The Content of CpG-DNA in Antigen-CpG Conjugate Vaccines Determines Their Cross-Presentation Activity.

Cross-presentation, the process that facilitates display of exogenous antigens on MHC-I molecules, is a crucial step in the cascade of CD8 T cell activation. Potentiation of cross-presentation therefore represents an essential design criterion for development of subunit vaccines that target the induction of CD8 T cell immunity. Covalent conjugation of CpG-DNA to antigenic proteins has shown the potential to promote cross-presentation and has attracted great interest as a promising approach for vaccine development. However, heterogeneous product mixtures that result from typical conjugation schemes precluded identification of active conjugate species and impeded optimization of cross-presentation activity. In this report, we explore the effect of molecular composition of antigen-CpG conjugates on their cross-presentation activity using model Ovalbumin (OVA)-CpG conjugates. We developed a method to generate antigen-CpG conjugates with defined molecular compositions and leveraged this method to produce a series of OVA-CpG conjugates with one, two, and three CpG molecules linked to OVA. We observed that conjugates containing one CpG per OVA enhanced cross-presentation by 4-fold compared to native OVA, while conjugates with higher contents of CpG provided no cross-presentation enhancement. These differences are likely due to enhanced aggregation propensity observed for conjugates that carry more than one CpG per OVA. Our findings suggest that tuning molecular composition of antigen-CpG conjugates to maintain physical stability may be essential for achieving potent cross-presentation activity. Our method to generate defined conjugates could facilitate such molecular tuning and may be useful for continued development of antigen-CpG vaccines.