Induction of abundant antibody formation with a protein-cellulose complex in mice

Journal oflmmunologicalMethods, 87 (1986) 161-167

161

Elsevier JIM03811

Induction of abundant antibody formation with a protein-cellulose complex in mice A.E. Gurvich and Asiya Korukova Antibody Chemistry and Biosynthesis Laboratory, Gamaleya Institute of Epidemiolo~v and Microbiology, U.S.S.R. Academy of Medical Science, Moscow, U.S.S.R. (Received 24 July 1985, accepted 30 October 1985)

A method is described for immunizing mice with a protein-cellulose complex obtained by covalent coupling of antigenic protein molecules to suspended cellulose particles. Primary immunization of the animals with the complex led to a pronounced immune response persisting for 20 30 days. Subsequent administration of the same antigen in a soluble form resulted in extremely active antibody formation equal to or better than that induced with Freund's complete adjuvant. In BALB/c mice the antibody response was about ten times greater than in the C57BL/6 strain. Key words: Antibody response," Protein cellulose complex

1. Introduction

2. Materials and methods

Investigators engaged in experimental work are often confronted with the necessity of potentiating the action of various immunogens. At present different adjuvants are widely used for this purpose (Tolles and Paraf, 1973; Liashenko and Vorobyev, 1982). However, immunization of humans using existing adjuvant methods may produce adverse sequelae and may be rather ineffective (World Health Organization, 1976). Difficulties also often arise in the immunization of animals carried out by these methods (for instance, if it is necessary to obtain potent antisera to highly toxic substances). Novel approaches are, therefore, under constant investigation, for example the use of antigens coupled to polyelectrolytes (Petrov et al., 1983), or the procedures developed in our laboratory for the immunization of animals (Olovnikov and Gurvich, 1966; Orlov and Gurvich, 1969; Korukova et al., 1985).

2.1. Mice Inbred BALB/c and C57BL/6 mice were obtained from the Stolbovaya animal breeding unit. They were immediately injected with smallpox vaccine to protect against ectromelia and used at least 1 month after vaccination. 2.2. Antigens Horse gamma globulin (HGG; Miles Lab.) was used as the antigen; in some experiments bovine gamma globulin (BGG; Mechnikov Institute, U.S.S.R.) or ovalbumin (OA; Oline Chemical Reagents, U.S.S.R.) were employed. 2.3. Protein-cellulose complex An activated dialdehyde cellulose suspension (CS) and a protein-cellulose complex (CS-HGG) were prepared as described previously (Lechtzind and Gurvich, 1981; Gurvich and Lechtzind, 1982).

0022-1759/86/$03.50 ~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)

162

Four g of cotton were minced with scissors and placed into 80 ml of 25% N H 4 O H . After 2 - 3 h, Cu(OH)2, 4.9 g, was carefully stirred in; simultaneously and again after 1 h 25% N H 4 O H was added to the mixture, each time in a volume of 40 ml. On the next day the mixture was diluted with 25% N H 4 O H to 400 ml, stirred well and poured into a vessel containing 4 1 of distilled water. Concentrated H2SO 4 was added dropwise with stirring to precipitate the cellulose. Then cellulose was washed by centrifugation in distilled water until the supernatant was free of SO 2 ions, resuspended in 1 liter of distilled water, and, upon addition of N a I O 4, 2.14 g, incubated in darkness for 24 h. The preparation was washed in distilled water and a sample removed to determine its dry weight and the amount of aldehyde groups (Szabolcs, 1961), which was usually about 10-30 m g / g cellulose. After autoclaving, the activated cellulose suspension had a prolonged shelf-life under sterile conditions. A portion of the dialdehyde cellulose suspension (dry weight, 1 g) was washed 3 times with 0.1 M carbonate-bicarbonate buffer (CBB), p H 9, and resuspended in 200 ml of CBB. The protein ( H G G ) , 100 rag, was dissolved in 10 ml of CBB and dialyzed for 48 h against two 500 ml changes of the same buffer. The dialyzed protein solution was added to 200 ml of the dialdehyde cellulose suspension and the mixture stirred continuously at 4°C for 48-72 h. Then it was centrifuged and the amount of uncoupled protein in the supernatant was measured spectrophotometrically. The pellet was washed 3 times with saline. The unreacted aldehyde groups were reduced with N a B H 4. For this purpose the cellulose particles pelleted upon covalent coupling to protein ( C S - H G G ) were resuspended in 200 ml of saline, 300 mg of dry powdered N a B H 4 were added to the suspension and it was allowed to stand at room temperature with occasional mixing for 1 h. Then the C S - H G G complex was washed 5 times in saline to remove N a B H 4. During centrifugation it was necessary to avoid losses due to floating cellulose particles. The pellet was resuspended in saline to a total volume of 100 ml. The preparation was stored at 4°C in the presence of 0.1% sodium azide and used for immunizing mice and for quantitative measurements of antibody in sera. Before use a portion of

C S - H G G was washed several times in saline. To characterize the preparation, its dry weight was determined and the content of immobilized protein was assayed by the bromphenol blue method (Gurvich, 1955).

