Hepatitis B virus (HBV) vaccination is recommended for individuals with hepatitis C virus (HCV) infection given their shared risk factors and increased liver-related morbidity and mortality upon super-infection. Vaccine responses in this setting are often blunted, with poor response rates to HBV vaccinations in chronically HCV-infected individuals compared to healthy subjects. In this study, we investigated the role of T cell immunoglobulin mucin domain-3 (Tim-3)-mediated immune regulation in HBV vaccine responses during HCV infection. We found that Tim-3, a marker for T cell exhaustion, was over-expressed on monocytes, leading to a differential regulation of IL-12/IL-23 production which in turn TH17 cell accumulation, in HCV-infected HBV vaccine non-responders compared to HCV-infected HBV vaccine responders or healthy subjects (HS). Importantly, ex vivo blockade of Tim-3 signaling corrected the imbalance of IL-12/IL-23 as well as the IL-17 bias observed in HBV vaccine non-responders during HCV infection. These results suggest that Tim-3-mediated dysregulation of innate to adaptive immune responses is involved in HBV vaccine failure in individuals with chronic HCV infection, raising the possibility that blocking this negative signaling pathway might improve the success rate of HBV immunization in the setting of chronic viral infection.

Hepatitis B virus (HBV) vaccination is recommended for individuals with hepatitis C virus (HCV) infection given their shared risk factors and increased liver-related morbidity and mortality upon super-infection. Vaccine responses in this setting are often blunted, with poor response rates to HBV vaccinations in chronically HCV-infected individuals compared to healthy subjects. In this study, we investigated the role of T cell immunoglobulin mucin domain-3 (Tim-3)-mediated immune regulation in HBV vaccine responses during HCV infection. We found that Tim-3, a marker for T cell exhaustion, was over-expressed on monocytes, leading to a differential regulation of IL-12/IL-23 production which in turn TH17 cell accumulation, in HCV-infected HBV vaccine non-responders compared to HCV-infected HBV vaccine responders or healthy subjects (HS). Importantly, ex vivo blockade of Tim-3 signaling corrected the imbalance of IL-12/IL-23 as well as the IL-17 bias observed in HBV vaccine non-responders during HCV infection. These results suggest that Tim-3-mediated dysregulation of innate to adaptive immune responses is involved in HBV vaccine failure in individuals with chronic HCV infection, raising the possibility that blocking this negative signaling pathway might improve the success rate of HBV immunization in the setting of chronic viral infection.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and chronic hepatitis B virus (HBV) infection is the major risk factor of HCC. The virus encodes HBV X (HBx) protein that plays a critical role in the development of HCC. Studies have revealed numerous HBx-altered genes and signalling pathways that heavily contribute to tumourigenesis of non-tumour hepatocytes. However, the role of HBx in regulating other critical gene regulators such as microRNAs is poorly understood, which impedes the exploration of a complete HBx-associated carcinogenic network. Besides, critical microRNAs that drive the transformation of non-tumour hepatocytes are yet to be identified. Here, we overexpressed C-terminal truncated HBx protein in a non-tumour hepatocyte cell line MIHA, and measured a panel of cancer-associated miRNAs. We observed that oncogenic miR-21 was upregulated upon ectopic expression of this viral protein variant. HBx-miR-21 pathway was prevalent in HCC cells as inhibition of HBx in Hep3B and PLC/PRF/5 cells significantly suppressed miR-21 expression. Subsequently, we showed that the upregulation of miR-21 was mediated by HBx-induced interleukin-6 pathway followed by activation of STAT3 transcriptional factor. The high dependency of miR-21 expression to HBx protein suggested a unique viral oncogenic pathway that could aberrantly affect a network of gene expression. Importantly, miR-21 was essential in the HBx-induced transformation of non-tumour hepatocytes. Inhibition of miR-21 effectively attenuated anchorage-independent colony formation and subcutaneous tumour growth of MIHA cells. Our study suggested that overexpression of miR-21 was critical to promote early carcinogenesis of hepatocytes upon HBV infection.

