Enhancing immunogenicity of HPV16 E7 DNA vaccine by conjugating codon-optimized GM-CSF to HPV16 E7 DNA.

OBJECTIVE: To generate immunity against human papillomavirus (HPV), the use of a recombinant DNA vaccine to carry an appropriate target gene is a promising and cost-effective approach. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that enhances the efficacy of vaccines by promoting the development and prolongation of humoral and cellular immunity. In this study, we linked codon-optimized GM-CSF (cGM-CSF) to the HPV16 E7 sequence as fused protein and evaluated the immunogenic potential of this DNA vaccine. MATERIALS AND METHODS: We have demonstrated that cGM-CSF enhanced immunity against tumor challenges by generating and promoting the proliferation of HPV16 E7-specific CD8+ T cells, which secrete IFN-γ in the murine model. In this study, we aimed to evaluate the immunogenic potential of DNA vaccine that constructed by linking codon-optimized GM-CSF to HPV16 E7 sequence in the animal model. We study the half-life of RNA decay and cellular location of HPV16 E7 by Q-PCR and Western blot. We also assess immune response in the animal model by flow cytometry and ELISA. RESULTS: The cGM-CSF-E7 sequence increased and extended the expression of E7 mRNA, in comparison with the E7 sequence alone. Mice vaccinated with the cGM-CSF-E7 DNA vaccine exhibited a slower rate of tumor growth than those vaccinated with the unconjugated E7 DNA vaccine. We also found that the CD4 and CD8+ T cells from these mice showed strong secretion of IFN-γ. CONCLUSION: Through in vivo antibody depletion experiments, we demonstrated that both CD4+ and CD8+ T cells play an important role in the suppression of tumor growth.

Stevens-Johnson syndrome and toxic epidermal necrolysis: risk factors, causality assessment and potential prevention strategies.

Introduction: The clinical manifestations of cutaneous adverse drug reactions are variable with different severity. Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening severe cutaneous adverse reactions (SCARs) majorly caused by drugs and mediated by cytotoxic T cells.Areas covered: In this review, we focus on risk factors that contribute to the development of SJS/TEN and review the updated immune mechanism, preventive strategies as well as current therapeutic approaches for SJS/TEN.Expert opinion: The progress of SJS/TEN researches reveals that cytotoxic T cells majorly activated by drug interacted with the human leukocyte antigen (HLA) and T cell receptors play an important role for the immune mechanism of SJS/TEN. Several clinical assessment tools and in vitro drug-T cells activation tests have been developed to identify the causality of SJS/TEN. New therapeutic approaches and biologics such as TNF-alpha antagonist have been conducted to improve the prognosis of SJS/TEN.

Highly efficient and tumor-selective nanoparticles for dual-targeted immunogene therapy against cancer.

While immunotherapy holds great promise for combating cancer, the limited efficacy due to an immunosuppressive tumor microenvironment and systemic toxicity hinder the broader application of cancer immunotherapy. Here, we report a combinatorial immunotherapy approach that uses a highly efficient and tumor-selective gene carrier to improve anticancer efficacy and circumvent the systemic toxicity. In this study, we engineered tumor-targeted lipid-dendrimer-calcium-phosphate (TT-LDCP) nanoparticles (NPs) with thymine-functionalized dendrimers that exhibit not only enhanced gene delivery capacity but also immune adjuvant properties by activating the stimulator of interferon genes (STING)-cGAS pathway. TT-LDCP NPs delivered siRNA against immune checkpoint ligand PD-L1 and immunostimulatory IL-2-encoding plasmid DNA to hepatocellular carcinoma (HCC), increased tumoral infiltration and activation of CD8+ T cells, augmented the efficacy of cancer vaccine immunotherapy, and suppressed HCC progression. Our work presents nanotechnology-enabled dual delivery of siRNA and plasmid DNA that selectively targets and reprograms the immunosuppressive tumor microenvironment to improve cancer immunotherapy.

Delivery of nitric oxide with a nanocarrier promotes tumour vessel normalization and potentiates anti-cancer therapies.

Abnormal tumour vasculature has a significant impact on tumour progression and response to therapy. Nitric oxide (NO) regulates angiogenesis and maintains vascular homeostasis and, thus, can be delivered to normalize tumour vasculature. However, a NO-delivery system with a prolonged half-life and a sustained release mechanism is currently lacking. Here we report the development of NanoNO, a nanoscale carrier that enables sustained NO release to efficiently deliver NO into hepatocellular carcinoma. Low-dose NanoNO normalizes tumour vessels and improves the delivery and effectiveness of chemotherapeutics and tumour necrosis factor-related, apoptosis-inducing, ligand-based therapy in both primary tumours and metastases. Furthermore, low-dose NanoNO reprogrammes the immunosuppressive tumour microenvironment toward an immunostimulatory phenotype, thereby improving the efficacy of cancer vaccine immunotherapy. Our findings demonstrate the ability of nanoscale NO delivery to efficiently reprogramme tumour vasculature and immune microenvironments to overcome resistance to cancer therapy, resulting in a therapeutic benefit.

