Nasal and parenteral immunizations with diphtheria toxoid using monoglyceride/fatty acid lipid suspensions as adjuvants Ulf SchroÈ der*, Stefan B. Svenson Department of Veterinary Bacteriology, Swedish University of Agricultural Sciences, P.O. Box 583, S-751 23 Uppsala, Sweden Received 21 April 1998; received in revised form 24 September 1998; accepted 20 October 1998 Abstract A novel suspension system was developed where monoglycerides were formulated together with fatty acids and subsequently admixed with antigens. In the present study, diphtheria toxoid was used as a model antigen primarily due to its weak immunological properties as well as to its importance as a future human vaccine for mucosal, particularly nasal immunization. The formulations were administered parenterally and/or nasally to mice whereafter the immune response was determined. In the present study, we have shown that mono-olein/oleic acid vesicles enhance the immunogenicity of admixed diphtheria toxoid in mice to the same level as Alum adsorbed (or Freund's complete adjuvant) when administered parenterally or nasally. It was also shown that the immunogenicity was linked to the length of the acyl chain of the lipids, where shorter acyl chains resulted in reduced titers. Furthermore, shorter acyl chains also gave rise to more pronounced toxic reactions at the injections sites, such as necrosis and alopeci, both of which were lacking when the optimal formulation consisting of mono-olein and oleic acid was used. Thus, this lipid matrix has in our view a great potential as an immunological adjuvant with an exceptionally simple and ecient preparation procedure without organic solvents and with low cost endogenous lipid based raw materials. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Adjuvants; Intranasal; Vaccination 1. Introduction Lipid matrices have been evaluated for a long time as vehicles for delivery of biologically active sub- stances. The use of liposomes is now well established with regard to the price of the phospholipids, entrap- ment eciency, production costs and procedures. However, the technique still has problems, and there has been a search for better systems that ful®l the demands regarding biocompatability and biodegrad- ability. Other lipid based systems have been evaluated, such as micelles [1, 2], where signi®cant uptake of nasally administered insulin has been obtained in ani- mal models using mixed micelles made from simple lipids such as sodium glycocholate/linoleic acid [3]. Immunological adjuvants now under evaluation for possible human use are dominated by systems that form particles; i.e. microspheres, liposomes and iscoms. Furthermore, a number of other adjuvants, such as MPL or charged polymers, are most probably forming particles or aggregates when mixed with a protein anti- gen. Almost all particulate systems now under evalu- ation depend on entrapment or adsorption of the antigens to the matrix [4]. One important aspect of the use of liposomes is as immunological adjuvants. As such, the liposomes have been extensively evaluated for over twenty years, partly as substitutes to the alum salts and also as stronger adjuvants to be used together with peptides and other weak antigens. Furthermore, the possibility of mucosally administered antigens has received a great deal of attention, and several papers describe the use of nasal administration of dierent antigens incor- porated into liposomes with encouraging results such as protective immunity in animal models [5, 6]. Also, there are reports on the use of liposomes or lipid sus- pensions where mixtures of nasally administered anti- gens and lipids enhance the immune response [7± 10, 15]. Vaccine 17 (1999) 2096±2103 0264-410X/99/$19.00 # 1999 Elsevier Science Ltd. All rights reserved. PII: S0264-410X(98)00408-3 * Corresponding author. Tel.: +46-70-511-3981; fax: +46-8-445- 3075; e-mail: ulf.schroder@bmc.uu.se.