Prophylactic and therapeutic potential of synthetic peptides against enterovirus 71 (EV71)

Ph.DDOCTOR OF PHILOSOPH

Exploring the structural integrity of a picornavirus capsid

Expected release date-May 202

Application of built-in adjuvants for epitope-based vaccines

Several studies have shown that epitope vaccines exhibit substantial advantages over conventional vaccines. However, epitope vaccines are associated with limited immunity, which can be overcome by conjugating antigenic epitopes with built-in adjuvants (e.g., some carrier proteins or new biomaterials) with special properties, including immunologic specificity, good biosecurity and biocompatibility, and the ability to vastly improve the immune response of epitope vaccines. When designing epitope vaccines, the following types of built-in adjuvants are typically considered: (1) pattern recognition receptor ligands (i.e., toll-like receptors); (2) virus-like particle carrier platforms; (3) bacterial toxin proteins; and (4) novel potential delivery systems (e.g., self-assembled peptide nanoparticles, lipid core peptides, and polymeric or inorganic nanoparticles). This review primarily discusses the current and prospective applications of these built-in adjuvants (i.e., biological carriers) to provide some references for the future design of epitope-based vaccines

Development of immunoassays for the investigation of peptide antibody interactions in array format

Antibodies perform a variety of important tasks in the immune system to protect us from diseases. They are trained to specifically target pathogens, which might be toxins, bacteria or viruses. However, they can also harm us by erroneously mark endogenous structures for degradation, which contributes to autoimmune diseases. Studying the immune response by determining, which antibodies are amplified during a successful immune reaction, and which part of the pathogen neutralizing antibodies are binding to, would help to develop new vaccine candidates and contribute to the common knowledge of the underlying mechanism. In addition to the information, what antibodies specifically bind to, it is of interest, in which concentration they are available in the blood and with which affinity they bind their target. Peptide arrays provide an ideal platform for the study of antibody-protein interactions, with the peptides mimicking antibody targets. Using a minute sample volume, several thousand peptides can be examined simultaneously. To study identified antibody-peptide interactions in terms of kinetics, on-line measurements in microfluidic channels, so called continuous flow assays, have to be performed. This work is divided into two main parts, covering the screening for epitopes and the development of an experimental setup to characterize the peptide-epitope interaction. In the first part, the immune response of healthy European individuals towards the tetanus, diphtheria and pertussis vaccination and two established epitopes (PALT(A)xET and PEFxGSxP) originating from enterovirus and probably Staphylococcus aureus, was investigated using peptide arrays. Vaccination specific antibodies were identified by determining their linear epitope in amino acid resolution. Therefore, the linear amino acid sequence of tetanus, diphtheria and pertussis toxin was synthesized in fragments (peptides) in array format onto a solid substrate and incubated with serum of 19 healthy European individuals. For the tetanus toxin, an epitope was identified (929ExxEVIVxK937), which was targeted by 8 out of 19 individuals. The amino acids crucial for antibody binding (the `antibody fingerprint\u27), were determined for this epitope, by substitution analysis. In a substitution, every amino acid is substituted by all of the other 19 amino acids while the rest of the sequence remains conserved. The antibody fingerprint can be determined, since the substitution of crucial amino acids leads to the loss of antibody binding. The `antibody fingerprints\u27 were found to be strongly conserved among individuals for the three investigated linear epitopes (929ExxEVIVxK937, PALT(A)xET and PEFxGSxP). Further, it was further verified by affinity batch chromatography and ELISA, that antibodies binding the identified epitope originating from the sequence of the tetanus toxin, also bind to the native tetanus toxin. This indicates, that the identified epitope is tetanus specific. For the mapping of the diphtheria toxin linear and cyclic peptides of different length were applied. Five prominent regions were identified in the mapping of cyclic peptides using pooled sera. For the pertussis toxin, three regions of interest could be identified, which also were reported in literature for their ability to induce toxin recognizing antibodies in rodents. However, no distinct antibody fingerprints could be determined for a variety of peptides covering the identified regions. Altogether, the homology in between fingerprints of different individuals targeting the same epitope (929ExxEVIVxK937, PALT(A)xET and PEFxGSxP) is striking, given the randomness of antibody formation. In the second part, an experimental setup was realized to incubate peptide arrays in an automated fashion in microfluidic channels to perform continuous flow assays. The interactions of fluorescently labelled antibodies with the peptides can be characterized via the detection of fluorescence using an epi-fluorescence setup design. For this purpose, fluorescence images are taken at short intervals and the increase of the fluorescence signal over time is determined. The fluorescence intensity of the fluorophore DL550 was found to be temperature dependent under assay conditions. However, for a constant temperature (24 +/- 0.1°C), the intensity can be approximated as being stable for 50 min of continuous exposure. For the fast production of peptide arrays needed during preliminary experiments, spotting of pre-synthesized peptides (bearing a C-terminal cysteine) on 3D-Maleimide surfaces was established. To have an additional label free imaging method at hand, VSI was established for the quality control of peptide arrays. Height profiles obtained by VSI did not significantly deviate from AFM measurements. Spots down to approximately 1 nm height can be resolved on a comparable large field of view of (1.74 x 1.31) mm within a minute. Two different approaches for the integration of peptide arrays into a microfluidic channel have been investigated. Molding channel structures in PDMS is a fast technique, but the channel design was determined to be restricted to have a minimum distance between channels to prevent leakage. By adding up to 2% of graphene particles to the PDMS prior to polymerization, the background fluorescence of PDMS was reduced by approximately 70%. The fabricated channel proved to be leak-tight up to a flow of 3 ml/min when screwed to a torque of 10 N/cm. However, the clamping led to a deformation of the PDMS channels. This results in non-homogeneous channel cross sections and is thus not apt for a concentration analysis, which requires a constant channel height. The second type of channel fabrication investigated is adhesive layer bonding using Ordyl SY355. It is the first approach to bond a glass substrate to a functionalized surface, used for micro array fabrication. This method has the potential to make microarrays available to other microsystem technologies. Fluidic in- and outlets are laser drilled into a glass substrate and the adhesive layer is applied and structured lithographically to form the walls of the channel. The parameter window for this lithographic process was determined to be quite broad with exposure doses ranging from (150-220) mJ/cm² and bonding pressures ranging from (220-735) N/cm². Process temperatures over 100°C were found to be critical, since damage of the functionalized surface occurred. The magnitude of fracture strength of the bond was estimated by random pulling tests to be about 305 +/- 50 N/cm². This results in a theoretical pressure resistance of ~ 4.9 bar and proved to be leak proof for a flow rate of up to 750 mL/min. The designed experimental setup, including the integration of peptide arrays into microfluidic channels, was shown to fulfill the requirements for performing continuous flow assays on peptide arrays. By conducting the calibration of fluorescence signal intensity to antibody concentration in a future project, the setup will be ready to further determine antibody titers

