THE VLP PRESENTATION SYSTEM
In the life cycle of the influenza virus, formation of the viral particle involves the budding of a portion of the host plasma
membrane saturated with the viral surface proteins. HA is the major surface protein of the influenza virus with a transmembrane
domain that spans through the viral envelope. The authors demonstrated that transient expression of the HA surface protein
in plant cells results in the formation of VLPs budding from microdomains of the plasma membrane saturated with recombinant
HA.
Figure 2: Structural characteristics of influenza virus and viruslike particles, including (a) a comparison of surface attributes,
(b) a cross-section showing internal distinctions, and (c) transmission electron microscopy images of influenza viral particles
and influenza viruslike particles.
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At maturity, these VLPs have morphological features similar to those of the influenza virus, but only contain its immunogenic
determinants, with none of the viral RNA, which is packaged within the enveloped particles of true viruses. Compared with
isolated soluble antigens, antigens at the surface of VLPs have the double advantage of adopting a conformation that is identical
to those found on the cognate virus, and of being presented in a multivalent structure similar to that of the native virus.
These features enable VLPs to achieve enhanced stimulation of the immune response with balanced humoral and cellular components.
This benefit of VLPs over soluble recombinant antigens is believed to be particularly strong in vaccine products against enveloped
viruses because VLPs present the surface antigens in their natural, membrane-bound state (4). As the influenza viral particle,
influenza VLPs are enveloped, pleiomorphic, and 100 to 150 nm in size (see Figure 2).
THE MANUFACTURING OF INFLUENZA VLPS
HA expression plasmid, master, and working cell bank of transformed Agrobacteria
The recombinant influenza HA gene is built in such a way that native HA is expressed, thus yielding a mature protein product
identical to the wild type influenza HA protein. Expression is maximized by the integration of elements that result in high
transcription of mRNA and hypertranslation of the product. In addition to these elements, a suppressor of silencing is coexpressed
with the HA protein to counteract specific HA mRNA degradation (i.e., mRNA silencing) that is triggered by the significant
accumulation of HA mRNA in plant cells.
Before their transfer into A. tumefaciens, the plasmids are fully sequenced to ensure sequence integrity. Upon confirmation of integrity, plasmids are introduced into
A. tumefaciens, selected clones are grown in a master cell bank (MCB) that generates the working cell bank (WCB). The MCB and WCB are tested
for purity and plasmid integrity.
A liquid culture of the bacterial vector is produced from the WCB and used to prepare the A. tumefaciens inoculum for infiltration.
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