Key words: foot-and-mouth disease; single emulsion vaccine, saponin, immune response, emergency vaccines.

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1 255 Appendix 32 Enhancement of the immune response induced by the inclusion of saponin in oil adjuvanted vaccines against foot-and-mouth disease E. Smitsaart (1), N. Mattion (1) ; J.L. Filippi (1) ; B. Robiolo (2) ; O. Periolo (2) ; J. La Torre (2) and R.C. Bellinzoni (1) (1) Biogenesis S.A. Ruta Panamericana km 38,2. (B1619IEA) Garín. (Buenos Aires). esmitsaart@biogenesis.com.ar. (2) CEVAN- CONICET. Serrano 669, (1414 ) Buenos Aires. (Argentina) ABSTRACT The immunogenicity of oil formulations that include saponin as an immunomodulator in cattle, swine and goats was evaluated. The specific immune response was examined by the detection of total antibodies (Ab) against each one of the viral strains in the vaccine by Liquid-Phase ELISA. The titres of Ab were related to the Expected Protection Percentage (EPP) and in each experimental group the proportion of individuals with Ab levels compatible with protection (EPP>81%) was determined. The specific titres of IgG1 and IgG2 isotypes in vaccinated cattle were measured by ELISA. The induction of Ab anti-viaa and against the polymerase 3D was evaluated by agar gel immunodiffusion and ELISA, respectively. The results indicate that oil vaccines with saponin induced 10 times higher total specific Ab levels, for each one of the viral strains at 30 days postvaccination (DPV) than formulations lacking saponin. In addition, the inclusion of this immunomodulator in formulations with reduced antigen payload, elicited at 30 DPV, significantly higher total antibody protection levels (P<0.005) than those obtained with formulations with high 140S antigen payload, and absence of the immunomodulator. The IgG isotype profiles induced in cattle by these vaccines indicated that both IgG1 and IgG2 were higher in formulations with saponin. In addition, these formulations did not elicited antibodies to non structural proteins in vaccinated animals at 30 DPV. High antibody levels were also induced in pigs and goats after vaccination with vaccines containing saponin. Furthermore, high and persistent immune response induced by this formulation in calves with maternal antibodies was also detected. No adverse effects were shown in the vaccinated animals of any species. The adjuvant effect of the formulations containing saponin with no adverse reactions suggests its use as alternative for emergency vaccination and for prophylactic campaigns. Key words: foot-and-mouth disease; single emulsion vaccine, saponin, immune response, emergency vaccines.

2 256 INTRODUCTION Either for prophylactic campaigns or for emergency vaccination, immunogens of rapid, persistent and of high potency capable to be applied in all susceptible species are required. Saponin has been extensively used incorporated into the aluminum hydroxide adjuvant for immunization of susceptible species (1). However, it is well known that these vaccines are ineffective in inducing adequate immunity in pigs (2). Oil based vaccines have proved to give a longer duration of immunity, negligible interference with maternal antibodies and ability to induce early protection both in cattle and pigs (3,4,5). The aim of this study was to evaluate the efficacy of the saponin incorporated into the aqueous phase of water-in-oil single emulsion oil adjuvanted vaccines as possible candidates for emergency or prophylactic vaccines. Both tolerance and immune response following vaccination of cattle, pigs and goats were examined. The immune response was evaluated by assessing both total and subclass IgG specific antibodies to FMDV. MATERIALS AND METHODS Animals: One hundred and nine (109) Hereford breed cattle aged months free of FMD antibodies were used. They belonged to Patagonia region (south paralell 42 ) of Argentina. Twenty-two Duroc Jersey 60-day-old pigs and thirty (30) 30-day-old goats free of Ab of FMDV were also used. Thirty-five (35) calves born from vaccinated dams aged 30 to 90 days were also used. Five non-vaccinated animals served as controls on each experiment. Animals were kept during the whole experiment under grazing conditions on controlled pens. Vaccines and vaccination: FMDV antigen of each of the four FMDV vaccine strains (O1 Caseros, A Argentina A79, A Argentina 87 and C3 Argentina 85) were propagated in BHK21 cell suspension cultures and inactivated with binary etilenimine. Antigen were concentrated and partially purified with 7% (w/v) polyethylene glycol Vaccines were prepared as water-in-oil single emulsion. Vaccines differed in their antigen and saponin content, both components included in the aqueous phase of the oil vaccines (Table 1). Vaccines were prepared individually for each trial. All animals were immunized by the intramuscular route with 2.0 ml dose, except goats vaccinated with vaccine N that received 1.0 ml dose. Detection of total antibodies (Ab) against FMDV: The Ab titres against each one of the viral strains in the vaccine was examined by Liquid-Phase ELISA (6). The titres of Ab were related to the Expected Protection Percentage (EPP) according to the Logist Transformation (7). In addition, the proportion of individuals with Ab levels compatible with protection (EPP>81%) was determined in each experimental group. Detection of IgG1 e IgG2 isotypes against FMDV in vaccinated cattle: IgG1 and IgG2 Ab titres against FMDV O strain were examined on serum samples from cattle of Trial 2 according to the method previously described (8). Detection of antibodies against VIAA and 3D protein: VIAA Ab and anti-3d Ab were examined on serum samples collected at 35 DPV from cattle of Trial 2 and from pigs at 30 DPV. Both assays were performed at Virology Institute (CICVyA-INTA) (9,10).

