Layer-by-layer nanoparticle encapsulating all-trans retinoic acid and CpG as a mucosal adjuvant targeting colorectal cancer.

Colorectal cancer (CRC) ranks among the most prevalent cancers globally, demanding innovative therapeutic strategies. Immunotherapy, a promising avenue, employs cancer vaccines to activate the immune system against tumors. However, conventional approaches fall short of eliciting robust responses within the gastrointestinal (GI) tract, where CRC originates. Harnessing the potential of all-trans retinoic acid (ATRA) and cytosine-phosphorothioate-guanine (CpG), we developed layered nanoparticles using a layer-by-layer assembly method to co-deliver these agents. ATRA, crucial for gut immunity, was efficiently encapsulated alongside CpG within these nanoparticles. Administering these ATRA@CpG-NPs, combined with ovalbumin peptide (OVA), effectively inhibited orthotopic CRC growth in mice. Our approach leveraged the inherent benefits of ATRA and CpG, demonstrating superior efficacy in activating dendritic cells, imprinting T cells with gut-homing receptors, and inhibiting tumor growth. This mucosal adjuvant presents a promising strategy for CRC immunotherapy, showcasing the potential for targeting gut-associated immune responses in combating colorectal malignancies.

Mn2+/CpG Oligodeoxynucleotides Codecorated Black Phosphorus Nanosheet Platform for Enhanced Antitumor Potency in Multimodal Therapy.

Manganese ions (Mn2+)-coordinated nanoparticles have emerged as a promising class of antitumor nanotherapeutics, capable of simultaneously disrupting the immunosuppressive tumor microenvironment (TME) and triggering the stimulator of interferon genes (STING) pathway-dependent antitumor immunity. However, the activation of STING signaling by Mn2+-based monotherapies is suboptimal for comprehensive stimulation of antigen presenting cells and reversal of immunosuppression in the TME. Here, we report the design of a Mn2+/CpG oligodeoxynucleotides (ODNs) codecorated black phosphorus nanosheet (BPNS@Mn2+/CpG) platform based on the Mn2+ modification of BPNS and subsequent adsorption of synthetic CpG ODNs. The coordination of Mn2+ significantly improved the stability of BPNS and the adsorption of CpG ODNs. The acidic TME and endosomal compartments can disrupt the Mn2+ coordination, triggering pH-responsive release of CpG ODNs and Mn2+ to effectively activate the Toll-like receptor 9 and STING pathways. As a result, M2-type macrophages and immature dendritic cells were strongly stimulated in the TME, thereby increasing T lymphocyte infiltration and reversing the immunosuppression within the TME. Phototherapy and chemodynamic therapy, utilizing the BPNS@Mn2+/CpG platform, have demonstrated efficacy in inducing immunogenic cell death upon 808 nm laser irradiation. Importantly, the treatment of BPNS@Mn2+/CpG with laser irradiation exhibited significant therapeutic efficacy against the irradiated primary tumor and effectively suppressed the growth of nonirradiated distant tumor. Moreover, it induced a robust immune memory, providing long-lasting protection against tumor recurrence. This study demonstrated the enhanced antitumor potency of BPNS@Mn2+/CpG in multimodal therapy, and its proof-of-concept application as a metal ion-modified BPNS material for effective DNA/drug delivery and immunotherapy.

Comparative study of the impact of dietary supplementation with different types of CpG oligodeoxynucleotides (CpG ODNs) on enhancing intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles in Pacific white shrimp (Litopenaeus vannamei).

The CpG oligodeoxynucleotides (CpG ODNs) reportedly possess the capacity to strengthen immunity in mammals. This experiment was conducted to evaluate the impact of dietary supplementation with 17 types of CpG ODNs on intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles of the shrimp Litopenaeus vannamei. Diets including 50 mg kg-1 CpG ODNs wrapped in egg whites were prepared and divided into 17 different groups, with 2 control groups (normal feed and feed with egg whites). These CpG ODNs supplemented diets and the control diets were fed to L. vannamei (5.15 ± 0.54 g) three times daily at 5%-8% shrimp body weight for three weeks. The results of consecutive detection of intestinal microbiota by 16S rDNA sequencing indicated that 11 of the 17 types of CpG ODNs significantly enhanced intestinal microbiota diversity, increased the populations of several probiotic bacteria, and activated possible mechanisms relevant to diseases. The immune-related genes expression and antioxidant capacity in hepatopancreas further demonstrated that the 11 types of CpG ODNs effectively improved the innate immunity of shrimp. Additionally, histology results showed that the CpG ODNs in the experiment did not damage the tissue structure of hepatopancreas. The results suggest that CpG ODNs could be used as a trace supplement to improve the intestinal health and immunity of shrimp.

