Incorporation of CpG Oligodeoxynucleotides into ?2-Macroglobulin: Development of a Novel Vaccine Adjuvant Delivery Mechanism

by Anderson, Ryan Berger

Bacterial DNA is immunostimulatory, and the motifs responsible for this activity are unmethylated CpG dinucleotides. Following cellular uptake, CpG-containing oligodeoxynucleotides (CpG ODN) are trafficked to the endosome where they bind Toll-like receptor 9 (TLR9) to initiate a signaling cascade that culminates in the release of numerous pro-inflammatory cytokines. Because of their immunostimulatory properties, CpG ODN are being clinically evaluated as treatments and vaccine adjuvants for infectious diseases, cancer, and allergic disorders.

?2-Macroglobulin (?2M) is a human plasma protein that binds and modulates the activity of a variety of cytokines, growth factors, enzymes, and antigens. Upon proteolytic activation, ?2M is converted to its receptor recognized form, ?2M*, and rapidly binds to and is internalized by immune competent cells expressing the ?2M* endocytic receptor, LRP, and is then trafficked to the endosome. Based on these interactions, ?2M seems to play an important role at sites of infection and inflammation by controlling the level of proteinase activity, modulating cytokine signals, and enhancing antigen processing for the adaptive immune response.

Here, we report the first evidence that ?2M* binds and forms stable complexes with nucleic acids. We have characterized the mechanisms and stoichiometry of this interaction, examined the pH and temperature stability of these complexes, and identified structural variables in the nucleic acids, namely length, base composition, and chemical modifications, that affect the nature of this interaction. We hypothesized that CpG ODN incorporation into ?2M* may alter their immunostimulatory properties. Murine

macrophages (M?s) treated with ?2M*-ODN complexes respond more rapidly and produce a greater cytokine response than those treated with free CpG ODN alone. Treating human PBMCs with ?2M*-ODN complexes likewise demonstrated their enhanced ability to elicit immune responses. This was due to more rapid uptake and CpG ODN protection from degradation by extracellular nucleases. Co-incorporation of both protein ligands and CpG ODN into ?2M* yields ternary complexes; these may permit the simultaneous delivery of both protein antigens and adjuvants to immune competent cells, potentially greatly enhancing the adaptive immune response and protective immunity.

Based on the findings that incorporation into ?2M* confers enhanced immunostimulatory activity of CpG ODN, this technology may be exploited to improve CpG ODN-based therapeutics by increasing efficacy, minimizing side effects, reducing dosing requirements, and reducing cost.