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•Phase 3 trial of investigational hepatitis B vaccine in adults 40–70years old.•2 doses of HBsAg-1018 demonstrated superior seroprotection to 3 doses of HBsAg-Eng.•HBsAg-1018 induced earlier seroprotection than HBsAg-Eng.•The safety profile of HBsAg-1018 was similar to that of HBsAg-Eng. The currently licensed hepatitis B vaccines have limitations including hyporesponsiveness in older adults, poor compliance, and the extended time for most persons to develop seroprotection (e.g. >6months). A vaccine containing HBsAg combined with a Toll-like receptor 9 agonist adjuvant (HBsAg-1018) has been developed to overcome these limitations. A Phase 3, multicenter, randomized, subject- and observer-blinded, active-controlled trial was conducted among healthy subjects 40–70years of age comparing the immunogenicity and safety of two doses of HBsAg-1018 at 0 and 4weeks to three doses of licensed hepatitis B vaccine, HBsAg-Eng, at 0, 4, and 24weeks. The primary immunogenicity endpoint was noninferiority of the seroprotection rate (SPR; % with anti-HBs≥10mIU/mL) of HBsAg-1018 compared to the SPR of HBsAg-Eng at 8 weeks following the last dose of vaccine. Conditional upon meeting noninferiority, superiority of HBsAg-1018 over HBsAg-Eng was assessed. Safety was compared between the two vaccines. At the primary endpoint, the SPR for the HBsAg-1018 group (90.0%) was superior to the SPR for the HBsAg-Eng group (70.5%) with an SPR difference of 19.5% (95% CI, 14.7%, 24.7%). At week 28 when the SPR peaked in the HBsAg-Eng group (72.8%), the SPR in the HBsAg-1018 group (94.8%) was significantly higher than in the HBsAg-Eng group. The SPR in the HBsAg-1018 group was significantly higher than in the HBsAg-Eng group at each study visit from week 4 through week 52. The safety profiles for the two vaccines were similar. When compared to the HBsAg-Eng three-dose regimen given at 0, 1, and 6months, HBsAg-1018 demonstrated superior seroprotection with only two doses at 0 and 1month. The safety profile of HBsAg-1018 was comparable to that of the licensed vaccine, HBsAg-Eng. HBsAg-1018 would provide a significant public health contribution toward the prevention of hepatitis B infection.

Crystal structures of three forms of Toll-like receptor (TLR) 9 — unliganded or bound either to immune stimulatory CpG-containing DNA or inhibitory DNA — together reveal the molecular basis of TLR9 activation. DNA recognition by TLR9 The nucleotide-sensing Toll-like receptor TLR9 triggers innate immune responses after recognizing microbial DNA containing CpG motifs. Toshiyuki Shimizu and colleagues now show exactly how TLR9 recognizes this immune stimulatory DNA. They provide crystal structures of three forms of TLR9: unliganded or bound either to immune stimulatory CpG DNA or inhibitory DNA, which together reveal the molecular basis of TLR9 activation. These findings should contribute to the development of anti-virals and other therapeutic agents that target TLR9. Innate immunity serves as the first line of defence against invading pathogens such as bacteria and viruses 1 . Toll-like receptors (TLRs) are examples of innate immune receptors, which sense specific molecular patterns from pathogens and activate immune responses 2 . TLR9 recognizes bacterial and viral DNA containing the cytosine–phosphate–guanine (CpG) dideoxynucleotide motif 3 , 4 . The molecular basis by which CpG-containing DNA (CpG-DNA) elicits immunostimulatory activity via TLR9 remains to be elucidated. Here we show the crystal structures of three forms of TLR9: unliganded, bound to agonistic CpG-DNA, and bound to inhibitory DNA (iDNA). Agonistic-CpG-DNA-bound TLR9 formed a symmetric TLR9–CpG-DNA complex with 2:2 stoichiometry, whereas iDNA-bound TLR9 was a monomer. CpG-DNA was recognized by both protomers in the dimer, in particular by the amino-terminal fragment (LRRNT–LRR10) from one protomer and the carboxy-terminal fragment (LRR20–LRR22) from the other. The iDNA, which formed a stem-loop structure suitable for binding by intramolecular base pairing, bound to the concave surface from LRR2–LRR10. This structure serves as an important basis for improving our understanding of the functional mechanisms of TLR9.