Vaccination for hepatitis B virus (HBV) in the setting of hepatitis C virus (HCV) infection is recommended, but responses to vaccination are blunted when compared to uninfected populations. The mechanism for this failure of immune response in HCV-infected subjects remains unknown but is thought to be a result of lymphocyte dysfunction during chronic viral infection. We have recently demonstrated that PD-1, a novel negative immunomodulator for T cell receptor (TCR) signaling, is involved in T and B lymphocyte dysregulation during chronic HCV infection. In this report, we further investigated the role of the PD-1 pathway in regulation of CD4+ T cell responses to HBV vaccination in HCV-infected individuals. In a prospective HCV infected cohort, a poor response rate to HBV vaccination as assayed by seroconversion was observed in HCV-infected subjects (53%), while a high response rate was observed in healthy or spontaneously HCV-resolved individuals (94%). CD4+ T cell responses to ex vivo stimulations of anti-CD3/CD28 antibodies or hepatitis B surface antigen (HBsAg) were found to be lower in HBV vaccine non-responders compared to those responders in HCV-infected individuals who had received a series of HBV immunizations. PD-1 expression on CD4+ T cells was detected at relatively higher levels in these HBV vaccine non-responders than those who responded, and this was inversely associated with the cell activation status. Importantly, blocking the PD-1 pathway improved T cell activation and proliferation in response to ex vivo HBsAg or anti-CD3/CD28 stimulation in HBV vaccine non-responders. These results suggest that PD-1 signaling may be involved in impairing CD4+ T cell responses to HBV vaccination in subjects with HCV infection, and raise the possibility that blocking this negative signaling pathway might improve success rates of immunization in the setting of chronic viral infection.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. It mainly infects young adults and has a mortality of up to 25% in pregnant women. Although hepatitis E is only sporadic in industrialized countries including the United States, a relative high seroprevalence rate has been reported in healthy individuals. Evidence suggests that there exist animal reservoirs for HEV and HEV transmission is zoonotic. Animal strains of HEV, swine HEV and avian HEV have been identified from a pig and a chicken, respectively, in the United States. Studies showed that swine HEV and avian HEV are genetically and antigenically related to human HEV, and that pigs and chickens are useful animal models to study HEV replication, pathogenesis and cross-species infection. The objectives of this dissertation were to genetically characterize both avian HEV and swine HEV, to determine their serological and molecular epidemiology in the United States, to assess the ability of avian HEV cross-species infection in non-human primates, to determine the full-length genomic sequence and genome organization, and to construct an infectious cDNA clone of avian HEV. The prevalence of swine HEV infections in US swine herds and the heterogeneity of swine HEV isolates from different geographic regions of the United States were determined. We found that 35% pigs and 54% swine herds were positive for swine HEV RNA. Partial capsid gene region of twenty-seven US swine HEV isolates was sequenced and was showed to share 88%-100% nucleotide sequence identity to each other and 89-98% identity with the prototype US swine HEV, but only <79% identity with Taiwanese swine HEV isolates and most known human strains of HEV worldwide. All US swine HEV isolates belong to the same genotype 3 with the prototype US swine HEV and the two US strains of human HEV. Similarly, the prevalence of avian HEV infections in US chicken flocks and the heterogeneity of avian HEV isolates were also determined. Helicase gene region of eleven field isolates of avian HEV from chickens with hepatitis-splenomegaly (HS) syndrome was sequenced and was found to share 78-100% nucleotide sequence identities with each other, 79-88% identities with the prototype avian HEV, 76-80% identities with Australian chicken big liver and spleen disease virus (BLSV), and 56-61% identities with other known strains of mammalian HEV. A relative high prevalence of anti-avian HEV antibodies was found in apparently healthy chicken flocks in 5 states. Like swine HEV, the seropositivity of avian HEV in adult chickens was higher than that in young chickens. To genetically characterize the avian HEV genome, we determined the full-length genomic sequence of avian HEV, which is 6,654 bp in length excluding the poly (A) tail, and 600 bp shorter than that of mammalian