Hepatitis B virus (HBV) is a life-threatening pathogen with major economic significance. Acute infection in adults is common, albeit usually self-limiting. Importantly, infection in infants typically results in chronic infection and increased incidence of hepatocellular carcinoma (HCC). Furthermore, the infectious carrier state is perpetuated in chronically infected individuals. Successful immuno-therapeutic vaccination would reduce the incidence of chronic infection and of HCC as well as reduce transmission of the disease. Recovery from acute and chronic HBV infection typically occurs in the presence of robust antigen-specific humoral and cellular immune responses (CMI), whereas these responses are low or absent in chronically HBV-infected individuals. Therefore, it was hypothesised that effective stimulation of both humoral and CMI responses, in conjunction with currently available antiviral therapies, may contribute significantly to development of vaccines for treatment of chronic HBV infection. The duck hepatitis B virus (DHBV) model of HBV infection was used to test novel vaccine strategies that could complement existing antiviral therapeutic approaches to treat chronically HBV-infected humans. To this end, three separate vaccine studies were conducted to investigate potential therapeutic regimes. Methods to assess the efficacies of the vaccine strategies included immunoperoxidase detection of viral antigen and immune cell markers within the liver and development of sensitive assays to monitor levels of DHBV DNA, duck hepatitis B virus surface antigen (DHBsAg), antibodies to duck hepatitis B core (anti-DHBc) and surface antigens (anti-DHBs) in serum were developed and validated which allowed monitoring of the kinetics of the humoral immune response following vaccination and the course and outcome of experimental DHBV infection. The first vaccine study tested the protective efficacy of DNA vaccines encoding either the small form of DHBsAg (DHBs) protein or the larger antigen (DHBpre-S/S), These were administered to ducks at day 4 and 14 of age. On the same day as the second vaccination, ducks were challenged intravenously with DHBV. Immunoperoxidase staining of biopsy tissue collected at day 4 p.i. showed significant decreases in the number of DHBV infected hepatocytes in ducks receiving the DNA vaccines compared to the mock-vaccinated control ducks. Significant protection against development of chronic DHBV infection was observed in ducks vaccinated with DNA vaccines expressing either pre-S/S or S protein. Although anti-DHBs antibodies were not detected prior to DHBV challenge, the decrease in the percentages of DHBV-infected hepatocytes at day 4 p.i is suggestive that neutralisation of the inoculum by low-level anti-DHBs antibodies in cohort with CMI responses induced by vaccination were the most probable mechanisms of action. The second vaccine study examined the protective efficacy of a novel whole-cell vaccine that expressed the DHBV core antigen (DHBcAg). Ducks were vaccinated on day 4 and 14 of age and DHBV challenge was administered 4 days later. Detectable anti-DHBc antibodies were generated as soon as 4 days after the initial vaccination suggesting that this regimen elicited increased immunogenicity than vaccination with DNA vaccines alone. In contrast to the first vaccine study with DNA vaccines expressing DHBsAg, no significant differences in the percentage of DHBV-infected hepatocytes were observed in biopsy tissue collected at day 4 p.i.This finding is confirmation that anti-DHBc antibodies were not neutralising to the initial DHBV inoculum. However, significant protection against development of chronic DHBV infection was observed in the whole-cell vaccinated ducks suggesting that the mechanism of protection was consistent immune-mediated killing of DHBV-infected hepatocytes following CMI responses to determinants of DHBcAg. The final vaccine study involved a combination strategy of antiviral drug Entecavir (ETV) and prime-boost vaccination with DNA vaccines and recombinant fowlpoxvirus (rFPV) expressing DHBV antigens. Immediately following DHBV infection, ducks were dosed by oral gavage with the antiviral drug Entecavir (ETV) and at the same time received the priming DNA vaccines encoding DHBV antigens. Seven days later the boosting vaccination consisting of recombinant fowlpox viruses (rFPV) also expressing DHBV antigens was administered. Extraordinary protection was observed, with 100% of ducks given combination therapy rapidly resolved their DHBV infection while 100% of non-treated ducks developed chronic infection. It was concluded that protection resulted from a combination of at least three factors. First, reduction and control of DHBV levels with the aid of ETV; secondly, stimulation of surface antigen-specific humoral immune responses resulting in neutralisation of newly produced virions; and finally, the combined up-regulation of CMI responses against DHBV core and surface antigens, resulting in elimination of infected hepatocytes. The four manuscripts that comprise this thesis provide insights into the viral kinetics and immune responses that follow DHBV infection and/or vaccination of ducks. The results provide new directions for future vaccine studies aimed at developing effective treatments for chronic HBV infection. