Streptococcus ovis sp. nov., isolated from sheep

Seven strains of an unknown Gram-positive catalase-negative chain-forming coccus-shaped organism isolated from clinical specimens from sheep were characterized by phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies demonstrated that the bacterium represents a new sub-line within the genus Streptococcus. The unknown bacterium was readily distinguished from recognized streptococcal species by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Streptococcus ovis sp. nov. The type strain of Streptococcus ovis is CCUG 39485T (= LMG 19174T).

Genetic expression of an amyloid peptide fragment and analysis of formylated products

The model amyloid peptide AAKLVFF was expressed as a His-tagged fusion protein with the immunoglobulin-binding domain B1 of streptococcal protein G (GB1), a small (56 residues), stable, single-domain protein. It is shown that expression of this model amyloid peptide is possible and is not hindered by aggregation. Formylation side reactions during the CNBr cleavage are investigated via synthesis of selectively formylated peptides.

Technologies for biological containment of GM and Non-GM crops

International Perspective The development of GM technology continues to expand into increasing numbers of crops and conferred traits. Inevitably, the focus remains on the major field crops of soybean, maize, cotton, oilseed rape and potato with introduced genes conferring herbicide tolerance and/or pest resistance. Although there are comparatively few GM crops that have been commercialised to date, GM versions of 172 plant species have been grown in field trials in 31 countries. European Crops with Containment Issues Of the 20 main crops in the EU there are four for which GM varieties are commercially available (cotton, maize for animal feed and forage, and oilseed rape). Fourteen have GM varieties in field trials (bread wheat, barley, durum wheat, sunflower, oats, potatoes, sugar beet, grapes, alfalfa, olives, field peas, clover, apples, rice) and two have GM varieties still in development (rye, triticale). Many of these crops have hybridisation potential with wild and weedy relatives in the European flora (bread wheat, barley, oilseed rape, durum wheat, oats, sugar beet and grapes), with escapes (sunflower); and all have potential to cross-pollinate fields non-GM crops. Several fodder crops, forestry trees, grasses and ornamentals have varieties in field trials and these too may hybridise with wild relatives in the European flora (alfalfa, clover, lupin, silver birch, sweet chestnut, Norway spruce, Scots pine, poplar, elm, Agrostis canina, A. stolonifera, Festuca arundinacea, Lolium perenne, L. multiflorum, statice and rose). All these crops will require containment strategies to be in place if it is deemed necessary to prevent transgene movement to wild relatives and non-GM crops. Current Containment Strategies A wide variety of GM containment strategies are currently under development, with a particular focus on crops expressing pharmaceutical products. Physical containment in greenhouses and growth rooms is suitable for some crops (tomatoes, lettuce) and for research purposes. Aquatic bioreactors of some non-crop species (algae, moss, and duckweed) expressing pharmaceutical products have been adopted by some biotechnology companies. There are obvious limitations of the scale of physical containment strategies, addressed in part by the development of large underground facilities in the US and Canada. The additional resources required to grow plants underground incurs high costs that in the long term may negate any advantage of GM for commercial productioNatural genetic containment has been adopted by some companies through the selection of either non-food/feed crops (algae, moss, duckweed) as bio-pharming platforms or organisms with no wild relatives present in the local flora (safflower in the Americas). The expression of pharmaceutical products in leafy crops (tobacco, alfalfa, lettuce, spinach) enables growth and harvesting prior to and in the absence of flowering. Transgenically controlled containment strategies range in their approach and degree of development. Plastid transformation is relatively well developed but is not suited to all traits or crops and does not offer complete containment. Male sterility is well developed across a range of plants but has limitations in its application for fruit/seed bearing crops. It has been adopted in some commercial lines of oilseed rape despite not preventing escape via seed. Conditional lethality can be used to prevent flowering or seed development following the application of a chemical inducer, but requires 100% induction of the trait and sufficient application of the inducer to all plants. Equally, inducible expression of the GM trait requires equally stringent application conditions. Such a method will contain the trait but will allow the escape of a non-functioning transgene. Seed lethality (‘terminator’ technology) is the only strategy at present that prevents transgene movement via seed, but due to public opinion against the concept it has never been trialled in the field and is no longer under commercial development. Methods to control flowering and fruit development such as apomixis and cleistogamy will prevent crop-to-wild and wild-to-crop pollination, but in nature both of these strategies are complex and leaky. None of the genes controlling these traits have as yet been identified or characterised and therefore have not been transgenically introduced into crop species. Neither of these strategies will prevent transgene escape via seed and any feral apomicts that form are arguably more likely to become invasives. Transgene mitigation reduces the fitness of initial hybrids and so prevents stable introgression of transgenes into wild populations. However, it does not prevent initial formation of hybrids or spread to non-GM crops. Such strategies could be detrimental to wild populations and have not yet been demonstrated in the field. Similarly, auxotrophy prevents persistence of escapes and hybrids containing the transgene in an uncontrolled environment, but does not prevent transgene movement from the crop. Recoverable block of function, intein trans-splicing and transgene excision all use recombinases to modify the transgene in planta either to induce expression or to prevent it. All require optimal conditions and 100% accuracy to function and none have been tested under field conditions as yet. All will contain the GM trait but all will allow some non-native DNA to escape to wild populations or to non-GM crops. There are particular issues with GM trees and grasses as both are largely undomesticated, wind pollinated and perennial, thus providing many opportunities for hybridisation. Some species of both trees and grass are also capable of vegetative propagation without sexual reproduction. There are additional concerns regarding the weedy nature of many grass species and the long-term stability of GM traits across the life span of trees. Transgene stability and conferred sterility are difficult to trial in trees as most field trials are only conducted during the juvenile phase of tree growth. Bio-pharming of pharmaceutical and industrial compounds in plants Bio-pharming of pharmaceutical and industrial compounds in plants offers an attractive alternative to mammalian-based pharmaceutical and vaccine production. Several plantbased products are already on the market (Prodigene’s avidin, β-glucuronidase, trypsin generated in GM maize; Ventria’s lactoferrin generated in GM rice). Numerous products are in clinical trials (collagen, antibodies against tooth decay and non-Hodgkin’s lymphoma from tobacco; human gastric lipase, therapeutic enzymes, dietary supplements from maize; Hepatitis B and Norwalk virus vaccines from potato; rabies vaccines from spinach; dietary supplements from Arabidopsis). The initial production platforms for plant-based pharmaceuticals were selected from conventional crops, largely because an established knowledge base already existed. Tobacco and other leafy crops such as alfalfa, lettuce and spinach are widely used as leaves can be harvested and no flowering is required. Many of these crops can be grown in contained greenhouses. Potato is also widely used and can also be grown in contained conditions. The introduction of morphological markers may aid in the recognition and traceability of crops expressing pharmaceutical products. Plant cells or plant parts may be transformed and maintained in culture to produce recombinant products in a contained environment. Plant cells in suspension or in vitro, roots, root cells and guttation fluid from leaves may be engineered to secrete proteins that may be harvested in a continuous, non-destructive manner. Most strategies in this category remain developmental and have not been commercially adopted at present. Transient expression produces GM compounds from non-GM plants via the utilisation of bacterial or viral vectors. These vectors introduce the trait into specific tissues of whole plants or plant parts, but do not insert them into the heritable genome. There are some limitations of scale and the field release of such crops will require the regulation of the vector. However, several companies have several transiently expressed products in clinical and pre-clinical trials from crops raised in physical containment.

