Influenza A viruses (IAVs) cause seasonal influenza epidemics and pose significant threats to public health. However, seasonal influenza vaccines often elicit strain-specific immune responses and confer little protection against mismatched strains. There is an urgent need to develop universal influenza vaccines against emerging and potentially re-emerging influenza virus infections. Multiepitope vaccines combining multiple conserved epitopes can induce more robust and broader immune responses and provide a potential solution.

Here, we demonstrated that an HA chimeric multiepitope nanoparticle vaccine, delivered intranasally conferred broad protection against challenges with various influenza viruses in mice. The nanoparticle vaccine co-expresses the ectodomain of haemagglutinin (H), three repeated highly conserved ectodomains of matrix protein 2 (M), and the M-cell-targeting ligand Co4B (C) in a baculovirus-insect cell system. These elements (C, H and M) were presented on the surface of self-assembling ferritin (f) in tandem to generate a nanoparticle denoted as CHM-f. Intranasal vaccination with CHM-f nanoparticles elicited robust humoral and cellular immune responses, conferring complete protection against a variety of IAVs, including the A/PR8/34 H1N1 strain, the swine flu H3N2 strain, the avian flu H5N8 strain, and H9N2. When CHM-f nanoparticles adjuvanted with CpG IAMA-002, the weight loss protective effect, cellular immune responses and mucosal IgA responses were significantly augmented. Compared with controls, mice immunized with CHM-f nanoparticles with or without CpG IAMA-002 showed significant reductions in weight loss, lung viral titres and pathological changes.

These results suggest that CHM-f nanoparticle with or without CpG IAMA-002 is a promising candidate as a universal influenza vaccine.

In vitro systems based on human peripheral blood mononuclear cells (PBMCs) can bridge the gap between preclinical and clinical vaccine evaluation but have so far mainly been exploited to assess vaccine effects on antigen-presenting cells and T cells. Our study aimed to assess whether B cells present in PBMCs also respond to vaccines and reflect the effects of different vaccine formulations and adjuvants. We stimulated PBMCs with whole inactivated virus (WIV) or split virus (SIV) H5N1 influenza vaccine, with or without the addition of the adjuvant cytosine phosphoguanine (CpG) ODN 2395, and collected the cells and supernatants at different timepoints. B cell subsets were measured by flow cytometry, immunoglobulin (IgG) levels by ELISA, B cell-related genes by qPCR, and cytokine levels by intracellular staining. B cells differentiated more readily to plasmablasts and plasma cells and produced more IgG when PBMC cultures were stimulated with WIV than when stimulated with SIV. In line, PRDM1, XBP1, and AICDA, genes associated with the differentiation of B cells to antibody-secreting cells, were expressed at higher levels in WIV- than in SIV-stimulated PBMCs. The combination of WIV and CpG consistently induced the highest levels of antibody-secreting cell differentiation, IgG production, and B-cells secreting IL-6 and IL-10. Taken together, B cells in human PBMC cultures show distinct responses to different types of vaccines and vaccine/CpG combinations. This underlines the suitability of unfractionated PBMCs for evaluating vaccine effects on different types of human immune cells before running costly clinical trials.

DNA sensors generally initiate innate immune responses through the production of type I interferons. While extensively studied for host defense against invading pathogens, emerging evidence highlights the involvement of DNA sensors in metabolic and cardiovascular diseases. Elevated levels of modified, damaged, or ectopically localized self-DNA and non-self-DNA have been observed in patients and animal models with obesity, diabetes, fatty liver disease, and cardiovascular disease. The accumulation of cytosolic DNA aberrantly activates DNA signaling pathways, driving the pathological progression of these disorders. This review highlights the roles of specific DNA sensors, such as cyclic AMP-GMP synthase and stimulator of interferon genes (cGAS-STING), absent in melanoma 2 (AIM2), toll-like receptor 9 (TLR9), interferon gamma-inducible protein 16 (IFI16), DNA-dependent protein kinase (DNA-PK), and DEAD-box helicase 41 (DDX41) in various metabolic disorders. We explore how DNA signaling pathways in both immune and non-immune cells contribute to the development of these diseases. Furthermore, we discuss the intricate interplay between metabolic stress and immune responses, offering insights into potential therapeutic targets for managing metabolic and cardiovascular disorders. Understanding the mechanisms of DNA sensor signaling in these contexts provides a foundation for developing novel interventions aimed at mitigating the impact of these pervasive health issues.

Cancer immunotherapy has emerged as a promising strategy to treat cancer patients. Among the wide range of immunological approaches, cancer vaccines have been investigated to activate and expand tumor-reactive T cells. However, most cancer vaccines have not shown significant clinical benefit as monotherapies. This is likely due to the antigen targets of vaccines, "self" proteins to which there is tolerance, as well as to the immunosuppressive tumor microenvironment. To help circumvent immune tolerance and generate effective immune responses, adjuvants for cancer vaccines are necessary. One representative adjuvant family is Toll-Like receptor (TLR) agonists, synthetic molecules that stimulate TLRs. TLRs are the largest family of pattern recognition receptors (PRRs) that serve as the sensors of pathogens or cellular damage. They recognize conserved foreign molecules from pathogens or internal molecules from cellular damage and propel innate immune responses. When used with vaccines, activation of TLRs signals an innate damage response that can facilitate the development of a strong adaptive immune response against the target antigen. The ability of TLR agonists to modulate innate immune responses has positioned them to serve as adjuvants for vaccines targeting infectious diseases and cancers. This review provides a summary of various TLRs, including their expression patterns, their functions in the immune system, as well as their ligands and synthetic molecules developed as TLR agonists. In addition, it presents a comprehensive overview of recent strategies employing different TLR agonists as adjuvants in cancer vaccine development, both in pre-clinical models and ongoing clinical trials.

