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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.

Mosquito-borne viruses, including dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), are rapidly emerging, global pathogens. Though the number of people impacted by each virus varies, there have been thousands to millions of people infected. The focus of this thesis work centers around USUV and WNV; both have RNA genomes and belong to the Flaviviridae virus family. Both WNV and USUV were initially isolated in Africa and have since spread to Europe; interestingly, WNV has also spread globally and is considered endemic in the Americas. Similar to other flaviviruses, USUV and WNV are maintained in a mosquito vector-avian host transmission cycle, with spillover infection into humans. Human infections of WNV and USUV are usually asymptomatic, but in severe cases can cause neuroinvasive disease. WNV and USUV belong to the JEV serocomplex group, which indicates that antibodies produced against these viruses share a common antigen; the common antigen is hypothesized to be the envelope (E) protein on the outside of the virion. Neutralizing antibodies against both WNV and USUV have been found in birds and humans across Europe. In vitro cross-neutralization of WNV and USUV has been modeled experimentally and been observed in clinical settings. The neutralizing antibody response generated against WNV has been studied extensively in mouse models; however, there are few studies which examine the neutralizing antibody response generated against USUV. Whether prior WNV exposure protects against USUV disease is also unknown. The main goal of this thesis was to characterize how a primary flavivirus exposure would influence a secondary flavivirus exposure; specifically, we wanted to observe if WNV exposure would protect against USUV disease in vivo and generate a cross-neutralizing antibody response in vitro. For the WNV exposure, we used an attenuated vaccine strain of WNV that contains the WNV E gene (D2/WN-V3) developed by our collaborators. We hypothesized that treatment with D2/WN-V3 would protect against USUV infection. Two in vivo models were used: CD-1 mice and interferon alpha-beta receptor 1 deficient (Ifnar1-/-) mice. We discovered that sera from mice vaccinated with D2/WN-V3 neutralized both WNV and USUV in vitro. In the Ifnar1-/- model, we observed that vaccinated mice had higher survival rates and lower USUV viremia levels after USUV challenge. This work helps characterize the consequences of flavivirus antibody cross-neutralization in vitro and cross-protection in vivo. As the flavivirus field moves toward the goal of creating a pan-flavivirus vaccine, both cross-reactive antibodies and cross-protection need to be considered. / Master of Science / West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that were originally isolated in Africa during the 20th century. Both viruses are maintained through a transmission cycle between mosquito vectors and avian hosts. Mosquitos transfer the infectious agent (WNV or USUV) through feeding on a bird (usually a passerine species); once in the bird, the virus can replicate to high levels. Human infections of WNV and USUV from mosquitos can also occur, with symptoms ranging from mild febrile illness to severe encephalitis or meningitis. Over the past few decades, WNV and USUV have spread to Europe, most likely through infected migratory birds. Interestingly, mosquito surveillance studies in mainland Europe have found mosquitos that tested positive for both USUV and WNV. In Europe, antibodies for both viruses have been found in humans and birds, indicating a previous exposure to WNV, USUV, or both. The neutralizing antibody response is a critical immune defense against viral infections. Neutralizing antibodies bind strongly to the outside of the virion (virus particle), preventing the virion from interacting with and infecting the host cell. For WNV and USUV, one of the targets that neutralizing antibodies bind to is the outer envelope (E) protein of the virion. In clinical settings and experimental studies, cross-neutralization of WNV and USUV has been documented. During cross-neutralization, a serum sample containing neutralizing antibodies against WNV can also neutralize USUV, and vice versa. Although the neutralizing response against WNV has been characterized in humans and lab animal models such as mice, there is little research regarding the neutralizing response against USUV. Importantly, whether prior WNV exposure provides protection against USUV infection is currently unknown. The main goal of this thesis was to characterize the disease outcome and neutralizing response against USUV after a WNV exposure. For the WNV exposure, we used a vaccine strain of WNV that contains the E gene (D2/WN-V3) developed by our collaborators. We predicted that vaccinated mice would avoid USUV clinical signs of disease and generate neutralizing responses to WNV and USUV. To do this work, we used two laboratory mouse models: mice with an intact immune response system (CD-1) and mice with a stunted immune response (Ifnar1-/-). We discovered that serum from vaccinated mice did cross-neutralize WNV and USUV. In the Ifnar1-/- model, vaccinated mice had higher survival rates and lower levels of virus in blood after USUV infection compared to unvaccinated mice. Ultimately, this work highlights the importance of characterizing the immune response against similar viruses and will inform the development of human vaccines for both viruses.

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