Teacher Victimization in Authoritative School Environments.

Background: Victimization in schools is not limited to students. Teachers increasingly face threats and attacks from their students. An authoritative school environment, characterized by high structure and support, has been associated with lower rates of victimization.

A 5' Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo.

We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5' UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro.

Enhanced neutralising antibody response to bovine viral diarrhoea virus (BVDV) induced by DNA vaccination in calves.

DNA vaccination is effective in inducing potent immunity in mice; however it appears to be less so in large animals. Increasing the dose of DNA plasmid to activate innate immunity has been shown to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection.

A noncytolytic α toxin recombinant protein protects turkeys against Clostridium septicum challenge.

Clostridium septicum and its associated cytolytic α toxin, along with several other clostridial species, has been implicated as the causative agent of gangrenous dermatitis. A recombinant noncytolytic C. septicum α toxin (NCAT) peptide was developed for use as a vaccine and demonstrated to be safe at concentrations as high as 1 mg/ml.

Development of antibiotic-free selection system for safer DNA vaccination.

The use of antibiotic-resistance markers in DNA vaccines is discouraged by regulatory agencies due to various theoretical safety concerns. This chapter presents methodologies for the design and cloning of synthetic antigen genes into RNA-OUT encoding antibiotic-free DNA vaccine vectors that are additionally optimized to improve protein expression, and immunogenicity, compared to alternative kanamycin-resistant vectors. First, antigen targeting considerations are discussed in the context of immune response customization through MHC class I or class II directed antigen presentation; the example NTC868 series RNA-OUT vector system allows simultaneous cloning into multiple vectors that feature various transgene intracellular targeting destinations.

Thermostable tag (TST) protein expression system: engineering thermotolerant recombinant proteins and vaccines.

Methods to increase temperature stability of vaccines and adjuvants are needed to reduce dependence on cold chain storage. We report herein creation and application of pVEX expression vectors to improve vaccine and adjuvant manufacture and thermostability. Defined media fermentation yields of 6g/L thermostable toll-like receptor 5 agonist flagellin were obtained using an IPTG inducible pVEX-flagellin expression vector.

Coexpressed RIG-I agonist enhances humoral immune response to influenza virus DNA vaccine.

Increasing levels of plasmid vector-mediated activation of innate immune signaling pathways is an approach to improve DNA vaccine-induced adaptive immunity for infectious disease and cancer applications. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA (dsRNA) pattern receptor required for innate immune activation in response to viral infection. Activation of RIG-I leads to type I interferon (IFN) and inflammatory cytokine production through interferon promoter stimulator 1 (IPS-1)-mediated activation of interferon regulatory factor 3 (IRF3) and NF-κB signaling.

Plasmid DNA fermentation strain and process-specific effects on vector yield, quality, and transgene expression.

Industrial plasmid DNA manufacturing processes are needed to meet the quality, economy, and scale requirements projected for future commercial products. We report development of a modified plasmid fermentation copy number induction profile that increases gene vaccination/therapy vector yields up to 2,600 mg/L. We determined that, in contrast to recombinant protein production, secretion of the metabolic byproduct acetate into the media had only a minor negative effect on plasmid replication.

Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields.

Background: For safety considerations, regulatory agencies recommend the elimination of antibiotic resistance markers and non-essential sequences from plasmid DNA-based gene medicines. In the present study, we analyzed antibiotic-free (AF) vector design criteria impacting upon bacterial production and mammalian transgene expression.

Methods: Both CMV-HTLV-I R RNA Pol II promoter (protein transgene) and murine U6 RNA Pol III promoter (RNA transgene) vector designs were studied.

Vector insert-targeted integrative antisense expression system for plasmid stabilization.

Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity.

Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system.

To ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. Here, we describe the development and application of a novel antibiotic-free selection system. Vectors incorporate and express a 150 bp RNA-OUT antisense RNA.

Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification.

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm.

Strain engineering by genome mass transfer: efficient chromosomal trait transfer method utilizing donor genomic DNA and recipient recombineering hosts.

Strain engineering, like cloning, is a fundamental technology used to confer new traits onto existing strains. While effective methods for trait development through gene modification within strains have been developed, methods for trait transfer between Escherichia coli strains to create complex strains are needed. We report herein the development of genome mass transfer (GMT), a broadly applicable new strain engineering methodology enabling rapid trait transfer from a donor strain into a recombineering gene-expressing recipient strain.

Generic plasmid DNA production platform incorporating low metabolic burden seed-stock and fed-batch fermentation processes.

DNA vaccines have tremendous potential for rapid deployment in pandemic applications, wherein a new antigen is "plugged" into a validated vector, and rapidly produced in a validated, fermentation-purification process. For this application, it is essential that the vector and fermentation process function with a variety of different antigen genes. However, many antigen genes are unpredictably "toxic" or otherwise low yielding in standard fermentation processes.

pDNAVACCultra vector family: high throughput intracellular targeting DNA vaccine plasmids.

DNA vaccines have the potential to provide a safe route for protective immunity to neoplasms and infectious agents. However, current DNA vaccine plasmids are not optimal with additional non-essential DNA, nor do they facilitate controlled or flexible targeting of antigens to various intracellular destinations. A family of DNA vaccine vectors, optimized and minimized to comply with FDA guidelines regarding content and elimination of extraneous materials, was constructed.