Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

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.

Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortus Delta wbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. Delta wbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence. (C) 2010 Elsevier Ltd. All rights reserved.

Humoral and cell-mediated immune responses to different doses of attenuated vaccine against avian infectious bronchitis virus

The antibody and cellular immune responses against infectious bronchitis virus (IBV) were evaluated at mucosal sites of chickens after immunization with various doses of an attenuated vaccine at 1 day of age. The correlation of these immune responses with protection of tracheal tissues was evaluated after experimental infection of these birds. Significantly reduced tracheal pathologic effects, measured according to ciliostasis and histology lesions, and reduced viral load were observed only in the full-dose vaccinated group at 5 days post-infection (dpi), while incomplete protection was observed for the subdose vaccinated groups. Moreover, birds of vaccinated groups, especially with full dose, developed higher levels of lachrymal IBV-specific IgG and IgA and increased the expression of cell-mediated immunity (CMI) genes, such as gamma interferon (IFNγ), CD8+ T cell marker, and granzyme homolog A more rapidly. In addition, these humoral and cellular immune responses evaluated at mucosal sites correlated significantly with tracheal protection against homologous IBV challenge in a vaccine dose-dependent manner. The results indicate that IgG, IgA and CD8+ T cell responses developed at mucosal sites after IBV vaccination of day-old chicks, could be taken as good correlates of protection against this virus. © 2013, Mary Ann Liebert, Inc.

Oral immunization with attenuated Salmonella vaccine expressing Escherichia coli O157:H7 intimin gamma triggers both systemic and mucosal humoral immunity in mice

Human infections with EHEC such as O157:H7 have been a great concern for worldwide food-industry surveillance. This pathogen is commonly associated with bloody diarrhea that can evolve to the life-threatening hemolytic uremic syndrome. Animals are the natural reservoir where this pathogen remains asymptomatically, in steps of ingestion and colonization of the bowel. The bacterium is shed in the feces, contaminating the surroundings, including water and food that are directed for human consumption. A major player in this colonization process is intimin, an outer membrane adhesion molecule encoded by the E. coli attachment and effacement (eae) gene that has been shown to be essential for intimate bacterial attachment to eukaryotic host cells. In an attempt to reduce the colonization of animal reservoirs with EHEC O157:H7, we designed a vaccine model to induce an immune response against intimin gamma. The model is based on its recombinant expression in attenuated Salmonella, used as a suitable vaccine vector because of its recognized ability to deliver recombinant antigens and to elicit all forms of immunity: mucosal, systemic, and humoral responses. To test this model, mice were orally immunized with a S. enterica serovar Typhimurium strain carrying the pYA3137eaeA vector, and challenged with E. coli O157:H7. Here we show that immunization induced the production of high levels of specific IgG and IgA antibodies and promoted reduction in the fecal shedding of EHEC after challenge. The live recombinant vaccine reported herein may contribute to the efforts of reducing animal intestinal mucosa colonization.

An attenuated Salmonella enterica serovar Typhimurium strain lacking the ZnuABC transporter induces protection in a mouse intestinal model of Salmonella infection

Salmonella enterica serovar Typhimurium has long been recognised as a zoonotic pathogen of economic significance in animals and humans. Attempts to protect humans and livestock may be based on immunization with vaccines aimed to induce a protective response. We recently demonstrated that the oral administration of a Salmonella enterica serovar Typhimurium strain unable to synthesize the zinc transporter ZnuABC is able to protect mice against systemic salmonellosis induced by a virulent homologous challenge. This finding suggested that this mutant strain could represent an interesting candidate vaccine for mucosal delivery. In this study, the protective effect of this Salmonella strain was tested in a streptomycin-pretreated mouse model of salmonellosis that is distinguished by the capability of evoking typhlitis and colitis. The here reported results demonstrate that mice immunized with Salmonella enterica serovar Typhimurium (S. Typhimurium) SA186 survive to the intestinal challenge and, compared to control mice, show a reduced number of virulent bacteria in the gut, with milder signs of inflammation. This study demonstrates that the oral administration a of S. Typhimurium strain lacking ZnuABC is able to elicit an effective immune response which protects mice against intestinal S. Typhimurium infection. These results, collectively, suggest that the streptomycin-pretreated mouse model of S. typhimurium infection can represent a valuable tool to screen S. typhimurium attenuated mutant strains and potentially help to assess their protective efficacy as potential live vaccines.

