Coadministration of antigen-conjugated and free CpG: Effects of in vitro and in vivo interactions in a murine model

• Free and antigen-conjugated CpG oligonucleotides both activate cells via TLR9 but compete for cellular uptake.
• Co-administration of these two forms adversely effects immunogenicity.
• Not only activating, but also control or inhibitory oligonucleotides showed competitive effects in vitro.

CpG oligodeoxynucleotides (CpG) are widely studied as promising adjuvants in vaccines against a range of diseases including infection, cancer or allergy. Conjugating antigen to CpG has been shown to potentiate the adjuvant effect via enhancing antigen uptake and danger signaling by the very same cell. In the present study, using biotinylated CpG and streptavidin as a model system, we demonstrate that CpG motif containing free and antigen-conjugated oligonucleotides do not compete in terms of cell activation via TLR9, but do compete for cellular uptake. Antigen-conjugated CpG enhances cellular association and uptake of the antigen by antigen-presenting cells (APC) and T cells. Free CpG efficiently competes with antigen-CpG conjugates in BMDC and T cells, but shows weak or no competition in B cells that have higher TLR9 expression. Vaccination with antigen-conjugated CpG or with a mixture of antigen and CpG elevates the level of antigen-specific antibodies but co-administration of CpG-antigen conjugates and free CpG adversely effects immunogenicity. These observations may help optimize CpG-based vaccine formulation.