Unmethylated cytosine-phosphate-guanine (CpG) motifs are often found in bacteria and viruses, but are rare in mammals. In mammals, CpG oligodeoxynucleotides (CpG ODN) stimulate the innate immune system via toll-like receptor 9 (TLR9). However, TLR9 is absent in birds; instead, TLR21 serves as the receptor for CpG ODN. While CpG ODN induce behavioral and physiological changes in mammals, there is limited research on their effects on behavioral and physiological parameters in birds. The aim of the present study was to determine whether intraperitoneal injection of K3, a synthetic class B CpG ODN, affected food intake, voluntary activity, cloacal temperature, blood constituents, and feed passage from the crop in chicks (Gallus gallus). Additionally, the effects of K3 (GC), which contains GpC motifs instead of CpG motifs, were investigated to determine the importance of these CpG motifs. Intraperitoneal injection of K3 significantly increased the mRNA expression of interleukin-1β, interleukin-6, interleukin-8, and interferon-γ in the spleen. These changes were not observed with K3 (GC) administration. Intraperitoneal injection of K3 significantly decreased food intake but did not affect voluntary activity. K3 also significantly increased cloacal temperature, tended to increase plasma glucose and corticosterone concentrations and significantly decreased feed passage from the crop. In contrast, K3 (GC) showed no effects on these parameters. These results demonstrate that class B CpG ODN is associated with anorexia, hyperthermia, and reduced feed passage through the digestive tract in chicks during bacterial and viral infections.

The identification of adjuvants to improve vaccination efficacy is a major unmet need. One approach is to augment the functionality of dendritic cells (DCs) by using Toll-like receptor-9 (TLR9) agonists such as cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) as adjuvants. Another approach is adjuvant selection based on production of bioactive interleukin-12 (IL-12). We report a D-peptide isomer, designated D-15800, that induces monocyte differentiation to the DC phenotype in vitro and more effectively stimulates IL-12p70 production upon T cell receptor (TCR) activation than the L-isomer. In the absence of TCR activation and either IL-12p70 or interleukin-2 production, only D-15800 activates CD4+ T and natural killer cells. In the presence of CpG ODN, D-15800 synergistically enhances production of interferon-alpha (IFN-α). Taken together with its biostability in human serum and depot retention upon injection, co-delivery of D-15800 with TLR9 agonists could serve to improve vaccine efficacy.

Unmethylated cytosine-guanine oligodeoxynucleotides (CpG ODNs) have a storied history as agonists for Toll-like receptor 9 (TLR9). CpG ODNs have shown promising antitumor effects in preclinical studies by inducing potent proinflammatory immune responses. However, clinical success has been hindered by inconsistent efficacy and immune-related toxicities caused by systemic exposure to CpG ODNs. We previously identified that glatiramer acetate (GA), an FDA-approved, lysine-rich polypeptide, could complex class B CpG into cationic nanoparticles which persist at the intratumoral injection site while mitigating the induction of systemic proinflammatory cytokines in mouse tumor models. To extend GA applications across subtypes of CpG ODN (class A, B, and C), we evaluated physiochemical properties and identified the immunological signaling of GA and its complexes with different classes of CpG ODNs. We compared the physiochemical characteristics of three types of GA-CpG nanoparticles, followed by assessments of cell uptake efficiency and endolysosomal trafficking. We then performed successive in vitro and in vivo assays to evaluate immunological discrepancies. Complexation with GA preserved the immunological activity of CpG ODN subtypes while encapsulating them into cationic spherical nanoparticles. GA improved the cellular uptake of CpG ODNs, generally increased retention in early endosomes, and amplified immunological responses. A subsequent in vivo experiment confirmed the achievement of potent tumor suppression while mitigating systemic immune-related toxicities. Together, these data help elucidate the noncanonical role of GA to serve as a nucleic acid delivery scaffold that can improve the efficacy and safety of CpG adjuvant for clinical cancer immunotherapy.

Human cells contain two types of adenosine deaminases (ADA) each with unique properties: ADA1, which is present in all cells where it modulates intracellular functions and extracellular signaling, and ADA2, which is secreted by immune cells. The exact intracellular functions of ADA2 remain undetermined and less defined than those of ADA1. ADA2 has distinct characteristics, such as low adenosine affinity, heparin-binding ability, and putative lysosomal entry. Here, we confirm that ADA2 is a lysosomal protein that binds toll-like receptor 9 (TLR9) agonists, specifically CpG oligodeoxynucleotides (CpG ODNs). We show that interferon-alpha (IFN-α) is secreted in response to TLR9 activation by CpG ODNs and natural DNA and markedly increases when ADA2 expression is downregulated in plasmacytoid dendritic cells (pDCs). Additionally, the pretreatment of pDCs with RNA further stimulates IFN-α secretion by pDCs after activation with CpG ODNs. Our findings indicate that ADA2 regulates TLR9 responses to DNA in activated pDCs. In conclusion, decreasing ADA2 expression or blocking it with specific oligonucleotides can enhance IFN-α secretion from pDCs, improving immune responses against intracellular infections and cancer.

Plasmacytoid dendritic cells (pDCs) are multifaceted immune cells executing various innate immunological functions. Their first line of defence consists in type I interferons (I-IFN) production upon nucleic acids sensing through endosomal Toll-like receptor (TLR) 7- and 9-dependent signalling pathways. Type I IFNs are a class of proinflammatory cytokines that have context-dependent functions on cancer immunosurveillance and immunoediting. In the last few years, different studies have reported that pDCs are also able to sense cytosolic DNA through cGAS-STING (stimulator of interferon genes) pathway eliciting a potent I-IFN production independently of TLR7/9. Human pDCs are also endowed with direct effector functions via the upregulation of TRAIL and production of granzyme B, the latter modulated by cytokines abundant in cancer tissues. pDCs have been detected in a wide variety of human malignant neoplasms, including virus-associated cancers, recruited by chemotactic stimuli. Although the role of pDCs in cancer immune surveillance is still uncompletely understood, their spontaneous activation has been rarely documented; moreover, their presence in the tumor microenvironment (TME) has been associated with a tolerogenic phenotype induced by immunosuppressive cytokines or oncometabolites. Currently tested treatment options can lead to pDCs activation and disruption of the immunosuppressive TME, providing a relevant clinical benefit. On the contrary, the antibody-drug conjugates targeting BDCA-2 on immunosuppressive tumor-associated pDCs (TA-pDCs) could be proposed as novel immunomodulatory therapies to achieve disease control in patients with advance stage hematologic malignancies or solid tumors. This Review integrate recent evidence on the biology of pDCs and their pharmacological modulation, suggesting their relevant role at the forefront of cancer immunity.

