Search

Background: Mitochondria are semiautonomous cell organelles having its own nucleic acid. Mitochondrial DNA (Mt-DNA) remain in hypomethylated (CpG) state and impose an immunogenic response by binding to the toll-like receptor (TLR-9) through the NF-kB pathway. Innate immune cells recognize the hypomethylated pattern of mt-DNA and quickly trigger the innate immune response. The immunomodulatory effects of urinary mt-DNA derived from renal transplant recipients with COVID-19-associated acute kidney injury (AKI) have not been studied. Materials and Methods: Healthy donor peripheral blood mononuclear cell (PBMC) was cultured with the urinary Mt-DNA derived from the renal transplant recipients, who previously developed SARS-CoV-2 infection associated AKI. Cell activation was measured by the flow cytometry. In cell pellets, interleukin IL-6, IL-10, and Myd88, TLR-9 mRNA transcript expression was measured by the reverse transcription polymerase chain reaction. The IL-6 and IL-10 cytokine levels were measured by the enzyme-linked immunosorbent assay in culture supernatants. Results: The urinary mitochondrial DNA (umt-DNA) significantly induces the activation of > 75% of PBMCs. The m-RNA transcript expression of the inflammatory gene in control versus umt-DNA treated PBMCs was for IL-6 (0.99 ± 0.05 vs. 2.18 ± 1.15 au; P = 0.004), MYD88 was (1.00 ± 0.05 vs. 1.55 ± 0.31; P < 0.001), TLR-9 (1.00 ± 0.05 vs. 3.33 ± 1.37 au; P < 0.001) was upregulated, and the IL-10 (1.00 ± 0.13 vs. −1.73 ± 0.58; P < 0.001) level was downregulated. However, in PBMC culture supernatants, IL-6 level in control versus umt-DNA-treated groups were (37.50 ± 13.79 vs. 186.9 ± 15.50 pg/mL; P < 0.001), which was significantly higher in umt-DNA-treated groups and the IL-10 (8.80 ± 2.16 vs. 7.60 ± 3.12 pg/mL; P = 0.32) level was similar between the control- and umt-DNA-treated groups. Conclusions: Urinary Mt-DNA significantly induces the inflammatory cytokine IL-6 secretion from the PBMCs through the Myd88-dependent pathway.

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

Background: Mitochondria are semiautonomous cell organelles having its own nucleic acid. Mitochondrial DNA (Mt‑DNA) remain in hypomethylated (CpG) state and impose an immunogenic response by binding to the toll‑like receptor (TLR‑9) through the NF‑kB pathway. Innate immune cells recognize the hypomethylated pattern of mt‑DNA and quickly trigger the innate immune response. The immunomodulatory effects of urinary mt‑DNA derived from renal transplant recipients with COVID‑19‑associated acute kidney injury (AKI) have not been studied. Materials and Methods: Healthy donor peripheral blood mononuclear cell (PBMC) was cultured with the urinary Mt‑DNA derived from the renal transplant recipients, who previously developed SARS‑CoV‑2 infection associated AKI. Cell activation was measured by the flow cytometry. In cell pellets, interleukin IL‑6, IL‑10, and Myd88, TLR‑9 mRNA transcript expression was measured by the reverse transcription polymerase chain reaction. The IL‑6 and IL‑10 cytokine levels were measured by the enzyme‑linked immunosorbent assay in culture supernatants. Results: The urinary mitochondrial DNA (umt‑DNA) significantly induces the activation of > 75% of PBMCs. The m‑RNA transcript expression of the inflammatory gene in control versus umt‑DNA treated PBMCs was for IL‑6 (0.99 ± 0.05 vs. 2.18 ± 1.15 au; P = 0.004), MYD88 was (1.00 ± 0.05 vs. 1.55 ± 0.31; P < 0.001), TLR‑9 (1.00 ± 0.05 vs. 3.33 ± 1.37 au; P < 0.001) was upregulated, and the IL‑10 (1.00 ± 0.13 vs. −1.73 ± 0.58; P < 0.001) level was downregulated. However, in PBMC culture supernatants, IL‑6 level in control versus umt‑DNA‑treated groups were (37.50 ± 13.79 vs. 186.9 ± 15.50 pg/mL; P < 0.001), which was significantly higher in umt‑DNA‑treated groups and the IL‑10 (8.80 ± 2.16 vs. 7.60 ± 3.12 pg/mL; P = 0.32) level was similar between the control‑ and umt‑DNA‑treated groups. Conclusions: Urinary Mt‑DNA significantly induces the inflammatory cytokine IL‑6 secretion from the PBMCs through the Myd88‑dependent pathway. [ABSTRACT FROM AUTHOR]

