In the post smallpox era, vaccinia virus (VACV) has emerged as an important candidate vaccine vector. As yet, the binding receptors and entry mechanisms utilised by the two infectious forms, IMV and EEV, in dendritic cells (DCs) are unknown. We have investigated the interactions between VACV and C-type lectin receptors (CLRs) that are known to be utilised by many other viruses for binding and entry in DCs. Using a variety of CLR ligands and inhibitors we were unable to inhibit IMV or EEV binding to MDDCs and we conclude that they do not bind to CLRs. We have also investigated VACV entry in MDDCs and show that both IMV and EEV enter MDDCs via an endocytic pathway. Using a variety of drugs that inhibit cellular processes we found IMV and EEV entry to be actin- and calcium-dependent. EEV entry was also cholesterol- and energy-dependent, whereas IMV entry was only partially dependent on these factors. Both IMV and EEV colocalised with endolysosomal markers. This data suggests that EEV may enter DCs via caveolin-mediated endocytosis whereas IMV entry can occur via multiple complementary mechanisms, including endocytosis and fusion. Macropinocytosis may also constitute a minor route of entry for IMV as entry was partially inhibited by dimethyl amiloride and the virus colocalised with dextran. Finally we have provided a comprehensive flow cytometric analysis of Toll-like receptor (TLR) expression at the protein level in MDDCs and monocyte-derived Langerhans cells (MDLCs) as models for different myeloid DC subsets. We found TLR expression to be cell type-specific and MDDCs expressed the full repertoire of TLRs 1-9, including small amounts of TLR8 and TLR9 on the cell surface. The expression of these TLRs that recognise nucleic acids on the surface of cells may constitute an early warning system for signalling the presence of viral invaders that would normally subvert the function of DCs. We also found TLR expression in mature cells to be dependent on the nature of the maturation stimulus (lipopolysaccharide versus cytokine/prostaglandin cocktail) and VACV infection induced profound down-regulation of all TLRs. These findings will have important implications for the rational design of VACV-vectored vaccines.

Vaccinia virus (VACV), the prototypic poxvirus, undergoes a complex life cycle, with multiple stages that are not yet fully understood. This work studied two cellular proteins which had previously been identified by siRNA screens as playing proviral roles in the replication cycle of VACV: the dual specificity mitogen-activated protein kinase kinase 3 (MKK3) and vacuolar protein sorting 52 (Vps52). MKK3 is an upstream regulator in the p38 pathway which, along with MKK6, phosphorylates and therefore activates p38. In HeLa cell cultures, siRNA depletion experiments confirmed that MKK3 supported VACV replication. MKK3 knockdown reduced production of both early and late-class VACV proteins, suggesting that it facilitates viral gene expression. However, this difference did not translate to an in vivo model, as comparison between wild type and MKK3 knockout mice infected with VACV revealed no significant differences in virus replication or overall disease. The Golgi-associated retrograde protein complex (GARP) is composed of four large heteromeric proteins: Vps51, Vps52, Vps53 and Vps54, and plays a key role in retrograde transport from endosomes to the TGN. The effects of loss of GARP function were investigated using three techniques: mouse embryonic fibroblasts (MEFs) containing the hypomorphic Vps54 “wobbler” mutation, Vps52-targetting siRNA in HeLa cells and pharmacological inhibition of retrograde transport using the drug Retro-2. GARP loss resulted in a marked reduction in VACV spread due to a reduction specifically in “double wrapped” extracellular enveloped virion (EEV) production. Investigation of the mechanism by which GARP facilitates EEV production revealed a disruption of the VACV morphogenesis pathway prior to the double wrapping event, resulting in mislocalisation and aggregation of the viral membrane protein B5 within the cytoplasm. The effects of GARP loss translated to an in vivo model, as mice infected with VACV and treated with Retro-2 exhibited reduced viral replication and overall disease. These results identify GARP as a pro-viral host complex required for EEV production, and suggest that cellular retrograde transport pathways are required for double-wrapping of VACV virions. Overall, the study illustrates both the potential pitfalls of carrying out genetic screens in a transformed cell line and the power of such studies to nevertheless identify novel features of virus biology as well as druggable targets for antiviral intervention.

