CpG Oligodeoxynucleotides Research Articles

Validation of Multi-epitope Peptides Encapsulated in PLGA Nanoparticles Against Influenza A Virus.

Influenza is a highly contagious respiratory disease which poses a serious threat to public health globally, causing severe diseases in 3-5 million humans and resulting in 650,000 deaths annually. The current licensed seasonal influenza vaccines lacked cross-reactivity against novel emerging influenza strains as they conferred limited neutralising capabilities. To address the issue, we designed a multi-epitope peptide-based vaccine delivered by the self-adjuvanting PLGA nanoparticles against influenza infections. A total of six conserved peptides representing B- and T-cell epitopes of Influenza A were identified and they were formulated in either incomplete Freund's adjuvant containing CpG ODN 1826 or being encapsulated in PLGA nanoparticles for the evaluation of immunogenicity in BALB/c mice. The self-adjuvanting PLGA nanoparticles encapsulating the six conserved peptides were capable of eliciting the highest levels of IgG and IFN- γ producing cells. In addition, the immunogenicity of the six peptides encapsulated in PLGA nanoparticles showed greater humoral and cellular mediated immune responses elicited by the mixture of six naked peptides formulated in incomplete Freund's adjuvant containing CpG ODN 1826 in the immunized mice. Peptide 3 from the mixture of six peptides was found to exert necrotic effect on CD3+ T-cells and this finding indicated that peptide 3 should be removed from the nanovaccine formulation. The study demonstrated the self-adjuvanting properties of the PLGA nanoparticles as a delivery system without the need for incorporation of toxic and costly conventional adjuvants in multi-epitope peptide-based vaccines.

Programming DNA Circuits for Controlled Immunostimulation through CpG Oligodeoxynucleotide Delivery.

Herein, we present a DNA circuit programmed for the delivery of CpG oligodeoxynucleotides (CpG ODNs) with the pharmacological immunostimulation function. The circuit employs a complementary DNA (cDNA) strand to deactivate the biological function of CpG ODNs via hybridization, while T7 exonuclease mediates the activation by hydrolyzing the cDNA and releasing the CpG ODN as an active moiety. We investigated the influence of several factors on the kinetic profile and temporal behavior of the circuit. These include the design of the cDNA strand, the concentration of the DNA duplex, and the concentration of T7 exonuclease. The DNA circuit's in vitro activation resulted in toll-like receptor 9 stimulation in the HEK-engineered cell line, as well as tumor necrosis factor-alpha release by J774A.1 macrophages. By programming the DNA circuit to control the release of the CpG ODN, we achieved an altered pharmacological profile with acute and potent immunostimulation, in comparison to a system without controlled CpG ODN release, which exhibited a slow and delayed response. Our findings demonstrate the potential of DNA circuits in controlling the pharmacological activity of DNA strands for controlled drug delivery.

CpG ODN 2102 promotes antibacterial immune responses and enhances vaccine-induced protection in golden pompano (Trachinotusovatus).

CpG oligodeoxynucleotides (ODNs) are oligodeoxynucleotides containing CpG motifs and can be recognized by toll-like receptor 9 (TLR9), activating the host's immune responses. In this study, ten different CpG ODNs were designed and synthesized to study the antibacterial immune responses of CpG ODNs in golden pompano (Trachinotus ovatus). Results showed that CpG ODN 2102 significantly improved the immunity of golden pompano against bacteria. Besides, CpG ODN 2102 promoted the proliferation of head kidney lymphocytes and activated the head kidney macrophages. When TLR9-specific small interfering RNA (siRNA) was used to interfere with TLR9 expression, the immune responses were decreased. Moreover, the expression levels of myeloid differentiation primary response 88 (Myd88), p65, tumor necrosis factor receptor-associated factor 6 (TRAF6), and tumor necrosis factor-alpha (TNF-α) in the TLR9-knockdown golden pompano kidney (GPK) cells were significantly reduced. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) promoter activity of the TLR9-knockdown GPK cells was also significantly reduced. In vivo, the antibacterial immune effects induced by CpG ODN 2102 in golden pompano were mostly abolished when TLR9 expression was knocked down. These results suggested that TLR9 was involved in the immune responses induced by CpG ODN 2102. CpG ODN 2102 also enhanced the protective effect of the Vibrio harveyi vaccine pCTssJ, where the survival rate of golden pompano was significantly improved by 20%. In addition, CpG ODN 2102 enhanced the messenger RNA (mRNA) expression levels of TLR9, Myxovirus resistance (Mx), interferon γ (IFN-γ), TNF-α, interleukin (IL)-1β, IL-8, major histocompatibility complex class (MHC) Iα, MHC IIα, Immunoglobulin D (IgD), and IgM. Therefore, TLR9 was involved in the antibacterial immune responses induced by CpG ODN 2102 and CpG ODN 2102 possessed adjuvant immune effects. These results enlarged our knowledge of the antibacterial immunity of fish TLRs signaling pathway and had important implications for exploring natural antibacterial molecules in fish and developing new vaccine adjuvants.