Biological safety concepts of genetically modified live bacterial vaccines

Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment includes knowledge of the precise function and genetic location of the genes to be mutated, their genetic stability, potential reversion mechanisms, possible recombination events with dormant genes, gene transfer to other organisms as well as gene acquisition from other organisms by phage transduction, transposition or plasmid transfer and cis- or trans-complementation. For this, GMOs that are constructed with modern techniques of genetic engineering display a significant advantage over random mutagenesis derived live organisms. The selection of suitable GMO candidate strains can be made under in vitro conditions using basic knowledge on molecular mechanisms of pathogenicity of the corresponding bacterial species rather than by in vivo testing of large numbers of random mutants. This leads to a more targeted safety testing on volunteers and to a reduction in the use of animal experimentation.

Respiratory syncytial virus (RSV) SH and G proteins are not essential for viral replication in vitro: Clinical evaluation and molecular characterization of a cold-passaged, attenuated RSV subgroup B mutant

A live, cold-passaged (cp) candidate vaccine virus, designated respiratory syncytial virus (RSV) B1 cp-52/2B5 (cp-52), replicated efficiently in Vero cells, but was found to be overattenuated for RSV-seronegative infants and children. Sequence analysis of reverse-transcription–PCR-amplified fragments of this mutant revealed a large deletion spanning most of the coding sequences for the small hydrophobic (SH) and attachment (G) proteins. Northern blot analysis of cp-52 detected multiple unique read-through mRNAs containing SH and G sequences, consistent with a deletion mutation spanning the SH:G gene junction. Immunological studies confirmed that an intact G glycoprotein was not produced by the cp-52 virus. Nonetheless, cp-52 was infectious and replicated to high titer in tissue culture despite the absence of the viral surface SH and G glycoproteins. Thus, our characterization of this negative-strand RNA virus identified a novel replication-competent deletion mutant lacking two of its three surface glycoproteins. The requirement of SH and G for efficient replication in vivo suggests that selective deletion of one or both of these RSV genes may provide an alternative or additive strategy for developing an optimally attenuated vaccine candidate.

Sleeper [for laptop computer and live performer]

Sleeper is an 18'00" musical work for live performer and laptop computer which exists as both a live performance work and a recorded work for audio CD. The work has been presented at a range of international performance events and survey exhibitions. These include the 2003 International Computer Music Conference (Singapore) where it was selected for CD publication, Variable Resistance (San Francisco Museum of Modern Art, USA), and i.audio, a survey of experimental sound at the Performance Space, Sydney. The source sound materials are drawn from field recordings made in acoustically resonant spaces in the Australian urban environment, amplified and acoustic instruments, radio signals, and sound synthesis procedures. The processing techniques blur the boundaries between, and exploit, the perceptual ambiguities of de-contextualised and processed sound. The work thus challenges the arbitrary distinctions between sound, noise and music and attempts to reveal the inherent musicality in so-called non-musical materials via digitally re-processed location audio. Thematically the work investigates Paul Virilio’s theory that technology ‘collapses space’ via the relationship of technology to speed. Technically this is explored through the design of a music composition process that draws upon spatially and temporally dispersed sound materials treated using digital audio processing technologies. One of the contributions to knowledge in this work is a demonstration of how disparate materials may be employed within a compositional process to produce music through the establishment of musically meaningful morphological, spectral and pitch relationships. This is achieved through the design of novel digital audio processing networks and a software performance interface. The work explores, tests and extends the music perception theories of ‘reduced listening’ (Schaeffer, 1967) and ‘surrogacy’ (Smalley, 1997), by demonstrating how, through specific audio processing techniques, sounds may shifted away from ‘causal’ listening contexts towards abstract aesthetic listening contexts. In doing so, it demonstrates how various time and frequency domain processing techniques may be used to achieve this shift.

Equal employment opportunity and diversity management : can either live up to the promise of achieving gender equity?

This paper examines the extent to which women movement into management positions. Like many other countries, this progress in Australia is slow. The paper includes discussion of the theoretical explanations for this and the extent to which these are borne out in Australia. We are aware this group represents only a minority of Australian women workers, and there are many other groups of women workers for whom constraints to women’s access to senior management may not be the most pressing issue. We have, however, chosen to focus on women in management in this paper, as while there was considerable research and public policy attention directed towards this group in the 1980s and early 1990s, over the past decade there seems to have been a reluctance to continue to address this group, despite the numerical evidence that women continue to be disproportionately represented in senior management positions. We believe it’s timely to refocus on women in management.