Caste, livelihoods and livestock: an exploration of the uptake of livestock vaccination adoption among poor farmers in India

The study explores the uptake of livestock vaccination among poor farming communities in Tamil Nadu State, India by revisiting innovation diffusion theory. Overall, 601 farmers participated in the study. We found the adoption of particular vaccines was strongly influenced by socio-cultural grouping i.e. caste, rather than other factors such as income, age, education-level or gender. Adoption was also related to specific knowledge frames regarding disease causality, rather than any wider ethno-veterinary beliefs. Thus, the adoption of livestock vaccination is unlikely to improve without knowledge transfer activities, which acknowledge both social divisions and local epistemologies regarding animal health. Copyright © 2010 John Wiley & Sons, Ltd.

Vaccine glycoproteins tagged with the human Fc domain as second generation vaccine candidates

Traditional vaccines such as inactivated or live attenuated vaccines, are gradually giving way to more biochemically defined vaccines that are most often based on a recombinant antigen known to possess neutralizing epitopes. Such vaccines can offer improvements in speed, safety and manufacturing process but an inevitable consequence of their high degree of purification is that immunogenicity is reduced through the lack of the innate triggering molecules present in more complex preparations. Targeting recombinant vaccines to antigen presenting cells (APCs) such as dendritic cells however can improve immunogenicity by ensuring that antigen processing is as efficient as possible. Immune complexes, one of a number of routes of APC targeting, are mimicked by a recombinant approach, crystallizable fragment (Fc) fusion proteins, in which the target immunogen is linked directly to an antibody effector domain capable of interaction with receptors, FcR, on the APC cell surface. A number of virus Fc fusion proteins have been expressed in insect cells using the baculovirus expression system and shown to be efficiently produced and purified. Their use for immunization next to non-Fc tagged equivalents shows that they are powerfully immunogenic in the absence of added adjuvant and that immune stimulation is the result of the Fc-FcR interaction.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