HEVs. Avian HEV has similar genomic organization with human and swine HEVs, but shared only about 50% nucleotide sequence identity with mammalian HEVs in the complete genome. Significant genetic variations such as deletions and insertions, particularly in the ORF1 of avian HEV, were observed, but motifs in the putative functional domains of the ORF1 were relatively conserved between avian HEV and mammalian HEVs. Phylogenetic analyses based on the full-length genomic sequence revealed that avian HEV represents a branch distinct from human and swine HEVs. Since swine HEV infects non-human primates and possibly humans, the ability of avian HEV cross-species infection in non-human primates was also assessed. However, unlike swine HEV, avian HEV failed to infect two rhesus monkeys under experimental conditions. With the availability of the complete genome sequence of avian HEV, we constructed three full-length cDNA clones of avian HEV and tested their infectivity by in vitro transfection of the LMH chicken liver cells and by in vivo intrahepatic inoculation of specific-pathogen-free (SPF) chickens. The results showed that all 3 cDNA clones of avian HEV were infectious both in vitro and in vivo, as the capped RNA transcripts from each of the clones were replication-competent in transfected LMH cells and developed active infection in inoculated SPF chickens. In summary, avian HEV and swine HEV infections are enzootic in chicken flocks and in swine herds in the United States, respectively. Like human HEV, swine HEV and avian HEV isolates from different geographic regions are also genetically heterogenic. Complete genomic sequence analyses showed that avian HEV is related to, but distinct from, human and swine HEVs. Unlike swine HEV, avian HEV is probably not transmissible to non-human primates. Infectious cDNA clones of avian HEV have been successfully constructed. The availability of the infectious clones for a chicken strain of HEV now affords us an opportunity to study the mechanisms of HEV replication, pathogenesis and cross-species infection. / Ph. D.

The purpose of this thesis is to scope the literature to understand how people who use injection drug (PWIDs) experience access to hepatitis C (HCV) testing and diagnosis. The design was a scoping review methodology, guided by Arksey and O’Malley, JBI, and PRISMA-ScR guidelines. A search was conducted through seven electronic databases using a peer-reviewed search strategy. Five studies were yielded through two-level screenings. The extracted data were synthesized using conventional data analysis and reported using tables and narrative summaries. Four categories were found: Awareness and Knowledge, Stigma, Healthcare Service, and Psychological Responses. The studies were conducted in Australia, UK, and US published between 2014 to 2018. A total of 19 participant characteristics were extracted to contextualize their experiences. The World Health Organization’s definition of accessibility should be defined through the guidance of the constructed truths of the individuals in the current context. The lack of demographic data and connection to client quotes further exacerbates the inequities among the population through overlooking their intragroup identities.

Una correcta inmunización de la población reclusa minimiza el riesgo de transmisión de enfermedades vacunables en las prisiones. El indudable beneficio de la vacunación como medida de salud pública en términos de eficacia y coste-efectividad tiene particular interés en el ambiente carcelario. La población reclusa reúne características que incrementan su vulnerabilidad para padecer enfermedades transmisibles, algunas de ellas prevenibles mediante inmunización. Su paso por la cárcel representa para este grupo de población una excelente oportunidad para entrar en contacto con el sistema de salud ya que es un colectivo difícilmente accesible en la comunidad. La importancia de la vacunación en las prisiones reporta un beneficio a la sociedad ya que su contacto con el exterior es frecuente. Ser una comunidad cerrada facilita la puesta en marcha de actividades de inmunización a cargo de los sanitarios de las instituciones penitenciarias. El objetivo de este estudio fue evaluar la cobertura vacunal en presos de larga duración del sistema penitenciario español, mediante un estudio longitudinal retrospectivo. Fueron seleccionados 1005 internos que permanecieron ingresados entre 2008 y 2018 en tres cárceles españolas. Se evaluó su nivel de inmunización frente a la hepatitis A, hepatitis B, tétanos, difteria, neumococo e influenza estacional. Se valoró también el estado vacunal de los presos con diagnóstico serológico de HBV, HCV y HIV positivos.