2.4. Immunization of mice For priming the mice, 0.2 ml of the antigen was injected subcutaneously into the flank. The antigen was administered (a) in saline; (b) as immobilized in the C S - H G G complex; and (c) in a 1 : 1 m i x t u r e with F r e u n d ' s c o m p l e t e a d j u v a n t (Calbiochem). The dose of C S - H G G was chosen so as to provide the required amount of immobilized protein. In some experiments C S - H G G was injected subcutaneously into the intermaxillary space or intraperitoneally. Before use C S - H G G was washed in saline and supplemented with antibiotics: penicillin, 250 U / m l , and streptomycin, 250 ~tg/ml. Booster doses of 0.2 ml of antigen ( H G G , B G G or OA) in saline were administered intravenously into the retro-or~ital sinus. 2.5. Measurements of antibody in sera The absolute antibody content in sera was estimated by the increment in the amount of protein bound to the C S - H G G immunoabsorbent (Gurvich and Kuzovleva, 1968). 2.6. Assay for antibody-forming cells Antibody-forming cells (AFC) were assayed by Jerne's method using sheep red blood cells (SRBC) sensitized with H G G or B G G via CrC13 (Kishimoto et al., 1968). Since the direct assay revealed very few AFC, we employed the indirect version based on the development of plaques by rabbit antiserum to murine gamma globulin (Dresser and Wortis, 1965). Before use the antiserum was absorbed with C S - H G G to remove antibodies crossreacting with H G G . The specificity of passive local hemolysis was confirmed by the fact that in the presence of 10 /,g of H G G plaque formation was suppressed by 98%. 2.7. Statistics For statistical analysis of the experimental data the geometric mean and its standard deviation were calculated.

163 m a r r o w ( 2 6 2 / 1 0 6 cells), thus i n d i c a t i n g a highly active i m m u n e response.

3. Results

O u r e x p e r i m e n t s showed that a d m i n i s t r a t i o n of H G G in saline elicited very weak, if any, a n t i b o d y p r o d u c t i o n in B A L B / c a n d C 5 7 B L / 6 mice. Imm u n i z a t i o n with the protein-cellulose C S - H G G c o m p l e x was c o n s i d e r a b l y m o r e effective. A n t i b o d y f o r m a t i o n was especially intense when the a n i m a l s were injected first with C S - H G G a n d then, after a certain p e r i o d of time, with soluble HGG.

3.1. Primary immune response in mice immunized by a single CS-HGG injection As shown in T a b l e I, B A L B / c mice showed evidence of a p r i m a r y i m m u n e r e s p o n s e in different l y m p h o i d o r g a n s after an injection of C S - H G G . A F C c o u n t s in the spleen a n d regional s u b m a x i l lary l y m p h n o d e s slowly rose to a p e a k b y d a y 17 20 a n d then g r a d u a l l y decreased up to d a y 30. A l t h o u g h the a b s o l u t e A F C n u m b e r at the p e a k of the p r i m a r y i m m u n e response was a b o u t twice as high in the spleen as in the regional l y m p h nodes, the A F C c o u n t p e r 10 6 cells was a p p r o x i m a t e l y 4 times greater at the l a t t e r sites. Similar changes were o b s e r v e d in o t h e r l y m p h n o d e s (axillary, inguinal a n d popliteal). O n d a y 20 a c o n s i d e r a b l e n u m b e r of A F C were also f o u n d in the b o n e

3.2. Secondary immune response to soluble HGG in mice primed with the CS-HGG complex A n intravenous injection of soluble H G G ind u c e d a very intense s e c o n d a r y i m m u n e response in B A L B / c mice p r i m e d with C S - H G G . The level of a n t i b o d y f o r m a t i o n d e p e n d e d on a n u m b e r of factors.

