subsequently 5 cycles of alternate washing of the gel with 0.1 M barbital-HC1 buffer, pH 8.3, and 0.2 M glycine-HC1 buffer, pH 2.8, were performed. Immunization procedure. The immunoadsorbent beads were diluted to a 33% suspension in PBS. Three ml were inoculated intramuscularly in each rabbit. Alternatively equal volumes of Freund's complete adjuvant (FCA) immunogens were used. At 4--6 weeks after the primary immunization a second inoculation was given intramuscularly. Occasionally individual rabbits died after heart puncture. However, no adverse effects of ABI were observed. Immunofluorescence (IF). The indirect IF test was performed according to Nairn (1976) as previously described (Johansson and Wadell, 1978). The ABI procedures could possibly give rise to antibodies against Sepharose. A b o u t 1000 Sepharose beads Were therefore incubated with specific sera and conjugate and washed 5 times in 0.65 M NaC1. One drop containing around 100 beads was analyzed. Sheep anti-rabbit IgG conjugated with fluorescein-isothiocyanate (SBL, Stockholm, Sweden), F/P molar ratio of 3.9, was used in a dilution of 1 : 12. Four-fold serum dilutions were used to determine the end-point titers which were defined as the highest serum dilution which gave a clear-cut fluorescence. A Standard Universal Microscope (Carl Zeiss, Oberkochen, Germany) equipped with a HBO 200 mercury lamp with exciter filters BG12/KP490 and barrier filters Nos. 41 and 44 was used. The total magnification of the optical system was 40 X 10. Immuno-electro-osmophoresis (IEOP). The IEOP tests were performed according to Grauballe et al. (1977). Type-specific anti-adeno sera. Preparation of type-specific antisera were performed by adsorption of sera obtained by ABI on immobilized soluble viral c o m p o n e n t s from heterologous adenovirus types (Johansson and Wadell, 1978). RESULTS

Elution of adenovirus antigens from the solid phase Soluble viral components of enteric adenovirus 1 reacted strongly with an immunoadsorbent containing group-specific anti-adenovirus antibodies. No adenovirus-specific antigen was detected after elution with 0.2 M glycineHC1 buffer, pH 2.8. Furthermore immunization of two rabbits with a corresponding eluate failed to induce adenovirus-specific antibodies. However, adenovirus-specific antigens were eluted with 8 M urea. The antigens recovered displayed group-specific but n o t type-specific reactivity indicating

144 l.F.-tifer

512-

o

256-

x

12864321680

10

20

30

, 40

, SO

, 60

~

Doys

Fig. 1. A n t i b o d y titers d e t e r m i n e d by i m m u n o f l u o r e s c e n c e after i m m u n i z a t i o n with affinity b e a d s w i t h o u t F r e u n d ' s c o m p l e t e a d j u v a n t with Ad 8 (o o) and Ad 31 (X X), respectively. S e c o n d a r y i m m u n i z a t i o n s o n day 32 and 27, respectively, as i n d i c a t e d by arrows.

that this procedure could not be relied on for preparing type-specific determinants.

Affinity bead immunization (ABI) with different adenoviruses Antibody titer rises. The antibody obtained after immunization was measured by immunofluorescence titration of sera obtained on consecutive bleedings. The endpoint titers were determined on diploid cell cultures infected with homologous virus (Fig. 1). The increase in antibody titer of sera taken 10 days after the first immunization was significant. The second dose, given intramuscularly 4--5 weeks after the first dose, hardly affected specific antibody titer.

TABLE 1 HOMOLOGOUS AND HETEROLOGOUS A B S O R B E D R A B B I T - A N T I - A d 8 SERUM.

