Immune Response and Protection of mRNA Vaccine Against the SARS-CoV-2 Omicron-XEC Variant

The XEC-S-mRNA vaccine was constructed to encode the ectodomain of the S protein of Omicron-XEC with a HexaPro sequence, an N-terminal tissue plasminogen activator (tPA) signal peptide, a C-terminal foldon trimeric motif and a His₆ tag, followed by synthesis using a T7 transcription kit (Figure 1A). The synthesized mRNA was capped at the N-terminus, tailed with a poly(A) at the C-terminus, and then encapsulated with lipid nanoparticles (LNPs) to increase its stability during delivery to target cells or animals (Figure 1A).

The particle size and stability of the LNP-encapsulated XEC-S-mRNA at different temperatures were characterized using a DynaPro NanoStar II Light Scattering Detector (DLS) instrument. Overall, this mRNA was stable at all temperatures tested (i.e., 4 °C, 25 °C, and 37 °C) during the 7-day test period, with a particle size between 88 and 96 nm (Figure 1B). By comparison, the LNPs alone, without mRNA, had a slightly decreased particle size, and were not stable at 37 °C (Figure 1C). Similar sizes of XEC-S-mRNA encapsulated in LNPs and the LNP-alone control are shown in the histogram figures (Figure 1D,E). Flow cytometry analysis further identified the protein expression of His₆-tagged XEC-S-mRNA in target HEK293T cells (Figure 1F). These data confirm the stability and successful antigen expression of the synthesized XEC-S-mRNA vaccine.

To investigate the humoral immune responses induced by the XEC-S-mRNA vaccine, BALB/c-based K18-hACE2 (hereinafter BALB/c-hACE2) transgenic mice were intradermally (i.d.) immunized with either the LNP-encapsulated XEC-S-mRNA or the LNP control three times at 3-week intervals (based on our previously optimized protocol) [27]. Sera collected 10 days after the last dose were tested for S-specific antibody responses and neutralizing activity against multiple recent Omicron subvariants of SARS-CoV-2 (Figure 2A).

As expected, high-titer IgG antibodies specific to the S protein of Omicron-XEC were observed in the XEC-S-mRNA-immunized mice (Figure 2B). The XEC-S-mRNA also induced similarly high-titer S-specific IgG1 subtype (Figure 2C) and favorable IgG2a subtype (Figure 2D) antibodies.

These antibodies potently neutralized the infection of all the pseudotyped Omicron subvariants of SARS-CoV-2 tested, including the early and recent epidemic strains of KP.2, KP.3, XEC, NB.1.8.1 and XFG (Figure 3A–E). The neutralizing activity of the serum was confirmed by a live virus neutralization assay to neutralize the KP.2 and KP.3 strains (Figure 3F,G). These data indicate the ability of the XEC-S-mRNA vaccine to elicit highly potent and broad neutralizing antibodies against multiple strains of the SARS-CoV-2 Omicron variant.

The protective efficacy of XEC-S-mRNA was assessed in the immunized BALB/c-hACE2 transgenic mice 9 weeks after the last vaccination. The mice were intranasally (i.n.) challenged with the Omicron-KP.3 subvariant of SARS-CoV-2, and the viral titers in the lungs and trachea were evaluated 5 days post-infection (Figure 2A). Viral titers in the mice immunized with the XEC-S-mRNA vaccine were below the detection limit in both the lungs (Figure 4A) and the trachea (Figure 4B), and were significantly lower than those in the control mice receiving only LNPs. These data demonstrate the efficacy of the XEC-S-mRNA vaccine in cross-protecting mice against an Omicron-KP.3 challenge.