3.2.1. Influence of the amount of antigen administered as CS-HGG on the efficacy of priming. B A L B / c mice were given C S - H G G either in a single injection or in d i v i d e d doses (3 injections a week apart). It t r a n s p i r e d that the subsequent adm i n i s t r a t i o n of soluble H G G could elicit active a n t i b o d y p r o d u c t i o n only if the single p r i m i n g dose of C S - H G G c o n t a i n e d at least 1 0 - 1 0 0 / ~ g of p r o t e i n (Fig. 1). The effective p r i m i n g dose could be greatly r e d u c e d if d i v i d e d into 3 injections given at 7 - d a y

10 .4

10 3

TABLE I PRIMARY IMMUNE RESPONSE IN BALB/c MICE INJECTED WITH CS-HGG Mice were injected subcutaneously into the intermaxillary space with 0.2 ml of the CS-HGG suspension containing 100 ~g of immobilized protein. Groups of 4 mice each were sacrificed on different days and pooled organ suspensions were prepared for AFC assays.

10 2

Day after AFC count/106cells CS-HGG Spleen Lymph nodes injection Submaxillary Axillary Inguinal and popliteal

101

10th 13th 17th 20th 24th 28th

87 232 516 302 85 22

126 1085 1580 2037 174 100

273 118 108 6 -

296 242 97 0.4 -

I~ I

10 0

I

I

101

10 2

Fig. 1. The effect of the dose of immobilized antigen used for priming with CS-HGG on the intensity of the secondary immune response. BALB/c mice received subcutaneously into the flank different doses of protein in CS-HGG; the antigen was administered in a single dose (e) or in 3 divided doses a week apart (O). Two months later 10 /~g of soluble HGG were injected intravenously and on day 4 the spleens were assayed for AFC. Abscissa: dose of CS-HGG (in ~tg of protein) used for priming; ordinate: AFC count/10 6 spleen cells.

164 intervals. It m a y b e seen in Fig. 1 t h a t the seco n d a r y i m m u n e r e s p o n s e in m i c e w h i c h r e c e i v e d 3 C S - H G G i n j e c t i o n s , e a c h w i t h 0.5 fig o f i m m o b i lized a n t i g e n (i.e., a t o t a l a m o u n t of 1.5 fig), was j u s t as p r o n o u n c e d as a f t e r a single a d m i n i s t r a t i o n o f 100 f g in the f o r m of the C S - H G G c o m p l e x . A single i n j e c t i o n o f C S - H G G w i t h 1.5 f g o f p r o t e i n was ineffective.

3.2.2. Effect of the interual between priming with CS-HGG and the injection of soluble antigen. E a r l y a d m i n i s t r a t i o n of s o l u b l e H G G (7 d a y s a f t e r p r i m i n g w i t h C S - H G G ) failed to i n d u c e a n t i b o d y f o r m a t i o n in B A L B / c m i c e (Fig. 2). T h e i n t e n s i t y o f the i m m u n e r e s p o n s e i n c r e a s e d w i t h the i n t e r v a l b e t w e e n the i n j e c t i o n s o f C S - H G G a n d of s o l u b l e antigen. The response reached a maximum when the i n t e r v a l was o n e o r m o r e m o n t h s . E v e n if s o l u b l e H G G was a d m i n i s t e r e d 1 y e a r after p r i m -

TABLE I1 RELATIONSHIP BETWEEN THE BOOSTER ANTIGEN DOSE AND THE INTENSITY OF THE SECONDARY IMMUNE RESPONSE IN MICE PRIMED WITH CS-HGG BALB/c mice were injected subcutaneously into the flank with 0.2 ml of the CS-HGG suspension containing 100 fig of immobilized protein. Three months later a soluble HGG was administered intravenously and on day 5 AFC were counted in the spleens. HGG dose (~g)

AFC count/106 spleen cells

10 l 0,1 0~

4978 (4517 5486) 2713 (2201-3 345) 1 062 (904-1 249) 10 (7-14)

Spleen AFC count 1 month after the CS-HGG injection in animals which did not receive a booster dose of soluble HGG.

ing there was a n intense, t h o u g h s u b m a x i m a l response.

3.2.3. Effect of the dose of soluble antigen in mice primed with CS-HGG. T a b l e II d e m o n s t r a t e s the

104

r e l a t i o n s h i p b e t w e e n spleen A F C c o u n t s a n d the T

10 `4

103 103

102

102

~ 101 I

I

I

I

I

7

14

30

60

90

Fig. 2. Intensity of the secondary immune response as affected by the interval between priming with CS-HGG and the administration of soluble antigen. BALB/c mice were injected subcutaneously into the flank with 0.2 ml of the CS-HGG suspension containing 100 /Lg of immobilized protein. At different times after priming 10 /~g of soluble HGG were administered intravenously and on day 4 (O) or day 5 (O) the spleens were assayed for AFC. Abscissa: interval (in days) between priming and the booster injection; ordinate: AFC count/106 spleen cells.