abstract: The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need to understand vaccination from vaccine design to prediction of vaccine efficacy using mathematical models. Post-exposure vaccination with VACV has been suggested to be effective if administered within four days of smallpox exposure although this has not been definitively studied in humans. The first and second chapters analyze post-exposure prophylaxis of VACV in an animal model using v50ΔB13RMγ, a recombinant VACV expressing murine interferon gamma (IFN-γ) also known as type II IFN. While untreated animals infected with wild type VACV die by 10 days post-infection (dpi), animals treated with v50ΔB13RMγ 1 dpi had decreased morbidity and 100% survival. Despite these differences, the viral load was similar in both groups suggesting that v50ΔB13RMγ acts as an immunoregulator rather than as an antiviral. One of the main characteristics of VACV is its resistance to type I IFN, an effect primarily mediated by the E3L protein, which has a Z-DNA binding domain and a double-stranded RNA (dsRNA) binding domain. In the third chapter a VACV that independently expresses both domains of E3L was engineered and compared to wild type in cells in culture. The dual expression virus was unable to replicate in the JC murine cell line where both domains are needed together for replication. Moreover, phosphorylation of the dsRNA dependent protein kinase (PKR) was observed at late times post-infection which indicates that both domains need to be linked together in order to block the IFN response. Because smallpox has already been eradicated, the utility of mathematical modeling as a tool for predicting disease spread and vaccine efficacy was explored in the last chapter using dengue as a disease model. Current modeling approaches were reviewed and the 2000-2001 dengue outbreak in a Peruvian region was analyzed. This last section highlights the importance of interdisciplinary collaboration and how it benefits research on infectious diseases. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2011

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

abstract: Since the molecular biology revolution in the 1980s, ease of gene editing had led to the resurgence of Oncolytic Virotherapy. Countless viruses have been engineered yet only three are approved for clinical use worldwide, with only one being approved by the U.S Food and Drug Administration (FDA). Vaccinia virus (VACV) has a large genome, contains many immune evasion genes and has been thoroughly studied, making it a popular candidate for an oncolytic platform. VACV mutants with deletions in the E3 immune evasion protein have been shown to have oncolytic efficacy but the mechanism of tumor selectivity has not been fully elucidated. These mutants have been shown to be regulated by the necroptosis pathway, a pathway that has been shown to be deficient in certain cancers. Using a pan-cancer screening method that combines dye exclusion assays, western blot analysis, and viral growth curve, the role of necroptosis in regulating VACV replication and oncolytic efficacy in cancer was further characterized. Results demonstrate a preliminary correlation between necroptosis, viral replication, and oncolytic efficacy. This correlation is clearest in breast cancer and melanomas yet may apply to other cancer subgroups. This data was also used to guide the development of a receptor-interacting protein kinase 3 (RIP3) matched pair mouse model in the E0771 mouse breast cancer line which can be used to further study the role of necroptosis and oncolytic efficacy in vivo. Understanding the contribution necroptosis plays in oncolytic efficacy can guide to design enhance the design of clinical trials to test VACV E3L mutants and may lead to better efficacy in humans and an improvement in clinical oncology. / Dissertation/Thesis / Masters Thesis Biology 2020

