The transfer of hereditary information can be achieved by means that employ viral or nonviral vectors. gene transfer into somatic cells (6, 10, 15, 36, 39, 42). The transfer of genetic information can be achieved by means that employ viral or nonviral vectors. Both types have problems that limit their potential application in the vaccination process. Viral vectors may possibly be infectious, integrate and disrupt the DNA of normal cells, or induce antivector immune responses, whereas nonviral pDNA vectors have the advantage of being simple and safe, and generally lack immunogenic components, but are readily degraded in MK7622 vivo (12). pDNA-based immunization is relatively inefficient and depends, among other things, on the frequency of CpG motifs and the ability of a very small amount of the pDNA administered, or the protein that it encodes, to be taken up by costimulatory antigen-presenting cells, survive degradation in the lysosomes, and generate the antigen of interest (15, 26, 34, 44). Furthermore, the relatively poor immune response induced by pDNA vaccination in primates and humans is a major problem (11). Several strategies have been used to increase the pDNA delivery rate and to enhance the immune response to encoded gene products of interest. These strategies include modification of the mode of delivery, targeting of the antigens, and coadministration of immunostimulatory MK7622 genes or DNA sequences (3, 5, 8, 11, 12, 15, 20, 25, 26, 34, 41, 43). Administration of Schiff-base-forming drugs, such as tucaresol, to animals has been reported to potentiate the immune response (30). In this study we investigated the possibility of enhancing immune responses following pDNA injection by combining this mode of immunization with systemic costimulation provided by tucaresol. We GNG12 detected significant enhancement of antigen-specific humoral and cellular immune responses. Whereas coadministration of plasmids encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) and gamma interferon (IFN-) was able to enhance antigen-specific antibody and T-cell responses, respectively, tucaresol was able to exert both effects simultaneously, with levels of induction comparable to or even better than that of either of these potent cytokines. MATERIALS AND METHODS Plasmid construction and testing. All genes were inserted into the pCDNA3 vector (Invitrogen BV, Groningen, The Netherlands). Genes used in this study included the Epstein-Barr virus (EBV) nuclear antigen 4 (EBNA-4) and mycobacterial heat shock protein 65 (Mhsp65) genes, which were used as antigens, and mouse GM-CSF and IFN-, which were chosen as immunostimulatory cytokines. Details about the subcloning and testing of these plasmids have been published elsewhere (3, 5). Mice. HLA-A?0201/Kb transgenic mice (kindly provided by L. Sherman, Scripps Laboratories, San Diego, Calif.) used in this study have been described previously (40). This strain was used to enable the measurement of the cytotoxic T-cell response to a defined T-cell epitope restricted by HLA-A2 (4, 5). The surface expression of HLA-A?0201/Kb was confirmed by using an HLA-A?0201-specific fluorescein isothiocyanate-conjugated monoclonal antibody (One Lambda, Canoga Park, Calif.) and assessed by flow cytometry using FACScan (Becton Dickinson & Co., Mountain View, Calif.). ACA (H-2f) mice were purchased from Jackson Laboratory, Bar Harbor, Maine. This strain was used because it was previously used successfully for measurement of the immune response to EBNA-4 induced by DNA immunization (3). These mice were propagated and maintained in our specific-pathogen-free environment in the Microbiology and Tumor Biology Center (MTC) animal house MK7622 at the Karolinska Institute. Immunization. DNA immunization was accomplished by intramuscular (i.m.) immunization. Mice were injected in the regenerating tibialis-anterior muscle according to the work of Davis et al. (9) and others (3, 5, 14) by using 20 g of pDNA/100 l of phosphate buffered-saline (PBS)/muscle. Mice received either a control plasmid (P3), a plasmid encoding EBNA-4 plus the control plasmid P3 (E4), a plasmid containing an Mhsp65 gene plus the control plasmid P3 (P3M.65), P3M.65 plus GM-CSF expression plasmids (P3M.65 G), or P3M.65 plus IFN- expression plasmids (P3M.65 ). Plasmids were mixed in equal molar quantities. Mice treated with tucaresol [4-(2-formyl-3-hydroxy-phenoxymethyl) benzoic acid; kindly provided by John Rhodes, Glaxo SmithKline, Stevenage, United Kingdom] were immunized with E4 or P3M.65 plasmids (E4, T and P3M.65, T, respectively). Tucaresol was injected subcutaneously (s.c.) separately from the DNA in the flank opposite the site of DNA injection. Different schedules of tucaresol injection were used, as follows: (i) one single injection of 1 1 mg of tucaresol/100 l of PBS at the same time as the DNA injection (experiments in Fig. ?Fig.11 to ?to44 and four out of six experiments reported.