Baculovirus display of single chain antibody (scFv) using a novel signal peptide

Background: Cells permissive to virus can become refractory to viral replication upon intracellular expression of single chain fragment variable (scFv) antibodies directed towards viral structural or regulatory proteins, or virus-coded enzymes. For example, an intrabody derived from MH-SVM33, a monoclonal antibody against a conserved C-terminal epitope of the HIV-1 matrix protein (MAp17), was found to exert an inhibitory effect on HIV-1 replication.<p></p> Results: Two versions of MH-SVM33-derived scFv were constructed in recombinant baculoviruses (BVs) and expressed in BV-infected Sf9 cells, N-myristoylation-competent scFvG2/p17 and N-myristoylation-incompetent scFvE2/p17 protein, both carrying a C-terminal HA tag. ScFvG2/p17 expression resulted in an insoluble, membrane-associated protein, whereas scFvE2/p17 was recovered in both soluble and membrane-incorporated forms. When coexpressed with the HIV-1 Pr55Gag precursor, scFvG2/p17 and scFvE2/p17 did not show any detectable negative effect on virus-like particle (VLP) assembly and egress, and both failed to be encapsidated in VLP. However, soluble scFvE2/p17 isolated from Sf9 cell lysates was capable of binding to its specific antigen, in the form of a synthetic p17 peptide or as Gag polyprotein-embedded epitope. Significant amounts of scFvE2/p17 were released in the extracellular medium of BV-infected cells in high-molecular weight, pelletable form. This particulate form corresponded to BV particles displaying scFvE2/p17 molecules, inserted into the BV envelope via the scFv N-terminal region. The BV-displayed scFvE2/p17 molecules were found to be immunologically functional, as they reacted with the C-terminal epitope of MAp17. Fusion of the N-terminal 18 amino acid residues from the scFvE2/p17 sequence (N18E2) to another scFv recognizing CD147 (scFv-M6-1B9) conferred the property of BV-display to the resulting chimeric scFv-N18E2/M6.<p></p> Conclusion: Expression of scFvE2/p17 in insect cells using a BV vector resulted in baculoviral progeny displaying scFvE2/p17. The function required for BV envelope incorporation was carried by the N-terminal octadecapeptide of scFvE2/p17, which acted as a signal peptide for BV display. Fusion of this peptide to the N-terminus of scFv molecules of interest could be applied as a general method for BV-display of scFv in a GP64- and VSV-G-independent manner.<p></p&gt

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

BACKGROUND: Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1. RESULTS: A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank(GAG)1D4 (16.5 kDa) was isolated. Ank(GAG)1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K(d) ~ 1 muM, and the Ank(GAG)1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank(GAG)1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank(GAG)1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank(GAG)1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank(GAG)1D4-CA with the Gag assembly and budding pathway. CONCLUSIONS: The resistance of Ank(GAG)1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank(GAG)1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules

Remodeling of the actin network associated with the non-structural protein 1 (NS1) of West Nile virus and formation of NS1-containing tunneling nanotubes

The cellular response to the recombinant NS1 protein of West Nile virus (NS1WNV) was studied using three different cell types: Vero E6 simian epithelial cells, SH-SY5Y human neuroblastoma cells, and U-87MG human astrocytoma cells. Cells were exposed to two different forms of NS1WNV: (i) the exogenous secreted form, sNS1WNV, added to the extracellular milieu; and (ii) the endogenous NS1WNV, the intracellular form expressed in plasmid-transfected cells. The cell attachment and uptake of sNS1WNV varied with the cell type and were only detectable in Vero E6 and SH-SY5Y cells. Addition of sNS1WNV to the cell culture medium resulted in significant remodeling of the actin filament network in Vero E6 cells. This effect was not observed in SH-SY5Y and U-87MG cells, implying that the cellular uptake of sNS1WNV and actin network remodeling were dependent on cell type. In the three cell types, NS1WNV-expressing cells formed filamentous projections reminiscent of tunneling nanotubes (TNTs). These TNT-like projections were found to contain actin and NS1WNV proteins. Interestingly, similar actin-rich, TNT-like filaments containing NS1WNV and the viral envelope glycoprotein EWNV were also observed in WNV-infected Vero E6 cells

Interférences avec l'assemblage et la maturation du Virus de l'Immunodeficience Humaine (VIH) par des protéines à motifs répétés Ankyrines

