Bruno You

Characterization of the lipid envelope of exosome encapsulated HEV particles protected from the immune response

The hepatitis E virus (HEV) is the most common cause of acute hepatitis worldwide. Although HEV is a small, naked RNA virus, HEV particles become associated with lipids in the blood of infected patients and in the supernatant of culture systems. The egress of these particles from cells implies the exocytosis pathway but the question of the role of the resulting HEV RNA containing exosomes and the nature of the lipids they contain has not been fully addressed. We determined the lipid proportions of exosomes from uninfected and HEV-infected cells and their role in HEV spreading. We cultured a suitable HEV strain on HepG2/C3A cells and analyzed the population of exosomes containing HEV RNA using lipidomics methods and electron microscopy. We also quantified HEV infectivity using an infectivity endpoint method based on HEV RNA quantification to calculate the tissue culture infectious dose 50. Exosomes produced by HEV-infected HepG2/C3A cells contained encapsidated HEV RNA. These HEV RNA-containing exosomes were infectious but ten times less than stools. HEV from stools, but not exosome-associated HEV from culture supernatant, was neutralized by anti-HEV antibodies in a dose-dependent manner. HEV infection did not influence the morphology or lipid proportions of the bulk of exosomes. These exosomes contained significantly more cholesterol, phosphatidylserine, sphingomyelin and ceramides than the parent cells, but less phosphoinositides and polyunsaturated fatty acids. Exosomes play a major role in HEV egress but HEV infection does not modify the characteristics of the bulk of exosomes produced by infected cells. PS and cholesterol enriched in these vesicles could then be critical for HEV entry. HEV particles in exosomes are protected from the immune response which could lead to the wide circulation of HEV in its host.

In vitro infectivity assay for prion titration for application to the evaluation of the prion removal capacity of biological products manufacturing processes.

Prions can be detected and quantified currently by using either immunoassays such as Western-blot, ELISA or conformation dependent immunoassay, or an infectivity assay in laboratory animals (bioassay). While immunoassays are inexpensive and rapid, they are based on the detection of PrP(Sc), the abnormal isoform of the prion protein, a surrogate marker for prion infectivity. The bioassay is considered the gold-standard analytical method for measuring prion infectivity, but it is very costly and time-consuming, involving the destruction of large numbers of animals. The use of the transgenic MovS6 cell line is described for the development of an in vitro tissue culture infectivity assay (TCIA) for prion detection and quantitation. Compared to a bioassay, the TCIA is rapid ( approximately 8 weeks), easy to implement, much less expensive, and requires far fewer animals. After titrating concomitantly a prion-infected brain homogenate sample by Western-blot, TCIA and bioassay, data show that the sensitivity of the TCIA is close to that of the bioassay, since 1 TCID(50) corresponds to 4 ID(50), and 80-fold more sensitive than the Western-blot. The application of the TCIA to the evaluation of prion removal in biological products manufacturing processes is described using a 15 nm-nanofiltration step of human albumin as a model.

Decontamination of prions in a plasma product manufacturing environment

The high resistance of prions to inactivating treatments requires the proper management of decontaminating procedures of equipment in contact with materials of human or animal origin destined for medical purposes. Sodium hydroxide (NaOH) is widely used today for this purpose as it inactivates a wide variety of pathogens including prions.