What have dendritic cells ever done for adjuvant design? Cellular and molecular methods for the rational development of vaccine adjuvants

Our new molecular understanding of immune priming states that dendritic cell activation is absolutely pivotal for expansion and differentiation of naïve T lymphocytes, and it follows that understanding DC activation is essential to understand and design vaccine adjuvants. This chapter describes how dendritic cells can be used as a core tool to provide detailed quantitative and predictive immunomics information about how adjuvants function. The role of distinct antigen, costimulation, and differentiation signals from activated DC in priming is explained. Four categories of input signals which control DC activation – direct pathogen detection, sensing of injury or cell death, indirect activation via endogenous proinflammatory mediators, and feedback from activated T cells – are compared and contrasted. Practical methods for studying adjuvants using DC are summarised and the importance of DC subset choice, simulating T cell feedback, and use of knockout cells is highlighted. Finally, five case studies are examined that illustrate the benefit of DC activation analysis for understanding vaccine adjuvant function.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen

Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.

Highly potent delivery method of gp160 envelope vaccine combining lentivirus-like particles and DNA electrotransfer

Particulate antigen assemblies in the nanometer range and DNA plasmids are particularly interesting for designing vaccines. We hypothesised that a combination of these approaches could result in a new delivery method of gp160 envelope HIV-1 vaccine which could combine the potency of virus-like particles (VLPs) and the simplicity of use of DNA vaccines. Characterisation of lentivirus-like particles (lentiVLPs) by western blot, dynamic light scattering and electron microscopy revealed that their protein pattern, size and structure make them promising candidates for HIV-1 vaccines. Although all particles were similar with regard to size and distribution, they clearly differed in p24 capsid protein content suggesting that Rev may be required for particle maturation and Gag processing. In vivo, lentiVLP pseudotyping with the gp160 envelope or with a combination of gp160 and VSV-G envelopes did not influence the magnitude of the immune response but the combination of lentiVLPs with Alum adjuvant resulted in a more potent response. Interestingly, the strongest immune response was obtained when plasmids encoding lentiVLPs were co-delivered to mice muscles by electrotransfer, suggesting that lentiVLPs were efficiently produced in vivo or the packaging genes mediate an adjuvant effect. DNA electrotransfer of plasmids encoding lentivirus-like particles offers many advantages and appears therefore as a promising delivery method of HIV-1 vaccines. Keywords:VLP, Electroporation, Electrotransfer, HIV vaccine, DNA vaccine

A laminated polymer film formulation for enteric delivery of live vaccine and probiotic bacteria

Live bacterial cells (LBC) are administered orally as attenuated vaccines, to deliver biopharmaceutical agents, and as probiotics to improve gastrointestinal health. However, LBC present unique formulation challenges and must survive gastrointestinal antimicrobial defenses including gastric acid after administration. We present a simple new formulation concept, termed Polymer Film Laminate (PFL). LBC are ambient dried onto cast acid-resistant enteric polymer films that are then laminated together to produce a solid oral dosage form. LBC of a model live bacterial vaccine and a probiotic were dried directly onto a cast film of enteric polymer. The effectiveness at protecting dried cells in a simulated gastric fluid (pH 2.0) depended on the composition of enteric polymer film used, with a blend of ethylcellulose plus Eudragit L100 55 providing greater protection from acid than Eudragit alone. However, although PFL made from blended polymers films completely released low molecular weight dye into intestinal conditions (pH 7.0), they failed to release LBC. In contrast, PFL made from Eudragit alone successfully protected dried probiotic or vaccine LBC from simulated gastric fluid for 2h, and subsequently released all viable cells within 60min of transfer into simulated intestinal fluid. Release kinetics could be controlled by modifying the lamination method.

Exploring farmer preferences for contagious bovine pleuropneumonia vaccination: a case study of Narok District, Kenya

Contagious bovine pleuropneumonia (CBPP) is an economically important disease in most of sub-Saharan Africa. A conjoint analysis and ordered probit regression models were used to measure the preferences of farmers for CBPP vaccine and vaccination attributes. This was with regard to inclusion or not of an indicator in the vaccine, vaccine safety, vaccine stability as well as frequency of vaccination, vaccine administration and the nature of vaccination. The analysis was carried out in 190 households in Narok District of Kenya between October and December 2006 using structured questionnaires, 16 attribute profiles and a five-point Likert scale. The factors affecting attribute valuation were shown through a two-way location interaction model. The study also demonstrated the relative importance (RI) of attributes and the compensation value of attribute levels. The attribute coefficient estimates showed that farmers prefer a vaccine that has an indicator, is 100% safe and is administered by the government (p<0.0001). The preferences for the vaccine attributes were consistent with expectations. Preferences for stability, frequency of vaccination and nature of vaccination differed amongst farmers (p>0.05). While inclusion of an indicator in the vaccine was the most important attribute (RI=43.6%), price was the least important (RI=0.5%). Of the 22 household factors considered, 15 affected attribute valuation. The compensation values for a change from non inclusion to inclusion of an indicator, 95-100% safety, 2h to greater than 2h stability and from compulsory to elective vaccination were positive while those for a change from annual to biannual vaccination and from government to private administration were negative. The study concluded that the farmers in Narok District had preferences for specific vaccine and vaccination attributes. These preferences were conditioned by various household characteristics and disease risk factors. On average the farmers would need to be compensated or persuaded to accept biannual and private vaccination against CBPP. There is need for consideration of farmer preferences for vaccine attribute levels during vaccine formulations and farmer preferences for vaccination attribute levels when designing delivery of vaccines.