Engineered Toll-like Receptor Nanoagonist Binding to Extracellular Matrix Elicits Safe and Robust Antitumor Immunity.

Cancer immunotherapy, such as the Toll-like receptor (TLR) agonist including CpG oligodeoxynucleotide, has shown potency in clinical settings. However, it is still confronted with multiple challenges, which include the limited efficacy and severe adverse events caused by the rapid clearance and systemic diffusion of CpG. Here we report an improved CpG-based immunotherapy approach composed of a synthetic extracellular matrix (ECM)-anchored DNA/peptide hybrid nanoagonist (EaCpG) via (1) a tailor designed DNA template that encodes tetramer CpG and additional short DNA moieties, (2) generation of elongated multimeric CpG through rolling circle amplification (RCA), (3) self-assembly of densely packaged CpG particles composed of tandem CpG building blocks and magnesium pyrophosphate, and (4) incorporation of multiple copies of ECM binding peptide through hybridization to short DNA moieties. The structurally well-defined EaCpG shows dramatically increased intratumoral retention and marginal systemic dissemination through peritumoral administration, leading to potent antitumor immune response and subsequent tumor elimination, with minimal treatment-related toxicity. Combined with conventional standard-of-care therapies, peritumor administration of EaCpG generates systemic immune responses that lead to a curative abscopal effect on distant untreated tumors in multiple cancer models, which is superior to the unmodified CpG. Taken together, EaCpG provides a facile and generalizable strategy to simultaneously potentiate the potency and safety of CpG for combinational cancer immunotherapies.

Optimized Cationic Lipid-assisted Nanoparticle for Delivering CpG Oligodeoxynucleotides to Treat Hepatitis B Virus Infection.

PURPOSE: Hepatitis B virus (HBV) infection is such a global health problem that hundreds of millions of people are HBV carriers. Current anti-viral agents can inhibit HBV replication, but can hardly eradicate HBV. Cytosine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODNs) are an adjuvant that can activate plasmacytoid dendritic cells (pDCs) and conventional dendritic cells (cDCs) to induce therapeutic immunity for HBV eradication. However, efficient delivery of CpG ODNs into pDCs and cDCs remains a challenge. In this study, we constructed a series of cationic lipid-assisted nanoparticles (CLANs) using different cationic lipids to screen an optimal nanoparticle for delivering CpG ODNs into pDCs and cDCs. METHODS: We constructed different CLANCpG using six cationic lipids and analyzed the cellular uptake of different CLANCpG by pDCs and cDCs in vitro and in vivo, and further analyzed the efficiency of different CLANCpG for activating pDCs and cDCs in both wild type mice and HBV-carrier mice. RESULTS: We found that CLAN fabricated with 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP) showed the highest efficiency for delivering CpG ODNs into pDCs and cDCs, resulting in strong therapeutic immunity in HBV-carrier mice. By using CLANCpG as an immune adjuvant in combination with the injection of recombinant hepatitis B surface antigen (rHBsAg), HBV was successfully eradicated and the chronic liver inflammation in HBV-carrier mice was reduced. CONCLUSION: We screened an optimized CLAN fabricated with DOTAP for efficient delivery of CpG ODNs to pDCs and cDCs, which can act as a therapeutic vaccine adjuvant for treating HBV infection.

Combinational therapy with Myc decoy oligodeoxynucleotides encapsulated in nanocarrier and X-irradiation on breast cancer cells.