Objetivo: El personal sanitario es un colectivo profesional expuesto a riesgos ocupacionales. Los objetivos de esta tesis fueron analizar la ausencia de seroprotección frente al sarampión, la rubeola, la parotiditis, la varicela, la hepatitis A y la hepatitis B de los trabajadores sanitarios vinculados a dos Departamentos de Salud de la Comunidad Valenciana, considerando edad, sexo, categoría profesional, riesgo laboral y área de desempeño e identificar, visualizar y describir patrones de coocurrencia de la ausencia de seroprotección al sarampión, la rubeola, la parotiditis, la varicela, la hepatitis A y la hepatitis B, junto con el sexo, la edad, la categoría profesional, el riesgo en el puesto de trabajo y el área de desempeño profesional. Métodos: Estudio observacional transversal. Datos recogidos de informes de vigilancia de la salud, que incluyen resultados serológicos de 2674 trabajadores. Las variables sociodemográficas fueron: sexo, grupos de edad (18-34; 35-49; >50 años), clasificación del servicio (riesgo/no riesgo), categoría profesional, área de desempeño profesional (atención primaria/atención hospitalaria); la variable dependiente fue ausencia de inmunización frente a sarampión, rubeola y parotiditis, varicela, hepatitis A y hepatitis B identificada por ausencia de niveles serológicos. Se realizó un análisis de regresión logística. Para valorar la coocurrencia de sarampión, rubeola, parotiditis, varicela, hepatitis A y hepatitis B se consideró un análisis de redes. Resultados: La prevalencia de no inmunización fue del 7,8%, 3,7%, 16,1%, 2,7%, 54,4% y 13,1% para el sarampión, la rubeola, la parotiditis, la varicela, la hepatitis A y la hepatitis B respectivamente. El análisis ajustado mediante regresión logística mostró diferencias significativas en la prevalencia de no inmunización a sarampión en función de grupos de edad (ORa 11,8 y ORa 5,8) y área de desempeño (ORa 0,5), frente a parotiditis en función de grupos de edad (ORa 4,9 y ORa 3,6) y categoría profesional 1 (médicos, farmacéuticos, odontólogos y psicólogos) (ORa 0,6), frente a rubeola en función del sexo (ORa 4,6), frente a varicela en función de la clasificación del servicio (ORa 2,3), frente a hepatitis A en función de grupos de edad (ORa 1,9 y ORa 1,5) y sexo (ORa 1,2) y frente a hepatitis B en función de grupos de edad (ORa 0,3 y ORa 0,5), sexo (ORa 1,4), área de desempeño (ORa 0,3) y categorías profesionales 1 (médicos, farmacéuticos, odontólogos y psicólogos) (ORa 0,2), 2 (enfermería, fisioterapeutas, nutricionistas y optometristas) (ORa 0,2) y 3 (técnicos en cuidados auxiliares de enfermería y técnicos especialistas sanitarios) (ORa 0,3). Considerando el efecto del sexo, la edad, categoría profesional, servicio de riesgo y área de desempeño, se observó una asociación positiva entre sarampión-rubeola, sarampión-parotiditis y rubeola-parotiditis, y una asociación positiva débil entre hepatitis A-hepatitis B en la comparación de las categorías profesionales 2 (enfermería, fisioterapeutas, nutricionistas y optometristas) y 4 (otros grupos profesionales). Conclusiones: Es necesario potenciar la vacunación entre sanitarios, especialmente en aquellos que ejercen sus funciones en áreas de riesgo y entre el personal de menor edad. La vacunación debe promoverse dentro de políticas de seguridad laboral.

