A SARS-CoV-2 and influenza double hit vaccine based on RBD-conjugated inactivated influenza A virus

To construct the Flu-RBD VLP vaccine, RBD antigen was modified with azido–polyethylene glycol–N-hydroxysuccinimidyl ester (AZO-PEG-NHS). SDS–polyacrylamide gel electrophoresis revealed the successfully conjugation of RBD with AZO-PEG-NHS (RBD-PEG-AZO) who ran more slowly than free RBD (fig. S1). Fourier transform infrared spectroscopy (FTIR) analysis further demonstrated that the successfully preparation of RBD-PEG-AZO as indicated by the presence of -N≡N stretching vibration located at 2161 cm⁻¹ (fig. S2). Meanwhile, Flu was conjugated with dibenzocyclooctyne (DBCO)-PEG-NHS to synthesize DBCO-PEG-Flu, which demonstrated by the FTIR analysis (fig. S3). Through the bio-orthogonal click reaction of DBCO with AZO, RBD was anchored on the surface of Flu successfully; in this way, a Flu-RBD vaccine with double-protecting ability was fabricated (Fig. 2A). Immunoblotting analysis showed that RBD band was presented in Flu-RBD and RBD lysates rather than in Flu lysate (Fig. 2B). Likewise, HA band, the marker of Flu, was observed in both Flu-RBD and Flu lysates but in free RBD (Fig. 2B). In addition, distinct immunogold nanoparticles against RBD antigen were found on the surface of Flu (Fig. 2C), indicative of the conjugation of RBD with Flu. FTIR results further demonstrated the successful preparation of Flu-RBD (fig. S3). In addition, we found that RBD modification caused a slight increase in the average diameter of Flu-RBD as evidenced by nanoparticle tracking analysis (NTA) (Fig. 2D). The reaction efficiency of RBD with Flu was calculated to be 71%, indicating that there is 0.71-μg RBD presented per 10¹⁰ Flu particles. Theoretically, approximately 1669 RBD molecules were bound to each individual Flu. Moreover, this conjugation strategy of RBD on Flu has little effect on the conformation and binding sites of RBD (fig. S4), ensuring its immunogenic profile as an antigen. Collectively, these results confirmed the successful synthesis of Flu-RBD VLP vaccine.

To be effective vaccines, the platform should promote the trafficking of antigen from injection site to draining LNs (28). Toward this end, free RBD and Flu-RBD were labeled with Rhodamine (RhB) to evaluate their microscopic distribution in vivo. Both were intramuscularly (i.m.) injected into the right hind limb of mice. The proximal and distal LNs from injection sites, including right and left inguinal LNs as well as cervical LNs, were collected. As illustrated in Fig. 2 (E and F), ex vivo fluorescence imaging and analysis of LNs showed that mice received with Flu-RBD were sacrificed 24 hours postinjection exhibited the greatest integrated density. These proportions in draining LNs at 24 hours after treatment with Flu-RBD were significantly higher than those treated with free RBD. These results suggested that Flu core improves the retention and distribution of RBD in both superficial and cervical LNs. In addition, we moved on to evaluate whether Flu-RBD could be delivered into major organs of animals after i.m. injection. As shown in fig. S5, Flu-RBD-RhB was discovered in the spleen, liver, kidneys, lung, and heart, which in line with the previous report that the intramuscularly injected nanoparticles could be absorbed into the bloodstream quickly (29). Compared to Flu-RBD-RhB, lower RBD signals were observed in the mice treated RBD-RhB group due to the rapid clearance of free RBD in vivo.

Efficient antigen uptake by antigen-presenting cells (APCs) is the key for vaccines to trigger immune response (19). Before assessing the internalization of Flu-RBD by APCs, we found that the proportion of Flu-RBD–positive lymphocytes in LNs were significantly higher than those in the LNs treated with free RBD (fig. S6A), indicating that Flu-RBD promoted internalization into LNs. Compared to free RBD, more CD11c⁺ F4/80⁻ APCs were found to colocalize with Flu-RBD–positive lymphocytes (fig. S6B). The weighted colocalization coefficient of Flu-RBD with CD11c⁺ F4/80⁻ APCs was determined to be 0.37, which decreases to 0.24 for RBD group (fig. S6, C and D). Furthermore, significantly more CD11b⁺ APCs were found in the LNs injected with Flu-RBD but free RBD (fig. S7A). Moreover, Flu significantly boosted the uptake of RBD by CD11b⁺ APCs (fig. S7B). These results suggested that Flu was an efficient platform to overcome the limitations of RBD in vivo and promote the internalization of RBD by APCs, which could be of great interest for improving the functional immunogenicity of RBD.