Potential threat of smallpox bioterrorism and concerns related to the adverse effects of currently licensed live-virus vaccines suggest the need to develop novel vaccines with better efficacy against smallpox. Use of DNA vaccines containing specific antigen-encoding plasmids prevents the risks associated with live-virus vaccines, offering a promising alternative to conventional smallpox vaccines. In this study, we investigated the efficiency of toll-like receptor (TLR) ligands in enhancing the immunogenicity of smallpox DNA vaccines. BALB/c mice were immunized with a DNA vaccine encoding the vaccinia virus L1R protein, along with the cytosine-phosphate-guanine (CpG) motif as a vaccine adjuvant, and their immune response was analyzed. Administration of B-type CpG oligodeoxynucleotides (ODNs) as TLR9 ligands 24 h after DNA vaccination enhanced the Th2-biased L1R-specific antibody immunity in mice. Moreover, B-type CpG ODNs improved the protective effects of the DNA vaccine against the lethal Orthopoxvirus challenge. Therefore, use of L1R DNA vaccines with CpG ODNs as adjuvants is a promising approach to achieve effective immunogenicity against smallpox infection.

Probiotics are widely used in food for their health benefits to the host. Inactivated probiotics also reportedly improve the intestinal environment and immune regulation. Our previous studies showed that heat-killed Lacticaseibacillus paracasei MCC1849 (hk-MCC1849) effectively induced IL-12 production in mouse spleen cells and significantly reduced cold symptoms in clinical trial subjects. To further elucidate the mechanism of host immune regulation by hk-MCC1849, human peripheral blood mononuclear cells (PBMCs) were cocultured with hk-MCC1849. The Toll-like receptor 9 ligands CpG-ODN 2216 and hk-MCC1849 and the heat-killed Lacticaseibacillus rhamnosus ATCC53103 were used as positive and negative controls, respectively. The results showed that, compared with the control, hk-MCC1849 significantly increased the expression of the plasmacytoid dendritic cell (pDC) marker CD86 (p < .0001) and the pDC marker HLA-DR (p < .001) in PBMCs. The expression levels of the IL-12p40, IFNα, IFNα1, IFNγ, and ISG15 genes were significantly increased after coculture with hk-MCC1849 (p < .05, p < .05, p < .05, p < .05, and p < .05, respectively, vs. control). Furthermore, to confirm whether hk-MCC1849 directly interacted with pDCs, DCs were enriched with PBMCs following 24 h of coculture with hk-MCC1849. Phagocytosis of fluorescently labeled hk-MCC1849 by pDCs was observed, and there were significant increases in CD86 (p < .05) and HLA-DR (p < .0001) expression in pDCs. These results suggest that hk-MCC1849 exerts a potential immunomodulatory effect on the host through the activation of peripheral pDCs.

Unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs), which were therapeutic DNA with high immunostimulatory activity, have been applied in widespread applications from basic research to clinics as therapeutic agents for cancer immunotherapy, viral infection, allergic diseases and asthma since their discovery in 1995. The major factors to consider for clinical translation using CpG motifs are the protection of CpG ODNs from DNase degradation and the delivery of CpG ODNs to the Toll-like receptor-9 expressed human B-cells and plasmacytoid dendritic cells. Therefore, great efforts have been devoted to the advances of efficient delivery systems for CpG ODNs. In this review, we outline new horizons and recent developments in this field, providing a comprehensive summary of the nanoparticle-based CpG delivery systems developed to improve the efficacy of CpG-mediated immune responses, including DNA nanostructures, inorganic nanoparticles, polymer nanoparticles, metal-organic-frameworks, lipid-based nanosystems, proteins and peptides, as well as exosomes and cell membrane nanoparticles. Moreover, future challenges in the establishment of CpG delivery systems for immunotherapeutic applications are discussed. We expect that the continuously growing interest in the development of CpG-based immunotherapy will certainly fuel the excitement and stimulation in medicine research.

Immunotherapy has shifted the treatment paradigm for many types of cancer. Unfortunately, the most commonly used immunotherapies, such as immune checkpoint inhibitors (ICI), have yielded limited benefit for most types of soft tissue sarcoma (STS). Radiotherapy (RT) is a mainstay of sarcoma therapy and can induce immune modulatory effects. Combining immunotherapy and RT in STS may be a promising strategy to improve sarcoma response to RT and increase the efficacy of immunotherapy. Most combination strategies have employed immunotherapies, such as ICI, that derepress immune suppressive networks. These have yielded only modest results, possibly due to the limited immune stimulatory effects of RT. Combining RT with immune stimulatory agents has yielded promising preclinical and clinical results but can be limited by the toxic nature of systemic administration of immune stimulants. Using intralesional immune stimulants may generate stronger RT immune modulation and less systemic toxicity, which may be a feasible strategy in accessible tumors such as STS. In this review, we summarize the immune modulatory effects of RT, the mechanism of action of various immune stimulants, including toll-like receptor agonists, and data for combinatorial strategies utilizing these agents.