Introduction: Respiratory syncytial virus (RSV) vaccines targeting the fusion glycoprotein (F protein) are highly effective clinically in preventing RSV challenges. The attachment glycoprotein (G protein) is a potentially effective vaccine antigen candidate, as it is important for cell adhesion during infection. However, vaccine-associated enhanced diseases in mice, such as eosinophilic lung inflammation following RSV challenge, are a concern with G protein vaccines. This study aimed to design an effective G protein vaccine with enhanced safety and efficacy by evaluating the efficacy and adverse reactions of vaccines composed of different recombinant G proteins and adjuvants in mice. Methods: Mice were subcutaneously immunized with glycosylated G protein expressed in mammalian cells (mG), non-glycosylated G protein expressed in Escherichia coli (eG), or F protein with or without aluminum salts (alum), CpG oligodeoxynucleotide (CpG ODN), or AddaVax. After vaccination, the levels of G-specific antibody and T-cell responses were measured. The immunized mice were challenged with RSV and examined for the viral load in the lungs and nasal turbinates, lung-infiltrating cells, and lung pathology. Results: mG with any adjuvant was ineffective at inducing G-specific antibodies and had difficulty achieving both protection against RSV challenge and eosinophilia suppression. In particular, mG+CpG ODN induced G-specific T helper 1 (Th1) cells but only a few G-specific antibodies and did not protect against RSV challenge. However, eG+CpG ODN induced high levels of G-specific antibodies and Th1 cells and protected against RSV challenge without inducing pulmonary inflammation. Moreover, the combination vaccine of eG+F+CpG ODN showed greater protection against upper respiratory tract RSV challenge than using each single antigen vaccine alone. Discussion: These results indicate that the efficacy of recombinant G protein vaccines can be enhanced without inducing adverse reactions by using appropriate antigens and adjuvants, and their efficacy is further enhanced in the combination vaccine with F protein. These data provide valuable information for the clinical application of G protein vaccines. [ABSTRACT FROM AUTHOR]

IntroductionRespiratory syncytial virus (RSV) vaccines targeting the fusion glycoprotein (F protein) are highly effective clinically in preventing RSV challenges. The attachment glycoprotein (G protein) is a potentially effective vaccine antigen candidate, as it is important for cell adhesion during infection. However, vaccine-associated enhanced diseases in mice, such as eosinophilic lung inflammation following RSV challenge, are a concern with G protein vaccines. This study aimed to design an effective G protein vaccine with enhanced safety and efficacy by evaluating the efficacy and adverse reactions of vaccines composed of different recombinant G proteins and adjuvants in mice.MethodsMice were subcutaneously immunized with glycosylated G protein expressed in mammalian cells (mG), non-glycosylated G protein expressed in Escherichia coli (eG), or F protein with or without aluminum salts (alum), CpG oligodeoxynucleotide (CpG ODN), or AddaVax. After vaccination, the levels of G-specific antibody and T-cell responses were measured. The immunized mice were challenged with RSV and examined for the viral load in the lungs and nasal turbinates, lung-infiltrating cells, and lung pathology.ResultsmG with any adjuvant was ineffective at inducing G-specific antibodies and had difficulty achieving both protection against RSV challenge and eosinophilia suppression. In particular, mG+CpG ODN induced G-specific T helper 1 (Th1) cells but only a few G-specific antibodies and did not protect against RSV challenge. However, eG+CpG ODN induced high levels of G-specific antibodies and Th1 cells and protected against RSV challenge without inducing pulmonary inflammation. Moreover, the combination vaccine of eG+F+CpG ODN showed greater protection against upper respiratory tract RSV challenge than using each single antigen vaccine alone.DiscussionThese results indicate that the efficacy of recombinant G protein vaccines can be enhanced without inducing adverse reactions by using appropriate antigens and adjuvants, and their efficacy is further enhanced in the combination vaccine with F protein. These data provide valuable information for the clinical application of G protein vaccines.

Key points TLR9 is not only a prognostic biomarker but also a potential target for B-ALL therapy. C-MYC-mediated BAX activation leads to CpG 685-induced apoptosis in B-ALL.

