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.