Additional use of α-IgM antibodies potentiates CpG ODN2006-induced B cell activation by targeting mainly naïve and marginal zone-like B cells.

CpG ODN2006 is widely used as a potent B cell stimulant in vitro and in vivo. However, it shows a deficit in targeting naïve B cells in vitro. In this study, we investigated whether α-IgM can support ODN2006-induced effects on B cells to obtain enhanced activation with focus on different B cell subsets. Our results delineated robust B cell activation, shown by increased activation marker expression and cytokine secretion by each agent alone, and further augmented when used in combination. Interestingly, α-IgM targeted mainly naïve and marginal zone-like B cells, thus complementing the pronounced effects of ODN2006 on memory B cells and achieving optimal activation for all B cell subsets. Taken together, combining ODN2006 and α-IgM is beneficial for in vitro activation including all B cell subsets. Furthermore, our results suggest that α-IgM could enhance efficacy of ODN2006 in vivo with further need of investigation.

Improved biodistribution and enhanced immune response of subunit vaccine using a nanostructure formed by self-assembly of ascorbyl palmitate.

New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.

An Open-Label Study of Subcutaneous CpG Oligodeoxynucleotide (PF03512676) in Combination with Trastuzumab in Patients with Metastatic HER2+ Breast Cancer.

OBJECTIVES: CpG ODN is a Toll-like receptor 9 agonist with immunotherapeutic potential for many cancer types, including aggressive breast cancers. There is strong interest in utilizing CpG ODN as an adjuvant to improve clinical efficacy of current treatments and immunogenicity of breast cancers not traditionally responsive to active immunotherapy, such as those that are human epidermal growth factor receptor 2 (HER2)-positive. This study aimed to study the efficacy and safety of combination CpG ODN plus anti-HER2 antibody trastuzumab treatment in patients with advanced/metastatic breast cancer. METHODS: This single-arm, open-label phase II clinical trial treated patients (n = 6) with advanced/metastatic HER2-positive breast cancer with weekly subcutaneous CpG ODN and trastuzumab. Patients may have received any number of prior therapies to be enrolled (most enrolled at median 1 prior line of chemotherapy). Peripheral blood was collected at baseline and weeks 2, 6, 12, and 18 for immune analyses. Six patients were enrolled and 50% achieved stable disease (SD) response. RESULTS: Median PFS was 8.3 months. Three of the six patients enrolled opted to stop treatment due to tolerability issues. Multiplex assay for cytokine measurements revealed significantly higher VEGF-D levels at week 2 compared to baseline. Peripheral blood mononuclear cells analyzed by flow cytometry showed a significant increase in monocytic MDSC between weeks 6 and 12. Patients with progressive disease tended to have higher levels of week 6 monocytic MDSC and PD-1+ T cells than patients with SD. NK cell populations did not significantly change throughout treatment. CONCLUSIONS: CpG ODN and trastuzumab treatment of metastatic HER2 + breast cancer was safe but was not tolerable for all patients. This combination did induce potentially predictive immune profile changes in treated patients with metastatic HER2 + breast cancer, the significance of which needs to be further explored.

Why was the study done? Breast cancer that has metastasized (moved outside of the breast and local lymph nodes) is currently considered incurable and can be difficult to treat. Treatments that can stimulate the immune system to recognize cancer cells have been found to be useful for many types of cancers, including some types of breast cancers. This study tested a new immune stimulator (CpG ODN) in combination with a currently on-the-market antibody treatment for breast cancer (trastuzumab). What did the researchers do? The research team enrolled patients who had metastatic breast cancer and treated them all with a combination of trastuzumab and CpG ODN for 12 weeks. These patients were monitored for any side effects/toxicity, monitored for how long their breast cancer responded to this treatment, and monitored for how long they lived after beginning this treatment. Patients also had their blood drawn at different time points to observe how their immune cells and immune proteins (e.g. cytokines) changed on treatment. What did the researchers find? The research team enrolled six patients and found that the treatment was safe and that 50% of the patients treated did not have any breast cancer growth when given CpG ODN plus trastuzumab. Looking at the immune cells in the patient blood samples, some cells that are known to decrease the immune response to cancers (myeloid-derived suppressor cells) did increase towards the end of treatment. What do the findings mean? Overall, CpG ODN and trastuzumab treatment was found to be safe and potentially effective in preventing breast cancer growth.

Depletion of regulatory T cells enhancing the anti-tumor effect of in situ vaccination in solid tumors.

