Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines

The use of DNA to deliver vaccine antigens offers many advantages, including ease of manufacture and cost. However, most DNA vaccines are plasmids and must be grown in bacterial culture, necessitating elements which are either unnecessary for effective gene delivery (e.g. bacterial origins of replication) or undesirable (e.g. antibiotic resistance genes). Removing these elements may improve the safety profile of DNA for the delivery of vaccines. Here we describe a novel, double-stranded, linear DNA construct produced by an enzymatic process that solely encodes an antigen expression cassette, comprising antigen, promoter, polyA tail and telomeric ends. We compared these constructs (called ‘Doggybones’ because of their shape) with conventional plasmid DNA. Using luciferase-expressing constructs, we demonstrated that expression levels were equivalent between Doggybones and plasmids both in vitro and in vivo. When mice were immunized with DNA constructs expressing the HIV envelope protein gp140, equivalent humoral and cellular responses were induced. Immunizations with either construct type expressing haemagluttinin were protective against H1N1 influenza challenge. This is the first example of an effective DNA vaccine which can be produced on a large scale by enzymatic processes

Design and evaluation of the immunogenicity and efficacy of a biomimetic particulate formulation of viral antigens

Subunit viral vaccines are typically not as efficient as live attenuated or inactivated vaccines at inducing protective immune responses. This paper describes an alternative 'biomimetic' technology; whereby viral antigens were formulated around a polymeric shell in a rationally arranged fashion with a surface glycoprotein coated on to the surface and non-structural antigen and adjuvant encapsulated. We evaluated this model using BVDV E2 and NS3 proteins formulated in poly-(D, L-lactic-co-glycolic acid) (PLGA) nanoparticles adjuvanted with polyinosinic:polycytidylic acid (poly(I:C) as an adjuvant (Vaccine-NP). This Vaccine-NP was compared to ovalbumin and poly(I:C) formulated in a similar manner (Control-NP) and a commercial adjuvanted inactivated BVDV vaccine (IAV), all inoculated subcutaneously and boosted prior to BVDV-1 challenge. Significant virus-neutralizing activity, and E2 and NS3 specific antibodies were observed in both Vaccine-NP and IAV groups following the booster immunisation. IFN-γ responses were observed in ex vivo PBMC stimulated with E2 and NS3 proteins in both vaccinated groups. We observed that the protection afforded by the particulate vaccine was comparable to the licenced IAV formulation. In conclusion, the biomimetic particulates showed a promising immunogenicity and efficacy profile that may be improved by virtue of being a customisable mode of delivery

Second best toll and capacity optimisation in network: solution algorithm and policy implications

This paper looks at the first and second-best jointly optimal toll and road capacity investment problems from both policy and technical oriented perspectives. On the technical side, the paper investigates the applicability of the constraint cutting algorithm for solving the second-best problem under elastic demand which is formulated as a bilevel programming problem. The approach is shown to perform well despite several problems encountered by our previous work in Shepherd and Sumalee (2004). The paper then applies the algorithm to a small sized network to investigate the policy implications of the first and second-best cases. This policy analysis demonstrates that the joint first best structure is to invest in the most direct routes while reducing capacities elsewhere. Whilst unrealistic this acts as a useful benchmark. The results also show that certain second best policies can achieve a high proportion of the first best benefits while in general generating a revenue surplus. We also show that unless costs of capacity are known to be low then second best tolls will be affected and so should be analysed in conjunction with investments in the network

Early Ultraviolet Observations of Type IIn Supernovae Constrain the Asphericity of Their Circumstellar Material