The potential role of CpG oligodeoxynucleotides on diabetic cardiac autonomic neuropathy mediated by P2Y12 receptor in rat stellate ganglia.

Cardiac autonomic neuropathy has a high prevalence in type 2 diabetes, which increases the risk of cardiovascular system disorders. CpG oligodeoxynucleotide (CpG-ODN), a Toll-like receptor 9 (TLR9) ligand, has been shown to have cardioprotection and cellular protection. Our previous work showed that P2Y12 in stellate ganglia (SG) is involved in the process of diabetic cardiac autonomic neuropathy (DCAN). Here, we aim to investigate whether CpG-ODN 1826 plays a protective role in DCAN and whether this beneficial protection involves regulation of the P2Y12-mediated cardiac sympathetic injury. Our results revealed that CpG-ODN 1826 activated TLR9 receptor, improved the abnormal blood pressure (BP), heart rate (HR), heart rate variability (HRV) and sympathetic nerve discharge (SND) activity in diabetic rats and reduced the up-regulated NF-κB, P2Y12 receptor, TNF-α and IL-1β in SG. Meanwhile, CpG-ODN 1826 significantly decreased the elevated ATP, nuclear receptor coactivator 4 (NCOA4), iron, ROS and MDA levels and increased GPX4 and GSH levels. In addition, CpG-ODN 1826 contributes to maintain normalization of mitochondrial structure in SG. Overall, CpG-ODN 1826 alleviates the sympathetic excitation and abnormal neuron-glial signal communication via activating TLR9 receptors to achieve a balance of autonomic activity and relieve the DCAN in rats. The mechanism may involve the regulation of P2Y12 receptor in SG by reducing ATP release and NF-κB expression, which counteract neuroinflammation and ferroptosis mediated by activated P2Y12 in SG.

AMPLIFY-201, a first-in-human safety and efficacy trial of adjuvant ELI-002 2P immunotherapy for patients with high-relapse risk with KRAS G12D- or G12R-mutated pancreatic and colorectal cancer.

2528 Background: RAS mutations occur in 25% of solid tumors with G12D being the most frequent variant. ELI-002 2P is a vaccine comprised of lymph-node targeted Amphiphile (Amph)-modified G12D and G12R mutant KRAS peptides with an Amph-modified CpG oligonucleotide adjuvant designed to expand polyfunctional mutant KRAS-specific T cells. Preclinical amphiphile data (relative to non-amphiphile controls) showed increased immunogenicity, tumor clearance and survival in mouse models. Methods: This first-in-human multicenter phase I trial assessed safety, immunogenicity and antitumor activity using a novel adjuvant trial design in patients (pts) with minimal residual disease (MRD) following standard locoregional treatment. Eligibility: elevated circulating tumor DNA (ctDNA) and/or serum tumor biomarker elevation (CA19-9/CEA), and KRAS/NRAS mutation. In a 3+3 dose escalation, n=22 received a subcutaneous course of 6 prime and 4 booster injections of fixed dose Amph-peptides (1.4 mg), admixed with escalating Amph-CpG-7909 (Table). Dose escalation based on observed safety in first 28 days. Safety, antitumor activity including biomarker reduction/clearance and relapse free survival using immune Response Evaluation Criteria in Solid Tumors (iRECIST) were assessed. Immunogenicity assessed by direct ex vivo Fluorospot and intracellular cytokine staining of peripheral blood mononuclear cells (T-cell responders = ≥ 2-fold increase over baseline) and by tumor CD3 immunohistochemistry in subset who underwent a biopsy. Results: No dose-limiting toxicities, treatment related SAEs or cytokine release syndrome were observed, and no maximum tolerated dose was identified. Safety: all grade 1, fatigue (19%), headache (19%), and injection site reaction (9.5%). Biomarker reduction was observed in 15/19 (79%) and in 5/5 at highest doses; clearance of MRD was observed in 4/19 (21% - n=2 pancreas, n=2 colorectal). Polyfunctional mKRAS-specific T cell responses observed in 80% of pts (n=12/15), with both CD8+ and CD4+ T cell responses for most, and CD3+ T cells observed on biopsies. The recommended phase 2 dose (RP2D) is 10.0 mg Amph-CpG-7909. Conclusions: ELI-002 2P, evaluated in a novel MRD+ trial, was safe with ctDNA and serum tumor biomarker reduction and clearance and notable immune responses. The RP2D is the start dose for a phase 1/randomized phase 2 study evaluating a new seven peptide formulation for G12 D, R, V, S, A, C, and G13D mutated solid tumors (NCT05726864). Clinical trial information: NCT04853017 . [Table: see text]

