Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L-1 in shake flasks using the OVA expression cassette containing a tac promoter (Ptac) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (PP5 combined with Ptac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L-1, respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L-1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

Evaluation of expression systems for recombinant protein production in chicken egg bioreactors.

Chicken eggs have gained attention as excellent bioreactors because of their genetic modifications. However, the development of chicken egg bioreactors requires a long time from the construction of the production system to the evaluation of the products. Therefore, in this study, a chicken cell line producing ovalbumin (OVA) was established and constructed a system for the rapid evaluation of the production system. First, the EF1α promoter was knocked in upstream of the OVA locus in chicken DF-1 cells for continuous OVA expression. Furthermore, an ideal position at the OVA locus for the insertion of useful protein genes to maximize recombinant protein yield was analyzed and identified. The knocking in the EF1α promoter upstream of exon1 yielded the maximum production of OVA protein was achieved. In addition, Linking a recombinant hFGF2 cDNA to the 5' side of the OVA was found to increase production efficiency. Therefore, an OVA-expressing cell line and an evaluation system for proteins in chicken egg bioreactors was established. The findings may improve the efficiency of chicken expression systems and expand their applications in protein production.

pH-Sensitive branched ß-glucan-modified liposomes for activation of antigen presenting cells and induction of antitumor immunity.

Induction of cellular immunity is important for effective cancer immunotherapy. Although various antigen carriers for cancer immunotherapy have been developed to date, balancing efficient antigen delivery to antigen presenting cells (APCs) and their activation via innate immune receptors, both of which are crucially important for the induction of strong cellular immunity, remains challenging. For this study, branched ß-glucan was selected as an intrinsically immunity-stimulating and biocompatible material. It was engineered to develop multifunctional liposomal cancer vaccines capable of efficient interactions with APCs and subsequent activation of the cells. Hydroxy groups of branched ß-glucan (Aquaß) were modified with 3-methylglutaric acid ester and decyl groups, respectively, to provide pH-sensitivity and anchoring capability to the liposomal membrane. The modification efficiency of Aquaß derivatives to the liposomes was significantly high compared with linear ß-glucan (curdlan) derivatives. Aquaß derivative-modified liposomes released their contents in response to weakly acidic pH. As a model antigenic protein, ovalbumin (OVA)-loaded liposomes modified with Aquaß derivatives interacted efficiently with dendritic cells, and induced inflammatory cytokine secretion from the cells. Subcutaneous administration of Aquaß derivative-modified liposomes suppressed the growth of the E.G7-OVA tumor significantly compared with curdlan derivative-modified liposomes. Aquaß derivative-modified liposomes induced the increase of CD8+ T cells, and polarized macrophages to the antitumor M1-phenotype within the tumor microenvironment. Therefore, pH-sensitive Aquaß derivatives can be promising materials for liposomal antigen delivery systems to induce antitumor immune responses efficiently.

Development and in vitro evaluation of recombinant chicken promoters to efficiently drive transgene expression in chicken oviduct cells.

Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in the chicken oviduct. Although a reconstructed chicken ovalbumin promoter that links an ovalbumin promoter and estrogen-responsive enhancer element (ERE) is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. We newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. Putative enhancer fragments of the genes, such as ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), were placed at the 5`-flanking region of the 2.8-kb ovalbumin promoter. Basal promoter fragments of the genes, namely, pTF, lysozyme (pLYZ), and ovomucoid (pOVM), were placed at the 3`-flanking region of the 1.6-kb ovalbumin ERE. The recombinant promoters cloned into each reporter vector were evaluated using a dual luciferase assay in human and chicken somatic cells, and LMH/2A cells treated with 0-1,000 nM estrogen, and cultured primary chicken oviduct cells. The recombinant promoters with linking ovalbumin and TF, OVOA, pOVM, and pLYZ regulatory regions had 2.1- to 19.5-fold (P < 0.05) higher luciferase activity than the reconstructed ovalbumin promoter in chicken oviduct cells. Therefore, recombinant promoters may be used to efficiently drive transgene expression in transgenic chickens.