Newborns and young infants suffer increased infectious morbidity and mortality as compared to older children and adults. Morbidity and mortality due to infection are highest during the first weeks of life, decreasing over several years. Furthermore, most vaccines are not administered around birth, but over the first few years of life. A more complete understanding of the ontogeny of the immune system over the first years of life is thus urgently needed. Here, we applied the most comprehensive analysis focused on the innate immune response following TLR stimulation over the first 2 years of life in the largest such longitudinal cohort studied to-date (35 subjects). We found that innate TLR responses (i) known to support Th17 adaptive immune responses (IL-23, IL-6) peaked around birth and declined over the following 2 years only to increase again by adulthood; (ii) potentially supporting antiviral defense (IFN-α) reached adult level function by 1 year of age; (iii) known to support Th1 type immunity (IL-12p70, IFN-γ) slowly rose from a low at birth but remained far below adult responses even at 2 years of age; (iv) inducing IL-10 production steadily declined from a high around birth to adult levels by 1 or 2 years of age, and; (v) leading to production of TNF-α or IL-1β varied by stimuli. Our data contradict the notion of a linear progression from an 'immature' neonatal to a 'mature' adult pattern, but instead indicate the existence of qualitative and quantitative age-specific changes in innate immune reactivity in response to TLR stimulation.

Macrophages enforce antitumor immunity by engulfing and killing tumor cells. Although these functions are determined by a balance of stimulatory and inhibitory signals, the role of macrophage metabolism is unknown. Here, we study the capacity of macrophages to circumvent inhibitory activity mediated by CD47 on cancer cells. We show that stimulation with a CpG oligodeoxynucleotide, a Toll-like receptor 9 agonist, evokes changes in the central carbon metabolism of macrophages that enable antitumor activity, including engulfment of CD47 + cancer cells. CpG activation engenders a metabolic state that requires fatty acid oxidation and shunting of tricarboxylic acid cycle intermediates for de novo lipid biosynthesis. This integration of metabolic inputs is underpinned by carnitine palmitoyltransferase 1A and adenosine tri-phosphate citrate lyase, which, together, impart macrophages with antitumor potential capable of overcoming inhibitory CD47 on cancer cells. Our findings identify central carbon metabolism to be a novel determinant and potential therapeutic target for stimulating antitumor activity by macrophages. Macrophages have important antitumor functions but can be evaded by tumor-expressed CD47-dependent ‘don’t-eat-me’ signals. Beatty and colleagues demonstrate that the Toll-like receptor 9 ligand CpG metabolically reprograms macrophages to overcome don’t-eat-me signals.

Over the past decade, tremendous progress has taken place in tumour immunotherapy, relying on the fast development of combination therapy strategies that target multiple immunosuppressive signaling pathways in the immune system of cancer patients to achieve a high response rate in clinical practice. Toll-like receptor 9 (TLR9) agonists have been extensively investigated as therapeutics in monotherapy or combination therapies for the treatment of cancer, infectious diseases and allergies. TLR9 agonists monotherapy shows limited efficacy in cancer patients; whereas, in combination with other therapies including antigen vaccines, radiotherapies, chemotherapies and immunotherapies exhibit great potential. Synthetic unmethylated CpG oligodeoxynucleotide (ODN), a commonly used agonist for TLR9, stimulate various antigen-presenting cells in the tumour microenvironment, which can initiate innate and adaptive immune responses. Novel combination therapy approaches, which co-deliver immunostimulatory CpG-ODN with other therapeutics, have been tested in animal models and early human clinical trials to induce anti-tumour immune responses. In this review, we describe the basic understanding of TLR9 signaling pathway; the delivery methods in most studies; discuss the key challenges of each of the above mentioned TLR9 agonist-based combination immunotherapies and provide an overview of the ongoing clinical trial results from CpG-ODN based combination therapies in cancer patients.

Purpose The immune modulatory oligonucleotide IMO-2055 (EMD 1201081) is a phosphorothioate oligodeoxynucleotide agonist of Toll-like receptor 9. In preclinical studies, IMO-2055 was shown to activate natural killer cells and to support the antitumor activity of monoclonal antibodies. This phase 1b, open-label, 3 + 3 dose-escalation trial was performed to determine the recommended phase 2 dose of IMO-2055 combined with FOLFIRI/cetuximab in patients with previously treated, advanced/metastatic colorectal cancer (NCT00719199). Methods Patients received 14-day cycles of cetuximab (days 1/8; 400 mg/m 2 day 1 cycle 1, 250 mg/m 2 for subsequent days/cycles), irinotecan (day 1; 180 mg/m 2 ), folinic acid (day 1; 400 mg/m 2 racemic or 200 mg/m 2 l -form), 5-fluorouracil (day 1; 400 mg/m 2 intravenous bolus, followed by 2,400 mg/m 2 as 46-h infusion), and escalating IMO-2055 doses (days 1/8; 0.16, 0.32, 0.48 mg/kg). Fifteen patients received IMO-2055, including six, three, and six patients who were treated at the dose levels 0.16, 0.32, and 0.48 mg/kg, respectively. Results One dose-limiting toxicity was observed (grade 3 fatigue; at dose level 0.16 mg/kg). The most common adverse events were injection site reactions, diarrhea, fatigue, hypomagnesemia, and stomatitis. One patient achieved a confirmed partial response; 12 had stable disease, including five with stable disease ≥4.0 months. Conclusions IMO-2055 combined with FOLFIRI/cetuximab was well tolerated at all dose levels tested. IMO-2055 0.48 mg/kg was considered as the recommended phase 2 dose.

