Rapid response pipeline for stabilized subunit vaccines

The Coalition for Epidemic Preparedness Innovations (CEPI) have recently put out a call for proposals aimed at platform technologies that can enable rapid vaccine development for novel or previously unrecognized viruses. We have proposed a streamlined process for the generation of stabilized subunit vaccines. This project brings together unique proprietary recombinant technology for generating stabilized subunit vaccines (the molecular clamp), a highly skilled team from some of Australia’s leading scientific organizations and world-class facilities. Molecular clamp is a broadly applicable platform technology that facilitates expression of recombinant viral glycoproteins in subunit form without loss of native antigenicity. The molecular clamp imparts superior stability over alternative trimerization domains, efficiently stabilizing soluble viral fusion proteins in their native trimeric \u27pre-fusion\u27 form. This form is equivalent to that expressed on the virion surface and the principle target for a protective neutralizing antibody response. Through stabilization of the pre-fusion form, the molecular clamp promotes the production of highly neutralizing and broadly cross-reactive antibodies. Importantly, the molecular clamp does not required prior knowledge of a proteins quaternary structure. Please click Additional Files below to see the full abstract

Yangtse River sediments and erosion rates from source to sink traced with cosmogenic 10 Be: Sediments from major rivers

Estimates of regional erosion and sediment mixing from different sources in the Yangtse River system are presented, based on sand samples collected from major tributaries and the trunk stream, at 23 sites between western Sichuan and the Yangtse Delta. Mixing is estimated from concentrations of Mg, Ca, Sr, Ti, Mn and Fe, which are substantially higher in sand from major tributaries in the western Yangtse River catchment than from tributaries in the eastern catchment. Intermediate concentrations occur in sand from the Yangtse Delta, both for modern samples from the surface and for early Holocene samples from drill holes. Mixing ratios indicate that 35 ± 5% of sand in the delta came from eastern sources. A similar result was obtained using cosmogenic 10Be in quartz grains as a tracer of mixing. Regional erosion rate estimated from 10Be in sand grains from high mountain catchments of the western Yangtse River are mostly similar to rates based on sediment gauging but are sometimes higher, and range to over 700 m Ma- 1, while 10Be measured at upper Yangtse River tributaries on the northeast Tibetan plateau gave rates of 20-30 m Ma- 1. For the eastern catchments, 10Be measurements from quartz sand and sediment gauging both gave rates of 30-70 m Ma- 1. Eroding at this rate, the eastern catchments could not supply more than 20% of the sediment in the delta, in contrast with 35% estimated from geochemical fingerprints. The relative input from eastern sources may have been higher in Late Pleistocene times, under a different climatic regime, and reworking of Pleistocene deposits may still be in progress

A coupled drug kinetics-cell cycle model to analyse the response of human cells to intervention by topotecan

A model describing the response of the growth of single human cells in the absence and presence of the anti-cancer agent topotecan (TPT) is presented. The model includes a novel coupling of both the kinetics of TPT and cell cycle responses to the agent. By linking the models in this way, rather than using separate (disjoint) approaches, it is possible to illustrate how the drug perturbs the cell cycle. The model is compared to experimental in vitro cell cycle response data (comprising single cell descriptors for molecular and behavioural events), showing good qualitative agreement for a range of TPT dose levels

Structurally confined influenza subunit vaccines in the prefusion conformation elicit a potent neutralizing antibody response

Effective vaccination against influenza viruses remains a significant global challenge. Despite ongoing efforts, continual antigenic changes in circulating viruses requires constant update of existing vaccine approaches. Furthermore, the majority of current licensed vaccines are derivatives of live virus and are inherently time consuming to produce and limit the potential response time to counter a new virus strain. However, the combined advances in subunit vaccine production and structural determination of critical neutralizing epitopes within influenza hemagglutinin (HA) provide the groundwork for the next generation of influenza vaccines which have the potential to overcome these limitations. In an effort to expand on these findings we have compared the effectiveness of both prefusion and postfusion forms of recombinant influenza hemagglutinin (rHA) as subunit vaccines. Using a novel stabilization tag to confine rHA in the prefusion conformation we demonstrated that while both HA conformations elicit anti-HA responses in mice, a neutralizing response (PRNT50 1:36000) is only observed for prefusion rHA. Using rHAs from a range of influenza subtypes and domain specific constructs together with a large panel of structurally defined antibodies we also examined the epitope specificity and cross-reactivity of the prefusion specific neutralizing response. Interestingly, a similar conformation dependence has been reported for respiratory syncytial virus1, 2, suggesting a universal strategy for the generation of potent subunit vaccines to target enveloped viruses. 1. Magro, M. et al. Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention. Proc Natl Acad Sci U S A 109, 3089-3094 (2012). 2. McLellan, J.S. et al. Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus. Science 342, 592-598 (2013)

