Research Progress on CpG ODN Vaccine Adjuvants

CpG oligodeoxynucleotides (CpG ODN) are immunomodulatory synthetic oligonucleotides that specifically stimulate Toll-like receptor 9 (TLR9), inducing cellular and humoral immune responses to enhance the body's immune defense. After infection, pathogen-expressed evolutionarily conserved molecular structures can trigger innate immune responses. These structures are known as pathogen-associated molecular patterns (PAMPs) and are recognized by host TLRs. PAMPs such as lipopolysaccharides and lipoproteins are recognized by TLR4 and TLR2; bacterial flagellin by TLR5; double-stranded RNA (dsRNA) by TLR3; single-stranded RNA (ssRNA) by TLR7/8; and unmethylated CpG motifs present in bacterial and viral DNA by TLR9.

TLR activation initiates innate immune responses characterized by the production of pro-inflammatory cytokines, chemokines, type I interferons (IFNs), and antimicrobial peptides. Subsequently, the host develops adaptive immune responses, generating high-affinity antibodies and cytotoxic T cells that provide bactericidal immunity and maintain immune memory to prevent subsequent infections. Due to their ability to effectively induce innate immune responses and contribute to adaptive immunity, CpG ODNs have attracted significant attention as potential vaccine adjuvants.

Types of CpG ODNs

Based on chemical structure and biological activity, CpG ODNs can be classified into three types: A, B, and C. Among these, B-class CpG ODNs are the earliest discovered and most extensively studied. They consist of one or more CpG motifs, typically placed on a phosphorothioate backbone. Compared to natural phosphodiester nucleotides, this backbone enhances resistance to nuclease digestion, providing a longer in vivo half-life (30-60 minutes vs. 5-10 minutes for phosphodiesters). B-class CpG ODNs trigger plasmacytoid dendritic cell (pDC) differentiation and TNF-α production, while stimulating B-cell proliferation and IgM secretion.

A-class CpG ODNs contain a palindromic sequence with a central CpG dinucleotide, flanked by poly-G tails, and a partially phosphorothioate-modified backbone. These CpG ODNs form higher-order structures through palindromic sequences and poly-G, enabling pDC activation and robust type I IFN induction, but exhibit weak B-cell activity. The differing activities of B- and A-class CpG ODNs are largely due to differences in the retention time of CpG/TLR9 complexes in pDC endosomes. B-class CpG ODNs rapidly transit from early to late endosomes, whereas A-class CpG ODNs are retained longer in early endosomes. Due to their tendency to form complex multimers in solution, clinical development of A-class CpG ODNs has been slow. Recent research has focused on packaging them into stable virus-like particles (VLPs) for adjuvant use.

C-class CpG ODNs resemble B-class in being fully phosphorothioate-modified but share A-class features by incorporating palindromic CpG motifs, allowing stem-loop or dimer formation. They exhibit combined A- and B-class activities, activating both pDCs and B cells.

The immunostimulatory effects of different CpG motifs vary across species, exhibiting sequence- and species-specificity. Additionally, core sequences, palindromic structures, and other features significantly influence CpG ODN activity.

Species-Specificity of CpG ODNs

It is now established that TLR9-mediated recognition of CpG ODNs represents an innate immune mechanism for vertebrate recognition of pathogenic microorganisms. Species divergence, TLR9 variations, and natural selection have led to species-specific recognition of microbial CpG ODNs by respective TLR9 variants. For example, human and mouse TLR9 share only 76% amino acid homology, explaining the existence of species-specific CpG motifs and recognition mechanisms.

Early studies identified GACGTT and AACGTT as highly mouse-specific CpG motifs with weak activity in human immune cells. Nevertheless, Dynavax demonstrated in Phase III trials that CpG-1018 (containing AACGTT) exhibits strong adjuvant activity in hepatitis B vaccines. However, a major drawback for clinical use is the high dose required (3 mg per vaccine dose).

Later, Coley's identified GTCGTT as a cross-species immunostimulatory motif active in humans, mice, sheep, horses, pigs, dogs, and rabbits. Its core human CpG motif (TCGT) confers strong activity in human immune cells. Coley's developed CpG-7909 based on GTCGTT, which demonstrated potent adjuvant activity in hepatitis B vaccine Phase I/II trials at doses of 0.125～1 mg per dose. Compared to CpG-1018, this sequence offers not only superior activity but also lower, more economical, and safer dosing.

Furthermore, CpG ODN effector cells exhibit species-specificity: in mice, they include monocytes/macrophages, dendritic cells, and B cells; in humans, pDCs and B cells. This suggests that animal model results may not always translate to human clinical outcomes. However, clinical studies confirm CpG ODNs as effective vaccine adjuvants. While human conventional dendritic cells lack TLR9 expression, CpG-activated pDCs and B cells provide robust antigen presentation. Conversely, in mice, CpG ODNs activate monocytes/macrophages to produce excessive TNF-α, causing severe side effects. Since these cells do not express TLR9 in humans, CpG ODNs may offer better safety profiles in clinical applications.

Clinical Applications of CpG ODN Adjuvants

Currently approved or clinically trialed CpG ODNs include CpG7909 (also called CpG2006 or CpG-2006), ISS-1018, and CpG684. Dynavax's hepatitis B vaccine (HeplisavⓇ), incorporating ISS-1018, was approved in the US in September 2017. Over 100 CpG-adjuvanted therapeutic and prophylactic vaccines have entered clinical trials, with more than 30 using CpG7909. In prophylactic vaccines, adjuvant formulations include CpG alone, CpG combined with aluminum hydroxide, or MF59. Clinical CpG doses range from 100 to 3,000 μg per dose, typically 250-1,000 μg, with the highest dose (3,000 μg/dose) used in HeplisavⓇ.

CpG adjuvants significantly enhance immunogenicity and accelerate immune responses in aluminum-adjuvanted vaccines against hepatitis B, anthrax, and influenza. However, they may cause mild-to-moderate adverse effects, including injection-site pain, swelling, induration, erythema, and systemic flu-like symptoms, which typically resolve within days and are likely due to their immunostimulatory properties.

Conclusion

Over the past 20–30 years, extensive animal and clinical studies have demonstrated CpG ODNs as promising novel vaccine adjuvants for human use. Compared to existing adjuvants, CpG ODNs have a clearer mechanism of action, stronger adjuvant activity (particularly for cellular immunity), and can be chemically synthesized at scale with ideal cost and quality control. As research on the safety and efficacy of diverse CpG ODN adjuvants continues to expand and accumulate, improved understanding among researchers and regulators will pave the way for CpG ODNs to become an ideal new-generation human vaccine adjuvant.