CuMVTT-VLPs constitute an efficient platform for
genetically fusing the receptor-binding domain (RBM)
Our first attempt to generate a COVID-19 vaccine using
CuMVTT VLPs platform, utilized recombinant RBD which was
chemically coupled to the VLPs using SMPH cross-linker (19). This method
resulted in a vaccine candidate that binds ACE2 and induces high levels
of RBD-specific antibodies which were able to strongly inhibit RBD
binding to ACE2 and neutralize SARS-CoV-2/ABS/NL20 virus (20).
In an attempt to produce a more readily scalable vaccine-candidate with
better yield, we genetically fused RBM domain into
CuMVTT to produce a mosaic vaccine as illustrated in
Figure 1A. The mosaic vaccine (mCuMVTT-RBM) consists of
both unmodified and a genetically modified monomer spontaneously
assembling in E. coli to form VLPs. The genetically modified
monomer displays RBM domain on the exterior surface.
mCuMVTT-RBM was expressed and produced in E.
coli. In contrast, incorporating either the RBD or RBM into all VLP
coat protein subunits was not successful and resulted in formation of
coat protein aggregates and insoluble VLPs (data not shown).
Purification of mCuMVTT-RBM was carried out by
ultracentrifugation using a 20-60% sucrose gradient (Fig. 1B). We have
shown previously that using E . coli as an expression
system facilitates packaging of prokaryotic ssRNA which serves as a
TLR7/9 ligand and results in an enhanced immune response (16, 21) (Fig.
1C). The final product mCuMVTT-RBM consists of an
unmodified VLP coat protein monomer of ~28 kDA in size
while the genetically modified one is ~42 kDA as shown
in the SDS-PAGE (Fig. 1D). Densitometric analysis suggested 40-50%
incorporation of coat protein-RBM fusion molecules into the VLPs. As
each VLP contains 180 capsid proteins, 40-50% RBM means that each VLP
has about 70-90 RBM antigens. Electron microscopy confirmed the
successful assembly of icosahedral mCuMVTT-RBM intoT=3 particles with no sign of aggregation or malformation of the
particles (Fig. 1E). Dynamic light scattering revealed a uniform and
homogenous peak of hydrodynamic diameter (DH) of ~94nm
(Fig. 1F).
An ideal vaccine would have a long shelf life at ambient temperatures,
nevertheless many commercial vaccines require storage temperatures
between 2-4°C (22). Whilst maintaining a vaccine in a cold chain below
freezing temperature can be difficult in developed countries, it is
particularly challenging and sometime prohibitive for developing
countries (23). Accordingly, we tested the stability of our vaccine
candidate. The results indicated that the vaccine is stable for 14
months at 4°C and for approximately 1 month at RT (Supplementary Fig.1).
As mentioned above, RBM is the part of the RBD, which is responsible for
the binding of the virus to the receptor ACE2. It is also the major site
of neutralizing antibody epitopes. To confirm the native confirmation of
RBM in the context of the mosaic fusion VLP, we tested whether the
vaccine was able to bind to ACE2. To this end, the human receptor ACE2
was coated onto an ELISA plate. The candidate vaccine or a control
CuMVTT VLP without an RBM insertion were then added and
anti-CuMVTT secondary antibodies used to detect receptor
bound VLPs. The results confirmed that mCuMVTT-RBM
vaccine can bind to ACE2 receptor indicating that RBM exhibits the right
native conformation(s) on the surface of the VLPs. The control did not
show any binding (Fig. 1G).