In this study, RBD was used as the vaccine antigen. A monomeric version of RBD preceded by SARS-CoV-2 spike signal peptide for secretion and a C-terminal hexahistidine (6xHis)-Tag was expressed after plasmid transfection in HEK-293 cells. The RBD segment selected in this study (residues 319-541) contains eight predicted immunodominant CD4⁺ T-cell epitopes and 17 predicted CD8⁺ T-cell epitopes in addition to B-cell epitope motifs (23).

Recombinant RBD was purified from cell culture supernatant through a single-step Ni-NTA affinity chromatography. SARS-CoV-2 RBD protein had high expression with remarkable purity ( Figure 1A ). Noteworthy, purified RBD was recognized by polyclonal antibodies in sera from a convalescent patient infected with SARS-CoV-2 ( Figure 1B ). Analytical evaluation by size exclusion chromatography revealed that the recombinant protein is monodispersed. The sample eluted as a single peak with an apparent molecular weight of 40.3 kDa, representing >95% of the sample ( Figure 1C ). Endotoxin levels measured in the purified protein were ≤1.25 EU/mg; this value is significantly lower than the maximum recommended endotoxin level for recombinant subunit human vaccines (24).

Purified RBD was also assessed for its direct binding to the human ACE2 receptor in ACE2-expressing HEK-293T cells by flow cytometry. Strong binding of recombinant RBD to hACE2-HEK-293T cells was observed (>90%) ( Figure 1D ). This result confirms that purified RBD binds to cell-associated hACE2 receptor, suggesting that it has a correct folding.

The immunogenicity of a variety of vaccine formulations comprising recombinant RBD with different adjuvants was evaluated in mice. We assessed formulations containing approved adjuvants for human use, including aluminum hydroxide, AddaS03 (similar to AS03), or AddaVax (similar to MF59), and the combination of alum and U-Omp19, a novel adjuvant developed in our laboratory that demonstrated vaccine adjuvant properties when co-administered with different Ags at pre-clinical stages.

Recombinant RBD was formulated with aluminum hydroxide (-alum-Alhydrogel 2%) alone, alum plus U-Omp19, AddaS03, or AddaVax. BALB/c mice received two doses at day 0 and 14 via intramuscular (i.m.) route ( Figure 2A ). After the first dose, animals immunized with RBD+alum or RBD+alum+U-Omp19 produced a specific anti-RBD IgG response in serum (GMT at day 14: 4,222 and 5,572, respectively, Figure 2B ). Anti-RBD IgG titers increased after the second dose reaching a plateau (GMT at day 44: 215,269 and 323,050, respectively, Figure 2B ). The groups of animals immunized with formulations containing AddaVax or AddaS03 as adjuvants failed to induce specific antibodies after the first dose ( Figure 2B ) but showed a significant anti-RBD IgG response after two doses (GMT at day 44: 337,794 and 215,269, respectively). RBD-specific IgG subclasses were evaluated 1 month after last immunization, demonstrating that all vaccine formulations induced higher titers of IgG1 than IgG2a in serum of mice ( Figure 2C ). Ag-specific-IgA at the low respiratory tract has an important role to control virus dissemination (25). Interestingly, levels of RBD-specific IgA in the bronchoalveolar lavage (BAL) of mice were higher in the group that received RBD+alum+U-Omp19 than in groups that received AddaVax or AddaS03 ( Figure 2D ). Besides, anti-RBD IgA was measured in serum samples revealing that groups containing alum or alum plus U-Omp19 as adjuvants induced significant higher levels compared to phosphate-buffered saline (PBS) or groups containing AddaVax or AddaS03 as adjuvants ( Figure 2E ).

Next, the neutralization capacity of the vaccine-induced antibodies was evaluated using an HIV-based pseudovirus neutralization assay (PsVNA). All vaccine formulations induced serum-neutralizing antibodies against the SARS-CoV-2 spike pseudotyped virus ( Figure 3A ). Remarkably, immunization with two doses of the formulation containing alum plus U-Omp19 as adjuvants induced a 10-fold increase in the neutralization titer (GMT, 325.1; 95%CI, 103.8–1,018) compared to the groups immunized with alum alone as adjuvant, AddaVax, or AddaS03 (GMT alum+RBD group: 34.2; 95%CI, 3.79–308.5, Figure 3A ). This increment was statistically significant (p = 0.0257 vs. RBD+alum, p = 0.0259 vs. AddaVax, and p =0.022 vs. AddaS03). AddaVax or AddaS03 as adjuvants induced a titer of neutralizing antibodies similar to alum alone (GMT AddaVax, 69.83; 95%CI, 33.08–147.4; GMT AddaS03, 45; 95%CI, 45–45, Figure 3B ).