A infecção pelo HEV é reconhecida como um considerável problema de saúde pública em diversas regiões do mundo. Embora caracterizada como uma infecção benigna com um curso evolutivo autolimitado, recentes estudos têm mostrado sua evolução para cronicidade em indivíduos imunocomprometidos. Além disso, tem sido verificado que nesses indivíduos a infecção crônica pelo HEV pode evoluir para fibrose hepática progressiva, culminando com o desenvolvimento de cirrose. Não existem dados acerca da prevalência da infecção pelo HEV em pacientes infectados pelo HIV no Brasil, onde a circulação deste vírus tem sido demonstrada em diversos grupos de indivíduos imunocompetentes e, até mesmo, em alguns animais provenientes de diferentes regiões do país. Com base nisso, este trabalho teve como objetivo estimar a prevalência de marcadores sorológicos e moleculares da infecção pelo HEV, bem como a padronização de uma PCR em tempo real para a detecção e quantificação da carga viral do HEV na população de soropositivos da cidade de São Paulo. Foram incluídos neste estudo soro e plasma de pacientes infectados pelo HIV (n=354), que foram divididos em grupos de acordo com a presença ou ausência de coinfecção pelos vírus das hepatites B (HBV) e C (HCV). Essas amostras foram coletadas entre 2007 e 2013. Anticorpos anti-HEV IgM e IgG foram pesquisados pela técnica de ELISA (RecomWell HEV IgM/ IgG - MIKROGEN®), e, em alguns casos, confirmados por Immunoblotting (RecomLine HEV IgM/ IgG - MIKROGEN®). Todas as amostras foram submetidas à pesquisa de HEV RNA através da PCR em tempo real padronizada. Cerca de 72% dos indivíduos avaliados pertenciam ao sexo masculino. A média de idade entre a população analisada foi de 48,4 anos. Os anticorpos anti-HEV IgM e IgG foram encontrados em 1,4% e 10,7% dos indivíduos dessa população, respectivamente. Apenas dois pacientes apresentaram positividade simultânea para anti-HEV IgM e IgG. Não houve diferença estatisticamente relevante quanto à presença de marcadores sorológicos nos grupos de estudo. Além disso, foi detectado o HEV RNA em 10,7% das amostras analisadas, entre as quais, seis apresentaram simultaneamente algum marcador sorológico (5 anti-HEV IgG e 1 IgM). A presença deste marcador foi predominante no grupo de pacientes com coinfecção pelo HCV. Através deste trabalho pôde-se constatar, portanto, que o HEV é circulante entre a população de infectados pelo HIV em São Paulo, e que o seguimento desses pacientes se faz necessário dado a possibilidade de progressão para infecção crônica e cirrose / HEV infection is recognized as a significant public health problem in different world regions. Although initially characterized as a benign infection with selflimited course, recent studies have showing its evolution to chronicity in immunocompromised individuals. Furthermore, in these individuals the chronic infection can develop progressive liver fibrosis leading to cirrhosis. There are no data regarding prevalence of HEV infections in HIV- infected patients in Brazil, where the circulation of this virus has been demonstrated in different individuals groups and in some animals from different regions of the country. Based on this, this study aimed to assess the prevalence of serological and molecular makers of HEV infection and the standardization of real-time PCR for the detection and quantification of HEV viral load in HIV-infected individuals in São Paulo. Serum and plasma samples of HIV-infected patients (n=354), collected between 2007 and 2013, were included and organized in groups of co-infection (HIV/ HBV, HIV/HCV and HIV/ HBV/ HCV) and HIV mono-infection. Antibodies anti-HEV IgM and IgG were detected by ELISA (RecomWell HEV IgM/ IgG - MIKROGEN®), and in some cases confirmed by immunoblotting (RecomLine HEV IgM/ IgG - MIKROGEN®). All samples were submitted to research HEV RNA by real-time PCR. About 72% of the patients were male. The mean age of this population was 48.4 years. The anti-HEV IgM and IgG antibodies were found in 1.4% and 10.7%, respectively. Only two patients presented simultaneous anti-HEV IgM and anti- HEV IgG. There was no statistically significant difference in the presence of serological makers among the HIV infection groups. In addition, HEV RNA was detected in 10.7% of samples and six of these samples presented simultaneously a serological maker (5 anti-HEV IgG and 1 IgM). The presence of this maker was more frequent in the co-infection HIV/ HCV group. Through this work, we observed that HEV is circulating among the HIV-infected population in São Paulo, and the monitoring these patients is necessary because of the possibility progression to chronic infection and cirrhosis

