CpG Oligodeoxynucleotides Research Articles

CpG ODN enhances the efficacy of F protein vaccine against respiratory syncytial virus infection in the upper respiratory tract via CD4+ T cells

Respiratory syncytial virus (RSV) is a leading cause of severe respiratory illness worldwide, particularly in infants and older adults. Vaccines targeting the fusion glycoprotein (F protein) —one of the surface antigens of RSV— are highly effective in preventing RSV-associated severe lower respiratory tract disease. However, the efficacy of these vaccines against upper respiratory tract challenge needs improvement. Here, we aimed to examine the efficacy of F protein vaccines with or without CpG oligodeoxynucleotide (CpG ODN) as an adjuvant in the upper and lower respiratory tracts in mice. F + CpG ODN induced higher levels of F-specific IgG than that induced by F alone; however, levels of neutralizing antibodies did not increase compared to those induced by F alone. F + CpG ODN induced T helper 1 (Th1) cells while F alone induced T helper 2 (Th2) cells. Moreover, F + CpG ODN improved the protection against RSV challenge in the upper respiratory tract compared to F alone, which was largely dependent on CD4+ T cells. Meanwhile, both F + CpG ODN and F alone protected the lower respiratory tract. In conclusion, we demonstrated that induction of F-specific Th1 cells is an effective strategy to prevent RSV challenge in the upper respiratory tract in F protein vaccines. These data support the development of novel F protein vaccines.

Comprehensive evaluation of dietary tandem CpG oligodeoxynucleotides on enhancement of antioxidant capacity, immunological parameters, and intestinal microbiota in white shrimp (Litopenaeus vannamei)

CpG oligodeoxynucleotides (CpG ODNs) has been considered as a promising immunologic stimulant to improve immunity in poultry and aquaculture when challenged with diseases outbreak and cultivation environment deterioration. In this study, a comprehensive evaluation of CpG ODNs used in white shrimps (Litopenaeus vannamei) was conducted. Eight tandem CpG ODNs were added to the basic diet with a concentration of 50 mg kg−1 to investigate their effects on antioxidant capacity, immunological parameters, and intestinal microbiota in white shrimp, when the conventional immunologic stimulants (vitamin C and β-glucan) were set as control. After two weeks, the results indicated that shrimps fed with tandem CpG ODNs (consisting of CpG 2216, 2006, 8954, 1826, and M362) exhibited the highest expression level (3.68–7.92-fold change, p < 0.05) of IL-8, TOR and proPO in hepatopancreas and intestine, better enhancement of antioxidant enzymes activity (GST with 4 times and LZM with 6.78 times than control, p < 0.05), and the relative abundance of pathogenic bacteria like Vibrio reduced. The observation of intestinal and hepatopancreas histologic sections showed that the dietary immune stimulant did not cause structural changes to the vital organs of shrimps. Therefore, the results demonstrated that dietary with tandem CpG ODNs can significantly enhance the antioxidant capacity, gut microbiota, and innate immunity of shrimps.

DNA Hydrogels as Functional Materials and Their Biomedical Applications

AbstractWith the remarkable development of DNA nanotechnology, interest in DNA molecules has expanded beyond its biological role to building blocks in materials science. As a unique branch of DNA‐based materials, DNA hydrogels have exhibited many fascinating characteristics, including broad biocompatibility, precise programmability, convenient modification, and tunable mechanical properties, which make DNA hydrogels ideal biomaterials. Moreover, by combining with functional nucleic acids, such as aptamers, i‐motif nanostructures, CpG oligodeoxynucleotides, and DNAzymes, DNA hydrogels can be further tailored to provide additional target recognition, therapeutic potential, and catalytic activities, allowing them to play important roles in biosensing and medical applications. This review, aims to provide readers with an up‐to‐date overview of the important developments of biomedical DNA hydrogels. First, it introduces different synthetic strategies of hydrogels that utilize DNA as building materials and functional units within the hydrogel networks and discuss their advantages in biomedical applications. Subsequently, new approaches and applications of biomedical DNA hydrogels in the recent years are highlighted, such as therapeutic systems, cell culture platforms, tissue engineering materials, and biosensors. Finally, future perspectives and remaining challenges of DNA hydrogels in biomedicine are presented.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

Three-Dimensional Modeling of CpG DNA Binding with Matrix Lumican Shows Leucine-Rich Repeat Motif Involvement as in TLR9-CpG DNA Interactions.

