Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

Hepatitis C virus (HCV) infects ～2% of the world's population. It is estimated that there are more than 500,000 new infections annually in Egypt, the country with the highest HCV prevalence. An effective vaccine would help control this expanding global health burden. HCV is highly variable, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human mAbs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies to HCV.     

Ligand-induced and nonfusogenic dissolution of a viral membrane

Hitherto, all enveloped viruses were thought to shed their lipid membrane during entry into cells by membrane fusion. The extracellular form of Vaccinia virus has two lipid envelopes surrounding the virus core, and consequently a single fusion event will not deliver a naked core into the cell. Here we report a previously underscribed mechanism in which the outer viral membrane is disrupted by a ligand-induced nonfusogenic reaction, followed by the fusion of the inner viral membrane with the plasma membrane and penetration of the virus core into the cytoplasm. The dissolution of the outer envelope depends on interactions with cellular polyanionic molecules and requires the virus glycoproteins A34 and B5. This discovery represents a remarkable example of how viruses manipulate biological membranes, solves the topological problem of how a double-enveloped virus enters cells, reveals a new effect of polyanions on viruses, and provides a therapeutic approach for treatment of poxvirus infections, such as smallpox.     

Antibodies against viruses: passive and active immunization

Antibodies, through passive or active immunization, play a central role in prophylaxis against many infectious agents. While neutralization is a primary function of antibodies in protection against most viruses, the relative contribution of Fc-dependent and complement-dependent anti-viral activities of antibodies was found to vary between different viruses in recent studies. The multiple hit model explains how antibodies neutralize viruses, and recent data on the stoichiometry of antibody neutralization suggest that the organization of viral surface proteins on viruses, in addition to virus size, influences the level of antibody occupancy required for neutralization. These new findings will improve our strategies in therapeutic antibody engineering and rational vaccine design.     

The use of microelectrode array (MEA) to study rat peritoneal mast cell activation

We performed this study to demonstrate the applicability of the microelectrode array (MEA) to study electrophysiological changes of rat peritoneal mast cells in the presence of compound 48/80 under normal, Ca(2+)-free, Ca(2+)-free with EDTA, and Cl(-)-free conditions. The use of high extracellular K(+) (KCl, 150 mM), charybdotoxin (ChTX, 100 nM), and Cl(-)-free containing ChTX buffers verified that the hyperpolarizing signal was due to the activation of mainly K(+) and, to a lesser extent, Cl(-) channels. Compound 48/80 concentration-dependently shortened the latent periods (the onset of response) and increased both the spatial (the K(+) and Cl(-) hyperpolarizing field potentials, HFP) and temporal measurements (the duration of response). Ca(2+)-free buffer had no effect on the latent period of compound 48/80 but increased the HFP at high concentrations. The latent period increased while the HFP diminished when cells were equilibrated in Ca(2+)-free buffer containing EDTA. Durations of the HFP were generally longer when cells were in either Ca(2+)-free or Ca(2+)-free containing EDTA buffers than when cells were in normal buffer. The EC(50) values confirmed that effects were only affected in Ca(2+)-free buffer containing EDTA but not in Ca(2+)-free or Cl(-)-free buffers, further reinforcing the hypothesis that the presence of Ca(2+) is not essential to the action of compound 48/80. The present study is the first application of MEA to study rat peritoneal mast cells, and our results indicate that it could be of value in future pharmacological research on other non-excitable cells.     

The feasibility of task-sharing the identification,emergency treatment,and referral for women with pre-eclampsia by community health workers in India

Background

Hypertensive disorders are the second highest direct obstetric cause of maternal death after haemorrhage, accounting for 14% of maternal deaths globally. Pregnancy hypertension contributes to maternal deaths, particularly in low- and middle-income countries, due to a scarcity of doctors providing evidence-based emergency obstetric care. Task-sharing some obstetric responsibilities may help to reduce the mortality rates. This study was conducted to assess acceptability by the community and other healthcare providers, for task-sharing by community health workers (CHW) in the identification and initial care in hypertensive disorders in pregnancy.

Methods

This study was conducted in two districts of Karnataka state in south India. A total of 14 focus group discussions were convened with various community representatives: women of reproductive age (N = 6), male decision-makers (N = 2), female decision-makers (N = 3), and community leaders (N = 3). One-to-one interviews were held with medical officers (N = 2), private healthcare OBGYN specialists (N = 2), senior health administrators (N = 2), Taluka (county) health officers (N = 2), and obstetricians (N = 4). All data collection was facilitated by local researchers familiar with the setting and language. Data were subsequently transcribed, translated and analysed thematically using NVivo 10 software.

Results

There was strong community support for home visits by CHW to measure the blood pressure of pregnant women; however, respondents were concerned about their knowledge, training and effectiveness. The treatment with oral antihypertensive agents and magnesium sulphate in emergencies was accepted by community representatives but medical practitioners and health administrators had reservations, and insisted on emergency transport to a higher facility. The most important barriers for task-sharing were concerns regarding insufficient training, limited availability of medications, the questionable validity of blood pressure devices, and the ability of CHW to correctly diagnose and intervene in cases of hypertensive disorders of pregnancy.