Chemodynamic therapy (CDT) shows immense potential in cancer treatment as it not only directly kills tumor cells but also induces anti-tumor immune responses. However, the efficacy of CDT is hampered by challenges in targeting CDT catalysts specifically to tumors using nanomaterials, along with the limitations of low H2 O2 levels and short catalyst duration within the tumor microenvironment. In this study, DNA adjuvant hydrogel to arrange a glucose oxidase-ferrocene cascade for continuously generating reactive oxygen species (ROS) from glucose in situ for tumor CDT combined with immunotherapy is employed. By precisely tuning the catalyst spacing with DNA double helix, ROS production efficiency is elevated by up to nine times compared to free catalysts, resulting in stronger immunogenetic cell death. Upon intratumoral injection, the DNA hydrogel system elicited potent anti-tumor immune responses, thereby effectively inhibiting established tumors and rejecting re-challenged tumors. This work offers a novel platform for integrated CDT and immunotherapy in cancer treatment.

Vaccination is a valid strategy to prevent and control newly emerging and reemerging infectious diseases in humans and animals. However, synthetic and recombinant antigens are poor immunogenic to stimulate efficient and protective host immune response. Immunostimulants are indispensable factors of vaccines, which can promote to trigger fast, robust, and long-lasting immune responses. Importantly, immunotherapy with immunostimulants is increasing proved to be an effective and promising treatment of cancer, which could enhance the function of the immune system against tumor cells. Pattern recognition receptors (PRRs) play vital roles in inflammation and are central to innate and adaptive immune responses. Toll-like receptors (TLRs)-targeting immunostimulants have become one of the hotspots in adjuvant research and cancer therapy. Bacterial-origin immunoreactive molecules are usually the ligands of PRRs, which could be fast recognized by PRRs and activate immune response to eliminate pathogens. Varieties of bacterial immunoreactive molecules and bacterial component-mimicking molecules have been successfully used in vaccines and clinical therapy so far. This work provides a comprehensive review of the development, current state, mechanisms, and applications of bacterial-origin immunostimulants. The exploration of bacterial immunoreactive molecules, along with their corresponding mechanisms, holds immense significance in deepening our understanding of bacterial pathogenicity and in the development of promising immunostimulants.

CpG oligodeoxynucleotides (CpG ODNs) boost the humoral and cellular immune responses to antigens through interaction with Toll-like receptor 9 (TLR9). These CpG ODNs have been extensively utilized in human vaccines. In our study, we evaluated five B-type CpG ODNs that have stimulatory effects on pigs by measuring the proliferation of porcine peripheral blood mononuclear cells (PBMCs) and assessing interferon gamma (IFN-γ) secretion. Furthermore, this study examined the immunoenhancing effects of the MF59 and CpG ODNs compound adjuvant in mouse and piglet models of porcine epidemic diarrhea virus (PEDV) subunit vaccine administration. The in vitro screening revealed that the CpG ODN named CpG5 significantly stimulated the proliferation of porcine PBMCs and elevated IFN-γ secretion levels. In the mouse vaccination model, CpG5 compound adjuvant significantly bolstered the humoral and cellular immune responses to the PEDV subunit vaccines, leading to Th1 immune responses characterized by increased IFN-γ and IgG2a levels. In piglets, the neutralizing antibody titer was significantly enhanced with CpG5 compound adjuvant, alongside a considerable increase in CD8+ T lymphocytes proportion. The combination of MF59 adjuvant and CpG5 exhibits a synergistic effect, resulting in an earlier, more intense, and long-lasting immune response in subunit vaccines for PEDV. This combination holds significant promise as a robust candidate for the development of vaccine adjuvant.

Injured tissue predisposes the subject to local and systemic infection. We studied injury-induced immune dysfunction seeking novel means to reverse such predisposition.

Injury mobilizes primitive "DANGER signals" [danger-associated molecular patterns (DAMPs)] activating innate immunocyte (neutrophils, PMN) signaling and function. Mitochondrial formyl peptides activate G -protein coupled receptors (GPCR) like formyl peptide receptor-1. Mitochondrial DNA and heme activate toll-like receptors (TLR9 and TLR2/4). GPCR kinases (GRKs) can regulate GPCR activation.

We studied human and mouse PMN signaling elicited by mitochondrial DAMPs (GPCR surface expression; protein phosphorylation, or acetylation; Ca 2+ flux) and antimicrobial functions [cytoskeletal reorganization, chemotaxis (CTX), phagocytosis, bacterial killing] in cellular systems and clinical injury samples. Predicted rescue therapies were assessed in cell systems and mouse injury-dependent pneumonia models.

Mitochondrial formyl peptides activate GRK2, internalizing GPCRs and suppressing CTX. Mitochondrial DNA suppresses CTX, phagocytosis, and killing through TLR9 through a novel noncanonical mechanism that lacks GPCR endocytosis. Heme also activates GRK2. GRK2 inhibitors like paroxetine restore functions. GRK2 activation through TLR9 prevented actin reorganization, implicating histone deacetylases (HDACs). Actin polymerization, CTX, bacterial phagocytosis, and killing were also rescued, therefore, by the HDAC inhibitor valproate. Trauma repository PMN showed GRK2 activation and cortactin deacetylation, which varied with severity and was most marked in patients developing infections. Either GRK2 or HDAC inhibition prevented loss of mouse lung bacterial clearance, but only the combination rescued clearance when given postinjury.

Tissue injury-derived DAMPs suppress antimicrobial immunity through canonical GRK2 activation and a novel TLR-activated GRK2-pathway impairing cytoskeletal organization. Simultaneous GRK2/HDAC inhibition rescues susceptibility to infection after tissue injury.