Lumican is an extracellular matrix proteoglycan known to regulate toll-like receptor (TLR) signaling in innate immune cells. In experimental settings, lumican suppresses TLR9 signaling by binding to and sequestering its synthetic ligand, CpG-DNA, in non-signal permissive endosomes. However, the molecular details of lumican interactions with CpG-DNA are obscure. Here, the 3-D structure of the 22 base-long CpG-DNA (CpG ODN_2395) bound to lumican or TLR9 were modeled using homology modeling and docking methods. Some of the TLR9-CpG ODN_2395 features predicted by our model are consistent with the previously reported TLR9-CpG DNA crystal structure, substantiating our current analysis. Our modeling indicated a smaller buried surface area for lumican-CpG ODN_2395 (1803 Å2) compared to that of TLR9-CpG ODN_2395 (2094 Å2), implying a potentially lower binding strength for lumican and CpG-DNA than TLR9 and CpG-DNA. The docking analysis identified 32 amino acids in lumican LRR1-11 interacting with CpG ODN_2395, primarily through hydrogen bonding, salt-bridges, and hydrophobic interactions. Our study provides molecular insights into lumican and CpG-DNA interactions that may lead to molecular targets for modulating TLR9-mediated inflammation and autoimmunity.

Small EV-based delivery of CpG ODNs for melanoma postsurgical immunotherapy

Blocking programmed cell death protein 1 (PD-1) is an effective therapeutic strategy for melanoma. However, patients often develop tumor recurrence postoperatively due to the low response rate to the anti-PD-1 antibody (aPD-1). In this study, we developed an in situ sprayable fibrin gel that contains cytosine-guanine oligodeoxynucleotides (CpG ODNs)-modified ovalbumin (OVA) antigen-expressing bone marrow dendritic cell (DC)-derived small extracellular vesicles (DC-sEVs) and aPD-1. CpG ODNs can activate DCs, which have potent immunostimulatory effects, by stimulating both the maturation and activation of tumor-infiltrating dendritic cells (TIDCs) and DCs in tumor-draining lymph nodes (TDLNs). In addition, DC-sEVs can deliver OVA to the same DCs, leading to the specific expression of tumor antigens by antigen-presenting cells (APCs). In brief, the unique synergistic combination of aPD-1 and colocalized delivery of immune adjuvants and tumor antigens enhances antitumor T-cell immunity, not only in the tumor microenvironment (TME) but also in TDLNs. This effectively attenuates local tumor recurrence and metastasis. Our results suggest that dual activation by CpG ODNs prolongs the survival of mice and decreases the recurrence rate in an incomplete tumor resection model, providing a promising approach to prevent B16-F10-OVA melanoma tumor recurrence and metastasis.

The effect of Toll-like receptor agonists on the immunogenicity of MVA-SARS-2-S vaccine after intranasal administration in mice.

