Synthesis and characterisation of conjugates
Conjugation of CpG to antigen as a means for co-delivery has been shown to enhance cellular immune responses compared to delivery as a mixture. The ODN CpG however can be conjugated via its 3’ or 5’ end, and blocking of the 5’ end has been described to reduce its immunostimmulatory properties in splenocytes. To determine whether the end of CpG conjugated to OVA effects the induction of a cellular immune response by an immunotherapeutic vaccine, CpG modified with an amine group on either the 3’ end or the 5’ end were conjugated to the model tumour antigen OVA. OVA was first purified from aggregates by preparative size-exclusion chromatography (SEC) and then conjugated onto CpG using a bis-aryl-hydrazone …show more content…
The yield of each conjugate obtained was calculated based on the protein recovery, both 3’ and 5’ CpG-OVA conjugates generated using the aromatic hydrazine linker had a similar yield, 64 % for the 5’ CpG-OVA and 67 % for the 3’ CpG-OVA conjugate. The 3’ CpG-OVA and 5’ CpG-OVA conjugates were characterised via analytical SEC. Figure 2a shows the elution profile for a mixture of CpG and OVA, OVA as the larger molecule eluted at 8.7±0.1 min after injection and CpG eluted after 9.4±0.1 min. The CpG-OVA conjugates shown in Figure 2b and c displayed a larger molecular weight than OVA, confirming the conjugation process, the elution profiles identified different populations of conjugates which implies a heterogeneous conjugation ratio of CpG to OVA. The 3’ CpG-OVA conjugate in Figure 2b and the 5’ CpG-OVA conjugate in Figure 2c showed similar elution profiles, suggesting the distribution of conjugation ratios with an average of 2.8±0.3 CpG per OVA was comparable between the conjugates. A small peak at 9.5 min in the conjugate elution profiles indicated small amounts of residual unconjugated CpG that could not be removed by the previous purification step. The residual unconjugated CpG was calculated to be on average 3.75 …show more content…
Blocking of the 5’ end of CpG as is the case in conjugation to an antigen has been shown to lower the activity of NF-κB in previous studies. This was tested in J774 macrophage, 293XL and Human Embryonic Kidney 293 cell lines expressing TLR9 9–11,13–15. However, to induce T-cell activation and proliferation, not the total amount of NK-κB produced following TLR9 activation but upregulation of co-stimulatory markers on DCs that bind to T-cells as well as presentation of antigen on MHC-I and MHC-II is critical. Only one study was found that looked at DC activation with CpG conjugated via the 3’ and 5’ end. Beaudette et al conjugated CpG via the 3’ and 5’ end to a nanoparticle and reported a non-significant, on average 10 % increased CD86 expression and IL-12 production in bone marrow-derived dendritic cells (BMDC) stimulated with 3’ conjugated CpG compared to 5’ conjugated CpG 33. In order to directly compare and evaluate whether blocking of the 5’ end of CpGs through conjugation effects the activation of DCs and their ability to upregulate activation markers, BMDCs were pulsed with the 3’ and 5’ CpG-OVA conjugates for 24 h and the level of MHC-II, CD86 and CD40 upregulation was measured. Figure 3a shows the gating strategy following flow cytometry of CD11c+ BMDCs. Figure 3b shows that the activation markers CD86 and CD40 were upregulated following incubation with a
Conjugation of CpG to antigen as a means for co-delivery has been shown to enhance cellular immune responses compared to delivery as a mixture. The ODN CpG however can be conjugated via its 3’ or 5’ end, and blocking of the 5’ end has been described to reduce its immunostimmulatory properties in splenocytes. To determine whether the end of CpG conjugated to OVA effects the induction of a cellular immune response by an immunotherapeutic vaccine, CpG modified with an amine group on either the 3’ end or the 5’ end were conjugated to the model tumour antigen OVA. OVA was first purified from aggregates by preparative size-exclusion chromatography (SEC) and then conjugated onto CpG using a bis-aryl-hydrazone …show more content…
The yield of each conjugate obtained was calculated based on the protein recovery, both 3’ and 5’ CpG-OVA conjugates generated using the aromatic hydrazine linker had a similar yield, 64 % for the 5’ CpG-OVA and 67 % for the 3’ CpG-OVA conjugate. The 3’ CpG-OVA and 5’ CpG-OVA conjugates were characterised via analytical SEC. Figure 2a shows the elution profile for a mixture of CpG and OVA, OVA as the larger molecule eluted at 8.7±0.1 min after injection and CpG eluted after 9.4±0.1 min. The CpG-OVA conjugates shown in Figure 2b and c displayed a larger molecular weight than OVA, confirming the conjugation process, the elution profiles identified different populations of conjugates which implies a heterogeneous conjugation ratio of CpG to OVA. The 3’ CpG-OVA conjugate in Figure 2b and the 5’ CpG-OVA conjugate in Figure 2c showed similar elution profiles, suggesting the distribution of conjugation ratios with an average of 2.8±0.3 CpG per OVA was comparable between the conjugates. A small peak at 9.5 min in the conjugate elution profiles indicated small amounts of residual unconjugated CpG that could not be removed by the previous purification step. The residual unconjugated CpG was calculated to be on average 3.75 …show more content…
Blocking of the 5’ end of CpG as is the case in conjugation to an antigen has been shown to lower the activity of NF-κB in previous studies. This was tested in J774 macrophage, 293XL and Human Embryonic Kidney 293 cell lines expressing TLR9 9–11,13–15. However, to induce T-cell activation and proliferation, not the total amount of NK-κB produced following TLR9 activation but upregulation of co-stimulatory markers on DCs that bind to T-cells as well as presentation of antigen on MHC-I and MHC-II is critical. Only one study was found that looked at DC activation with CpG conjugated via the 3’ and 5’ end. Beaudette et al conjugated CpG via the 3’ and 5’ end to a nanoparticle and reported a non-significant, on average 10 % increased CD86 expression and IL-12 production in bone marrow-derived dendritic cells (BMDC) stimulated with 3’ conjugated CpG compared to 5’ conjugated CpG 33. In order to directly compare and evaluate whether blocking of the 5’ end of CpGs through conjugation effects the activation of DCs and their ability to upregulate activation markers, BMDCs were pulsed with the 3’ and 5’ CpG-OVA conjugates for 24 h and the level of MHC-II, CD86 and CD40 upregulation was measured. Figure 3a shows the gating strategy following flow cytometry of CD11c+ BMDCs. Figure 3b shows that the activation markers CD86 and CD40 were upregulated following incubation with a