In a separate experiment, BALB/c mice were immunized with either the XEC-S-mRNA vaccine or LNP control and boosted at 3 and 6 weeks (Figure 5A). To determine whether XEC-S-mRNA-induced neutralizing antibodies were important in protecting against SARS-CoV-2 Omicron infection, the mice were boosted again at 22 weeks (with the goal of obtaining high-titer serum neutralizing antibodies for challenge studies), and the pooled sera were intraperitoneally (i.p.) injected into naïve C57BL/6-based K18-hACE2 (B6-hACE2) transgenic mice (with the goal of validating the protective efficacy in another mouse strain) (Figure 5A). This was followed by the challenge of the B6-hACE2 mice with the Omicron-KP.3 subvariant 6 h post-serum injection, with measurement of viral titers in the lungs and trachea performed 5 days after the challenge (Figure 5A). Indeed, the pooled sera from the XEC-S-mRNA-immunized BALB/c mice exhibited potent neutralizing activity against infection from both the pseudotyped (Figure 5B) and live virus (Figure 5C) of the Omicron-KP.3 subvariant of SARS-CoV-2. As expected, B6-hACE2 mice receiving these immune sera presented significantly lower viral titers in the lungs (Figure 5D) and trachea (Figure 5E) compared to B6-hACE2 mice receiving sera from BALB/c mice injected with control LNPs alone (Figure 5), with the former values both being below the detection limit. These results show that serum antibodies elicited by the XEC-S-mRNA vaccine are critical for passively protecting naïve mice from a SARS-CoV-2 Omicron-KP.3 challenge.

The ability of the XEC-S-mRNA vaccine to induce durable cellular immune responses was evaluated in immunized BALB/c mice 32 weeks after the immunization, and splenocytes were isolated for this test using a multiplex assay (Figure 5A). Notably, high-levels of GM-CSF, IFN-γ, TNF-α, and IL-4 cytokines were observed in the mice immunized with the XEC-S-mRNA, whereas only background levels of such cytokines were found in the control mice receiving LNPs alone (Figure 6A–D). These data reveal that the XEC-S-mRNA elicited effective and long-term cellular immune responses in the immunized mice.

HEK293T cells expressing SARS-CoV-2 receptor human ACE2 (hACE2/293T, Laboratory stock) and HEK293T cells (ATCC, Manassas, VA, USA) were diluted in Dulbecco’s Modified Eagle Medium (DMEM) cell culture medium containing 1% Penicillin-Streptomycin solution (Corning, New York, NY, USA) and 10% Fetal Bovine Serum (FBS) (R&D Systems, Minneapolis, MN, USA), and cultured in a 37 °C cell culture incubator supplied with 5% CO₂. HEK293F cells (Thermo Fisher Scientific, Waltham, MA, USA) were diluted in a serum-free medium (ESF SFM) (Expression Systems, Davis, CA, USA) containing 1% Antibiotic-Antimycotic solution (Corning), and cultured in a 37 °C cell culture incubator supplied with 8% CO₂ and subjected to constant rotation at 120 rpm.

Male and female BALB/c mice, BALB/c mice expressing human ACE2 (i.e., BALB/c-K18-hACE2 or BALB/c-hACE2), and C57BL/6 mice expressing human ACE2 (i.e., B6-K18-hACE2 or B6-hACE2) were initially obtained from the Jackson Laboratory, then bred in our animal facilities until use. The mice were randomly allocated to various groups (5 mice in each group) for the immunization and/or challenge experiments described below.

The template for mRNA synthesis was designed and constructed as described below. The S gene of the SARS-CoV-2 Omicron-XEC subvariant (GISAID number: EPI_ISL_19612171) was codon-optimized and synthesized by GenScript (Piscataway, NJ, USA), which consists of a HexaPro sequence (e.g., the furin cleavage site mutation and six proline amino acid substitutions). The synthesized XEC-S gene, together with an N-terminal signal peptide (tPA), a C-terminal trimeric foldon and 6× His tag sequences, was amplified via PCR and ligated with an enzyme-digested pCAGGS expression vector using Seamless Cloning reagent (Vazyme International LLC, Woburn, MA, USA). A positive XEC-S recombinant plasmid was verified by Sanger DNA sequencing (Psomagen, Rockville, MD, USA).

