Vaccine 32 (2014) 3460–3467 Contents lists available at ScienceDirect Vaccine j o ur na l ho me page: www.elsevier.com/locate/vaccine Combination of MIDGE-Th1 DNA vaccines with the cationic lipid SAINT-18: Studies on formulation, biodistribution and vector clearance Anne Endmann a,∗ , Detlef Oswald a , Oliver Riede a , Eduard G. Talman b , Roelien E. Vos b , Matthias Schroff a , Christiane Kleuss a , Marcel H.J. Ruiters b,c , Christiane Juhls a a MOLOGEN AG, Fabeckstraße 30, 14195 Berlin, Germany b Synvolux Therapeutics B.V., L.J. Zielstraweg 1, 9713 GX Groningen, The Netherlands c Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands a r t i c l e i n f o Article history: Received 19 December 2013 Received in revised form 5 March 2014 Accepted 13 March 2014 Available online 26 March 2014 Keywords: MIDGE-Th1 DNA vaccine Biodistribution SAINT-18 Transfection reagent Cationic lipid a b s t r a c t We have previously shown that the combination of MIDGE-Th1 DNA vectors with the cationic lipid SAINT- 18 increases the immune response to the encoded antigen in mice. Here, we report on experiments to further optimize and characterize this approach. We evaluated different formulations of MIDGE-Th1 vec- tors with SAINT-18 by assessing their influence on the transfection efficiency in cell culture and on the immune response in mice. We found that high amounts of SAINT-18 in formulations with a w/w ratio MIDGE Th1/SAINT-18 of 1:4.8 are beneficial for cell transfection in vitro. In contrast, the formulation of HBsAg-encoding MIDGE-Th1 DNA vectors with the lowest amount of SAINT-18 (w/w ratio MIDGE Th1/SAINT-18 of 1:0.5) resulted in the highest serum IgG1 and IgG2a levels after intradermal immuniza- tion of mice. Consequently, latter formulation was selected for a comparative biodistribution study in rats. Following intradermal administration of both naked and formulated MIDGE-Th1 DNA, the vectors localized primarily at the site of injection. Vector DNA levels decreased substantially over the two months duration of the study. When administered in combination with SAINT-18, the vectors were found in sig- nificantly higher amounts in draining lymph nodes in comparison to administration of naked MIDGE-Th1 DNA. We propose that the high immune responses induced by MIDGE-Th1/SAINT-18 lipoplexes are medi- ated by enhanced transfection of cells in vivo, resulting in stronger antigen expression and presentation. Importantly, the combination of MIDGE-Th1 vectors with SAINT-18 was well tolerated in mice and rats and is expected to be safe in human clinical applications. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction DNA vaccines have been described to induce both relevant cell- mediated and humoral immune responses against numerous viral, bacterial and parasitic pathogens in a number of animal models [1,2] but less so in clinical trials. Their success has been hampered mainly by low immunogenicity in humans [3]. In order to improve the immunogenicity of DNA vaccines, a variety of strategies is being applied, primarily addressing delivery systems to increase the DNA uptake into target cells by physical [4,5] or chemical meth- ods [6]. Complexes of DNA with cationic lipids or polymers have ∗ Corresponding author. Tel.: +49 30 841788 0; fax: +49 30 841788 50. E-mail address: endmann@mologen.com (A. Endmann). been reported to improve immune responses by facilitating the transfer of DNA vectors across membranes and protecting the DNA from degradation by nucleases [7]. We have developed DNA vectors exclusively comprising the expression cassette and having a reduced size in comparison to plasmids, the Minimalistic Immunogenically Defined Gene Expression (MIDGE) vectors [8]. MIDGE-Th1 vectors are linear double-stranded DNA molecules, which are closed with single- stranded hairpin loops at both ends and contain a peptide nuclear localization sequence covalently bound to one of the loops. Immunization with MIDGE-Th1 vectors induced strong humoral and cellular immune responses [9–11] which were significantly increased by formulation with the cationic lipid SAINT-18 [12]. A SAINT-18 molecule contains two hydrophobic tails and one cationic pyridinium head group and builds complexes with the negatively http://dx.doi.org/10.1016/j.vaccine.2014.03.048 0264-410X/© 2014 Elsevier Ltd. All rights reserved.