Among several types of CpG-ODNs, A/D-type CpG-ODNs have potent adjuvant activity to induce Th-1 immune responses, but exhibit a propensity to aggregate. For the clinical application of A/D-type CpG-ODNs, it is necessary to control such aggregation and obtain a comprehensive understanding of the relationship between their structure and the immune responses. This study revealed that a representative A/D-type CpG ODN, D35, adopted a single-stranded structure in water, while it assembled into aggregates in response to Na+ ions. From polyacrylamide gel electrophoresis and circular dichroism analyses, D35 adopted a homodimeric form (duplex) via palindromic sequences in low-Na+-concentration conditions (10-50 mM NaCl). After replacement of the solution with PBS, quadruplexes began to form in a manner coordinated by Na+, resulting in large aggregates. The duplexes and small aggregates prepared in 50 mM NaCl showed not only high cellular uptake but also high affinity to Toll-like receptor 9 (TLR9) proteins, leading to the production of a large amount of interferon-α for peripheral blood mononuclear cells. The much larger aggregates prepared in 100 mM NaCl were incorporated into cells at a high level, but showed a low ability to induce cytokine production. This suggests that the large aggregates have difficulty inducing TLR9 dimerization, resulting in loss of the stimulation of the cells. We thus succeeded in inducing adequate innate immunity in vitro by controlling and adjusting the formation of D35 aggregates. Therefore, the findings in this study for D35 ODNs could be a vital research foundation for in vivo applications.

Mucosal vaccination appears to be suitable to protect against SARS-CoV-2 infection. In this study, we tested an intranasal mucosal vaccine candidate for COVID-19 that consisted of a cationic liposome containing a trimeric SARS-CoV-2 spike protein and CpG-ODNs, a Toll-like receptor 9 agonist, as an adjuvant. In vitro and in vivo experiments indicated the absence of toxicity following the intranasal administration of this vaccine formulation. First, we found that subcutaneous or intranasal vaccination protected hACE-2 transgenic mice from infection with the wild-type (Wuhan) SARS-CoV-2 strain, as shown by weight loss and mortality indicators. However, when compared with subcutaneous administration, the intranasal route was more effective in the pulmonary clearance of the virus and induced higher neutralizing antibodies and anti-S IgA titers. In addition, the intranasal vaccination afforded protection against gamma, delta, and omicron virus variants of concern. Furthermore, the intranasal vaccine formulation was superior to intramuscular vaccination with a recombinant, replication-deficient chimpanzee adenovirus vector encoding the SARS-CoV-2 spike glycoprotein (Oxford/AstraZeneca) in terms of virus lung clearance and production of neutralizing antibodies in serum and bronchial alveolar lavage (BAL). Finally, the intranasal liposomal formulation boosted heterologous immunity induced by previous intramuscular vaccination with the Oxford/AstraZeneca vaccine, which was more robust than homologous immunity.

Single-stranded phosphorothioate oligonucleotides (PS-oligos) can activate TLR9, leading to an innate immune response. This can occur with PS-oligos containing unmethylated CpG sites, the canonical motif, or PS-oligos that do not contain those motifs (non-CpG). Structural evidence shows that TLR9 contains two PS-oligo binding sites, and recent data suggest that synergistic cooperative activation of TLR9 can be achieved by adding two separate PS-oligos to cells, each engaging with a separate site on TLR9 to enhance TLR9 activation as a pair. Here, we demonstrate and characterize this cooperativity phenomenon using PS-oligos in human cell lines, and we introduce several novel PS-oligo pairs (CpG and non-CpG pairs) that show cooperative activation. Indeed, we find that cooperative PS-oligos likely bind at different sites on TLR9. Interestingly, we find that PS-oligos that generate little TLR9 activation on their own can prime TLR9 to be activated by other PS-oligos. Finally, we determine that previous models of TLR9 activation cannot be used to fully explain data from systems using human TLR9 and PS-oligos. Overall, we reveal new details of TLR9 activation, but we also find that more work needs to be done to determine where certain PS-oligos are binding to TLR9.

Toll like receptor 9 (TLR9) has been considered as a crucial intracellular pattern recognition receptor in the immune system, which can directly or indirectly mediate innate and adaptive immune responses by recognizing CpG DNA in endosomes to initiate its downstream signaling. However, TLR9 can also be expressed on the membrane surface of some immune and non-immune cells, called surface TLR9 (sTLR9), which covers the TLR9 and its immunomodulatory role with a mysterious veil. In this review, we mainly focus on the sTLR9 expressed on neutrophils, B cells and erythrocytes, and its immunomodulatory roles displayed alone or in coordination with endosomal TLR9 (eTLR9), providing a theoretical reference for the application of its modulators.

Immune evasion caused by the paucity of MHCI is a prominent characteristic of pancreatic adenocarcinoma (PAAD), which is thought to underlie dysfunctional even absent adaptive T cell immunity and is responsible for ineffective immunotherapy. Here, we report a ROS-responsive DNA nano-orchestrator to cascade reverse MHC I-associated immune evasion and boost anti-tumor T cell stimulation, stimulating the activation of tumoricidal immunity against PAAD. Chloroquine phosphate (CQP) as an autophagy inhibitor was first encapsulated with ferritin, and via DNA modular self-assembly technology, the generated ferritin nanocores (FNC) were then caged into ROS-responsive CpG-DNA nanoframe. After systemic injection, the FNC-laden DNA nanoframe (FNC@NF) was passively enriched in tumor tissues in which the DNA nanoframe was cleaved upon the ROS stimulation. Oligodeoxynucleotide (ODN) with CpG motifs was detached and functioned as a TLR9 agonist. The liberated FNC was then endocytosed in an actively targeted manner by binding to transferrin receptor 1. In the lysosome, CQP was burst released from FNC due to acid-triggering. Through CQP-mediated autophagy abrogation, MHC-I molecules were preserved. We demonstrated that cascade inhibiting autophagy and boosting TLR9 stimulation via our proposed DNA-based hybrid nanosystem restored MHC I on the tumor cell surface and reshaped the antigen presentation of DCs, and ultimately reversed immune evasion and synergistically reinforced the activation of cytotoxic T cells against PAAD cells. In sum, our work provides an alternative strategy for cascade reversing immune evasion and boosting anti-tumor T cell stimulation and holds great potential for pancreatic cancer immunotherapy. STATEMENT OF SIGNIFICANCE: A DNA nano-orchestrator was created by sequentially assembling chloroquine phosphate-laden ferritin nanocores with ROS-responsive CpG-DNA nanoframe. Through cascade inhibiting autophagy and boosting TLR9 stimulation, the nano-orchestrator efficiently reversed MHC I-associated immune evasion and augmented anti-tumor T cell stimulation, which ultimately activated tumoricidal immunity against pancreatic adenocarcinoma.

