Functional characterization of AF-04, an afucosylated anti-MARV GP antibody.

Marburg virus (MARV), one member of the Filoviridae family, cause sporadic outbreaks of hemorrhagic fever with high mortality rates. No countermeasures are currently available for the prevention or treatment of MARV infection. Monoclonal antibodies (mAbs) are promising candidates to display high neutralizing activity against MARV infection in vitro and in vivo. Recently, growing evidence has shown that immune effector function including antibody-dependent cell-mediated cytotoxicity (ADCC) is also required for in vivo efficacy of a panel of antibodies. Glyco-engineered methods are widely utilized to augment ADCC function of mAbs. In this study, we generated a fucose-knockout MARV GP-specific mAb named AF-04 and showed that afucosylation dramatically increased its binding affinity to polymorphic FcγRIIIa (F176/V176) compared with the parental AF-03. Accordingly, AF-04-mediated NK cell activation and NFAT expression downstream of FcγRIIIa in effector cells were also augmented. In conclusion, this work demonstrates that AF-04 represents a novel avenue for the treatment of MARV-caused disease.

A novel MARV glycoprotein-specific antibody with potentials of broad-spectrum neutralization to filovirus.

Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.

Development of novel monoclonal antibodies for blocking NF-κB activation induced by CD2v protein in African swine fever virus.

Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.

pHLIP-fused PD-L1 engages avelumab to elicit NK cytotoxicity under acidic conditions.

Natural killer (NK) cells represent key player in immune surveillance to eliminate transformed or malignant cells. One of mechanisms of action of NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC) by recognizing tumor antigens on the surface of cancer cells. However, the heterogeneity of tumor antigens and the scarcity of membrane surface targets significantly restrict this strategy. Recently, we constructed a new cargo by tethering a low pH insertion peptide (pHLIP) to the C terminus of the ectodomain of programed death ligand-1 (PD-L1) and demonstrated its ability to modulate immune responses. Herein, the potential application of PD-L1-pHLIP in cancer therapy was determined. pHLIP tethering had no effect on the binding capacity of PD-L1 protein to an anti-PD-L1 antibody (i.e. avelumab). Association of pHLIP rendered PD-L1 segment display on the surface of cellular membrane in the acidic buffer instead of the neutral solution. Importantly, plate-coated or beads-coupled PD-L1-pHLIP enable robust activation and expression of cytotoxic mediators of NK cells via engaging avelumab. Overall, this work provides proof of concept that recombinant PD-L1 protein decorated on the cellular membrane driven by pHLIP in combination with appropriate monoclonal antibody has potentials to elicit NK cytotoxicity, which may represent a novel and promising therapeutic avenue in cancer.

Investigation on the antitumor effects of paeonol against renal cell carcinoma based on network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Renal cell carcinoma (RCC) is the most common cancer of the urinary system, the current treatments for RCC are unsatisfactory. Paeonol is the main pharmacologically active ingredient of the traditional Chinese medicine (TCM) moutan cortex (Paeonia suffruticosa Andrews) and Paeonia albiflora Pall, and has been used in TCM to treat various diseases including cancer. However, the underlying therapeutic mechanisms of paeonol in RCC have not been investigated yet. AIM OF THE STUDY: This study aimed to explore the potential antitumor effects and mechanisms of paeonol on RCC based on network pharmacology and experimental validation. MATERIALS AND METHODS: Network pharmacological analysis was performed to predict the potential targets and mechanism of paeonol against RCC. The antitumor effects and the priority targets of paeonol against RCC were further assessed by in vitro experiments. RESULTS: 104 intersection targets shared by paeonol and RCC were collected, 15 hub genes were obtained, among these genes, VEGFA expression was higher in RCC, and the higher expression of IL-6 or lower expression of AKT1, JUN, MAPK1, and MAPK8 were correlated to the shorter overall survival (OS) in RCC patients. GO and KEGG analyses suggested that the genes were mainly enriched in the positive regulation of cell death and apoptosis pathway. In vitro experiments showed that paeonol inhibited 786-O cell proliferation, migration, invasion, and promoted apoptosis. When 786-O cells were treated with paeonol, the expression of Bax increased while Bcl-2 and VEGFA decreased. CONCLUSION: The present study demonstrated that paeonol might play an essential role in RCC by regulating cell proliferation, apoptosis, metastasis, and invasion through the Bcl-2/Bax signaling pathway and VEGFA, providing a theoretical and experimental scientific basis for future investigations of the antitumor effects of paeonol against RCC.