Human p-glycoprotein (P-gp) is an ATP-binding cassette transporter that actively transports a diverse set of substrates at the plasma membrane. Specifically, P-gp is expressed most highly at important blood tissue barriers on the lumenal side of endothelial cells and secretory tissues asymmetrically where it provides generalized protection against xenobiotics due to its promiscuous substrate binding pocket. Substrates typically interact with P-gp within the inner leaflet of the plasma membrane before being effluxed through large conformation changes driven by ATP binding and hydrolysis. Since many small molecule drugs are substrates of P-gp and P-gp has the ability to transport chemically and structurally diverse molecules, delivery of bioavailable small molecule therapies and treatment of diseases beyond blood-tissue barriers may be difficult. In cancer, expression of P-gp may confer a multidrug resistance phenotype due to upregulation of the MDR1 gene, which encodes P-gp, in response to treatment with chemotherapies. Treatments of diseases beyond blood-tissue barriers and some cancers may be more complex given the protective role of P-gp coupled with it promiscuous substrate binding site.<br>Many studies of P-gp have been centered around understanding the structure function relationship of how P-gp effluxes small molecules across the plasma membrane. Here we have used a transient Vaccinia virus expression system to rapidly express many mutants of P-gp in human cells for analysis. Transient expression using the Vaccinia system was optimized to produce a large amount of protein while avoiding significant cell death. Optimization of the Vaccinia expression system has also helped to show that changes in P-gp surface expression are not correlated to changes in substrate accumulation within cells expressing P-gp, a topic that has yet to be addressed within the field of P-gp study. Reduced surface expression of P-gp to 68% maintained the same level of reduced cellular accumulation of two substrates, calcein-AM and rhodamine 123, relative to a WT P-gp control. Further study of P-gp mutations revealed a Y998A mutation had a 90% reduction of surface expression but the same reduction of cellular accumulation of rhodamine 123 further supporting that changes in surface expression do not correlate to changes in substrate transport.<br>We then sought to demonstrate how flexibility in transmembrane helix (TMH) 12 of P-gp affected overall stability and transport ability in vitro. TMH 12 in inward facing conformations shows a region of decreased hydrogen bonding in the backbone of the helix leading to a “kink” present in many crystal structures of C. elegans and mouse P-gp as well as in an occluded structure of human P-gp. Outward facing crystal structures of C. elegans, mouse, and human P-gp show TMH 12 where the backbone of the helix is fully hydrogen bonded and ordered. The change in hydrogen bonding pattern and the presence of the kink in TMH 12 suggest the importance of flexibility in the function of TMH 12. Clustal Omega was used to align the primary structure of P-gp between 8 species and a conserved sequence of 996-PDYAKA-1001 was identified aligning with the kink observed in crystallographic data. The kinked nature of this region led to our development of a rigid poly-alanine mutation and a flexible poly-glycine mutation based on the propensity of these amnio acids to form helices. The more flexible poly-glycine mutation obtained no significant transport while the poly-alanine mutation maintained some ability to transport fluorescent substrate relative to a WT control. Crosslinking of the nucleotide binding domains (NBDs) revealed a decrease of NBD dimerization likely correlating to decreased transport. Thus, some degree of flexibility within the kink region is critical for substrate transport as rigid and flexible mutations of this region abrogate transport of fluorescent substrates.<br>While the substrate binding pocket it located towards the interior of P-gp within the lipid bilayer, it has been theorized that substrates may interact with P-gp at the lipid-protein interface of the inner leaflet near portals for substrate entry formed by pairs of helices either side of the protein. To test this hypothesis, aromatic residues on TMH 12 and adjacent elbow helix 2 near the interface region of the inner leaflet, that have also been observed to interact with a cyclic peptide in a crystal structure of P-gp, were mutated to alanine. Y998, on TMH 12, was shown to interact with the cyclic peptide and is ideally located at the protein-lipid interface near a surface formed by elbow helix 2 and TMH 9 and was observed to have the largest effect on substrate accumulation. Accumulation of fluorescent substrates, relative to WT P-gp, was increased though not all substrates were affected similarly. No increase of accumulation was observed with rhodamine 123 while accumulation of BD-prazosin increased 65% relative to WT P-gp. It is to be expected that the large diversity of substrates recognized by P-gp would interact preferentially with carrying residues at the protein-lipid interface similar to observations of substrate binding at the substrate binding pocket. Variability in accumulation signifies that substrates do interact with P-gp at the lipid-protein interface and substrates interact differently at this interface similarly to substrate interaction at the substrate biding pocket.<br>

Indiana University-Purdue University Indianapolis (IUPUI) / Inflammatory immune responses are regulated by T cell subsets that secrete specific panels of cytokines. While CD8+ T cells that secrete IFN- and cytotoxic molecules (Tc1 cells) are known to mediate antiviral immunity, IL-17-secreting CD8+ T (Tc17) cells have only recently been described and the development and function of these cells has not been clearly examined. Using in vitro T cell cultures and mice deficient in transcription factors regulating lineage development, we defined Tc17 development and function. Similar to IL-17 secretion from CD4 T cells, IL-17 secretion from Tc17 cells is dependent on the transcription factor Stat3 and inhibited by Stat1. Expression of transcription factors important for Tc1 function, T-bet and Eomesodermin (Eomes), is reduced in Tc17 cells and consistent with this, Tc17 cells are non-cytotoxic in vitro. However, Tc17 cells are unstable and switch to cytotoxic IFN- producing cells when exposed to a Tc1 inducing cytokine, IL-12. Overexpression of the lineage promoting transcription factors T-bet and Eomes is unable to induce a Tc1 phenotype in Tc17 cells and Stat3 is also unable to switch Tc1 cells into Tc17 cells, suggesting additional signals are involved in CD8 T cell lineage commitment. In vivo, Tc17 cells are induced by vaccinia virus, dependant on Stat3, and are capable of mediating antiviral immunity. Tc17 cells acquire an IFN--secreting phenotype after encounter with virus in vivo, however, viral clearance by Tc17 cells is independent of IFN-. Instead, viral clearance is correlated with a gain in T-bet expression and cytotoxic function in Tc17 cells which have encountered virus. The development of anti-viral activity independent of IFN-, suggests that Tc17 cells may mediate anti-viral immunity through novel mechanisms that depend on the ability of Tc17 cells to acquire other phenotypes.

