Archives of Virology

Archives of Virology 55, 275--285 (1977)

© by Springer-Verlag 1977

Potentiation of FMD Vaccines With Polyeationie-Nueleie Acid Complexes By R. F. MAES, A. ~rlEIRA, I, GOMES, P. AUGE DE MELLO, C. BERNAL LOrEZ, and K. COSTA FREITAS Centre Pan-Americano de Febre Aftosa, Rio de Janeiro, Brazil With 4 ]?igures Accepted June 23, 1977

Summary The effect of various polycations on the immune response potentiated with poly I : C was studied. I t was found that low molecular weight polycations had no potentiating effect. Polylysine was ineffective whereas protamine was superior to tysozyme, poly-arginine, poly-histidine, D E A E - D e x t r a n and histone. A foot-and-mouth disease trivalent vaccine composed of strains A24 Cruzeiro, 01 Caseros and C2 Resende elicited no immune response in swine when adjuvanted with aluminium hydroxide but was effective when emulsified in oil. I n genera], the immune response was potentiated ten-fold when the emulsion contained poly I:C. The antibody production was in most cases further potentiated b y a factor of ten when the mlcleic acid double-strand was complexed with i : 10 (w/w) DEAE-Dextran. Protamine was as effective, or perhaps even more, than DEAEDextran to this effect. Guinea pigs vaccinated with a water-in-oil emulsion type monovalent Ca vaccine showed an increase in antibody production when the vaccine contained poly I : C or poly I : C complexed with 1 : 10 (w/w) protamine.

Introduction The effect of polybasic substances on the biologic properties of the nucleic acids to which they are bound (1) is variable. For example D E A E - D e x t r a n enhances the infectivity of viral nucleic acid (2), and potentiates the production of interferon induced b y poly I : C (3, 4). In the latter case, certain combinations of the nucleic acid and polyeation have been shown to be inhibitory (3). Since methylated albumin has been shown (5) to improve the immunologic response obtained after injection of ribonuclcotide homopolymers, it was deemed useful to pursue such investigations. The aim of our experiments was to assess the influence of

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various polycations on the p o t e n t i a t i o n of the i m m u n e m e c h a n i s m o b t a i n e d t h r o u g h poly I : C so as to be able to e v e n t u a l l y select a p o l y e a t i o n of reduced toxicity a n d i m m u n o g e n i e i t y . F o o t - a n d - m o u t h disease virus (FMDV) is a weak a n t i g e n for swine (6). Vaccines a d j u v a n t e d with a l u m i n i u m hydroxide are n o t very effective in elieiting an i m m u n e response, while water-in-oil emulsion type vaccines elicit a n i m m u n e response b u t have sometimes the side effect of producing u n c o n t r o l l a b l e sterile abscesses a t the site of inoculation. The i m p r o v e m e n t of ~he q u a l i t y of F M D V vaccines for this a n i m a l species is therefore desirable. This p r o m p t e d us to investigate the p o t e n t i a l practical applications of the p o t e n t i a t i o n b y polybasie substances of the i m m u n e response b y e x a m i n i n g their effects in swine v a c c i n a t e d with F M D V when the a n t i g e n is a d j u v a n t e d either b y A1 (OI-I)a or b y incorporation into a water-in-oil emulsion.

Materials and Methods

WgT'use8

Virus O1 Caseros (7 passages in BItK21/13 cells), A24 Cruzeiro (5 passages) and C:~ t~esende (6 passages) were inactivated with 0.05 per cent Acetyl-ethylene-imine (AEI) (Dow Chemical) at 37 ° C (7). The initial infectious titres in suckling mice of the three virus types were i07-1/ml for virus O1, 107-3/mt for virus A~4 and 10s-°/ml for virus Ca. The complement fixation (CF) titres of the inactivated viruses were 1 : 20 for virus A24 and 1 : 25 for viruses Cs and O1. Vaccines

Aluminium Hydroxide Adjuvant For the preparation of the A1 (OI-t)~ vaccine, the three viral suspensions were mixed together in equal parts and the mixture later added to an equal volume of an A1 (OH)a suspension. The final concentration of the ahuninium hydroxide was equivalent to 2.0 per cent (g/100 ml) of dried aluminium oxide. Water-in-Oil Emulsion Type Vaccine For the emulsion type vaccine, the trivalent viral suspension was mixed in equal parts with light mineral oil containing 10 per cent arlaeel and emulsified in a blender. Chemicals

