Generation of a neutralizing antibody against RD114-pseudotyped viral vectors

The feline endogenous RD114 glycoprotein has proved to be an attractive envelope to pseudotype both retroviral and lentiviral vectors. As a surface protein, its detection on packaging cells as well as viral particles would be useful in different fields of its use. To address this, we generated a monoclonal antibody against RD114 by immunization of rats, termed 22F10. Once seroconversion was confirmed, purified 22F10 was cloned into murine Fc and characterized with a binding affinity of 10nM. The antibody was used to detect RD114 and its variant envelopes on different stable viral packaging cell lines (FLYRD18 and WinPac-RD). 22F10 was also shown to prevent the infections of different strains of RD-pseudotyped vectors but not related envelope glycoproteins by blocking cell surface receptor binding. We are the first to report the neutralization of viral particles by a monoclonal αRD114 antibody

WHO collaborative study to assess the suitability of the 1st International Standard and the 1st International Reference Panel for antibodies to Ebola virus

A WHO international collaborative study was undertaken to evaluate preparations of Ebola virus disease (EVD) convalescent plasmas for their suitability to serve as the WHO 1st International Standard (IS) and the WHO 1st International Reference Panel (IRP) for Ebola virus antibodies for use in the standardization and control of assays. The study involved participants testing the convalescent plasma sample preparations and additional monoclonal antibody samples in a blinded manner alongside the WHO International Reference Reagent (NIBSC code 15/220) using anti-EBOV assays established in their laboratories. The candidate 1st IS for Ebola virus antibodies (study sample code 92, NIBSC 15/262) consists of ampoules containing the freeze-dried equivalent of 0.5 mL pooled convalescent plasma obtained from six Sierra Leone patients recovered from EVD. The candidate 1st IRP of anti-Ebola virus convalescent plasmas (NIBSC 16/344) consists of freeze-dried preparations of single donations of convalescent plasma obtained from four patients and one healthy blood donor. Each panel member is an ampoule containing the equivalent of 0.25mL plasma. All convalescent plasmas are confirmed PCR-negative for Ebola virus and underwent, along with the negative plasma, solvent detergent (SD) treatment prior to their development into candidate WHO biological reference materials. In this collaborative study, 17 laboratories from 4 countries used a range of live Ebola virus neutralization assays, pseudotyped virus neutralisation assays and enzyme immunoassays to test the collaborative study samples. Surface plasmon resonance and Western blot assessments were also undertaken. The study found that the candidate International Standard has the highest absolute titre among the convalescent plasma samples, although the geometric mean titres of all the convalescent plasmas fall within ~5-fold of each other. The potencies of three of the convalescent samples fall near the detection limit of some assays. This study also demonstrated that the agreement between laboratories for potencies relative to the candidate International Standard represents an improvement compared to the agreement in absolute titres; however, there is poor agreement between relative potencies for some assays. The results obtained from accelerated thermal degradation studies at 1year indicate that the candidate IS is stable and suitable for long-term use. The results of the collaborative study indicate the suitability of the candidates to serve as WHO reference materials and it is proposed that 15/262 is established as the WHO 1st IS for EBOV antibodies with an assigned potency of 1.5 IU/mL when reconstituted as directed in the instructions for use. It is also proposed that 16/344 is established as the WHO 1st IRP of anti-EBOV convalescent plasmas with panel member code 95 (NIBSC 15/280) assigned a unitage of 1.1 IU/mL when reconstituted as directed in the instructions for use. The other panel members have not been assigned a unitage. The implementation and use by laboratories of the proposed WHO reference materials for EBOV antibodies will facilitate the characterization of the factors that contribute to assay variability and standardization of results across assays and laboratorie

A bipartite structural organization defines the SERINC family of HIV-1 restriction factors

The human integral membrane protein SERINC5 potently restricts HIV-1 infectivity and sensitizes the virus to antibody-mediated neutralization. Here, using cryo-EM, we determine the structures of human SERINC5 and its orthologue from Drosophila melanogaster at subnanometer and near-atomic resolution, respectively. The structures reveal a novel fold comprised of ten transmembrane helices organized into two subdomains and bisected by a long diagonal helix. A lipid binding groove and clusters of conserved residues highlight potential functional sites. A structure-based mutagenesis scan identified surface-exposed regions and the interface between the subdomains of SERINC5 as critical for HIV-1-restriction activity. The same regions are also important for viral sensitization to neutralizing antibodies, directly linking the antiviral activity of SERINC5 with remodeling of the HIV-1 envelope glycoprotein

Small molecules targeted to the microtubule–Hec1 interaction inhibit cancer cell growth through microtubule stabilization