The Myc gene is the essential oncogene in triple-negative breast cancer (TNBC). This study investigates the synergistic effects of combining Myc decoy oligodeoxynucleotides-encapsulated niosomes-selenium hybrid nanocarriers with X-irradiation exposure on the MDA-MB-468 cell line. Decoy and scramble ODNs for Myc transcription factor were designed and synthesized based on promoter sequences of the Bcl2 gene. The nanocarriers were synthesized by loading Myc ODNs and selenium into chitosan (Chi-Se-DEC), which was then encapsulated in niosome-nanocarriers (NISM@Chi-Se-DEC). FT-IR, DLS, FESEM, and hemolysis tests were applied to confirm its characterization and physicochemical properties. Moreover, cellular uptake, cellular toxicity, apoptosis, cell cycle, and scratch repair assays were performed to evaluate its anticancer effects on cancer cells. All anticancer assessments were repeated under X-ray irradiation conditions (fractionated 2Gy). Physicochemical characteristics of niosomes containing SeNPs and ODNs showed that it is synthesized appropriately. It revealed that the anticancer effect of NISM@Chi-Se-DEC can be significantly improved in combination with X-ray irradiation treatment. It can be concluded that NISM@Chi-Se-DEC nanocarriers have the potential as a therapeutic agent for cancer treatment, particularly in combination with radiation therapy and in-vivo experiments are necessary to confirm the efficacy of this nano-drug.

Non-Modified CpG Oligodeoxynucleotide Forming Guanine-Quadruplex Structure Complexes with ε-Poly-L-Lysine Induce Antibody Production as Vaccine Adjuvants.

Unmethylated cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) induce inflammatory cytokines and type I interferons (IFNs) to activate the immune system. To apply CpG ODNs as vaccine adjuvants, the cellular uptake and stability of phosphodiester-based, non-modified ODNs require further improvement. Previously developed new CpG ODNs forming guanine-quadruplex (G4) structures showed higher nuclease resistance and cellular uptake than linear CpG ODNs; however, the complex formation of G4-CpG ODNs with antigen proteins is necessary for their application as vaccine adjuvants. In this study, we utilized a cationic polymer, ε-poly-L-lysine (ε-PLL), as a carrier for G4-CpG ODNs and antigen. The ε-PLL/G4-CpG ODN complex exhibited enhanced stability against nucleases. Cellular uptake of the ε-PLL/G4-CpG ODN complex positively correlated with the N/P ratio. In comparison to naked G4-CpG ODNs, the ε-PLL/G4-CpG ODN complex induced extremely high levels of interleukin (IL)-6, IL-12, and IFN-ß. Relative immune cytokine production was successfully tuned by N/P ratio modification. Mice with the ε-PLL/G4-CpG ODN/ovalbumin (OVA) complex showed increased OVA-specific immunoglobulin (Ig)G, IgG1, and IgG2c levels, whereas total IgE levels did not increase and weight gain rates were not affected. Therefore, ε-PLL can serve as a safe and effective phosphodiester-based, non-modified CpG ODN delivery system, and the ε-PLL/G4-CpG ODN/antigen complex is a highly promising candidate for vaccine adjuvants and can be further used in clinical research.

Image-guided in situ cancer vaccination with combination of multi-functional nano-adjuvant and an irreversible electroporation technique.

Cancer immunotherapy is a next-generation treatment strategy; however, its side effects limit its clinical translation. Here, a novel combination of a multi-functional nano-adjuvant (M-NA) prepared with an iron oxide/gold core and a cationic polymer shell via multilayer synthesis with CpG oligodeoxynucleotide (CpG-ODN) electrostatically complexed on its surface, and irreversible electroporation (IRE) technique was developed for effective image-guided in situ cancer vaccination. The M-NA can be retained long-term in the dense tumoral extracellular matrix after intratumoral injection and internalized by antigen-presenting cells (APCs). The IRE can induce immunogenic cell death. Indeed, in a mouse tumor model, the M-NA showed longer tumor retention time than free CpG-ODN. Compared with other treatments, the combined treatment significantly inhibited tumor growth with 100% survival rate for ∼60 days. The therapy induced the activation of cytotoxic lymphocytes and the maturation of APCs in vivo. This treatment could be effective in image-guided local cancer immunotherapy.

In situ photothermal nano-vaccine based on tumor cell membrane-coated black phosphorus-Au for photo-immunotherapy of metastatic breast tumors.