IF

TITERS

OF

N O N - A B S O R B E D AND

The s e r u m was o b t a i n e d by ABI and a b s o r b e d with A d 9 soluble c o m p o n e n t s i m m o bilized o n solid phase. Rabbit-anti-Ad 8 serum

Non-absorbed Absorbed

Adenovirus types 2 (III) a

3 (I)

8 (II)

9 (II)

32

64

512

512

--

512

8

_ b

a R o m a n n u m e r a l s d e n o t e s u b g r o u p d e s i g n a t i o n according to R o s e n (1960). b D e n o t e s a t i t e r o f less t h a n 2.

145

Specificity The relative degree of homologous and heterologous reactivity in an antiserum against Ad 8 drawn 1 week after the secondary immunization (day 32) was determined (Table 1). Ad 8 and Ad 9 are closely related (Hierholzer and Dowdle, 1970). However, absorption of this serum with soluble virus c o m p o n e n t s of Ad 9 coupled to CNBr-activated Sepharose 4B, rendered it discriminating between these serotypes (Table 1). It was therefore considered to be strictly type-specific. The reactivity of antisera against enteric adenovirus obtained by ABI was assayed by IEOP (Fig. 2). Further work to prepare type-specific sera for typing of enteric adenoviruses by IEOP is in progress. Occasionally rocket- or curtain-like precipitates were noted in IEOP of sera obtained by ABI (Fig. 2). These precipitations were eliminated after absorption with Sepharose 4B and were interpreted to be due to antibodies against agarose. Sera obtained after ABI occasionally gave clear-cut fluorescence in low dilutions, when tested by indirect IF technique on Sepharose 4B beads. With ABI, antibodies against blue dextran , bovine serum or host cell components could n o t be detected by CF or by immune fluorescence. Anti-

t

Fig. 2. T h e reactivity of a n t i s e r u m against e n t e r i c a d e n o v i r u s t y p e 1 o b t a i n e d b y ABI assayed b y IEOP. T h e a n t i g e n s in t h e u p p e r wells are f r o m left t o right: e n t e r i c a d e n o virus 1, Ad 31 a n d e n t e r i c a d e n o v i r u s t y p e 1. T h e 3 l o w e r wells c o n t a i n s e r u m against e n t e r i c a d e n o v i r u s t y p e 1 o b t a i n e d b y ABI. T h e a r r o w i n d i c a t e s a weak p r e c i p i t a t e i n t e r p r e t e d to result f r o m a r e a c t i o n b e t w e e n anti-agarose a n t i b o d i e s a n d t h e gel.

146 b o d i e s a g a i n s t b o v i n e s e r u m o r h o s t cell c o m p o n e n t s f r e q u e n t l y c a u s e n o n s p e c i f i c s t a i n i n g i n t h e I F t e s t ( J o h a n s s o n e t al., 1 9 7 6 ) .

Comparison o f A B I with other immunization schedules A d 3 3 w a s u s e d t o c o m p a r e t h e e f f i c i e n c y o f i m m u n i z a t i o n w i t h viral s u b u n i t s in P B S w i t h A B I { T a b l e 2). A f t e r A B I w i t h a h i g h d o s e o f i m m u n o g e n ( 5 0 tLg) t h e a n t i b o d y r e s p o n s e w a s n o t i n f e r i o r t o t h a t o b t a i n e d b y i n t r a m u s c u l a r a d m i n i s t r a t i o n o f v i r al s u b u n i t s i n P B S . A B I in t h e p r e s e n c e o f F C A e n h a n c e d t h e i m m u n e r e s p o n s e of the rabbits. However, the response to secondary immunization after ABI in t h e p r e s e n c e o f F C A w a s m o d e r a t e c o m p a r e d t o t h e b o o s t e r r e s p o n s e o b t a i n e d a f t e r a s e c o n d a r y i m m u n i z a t i o n w i t h v i r a l s u b u n i t s in t h e p r e s e n c e of FCA. The comparison of the immune response obtained after immunization with m i n u t e a m o u n t s o f i m m u n o g e n (50 ng) revealed t h a t significant antibody titers could be obtained by ABI without FCA. Furthermore the i m m u n e r e s p o n s e o b t a i n e d a f t e r A B I in t h e p r e s e n c e o f F C A w a s c o m p a r a b l e