CD 1 mice were administered two doses of Flu-RBD at an interval of 1 week, while others immunized with two doses of phosphate-buffered saline (PBS) or Flu or RBD or a mixture of Flu with RBD (Flu-M-RBD) at the same interval (Fig. 3A). As corresponding antibody responses illustrated in Fig. 3B, Flu-RBD elicited the highest level of RBD-specific IgG antibody titer in murine serum. We further evaluated the RBD-specific IgG subclass antibody responses. As shown in Fig. 3C, both free RBD and Flu-M-RBD induced a T helper cell (T_H2)–biased humoral response as validated by a primary production of IgG1 antibody, whereas Flu-RBD vaccine induced a more balanced T_H1/T_H2-type response with substantially increased levels of IgG2a. Furthermore, Flu-RBD elicited a higher RBD-specific IgG titer than RBD adjuvanted with aluminum (Alum) (Fig. 3D), which could be attributed to enhanced uptake of RBD by APCs. Notably, RBD with Alum induced a T_H2-mediated humoral response as compared with Flu-RBD (Fig. 3C). After restimulation with a RBD peptide pool, SARS-CoV-2–specific CD4⁺ and CD8⁺ T cell responses in splenocytes were determined. Compared to RBD or Flu-M-RBD, Flu-RBD triggered a higher population of interferon-γ (IFN-γ) (T_H1) generating CD4⁺ and CD8⁺ T cells instead of interleukin-4 (IL-4) (T_H2) or IL-17a (T_H17) generating CD4⁺ and CD8⁺ T cells (Fig. 3, E and F). These findings indicated that Flu-RBD elicited T_H1-biased cellular immune responses. In addition, we found that Flu-RBD induced a higher T_H1/T_H2 ratio of produced CD4⁺ and CD8⁺ T cells than RBD with Alum (fig. S8). These results are extraordinary as the primary safety consideration when designing SARS-CoV-2 vaccines is inducing a robust T_H1-biased immune response, while avoiding a T_H2-type immune response that may evoke vaccine-associated enhanced respiratory diseases (30). Furthermore, we observed that Flu-RBD vaccinations induced a higher frequency of CD4⁺ and CD8⁺ T cells expressing IL-17a than free RBD or RBD with Alum vaccinations, indicating that a higher T_H17 immune response was generated (Fig. 3, E and F, and fig. S8).

To compare the immunogenicity of Flu-RBD with the clinical standard mRNA vaccine, we encoded the full-length of spike (S) protein with the same sequence as the Pfizer BNT162b2 (31, 32). According to Pfizer’s reports, the prepared mRNA was formulated in lipid nanoparticles to synthesize mRNA-lipid nanoparticles (LNP@mRNA) (33, 34). In addition, commercial Lipofectamine 3000 (Lipo3000) was used to deliver mRNA (Lipo3000@mRNA) as a control. Their cellular translation in vitro was verified by immunoblotting (fig. S9, A and B), where LNP@mRNA showed a higher S protein expression than Lipo3000@mRNA in human embryonic kidney (HEK) 293 cells. Immunogenicity comparisons of Flu-RBD vaccine with mRNA vaccines in mice were performed. Antibody titers revealed that Flu-RBD induced a higher level of RBD-specific IgG antibody and a higher IgG2a/IgG1 ratio in mouse sera than LNP@mRNA or Lipo3000@mRNA vaccinations (fig. S9, C and D). Flow cytometry results further demonstrated that LNP@mRNA or Lipo3000@mRNA vaccinations were unable to elicit a T_H1-skewing immune response in splenocytes as observed in Flu-RBD vaccinations (fig. S9, E and F). We further assessed the ability of Flu-RBD to clear inhaled SARS-CoV-2 Delta pseudovirus with a red fluorescent protein (RFP) reporter in mice and compared it with LNP@mRNA or LNP@mRNA plus Fluzone Quadrivalent influenza vaccine (FluZ) group. As shown in fig. S9 (G to I), mice vaccinated with Flu-RBD showed significantly greater viral clearance than those vaccinated with LNP@mRNA or LNP@mRNA plus FluZ, suggesting that Flu-RBD provided more efficient protection against viral infection.

