Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that mediate host-responses to pathogens. In mammals, TLR ligands promote cellular activation and the production of cytokines. Several TLR ligands have been employed prophylactically for the control of bacterial or viral diseases in the mouse model. However, the TLR-mediated responses in chickens have not been well described. Importantly, the utility of TLR agonists for the control of viral pathogens, such as avian influenza virus (AIV), has not been fully explored in chickens. To this end, the studies described in this thesis characterized the kinetics of in vivo responses in chickens to the TLR4 ligand lipopolysaccharide (LPS) and the TLR21 ligand CpG ODN. It was demonstrated that both of these ligands induced the up-regulation of several immune system genes in the spleen, including those associated with pro-inflammatory and antiviral responses, as well antigen presentation. By harnessing the immunostimulatory properties of TLR ligands, it was also demonstrated that the prophylactic administration of either poly I:C (a TLR3 ligand), LPS or CpG ODN may confer immunity to a low pathogenic avian influenza virus, as determined by a reduction in both oropharyngeal and cloacal virus shedding in infected birds. Furthermore, transcriptional analysis of genes in the spleen and lungs identified interleukin (IL)-8, interferon (IFN)-α and IFN-γ as correlates of immunity. In conclusion, TLR ligands may modulate several aspects of the chicken immune system to induce an anti-viral state, thereby conferring immunity to AIV.

Cold-adapted (ca) live attenuated influenza vaccines (LAIVs) have been introduced as alternatives to existing inactivated influenza vaccines. The influenza A components of the FDA-approved ca LAIVs (Flumist®; Medimmune) have common internal genes derived from the donor strain A/Ann Arbor/6/60 ca and surface genes derived from current wild-type (wt) epidemic strains. The aim of this thesis was to investigate determinants of immunogenicity for reassortants of A/Ann Arbor/6/60 ca, using a range of immunological assays, including recently developed MHC tetramer techniques. From the study, the extent of viral replication in the respiratory tract of mice, the primary site of inoculation, was a key factor in determining ca vaccine immunogenicity. Replication was shown to be influenced by both viral surface Ags and the host MHC. The H3 ca reassortants CR6, CR18, CR29 and CR6-35* exhibited greater replication efficiency (as determined by their PFU:HAU ratios) than the H1 ca reassortants CR35 and CR6-35. The H3 ca reassortant CR6 caused a 3.79% loss in body weight but no losses were observed for the H1 ca reassortant CR35 and the ca H2N2 donor strain A/Ann Arbor/6/60 ca. Higher HI responses were detected after 3 weeks in groups infected with the H3 ca reassortant CR6 (GMT 80) than with the H1 reassortant CR35 (GMT 10) and the H2 ca donor strain A/Ann Arbor/6/60 ca (GMT 13). Recently developed techniques were used to evaluate specific T-cell response to ca LAIVs. Fluorescent-labelled tetramer is the key reagent for use in tetramer-based flow cytometry assays. The NP366-374 peptide of influenza A viruses comprises an immunodominant epitope that is highly conserved between subtypes. Tetramers developed for A/PR/8/34 (H1N1) were able to detect NP-specific cytotoxic T lymphocytes (CTLs) induced by A/Ann Arbor /6/60 ca (H2N2). An attempt to prepare the A/Ann Arbor/6/60 ca-specific-NP-tetramer is described. H-2Db monomers were successfully refolded with the peptide, but only 20% were able to form tetramers through biotin-streptavidin linkage, resulting in a poor capacity to stain. By contrast, an IFN-γ ICC assay developed in parallel demonstrated that peptide NP366-374 was able to restimulate A/Ann Arbor/6/60 NP ca-specific CTLs and secrete IFN-γ when tested in vitro. Specific-B and T cell responses induced in the lungs in response to infection by ca reassortants exhibited great variability that was determined by the growth characteristics of different viruses. Type I (CTL) responses were induced by low yielding ca reassortants, such as CR35 (H1N1). Viruses with enhanced growth characteristics, such as CR6 (H3N2), produced higher Type II (HA-specific Ab) responses. In addition, host factors, such as MHC type, were found to play an important role in responses to the same viruses. Susceptible mouse strains, such as C57BL/6, showed higher CTL but lower serum Ab responses than more resistant strains, such as BALB/c. Throughout this PhD project, a fine balance between the humoral and CMI, local and systemic immune responses induced by ca LAIVs was demonstrated. The need to assess local immune responses, in addition to serum antibody levels, for the evaluation of vaccine efficacy was an important conclusion of the thesis.

