Gram-negative bacteria inhabit a broad range of ecological niches. For Escherichia coli, this includes river water as well as humans and animals where it can be both a commensal and a pathogen1–3. Intricate regulatory mechanisms ensure bacteria have the right complement of β-barrel outer membrane proteins (OMPs) to enable adaptation to a particular habitat4,5. Yet no mechanism is known for replacing OMPs in the outer membrane (OM), a biological enigma further confounded by the lack of an energy source and the high stability6 and abundance of OMPs5. Here, we uncover the process underpinning OMP turnover in E. coli and show it to be passive and binary in nature wherein old OMPs are displaced to the poles of growing cells as new OMPs take their place. Using fluorescent colicins as OMP-specific probes, in combination with ensemble and single-molecule fluorescence microscopy in vivo and in vitro, as well as molecular dynamics (MD) simulations, we established the mechanism for binary OMP partitioning. OMPs clustered to form islands of ~0.5 μm diameter where their diffusion was restricted by promiscuous interactions with other OMPs. OMP islands were distributed throughout the cell and contained the Bam complex, which catalyses the insertion of OMPs in the OM7,8. However, OMP biogenesis occurred as a gradient that was highest at mid-cell but largely absent at cell poles. The cumulative effect is to push old OMP islands towards the poles of growing cells, leading to a binary distribution when cells divide. Hence the OM of a Gram-negative bacterium is a spatially and temporally organised structure and this organisation lies at the heart of how OMPs are turned over in the membrane.

The failure of traditional protein-based vaccines to prevent infection by viruses such as HIV or hepatitis C highlights the need for novel vaccine strategies. DNA vaccines have shown promise in small animal models, and are effective at generating anti-viral T cell-mediated immune responses; however, they have proved to be poorly immunogenic in clinical trials. We propose that the induction of necrosis will enhance the immune response to vaccine antigens encoded by DNA vaccines, as necrotic cells are known to release a range of intracellular factors that lead to dendritic cell (DC) activation and enhanced cross-presentation of antigen. Here we provide evidence that induction of cell death in DNA vaccine-targeted cells provides an adjuvant effect following intradermal vaccination of mice; however, this enhancement of the immune response is dependent on both the mechanism and timing of cell death after antigen expression. We report that a DNA vaccine encoding the cytolytic protein, perforin, resulted in DC activation, enhanced broad and multifunctional CD8 T-cell responses to the HIV-1 antigen GAG and reduced viral load following challenge with a chimeric virus, EcoHIV, compared with the canonical GAG DNA vaccine. This effect was not observed for a DNA vaccine encoding an apoptosis-inducing toxin, DTa, or when the level of perforin expression was increased to induce cell death sooner after vaccination. Thus, inducing lytic cell death following a threshold level of expression of a viral antigen can improve the immunogenicity of DNA vaccines, whereas apoptotic cell death has an inhibitory effect on the immune response.

There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multiantigen" vaccine that elicits robust CMI. IMPORTANCESince their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans. G lobal efforts to generate an effective vaccine for hepatitis C virus (HCV) have been hampered, since correlates of sterilizing immunity have not been identified and traditional vaccine strategies have proved to be ineffective (1). Approximately 230 million individuals are infected, many of whom will develop serious liver disease, and since current treatment is costly and often results in serious side effects, this has limited the number of patients who are treated (2, 3). Much of the global effort to develop an effective HCV vaccine/treatment has been directed toward genotype 1 (gt1) (4, 5); however, gt3 is ...

Currently, no vaccine is available against hepatitis C virus (HCV), and although DNA vaccines have considerable potential, this has not been realised. Previously, the efficacy of DNA vaccines for human immunodeficiency virus (HIV) and HCV was shown to be enhanced by including the gene for a cytolytic protein, viz. perforin. In this study, we examined the mechanism of cell death by this bicistronic DNA vaccine, which encoded the HCV non-structural protein 3 (NS3) under the control of the CMV promoter and perforin is controlled by the SV40 promoter. Compared with a canonical DNA vaccine and a bicistronic DNA vaccine encoding NS3 and the proapoptotic gene NSP4, the perforin-containing vaccine elicited enhanced cell-mediated immune responses against the NS3 protein in vaccinated mice and pigs, as determined by ELISpot and intracellular cytokine staining, whereas a mouse challenge model suggested that the immunity was CD8(+) T-cell-dependent. The results of the study showed that the inclusion of perforin in the DNA vaccine altered the fate of NS3-positive cells from apoptosis to necrosis, and this resulted in more robust immune responses in mice and pigs, the latter of which represents an accepted large animal model in which to test vaccine efficacy.