Le but de ce travail est de découvrir des nouvelles protéines suceptibles d'interférer avec le cycle vital du virus HIV. De par leur repliement, les protéines à motifs ankyrines peuvent constituer une ossature protéique trés bien adaptée à cet objectif. Plusieurs interacteurs spécifiques de la protéine MA-CA du HIV ont été sélectionnées par exposition sur phage à partir d'une bibliothèque de variants d'ankyrines. Trois protéines isolées ont été produites à partir de clones ayant une forte activité de liaison. Le meilleur interacteur protéique (1D4) interagit avec un épitope situié sur le domaine CA. La constante de dissociation entre 1D4 et la protéine HACA a été déterminée par Calorimétrie de Titrage Isotherme (ou ITC) et est égale à 0,45M. La protéine 1D4 n'a pas d'effet détectable sur la maturation virale suivie par une technique ELISA de dosage de la Protease du HIV. En revanche, cette protéine interfere avec l'assemblage viral dans des cellules supT1 qui exprime de façon stable la protéine 1D4 sous forme myristoylée. Ce resultat ouvre une perspective d'appoche pour interferer avec le cycle vital du HIV.Presently, the standard regimen for antiretroviral treatment is highly active antiretroviral therapy (HAART). However, this strategy inherits the well-known side effects and is prone to promote the HIV drug-resistant strains. As a consequence, gene therapy has been introduced as an alternative approach. In this study, we aimed to discover the novel protein-based agents for intervening viral replication by gene targeting procedure. Regarding the efficient folding dynamic in cytoplasm, ankyrin repeat protein was considered to be a candidate scaffold. Several engineered ankyrin binders specific to HIV MA-CA domain were successfully retrieved from the ankyrin-displayed phage library. Three positive clones with high binding activity by ELISA were selected for further analyzing their binding property in soluble form. The best binder, 1D4, recognized its epitope located on CA domain as shown by Western immunobloting and ELISA. The affinity of 1D4 against H6MA-CA was 0.45 μM with one to two moles of target molecule determined by isothermal titration calorimetry (ITC). Although 1D4 exhibited no effect on viral maturation as verified by an ELISA based HIV protease assay technique, it disturbed the viral assembly process in Sup-T1 cells which stably expressed the myristoylated 1D4. This finding has provided a concrete prospect for HIV life cycle interruption by stem cell gene therapy in the future

The interference of human immunodeficiency virus assembly and maturation by ankyrin repeat proteins

Le but de ce travail est de découvrir des nouvelles protéines suceptibles d'interférer avec le cycle vital du virus HIV. De par leur repliement, les protéines à motifs ankyrines peuvent constituer une ossature protéique trés bien adaptée à cet objectif. Plusieurs interacteurs spécifiques de la protéine MA-CA du HIV ont été sélectionnées par exposition sur phage à partir d'une bibliothèque de variants d'ankyrines. Trois protéines isolées ont été produites à partir de clones ayant une forte activité de liaison. Le meilleur interacteur protéique (1D4) interagit avec un épitope situié sur le domaine CA. La constante de dissociation entre 1D4 et la protéine HACA a été déterminée par Calorimétrie de Titrage Isotherme (ou ITC) et est égale à 0,45 M. La protéine 1D4 n'a pas d'effet détectable sur la maturation virale suivie par une technique ELISA de dosage de la Protease du HIV. En revanche, cette protéine interfere avec l'assemblage viral dans des cellules supT1 qui exprime de façon stable la protéine 1D4 sous forme myristoylée. Ce resultat ouvre une perspective d'appoche pour interferer avec le cycle vital du HIV.Presently, the standard regimen for antiretroviral treatment is highly active antiretroviral therapy (HAART). However, this strategy inherits the well-known side effects and is prone to promote the HIV drug-resistant strains. As a consequence, gene therapy has been introduced as an alternative approach. In this study, we aimed to discover the novel protein-based agents for intervening viral replication by gene targeting procedure. Regarding the efficient folding dynamic in cytoplasm, ankyrin repeat protein was considered to be a candidate scaffold. Several engineered ankyrin binders specific to HIV MA-CA domain were successfully retrieved from the ankyrin-displayed phage library. Three positive clones with high binding activity by ELISA were selected for further analyzing their binding property in soluble form. The best binder, 1D4, recognized its epitope located on CA domain as shown by Western immunobloting and ELISA. The affinity of 1D4 against H6MA-CA was 0.45 M with one to two moles of target molecule determined by isothermal titration calorimetry (ITC). Although 1D4 exhibited no effect on viral maturation as verified by an ELISA based HIV protease assay technique, it disturbed the viral assembly process in Sup-T1 cells which stably expressed the myristoylated 1D4. This finding has provided a concrete prospect for HIV life cycle interruption by stem cell gene therapy in the future.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Development of a one-step immunochromatographic strip test for the rapid detection of nevirapine (NVP), a commonly used antiretroviral drug for the treatment of HIV/AIDS