Cancer vaccines which can activate antitumor immune response have great potential for metastatic tumors treatment. However, clinical translation of cancer vaccines remained challenging due to weak tumor antigen immunogenicity, inefficient in vivo delivery, and immunosuppressive tumor microenvironment. Nanomaterials-based photothermal treatment (PTT) triggers immunogenic cell death while providing in situ tumor-associated antigens for subsequent anti-tumor immunity. Here, an in situ photothermal nano-vaccine (designated as BCNCCM) based on cancer cell membrane (CCM) was explored by co-encapsulating immune adjuvant CpG oligodeoxynucleotide (ODN) loaded black phosphorus-Au (BP-Au) nanosheets together with an indoleamine 2,3-dioxygenase (IDO) inhibitor (NLG919) by CCM, for the elimination of primary and metastatic breast tumors. The nano-vaccine could be delivered to tumor site selectively by CCM targeting and exhibit vaccine-like functions through the combined effect of in situ generated tumor-associate agents after PTT and immune adjuvant CpG, resulting in trigger of tumor-specific immunity. Furthermore, tumor inhibition was enhanced owing to the reversed immunosuppressive microenvironment mediated by IDO inhibitors. The nano-vaccine not only had good therapeutic effect on primary and metastatic tumors, but also could prevent tumor recurrence by producing systemic immune memory. Therefore, the photothermal nano-vaccine which coordinate in situ vaccine-like function and immune modulation may be a promising stragegy for photo-immunotherapy of metastatic tumors.

CpG ODN (K3)-toll-like receptor 9 agonist-induces Th1-type immune response and enhances cytotoxic activity in advanced lung cancer patients: a phase I study.

BACKGROUND: Cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) (K3)-a novel synthetic single-stranded DNA immune adjuvant for cancer immunotherapy-induces a potential Th1-type immune response against cancer cells. We conducted a phase I study of CpG ODN (K3) in patients with lung cancer to assess its safety and patients' immune responses. METHODS: The primary endpoint was the proportion of dose-limiting toxicities (DLTs) at each dose level. Secondary endpoints included safety profile, an immune response, including dynamic changes in immune cell and cytokine production, and progression-free survival (PFS). In a 3 + 3 dose-escalation design, the dosage levels for CpG ODN (K3) were 5 or 10 mg/body via subcutaneous injection and 0.2 mg/kg via intravenous administration on days 1, 8, 15, and 29. RESULTS: Nine patients (eight non-small-cell lung cancer; one small-cell lung cancer) were enrolled. We found no DLTs at any dose level and observed no serious treatment-related adverse events. The median observation period after registration was 55 days (range: 46-181 days). Serum IFN-α2 levels, but not inflammatory cytokines, increased in six patients after the third administration of CpG ODN (K3) (mean value: from 2.67 pg/mL to 3.61 pg/mL after 24 hours). Serum IFN-γ (mean value, from 9.07 pg/mL to 12.7 pg/m after 24 hours) and CXCL10 levels (mean value, from 351 pg/mL to 676 pg/mL after 24 hours) also increased in eight patients after the third administration. During the treatment course, the percentage of T-bet-expressing CD8+ T cells gradually increased (mean, 49.8% at baseline and 59.1% at day 29, p = 0.0273). Interestingly, both T-bet-expressing effector memory (mean, 52.7% at baseline and 63.7% at day 29, p = 0.0195) and terminally differentiated effector memory (mean, 82.3% at baseline and 90.0% at day 29, p = 0.0039) CD8+ T cells significantly increased. The median PFS was 398 days. CONCLUSIONS: This is the first clinical study showing that CpG ODN (K3) activated innate immunity and elicited Th1-type adaptive immune response and cytotoxic activity in cancer patients. CpG ODN (K3) was well tolerated at the dose settings tested, although the maximum tolerated dose was not determined. TRIAL REGISTRATION: UMIN-CTR number 000023276. Registered 1 September 2016, https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000026649.

Synergy of a STING agonist and an IL-2 superkine in cancer immunotherapy against MHC I-deficient and MHC I+ tumors.