Background: The prognosis of B-cell acute lymphoblastic leukemia (B-ALL) has improved significantly with current first-line therapy, although the recurrence of B-ALL is still a problem. Toll-like receptor 9 (TLR9) agonists have shown good safety and efficiency as immune adjuvants. Apart from their immune regulatory effect, the direct effect of TLR9 agonists on cancer cells with TLR9 expression cannot be ignored. However, the direct effect of TLR9 agonists on B-ALL remains unknown. Methods: We discussed the relationship between TLR9 expression and the clinical characteristics of B-ALL and explored whether CpG 685 exerts direct apoptotic effect on B-ALL without inhibiting normal B-cell function. By using western blot, co-immunoprecipitation, immunofluorescence co-localization, and chromatin immunoprecipitation, we explored the mechanism of the apoptosis-inducing effect of CpG 685 in treating B-ALL cells. By exploring the mechanism of CpG 685 on B-ALL, the predictive biomarkers of the efficacy of CpG 685 in treating B-ALL were explored. These efficiencies were also confirmed in mouse model as well as clinical samples. Results: High expression of TLR9 in B-ALL patients showed good prognosis. C-MYC-induced BAX activation was the key to the effect of CpG oligodeoxynucleotides against B-ALL. C-MYC overexpression promoted P53 stabilization, enhanced Bcl-2 associated X-protein (BAX) activation, and mediated transcription of the BAX gene. Moreover, combination therapy using CpG 685 and imatinib, a BCR-ABL kinase inhibitor, could reverse resistance to CpG 685 or imatinib alone by promoting BAX activation and overcoming BCR-ABL1-independent PI3K/AKT activation. Conclusion: TLR9 is not only a prognostic biomarker but also a potential target for B-ALL therapy. CpG 685 monotherapy might be applicable to Ph− B-ALL patients with C-MYC overexpression and without BAX deletion. CpG 685 may also serve as an effective combinational therapy against Ph+ B-ALL. Key points: TLR9 is not only a prognostic biomarker but also a potential target for B-ALL therapy. C-MYC-mediated BAX activation leads to CpG 685-induced apoptosis in B-ALL. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) poses a significant public health challenge due to the lack of an approved human vaccine. We attempted to enhance the efficacy of virus-like particle vaccines expressing the Leishmania donovani promastigote surface antigen (LdPSA-VLP) by adjuvanting with CpG oligodeoxynucleotide (CpG-ODN). Here, adjuvanted vaccine-induced immune responses and their efficacies in mice challenged with mCherry-expressing L. donovani promastigotes were evaluated. Adjuvanted LdPSA-VLP vaccination significantly elevated parasite-specific IgG, IgG1, IgG2a, and IgG2b serum antibody levels. Additionally, vaccinated mice exhibited enhanced germinal center B cells and splenic T cell activities, compared to unimmunized mice. Importantly, adjuvanted LdPSA-VLPs reduced the levels of inflammatory cytokines IFN-γ and IL-6 in visceral organs, leading to decreased total parasite burden and protection against L. donovani challenge. Our findings indicate that CpG-ODN enhanced the protection conferred by LdPSA-VLPs, offering a promising step toward effective VL vaccine development., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

One characteristic of tumor-associated CD4+Foxp3+ regulatory T cells (Tregs) is the high expression of tumor necrosis factor receptor type II (TNFR2), a receptor that mediates the decisive effect of tumor necrosis factor (TNF) in the activation and expansion of Tregs. There is increasing evidence that inhibition of TNFR2 can enhance anti-tumor immune responses. Therefore, we screened Chinese herbal extracts for their capacity to block TNF-TNFR2 interaction. The results showed that the treatment with a Chinese herb extract could inhibit TNFR2-induced biological responses in vitro, including the proliferation of TNFR2+ Tregs. Our subsequent study led to the identification of flavonoid compound scutellarin was responsible for the activity. Our results showed that scutellarin is able to disrupt the interaction of TNF-TNFR2 and inhibited the phosphorylation of p38 MAPK, a down-stream signaling component of TNFR2. Importantly, in vivo scutellarin treatment markedly enhanced the efficacy of tumor immunotherapy with CpG oligodeoxynucleotide in mouse CT26 colon cancer model. This effect of scutellarin was associated with the reduction of the number of tumor-infiltrating TNFR2-expressing Tregs and increased tumor infiltration of interferon-γ–producing CD8+ T cells. Our result also suggests that scutellarin or its analogs may be used as an adjuvant to enhance the anti-tumor effect of immunotherapeutic agent by eliminating TNFR2+ Treg activity.