An evidence-based review on the likely economic and environmental impact of genetically modified cereals and oilseeds for UK agriculture

An evidence-based review of the potential impact that the introduction of genetically-modified (GM) cereal and oilseed crops could have for the UK was carried out. The inter-disciplinary research project addressed the key research questions using scenarios for the uptake, or not, of GM technologies. This was followed by an extensive literature review, stakeholder consultation and financial modelling. The world area of canola, oilseed rape (OSR) low in both erucic acid in the oil and glucosinolates in the meal, was 34M ha in 2012 of which 27% was GM; Canada is the lead producer but it is also grown in the USA, Australia and Chile. Farm level effects of adopting GM OSR include: lower production costs; higher yields and profits; and ease of farm management. Growing GM OSR instead of conventional OSR reduces both herbicide usage and environmental impact. Some 170M ha of maize was grown in the world in 2011 of which 28% was GM; the main producers are the USA, China and Brazil. Spain is the main EU producer of GM maize although it is also grown widely in Portugal. Insect resistant (IR) and herbicide tolerant (HT) are the GM maize traits currently available commercially. Farm level benefits of adopting GM maize are lower costs of production through reduced use of pesticides and higher profits. GM maize adoption results in less pesticide usage than on conventional counterpart crops leading to less residues in food and animal feed and allowing increasing diversity of bees and other pollinators. In the EU, well-tried coexistence measures for growing GM crops in the proximity of conventional crops have avoided gene flow issues. Scientific evidence so far seems to indicate that there has been no environmental damage from growing GM crops. They may possibly even be beneficial to the environment as they result in less pesticides and herbicides being applied and improved carbon sequestration from less tillage. A review of work on GM cereals relevant for the UK found input trait work on: herbicide and pathogen tolerance; abiotic stress such as from drought or salinity; and yield traits under different field conditions. For output traits, work has mainly focussed on modifying the nutritional components of cereals and in connection with various enzymes, diagnostics and vaccines. Scrutiny of applications submitted for field trial testing of GM cereals found around 9000 applications in the USA, 15 in Australia and 10 in the EU since 1996. There have also been many patent applications and granted patents for GM cereals in the USA for both input and output traits;an indication of the scale of such work is the fact that in a 6 week period in the spring of 2013, 12 patents were granted relating to GM cereals. A dynamic financial model has enabled us to better understand and examine the likely performance of Bt maize and HT OSR for the south of the UK, if cultivation is permitted in the future. It was found that for continuous growing of Bt maize and HT OSR, unless there was pest pressure for the former and weed pressure for the latter, the seed premia and likely coexistence costs for a buffer zone between other crops would reduce the financial returns for the GM crops compared with their conventional counterparts. When modelling HT OSR in a four crop rotation, it was found that gross margins increased significantly at the higher levels of such pest or weed pressure, particularly for farm businesses with larger fields where coexistence costs would be scaled down. The impact of the supply of UK-produced GM crops on the wider supply chain was examined through an extensive literature review and widespread stakeholder consultation with the feed supply chain. The animal feed sector would benefit from cheaper supplies of raw materials if GM crops were grown and, in the future, they might also benefit from crops with enhanced nutritional profile (such as having higher protein levels) becoming available. This would also be beneficial to livestock producers enabling lower production costs and higher margins. Whilst coexistence measures would result in increased costs, it is unlikely that these would cause substantial changes in the feed chain structure. Retailers were not concerned about a future increase in the amount of animal feed coming from GM crops. To conclude, we (the project team) feel that the adoption of currently available and appropriate GM crops in the UK in the years ahead would benefit farmers, consumers and the feed chain without causing environmental damage. Furthermore, unless British farmers are allowed to grow GM crops in the future, the competitiveness of farming in the UK is likely to decline relative to that globally.

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design

Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

Modulation of potassium channels inhibits bunyavirus infection

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, twin-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.