The mRNA vaccine was synthesized as described below. After linearizing the aforementioned XEC-S recombinant plasmid, the mRNA was synthesized in vitro using a T7 Transcription Kit (MEGAscript™, Thermo Fisher Scientific), according to the manufacturer’s instructions. This was performed using a naturally occurring nucleotide, Pseudo-UTP (APExBIO, Houston, TX, USA), which incorporates pseudouridine as a substitute for uridine, together with three regular nucleotides, including ATP, GTP, and CTP. The ScriptCap™ Cap 1 Capping System (CELLSCRIPT, Madison, WI, USA) and an A-Plus™ Poly(A) Polymerase Tailing Kit (CELLSCRIPT) were then utilized to add the Cap1 structure and a poly(A) tail to the 5′ and 3′ ends of the synthesized mRNA, respectively. The mRNA products were purified using a Monarch^® Spin RNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA) and stored at −80 °C until further use.

The LNP was formulated by dissolving IM21.7c (PolyPlus, Berkeley, CA, USA), SM102 (Cayman Chemical, Ann Arbor, MI, USA), DSPC (Avanti Research, Foster, CA, USA), cholesterol (Sigma-Aldrich, St. Louis, MO, USA), and DMG-PEG2000 (Cayman Chemical) at a ratio of 20:30:10:38.5:1.5 in anhydrous ethanol. The above-synthesized mRNA was dissolved in 10 mM of sodium acetate (pH 4). The LNP and XEC-S mRNA were mixed at a volume ratio of 1:3 using a NanoAssemblr™ Ignite System (Cytiva, Marlborough, MA, USA) for nanoparticle formulation. The XEC-S mRNA-LNP formulation was then diluted with a 30-fold volume of 1× phosphate buffer saline (PBS) (pH 7.4) and concentrated with Amicon Ultra Centrifugal Filter (10 kDa MWCO; MilliporeSigma, Burlington, MA, USA). The particle size of the formulated mRNA-LNP was measured using the DLS Detector (Wyatt Technology, Santa Barbara, CA, USA).

To test the expression of the His-tagged, mRNA-encoded protein, HEK293T cells were seeded onto a 6-well cell culture plate and cultured overnight until the cells reached 75% confluency. The cells were then incubated with either 4 µg of the XEC-S mRNA-LNPs or the control LNPs. After 24 h, Brefeldin A (Biolegend, SanDiego, CA, USA) was added to the cells at a final concentration of 2.5 µg/mL, with the goal to block the secretion of protein. The cells were continued to culture for another 24 h, followed by detachment with trypsin and two washes with 1× PBS. The cells were stained with eFluor 780 Fixable Viability Dye (Thermo Fisher Scientific) at 4 °C for 20 min, then fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences, Bedford, MA, USA). The fixed and permeabilized cells were further stained with an anti-His-FITC antibody (1:100 dilution; Thermo Fisher Scientific) at room temperature for 30 min. After the staining, the cells were analyzed for protein expression using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Brea, CA, USA). The resulting data was analyzed using FlowJo software v7.6.

HEK293F cells were transfected by a sequence-verified recombinant His-tagged plasmid encoding S protein of a SARS-CoV-2 Omicron-XEC subvariant (e.g., XEC-S protein) using PEI MAX (Linear Polyethylenimine Hydrochloride, M.W 40,000) (Kyfora Bio, Horsham, PA, USA), with a DNA: PEI MAX weight ratio of 1:3. Five days after transfection, the culture supernatant containing the expressed protein was collected by centrifugation at 6000× g for 20 min. The expressed protein was purified using Ni-NTA Superflow Resin (Qiagen, Germantown, MD, USA), followed by dialysis against 1× PBS (pH 7.4) using Amicon Ultra Centrifugal Filter (50 kDa MWCO, MilliporeSigma).

