Generation of monoclonal antibodies against native viral proteins using antigen-expressing mammalian cells for mouse immunization

Due to their rising incidence and progressive geographical spread, infections with mosquito-borne viruses, such as dengue (DENV), chikungunya and zika virus, have developed into major public health challenges. Since all of these viruses may cause similar symptoms and can occur in concurrent epidemics, tools for their differential diagnosis and epidemiological monitoring are of urgent need.; Here we report the application of a novel strategy to rapidly generate monoclonal antibodies (mAbs) against native viral antigens, exemplified for the DENV nonstructural glycoprotein 1 (NS1). The described system is based on the immunization of mice with transfected mammalian cells expressing the target antigens in multiple displays on their cell surface and thereby presenting them efficiently to the host immune system in their native conformation. By applying this cell-based approach to the DENV NS1 protein of serotypes 1 (D1NS1) and 4 (D4NS1), we were able to rapidly generate panels of DENV NS1 serotype cross-reactive, as well as D1NS1- and D4NS1 serotype-specific mAbs. Our data show that the generated mAbs were capable of recognizing the endogenous NS1 protein in DENV-containing biological samples.; The use of this novel immunization strategy, allows for a fast and efficient generation of hybridoma cell lines, producing mAbs against native viral antigens. Envisaged applications of the mAbs include the development of test platforms enabling a differentiation of the DENV serotypes and high resolution immunotyping for epidemiological studies

Healthcare Access and Quality Index based on mortality from causes amenable to personal health care in 195 countries and territories, 1990-2015 : a novel analysis from the Global Burden of Disease Study 2015

Background National levels of personal health-care access and quality can be approximated by measuring mortality rates from causes that should not be fatal in the presence of effective medical care (ie, amenable mortality). Previous analyses of mortality amenable to health care only focused on high-income countries and faced several methodological challenges. In the present analysis, we use the highly standardised cause of death and risk factor estimates generated through the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) to improve and expand the quantification of personal health-care access and quality for 195 countries and territories from 1990 to 2015. Methods We mapped the most widely used list of causes amenable to personal health care developed by Nolte and McKee to 32 GBD causes. We accounted for variations in cause of death certification and misclassifications through the extensive data standardisation processes and redistribution algorithms developed for GBD. To isolate the effects of personal health-care access and quality, we risk-standardised cause-specific mortality rates for each geography-year by removing the joint effects of local environmental and behavioural risks, and adding back the global levels of risk exposure as estimated for GBD 2015. We employed principal component analysis to create a single, interpretable summary measure-the Healthcare Quality and Access (HAQ) Index-on a scale of 0 to 100. The HAQ Index showed strong convergence validity as compared with other health-system indicators, including health expenditure per capita (r= 0.88), an index of 11 universal health coverage interventions (r= 0.83), and human resources for health per 1000 (r= 0.77). We used free disposal hull analysis with bootstrapping to produce a frontier based on the relationship between the HAQ Index and the Socio-demographic Index (SDI), a measure of overall development consisting of income per capita, average years of education, and total fertility rates. This frontier allowed us to better quantify the maximum levels of personal health-care access and quality achieved across the development spectrum, and pinpoint geographies where gaps between observed and potential levels have narrowed or widened over time. Findings Between 1990 and 2015, nearly all countries and territories saw their HAQ Index values improve; nonetheless, the difference between the highest and lowest observed HAQ Index was larger in 2015 than in 1990, ranging from 28.6 to 94.6. Of 195 geographies, 167 had statistically significant increases in HAQ Index levels since 1990, with South Korea, Turkey, Peru, China, and the Maldives recording among the largest gains by 2015. Performance on the HAQ Index and individual causes showed distinct patterns by region and level of development, yet substantial heterogeneities emerged for several causes, including cancers in highest-SDI countries; chronic kidney disease, diabetes, diarrhoeal diseases, and lower respiratory infections among middle-SDI countries; and measles and tetanus among lowest-SDI countries. While the global HAQ Index average rose from 40.7 (95% uncertainty interval, 39.0-42.8) in 1990 to 53.7 (52.2-55.4) in 2015, far less progress occurred in narrowing the gap between observed HAQ Index values and maximum levels achieved; at the global level, the difference between the observed and frontier HAQ Index only decreased from 21.2 in 1990 to 20.1 in 2015. If every country and territory had achieved the highest observed HAQ Index by their corresponding level of SDI, the global average would have been 73.8 in 2015. Several countries, particularly in eastern and western sub-Saharan Africa, reached HAQ Index values similar to or beyond their development levels, whereas others, namely in southern sub-Saharan Africa, the Middle East, and south Asia, lagged behind what geographies of similar development attained between 1990 and 2015. Interpretation This novel extension of the GBD Study shows the untapped potential for personal health-care access and quality improvement across the development spectrum. Amid substantive advances in personal health care at the national level, heterogeneous patterns for individual causes in given countries or territories suggest that few places have consistently achieved optimal health-care access and quality across health-system functions and therapeutic areas. This is especially evident in middle-SDI countries, many of which have recently undergone or are currently experiencing epidemiological transitions. The HAQ Index, if paired with other measures of health-systemcharacteristics such as intervention coverage, could provide a robust avenue for tracking progress on universal health coverage and identifying local priorities for strengthening personal health-care quality and access throughout the world. Copyright (C) The Author(s). Published by Elsevier Ltd.Peer reviewe