The Serpin-like Loop Insertion of Ovalbumin Increases the Stability and Decreases the OVA 323-339 Epitope Processing Efficiency.

Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possesses a protease-sensitive reactive center loop that lies adjacent to the OVA 323-339 CD4+ T-cell epitope. We took advantage of the previously described single-substitution variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins, to study how changes in loop size and protein stability influence the processing and presentation of the OVA 323-339 epitope. We observed that the OVA R339T loop insertion increases the stability and protease resistance, resulting in the reduced presentation of the OVA 323-339 epitope in vitro. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools.

Molecular tracking devices quantify antigen distribution and archiving in the murine lymph node.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a 'molecular tracking device' to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

The lymphatic system is a network of ducts that transports fluid, proteins, and immune cells from different organs around the body. Lymph nodes provide pit stops at hundreds of points along this network where immune cells reside, and lymph fluid can be filtered and cleaned. When pathogens, such as viruses or bacteria, enter the body during an infection, fragments of their proteins can get swept into the lymph nodes. These pathogenic proteins or protein fragments activate resident immune cells and kickstart the immune response. Vaccines are designed to mimic this process by introducing isolated pathogenic proteins in a controlled way to stimulate similar immune reactions in lymph nodes. Once an infection has been cleared by the immune system, or a vaccination has triggered the immune system, most pathogenic proteins get cleared away. However, a small number of pathogenic proteins remain in the lymph nodes to enable immune cells to respond more strongly and quickly the next time they see the same pathogen. Yet it is largely unclear how much protein remains for training and how or where it is all stored. Current techniques are not sensitive or long-lived enough to accurately detect and track these small protein deposits over time. Walsh, Sheridan, Lucas, et al. have addressed this problem by developing biological tags that can be attached to the pathogenic proteins so they can be traced. These tags were designed so the body cannot easily break them down, helping them last as long as the proteins they are attached to. Walsh, Sheridan, Lucas et al. tested whether vaccinating mice with the tagged proteins allowed the proteins to be tracked. The method they used was designed to identify individual cell types based on their genetic information along with the tag. This allowed them to accurately map the complex network of cells involved in storing and retrieving archived protein fragments, as well as those involved in training new immune cells to recognize them. These results provide important insights into the protein archiving system that is involved in enhancing immune memory. This may help guide the development of new vaccination strategies that can manipulate how proteins are archived to establish more durable immune protection. The biological tags developed could also be used to track therapeutic proteins, allowing scientists to determine how long cancer drugs, antibody therapies or COVID19 anti-viral agents remain in the body. This information could then be used by doctors to plan specific and personalized treatment timetables for patients.

Site-specific antigen-adjuvant conjugation using cell-free protein synthesis enhances antigen presentation and CD8+ T-cell response.

Antigen-adjuvant conjugation is known to enhance antigen-specific T-cell production in vaccine models, but scalable methods are required to generate site-specific conjugation for clinical translation of this technique. We report the use of the cell-free protein synthesis (CFPS) platform as a rapid method to produce large quantities (> 100 mg/L) of a model antigen, ovalbumin (OVA), with site-specific incorporation of p-azidomethyl-L-phenylalanine (pAMF) at two solvent-exposed sites away from immunodominant epitopes. Using copper-free click chemistry, we conjugated CpG oligodeoxynucleotide toll-like receptor 9 (TLR9) agonists to the pAMF sites on the mutant OVA protein. The OVA-CpG conjugates demonstrate enhanced antigen presentation in vitro and increased antigen-specific CD8+ T-cell production in vivo. Moreover, OVA-CpG conjugation reduced the dose of CpG needed to invoke antigen-specific T-cell production tenfold. These results highlight how site-specific conjugation and CFPS technology can be implemented to produce large quantities of covalently-linked antigen-adjuvant conjugates for use in clinical vaccines.

