Phase 1b/2a randomized study of heterologous ChAdOx1-HBV/MVA-HBV therapeutic vaccination (VTP-300) as monotherapy and combined with low-dose nivolumab in virally-suppressed patients with chronic hepatitis B.

BACKGROUND AND AIM: The induction of effective CD8+ T cells is thought to play a critical role in the functional cure of chronic hepatitis B (CHB). Additionally, the use of checkpoint inhibitors is being evaluated to overcome T cell dysfunction during CHB. APPROACH AND RESULTS: A chimpanzee adenoviral vector (ChAdOx1-HBV) and a Modified vaccinia Ankara boost (MVA-HBV) encoding the inactivated polymerase, core, and S region from a consensus genotype C HBV were studied. The trial enrolled 55 patients with virally-suppressed CHB virus infection and HBsAg <4,000 IU/mL Group 1 received MVA-HBV intramuscularly (IM) on Day 0 and 28, Group 2 received ChAdOx1-HBV on Day 0/MVA-HBV on Day 28 (VTP-300), Group 3 received VTP-300 + low-dose nivolumab (LDN) on Day 28, and Group 4 received VTP-300 plus LDN with both injections. VTP-300 alone and in combination with LDN was well tolerated with no treatment-related serious adverse events. Reductions of HBsAg were demonstrated in the VTP-300 group 2: 3 of 18 patients with starting HBsAg < 50 IU/ml had durable log10 declines > 0.7 log10 2 months post last-dose. Group 3 (N=18) had reductions in HBsAg of 0.76 log10 and 0.80 log10 3 (p<0.001) at 2 and 7 months post last dose. Two developed persistent non-detectable HBsAg levels. CD4+ and CD8+ antigen-specific T cell responses were generated and there was a correlation between IFN-y ELISpot response and HBsAg decline in Group 2. CONCLUSIONS: VTP-300 induced CD4+ and CD8+ T cells and lowered HBsAg in a subset of patients with baseline values below 100 IU/ml. The addition of LDN resulted in significant reduction in surface antigen. VTP-300 is a promising immunotherapeutic to move forward alone or in combination therapies. IMPACT AND IMPLICATIONS: The induction of potent, durable CD8+ T cells may be critical to achieving a functional cure in chronic hepatitis B virus infection. A prime-boost immunotherapeutic consisting of an adenoviral-vector encoding hepatitis B antigens followed by a pox virus boost was shown to induce CD8+ T cells and to lower HBsAg in CHB patients, either alone or more impactfully when administered in conjunction with a checkpoint inhibitor. The use of immunotherapeutics CLINTRIALS: NCT047789.

A comparison of a prototype electromyograph vs. a mechanomyograph and an acceleromyograph for assessment of neuromuscular blockade.

The extent of neuromuscular blockade during anaesthesia is frequently measured using a train-of-four stimulus. Various monitors have been used to quantify the train-of-four, including mechanomyography, acceleromyography and electromyography. Mechanomyography is often considered to be the laboratory gold standard of measurement, but is not commercially available and has rarely been used in clinical practice. Acceleromyography is currently the most commonly used monitor in the clinical setting, whereas electromyography is not widely available. We compared a prototype electromyograph with a newly constructed mechanomyograph and a commercially available acceleromyograph monitor in 43 anesthetised patients. The mean difference (bias; 95% limits of agreement) in train-of-four ratios was 4.7 (-25.2 to 34.6) for mechanomyography vs. electromyography; 14.9 (-13.0 to 42.8) for acceleromyography vs. electromyography; and 9.8 (-31.8 to 51.3) for acceleromyography vs. mechanomyography. The mean difference (95% limits of agreement) in train-of-four ratios between opposite arms when using electromyography was -0.7 (-20.7 to 19.3). There were significantly more acceleromyography train-of-four values > 1.0 (23%) compared with electromyography or mechanomography (2-4%; p < 0.0001). Electromyography most closely resembled mechanomyographic assessment of neuromuscular blockade, whereas acceleromyography frequently produced train-of-four ratio values > 1.0, complicating the interpretation of acceleromyography results in the clinical setting.

Facilitated self-reported anaesthetic medication errors before and after implementation of a safety bundle and barcode-based safety system.