Highly expressed in cancer protein 1 (Hec1) is a subunit of the kinetochore (KT)-associated Ndc80 complex, which ensures proper segregation of sister chromatids at mitosis by mediating the interaction between KTs and microtubules (MTs). HEC1 mRNA and protein are highly expressed in many malignancies as part of a signature of chromosome instability. These properties render Hec1 a promising molecular target for developing therapeutic drugs that exert their anticancer activities by producing massive chromosome aneuploidy. A virtual screening study aimed at identifying small molecules able to bind at the Hec1–MT interaction domain identified one positive hit compound and two analogs of the hit with high cytotoxic, pro-apoptotic and anti-mitotic activities. The most cytotoxic analog (SM15) was shown to produce chromosome segregation defects in cancer cells by inhibiting the correction of erroneous KT–MT interactions. Live cell imaging of treated cells demonstrated that mitotic arrest and segregation abnormalities lead to cell death through mitotic catastrophe and that cell death occurred also from interphase. Importantly, SM15 was shown to be more effective in inducing apoptotic cell death in cancer cells as compared to normal ones and effectively reduced tumor growth in a mouse xenograft model. Mechanistically, cold-induced MT depolymerization experiments demonstrated a hyper-stabilization of both mitotic and interphase MTs. Molecular dynamics simulations corroborate this finding by showing that SM15 can bind the MT surface independently from Hec1 and acts as a stabilizer of both MTs and KT–MT interactions. Overall, our studies represent a clear proof of principle that MT-Hec1-interacting compounds may represent novel powerful anticancer agents

Tetherin Restricts Productive HIV-1 Cell-to-Cell Transmission

The IFN-inducible antiviral protein tetherin (or BST-2/CD317/HM1.24) impairs release of mature HIV-1 particles from infected cells. HIV-1 Vpu antagonizes the effect of tetherin. The fate of virions trapped at the cell surface remains poorly understood. Here, we asked whether tetherin impairs HIV cell-to-cell transmission, a major means of viral spread. Tetherin-positive or -negative cells, infected with wild-type or ΔVpu HIV, were used as donor cells and cocultivated with target lymphocytes. We show that tetherin inhibits productive cell-to-cell transmission of ΔVpu to targets and impairs that of WT HIV. Tetherin accumulates with Gag at the contact zone between infected and target cells, but does not prevent the formation of virological synapses. In the presence of tetherin, viruses are then mostly transferred to targets as abnormally large patches. These viral aggregates do not efficiently promote infection after transfer, because they accumulate at the surface of target cells and are impaired in their fusion capacities. Tetherin, by imprinting virions in donor cells, is the first example of a surface restriction factor limiting viral cell-to-cell spread

A cost-effectiveness analysis of seminatural wetlands and activated sludge wastewater-treatment

A cost-effectiveness analysis was performed to evaluate the competitiveness of seminatural Free Water Surface (FWS) wetlands compared to traditional wastewater-treatment plants. Six scenarios of the service costs of three FWS wetlands and three different wastewater-treatment plants based on active sludge processes were compared. The six scenarios were all equally effective in their wastewater-treatment capacity. The service costs were estimated using real accounting data from an experimental wetland and by means of a market survey. Some assumptions had to be made to perform the analysis. A reference wastewater situation was established to solve the problem of the different levels of dilution that characterize the inflow water of the different systems; the land purchase cost was excluded from the analysis, considering the use of public land as shared social services, and an equal life span for both seminatural and traditional wastewater-treatment plants was set. The results suggest that seminatural systems are competitive with traditional biotechnological systems, with an average service cost improvement of 2.1-fold to 8-fold, according to the specific solution and discount rate. The main improvement factor was the lower maintenance cost of the seminatural systems, due to the self-regulating, low artificial energy inputs and the absence of waste to be disposed. In this work, only the waste-treatment capacity of wetlands was considered as a parameter for the economic competitiveness analysis. Other goods/services and environmental benefits provided by FWS wetlands were not considered

Ecdysteroid Derivatives that Reverse P-Glycoprotein-Mediated Drug Resistance

The expression of multidrug resistance P-glycoprotein (P-gp) by cancer cells represents one of the major drawbacks to successful cancer therapy. Accordingly, the development of drugs that inhibit the activity of this transporter remains a major challenge in cancer drug discovery. In this context, several new ecdysteroid derivatives have been synthesized and evaluated as P-gp inhibitors. Two of them (compounds 9 and 14) were able to resensitize CEMVbl100 and LoVoDoxo resistant cell lines to vinblastine and doxorubicin, respectively. Indeed, both compounds 9 and 14 increased the cellular accumulation of rhodamine 123 in cells expressing P-gp and stimulated basal P-glycoprotein-ATPase activity at a 1 \u3bcM concentration, demonstrating their interference with the transport of other substrates in a competitive mode. Moreover, in a medulloblastoma cell line (DAOY), compounds 9 and 14 reduced the side population representing cancer stem cells, which are characterized by a high expression of ABC drug transporters. Further, in DAOY cells, the same two compounds synergized with cisplatin and vincristine, two drugs used commonly in the therapy of medulloblastoma. Molecular docking studies on the homology-modeled structure of the human P-glycoprotein provided a rationale for the biological results, validating the binding mode within the receptor site, in accordance with lipophilicity data and observed structure-activity relationship information. Altogether, the present results endorse these derivatives as promising P-gp inhibitors, and they may serve as candidates to reverse drug resistance in cancer cells