Appropriate aglycone modification significantly expands the glycan substrate acceptability of α1,6-fucosyltransferase (FUT8).

The α1,6-fucosyltransferase, FUT8, is the sole enzyme catalyzing the core-fucosylation of N-glycoproteins in mammalian systems. Previous studies using free N-glycans as acceptor substrates indicated that a terminal ß1,2-GlcNAc moiety on the Man-α1,3-Man arm of N-glycan substrates is required for efficient FUT8-catalyzed core-fucosylation. In contrast, we recently demonstrated that, in a proper protein context, FUT8 could also fucosylate Man5GlcNAc2 without a GlcNAc at the non-reducing end. We describe here a further study of the substrate specificity of FUT8 using a range of N-glycans containing different aglycones. We found that FUT8 could fucosylate most of high-mannose and complex-type N-glycans, including highly branched N-glycans from chicken ovalbumin, when the aglycone moiety is modified with a 9-fluorenylmethyloxycarbonyl (Fmoc) moiety or in a suitable peptide/protein context, even if they lack the terminal GlcNAc moiety on the Man-α1,3-Man arm. FUT8 could also fucosylate paucimannose structures when they are on glycoprotein substrates. Such core-fucosylated paucimannosylation is a prominent feature of lysosomal proteins of human neutrophils and several types of cancers. We also found that sialylation of N-glycans significantly reduced their activity as a substrate of FUT8. Kinetic analysis demonstrated that Fmoc aglycone modification could either improve the turnover rate or decrease the KM value depending on the nature of the substrates, thus significantly enhancing the overall efficiency of FUT8 catalyzed fucosylation. Our results indicate that an appropriate aglycone context of N-glycans could significantly broaden the acceptor substrate specificity of FUT8 beyond what has previously been thought.

CD8+ T-Lymphocyte-Driven Limbic Encephalitis Results in Temporal Lobe Epilepsy.

OBJECTIVE: Limbic encephalitis (LE) comprises a spectrum of inflammatory changes in affected brain structures including the presence of autoantibodies and lymphoid cells. However, the potential of distinct lymphocyte subsets alone to elicit key clinicopathological sequelae of LE potentially inducing temporal lobe epilepsy (TLE) with chronic spontaneous seizures and hippocampal sclerosis (HS) is unresolved. METHODS: Here, we scrutinized pathogenic consequences emerging from CD8+ T cells targeting hippocampal neurons by recombinant adeno-associated virus-mediated expression of the model-autoantigen ovalbumin (OVA) in CA1 neurons of OT-I/RAG1-/- mice (termed "OVA-CD8+ LE model"). RESULTS: Viral-mediated antigen transfer caused dense CD8+ T cell infiltrates confined to the hippocampal formation starting on day 5 after virus transduction. Flow cytometry indicated priming of CD8+ T cells in brain-draining lymph nodes preceding hippocampal invasion. At the acute model stage, the inflammatory process was accompanied by frequent seizure activity and impairment of hippocampal memory skills. Magnetic resonance imaging scans at day 7 of the OVA-CD8+ LE model revealed hippocampal edema and blood-brain barrier disruption that converted into atrophy until day 40. CD8+ T cells specifically targeted OVA-expressing, SIINFEKL-H-2Kb -positive CA1 neurons and caused segmental apoptotic neurodegeneration, astrogliosis, and microglial activation. At the chronic model stage, mice exhibited spontaneous recurrent seizures and persisting memory deficits, and the sclerotic hippocampus was populated with CD8+ T cells escorted by NK cells. INTERPRETATION: These data indicate that a CD8+ T-cell-initiated attack of distinct hippocampal neurons is sufficient to induce LE converting into TLE-HS. Intriguingly, the role of CD8+ T cells exceeds neurotoxic effects and points to their major pathogenic role in TLE following LE. ANN NEUROL 2021;89:666-685.