Numerous toxins and pathogens gain entry into mammalian cells using cell surface glycans. The Iyer group at Georgia State University is working on the development of glycoconjugates for the accurate detection of infectious agents. In this thesis, I have focused on the development of glycans to detect influenza virus and norovirus. In the first section, I have focused on influenza viruses. A panel of synthetic glycans was synthesized as receptor mimics for the specific capture of influenza viruses. The synthetic glycans were printed onto commercial glass slides using a free amine at the end of a spacer to generate a small focused microarray. This glycan printed microarray was evaluated for its ability to capture three strains of influenza viruses. The analytical limit of detection is ~10 pfu/ml, (plaque forming units/milliliter) which is clinical relevant as 102 viral particles are typically required to cause infection. We also tested the drug susceptibility of current antivirals, Zanamivir and Ostelamivir using the microarray and determined the feasibility of this system to determine antiviral resistance for different strains. In addition to optical detection, I developed an electrochemical assay to rapidly detect influenza viruses. Here, we utilized an unique property of influenza viral surface enzyme, Neuraminidase (NA), which cleaves terminal N-Acetyl Neuraminic acid (sialic acid) from cell surfaces and proteins. We designed an electrochemical assay that uses glucose bearing sialic acid substrates. Glucose is released when exposed to viral NA or intact viruses. The released glucose can be detected using repurposed glucose meters. Thus, personal glucose meters that were designed to assist diabetics and prediabetics monitor blood glucose can potentially be used to detect pathogens. Using this approach, we have detected 19 unique strains of influenza viruses. We also demonstrated drug susceptibility using this assay. The limit of detection of this assay is 102 pfu/sample, which is clinically relevant. The results were validated plaque assays and polymerase chain reaction (PCR). In the second part of this thesis, I focused on norovirus detection. I developed a focused glycan microarray that comprised of a library of histo blood group antigens (HBGAs). The HBGAs were attached to a carrier protein and printed onto activated glass slides. A panel of norovirus virus like particles (VLPs) and strains that included different genogroups was exposed to the microarray. We found that different VLPs and strains give rise to unique binding patterns. When the binding pattern of VLPs for a particular strain were compared to the corresponding intact virus, the binding patterns didn't match well, presumably because the virus does not recognize the same antibody as the VLPs. Unfortunately, antibodies for the virus cannot be generated because the virus cannot be grown in a laboratory setting. Indeed, all norovirus samples are obtained from human challenge studies. I also used surface plasmon resonance (SPR) studies in an effort to determine the binding affinities. Divalent biotinylated H type glycans were synthesized and their binding affinities with different VLPs and viral strains were determined. Initial studies suggest that the binding affinities are strain specific. These results demonstrate that glycans can be used to capture and isolate norovirus, although more research is required to develop glycan based norovirus detection kits.

Deeper understanding and technological progress in materials physics demand exploration of soft and hard matter at their relevant length scales. This research focuses on the nanometer length scale investigation of structural changes required for membrane fusion in virus nanoparticles and nano-spectroscopic investigation of layered material surfaces implementing scattering type scanning near-field optical microscopy (s-SNOM). Spectroscopy and imaging experiments were deployed to investigate the chemical and structural modifications of the viral protein and lipid bilayer under various environmental pH variations. It has been shown that breakage of viral membrane could occur even without the presence of a targeting membrane, if the environment pH is lowered. This is in contrary to the current viral fusion model, which requires virus binding to a host cell membrane for forming the fusion pore to release the viral genome. The fusion inhibitor compound 136 can effectively prevent the membrane breakage induced by low pH. The chemical surface stability and degradation of black phosphorus (BP) under ambient conditions have been studied using s-SNOM. We found that the degraded area and volume on the surface of black phosphorus increase with time slowly at the start of degradation and enlarge rapidly (roughly exponentially) afterward and reach saturation growth following S-shaped growth curve (sigmoid growth curve). The theoretical model presented suggests that the degraded sites in the adjacent surrounding causes the experimentally observed exponential growth of degraded area at the initial stage. By studying the BP surfaces coated by Al2O3, boron nitride (BN) and hybrid BN/Al2O3 layers through the period up to 6 months, it has been concluded that ~5 nm thin hybrid layer of BN/Al2O3 helps the surface passivation of BP flakes of thickness ~30 nm. This is supported by the electrical characterization results of BP field effect transistor coated with a BN/Al2O3 layer. We have performed infrared nano-spectroscopy on muscovite mica exfoliated on silicon and silicon dioxide substrates. We show that the near-field profile in s-SNOM can penetrate down to several hundreds of nanometers and enable spectroscopy of buried structures. We found spectral broadening of mica as its thickness increases revealing clearly the effect of size on the absorption response.