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Hepatitis B virus (HBV) is a life-threatening pathogen with major economic significance. Acute infection in adults is common, albeit usually self-limiting. Importantly, infection in infants typically results in chronic infection and increased incidence of hepatocellular carcinoma (HCC). Furthermore, the infectious carrier state is perpetuated in chronically infected individuals. Successful immuno-therapeutic vaccination would reduce the incidence of chronic infection and of HCC as well as reduce transmission of the disease. Recovery from acute and chronic HBV infection typically occurs in the presence of robust antigen-specific humoral and cellular immune responses (CMI), whereas these responses are low or absent in chronically HBV-infected individuals. Therefore, it was hypothesised that effective stimulation of both humoral and CMI responses, in conjunction with currently available antiviral therapies, may contribute significantly to development of vaccines for treatment of chronic HBV infection. The duck hepatitis B virus (DHBV) model of HBV infection was used to test novel vaccine strategies that could complement existing antiviral therapeutic approaches to treat chronically HBV-infected humans. To this end, three separate vaccine studies were conducted to investigate potential therapeutic regimes. Methods to assess the efficacies of the vaccine strategies included immunoperoxidase detection of viral antigen and immune cell markers within the liver and development of sensitive assays to monitor levels of DHBV DNA, duck hepatitis B virus surface antigen (DHBsAg), antibodies to duck hepatitis B core (anti-DHBc) and surface antigens (anti-DHBs) in serum were developed and validated which allowed monitoring of the kinetics of the humoral immune response following vaccination and the course and outcome of experimental DHBV infection. The first vaccine study tested the protective efficacy of DNA vaccines encoding either the small form of DHBsAg (DHBs) protein or the larger antigen (DHBpre-S/S), These were administered to ducks at day 4 and 14 of age. On the same day as the second vaccination, ducks were challenged intravenously with DHBV. Immunoperoxidase staining of biopsy tissue collected at day 4 p.i. showed significant decreases in the number of DHBV infected hepatocytes in ducks receiving the DNA vaccines compared to the mock-vaccinated control ducks. Significant protection against development of chronic DHBV infection was observed in ducks vaccinated with DNA vaccines expressing either pre-S/S or S protein. Although anti-DHBs antibodies were not detected prior to DHBV challenge, the decrease in the percentages of DHBV-infected hepatocytes at day 4 p.i is suggestive that neutralisation of the inoculum by low-level anti-DHBs antibodies in cohort with CMI responses induced by vaccination were the most probable mechanisms of action. The second vaccine study examined the protective efficacy of a novel whole-cell vaccine that expressed the DHBV core antigen (DHBcAg). Ducks were vaccinated on day 4 and 14 of age and DHBV challenge was administered 4 days later. Detectable anti-DHBc antibodies were generated as soon as 4 days after the initial vaccination suggesting that this regimen elicited increased immunogenicity than vaccination with DNA vaccines alone. In contrast to the first vaccine study with DNA vaccines expressing DHBsAg, no significant differences in the percentage of DHBV-infected hepatocytes were observed in biopsy tissue collected at day 4 p.i.This finding is confirmation that anti-DHBc antibodies were not neutralising to the initial DHBV inoculum. However, significant protection against development of chronic DHBV infection was observed in the whole-cell vaccinated ducks suggesting that the mechanism of protection was consistent immune-mediated killing of DHBV-infected hepatocytes following CMI responses to determinants of DHBcAg. The final vaccine study involved a combination strategy of antiviral drug Entecavir (ETV) and prime-boost vaccination with DNA vaccines and recombinant fowlpoxvirus (rFPV) expressing DHBV antigens. Immediately following DHBV infection, ducks were dosed by oral gavage with the antiviral drug Entecavir (ETV) and at the same time received the priming DNA vaccines encoding DHBV antigens. Seven days later the boosting vaccination consisting of recombinant fowlpox viruses (rFPV) also expressing DHBV antigens was administered. Extraordinary protection was observed, with 100% of ducks given combination therapy rapidly resolved their DHBV infection while 100% of non-treated ducks developed chronic infection. It was concluded that protection resulted from a combination of at least three factors. First, reduction and control of DHBV levels with the aid of ETV; secondly, stimulation of surface antigen-specific humoral immune responses resulting in neutralisation of newly produced virions; and finally, the combined up-regulation of CMI responses against DHBV core and surface antigens, resulting in elimination of infected hepatocytes. The four manuscripts that comprise this thesis provide insights into the viral kinetics and immune responses that follow DHBV infection and/or vaccination of ducks. The results provide new directions for future vaccine studies aimed at developing effective treatments for chronic HBV infection. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Thesis (M.D.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 102-116).

