Commonly subunit-based vaccine requires the additionof an adju­vant. To overcome this challenge, new adjuvant strategies are beingdeveloped worldwide in experimental models or in human clinical trials. In thiscontext, we formulated a TLR-9 agonist, CpG-ODN and a model antigen, OVA, witha nanostructure (Coa-ASC16) formed by self-assembly of 6-O-ascorbyl palmitate(OCC). This nanoformu­lation elicited superior adaptive immune responses thansoluble for­mulation of OVA/CpG-ODN (OC). In addition, Coa-ASC16 creates adepot of antigen and CpG-ODN at the injection site. However, de­tails about invivo mechanisms that dictate the priming of vaccine-in­duced immunity arelacking. Here, we investigate the early events in the proximal-draining lymphnodes (LN) of the vaccine injection site. Mice were subcutaneously immunizedwith formulations containing fluorescent-dye labeled OVA and CpG-ODN. Then, theavailability of both molecules contained in the whole LN was measured withOdyssey® CLx at several time points post-immunization (p.i.). In addition, wedetermined, by flow cytometry, the uptake of both mol­ecules from LN bydendritic cells (DC). OVA signal was OC>OCC (p<0.001) and OCC>OC(p<0.0001) at 20 min and 24h p.i. respec­tively; CpG-ODN signal wasOC>OCC (p<0.0001) and OCC>OC (p<0.0001) at 20 min and 24h p.i.respectively. 72 hs after immu­nization, we analyzed single cell suspension ofLN and observed that Coa-ASC16 impacts on antigen uptake, mice immunized with OCCshowed higher numbers of OVA+CD11c+DC, CpG-ODN+C­D11c+DC andOVA+CpG-ODN+CD11c+DC compared with those mice immunized with OC (p<0.001).Conclusion: The nanoformula­tion of vaccine modifies the kinetics of antigenand immunostimulant availability in the LN and improves co-uptake of bothmolecules by DCs despite the fact that our system does not chemically link OVAto CpG-ODN. Understanding of the impact of vaccine formulation on earlyresponse might have a significant impact for rational vaccine design.