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Monday, August 13, 2018 9:55:34 PM
.
RESULTS
Physical and Biochemical Analysis of the Composition
of RSV Virosomes
Virosomes were prepared from purified RSV virus, strain A2, as described in Materials and Methods. Briefly, purified RSV virus was solubilized with DCPC, the viral nucleocapsid was removed by ultracentrifugation, the supernatant was added to a dry lipid film consisting of DOPC, DOPE, cholesterol and 3D-PHAD®, the film was solubilized in the DCPC-
containing supernatant and, after sterile filtration, the final
mixture was dialyzed.
The formation of virosomes was analyzed by equilibrium
sucrose density-gradient centrifugation (Fig. 1). Figure 2shows
the results for a typical virosome preparation. Protein and
phosphate were found to co-migrate in a single peak in the
gradient, indicating successful reconstitution of the viral enve-
lope. The absence of phosphate outside the virosome peak
indicates that DOPC, DOPE and 3D-PHAD® (which all
have phosphate groups) were essentially quantitatively associ-
ated with the virosomal membrane. Some non-incorporated
protein was present in fraction 3 of the gradient.
The protein composition of the virosomes was analyzed by
SDS-PAGE. In unfractionated virosome preparations, the F
and G membrane proteins were found (Fig. 3a), along with
the viral matrix (M), and phosphoprotein (P). G, uncleaved or
non-reduced F (F0), and its F1 subunit were identified by blot
(not shown); the F1 subunit has an apparent molecular weight
of 54 kDa, and the G-protein was around 120 kDa in size,
corresponding to the sizes of F and G reported in the literature
(39–41). On the gel, the position of uncleaved F (F0) overlaps
with that of G (Fig. 3a).In the virosome fraction of the gradi-
ent, F, G and M were present. The non-incorporated protein
consisted mainly of M and P, most likely from residual nucle-
ocapsid (Fig. 3b). Semi-quantitative analysis of M and P band
intensity of fraction 3 and 8 by ImageJ revealedthat 46% of M
and 40% of P are present in virosome fraction 8.
The incorporation of lipids and adjuvant into the
virosomes was analyzed by thin-layer chromatography
(TLC). The results of the TLC analysis confirmed that
DOPC, DOPE and 3D-PHAD® were present in the
virosome preparation (Fig. 3c). To roughly quantify the
amount of incorporated lipids and adjuvant, the intensities
of the spots observed after TLC analysis of the virosomal
lipids, shown in Fig. 3c, were determined using ImageJ anal-
ysis. The relative recoveries of DOPE and DOPC in the
virosomes were essentially equal and represented approxi-
mately half of the amounts that were initially added, indicat-
ing a somewhat lower recovery than that of the viral protein
(64%). However, the total quantity of virosome-associated ad-
juvant 3D-PHAD® was found to be less than 10% of the
amount initially added. It therefore appeared that 3D-
PHAD® was specifically lost during the production proce
(PDF) Development of a Virosomal RSV Vaccine.... Available from: https://www.researchgate.net/publication/326166555_Development_of_a_Virosomal_RSV_Vaccine_Containing_3D-PHADR_Adjuvant_Formulation_Composition_and_Long-Term_Stability [accessed Aug 13 2018].
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