Search Results (4)

The combination of supported lipid bilayers (SLBs) with the Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) has been proven to be a powerful tool to simultaneously monitor mass and viscoelastic changes related to membrane binding-events. In this work, the above methodology is employed for the study of the interaction of the Early Endosomal Antigen 1 (EEA1) to a model lipid bilayer that mimics the early endosome (EE) membrane, focusing on the membrane composition. Starting with the formation of a lipid bilayer through the vesicles fusion technique, we investigated the formation of SLBs that incorporate phosphatidylinositol 3-phosphate (PI(3)P), a key component for EEA1 binding, in combination with other lipids, e.g., (1,2-dioleoyl-sn-glycero-3)-phosphocholine (DOPC), -phosphoserine (DOPS), -phosphoethanolamine (DOPE), and cholesterol (Chol). The interaction of the full-length coiled-coil EEA1 to the formed SLBs was further studied in real time with the QCM-D and characterized with respect to the lipid composition and pH. Our findings confirm that PI(3)P is essential for the EEA1–membrane interaction, while it was shown that Chol and phosphatidylserine greatly influence the binding event. In fact, including 30% Chol in a PI(3)P (3%):PS (6%) SLB resulted in almost double EEA1 binding than in the absence of Chol. Moreover, we employed the QCM-viscoelastic model available to analyze the QCM-D data with emphasis on the study of the protein conformation. Our results showed that, in our in vitro system, EEA1 is not fully extended and/or highly packed, but is mainly in a bent, distorted conformation with an average size close to 100 nm. This study complements previous works employing in vitro assays, also demonstrating the ability to reconstitute more complex biomimetic EE membranes containing inositol phospholipids on a QCM surface for the study of EEA1 binding. Full article

Resveratrol, a polyphenolic molecule found in edible fruits and vegetables, shows a wide range of beneficial effects on human health, including anti-microbial, anti-inflammatory, anti-cancer, and anti-aging properties. Due to its poor water solubility and high liposome-water partition coefficient, the biomembrane seems to be the main target of resveratrol, although the mode of interaction with membrane lipids and its location within the cell membrane are still unclear. In this study, using electrophysiological measurements, we study the interaction of resveratrol with planar lipid membranes (PLMs) of different composition. We found that resveratrol incorporates into palmitoyl-oleoyl-phosphatidylcholine (POPC) and POPC:Ch PLMs and forms conductive units unlike those found in dioleoyl-phosphatidylserine (DOPS):dioleoyl-phosphatidylethanolamine (DOPE) PLMs. The variation of the biophysical parameters of PLMs in the presence of resveratrol provides information on its location within a lipid double layer, thus contributing to an understanding of its mechanism of action. Full article

Zika virus (ZIKV) is a flavivirus similar to Dengue virus (DENV) in terms of transmission and clinical manifestations, and usually both viruses are found to co-circulate. ZIKV is usually transmitted by mosquitoes bites, but may also be transmitted by blood transfusion, via the maternal–foetal route, and sexually. After 2015, when the most extensive outbreak of ZIKV had occurred in Brazil and subsequently spread throughout the rest of South America, it became evident that ZIKV infection during the first trimester of pregnancy was associated with microcephaly and other neurological complications in newborns. As a result, the development of a vaccine against ZIKV became an urgent goal. A major issue with DENV vaccines, and therefore likely also with ZIKV vaccines, is the induction of antibodies that fail to neutralize the virus properly and cause antibody-dependent enhancement (ADE) of the infection instead. It has previously been shown that antibodies against the third domain of the envelope protein (EDIII) induces optimally neutralizing antibodies with no evidence for ADE for other viral strains. Therefore, we generated a ZIKV vaccine based on the EDIII domain displayed on the immunologically optimized Cucumber mosaic virus (CuMVtt) derived virus-like particles (VLPs) formulated in dioleoyl phosphatidylserine (DOPS) as adjuvant. The vaccine induced high levels of specific IgG after a single injection. The antibodies were able to neutralise ZIKV without enhancing infection by DENV in vitro. Thus, the here described vaccine based on EDIII displayed on VLPs was able to stimulate production of antibodies specifically neutralizing ZIKV without potentially enhancing disease caused by DENV. Full article

Vaccination remains the most effective and essential prophylactic tool against infectious diseases. Enormous efforts have been made to develop effective vaccines against malaria but successes remain so far limited. Novel adjuvants may offer a significant advantage in the development of malaria vaccines, in particular if combined with inherently immunogenic platforms, such as virus-like particles (VLP). Dioleoyl phosphatidylserine (DOPS), which is expressed on the outer surface of apoptotic cells, represents a novel adjuvant candidate that may confer significant advantage over existing adjuvants, such as alum. In the current study we assessed the potential of DOPS to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using Plasmodium falciparum thrombospondin-related adhesive protein (TRAP) as a target antigen. TRAP was chemically coupled to VLPs derived from the cucumber mosaic virus fused to a universal T cell epitope of tetanus toxin (CuMVtt). Mice were immunized with TRAP alone or formulated in alum or DOPS and compared to TRAP coupled to CuMVtt formulated in PBS or DOPS. Induced immune responses, in particular T cell responses, were assessed as the major protective effector cell population induced by TRAP. The protective capacity of the various formulations was assessed using a transgenic Plasmodium berghei expressing PfTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral and T cell immunogenicity for PfTRAP compared to the antigen alone. Display on VLPs, in particular if formulated with DOPS, induced the strongest and most protective immune response. Thus, the combination of VLP with DOPS may harness properties of both immunogenic components and optimally enhance induction of protective immune responses. Full article