TABLE 2 COMPARISON OF ABI WITH OTHER IMMUNIZATION SCHEDULES Immunogen dose

Administration

Days after immunization 0

50 pg

IM b IM + FCA d ABI f

42 c 4c 4 ~2 2 --

50 ng

10

17

28

49 a

8 16 256 128 8

32 32 512 1024 32

64 32 1024 512 64 64 256 128

128 64 e 8192 256 128 512 512

4096 128 128 512 256

32

64

32

128 128

128 64

64 128

256 256 256

256 256 512

128 128 1024

8

8

ABI + FCA

-8

128 128

512 256

IM

2 8 8 2 4 8 4 2

2 8 8 8 8 16 8 4

IM + FCA ABI ABI + FCA

16 16 e 8 8 8 e 64 32 32

a Secondary immunizations given on day 37. b IM = intramuscular inoculation of virus fragments in PBS. c Pair of rabbits were immunized according to each schedule. d FCA = Freund's complete adjuvant. e Died. f Inoculation of affinity beads intramuscularly.

70 64 64

147 TABLE 3 DETERMINATION OF THE RATIO BETWEEN HOMOLOGOUS AND HETEROL O G O U S IF T I T E R S O F A N T I - A D E N O V I R U S T Y P E 33 S E R A O B T A I N E D A F T E R DIFFERENT IMMUNIZATION PROCEDURES

Rabbit-anti-adenovirus type 33 serum obtained after immunization with ABI (+FCA) Virus fragments in PBS (+FCA} Intact virions (+FCA)

Ratio: homologous over heterologous titers

Adenovirus types 2 (III)

3 (I)

8 (II)

33 (II)

128 512

128 1024

256 1024

1 024 4 096

8 8

2048

2048

2048

16 384

8

with or even superior to that obtained by immunization with viral subunits in the presence of FCA. The relative a n t i b o d y response obtained against type-specific and groupspecific determinants after ABI and other immunization procedures were compared. The ratio between homologous and heterologous reactivity was found to be the same, independent of the immunization procedure. This means that binding of viral subunits to the affinity beads via group-specific determinants had no detectable effect on the reactivity of sera obtained after ABI (Table 3). DISCUSSION Immunization with immune precipitates has been extensively used for preparation of monspecific antisera (Laron and Assa, 1962; Smith et al., 1964; Shivers and James, 1967; Nansen et al., 1971; Vestergaard, 1975; Grauballe et al., 1977). However, this technique has certain limitations. The available a m o u n t of immunogen can be limited in a procedure, which requires precipitation of antigen-antibody complexes in agarose. Furthermore complete removal of contaminating antigens is difficult to achieve. Affinity bead immunization (ABI) is an application of affinity chromatography, similar to immunization with immune precipitates. ABI has, however, the following advantages: (1) minute amounts of immunogen may be used; (2) immunogens may be isolated directly from complex antigen preparations o f large volume; (3) the beads can be efficiently washed with buffers of a wide pH range, which allows extensive purification of the immunogen; (4) the size of the beads can be varied which enables studies of the effect of intracellular processing of the immunogen. We illustrate here the usefulness of ABI by describing the properties of specific antisera against Ad 8 and adenoviruses associated with infantile gastroenteritis obtained by this immunization procedure. These have clinical