Nano-particulate Toll-like Receptor 9 Agonist Potentiates the Antitumor Activity of Anti-Glypican-1 Antibody.

Monoclonal antibodies (mAbs) that target tumor antigens have recently been developed. Their antitumor activity is mainly achieved through antibody-dependent cellular cytotoxicity (ADCC) via effector cells such as tumor-infiltrated macrophages and natural killer (NK) cells. CpG oligodeoxynucleotides (ODNs) have potent antitumor activity and are considered to increase the tumor infiltration of macrophages and NK cells; however, a completely solubilized novel CpG-schizophyllan (SPG) complex, K3-SPG, displays more potent antitumor activity. We recently reported the significant antitumor activity of anti-glypican-1 (GPC1) mAb against GPC1-positive esophageal squamous cell carcinoma (ESCC) via ADCC. The aim of this study was to evaluate the potential synergistic antitumor activity of anti-GPC1 mAb and K3-SPG and elucidate the underlying mechanisms using a xenograft model of GPC1-positive human ESCC cells. The established human esophageal cancer cell line TE14 was subcutaneously injected into SCID mice. Xenograft mice were treated with anti-GPC1 mAb, K3-SPG, or their combination. Antitumor activity was evaluated by measuring the tumor volume. For FACS analysis, agents were administrated, and tumors were resected 1 day after the final treatment. Anti-GPC1 mAb or K3-SPG monotherapy showed dose-dependent antitumor activity, and combination therapy with anti-GPC1 mAb and K3-SPG showed antitumor activity (p=0.0859). Flow cytometry revealed significantly increased numbers of macrophages (p=0.0133) and of the ratio of activated NK cells/total NK cells (p=0.0058) following K3-SPG or combination therapy. Combination therapy with K3-SPG and anti-GPC1 mAb or another antitumor mAb may represent a new cancer treatment option acting via ADCC.

Tumor microenvironment-responsive spherical nucleic acid nanoparticles for enhanced chemo-immunotherapy

Certain chemotherapeutics can induce tumor cells’ immunogenic cell death (ICD), release tumor antigens, and thereby trigger personalized antitumor immune responses. Co-delivery of adjuvants using nanocarriers could amplify the ICD-induced tumor-specific immunity achieving a synergistic chemo-immunotherapeutic effect. However, complicated preparation, low drug loading efficiency, and potential carrier-associated toxicity are the major challenges that limited its clinical applications. Herein, a carrier-free core–shell nanoparticle (MPLA-CpG-sMMP9-DOX, MCMD NPs) was constructed by facile self-assembly of spherical nucleic acids (SNA) with two adjuvants of CpG ODN and monophosphoryl lipid A (MPLA) as a core and doxorubicin (DOX) radially around the dual-adjuvants SNA as a shell. The results demonstrated that MCMD NPs could enhance drugs accumulation in tumors, and release DOX upon enzymatic degradation of matrix metalloproteinase-9 (MMP-9) peptide in the tumor microenvironment (TME), which enhanced the direct-killing effect of DOX on tumor cells. The core of MPLA-CpG SNA efficiently boosted the ICD-induced antitumor immune response to further attack tumor cells. Thus, MCMD NPs achieved a synergistic therapeutic effect of chemo-immunotherapy with reduced off-target toxicity. This study provided an efficient strategy for the development of a carrier-free nano-delivery system for enhanced cancer chemo-immunotherapy.

