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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23800号 / 医博第4846号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 高橋 淳, 教授 髙折 晃史, 教授 辻川 明孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Salmonella enterica serotype Typhimurium is a rod-shaped, motile, Gram-negative bacterium that causes gastroenteritis in immunocompetent individuals. S. Typhimurium produces an extracellular protein termed curli, a bacterial amyloid with a cross beta-sheet tertiary structure that is common across all amyloids. Curli formation is critical for biofilm formation by enteric pathogens such as S. Typhimurium and E. coli. Curli expression requires the production of multiple proteins, which are encoded by two operons known as csgBAC and csgDEFG. Curli production can be induced in vitro by low temperature and low osmolarity, which is evident by growth on T-medium plates for 72 hours at 28oC. Earlier studies have shown that curli is expressed in sepsis patients with E. coli, as well as in mice after S. Typhimurium infection. This is evidenced by the production of antibodies to CsgA, the major subunit of curli. Our lab has shown that curli fibers are recognized by the TLR2/TLR1 complex of the innate immune system during infection. Infection with curli expressing bacteria causes elevated levels of proinflammatory cytokines, nitric oxide, and autoantibodies. Nonetheless, the details of curli expression in vivo during bacterial infection remain unknown. The focus of these studies was to elucidate the location where bacteria expresses curli in vivo during infection. Initially, we used S. Typhimurium strains carrying plasmids with csgB and csgD promoter regions fused to the gfp gene to study curli expression in vivo by use of flow cytometry. Unfortunately, we were unable to determine curli expression with this model, due to the diminished fluorescence intensity of GFP under anaerobic conditions in the gastrointestinal tract. As the question of curli expression in vivo was left unanswered, we next used a long-term infection model of S. Typhimurium with the goal of determining seroconversion to curli as well as the location and timing of curli expression. Using CBA/J mice infected with wild-type S. Typhimurium or a curli mutant strain, we were able to identify seroconversion to CsgA in the mice infected with the wild-type strain through ELISA and western blot analysis. We were also able to identify autoantibody production in mice infected with the wild-type strain through ELISA. However, we were unable to determine curli expression in the feces of mice either by western blot or qPCR data. We were also able to identify autoantibody production in mice infected with the wild-type strain through an anti-double stranded DNA ELISA. Preliminary findings lead us to hypothesize that curli expression may occur very early on in infection, and may be expressed inside cells such as macrophages. Overall, our results partially elucidate curli expression in vivo, although more research is needed in order to answer our remaining questions regarding location and timing of expression. / Biomedical Sciences

Herpes simplex virus is man's oldest viral enemy. Infections result in symptoms ranging from mild skin lesions to deadly herpes simplex encephalitis, making HSV one of the most costly of viral diseases to treat. Thus the development of a vaccine is imperative. To this end, several vaccine strategies have been utilized to generate immunity to HSV in rodents. These include the use of recombinant DNA, recombinant adenoviruses, and dendritic cells transduced with either of the former and re-introduced to the host to induce immunity. In this study, different aspects of these vaccine types were examined. Antibody and cytotoxic T-cell (CTL) responses to a DNA vaccine encoding gB of HSV-1 (gB-DNA) were evaluated. This resulted in variable long lived antibody responses to a wide range of dosages and CTL responses which followed dose-response relationships. An adenovirus expressing gB of HSV -1 (AdgB) which is able to generate IgA responses (Gallichan et al., 1993) was utilized to determine the best method of mucosal administration to optimize these responses. It was suggested in a previous report that nasal associated lymphoid tissue (NALT) was the desired target for inducing lgA responses (Heritage et al., 1997). Accordingly, the hypothesis was formulated that NALT can produce IgA responses similar to those produced from a combination of inductive sites. To test this hypothesis, mice were immunized either awake or asleep with AdgB assuming that awake delivery restricts induction to the NALT, whereas asleep administration disseminates AdgB throughout the respiratory system. The results demonstrate participation of lower airways in the induction of immunity is desirable for generating IgA responses. Lastly, dendritic cells transduced with AdgB were assessed for their ability to generate systemic and mucosal antibody responses, resulting in the inability to generate IgA, but the ability to generate systemic antibody responses. / Thesis / Master of Science (MS)

Lines of White Leghorn chickens selected > 20 generations for (HA) and against (LA) antibody response to SRBC injected i.v. from 41 to 51 days of age, are now known to have diverged in primary antibody response to SRBC. Experiments described in this dissertation were designed to further evaluate the immune competence of these lines as influenced by age, diet, and a disease agent. A crossing experiment was also conducted to further describe the mode of inheritance of such competence. Humoral immunocompetence was evaluated by primary, memory, and maternal antibody responses to SRBC. Primary antibody response, measured 5, 10, and 20 days after inoculation with SRBC was greater in HA than LA chicks inoculated at 14, 21, and 28 days of age. In chicks injected at 7 days of age, a higher frequency of responders was observed for HA than LA chicks suggesting an earlier onset of immunocompetence in the high than low antibody line. Immunological memory antibody responses (secondary and tertiary) was studied in parallel experiments on two groups of chicks hatched at a 14-day interval. Chicks in both hatches were from the same matings of parental Lines HA and LA. Memory responses were evident in chicks at 14 days of age. Antibody responses to a second and third inoculation with SRBC were similar for both lines suggesting that genetic factors that influence primary and memory responses are not the same. The responses of LA chicks to repeat inoculations with SRBC were anamnestic whereas those of HA chicks initially inoculated at 28 days of age were not anamnestic. This study did not establish any major influence of nutrient density on either primary or memory immune responses even though the higher nutrient density diet improved growth performance. Assays in chicks indicated that maternal antibodies were transferred earlier into eggs laid by HA hens than in those of LA hens ( 7 to 9 days vs 10 to 12 days after inoculation) regardless of dosage administered. Response patterns whether assessed in terms of frequency of detection or magnitude of response showed divergence between the lines. Chicks of parental, reciprocal F , F , and backcrosses of 1 2 mating combinations of Lines HA and LA were injected with SRBC at 36 days of age. Contrasts between parental lines for antibody titers measured 5 and 12 days later showed higher antibody titers in HA than LA chicks. Sex-linked effects were evident because reciprocal contrasts for F crosses, individual heterosis, and 1 maternal heterosis were sex dependent. Response to marble spleen disease virus ( MSDV) measured 6 days after inoculation of chicks from parental, reciprocal F1, F2, and backcross matings of the lines, indicated that the mode of inheritance of spleen weight differed after infection. In the infected chicks, parental contrasts for absolute and relative spleen weights showed greater resistance to MSDV in LA than HA chicks. No other genetic effect was consistently important after infection. / Ph. D.