© 2020. The American Astronomical Society. All rights reserved.. We present a survey of the early evolution of 12 Type IIn supernovae (SNe IIn) at ultraviolet and visible light wavelengths. We use this survey to constrain the geometry of the circumstellar material (CSM) surrounding SN IIn explosions, which may shed light on their progenitor diversity. In order to distinguish between aspherical and spherical CSM, we estimate the blackbody radius temporal evolution of the SNe IIn of our sample, following the method introduced by Soumagnac et al. We find that higher-luminosity objects tend to show evidence for aspherical CSM. Depending on whether this correlation is due to physical reasons or to some selection bias, we derive a lower limit between 35% and 66% for the fraction of SNe IIn showing evidence for aspherical CSM. This result suggests that asphericity of the CSM surrounding SNe IIn is common - consistent with data from resolved images of stars undergoing considerable mass loss. It should be taken into account for more realistic modeling of these events

Particle emission characteristics of a gas turbine with a double annular combustor

The total climate, air quality and health impact of aircraft black carbon (BC) emissions depends on quantity (mass and number concentration), as well as morphology (fractal dimension and surface area) of emitted BC aggregates. This study examines multiple BC emission metrics from a gas turbine with a double annular combustor, CFM56-5B4-2P. As a part of the SAMPLE III.2 campaign, concurrent measurements of particle mobility, particle mass, particle number concentration and mass concentration, as well as collection of transmission electron microscopy (TEM) samples, allowed for characterization of the BC emissions. Mass- and number-based emission indices were strongly influenced by thrust setting during pilot combustion and ranged from <1 to 208 mg/kg-fuel and 3×1012 to 3×1016 particles/kg-fuel, respectively. Mobility measurements indicated that mean diameters ranged from 7-44 nm with a strong dependence on thrust during pilot-only combustion. Using aggregation and sintering theory with empirical effective density relationships, a power law relationship between primary particle diameter and mobility diameter is presented. Mean primary particle diameter ranged from 6-19 nm, however, laser induced incandescence (LII) and mass-mobility calculated primary particle diameters demonstrated opposite trends with thrust setting. Similarly, mass-mobility-calculated aggregate mass specific surface area and LII-measured surface area were not in agreement, indicating both methods need further development and validation before use as quantitative indicators of primary particle diameter and mass-specific surface area.The authors express their gratitude to a number of people and organizations in helping to plan, conduct, finance and provide instruments for this measurement campaign. The 537 European Aviation Safety Agency (EASA) funded the SAMPLE III SC02 campaign (EASA.2010.FC.10, Specific Contract No: SC02). The Federal Office of Civil Aviation, Switzerland (FOCA) was critical in for providing additional financial support and arranging facilities which made this study possible. We also thank the SR Technics test bed staff, including Frithjof Siegerist, for operating the engines and enabling access to the test facility. We thank AVL, Cambustion, Grimm & TSI supplying both instruments and expertise.This is the author accepted manuscript. The final version is available from Taylor & Francis via http://dx.doi.org/10.1080/02786826.2015.107845

Increased myofibroblasts in the small airways, and relationship to remodelling and functional changes in smokers and COPD patients: potential role of epithelial-mesenchymal transition

Introduction: Previous reports have shown epithelial–mesenchymal transition (EMT) as an active processthat contributes to small airway fibrotic pathology. Myofibroblasts are highly active pro-fibrotic cells thatsecrete excessive and altered extracellular matrix (ECM). Here we relate small airway myofibroblastpresence with airway remodelling, physiology and EMT activity in smokers and COPD patients.Methods: Lung resections from nonsmoker controls, normal lung function smokers and COPD currentand ex-smokers were stained with anti-human α-smooth muscle actin (SMA), collagen 1 and fibronectin.αSMA+ cells were computed in reticular basement membrane (Rbm), lamina propria and adventitia andpresented per mm of Rbm and mm2 of lamina propria. Collagen-1 and fibronectin are presented as apercentage change from normal. All analyses including airway thickness were measured using Image-proplus 7.0.Results: We found an increase in subepithelial lamina propria (especially) and adventitia thickness in allpathological groups compared to nonsmoker controls. Increases in αSMA+ myofibroblasts were observedin subepithelial Rbm, lamina propria and adventitia in both the smoker and COPD groups compared tononsmoker controls. Furthermore, the increase in the myofibroblast population in the lamina propria wasstrongly associated with decrease in lung function, lamina propria thickening, increase in ECM proteindeposition, and finally EMT activity in epithelial cells.Conclusions: This is the first systematic characterisation of small airway myofibroblasts in COPD based ontheir localisation, with statistically significant correlations between them and other pan-airway structural,lung function and ECM protein changes. Finally, we suggest that EMT may be involved in such changes

A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy

No exit from the euro-rescuing trap?

