CpG Adjuvant in Allergen-Specific Immunotherapy: Finding the Sweet Spot for the Induction of Immune Tolerance

Abstract

Prevalence and incidence of IgE-mediated allergic diseases have increased over the past years in developed and developing countries. Allergen-specific immunotherapy (AIT) is currently the only curative treatment available for allergic diseases that has long-term efficacy. Although AIT has been proven successful as an immunomodulatory therapy since its beginnings, it still faces several unmet needs and challenges today. For instance, some patients can experience severe side effects, others are non-responders, and prolonged treatment schedules can lead to lack of patient adherence and therapy discontinuation. A common strategy to improve AIT relies on the use of adjuvants and immune modulators to boost its effects and improve its safety. Among the adjuvants tested for their clinical efficacy, CpG oligodeoxynucleotide (CpG-ODN) was investigated with limited success and without reaching phase III trials for clinical allergy treatment. However, recently discovered immune tolerance-promoting properties of CpG-ODN place this adjuvant again in a prominent position as an immune modulator for the treatment of allergic diseases. Indeed, it has been shown that the CpG-ODN dose and concentration are crucial in promoting immune regulation through the recruitment of pDCs. While low doses induce an inflammatory response, high doses of CpG-ODN trigger a tolerogenic response that can reverse a pre-established allergic milieu. Consistently, CpG-ODN has also been found to stimulate IL-10 producing B cells, so-called B regulatory cells (Bregs). Accordingly, CpG-ODN has shown its capacity to prevent and revert allergic reactions in several animal models showing its potential as both preventive and active treatment for IgE-mediated allergy. In this review, we describe how CpG-ODN-based therapies for allergic diseases, despite having shown limited success in the past, can still be exploited further as an adjuvant or immune modulator in the context of AIT and deserves additional attention. Here, we discuss the past and current knowledge, which highlights CpG-ODN as a potential adjuvant to be reevaluated for the enhancement of AIT when used in appropriate conditions and formulations.

Keywords: CpG-oligonucleotides; allergen-specific immunotherapy; allergy; immune tolerance; tolerance induction.

Figure 1

Toll-like receptor 9 (TLR9) signaling pathway(s) upon CpG oligodeoxynucleotides (CpG-ODN) stimulation: CpG-ODN is internalized through endosomes where it interacts with the endosomic domain of TLR9. In the subsequent signaling cascade, the cytosolic domain of TLR9 interacts with the adaptor proteins MyD88 or TRIF in the cytosol. Depending on the adapter engaged, a different signaling cascade occurs. MyD88 signaling (red) is triggered when TLR9 is agonized by a low CpG-ODN concentration. This cascade leads to the induction of the canonical and non-canonical NF-κB pathways, AP1 and IRF7 transcription factors, inducing inflammatory mediators such as TNF-α, IL-12, IL-1β, IL-6, and IL-23. TRIF signaling (green) is triggered when TLR9 is agonized by a high concentration of CpG-ODN. In contrast to the MyD88 signaling pathway, the TRIF cascade leads to the activation of the non-canonical NF-κB and IRF3. These transcription factors will induce the production of type 1 IFN and anti-inflammatory molecules such as TGF-β and IDO. When LPS is present (orange), TLR4 and TLR9 signals are combined to engage MyD88 leading to an enhanced inflammatory reaction by the increased production of IL-12 and TNF-α.

Figure 2

Effects of CpG oligodeoxynucleotides (CpG-ODN) on the immune system and inhibition mechanisms of the Th2 response: In an allergic immune environment (orange and red), allergens skew APCs toward the production of cytokines such as IL-4, prompting naïve T cells into Th2 cells. Th2 cells produce Th2 cytokines (IL-4, IL-5, IL-13) and induce B cells to produce immunoglobulin E (IgE), causing the hallmarks of the allergic disease such as increased epithelial permeability, effector cell activation, anaphylaxis, smooth muscle contraction, mucus hypersecretion etc. CpG-ODN can have different effects on APCs. On one side, it can promote a Th1-like response (blue) by skewing APC to produce IL-12 and then derive naïve T cells to IFN-γ producing Th1 cells. Th1 cells induce B cells to produce neutralizing antibodies such as IgG2a and IgG3. On the other side, CpG-ODN can skew APCs classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs) toward a tolerogenic phenotype (green). Tolerogenic APCs, cDCs and pDCs produce immune regulatory soluble factors such as TGF-β and indoleamine 2,3-dioxygenase (IDO), and express immune modulatory surface molecules like programmed death ligands 1 (PD-L1) and PD-L2. Tolerogenic APCscDCs and pDCs derive naïve T cells to Tregs, initiating an immune regulatory response. CpG-ODN can also engage directly B cells and skew them into a regulatory phenotype (Bregs). Bregs secrete the regulatory cytokines IL-10 and IL-35, as well as neutralizing IgG isotypes. The Th1 response can inhibit Th2 and allergy responses through the production of interferon-γ (IFN-γ) and neutralizing antibodies. However, Treg and Breg cells utilize a wide range of regulatory molecules from soluble IL-10 and IL-35, to the immune checkpoints CTLA-4 and PD-1 to suppress both Th2 and Th1 responses. Solid lines indicate induction, dotted lines indicate inhibition. *IgG4 refers to humans only.

Figure 3

Dual dose effect of CpG oligodeoxynucleotides (CpG-ODN) in AIT: CpG-ODN at low doses triggers immune activation and inflammation, while at high doses it induces immune regulation and tolerance. Clinical (downward-pointing triangles) and pre-clinical studies (upward-pointing triangles) are placed on the X axis (CpG dose in mg/kg body weight, Log10 scale) according to the CpG-ODN dose equivalent used in each study. Unsuccessful or suspended clinical trials and drugs (red triangles) fall under the immune activation/inflammation range of CpG-ODN dosage, whereas most of the successful (green triangles) pre-clinical research studies fall under the regulation/tolerance range. The orange dotted line (Jackson et al., 2018) indicates the dose of B-class CpG-ODN employed to boost the immune response in an FDA-approved vaccine for hepatitis B virus infection. The purple dotted line (Zent et al., 2012) indicates the maximum dose of B-class CpG-ODN that was well-tolerated in humans. Open triangles indicate non-type B CpG-ODN. The table on the right indicates the approximate dose equivalent of CpG-ODN used in each study. *For clinical trials, an average body weight of 75 kg was considered to calculate the CpG-ODN dose. For pre-clinical murine studies, an average body weight of 20 g was considered. IP, Intraperitoneal; NI, Nasal instillation; IT, Intratracheal instillation; ID, intradermal; IM, intramuscular; OG, oral gavage.