RESEARCH PAPER Sytematic Investigation of the Role of Surfactant Composition and Choice of oil: Design of a Nanoemulsion-Based Adjuvant Inducing Concomitant Humoral and CD4 + T-Cell Responses Signe Tandrup Schmidt 1,2 & Malene Aaby Neustrup 1 & Stine Harloff-Helleberg 1 & Karen Smith Korsholm 2 & Thomas Rades 1 & Peter Andersen 2 & Dennis Christensen 2 & Camilla Foged 1 Received: 11 November 2016 /Accepted: 11 May 2017 # Springer Science+Business Media New York 2017 ABSTRACT Purpose Induction of cell-mediated immune (CMI) responses is crucial for vaccine-mediated protection against difficult vac- cine targets, e.g., Chlamydia trachomatis (Ct). Adjuvants are included in subunit vaccines to potentiate immune responses, but many marketed adjuvants stimulate predominantly hu- moral immune responses. Therefore, there is an unmet med- ical need for new adjuvants, which potentiate humoral and CMI responses. The purpose was to design an oil-in-water nanoemulsion adjuvant containing a synthetic CMI- inducing mycobacterial monomycoloyl glycerol (MMG) ana- logue to concomitantly induce humoral and CMI responses. Methods The influence of emulsion composition was analyzed using a systematic approach. Three factors were varied: i) satu- ration of the oil phase, ii) type and saturation of the applied surfactant mixture, and iii) surfactant mixture net charge. Results The emulsions were colloidally stable with a droplet diameter of 150–250 nm, and the zeta-potential correlated closely with the net charge of the surfactant mixture. Only cationic emulsions containing the unsaturated surfactant mix- ture induced concomitant humoral and CMI responses upon immunization of mice with a Ct antigen, and the responses were enhanced when squalene was applied as the oil phase. In contrast, emulsions with neutral and net negative zeta- potentials did not induce CMI responses. The saturation de- gree of the oil phase did not influence the adjuvanticity. Conclusion Cationic, MMG analogue-containing nanoemulsions are potential adjuvants for vaccines against path- ogens for which both humoral and CMI responses are needed. KEY WORDS adjuvant . drug delivery . emulsion . immune response . vaccine ABBREVIATIONS CAF Cationic adjuvant formulation CMI Cell-mediated immunity Cryo-TEM Cryo-transmission electron microscopy Ct Chlamydia trachomatis DDA Dimethyldioctadecylammonium bromide DODAC Dioleoyldimethylammonium chloride DOPE Dioleoylphosphoethanolamine DSPE Distearoylphosphoethanolamine GLA Glucopyranosyl lipid A GRAS Generally regarded as safe HA Hemagglutinin HEPES 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid HLB Hydrophile-lipophile balance HRP Horseradish peroxidase HSM High shear mixing IFN Interferon IL Interleukin LN Lymph node MHC Major histocompatibility complex MMG-1 Monomycoloyl glycerol MOMP Major outer membrane protein Mtb Mycobacterium tuberculosis o/w Oil-in-water PDI Polydispersity index Dennis Christensen and Camilla Foged shared senior authorship. Electronic supplementary material The online version of this article (doi:10.1007/s11095-017-2180-9) contains supplementary material, which is available to authorized users. * Camilla Foged camilla.foged@sund.ku.dk 1 Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen Ø, Denmark 2 Statens Serum Institut, Department of Infectious Disease Immunology, Artillerivej 5, 2300 Copenhagen S, Denmark Pharm Res DOI 10.1007/s11095-017-2180-9