Lipooligosaccharide (LOS) is the major outer membrane component of many Gram-negative bacteria inhabiting the mucosal membranes, including pathogenic species of <I>Haemophilus</I> and <I>Neisseria</I>. LOS phase variation is one mechanism by which some of these bacteria avoid the host immune response. To better understand LOS phase variation as a virulence mechanism of <I>H. somnus</I>, knowledge of the antigenic diversity of LOS epitopes must be increased. Monoclonal antibodies (MAbs) to <I>H. somnus</I> LOS were produced and used with cross-reacting MAbs to <I>H. aegyptius</I> LOS (MAb 5F5) and <I>Neisseria</I> <I>gonorrhoeae</I> LOS (MAb 3F11) in an ELISA to investigate LOS heterogeneity among forty-five strains of <I>H. somnus</I>. Using three MAbs, thirty-nine of these <I>H. somnus</I> strains were grouped into six antigenic types. Three groups, associated solely with the cross-reacting MAbs 5F5 and 3F11, included the majority (76%) of <I>H. somnus</I> strains. The anti-<I>H. somnus</I> LOS MAb 5D7 recognized a low frequency epitope associated with each of the remaining three groups, which included 11% of the <I>H. somnus</I> strains. Six strains (13%) were not recognized by any of these MAbs. Inhibition ELISA experiments showed that the MAb 5F5 epitope contained phosphocholine (PCho) and this epitope was present in 56% of the strains tested. The MAb 5F5 epitope is phase variable in <I>H. somnus</I> LOS. How PCho negative variants could allow for systemic infection after initial colonization of the mucosa by PCho positive variants is discussed. / Master of Science

Pandemics are periodic events characterized by rapid and widespread transmission of infectious disease affecting a significant proportion of the population over a large geographical area. Zoonotic strains of influenza viruses and coronaviruses have both caused pandemics in the recent past. Although vaccination is the often the most effective way to prevent infection or serious outcomes of infection, vaccine development, production, distribution, and deployment are all time- consuming and logistically challenges processes. Alternative readily deployed approaches must be quickly executed to mitigate the toll of future pandemics, especially during the early phases. The work described in this thesis describes some of these approaches. Firstly, I describe the process by which I performed genome-wide CRISPR-Cas9 knockout screens using SARS-CoV-2 variants of concern to discover crucial host factors as targets for broad-acting antivirals. I found that all variants rely on similar host pathways to replicate in the glial cell line used for the screen. I identified BCL-xL, a regulator of apoptosis, as a potential target for a broad- acting antiviral. I show that chemical inhibition of BCL-xL results in accelerated cell death in infected cells in vitro, but improved clinical signs and disease mortality of SARS-CoV-2 in our murine infection model. Secondly, I describe a unique mechanism for cooperative antiviral combination therapy. I demonstrate that chemical inhibition of neuraminidase by oseltamivir improved immune effector cell activation by hemagglutinin stalk-binding antibodies. Combination therapy of oseltamivir and stalk-binding antibodies also improved clinical signs and disease mortality of influenza in our murine infection model compared to monotherapy in both prophylactic and therapeutic contexts. Finally, I show that non-pharmaceutical public health interventions used to restrict the spread of COVID-19 were also effective against several other infectious diseases. I used an interrupted time- series analysis on Ontario public health administrative data during the early COVID-19 pandemic period and found a drastic and sustained decline in outpatient visits for diseases that are typically caused by viruses that transmit by droplet or aerosol. The three projects described in this thesis outlines broadly-protective and distinct strategies to curb the spread of novel respiratory viruses. These new tools may be leveraged to improve the response and to mitigate the burden of future pandemics. / Thesis / Doctor of Philosophy (PhD) / Most pandemics in recent history have been caused by viruses that infect the respiratory tract. Vaccination is often the best way to prevent the spread of these pandemic viruses, but making these vaccines takes time. Vaccines also work less well in the very young, the elderly, and those with a compromised immune system. These people are often also the most vulnerable to severe disease. My work describes three novel approaches to help combat the next pandemic, especially during the early phases when vaccines are still being developed, or for the segments of the population that respond poorly to vaccination. These include discovering and using new drugs that work against a wide range of viruses, using combinations of previously-discovered antiviral drugs, and using non-pharmaceutical methods such as physical distancing and wearing masks.