The emergence of immune checkpoint inhibitors (ICIs) has revolutionized the clinical treatment for tumor. However, the low response rate of ICIs remains the major obstacle for curing patients and effective approaches for patients with primary or secondary resistance to ICIs remain lacking. In this study, immune stimulating agent unmethylated CG-enriched (CpG) oligodeoxynucleotide (ODN) was locally injected into the tumor to trigger a robust immune response to eradicate cancer cells, while anti-CD25 antibody was applied to remove immunosuppressive regulatory T cells, which further enhanced the host immune activity to attack tumor systematically. The combination of CpG and anti-CD25 antibody obtained notable regression in mouse melanoma model. Furthermore, rechallenge of tumor cells in the xenograft model has resulted in smaller tumor volume, which demonstrated that the combinational treatment enhanced the activity of memory T cells. Remarkably, this combinational therapy presented significant efficacy on multiple types of tumors as well and was able to prevent relapse of tumor partially. Taken together, our combinational immunotherapy provides a new avenue to enhance the clinical outcomes of patients who are insensitive or resistant to ICIs treatments.

The orientation of CpG conjugation on aluminum oxyhydroxide nanoparticles determines the immunostimulatory effects of combination adjuvants.

In subunit vaccines, aluminum salts (Alum) are commonly used as adjuvants, but with limited cellular immune responses. To overcome this limitation, CpG oligodeoxynucleotides (ODNs) have been used in combination with Alum. However, current combined usage of Alum and CpG is limited to linear mixtures, and the underlying interaction mechanism between CpG and Alum is not well understood. Thus, we propose to chemically conjugate Alum nanoparticles and CpG (with 5' or 3' end exposed) to design combination adjuvants. Our study demonstrates that compared to the 3'-end exposure, the 5'-end exposure of CpG in combination adjuvants (Al-CpG-5') enhances the activation of bone-marrow derived dendritic cells (BMDCs) and promotes Th1 and Th2 cytokine secretion. We used the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen (HBsAg) as model antigens to demonstrate that Al-CpG-5' enhanced antigen-specific antibody production and upregulated cytotoxic T lymphocyte markers. Additionally, Al-CpG-5' allows for coordinated adaptive immune responses even at lower doses of both CpG ODNs and HBsAg antigens, and enhances lymph node transport of antigens and activation of dendritic cells, promoting Tfh cell differentiation and B cell activation. Our novel Alum-CPG strategy points the way towards broadening the use of nanoadjuvants for both prophylactic and therapeutic vaccines.

Vaccination with a combination of STING agonist-loaded lipid nanoparticles and CpG-ODNs protects against lung metastasis via the induction of CD11bhighCD27low memory-like NK cells.

BACKGROUND: Natural killer (NK) cells are effective in attacking tumor cells that escape T cell attack. Memory NK cells are believed to function as potent effector cells in cancer immunotherapy. However, knowledge of their induction, identification, and potential in vivo is limited. Herein, we report on the induction and identification of memory-like NK cells via the action of a combination of a stimulator of interferon genes (STING) agonist loaded into lipid nanoparticles (STING-LNPs) and cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG-ODNs), and the potential of the inducted memory-like NK cells to prevent melanoma lung metastasis. METHODS: The antitumor effects of either the STING-LNPs, CpG-ODNs, or the combination therapy were evaluated using a B16-F10 lung metastasis model. The effect of the combined treatment was evaluated by measuring cytokine production. The induction of memory-like NK cells was demonstrated via flow cytometry and confirmed through their preventative effect. RESULTS: The combination of STING-LNPs and CpG-ODNs tended to enhance the production of interleukin 12 (IL-12) and IL-18, and exerted a therapeutic effect against B16-F10 lung metastasis. The combination therapy increased the population of CD11bhighCD27low NK cells. Although monotherapies failed to show preventative effects, the combination therapy induced a surprisingly strong preventative effect, which indicates that CD11bhighCD27low cells could be a phenotype of memory-like NK cells. CONCLUSION: As far as could be ascertained, this is the first report of the in vivo induction, identification, and confirmation of a phenotype of the memory-like NK cells through a prophylactic effect via the use of an immunotherapeutic drug. Our findings provide novel insights into the in vivo induction of CD11bhighCD27low memory-like NK cells thus paving the way for the development of efficient immunotherapies.

Evaluation of different types of adjuvants in a malaria transmission-blocking vaccine.