Currently, high-performance liquid chromatographic (HPLC) methods are mainly used to measure anfiretroviral plasma concentrations in HIV-infected patients. Although the utility of routine therapeutic drug monitoring (TDM) as an additional toot to optimize long-term antiretroviral therapy is unclear, if TDM is to be widely used, the availability of simple, cheap and reliable methods for the measurement of antiretroviral drug levels are needed, particularly in resource-limited settings. In this study, an immunochromatograhic (IC) strip test to detect the presence of nevirapine (NVP) in body fluids has been developed. Antiserum to NVP was first raised in rabbits by immunization against NVP chemically conjugated with bovine serum albumin, and subsequently validated by Western immunoblotting and competitive indirect ELISA. The partially purified anti-NVP antibodies were conjugated with colloidal gold particles. The conjugation of the colloidal gold and polyclonal antibodies was monitored by UV-vis spectroscopy, while transmission electron microscopy images were used to characterize the particle size and shape of the conjugates, The resulting colloidal gold conjugates were used for the production of an IC strip test to detect nevirapine in human plasma. Preliminary assessment suggests no-cross reactivity of the NVP polyclonal antibodies but assessment of plasma samples from HIV-infected patients receiving HAART needs to be conducted. This assay could potentially be used for drug monitoring as part of the clinical care of HIV infected patients

Expression of Secreted Neutrophil Gelatinase-Associated Lipocalin in 293T Cell Using the Inducible Dual-Function System

Neutrophil gelatinase-associated lipocalin (NGAL) has emerged as a promising biomarker for the early prediction of acute kidney injury (AKI). The production of recombinant NGAL is considered to be necessary for the development of a detection method. This study intended to express the recombinant NGAL protein in 293T cell under the Tet-On inducible system and human serum albumin signal sequence (HSA-SS). The transfection efficiency and protein modulation were assessed by detecting the expression of the enhanced green fluorescent protein (EGFP) and secreted NGAL protein. Both proteins were detected only in the presence of a doxycycline (Dox) inducer. Cell toxicity was not found under any conditions. Moreover, a higher level of soluble NGAL protein in the supernatant secreted by HSA-SS compared with a native signal peptide (Nat-SS) was observed. In summary, this work successfully optimized the conditions for induction of NGAL expression. This system will provide as an efficient strategy to produce other recombinant proteins secreted from a mammalian cell

Current Peptide and Protein Candidates Challenging HIV Therapy beyond the Vaccine Era

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Bicistronic vector-based procedure to measure correlative expression and bacteriostatic activity of recombinant neutrophil gelatinase-associated lipocalin

AbstractNeutrophil gelatinase-associated lipocalin (NGAL) is a component of the human innate immune system that exerts bacteriostatic effects. Recent research suggests that NGAL is also a promising biomarker for tubular damage and aids in the early diagnosis of acute kidney injury. Therefore, the development of a method to efficiently produce recombinant NGAL has considerable clinical value. This study aimed to analyze the correlation between fluorescence reporter (enhanced green fluorescent protein, EGFP) intensity and NGAL levels, mediated by a bicistronic expression system based on internal ribosome entry sites (IRESs). During the 14-day culture period, EGFP intensity was positively correlated with NGAL concentrations. The purified protein also exerted bacteriostatic effects, interfering with bacterial iron uptake machinery during periods of iron starvation. The purified NGAL was also applied to generate a standard curve that could be used for enzyme-linked immunosorbent assay of samples containing unknown NGAL quantities. In conclusion, we successfully developed a procedure using EGFP and IRES-based vector to predict NGAL production levels and optimize protein harvest time. Our approach is also a simple and effective method for evaluating bioactivity