Generation of antibodies against foot-and-mouth-disease virus capsid protein VP4 using hepatitis B core VLPs as a Scaffold

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the eco-nomically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neu-tralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.</p

Readily available drugs and other interventions to potentially improve the efficacy of immune checkpoint blockade in cancer

To improve the efficacy of immune checkpoint inhibitors (ICIs) for cancer treatment, various strategies, including combination therapies with repurposed drugs, are being explored. Several readily available interventions with potential to enhance programmed death 1 (PD-1) blockade have been identified. However, these interventions often remain overlooked due to the lack of financial incentives for their development, making them financial orphans. This review summarizes current knowledge regarding off-label drugs, supplements, and other readily available interventions that could improve the efficacy of PD-1 blockade. The summary of each intervention includes the proposed mechanism of action for combination with checkpoint inhibitors and data from animal and human studies. Additionally, we include summaries of common interventions to be avoided by patients on PD-1 blockade. Finally, we present approaches for conducting further studies in patients, with the aim of expediting the clinical development of these interventions. We strive to increase awareness of readily available combination therapies that may advance cancer immunotherapy and help patients today

Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication

Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery. IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery

Picornavirus entry: membrane permeability induced by capsid protein VP4

Non-enveloped viruses such as picornaviruses must penetrate the host cell membrane without the advantage of membrane fusion. This process is thought to involve membrane permeabilisation but the mechanism remains poorly understood. During picornavirus cell entry, capsid protein VP4 is released from the virus and is implicated in the delivery of the viral genome into the cytoplasm. The studies described in this thesis were undertaken to improve our understanding of the role of VP4 in cell entry. The approach to investigate this used recombinant VP4 and liposome model membranes. Recombinant VP4 was shown to induce membrane permeability with characteristics similar to that induced by both model pore-forming peptides and infectious virus particles and was influenced by pH, membrane composition and VP4 myristoylation. Chemical crosslinking and dextran release studies demonstrated that VP4 formed a multimeric size-selective membrane pore. The VP4 pore complex was visualised by transmission electron microscopy, which confirmed the multimeric nature of the pore and showed a lumen diameter in agreement with the dextran release studies and consistent with the dimensions required for the passage of viral RNA. The structure of VP4 reconstituted in membrane-mimetic detergent micelles was analysed by circular dichroism spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. This showed VP4 contained predominantly alpha helical content and the acquired 2D NMR spectrum was typical of a membrane protein. The membrane permeability induced by synthetic peptides showed that VP4 activity maps to the N-terminal half of VP4. In addition, VP4 activity was enhanced by the presence of a peptide corresponding to the N-terminus of VP1, an additional capsid protein also implicated in cell entry. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family

Genetically Modified Insects and Disease Control: Report on 10th February 2015 public event

This reports describes an event we organised in February 2015 in central London on the use of genetically modified insects for disease control and summarises the audience responses received. The event provided an opportunity to meet scientists, policymakers and stakeholders involved in this area to discuss the potential risks and benefits of using GM insects as a disease control measure and debate how future policy decisions about it should be made.<br>The questionnaire responses indicated that the event succeeded at:<br>• increasing the audience’s level of understanding of the core technologies which make the production of GM insects possible;<br>• increasing the audience’s level of understanding of how GM insects could help to control animal and human disease;<br>• encouraging participants develop an opinion on whether GM insects should be used to control animal and human diseases;<br>• improving the audience’s understanding of the current UK regulatory system.<br>We also recorded information about the audience’s level of concern about GM and organisations involved in GM as well as their trust in regulatory systems designed to regulate research in GM

VP4-induced permeability is comparable to that of virus.

<p>Carboxyfluorescein-containing liposomes were mixed with VP4His at 5 µM (equivalent to approximately 5 µg/assay) or 1 µg HRV16 (equivalent to 50 ng VP4/assay) and membrane permeability detected by fluorescence measurements recorded every 30 seconds. Assays were conducted at 25°C (<b>A</b>) or 37°C (<b>B</b>). Only a minority proportion of recombinant protein is thought to take part in the reaction. Data is presented as % of total end-point release observed by lysis of liposomes by addition of detergent. Data shown is representative of multiple experiments (n>3).</p

Recombinant VP4 induces dose-dependent membrane permeability.

<p>Liposomes containing carboxyfluorescein (CF) at self-quenching concentration, were mixed with VP4His at the indicated final concentrations. Membrane permeability resulting in leakage and dequenching of CF was detected by fluorescence measurements (excitation 492 nm/emission 512 nm) recorded every 30 seconds. Data shown is representative of multiple experiments (n>3). The end point fluorescent signal induced by 5000 nM VP4His was equivalent to 70–80% of the total release induced by addition of 0.5% Triton X-100.</p

Purity and concentration of recombinant VP4 assessed by SDS-PAGE.

<p>Concentration of purified VP4His estimated by protein assay was confirmed by comparison with known quantities of native VP4 in preparations of purified virus. HRV16 (3 or 1 µg, equivalent to 0.15 or 0.05 µg VP4 respectively) and VP4His at amounts indicated, were subjected to SDS-PAGE and visualized by silver staining (<b>A</b>) or western blot using antisera to VP4 (<b>B</b>). Molecular mass markers (in kilodaltons) are indicated on the left. Arrows show expected position of the indicated viral proteins. The migration of VP4His appears slower with increasing concentration as a result of the increasing concentration of DMSO in these samples. The migration of VP4His was not altered when diluted in a constant concentration of DMSO (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004294#ppat.1004294.s001" target="_blank">figure S1</a>).</p

VP4-induced permeability is size-selective.

<p>Liposomes containing FITC-labelled dextrans of 4 kD (FD4), 10 kD (FD10), 70 kD (FD70) or 250 kD (FD250) were mixed with 5 µM VP4His (<b>A</b>) or 10 µM melittin (<b>B</b>). Release of dextrans was quantified by pelleting the liposomes and measuring the fluorescence in the supernatant. Data is presented as percentage of total release observed by lysis of liposomes by addition of detergent. Error bars represent standard error of the mean (n = 3) and asterisks indicate statistical significance calculated by one way Anova (p*<0.05). Data is representative of multiple independent experiments.</p