Modified Vaccinia virus Ankara (MVA) represents a promising vaccine vector for respiratory administration to induce protective lung immunity including tertiary lymphoid structure, the bronchus-associated lymphoid tissue (BALT). However, MVA expressing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike protein (MVA-SARS-2-S) required prime-boost administration to induce high titers of anti-Spike antibodies in serum and bronchoalveolar lavage (BAL). As the addition of adjuvants enables efficient tailoring of the immune responses even to live vaccines, we tested whether Toll-like receptor (TLR)-agonists affect immune responses induced by a single dose of intranasally applied MVA-SARS-2-S. We intranasally immunized C57BL/6 mice with MVA-SARS-2-S vaccine in the presence of either TLR3 agonist polyinosinic polycytidylic acid [poly(I:C)], TLR4 agonist bacterial lipopolysaccharide (LPS) from Escherichia coli, or TLR9 agonist CpG oligodeoxynucleotide (CpG ODN) 1826. At different time-points after immunization, we analyzed induced immune responses using flow cytometry, immunofluorescent microscopy, and ELISA. TLR agonists had profound effects on MVA-SARS-2-S-induced immune responses. At day 1 post intranasal application, the TLR4 agonist significantly affected MVA-induced activation of dendritic cells (DCs) within the draining bronchial lymph nodes, increasing the ratio of CD11b+CD86+ to CD103+CD86+ DCs. Nevertheless, the number of Spike-specific CD8+ T cells within the lungs at day 12 after vaccination was increased in mice that received MVA-SARS-2-S co-administered with TLR3 but not TLR4 agonists. TLR9 agonist did neither significantly affect MVA-induced DC activation nor the induction of Spike-specific CD8+ T cells but reduced both number and size of bronchus-associated lymphoid tissue. Surprisingly, the addition of all TLR agonists failed to boost the levels of Spike-specific antibodies in serum and bronchoalveolar lavage. Our study indicates a potential role of TLR-agonists as a tool to modulate immune responses to live vector vaccines. Particularly TLR3 agonists hold a promise to potentiate MVA-induced cellular immune responses. On the other hand, additional research is necessary to identify optimal combinations of agonists that could enhance MVA-induced humoral responses.

A Recombinant Protein XBB.1.5 RBD/Alum/CpG Vaccine Elicits High Neutralizing Antibody Titers against Omicron Subvariants of SARS-CoV-2.

(1) Background: We previously reported the development of a recombinant protein SARS-CoV-2 vaccine, consisting of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, adjuvanted with aluminum hydroxide (alum) and CpG oligonucleotides. In mice and non-human primates, our wild-type (WT) RBD vaccine induced high neutralizing antibody titers against the WT isolate of the virus, and, with partners in India and Indonesia, it was later developed into two closely resembling human vaccines, Corbevax and Indovac. Here, we describe the development and characterization of a next-generation vaccine adapted to the recently emerging XBB variants of SARS-CoV-2. (2) Methods: We conducted preclinical studies in mice using a novel yeast-produced SARS-CoV-2 XBB.1.5 RBD subunit vaccine candidate formulated with alum and CpG. We examined the neutralization profile of sera obtained from mice vaccinated twice intramuscularly at a 21-day interval with the XBB.1.5-based RBD vaccine, against WT, Beta, Delta, BA.4, BQ.1.1, BA.2.75.2, XBB.1.16, XBB.1.5, and EG.5.1 SARS-CoV-2 pseudoviruses. (3) Results: The XBB.1.5 RBD/CpG/alum vaccine elicited a robust antibody response in mice. Furthermore, the serum from vaccinated mice demonstrated potent neutralization against the XBB.1.5 pseudovirus as well as several other Omicron pseudoviruses. However, regardless of the high antibody cross-reactivity with ELISA, the anti-XBB.1.5 RBD antigen serum showed low neutralizing titers against the WT and Delta virus variants. (4) Conclusions: Whereas we observed modest cross-neutralization against Omicron subvariants with the sera from mice vaccinated with the WT RBD/CpG/Alum vaccine or with the BA.4/5-based vaccine, the sera raised against the XBB.1.5 RBD showed robust cross-neutralization. These findings underscore the imminent opportunity for an updated vaccine formulation utilizing the XBB.1.5 RBD antigen.

Multifunctional magnetic nanoparticles elicit anti-tumor immunity in a mouse melanoma model

Immunotherapy has emerged as a promising strategy to eradicate cancer cells. Particularly, the development of cancer vaccines to induce a potent and sustained antigen-specific T cell response has become a center of attention. Herein, we describe a novel immunotherapy based on magnetic nanoparticles (MNP) covalently modified with the OVA254-267 antigen and a CpG oligonucleotide via disulfide bonds. The MNP-CpG-COVA significantly enhances dendritic cell activation and CD8+ T cell antitumoral response against B16-OVA melanoma cells in vitro. Notably, the immune response induced by the covalently modified MNP is more potent and sustained over time than that triggered by the free components, highlighting the advantage of nanoformulations in immunotherapies. What is more, the nanoparticles are stable in the blood after in vivo administration and induce potent levels of systemic tumor-specific effector CD8 + T cells. Overall, our findings highlight the potential of covalently functionalized MNP to induce robust immune responses against mouse melanoma.