Protamine free base, grade IV; Spermine free base; poly-l-lysine.HBr, type I (tool. wt. 195,000) ; /cIarmine.HC1 ; Neomycine sulfate; Lysozyme, egg white, grade I (tool. wt. 14,000); poly-l-ornithine.HBr (tool. wt. 100,000); histone type II~A from calf thymus; poly-eytidylic acid (C), poly-inosinic acid (I), poly-adenylic acid (A) and poly-uridylie acid (U), were purchased from Sigma (Milwaukee). I-Iexadimethrine bromide (Polybrene) was a gift from Abbott Laboratories; poly-l-arginine (tool. wt. 15,000) was purchased from NBC and DEAE-Dextran (mol. wt. 2 × t06) was purchased from Pharmacia. Complex ~ o r m a t i o n

The individual polymers were solubilized in a solution of 0.15 molar NaC1, 0.02 molar Tris.ttC1 pI-I 7.2 and 1.2 × 10 -s molar EDTA. Complex formation and double-strand formation were performed as described previously (3). Antibody Assays

Serum protection (SP) tests in suckling mice were performed according to the technique described by C u ~ I ~ et al. (8).

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Mouse Vaccination Inactivated type A~4 strain Cruzeiro was inoculated intraperitonealty (0.2 or 0.4 ml). Five mice were used per sample and time. The mice were bled through decapitation and a serum-neutralization test performed on the pooled serum: serial two-fold dilutions of the antiserum were made in Eagle's medium without serum, to which 500 TCDs0/mt of homologous virus were added. After one hour incubation at 37 ° C, 0.2 ml of the mixture were inoculated into tissue culture tubes containing B H K cells. Six tubes were inoculated per dilution. After 7"2 hours incubation at 37 o C, the tubes containing destroyed cells were recorded, and the dilution of serum where 50 per cent viral inhibition would have occurred was calculated. Virus C~ t~esende was adjuvanted with A1 (OH)a before inoculation. Guinea Pig Vaccination A monovalent C water-in-oil emulsion type vaccine was inoculated subcutaneously (0.05 to 0.2 mI) into guinea pigs (6 animals per sample). P o l y I : C and Poly I :C complexed with 1:10 protamine (w/w) were incorporated in the vaccines at a concentration of 100 ~g/ml. Blood was obtained through heart puncture on the 28th day after vaccination (DPV) and antibody titres measured as described. Swine Landrace crossbred swine, 3 - - 4 months old, weighing 30--35 kilos at the beginning of the experiment, were used, Five groups of 4 swine were inoculated with 2 ml and 6 ml doses of the water-in-oil emulsion type vaccine containing the different potentiators. Four groups of 4 swine were inoculated with 2 ml and 6 ml doses of the A1 (OH)3 type vaccine containing the different potentiators. Blood samples withdrawn at day 0 and 30 after inoculation were assayed for neutralizing antibodies. For the analysis of the secondary immune response of swine, previously immunized animals (8 per group) were inoculated with a 6 ml dose of a monovalent virus type C8 oil vaccine. The vaccine contained 100 ~g/ml poly (I:C). Blood was withdrawn every month after inoculation.

Results Mouse I m m u n i s a t i o n Effect of Various D E A E - D e x t r a n - P o l y I : C Complexes As shown in a n earlier p u b l i c a t i o n (3), the mode of complexing of the polycation with the nucleic-acid plays a n i m p o r t a n t role in the biological effectiveness of the preparation. One can either first form a nucleic acid d o u b l e - s t r a n d a n d thereafter b i n d it to D E A E - D e x t r a n , or else one m a y first b i n d each nucleic acid s t r a n d to the p o l y e a t i o n a n d only thereafter form the nucleic acid double-strand. Virus type A24 (CF: 1 : 12) i n a c t i v a t e d with A E I , was inoculated i n t r a p e r i t o n e ally i n mice. The viral suspension was s u p p l e m e n t e d with t : C (20 ~xg/0.2 ml/mouse) which was complexed either with 1 : 20 (w/w) D E A E - D e x t r a n after d o u b l e - s t r a n d f o r m a t i o n (I : C), or with a n equal a m o u n t of D E A E - D e x t r a n (w/w) before doubles t r a n d f o r m a t i o n (I) : (C) (3). Controls composed of the viral suspension c o n t a i n i n g 20 ~zg D E A E - D e x t r a n / 0 . 2 ml were inoculated likewise. F i g u r e 1 shows t h a t only the (I :C) c o m b i n a t i o n induced the f o r m a t i o n of r e l a t i v e l y large a m o u n t s of antibodies. P o l y (I):(C) a n d poly I :C alone had no a d j u v a n t effect a n d D E A E - D e x t r a n alone appeared to be ineffective or perhaps even inhibitory. This lack of effect will be confirmed in experiments with pigs.