An integrated bioinformatics and computational biophysics approach to enterovirus surveillance and research

This PhD thesis examines the integration of complex computational methodologies with the surveillance and research of a genus of viruses implicated in a wide variety of clinical conditions, ranging from asymptomatic infection to death. These viruses, known as the enteroviruses, are some of the most studied viruses in history and as a result are represented by a vast body of literature. The fact that enterovirus research and surveillance rests upon such an extensive foundation of published material, makes enteroviruses a perfect candidate for the experimental application of modern computational methods, or in-silico experimentation. The hypothesis that computational power currently available can be utilised for multiple stages of virus study incorporating identification, epidemiology and atomic structure prediction forms the basis of this thesis. Fundamental to the understanding of virus behaviour is the determination of molecular structure and function, a fact which applies not only to viruses, but to biological entities in general. Extensive work was performed during the course of this thesis in adapting classical molecular dynamics techniques to the large scale simulation of a prototype poliovirus, using millions of simulated atoms. The successful application of these techniques has resulted in microsecond-timescale, atomistic simulations of complete virus particles. These simulations represent the first published instance of the simulation of a biologically complete pathogenic microorganism, incorporating the encoding genetic information. This thesis also examines the use of bioinformatics methods in the development and application of an advanced quantitative multiplex real-time reverse-transcription polymerase chain reaction (qRT-PCR) methodology, for the primary screening of samples from patients suffering acute flaccid paralysis (AFP), which is one of the most debilitating presentations of enterovirus infection. The application of this novel qRT-PCR method reduces the initial screening time of samples derived from a symptomatic patient from 4-5 days using virus culture, to four hours using the novel qRT-PCR. This novel qRT-PCR method can be rapidly scaled-up in response to an outbreak situation. The ability to screen large numbers of samples during an outbreak situation is important and is hampered when using virus culture methods exclusively. In Australia and the Western Pacific region over the last decade, the rate at which non-polio enteroviruses in cases of AFP have been identified, is on average 18%. With the introduction of PCR screening methods, a number of non-cultivable enteroviruses were identified, along with newly described and a previously undescribed enterovirus. Little is known about these newly described and novel enteroviruses. This thesis aimed to investigate the identification of viruses that may represent a significant public health threat and to then use their genetic sequence information to recreate major virus structural components in-silico. This reconstruction process was achieved by exploiting advances in comparative protein modelling and molecular dynamics simulation methods. In order to apply these methods to the reconstruction of previously undescribed viruses for which no structural data exist, validation of different comparative protein modelling techniques was required. The predictive in-silico methods generated reliable atomic coordinates, representing structures suitable for the reconstruction of virus capsid models for further study

