Avian influenza

Non peer reviewe

The design, development and characterization of a self-replicating DNA expression technology

High quality T-cell immunogenicity can be an elusive type of immunity to generate and one that is often sought after by virologists, immunologists and cancer researchers alike. When T-cell immunity is generated using current methodologies the quality and magnitude of the immunological response achieved is often weak and unable to create protective immunity. Among current methods, DNA vaccines, generate highly specific T-cell immunity towards targeted antigens, and do not suffer from issues like misdirected vector targeted immunity, like viral based vectors. DNA vaccines, however, face a variety of their own weaknesses. These include, inefficient delivery, high biological loss inside the body, and the inability to counteract or avoid immediate innate cellular defence mechanisms, which limit their ability to persist inside a host cell. For these reasons, DNA vaccines are usually combined with more conventional viral vaccines in what is known as a DNA prime and viral boost regiment strategy. Combining them works well and results in improved immunity towards targeted antigens that is superior to what is obtained when either DNA or recombinant vaccines are used alone. To address many of the core issues faced by DNA vaccines, I report here on the design, development and characterization of a self-replication DNA gene expression technology. This novel DNA expression system employs a form of DNA replication (known as rolling circle replication) to generate a self-replicating DNA amplicon that can amplify its own copy number and the relative localised levels of antigen expression inside transfected mammalian cells in tissue culture and within Balb/cJ mice. These capabilities help effectively mitigate many of the core issues faced by DNA vaccines. The technology developed was shown to significantly increase gene expression for eGFP and Luciferase reporter genes, with an overall average increase in expression of approximately two-fold by 48 h post transfection in HeLa S3 cells. More specifically, an increase of at least two-fold in the absolute maximum level of the gene of interest per cell was also observed. Such localised doubling in antigen expression, at the cellular level, is believed to enhance innate immune activation and improve the overall immune response. Experimental results indicated that gene expression levels by this technology is non static in nature and appears to increase in magnitude within affected cells over time as was hypothesised. This provided strong evidence that the replication technology appears to be functioning as was expected and was able to demonstrate the ability to elevate antigen expression over time, potentially starting from extremely low and otherwise ineffective starting concentrations. This ability has potential to effectively mitigate many of the issues associated DNA vaccines such as low and ineffective delivery. This capability was observed in tissue culture as a steady increase in reporter gene expression levels across the entire range of DNA transfection levels. Furthermore, the increases in gene expression were observed to continue to amplify over time, eliminating the presence of weakly fluorescing cells in tissue culture. By 11 days post transfection, every observable cell transfected with the replication expression system, was observed to have extremely high levels of fluorescence. With recorded fluorescence levels being as bright or brighter than the highest levels obtained under normal transfections with no replicative plasmids (~48-72 h). Unique cellular responses to the presence of the replicating gene expression technology were also observed. These included an apparent slowdown in cellular metabolic activity and growth among cells transfected with replicating vectors. This was observed as a decrease in cellular division and total cell number by ~50%, by 48 h post transfection. This was accompanied by significant increases in cell size, internal cellular granularity, and gene-of-interest expression per cell. These changes were observed among all cells regardless of their relative DNA transfection level. This was demonstrated by assessment of the change in the range, mean, median, skewness and standard deviation of the cellular distribution curves for eGFP expression, cell size and internal cellular granularity. These observations provided further evidence of the dynamically changing and active nature of this technology. This also provided evidence that the replicating gene expression technology has a definitively different kind of cellular impact and effect on transfected cells compared to non-replicating DNA expression systems. Pilot studies to test the technology in Balb/cJ mice indicated, the technology appears to be functional within this animal model and was able to increase gene of interest (eGFP) expression levels compared to an equivalent non-replicating DNA expression vector control. Furthermore, these animal experiments also demonstrated significant increases in the maximum possible level of expression achieved within localised ‘hot spots' of muscle fibre bundles. This effect appeared to increase following transient addition of additional replication associated protein (Rep), giving further evidence this technology appears to be functional within the Balb/cJ animal model. Suggesting that the rate at which the replication amplification process occurs, may also be manipulated by adjusting Rep concentration. Finally, an antiviral response gene array was run to look for evidence that the replicating gene expression technology could increases antiviral response gene activation, to possibly improve T-cell activation and immunity. The array provided evidence improved antiviral response gene activation was occurring however the data was inconclusive in nature and further investigation is needed to verify these preliminary findings. The array also showed significant evidence of Rep induced Caspase 10 (CASP10), gene suppression. This suggests that Rep may play a role in the survival and virulence ofBFDV by acting as a suppressor of cellular apoptosis in a concentration-dependant manner and is worth investigating further

The Development and application of pseudovirus based cell entry assays for emerging bat viruses

