ABSTRACT
Among several types of CpG-ODNs, A/D-type CpG-ODNs have potent adjuvant activity to induce Th-1 immune responses, but exhibit a propensity to aggregate. For the clinical application of A/D-type CpG-ODNs, it is necessary to control such aggregation and obtain a comprehensive understanding of the relationship between their structure and the immune responses. This study revealed that a representative A/D-type CpG ODN, D35, adopted a single-stranded structure in water, while it assembled into aggregates in response to Na+ ions. From polyacrylamide gel electrophoresis and circular dichroism analyses, D35 adopted a homodimeric form (duplex) via palindromic sequences in low-Na+-concentration conditions (10-50 mM NaCl). After replacement of the solution with PBS, quadruplexes began to form in a manner coordinated by Na+, resulting in large aggregates. The duplexes and small aggregates prepared in 50 mM NaCl showed not only high cellular uptake but also high affinity to Toll-like receptor 9 (TLR9) proteins, leading to the production of a large amount of interferon-α for peripheral blood mononuclear cells. The much larger aggregates prepared in 100 mM NaCl were incorporated into cells at a high level, but showed a low ability to induce cytokine production. This suggests that the large aggregates have difficulty inducing TLR9 dimerization, resulting in loss of the stimulation of the cells. We thus succeeded in inducing adequate innate immunity in vitro by controlling and adjusting the formation of D35 aggregates. Therefore, the findings in this study for D35 ODNs could be a vital research foundation for in vivo applications.
ABSTRACT
Recent studies have shown the potent efficacy of peptide-based vaccines for cancer immunotherapy. Immunological performance is optimized through the co-delivery of adjuvant and antigenic peptide molecules to antigen-presenting cells simultaneously. In our previous study, we showed that a conjugate consisting of 40-mer CpG-DNA and an antigenic ovalbumin peptide through disulfide bonding could efficiently induce ovalbumin-specific cytotoxic T lymphocyte (CTL) responses in vivo. In this study, based on the conjugation design, we prepared a conjugate consisting of 30-mer CpG-DNA (CpG30) and a cancer antigenic peptide of Tyrosinase-related protein 2 (TRP2180-188) using a cysteine residue attached at the N-terminus of TRP2180-188. However, the immunization of mice with this conjugate did not induce efficient TRP2180-188-specific immune responses. It was thought that the resultant peptide (10-mer) cleaved from the conjugate might be too long to fit into the H-2Kb molecule because the optimal length for binding to it is 8-9 amino acids. We newly designed a conjugate consisting of CpG30 and the C-TRP2181-188 peptide (9-mer), in which the N-terminal serine residue of TRP2180-188 is replaced by a cysteine. By adjusting the peptide length, we succeeded in inducing strong TRP2180-188 peptide-specific CTL activity upon immunization with the CpG30-C-TRP2181-188 conjugate. Furthermore, various CpG30-C-TRP2181-188 conjugates having other CpG-DNA sequences or cysteine analogues also induced the same level of CTL activity. Therefore, CpG-C-peptide conjugates prepared by replacement of the amino acid residue at the N-terminus with a cysteine residue could be a new and effective platform for peptide vaccines for targeting specific antigens of cancers and infectious diseases.