Synergistic immunostimulation for tumor sensitization with a biomineralized DNA sponge

The activation of stimulator of interferon genes (STING) plays a key role for provoking effective innate and adaptive immunity, yet is constrained by the limited clinical translation of STING agonists. Herein, we construct a smart DNA sponge to deliver STING agonists for robust cancer immunotherapy via the synergistic activation of innate immune pathways. The compact DNA sponge (Mn-CpG), assembling through a simple rolling circle amplification reaction, is encoded with numerous STING agonist (Mn2+) and toll-like receptor 9 (TLR9) agonist (CpG oligodeoxynucleotide) and allows their on-demand cellular delivery We show that Mn-CpG sponge can trigger the synergistic type I interferon response and robust T cell priming through a convenient coordination of different signaling pathways. In a poorly immunogenic breast cancer model, the method achieves the boosted immune responses with significantly inhibited tumor growth and metastasis.

Aminated yeast β-D-glucan for macrophage-targeted delivery of CpG oligodeoxynucleotides and synergistically enhanced cancer immunotherapy

CpG oligodeoxynucleotides (CpG ODNs) activate immune system and show strong potential in cancer immunotherapy. However, therapeutic efficacy of CpG ODNs is hampered due to rapid nuclease degradation and insufficient cellular uptake. Delivery of CpG ODNs into antigen presenting cells (APCs) is vital to enhance their therapeutic efficacy. Herein, we developed a super-convenient yet efficient strategy for macrophage-targeted delivery of CpG ODNs and synergistically enhanced cancer immunotherapy. Aminated yeast β-D-glucan (NH2-Glu) was simply synthesized through functionalization of β-D-glucan with DETA, which exhibited a dendrimer-like shape with size of about 80 nm. NH2-Glu complexed negatively-charged CpG ODNs. The as-prepared NH2-Glu/CpG complexes were positively charged, uniformly dispersed and exhibited good stability against nuclease degradation. Due to the specific recognition with dectin-1 expressed on macrophages, NH2-Glu/CpG complexes targeted macrophage and exhibited significantly enhanced cellular uptake due to dectin-1-mediated endocytosis. NH2-Glu/CpG complexes showed potent immunostimulatory activity. Contributed by the inherent immunostimulatory and antitumor activity, yeast β-D-glucan functioned synergistically with CpG ODNs in inducing antitumor immunity. NH2-Glu/CpG complexes remarkably inhibited tumor growth without causing toxic effect. In summary, this work provides a facile yet efficient macrophage-targeted CpG ODNs delivery system for cancer immunotherapy.

Cloning, expression, purification and characterization of the recombinant nucleocapsid protein from SARS-CoV-2 and its combination with a CpG ODN-39M

Several studies have reported the ability of the nucleocapsid (N) protein from the SARS-CoV-2 virus to interact with RNA in vitro. Nevertheless, its capacity to interact with deoxyribonucleic acid has yet to be widely described. In the present work, the nucleocapsid protein from the SARS-CoV-2 Delta variant was obtained in E. coli. The recombinant protein was purified, immune identified and incubated with different quantities of the ODN-39M, a CpG ODN with adjuvant properties. As a result, a curve of precipitation was obtained. The analysis by agarose gel electrophoresis of the mixtures revealed that ODN-39M retarded its mobility, whereas the protein increased its migration. When a sample of N+ODN-39M was crosslinked and analyzed by SDS-PAGE, high molecular weight aggregates were detected. Accordingly, upon analysis by Transmission Electron Microscopy, particles of approximately 20 nm were visualized in both samples of N protein, with and without ODN-39M. However, aggregates of particles were abundant in the mixture. Notably, such structures kept the ability of N protein to be recognized by human sera of COVID-19 convalescent donors. We can assert that nucleocapsid protein can bind to deoxyribonucleic acid. Such interaction changed the protein conformation, contributing to the previously reported immunogenicity in mice of N+ODN-39M complexes. Keywords: Nucleocapsid, SARS-CoV-2, ODN-39M, Particles

Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial immunometabolic modulation.

