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Current approaches in the development of vaccines against infectious viral diseases
Vargová, Soňa ; Malý, Petr (advisor) ; Osička, Radim (referee)
Vaccination remains one of the most successful biomedical interventions for preventing viral diseases. While early vaccines were developed by attenuating the infectious agent in cell cultures or by inactivation, new delivery platforms are on the rise thanks to the advent of genetic engineering. The COVID-19 pandemic stimulated the rapid adoption and a massive deployment of these platforms. Viral vector vaccines elicit antigen expression within cells and induce a robust cytotoxic T cell response, unlike protein subunit vaccines conferring mainly humoral immunity. mRNA vaccines also deliver the antigen inside the cells while offering more manageable and faster manufacturing possibilities. Unlike DNA-based vaccines, mRNA does not enter the nucleus, and thus, the probability of disrupting gene expression in the recipient cell is diminished. This thesis aims to offer an overview of current approaches in vaccinology and discuss the various platforms in use. The thesis will also present recent advances in the development of prophylactic vaccines against infections with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) and also will focus on a recently proposed strategy for vaccine development based on non-cognate ligands mimicking epitopes recognised by broadly neutralising antibodies...
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Novel binding proteins derived from small protein domains targeting diagnostically important molecules
Vaňková, Lucie ; Malý, Petr (advisor) ; Brynda, Jiří (referee) ; Trefil, Pavel (referee)
The rapid development of the gene engineering techniques, especially methods for in vitro directed evolution and combinatorial mutagenesis, has triggered the generation of new binding agents to almost any antigen of interest as an alternative to broadly used antibodies. These so-called non-Ig scaffolds are often derived from proteins with useful biophysical properties. While the therapeutic market is still dominated by monoclonal antibodies, the easy option of desired customization of non-Ig binders by conventional methods of gene engineering predestine them largely for the use in the diagnostic area. The ABD scaffold, derived from a three-helix bundle of albumin-binding domain of streptococcal protein G, represents one of the small non-Ig scaffolds. In our laboratory, we have established a highly complex combinatorial library developed on the ABD scaffold. This ABD scaffold-derived library was used to generate unique binders of human prostate cancer (PCa) biomarkers PSP94, KLK2, KLK11 for the more precise diagnosis of PCa. The second part of the thesis describes the generation of ABD-derived binders selectively recognizing different phenotypes of circulating tumor cells as a binding component of the cell capture zone of microfluidic chip for lung adenocarcinoma diagnosis. Beside this already...
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Preparation and Characterization of Protein Binders Mimicking Epitopes of HIV-1 Neutralizing Antibodies
Šulc, Josef ; Malý, Petr (advisor) ; Novotný, Marian (referee)
For three decades, the ongoing HIV pandemic has taken the lives of tens of millions of people. Still, more tens of millions are fighting this incurable disease today. Current failures in combating this global problem are caused mainly by the virus's extreme ability of mutation, its very effective molecular shield which repels the immune system's attacks, and its immense variability. A breakthrough, achieved relatively recently, is the discovery of the so-called broadly neutralizing antibodies against HIV-1, which carry a very efficient and broad neutralizing response. So far, it's not known how to elucidate the production of these antibodies in the infected hosts to quell or altogether eliminate the virus. This work deals with experimental results, which led to both in vivo and in vitro proof-of-concept of the so-called protein mimetics, the ability to imitate viral surface epitopes, and therefore stimulate an efficient immune response carried by targeted broadly neutralizing antibodies. This effect is mediated by recombinant binding proteins, based on the Myomedin scaffold. This work describes the selection and characterization of these binding proteins mimicking the epitopes of one of the most effective broadly neutralizing antibodies, 10E8. It shows that the binding affinities of selected...
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Novel binding proteins derived from small protein domains targeting diagnostically important molecules
Vaňková, Lucie ; Malý, Petr (advisor) ; Brynda, Jiří (referee) ; Trefil, Pavel (referee)
The rapid development of the gene engineering techniques, especially methods for in vitro directed evolution and combinatorial mutagenesis, has triggered the generation of new binding agents to almost any antigen of interest as an alternative to broadly used antibodies. These so-called non-Ig scaffolds are often derived from proteins with useful biophysical properties. While the therapeutic market is still dominated by monoclonal antibodies, the easy option of desired customization of non-Ig binders by conventional methods of gene engineering predestine them largely for the use in the diagnostic area. The ABD scaffold, derived from a three-helix bundle of albumin-binding domain of streptococcal protein G, represents one of the small non-Ig scaffolds. In our laboratory, we have established a highly complex combinatorial library developed on the ABD scaffold. This ABD scaffold-derived library was used to generate unique binders of human prostate cancer (PCa) biomarkers PSP94, KLK2, KLK11 for the more precise diagnosis of PCa. The second part of the thesis describes the generation of ABD-derived binders selectively recognizing different phenotypes of circulating tumor cells as a binding component of the cell capture zone of microfluidic chip for lung adenocarcinoma diagnosis. Beside this already...
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Protein domains utilizable for development of binding molecules
Dobešová, Petra ; Malý, Petr (advisor) ; Veverka, Václav (referee)
Small protein domains represent basic building blocks of naturally occurring proteins. Many of them exhibit excellent stability, lack disulfide bonds and their structures, therefore, represent attractive tools for generation of artificial binding molecules. First step in the production of novel binding proteins is the definition of a basic domain structure, called "scaffold", which is identified using in silico approaches, resulting in discovery of mutable amino acid residues. Then, randomization of such residues leads to design of a highly complex combinatorial library as a key tool for targeted selection of protein variants. Based on chosen selection approach, the particular protein variants can be tested for their ability to recognize the target molecule with high specificity and binding affinity. Small binding proteins lack post-translation modifications, exhibit thermal stability, are resistant to many organic solvents and can be produced on a mass scale in bacterial host cells. In addition, they can be easily modified and used in vivo with excellent tissue penetration. Due to these beneficial properties, small artificial binding proteins are extraordinary useful biotechnological tools and represent a promising alternative to monoclonal antibodies. The aim of this work is to summarize our knowledge on...
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