Excerpt: The title and conclusions of the study recently published by Jones et al. (1) concluded that monkeys were protected from dying from a form of simian-human immunodeficiency virus (SHIV) by an psoralen- and ultraviolet-inactivated vaccinia virus in a multi-envelope DNA-VV-protein (DVP). However, the findings in the study are more equivocal than indicated by the title because the effectiveness of the modified vaccinia virus was not decisively demonstrated.

CHAPTER 1 Aggregation of receptors for IgE (Fc RI) causes mast cells and basophils to release preformed contents of granules, including histamine and a variety of enzymes. This process, called degranulation plays a central role in allergic reactions. Methods to study this process are to create multivalent ligands which can interact with the receptors and, in turn, lead to aggregation of the receptors. We prepared a series of fluorophore-labeled divalent and trivalent antigens to study the degranulation of mast cells. Trivalent antigens proved to be much better stimulators for degranulation of mast cells than divalent antigens. These results indicate that aggregates formed by trivalent antigens are more complicated than those of divalent antigens. CHAPTER 2 Membrane-active antibiotics include antimicrobial peptides (AMPs) and a class of amphiphilic steroids termed ceragenins. Recent studies of membrane-active antibiotics show that cationic, facially amphiphilic molecules could disrupt bacterial membranes. It was found recently that some antibiotics, including AMPs and ceragenins, may share both antibacterial and antiviral activity. We prepared a series of ceragenins to optimize the antiviral activity of ceragenins against vaccinia virus (VV). The results show that ceragenins exhibit potent activity against VV, protect keratinocytes against VV-mediated cell death, and preferentially target the virus. It also shows that antibacterial and antiviral activities do not correlate with each other. Although ceragenins show good antiviral activity against VV, the mechanism for this activity still remains unclear.