Pneumonia is a worldwide threat, making discovery of novel means to combat lower respiratory tract infection an urgent need. Manipulating the lungs' intrinsic host defenses by therapeutic delivery of certain pathogen-associated molecular patterns protects mice against pneumonia in a reactive oxygen species (ROS)-dependent manner. Here we show that antimicrobial ROS are induced from lung epithelial cells by interactions of CpG oligodeoxynucleotides (ODN) with mitochondrial voltage-dependent anion channel 1 (VDAC1). The ODN-VDAC1 interaction alters cellular ATP/ADP/AMP localization, increases delivery of electrons to the electron transport chain (ETC), increases mitochondrial membrane potential (ΔΨm), differentially modulates ETC complex activities and consequently results in leak of electrons from ETC complex III and superoxide formation. The ODN-induced mitochondrial ROS yield protective antibacterial effects. Together, these studies identify a therapeutic metabolic manipulation strategy to broadly protect against pneumonia without reliance on antibiotics.

Prominence of Bioresponsive DNA Nanococoons in Tackling Post-Surgery Cancer Recurrence

Post-surgery cancer recurrence is one of the reasons for increased cancer cases. The effective usage of the enhanced permeability and retention effect of a nanocarrier infused with the bioresponsive release mechanism of checkpoint inhibitors (aPD1 and aCTLA4) can become a boon to mankind. DNA nanococoons (DNCs) comprising cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs) with potent immunostimulatory effects can significantly enhance anti-cancer activity. Triglycerylmonostearate (TGMS) with enzymatic cleavage potential at the wound sites of tumor resection, upon caging with restriction enzyme (HhaI) followed by attaching to DNCs, makes the immunotherapy bioresponsive. Hhal-TGMS-DNCs-aPD1 triggered by the inflammation at the wound site undergoes enzymatic cleavage, releases the restriction enzyme, converts DNCs to CpG ODNs sequentially and with sustained aPD1 release exerts an appreciable anti-cancer effect.

Inhalable pH-responsive DNA tetrahedron nanoplatform for boosting anti-tumor immune responses against metastatic lung cancer

Despite advancements in the treatment of pulmonary cancer, the existence of mucosal barriers in lung still hampered the penetration and diffusion of therapeutic agents and greatly limited the therapeutic benefits. In this work, we reported a novel inhalable pH-responsive tetrahedral DNA nanomachines with simultaneous delivery of immunomodulatory CpG oligonucleotide and PD-L1-targeting antagonistic DNA aptamer (CP@TDN) for efficient treatment of pulmonary metastatic cancer. By precisely controlling the ratios of CpG and PD-L1 aptamer, the obtained CP@TDN could specifically release PD-L1 aptamer to block PD-1/PD-L1 immune checkpoint axis in acidic tumor microenvironment, followed by endocytosis by antigen-presenting cells to generate anti-tumor immune activation and secretion of anti-tumor cytokines. Moreover, inhalation delivery of CP@TDN showed highly-efficient lung deposition with greatly enhanced intratumoral accumulation, ascribing to the DNA tetrahedron-mediated penetration of pulmonary mucosa. Resultantly, CP@TDN could significantly inhibit the growth of metastatic orthotopic lung tumors via the induction of robust antitumor responses. Therefore, our work presents an attractive approach by virtue of biocompatible DNA tetrahedron as the inhalation delivery system for effective treatment of metastatic lung cancer.

Morphology Dictated Immune Activation with Framework Nucleic Acids.

Framework nucleic acids (FNAs) of various morphologies, designed using the precise and programmable Watson-Crick base pairing, serve as carriers for biomolecule delivery in biology and biomedicine. However, the impact of their shape, size, concentration, and the spatial presentation of cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) on immune activation remains incompletely understood. In this study, representative FNAs with varying morphologies are synthesized to explore their immunological responses. Low concentrations (50 nM) of all FNAs elicited no immunostimulation, while high concentrations of elongated DNA nanostrings and tetrahedrons triggered strong activation due to their larger size and increased cellular uptake, indicating that the innate immune responses of FNAs depend on both dose and morphology. Notably, CpG ODNs' immune response can be programmed by FNAs through regulating the spatial distance, with optimal spacing of 7-8 nm eliciting the highest immunostimulation. These findings demonstrate FNAs' potential as a designable tool to study nucleic acid morphology's impact on biological responses and provide a strategy for future CpG-mediated immune activation carrier design.