Abstract Background Currently, anticancer immunotherapy based on PD‐1/PD‐L1 blockade with immune checkpoint inhibitors (ICIs) is being used as a standard therapy for non‐small cell lung cancer (NSCLC). However, more effective treatments are required as these tumors are often resistant and refractory. Here, we aimed to determine the effects of immunomodulatory oligodeoxynucleotides (ODNs) in terms of the presence or absence of CpG motifs and the number of consecutive guanosines. Methods Western blots were used to measure the molecules which regulate the expression of PD‐L1 in human lung cancer cell lines after incubation with several cytokines and ODNs. The expression of PD‐L1 and β2‐microglobulin (β2‐MG) on A549 cells, and IFN‐γ‐induced apoptosis with ODNs were examined by flow cytometry. The relationship between IFN‐γ receptor and ODN was analyzed by ELISA and immunofluorescence chemistry. Results Our results verified that A‐CpG ODNs suppress the upregulation of IFN‐γ‐induced PD‐L1 and β2‐MG expression. In addition, we found that ODNs with six or more consecutive guanosines (ODNs with poly‐G sequences) may competitively inhibit the IFN‐γ receptor and abolish the effect of IFN‐γ, thereby suppressing apoptosis and indoleamine 2,3‐dioxygenase 1 expression in human lung cancer cells. The tumor microenvironment regulates whether this action will promote or suppress tumor immunity. Thus, in immunotherapy with CpG ODNs, it is essential to consider the effect of ODNs with poly‐G sequences. Conclusions This study suggests that ODNs containing six or more consecutive guanosines may inhibit the binding of IFN‐γ to IFN‐γ receptor. However, it does not directly show that ODNs containing six or more consecutive guanosines competitively inhibit the IFN‐γ receptor, and further studies are warranted to confirm this finding. Key points Significant findings of the study: Oligodeoxynucleotides with a contiguous sequence of six or more guanosines may competitively inhibit the IFN‐γ receptor and abolish the action of IFN‐γ. This may suppress IFN‐γ‐induced apoptosis and indoleamine‐2,3‐dioxygenase‐1 expression in human lung cancer cells. What this study adds: A‐CpG and poly‐G ODN may overcome tolerance if the cause of ICI tolerance is high IDO expression. However, IFN‐γ also has the effect of suppressing apoptosis of cancer cells, and it is necessary to identify the cause of resistance.

Considering the potent immune stimulation by CpG oligodeoxynucleotides (CpGs), the development of CpG carriers is a prerequisite for efficient cancer immunotherapy. In this study, we conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[hydroxyl succinimidyl (polyethylene glycol)] (DSPE-PEG-NHS) with polyethylenimine (PEI) to develop a PEI-PEG-DSPE conjugate that can serve as a biocompatible and efficient CpG carrier. Five types of PEIPEG-DSPE conjugates were developed, each with different molecular weights of PEI and different degrees of DSPEPEG modification, and all exhibited significantly lower cytotoxicity. In particular, compared to CpG delivery via natural PEI, delivery with PEI (25 kDa)-PEG-DSPE and DSPE-PEG-NHS/(amine groups of PEI) at a molar ratio of 0.1 resulted in a higher uptake of CpGs into RAW264.7 cells, probably because of the presence of a hydrophobic lipid moiety. In addition, PEI-PEG-DSPE/CpG complexes triggered significant cytokine secretion (TNF-α) from RAW264.7 cells, comparable to that triggered by PEI/CpG complexes. Thus, PEI-PEG-DSPE conjugates could serve as biocompatible and efficient carriers of the immune stimulator CpG to the macrophages. [ABSTRACT FROM AUTHOR]

Guoqun Wang,1 Jiaojiao Zhao,2 Meiling Zhang,1 Qian Wang,1 Bo Chen,3 Yayi Hou,2,4 Kaihua Lu11Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, People’s Republic of China; 2The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, People’s Republic of China; 3Institute of Materials Science and Devices, Suzhou University of Science and Technology, Suzhou 215009, People’s Republic of China; 4Jiangsu Key Laboratory of Molecular Medicine, Nanjing University, Nanjing 210093, People’s Republic of ChinaPurpose: Drug resistance is a major challenge for epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) treatment of lung cancer. Ferumoxytol (FMT) drives macrophage (MΦ) transformation towards a M1-like phenotype and thereby inhibits tumor growth. CpG oligodeoxynucleotide 2395 (CpG), a toll-like receptor 9 (TLR9) agonist, is an effective therapeutic agent to induce anticancer immune responses. Herein, the effect of co-administered FMT and CpG on MΦ activation for treating non-small cell lung cancer (NSCLC) was explored.Methods: The mRNA expression levels of M1-like genes in RAW 264.7 MΦ cells stimulated by FMT, CpG and FMT and CpG (FMT/CpG) were evaluated by quantitative reverse transcription PCR (qRT-PCR). Then, the effects of FMT/CpG-pretreated MΦ supernatant on apoptosis and proliferation of H1975 cells were detected by flow cytometry, and the expression of EGFR and its downstream signaling pathway in H1975 cells were explored by western blotting. Finally, a H1975 cell xenograft mouse model was used to study the anti-tumor effect of the combination of FMT and CpG in vivo.Results: FMT and CpG synergistically enhanced M1-like gene expression in MΦ, including tumor necrosis factor-α, interleukin (IL)-12, IL-1α, IL-1β, IL-6 and inducible nitric oxide synthase (iNOS). FMT/CpG-pretreated MΦ supernatant inhibited proliferation and induced apoptosis of H1975 cells, accompanied by down-regulation of cell cycle-associated proteins and up-regulation of apoptosis-related proteins. Further studies indicated that the FMT/CpG-pretreated MΦ supernatant suppressed p-EGFR and its downstream AKT/mammalian target of rapamycin signaling pathway in H1975 cells. Furthermore, FMT/CpG suppressed tumor growth in mice accompanied by a decline in the EGFR-positive tumor cell fraction and increased M1 phenotype macrophage infiltration.Conclusion: FMT acted synergistically with CpG to activate MΦ for suppressed proliferation and promoted apoptosis of NSCLC cells via EGFR signaling. Thus, combining FMT and CpG is an effective strategy for the treatment of NSCLC with EGFRL858R/T790M, mutation.Keywords: ferumoxytol, CpG oligodeoxynucleotide, macrophages, non-small cell lung cancer, epidermal growth factor receptor