Recent years have seen the emergence of two serious coronavirus pathogens with the emergence of the MERS-CoV still an ongoing concern. In addition, the recent unprecedented Ebola outbreak has claimed more than 10,000 lives and affected the lives of countless more. All three of these viruses have been linked through differing strands of research to the second largest mammalian family, Chiroptera, the bats. Bats are among the most diverse a widespread of all mammalian species and have become subject of intensive research in recent years as various bats species have been linked to a number of severe viral outbreaks. In the studies described in this thesis attempts were made to develop, pseudotyped viruses (PV) bearing the glycoprotein of a number of highly pathogenic viruses including MERS-CoV, Ebolavirus, Marburgvirus and SARS-CoV coupled with envelope-defective Human Immunodeficiency Virus and envelope-defective Murine Leukemia Virus. These tools were then used to examine the potential for cross reactivity among related coronaviruses and a number of computational tools were employed to investigate the phenomenon and attempt to develop a better understanding of the antigenic regions that are responsible for the observed cross reactivity. The next stage in the thesis involved attempts to develop a novel form of multiplex assay that made use of PV to attempt to make serological screening of bat specimens more feasible and efficient. The novel bat-borne influenza A haemagglutinin H17 was then successfully incorporated into the PV system and screening of this PV against a number of cell lines led to improved understanding of the viral tropism and the role protease plays in this tropism. The final set of experiments carried out in this thesis involved a combination of computational biology and PV based protocols to both predict patterns of viral evolution through selection analysis and to then test these predictions in the PV framework. This study lead to the generation of a number of mutant MERS-CoV spike proteins and Ebolavirus glycoproteins. These were then incorporated into the PV system and the effects of these mutations of PV production and serum neutralization were investigated

Medical Microbiology, Virology & Immunology

УЧЕБНЫЕ ПОСОБИЯМИКРОБИОЛОГИЯВИРУСОЛОГИЯИММУНОЛОГИЯАЛЛЕРГОЛОГИЯ И ИММУНОЛОГИЯВ пособие включены разделы по общей микробиологии и медицинской иммунологии

An in-silico study: Investigating small molecule modulators of bio-molecular interactions

Small molecule inhibitors are commonly used to target protein targets that assist in the spread of diseases such as AIDS, cancer and deadly forms of influenza. Despite drug companies spending millions on R&D, the number of drugs that pass clinical trials is limited due to difficulties in engineering optimal non-covalent interactions. As many protein targets have the ability to rapidly evolve resistance, there is an urgent need for methods that rapidly identify effective new compounds. The thermodynamic driving force behind most biochemical reactions is known as the Gibbs free energy and it contains opposing dynamic and structural components that are known as the entropy (ΔS°) and enthalpy (ΔH°) respectively. ΔG° = ΔH° - TΔS°. Traditionally, drug design focussed on complementing the shape of an inhibitor to the binding cavity to optimise ΔG° favourability. However, this approach neglects the entropic contribution and phenomena such as Entropy-Enthalpy Compensation (EEC) often result in favourable bonding interactions not improving ΔG°, due to entropic unfavorability. Similarly, attempts to optimise inhibitor entropy can also have unpredictable results. Experimental methods such as ITC report on global thermodynamics, but have difficulties identifying the underlying molecular rationale for measured values. However, computational techniques do not suffer from the same limitations. MUP-I can promiscuously bind panels of hydrophobic ligands that possess incremental structural differences. Thus, small perturbations to the system can be studied through various in silico approaches. This work analyses the trends exhibited across these panels by examining the dynamic component via the calculation of per-unit entropies of protein, ligand and solvent. Two new methods were developed to assess the translational and rotational contributions to TΔS°, and a protocol created to study ligand internalisation. Synthesising this information with structural data obtained from spatial data on the binding cavity, intermolecular contacts and H-bond analysis allowed detailed molecular rationale for the global thermodynamic signatures to be derived

Molecular Dynamics for Synthetic Biology

Synthetic biology is the field concerned with the design, engineering, and construction of organisms and biomolecules. Biomolecules such as proteins are nature's nano-bots, and provide both a shortcut to the construction of nano-scale tools and insight into the design of abiotic nanotechnology. A fundamental technique in protein engineering is protein fusion, the concatenation of two proteins so that they form domains of a new protein. The resulting fusion protein generally retains both functions, especially when a linker sequence is introduced between the two domains to allow them to fold independently. Fusion proteins can have features absent from all of their components; for example, FRET biosensors are fusion proteins of two fluorescent proteins with a binding domain. When the binding domain forms a complex with a ligand, its dynamics translate the concentration of the ligand to the ratio of fluorescence intensities via FRET. Despite these successes, protein engineering remains laborious and expensive. Computer modelling has the potential to improve the situation by enabling some design work to occur virtually. Synthetic biologists commonly use fast, heuristic structure prediction tools like ROSETTA, I-TASSER and FoldX, despite their inaccuracy. By contrast, molecular dynamics with modern force fields has proven itself accurate, but sampling sufficiently to solve problems accurately and quickly enough to be relevant to experimenters remains challenging. In this thesis, I introduce molecular dynamics to a structural biology audience, and discuss the challenges and theory behind the technique. With this knowledge, I introduce synthetic biology through a review of fluorescent sensors. I then develop a simple computational tool, Rangefinder, for the design of one variety of these sensors, and demonstrate its ability to predict sensor performance experimentally. I demonstrate the importance of the choice of linker with yet another sensor whose performance depends critically thereon. In chapter 6, I investigate the structure of a conserved, repeating linker sequence connecting two domains of the malaria circumsporozoite protein. Finally, I develop a multi-scale enhanced sampling molecular dynamics approach to predicting the structure and dynamics of fusion proteins. It is my hope that this work contributes to the structural biology community's understanding of molecular dynamics and inspires new techniques developed for protein engineering

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

Pharmacokinetics of Extended vs Intermittent Intravenous Infusion of Meropenem in Critically Ill Patients Receiving Continuous Veno-Venous Haemofiltration

peer reviewe