Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants

Abstract

A principal barrier to the development of effective vaccines is the availability of adjuvants and formulations that can elicit both effector and long-lived memory CD4 and CD8 T cells. Cellular immunity is the presumptive immune correlate of protection against intracellular pathogens: a group composed of bacteria, viruses and protozoans that is responsible for a staggering level of morbidity and mortality on a global scale. T-cell immunity is also correlated with clinical benefit in cancer, and the development of therapeutic strategies to harness the immune system to treat diverse malignancies is currently undergoing a renaissance. Cyclic dinucleotides (CDNs) are ubiquitous small molecule second messengers synthesized by bacteria that regulate diverse processes and are a relatively new class of adjuvants that have been shown to increase vaccine potency. CDNs activate innate immunity by directly binding the endoplasmic reticulum-resident receptor STING (stimulator of interferon genes), activating a signaling pathway that induces the expression of interferon-β (IFN-β) and also nuclear factor-κB (NF-κB) dependent inflammatory cytokines. The STING signaling pathway has emerged as a central Toll-like receptor (TLR) independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by bacteria, or, as shown recently, through binding of a structurally distinct CDN produced by a host cell receptor in response to binding cytosolic double-stranded (ds)DNA. Although this relatively new class of adjuvants has to date only been evaluated in mice, newly available CDN-STING cocrystal structures will likely intensify efforts in this field towards further development and evaluation in human trials both in preventive vaccine and immunotherapy settings.

Keywords: STING; adjuvant; cellular immunity; cyclic dinucleotides; cytosolic nucleic acid sensing; formulation; innate immunity; stimulator of interferon genes; vaccine.

Figure 1.

STING (stimulator of interferon genes) is a central sensor of cytosolic nucleic acids. STING is a MyD88-independent host cell defense factor that senses cytosolic nucleic acids, and in response activates and TBK-1/IRF-3 signaling cascades, inducing the expression of pro-inflammatory cytokines and IFN-β. The activating ligands for STING are CDNs, second messengers that are produced by bacteria or by cellular cGAS in response to binding cytosolic dsDNA. CDNs produced by bacteria and cGAS are structurally distinct. Less well studied, activation of STING by CDNs has also been shown to induce the expression of STAT6-dependent Th2 cytokines. AMP, adenosine monophosphate; CDN, cyclic dinucleotide; cGAS, cyclic GMP–AMP synthase; ds, double-stranded; GMP, guanosine monophosphate; IFN-β, interferon-β; IRF-3, interferon regulatory factor 3; NF-κB, nuclear factor κB; STING, stimulator of interferon genes; TBK-1, TANK-binding kinase 1.

Figure 2.

STING activating ligands and CDN synthesis: (A) ‘canonical’ cyclic-di-GMP (cyclic[G(3′,5′)pG(3′,5′)p]) produced by bacteria; (B) ‘noncanonical’ cyclic-GMP-AMP cyclic[G(2′,5′)pA(3′,5′)p] produced by cellular cGAS; (C) DMXAA, a small molecule structurally unrelated to CDNs; and (D) schematic representation for synthesis of dioxo- and dithio-CDNs using phosphoramidite and H-phosphonate methods for the dimerization and cyclization steps, respectively. Full sequence requires 10 steps from commercially available starting material. AMP, adenosine monophosphate; CDN, cyclic dinucleotide; DMXAA, 5,6-dimethylxanthenone-4-acetic acid; cGAS, GMP–AMP synthase; GMP, guanosine monophosphate.

Figure 3.

CDNs promote priming of robust antigen-specific CD8⁺ T-cell immunity. C57BL/6 mice (n = 3) were vaccinated twice at a 3-week interval with 50 µg cyclic di-GMP coformulated with 10 µg OVA protein and 2% squalene-in water emulsion (Addavax). Mice were injected by subcutaneous (base of the tail) or intramuscular routes. OVA_257-264-specific CD8⁺ T-cell responses were measured in the spleen at the peak of the secondary response at 6 days post boost by intracellular cytokine staining for IFN-γ and TNF-α cytokines. Shown are representative FACS plots, each containing the mean and standard deviation of responses for the group of mice. CDN, cyclic dinucleotide; FACS, GMP, guanosine monophosphate; IFN-γ, interferon-γ; OVA, ovalbumin; TNF-α, tumour necrosis factor-α.