A infecção pelo HEV é reconhecida como um considerável problema de saúde pública em diversas regiões do mundo. Embora caracterizada como uma infecção benigna com um curso evolutivo autolimitado, recentes estudos têm mostrado sua evolução para cronicidade em indivíduos imunocomprometidos. Além disso, tem sido verificado que nesses indivíduos a infecção crônica pelo HEV pode evoluir para fibrose hepática progressiva, culminando com o desenvolvimento de cirrose. Não existem dados acerca da prevalência da infecção pelo HEV em pacientes infectados pelo HIV no Brasil, onde a circulação deste vírus tem sido demonstrada em diversos grupos de indivíduos imunocompetentes e, até mesmo, em alguns animais provenientes de diferentes regiões do país. Com base nisso, este trabalho teve como objetivo estimar a prevalência de marcadores sorológicos e moleculares da infecção pelo HEV, bem como a padronização de uma PCR em tempo real para a detecção e quantificação da carga viral do HEV na população de soropositivos da cidade de São Paulo. Foram incluídos neste estudo soro e plasma de pacientes infectados pelo HIV (n=354), que foram divididos em grupos de acordo com a presença ou ausência de coinfecção pelos vírus das hepatites B (HBV) e C (HCV). Essas amostras foram coletadas entre 2007 e 2013. Anticorpos anti-HEV IgM e IgG foram pesquisados pela técnica de ELISA (RecomWell HEV IgM/ IgG - MIKROGEN®), e, em alguns casos, confirmados por Immunoblotting (RecomLine HEV IgM/ IgG - MIKROGEN®). Todas as amostras foram submetidas à pesquisa de HEV RNA através da PCR em tempo real padronizada. Cerca de 72% dos indivíduos avaliados pertenciam ao sexo masculino. A média de idade entre a população analisada foi de 48,4 anos. Os anticorpos anti-HEV IgM e IgG foram encontrados em 1,4% e 10,7% dos indivíduos dessa população, respectivamente. Apenas dois pacientes apresentaram positividade simultânea para anti-HEV IgM e IgG. Não houve diferença estatisticamente relevante quanto à presença de marcadores sorológicos nos grupos de estudo. Além disso, foi detectado o HEV RNA em 10,7% das amostras analisadas, entre as quais, seis apresentaram simultaneamente algum marcador sorológico (5 anti-HEV IgG e 1 IgM). A presença deste marcador foi predominante no grupo de pacientes com coinfecção pelo HCV. Através deste trabalho pôde-se constatar, portanto, que o HEV é circulante entre a população de infectados pelo HIV em São Paulo, e que o seguimento desses pacientes se faz necessário dado a possibilidade de progressão para infecção crônica e cirrose / HEV infection is recognized as a significant public health problem in different world regions. Although initially characterized as a benign infection with selflimited course, recent studies have showing its evolution to chronicity in immunocompromised individuals. Furthermore, in these individuals the chronic infection can develop progressive liver fibrosis leading to cirrhosis. There are no data regarding prevalence of HEV infections in HIV- infected patients in Brazil, where the circulation of this virus has been demonstrated in different individuals groups and in some animals from different regions of the country. Based on this, this study aimed to assess the prevalence of serological and molecular makers of HEV infection and the standardization of real-time PCR for the detection and quantification of HEV viral load in HIV-infected individuals in São Paulo. Serum and plasma samples of HIV-infected patients (n=354), collected between 2007 and 2013, were included and organized in groups of co-infection (HIV/ HBV, HIV/HCV and HIV/ HBV/ HCV) and HIV mono-infection. Antibodies anti-HEV IgM and IgG were detected by ELISA (RecomWell HEV IgM/ IgG - MIKROGEN®), and in some cases confirmed by immunoblotting (RecomLine HEV IgM/ IgG - MIKROGEN®). All samples were submitted to research HEV RNA by real-time PCR. About 72% of the patients were male. The mean age of this population was 48.4 years. The anti-HEV IgM and IgG antibodies were found in 1.4% and 10.7%, respectively. Only two patients presented simultaneous anti-HEV IgM and anti- HEV IgG. There was no statistically significant difference in the presence of serological makers among the HIV infection groups. In addition, HEV RNA was detected in 10.7% of samples and six of these samples presented simultaneously a serological maker (5 anti-HEV IgG and 1 IgM). The presence of this maker was more frequent in the co-infection HIV/ HCV group. Through this work, we observed that HEV is circulating among the HIV-infected population in São Paulo, and the monitoring these patients is necessary because of the possibility progression to chronic infection and cirrhosis