Current influenza vaccines are generally effective against highly similar (homologous) strains, but their effectiveness decreases markedly against antigenically mismatched (heterologous) strains. One way of developing a universal influenza vaccine with a broader spectrum of protection is to use appropriate vaccine adjuvants to improve a vaccine's effectiveness and change its immune properties. Oligodeoxynucleotides (ODNs) with unmethylated cytosine-phosphate-guanine (CpG) motifs (CpG ODNs), which are Toll-like-receptor 9 (TLR9) agonists, are among the most promising adjuvants and are already being used in humans. However, the development of novel delivery vehicles to improve adjuvant effects in vivo is highly desirable. Here, we assessed the potential of lipid nanoparticles (LNPs) as CpG ODN delivery vehicles in mice to augment the vaccine adjuvant effects of CpG ODN and enhance the protective spectrum of conventional influenza split vaccine (SV). In vitro, compared with CpG ODN, LNPs containing CpG ODNs (LNP-CpGs) induced significantly greater production of cytokines such as IL-12 p40 and IFN-α by mouse dendritic cells (DCs) and significantly greater expression of the co-stimulatory molecules CD80 and CD86 on DCs. In addition, after subcutaneous administration in mice, compared with CpG ODN, LNP-CpGs enhanced the expression of CD80 and CD86 on plasmacytoid DCs in draining lymph nodes. LNP-CpGs given with SV from H1N1 influenza A virus improved T-cell responses and gave a stronger not only SV-specific but also heterologous-virus-strain-specific IgG2c response than CpG ODN. Furthermore, immunization with SV plus LNP-CpGs protected against not only homologous strain challenge but also heterologous and heterosubtypic strain challenge, whereas immunization with SV plus CpG ODNs protected against homologous strain challenge only. We therefore demonstrated that LNP-CpGs improved the adjuvant effects of CpG ODN and broadened the protective spectrum of SV against influenza virus. We expect that this strategy will be useful in developing adjuvant delivery vehicles and universal influenza vaccines.

The G-quadruplex (GQ) structure has potential applications in nucleic acid drug delivery because of its superior stability. In this study, we added one G-tract (five guanines) to an unmethylated phosphodiester-linked cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), a potential immune adjuvant, to construct a GQ-structured CpG ODN with precise structural properties, increased biological stability, and efficient delivery to Toll-like receptor 9 (TLR9)-positive immune cells. A G-tract was added to phosphodiester-backboned CpG1668 at the 5′-end [1668(5′-G5)], 3′-end [1668(3′-G5)], or within the sequence [1668(mid-G5)]. Circular dichroism analysis showed that all CpG ODNs with a G-tract formed parallel GQ structures, irrespective of its position. Electrophoresis showed that 1668(5′-G5) formed a GQ dimer, whereas others remained GQ monomers. GQ-structured CpG ODNs induced greater tumor necrosis factor-α and interleukin-6 secretion from TLR9-positive mouse macrophage-like RAW264.7 cells than single-stranded CpG ODNs, with the highest for 1668(3′-G5). GQ structuration increased CpG ODN uptake by RAW264.7 cells, and 1668(3′-G5) decomposed more slowly in serum than 1668(5′-G5). Thus, GQ formation with one G-tract is a simple and efficient strategy for CpG ODN delivery to TLR9-positive cells, and addition of a G-tract to the 3′-end is effective in obtaining monomeric GQ-structured CpG ODN with high biological stability and immunostimulatory activity. [ABSTRACT FROM AUTHOR]