I

I

I

I

I

t

I

I

0

1

2

3

4

5

6

7

Fig. 3. Time course of the secondary immune response in mice primed with CS-HGG. BALB/c animals were injected subcutaneously into the flank with 0.2 ml of the CS-HGG suspension containing 100 ~g of immobilized protein. Fifty days later 10 ~g of soluble HGG were administered intravenously and then the spleens were assayed for AFC on different days. Abscissa: days after the booster injection; ordinate: AFC count/106 spleen cells.

165 T A B L E III SPECIFICITY OF SPLEEN A F C IN B A L B / c MICE A D M I N I S T E R E D D I F F E R E N T PROTEINS A F T E R PRIMING WITH CS-HGG Mice were injected subcutaneously into the flank with 0.2 ml of the C S - H G G suspension containing 100 ~g of immobilized protein. One m o n t h later a solution of H G G , B G G or OA was administered intravenously and on day 4 the spleens were assayed for A F C using two types of sensitized SRBC. Protein used

A F C c o u n t / 1 0 6 spleen cells

for boosting

Anti-HGG

Anti-BGG

4587 (3729-5644) 115 (71-188) 90 (65-125)

719 (502-1013) 27 (16-45) 6 (4.2-8.5)

(lO ~g) HGG BGG OA

dose of soluble antigen injected into B A L B / c mice 3 months after priming with C S - H G G . It may be seen that even 0.1 /~g of soluble H G G induced significant antibody production. As a standard booster dose, we used 10 /tg, since some of the mice died immediately u p o n receiving intravenously 100/~g of H G G . 3.2.4. Time course of the secondary immune response to soluble HGG in mice primed with CS-HGG. The curve showing spleen AFC counts at various times after intravenous administration of H G G in B A L B / c mice primed with C S - H G G (Fig. 3) was similar to the usual time course of the primary immune response to SRBC. In both cases A F C

increased exponentially within the first 4 days, a peak was reached on day 4 or 5 and then spleen A F C counts declined rapidly over 3 days. The only difference was that the immune response to H G G was more intense under the above conditions, though practically absent in mice which had not been primed with C S - H G G . Moreover, the great majority of cells responding to H G G formed 7S antibody and could only be detected by the indirect method. 3.2.5. Specificity of the secondary immune response in mice primed with CS-HGG. In our experiments B A L B / c mice primed with C S - H G G were given after 1 month one of the following 3 antigens in solution: H G G , cross-reactive B G G or unrelated OA. For detecting AFC, SRBC were sensitized either with H G G or BGG. As shown in Table III, administration of B G G or OA failed to elicit the appearance of a significant number of cells producing antibody to H G G or BGG. In contrast, the animals injected with H G G were characterized by numerous cells forming antibody to this protein and high anti-BGG AFC counts. Presumably this was a reflection of common antigenic determinants on H G G and B G G molecules.

3.3. Intensity of the secondary immune response as affected by different methods of priming Soluble H G G used for priming via the subcutaneous or intraperitoneal route was a weak immunogen for B A L B / c mice (Table IV). In con-

T A B L E IV I N T E N S I T Y OF T H E S E C O N D A R Y I M M U N E RESPONSE IN B A L B / c MICE AS A F U N C T I O N OF T H E P R I M I N G METHOD Two months after the use of one of the above methods mice were injected intravenously with 10 ~tg of soluble H G G and on day 4 (subcutaneous priming) or day 5 (intraperitoneal priming) A F C were counted. Priming (100 p.g of protein) State of antigen

Route

AFC count/106 spleen cells

HGG FCA a + HGG CS-HGG

Solution Water-in-oil emulsion Complex

Subcutaneous into the flank

16 (9-29) 211 (142-313) 5 282 (4 748-5 875)

HGG FCA + HGG CS + H G G CS-HGG

Solution Water-in-oil emulsion Mixture Complex

Intraperitoneal

112 2 333 93 6 789

a Freund's complete adjuvant.