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Fig. l. l~]ffeet of different poly I :C + DEAE-Dcxtran combinations on neutralizing antibodies production in mice - - • virus A~4, alone in aqueous solution = control; = + - - - ~ 20 ~xg DEAEDextran/mouse; • . . . . . • 20 ~g I: C/mouse; • . . . . . . . • 20 ~g I : C plus 20 ~g DEAEDextran, added to the nucleic acid polymers before double-strand formation; o o 20 Izg I :C plus 1 ~g DEAE-I)extran, added after double-strand formation •

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Effect of Different Polycations Having thus established that I : C coated with a polycation after double-strand formation is potentiating the production of neutrMizing antibodies, we tried to ascertain the effect of various other polyeations in the same system. In order to evaluate more easily the quality of the inducers, the antigenic mass of the protein was lowered (CF: 1 : 7) and the amount of I : C inoculated was reduced by half. Virus A24 was inoculated intraperitoneally in 0.2 ml doses. The vaccine alone or injected together with (I:C) plus spermine or A : U plus DEAE-Dextran produced no detectable response at all under conditions of low antigenic mass (not shown in Fig. 2). A response was observed however when the vaccine dose contained 10 Exg I : C coated with 0.5 ptg of various polycations. Figure 2 shows that protamine, poly-l-ornithine and lysozyme were better than the other polyeations tested, with large amounts of antibodies being produced on day 28 post inoculation. Production of antibodies on day 4 was noticed also. This biphasic response is characteristic of IgM-antibodies produced on day 4 and IgG antibodies appearing at later times. DEAE-Dextran had an effect similar to historic, whereas polybrene seemed to induce the production of antibodies on day 4 only. Polylysine, harmine and neomycine triggered likewise a small antigenic response appearing on the 4th day after inoculation. They are omitted from the figure. Effect of Protamine Alone The stimulating effect of protamine alone on the mouse immune mechanism was also examined. For this purpose, virus C3 Resende was mixed with aluminium hydroxide and this vaccine (CF: 1 : 45) was supplemented with 10 ~xg poly I : C plus 1 i~g protamine or l0 ~xg protamine alone. I t can be seen in Figure 3 that protamine alone added to an aluminium hydroxide vaccine has a stimulating

Potentiation

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F i g . 2. E f f e c t o n t h e a n t i b o d y p r o d u c t i o n of d i f f e r e n t p o l y c a t i o n s c o m p l e x e d w i t h I : C. F i v e m i c e w e r e i n o c u l a t e d i n t r a p e r i t o n e a l l y p e r t i m e w i t h v i r u s Ae4 i n t h e p r e s e n c e of 10 F-g I :C c o a t e d w i t h 0.5 F g p o l y e a t i o n a f t e r d o u b l e - s t r a n d f o r m a t i o n . N o r e s p o n s e was detectable when the virus alone was inoculated (not shown in the Figure) Left: • ....... • Polybrene, •• Lysozyme, • ..... t Protamine; Right: • ..... • His~one, A • DEAE-Dextran, • ........ • Poly-l-ornithine