BACKGROUND: Anaesthetic medication administration errors are a significant threat to patient safety. In 2002, we began collecting data about the rate and nature of anaesthetic medication errors and implemented a variety of measures to reduce errors. METHODS: Facilitated self-reporting of errors was carried out in 2002-2003. Subsequently, a medication safety bundle including 'smart' infusion pumps were implemented. During 2014 facilitated self-reporting commenced again. A barcode-based medication safety system was then implemented and the facilitated self-reporting was continued through 2015. RESULTS: During 2002-2003, a total of 11 709 paper forms were returned. There were 73 reports of errors (0.62% of anaesthetics) and 27 reports of intercepted errors (0.23%). During 2014, 14 572 computerised forms were completed. There were 57 reports of errors (0.39%) and 11 reports of intercepted errors (0.075%). Errors associated with medication infusions were reduced in comparison with those recorded in 2002-2003 (P<0.001). The rate of syringe swap error was also reduced (P=0.001). The reduction in error rate between 2002-2003 and 2014 was statistically significant (P=0.0076 and P=0.001 for errors and intercepted errors, respectively). From December 2014 through December 2015, 24 264 computerised forms were completed after implementation of a barcode-based medication safety system. There were 56 reports of errors (0.23%) and six reports of intercepted errors (0.025%). Vial swap errors in 2014-2015 were significantly reduced compared with those in 2014 (P=0.004). The reduction in error rate after implementation of the barcode-based medication safety system was statistically significant (P=0.0045 and P=0.021 for errors and intercepted errors, respectively). CONCLUSIONS: Reforms intended to reduce medication errors were associated with substantial improvement.

DNA vaccination against respiratory influenza virus infection.

DNA vaccination using plasmid encoding the hemagglutinin (HA) gene of influenza A/PR/8/34 virus to induce long-lasting protective immunity against respiratory infection was evaluated in this study. Using liposomes as carriers, the efficacy of DNA vaccines was determined using a lethal influenza infection model in mice. Mice immunized intranasally or intramuscularly with liposome-encapsulated pCI plasmid encoding HA (pCI-HA10) were completely protected against an intranasal 5 LD(50) influenza virus challenge. Mice immunized with liposome-encapsulated pCI-HA10, but not naked pCI-HA10, by intranasal administration were found to produce high titers of serum IgA. These results suggest DNA vaccines encapsulated in liposomes are efficacious in inducing complete protective immunity against respiratory influenza virus infection.

In vivo studies of gene expression via transient transgenesis using lipid-DNA delivery.

As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.

Aerosol delivery of lipid:DNA complexes to lungs of rhesus monkeys.

PURPOSE: The potential use of aerosol delivery for non-viral gene therapy was tested by nebulization of lipid:DNA complexes to the lungs of rhesus monkeys. METHODS: Four female rhesus monkeys were dosed with lipid:DNA formulations via aerosol inhalation, where the DNA coded for the human Cystic Fibrosis Transmembrane Conductance Regulator (hCFTR) protein. Delivery of DNA was determined in lung samples by polymerase chain reaction (PCR) by qualitative and quantitative methods. Transgene specific messenger RNA was measured by reverse transcriptase PCR (RT-PCR) and protein expression and localization were evaluated by immunohistochemistry (IHC). RESULTS: Approximately four mg of DNA, complexed with cationic lipid 1.2-dimyristoyl-sn-glycero-3-ethylphosphatidylcholine (EDMPC) and cholesterol were delivered to the lungs of animals by airjet nebulizer. Three days after dosing, tissue samples from the lung were collected and shown to have vector specific DNA, RNA and the presence of CFFR protein. Specifically, the hCFTR protein was distributed widely, although non-uniformly, throughout airway epithelium being located on the apical surface of epithelial cells. Importantly, no adverse clinical effects were observed and the lungs showed no histological abnormalities or signs of acute inflammation. CONCLUSIONS: This study shows that lipid:DNA formulations based on EDMPC and cholesterol can be administered to primates by nebulization resulting in measurable expression of the hCFTR protein. The absence of inflammation is also encouraging and such systems may have utility for delivery of genes to the lungs for the treatment of a variety of pulmonary diseases including cystic fibrosis.

The degA gene product accelerates degradation of Bacillus subtilis phosphoribosylpyrophosphate amidotransferase in Escherichia coli.