A pre-fusion, trimeric subunit influenza HA-based vaccine elicits cross-protection between highly divergent influenza A viruses

Despite our best efforts to vaccinate against influenza viruses they remain a major cause of morbidity and mortality worldwide, resulting in 3-5 million severe infections and more than 250,000 deaths annually. Constant antigenic changes in circulating viruses means current vaccines must be updated and re-administered annually. This approach is time-consuming and expensive, and is often hindered by mismatches between circulating and vaccine strains. Strain mismatch can contribute to insufficient vaccine efficacy, which has ranged from just 10-60% over the last decade. Furthermore, recent sporadic zoonotic outbreaks of novel highly pathogenic viruses from avian species, to which current vaccines provide no immunity, have been observed, with fatality rates around 40%. This raises serious concerns of a global pandemic with the potential to spread rapidly before a vaccine can be manufactured. Novel approaches to influenza vaccination are clearly needed in order to overcome these limitations with “universal” flu vaccines being the holy grail. We have stabilized recombinant influenza haemagglutinin (rHA) in its native, pre-fusion conformation by the addition of a novel “clamp” stabilization motif to enhance subunit vaccine potency and breadth of protection. Immunisation of mice with clamp-stabilized prefusion rHA elicited a potent neutralizing antibody response (~4-fold improvement over current vaccines). Most importantly, antibodies elicited upon immunisation with clamp-stabilised prefusion rHA showed an 80-fold increase in cross-reactivity to rHA derived from a divergent, highly pathogenic avian virus (H5N1) when compared to the current influenza vaccines. We have also shown that vaccination with clamp-stabilisted rHA based on the H3 subtype (group 2) is capable of providing cross-protection to a challenge with a highly-divergent group 1 virus (H1N1). Ultimately, this approach could represent a potential universal influenza vaccine, providing enhanced cross-protection against both group 1 and 2 seasonal influenza virus strains while simultaneously providing an increased cross-reactive humoral immune response to potential zoonotic pandemic strains. Please click Additional Files below to see the full abstract

Geographic patterns of koala retrovirus genetic diversity, endogenization, and subtype distributions

Koala retrovirus (KoRV) subtype A (KoRV-A) is currently in transition from exogenous virus to endogenous viral element, providing an ideal system to elucidate retroviral–host coevolution. We characterized KoRV geography using fecal DNA from 192 samples across 20 populations throughout the koala’s range. We reveal an abrupt change in KoRV genetics and incidence at the Victoria/New South Wales state border. In northern koalas, pol gene copies were ubiquitously present at above five per cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers below one, while the env gene was detected in all animals and in a majority at copy numbers above one per cell. These results suggest that southern koalas carry partial endogenous KoRV-like sequences. Deep sequencing of the env hypervariable region revealed three putatively endogenous KoRV-A sequences in northern koalas and a single, distinct sequence present in all southern koalas. Among northern populations, env sequence diversity decreased with distance from the equator, suggesting infectious KoRV-A invaded the koala genome in northern Australia and then spread south. The exogenous KoRV subtypes (B to K), two novel subtypes, and intermediate subtypes were detected in all northern koala populations but were strikingly absent from all southern animals tested. Apart from KoRV subtype D, these exogenous subtypes were generally locally prevalent but geographically restricted, producing KoRV genetic differentiation among northern populations. This suggests that sporadic evolution and local transmission of the exogenous subtypes have occurred within northern Australia, but this has not extended into animals within southern Australia

