Rapid development of an updated mRNA vaccine against the SARS-CoV-2 Omicron variant

Dear Editor, Since the declaration of public Health Emergency of International Concern (PHEIC) by the WHO, the COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to over 300 million confirmed cases with more than 5 million deaths in the past 2 years. On top of that, SARS-CoV-2 continues evolving into many variants, and many of these variants with evidence to enhance viral transmissibility, adaptiveness, infectivity, and/or to escape from host immune response are classified as variants of concerns (VOC). Since the outbreak of the pandemic, five VOCs, including Alpha, Beta, Gamma, Delta, and Omicron have been verified by the WHO. The newest SARS-CoV-2 VOC, Omicron (also known as B.1.1.529) designated by WHO was first reported in South Africa in November 2021. In a few weeks, Omicron has thrived throughout the world and became the predominantly circulating strain in most continents. Remarkably, Omicron carries an unprecedented number of mutations/deletions/insertion (over 30) in the spike (S) protein as well as the receptor binding domain (RBD), the main target of the host immune responses and vaccine development. Many of these mutations, e.g., K417Y, E484A, N501Y, D614G, P681H, have been identified in other VOCs and are predicted to affect neutralization epitopes. Indeed, accumulated evidence has demonstrated that the Omicron variant can largely escape from vaccination, convalescent sera and most approved monoclonal antibodies. For example, the most potent mRNA vaccines, BNT162b2 from Pfizer-BioNTech and mRNA-1273 from Moderna, also showed significant reduction in neutralization antibody titers against the Omicron variant. Previously, we have developed a Lipid nanoparticle (LNP)encapsulated mRNA vaccine ARCoV, which is at the final stage of a multi-regional phase 3 clinical trial (NCT04847102). Distinct from BNT162b2 and mRNA-1273, the two full-length S proteins-based mRNA vaccines, ARCoV encodes the RBD of the wild-type (WT) SARSCoV-2 S protein. As there are 15 variant mutations in Omicron RBD (Fig. 1a), it is interesting to assess the neutralizing activity of serum samples from vaccinees of ARCoV against the Omicron variant. To do so, a panel of serum samples (n= 11) from participants in the phase 1 clinical trial were analyzed for their neutralizing Ab titers using VSV-based pseudovirus. All samples were collected on day 14 post two-dose immunization with 15 μg of ARCoV. As shown in Fig. 1b, most samples (8/11, 72.7%) retained low but detectable neutralization activity against Omicron, with a 47-fold reduction in geometric mean titers (GMTs) against Omicron compared to the WT strain (GMT 1440.87 to 30.67). This observation is consistent with other studies with convalescent or vaccinee sera, suggesting the immune escape capability of Omicron. A third dose of mRNA vaccination (booster) has been widely used and well evidenced to induce more robust antibody response and improve vaccine efficacy against VOCs. To make sure the potential impact of a homologous booster of ARCoV, groups of 8–9-month female BALB/c mice that have received two doses of ARCoV were further boosted with a third dose ARCoV at day 300 post prime immunization (Fig. 1c). Remarkably, a booster immunization readily induced the production of neutralization antibody, and the GMTs against WT and Omicron increased to 28387.66 and 17206.32, respectively. Interestingly, only 1.65-fold reduction in GMTs against WT and Omicron was observed, and the difference is not statistically significant (Fig. 1c). This result highlights the invaluable benefits of a homologous booster vaccination and supports further validation in clinical trials. Considering the reduced neutralizing activities of sera from ARCoV vaccinees against Omicron variant, a new mRNA vaccine that directly targets the Omicron RBD will be critical. We therefore took an immediate action to start the effort of developing an Omicron mRNA vaccine based on our established LNP-mRNA vaccine platform. Briefly, the mRNAs encoding the Omicron RBD (Fig. 1d) were prepared and processed into the final LNP formulation as previously described. The most potent two mRNA constructs, named Omicron/1 and Omicron/2, were selected from a total of 18 mRNAs with different untranslated regions (UTRs) and codon optimizations based on their in vitro expression levels detected by enzyme linked immunosorbent assay (ELISA) (Supplementary information, Fig. S1). Western blotting results demonstrated that recombinant Omicron RBD proteins were expressed and secreted in the supernatants of Huh-7 cells transfected with both mRNAs (Fig. 1e). Immunofluorescence staining results further confirmed that both RBD proteins could be recognized by an Omicron RBD-reactive monoclonal antibody (Supplementary information, Fig. S2). Upon intravenous injection, both LNP-formulated mRNAs, ARCoV-Omicrons, are potent in producing Omicron RBDs in mouse sera (Fig. 1f). After two doses of intramuscularly immunizations at 7-day interval, robust IgG antibodies as well as neutralization antibodies were readily induced by the two ARCoV-Omicrons (Fig. 1g, h) at 14 days post initial immunization. The antibody titers induced by ARCoVOmicrons were comparable to the original mRNA vaccine ARCoV. It should be mentioned that a 0,7-immunization schedule was utilized in this study to accelerate research and development process, and a more robust antibody response can be expected with a regular 0,14-immunization schedule. The final clinical grade mRNA vaccine is currently being manufactured in a Good Manufacturing Practice (GMP) factory. Overall, our data presented here clearly demonstrate that a third dose of ARCoV would probably lead to a sharp increasement in neutralization antibodies not only against the WT SARS-CoV-2 but also the newly Omicron variant. Homologous booster vaccination with ARCoV represents a rational strategy in response to the Omicron emergency. More importantly, the continuously evolving SARS-CoV-2 calls for the most flexible and deployable