IL-17A/F enable cholangiocytes to restrict T cell-driven experimental cholangitis by upregulating PD-L1 expression.

BACKGROUND & AIMS: IL-17A-producing T cells are present in autoimmune cholestatic liver diseases; however, little is known about the contribution of IL-17 to periductal immune responses. Herein, we investigated the role of IL-17 produced by antigen-specific CD8+ T cells in a mouse model of cholangitis and in vitro in human cholangiocyte organoids. METHODS: K14-OVAp mice express a major histocompatibility complex I-restricted ovalbumin (OVA) peptide sequence (SIINFEKL) on cholangiocytes. Cholangitis was induced by the adoptive transfer of transgenic OVA-specific ovalbumin transgene (OT)-1 CD8+ T cells that either had OT-1wt or lacked IL-17A/F (OT-1IL17ko). The response of mouse and human cholangiocytes/organoids to IL-17A was assessed in vitro. RESULTS: Transfer of OVA-specific OT-1IL17ko cells significantly aggravated periductal inflammation in K14-OVAp recipient mice compared with transfer of OT-1wt T cells. OT-1IL17ko T cells were highly activated in the liver and displayed increased cytotoxicity and proliferation. IL-17A/F produced by transferred OT-1wt CD8+ T cells induced upregulation of the inhibitory molecule programmed cell death ligand 1 (PD-L1) on cholangiocytes, restricting cholangitis by limiting cytotoxicity and proliferation of transferred cells. In contrast, OT-1IL17ko T cells failed to induce PD-L1 on cholangiocytes, resulting in uncontrolled expansion of cytotoxic CD8+ T cells and aggravated cholangitis. Blockade of PD-L1 after transfer of OT-1wt T cells with anti-PD-L1 antibody also resulted in aggravated cholangitis. Using human cholangiocyte organoids, we were able to confirm that IL-17A induces PD-L1 expression in cholangiocytes. CONCLUSIONS: We demonstrate that by upregulating PD-L1 on cholangiocytes, IL-17 has an important role in restricting cholangitis and protecting against CD8+ T cell-mediated inflammatory bile duct injury. Caution should be exercised when targeting IL-17 for the treatment of cholangitis. LAY SUMMARY: IL-17 is assumed to be a driver of inflammation in several autoimmune diseases, such as psoriasis. IL-17 is also present in inflammatory diseases of the bile duct, but its role in these conditions is not clear, as the effects of IL-17 depend on the context of its expression. Herein, we investigated the role of IL-17 in an experimental autoimmune cholangitis mouse model, and we identified an important protective effect of IL-17 on cholangiocytes, enabling them to downregulate bile duct inflammation via checkpoint inhibitor PD-L1.

Cutting Edge: Tissue Antigen Expression Levels Fine-Tune T Cell Differentiation Decisions In Vivo.

Immune homeostasis in peripheral tissues is, to a large degree, maintained by the differentiation and action of regulatory T cells (Treg) specific for tissue Ags. Using a novel mouse model, we have studied the differentiation of naive CD4+ T cells into Foxp3+ Treg in response to a cutaneous Ag (OVA). We found that expression of OVA resulted in fatal autoimmunity and in prevention of peripheral Treg generation. Inhibiting mTOR activity with rapamycin rescued the generation of Foxp3+ T cells. When we varied the level of Ag expression to modulate TCR signaling, we found that low Ag concentrations promoted the generation of Foxp3+ T cells, whereas high levels expanded effector T cells and caused severe autoimmunity. Our findings indicate that the expression level of tissue Ag is a key determinant of the balance between tissue-reactive effector and peripheral Foxp3+ T cells, which determines the choice between tolerance and autoimmunity.

Polyglutamine-Related Aggregates Can Serve as a Potent Antigen Source for Cross-Presentation by Dendritic Cells.