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280

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effect on t h e i m m u n e m e c h a n i s m reminiscent of the one o b s e r v e d when D E A E D e x t r a n is used as a p o t e n t i a t o r (9). P o l y I : C plus p r o t a m i n e gave, as noticed previously, a b u r s t of a n t i b o d i e s on d a y 4, a n d a g a i n on d a y 28 a s s u m e d to be r e s p e c t i v e l y I g M a n d I g G responses. Guinea Pig t m m u n i s a t i o n The p o t e n t i a t i o n of t h e i m m u n e response w i t h a p o l y (I : C)-protamine complex was f u r t h e r e s t a b l i s h e d in guinea pigs. A m o n o v a l e n t water-in-off emulsion t y p e Ca vaccine was p r e p a r e d . The vaccine was s u p p l e m e n t e d before emulsification w i t h either 100 ~g/ml p o l y I : C o1" 100 ~ g / m l p o l y ( I : C ) - p r o t a m i n e . N o n - s u p p l e m e n t e d vaccine was used as control. The vaccines were i n o c u l a t e d in doses ranging f r o m 0.05 ml to 0.2 ml in six a n i m a l s per dose. Table 1 r e p o r t s t h e results o b t a i n e d . I t can be seen t h a t the control vaccine was consisting]y good a t a dose of 0.2 ml. This dose could be r e d u c e d to 0.1 ml when 10 ~g of p o l y I : C are included in the vaccine dose. A lowering of t h e dose to 0.05 ml a n d 5 ~g I : C / a n i m a l d i d n o t allow a clear cut d i s c r i m i n a t i o n b e t w e e n the vaccine a d j u v a n t e d b y p o l y I : C a n d the one a d j u v a n t e d b y p o l y (I:C) plus p r o t a m i n e . B o t h p r o d u c e d v e r y similar results. The I : C plus p r o t a m i n e p r e p a r a tion was s l i g h t l y more effective since the serum p r o t e c t i o n indices for this group were all g r e a t e r t h a n 2.16. Table 1. Serum protection indices (loglo) 30 days a/ter primary vaccination o/guinea pigs with various doses o] a virus Ca water-in-oil emulsion type vaccine Amount

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2.23 2.23 4.23 2.23 2.57 0.0

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3.90 3.57 3.73 3.50 3.66 3.93

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>2.16 3.50 >2.16 2.93 2.33 2.66

3.16 > 4 . t6 2.50 3.83 4.16 1.93

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Antibody Production in Swine The i m m u n e response of swine u p o n a d m i n i s t r a t i o n of F M D vaccine varies c o n s i d e r a b l y on a n i n d i v i d u a l basis. Since only a l i m i t e d n u m b e r of a n i m a l s was

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available for e x p e r i m e n t a t i o n , a n d being restrained also i n a practical w a y in the n u m b e r of a n i m a l s t h a t could be analyzed, a choice in the e x p e r i m e n t a l design applied h a d to be made. To o b t a i n a n overall picture t h a t was representative of the effects expected, we used three types of vaccine a n d inoculated t h e m a t two different doses. A n i m a l s were bled at d a y 0, 15, 30 a n d 60 after inoculation. The results o b t a i n e d were all i n t e r n a l l y consistent a n d only those o b t a i n e d on d a y 30 are shown. The i n d i v i d u a l values o b t a i n e d are t a b u l a t e d , allowing the reader a b e t t e r e v a l u a t i o n of the effects observed. Studies W i t h I : C Plus D E A E - D e x t r a n A l m n i n i u m hydroxide vaccines: Antibodies were n o t detected u p to 60 days post i n o c u l a t i o n (I)PI) i n swine inoculated with a vaccine composed of the a n t i g e n mixed with a l u m i n i u m hydroxide. The a d d i t i o n of I : C (600 ~g/vaceine dose), regardless whether complexed with D E A E - D e x t r a n or not, did n o t produce a n y response. Vv~ater-in-oil emulsion vaccines: W h e n the antigens -were incorporated into a water-in-oil emulsion, t h e y induced the f o r m a t i o n of antibodies (Table 2). A n t i g e n A24 was good, a n t i g e n 01

Table 2. Serum protection indices obtained 30 days a/ter primary vaccination o] swine with a trivalent water-in-oil emulsion type vaccine. The indices obtained on day 0 were substracted ]rom those obtained on day 30, except/or vaccine A~4 2 ml dose Vaccine Ca

Vaccine Ol

Vaccine A24

Vaccine Vaccine C~ 01

0 0.85 0 0

1.96 0.87 ~