Sequence alignment and structural modeling
Structural modeling was built using SWISS-MODEL 1 (https://swissmodel.expasy.org) with 6 ZGE 2 as the template model and visualized with PyMOL (v2.5.0). All mutations on the spike protein were compared with the reference sequence (NC_045512).

mRNA preparation and characterization
All optimized DNA sequences encoding Omicron RBD were synthesized (Tsingke Biotechnology) and cloned into the plasmid vectors as described previously. 3 The mRNAs were produced in vitro using T7 RNA polymerase-mediated transcription from linearized DNA templates.
All Omicron RBD-encoding mRNAs (5 μg) were transfected into HEK-293T, Huh-7 or Vero cells using Lipofectamine TM 2000 (Thermo Fisher Scientific) following the manufacturer's instruction, and the supernatant and lysates were collected and subjected to ELISA, Western blotting, and immunofluorescent staining assays, respectively.
The Omicron RBD concentration was measured by ELISA as previously described. 3 Breifly, 96-well microtiter plates coated with human ACE2 (Sino Biological) were incubated with culture supernatants or mouse sera in serial dilutions.
The absorbance at 450/620 nm was measured and accurate quantification were conducted using SpectraMax iD3 (Molecular Devices).

LNP-mRNA formulation
Lipid-nanoparticle (LNP) formulations were prepared using the same procedure for ARCoV. 3 Briefly, lipids were dissolved in ethanol containing an ionizable lipid, 1, . The final formulations were tested for mRNA purity, mRNA encapsulation efficiency, particle size, and size distribution with the same release specification as ARCoV. 3

Omicron RBD expression in vivo
Groups of 6-8 weeks female ICR mice (n=5) were intravenously inoculated with ARCoV-Omicrons (1 mg/kg), and PBS (n=4) was used as Placebo. Sera were collected 6 hours post injection, and Omicron RBD concentration was determined by ELISA as described above.

Mouse vaccination
For ARCoV booster immunization, groups of female BALB/c mice aged 8-9 months (n=3) were intramuscularly immunized with 10 μg ARCoV at day 0, 14, and boosted at day 300. The blood samples were collected before and 14 days after the booster dose for detection of neutralizing antibodies against Wild-type and Omicron SARS-CoV-2.
Blood was collected at 14 days after initial immunization for Omicron specific IgG and pseudovirus-based neutralizing antibodies as described below.

Omicron specific IgG and neutralization antibody detection
The Omicron RBD-specific IgG antibody titers were detected by a modified ELISA as previously described. 3 The VSV-based pseudovirus neutralization assay was performed as previously described. 3 Briefly, heat-inactivated serum samples in serial dilutions were mixed with pseudoviruses carrying the spike of SARS-CoV-2 Wild-type or Omicron variant (Vazym, China) before adding to Huh-7 cells in 96-well white plate.

Statistical analysis
Data were presented as the mean ±SEM. The difference between any two groups were determined by unpaired parametric t-test or one-way ANOVA with multiple comparisons tests depending on the distribution of the data. All graphs were generated with GraphPad Prism version 9.0 software.