Traditional vaccine strategies are inefficient against challenge with complex pathogens including HIV; therefore, novel vaccine technologies are required. DNA vaccines are attractive as they are relatively cheap and easy to manufacture, but a major limitation has been their lack of immunogenicity in humans, which may be overcome with the incorporation of an adjuvant. HSP70 is a recognised damage-associated molecular pattern, which is a potential adjuvant. We investigated the immunogenicity of a DNA vaccine encoding HIV gag and HSP70; the latter was genetically modified to produce cytoplasmic, secreted or membrane-bound HSP70, the expression of which was controlled by an independent promoter. The DNA was administered to C57BL/6 mice to evaluate gag-specific T-cell responses. Our results demonstrated the ability of membrane-bound and secreted HSP70 to significantly enhance gag-specific T-cell responses and increase the breadth of T-cell responses to include subdominant epitopes. Membrane-bound or secreted HSP70 also significantly improved the multifunctionality of HIV-specific T cells and T-cell proliferation, which is important for maintaining T-cell integrity. Most importantly, the inclusion of membrane-bound HSP70, secreted HSP70 or a combination significantly increased protection in mice challenged with EcoHIV, a chimeric virus that replicates in mouse leukocytes in vivo.Keywords: Adjuvant r DNA vaccines r HSP70 Additional supporting information may be found in the online version of this article at the publisher's web-site Correspondence: Prof. Eric J. Gowans e-mail: eric.gowans@adelaide.edu.au IntroductionTraditional vaccine strategies are unable to elicit protection against complex pathogens such as HIV and HCV [1,2], and consequently, novel strategies are necessary. Current vaccine candidates include DNA vaccines, as they are stable, easy to manufacture and C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2014. 44: 1992 Immunity to infection 1993 can induce humoral and cell-mediated immune responses. Four DNA vaccines have been licensed for veterinary use, emphasising the potential of this approach [3]. In human clinical trials, DNA vaccines have demonstrated a good safety profile, but are limited by poor immune responses [4]. This limitation may be overcome with the addition of an adjuvant [5]. Damage-associated molecular patterns (DAMPs) are hostderived molecules that are released from cells as a result of stress, damage or necrosis [6]. Once released into the extracellular milieu, DAMPs bind to pattern-recognition receptors (PRRs) on dendritic cells (DCs) [7], resulting in DC activation and maturation. DC activation is important for upregulation of costimulatory molecule expression, migration to the draining LNs (DLNs) and T-cell activation [8]. DCs are crucial for vaccine efficacy due to their ability to cross-present exogenous protein via the MHC class I pathway and activate naive CD8 + T cells [9].Molecules classified as DAMPs include HMGB1, uric acid and HSPs [10]...

There has been a decline in the proportion of clinical academics compared with full-time clinicians, since 2004. A Working Party was established to help develop and implement a model for the training of clinical academics. After a highly successful first summit in 2014 that summarised the challenges faced by clinical academics in Australia and New Zealand, a second summit was convened late in 2015 to report on progress and to identify key areas for further action. The second summit provided survey results that identified the varied training pathways currently offered to clinical academics and the institutions willing to be involved in developing improved pathways. A literature review also described the contributions that clinical academics make to the health sector and the challenges faced by this workforce sector. Current training pathways created for clinical academics by Australasian institutions were presented as examples of what can be done. The perspectives of government and research organisations presented at the summit helped define how key stakeholders can contribute. Following the summit, there was a strong commitment to continue to work towards developing a sustainable and defined training pathway for clinical academics. The need for a coordinated and integrated approach was highlighted. Some key objectives were agreed upon for the next phase, including identifying and engaging key advocates within government and leading institutions; publishing and profiling the contributions of successful clinical academics to healthcare outcomes; defining the stages of a clinical academic training pathway; and establishing a mentoring programme for training clinical academics.

DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans.

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