Discovery of frameshifting in Alphavirus 6K resolves a 20-year enigma.

BACKGROUND: The genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet--a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood. RESULTS: We report that ribosomal -1 frameshifting, with an estimated efficiency of approximately 10-18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; approximately 8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species. CONCLUSION: Our results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms

Subsets of migrating intestinal dendritic cells

Dendritic cells (DCs) in the intestine are heterogeneous. Phenotypically different populations of conventional DCs have been identified in the intestinal lamina propria, Peyer's patches, and in the draining mesenteric lymph nodes, to which these DCs constitutively migrate. Markers used to identify these populations include major histocompatibility complex class II, CD11c, CD8α, CD11b, and CD103. Extensive studies in rats, summarized here, which involved collection of migrating DCs by thoracic duct cannulation after mesenteric lymphadenectomy, have clearly demonstrated that the subsets of migrating intestinal lymph DCs have different functional properties. The subsets might play different roles in the induction of oral tolerance and in driving systemic immune responses after vaccination or intestinal stimulation with Toll-like receptor ligands. The use of these surgical techniques allows investigation of the functions of purified subsets of migrating DCs. However, in the rat, these studies are limited by the range of available reagents and are difficult to compare with data from other species in this fast-moving field. Recent refinements have enabled the collection of migrating intestinal DCs from mice; our initial results are described here. We believe that these studies will generate exciting data and have the potential to resolve important questions about the functions of migrating intestinal DC subsets

Type I IFN controls chikungunya virus via its action on nonhematopoietic cells

Chikungunya virus (CHIKV) is the causative agent of an outbreak that began in La Réunion in 2005 and remains a major public health concern in India, Southeast Asia, and southern Europe. CHIKV is transmitted to humans by mosquitoes and the associated disease is characterized by fever, myalgia, arthralgia, and rash. As viral load in infected patients declines before the appearance of neutralizing antibodies, we studied the role of type I interferon (IFN) in CHIKV pathogenesis. Based on human studies and mouse experimentation, we show that CHIKV does not directly stimulate type I IFN production in immune cells. Instead, infected nonhematopoietic cells sense viral RNA in a Cardif-dependent manner and participate in the control of infection through their production of type I IFNs. Although the Cardif signaling pathway contributes to the immune response, we also find evidence for a MyD88-dependent sensor that is critical for preventing viral dissemination. Moreover, we demonstrate that IFN-α/β receptor (IFNAR) expression is required in the periphery but not on immune cells, as IFNAR−/−→WT bone marrow chimeras are capable of clearing the infection, whereas WT→IFNAR−/− chimeras succumb. This study defines an essential role for type I IFN, produced via cooperation between multiple host sensors and acting directly on nonhematopoietic cells, in the control of CHIKV

Intradermal Electroporation of Naked Replicon RNA Elicits Strong Immune Responses

RNA-based vaccines represent an interesting immunization modality, but suffer from poor stability and a lack of efficient and clinically feasible delivery technologies. This study evaluates the immunogenic potential of naked in vitro transcribed Semliki Forest virus replicon RNA (RREP) delivered intradermally in combination with electroporation. Replicon-immunized mice showed a strong cellular and humoral response, contrary to mice immunized with regular mRNA. RREP-elicited induction of interferon-γ secreting CD8+ T cells and antibody responses were significantly increased by electroporation. CD8+ T cell responses remained substantial five weeks post vaccination, and antigen-specific CD8+ T cells with phenotypic characteristics of both effector and central memory cells were identified. The immune response during the contraction phase was further increased by a booster immunization, and the proportion of effector memory cells increased significantly. These results demonstrate that naked RREP delivered via intradermal electroporation constitute an immunogenic, safe and attractive alternative immunization strategy to DNA-based vaccines

Exploiting the Role of Endogenous Lymphoid-Resident Dendritic Cells in the Priming of NKT Cells and CD8+ T Cells to Dendritic Cell-Based Vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d−/− BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose

Geographical Fire Research in Australia: Review and Prospects

\u27You live in the bush. You live by the rules of the bush, and that\u27s it.\u27 These were the reflective words of Mrs Dunlop upon seeing the blackened rubble of her home, which made headline news the morning after the first, and most destructive, fire front tore through the Blue Mountains in New South Wales on 17 October 2013 (Partridge and Levy, 2013). While seemingly a simple statement, it goes right to the heart of heated public and political debates - past and present - over who belongs where and why in the fire-prone landscapes that surround Australia\u27s cities. Bushfire is a constant and ongoing part of Australian history, ecology and culture. The love of a sunburnt country, the beauty and terror of fire, and the filmy veil of post-fire greenness described in the century-old poem \u27Core of My Heart\u27 (Mackellar, 1908) are still apt depictions of Australian identity today. Yet longer fire seasons and an increase in extreme fire weather days with climate change add both uncertainty and urgency to Australia\u27s ability to coexist with fire in the future (Head et al., 2013)

Intestinal Dendritic Cells in Health and Gut Inflammation

Broad Medical Research Programme, Crohn’s in Childhood Research Association and a MRC Doctoral Training Award

Play for life

The Australian Sports Commission Active After-School Communities program and national ‘Play for Life’ campaign aim to improve children’s health by inspiring a lifelong love of sport, explains Danielle Fleeton

Genetic vaccination against acute viral disease

This thesis describes the development of recombinant vaccines based on the Semliki Forest virus (SFV) expression system. Immunisation of mice with recombinant virus particles, a layered DNA/RNA plasmid vector, and recombinant self-replicating RNA were carried out and the protective effect of these recombinant vaccines against viral challenge were examined. The construction of a full-length infectious clone formed the basis for the SFV expression system which has previously been described. In the expression system, genes coding for the SFV structural proteins are replaced with a multiple cloning site where the gene coding for a foreign antigen can be inserted. Transfection of cells with RNA transcribed in vitro from such vectors results in transient high level production of the antigen followed by death of the cells as a result of the induction of apoptosis. Recombinant virus particles can be made by co-transfecting cells with expression vector RNA and a defective helper RNA coding for the SFV structural proteins. Recombinant SFV (rSFV) particles lack the genes for the structural proteins and thus undergo only a single round of non-productive infection after entry into a cell. Mice immunised with rSFV encoding influenza A virus (FLU) nucleoprotein (NP) or E.coli LacZ protein were shown to develop antigen-specific IgG and CTL responses that persisted for over one year. Humoral and cellular immune responses could be induced in mice following vaccination by peripheral and mucosal routes. Examination of IgG1/IgG2a antibody isotypes indicated that predominantly a T-helper type 1 response was induced. Importantly, this study showed that development of immune responses against the vector itself did not significantly inhibit responses following booster immunisations with rSFV. This is important for recombinant vaccine design as the use of many recombinant virus vaccines is hampered due to pre-existing immunity to the vectors. Expression of envelope proteins, prME, and a nonstructural protein, NS1, of the tick-borne flavivirus, louping ill virus (LIV). from rSFV in cell culture was characterised. both proteins were shown to be correctly processed and secreted from transfected cells. Humoral and T-cell proliferative responses were induced in mice immunised with rSFV particles encoding LIV antigens. Mice vaccinated with rSFV` particles coding for FLU or LIV antigens were protected from challenge with FLU or from challenge with two strains of LIV. the prototype strain LI/31, and LI/l, a naturally occurring antibody escape variant. A layered DNA/RNA vector based on the SFV replicon (pBK-SFV) was developed. A cytomegalovirus (CMV) promoter drives production, not of the antigen-encoding gene, but of the recombinant SFV RNA. In comparison to a conventional DNA vector coding for the same antigen, vaccination of mice with pBK-SFV DNA was shown to elicit stronger humoral and cellular immune responses. Mice vaccinated with pBK-SFV` encoding FLU antigens were shown to be protected against influenza A virus challenge. In order to compare the protective efficacy of rSFV vaccines, a triple challenge model based on three strains of LIV with graded virulence was established. Results from challenge experiments showed that rSFV particles induced better protective immunity than plasmid vaccines (conventional or SFV-based) or the commercially available LIV inactivated vaccine. Antigen-specific humoral and cellular immune responses were also elicited in mice following immunisation with naked rSFV RNA encoding FLU, LIV, or respiratory syncytial virus (RSV) antigens. Mice vaccinated with rSFV RNA were protected from challenge with FLU. LIV or RSV. Studies showed that bone marrow derived dendritic cells (BMDDC) from mice are unable to be productively infected by rSFV particles encoding GFP (green fluorescent protein) as de novo synthesis of GFP could not be detected in these cells. However, RT-PCR analysis of rSFV` infected BMDDC showed that negative strand RNA intermediates of rSFV infection could be detected. indicating that rSFV RNA replication had been initiated in these cells. rnRNA coding for interferon-[alpha]-induced antiviral proteins and the cytokines TNF-[alpha] and IL-12 rnRNA were also upregulated in these cells. This suggests that cytokine signals required for DC maturation and activation were turned on as a result of the initiation of viral RNA replication. Uptake of antigen (GFP) by BMDDC from rSFV infected syngeneic B 16 cells was shown by confocal microscopy. Thus, induction of apoptosis in rSFV transfected cells could facilitate antigen transfer to professional antigen presenting cells for subsequent cross-priming, and might thereby explain the efficient immune responses induced following immunisation with rSFV vaccines