Diverse pathogen-associated molecular patterns affect transcription of genes in the toll-like receptor signaling pathway in goat blood

Pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), peptidoglycan (PGN), Polyinosinic-polycytidylic acid (poly I:C), and CpG Oligodeoxynucleotides (ODN) are recognized by Toll-like receptors (TLR). This study aimed to investigate the effect of diverse PAMPs on the transcription of TLR signaling pathway genes in goat blood. Whole blood was collected from 3 female BoerXSpanish goats and treated with the following PAMPs: 10 µg/ml LPS, PGN, CpG ODN (2216), CpG ODN (2006), and 12.5 µg/ml Poly I:C. Blood-treated PBS served as a control. The expression of 84 genes in the human TLR signaling pathway RT2 PCR Array (Qiagen) was evaluated using real-time PCR. Treatment with PBS affected the expression of 74 genes, Poly I:C affected the expression of 40 genes, t ODN 2006 affected the expression of 50 genes, ODN 2216 affected the expression of 52 genes, LPS affected the expression of 49 genes, while PGN affected the expression of 49 genes. Our results show that PAMPs modulated and increased the expression of genes in the TLR signaling pathway. These results highlight important insights into how the host responds to different pathogens and may help design adjuvants for therapeutics and vaccines that target different.

A combination of genetically engineered oncolytic virus and melittin-CpG for cancer viro-chemo-immunotherapy

BackgroundImmunotherapy has emerged as an efficient therapeutic approach for cancer management. However, stimulation of host immune system against cancer cells often fails to achieve promising clinical outcomes mainly owing to the immunosuppressive characteristics of the tumor microenvironment (TME). Combination therapeutics that can trigger sustained immunogenic cell death (ICD) have provided new opportunities for cancer treatment.MethodsIn this study, we designed and applied an ICD inducer regimen, including a genetically engineered oncolytic virus (miRNA-modified coxsackieviruses B3, miR-CVB3), a pore-forming lytic peptide (melittin, found in bee venom), and a synthetic toll-like receptor 9 ligand (CpG oligodeoxynucleotides), for breast cancer and melanoma treatment. We compared the anti-tumor efficacy of miR-CVB3 and CpG-melittin (CpGMel) alone and in combination (miR-CVB3 + CpGMel) and investigated possible mechanisms involved.ResultsWe demonstrated that miR-CVB3 + CpGMel had no major impact on viral growth, while enhancing the cellular uptake of CpGMel in vitro. We further showed that combination therapy led to significant increases in tumor cell death and release of damage-associated molecular patterns compared with individual treatment. In vivo studies in 4T1 tumor-bearing Balb/c mice revealed that both primary and distant tumors were significantly suppressed, and the survival rate was significantly prolonged after administration of miR-CVB3 + CpGMel compared with single treatment. This anti-tumor effect was accompanied by increased ICD and immune cell infiltration into the TME. Safety analysis showed no significant pathological abnormalities in Balb/c mice. Furthermore, the developed therapeutic regimen also demonstrated a great anti-tumor activity in B16F10 melanoma tumor-bearing C57BL/6 J mice.ConclusionsOverall, our findings indicate that although single treatment using miR-CVB3 or CpGMel can efficiently delay tumor growth, combining oncolytic virus-based therapy can generate even stronger anti-tumor immunity, leading to a greater reduction in tumor size.

Preclinical Development and Validation of Translational Temperature Sensitive Iodized Oil Emulsion Mediated Transcatheter Arterial Chemo-Immuno-Embolization for the Treatment of Hepatocellular Carcinoma.