Thesis (M.D.S.)--University of Hong Kong, 1996. / Includes bibliographical references (leaves 144-167).

Source: Masters Abstracts International, Volume: 46-04, page: 2101. Adviser: Lu-Yu Hwang. Includes bibliographical references.

Thesis (M.S.)--University of Texas Health Science Center at Houston, School of Public Health, 2008. / Source: Masters Abstracts International, Volume: 46-04, page: 2101. Adviser: Lu-Yu Hwang. Includes bibliographical references.

Thesis (M.D.S.)--University of Hong Kong, 1996. / Includes bibliographical references (leaves 144-167). Also available in print.

Thesis (M.D.)--University of Hong Kong, 1996. / Includes bibliographical references (leaf 102-116). Also available in print.

Lamivudin war der erste Inhibitor der Hepatitis-B-Virus(HBV)-Polymerase, der für die Therapie der chronischen HBV-Infektion in Deutschland zugelassen wurde und seitdem zu einer signifikanten Senkung der HBV-assoziierten Letalität beigetragen hat. Aufgrund des hohen Resistenzrisikos unter Lamivudin wird der Einsatz der Substanz jedoch in den aktuellen HBV-Leitlinien nicht mehr empfohlen. Stattdessen sollen hochpotente Nukleos(t)id-Analoga wie Entecavir oder Tenofovir angewendet werden, die jedoch gerade in den ärmeren Endemiegebieten nur selten verfügbar sind. Zudem gibt es auch in den westlichen Industrieländern Patienten, die seit Jahren resistenzfrei mit Lamivudin behandelt wurden. Ob diese von einer prophylaktischen Therapieumstellung profitieren, ist bislang unklar. Ziel dieser Studie war es, die Langzeiteffektivität der Lamivudin-Therapie sowie die Häufigkeit und die prädiktiven Faktoren der Lamivudin-Resistenz zu ermitteln. In einer retrospektiven Untersuchung wurden hierzu die Verläufe von 147 HBeAg-positiven und 80 HBeAg-negativen Patienten aus 3 hepatologischen Zentren in Deutschland analysiert. Zur Sicherung der Lamivudin-Resistenz erfolgte eine genetische Resistenztestung. Zusätzlich wurde der klinische Nutzen einer hoch-sensitiven Real-Time-PCR zur Bestimmung der HBV-DNA evaluiert. Im Langzeitverlauf zeigte sich bei einer bestimmten Subgruppe von Patienten ein gutes virologisches, serologisches und biochemisches Ansprechen. Die vorliegende Studie bestätigte jedoch auch das hohe Resistenzrisiko unter einer Langzeittherapie mit Lamivudin, welches nach 7 Jahren 55% erreichte. Maßgeblicher prädiktiver Faktor für eine spätere Resistenzentwicklung war das virologische Ansprechen im Therapieverlauf. Entgegen den Ergebnissen früherer Studien stellte die Höhe der HBV-DNA zum Zeitpunkt des Therapiebeginns keinen prädiktiven Faktor der Resistenzentwicklung dar, sondern beeinflusste die Wahrscheinlichkeit des virologischen Ansprechens. Der Einsatz hoch-sensitiver Verfahren zur Bestimmung der HBV-DNA zur Therapiekontrolle oder Prädiktion der Lamivudin-Resistenz ist unseren Beobachtungen nach nicht erforderlich. Bei HBeAg-positiven Patienten hatte zudem ein HBeAg-Verlust einen günstigen Einfluss auf die Resistenzwahrscheinlichkeit. Auffällig war außerdem, dass HBeAg-positive Patienten nach einem virologischen Ansprechen nur noch ein sehr geringes Resistenzrisiko aufwiesen, während bei HBeAg-negativen Patienten trotz eines virologischen Ansprechens im gesamten Therapiezeitraum neue Fälle von Lamivudin-Resistenz beobachtet wurden. Auch durch die Kombination verschiedener Merkmale, die die Resistenzwahrscheinlichkeit nachweislich beeinflussten, konnte letztlich keine Subgruppe identifiziert werden, die im Verlauf resistenzfrei blieb oder nur ein sehr geringes Resistenzrisiko aufwies. Zusätzlich wurden diverse weitere Wirts-, virale und therapieassoziierte Charakteristika untersucht, die jedoch keinen prädiktiven Wert in Hinblick auf eine Resistenzentwicklung aufwiesen. Insbesondere natürlich vorkommende HBV-Mutationen, die mit einer Lamivudin-Resistenz assoziiert sind, wurden bei 9% der unvorbehandelten Patienten nachgewiesen, ohne dass dieser Befund einen signifikanten Einfluss auf das virologische Ansprechen oder die Resistenzwahrscheinlichkeit zeigte. Zusammenfassend ist festzustellen, dass wir in unserer Studie Parameter definieren konnten, die mit einem Langzeitansprechen assoziiert sind und damit die Grundlage einer individualisierten Therapie darstellen könnten. Die Ergebnisse sind vor allem für Länder mit eingeschränkten Therapiemöglichkeiten von Relevanz. Sofern höher potentere Nukleos(t)idanaloga zur Verfügung stehen, sollte in Anbetracht des hohen Resistenzrisikos allerdings von einer Therapie mit Lamivudin abgesehen werden. Liegt bereits eine langjährige Lamivudin-Therapie ohne Zeichen einer Resistenzentwicklung vor, so erscheint die Fortsetzung jedoch gerechtfertigt. Dennoch sind regelmäßige Kontrollen erforderlich, da Lamivudin-Resistenzen auch nach mehr als 10 Jahren auftreten können.