Cyclic dinucleotides (CDN) and Toll-like receptor (TLR) ligands mobilize antitumor responses by natural killer (NK) cells and T cells, potentially serving as complementary therapies to immune checkpoint therapy. In the clinic thus far, however, CDN therapy targeting stimulator of interferon genes (STING) protein has yielded mixed results, perhaps because it initiates responses potently but does not provide signals to sustain activation and proliferation of activated cytotoxic lymphocytes. To improve efficacy, we combined CDN with a half life-extended interleukin-2 (IL-2) superkine, H9-MSA (mouse serum albumin). CDN/H9-MSA therapy induced dramatic long-term remissions of the most difficult to treat major histocompatibility complex class I (MHC I)­deficient and MHC I+ tumor transplant models. H9-MSA combined with CpG oligonucleotide also induced potent responses. Mechanistically, tumor elimination required CD8 T cells and not NK cells in the case of MHC I+ tumors and NK cells but not CD8 T cells in the case of MHC-deficient tumors. Furthermore, combination therapy resulted in more prolonged and more intense NK cell activation, cytotoxicity, and expression of cytotoxic effector molecules in comparison with monotherapy. Remarkably, in a primary autochthonous sarcoma model that is refractory to PD-1 checkpoint therapy, the combination of CDN/H9-MSA with checkpoint therapy yielded long-term remissions in the majority of the animals, mediated by T cells and NK cells. This combination therapy has the potential to activate responses in tumors resistant to current therapies and prevent MHC I loss accompanying acquired resistance of tumors to checkpoint therapy.

Development of Hydrophobic Interaction-based DNA Supramolecules as Efficient Delivery Carriers of CpG DNA to Immune cells.

Unmethylated cytosine-phosphate-guanine (CpG) DNA stimulates mammalian immune cells through recognition by Toll-like receptor 9 (TLR9). Therefore, CpG DNA is expected to be an effective adjuvant for the treatment of immune and allergic diseases. However, challenges, such as low stability against DNase and low delivery efficiency for immune cells, still need to be resolved for the application of CpG DNA. To overcome these challenges, we developed DNA supramolecules consisting of long single-stranded DNA (lss-DNA) synthesized using rolling circle amplification (RCA) and cholesterol-modified DNA (chol-DNA). Lss-DNAs containing multiple CpG motifs were annealed with complementary chol-DNAs to form DNA supramolecules through hydrophobic interactions. Transmission electron microscopy revealed that lss-DNA mixed with chol-DNA formed micrometer-sized DNA supramolecules. The formation of DNA supramolecules increased their stability against DNase compared to lss DNA, which was evaluated using FBS. Furthermore, DNA supramolecules induced three-times higher TNF-α release from RAW264.7 cells than lss-DNA alone. These results demonstrate that DNA supramolecules are efficient delivery carriers of CpG DNA to immune cells.

Aptamer-Aptamer Chimera for Targeted Delivery and ATP-Responsive Release of Doxorubicin into Cancer Cells.

Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5'-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411-ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411-ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin.

Intelligent Tumor Microenvironment-Activated Multifunctional Nanoplatform Coupled with Turn-on and Always-on Fluorescence Probes for Imaging-Guided Cancer Treatment.

Intrinsic tumor microenvironment (TME)-related therapeutic resistance and nontumor-specific imaging have limited the application of imaging-guided cancer therapy. Herein, a TME-responsive MnO2-based nanoplatform coupled with turn-on and always-on fluorescence probes was designed through a facile biomineralization method for imaging-guided photodynamic/chemodynamic/photothermal therapy (PDT/CDT/PTT). After the tumor-targeting delivery of the AuNCs@MnO2-ICG@AS1411 (AMIT) nanoplatform via aptamer AS1411, the TME-responsive dissociation of MnO2 generated sufficient O2 and Mn2+ with the consumption of GSH for improving PDT efficacy and Fenton-like reaction-mediated CDT. Simultaneously, the released small-sized ICG and AuNCs facilitated PDT and PTT efficacy via the deep tumor penetration. Moreover, the turn-on fluorescence of AuNCs revealed the real-time TME-responsive MnO2 degradation process, and the always-on ICG fluorescence enabled the in situ monitoring of the payload distribution in vitro and in vivo. The AMIT NPs also provided magnetic resonance and thermal imaging guidance for the enhanced PDT, CDT, and PTT. Therefore, this all-in-one nanosystem provides a simple and versatile strategy for multiple imaging-guided theranostic applications.

Nucleic acid vaccines and CpG oligodeoxynucleotides for allergen immunotherapy.