Herein a practical strategy for augmenting immune activation in transcatheter arterial chemoembolization (TACE) of hepatocellular carcinoma (HCC) is presented. Pluronic F127 (PF127) is incorporated with Lipiodol (LPD) to achieve safe and effective delivery of therapeutic agents during transcatheter intra-arterial (IA)local delivery. Enhanced emulsion stability, IA infusion, embolic effect, safety, pharmacokinetics, and tumor response of Doxorubicin loaded PF127-LPD (Dox-PF127-LPD) for TACE in both in vitro and in vivo preclinical VX2 liver cancer rabbit model and N1S1 HCC rat model are demonstrated. Then, transcatheter arterial chemo-immuno-embolization (TACIE) combining TACE and local delivery of immune adjuvant (TLR9 agonist CpG oligodeoxynucleotide) is successfully performed using CpG-loaded Dox-PF127-LPD. Concurrent and safe local delivery of CpG and TACE during TACIE demonstrate leveraged TACE-induced immunogenic tumor microenvironment and augment systemic anti-tumor immunity in syngeneic N1S1 HCC rat model. Finally, the broad utility and enhanced therapeutic efficacy of TACIE are validated in the diethylnitrosamine-induced rat HCC model. TACIE using clinically established protocols and materials shall be a convenient and powerful therapeutic approach that can be translated to patients with HCC. The robust anti-cancer immunity and tumor regression of TACIE, along with its favorable safety profile, indicate its potential as a novel localized combination immunotherapy for HCC treatment.

CD16 CAR-T cells enhance antitumor activity of CpG ODN-loaded nanoparticle-adjuvanted tumor antigen-derived vaccinevia ADCC approach

BackgroundCombinatorial immunotherapy strategies for enhancing the responsiveness of immune system have shown great promise for cancer therapy. Engineered nanoformulation incorporated toll-like receptor (TLR) 9 agonist CpG ODN has shown more positive results in suppressing tumor growth and can significantly enhance other immunotherapy activity with combinatorial effects due to the innate and adaptive immunostimulatory effects of CpG.ResultsIn the present work, protamine sulfate (PS) and carboxymethyl β-glucan (CMG) were used as nanomaterials to form nanoparticles through a self-assembly approach for CpG ODN encapsulation to generate CpG ODN-loaded nano-adjuvant (CNPs), which was subsequently mixed with the mixture of mouse melanoma-derived antigens of tumor cell lysates (TCL) and neoantigens to develop vaccine for anti-tumor immunotherapy. The obtained results showed that CNPs was able to effectively deliver CpG ODN into murine bone marrow-derived dendritic cells (DC) in vitro, and remarkably stimulate the maturation of DC cells with proinflammatory cytokine secretion. In addition, in vivo analysis showed that CNPs enhanced anti-tumor activity of PD1 antibody and CNPs-adjuvanted vaccine based on the mixture antigens of melanoma TCL and melanoma-specific neoantigen could not only induce anti-melanoma cellular immune responses, but also elicit melanoma specific humoral immune responses, which significantly inhibited xenograft tumor growth. Furthermore, CD16 CAR-T cells were generated by expressing CD16-CAR in CD3+CD8+ murine T cells.ConclusionOur results eventually showed that anti-melanoma antibodies induced by CNPs-adjuvanted TCL vaccines were able to collaborate with CD16-CAR-T cells to generate an enhanced targeted anti-tumor effects through ADCC (antibody dependent cell cytotoxicity) approach. CD16 CAR-T cells has thus a great potential to be an universal promising strategy targeting on solid tumor synergistic immunotherapy via co-operation with TCL-based vaccine.

Comparison of the Immune Effects of an mRNA Vaccine and a Subunit Vaccine against Herpes Zoster Administered by Different Injection Methods.

Previous studies have shown that the herpes zoster subunit vaccine Shingrix™ performs well in clinical trials. However, the key ingredient in its adjuvant, QS21, is extracted from rare plants in South America, so vaccine production is limited. Compared with subunit vaccines, mRNA vaccines have the advantages of faster production and not requiring adjuvants, but currently, there is no authorized mRNA vaccine for herpes zoster. Therefore, this study focused on herpes zoster subunit and mRNA vaccines. We prepared a herpes zoster mRNA vaccine and compared the effects of vaccine type, immunization route, and adjuvant use on vaccine immunological efficacy. The mRNA vaccine was injected directly into mice via subcutaneous or intramuscular injection. The subunit vaccine was mixed with adjuvants before immunization. The adjuvants include B2Q or alum. B2Q is BW006S + 2395S + QS21. BW006S and 2395S are phosphodiester CpG oligodeoxynucleotides (CpG ODNs). Then, we compared the cell-mediated immunity (CIM) and humoral immunity levels of the different groups of mice. The results showed that the immune responses of mice inoculated with the mRNA vaccine prepared in this study were not significantly different from those of mice inoculated with the protein subunit vaccine supplemented with the B2Q. The mRNA vaccine-induced immune responses following subcutaneous or intramuscular injection, and the different immunization routes did not lead to significant differences in immune response intensity. Similar results were also observed for the protein subunit vaccine adjuvanted with B2Q but not alum. The above results suggest that our experiment can provide a reference for the preparation of mRNA vaccines against herpes zoster and has certain reference significance for the selection of the immunization route; that is, there is no significant difference in the immune response caused by subcutaneous versus an intramuscular injection, so the injection route can be determined according to the actual situation of individuals.