Protective MHC class I-dependent immune responses require an overlap between repertoires of proteins directly presented on target cells and cross-presented by professional APC, specifically dendritic cells. How stable proteins that rely on defective ribosomal proteins for direct presentation are captured for cell-to-cell transfer remains enigmatic. In this study, we address this issue using a combination of in vitro (C57BL/6-derived mouse cell lines) and in vivo (C57BL/6 mouse strains) approaches involving stable and unstable versions of OVA model Ags displaying defective ribosomal protein-dependent and -independent Ag presentation, respectively. Apoptosis, but not necrosis, of donor cells was found associated with robust global protein aggregate formation and captured stable proteins permissive for cross-presentation. Potency of aggregates to serve as Ag source was directly demonstrated using polyglutamine-equipped model substrates. Collectively, our data implicate global protein aggregation in apoptotic cells as a mechanism that ensures the overlap between MHC class I epitopes presented directly or cross-presented by APC and demonstrate the unusual ability of dendritic cells to process stable protein aggregates.

Vesicular Stomatitis Virus Encoding a Destabilized Tumor Antigen Improves Activation of Anti-tumor T Cell Responses.

Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and T cell activation against a vesicular stomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) in vitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I T cells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 T cell response in vivo without altering the anti-viral CD8 T cell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.

The adjuvant effect of melanin is superior to incomplete Freund's adjuvant in subunit/peptide vaccines in mice.

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

Cholinergic neuroplasticity in asthma driven by TrkB signaling.

Parasympathetic neurons in the airways control bronchomotor tone. Increased activity of cholinergic neurons are mediators of airway hyperresponsiveness (AHR) in asthma, however, mechanisms are not elucidated. We describe remodeling of the cholinergic neuronal network in asthmatic airways driven by brain-derived neurotrophic factor (BDNF) and Tropomyosin receptor kinase B (TrkB). Human bronchial biopsies were stained for cholinergic marker vesicular acetylcholine transporter (VAChT). Human lung gene expression and single nucleotide polymorphisms (SNP) in neuroplasticity-related genes were compared between asthma and healthy patients. Wild-type (WT) and mutated TrkB knock-in mice (Ntrk2tm1Ddg/J) with impaired BDNF signaling were chronically exposed to ovalbumin (OVA). Neuronal VAChT staining and airway narrowing in response to electrical field stimulation in precision cut lung slices (PCLS) were assessed. Increased cholinergic fibers in asthmatic airway biopsies was found, paralleled by increased TrkB gene expression in human lung tissue, and SNPs in the NTRK2 [TrkB] and BDNF genes linked to asthma. Chronic allergen exposure in mice resulted in increased density of cholinergic nerves, which was prevented by inhibiting TrkB. Increased nerve density resulted in AHR in vivo and in increased nerve-dependent airway reactivity in lung slices mediated via TrkB. These findings show cholinergic neuroplasticity in asthma driven by TrkB signaling and suggest that the BDNF-TrkB pathway may be a potential target.

Measles Vaccines Designed for Enhanced CD8+ T Cell Activation.

Priming and activation of CD8+ T cell responses is crucial to achieve anti-viral and anti-tumor immunity. Live attenuated measles vaccine strains have been used successfully for immunization for decades and are currently investigated in trials of oncolytic virotherapy. The available reverse genetics systems allow for insertion of additional genes, including heterologous antigens. Here, we designed recombinant measles vaccine vectors for priming and activation of antigen-specific CD8+ T cells. For proof-of-concept, we used cytotoxic T lymphocyte (CTL) lines specific for the melanoma-associated differentiation antigen tyrosinase-related protein-2 (TRP-2), or the model antigen chicken ovalbumin (OVA), respectively. We generated recombinant measles vaccine vectors with TRP-2 and OVA epitope cassette variants for expression of the full-length antigen or the respective immunodominant CD8+ epitope, with additional variants mediating secretion or proteasomal degradation of the epitope. We show that these recombinant measles virus vectors mediate varying levels of MHC class I (MHC-I)-restricted epitope presentation, leading to activation of cognate CTLs, as indicated by secretion of interferon-gamma (IFNγ) in vitro. Importantly, the recombinant OVA vaccines also mediate priming of naïve OT-I CD8+ T cells by dendritic cells. While all vaccine variants can prime and activate cognate T cells, IFNγ release was enhanced using a secreted epitope variant and a variant with epitope strings targeted to the proteasome. The principles presented in this study will facilitate the design of recombinant vaccines to elicit CD8+ responses against pathogens and tumor antigens.