Vaccines comprising innovative adjuvants are rapidly reaching advanced translational stages, such as the authorized nanotechnology adjuvants in mRNA vaccines against COVID-19 worldwide, offering new strategies to effectively combat diseases threatening human health. Adjuvants are vital ingredients in vaccines, which can augment the degree, extensiveness, and longevity of antigen specific immune response. The advances in the modulation of physicochemical properties of nanoplatforms elevate the capability of adjuvants in initiating the innate immune system and adaptive immunity, offering immense potential for developing vaccines against hard-to-target infectious diseases and cancer. In this review, we provide an essential introduction of the basic principles of prophylactic and therapeutic vaccination, key roles of adjuvants in augmenting and shaping immunity to achieve desired outcomes and effectiveness, and the physiochemical properties and action mechanisms of clinically approved adjuvants for humans. We particularly focus on the preclinical and clinical progress of highly immunogenic emerging nanotechnology adjuvants formulated in vaccines for cancer treatment or infectious disease prevention. We deliberate on how the immune system can sense and respond to the physicochemical cues (e.g., chirality, deformability, solubility, topology, and chemical structures) of nanotechnology adjuvants incorporated in the vaccines. Finally, we propose possible strategies to accelerate the clinical implementation of nanotechnology adjuvanted vaccines, such as in-depth elucidation of nano-immuno interactions, antigen identification and optimization by the deployment of high-dimensional multiomics analysis approaches, encouraging close collaborations among scientists from different scientific disciplines and aggressive exploration of novel nanotechnologies.

The emerging virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2 virus), agent of COVID-19, appeared in December 2019 in Wuhan, China, and became a serious threat to global health and public safety. Many COVID-19 vaccines have been approved and licensed around the world. Most of the developed vaccines include S protein and induce an antibody-based immune response. Additionally, T-cell response to the SARS-CoV-2 antigens could be beneficial for combating the infection. The type of immune response is greatly dependent not only on the antigen, but also on adjuvants used in vaccine formulation. Here, we compared the effect of four different adjuvants (AddaS03, Alhydrogel/MPLA, Alhydrogel/ODN2395, Quil A) on the immunogenicity of a mixture of recombinant RBD and N SARS-CoV-2 proteins. We have analyzed the antibody and T-cell response specific to RBD and N proteins and assessed the impact of adjuvants on virus neutralization. Our results clearly indicated that Alhydrogel/MPLA and Alhydrogel/ODN2395 adjuvants elicited the higher titers of specific and cross-reactive antibodies to S protein variants from various SARS-CoV-2 and SARS-CoV-1 strains. Moreover, Alhydrogel/ODN2395 stimulated high cellular response to both antigens, as assessed by IFN-γ production. Importantly, sera collected from mice immunized with RBD/N cocktail in combination with these adjuvants exhibited neutralizing activity against the authentic SARS-CoV-2 virus as well as particles pseudotyped with S protein from various virus variants. The results from our study demonstrate the immunogenic potential of RBD and N antigens and point out the importance of adjuvants selection in vaccine formulation in order to enhance the immunological response. IMPORTANCE Although several COVID-19 vaccines have been approved worldwide, continuous emergence of new SARS-CoV-2 variants calls for new efficient vaccines against them, providing long-lasting immunity. As the immune response after vaccination is dependent not only on antigen used, but also on other vaccine components, e.g., adjuvants, the purpose of this work was to study the effect of different adjuvants on the immunogenicity of RBD/N SARS-CoV-2 cocktail proteins. In this work, it has been shown that immunization with both antigens plus the different adjuvants studied elicited higher Th1 and Th2 responses against RBD and N, which contributed to higher neutralization of the virus. The obtained results can be used for design of new vaccines, not only against SARS-CoV-2, but also against other important viral pathogens.

Glucose-regulated protein-78 (Grp78) is an endoplasmic reticulum chaperone, which is secreted by cells and associates with cell surfaces, where it functions as a receptor for activated α2 -macroglobulin (α2 M) and tissue-type plasminogen activator (tPA). In macrophages, α2 M and tPA also bind to the transmembrane receptor, LDL receptor-related protein-1 (LRP1), activating a cell-signaling receptor assembly that includes the NMDA receptor (NMDA-R) to suppress innate immunity. Herein, we demonstrate that an antibody targeting Grp78 (N88) inhibits NFκB activation and expression of proinflammatory cytokines in bone marrow-derived macrophages (BMDMs) treated with the toll-like receptor-4 (TLR4) ligand, lipopolysaccharide, or with agonists that activate TLR2, TLR7, or TLR9. Pharmacologic inhibition of the NMDA-R or deletion of the gene encoding LRP1 (Lrp1) in BMDMs neutralizes the activity of N88. The fibrinolysis protease inhibitor, plasminogen activator inhibitor-1 (PAI1), has been implicated in diverse diseases including metabolic syndrome, cardiovascular disease, and type 2 diabetes. Deletion of Lrp1 independently increased expression of PAI1 and PAI2 in BMDMs, as did treatment of wild-type BMDMs with TLR agonists. tPA, α2 M, and N88 inhibited expression of PAI1 and PAI2 in BMDMs treated with TLR-activating agents. Inhibiting Src family kinases blocked the ability of both N88 and tPA to function as anti-inflammatory agents, suggesting that the cell-signaling pathway activated by tPA and N88, downstream of LRP1 and the NMDA-R, may be equivalent. We conclude that targeting cell-surface Grp78 may be effective in suppressing innate immunity by a mechanism that requires LRP1 and the NMDA-R.

