Final Activity Report Summary - BIOTHERAPEUTIC ODN (Use of CpG oligodeoxynucleotides to improve the immunogenicity of vaccines) The specific aim of the present project is to develop novel strategies to improve the immunogenicity of vaccines targeting bioterror pathogens, focusing on AVA (anthrax vaccine adsorbed) and rPA (recombinant protective antigen) as the components of the vaccine formulation. In a study using six different TLR ligands to address contrasting and synergistic effects of cell surface expressed and endosome associated TLR ligand mixtures revealed that when TLR7 ligand (R-848), is mixed with either one of the TLR2 (PGN), TLR3 (pI:C), TLR4 (LPS), or TLR9 (CpG DNA) ligands and spleen cells in culture were treated, they induced significantly high IL6 and IFNg production even at very low combination doses, (compared to their alone stimulation levels), indicating that a synergistic Th1-mediated immune response is generated. The mRNA expression levels of TLR genes following ligand combination treatments by RT-PCR showed that TLR9 plus either of the TLR2, 3, 4, and 7 ligand combinations are significantly upregulating only tlr7 and tlr9 genes whereas tlr2, tlr3, tlr4, tlr5,and tlr6 genes were significantly downregulated. A considerable amount of effort was put to identify immunostimulatory potential of four different polysaccharides (PS) isolated from edible mushrooms. The most potent candidate was selected to achieve PS:TLR ligand complexations to assess their controlled delivery, adjuvant stability and their synergistic immunostimulatory effects in culture. Polysaccharides mainly act on innate immune system through TLR2/6 (similar to peptidoglycan, and zymosan) but not through TLR4 (lipopolysaccharide). So, we did check the pro-inflammatory cytokines TNFa and IL6 which were primarily secreted from immune cells, after they are exposed to polysaccharides. IL-6 secreted from macrophages in response to four different PS demonstrated that PS2 and PS4 released high levels cytokine even at very low doses by 24 or 42 hours of incubation. The dose and time-dependent stimulation assays demonstrated that PS2 and PS4 are the strongest inducers and cells respond to these stimulants as low as 0,02 µg/ml concentration and as early as 6 h post stimulation It peaked around 12 h post-treatment and induction stayed at this level up to 72 h. To determine the bactericidal effect of these polysaccharide-based carriers, nitric oxide levels were also determined. After 24h stimulation of mouse macrophage cell line (RAW264.7) NO levels reached peak levels with PS4. Finally the most potent PS extract, PS4, was selected for complexation with the three nucleic acid TLR ligand. These complexes were incubated with murine macrophages at different dilutions. PS4 plus R848 or pI:C even at very low doses showed significantly high IL-6 production compared with their alone treatments. As planed originally, we then focused on the preparation of different liposomes having various physicochemical and charge properties, and studied nucleic acid TLR ligand encapsulation including single or multiple TLR ligands, and assessed their immunostimulatory / adjuvant potentials. The major goal of this study is to reveal the type of liposome encapsulating different TLR ligand(s) that yields the strongest activation on spleen cells. Different endosome-associated TLR ligands (K-ODN, D-ODN and pIC) were incorporated within neutral, anionic, cationic, stealth and cationic stealth liposomes. Data implicated that adjuvanticity of TLR ligands can be achieved when they are encapsulated within proper liposome types expressing proper lammelarity, size and charge. Our findings suggested that encapsulating K type ODN in positively charged liposome augmented its immunogenicity while neutral or anionic charges are not contributing to stimulatory action if not suppressing. D ODN encapsulation with cationic moiety is detrimental and anionic or neutral charge significantly contributes to immune activity. Finally, vaccination studies in mice to determine anti-rPA response by using the most effective liposomal formulation against bioterror agents was undertaken. We have planned to analyse IgG titers from mice serum at the end of priming and boosting. There were eight treatment groups. Among these the most promising was observed to be the mice group treated with the (CpG+rPA) co-encapsulated liposome formulation. The ability of the immune cells to induce antigen specific IFNg and the ratio of IgG2a/IgG1>1 is a strong indication of this event. After the booster injection Ig and IFNg data revealed that only liposoems coencapsulating (rPA+CpG) strongly supported these features.