Having demonstrated that Flu-RBD has the capacity to induce RBD-specific antibody response, we next studied whether Flu-RBD preserved the immunogenicity as a flu vaccine. As shown in Fig. 3G, Flu-RBD induced comparable HA antibody titer to that of Flu or Flu-M-RBD group, as well as resulted in a similar T_H1-skewing immune response by a predominant induction of IgG2a antibody rather than IgG1 antibody (Fig. 3H). Restimulation by a HA peptide pool induced a higher population of IFN-γ⁺ CD4 T cells and IFN-γ⁺ CD8 T cells than IL-4⁺ or IL-17a⁺ CD4 and CD8 T cells for Flu-RBD vaccinations (Fig. 3, I and J), indicative of a robust production of T_H1-biased immune response. In addition, a higher level of IL-17a⁺ CD4 and CD8 T cells was observed in Flu-RBD group relative to free Flu group. These results demonstrated that the modification of RBD antigen did not compromise the efficacy of Flu vaccine element. Furthermore, we found that Flu-RBD produced some amount of both RBD-specific and HA-specific IgA Abs in bronchoalveolar lavage fluid (BALF) of mice (fig. S10), indicating that Flu-RBD has potential to provide protection at or near the site of SARS-CoV-2 infection.

Dendritic cells (DCs) are potent APCs and their activation prompts the priming of immunity mediators for B cells and T cells (35). To assess DC activation in response to our Flu-RBD vaccine, murine splenocytes and lymphocytes were isolated from mice that received two vaccinations. A higher percentage of CD86⁺, CD40⁺, and CD80⁺ cells presented in both splenocytes and lymphocytes vaccinated with Flu-RBD group (figs. S11 and S12). Free RBD vaccinations significantly up-regulated the expression of CD40⁺ compared to Flu vaccinations, whereas the frequency of cells expressing CD80⁺ was drastically increased in Flu-vaccinated mice. Flu-RBD vaccinations induced the highest percentage of CD40⁺ cells and a comparable percentage of CD80⁺ cells with Flu vaccinations in both splenocytes and lymphocytes, suggesting a high degree of DC activation.

Cellular immune responses were evaluated in splenocytes from vaccinated mice via induction of IFN-γ secretion after exposure to RBD and HA peptide pools (Fig. 4A). Flu-RBD vaccinations induced significantly higher levels of IFN-γ secretion relative to RBD, Flu, or Flu-M-RBD group, eliciting approximately 300 spot-forming units (SFU) per million splenocytes (Fig. 4, B and C). Like results in IFN-γ enzyme-linked immunosorbent spot (ELISpot) assay, Flu-RBD vaccinations stimulated the highest levels of IL-6 and tumor necrosis factor–α (TNF-α) secretion, whereas in RBD, Flu, and Flu-M-RBD groups, relatively lower levels of IL-6 and TNF-α were discovered (Fig. 4, D and E). The higher level of IL-6 may contribute to the induction of a T_H17 immune response for Flu-RBD group (36). These results indicated that Flu-RBD vaccines induced a robust cellular immune response, which could protect the hosts from viral replication.

The Syrian golden hamster model resembles severe clinical diseases of COVID-19 observed in humans, with rapid weight loss and severe lung pathology (37–39), which was used to assess the protection potency of Flu-RBD vaccine. After two doses of vaccinations, Syrian golden hamsters were challenged with live SARS-CoV-2 via intranasal route (Fig. 5A). Viral RNA levels in bronchoalveolar lavage (BAL) and oral swabs (OS) were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR; Fig. 5, B and C). At 2 days postinoculation (dpi), all immunized groups of PBS, Flu, Flu-M-RBD, and Flu-RBD showed high levels of RNA copies with median peaks of 6.772, 6.512, 6.318, and 6.122 log₁₀ RNA copies/ml, respectively (Fig. 5C). Notably, peak RNA level in Flu-RBD–immunized animals was markedly decreased with a median of 3.702 log₁₀ RNA copies/ml in OS on 7 dpi, indicative of the efficient protection medicated by Flu-RBD. In accordance with OS results, fewer copies of viral RNA with 2.568 log₁₀ RNA copies/ml were tested in the BAL of Flu-RBD–vaccinated hamsters, much lower than that of PBS (5.916)–, Flu (5.906)–, and Flu-M-RBD (5.526)–vaccinated hamsters (Fig. 5B). Furthermore, RBD-specific IgG antibody titer was increased after SARS-CoV-2 infection in hamsters vaccinated with Flu-RBD (Fig. 5D). However, no significant increase in IgG antibody titer was observed for the hamsters who vaccinated with Flu-M-RBD (fig. S13). We further found that Flu-RBD vaccinations elicited 1.35 log₁₀ fold higher median enzyme-linked immunosorbent assay (ELISA) titers in response to RBD than Flu-M-RBD at 7 dpi (Fig. 5E). Moreover, Flu-RBD induced comparable HA-specific antibody titers with free Flu or Flu-M-RBD (Fig. 5F). Clinical chemistry and hematological parameters of hamsters vaccinated with Flu-RBD remained within normal ranges (fig. S14).