To assess the functionality of vaccine-elicited antibodies against the ancestral SARS-CoV-2 (Wuhan reference strain), neutralization assay with sera from immunized animals was performed. Similar to the results obtained using the pseudovirus system, 1 month after the second dose, RBD+alum+U-Omp19 immunized mice had significant higher virus neutralization antibody titers in serum ( Figure 3C ; GMT, 612.1; 95%CI, 87.80–4267) than mice immunized with RBD+alum ( Figure 3C ; GMT, 140; 95%CI, 16.34–1,199) or plus commercial adjuvants (AddaVax or AddaS03). These results further confirm the data obtained by PsVNA.

As SARS-CoV-2 initially infects the upper respiratory tract, its first interactions with the immune system must occur predominantly at the respiratory mucosal surfaces. Mucosal responses may be crucial to stop person to person transmission of this virus. Thus, examination of neutralizing activity in BAL was performed using pseudotyped virus system. Adding U-Omp19 as an adjuvant to the formulation induced a slight but not significant increase in ancestral virus neutralization in the BAL of mice compared with the vaccine adjuvanted with alum alone, AddaVax, or AddaS03 ( Figure 3D ).

Duration of vaccine immunity is key to estimate how long protection lasts. To this effect, we evaluated the level of total antibodies over 175 days after prime immunization with the vaccine formulation containing U-Omp19 and alum as adjuvants. Interestingly, titers of anti-RBD IgG antibodies remained stable at least 5–6 months after i.m. immunization of mice with this formulation ( Figure 4A ). Remarkably, neutralizing capacity of the antibodies remained stable till day 175 post prime immunization ( Figure 4B ).

To adequately address the public health impact that newly emerging COVID-19 variants present, there is a need for vaccine-elicited antibodies that can cross-neutralize different SARS-CoV-2 variants. Thus, neutralization activity of sera against prevalent circulating variants of SARS-CoV-2 in our region, namely, alpha (B.1.1.7, first identified in UK), gamma (P.1, first identified in Manaos, Brazil), and lambda (C.37, first identified in Peru), was evaluated and compared to neutralizing activity against ancestral reference strain. In particular, gamma and lambda variants have been shown to partially escape neutralization by antibodies triggered by previously circulating variants or vaccine induced antibodies (26, 27). Noteworthy, antibodies induced after vaccination with the formulation containing U-Omp19 not only neutralize the ancestral virus but also cross-neutralized alpha, lambda, and gamma variants ( Figure 5 ). In contrast, antibodies produced by mice immunized with RBD+alum could neutralize the ancestral SARS-CoV-2 and alpha variant but showed significantly lower neutralizing activity against gamma and lambda variants ( Figure 5 ). AddaVax and AddaS03 adjuvanted formulations induced similar neutralizing antibody titers against the ancestral, gamma, and lambda variants ( Figure 5 ).

Altogether these results demonstrate that addition of U-Omp19 to the alum plus RBD vaccine formulation increases virus neutralizing antibodies, specific IgA in BAL, and neutralizing antibodies of the virus in BAL. Neutralizing antibodies are proposed as the best correlate of protection; thus, we focused the next studies on the vaccine formulation containing U-Omp19 as adjuvant.

In addition to memory B cells and neutralizing antibodies, induction of specific T-cell immune responses could have a role in protection against SARS-CoV-2 infection (28).

To determine T-cell-mediated immune responses, splenocytes and lung cells from RBD+alum or RBD+alum+U-Omp19 immunized mice were stimulated with RBD or medium alone, and then, cytokines levels in the supernatants were measured. Both formulations were able to induce Ag-specific cytokine secretion at spleen ( Figure 6A ). Importantly, the levels of interferon (IFN)-γ were higher than that of interleukin (IL)-5 at the spleens of both vaccine formulations. In the lungs, immunization with RBD+alum+U-Omp19 promoted a significant increment in IFN-γ secretion compared with the formulation containing RBD+alum ( Figure 6B ). However, the alum-adjuvanted vaccine elicited a higher amount of IL-5 in the lung ( Figure 6B ). These results suggest that U-Omp19 as adjuvant promotes a specific T-cell response biased to a Th1 profile in the lung.

