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

Abstract

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.

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

“…To demonstrate the applicability of CFPS and to provide an appropriate comparison to previous antigen-adjuvant co-localization experiments [9][10][11][12][13][14][15] , we used a model antigen, OVA. Noting that a pioneering study of non-specific conjugation of CpG to OVA at a ≥ 2:1 ratio induced aggregation and reduced antigen presentation 11 , we introduced two site-specific conjugatable handles on one OVA protein to overcome this limitation of non-specific conjugation. We selected the human-mouse cross-reactive CpG1018 as a clinically relevant TLR9 agonist and appended a commercially available 5′-thiol handle for conjugation 6 .…”

“…Preparative anion exchange chromatography using a 0-1 M NaCl gradient in a method previously described by the Chertok group was shown to remove unreacted OVA-2pAMF from the reaction mixture; however, components containing one and two CpG/OVA eluted together (Fig. S6) 11 . With the limited benefit to this purification strategy, we opted to use the unpurified heterogeneous mixtures in future studies; thus, site-specific OVA-CpG conjugates containing 1.2-1.5 CpG/OVA were achieved for use in subsequent experimentation.…”

“…Oligodeoxynucleotides, such as unmethylated CpG, activate the innate immune system by binding endosomal toll-like receptor 9 (TLR9) to enhance cross-presentation of antigenic components 16 . TLR9 agonists conjugated to ovalbumin (OVA) were shown to enhance both cross-presentation and antigen-specific CD8 + T-cell production 9,11 . However, these systems were limited by poor control over the modification site and aggregation resulting from over-conjugation of ODN adjuvants.…”

Better Adjuvants for Better Vaccines: Progress in Adjuvant Delivery Systems, Modifications, and Adjuvant–Antigen Codelivery

“…The codelivery system showed a better immune response than the simple mix of adjuvant-antigen [122]. Cross presentation of the OVA antigen was significantly enhanced by chemical coupling of a CpG molecule [123]; however, this cross-presentation did not increase with the number of coupled CpG molecules. For HA antigen displayed on the surface of ferritin and then coupled with TLR9 agonist CpG or TLR7/8 agonist 3M012 [124], the dosage of chemically coupled adjuvants was reduced by 5000-fold while achieving the same immune enhancement effect compared to noncoupled adjuvants [125].…”

Unmethylated Cytosine phosphate guanine DNA (CpG-DNA) exacerbates liver Kupffer cells (KCs) inflammation through Toll-like receptor 9 (TLR9) in diabetic rats

“…Unmethylated CpG-DNA is the speci c component of bacterial DNA containing CpG motifs called pathogen associated molecular pattern (PAMP) [5]. The length, number and lateral sequence of unmethylated CpG-DNA that differ from different microbiota and determine their immunocompetence [6]. Pathogens such as Clostridia and Escherichia coli express higher unmethylated CpG-DNA that had stronger immunocompetence compared to the probiotics [7].…”