Vaccine 27 (2009) 5589–5598 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine Expression of tak1 and tram induces synergistic pro-inflammatory signalling and adjuvants DNA vaccines Karen Colbjørn Larsen a,∗ , Alexandra J. Spencer a , Anna L. Goodman a , Ashley Gilchrist b , Julie Furze a , Christine S. Rollier a , Endre Kiss-Toth c , Sarah C. Gilbert a , Migena Bregu a , Elizabeth J. Soilleux b , Adrian V.S. Hill a , David H. Wyllie a a The Jenner Institute, University of Oxford, Oxford OX3 7DQ, United Kingdom b Department of Cellular Pathology, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom c Cardiovascular Research Unit, University of Sheffield, Sheffield S10 2RX, United Kingdom article info Article history: Received 6 February 2009 Received in revised form 21 May 2009 Accepted 13 July 2009 Available online 29 July 2009 Keywords: DNA vaccine Adjuvant tak1 tram cxcl2 abstract Improving vaccine immunogenicity remains a major challenge in the fight against developing country diseases like malaria and AIDS. We describe a novel strategy to identify new DNA vaccine adjuvants. We have screened components of the Toll-like receptor signalling pathways for their ability to activate pro-inflammatory target genes in transient transfection assays and assessed in vivo adjuvant activity by expressing the activators from the DNA backbone of vaccines. We find that a robust increase in the immune response necessitates co-expression of two activators. Accordingly, the combination of tak1 and tram elicits synergistic reporter activation in transient transfection assays. In a mouse model this combination, but not the individual molecules, induced approximately twofold increases in CD8+ T-cell immune responses. These results indicate that optimal immunogenicity may require activation of distinct innate immune signalling pathways. Thus this strategy offers a novel route to the discovery of a new generation of adjuvants. © 2009 Elsevier Ltd. All rights reserved. 1. Introduction DNA vaccines have many advantages, compared with other types of vaccines, including ease and low cost of production and manufacture, good safety, and preclinical efficacy [1]. Several DNA vaccines are approved for use in animals [2–4] and human clinical trials aiming to elicit cell-mediated immunity using intra-muscular (i.m.) administration of DNA vaccines are ongoing for several dis- eases including HIV and SARS [5–9]. Since the protection afforded by DNA vaccines in animal models has not easily translated into pri- mates, attempts have been made to increase DNA vaccine efficacy by engineering the vector to produce molecular adjuvants, such as cytokines and co-stimulatory molecules [10,11]. Toll-like receptors (TLRs) and other pattern-recognition recep- tors recognise intra- and extra-cellular pathogens to provide the first line of defence in the innate immune response [12]. TLRs are Abbreviations: APC, antigen presenting cell; i.m., intra-muscular; LPS, lipopolysaccharides; TLR, Toll-like receptor; TIR, Toll-interleukin1 receptor; IFN, interferon; TLR-SM, TLR-signalling molecule; TIP, Tuberculosis epitope, Immunode- ficiency virus epitope, and Plasmodium epitope; SFC, spot forming cells. ∗ Corresponding author. Tel.: +44 1865 617629; fax: +44 1865 617608. E-mail address: karen.colbjorn@googlemail.com (K.C. Larsen). transmembrane receptors, located at the cell surface or in endo- somes. Initiation of signalling is dependent on the recruitment of cytosolic adaptor proteins, containing Toll-interleukin1 receptor (TIR) domains, to the intracellular TIR domain of the TLR. There are four activating adapters: Myd88, TIRAP (MAL), TRIF (TICAM1), and TRAM (TICAM2) [12]. Of these, Myd88 is the most versa- tile and is used by all TLRs except for TLR3 [13], while TRAM is unique as it is only involved in Myd88-independent TLR4 sig- nalling where it recruits TRIF to the receptor [14]. Through these TIR-domain containing adaptor proteins signalling cascades are initiated in which series of phosphorylation and ubiquitination events culminate in increased transcription of pro-inflammatory genes and type I interferons (IFNs). The two major groups of tran- scription factors inducing pro-inflammatory gene expression upon TLR stimulation are AP-1 and NFB transcription factors. They are both activated through all the TLRs and by numerous TLR agonists [15]. TGF- activated kinase 1 (TAK1 or MAP3K7) is an essential kinase in Myd88 dependent TLR-signalling pathways, which medi- ates activation of AP-1 and NFB transcription factors critical for pro-inflammatory gene expression [16–19]. TAK1 induced AP-1 transcription factor activation is mediated through the MAP kinase signalling pathways [16,20], while NFB transcription factor acti- vation depends on phosphorylation of the IKK complex by TAK1 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.07.025