Novel Adjuvant S-540956 Targets Lymph Nodes and Reduces Genital Recurrences and Vaginal Shedding of HSV-2 DNA When Administered with HSV-2 Glycoprotein D as a Therapeutic Vaccine in Guinea Pigs.

Herpes simplex virus type 2 (HSV-2) is a leading cause of genital ulcer disease and a major risk factor for acquisition and transmission of HIV. Frequent recurrent genital lesions and concerns about transmitting infection to intimate partners affect the quality of life of infected individuals. Therapeutic vaccines are urgently needed to reduce the frequency of genital lesions and transmission. S-540956 is a novel vaccine adjuvant that contains CpG oligonucleotide ODN2006 annealed to its complementary sequence and conjugated to a lipid that targets the adjuvant to lymph nodes. Our primary goal was to compare S-540956 administered with HSV-2 glycoprotein D (gD2) with no treatment in a guinea pig model of recurrent genital herpes (studies 1 and 2). Our secondary goals were to compare S-540956 with oligonucleotide ODN2006 (study1) or glucopyranosyl lipid A in a stable oil-in-water nano-emulsion (GLA-SE) (study 2). gD2/S-540956 reduced the number of days with recurrent genital lesions by 56%, vaginal shedding of HSV-2 DNA by 49%, and both combined by 54% compared to PBS, and was more efficacious than the two other adjuvants. Our results indicate that S-540956 has great potential as an adjuvant for a therapeutic vaccine for genital herpes, and merits further evaluation with the addition of potent T cell immunogens.

Construction of pH-Sensitive Nanovaccines Encapsulating Tumor Cell Lysates and Immune Adjuvants for Breast Cancer Therapy.

The current immunotherapy strategies for triple negative breast cancer (TNBC) are greatly limited due to the immunosuppressive tumor microenvironment (TME). Immunization with cancer vaccines composed of tumor cell lysates (TCL) can induce an effective antitumor immune response. However, this approach also has the disadvantages of inefficient antigen delivery to the tumor tissues and the limited immune response elicited by single-antigen vaccines. To overcome these limitations, a pH-sensitive nanocalcium carbonate (CaCO3 ) carrier loaded with TCL and immune adjuvant CpG (CpG oligodeoxynucleotide 1826) is herein constructed for TNBC immunotherapy. This tailor-made nanovaccine, termed CaCO3 @TCL/CpG, not only neutralizes the acidic TME through the consumption of lactate by CaCO3 , which increases the proportion of the M1/M2 macrophages and promotes infiltration of effector immune cells but also activates the dendritic cells in the tumor tissues and recruits cytotoxic T cells to further kill the tumor cells. In vivo fluorescence imaging study shows that the pegylated nanovaccine could stay longer in the blood circulation and extravasate preferentially into tumor site. Besides, the nanovaccine exhibits high cytotoxicity in 4T1 cells and significantly inhibits tumor growth of tumor-bearing mice. Overall, this pH-sensitive nanovaccine is a promising nanoplatform for enhanced immunotherapy of TNBC.