148 relevance. In cases of keratitis it is essential to be able to distinguish between herpes simplex and Ad 8. This can be achieved by use of an imprint technique followed by i m m u n o f l u o r e s c e n c e analysis, provided specific sera are available. Adenovirus particles are usually preferred as immunogens (Johansson and Wadell, 1978). Conventional purification procedure for virions can be applied to Ad 8 only with difficulty, since this virus replicates to m o d e r a t e titers and virions are released f r o m the cell early. We have therefore chosen to use ABI, in order to be able to use crude virus suspensions. Enteric adenoviruses can only exceptionally be replicated in tissue cultures. Consequently it is for practical purposes impossible to follow conventional procedures for propagation and purification of viral immunogens. Conventional typing by neutralization tests cannot be performed. Direct typing of the viruses in the specimen obtained from the patient by, e.g., immunoelectro-osmophoresis (IEOP) is, however, feasible. This approach requires preparation of type-specific sera. All mammalian adenoviruses share group-specific antigenic determinants. The ratio o f type-specific to group-specific antibodies induced with virus types belonging to Rosen's 3 subgroups varies (Wadell and N orrby, 1969). Sera against adenovirus types belonging t o subgroup III, e.g., adenovirus t ype 2 and 5, have been shown to contain a large port i on of group-specific antibodies (Wadell and N or r by, 1969; Willcox and Mautner, 1976). We have f o u n d it feasible to use the IgG fraction of a rabbit-anti-Ad 2 virion serum coupled to a solid phase (CNBr-activated Sepharose 4B beads). Viral subunits of adenoviruses are p r o d u c e d in 10-fold excess over intact virus particles (White et al., 1969), and are consequently well suited for binding to the solid phase via the group-specific antigenic determinants. ABI compares favorably with conventional immunization procedures. This is particularly evident after immunization with minute am ount s of antigens isolated f r o m crude preparations. ABI should also be well suited to preparation o f monospecific antisera against o t h e r viruses unable to replicate in vitro, such as the viruses causing infectious hepatitis. REFERENCES Belian, W., B. B6thig and R. Wigand, 1968, Z. Med. Mikrobiol. Immunol. 154, 196. Grauballe, P.C., J. Genner, A. Meyling and A. Hornsleth, 1977, J. Gen. Virol. 35, 203. Heinzel, W., I. Rahimi-Laridjani and H. Grimminger, 1976, J. Immunol. Methods 9,337. Hierholzer, J.C. and W.R. Dowdle, 1970, J. Virol. 6,782. Johansson, M.E. and G. Wadell, 1978, J. Immunol. Methods 19,259. Johansson, M.E., N.R. Bergqvist and M. Grandien, 1976, J. Immunol. Methods 11,265. Laron, Z. and S. Assa, 1962, Nature 194,491. Lowry, O.H., N.J. Rosenbrough, A.L. Farr and R.J. Randall, 1951, J. Biol. Chem. 193, 265. Moritsugu, Y., 1969, Jpn. J. Med. Sci. Biol. 22,279. Nairn, R.C., 1976, Fluorescent Protein Staining, 4th ed. (Churchill, Livingstone). Nansen, P., T. Flagstad and K.B. Pedersen, 1971, Acta Pathol. Microbiol. Scand. Sect. B 79,459.

149 Norrby, E., 1969, J. Gen. Virol. 5,221. Pharmacia Fine Chemicals, Uppsala, Sweden. Information booklets, 1974, Affinity Chromatography. Rosen, L., 1960, Am. J. Hyg. 71,120. Shivers, C.A. and J.M. James, 1967, Immunology 13,547. Smith, H., R.C. Gallop and B.T. Tozer, 1964, Immunology 7,111. Thorell, J.J. and B.G. Johansson, 1971, Biochim. Biophys. Acta 251,363. Vestergaard, B.F., 1975, Scand. J. Immunol. 4 (Suppl. 2), 203. Wadell, G., 1979, Intervirology 11, 47. Wadell, G. and E. Norrby, 1969, J. Virol. 4,671. Werblin, T.P., Y.T. Kim, F. Quagliata and G.W. Siskind, 1973, Immunology 24,447. White, D.O., M.D. Scharff and J.V. Maizel, Jr., 1969, Virology 38,395. Willcox, N. and V. Mautner, 1976, J. Immunol. 116, 19. Winberg, G. and G. Wadell, 1977, J. Virol. 22,389.