Effect of Multiple Vaccinations with Tumor Cell-Based Vaccine with Codon-Modified GM-CSF on Tumor Growth in a Mouse Model.

Ectopic expression of codon-modified granulocyte-macrophage colony-stimulating factor (cGM-CSF) in TC-1 cells (TC-1/cGM-CSF), a model cell line for human papillomavirus (HPV)-infected cervical cancer cells, increased the expression level of GM-CSF and improved the efficacy of tumor cell-based vaccines in a cervical cancer mouse model. The number of vaccine doses required to induce a long-term immune response in a cervical cancer mouse model is poorly understood. Here, we investigated one, three, and five doses of the irradiated TC-1/cGM-CSF vaccine to determine which dose was effective in inducing a greater immune response and the suppression of tumors. Our findings showed that three doses of irradiated TC-1/cGM-CSF vaccine elicited slower tumor growth rates and enhanced survival rates compared with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Consistently, mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine exhibited stronger interferon gamma (IFN-γ) production in HPV E7-specific CD8⁺ T cells and CD4⁺ T cells. A higher percentage of natural killer cells and interferon-producing killer dendritic cells (IKDCs) appeared in the splenocytes of the mice vaccinated with three doses of irradiated TC-1/cGM-CSF vaccine compared with those of the mice vaccinated with one dose or five doses of irradiated TC-1/cGM-CSF vaccine. Our findings demonstrate that single or multiple vaccinations, such as five doses, with irradiated TC-1/cGM-CSF vaccine suppressed the immune response, whereas three doses of irradiated TC-1/cGM-CSF vaccine elicited a greater immune response and subsequent tumor suppression.

Determination of Aqueous Two-Phase System Binodals and Tie-Lines by Electrowetting-on-Dielectric Droplet Manipulation.

Handling the aqueous two-phase systems (ATPSs) formed by liquid-liquid phase separation (LLPS) relies on the accurate construction of binodal curves and tie-lines, which delineate the polymer concentrations required for phase separation and depict the properties of the resulting phases, respectively. Various techniques to determine the binodal curves and tie-lines of ATPSs exist, but most rely on manually pipetting relatively large volumes of fluids in a slow and tedious manner. We describe a method to determine ATPS binodals and tie-lines that overcomes these disadvantages: microscale droplet manipulation by electrowetting-on-dielectric (EWOD). EWOD enables automated handling of droplets in an optically transparent platform that allows for in situ droplet observation. Separated phases are clearly visible, and the volumes of each phase are readily determined. Additionally, in considering the molecular crowding present in living cells, this work examines the role of a macromolecule in prompting LLPS. These results show that EWOD-driven droplet manipulation effectively interrogates the phase dynamics of ATPSs and macromolecular crowding in LLPS.

A multifunctional nanocarrier for efficient TRAIL-based gene therapy against hepatocellular carcinoma with desmoplasia in mice.

The anticancer efficacy of TNF-related apoptosis-inducing ligand (TRAIL)-based therapy is limited because of systemic toxicity, poor bioavailability, and development of TRAIL resistance. We developed a tumor-targeted LCPP (lipid/calcium/phosphate/protamine) nanoparticle (NP) to deliver TRAIL plasmid DNA (pDNA) into hepatocellular carcinoma (HCC) cells in a mouse model of HCC. TRAIL pDNA was encapsulated in a pH stimuli-responsive calcium phosphate (CaP) core, and protamine was added to facilitate nuclear delivery of pDNA. In addition, intracellular release of Ca2+ from the CaP core overcame TRAIL resistance by calcium influx-dependent DR5 up-regulation. TRAIL expression also attenuated fibrosis in liver tissues surrounding HCCs by reverting activated hepatic stellate cells (HSCs) to a quiescent state or by directly inducing apoptosis in activated HSCs. CONCLUSION: TRAIL pDNA delivered by HCC-targeted LCPP NPs in combination with conventional sorafenib treatment attenuated HCC progression as well as liver fibrosis. Overall, our study presents an effective TRAIL-based cancer therapy that could be developed for clinical applications. (Hepatology 2018;67:899-913).