‘Live your liberation, don’t lobby for it’ : Australian queer student activists’ perspectives of same-sex marriage

One topic covered in Australian queer university student print media is the legalisation of same-sex marriage. The legalisation of same-sex marriage is currently generating much debate in Western queer communities. Same-sex marriage is legalised in some countries such as, Canada, Spain, the Netherlands and Belgium. It has been outlawed in Australia and most states in the US. Campaigns continue to reverse these restrictions. Other countries, such as the UK and New Zealand allow same-sex civil unions, providing couples with the rights afforded to married couples. There is a range of research documenting queer communities’ attitudes towards this issue (for example Lannutti 2005; Clarke, Burgoyne and Burns 2006; Yep, Lovaas and Elia 2003; Wolfson 1993; Egan and Sherrill 2005). These studies document broad community views as well as those of community sub-sections. For example, Yip (2004) looks at the views of gay and lesbian Christians on same-sex marriage and Lahey and Alderson (2004) document the experiences of same-sex couples who have gotten married or who are waiting to get married. Philosophical analyses consider the legalisation of same-sex marriage in relation to, for example, liberalism, equal rights, liberation, queer theory, citizenship, history, activism, religious discourse and feminism (Ferguson 2007; Jordan 2005; Josephson 2005; Lipton 2006; Sullivan and Chauncey 2005; Riggs 2007). This paper explores Australian queer university student activist media’s representation of same-sex marriage, and the debates surrounding its legalisation. It examines a selection of queer student media from four metropolitan Australian universities, and the 2003 and 2004 editions of national queer student publication, Querelle. This paper uses discourse analysis of queer student activists’ media representations of marriage to investigate this issue in one specific context – metropolitan Australian universities. This paper thus contributes to the history of queer activism, documenting what one group of young people say about the legalisation of same-sex marriage, and furthers research on queer perspectives of marriage and same-sex relationships.

A brush with the real world : the future of inertial motion capture in live performance

3D Motion capture is a medium that plots motion, typically human motion, converting it into a form that can be represented digitally. It is a fast evolving field and recent inertial technology may provide new artistic possibilities for its use in live performance. Although not often used in this context, motion capture has a combination of attributes that can provide unique forms of collaboration with performance arts. The inertial motion capture suit used for this study has orientation sensors placed at strategic points on the body to map body motion. Its portability, real-time performance, ease of use, and its immunity from line-of-sight problems inherent in optical systems suggest it would work well as a live performance technology. Many animation techniques can be used in real-time. This research examines a broad cross-section of these techniques using four practice-led cases to assess the suitability of inertial motion capture to live performance. Although each case explores different visual possibilities, all make use of the performativity of the medium, using either an improvisational format or interactivity among stage, audience and screen that would be difficult to emulate any other way. A real-time environment is not capable of reproducing the depth and sophistication of animation people have come to expect through media. These environments take many hours to render. In time the combination of what can be produced in real-time and the tools available in a 3D environment will no doubt create their own tree of aesthetic directions in live performance. The case study looks at the potential of interactivity that this technology offers.

Inertial motion capture and live performance (with a focus on dance)

3D Motion capture is a fast evolving field and recent inertial technology may expand the artistic possibilities for its use in live performance. Inertial motion capture has three attributes that make it suitable for use with live performance; it is portable, easy to use and can operate in real-time. Using four projects, this paper discusses the suitability of inertial motion capture to live performance with a particular emphasis on dance. Dance is an artistic application of human movement and motion capture is the means to record human movement as digital data. As such, dance is clearly a field in which the use of real-time motion capture is likely to become more common, particularly as projected visual effects including real-time video are already often used in dance performances. Understandably, animation generated in real-time using motion capture is not as extensive or as clean as the highly mediated animation used in movies and games, but the quality is still impressive and the ‘liveness’ of the animation has compensating features that offer new ways of communicating with an audience.

Performing composition : developing a computer assisted composition system through live coding performance

This thesis maps the author's journey from a music composition practice to a composition and performance practice. The work involves the development of a software library for the purpose of encapsulating compositional ideas in software, and realising these ideas in performance through a live coding computer music practice. The thesis examines what artistic practice emerges through live coding and software development, and does this permit a blurring between the activities of music composition and performance. The role that software design plays in affecting musical outcomes is considered to gain an insight into how software development contributes to artistic development. The relationship between music composition and performance is also examined to identify the means by which engaging in live coding and software development can bring these activities together. The thesis, situated within the discourse of practice led research, documents a journey which uses the experience of software development and performance as a means to guide the direction of the research. The journey serves as an experiment for the author in engaging an hitherto unfamiliar musical practice, and as a roadmap for others seeking to modify or broaden their artistic practice.