Background: Currently, anticancer immunotherapy based on PD‐1/PD‐L1 blockade with immune checkpoint inhibitors (ICIs) is being used as a standard therapy for non‐small cell lung cancer (NSCLC). However, more effective treatments are required as these tumors are often resistant and refractory. Here, we aimed to determine the effects of immunomodulatory oligodeoxynucleotides (ODNs) in terms of the presence or absence of CpG motifs and the number of consecutive guanosines. Methods: Western blots were used to measure the molecules which regulate the expression of PD‐L1 in human lung cancer cell lines after incubation with several cytokines and ODNs. The expression of PD‐L1 and β2‐microglobulin (β2‐MG) on A549 cells, and IFN‐γ‐induced apoptosis with ODNs were examined by flow cytometry. The relationship between IFN‐γ receptor and ODN was analyzed by ELISA and immunofluorescence chemistry. Results: Our results verified that A‐CpG ODNs suppress the upregulation of IFN‐γ‐induced PD‐L1 and β2‐MG expression. In addition, we found that ODNs with six or more consecutive guanosines (ODNs with poly‐G sequences) may competitively inhibit the IFN‐γ receptor and abolish the effect of IFN‐γ, thereby suppressing apoptosis and indoleamine 2,3‐dioxygenase 1 expression in human lung cancer cells. The tumor microenvironment regulates whether this action will promote or suppress tumor immunity. Thus, in immunotherapy with CpG ODNs, it is essential to consider the effect of ODNs with poly‐G sequences. Conclusions: This study suggests that ODNs containing six or more consecutive guanosines may inhibit the binding of IFN‐γ to IFN‐γ receptor. However, it does not directly show that ODNs containing six or more consecutive guanosines competitively inhibit the IFN‐γ receptor, and further studies are warranted to confirm this finding. Key points: Significant findings of the study: Oligodeoxynucleotides with a contiguous sequence of six or more guanosines may competitively inhibit the IFN‐γ receptor and abolish the action of IFN‐γ. This may suppress IFN‐γ‐induced apoptosis and indoleamine‐2,3‐dioxygenase‐1 expression in human lung cancer cells.What this study adds: A‐CpG and poly‐G ODN may overcome tolerance if the cause of ICI tolerance is high IDO expression. However, IFN‐γ also has the effect of suppressing apoptosis of cancer cells, and it is necessary to identify the cause of resistance. [ABSTRACT FROM AUTHOR]

Nobutaka Hanagata,1,2 Xianglan Li,1 Min-Hua Chen,1 Jie Li,1 Shinya Hattori1 1Nanotechnology Innovation Station, National Institute for Materials Science, Tsukuba, 2Graduate School of Life Science, Hokkaido University, Sapporo, Japan Abstract: Conventional class B cytosine-guanine (CpG) (CpG-B) oligodeoxynucleotide (ODNs) consisting of a single-stranded (ss) phosphorothioate (PT) backbone (ss CpG-B-PT) is converted from a proinflammatory cytokine inducer to a type-I interferon (IFN) inducer when complexed with cationic materials. In this study, we designed ss CpG-B and double-stranded (ds) CpG-B ODNs with a phosphodiester (PD) backbone (ss CpG-B-PD and ds CpG-B-PD, respectively) that became type-I IFN inducers upon complexation with Lipofectamine 2000 (Lipo), a cationic liposome. The ds CpG-B-PD complex induced higher IFN-β expression in mouse macrophage-like RAW264 cells than ss CpG-B-PD and ss CpG-B-PT complexes. The fold induction of IFN-β increased with the number of CpG motifs in ds CpG-B-PD, and a complex of ds CpG-B-PD consisting of 72 base pairs with nine CpG motifs (ds CpG-B72-PD) and Lipo showed the highest capacity to induce IFN-β. The materials and method used for complexation influenced the degree of IFN-β induction: ds CpG-B72-PD entrapped by calcium phosphate (CaP) (ds CpG-B72-PD/CaP) showed a higher induction capacity than ds CpG-B72-PD adsorbed onto the CaP surface. Entrapment of ds CpG-B72-PD by CaP also enhanced the induction of the proinflammatory cytokine interleukin-12. Vaccinating mice with ds CpG-B72-PD/CaP in conjunction with ovalbumin (OVA) increased the ratios of OVA-specific CD8+ T cells to total CD8+ T cells in peripheral blood and of OVA-specific IgG2a associated with helper T (Th)1 cells to OVA-specific IgG1 associated with Th2 cells. These results indicate that ds CpG-B72-PD/CaP is an effective vaccine adjuvant that can activate both cellular and Th1-type humoral immune responses. Keywords: CpG oligodeoxynucleotide, calcium phosphate, vaccine adjuvant, cellular immunity, humoral immunity