Global, regional, and national age-sex-specific mortality and life expectancy, 1950–2017: a systematic analysis for the Global Burden of Disease Study 2017

© 2018 The Author(s). Background: Assessments of age-specifc mortality and life expectancy have been done by the UN Population Division, Department of Economics and Social Afairs (UNPOP), the United States Census Bureau, WHO, and as part of previous iterations of the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD). Previous iterations of the GBD used population estimates from UNPOP, which were not derived in a way that was internally consistent with the estimates of the numbers of deaths in the GBD. The present iteration of the GBD, GBD 2017, improves on previous assessments and provides timely estimates of the mortality experience of populations globally. Methods: The GBD uses all available data to produce estimates of mortality rates between 1950 and 2017 for 23 age groups, both sexes, and 918 locations, including 195 countries and territories and subnational locations for 16 countries. Data used include vital registration systems, sample registration systems, household surveys (complete birth histories, summary birth histories, sibling histories), censuses (summary birth histories, household deaths), and Demographic Surveillance Sites. In total, this analysis used 8259 data sources. Estimates of the probability of death between birth and the age of 5 years and between ages 15 and 60 years are generated and then input into a model life table system to produce complete life tables for all locations and years. Fatal discontinuities and mortality due to HIV/AIDS are analysed separately and then incorporated into the estimation. We analyse the relationship between age-specifc mortality and development status using the Socio-demographic Index, a composite measure based on fertility under the age of 25 years, education, and income. There are four main methodological improvements in GBD 2017 compared with GBD 2016: 622 additional data sources have been incorporated; new estimates of population, generated by the GBD study, are used; statistical methods used in diferent components of the analysis have been further standardised and improved; and the analysis has been extended backwards in time by two decades to start in 1950. Findings: Globally, 18·7% (95% uncertainty interval 18·4-19·0) of deaths were registered in 1950 and that proportion has been steadily increasing since, with 58·8% (58·2-59·3) of all deaths being registered in 2015. At the global level, between 1950 and 2017, life expectancy increased from 48·1 years (46·5-49·6) to 70·5 years (70·1-70·8) for men and from 52·9 years (51·7-54·0) to 75·6 years (75·3-75·9) for women. Despite this overall progress, there remains substantial variation in life expectancy at birth in 2017, which ranges from 49·1 years (46·5-51·7) for men in the Central African Republic to 87·6 years (86·9-88·1) among women in Singapore. The greatest progress across age groups was for children younger than 5 years; under-5 mortality dropped from 216·0 deaths (196·3-238·1) per 1000 livebirths in 1950 to 38·9 deaths (35·6-42·83) per 1000 livebirths in 2017, with huge reductions across countries. Nevertheless, there were still 5·4 million (5·2-5·6) deaths among children younger than 5 years in the world in 2017. Progress has been less pronounced and more variable for adults, especially for adult males, who had stagnant or increasing mortality rates in several countries. The gap between male and female life expectancy between 1950 and 2017, while relatively stable at the global level, shows distinctive patterns across super-regions and has consistently been the largest in central Europe, eastern Europe, and central Asia, and smallest in south Asia. Performance was also variable across countries and time in observed mortality rates compared with those expected on the basis of development. Interpretation: This analysis of age-sex-specifc mortality shows that there are remarkably complex patterns in population mortality across countries. The fndings of this study highlight global successes, such as the large decline in under-5 mortality, which refects signifcant local, national, and global commitment and investment over several decades. However, they also bring attention to mortality patterns that are a cause for concern, particularly among adult men and, to a lesser extent, women, whose mortality rates have stagnated in many countries over the time period of this study, and in some cases are increasing

