Pharmaceutical Research

, Volume 34, Issue 8, pp 1716–1727 | Cite as

Systematic 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
  • Malene Aaby Neustrup
  • Stine Harloff-Helleberg
  • Karen Smith Korsholm
  • Thomas Rades
  • Peter Andersen
  • Dennis Christensen
  • Camilla Foged
Research Paper



Induction of cell-mediated immune (CMI) responses is crucial for vaccine-mediated protection against difficult vaccine targets, e.g., Chlamydia trachomatis (Ct). Adjuvants are included in subunit vaccines to potentiate immune responses, but many marketed adjuvants stimulate predominantly humoral immune responses. Therefore, there is an unmet medical 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) analogue to concomitantly induce humoral and CMI responses.


The influence of emulsion composition was analyzed using a systematic approach. Three factors were varied: i) saturation of the oil phase, ii) type and saturation of the applied surfactant mixture, and iii) surfactant mixture net charge.


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 mixture 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 degree of the oil phase did not influence the adjuvanticity.


Cationic, MMG analogue-containing nanoemulsions are potential adjuvants for vaccines against pathogens for which both humoral and CMI responses are needed.

Key Words

adjuvant drug delivery emulsion immune response vaccine 



Cationic adjuvant formulation


Cell-mediated immunity


Cryo-transmission electron microscopy


Chlamydia trachomatis


Dimethyldioctadecylammonium bromide


Dioleoyldimethylammonium chloride






Glucopyranosyl lipid A


Generally regarded as safe




4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid


Hydrophile-lipophile balance


Horseradish peroxidase


High shear mixing






Lymph node


Major histocompatibility complex


Monomycoloyl glycerol


Major outer membrane protein


Mycobacterium tuberculosis




Polydispersity index






Respiratory syncytial virus


Room temperature






Trivalent influenza vaccine








Intensity-weighted average hydrodynamic diameter


Acknowledgments and Disclosures

The work was funded by University of Copenhagen (STS) and Statens Serum Institut. Additional funding was provided by Innovation Fund Denmark [GeniVac (069–2011-1) and Centre for Nano-vaccine (grant number 09–067052)] and the European Commission through the ADITEC consortium contract (FP7-HEALTH-2011.1.4–4-280,873). The funding sources had no involvement in the study design; in the collection, analysis and interpretation of the data; in the writing of the report; nor in the decision to submit the paper for publication. We wish to thank the staff of the adjuvant group at INFIMM, SSI, in particular Janne Rabech and Rune Fledelius Jensen. We are grateful to Petra Alexandra Priemel, Department of Pharmacy, University of Copenhagen for technical assistance with the polarized light microscopy, and The Core Facility for Integrated Microscopy, Faculty of Health and Medical Sciences, University of Copenhagen. Karen Smith Korsholm, Peter Andersen and Dennis Christensen are employed by Statens Serum Institut, a nonprofit government research facility, which holds patents on the cationic liposomal adjuvants (CAFs).

Supplementary material

11095_2017_2180_MOESM1_ESM.pptx (104 kb)
Supplementary data, Figure 1 Gating strategy for the assessment of cytokine producing T cells by using flow cytometry. Single cells were further gated into lymphocytes, from which CD4+ T-cell were identified. The expression of IFN-γ, IL-17a, IL-2 and TNF-α was subsequently determined. The sample show splenocytes from a mouse immunized with CAF04-adjuvanted OVA, which were restimulated with CTH522. (PPTX 104 kb)
11095_2017_2180_MOESM2_ESM.pptx (87 kb)
Supplementary data, Figure 2 Hydrodynamic diameter of emulsion droplets measured during optimization of process parameters for high pressure homogenization, performed on emulsions composed of squalene, Span 60 and Tween 60. The z-average of the pre-emulsion was approx. 530 nm (data not shown). Data points represent mean values ± SD (n = 1, three technical replicates). (PPTX 86 kb)


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • 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
  1. 1.Department of Pharmacy, Faculty of Health and Medical SciencesUniversity of CopenhagenCopenhagen ØDenmark
  2. 2.Statens Serum Institut, Department of Infectious Disease ImmunologyCopenhagen SDenmark

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