CpG oligodeoxynucleotides with double stem-loops show strong immunostimulatory activity

Area of Science:

• Immunology
• Molecular Biology
• Cancer Research

Background:

• Toll-like receptor 9 (TLR9) recognizes CpG oligonucleotides (ODNs).
• Understanding TLR9-CpG ODN interactions is crucial for developing immunotherapies.
• Designing novel CpG ODNs with enhanced properties is an ongoing research area.

Purpose of the Study:

• To design and synthesize novel CpG ODNs with double stem-loop secondary structures.
• To evaluate the immunostimulatory activity and immune cell activation potential of these novel CpG ODNs.
• To assess the in vivo efficacy of the lead CpG ODNs against breast cancer.

Main Methods:

• Secondary structure prediction using the mfold web server.
• Screening of CpG ODNs using human peripheral blood mononuclear cell (PBMC) proliferation and interferon (IFN) production assays.
• Flow cytometry analysis for immune cell subset activation (B cells, NK cells, mononuclear cells, T cells) and co-stimulatory molecule expression (CD80, CD86, HLA-DR).
• Assessment of PBMC/splenocyte proliferation from diverse vertebrate species.
• In vivo anti-breast cancer efficacy study in a mouse model via peritumoral injection.

Main Results:

• Two designed CpG ODNs, DSL01 (Class B) and D-SL03 (Class C), exhibited significant immunostimulatory activity.
• Both DSL01 and D-SL03 activated human B cells, NK cells, mononuclear cells, and T cells, upregulating CD80, CD86, and HLA-DR.
• These CpG ODNs potently stimulated PBMC/splenocyte proliferation across various vertebrate species.
• DSL01 and D-SL03 demonstrated anti-breast cancer effects in mice when administered peritumorally.

Conclusions:

• Novel double stem-loop CpG ODNs (DSL01 and D-SL03) possess potent immunostimulatory properties.
• These CpG ODNs effectively activate key immune cells and exhibit cross-species activity.
• The designed CpG ODNs show promise as potential immunotherapeutics for breast cancer.