Histopathological examinations revealed that the hamsters received PBS or Flu vaccinations had severe pulmonary lesions with multifocal necrotizing bronchiolitis, remarkable inflammatory infiltrates in the alveolar walls and air spaces, as well as pulmonary edema as compared to sham hamsters’ lung (Fig. 5, G and H). This pattern with dim amelioration was seen in the hamsters vaccinated with Flu-M-RBD. Comparatively, the hamsters vaccinated with Flu-RBD had obviously alleviated pneumonia symptoms. The resolution of pulmonary alveolus was improved with extended across larger areas, and a significant reduction in the number of polymorphonuclear and neutrophils was observed. Masson trichrome staining and Ashcroft score results revealed that Flu-RBD immunizations could diminish lung fibrosis with the preservation of alveolar epithelial structures as compared to Flu or Flu-M-RBD vaccination (Fig. 5, I and J).

We further assessed the local distribution of SARS-CoV-2 within lung and found that SARS nucleocapsid (SARS-N) protein was prevalent in epithelial cells lining the bronchioles and alveoli (Fig. 6A). Frequently, the SARS-N–positive cells were found to colocalize with pan-cytokeratin marker, indicating that they mainly infected alveolar epithelial cells (Fig. 6B) (40). In addition, the cells infected with foci of the virus were accompanied by large inflammatory infiltrates of activated ionized calcium binding adaptor (Iba-1⁺) or CD206⁺ cells (Fig. 6C). In situ RNA hybridization (RNAscope) analysis showed that the lowest levels of both positive-sense and negative-sense viral RNA were presented in Flu-RBD immunization group rather than Flu or Flu-M-RBD group (Fig. 6D). In addition, we found that vaccinated hamsters with Flu-RBD significantly reduced inflammation infiltrates as reflected by less myeloperoxidase (MPO)–positive neutrophils in lung as compared to Flu or Flu-M-RBD vaccinations (Fig. 6E). Diffuse expression of CD3⁺ T lymphocytes was evaluated in lungs (Fig. 6F), showing no notable changes among PBS, Flu, Flu-M-RBD, and Flu-RBD groups (Fig. 6G). We further discovered the obvious down-regulation of MX1, an IFN-induced antiviral protein against a wide range of RNA viruses (41), in the hamsters vaccinated with Flu-RBD (Fig. 6, F and G), confirming the reduction of virus replication due to highly potent neutralizing antibody, in keeping with the lowest level of SARS-N expression in the animals immunized with Flu-RBD (Fig. 6G). Collectively, these results indicated that Flu-RBD immunizations effectively protected hamster against SARS-CoV-2 infection.

SARS-CoV-2 Delta (B.1.617.2) variant has been found to be more highly fusogenic and pathogenic than ancestral SARS-CoV-2 (42–44), which was used to evaluate the cross-protection activity of Flu-RBD vaccine against SARS-CoV-2 variants. Seven days after two immunizations, the mice were challenged with RFP and luciferase-expressing SARS-CoV-2 Delta pseudovirus via intranasal route (Fig. 7A). Ex vivo imaging results in Fig. 7B revealed that the bright RFP signal was observed in the mice vaccinated with RBD or Flu-M-RBD group. In contrast, Flu-RBD vaccinations remarkably decreased the RFP signal in lung (Fig. 7, B and C), showing a stronger cross-variant neutralization ability. Confocal imaging of the whole lungs revealed that, in the Flu-RBD group, a lower amount of SARS-CoV-2 Delta pseudovirus was distributed in both the trachea/bronchioles and parenchyma compared to the free RBD or Flu-M-RBD vaccination groups (Fig. 7, D to F). We further evaluated the neutralization properties of sera from vaccinated mice using A549 cells expressing ACE2. As illustrated in Fig. 7G, the sera collected from Flu-RBD group efficiently blocked the infection of A549 cells expressing ACE2 by SARS-CoV-2 Delta pseudovirus. The half-maximal inhibitory dilution of sera from Flu-RBD vaccinations was determined to be 0.0085, significantly lower than that of RBD vaccinations (0.127) and Flu-M-RBD vaccinations (0.0665). Flow cytometry analysis further demonstrated that the sera (0.5 dilution) stemmed from mice with Flu-RBD vaccinations could efficiently prevent SARS-CoV-2 Delta pseudovirus entry into A549 cells (Fig. 7H). Collectively, these compound datasets demonstrated that Flu-RBD shows a high potent neutralization effect against the continued emergence of SARS-CoV-2 variants.