The immune enhancement effect of CpG-ODNs on the vaccine of inactivated Vibrio harveyi in tiger puffer (Takifugu rubripes)

Vibrio harveyi is an opportunistic pathogen among aquatic organisms, which can cause disease and even death of the cultured tiger puffer (Takifugu rubripes). Vaccination is one of the most promising strategies to prevent infection, and potent adjuvant systems are essential to enhance the immunogenicity of inactivated vaccines. CpG-oligodeoxynucleotides (CpG-ODNs) are novel immunopotentiators to induce strong humoral and cellular immunity. In the present study, the survival rate and immune response in the vaccinated individuals of T. rubripes were examined after the treatment with CpG-ODNs to verify its immune enhancement effect as an adjuvant against V. harveyi. After the V. harveyi challenge, the survival rate of puffers in the vaccine group (vaccinated with inactivated Vibrio harveyi), CpG + vaccine group (vaccinated inactivated Vibrio harveyi and treated with CpG), CpG group (treated with CpG), and control group (treated with PBS) was 53.33%, 60%, 36.67%, and 16.67%, respectively. The proliferation index of spleen lymphocytes in the vaccine group (2.13 ± 0.26), CpG + vaccine group (2.62 ± 0.67) and the CpG group (1.74 ± 0.16) were significantly higher than that in the PBS group (1.27 ± 0.18, p < 0.05), respectively. The concentration of cytokines (IL-1β, TNF-α, IL-6 and IL-8), IgM antibodies and non-specific enzymes (SOD and LZM) in the serum of puffers from vaccine group all increased significantly (p < 0.05) after the vaccination with inactivated V. harveyi. More noticeably, the concentration of IL-1β, TNF-α and IgM antibodies in the serum of CpG + vaccine group increased further (p < 0.05), which was 1.28-, 1.31-, and 1.18-fold of that in the vaccine group at 21 d post-vaccination. The results collectively indicated that the inactivated V. harveyi was able to induce both humoral and cellular immune responses of puffer to defend against infection, and CpG-ODNs could further enhance the immune protection of vaccine as a potential immunopotentiator.

B-Cell-Activating Factor Contributes to Elevation of the Content of Regulatory B Cells in Neonatal Sepsis.

We studied the role of B cell-activating factor (BAFF) in PI3K/AKT/mTOR signaling pathway in promoting proliferation and maintaining survival of regulatory B lymphocytes (Breg) in newborns with sepsis. The peripheral blood samples were collected from preterm neonates (n=40) diagnosed with sepsis on the day of diagnosis and on days 7, 14, and 21 after diagnosis, as well as from the matched preterm neonates without sepsis (n=40; control group). The peripheral blood mononuclear cells and B cells were isolated, cultured, and stimulated with LPS and immunostimulant CpG-oligodeoxynucleotide (CpG-ODN). Proliferation and differentiation of B-cells into CD19+CD24hiCD38hi Breg cells and the role of the PI3K/AKT/mTOR signaling pathway in these processes were studied by flow cytometry, real-time quantitative reverse transcription PCR (qRT-PCR), and Western blotting. BAFF levels in the peripheral blood of neonates with sepsis were significantly increased at one week after diagnosis in parallel with increasing trend of expression of BAFF receptor. When applied with LPS and CpG-ODN, BAFF promoted differentiation of B cells into CD19+CD24hiCD38hi Breg cells. Phosphorylation of 4E-BP1 factor and 70S6K kinase located downstream in PI3K/AKT/mTOR signaling pathway was significantly up-regulated when stimulated with BAFF in combination with LPS and CpG-ODN. Thus, increased level of BAFF activates PI3K/AKT/mTOR signaling pathway and induces in vitro differentiation of peripheral blood B cells into CD19+CD24hiCD38hi Breg cells.

Gold Nanorods and Polymer Micelles Mediated Dual TLR Stimulators Delivery System CPG@Au NRs/M‐R848 Regulate Macrophages Reprogramming and DC Maturation for Enhanced Photothermal Immunotherapy of Melanoma (Small Methods 5/2023)

Back Cover In article number 2201087, ZhiYong Qian and co-workers show that in the hybrid delivery systems [email protected] NRs/m-R848, immunogenic cell death (ICD) induced by Au NRs could synergistically produce an in-situ vaccination effect with CPG ODN and R848. By triggering dendritic cells maturation and macrophage reprogramming, this photothermal immunotherapy strategy holds great potential in generating systemic and lasting antitumor immunity by inhibiting tumor growth and metastases simultaneously.