Natural killer (NK) cells are lymphocytes of the innate immune system that play a major role in the destruction of both tumours and virally-infected cells. The cytotoxicity of NK cells is tightly controlled by signals received through activating and inhibitory receptors. NK cells express a variety of inhibitory receptors such as Ly49 receptors. Ly49 receptors bind to class I MHC molecules that expressed on normal cells. Using Ly49-deficient (NKCKD) mice we show that Ly49-KD NK cells successfully recognize and kill influenza virus-infected cells and that NKCKD mice exhibit better survival than wild-type mice. Moreover, influenza virus infection has a propensity to upregulate cell surface expression of MHC-I on murine lung epithelial cells in vivo. Significantly, we demonstrate increased lung damage of WT-mice versus NKCKD mice after influenza virus infection as determined by histological analyses. This data indicated that absence of Ly49 inhibitory NK receptors greatly enhances survival of infected mice.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19633号 / 医科博第71号 / 新制||医科||5(附属図書館) / 32669 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 小柳 義夫, 教授 斎藤 通紀, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The Influenza virus is a major human respiratory pathogen responsible for seasonal ‘flu’ outbreaks and sporadic global pandemics. The Influenza polymerase complex is necessary for viral RNA synthesis and full virulence and requires the assembly of three conserved subunits: PA, PB1 and PB2. A recombinant chimeric protein mimetic consisting of the N-terminus (20 amino acids) of PB1 fused to Maltose Binding Protein (MBP) and Tat Nuclear Localization Signal (NLS) was designed and purified with the aim of inhibiting the assembly of the polymerase by mimicking PB1. The cell-penetrating protein mimetic was shown to efficiently enter the cell nucleus and prevent assembly of the Influenza polymerase, thus inhibiting viral replication. When MDCK cells were incubated with the mimetic and subsequently challenged with Influenza A virus, viral replication decreased up to 98% at 50 µM. Using a nuclear extraction assay, the mimetic was shown to efficiently penetrate the plasma membrane and enter the host nucleus. GST pull-down assays showed that the mimetic interacts with PA. Molecular modeling was then employed to predict the improved hypothetical free energy of binding between PB1 and PA and determined two significant substitutions for PB1 threonine at position six: glutamic acid (T6E) and arginine (T6R). These mutations increased potency of the mimetic at 25 µM (71% for T6E and 77% for T6R compared to 36% for the native construct) and 12.5 µM (27% for T6E and 70% for T6R compared to 16% for the native construct), suggesting a more stable interaction with PA consistent with molecular modeling. Using various in vitro assays, the mimetic was shown to be non-toxic to host cells. Targeting critical protein-protein interactions using a peptide fused to a cell-penetrating carrier protein presents a novel and intriguing approach in designing anti-viral therapeutics. / Thesis / Master of Science in Medical Sciences (MSMS)

The COVID-19 pandemic has been an ongoing disaster that has devasted millions of lives. With the development of COVID-19 vaccines in late 2020, there was a potential for populations to gain artificial active immunity in order to prevent future outbreaks. However, despite successful clinical trials, millions of citizens have been hesitant to receive the COVID-19 vaccines (Khubchandani et al., 2021). Demographics of the most prominent US vaccine-hesitant populations consist of ethnic/racial minorities and Republicans groups (Khubchandani et al., 2021). Little information is known about COVID-19 vaccine hesitancy in colleges and universities. Colleges provide an elevated risk for infection through their communal residencies, the reemergence of campus activities, and continuous travel to home (Sharma et al., 2021). This study explored COVID-19 vaccine hesitancy in UCF college students and explored potential pathways to achieve higher vaccination rates. Potentially believed COVID-19 misinformation was also studied. A COVID-19 opinion survey was designed and distributed to the UCF college population. Two hypotheses were made for this study: (1) There is a significant effect on vaccination status among people of different political parties, field of study, living conditions, masking frequency, and scores on the knowledge-based questions portion. (2) There will be a significant effect on knowledge-based scores with political party and field of study. The results were analyzed using Chi-square, one-way ANOVA, or two-way ANOVA on SPSS. The results showed a significant effect on vaccination status in political parties, masking frequency in class, and scores on the knowledge-based survey questions. There was no significance with race/ethnicity and field of study. There was a significant effect on the knowledge-based survey questions with political party and field of study. Potential side effects and the vaccines being seen as ineffective were the top two reasons that students choose not to vaccinate.