Structural and Functional Studies on the Early Steps of Polyomavirus and Adenovirus Life Cycles

Polyoma- and adenoviruses are important human pathogens with worldwide significance. Both are non-enveloped DNA viruses, but differ significantly in terms of how they infect humans: Polyomaviruses (PyVs) cause persistent and occasionally fatal diseases involving multiple organs, while adenoviruses (HAdVs) are ubiquitous among societies worldwide and generally cause self-limiting diseases of mucous epithelial tissues that range from common cold symptoms to gastroenteritis and eye infections. The main focus of this dissertation is placed on the fundamental processes underlying the earliest steps of the viral life cycles of both viruses: cell attachment and entry. Most of the work is dedicated to the unraveling of the attachment and entry strategies of specific strains or serotypes, and a comparison of the factors that govern the differences between them. The cell entry of polyomaviruses is mediated by the interaction of the major capsid protein VP1 with ubiquitous surface glycolipids called gangliosides. I have investigated three well-known strains of the murine polyomavirus (MuPyV) that show remarkable differences in terms of pathogenicity and tissue tropism, but only differ by singular amino acid exchanges located within their ganglioside binding cavities. This work establishes the ganglioside GT1a as a novel functional receptor and discovers minimal changes in the receptor binding affinities among the three strains that presumably lead to the drastically altered in vivo behavior. Unlike PyVs, HAdVs generally employ a two-step mechanism mediated by distinct sets of capsid proteins to enter their target cells. The interaction of the C-terminal knob domain of the so-called fiber with a cellular primary attachment factor selects and tethers the viral particles to the host cell and facilitates cell entry mediated by the interaction of the viral penton base with a secondary entry receptor. These processes are assumed to contribute to the manifestation of viral tropism and host range. Here, the discovery and functional and structural characterization of novel primary attachment factors for two unrelated HAdV types is reported. The first type, HAdV-G52, is a rare and unique serotype that possesses two distinct fibers, which use completely different receptors: the long fiber recognizes the tight junction protein coxsackie- and adenovirus receptor (CAR), while the short fiber shows a specific preference for the glycan polysialic acid using a novel binding site on its fiber knob. The second type, HAdV-D36, is associated with obesity and has the unique ability to infect animals. This work demonstrates that HAdV D36 uses a yet unidentified protein for attachment, and at the same time possesses a specificity for CAR and a sialic acid variant that is presumably only found in animals. Both projects have implications for the infectious routes of the two viruses. An additional, third project presents the purification of the penton base protein of HAdV-D09 with the aim of structurally characterizing the interactions with its receptor counterpart, the integrin αvβ3, and unraveling the factors that dominate later phases of cell entry. The fourth part of this study addresses ways to interfere with adenoviral infections and to use adenoviruses as vectors for highly specific therapeutic applications. To this end, the development and evaluation of a series of second-generation inhibitors for the ocular pathogen HAdV-D37 is reported, whose design was inspired by its natural receptor, the sialic acid-containing glycan GD1a. Furthermore, this work sets the stage for the development of an HAdV-G52-based viral vector for the oncolytic treatment of somatic cancers expressing the tumor antigen polysialic acid. The last part of this dissertation describes ongoing work for the structural characterization of the adenoviral early gene product E4ORF1, a viral powerhouse protein that deregulates cell metabolism and polarity through various host interactions. The findings presented in this dissertation have implications for our general understanding of how the differences among virus entry strategies emerge on a structural level, and provide valuable information for the development of efficient antiviral strategies and safer virus-based drugs

Atherogenesis

This monograph will bring out the state-of-the-art advances in the dynamics of cholesterol transport and will address several important issues that pertain to oxidative stress and inflammation. The book is divided into three major sections. The book will offer insights into the roles of specific cytokines, inflammation, and oxidative stress in atherosclerosis and is intended for new researchers who are curious about atherosclerosis as well as for established senior researchers and clinicians who would be interested in novel findings that may link various aspects of the disease

Molecular Science for Drug Development and Biomedicine

With the avalanche of biological sequences generated in the postgenomic age, molecular science is facing an unprecedented challenge, i.e., how to timely utilize the huge amount of data to benefit human beings. Stimulated by such a challenge, a rapid development has taken place in molecular science, particularly in the areas associated with drug development and biomedicine, both experimental and theoretical. The current thematic issue was launched with the focus on the topic of “Molecular Science for Drug Development and Biomedicine”, in hopes to further stimulate more useful techniques and findings from various approaches of molecular science for drug development and biomedicine

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

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