The efficacy of a novel vaccine approach using tumor cells that ectopically express a codon-optimized murine GM-CSF in a murine tumor model.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a potent immunomodulatory cytokine that is known to facilitate vaccine efficacy by promoting the development and prolongation of both humoral and cellular immunity. Here, we investigated a novel vaccine approach using a human papillomavirus (HPV)-16 E6/E7-transformed cell line, TC-1, that ectopically expresses a codon-optimized 26-11-2015 murine GM-CSF (cGM-CSF). Ectopically expressing cGM-CSF in TC-1 (TC-1/cGM) cells significantly increased expression of a GM-CSF that was functionally identical to wt GM-CSF by 9-fold compared with ectopically expressed wild type GM-CSF in TC-1 cells (TC-1/wt). Mice vaccinated with irradiated TC-1/cGM cells exhibited enhanced survival compared with mice vaccinated with TC-1/wt cells when both groups were subsequently injected with live TC-1. Consistently, mice vaccinated with irradiated TC-1/cGM cells exhibited stronger IFN-γ production in HPV E7-specific CD8(+) T cells. More dendritic cells were recruited to the draining lymph nodes (dLNs) of mice vaccinated with TC-1/cGM cells than C-1/wt cells. Regarding dLN cell recall responses, both proliferation and IFN-γ production in the HPV E7-specific CD8(+) T cells were enhanced in mice that were vaccinated with TC-1/cGM cells. Our results demonstrate that a novel practical molecular strategy utilizing a codon-optimized GM-CSF gene overcomes the limitation and improves the efficacy of tumor cell-based vaccines.

Magnetic-composite-modified polycrystalline silicon nanowire field-effect transistor for vascular endothelial growth factor detection and cancer diagnosis.

This study proposes a vascular endothelial growth factor (VEGF) biosensor for diagnosing various stages of cervical carcinoma. In addition, VEGF concentrations at various stages of cancer therapy are determined and compared to data obtained by computed tomography (CT) and cancer antigen 125 (CA-125). The increase in VEGF concentrations during operations offers useful insight into dosage timing during cancer therapy. This biosensor uses Avastin as the biorecognition element for the potential cancer biomarker VEGF and is based on a n-type polycrystalline silicon nanowire field-effect transistor (poly-SiNW-FET). Magnetic nanoparticles with poly[aniline-co-N-(1-one-butyric acid) aniline]-Fe3O4 (SPAnH-Fe3O4) shell-core structures are used as carriers for Avastin loading and provide rapid purification due to their magnetic properties, which prevent the loss of bioactivity; furthermore, the high surface area of these structures increases the quantity of Avastin immobilized. Average concentrations in human blood for species that interfere with detection specificity are also evaluated. The detection range of the biosensor for serum samples covers the results expected from both healthy individuals and cancer patients.

The N-terminal amphipathic helix of the topological specificity factor MinE is associated with shaping membrane curvature.

Pole-to-pole oscillations of the Min proteins in Escherichia coli are required for the proper placement of the division septum. Direct interaction of MinE with the cell membrane is critical for the dynamic behavior of the Min system. In vitro, this MinE-membrane interaction led to membrane deformation; however, the underlying mechanism remained unclear. Here we report that MinE-induced membrane deformation involves the formation of an amphipathic helix of MinE(2-9), which, together with the adjacent basic residues, function as membrane anchors. Biochemical evidence suggested that the membrane association induces formation of the helix, with the helical face, consisting of A2, L3, and F6, inserted into the membrane. Insertion of this helix into the cell membrane can influence local membrane curvature and lead to drastic changes in membrane topology. Accordingly, MinE showed characteristic features of protein-induced membrane tubulation and lipid clustering in in vitro reconstituted systems. In conclusion, MinE shares common protein signatures with a group of membrane trafficking proteins in eukaryotic cells. These MinE signatures appear to affect membrane curvature.

Direct MinE-membrane interaction contributes to the proper localization of MinDE in E. coli.

Dynamic oscillation of the Min system in Escherichia coli determines the placement of the division plane at the midcell. In addition to stimulating MinD ATPase activity, we report here that MinE can directly interact with the membrane and this interaction contributes to the proper MinDE localization and dynamics. The N-terminal domain of MinE is involved in direct contact between MinE and the membranes that may subsequently be stabilized by the C-terminal domain of MinE. In an in vitro system, MinE caused liposome deformation into membrane tubules, a property similar to that previously reported for MinD. We isolated a mutant MinE containing residue substitutions in R10, K11 and K12 that was fully capable of stimulating MinD ATPase activity, but was deficient in membrane binding. Importantly, this mutant was unable to support normal MinDE localization and oscillation, suggesting that direct MinE interaction with the membrane is critical for the dynamic behavior of the Min system.