Adjuvants are critical components for vaccines, which enhance the strength and longevity of the antibody response and influence the types of immune response. Limited research has been conducted on the immunogenicity and protective efficacy of various adjuvants in malaria transmission-blocking vaccines (TBVs). In this study, we formulated a promising TBV candidate antigen, the P. berghei ookinete surface antigen PSOP25, with different types of adjuvants, including the TLR4 agonist monophosphoryl lipid A (MPLA), the TLR9 agonist cytosine phosphoguanosine oligodeoxynucleotides (CpG ODN 1826) (CpG), a saponin adjuvant QS-21, aluminum hydroxide (Alum), and two combination adjuvants MPLA + QS-21 and QS-21 + CpG. We demonstrated that adjuvanted vaccines results in elevated elicited antibody levels, increased proliferation of plasma cells, and efficient formation of germinal centers (GCs), leading to enhanced long-term protective immune responses. Furthermore, CpG group exhibited the most potent inhibition of ookinete formation and transmission-blocking activity. We found that the rPSOP25 with CpG adjuvant was more effective than MPLA, QS-21, MPLA + QS-21, QS-21 + CpG adjuvants in dendritic cells (DCs) activation and differentiation. Additionally, the CpG adjuvant elicited more rubust immune memory response than Alum adjuvant. CpG and QS-21 adjuvants could activate the Th1 response and promote the secretion of IFN-γ and TNF-α. PSOP25 induced a higher number of Tfh cells in splenocytes when combined with MPLA, CpG, and QS-21 + CpG; and there was no increase in these cell populations when PSOP25 was administered with Alum. In conclusion, CpG may confer enhanced efficacy for the rPSOP25 vaccine, as evidenced by the ability of the elicited antisera to induce protective immune responses and improved transmission-blocking activity.

A novel CpG ODN compound adjuvant enhances immune response to spike subunit vaccines of porcine epidemic diarrhea virus.

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

Liposomal co-encapsulation of a novel gemini lipopeptide and a CpG-ODN induces a strong Th1 response with the co-activation of a Th2/Th17 profile and high antibody levels.

A successful vaccine depends on its capacity to elicit a protective immune response against the target pathogen. The adjuvant used plays an important role in enhancing and directing the immune response. Liposomes are vaccine adjuvants that allow the co-encapsulation of antigens and immunostimulants. Our aim was to evaluate the adjuvanticity of a cationic liposome (Lip) formulated with a novel gemini lipopeptide (AG2-C16) alone or in combination with CpG-ODN as immunostimulants. To achieve this, we used the recombinant clumping factor of Staphylococcus aureus (rClfA) as a model antigen, in a murine model. We characterized the formulations by DLS, Cryo-SEM, and TEM, and analyzed the humoral and cellular immune responses induced in BALB/c and C57BL/6J mice injected with free rClfA and three formulations: Lip + CpG-ODN + rClfA, Lip + AG2-C16 + rClfA and Lip + AG2-C16 + CpG-ODN + rClfA. The addition of immunostimulants to the liposomes did not change the membrane diameter but affected their hydrodynamic diameter, z-potential, and homogeneity. All liposomal formulations were able to stimulate a specific humoral response, with high serum IgG, IgG1 and IgG2a or IgG2c titers in BALB/c or C57BL/6J mice, respectively. In addition, increased vaginal IgG levels were detected after injection, with no specific IgA. The cellular immunity induced by Lip + AG2-C16 + CpG-ODN + rClfA was characterized by a predominant Th1 profile, with the co-induction of Th2 and Th17 cells, and IFN-γ+ cytotoxic T cells. Furthermore, we studied the capacity of the different formulations to stimulate murine keratinocytes and fibroblasts in vitro. While no formulation activated keratinocytes, Lip + AG2-C16 + CpG-ODN increased the expression of CXCL9 in fibroblasts. These results suggest Lip + AG2-C16 + CpG-ODN as a promising adjuvant candidate to be used in vaccines against pathogens that require Th1/Th2/Th17 combined profiles, like S. aureus. Additionally, based on the IFN-γ+ cytotoxic T cells stimulation and the CXCL9 production by fibroblasts, we propose the use of this adjuvant formulation for the stimulation of a Th1 profile.

Effectiveness of dietary heat-killed Bacillus subtilis harboring plasmid containing 60 copies of CpG-ODN 1668 against Vibrio harveyi in Penaeus vannamei.