Programming Injectable DNA Hydrogels Yields Tumor Microenvironment-Activatable and Immune-Instructive Depots for Augmented Chemo-Immunotherapy.

Injectable hydrogels have attracted increasing attention for promoting systemic antitumor immune response through the co-delivery of chemotherapeutics and immunomodulators. However, the biosafety and bioactivity of conventional hydrogel depots are often impaired by insufficient possibilities for post-gelling injection and means for biofunction integration. Here, an unprecedented injectable stimuli-responsive immunomodulatory depot through programming a super-soft DNAhydrogel adjuvant is reported. This hydrogel system encoded with adenosine triphosphate aptamers can be intratumorally injected in a gel formulation and then undergoes significant molecular conformation change to stimulate the distinct release kinetics of co-encapsulated therapeutics. In this scenario, doxorubicinis first released to induce immunogenic cell death that intimately works together with the polymerized cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) in gel scaffold for effectively recruiting and activating dendritic cells. The polymerized CpG ODN not only enhances tumor immunogenicity but minimizes free CpG-induced splenomegaly. Furthermore, the subsequently released anti-programmed cell death protein ligand 1 (aPDL1) blocks the corresponding immune inhibitory checkpoint molecule on tumor cells to sensitize antitumor T-cell immunity. This work thus contributes to the first proof-of-concept demonstration of a programmable super-soft DNA hydrogel system that perfectly matches the synergistic therapeutic modalities based on chemotherapeutic toxicity, in situ vaccination, and immune checkpoint blockade.

Novel neoantigen peptides and tumor regression in the MC38 colorectal cancer model.

28 Background: Neoantigen vaccine is an emerging modality to treat cancer by unleashing cytotoxic T cells against tumor-specific mutant antigens. Neoantigens are de-novo changes in tumor cells and are hence more immunogenic and less susceptible to off-target toxicities. In building our own pipeline for neoantigen discovery, we used the mouse colon adenocarcinoma cell line MC38 as a validation model. Methods: We sequenced MC38 cells by whole exome and RNA sequencing to shortlist variants. Candidates were confirmed by sanger sequencing, then analyzed for binding affinity to MHC class I H2-Kb and H2-Db. We selected top 10 epitopes for facile modification to maximize immunogenicity by taking into account peptide characteristics, such as hydrophobicity. In-vivo immunogenicity was tested by subcutaneous injections of pooled peptides in C57BL/6J mice. Amongst the 10 peptides tested, we picked five that elicited greatest IFNγ secretion in re-stimulated splenocytes. Predicted binding affinity did not correlate with in-vivo immunogenicity. We inoculated MC38 cells into the left flank of C57BL/6J mice and started treatment four days later. Mice were injected twice weekly with PBS (group 1), CpG only (group 2), anti-PDL1 only (group 3), Peptide pool + CpG (group 4) and Peptide pool + CpG + anti-PDL1 (group 5) (n=8 per group). CpG ODN 1826 is a TLR9 agonist and acts as an adjuvant, while anti-PDL1 is an immune checkpoint inhibitor. We monitored tumor size, animal weights and food intake every 2-3 days. Results: In control group 1, tumor size exponentially increased by 36 folds on the 18th day of treatment. Groups 2, 3, 4 and 5 saw 16, 13, 8 and 5-fold increases in tumor size respectively, 21 days post-treatment. No tumors were found in 3/7 mice in group 5 upon sacrifice, suggesting tumor regression. Tumor control in groups 4 and 5 corroborated with significant IFNγ secretion from neoantigen re-stimulated splenocytes. We observed 38-45 fold expansion in the number of CD8+ KLRG1+ short-lived effector cells and significant increase in PD-1+ TIM3+ exhausted T cells in group 4 and 5 relative to control. Reps1 and Adpgk neoantigen peptides are widely reported and used in MC38 syngenic model. However, they were not included because both were undetected by sequencing. Cell line authentication by STR profiling confirmed that our cells matched that of MC38 from Kerafast. Conclusions: Using an in-house pipeline, we derived a set of novel neoantigen peptides that robustly controlled tumor growth in the MC38 tumor challenge model. In-silico binding predictions did not correlate with in-vivo patterns of IFNγ secretion. In-vivo tests for IFNγ induction after peptide vaccination is a better measure for potential in tumor control. Upregulation of T-cell exhaustion markers after peptide vaccination explains the improved response with anti-PDL1. Reps1 and Adpgk mutations were absent in our cells, which calls caution to researchers conducting similar research.