Maternal-derived antibodies (MDAs) are one of reasons why vaccination with the H9N2 inactivated whole virus (IWV) vaccine failed in poultry. Unmethylated CpG motif-containing oligodeoxynucleotides (CpG ODN) shows great potential to overcome MDAs interference in mammals, but whether it has similar characteristics in poultry is still unknown. In the present study, different classes and various copies of CpG ODN motifs were cloned into two different plasmids (pCDNA3.1 or T vector). Immunomodulatory activities and immunoadjuvant efficacy of these CpG ODN plasmids were tested in vitro and in vivo in the presence of passively transferred antibodies (PTAs) that were used to mimic MDAs. Results showed that the T vector enriched with 30 copies of CpG-A ODN and 20 copies of CpG-B ODN (T-CpG-AB) significantly up-regulated mRNA expression of chicken-interferon-α (ch-IFN-α), chicken-interferon-β (ch-IFN-β) and chicken-interleukin-12 protein 40 (ch-IL-12p40). When administered as adjuvant of the H9N2 IWV vaccine, the minimal dose of T-CpG-AB plasmid was 30 µg per one-day-old chicken, which could induce strong humoral immune responses in the presence of PTAs. Furthermore, T-CpG-AB plasmid-based vaccine triggered both strong humoral immune responses and cytokines expression in the presence of PTAs in chickens. Overall, our findings suggest that T-CpG-AB plasmid can be an excellent adjuvant candidate for the H9N2 IWV vaccine to overcome MDAs interference in chickens.

Toll-like receptors (TLRs), which serve as a bridge between innate and adaptive immunity, may be viable treatment targets. TLRs are the first line of defense against microbes and activate signaling cascades that induce immune and inflammatory responses. Patients with "hot" versus "cold" tumors may respond more favorably to immune checkpoint inhibition, and through their downstream effects, TLR agonists have the potential to convert "cold tumors" into "hot tumors" making TLRs in combination with immune checkpoint inhibitors, potential targets for cancer therapies. Imiquimod is a topical TLR7 agonist, approved by the FDA for antiviral and skin cancer treatments. Other TLR adjuvants are used in several vaccines including Nu Thrax, Heplisav, T-VEC, and Cervarix. Many TLR agonists are currently in development as both monotherapy and in combination with immune checkpoint inhibitors. In this review, we describe the TLR agonists that are being evaluated clinically as new therapies for solid tumors.

Three-dimensional wireframe DNA origami have programmable structural and sequence features that render them potentially suitable for prophylactic and therapeutic applications. However, their innate immunological properties, which stem from parameters including geometric shape and cytosine-phosphate-guanine dinucleotide (CpG) content, remain largely unknown. Here, we investigate the immunostimulatory properties of 3D wireframe DNA origami on the TLR9 pathway using both reporter cell lines and primary immune cells. Our results suggest that bare 3D polyhedral wireframe DNA origami induce minimal TLR9 activation despite the presence of numerous internal CpG dinucleotides. However, when displaying multivalent CpG-containing ssDNA oligos, wireframe DNA origami induce robust TLR9 pathway activation, along with enhancement of downstream immune response as evidenced by increases in Type I and Type III interferon (IFN) production in peripheral blood mononuclear cells. Further, we find that CpG copy number and spatial organization each contribute to the magnitude of TLR9 signaling and that NANP-attached CpGs do not require phosphorothioate stabilization to elicit signaling. These results suggest key design parameters for wireframe DNA origami that can be programmed to modulate immune pathway activation controllably for prophylactic and therapeutic applications.

Tumor immunotherapy is designed to control malignancies through the host immune response but requires circumventing tumor-dysregulated immunomodulation through immunostimulation, relieving immunorepression, or a combination of both approaches. Here we designed and characterized cavrotolimod (formerly AST-008), an immunostimulatory spherical nucleic acid (SNA) compound targeting Toll-like receptor 9 (TLR9). We assessed the safety and pharmacodynamic (PD) properties of cavrotolimod in healthy participants in a first-in-human Phase 1 study under protocol AST-008-101 (NCT03086278; https://clinicaltrials.gov/ct2/show/NCT03086278).

Healthy participants aged 18 to 40 years were enrolled to evaluate four dose levels of cavrotolimod across four cohorts. Each cohort included four participants, and all received a single subcutaneous dose of cavrotolimod. The dose levels were 5, 10, 12.5 and 18.8 µg/kg.

Cavrotolimod was well tolerated and elicited no serious adverse events or dose limiting toxicities at the doses tested. The results demonstrated that cavrotolimod is a potent innate immune activator, specifically stimulating Th1-type immune responses, and exhibits PD properties that may result in anti-tumor effects in patients with cancer. This study suggests that cavrotolimod is a promising clinical immunotherapy agent.

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.

Single-agent immunotherapy has shown remarkable efficacy in selected cancer entities and individual patients. However, most patients fail to respond. This is likely due to diverse immunosuppressive mechanisms acting in a concerted way to suppress the host anti-tumor immune response. Combination immunotherapy approaches that are effective in such poorly immunogenic tumors mostly rely on precise knowledge of antigenic determinants on tumor cells. Creating an antigen-agnostic combination immunotherapy that is effective in poorly immunogenic tumors for which an antigenic determinant is not known is a major challenge.

We use multiple cell line and poorly immunogenic syngeneic, autochthonous, and autologous mouse models to evaluate the efficacy of a novel combination immunotherapy named tripartite immunotherapy (TRI-IT). To elucidate TRI-ITs mechanism of action we use immune cell depletions and comprehensive tumor and immune infiltrate characterization by flow cytometry, RNA sequencing and diverse functional assays.

We show that combined adoptive cellular therapy (ACT) with lymphokine-activated killer cells, cytokine-induced killer cells, Vγ9Vδ2-T-cells (γδ-T-cells) and T-cells enriched for tumor recognition (CTLs) display synergistic antitumor effects, which are further enhanced by cotreatment with anti-PD1 antibodies. Most strikingly, the full TRI-IT protocol, a combination of this ACT with anti-PD1 antibodies, local immunotherapy of agonists against toll-like receptor 3, 7 and 9 and pre-ACT lymphodepletion, eradicates and induces durable anti-tumor immunity in a variety of poorly immunogenic syngeneic, autochthonous, as well as autologous humanized patient-derived models. Mechanistically, we show that TRI-IT coactivates adaptive cellular and humoral, as well as innate antitumor immune responses to mediate its antitumor effect without inducing off-target toxicity.

