Discussion:
In the current study, we investigated a novel VLP-based vaccine for COVID-19. Specifically, we have generated a mosaic VLP-vaccine using the plant-derived cucumber-mosaic VLPs (CuMVTT). Mosaic VLPs are well known in the field with GSKs RTS, S malaria vaccines as the most prominent member of the filed (34). The mosaic vaccine candidate consists of an unmodified monomer and a genetically modified monomer that incorporates the RBM domain of SARS-CoV-2. The RBM domain was chosen as the target epitope due to the fact that RBD/RBM are considered the immunological Achille’s heel of SARS-CoV-2 and unlike RBD, RBM domain does not show any glycosylation, likely facilitating protein-protein interaction with ACE2 (10, 35, 36). Furthermore, incorporating the whole RBD domain into CuMVTT did not allow the formation of VLPs most likely due to steric constraints. The used genetic fusion technique in this study facilitated the assembly ofT=3 icosahedral VLPs which is essential for effective induction of a humoral immune response (13). Using this technique, we have recently developed a vaccine against MERS-CoV by incorporating the RBM domain into CuMVTT. The developed vaccine induced antibodies that completely neutralized MERS-CoV/EMC/2012 isolate (manuscript in press). Furthermore, we have shown that fusing RBM domain into AP205-VLPs results in an effective vaccine which induced RBD and spike-antibodies and was capable of neutralizing the wild type virus SARS-CoV-2/ABS/NL20 (37). Development of AP205-RBM vaccine required a refolding process which typically results in lower amounts of correctly folded target protein, and may be distinguished from re-assembly processes used for HPV and  HEV vaccines (38). The simplicity of the downstream processing at industrial scale is therefore a major advantage of the current vaccine candidate.
Using CuMVTT-VLPs as a vaccine platform resulted in a soluble VLP. The SDS-PAGE analysis indicated 40-50% incorporation of RBM domain. Using Sandwich ELISAs, we have shown that the mCuMVTT-RBM vaccine is able to detect and bind to the viral receptor ACE2. This confirms that the RBM domain displayed and fused to the VLP, has the correct conformation which is essential for the induction of the appropriate neutralizing antibody response. Expression in E . coli facilitates the packaging of bacterial RNA which serves as TLR7/8 ligand. We have shown previously that VLP-based vaccines are capable of inducing IL-21 independent secondary plasma cells only in the presence of TLR7/8 agonist such as bacterial ssRNA (16, 21). Additionally, TLR7/8 agonist polarizes the immune response towards TH1 and cytotoxic T cells which is essential to avoid enhanced disease as shown in preclinical challenge models (9). A recent study has shown that IgA antibodies in serum, saliva as well as bronchoalveolar lavage dominated the early response against SARS-CoV-2 infection in comparison to IgG and IgM. Sterlin et al , have also shown that IgA serum are more potent in neutralizing wild type SARS-CoV-2 than IgG (39). RNA loaded VLPs may also induce IgA responses, again in a TLR7/8 dependent manner (16, 40). This appears particularly important for SARS-CoV-2 and other respiratory diseases-causing viruses, such as SARS-CoV-1 and MERS-CoV-2, as IgA may be able to neutralize the virus locally in the lung without causing inflammation, a feature that may be particularly critical in patients with high viral load (41). Thus, it is therefore of key importance that our newly developed vaccine is able to induce a significant increase in serum IgA levels. Whether the increased serum IgA levels in mice can translate to correspondingly high IgA levels in humans and in particular at mucosa sites needs to be confirmed.
We have also shown that mCuMVTT-RBM vaccine candidate is strongly immunogenic in mice and rabbits. The response was further augmented following the booster dose. Using a D0/28 vaccination regimen induced a better quality of RBD and spike-protein antibodies in comparison to D0/14 regimen. This may indicate that it takes longer to induce such high-affinity/avidity antibodies as demonstrated here by mCuMVTT-RBM.
It has been shown that the induced neutralizing antibody response in SARS-CoV-2 patients are of low and short duration (27, 32). This may be explained by coronaviruses morphological structure which are large particles with long spike proteins exhibiting RBD trimers spaced by 25 nm. Other viruses as well as virus-like particles (VLPs) are capable of inducing optimal and long-lived neutralizing antibodies thanks to the 180 monomers forming a repetitive surface structure with epitopes spaced by 5-10 nm (27). The induced antibodies using mCuMVTT-RBM vaccine candidate could be detected in a similar level 4 months following the priming boost in the immunized mice sera. Furthermore, the main goal of any-viral vaccine is the induction of neutralizing antibodies that can inhibit SARS-CoV-2 infection. Our test sera were probed for their ability to inhibit a cytopathic effect (CPE) of wild-type SARS-CoV-2 isolate on Vero cells. The neutralizing capability of the virus was further enhanced following a 3rd dose.
We have shown recently that N501Y mutation enhanced the binding affinity to ACE2 but did not significantly affect the recognition of RBD by convalescent sera, which was not the case for E484K mutation that resulted in abolished the recognition (42). mCuMVTT-RBM is shown here to induce antibodies of much higher affinity/avidity than SARS-CoV-2 typically does in humans. This increased affinity/avidity translates to increased cross-reactivity with SARS-CoV-2 VoC. Indeed, antibodies induced by the here presented vaccine candidate recognizes variant strains of concern from Brazil, UK and India with equal efficiency suggesting that our vaccine can protect against the new variants. In addition, the vaccine candidate may be stored at 4oC for at least a year, representing in addition to the very high production yields and immunogenicity, two additional key assets of mCuMVTT-RBM.
Collectively, we have shown in this study that this novel mosaic VLP-based vaccine can efficiently induce high specific anti-RBD and spike antibodies that effectively neutalize SARS-CoV-2 and highly cross-reacts with all emerging viral VoC tested. As COVID-19 continues to represents a global threat to human health, it seems rational to further develop this vaccine candidate.