3 257 RESULTS Immune response in vaccinated animals: Trial 1: Mean group Ab titres against each of the four FMDV strains induced by vaccine containing saponin (vaccines B, C, E, F, G ) were significantly higher (t student P<0.005) than those induced by vaccines lacking immunomodulator (vaccines A and D ). Vaccines with saponin induced an early immune response at 30 DPV with Ab levels equivalent to that obtained at 60 DPV with formulations lacking saponin. Furthermore, the inclusion of this immunomodulator in formulations with antigen payload of 9.7 µg/dose, induced Ab levels higher than those obtained with vaccines including 4 times higher 140S in absence of saponin (data not shown). The percentage of cattle with protected Ab is shown in Fig 1. Vaccines containing saponin conferred Ab titres compatible with protection at 30 DPV in the % of cattle, whereas those lacking saponin induced protected Ab levels in the 45-55% of the vaccinated cattle. Trial 2: the percentage of cattle with protective Ab titres is shown in Fig 2. At 35 DPV 100% of the cattle vaccinated with vaccines including saponin ( H and I ) achieved protective Ab, whereas 25-75% of the cattle vaccinated with vaccine lacking saponin ( J ) achieved protective Ab titres even in the presence of higher antigen mass content. Trial 3-4: High immune response was also induced by these formulations in pigs and goats (Table 2). Trial 5:Vaccines containing saponin induced positive immune response in calves in the presence of high levels of maternal antibodies (Fig 3). Calves vaccinated at 30 days of age showed at 150 DPV higher Ab titres than non-vaccinated controls. Percentage of calves with protective titres was higher than 80% at 150 DPV, regardless the age at which vaccination was applied (data not shown). Detection of IgG1 e IgG2 isotypes against FMDV in vaccinated cattle: IgG1 and IgG2 Ab titres against FMDV O type are shown in Table 3. High levels of IgG1 and IgG2 were induced by vaccines containing saponin, whereas low levels of both isotypes were elicited by vaccine lacking saponin in agreement with the titres of total Ab detected. Vaccine containing saponin and higher antigen payload (vaccine H ) induced higher IgG2 than that with lower 140S content (vaccine I ). Detection of antibodies against VIAA and 3D protein: Non detectable Ab against VIAA and 3D protein were detected in the serum samples assayed. DISCUSSION The immunomodulator effect of saponin included in the water phase of water-in-oil vaccines was evaluated in cattle, pigs and goats. Oil vaccines with saponin induced 10 times higher total specific Ab levels, for each one of the viral strains at 30 DPV than formulations lacking saponin. In addition, the inclusion of this immunomodulator in formulations with reduced antigen payload, elicited at 30 DPV, significantly higher total antibody protection levels (P<0.005) than those obtained with formulations with high 140S antigen payload, and absence of the immunomodulator. Moreover, the immune response obtained at 30 DPV with vaccines containing saponin was as high as that achieved at 60 DPV when vaccines lacking saponin were applied. No effect on the Ab response was detected with increasing amount of saponin per dose (2, 3 or 6 mg/dose) (Fig 1). It remains to be investigated the immunomodulator effect by using lower amount of saponin per dose. The IgG isotype profiles induced in cattle by these vaccines indicated that both IgG1 and IgG2 Ab titres were higher in formulations with saponin. Both isotypes were also induced in high levels by oil vaccines including other immunomodulators (Mycobacterium sp and Avridine ) (12).

