SEPPIC Vaccine Adjuvants for Poultry

L. DUPUIS, S. ASCARATEIL, J. AUCOUTURIER, AND V. GANNE

SEPPIC, 75321 Paris, Cedex 07, France

ABSTRACT: Two inactivated antigens (Newcastle and Pasteurella Mul-tocida) were formulated with different adjuvants and tested in two sep-arate experiments in poultry. Oil formulations constituting water in oil(W/O) or water in oil in water (W/O/W) emulsions were assessed for anti-body response, protection, local reactions, and vaccine physicochemicalparameters. Robust, efficacious, and safe formulations were obtainedwith W/O formulations whereas W/O/W was especially safe with main-tained efficacy. Results show that it is possible to improve traditionalTween Span formulations for safety and efficacy parameters by usingMontanideTM ISA 70 for W/O formulations and MontanideTM ISA 206for W/O/W when safety is the priority.

KEYWORDS: adjuvants; poultry vaccine; Montanide

INTRODUCTION

Inactivated antigens require an oily formulation for the production of effi-cacious vaccines. Two different animal experiments were carried out to testseveral oily adjuvants for poultry vaccines. Vaccine efficacy, safety, and for-mulation robustness were compared.

MATERIAL AND METHODS

Vaccination Experiment 1

ISA Brown layers aged 16 weeks and maintained under conventional con-ditions at the Ecole Nationale Veterinaire de Nantes (France) were inoculatedwith different vaccines. Antigen included in vaccinal preparations resultedfrom inactivation of a Pasteurella multocida strain (serotype 3) (PMS3). Pro-tection and safety were assessed.

Address for correspondence: Laurent Dupuis, SEPPIC, 75 quai d’Orsay, 75321 Paris, Cedex 07,France. Voice: +33-0-1-40-62-53-45; fax: +33-0-1-40-62-52-53.

email: laurent.dupuis@airliquide.com

Ann. N.Y. Acad. Sci. 1081: 202–205 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1373.024

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Vaccination Experiment 2

Five hundred SPF Leghorn chicks distributed in different cages were in-oculated with different vaccine formulations or different vaccine doses. Theantigen is an inactivated Newcastle Disease (ND) virus. Four-week-old chickswere vaccinated intramuscularly with either complete 0.5 mL doses or 1/25th,1/50th, or 1/100th fractional doses using micro-syringes. Three weeks aftervaccination, all of the vaccinated chicks and 10 SPF nonvaccinated controlsubjects were inoculated with 106 LD 50 of a virulent ND virus strain via a0.5 mL intramuscular injection on the left side of the breastbone. Protectionand safety were assessed.

Adjuvants

A classical Tween Span mineral oil water in oil (W/O) emulsion was com-pared with different SEPPIC adjuvants (TABLE 1). W/O emulsions were pre-pared under high shear (rotor stator equipment). One step process water in oilin water (W/O/W) emulsion was prepared under low shear.

RESULTS

Experiment 1 (PMS3)

Antibody titers after 28 and 56 days were similar for the three W/O formu-lations in intensity (7000) and significantly higher than the control (FIG. 1).Local reactions were detected at all injection sites for Tween Span formulationswhereas none were found in either the MontanideTM (Paris, France) ISA 70and ISA 775 adjuvant groups or with the control antigen alone (FIG. 2).

Experiment 2 (ND)

Approximately 100% protection was obtained for 0.5 mL doses whateverthe adjuvant system (FIG. 3). When the vaccine dose was reduced to 1/25th of

TABLE 1. MONTANIDETM SEPPIC adjuvants tested in poultry vaccines

ADJ/ANTIG Viscosity

MONTANIDETM Oil (w/w) Emulsion (mPa.s)

ISA 70 MINERAL 70/30 W/O 30ISA 775 MINERAL + 70/30 W/O 25

NONMINERALISA 206 MINERAL 50/50 W/O/W 10

204 ANNALS NEW YORK ACADEMY OF SCIENCES

FIGURE 1. PMS3 vaccine antibody titers.

the dose, protection with ISA 70 remained 100% but dropped down to 0% forISA 206 and to 40% for Tween Span formulation. ISA 70 conserved 100%protection for 1/50 and 1/100 volume doses, whereas ISA 206 and Tween Spanno longer induced a protection. No local reactions were observed with any ofthe vaccines.

FIGURE 2. PMS3 vaccine local reactions.

DUPUIS et al.: SEPPIC VACCINE ADJUVANTS FOR POULTRY 205

FIGURE 3. ND vaccine dose effect.

DISCUSSION AND CONCLUSION

Different oily adjuvants were compared with a bacterial and viral antigen inpoultry. Efficacy and safety were assessed and compared to classic water inmineral oil Tween Span formulation. The bacterial antigen, more immunogenicbut also reactogenic, proved that a selected W/O formulation could maintainthe same level of antibody response with disappearance of local reactions. Fora conventional viral antigen (ND), the same formulation proved its superiorityby keeping the same protection level even when the dose volume is reducedby 100 times. In this experiment, a W/O/W formulation was tested and gavea similar protection to the reference with the advantage of safety linked toaqueous fluid formulations. Furthermore, aqueous formulations can be usedto dilute lyophilized live vaccines and are easy to inject. High tech adjuvants(for W/O or W/O/W) can help to reformulate existing vaccines with a cost ef-fectiveness (reduction of the antigen load or vaccinal dose), a safety efficiency(when antigens are reactogenic or for sensitive animals), and an outstandingefficacy when a strong protection is needed. The possibility of adjusting ad-juvant formulation according to antigens for efficacy, safety, and stability ofthe vaccine even for high antigen ratio and stability disturbing antigens is aspecificity of the MontanideTM range.