(52-242) ( 2 1 1 7 - 2 571) (63-138) ( 5 1 0 4 - 9 030)

166

trast, priming with H G G as a protein-cellulose complex resulted in extremely active secondary antibody formation. This effect was only achieved if protein molecules were covalently coupled to cellulose. A solution of H G G mixed with a suspension of reduced dialdehyde cellulose was no more effective for priming the animals than soluble H G G alone. A comparison of immune responses in mice primed with C S - H G G or a mixture of H G G and Freund's complete adjuvant revealed the complex to be markedly superior both by the intraperitoneal and subcutaneous routes. To ascertain the importance of the nature of coupling between cellulose and the protein we also performed an experiment (data not shown) with another complex based on aminobenzyl cellulose and prepared by Gurvich and Kuzovleva's method (1968). Both complexes demonstrated a similar immunogenicity.

3. 4. Comparative intensity of the secondary immune response in B A L B / c and C57BL/6 mice primed with CS-HGG Our immunization schedule proved to be much more effective for B A L B / c mice than for the C57BL/6 strain (Table V). The intensity of the immune response as judged by spleen AFC counts in B A L B / c animals was consistent with the higher levels of antibody detected in their blood.

TABLE V COMPARATIVE INTENSITY OF ANTIBODY FORMATION IN BALB/c A N D C57BL/6 MICE Mice were injected subcutaneously into the flank with 0.2 ml of the CS-HGG suspension containing 100 ~tg of immobilized protein. Fifty days later 10 ,ttg of soluble H G G were administered intravenously; on day 4 the spleens were assayed for AFC and on day 7 the absolute antibody content was estimated in the sera. Strain

AFC count/10 s' spleen cells

Antibody content in sera (/~g/ml)

BALB/c C57BL/6

7415 (6490-8470) 204 (164 254)

2600 230

4. Discussion

Our experimental results indicate that immunization with the protein-cellulose complex was highly effective. This complex was prepared from non-metabolizable cellulose particles with a very large total surface area available for covalent immobilization of antigenic protein molecules. The nature of the covalent coupling seemed to be of little importance for immunization and in experiments with B A L B / c animals we observed similar immunogenicity of protein-cellulose complexes based either on dialdehyde cellulose or aminobenzyl cellulose. The serum of the animals immunized by our method contained up to 5 mg of antibody per ml and this level is comparable to the content of IgG in the serum of non-immunized mice. The bulk of this antibody was produced in the spleen and lymph nodes. The contribution of small granulomas developed subcutaneously at the sites of the injections of the complex, which only contained low AFC levels, was probably insignificant. These data are basically similar to the results obtained several years ago by Askonas and Humphrey (1958) and Askonas and White (1956) using Freund's adjuvant. A comparatively larger role for granulomas in antibody formation described by these authors may possibly be explained by a more pronounced local tissue reaction to Freund's adj uvant. The protein-cellulose complex is easily prepared. Careful attention should be paid to dialysis of the protein against CBB, since the preparation may contain low molecular weight inhibitors of coupling (amino acids, peptides and other substances which will compete with the reaction). The protein:cellulose ratios in the complexes to be used for immunization may vary within a very wide range. The method we have developed for immunizing mice seems to be especially promising when it is necessary to induce an active immune response to a low dose of antigen, in particular since small amounts of protein are, as a rule, coupled almost completely to activated cellulose. In such cases priming may be achieved by multiple injections because this schedule ensures high efficiency of small doses of immobilized antigen.

167

Protein-cellulose complexes will probably be useful for immunization with protein toxins, which are seldom used in their native state, and also for obtaining hybridomas.

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Korukova, A.A., O.S. Grigoryeva and A.E. Gurvich, 1985, Byull. Eksp. Biol. Med. 50, 44. Lechtzind, E.V. and A.E. Gurvich, 1981, Byull. Eksp. Biol. Med. 41, 68. Liashenko, V.A. and A.A. Vorobyev, 1982, The Molecular Foundation of Antigen Immunogenicity (Meditsina Publ., Moscow). Olovnikov, A.M. and A.E. Gurvich, 1966, Nature (London) 209, 417. Orlov, G.E. and A.E. Gurvich, 1969, Probl. Med. Khim. 15, 150. Petrov, R.V., R.M. Khaitov and R.I. Ataullakhanov, 1983, Immunogenetics and Artificial Antigens (Meditsina Publ., Moscow). Szabolcs, O., 1961, Papier 15, 41. Tolles, P. and A. Paraf, 1973, in: Chemical and Biological Basis of Adjuvants (Springer-Verlag, Berlin, Heidelberg, New York). World Health Organization, 1976, Immunological Adjuvants (WHO Scientific Group, Geneva).