NLRP6 Induces Lung Injury and Inflammation Early in Brucella and Influenza Coinfection.

(1) Background: With the resurgence of brucellosis epidemics in China in recent years, the chances of a brucella coinfection with other common respiratory pathogens, such as the influenza virus, have increased dramatically. However, little is known about the pathogenicity or the mechanisms of brucella and influenza coinfections. (2) Methods: To clarify the interventions in the early stages of lung damage due to brucella and influenza coinfections, we evaluated the effect of the coinfection on disease progression and mortality using a coinfection model in WT mice and NLRP6-/- mice, and we verified the function of NLRP6 in infection and proinflammation. (3) Results: The coinfection induced significant respiratory symptoms, weight loss, and a high mortality rate in WT mice. Influenza in the coinfection group significantly increased brucella proliferation in a synergistic manner. Meanwhile, a histological examination showed severe lung tissue destruction and excessive inflammatory responses in coinfected WT animals, and the expression of NLRP6 and IL-18 was dramatically increased in the lung tissues. Furthermore, NLRP6 deletion attenuated lung injuries and inflammation, a reduced bacterial load, and decreased IL-18 protein expression. (4) Conclusions: Our findings indicated that NLRP6 plays a critical role and might be a promising potential therapeutic target for brucella-influenza coinfections.

pH Low Insertion Peptide-Modified Programmed Cell Death-Ligand 1 Potently Suppresses T-Cell Activation Under Acidic Condition.

Programmed cell death-ligand 1 (PD-L1)/PD-1 axis is critical for maintenance of immune homeostasis by limiting overactivation of effector T-cell responses. The impairment of PD-L1/PD-1 signals play an important role in the pathogenesis of inflammatory diseases, making this pathway an ideal target for novel therapeutics to induce immune tolerance. Given weakly acidic environment as a putative hallmark of inflammation, in this study we designed a new cargo by linking the ectodomain of murine PD-L1 to the N terminus of pHLIPs, a low pH-responding and membrane-insertion peptide, and demonstrated its potent immune-suppressive activity. Specifically, PD-L1-pHLIP spanned the cellular membrane and perfectly recognized its ligand PD-1 in acidic buffer. Immobile PD-L1-pHLIP actively inhibited T-cell proliferation and IFN-γ production. Importantly, soluble PD-L1-pHLIP retained its function to dampen T-cell responses under acidic condition instead of neutral aqueous solution. Overall, these data suggest that PD-L1-pHLIP has potentials to be a novel therapeutic avenue for T-cell-mediated inflammatory diseases.

[Bioinformatic analysis of Eftud2 enhancing mouse macrophage function].

Objective To elucidate the mechanisms by which elongation factor Tu GTP binding domain containing 2 (Eftud2) enhances the immune function of murine macrophages by bioinformatics analysis. Methods The bone marrow-derived macrophages (BMDMs) of Eftud2 myeloid cell-specific knockout (MKO) mice (n=10) and wild-type (WT) littermates (n=10) were collected and stimulated by lipopolysaccharide (LPS) (100 ng/mL) for 2 hours. Bioinformatics analysis was conducted to examine the differences in gene expression and mRNA transcription levels. The the differences in gene expression and alternative splicing of mRNA transcription in BMDMs were analyzed by DEGseq and rMATS, respectively. The signaling pathways affected were clarified by Kyoto Encyclopedia of Genes and Genomes (KEGG) classification and enrichment methods. Results Compared with WT counterparts, the expression levels of IL-6, IL-1ß, TNF-α, and the genes related to immune response in MKO BMDMs were down-regulated following LPS stimulation. KEGG pathway analysis showed that the differently expressed genes in BMDMs and alternative splicing mainly affected the signal transduction and immune system-related metabolic pathways, and had a strong correlation with PI3K-AKT signaling pathway. The difference in alternative splicing also existed in ubiquitination and endocytosis. Compared with WT counterparts, there were 232 differences in alternative splicing in MKO BMDMs, among which 125 were skipping exons, accounting for the largest proportion. In addition, the analysis of alternative splicing differences also confirmed the previous experimental results, that is, Eftud2 could participate in the activation of inflammatory signaling pathways by enhancing the alternative splicing of key molecules such as MyD88 in TLR4-NF-κB signaling pathway, thereby augmenting the function of macrophages. Conclusion Eftud2 can promote the release of inflammatory cytokines in BMDMs by regulating gene expression and alternative splicing, and consequently enhance the immune function of macrophages.