B cell intrinsic Toll-like receptor 9 signaling regulates class switching to IgE

Abstract Chronic allergic diseases such as allergic asthma are driven by excessive production of allergen specific IgE. B cells produce IgE by class switch recombination (CSR) in response to T cell help and Th2 cytokines. However, development of allergic diseases can also be influenced by factors such as early-life exposure to microbial products. In vivo, stimulation of Toll-like receptor 9 (TLR9) with microbial DNA reduces IgE levels, largely thought to be a response to Th1 cytokines. However, the directeffects of TLR9 signaling on B cell class switching to IgE remain unclear. To delineate the B cell intrinsic effects of TLR9 stimulation, we treated mouse splenic B cells in vitrowith IL-4 and anti-CD40 antibody with or without a synthetic TLR9 agonist, CpG oligodeoxynucleotides (CpG). Compared to stimulation with IL-4 and anti-CD40 alone, the addition of CpG decreased IgE production. Using B cells from reporter mice that track germline transcription, a prerequisite for CSR, we found that CpG inhibited Iɛ but increased Iγ1 germline transcription. By Western blot analyses, we probed the expression of transcription factors known to negatively regulate Iɛ and positively regulate Iγ1 transcription. These transcription factors were synergistically upregulated in the presence of CpG with IL-4 and anti-CD40. Together, these data illustrate a molecular mechanism by which microbial products directly perturb the production of IgE and influence the development of allergic disease. R01 HL165120

Spike protein vaccine triggers systemic Th2 and Th17 inflammatory responses and pulmonary immunopathology following SARS-CoV-2 breakthrough infection

Abstract Vaccines have shown remarkable protection against COVID-19, but vaccination-enhanced respiratory disease (VAERD) following breakthrough infections poses safety concerns. SARS-CoV-2 Spike (SARS-2 S) protein subunit vaccines induced VAERD in hamsters, where aluminum adjuvant primed a Th2-biased immunization, resulting in elevated type 2 pulmonary inflammation in animals with breakthrough infections. To further understand the potential risks and immunopathogenesis of VAERD, we immunized K18-hACE2 mice with SARS-2 S protein in the presence of aluminum and CpG ODN adjuvants and infected them with a lethal dose of SARS-2. The vaccine induced robust antibody and T cell responses, significantly reducing viral titers and increasing host survival. However, following breakthrough infection, vaccinated animals exhibited severe airway immunopathology, with a dramatic increase of eosinophils and CD4 +T cells, and elevated Th2/Th17 cytokines in the lungs. Intracellular flow cytometric analysis showed a systemic Th2/Th17 inflammatory response, prominently in the lungs. Our data show that the aluminum/ODN adjuvants elicit strong protection against COVID-19 but prime a robust Th2/Th17 inflammatory response that may contribute to rapid onset of T cell-mediated pulmonary immunopathology following breakthrough infection. Our results highlight the need for additional studies to elucidate COVID-19-associated VAERD and to further improve vaccine formulations for broad protection and maximal safety. This research was supported in part by grants from the National Institutes of Health (P20 GM130555-6610 and R01 AI151139).

Self-assembled nanoparticles based on DNA origami and a nitrated T helper cell epitope as a platform for the development of personalized cancer vaccines.

Neoantigen vaccines constitute an emerging and promising cancer immunotherapy. However, not all neoantigens have anti-tumor activity, as poor CD4+ epitope recognition can lead to the lack of greatly limit the persistence of the CD8+ T cell response. Therefore, we designed a self-assembled nanoplatform hereinafter referred to as DNA-coupled nitrated T helper cell epitope nanoparticle (DCNP) based on DNA origami containing a nitrated CD4 + T cell epitope, which can facilitate the effective activation of neoantigen-specific CD8+ T cells. Moreover, we embedded the cytidine-phosphate-guanosine oligonucleotide (CpG ODN) motif sequence in the DNA skeleton to function as a built-in adjuvant to activate Toll-like receptor 9. DCNP can markedly improve adjuvant and neoantigen co-delivery to lymphoid organs and promote neoantigen presentation on dendritic cells. Moreover, DCNP induced robust, and long-lived neoantigen-specific CD8+ T cell responses that significantly delayed tumor growth. Further, these effects were largely dependent on the nitrated T cell epitope. Collectively, our findings indicate that DCNP is a promising platform that could improve the development of personalized therapeutic neoantigen vaccines for cancer immunotherapy.