Strength Training to Improve Performance in Athletes with Cerebral Palsy: Effects on Strength, Power, and Body Composition

Despite athletes with cerebral palsy (CP) comprising 20% of all Paralympians, their capacity to improve performance through training remains unknown. Sedentary individuals with CP commonly have low bone mineral density (BMD), increasing fracture risk. Long-term sport participation is suggested to protect against low BMD. Therefore, this thesis investigated the effects of strength training on performance in athletes with CP and the impact of long-term exercise participation on BMD of ambulatory adults with CP. Objectives were investigated through a single group pre-post repeated measures intervention study with waitlist control, and a cross-sectional cohort study. Eleven state to international level athletes with CP (28.2 (9.5) years; m=10, f=1; Gross Motor Function Classification System (GMFCS) I=8, II=3; unilateral=8, bilateral=1, ataxia=2) completed a 12-week supervised maximal strength training intervention. Strength and power were assessed via the isometric mid-thigh pull, unilateral one-repetition maximum chest press, countermovement jump (CMJ), reactive strength index modified (RSImod), and unilateral medicine ball shot-put. Main effects of time were determined via repeated measures ANOVA, with adaptation timeframe determined via planned comparisons between intervention weeks 1-6 and 7-12. Twenty-six adults with CP (30.2 (10.3) years, m=19; f=7; GMFCS I=15, II=9, III=2; unilateral=15, bilateral=6, quadriplegia=1, ataxia=4), including 13 athletes, completed the cross-sectional study. Dual-energy X-ray absorptiometry derived BMD was compared between those completing <150 min activity/ week (Low, n=10), and those who were active and commenced sport after (Post-16, n=6) or before (Pre-16, n=10) age 16. Between group differences were assessed via Kruskal-Wallis one-way analysis of variance due to uneven group sizes. Strength, CMJ height and RSImod improved significantly (13-40%, p<0.001-0.003), with most strength improvements achieved by intervention week 6. Whole body, spine and hip BMD Z-scores were significantly higher in Pre-16 vs. Low group (p<0.001-0.008). Maximal strength training can significantly improve strength and power of athletes with CP such that competition outcomes may be meaningfully affected. Athletes with CP adapt to training in timeframes typical of non-disabled athletes, contrasting with previous findings for sedentary individuals with CP. Long-term exercise commenced before age 16 protects against low BMD in adults with CP. To optimize lifelong health in those with CP, treatments and interventions promoting high-intensity activities producing aerobic and musculoskeletal adaptations should be prioritized from childhood and maintained throughout the lifespan