Site-specific antigen-adjuvant conjugation using cell-free protein synthesis enhances antigen presentation and CD8+ T-cell response

Abstract

Antigen-adjuvant conjugation is known to enhance antigen-specific T-cell production in vaccine models, but scalable methods are required to generate site-specific conjugation for clinical translation of this technique. We report the use of the cell-free protein synthesis (CFPS) platform as a rapid method to produce large quantities (> 100 mg/L) of a model antigen, ovalbumin (OVA), with site-specific incorporation of p-azidomethyl-l-phenylalanine (pAMF) at two solvent-exposed sites away from immunodominant epitopes. Using copper-free click chemistry, we conjugated CpG oligodeoxynucleotide toll-like receptor 9 (TLR9) agonists to the pAMF sites on the mutant OVA protein. The OVA-CpG conjugates demonstrate enhanced antigen presentation in vitro and increased antigen-specific CD8+ T-cell production in vivo. Moreover, OVA-CpG conjugation reduced the dose of CpG needed to invoke antigen-specific T-cell production tenfold. These results highlight how site-specific conjugation and CFPS technology can be implemented to produce large quantities of covalently-linked antigen-adjuvant conjugates for use in clinical vaccines.

Layer-by-layer nanoparticle encapsulating all-trans retinoic acid and CpG as a mucosal adjuvant targeting colorectal cancer

“…We hypothesized that DC activation by free OVA is overshadowed by the potent adjuvant. 31–33 Previous studies and our results have shown that free OVA antigen without adjuvant has poor DC activation efficacy. 34…”

“…We hypothesized that DC activation by free OVA is overshadowed by the potent adjuvant. [31][32][33] Previous studies and our results have shown that free OVA antigen without adjuvant has poor DC activation efficacy. 34 Activated BMDCs could secrete proinflammatory cytokines that recruit and activate T cells, instigating adaptive immune responses.…”

Enhancement of Immune Response of Bioconjugate Nanovaccine by Loading of CpG through Click Chemistry

“…Having confirmed the efficient lymph node drainage of CpG loaded onto the nanovaccine, we further analyzed the endocytic activity of DCs in different forms of CpG. DC2.4 is a mouse bone marrow dendritic cell line established with C57BL/6 mouse bone marrow cells transfected by the V-myc and V-raf genes and used to simulate the function of APC in several studies [ 30 , 38 ]. By stimulating DC2.4s with CpG Cy5 , NP-OPS+CpG Cy5 , or NP-OPS-CpG Cy5 , the amount of CpG phagocytosed by DC2.4s was analyzed by flow cytometry.…”

“…However, this compatibility method requires a large amount of CpG, which increases the cost and potential safety risks. Considering that CpG needs to enter antigen-presenting cells to perform its biological function, researchers also tried to improve the utilization of CpG by coupling it with antigens [ 30 , 31 ]. At present, many CpG and antigen connectors, such as NHS-PEG n -maleimide (SMCC) and the His-hydrophobic-His (HUH) superfamily of endonucleases, have been explored [ 32 , 33 , 34 ].…”