Genetic engineering of a Lemna isoleucine auxotroph.

Lemna, a member of the Lemnaceae or duckweed family, is a small aquatic plant that can be quickly transformed to produce recombinant proteins in a contained and controlled bioprocessing environment. The containment capability of Lemna has been further improved with the creation of an auxotroph platform that requires isoleucine supplementation for survival of transformed plant lines. Using an RNAi based approach, threonine deaminase (TD) expression was targeted and thus resulted in dramatically reduced expression of this key enzyme in the isoleucine biosynthesis pathway. Auxotrophic plants expressing RNAi for TD were generated in the presence of isoleucine and selected based on their inability to propagate without isoleucine supplementation. TD transcripts isolated from the superior auxotroph lines were shown to be less than 10% of wild type level and thus confirmed the auxotroph phenotype to be derived from the specific knock down of TD expression. When grown under optimal conditions with appropriate isoleucine supplementation, biomass accumulation of the auxotroph lines was equivalent to that of wild type plants. To demonstrate the application of this system for production of recombinant proteins, an avian influenza H5N1 hemagglutinin (HA) protein was expressed in the isoleucine auxotroph platform. The successful expression of H5N1 HA vaccine antigen, in the isoleucine auxotroph background demonstrates the applicability of using an auxotroph to express biotherapeutics and vaccines in a highly contained expression system.

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor.

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens.

A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (rLemna-HA). In Experiment 1, efficacy of rLemna-HA was tested on birds immunized with 0.2µg or 2.3 µg HA and challenged with 10(6) mean chicken embryo infectious doses (EID50) of homologous virus strain. Both dosages of rLemna-HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of rLemna-HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of rLemna-HA was tested on birds immunized with 0.9 µg or 2.2 µg HA and challenged with 10(6) EID50 of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9 µg HA and 2.2 µg HA, respectively. For each challenge virus, viral shedding titers from 2.2 µg HA vaccinated birds were significantly lower than those from 0.9µg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (rLemna-HA 0.9 µg HA) and 40% (rLemna-HA 2.2 µg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna-expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a good alternative for producing high quality antigen for an injectable vaccine against H5N1 HPAI viruses.

Junctional Tourniquet Training Experience.

Since 2009, out-of-hospital care of junctional hemorrhage bleeding from the trunk-appendage junctions has changed, in part, due to the newly available junctional tourniquets (JTs) that have been cleared by the US Food and Drug Administration. Given four new models of JT available in 2014, several military services have begun to acquire, train, or even use such JTs in care. The ability of users to be trained in JT use has been observed by multiple instructors. The experience of such instructors has been broad as a group, but their experience as individuals has been neither long nor deep. A gathering into one source of the collective experience of trainers of JT users could permit a collation of useful information to include lessons learned, tips in skill performance, identification of pitfalls of use to avoid, and strategies to optimize user learning. The purpose of the present review is to record the experiences of several medical personnel in their JT training of users to provide a guide for future trainers.

Evaluation of Contingency Telemedical Support to Improve Casualty Care at a Simulated Military Intermediate Resuscitation Facility: The EM-ANGEL Study.

OBJECTIVE: We sought to determine whether Contingency Telemedical Support (CTS) improves the success rate and efficiency of primary care providers performing critical actions during simulated combat trauma resuscitation. Critical actions included advanced airway, chest decompression, extremity hemorrhage control, hypothermia prevention, antibiotics and analgesics, and hypotensive resuscitation, among others. BACKGROUND: Recent studies report improved survival associated with skilled triage and treatment in the out-of-hospital/preoperative phase of combat casualty care. Historically, ground combat units are assigned primary care physicians and physician assistants as medical staff, due to resource limitations. Although they are recognized as optimal resuscitators, demand for military trauma surgeons and emergency physicians exceeds supply and is unlikely to improve in the near term. METHODS: A prospective trial of telemedical mentoring during a casualty resuscitation encounter was studied using a high-fidelity patient simulator (HFPS). Subjects were randomized and formed into experimental (CTS) or control teams. CTS team leaders were equipped with a headset/microphone interface and telementored by a combat-experienced emergency physician or trauma surgeon. A standardized, scripted clinical scenario and HFPS were used with 14 critical actions. At completion, subjects were surveyed. Statistical approach included contingency table analysis, two-tailed t-test, and correlation coefficient. This study was reviewed and approved by our institutional review board (IRB). RESULTS: Eighteen CTS teams and 16 control teams were studied. By intention-to-treat ITT analysis, 89% of CTS teams versus 56% of controls completed all life-threatening inventions (LSIs) (p<.01); 78% versus 19% completed all critical actions (p<.01); and 89% versus 56% established advanced airways within 8 minutes (p<.06). Average time to completion in minutes (95% confidence interval [CI] 95) was 12 minutes (10?14) for CTS versus 18 (16?20) for controls, with 75% of control teams not completing all critical actions. CONCLUSION: In this model, real-time telementoring of simulated trauma resuscitation was feasible and improved accuracy and efficiency of non?emergency-trained resuscitators. Clinical validation and replicated study of these findings for guiding remote damage control resuscitation are warranted.