To further evaluate the Th1/2 balance, IFN-γ- and IL-4-producing cells were measured by intracellular cytokine staining. Spleen cells from immunized mice were stimulated with a pool of SARS-CoV-2 RBD peptides to detect antigen-specific T-cell responses. Percentages of IFN-γ-producing CD4⁺ and CD8⁺ T cells were increased in both groups of mice while IL-4-producing CD4⁺ T cells were only increased after RBD+alum administration ( Figures 6C, D ). These results support the induction of Th1 and CD8⁺ T-cell immune responses after immunization with RBD adjuvanted with U-Omp19 in combination with alum.

Immunogenicity of vaccine formulations using alum alone or combining both adjuvants (alum and U-Omp19) with RBD as Ag was also evaluated in the C57BL/6 mouse strain. Vaccine formulations were administered following the same schedule used for BALB/c mice, two doses every 14 days.

Both vaccine formulations induced high anti-RBD IgG titers in sera ( Figure 7A ). Remarkably, anti-RBD IgA levels in the BAL of RBD+alum+U-Omp19 immunized mice were higher than in the RBD+alum immunized mice ( Figure 7B , p<0.05). There were no differences in specific IgG levels in the BAL between both groups ( Figure 7B ).

Formulation containing RBD+U-Omp19+alum induced higher neutralizing antibody titers than RBD+alum formulation ( Figure 7C ; GMT at 44 days post prime dose, 93.60; 95%CI, 19.36–452.5 and 37.47; 95%CI, 23.40–59.99, respectively).

The addition of U-Omp19 to RBD plus alum vaccine increased the neutralizing antibody titers against authentic ancestral virus. Four weeks after second dose, sera of mice immunized with RBD+alum+U-Omp19 produced a threefold increase in the viral neutralizing antibodies titer (GMT, 93.60; 95%CI, 19.36–452.5, Figure 7D ) compared with mice receiving RBD + alum ( Figure 7D ; GMT, 37.47; 95%CI, 23.40–59.99). These results validated the data obtained in BALB/c mice and further indicate that U-Omp19 can be used under different genetic backgrounds.

A persistent germinal center (GC) B cell response enables the generation of robust humoral immunity (29). Therefore, specific GC B cells were evaluated in spleens from vaccinated mice 1 month after second dose; Figure 8A shows the gating strategy used. There were no differences between groups in the total CG cells (B220⁺ CD19⁺ IgD⁻ CD95⁺ GL7⁺ cells) among spleen samples ( Figure 8B ). Of note, there were differences in the frequency of RBD⁺-specific GC cells, since mice immunized with RBD+alum+U-Omp19 increased the percentage of RBD⁺-specific GC cells in comparison with RBD+alum ( Figure 8C ). Besides, the percentages of RBD⁺-specific plasma blasts (B220⁺ CD19⁺ IgD⁻ CD138⁺ cells) were also higher in animals from RBD+alum+U-Omp19 than from RBD+alum ( Figure 8D ). These results indicate a better performance of the vaccine formulation containing U-Omp19 to induce specific GC and secretory B cells 1 month after immunization.

To determine vaccine efficacy, we used a severe disease model using K-18-hACE2 transgenic mice. Infection of transgenic mice with SARS-CoV-2 results in lung disease with signs of diffuse alveolar damage and variable spread to the central nervous system (30). The lethal dose 50% (LD50) is estimated to be 10 (4) plaque-forming units (PFU) (31). Vaccine formulation efficacy was evaluated in K18-hACE2 mice vaccinated with RBD+alum+U-Omp19 or PBS (control) and challenged intranasally with 2 × 10⁵ PFU of SARS-CoV-2. At day 5 post-infection, some animals were euthanized to assess the viral load in lungs and brains. The presence of the SARS-CoV-2 virus was not detected in the lungs, while very low virus titers were detected in the brains of animals vaccinated with RBD+alum+U-Omp19 ( Figure 9A ). In contrast, a high viral load was detected in the lungs and brains of animals immunized with PBS ( Figure 9A ). It is noteworthy that most of mice vaccinated with RBD+alum+U-Omp19 did not lose weight after challenge ( Figure 9B ). Thus, the RBD+alum+U-Omp19 vaccine induced protection against the experimental challenge with SARS-CoV-2.

