Spontaneous lipid vesicle fusion with electropermeabilized cells

AbstractFusion is obtained between electropermeabilized mammalian cells and intact large unilamellar lipid vesicles. This is monitored by a fluorescence assay. Prepulse contact is obtained by Ca2+ when negatively charged lipids are present in the liposomes. The mixing of the liposome content in the cell cytoplasm is observed under conditions preserving cell viability. Electric conditions are such that free liposomes are not affected by the external field. Therefore destabilization of only one of the two membranes of the partners is sufficient for fusion. The comparison between the efficiency of dye delivery for different liposome preparations (multilamellar vesicles, large unilamellar vesicles, small unilamellar vesicles) is indicative that more metastable liposomes are more fusable with electropulsated cells. This observation is discussed within the framework of the recent hypothesis that occurrence of a contact induced electrostatic destabilization of the plasma membrane is a key step in the exocytosis process

Production of high-quality SARS-CoV-2 antigens for vaccine development and serological assays implementation

The development of novel and/or improved vaccines as well as the establishment of tools to monitor vaccine responses are two key factors to control COVID-19 pandemic, often requiring the manufacturing of significant amounts of high-quality SARS-CoV-2 antigens. In this work, we produced SARS-CoV-2 Spike (S) and the receptor binding domain (RBD) proteins in human or insect cell lines to be further used in (i) the implementation of serological assays for detection of antibodies against SARS-CoV-2 virus in the Portuguese population, and (ii) the development of a virosome-based COVID-19 vaccine candidate. Please click Download on the upper right corner to see the full abstract

A novel inactivated virus system (InViS) for a fast and inexpensive assessment of viral disintegration.

The COVID-19 pandemic has caused considerable interest worldwide in antiviral surfaces, and there has been a dramatic increase in the research and development of innovative material systems to reduce virus transmission in the past few years. The International Organization for Standardization (ISO) norms 18,184 and 21,702 are two standard methods to characterize the antiviral properties of porous and non-porous surfaces. However, during the last years of the pandemic, a need for faster and inexpensive characterization of antiviral material was identified. Therefore, a complementary method based on an Inactivated Virus System (InViS) was developed to facilitate the early-stage development of antiviral technologies and quality surveillance of the production of antiviral materials safely and efficiently. The InViS is loaded with a self-quenched fluorescent dye that produces a measurable increase in fluorescence when the viral envelope disintegrates. In the present work, the sensitivity of InViS to viral disintegration by known antiviral agents is demonstrated and its potential to characterize novel materials and surfaces is explored. Finally, the InViS is used to determine the fate of viral particles within facemasks layers, rendering it an interesting tool to support the development of antiviral surface systems for technical and medical applications

Comparison of Closure of Gastric Perforation Ulcers With Biodegradable Lactide-Glycolide-Caprolactone or Omental Patches

Results of both methods of gastric closure (omental and biodegradable patch) were similar suggesting that a biodegradable patch glued to the outside of the stomach may be a viable alternative for closure of perforations of the digestive tract

Insect Cells for High-Yield Production of SARS-CoV-2 Spike Protein: Building a Virosome-Based COVID-19 Vaccine Candidate.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) homotrimeric spike (S) protein is responsible for mediating host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2) receptor, thus being a key viral antigen to target in a coronavirus disease 19 (COVID-19) vaccine. Despite the availability of COVID-19 vaccines, low vaccine coverage as well as unvaccinated and immune compromised subjects are contributing to the emergence of SARS-CoV-2 variants of concern. Therefore, continued development of novel and/or updated vaccines is essential for protecting against such new variants. In this study, we developed a scalable bioprocess using the insect cells-baculovirus expression vector system (IC-BEVS) to produce high-quality S protein, stabilized in its pre-fusion conformation, for inclusion in a virosome-based COVID-19 vaccine candidate. By exploring different bioprocess engineering strategies (i.e., signal peptides, baculovirus transfer vectors, cell lines, infection strategies and formulation buffers), we were able to obtain ~4 mg/L of purified S protein, which, to the best of our knowledge, is the highest value achieved to date using insect cells. In addition, the insect cell-derived S protein exhibited glycan processing similar to mammalian cells and mid-term stability upon storage (up to 90 days at -80 and 4 °C or after 5 freeze-thaw cycles). Noteworthy, antigenicity of S protein, either as single antigen or displayed on the surface of virosomes, was confirmed by ELISA, with binding of ACE2 receptor, pan-SARS antibody CR3022 and neutralizing antibodies to the various epitope clusters on the S protein. Binding capacity was also maintained on virosomes-S stored at 4 °C for 1 month. This work demonstrates the potential of using IC-BEVS to produce the highly glycosylated and complex S protein, without compromising its integrity and antigenicity, to be included in a virosome-based COVID-19 vaccine candidate