Das Hämagglutinin (HA) der Influenzaviren wird während der Assemblierung in Cholesterin- und Sphingolipid-reiche Domänen (Rafts) der Plasmamembran rekrutiert. Vorangehende Studien konnten mittels Fluoreszenzresonanzenergietransfer eine Raft-Integration nachweisen, die von zwei Raft-Zielsignalen abhängig war; zum einen von drei S-acylierten Cysteinen in der zytoplasmatischen Domäne und zum anderen von hydrophoben Aminosäuren (VIL) am Beginn der Transmembrandomäne (TMD). Zudem zeigte sich ein möglicher Einfluss des VIL-Motives auf den intrazellulären Proteintransport. Um diese Annahme zu bestätigen, wurden HA Mutanten in Zellen exprimiert und ihre Ankunft im medialen und trans-Golgi verfolgt. In dieser Arbeit konnte eine Beteiligung des VIL-Motives am Transport bestätigt werden, jedoch nicht der S-Acylierungen. Zudem wurde eine generelle Abhängigkeit des Transportes von der Sphingolipidsynthese beobachtet. Da sowohl die Cholesterinsynthese als auch die Sphingolipidsynthese für den Transport von HA benötigt werden, habe ich die Hypothese aufgestellt, dass das VIL-Motiv in der Lage sein könnte, mit Raft Lipiden zu interagieren. Ein Sequenzvergleich ergab, dass kein Sphingolipid-Bindemotiv vorhanden ist, jedoch ein potenzielles Cholesterin-Consensus-Motiv (CCM, W/Y-I/V/L-K/R). Dieses Motiv wurde nur in der Sequenz von Gruppe 1 jedoch nicht Gruppe 2 HAs gefunden und umfasst das Leucin des VIL Motives. Tatsächlich ist die Mutation des Leucins aber nicht des vorangehenden Isoleucins für den verzögerten Transport verantwortlich. Untersuchungen weiter Einzel- und Mehrfachmutanten konnten eine Abhängigkeit des intrazellulären Transportes von einer möglichen Cholesterinbindung verifizieren. Zudem konnte auch ein zunehmender Effekt auf die Kinetiken vom medialen Golgi zum TGN beobachtet werden, welcher auch die Oberflächenexpression negativ beeinflusste. FLIM-FRET Analysen zeigten zusätzlich eine reduzierte Raft Assoziation der CCMMutanten mit Rafts an der Plasmamembran. Daher kann man spekulieren, dass HA mit Cholesterin interagiert, wodurch sein intrazellulärer Transport durch den Golgi und die Assoziation mit Rafts gewährleistet wird. / During assembly the hemagglutinin (HA) of influenza viruses is recruited to cholesterol- and sphingolipid rich domains of the plasma membrane (lipid rafts). Preceding studies using fluorescence resonance energy transfer showed that lipid-raft integration is dependent on two raft-targeting signals, three S-acylated cysteines located in the cytoplasmic tail and hydrophobic amino acids (VIL) in the part of the transmembrane region (TMR). Furthermore, they gave rise to the assumption that at least the VIL motif might also be important for the intracellular transport of the protein along the exocytic pathway. To verify this assumption, HA mutants were transiently expressed in cells and their arrival in the medial and trans-Golgi compartment was quantified. The observation regarding the involvement of the VIL motif, but not the S-acylation, was verified and a general dependency of HA´s transport on sphingolipid synthesis was detected. Since both cholesterol and sphingolipid synthesis are needed for the transport of HA, I hypothesized that the VIL motif might be able to interact with raft lipids. Sequence alignment revealed no sphingolipid-binding motif, but a putative cholesterol consensus motif (CCM, W/Y-I/V/L-K/R). This CCM is found only in the sequence of group 1 but not group 2 HAs and includes the leucine of the VIL motif. Indeed, mutation of the leucine, but not of the preceding isoleucine is responsible for the delayed transport. Investigation of further single and multiple mutations in the CCM verified a dependency of HA´s intracellular transport on the putative cholesterol-binding motif. Additionally the effect on the kinetics increased from the medial Golgi to the TGN also negatively effecting surface expression. Analysis by FLIM-FRET furthermore displayed a reduced association of HA with mutations in the CCM with lipid rafts at the plasma membrane. Therefore, it is speculated that HA associates with cholesterol, an interaction that facilitates its intracellular transport through the Golgi and association with lipid rafts at the plasma membrane.