In der vorliegenden Arbeit wurden Tumorimpfstoffe auf der Basis des Plasmid-Vektors pCI, modified vaccinia virus Ankara (MVA) und MVA-infizierten dendritischen Zellen entwickelt und durch Sequenzierung, Western blotting und durchflußzytometrische Analyse überprüft. Die in vivo Wirksamkeit der Vakzinen wurde in verschiedenen Tumormodellen in C57BL/6 Mäusen verglichen. Die auf dem eukaryotischen Expressionsvektor pCI basierende DNA-Vakzinierung induzierte einen sehr wirksamen, antigenspezifischen und langfristigen Schutz vor Muzin, CEA oder beta-Galactosidase exprimierenden Tumoren. Eine MVA-Vakzinierung bietet in den in dieser Arbeit durchgeführten Tumormodellen keinen signifikanten Schutz vor Muzin oder beta-Galactosidase exprimierenden Tumoren. <br /> <br /> Sowohl humane, als auch murine in vitro generierte dendritische Zellen lassen sich mit MVA &ndash; im Vergleich zu anderen viralen Vektoren &ndash; sehr gut infizieren. Die Expressionsrate der eingefügten Gene ist aber gering im Vergleich zur Expression in permissiven Wirtszellen des Virus (embryonale Hühnerfibroblasten). Es konnte gezeigt werden, daß eine MVA-Infektion dendritischer Zellen ähnliche Auswirkungen auf den Reifezustand humaner und muriner dendritischer Zellen hat, wie eine Infektion mit replikationskompetenten Vakzinia-Stämmen, und außerdem die Hochregulation von CD40 während der terminalen Reifung von murinen dendritischen Zellen inhibiert wird. Die während der langfristigen in vitro Kultur auf CEF-Zellen entstandenen Deletionen im MVA Genom führten zu einer starken Attenuierung und dem Verlust einiger Gene, die immunmodulatorische Proteine kodieren, jedoch nicht zu einer Verminderung des zytopathischen Effekts in dendritischen Zellen. <br /> <br /> Die geringe Expressionsrate und die beobachtete Inhibition der Expression kostimulatorischer Moleküle auf dendritischen Zellen kann für eine wenig effektive Induktion einer Immunantwort in MVA vakzinierten Tieren durch cross priming oder die direkte Infektion antigenpräsentierender Zellen verantwortlich sein.<br /> <br /> Durch die Modifikation einer Methode zur intrazellulären IFN-gamma Färbung konnten in vakzinierten Mäusen tumorantigenspezifische CTL sensitiv und quantitativ detektiert werden. Die so bestimmte CTL-Frequenz, nicht jedoch die humorale Antwort, korrelierte mit der in vivo Wirksamkeit der verschiedenen Vakzinen: DNA vakzinierte Tiere entwickeln starke tumorantigenspezifische CTL-Antworten, wohingegen in MVA-vakzinierten Tieren überwiegend gegen virale Epitope gerichtete CD4 und CD8-T-Zellen detektiert wurden.<br /> <br /> Die Wirksamkeit der pCI-DNA-Vakzine spricht für die Weiterentwicklung in weiteren präklinischen Mausmodellen, beispielsweise unter Verwendung von MUC1 oder HLA-A2 transgenen Mäusen. Die Methoden zur Detektion Tumorantigen-spezifischer CTL in 96-Loch-Mikrotiterplatten können dabei zur systematischen Suche nach im Menschen immundominanten T-Zell-Epitopen im Muzin-Molekül genutzt werden. <br /> <br /> Der durchgeführte Vergleich der auf den Vektoren pCI und MVA basierenden Vakzinen und die Analyse neuerer Publikationen führen zu dem Ergebniss, daß vor allem DNA-Vakzinen in Zukunft eine wichtige Rolle bei der Entwicklung von aktiven Tumorimpfstoffen spielen werden. Rekombinante MVA-Viren, eventuell in Kombination mit DNA- oder anderen Vektoren, haben sich dagegen in zahlreichen Studien als wirksame Impfstoffe zur Kontrolle von durch Pathogene hervorgerufenen Infektionserkrankungen erwiesen. / In this study, tumor vaccines based on the plasmid pCI, the attenuated vaccinia virus strain modified vaccinia virus Ankara (MVA) and MVA-infected dendritic cells were constructed and characterized by sequencing, Western blot and flow cytometric analysis. The efficiency to induce tumor immunity in vivo was compared in several C57BL/6 mouse tumor models. Naked DNA Vaccination based on the eukaryotic expression vector pCI did induce very effective, antigen-specific and long-term protection against tumor cell lines expressing mucin, CEA or beta-Gal whereas MVA vaccination did not elicit protective immunity against Mucin or beta-Gal expressing tumors. MVA does infect human or murine in vitro generated dendritic cells very efficiently compared to other viral vectors, however expression levels of the inserted antigens in dendritic cells are significantly lower than in permissive host cells (chicken embryo fibroblasts). <br /> <br /> It could be shown that the effect of MVA infection on the maturation status of dendritic cells is similar to the effects described for dendritic cells infected with replication competent vaccinia strains. In addition it was shown that the upregulation of the important costimulatory molecule CD40 through LPS stimulation is strongly inhibited in MVA infected cells. During passage in tissue culture, MVA has accumulated a number of large deletions, including a number of immunomodulatory molecules and resulting in a strong attenuation. However the strong cytopathic effect on dendritic cells is maintained. <br /> <br /> The low level of expression and the effect on dendritic cell maturation may be responsible for the failure of MVA to induce tumor immunity through either cross presentation or direct infection of antigen presenting cells.<br /> <br /> To detect and quantify tumor-antigen-specific CTL a method based on intracellular IFN-gamma staining was modified and it could be shown that the cellular &ndash; but not the humoral &ndash; response does correlate with in vivo protection: DNA but not MVA vaccines do induce high levels of tumorantigen-specific CTL whereas MVA-vaccines do induce strong and long lasting CD4 and CD8-T-cell responses against vaccinia antigens. <br /> <br /> The excellent protection induced by pCI-DNA-vaccination in different tumor models does encourage us to further investigate the elicitation of tumor immunity in MUC1 or HLA-A2 transgenic mice. In mice transgenic for human MHC-I, the IFN-gamma staining protocol could be used to systematically screen for mucin T-cell epitopes that are relevant in humans.