In the last years much effortin vaccinology has focused on the new formulation strategies for subunit vaccines.We formulated OVA (antigen) and CpG-ODN (TLR-9 agonist) with a nanostructureformed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16). We havepreviously shown that this nanovaccine (OVA/CpG-ODN/Coa- ASC16) elicited anadaptive immune response superior to those induced by an aqueous formulation(OVA/CpG-ODN). However, we still do not know exactly the mechanisms of actionof Coa-ASC16. Hence, the aim of this work was to test the impact of thisnanoformu- lation on biodistribution of vaccine components and early immuneresponse. Methods: mice were s.c. immunized with OVA/CpG-ODN orOVA/CpG-ODN/Coa-ASC16. OVA and CpG-ODN were labeled with near-infraredfluorescent dye, and both signals were measured with an Odyssey® CLx at severaltime points post immunization (pi). Cytokines/chemokines were evaluated inplasma by a multiplex as- say at 1.5h pi. Results are indicated asOVA/CpG-ODN vs OVA/ CpG-ODN/Coa-ASC16. Liver: OVA signal was 1.2 x 107 vs 0.6 x 107

(p<0.01), 4.3 x 107 vs 1.0 x 107 (p<0.001) and 2.0 x 107 vs 0.8 x 107 (p<0.01) at 20 min, 2 and4h pi. No signal was observed in spleen and kidney in any of groups. Injectionsite: OVA signal was 1.6 x 106 vs 6.9 x 106 (p<0.001) and 0.05 x 106 vs 2.45 x 106 (p<0.05) at 0.3 and 5 days pi; for CpG-ODN there was no significantdifferences between both groups at any time. Lymph node: OVA signal was 1.8 x105 vs 0.3 x 105 (p<0.01),0.5 x 105 vs 4.5 x 105 (p<0.001)and 0.1 x 105 vs 2.8 x 105 (p<0.01)at 20 min, 2 and 24h pi; in contrast CpG- ODN signal was similar between twogroups at 20 min and 2h pi. In addition, soluble vaccine elicited higheramounts of systemic TNF-α and MCP-1 than nanovaccineimmunization (p<0.05). Conclusions: Coa-ASC16 retains antigen at theinjection site but not CpG-ODN, and promotes co-delivery of both molecules tolymph node without comitant induction of systemic inflammation.