Prophylactic and therapeutic benefits of a monoclonal antibody against the fusion protein of human metapneumovirus in a mouse model

ABSTRACT Human metapneumovirus (HMPV) is a recently described human pathogen of the pneumovirus subfamily within the paramyxovirus family. HMPV infection is prevalent worldwide and is associated with severe respiratory disease, particularly in infants. The HMPV fusion protein (F) amino acid sequence contains features characteristic of other paramyxovirus F proteins, including a putative cleavage site and potential N-linked glycosylation sites. Propagation of HMPV in cell culture requires exogenous trypsin, which cleaves the F protein, and HMPV, like several other pneumoviruses, is infectious in the absence of its attachment protein (G). However, little is known about HMPV F-promoted fusion, since the HMPV glycoproteins have yet to be analyzed separately from the virus. Using syncytium and luciferase reporter gene fusion assays, we determined the basic requirements for HMPV F protein-promoted fusion in transiently transfected cells. Our data indicate that proteolytic cleavage of the F protein is a stringent requirement for fusion and that the HMPV G protein does not significantly enhance fusion. Unexpectedly, we also found that fusion can be detected only when transfected cells are treated with trypsin and exposed to low pH, indicating that this viral fusion protein may function in a manner unique among the paramyxoviruses. We also analyzed the F protein cleavage site and three potential N-linked glycosylation sites by mutagenesis. Mutations in the cleavage site designed to facilitate endogenous cleavage did so with low efficiency, and our data suggest that all three N-glycosylation sites are utilized and that each affects cleavage and fusion to various degrees.

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Characterization and Evaluation of HGF-Loaded PLGA Nanoparticles in a CCl4-Induced Acute Liver Injury Mouse Model

Journal of Nanomaterials ◽

10.1155/2019/7936143 ◽

2019 ◽

Vol 2019 ◽

pp. 1-13

Author(s):

Chuxi Lin ◽

Xueer Wang ◽

Nuyun Liu ◽

Qing Peng ◽

Yang Li ◽

...

Keyword(s):

Liver Injury ◽

Mouse Model ◽

Acute Liver Injury ◽

Redox Homeostasis ◽

Plga Nanoparticles ◽

Physical Characteristics ◽

Sod Activity ◽

Therapeutic Benefits ◽

Antioxidant Proteins ◽

Drug Free

Liver injury can be caused by various harmful factors since the liver is considered the key organ for detoxifying endogenous and exogenous substances. Hepatocyte growth factor (HGF) can regulate redox homeostasis through the expression of antioxidant proteins when the liver is under injury. However, HGF is easily degraded. In this study, we produced three kinds of HGF-loaded poly(lactic-co-glycolic) acid (PLGA) nanoparticles with an initial addition of 2 μg HGF (NPs-HGF-2 μg), 4 μg HGF (NPs-HGF-4 μg), and drug-free nanoparticles (NPs) using the W/O/W emulsion-solvent evaporation method in accordance with our patent. The morphology and physical characteristics were analyzed, and effects of HGF-loaded PLGA nanoparticles on a CCl4-induced acute liver injury mouse model were investigated and compared with HGF solutions. We observed that the morphology and the physical characteristics of the nanoparticles were almost the same, with similar sizes, polydispersity, and zeta potential. HGF-loaded PLGA nanoparticles maintained higher HGF concentrations for a longer period of time in blood and liver tissues. HGF-loaded PLGA nanoparticles increased the SOD activity and GPX levels, decreased the MDA levels in the liver, reduced the necrotic areas of the liver, and decreased the levels of AST, ALT, ALP, T-BIL, BUN, and Scr in blood. In conclusion, our technique for preparing HGF-loaded PLGA nanoparticles was stable and the products were of good quality. HGF-loaded PLGA nanoparticles could provide greater therapeutic benefits on CCl4-induced acute liver injury, including antilipid peroxidation and a reduction in indicator enzymes of liver injury.

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Comparison of Three Complementary Analytical Techniques for the Evaluation of the Biosimilar Comparability of a Monoclonal Antibody and an Fc-Fusion Protein

Frontiers in Chemistry ◽

10.3389/fchem.2021.782099 ◽

2021 ◽

Vol 9 ◽

Author(s):

Alice Demelenne ◽

Arij Ben Yahia ◽

Delphine Lempereur ◽

Jacques Crommen ◽

Anne-Catherine Servais ◽

...

Keyword(s):

Monoclonal Antibody ◽

Fusion Protein ◽

Method Development ◽

Reference Product ◽

Degradation Products ◽

Stress Conditions ◽

Size Exclusion ◽

Mild Stress ◽

Molecular Weight Determination ◽

Fc Fusion Protein

In this work, a monoclonal antibody, adalimumab, and an Fc-fusion protein, etanercept, were studied and compared to one of their biosimilars. Samples submitted to stress conditions (agitation and high temperature) were used for method development. The developed methods were also applied to samples reduced by beta-mercaptoethanol to evaluate their capability to distinguish the expected species. Capillary gel electrophoresis (CGE), reversed-phase liquid chromatography (RPLC), and size-exclusion chromatography (SEC) methods coupled with UV detection were used to analyze the biopharmaceuticals. Their complementarity was investigated. For further molecular weight determination, SEC-multi angle light scattering and RPLC-quadrupole time-of-flight were occasionally used. For adalimumab, a larger amount of fragments and aggregates was observed in the biosimilar compared with the reference product. For etanercept, more related species were found in the reference product. Those three separation techniques showed good complementarity. Indeed, RPLC enabled the separation of hydrophilic and hydrophobic degradation products. CGE provided good selectivity for several adalimumab fragments, and SEC was useful for the analysis of aggregates and certain fragments that cannot be separated by the other approaches. Moreover, those formulations were submitted to mild stress conditions (30°C, 300 rpm for 4 h) that mimic shipping conditions. No additional peak was found under these conditions for the two studied biopharmaceuticals.

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