This paper attempts a normative assessment of the input and output-oriented legitimacy of the present euro-rescuing regime on the basis of policy analyses examining the causes of present crises, the available policy options, and the impact of the policies actually chosen. Concluding that the regime lacks input-oriented legitimacy and that its claim to output-oriented legitimacy is ambivalent at best, the paper explores potential – majoritarian or unilateral – exits from the present institutional constellation that is characterized by the synthesis of a non-democratic expertocracy and an extremely asymmetric intergovernmental bargaining system.Die hier präsentierte normative Bewertung der input- und outputorientierten Legitimität des gegenwärtigen Euro-Rettungs-Regimes stützt sich auf empirisch fundierte Aussagen zu den Ursachen der Eurokrise, den prinzipiell verfügbaren Politik-Optionen und den Wirkungen der gewählten Politik. Im Ergebnis wird eine inputorientierte Legitimation verneint, während die outputorientierte Bewertung höchst ambivalent erscheint. Im Schlussteil untersucht der Text mögliche – majoritäre oder einseitige – Auswege aus einer institutionellen Konstellation, die ein nicht demokratisches Expertenregime mit inem extrem asymmetrischen intergouvernementalen Verhandlungsregime verbindet

Betibeglogene Autotemcel Gene Therapy for Non-β⁰/β⁰ Genotype β-Thalassemia

BACKGROUND: Betibeglogene autotemcel (beti-cel) gene therapy for transfusion-dependent β-thalassemia contains autologous CD34+ hematopoietic stem cells and progenitor cells transduced with the BB305 lentiviral vector encoding the β-globin (βA-T87Q) gene. METHODS: In this open-label, phase 3 study, we evaluated the efficacy and safety of beti-cel in adult and pediatric patients with transfusion-dependent β-thalassemia and a non-β0/β0 genotype. Patients underwent myeloablation with busulfan (with doses adjusted on the basis of pharmacokinetic analysis) and received beti-cel intravenously. The primary end point was transfusion independence (i.e., a weighted average hemoglobin level of ≥9 g per deciliter without red-cell transfusions for ≥12 months). RESULTS: A total of 23 patients were enrolled and received treatment, with a median follow-up of 29.5 months (range, 13.0 to 48.2). Transfusion independence occurred in 20 of 22 patients who could be evaluated (91%), including 6 of 7 patients (86%) who were younger than 12 years of age. The average hemoglobin level during transfusion independence was 11.7 g per deciliter (range, 9.5 to 12.8). Twelve months after beti-cel infusion, the median level of gene therapy-derived adult hemoglobin (HbA) with a T87Q amino acid substitution (HbAT87Q) was 8.7 g per deciliter (range, 5.2 to 10.6) in patients who had transfusion independence. The safety profile of beti-cel was consistent with that of busulfan-based myeloablation. Four patients had at least one adverse event that was considered by the investigators to be related or possibly related to beti-cel; all events were nonserious except for thrombocytopenia (in 1 patient). No cases of cancer were observed. CONCLUSIONS: Treatment with beti-cel resulted in a sustained HbAT87Q level and a total hemoglobin level that was high enough to enable transfusion independence in most patients with a non-β0/β0 genotype, including those younger than 12 years of age. (Funded by Bluebird Bio; HGB-207 ClinicalTrials.gov number, NCT02906202.)