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Conjugation of CpG to an antigen induces a stronger immune response compared to that of the mixture. This study compares the in vitro immunostimulatory activity of CpG conjugated via either its 5' or 3' end to the model antigen ovalbumin (OVA). CpG modified with an amine at either the 5' or 3' end was conjugated to OVA via a stable bis-aryl hydrazone bond. Similar levels of CpG conjugation to OVA were observed for both conjugates on the basis of the absorbance at 360 nm for the formation of the bis-aryl hydrazone bond, which determined 2.8 ± 0.3 CpGs linked per OVA. Both the 5' and 3' CpG-OVA conjugates had similar size-exclusion chromatography elution profiles. The immunostimulatory properties of the conjugates were determined by dendritic cells (DCs) and T-cells isolated from mice. The activation of DCs was determined by the upregulation of activation markers CD86 and CD40. T-cells were co-cultured with stimulated DCs, and the immunogenicity was determined by measuring T-cell proliferation and interferon γ production. Both the CpG 5'- and 3'-linked conjugates induced the same level ( p &gt; 0.5) of DC activation markers, which were significantly higher than those of the untreated control. Similarly, T-cell assays showed no significant difference ( p &gt; 0.5) between the 5' and 3' conjugates with respect to T-cell proliferation and interferon γ production. The 5' and 3' conjugates induced T-cell activation significantly higher than the mixture of CpG and OVA. This study showed that the end at which CpG is conjugated to an antigen has no influence on the generation of a T-cell-based immune response in vitro.

Comparative Study of 5′- and 3′-Linked CpG–Antigen Conjugates for the Induction of Cellular Immune Responses