Purification of yellow fever virus produced in Vero cells for inactivated vaccine manufacture.

Yellow fever (YF) is a high-lethality viral disease, endemic in tropical regions of South America and Africa, with a population of over 900 million people under risk. A highly effective attenuated vaccine, produced in embryonated eggs, has been used for about 80 years. However, egg-based production limits manufacturing capacity, and vaccine shortage led to the emergency use of a fractional dose (1/5) by the WHO in an outbreak in Africa in 2016 and by Brazilian authorities during an outbreak in 2018. In addition, rare but fatal adverse events of this vaccine have been reported since 2001. These two aspects make clear the need for the development of a new vaccine. In an effort to develop an inactivated YF vaccine, Bio-Manguinhos/FIOCRUZ started developing a new vaccine based on the production of the attenuated 17DD virus in serum-free conditions in Vero cells propagated in bioreactors, followed by chromatography-based purification and ß-propiolactone inactivation. Virus purification was studied in this work. Capture was performed using an anion-exchange membrane adsorber (Sartobind® Q), resulting in a virus recovery of 80.2 ±â€¯4.8% and a residual DNA level of 1.3 ±â€¯1.6 ng/dose, thus in accordance with the recommendations of the WHO (<10 ng/dose). However, the level of host cell proteins (HCP) was still high for a human vaccine, so a second chromatography step was developed based on a multimodal resin (Capto™ Core 700). This step resulted in a virus recovery of 65.7 ±â€¯4.8% and decreased HCP levels to 345 ±â€¯25 ppm. The overall virus recovery in these chromatography steps was 52.7%. SDS-PAGE of the purified sample showed a band with molecular mass of 56 kDa, thus consistent with the virus envelope protein (E) and corresponding to 96.7% of identified proteins. A Western blot stained with an antibody against the E protein showed a single band, confirming the identity of the sample.

Dermatan sulfate is the predominant antithrombotic glycosaminoglycan in vessel walls: implications for a possible physiological function of heparin cofactor II.

The role of different glycosaminoglycan species from the vessel walls as physiological antithrombotic agents remains controversial. To further investigate this aspect we extracted glycosaminoglycans from human thoracic aorta and saphenous vein. The different species were highly purified and their anticoagulant and antithrombotic activities tested by in vitro and in vivo assays. We observed that dermatan sulfate is the major anticoagulant and antithrombotic among the vessel wall glycosaminoglycans while the bulk of heparan sulfate is a poorly sulfated glycosaminoglycan, devoid of anticoagulant and antithrombotic activities. Minor amounts of particular a heparan sulfate (< 5% of the total arterial glycosaminoglycans) with high anticoagulant activity were also observed, as assessed by its retention on an antithrombin-affinity column. Possibly, this anticoagulant heparan sulfate originates from the endothelial cells and may exert a significant physiological role due to its location in the interface between the vessel wall and the blood. In view of these results we discuss a possible balance between the two glycosaminoglycan-dependent anticoagulant pathways present in the vascular wall. One is based on antithrombin activation by the heparan sulfate expressed by the endothelial cells. The other, which may assume special relevance after vascular endothelial injury, is based on heparin cofactor II activation by the dermatan sulfate proteoglycans synthesized by cells from the subendothelial layer.

Increasing Vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium.

In this work, changes in Vero cell cultivation methods have been employed in order to improve cell growth conditions to obtain higher viable cell densities and to increase viral titers. The propagation of the 17DD yellow fever virus (YFV) in Vero cells grown on Cytodex I microcarriers was evaluated in 3-L bioreactor vessels. Prior to the current changes, Vero cells were repeatedly displaying insufficient microcarrier colonization. A modified cultivation process with four changes has resulted in higher cell densities and higher virus titers than previously observed for 17DD YFV.