Vaccine 21 (2003) 1548–1553 The role of lipopolysaccharide in T-cell responses following DNA vaccination William G. Hawkins, Jiri Trcka, Neil Segal, Nathalie E. Blachere, Jason S. Gold, Yoichi Moroi, Wilbur B. Bowne, Jonathan J. Lewis, Jedd D. Wolchok, Alan N. Houghton * Swim Across America Laboratory, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USA Received 22 June 2001; received in revised form 7 March 2002; accepted 26 March 2002 Abstract Bacterial products, including lipopolysaccharide (LPS), are potential impurities in plasmid DNA vaccines. LPS has immunostimulatory properties even at exceedingly low concentrations through activation of Toll-like receptor 4 (TLR4). The potency of T-cell responses after vaccination was tested with DNA containing high LPS or depleted of LPS in TLR4-competent and TLR4-deficient mice. CD8 + T-cell responses were readily induced in TLR4-deficient mice immunized with DNA depleted of LPS. LPS in DNA vaccines is not required for CD8 + T-cell responses. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Lipopolysaccharide; DNA vaccination; T-cell immunity 1. Introduction DNA vaccination induces T-cell immunity through en- dogenous expression of the antigens by host somatic cells, especially APCs [1,2]. Plasmid DNA contains potential ad- juvants in the forms of bacterial DNA and products [3–5]. Eukaryotic organisms have evolved receptors to recognize and respond to bacterial structures [6–8]. Toll receptors are a family of molecules that can recognize bacterial products and transduce signals to host cells [6,9–11]. Lipopolysaccharide (LPS) is a product of gram negative organisms that induces profound effects on APCs including maturation, upregulation of costimulatory molecules, and cytokine release [12]. Toll-like receptor 4 (TLR4) is part of a receptor complex for LPS. Mice that are deficient in TLR4, either through spontaneous mutations or targeted disruption of the gene, are markedly hyporesponsive to LPS [7,8]. Plas- mid DNA vaccines are prepared from gram negative bacteria and contamination with LPS could conceivably play a role in the potency of DNA vaccines for inducing T-cell responses Abbreviations: LPS, Lipopolysaccharide; TLR4, Toll-like receptor 4; IFN-, interferon gamma * Corresponding author. Tel.: +1-212-639-7595; fax: +1-212-794-4352. E-mail address: a-houghton@ski.mskcc.org (A.N. Houghton). in vivo by activating APCs. We assessed the requirements for LPS in T-cell responses induced by DNA vaccination. 2. Materials and methods 2.1. Mice C57BL/10J and C.C3HJ mice were obtained from Jack- son Laboratories (Bar Harbor, ME). C57BL/10ScN and BALB/cN mice were obtained from the National Can- cer Institute (Frederick, MD). Animals (2–4 months) were treated in accordance with the Animal Welfare Act and NIH Guidelines for the Care and Use of Laboratory Animals. 2.2. Plasmid DNA, bacterial strain and LPS testing The WRG7077BEN plasmid constructs, which contain a CMV promoter and a kanamycin-resistance gene, with or without ovalbumin or -galactosidase inserts, have been pre- viously described [13]. The vector without insert served as a control for all experiments. Plasmid DNA was expressed and amplified in E. coli strain DH5. DNA was purified using QIAGEN ® Maxi-kit or the Endofree TM QIAGEN ® 0264-410X/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S0264-410X(02)00676-X