Peripherally Administered Nanoparticles Target Monocytic Myeloid Cells, Secondary Lymphoid Organs and Tumors in Mice

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to similar to 50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma

Immunogenicity and Protective Capacity of a Virosomal Respiratory Syncytial Virus Vaccine Adjuvanted with Monophosphoryl Lipid A in Mice

Respiratory Syncytial Virus (RSV) is a major cause of viral brochiolitis in infants and young children and is also a significant problem in elderly and immuno-compromised adults. To date there is no efficacious and safe RSV vaccine, partially because of the outcome of a clinical trial in the 1960s with a formalin-inactivated RSV vaccine (FI-RSV). This vaccine caused enhanced respiratory disease upon exposure to the live virus, leading to increased morbidity and the death of two children. Subsequent analyses of this incident showed that FI-RSV induces a Th2-skewed immune response together with poorly neutralizing antibodies. As a new approach, we used reconstituted RSV viral envelopes, i.e. virosomes, with incorporated monophosphoryl lipid A (MPLA) adjuvant to enhance immunogenicity and to skew the immune response towards a Th1 phenotype. Incorporation of MPLA stimulated the overall immunogenicity of the virosomes compared to non-adjuvanted virosomes in mice. Intramuscular administration of the vaccine led to the induction of RSV-specific IgG2a levels similar to those induced by inoculation of the animals with live RSV. These antibodies were able to neutralize RSV in vitro. Furthermore, MPLA-adjuvanted RSV virosomes induced high amounts of IFNγ and low amounts of IL5 in both spleens and lungs of immunized and subsequently challenged animals, compared to levels of these cytokines in animals vaccinated with FI-RSV, indicating a Th1-skewed response. Mice vaccinated with RSV-MPLA virosomes were protected from live RSV challenge, clearing the inoculated virus without showing signs of lung pathology. Taken together, these data demonstrate that RSV-MPLA virosomes represent a safe and efficacious vaccine candidate which warrants further evaluation

Fusion membranaire induite par l'hémagglutinine du virus de la grippe

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Gene transfer mediated by cationic lipids: lack of a correlation between lipid mixing and transfection

AbstractComplexes of DNA with cationic lipids are used to transfect eukaryotic cells. The mechanism of transfection is unknown, but it has been suggested that the complexes are taken up into the cell by endocytosis, after which fusion of the cationic lipids with the membranes of intracellular vesicles would allow the DNA to escape into the cytoplasm. Here, we have compared transfection of CHO-K1 cells with lipid mixing measured by fluorescence assays, using liposomes or complexes with plasmid DNA of the cationic lipids 1,2 dioleolyl-3-N,N,N,-trimethylammonium-propane (DOTAP), N-[2,3-(dioleoyloxy)propyl]-N,N,N,-trimethylammonium (DOTMA), or combinations of these lipids with dioleoylphosphatidylethanolamine (DOPE), at various lipid/DNA charge ratios. Mixing of the lipids of the complexes or liposomes with cellular membranes occurred readily at 37°C, and was more efficient with liposomes than with complexes. Lipid mixing was inhibited at low temperatures (0–17°C), by the presence of NH4Cl in the medium, and by low extracellular pH, indicating the involvement of the endocytic pathway in entry. In the absence of DOPE, there was no correlation between the efficiency of lipid mixing and the efficiency of transfection. Moreover, although DOPE, which is thought to promote membrane fusion, enhanced transfection, it did not always enhance lipid mixing. Neither the size nor the zeta potential of the complexes were clearly associated with transfection efficiency. Therefore, although fusion between the lipids of the complexes and cellular membranes takes place, a step at a later stage in the transfection process determines the efficiency of transfection