PURPOSE OF REVIEW: Molecular forms of allergen-specific immunotherapy (AIT) are continuously emerging to improve the efficacy of the treatment, to shorten the duration of protocols and to prevent any side effects. The present review covers the recent progress in the development of AIT based on nucleic acid encoding allergens or CpG oligodeoxynucleotides (CpG-ODN). RECENT FINDINGS: Therapeutic vaccinations with plasmid deoxyribonucleic acid (DNA) encoding major shrimp Met e 1 or insect For t 2 allergen were effective for the treatment of food or insect bite allergy in respective animal models. DNA expressing hypoallergenic shrimp tropomyosin activated Foxp3+ T regulatory (Treg) cells whereas DNA encoding For t 2 down-regulated the expression of pruritus-inducing IL-31. Co-administrations of major cat allergen Fel d 1 with high doses of CpG-ODN reduced Th2 airway inflammation through tolerance induction mediated by GATA3+ Foxp3hi Treg cells as well as early anti-inflammatory TNF/TNFR2 signaling cascade. Non-canonical CpG-ODN derived from Cryptococcus neoformans as well as methylated CpG sites present in the genomic DNA from Bifidobacterium infantis mediated Th1 or Treg cell differentiation respectively. SUMMARY: Recent studies on plasmid DNA encoding allergens evidenced their therapeutic potential for the treatment of food allergy and atopic dermatitis. Unmethylated or methylated CpG-ODNs were shown to activate dose-dependent Treg/Th1 responses. Large clinical trials need to be conducted to confirm these promising preclinical data. Moreover, tremendous success of messenger ribonucleic acid (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 must encourage as well the re-exploration of mRNA vaccine platform for innovative AIT.

Difluoromethylornithine (DFMO), an Inhibitor of Polyamine Biosynthesis, and Antioxidant N-Acetylcysteine Potentiate Immune Response in Mice to the Recombinant Hepatitis C Virus NS5B Protein.

Hepatitis C virus (HCV) is one of the main triggers of chronic liver disease. Despite tremendous progress in the HCV field, there is still no vaccine against this virus. Potential vaccines can be based on its recombinant proteins. To increase the humoral and, especially, cellular immune response to them, more effective adjuvants are needed. Here, we evaluated a panel of compounds as potential adjuvants using the HCV NS5B protein as an immunogen. These compounds included inhibitors of polyamine biosynthesis and urea cycle, the mTOR pathway, antioxidants, and cellular receptors. A pronounced stimulation of cell proliferation and interferon-γ (IFN-γ) secretion in response to concanavalin A was shown for antioxidant N-acetylcysteine (NAC), polyamine biosynthesis inhibitor 2-difluoromethylornithine (DFMO), and TLR9 agonist CpG ODN 1826 (CpG). Their usage during the immunization of mice with the recombinant NS5B protein significantly increased antibody titers, enhanced lymphocyte proliferation and IFN-γ production. NAC and CpG decreased relative Treg numbers; CpG increased the number of myeloid-derived suppressor cells (MDSCs), whereas neither NAC nor DFMO affected MDSC counts. NAC and DFMO suppressed NO and interleukin 10 (IL-10) production by splenocytes, while DFMO increased the levels of IL-12. This is the first evidence of immunomodulatory activity of NAC and DFMO during prophylactic immunization against infectious diseases.

Adjuvanting a subunit COVID-19 vaccine to induce protective immunity.