Codon-optimized RBD containing spike signal peptide (residues 1-14) fused to the RBD domain (residues 319–541), and a C-terminal 6xHis tag was obtained. The protein was expressed from a pcDNA 3.1 plasmid in HEK 293 cells. Cells grown in monolayer were transfected with polyethylenimine (PEI), and 3 days after transfection, the supernatant was harvested and clarified by centrifugation at 1,500 × g for 15 min. The recombinant proteins were purified from supernatants by affinity chromatography with a Ni-agarose column (HisTrapTM HP, GE Healthcare, Chicago, IL), dialyzed against PBS, quantified, and stored at −80°C. LPS contamination from RBD was adsorbed with Sepharose–polymyxin B (Sigma Aldrich, St Louis, MO). Endotoxin determination was performed with a Limulus amebocyte chromogenic assay (Lonza, Basel, Switzerland).

RBD samples were run under reducing conditions by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Samples were mixed with Laemmli sample buffer with β-mercaptoethanol. The samples were incubated at 95°C for 5 min. Protein bands were visualized by staining with Coomassie blue R250. Bands were then transferred to a nitrocellulose membrane (GE, Healthcare, Chicago, IL), blocked with Tris-buffered saline (TBS)–Tween 0.05%, and incubated with human convalescent serum (1/100 dilution). An anti-human IRDye 800 (1/2,000 dilution) was used as a secondary antibody for infrared fluorescence detection on the Odyssey Imaging System.

SEC runs were performed on a Superdex 75 column by injecting 150 µg of RBD protein in 20 mM sodium phosphate buffer, pH 7.0 and 0.2M NaCl in the absence of DTT. Runs were performed at a flowrate of 0.4 ml/min. Apparent molecular weight was calculated by calibrating the column with molecular weight markers: bovine serum albumin (66.4 kDa), ovalbumin (44.3 kDa), papain (23 kDa), Ribonuclease A (13.7 kDa), and Aprotinin (6.5 kDa) with Vo = 8.1 ml and Vo+Vi = 19.5 ml.

Recombinant U-Omp19 was expressed in Escherichia coli cells and purified as previously described in Ref. 18. LPS contamination from RBD was adsorbed with sepharose–polymyxin B (Sigma Aldrich, St Louis, MO). Endotoxin determination was performed with a Limulus amebocyte chromogenic assay (Lonza, Basel, Switzerland). U-Omp19 preparations used contained <0.1 endotoxin units per milligram protein.

AddaVax or AddaS03 was purchased from InvivoGen (San Diego, CA, USA), and Alhydrogel 2% was kindly provided by CRODA (Newcastle, DE, USA). In vaccine formulations, Ag and U-Omp19 were absorbed to Alhydrogel (CRODA, Inc., Newcastle, DE, USA). Protein adsorption was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by Coomassie staining. Protein concentration was determined by the bicinchoninic acid method.

All experimental protocols with animals were conducted in strict accordance with international ethical standards for animal experimentation (Helsinki Declaration and its amendments, Amsterdam Protocol of welfare and animal protection and National Institutes of Health, USA NIH, guidelines). The protocols performed were also approved by the Institutional Committee for the use and care of experimental animals (CICUAE) from National University of San Martin (UNSAM) (01/2020).

Eight-week-old female BALB/c or C57BL/6 mice were obtained from IIB UNSAM animal facility. Four-week-old K18-hACE2 male and female mice were obtained from Jackson Laboratory.

Mice were intramuscularly (i.m) inoculated at day 0 and 14 with (i) RBD + Alhydrogel, (ii) RBD + Alhydrogel + U-Omp19, (iii) RBD + AddaVax, and (iv) RBD + AddaS03. The dose of RBD was 20 µg in the first inoculation and 10 µg in the second inoculation. Dose of adjuvants in the formulations were Alhydrogel, 500 µg; U-Omp19, 100 µg; AddaVax, 50 µl (InvivoGen); and AddaS03, 50 µl (InvivoGen). Blood samples were collected weekly to measure total and neutralizing antibody titers. Four weeks post prime immunization, animals were sacrificed, and the spleens, lungs, and bronchoalveolar lavages (BAL) were obtained.