Torque Teno Virus em amostras fezes de pacientes com gastroenterite : prevalência, distribuição por genogrupos e carga viral

Submitted by Anderson Silva ([email protected]) on 2012-10-23T13:30:15Z No. of bitstreams: 1 carlos_a_p_nascimento_ioc_bcm_0045_2011.pdf: 1006058 bytes, checksum: e670c764b773d1d5bd9fa107d877a76b (MD5)Made available in DSpace on 2012-10-23T13:30:15Z (GMT). No. of bitstreams: 1 carlos_a_p_nascimento_ioc_bcm_0045_2011.pdf: 1006058 bytes, checksum: e670c764b773d1d5bd9fa107d877a76b (MD5) Previous issue date: 2011CNPq FAPERJFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.O Torque teno virus (TTV) é um vírus de DNA do gênero Alphatorquevirus da família Anelloviridae. O TTV é altamente prevalente em populações de todo o mundo. Isolados foram classificados em cinco grupos filogenéticos (1-5) com grande distância genética entre eles. A presença do TTV já foi detectada nas fezes, porém, não se sabe se todos os cinco genogrupos do TTV são excretados nas fezes, e qual a distribuição do TTV entre os genogrupos. A fim de avaliar a presença, a diversidade genômica e a carga viral do TTV em fezes, 135 amostras de pacientes com gastroenterite foram analisadas. O DNA do TTV foi extraído de suspensão fecal e três diferentes métodos de reação em cadeia da polimerase (PCR), dois qualitativos e um quantitativo, foram avaliados. Nas amostras de fezes estudadas, 123 (91,1%) foram positivas em pelo menos um dos três métodos. O DNA do TTV pertencente aos genogrupos de 1 a 5 foi detectado em 37 (27,4%), 27 (20,0%), 57 (42,2%), 29 (21,5%) e 33 (24,4%) amostras, respectivamente. Co-infecções com dois, três, quatro e cinco genogrupos do TTV foram encontradas em 23 (17,0%), 15 (11,1%), 7 (5,2%) e 7 (5,2%) amostras fecais, respectivamente. Assim, 52 (38,5%) amostras continham mais de um genogrupo de TTV. A carga viral variou de 2,6 a 6,5 log de genoma equivalentes por grama de fezes. No entanto, variações de carga viral foram observadas em função do genogrupo detectado e do número de genogrupos presentes simultaneamente. Os resultados encontrados são os primeiros a mostrar a alta prevalência e a diversidade de TTV nas fezes humanas.Torque teno virus (TTV) is a DNA virus of the genus Alphatorquevirus of Anelloviridae family. The TTV is highly prevalent in populations from around the world. Isolates have been classified into at least five main phylogenetic groups (1-5) showing a large genetic distance between them. The presence of TTV has been detected in feces. However, are presently unknown whether all five TTV genogroups are excreted in feces and the genogroup distribution. To evaluate the presence and the genomic distribution of TTV DNA in feces, 135 samples of patients with gastroenteritis were analyzed. The DNA was extracted of fecal suspension and three different PCR methods, two qualitative and one quantitative, were used. One hundred and twenty three (91.1%) samples were positive with at least one method. The TTV DNA belonging to the genogroups 1 to 5 was detected in 37 (27.4%), 27 (20.0%), 57 (42.2%), 29 (21.5%) and 33 (24.4%) fecal samples, respectively. Coinfections with two, three, four and five TTV genogroups were found in 23 (17.0%), 15 (11.1%), 7 (5.2%) and 7 (5.2%) fecal samples, respectively. Thus, 52 (38.5%) samples contained more than one TTV genogroup. Viral loads ranged from 2.6 to 6.5 log genome equivalents per gram of feces. However, variations of viral load were noted depending on genogroup and number of coinfecting TTV genogroups. These results are the first to show high prevalence and the diversity of TTV isolates in human feces

Development of dengue virus serotype-specific NS1 capture assays for the rapid and highly sensitive identification of the infecting serotype in human sera

