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

Involvement of p38 MAPK in Synaptic Function and Dysfunction

Many studies have revealed a central role of p38 MAPK in neuronal plasticity and the regulation of long-term changes in synaptic efficacy, such as long-term potentiation (LTP) and long-term depression (LTD). However, p38 MAPK is classically known as a responsive element to stress stimuli, including neuroinflammation. Specific to the pathophysiology of Alzheimer's disease (AD), several studies have shown that the p38 MAPK cascade is activated either in response to the Aβ peptide or in the presence of tauopathies. Here, we describe the role of p38 MAPK in the regulation of synaptic plasticity and its implication in an animal model of neurodegeneration. In particular, recent evidence suggests the p38 MAPK α isoform as a potential neurotherapeutic target, and specific inhibitors have been developed and have proven to be effective in ameliorating synaptic and memory deficits in AD mouse models

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

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

The heptad repeat C domain of the respiratory syncytial virus fusion protein plays a key role in membrane fusion

Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. RSV F comprises a trimer of disulfide bonded F1 and F2 subunits that is present on the virion surface in a 'metastable' pre-fusion state. This pre-fusion form is readily triggered to undergo refolding to bring two heptad repeats (HRA and HRB) into close proximity to form a six-helix bundle that stabilizes the post-fusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75-97), an amphipathic α-helix that lies at the interface of the pre-fusion F trimer and is a major structural feature of the F2 subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Herein we propose a model in which HRC plays a stabilizing role within the globular head for the pre-fusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the post-fusion state.IMPORTANCERSV is recognized as the most important viral pathogen amongst pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of pre-fusion stabilized F protein based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here we present a comprehensive mutagenesis based study of a region of the RSV F2 subunit (amino acids 75 - 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the pre-fusion form

IBM, Elsevier Science, and Academic Freedom

Elsevier Science refused to publish a study of IBM workers that IBM sought to keep from public view. Occupational and environmental health (OEH) suffers from the absence of a level playing field on which science can thrive. Industry pays for a substantial portion of OEH research. Studies done by private consulting firms or academic institutions may be published if the results suit the sponsoring companies, or they may be censored. OEH journals often reflect the dominance of industry influence on research in the papers they publish, sometimes withdrawing or modifying papers in line with industry and advertising agendas. Although such practices are widely recognized, no fundamental change is supported by government and industry or by professional organizations

Dengue virus NS1 protein activates immune cells via TLR4 but not TLR2 or TLR6

The secreted hexameric form of the dengue virus (DENV) non-structural protein 1 (NS1) has recently been shown to elicit inflammatory cytokine release and disrupt endothelial cell monolayer integrity. This suggests that circulating NS1 contributes to the vascular leak that plays a major role in the pathology of dengue haemorrhagic fever and shock. Pathways activated by NS1 are thus of great interest as potential therapeutic targets. Recent works have separately implicated both toll-like receptor 4 (TLR4) and the TLR2/6 heterodimer in immune cell activation by NS1. Here we have used mouse gene knockout macrophages and antibodies blocking TLR function in human peripheral blood mononuclear cells to show that recombinant NS1, expressed and purified from eukaryotic cells, induces cytokine production via TLR4 but not TLR2/6. Furthermore, the commercial Escherichia coli-derived recombinant NS1 preparation used in other work to implicate TLR2/6 in the response is not correctly folded and appears to be contaminated by several microbial TLR ligands. Thus TLR4 remains a therapeutic target for DENV infections, with TLR4 antagonists holding promise for the treatment of dengue disease

First-in-Human Studies of MW01-6-189WH, a Brain-Penetrant, Antineuroinflammatory Small-Molecule Drug Candidate: Phase 1 Safety, Tolerability, Pharmacokinetic, and Pharmacodynamic Studies in Healthy Adult Volunteers

MW01-6-189WH (MW189) is a novel central nervous system-penetrant small-molecule drug candidate that selectively attenuates stressor-induced proinflammatory cytokine overproduction and is efficacious in intracerebral hemorrhage and traumatic brain injury animal models. We report first-in-human, randomized, double-blind, placebo-controlled phase 1 studies to evaluate the safety, tolerability, and pharmacokinetics (PK) of single and multiple ascending intravenous doses of MW189 in healthy adult volunteers. MW189 was safe and well tolerated in single and multiple doses up to 0.25 mg/kg, with no clinically significant concerns. The most common drug-related treatment-emergent adverse event was infusion-site reactions, likely related to drug solution acidity. No clinically concerning changes were seen in vital signs, electrocardiograms, physical or neurological examinations, or safety laboratory results. PK analysis showed dose-proportional increases in plasma concentrations of MW189 after single or multiple doses, with approximately linear kinetics and no significant drug accumulation. Steady state was achieved by dose 3 for all dosing cohorts. A pilot pharmacodynamic study administering low-dose endotoxin to induce a systemic inflammatory response was done to evaluate the effects of a single intravenous dose of MW189 on plasma cytokine levels. MW189 treatment resulted in lower levels of the proinflammatory cytokine TNF-α and higher levels of the anti-inflammatory cytokine IL-10 compared with placebo treatment. The outcomes are consistent with the pharmacological mechanism of MW189. Overall, the safety profile, PK properties, and pharmacodynamic effect support further development of MW189 for patients with acute brain injury

Evidence for Pervasive Adaptive Protein Evolution in Wild Mice

The relative contributions of neutral and adaptive substitutions to molecular evolution has been one of the most controversial issues in evolutionary biology for more than 40 years. The analysis of within-species nucleotide polymorphism and between-species divergence data supports a widespread role for adaptive protein evolution in certain taxa. For example, estimates of the proportion of adaptive amino acid substitutions (alpha) are 50% or more in enteric bacteria and Drosophila. In contrast, recent estimates of alpha for hominids have been at most 13%. Here, we estimate alpha for protein sequences of murid rodents based on nucleotide polymorphism data from multiple genes in a population of the house mouse subspecies Mus musculus castaneus, which inhabits the ancestral range of the Mus species complex and nucleotide divergence between M. m. castaneus and M. famulus or the rat. We estimate that 57% of amino acid substitutions in murids have been driven by positive selection. Hominids, therefore, are exceptional in having low apparent levels of adaptive protein evolution. The high frequency of adaptive amino acid substitutions in wild mice is consistent with their large effective population size, leading to effective natural selection at the molecular level. Effective natural selection also manifests itself as a paucity of effectively neutral nonsynonymous mutations in M. m. castaneus compared to humans