Eroding Australia: rates and processes from Bega Valley to Arnhem Land

Abstract: We report erosion rates determined from in situ produced cosmogenic 10 Be across a spectrum of Australian climatic zones, from the soil-mantled SE Australian escarpment through semi-arid bedrock ranges of southern and central Australia, to soil-mantled ridges at a monsoonal tropical site near the Arnhem escarpment. Climate has a major effect on the balance between erosion and transport and also on erosion rate: the highest rates, averaging 35 m Ma 21 , were from soil-mantled, transport-limited spurs in the humid temperate region around the base of the SE escarpment; the lowest, averaging about 1.5 m Ma 21 , were from the steep, weatheringlimited, rocky slopes of Kings Canyon and Mt Sonder in semi-arid central Australia. Between these extremes, other factors come into play including rock-type, slope, and recruitment of vegetation. We measured intermediate average erosion rates from rocky slopes in the semi-arid Flinders and MacDonnell ranges, and from soil-mantled sites at both semi-arid Tyler Pass in central Australia and the tropical monsoonal site. At soil-mantled sites in both the SE and tropical north, soil production generally declines exponentially with increasing soil thickness, although at the tropical site this relationship does not persist under thin soil thicknesses and the relationship here is 'humped'. Results from Tyler Pass show uniform soil thicknesses and soil production rates of about 6.5 m Ma 21 , supporting a longstanding hypothesis that equilibrium, soil-mantled hillslopes erode in concert with stream incision and form convex-up spurs of constant curvature. Moreover, weathering-limited slopes and spurs also occur in the same region: the average erosion rate for rocky sandstone spurs at Glen Helen is 7 m Ma 21 , similar to the Tyler Pass soil-mantled slopes, whereas the average rate for high, quartzite spurs at Mount Sonder is 1.8 m Ma 21 . The extremely low rates measured across bedrock-dominated landscapes suggest that the ridge-valley topography observed today is likely to have been shaped as long ago as the Late Miocene. These rates and processes quantified across different, undisturbed landscapes provide critical data for landscape evolution models

A stabilized subunit vaccine for ebola virus

The ongoing Ebola epidemic in West Africa has claimed over eleven thousand lives and has highlighted our unpreparedness to counter emerging viral epidemics. While two recombinant vaccines have shown promising results in clinical trials, we have developed an alternate subunit vaccine candidate that could be called upon in the event that problems are encountered with regard to safety or protection efficacy. Our subunit vaccine candidate is based on a soluble version of the recombinant Ebola glycoprotein (GP) stabilized in its pre-fusion conformation. This protein is recognized by the neutralizing monoclonal antibody KZ52 and all three ZMapp antibodies (currently employed as a therapeutic for clinical treatment), indicating both GP1/2 and glycan cap domains are available and are presented in the desired conformation. Immunization via NanopatchTM (NP) microneedle delivery and intradermal injection were compared in C57 black mice. We assessed the antibody response elicited in immunized mice against Ebola virus (Zaire strain) using facilities at CSIRO’s Australian Animal Health Laboratories in Geelong (AAHL). Promising plaque reduction neutralization titers (PRNT50 = 1/80 sera dilution) were demonstrated. Furthermore, we have shown this vaccine is thermostable, retaining significant antigenicity after extended incubation at 37°C, indicating this vaccine strategy may not require cold chain delivery. In addition, the absence of any replicative elements ensures that it is likely to have a safer profile than live recombinant vaccines

Reversing hydrology:quantifying the temporal aggregation effect of catchment rainfall estimation using sub-hourly data

Inferred rainfall sequences generated by a novel method of inverting a continuous time transfer function show a smoothed profile when compared to the observed rainfall however streamflow generated using the inferred rainfall is almost identical to that generated using the observed rainfall (Rt2 = 95%). This paper compares the inferred effective and observed effective rainfall in both time and frequency domains in order to confirm that, by using the dominant catchment dynamics in the inversion process, the main characteristics of catchment rainfall are being captured by the inferred effective rainfall estimates. Estimates of the resolution of the inferred effective rainfall are found in the time domain by comparison with aggregated sequences of observed effective rainfall, and in the frequency domain by comparing the amplitude spectra of observed and inferred effective rainfall. The resolution of the rainfall estimates is affected by the slow time constant of the catchment and the rainfall regime, but also by the goodness-of-fit of the model, which incorporates the amount of other disturbances in the data