Overall, TRI-IT is a novel, highly effective, antigen-agnostic, non-toxic combination immunotherapy. In this study, comprehensive insights into its preclinical efficacy, even in poorly immunogenic tumors, and mode of action are given, so that translation into clinical trials is the next step.

Synthetic or natural materials have been used as vaccines in cancer immunotherapy. However, using them as vaccines necessitates multiple injections or surgical implantations. To tackle such daunting challenges, we develop an injectable macroporous Bombyx mori (B. mori) silk fibroin (SF) microsphere loaded with antigens and immune adjuvants to suppress established tumors with only a single injection. SF microspheres can serve as a scaffold by injection and avoid surgical injury as seen in traditional scaffold vaccines. The macroporous structure of the vaccine facilitates the recruitment of immune cells and promotes the activation of dendritic cells (DCs), resulting in a favorable immune microenvironment that further induces strong humoral and cellular immunity. We have also modified the vaccine into a booster version by simply allowing the antigens to be adsorbed onto the SF microspheres. The booster vaccine highly efficiently suppresses tumor growth by improving the cytotoxic T lymphocyte (CTL) response. In general, these results demonstrate that the macroporous SF microspheres can serve as a facile platform for tumor vaccine therapy in the future. Since the SF microspheres are also potential scaffolds for tissue regeneration, their use as a vaccine platform will enable their applications in eradicating tumors while regenerating healthy tissue to heal the tumor-site cavity.

Recognition of pathogen-derived nucleic acids by host cells is an effective host strategy to detect pathogenic invasion and trigger immune responses. In the context of pathogen-specific pharmacology, there is a growing interest in mapping the interactions between pathogen-derived nucleic acids and host proteins. Insight into the principles of the structural and immunological mechanisms underlying such interactions and their roles in host defense is necessary to guide therapeutic intervention. Here, we discuss the newest advances in studies of molecular interactions involving pathogen nucleic acids and host factors, including their drug design, molecular structure and specific patterns. We observed that two groups of nucleic acid recognizing molecules, Toll-like receptors (TLRs) and the cytoplasmic retinoic acid-inducible gene (RIG)-I-like receptors (RLRs) form the backbone of host responses to pathogen nucleic acids, with additional support provided by absent in melanoma 2 (AIM2) and DNA-dependent activator of Interferons (IFNs)-regulatory factors (DAI) like cytosolic activity. We review the structural, immunological, and other biological aspects of these representative groups of molecules, especially in terms of their target specificity and affinity and challenges in leveraging host-pathogen protein-nucleic acid interactions (HP-PNI) in drug discovery.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in pigs of various ages, especially in suckling piglets, and there are no effective measures to prevent and control PDCoV currently. In this study, two adjuvants Al(OH)₃ and ODN2395 working through different mechanisms were used to prepare inactivated PDCoV vaccines, and the immune effects of PDCoV inactivated vaccines were assessed in mice. From the results, we found that both PDCoV/Al(OH)₃ vaccine and PDCoV/2395 vaccine could induce IgG and neutralizing antibodies with high levels in mice. At the same time, cytokines of IFN-γ, IL-4 and chemokine ligand of CXCL13 in serum were significantly increased after immunization, and reached the highest levels in PDCoV/2395 vaccine group, which suggested that PDCoV/2395 could promote the production of both Th1 and Th2 polarized cytokines. In addition, histopathological observations showed that vaccination helped mice resist PDCoV infection. These results indicated that both the two inactivated vaccines have good immune effects. Moreover, the PDCoV/2395 vaccine worked better than the PDCoV/Al(OH)₃ vaccine for PDCoV/2395 having the good ability to induce both humoral and cellular immunogenicity. The PDCoV/2395 inactivated vaccine developed in this study might be an effective tool for the prevention of PDCoV infection.

DNA origami (DO) nanotechnology enables the construction of precise nanostructures capable of functionalization with small molecule drugs, nucleic acids, and proteins, suggesting a promising platform for biomedical applications. Despite the potential for drug and vaccine delivery, the impact of DO vehicles on immunogenicity in vivo is not well understood. Here, two DO vehicles, a flat triangle and a nanorod, at varying concentrations are evaluated in vitro and with a repeated dosing regimen administered at a high dose in vivo to study early and late immunogenicity. The studies show normal CD11b⁺ myeloid cell populations preferentially internalize DO in vitro. DO structures distribute well systemically in vivo, elicit a modest pro-inflammatory immune response that diminishes over time and are nontoxic as shown by weight, histopathology, lack of cytokine storm, and a complete biochemistry panel at the day 10 end point. The results take critical steps to characterize the biological response to DO and suggest that DO vehicles represent a promising platform for drug delivery and vaccine development where immunogenicity should be a key consideration.

Cancer immunotherapy is a powerful treatment strategy that mobilizes the immune system to fight disease. Cancer vaccination is one form of cancer immunotherapy, where spatiotemporal control of the delivery of tumor-specific antigens, adjuvants, and/or cytokines has been key to successfully activating the immune system. Nanoscale materials that take advantage of chemistry to control the nanoscale structural arrangement, composition, and release of immunostimulatory components have shown significant promise in this regard. In this Outlook, we examine how the nanoscale structure, chemistry, and composition of immunostimulatory compounds can be modulated to maximize immune response and mitigate off-target effects, focusing on spherical nucleic acids as a model system. Furthermore, we emphasize how chemistry and materials science are driving the rational design and development of next-generation cancer vaccines. Finally, we identify gaps in the field that should be addressed moving forward and outline future directions to galvanize researchers from multiple disciplines to help realize the full potential of this form of cancer immunotherapy through chemistry and rational vaccinology.