Conclusion

Task-sharing to community-based health workers has potential to facilitate early diagnosis of the hypertensive disorders of pregnancy and assist in the provision of emergency care. We identified some facilitators and barriers for successful task-sharing of emergency obstetric care aimed at reducing mortality and morbidity due to hypertensive disorders of pregnancy.

     

To establish a pharmacological experimental platform for the study of cardiac hypoxia using the microelectrode array

IntroductionSimultaneous recording of electrical potentials from multiple cells may be useful for physiological and pharmacological research. The present study aimed to establish an in vitro cardiac hypoxia experimental platform on the microelectrode array (MEA).MethodsEmbryonic rat cardiac myocytes were cultured on the MEAs. Following ≥ 90 min of hypoxia, changes in lactate production (mM), pH, beat frequency (beats per min, bpm), extracellular action potential (exAP) amplitude, and propagation velocity between the normoxic and hypoxic cells were compared.ResultsUnder hypoxia, the beat frequency of cells increased and peaked at around 42.5 min (08.1 ± 3.2 bpm). The exAP amplitude reduced as soon as the cells were exposed to the hypoxic medium, and this reduction increased significantly after approximately 20 min of hypoxia. The propagation velocity of the hypoxic cells was significantly lower than that of the control throughout the entire 90+ min of hypoxia. The rate of depolarisation and Na⁺ signal gradually reduced over time, and these changes had a direct effect on the exAP duration.DiscussionThe extracellular electrophysiological measurements allow a partial reconstruction of the signal shape and time course of the underlying hypoxia-associated physiological changes. The present study showed that the cardiac myocyte-integrated MEA may be used as an experimental platform for the pharmacological studies of cardiovascular diseases in the future.     

Evaluation of a Series of Lipidated Tucaresol Adjuvants in a Hepatitis C Virus Vaccine Model

Hepatitis C virus (HCV) infections represent a global health challenge; however, developing a vaccine for treatment of HCV infection has remained difficult as heterogeneous HCV contains distinct genotypes, and each genotype contains various subtypes and different envelope glycoproteins. Currently, there is no effective preventive vaccine for achieving global control over HCV. In our efforts to improve upon current HCV vaccines we designed a synthetically accessible adjuvant platform, wherein we synthesized 11 novel lipidated tucaresol analogues to assess their immunological potential. Using a tucaresol-based adjuvant approach, truncated lipid-variants together with an engineered E1E2 antigen construct, namely E2ΔTM3, elicited antibody (Ab) responses that were significantly higher than tucaresol. In sum, antibody end-point titer values largely corroborated HCV neutralization data with a simplified lipidated tucaresol variant affording the highest end point titer and % neutralization. This study lays the groundwork for additional permutations in tucaresol adjuvant design, including the examination of other proteins in vaccine development.     

The exit of vaccinia virus from infected cells

Vaccinia virus (VACV) is the prototypic member of the Poxviridae a group of large DNA viruses that replicate in the cell cytoplasm. The entry and exit of VACV are complicated by the existence of two distinct forms of virus, intracellular mature virus (IMV) and extracellular enveloped virus (EEV), that are surrounded by different numbers of lipid membranes and have different surface proteins. Here the mechanisms used by these different forms of VACV to leave the infected cell are reviewed. Whereas some enveloped viruses complete virus assembly by budding through the plasma membrane, infectious poxvirus particles (IMV) are produced within the cytoplasm. These particles are either further enveloped by intracellular membranes to form intracellular enveloped virus (IEV) that are transported to the cell surface on microtubules and exposed on the cell surface by exocytosis, or are released after cell lysis. If the enveloped virion remains attached to the cell surface it is called cell-associated enveloped virus (CEV) and is propelled into surrounding cells by growing actin tails beneath the plasma membrane. Alternatively, the surface virion may be released as EEV.     

Functional convergence of a germline-encoded neutralizing antibody response in rhesus macaques immunized with HCV envelope glycoproteins

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Chronic Hepatitis C Virus Infection Breaks Tolerance and Drives Polyclonal Expansion of Autoreactive B Cells

Chronic Hepatitis C virus (HCV) infection has been linked with B cell lymphoproliferative disorders and several autoimmune-related diseases. The mechanisms of how chronic viral infection affects B cell development and predisposes the patients to autoimmune manifestations are poorly understood. In this study, we established an experimental system to probe the B cell responses and characterize the antibodies from chronic-HCV-infected individuals. We identified an unusual polyclonal expansion of the IgM memory B cell subset in some patients. This B cell subset is known to be tightly regulated, and autoreactive cells are eliminated by tolerance mechanisms. Genetic analysis of the immunoglobulin (Ig) heavy chain variable gene (V_H) sequences of the expanded cell population showed that the levels of somatic hypermutation (SHM) correlate with the extent of cell expansion in the patients and that the V_H genes exhibit signs of antigen-mediated selection. Functional analysis of the cloned B cell receptors demonstrated autoreactivity in some of the expanded IgM memory B cells in the patients which is not found in healthy donors. In summary, this study demonstrated that, in some patients, chronic HCV infection disrupts the tolerance mechanism that normally deletes autoreactive B cells, therefore increasing the risk of developing autoimmune antibodies. Long-term follow-up of this expanded B cell subset within the infected individuals will help determine whether these cells are predictors of more-serious clinical manifestations.