RBD-specific antibody responses (IgA, IgG, IgG1, and IgG2a) were evaluated by indirect ELISA. Ninety-six-well plates were coated with 0.1 µg/well of RBD in phosphate-buffered saline (PBS) overnight at 4°C. Plates were washed with PBS–Tween 0.05% and blocked with PBS–Tween 0.01% 1% non-fat milk for 1 h. Plates were then incubated with sera or BAL (diluted in PBS–Tween 0.01% containing 1% non-fat milk) for 1 h, and then plates, were washed and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgA, IgG (Sigma, St. Louis, MO, USA), IgG1, or IgG2a (Thermo Fisher Scientific, Waltham, MA) for 1 h at 37°C. Then, TMB (3,3í,5,5í-tetramethylbenzidine) was added, and reaction was stopped with 4 N H₂SO₄ and immediately read at 450 nm to collect end-point ELISA data. End-point cutoff values for serum titer determination were calculated as the mean specific optical density (OD) plus three standard deviations (SD) from sera of saline-immunized mice, and titers were established as the reciprocal of the last dilution yielding an OD higher than the cutoff.

Plasmid pCMV14-3X-Flag-SARS-CoV-2 S was a gift from Zhaohui Qian (Addgene plasmid # 145780) (68), psPAX2 was a gift from Didier Trono (Addgene plasmid # 12260), and pLB-GFP was a gift from Stephan Kissler (Addgene plasmid # 11619).

Human embryonic kidney cell line 293T expressing the SV40 T-antigen (HEK-293T, ATCC #CRL-11268) was kindly provided by Cecilia Frecha (Instituto de Medicina Traslacional e Ingeniería Biomédica, Hospital Italiano de Buenos Aires) and maintained in complete Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermofisher Scientific. Waltham, MA USA) containing 10% (vol/vol) fetal bovine serum (FBS, Internegocios, Buenos Aires, Argentina), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Gibco). For lentivirus production, HEK-293T cells were maintained in DMEM10 containing 100 µg/ml G418 (Sigma Aldrich, St Louis, MO). African green monkey kidney cell line Vero E6 ATCC #CRL-1586 were cultured at 37°C in 5% CO₂ in Dulbecco’s modified Eagle’s high glucose medium (Sigma Aldrich) supplemented with 5% fetal bovine serum (FBS) (Sigma Aldrich).

HEK-293T cells expressing the SARS-CoV-2 receptor protein ACE2 (HEK-hACE2) were established in our laboratory by lentivirus transduction. Clonal selection of HEK-hACE2 was achieved by limit dilution and selection with 200 µg/ml of hygromycin.

For SARS-CoV-2 pseudovirus production, 5 × 10⁶ HEK-293T cells were seeded in complete DMEM in 10-cm dishes and incubated 24 h at 37°C and 5% CO₂. Pseudoviruses were obtained by co-transfection with psPAX2, pCMV14-3xFlag SARS-CoV-2 S, and pLB GFP by using polyetherimide (PEI) (1:2, DNA/PEI). The supernatants were harvested at 72 h post-transfection and centrifuged at 3,000×g for 15 min at 4°C. Then, HEPES was added to a final concentration of 20 mM to the supernatant, which was then passed through a 0.45-μm filter. When necessary, pseudovirus suspensions were concentrated by an overnight centrifugation at 3,000×g at 4°C. Pseudovirus (PsV) was titrated in HEK-hACE2 cells and stored at −70°C until use.

HEK-hACE2 cells were seeded in 96-well plates in DMEM10 and incubated 24 h at 37°C and 5% CO2. SARS-CoV-2 PsV (500–800 focus-forming units, FFU) were preincubated with serially diluted sera for 1 h at 37°C. Then, PsV-sera mixture was added to HEK-hACE2 and centrifuged at 2,500 rpm for 1 h at 26°C. After a 72-h incubation at 37°C and 5% CO₂, cultures were fixed with 4% paraformaldehyde for 20 min. Transduced cells express GFP, and neutralization titer 50 (NT50) was defined as the reciprocal serum dilution that causes a 50% reduction of transduction efficiency.