Introduction: Respiratory syncytial virus (RSV) vaccines targeting the fusion glycoprotein (F protein) are highly effective clinically in preventing RSV challenges. The attachment glycoprotein (G protein) is a potentially effective vaccine antigen candidate, as it is important for cell adhesion during infection. However, vaccine-associated enhanced diseases in mice, such as eosinophilic lung inflammation following RSV challenge, are a concern with G protein vaccines. This study aimed to design an effective G protein vaccine with enhanced safety and efficacy by evaluating the efficacy and adverse reactions of vaccines composed of different recombinant G proteins and adjuvants in mice., Methods: Mice were subcutaneously immunized with glycosylated G protein expressed in mammalian cells (mG), non-glycosylated G protein expressed in Escherichia coli (eG), or F protein with or without aluminum salts (alum), CpG oligodeoxynucleotide (CpG ODN), or AddaVax. After vaccination, the levels of G-specific antibody and T-cell responses were measured. The immunized mice were challenged with RSV and examined for the viral load in the lungs and nasal turbinates, lung-infiltrating cells, and lung pathology., Results: mG with any adjuvant was ineffective at inducing G-specific antibodies and had difficulty achieving both protection against RSV challenge and eosinophilia suppression. In particular, mG+CpG ODN induced G-specific T helper 1 (Th1) cells but only a few G-specific antibodies and did not protect against RSV challenge. However, eG+CpG ODN induced high levels of G-specific antibodies and Th1 cells and protected against RSV challenge without inducing pulmonary inflammation. Moreover, the combination vaccine of eG+F+CpG ODN showed greater protection against upper respiratory tract RSV challenge than using each single antigen vaccine alone., Discussion: These results indicate that the efficacy of recombinant G protein vaccines can be enhanced without inducing adverse reactions by using appropriate antigens and adjuvants, and their efficacy is further enhanced in the combination vaccine with F protein. These data provide valuable information for the clinical application of G protein vaccines., Competing Interests: YY was employed by the Research Foundation for Microbial Diseases at the Osaka University. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kawahara, Shibata, Hirai and Yoshioka.)

Objective To investigate the clinical effect of topical application of CpG oligodeoxynucleotide( CpG-ODN) combined with OX40 monoclonal antibody in the treatment of hepatocellular carcinoma and its therapeutic effect on distant homologous tumors. Methods H22 single cell suspension was subcutaneously injected into the axilla of the four limbs of BALB/c male mice to establish a tumor-bearing model. After seven days, 30 mice with a similar tumor volume were screened out and randomly divided into model control group, CpG group, and CpG + OX40 group, with 10 mice in each group, and drugs were injected into the tumor in the left lower limb. Ten normal mice in the same batch were selected as normal control group. Tumor volume was calculated, ELISA was used to measure the serum levels of interleukin-12( IL-12) and interferon-γ( IFNγ), and flow cytometry was used to measure the percentage of CD8+T lymphocytes in the spleen. The three groups were compared in terms of survival after treatment. Repeated measures analysis of variance and one-way analysis of variance were used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The log-rank test was used to analyze survival rate for the model control group, CpG group, and CpG+ OX40 group, and survival curves were plotted. Results After treatment, the model control group had progressive growth of tumor, with significantly lower serum levels of IL-12 and IFNγ and percentage of spleen CD8+T lymphocytes than the normal control group( all P <0.05). Compared with the model control group, the CpG group and the CpG + OX40 group had a significant reduction in tumor volume after intervention( all P < 0.05). The CpG + OX40 group had a significantly lower growth rate of distant tumor than the CpG group and the model control group( all P < 0.05), but there was no significant difference in distant tumor volume between the CpG group and the model control group( P > 0.05). Compared with the model control group, the CpG group and the CpG + OX40 group had significant increases in serum levels of IL-12 and IFN-γ and percentage of spleen CD8+T lymphocytes( all P < 0.05), which were significantly higher in the CpG +OX40 group than in the CpG group( all P < 0.05). The CpG + OX40 group had a significantly higher survival rate than the model control group and the CpG group( both P < 0.05), while there was no significant difference between the CpG group and the model control group( P> 0.05). Conclusion OX40 monoclonal antibody combined with CpG-ODN can reduce liver tumor volume, effectively slow down the growth of distant tumors, and prolong the survival time of mice. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Thiolated CpG ODNs linked to HGNs via 'Au-S' bonding to form CpG-HGNs. • The synthesized HGNs presented good photothermal conversion effect. • The CpG-HGNs enhanced the immunostimulatory effects of CpG ODNs. • CpG-HGNs may potentially realize synergistic photothermal-immunotherapy. Abstract To realize efficiently cellular uptake and enhance the immunostimulation, thiolated cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) were self-assembled on hollow gold nanospheres (HGNs) to form CpG-HGNs. The cellular uptake of CpG-HGNs in immune cells was studied in RAW 264.7 cells. Due to the enhanced delivery efficiency, CpG-HGNs exhibited a higher immune stimulatory activity compared with CpG ODNs, resulting in a dramatically enhanced secretion of proinflammatory cytokines. In addition, CpG-HGNs showed low cytotoxicity in RAW 264.7 cells. CpG-HGNs could potentially realize synergistic photothermal therapy and immunotherapy in vivo. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract FDA approval of CpG oligodeoxynucleotide (CpG ODN) adjuvants for a human hepatitis B virus vaccine has been delayed until late 2017 because of concerns regarding the severe side effects, which may be attributed to the high dosage and systemic diffusion of this proinflammatory material. Considering that PLGA could provide shelter to resist nucleases in tissue and that cationic lipids could confine anionic oligonucleotides in the nanoparticles via electrostatic attraction to avoid systemic diffusion, we encapsulated a natural phosphodiester or the expensive phosphorothioate CpG ODNs in our previously reported hyaluronic acid-modified cationic lipid-PLGA hybrid nanoparticles and evaluated vaccine efficacy in a TC-1-grafted mouse model. Our results showed that together with Poly I:C, CpG ODN could promote the maturation of bone marrow-derived dendritic cells and the cross-presentation of exogenous antigens in vitro. For the coencapsulation with Poly I:C, in vivo studies showed that adjuvant effects on the vaccine efficacy of tumor depression, immune cell activation, and memory T-cell elevation of phosphodiester CpG ODNs were comparable to those of the phosphorothioate CpG ODNs at a low concentration (5 µg/dose). In conclusion, the combination of oligonucleotide adjuvants and synthetic particulate systems not only potentiated the immunogenicity of these nanoparticles but also made these adjuvants safer and more economical, which may be helpful for their wide application. [ABSTRACT FROM AUTHOR]