Targeted knock-in into the OVA locus of chicken cells using CRISPR/Cas9 system with homology-independent targeted integration.

It is anticipated that transgenic avian species will be used as living bioreactors for the production of biopharmaceutical proteins. Precise tissue-specific expression of exogenous genes is a major challenge for the development of avian bioreactors. No robust vector is currently available for highly efficient and specific expression. In recent years, genome-editing techniques such as the CRISPR/Cas9 system have emerged as efficient and user-friendly genetic modification tools. Here, to apply the CRISPR/Cas9 system for the development of transgenic chickens, guide RNA sequences (gRNAs) of the CRISPR/Cas9 system for the ovalbumin (OVA) locus were evaluated for the oviduct-specific expression of exogenous genes. An EGFP gene expression cassette was introduced into the OVA locus of chicken DF-1 and embryonic fibroblasts using the CRISPR/Cas9 system mediated by homology-independent targeted integration. For the knock-in cells, EGFP expression was successfully induced by activation of the endogenous OVA promoter using the dCas9-VPR transactivation system. The combination of gRNAs designed around the OVA TATA box was important to induce endogenous OVA gene expression with high efficiency. These methods provide a useful tool for studies on the creation of transgenic chicken bioreactors and the activation of tissue-specific promoters.

Adjuvant incorporated lipid nanoparticles for enhanced mRNA-mediated cancer immunotherapy.

For mRNA mediated cancer immunotherapy, Pam3 was incorporated as an adjuvant within lipid nanoparticles (LNPs) with OVA mRNA. The developed Pam3 incorporated LNPs showed successful expression of tumor antigens with enhanced immune stimulation. We demonstrated that the synergies of Pam3 LNPs could greatly improve the efficacy of tumor prevention by mRNA vaccines.

Effect of Blocking the OX40/OX40L Signaling Pathway by siRNA Interference on Animal Experimental Study of Allergic Rhinitis.

BACKGROUND: The identification of new approaches and intervention targets for the treatment of AR is urgently needed. We aimed to investigate the effect of blocking the OX40/OX40L signaling pathway by small interfering RNA (siRNA) on ovalbumin (OVA)-induced AR in a mouse model. METHODS: After establishment of the AR model, the mice were interfered by siRNA-OX40L (experimental group), siRNA-C (negative control group), or PBS (control group). Nose scratching, sneezing and nasal discharge were observed. OX40L mRNA and protein and the IL-5, TNF-α, regulatory T cell (Treg) -specific marker Foxp3, and eosinophil (EOS) levels were analyzed. RESULTS: The numbers of nose scratching and sneezing were significantly lower in the siRNA-OX40L-treated group (p <0.05). After the intervention of siRNA-OX40L, OX40L mRNA and protein levels were significantly inhibited (p <0.05), but the Foxp3 level was significantly increased in the experimental group (p <0.05). The IL-5 and TNF-α levels were significantly lower in the experimental group (p <0.05), and the reduction was more evident for the Th2-type cytokine IL-5 than for the Th1-type cytokine TNF-α. Few or no EOSs were found in the nasal mucosal epithelium of the experimental group (p <0.05), whereas EOS infiltration was significant in the other two groups. CONCLUSIONS: Blockage of the OX40/OX40L signaling pathway with siRNA-OX40L interference can inhibit allergic reactions and relieve allergic symptoms in AR mice. The underlying mechanism may be related to correcting Th2 immune deviation, inducing immune tolerance, and promoting Treg production.