A search for genes involved in the inactivation and degradation of enzymes in sporulating Bacillus subtilis led to identification of the B. subtilis degA gene, whose product stimulates degradation of B. subtilis glutamine phosphoribosylpyrophosphate amidotransferase in Escherichia coli cells. degA encodes a 36.7-kDa protein that has sequence similarity to several E. coli and B. subtilis regulatory proteins of the LacI class. B. subtilis degA::cat insertional inactivation mutants had no detectable defect in the inactivation or degradation of phosphoribosylpyrophosphate amidotransferase in glucose- or lysine-starved B. subtilis cells, however. We suggest that degA encodes either a novel protease or, more likely, a gene that stimulates production of such a protease.

Carbamoylphosphate synthetase from Pseudomonas aeruginosa. Subunit composition, kinetic analysis and regulation.

Carbamoylphosphate synthetase from Pseudomonas aeruginosa is subject to repression by pyrimidines and significant derepression by limitation of arginine or pyrimidines. Carbamoylphosphate synthetase was purified to homogeneity from a derepressed strain of P. aeruginosa. The molecular weight of the enzyme was estimated to be 168 000 by sucrose gradient ultracentrifugation. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed that the enzyme is composed of two non-identical subunits with molecular weights of 122 000 and 44 000. Cross-linking the enzyme prior to electrophoresis yielded an additional band corresponding to a molecular weight of 165 000, showing that the enzyme is composed of one of each subunit. The enzyme utilized either glutamine (Km 0.15 mM) or NH3 (Km 17 mM) and requires free Mg2+ for maximal activity with the optimal level between 4 mM and 10 mM. Hill plots of MgATP saturation data yielded coefficients of 1.2 and 1.4 at pH 8.0 and 8.5, respectively. A Hill equation was derived on the assumptions that MgATP binds at the same time to two distinct sub-sites as was shown to be the case for carbamoylphosphate synthetase from Escherichia coli and that these sub-sites are strictly non-interacting. The resulting theoretical Hill coefficients correspond very closely to the experimental coefficients. Carbamoylphosphate synthetase activity was subject to activation by ornithine and N-acetylornithine and feedback inhibition by UMP. Carbamoylphosphate synthetase from P. aeruginosa does not associate under all conditions examined, establishing that self-association does not play a role in regulation of enzyme activity as suggested by other workers for the enzyme from E. coli.

A regulatory gene (use) affecting the expression of pyrA and certain other pyrimidine genes.

The use-1 mutation in Salmonella typhimurium confers a complex and pleiotrophic phenotype which is primarily characterized as a temperature-dependent sensitivity to uracil. This sensitivity can be reversed by arginine or citrulline, but not by ornithine, suggesting that the use-1 mutation affects the synthesis or the activity (or both) of carbamoylphosphate synthetase or ornithine carbamoyltransferase (or both). Activity measurements showed that use-1 caused superrepression of both of these enzymes, especially when uracil was present in the medium. Dihydro-orotase and dihydro-orotate oxidase were also superrepressed, but aspartate carbamoyltransferase and orotate phosphoribosyltransferase were not. Lowered nucleotide triphosphate and guanosine tetra- and pentaphosphate pools in use-1 strains indicated that the mutation affected synthesis or breakdown of all of these phosphorylated compounds, but the UTP pool increased by a larger relative factor in use-1 strains in the presence of uracil. The uracil-sensitive phenotype of the use-1 mutation is a complex response to several environmental factors: temperature, aerobiosis, carbon sources, and uracil concentration. Uracil sensitivity was eliminated by alteration of one or more of these factors. Uracil sensitivity was suppressed by several genetic alterations. These include introduction into use-1 strains of a multi-copy ColE1 derivative which carries the structural gene(s) for carbamoylphosphate synthetase, episomes that carry use, mutations including argR and pyrH, and various unclassified intergenic suppressor mutations. These genetic changes increased significantly the expression of carbamoylphosphate synthetase or ornithine carbamoyltransferase (or both). The activity of use-1 is not known, but the facts that it altered expression of at least four unlinked genes (pyrA, pyrC, pyrD, and argI) and that the Escherichia coli F'133 complemented it establish it as a trans-acting regulatory factor.

Isolation and mapping of a uracil-sensitive mutant of Salmonella typhimurium.

A uracil-sensitive mutant of Salmonella typhimurium was isolated by diethyl sulfate mutagenesis and penicillin counterselection. This mutation identifies a new Salmonella gene that is well separated from the structural genes for arginine and pyrimidine biosynthesis. The use-1 mutation was located between the ilv gene cluster (isoleucine-valine operon) and hisR (structural gene for histidine tRNA) at 83 map units.