Dengue fever can be caused by one of four distinct dengue virus (DENV) serotypes that cocirculate in many parts of the world. Point of care serotype-specific nonstructural protein-1 (NS1) capture assays for the rapid serotyping of DENV in human sera would greatly support epidemiological surveillance and potentially also prognosis in individual patients. To ensure both serotype specificity and broad coverage of variants within serotypes, we have applied an innovative approach for the generation and selection of serotype-specific anti-NS1 mAbs. To elicit mAbs against conformational epitopes, NMRI mice were immunized with living HEK 293 transfectants expressing the native target Ags in multiple display on the cell surface. For each serotype, three different NS1 sequence variants were sequentially used for immunization of mice, hybridoma selection, and capture assay development, respectively. Selection of optimal combinations of capturing and detecting mAbs yielded highly sensitive and specific NS1 serotyping ELISAs (st-ELISAs) for the four serotypes. st-ELISA testing of 41 dengue patient sera showed a 100% concordance with the serotype determined by serotype-specific reverse transcriptase real-time quantitative PCR. The respective NS1 variants could be detected for ∼10 d after the onset of illness. Ab-dependent enhancement of DENV infections may be associated with a specific range of pre-existing anti-DENV serological Ab titers. Testing of patient sera with the developed st-ELISAs will not only be useful for epidemiological studies and surveillance, but it may also help to develop and validate assays that can distinguish protective versus enhancing Ab responses for risk assessment for the development of severe dengue disease in individual patients

Limited Genetic Diversity of Hepatitis B Virus in the General Population of the Offin River Valley in Ghana

Submitted by Sandra Infurna ([email protected]) on 2016-12-15T11:48:32Z No. of bitstreams: 1 christian_niel2_etal_IOC_2016.pdf: 987243 bytes, checksum: d8c9c3783e2ce56b496ca29fb7671316 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2016-12-15T11:58:38Z (GMT) No. of bitstreams: 1 christian_niel2_etal_IOC_2016.pdf: 987243 bytes, checksum: d8c9c3783e2ce56b496ca29fb7671316 (MD5)Made available in DSpace on 2016-12-15T11:58:38Z (GMT). No. of bitstreams: 1 christian_niel2_etal_IOC_2016.pdf: 987243 bytes, checksum: d8c9c3783e2ce56b496ca29fb7671316 (MD5) Previous issue date: 2016Swiss Tropical and Public Health Institute. Molecular Immunology. Basel, Switzerland / University of Basel. Basel, Switzerland / University of Ghana. Noguchi Memorial Institute for Medical Research. Legon, Ghana.Universidade Federal Fluminense. Instituto de Biologia. Laboratório de Virologia Molecular. Niterói, RJ, Brasil.Swiss Tropical and Public Health Institute. Molecular Immunology. Basel, Switzerland / University of Basel. Basel, Switzerland.University of Ghana. Noguchi Memorial Institute for Medical Research. Legon, Ghana.University of Ghana. Noguchi Memorial Institute for Medical Research. Legon, Ghana.Radford University College. Accra, Ghana.Universidade Federal Fluminense. Instituto de Biologia. Laboratório de Virologia Molecular. Niterói, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Virologia Molecular. Rio de Janeiro, RJ. Brasil.University of Ghana. Noguchi Memorial Institute for Medical Research. Legon, Ghana.Swiss Tropical and Public Health Institute. Molecular Immunology. Basel, Switzerland / University of Basel. Basel, Switzerland.Swiss Tropical and Public Health Institute. Molecular Immunology. Basel, Switzerland / University of Basel. Basel, Switzerland.Hepatitis B virus (HBV) infections account for approximately 780,000 deaths per year, most of which occur in the developing world. Co-infection with HBV and hepatitis delta virus (HDV) may lead to the most severe form of viral hepatitis. In Ghana, knowledge on the prevalence of HBV and HDV in the general population is scanty and the few genetic analyses of the prevailing HBV genotypes are dating back more than a decade. In the present study, 1,323 serum samples from individuals living in a rural area (Offin river valley) of Ghana were analyzed for the presence of the hepatitis B surface antigen (HBsAg). Positive sera were subsequently tested for the presence of anti-HDV antibodies. A total of 107 (8%) sera were HBsAg positive with an 8.4% prevalence of anti-HDV antibodies among the HBsAg positives. Phylogenetic analysis based on HBV pre-S/S sequences, attributed all 52 typable samples to genotype E. All belonged to serotype ayw4. While 19 sequences clustered with those from a number of African countries, the other 33 formed a separate cluster distinguished by an intergroup mean distance of 1.5% from the pan-African HBV/E cluster. Successful implementation of HBV vaccination in the region was reflected by the low HBsAg carrier rate of 1.8% among children ≤11 years