4 258 Correlation between high levels of IgG1 and protection against challenge has been previously demonstrated in cattle (8). The superiority of oil vaccines in conferring neutralizing Ab and protection against challenge in pigs has been well documented (2) Several authors have demonstrated significant correlation between Ab detected at 30 DPV and protection against challenge in pigs (11, 13, 14). In Trial 3, at 30 DPV high Ab levels were detected. However, no differences were observed between vaccines lacking and not the immunomodulator, probably due to the high antigen content into both experimental vaccines. The immune response in goats with these formulations was evident without causing untoward side effects. Furthermore, the Ab response was persistent, at 6 months post-vaccination Ab titres against each of the four vaccine strains were related to EPP above 93% (data not shown). The results obtaining in calves (Trial 5) added more evidence to the advantages of oil vaccines in conferring persisted and high immunity in the presence of maternal antibodies. None of the vaccines used caused neither general nor local untoward side effects. It is also desirable that immunogens to be used in prophylactic or emergency vaccination lack of antigenic components that could induce Ab against non-structural proteins that serve as infection indicators (10,15,16). The non-detectable Ab to 3D and VIAA suggests that even with vaccines containing high antigen payload, the concentration antigen process did not lead to non-structural protein concentration. The adjuvant effect of the formulations containing saponin without causing adverse reactions recommends its use as an alternative for emergency vaccination and for prophylactic campaigns as universal vaccine. Acknowledgements: The authors are grateful to Dr. D.Abaracón for his valuable suggestions and support and to M. Iglesias, C. Devicenzo and M. Rodríguez (CEVAN) for their technical assistance. REFERENCES 1-Barei, S et al. (1979). ZblVetB, 26: Cunliffe & Graves (1963) Can J Comp Med, 27: Doel, T R Rev Sci Tech Off Int Epiz. 1996; 15 (3): Salt, J; Barnett,P; Dani,P et al. (1998) Vaccine 7: Späth, E J A., Smitsaart, E N, Casaro, A P E et al. (1995) Vaccine 13, Robiolo, B, Grigera, P, Periolo, O, et al. (1995) Vaccine14: SENASA. Resolución 219/95, Boletín Oficial N 28125, Disposición Oficial N 03/96. 8-Capozzo, A V, Periolo, O, Robiolo, B et al. (1997) Vaccine 15: Alonso Fernández, A; Sondhal, et al. (1984) Serie de Manuales Técnicos N 6 CPFA, Río de Janeiro, OPS, OMS, O Donnell, V K; Boyle, D; Sproat, K et al. (1996) J Vet Diagn Invest 8: Augé de Mello, P; Gomes, I; AFernandez, A et al. (1978) Bltn Centro Panamericano Fiebre Aftosa Pérez Filgueira, D M, Wigdorovitz, A, Zamorano, P I et al. (1999) Vaccine 17: Anderson, E C; Masters, R C y Mowat, G N (1971) Res Vet Sci, 12: Mc Kercher, P y Bacharach, H L (1976) Can J Comp Med 40: Garland, AM In: Report of the 32nd Sess of the European Commission for the Control of FMD April Mackay, D, Forsyth, M et al. (1998) Vaccine 5:

5 259 Tables and Figures Table 1a: Animals free of FMDV antibodies. Experimental design and vaccines used. Trial Species Group N 1 Vaccine 140S 2 Saponine 3 Bleedings (DPV) 4 1 Cattle I 9 A 38.8 no 0, 30, 60 II 15 B III 9 C IV 9 D 9.7 no V 15 E VI 14 F VII 9 G Cattle I 10 H , 35 II 10 I 15 3 III 8 J 40 no 3 Pigs I 10 K 38 no 0, 30, 60 II 12 L Goats I 10 J 40 no 0, 30 II 10 M , 30, 60 III 10 N , 30, 60 Table 1b: Calves born from vaccinated dams. Experimental design and vaccine used. Trial Species Age group* N 1 Vaccine 140S 2 Saponine 3 Bleedings (DPV) 4 5 Calves 30 days 10 O , 30, 60, 90, 120, days 10 O days 10 O Number of animals in each group. 2- Antigen payload (µg of 140S particles/dose of the mixture of the 4 FMDV strains (O1 Caseros, A79, A87 y C85). 3-Saponin content (mg/dose). 4- Days post-vaccination. * Five day-old calves remained non-vaccinated as control of decay of maternal antibodies. Table 2: Immune response in pigs and goats following vaccination with polyvalent oil vaccines containing saponin Trial Vaccine 140S 1 Sapo- ELISA titres (log 10 ) %n 3 EPP 4 (species) (ug/ds) nin K (pigs) L J ND 100 ND 96 ND (goats) M N , Antigen payload (µg of 140S particles/dose of the mixture of the 4 FMDV strains (O1 Caseros, A79, A87 and C85). 2-Saponin content (mg/dose). 3- Percentage of animals with protective titres (for A79 strain log10> 1,74) 4- Expected Protection Percentage (EPP) 5- Days post-vaccination. ND: not done

6 260 Table 3: IgG1 and IgG2 isotype Ab titres against O type following vaccination with polyvalent oil vaccines containing saponin (Trial 2) Vac- 140S 1 Sapo ELISA titres (log 10 ) 3 IgG1 (log 10 ) 4 IgG2 (log 10 ) 5 cine (ug/ds) nin H 30 3 mg < I 15 3 mg < J < Antigen payload (µg of 140S particles/dose of the mixture of the 4 FMDV strains (O1 Caseros, A79, A87 and C85). 2-Saponin content (mg/dose) 3-Total Ab titres against O type. 4-IgG1 Ab titres against O type 5- IgG2 Ab titres against O type. 6-Days post-vaccination. Figure 1: Percentage of cattle with protective Ab titres following vaccination with oil vaccines. A y D : vaccines lacking saponin. B, C, E, F and G : vaccines including saponin. A, B, and C : vaccines containing 38,8 µg 140S /dose. D, E, F and G : vaccines containing 9.7 µg 140S/ dose. PERCENTAGE OF PROTECTED CATTLE-FMDV-A A B C D E F G 0 30DPV 60DPV

7 261 Figure 2: Percentage of cattle with Ab titres compatible with protection following vaccination (35 DPV) with oil vaccines containing and lacking saponin (Trial 2). PERCENTAGE OF PROTECTED CATTLE ug140s/dose- Sap 3 mg/dose 15 ug 140S/dose- Sap 3 mg/dose 40 ug 140S/dose- No sap A79 A87 O1 C85

8 262 Figure 3: Immune response of calves with maternal antibodies after vaccination. Polyvalent oil vaccine with saponin. Line indicates ELISA antibody titre related with 81% of protection after challenge (log ) 30 DAY-OLD CALVES ELISA TITERS (LOG 10) O1 C FMDV T DAYS POST-VACCINATION VACCINATED NON VACCINATED ELISA TITERS (LOG 10) O1 C FMDV 3,50 3,00 2,50 2,00 1,50 1,00 60 DAY-OLD CALVES T DAYS POST-VACCINATION VACCINATED NON VACCINATED 90 DAY-OLD CALVES ELISA TITERS (LOG 10) O1C FMDV 3,50 3,00 2,50 2,00 1,50 1,00 T DAYS POST-VACCINATION VACCINATED NON VACCINATED

Appendix 53 Addition of saponin to double oil emulsion FMD vaccines enhances specific antibody responses in cattle and pigs Introduction:

Appendix 53 Addition of saponin to double oil emulsion FMD vaccines enhances specific antibody responses in cattle and pigs Smitsaart E* 1, Espinoza AM 1, Sanguinetti R 2, Filippi J 1, Ham A 1, Bellinzoni