SARS-CoV-2 reference strain -ancestral- (hCoV-19/Argentina/PAIS-G0001/2020 GISAID Accession ID: EPI_ISL_499083) was obtained from Dr. Sandra Gallegos (InViV working group). SARS-CoV-2 Gamma P.1 (GISAID Accession ID: EPI_ISL_2756556) and alpha (GISAID Accession ID: EPI_ISL_2756558) were isolated in Instituto de Investigaciones Biomédicas en Retrovirus y SIDA (INBIRS, UBA-CONICET) from nasopharyngeal swabs of patients. SARS-CoV-2 Lambda C.37 (hCoV-19/Argentina/PAIS-A0612/2021 GISAID Accession ID: EPI_ISL_3320903) was isolated at INBIRS from a sample of nasopharyngeal swabs kindly transferred by Dr. Viegas and Proyecto PAIS. Virus was amplified in Vero E6 cells, and each stock was fully sequenced. Studies using SARS-CoV-2 were done in a Biosafety Level 3 laboratory, and the protocol was approved by the INBIRS Institutional Biosafety Committee.

Serum samples were heat inactivated at 56°C for 30 min. Serial dilutions were performed and then incubated for 1 h at 37°C in the presence of SARS-CoV-2 in DMEM 2% FBS. Fifty microliters of the mixtures were then added to Vero cells monolayers for an hour at 37°C (MOI = 0.004). Infectious media were removed and replaced for DMEM 2% FBS. After 72 h, cells were fixed with paraformaldehyde (PFA) 4% (4°C, 20 min) and stained with crystal violet solution in methanol. The cytopathic effect (CPE) of the virus on the cell monolayer was assessed visually, if even a minor damage to the monolayer (1–2 «plaques») was observed in the well; this well was considered as a well with a manifestation of CPE. Neutralization titer was defined as the highest serum dilution without any CPE in two of three replicable wells. Otherwise, plates were scanned for determination of media absorbance at 585 nm, and non-linear curves were fitted to obtain the titer corresponding to the 50% of neutralization (NT50). Neutralization assays to compare neutralization among different SARS-CoV-2 variants (alpha, gamma, and lambda) were performed in the same plate for each sample.

Four weeks after the second dose, mice were sacrificed to study cellular responses. Intracellular cytokine determination: splenocytes were cultured (4×10⁶ cells/well) in the presence of stimulus medium (complete medium supplemented with anti-CD28 and anti-CD49d) or Ag stimuli (stimulus medium + RBD-peptides + RBD protein) for 18 h. Next, brefeldin A was added for 5 h to the samples. After that, cells were washed, fixed, permeabilized, stained, and analyzed by flow cytometry. The cells were stained with Viability dye (Zombie Acqua), anti-mouse-CD8a Alexa Fluor 488, anti-mouse-CD4 Alexa Fluor 647, anti-IL-4 Brilliant Violet 421, and anti-IFN-γ PE (Biolegend, San Diego, CA).

Ag-specific B cells (plasmablasts and germinal center B cells) present in the spleens were determined by flow cytometry. Splenocytes were plated (2×10⁶ cells/well) and stained with Viability dye (Zombie Acqua), anti-B220 Alexa Fluor 594, anti-CD19 APC/Cy7, anti-CD138 Brilliant Violet 785, anti-IgD Brilliant Violet 605, anti-GL7 Alexa Fluor 488, and anti-CD95 PE (Biolegend, San Diego, CA). For Ag-specific detection, cells were also stained with fluorescent RBD (RBD conjugated to Alexa Fluor 647 succinimidil ester). Next, cells were washed, fixed, and analyzed by flow cytometry.

Four-week-old K18-hACE2 mice from Jackson Laboratory were used for evaluating vaccine efficacy. Mice were separated into two groups: (i) control, inoculated with PBS (n = 7), and (ii) vaccine, inoculated with RBD+alum+U-Omp19 (n = 8). Mice in each group included males and females. They were i.m. immunized at day 0 and 14 as described for immune assays. Four weeks post second vaccination, mice were challenged intranasally (i.n.) with 10⁵ PFU of SARS-CoV-2 strain WA1/2020 in each nare. Then, they were monitored daily for weight loss and signs of disease for 2 weeks post-challenge. Three mice per group were euthanized at day 5 post-challenge to evaluate organ viral loads, by plaque assay on Vero E6 cells.

Statistical analysis and plotting were performed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA). In experiments with more than two groups, data were analyzed using one-way ANOVA with a Bonferroni post-test. When necessary, a logarithmic transformation was applied prior to the analysis to obtain data with a normal distribution. In experiments with two groups, an unpaired t-test or Mann–Whitney U-test were used. A p-value <0.05 was considered significant. When bars were plotted, results were expressed as means ± SEM for each group.