The titer of specific antibodies (IgG, IgG1, and IgG2a) in sera of the immunized mice was determined using the ELISA technique. Half-area, 96-well ELISA microplates were coated with the above-purified His-tagged XEC-S protein (1 μg/mL) in a 50 mM carbonate coating buffer (pH 9.4) overnight at 4 °C. The coated plates were washed once with 1× PBS containing 0.1% Tween-20 (PBST) and blocked with 2% fat-free milk (Bio-Rad, Hercules, CA, USA) prepared in PBST at 37 °C for 1 h. After washing with PBST, the plates were incubated with serially diluted mouse sera at 37 °C for 2 h. The plates were then washed again with PBST 6 times and incubated at 37 °C for 1 h with horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (1:25,000 dilution; Thermo Fisher Scientific), anti-mouse-IgG1 antibody (1:25,000 dilution; Thermo Fisher Scientific), or anti-mouse-IgG2a antibody (1:2000 dilution; Thermo Fisher Scientific). After washing 6 times with PBST, the plates were incubated with the substrate TMB (3,3′,5,5′-Tetramethylbenzidine) (MilliporeSigma), before terminating the reaction with 1 N of sulfuric acid. Absorbance at 450 nm (OD₄₅₀) was acquired by reading the plates with a Cytation 7 microplate reader (BioTek Instruments, Winooski, VT, USA). The OD₄₅₀ values were analyzed via non-linear regression, and the titer of specific antibodies was calculated accordingly (the cutoff was set as four times the blank (e.g., 0.192)).

The pseudoviruses, including the KP.2, KP.3, XEC, NB.1.8.1, and XFG Omicron subvariants of SARS-CoV-2, were produced from HEK293T cells co-transfected with pLenti-CMV-LUC (Addgene, Watertwon, MA, USA) plasmid, psPAX2 (Addgene) plasmid, and each of the recombinant plasmids (pcDNA3.1 backbone) encoding-relevant S protein of the above SARS-CoV-2 Omicron subvariants, using the PEI MAX transfection reagent. The medium was replaced with a fresh culture medium (e.g., DMEM containing 10% FBS and a 1% Penicillin-Streptomycin solution) ~18 h post-transfection. The culture supernatant containing the produced pseudovirus was harvested 2 to 3 days after transfection by centrifugation at 2000 rpm for 5 min. For the pseudovirus neutralization assay, target cells (hACE2/293T) were seeded onto 96-well culture plates (2 × 10⁴ cells/100 µL per well) 4~6 h before the experiment. The mouse sera at serial dilutions were pre-incubated with the respective types of pseudoviruses collected above at 37 °C for 1 h, then 100 µL/well of the serum-pseudovirus mixture was added to the above pre-seeded target cells in 96-well plates. After 24 h, the cells were replenished with 80 µL of fresh culture medium, then they continued to culture for another 48 h. The cells were lysed with cell lysis buffer (Promega, Madison, WI, USA) at room temperature for 1 h, followed by the addition of luciferase substrate (Promega). The relative luciferase activity was immediately measured via a Cytation 7 imaging reader (BioTek Instruments). The NT₅₀ values (i.e., the serum dilution at which the pseudovirus was neutralized by 50%) were calculated as the pseudovirus neutralizing antibody titer using a method based on the Median Effect Principle.

A CPE-based neutralization method was used to evaluate neutralizing activity of the sera of mice immunized against KP.2 and KP.3 Omicron subvariants of live SARS-CoV-2. Target cells (Vero E6) were seeded onto 96-well culture plates 24 h before the experiment. The mouse sera at serial dilutions were pre-incubated with 100 TCID₅₀ (50% tissue culture infectious dose) per well of each virus at 37 °C for 1 h, and the mixture of sera and virus was added to the above pre-seeded target cells in 96-well plates. The cells continued to culture at 37 °C for 4–5 days and were then assessed for the presence of CPE using a microscope. The NT₅₀ values (e.g., the highest serum dilution at which at least 50% of the cells were inhibited from the CPE) were calculated as the live virus neutralizing antibody titer.

