Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

[[abstract]]Background: Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection. Principal Finding: In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10?5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225). Conclusion:These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification

Pilot Scale Production of Highly Efficacious and Stable Enterovirus 71 Vaccine Candidates

BACKGROUND: Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial. PRINCIPAL FINDING: In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates. CONCLUSION: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials

Executive Functions of Six-Year-Old Boys with Normal Birth Weight and Gestational Age

Impaired fetal development, reflected by low birth weight or prematurity, predicts an increased risk for psychopathology, especially attention deficit hyperactivity disorder (ADHD). Such effects cut across the normal range of birth weight and gestation. Despite the strength of existing epidemiological data, cognitive pathways that link fetal development to mental health are largely unknown. In this study we examined the relation of birth weight (>2500 g) and gestational age (37–41 weeks) within the normal range with specific executive functions in 195 Singaporean six-year-old boys of Chinese ethnicity. Birth weight adjusted for gestational age was used as indicator of fetal growth while gestational age was indicative of fetal maturity. Linear regression revealed that increased fetal growth within the normal range is associated with an improved ability to learn rules during the intra/extra-dimensional shift task and to retain visual information for short period of time during the delayed matching to sample task. Moreover, faster and consistent reaction times during the stop-signal task were observed among boys born at term, but with higher gestational age. Hence, even among boys born at term with normal birth weight, variations in fetal growth and maturity showed distinct effects on specific executive functions

An integrated holistic model of a complex process

Conducting experiments to understand and model a complex process or system is usually costly and time-consuming due to multistages, multivariables, and multidisciplinary issues involved in the complex process. To reduce the complexity, for a single experiment, experimenters often fix some variables and investigate the effects of a smaller subset of variables. If then, it is possible to build individual models for each subset of variables, but this only allows partial understanding of the whole process. In this paper, we propose a method for building a holistic model of a complex process using multiple partial models that are learned from multiple sub-experiments that focus on different variables or the same variables but with different variable ranges. Using the proposed holistic model, it should be possible to provide an initial understanding of the complex process involving all variables. The effectiveness of the proposed method is demonstrated using a real example from a buckypaper process.clos

Practice variation in late-preterm deliveries: a physician survey

OBJECTIVE: Late preterm (LPT) neonates account for over 70% of all preterm births in the US. Approximately 60% of LPT births are the result of non-spontaneous deliveries. The optimal timing of delivery for many obstetric conditions at LPT gestations is unclear, likely resulting in obstetric practice variation. The purpose of this study is to identify variation in the obstetrical management of LPT pregnancies. STUDY DESIGN: We surveyed obstetrical providers in NC identified from NC Medical Board and NC Obstetrical and Gynecological Society membership lists. Participants answered demographic questions and 6 multiple-choice vignettes on management of LPT pregnancies. RESULT: We obtained 215/859 (29%) completed surveys; 167 (78%) from Obstetrics/Gynecology, 27 (13%) from Maternal-Fetal Medicine, and 21 (10%) from Family Medicine physicians. Overall, we found more agreement on respondents’ management of chorioamnionitis (97% would proceed with delivery), mild preeclampsia (84% would delay delivery/expectantly manage), and fetal growth restriction (80% would delay delivery/expectantly manage). We found less agreement on the management of severe preeclampsia (71% would proceed with delivery), premature preterm rupture of membranes (69% would proceed with delivery), and placenta previa (67% would delay delivery/expectantly manage). Management of LPT pregnancies complicated by PPROM, FGR, and placenta previa vary by specialty. CONCLUSION: Obstetrical providers report practice variation in the management of LPT pregnancies. Variation might be influenced by provider specialty. The absence of widespread agreement on best practice might be a source of modifiable LPT birth

Factors associated with initiation and exclusive breastfeeding at hospital discharge: late preterm compared to 37 week gestation mother and infant cohort

<p>Abstract</p> <p>Background</p> <p>To investigate and examine the factors associated with initiation of, and exclusive breastfeeding at hospital discharge of, late preterm (34 ^0/7 - 36 ^6/7 weeks) compared to 37 week gestation (37 ^0/7 - 37 ^6/7 week) mother and baby pairs.</p> <p>Methods</p> <p>A retrospective population-based cohort study using a Perinatal National Minimum Data Set and clinical medical records review, at the Royal Hobart Hospital, Tasmania, Australia in 2006.</p> <p>Results</p> <p>Late preterm and 37 week gestation infants had low rates of initiation of breastfeeding within one hour of birth, 31 (21.1%) and 61 (41.5%) respectively. After multiple regression analysis, late preterm infants were less likely to initiate breastfeeding within one hour of birth (OR 0.3 95% CI 0.1, 0.7 p = 0.009) and were less likely to be discharged exclusively breastfeeding from hospital (OR 0.4 95% CI 0.1, 1.0 p = 0.04) compared to 37 week gestation infants.</p> <p>Conclusion</p> <p>A late preterm birth is predictive of breastfeeding failure, with late preterm infants at greater risk of not initiating breastfeeding and/or exclusively breastfeeding at hospital discharge, compared with those infants born at 37 weeks gestation. Stratifying breastfeeding outcomes by gestational age groups may help to identify those sub-populations at greatest risk of premature cessation of breastfeeding.</p