The cost of the purification process hinders the extensive use of cytosine phosphate guanosine-oligodeoxynucleotides (CpG-ODNs) for shrimp culture. Therefore, this study used a shuttle vector plasmid to carry 60 copies of CpG-ODN 1668 (pAD43-25_60CpG), which can replicate in Escherichia coli and Bacillus subtilis strain RIK1285. The first experiment used a reverse gavage procedure to deliver a substance (PBS [CK], pAD43-25 [P0], and pAD43-25_60CpG [P60], respectively) directly into the anterior midgut of Penaeus vannamei and transcriptome sequence analysis with a reference genome was performed to examine the expression of well-known immune-related genes. The results showed that the expression levels of immune-related genes in P60 group were significantly increased, particularly those associated with AMPs. In addition, using RT‒qPCR, the expression levels of AMP genes (LvALF, LvPEN-2, and LvPEN-3) in the P60 group may vary depending on the tissue and time point. The second experiment used dietary supplementation with three kinds of heat-killed B. subtilis (HKBS, HKBS-P0, and HKBS-P60) in 28 days of feeding experiments. The results showed that dietary supplementation with HKBS-P60 did not significantly improve shrimp growth performance and survival. However, on days 14 and 28 of the feeding regimens, alkaline phosphatase (AKP) and acid phosphatase (ACP) activity were considerably higher than in other treatments. In addition, following infection with Vibrio harveyi, AKP and ACP activity in the HKBS-P60 group was significantly higher than in other treatments, particularly at the early stage of bacterial infection. Moreover, HKBS-P60 was found to be better protected against V. harveyi infection with lower cumulative mortality (60%) compared to HKBS (90%) and HKBS-P0 (100%) at 7 days after infection. Overall, these findings confirmed that P60 could increase immunological responses in the shrimp midgut, and HKBS-P60 could be used as an effective tool to enhance the immune response and disease resistance in shrimp.

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.

Control of A/D type CpG-ODN aggregates to a suitable size for induction of strong immunostimulant activity.

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

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.

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-1ß 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.

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-1ß 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.

CpG ODN 1668 as TLR9 agonist mediates humpback grouper (Cromileptes altivelis) antibacterial immune responses.

Cromileptes altivelis (humpback grouper) is the main farmed species in the southern coastal area of China owing to its important economic value. Toll-like receptor 9 (TLR9) belongs to the toll-like receptor (TLR) family and functions as a pattern recognition receptor, recognising unmethylated oligodeoxynucleotides containing the CpG motif (CpG ODNs) in bacterial and viral genomes, thereby activating host immune response. In this study, the C. altivelis TLR9 (CaTLR9) ligand CpG ODN 1668 was screened and found to significantly enhance the antibacterial immunity of humpback grouper in vivo and head kidney lymphocytes (HKLs) in vitro. In addition, CpG ODN 1668 also promoted the cell proliferation and immune gene expression of HKLs and strengthened the phagocytosis activity of head kidney macrophages. However, when the CaTLR9 expression was knocked down in the humpback group, the expression levels of TLR9, myeloid differentiation factor 88 (Myd88), tumour necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-1ß (IL-1ß), IL-6, and IL-8 were significantly reduced, and the antibacterial immune effects induced by CpG ODN 1668 were mostly abolished. Therefore, CpG ODN 1668 induced antibacterial immune responses in a CaTLR9-dependent pathway. These results enhance the knowledge of the antibacterial immunity of fish TLR signalling pathways and have important implications for exploring natural antibacterial molecules in fish.

The Use of Chitosan Nanoparticles for Delivery of CpG ODN in Treatment of Allergic Balb/C Mice.

Background: This study aims to prepare high stability chitosan nanoparticles (CNP) and examine the ability of CNP in CpG-ODN delivery when treating allergic mice model. Methods: Preparation and characterization of CNP were performed by ionic gelation, dynamic light scattering, and zeta sizer. The CNP cytotoxicity and activation ability of CpG ODN delivered with CNP were tested using a cell counting kit-8 and Quanti blue method. Allergic mice were injected intraperitoneal with 10 ug ovalbumin on day 0 and 7, and then treated with intranasal CpG ODN/CpG ODN, delivered with CNP/CNP, on the third week three times per week for three weeks. The ELISA method measured cytokine and IgE profiles in the allergic mice's plasma and spleen. Results: CNP results have sizes 27.73 nm±3.67 dan 188.23 nm±53.47, spherical in shape and non-toxic, and did not alter the NF-κB activation of CpG ODN in RAW-blue cells. The application of CpG ODN delivered by chitosan nanoparticles shows no statistical difference between groups of IFN-γ, IL-10, and IL-13 in Balb/c mice's plasma and spleen, in contrast with IgE level. Conclusions: The results showed that using chitosan nanoparticles as a delivery system for CpG ODN has the potency to safely CpG ODN efficacy.

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)-1ß, 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.

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

BACKGROUND: Combinatorial 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. RESULTS: In the present work, protamine sulfate (PS) and carboxymethyl ß-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. CONCLUSION: Our 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.