Killing three birds with one stone: Tumor-membrane-decorated Prussian blue nanovaccines for synergistic management of skin tumors, radiation dermatitis and wounds

Unhealed wounds following the resection of malignant skin tumors, featuring incomplete tumor tissue resection, full-thickness cutaneous defects, wound infection and radiation dermatitis, represent the main causes of prolonged recovery time, poor prognosis, and elevated tumor recurrence. Herein, a tumor cell membrane-decorated Prussian blue (PB) nanovaccine, referred to as PBVac, was fabricated via camouflage of PB nanozymes with the tumor cell membrane as tumor antigens and CpG oligodeoxynucleotides (ODNs) as adjuvants. Reinforcement by the tumor cell membrane substantially enhanced the tumor targeting potential of PBVac, achieving tumor-selective targeting and therapeutic efficacy. The bioactivity of PB nanozymes conferred PBVac with remarkable photothermal activity, antibacterial and antioxidant effects, curing wounds and radiation dermatitis by stimulating angiogenesis, antioxidation and anti-infection. The enhanced antitumor immunity of PBVac conferred by improved antigenicity was also assessed in a tumor challenge assay. PBVac could provide tumor antigens and molecular adjuvants for the priming of an efficient adaptive immune response, suppressing tumor growth. The combined merits of PBVac (photothermal, antibacterial and antioxidant capacities, and tumor membrane antigen) make it a novel nanoplatform that could be utilized as a safe and efficient therapeutic for synergistic management of skin tumors, radiation dermatitis and wounds.

CpG ODN/Mangiferin Dual Delivery through Calcium Alginate Hydrogels Inhibits Immune-Mediated Osteoclastogenesis and Promotes Alveolar Bone Regeneration in Mice.

The immune system plays an important role in the skeletal system during bone repair and regeneration. The controlled release of biological factors from the immune system could facilitate and optimize the bone remodeling process through the regulation of the activities of bone cells. This study aimed to determine the effect of the controlled delivery of immunomodulatory biologicals on bone regeneration. Immunostimulatory cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) and glucosylxanthone Mangiferin (MAG)-embedded microbeads were incubated with P. gingivalis-challenged splenocytes, or co-cultured with RAW264.7 cells. The effect of CpG ODN/MAG-containing microbeads on bone regeneration was then tested in vivo in a mouse alveolar bone defect model. The results demonstrated that MAG significantly antagonized P. gingivalis proliferation and reduced the live/dead cell ratio. After the addition of CpG ODN + MAG microbeads, anti-inflammatory cytokines IL-10 and IL-4 were upregulated on day 2 but not day 4, whereas pro-inflammatory cytokine IL-1β responses showed no difference at both timepoints. RANKL production by splenocytes and TRAP+ cell formation of RAW264.7 cells were inhibited by the addition of CpG ODN + MAG microbeads. Alveolar bony defects, filled with CpG ODN + MAG microbeads, showed significantly increased new bone after 4 weeks. In summary, this study evaluated a new hydrogel-based regimen for the local delivery and controlled release of biologicals to repair and regenerate alveolar bony defects. The combined CpG ODN + MAG treatment may promote alveolar bone regeneration through the anti-microbial/anti-inflammatory effects and the inhibition of RANKL-mediated osteoclastogenesis.