Toll-like receptors (TLRs) are a family of pattern recognition receptors and part of the first line of defense against invading microbes. In humans, we know of 10 different TLRs, which are expressed to varying degrees in immune cell subsets. Engaging TLRs through their specific ligands leads to activation of the innate immune system and secondarily priming of the adaptive immune system. Because of these unique properties, TLR agonists have been investigated as immunotherapy in cancer treatment for many years, but in recent years there has also been growing interest in the use of TLR agonists in the context of human immunodeficiency virus type 1 (HIV-1) cure research. The primary obstacle to curing HIV-1 is the presence of a latent viral reservoir in transcriptionally silent immune cells. Due to the very limited transcription of the integrated HIV-1 proviruses, latently infected cells cannot be targeted and cleared by immune effector mechanisms. TLR agonists are very interesting in this context because of their potential dual effects as latency reverting agents (LRAs) and immune modulatory compounds. Here, we review preclinical and clinical data on the impact of TLR stimulation on HIV-1 latency as well as antiviral and HIV-1-specific immunity. We also focus on the promising role of TLR agonists in combination strategies in HIV-1 cure research. Different combinations of TLR agonists and broadly neutralizing antibodies or TLRs agonists as adjuvants in HIV-1 vaccines have shown very encouraging results in non-human primate experiments and these concepts are now moving into clinical testing.

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-a and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required.

CpG-oligodeoxynucleotides (CpG-ODNs) are potent immune stimuli currently under investigation as antimicrobial agents for different species. Toll-like receptor (TLR) 9 and TLR21 are the cellular receptors of CpG-ODN in mammals and chickens, respectively. The avian genomes lack TLR9 , whereas mammalian genomes lack TLR21 . Although fish contain both of these genes, the biological functions of fish TLR9 and TLR21 have not been investigated previously. In this study, we comparatively investigated zebrafish TLR9 (zebTLR9) and TLR21 (zebTLR21). The two TLRs have similar expression profiles in zebrafish. They are expressed during early development stages and are preferentially expressed in innate immune function-related organs in adult fish. Results from cell-based activation assays indicate that these two zebrafish TLRs are functional, responding to CpG-ODN but not to other TLR ligands. zebTLR9 broadly recognized CpG-ODN with different CpG motifs, but CpG-ODN with GACGTT or AACGTT had better activity to this TLR. In contrast, zebTLR21 responded preferentially to CpG-ODN with GTCGTT motifs. The distinctive ligand recognition profiles of these two TLRs were determined by their ectodomains. Activation of these two TLRs by CpG-ODN occurred inside the cells and was modulated by UNC93B1. The biological functions of these two TLRs were further investigated. The CpG-ODNs that activate both zebTLR9 and zebTLR21 were more potent than others that activate only zebTLR9 in the activation of cytokine productions and were more bactericidal in zebrafish. These results suggest that zebTLR9 and zebTLR21 cooperatively mediate the antimicrobial activities of CpG-ODN. Overall, this study provides a molecular basis for the activities of CpG-ODN in fish.

Hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are 2 similar diseases characterized by a cytokine storm, overwhelming inflammation, multiorgan dysfunction, and death. Animal models of HLH suggest that disease is driven by IFN-γ produced by CD8⁺ lymphocytes stimulated by persistent antigen exposure. In these models and patients with \"primary\" HLH, the antigen persists due to genetic defects, resulting in ineffective cytotoxic responses by CD8⁺ T cells and poor pathogen clearance. However, infectious triggers are often not identified in patients with MAS, and some patients with HLH or MAS lack defects in cytotoxic T cell killing. Herein, we show that repeated stimulation of TLR9 produced an HLH/MAS-like syndrome on a normal genetic background, without exogenous antigen. Like previous HLH models, TLR9-induced MAS was IFN-γ dependent; however, unlike other models, disease did not require lymphocytes. We further showed that IL-10 played a protective role in this model and that blocking IL-10 signaling led to the development of hemophagocytosis. IL-10 may therefore be an important target for the development of effective therapeutics for MAS. Our data provide insight into MAS-like syndromes in patients with inflammatory diseases in which there is chronic innate immune activation but no genetic defects in cytotoxic cell function.