Das Hämagglutinin (HA) des Influenzavirus ist post-translational durch S-Acylierung von drei Cysteinen modifiziert. Zwei davon befinden sich in seiner zytoplasmatischen Domäne (CD) und enthalten Palmitat und eines am Cytosol-zugewandten Ende der Transmembranregion (TMR) wird bevorzugt mit Stearat acyliert. Es wird vermutet, dass entweder die Aminosäureumgebung der Acylierungsstelle oder dessen Lage relativ zur Membran bestimmt welcher Fettsäuretyp angeheftet wird. Diese Acylierungstellen sind zudem essentiell für die Virusreplikation. Ob auch andere Aminosäuren der CD essentiell sind, ist nicht bekannt. Nach einem umfangreichen Sequenzvergleich zur Identifikation konservierter Aminosäuren wurden rekombinante Viren mit Aminosäureaustauschen in der Nähe der drei Acylierungstellen hergestellt. Diese Austausche enthielten Punktmutationen, Verschieben des TMR Cysteins in die CD sowie die Deletion der gesamten CD. Viren ohne CD und ein Austausch neben einem acylierten Cystein verhinderten die Virusreplikation. Eine konservative Substitution derselben Position, andere Austausche in TMR und CD sowie das Schieben des TMR-Cysteins in die CD dagegen beeinflussten das Viruswachstum nur schwach. Einige der mutierten Codons revertierten zur ursprünglichen oder einer neuen Aminosäure. Rekombinante Viren wurden in MDCK-Zellen und embryonierten Hühnereiern vermehrt und mittels Massenspektrometrie analysiert. Es wurden keine unteracylierten Peptide detektiert, und selbst die zwei Letalmutationen behielten die Acylierung. Punktmutationen beeinträchtigten nur mäßig den Stearat-Gehalt, wogegen die Verlagerung des TMR-Cysteins in die CD die Stearylierung praktisch eliminierte. Mehr Stearat wurde angeheftet, wenn humane Viren in Säugerzellen im Vergleich zu aviären Zellen angezüchtet wurden. Die Position einer Acylierungsstelle repräsentiert relativ zur TMR-Spanne das Hauptsignal der Stearylierung während der Sequenzkontext und der Zelltyp das Fettsäuremuster modulieren. / Influenza virus’s hemagglutinin (HA) is post-translationally modified by S-acylation of three cysteines. Two are located in its cytoplasmic tail (CT) and contain palmitate and one at the end of the transmembrane region (TMR) is acylated primarily with stearate. It is hypothesized that either the acylation site’s amino acid environment or its location relative to the membrane determines which type of fatty acid is attached. Additionally, these acylation sites are essential for virus replication. Whether other amino acids in the CT are required for virus replication, is not known. Based on a comprehensive sequence comparison to identify conserved amino acids, recombinant viruses with amino acid substitutions in the vicinity of HA’s acylation sites were created. These substitutions included point mutations, shifting of a TMR cysteine to the CT and the deletion of the entire tail. The truncated tail mutation and a substitution adjacent to an acylated cysteine disabled virus replication. In contrast, a conservative substitution at this position, other exchanges in TMR and CT and moving the TMR cysteine to the CT had only subtle effects on virus growth. Yet, some of the mutated codons reverted to the original or other amino acids. Recombinant viruses were propagated in MDCK cells and embryonated chicken eggs and analyzed by mass spectrometry. No under-acylated peptides were detected, even the two lethal mutations did not abolish acylation. Point mutations only moderately affected the stearate content, while relocating the TMR cysteine to the CT virtually eliminated attachment of stearate. More stearate was attached if human viruses were grown in mammalian compared to avian cells. Hence, the location of an acylation site relative to the TMR represents the principal signal for stearate attachment, while the sequence context and the cell type modulate the fatty acid pattern.