Gold nanoparticles (AuNPs) are successfully used as an adjuvant in the design of effective vaccines and in the preparation of high-affinity antibodies to haptens and complete antigens. Here, we assessed the adjuvant properties of AuNPs conjugated to a synthetic M2e peptide of the influenza A virus capsid. The resulting conjugate, a commercial influenza vaccine, and M2e in combination with different adjuvants were used to immunize laboratory mice. The highest titer was detected in the sera of mice immunized with two adjuvants: AuNPs and AuNP-conjugated CpG oligodeoxynucleotide 1826. With this combination, we also recorded increases in the respiratory activity of splenic lymphocytes, in the respiratory activity of peritoneal macrophages, and in the production of proinflammatory cytokines (IL-6 and IFN-γ). The results indicate that simultaneous immunization of the animals with two conjugates—M2e + AuNPs and CpG + AuNPs—activates antibody development. Therefore, the use of AuNPs as an antigen carrier leads to a complete and coordinated immune response from both cellular and humoral immunity. [ABSTRACT FROM AUTHOR]

Human respiratory syncytial virus (RSV) is themost significant cause of acute lower respiratory infection in children. However, there is no licensed vaccine available. Here, we investigated the effect of five or 20 copies of C-Class of CpG ODN (CpG-C) motif incorporated into a plasmid DNA vaccine encoding RSV fusion (F) glycoprotein on the vaccine-induced immune response. The addition of CpG-C motif enhanced serum binding and virus-neutralizing antibody responses in BALB/c mice immunized with the DNA vaccines. Moreover, mice vaccinated with CpG-modified vaccines, especially with the higher 20 copies, resulted in an enhanced shift toward a Th1-biased antibody and T-cell response, a decrease in pulmonary pathology and virus replication, and a decrease in weight loss after RSV challenge. This study suggests that CpG-C motif, cloned into the backbone of DNA vaccine encoding RSV F glycoprotein, functions as a built-in adjuvant capable of improving the efficacy of DNA vaccine against RSV infection. [ABSTRACT FROM AUTHOR]

Context:Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by autoreactive antibodies. Recent findings revealed the importance of innate immune responses, especially Toll-like receptors (TLRs) in the pathogenesis of SLE. Objective:In this study, the level of TLR9 expression on peripheral blood mononuclear cells (PBMCs) was analyzed. The levels of produced IFN-α were also measured in supernatant of PBMCs from SLE patients and healthy controls after stimulation with CpG ODN2216 which is a plasmocytoid dendritic cell (pDC)-specific TLR9 ligand. Materials and methods:TLR9 expression was analyzed by real-time polymerase chain reaction (PCR) and flow cytometry in 35 SLE patients and 38 healthy controls and IFN-α concentration was measured in supernatants using enzyme-linked immunosorbent assay (ELISA). Results:The results showed that the TLR9 expression in the mRNA and the protein level was significantly higher in PBMCs from SLE patients. However, IFN-α concentration in patients and controls significantly increased in response to CpG stimulation but this increase was significantly higher in healthy controls compared with SLE patients. Our results do not show any association between taking hydroxychloroquine and reduction in IFN-α production in SLE patients. Discussion and conclusions:Regarding the findings of the study, there is the possibility that TLR9 has played a role in SLE pathogenesis, and consequently it implies that TLRs can be considered to be the therapeutic targets for systemic autoimmunity. We may conclude that PBMCs in patients are functionally impaired in response to TLR ligationviainnate response stimulating pathogen-associated molecular patterns (PAMPs). [ABSTRACT FROM AUTHOR]