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).