Overcoming tumor cell chemoresistance using nanoparticles: lysosomes are beneficial for (stearoyl) gemcitabine-incorporated solid lipid nanoparticles.

Despite recent advances in targeted therapies and immunotherapies, chemotherapy using cytotoxic agents remains an indispensable modality in cancer treatment. Recently, there has been a growing emphasis in using nanomedicine in cancer chemotherapy, and several nanomedicines have already been used clinically to treat cancers. There is evidence that formulating small molecular cancer chemotherapeutic agents into nanomedicines significantly modifies their pharmacokinetics and often improves their efficacy. Importantly, cancer cells often develop resistance to chemotherapy, and formulating anticancer drugs into nanomedicines also helps overcome chemoresistance. In this review, we briefly describe the different classes of cancer chemotherapeutic agents, their mechanisms of action and resistance, and evidence of overcoming the resistance using nanomedicines. We then emphasize on gemcitabine and our experience in discovering the unique (stearoyl) gemcitabine solid lipid nanoparticles that are effective against tumor cells resistant to gemcitabine and elucidate the underlying mechanisms. It seems that lysosomes, which are an obstacle in the delivery of many drugs, are actually beneficial for our (stearoyl) gemcitabine solid lipid nanoparticles to overcome tumor cell resistance to gemcitabine.

Oral 4-(N)-stearoyl gemcitabine nanoparticles inhibit tumor growth in mouse models.

In spite of recent advances in targeted tumor therapy, systemic chemotherapy with cytotoxic agents remains a vital cancer treatment modality. Gemcitabine is a nucleoside analog commonly used in the treatment of various solid tumors, but an oral gemcitabine dosage form remain unavailable. Previously, we developed the 4-(N)-stearoyl gemcitabine solid lipid nanoparticles (GemC18-SLNs) by incorporating 4-(N)-stearoyl gemcitabine (GemC18), an amide prodrug of gemcitabine, into solid lipid nanoparticles. GemC18-SLNs, when administered intravenously, showed strong antitumor activity against various human and mouse tumors in mouse models. In the present study, we defined the plasma pharmacokinetics of gemcitabine when GemC18-SLNs were given orally to healthy mice and evaluated the antitumor activity of GemC18-SLNs when given orally in mouse models of lung cancer. In mice orally gavaged with GemC18-SLNs, plasma gemcitabine concentration followed an absorption phase and then clearance phase, with a Tmax of ~2 h. The absolute oral bioavailability of gemcitabine in the GemC18-SLNs was ~70% (based on AUC0-24 h values). In mice with pre-established tumors (i.e. mouse TC-1 or LLC lung cancer cells), oral GemC18-SLNs significantly inhibited the tumor growth and increased mouse survival time, as compared to the molar equivalent dose of gemcitabine hydrochloride or GemC18 in vegetable oil or in Tween 20. Immunohistostaining revealed that oral GemC18-SLNs also have significant antiproliferative, antiangiogenic, and proapoptotic activity in LLC tumors. Formulating a lipophilic amide prodrug of gemcitabine into solid lipid nanoparticles may represent a viable approach toward developing a safe and efficacious gemcitabine oral dosage form.

Applications of bacillus Calmette-Guerin and recombinant bacillus Calmette-Guerin in vaccine development and tumor immunotherapy.

Bacillus Calmette-Guerin (BCG) vaccines are attenuated live strains of Mycobacterium bovis and are among the most widely used vaccines in the world. BCG is proven to be effective in preventing severe infant meningitis and miliary tuberculosis. Intravesical instillation of BCG is also a standard treatment for non-muscle invasive bladder cancer. In the past few decades, recombinant BCG (rBCG) technology had been extensively applied to develop vaccine candidates for a variety of infectious diseases, including bacterial, viral, and parasite infections, and to improve the efficacy of BCG in bladder cancer therapy. This review is intended to show the vast applications of BCG and recombinant BCG (rBCG) in the prevention of infectious diseases and cancer immunotherapy, with a special emphasis on recent approaches and trends on both pre-clinical and clinical levels.