To evaluate the immune responses and protective efficacy induced by the mRNA vaccine, BALB/c-hACE2 transgenic mice (~20–24-week-old, female and male) were (i.d.) vaccinated with either the LNP-formulated XEC-S-mRNA vaccine (100 μL containing 10 μg mRNA per mouse) or control LNPs (100 μL per mouse) using an optimal three-dose scheme at three-week intervals. Ten days after the third injection, the sera were collected via facial bleeding and inactivated at 56 °C for 30 min before use. The immunized mice were (i.n.) challenged with the KP.3 live virus of the SARS-CoV-2 Omicron subvariant (10⁴ plaque-forming unit (PFU)/mouse) under anesthesia 9 weeks after the third dose of vaccination.

Immune sera for passive transfer were generated by immunizing ~9–11-week-old, male BALB/c mice with the LNP-formulated XEC-S-mRNA vaccine (10 μg/100 μL/mouse) or control LNPs (100 μL/mouse) via i.d. route at weeks 0, 3, 6 and 22. The serum samples were collected by facial bleeding 3 times (10, 17, and 28 days after the final immunization) and inactivated at 56 °C for 30 min. The pooled mouse immune sera were tested for neutralizing antibodies against infection from the Omicron-KP.3 pseudovirus and live virus. These pooled mouse immune sera were then passively transferred into ~10–12-week-old, naïve female and male B6-hACE2 mice (200 μL/mouse, 5 mice/group) via the intraperitoneal (i.p.) route. The mice receiving the pooled sera were further (i.n.) challenged with the KP.3 live virus of the SARS-CoV-2 Omicron subvariant (10⁴ PFU/mouse) 6 h post-transfer of the immune sera.

The mice challenged with the SARS-CoV-2 KP.3 virus described above were sacrificed on day 5 post-challenge, and lung and trachea tissue samples were collected for the measurement of viral titers using the plaque assay described below. The tissues were homogenized and added with serial dilutions to pre-seed Vero E6 cells, prior to incubation at 37 °C for 1 h. After removing the culture medium, an MEM culture medium containing 2% FBS and 1.25% carboxymethyl cellulose solution was added to cells, then the cells continued to culture at 37 °C for 4 days. After washing with PBS, the cells were fixed with 10% formaldehyde at 37 °C for 2 h and stained with 0.5% crystal violet, until the presence of plaques was observed. The viral titer in the tissues was calculated based on the number of plaques, which was shown as the PFU/mL of each tissue sample.

A multiplex assay was used to detect cytokine expression in immunized mouse splenocytes in response to the XEC-S protein stimulation. Ten weeks after the final immunization of the BALB/c mice with the LNP-formulated XEC-S-mRNA or control LNPs described above, the mice were euthanized, and the splenocytes were extracted for evaluation of cellular immune responses via the multiplex assay. The splenocytes isolated from the immunized or control mice were cultured in a RPMI 1640 medium, which contained 1% Penicillin-Streptomycin solution, 10% FBS, 1 mM of sodium pyruvate, 55 µM of β-Mercaptoethanol, and 1% non-essential amino acids (Sigma-Aldrich). The splenocytes were seeded onto 96-well, round-bottom culture plates (1 × 10⁶ cells/mL, 200 µL/well) and stimulated with the XEC-S protein of SARS-CoV-2 (5 μg/mL) for four days in a 37 °C cell culture incubator supplied with 5% CO₂. The cell culture supernatant was collected after centrifugation and cytokine expression was then measured using a Bio-Plex Pro Mouse Cytokine Assay kit (Bio-Rad). The results were analyzed with the Bio-Plex 200 System instrument (Bio-Rad).

An unpaired-t test was used to compare difference between the two groups. p < 0.05, p < 0.01, and p < 0.0001 are shown as *, **, and ****, respectively. All the statistical calculations were performed using the statistical software GraphPad Prism 10.