Mymetics Corp (MYMX)

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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].