Transmission of Hepatitis B and D Viruses in an African Rural Community

This study revealed that the prevalence of HBV and HDV in a rural area of Cameroon is extremely high, underlining the pressing need for the improvement of control strategies. Systematic serological and phylogenetic analyses of HBV sequences turned out to be useful tools to identify networks of virus transmission within and between households. The high HBsAg carriage rate found among children demonstrates that implementation of the HBV birth dose vaccine and improvement of vaccine coverage will be key elements in preventing both HBV and HDV infections. In addition, the high HBsAg carriage rate in adolescents and adults emphasizes the need for identification of chronically infected individuals and linkage to WHO-recommended treatment to prevent progression to liver cirrhosis and hepatocellular carcinoma.According to the World Health Organization (WHO), an estimated 257 million people worldwide are chronically infected with hepatitis B virus (HBV), with approximately 15 million of them being coinfected with hepatitis D virus (HDV). To investigate the prevalence and transmission of HBV and HDV within the general population of a rural village in Cameroon, we analyzed serum samples from most (401/448) of the villagers. HBV surface antigen (HBsAg) was detected in 54 (13.5%) of the 401 samples, with 15% of them also containing anti-HDV antibodies. Although Cameroon has integrated HBV vaccination into their Expanded Program on Immunization for newborns in 2005, an HBsAg carriage rate of 5% was found in children below the age of 5 years. Of the 54 HBsAg-positive samples, 49 HBV pre-S/S sequences (7 genotype A and 42 genotype E sequences) could be amplified by PCR. In spite of the extreme geographical restriction in the recruitment of study participants, a remarkable genetic diversity within HBV genotypes was observed. Phylogenetic analysis of the sequences obtained from PCR products combined with demographic information revealed that the presence of some genetic variants was restricted to members of one household, indicative of intrafamilial transmission, which appears to take place at least in part perinatally from mother to child. Other genetic variants were more widely distributed, reflecting horizontal interhousehold transmission. Data for two households with more than one HBV-HDV-coinfected individual indicate that the two viruses are not necessarily transmitted together, as family members with identical HBV sequences had different HDV statuses

Analysis of biliary MICRObiota in hepatoBILIOpancreatic diseases compared to healthy people [MICROBILIO]: Study protocol.

BackgroundThe performance of the microbiota is observed in several digestive tract diseases. Therefore, reaching the biliary microbiota may suggest ways for studies of biomarkers, diagnoses, tests and therapies in hepatobiliopancreatic diseases.MethodsBile samples will be collected in endoscopic retrograde cholangiopancreatography patients (case group) and living liver transplantation donors (control group). We will characterize the microbiome based on two types of sequence data: the V3/V4 regions of the 16S ribosomal RNA (rRNA) gene and total shotgun DNA. For 16S sequencing data a standard 16S processing pipeline based on the Amplicon Sequence Variant concept and the qiime2 software package will be employed; for shotgun data, for each sample we will assemble the reads and obtain and analyze metagenome-assembled genomes.ResultsThe primary expected results of the study is to characterize the specific composition of the biliary microbiota in situations of disease and health. In addition, it seeks to demonstrate the existence of changes in the case of illness and also possible disease biomarkers, diagnosis, interventions and therapies in hepatobiliopancreatic diseases.Trial registrationNCT04391426. Registered 18 May 2020, https://clinicaltrials.gov/ct2/show/NCT04391426

Study area.

<p>Map of Ghana with the Offin river valley and surrounding countries. The 13 study communities along the Offin River are indicated as multicolored dots: Bedomase (BDS); Krakrom (KKS); Kapro (KPS); Akomfore (AFS); Ntobroso (NBUS); Achiase (ABUS); Keniago (KGS); Tontonkrom (TNS); Dominase (BUDS); Wromanso (WMS); Nkotumso (NKS); Mfantseman (MFS); Pokukrom (PKS). The grey lines in the Ghana map indicate the borders of the Ghanaian regions. The background maps were created using the <i>ArcMap</i> program in <i>ArcGIS</i> v.10.0 and were modified with Adobe Photoshop CS6.</p

Age distribution and percentage of HBsAg carriers.

<p>A stacked graph of the total number of study participants for each age group (white bar) and of the corresponding number of HBsAg carriers (black bar) (left y-axis) is shown. The percentage of HBsAg carriers for each age group (right y-axis) is indicated by squares.</p