The development of a portfolio of COVID-19 vaccines to vaccinate the global population remains an urgent public health imperative1. Here we demonstrate the capacity of a subunit vaccine, comprising the SARS-CoV-2 spike protein receptor-binding domain displayed on an I53-50 protein nanoparticle scaffold (hereafter designated RBD-NP), to stimulate robust and durable neutralizing-antibody responses and protection against SARS-CoV-2 in rhesus macaques. We evaluated five adjuvants including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an α-tocopherol-containing oil-in-water emulsion; AS37, a Toll-like receptor 7 (TLR7) agonist adsorbed to alum; CpG1018-alum, a TLR9 agonist formulated in alum; and alum. RBD-NP immunization with AS03, CpG1018-alum, AS37 or alum induced substantial neutralizing-antibody and CD4 T cell responses, and conferred protection against SARS-CoV-2 infection in the pharynges, nares and bronchoalveolar lavage. The neutralizing-antibody response to live virus was maintained up to 180 days after vaccination with RBD-NP in AS03 (RBD-NP-AS03), and correlated with protection from infection. RBD-NP immunization cross-neutralized the B.1.1.7 SARS-CoV-2 variant efficiently but showed a reduced response against the B.1.351 variant. RBD-NP-AS03 produced a 4.5-fold reduction in neutralization of B.1.351 whereas the group immunized with RBD-NP-AS37 produced a 16-fold reduction in neutralization of B.1.351, suggesting differences in the breadth of the neutralizing-antibody response induced by these adjuvants. Furthermore, RBD-NP-AS03 was as immunogenic as a prefusion-stabilized spike immunogen (HexaPro) with AS03 adjuvant. These data highlight the efficacy of the adjuvanted RBD-NP vaccine in promoting protective immunity against SARS-CoV-2 and have led to phase I/II clinical trials of this vaccine (NCT04742738 and NCT04750343).

Site-specific antigen-adjuvant conjugation using cell-free protein synthesis enhances antigen presentation and CD8+ T-cell response.

Antigen-adjuvant conjugation is known to enhance antigen-specific T-cell production in vaccine models, but scalable methods are required to generate site-specific conjugation for clinical translation of this technique. We report the use of the cell-free protein synthesis (CFPS) platform as a rapid method to produce large quantities (> 100 mg/L) of a model antigen, ovalbumin (OVA), with site-specific incorporation of p-azidomethyl-L-phenylalanine (pAMF) at two solvent-exposed sites away from immunodominant epitopes. Using copper-free click chemistry, we conjugated CpG oligodeoxynucleotide toll-like receptor 9 (TLR9) agonists to the pAMF sites on the mutant OVA protein. The OVA-CpG conjugates demonstrate enhanced antigen presentation in vitro and increased antigen-specific CD8+ T-cell production in vivo. Moreover, OVA-CpG conjugation reduced the dose of CpG needed to invoke antigen-specific T-cell production tenfold. These results highlight how site-specific conjugation and CFPS technology can be implemented to produce large quantities of covalently-linked antigen-adjuvant conjugates for use in clinical vaccines.

Efficient Gene Suppression by DNA/DNA Double-Stranded Oligonucleotide In Vivo.

We recently reported the antisense properties of a DNA/RNA heteroduplex oligonucleotide consisting of a phosphorothioate DNA-gapmer antisense oligonucleotide (ASO) strand and its complementary phosphodiester RNA/phosphorothioate 2'-O-methyl RNA strand. When α-tocopherol was conjugated with the complementary strand, the heteroduplex oligonucleotide silenced the target RNA more efficiently in vivo than did the parent single-stranded ASO. In this study, we designed a new type of the heteroduplex oligonucleotide, in which the RNA portion of the complementary strand was replaced with phosphodiester DNA, yielding an ASO/DNA double-stranded structure. The ASO/DNA heteroduplex oligonucleotide showed similar activity and liver accumulation as did the original ASO/RNA design. Structure-activity relationship studies of the complementary DNA showed that optimal increases in the potency and the accumulation were seen when the flanks of the phosphodiester DNA complement were protected using 2'-O-methyl RNA and phosphorothioate modifications. Furthermore, evaluation of the degradation kinetics of the complementary strands revealed that the DNA-complementary strand as well as the RNA strand were completely cleaved in vivo. Our results expand the repertoire of chemical modifications that can be used with the heteroduplex oligonucleotide technology, providing greater design flexibility for future therapeutic applications.

CpG Oligonucleotides as Vaccine Adjuvants.

CpG Oligonucleotides (ODN) are immunomodulatory synthetic oligonucleotides specifically designed to stimulate Toll-like receptor 9. TLR9 is expressed on human plasmacytoid dendritic cells and B cells and triggers an innate immune response characterized by the production of Th1 and pro-inflammatory cytokines. This chapter reviews recent progress in understanding the mechanism of action of CpG ODN and provides an overview of human clinical trial results using CpG ODN to improve vaccines for the prevention/treatment of cancer, allergy, and infectious disease.