Last, we studied the protection efficacy of Flu-RBD vaccine against wild-type influenza A H1N1 inactivated virus with red fluorescence. Compared to PBS control, Flu-RBD vaccinations efficiently accelerated the clearance of H1N1 inactivated virus in mice (fig. S15), suggesting the preservation of the immunogenicity of Flu vaccine. Furthermore, we compared the neutralization activity of Flu-RBD with clinical standard vaccine LNP@mRNA plus FluZ and found that Flu-RBD vaccine’s protection efficacy against H1N1 inactivated virus was similar to that of the LNP@mRNA plus FluZ group, indicating the potential of Flu-RBD vaccine for preventing flu infection (fig. S15). We next assessed whether there is improved protection activity when using the booster immunization regimen. As illustrated in fig. S16, mice immunized with the booster regimen had substantially higher level of RBD-specific IgG and HA-specific IgG antibody titers in serum compared to the prime regimen. These findings suggest that a booster dose of Flu-RBD significantly augmented immune responses and protective activity against COVID-19 or influenza infection.

Before preparing Flu-RBD, RBD were reacted with AZO-PEG-NHS (PG2-AZNS-3k, NANOCS). Briefly, 10 μg of recombinant SARS-CoV-2 RBD protein (40592-V08B, Sino Biological) was reconstituted and dropped into AZO-PEG-NHS solution (2 mg/ml) and reacted for 24 hours at 4°C to form RBD-PEG-AZO. To remove unreacted AZO-PEG-NHS, the above mixture was dialyzed using Slide-A-Lyzer MINI Dialysis Units [10,000 molecular weight cut-off (MWCO)] and concentrated by Amicon Ultra-4 filter (10-kDa cutoff).

Meanwhile, inactivated influenza A (H1N1) virus (23-047-299, Thermo Fisher Scientific) defined as Flu was reacted with DBCO-PEG-NHS (PG2-DBNS-5k, NANOCS) at 4°C for 24 hours to generate DBCO-PEG-Flu. Unreacted DBCO-PEG-NHS removal was performed by centrifugation via Amicon Ultra-4 filter (100 kDa). To synthesize Flu-RBD, 10 μg of RBD-PEG-AZO was added into 10¹¹ of DBCO-PEG-Flu solution and reacted at 4°C for overnight. The resultant Flu-RBD was ultracentrifugated at 100,000g for 70 min at 4°C and washed with PBS twice (10 mM, pH 7.4) and resuspension in PBS buffer. To quantify the number of RBD protein on Flu-RBD, 10⁵ of Flu-RBD particles were lysed by radioimmunoprecipitation assay buffer and quantification via an ELISA assay (EH492RB, Thermo Fisher Scientific).

One hundred micrograms of NHS-RhB (186-1425, Thermo Fisher Scientific) was reacted with 10 μg of RBD in PBS buffer at 4°C overnight. Free NHS-RhB removal was performed by centrifugation and washed with PBS buffer three times via Amicon Ultra-0.5 filter (10-kDa cutoff). RBD-RhB was mixed with AZO-PEG-NHS to generate RBD-PEG-AZO-RhB and then reacted with DBCO-PEG-Flu to synthesize Flu-RBD-RhB according to above methods.

The S protein plasmid for in vitro transcription was created using the full-length DNA sequence of the trimerized S surface glycoprotein (Gene Bank: QHD43416.1; amino acids 1 to 1273) of the SARS-CoV-2 isolate Wuhan-Hu-1. The mRNA construct of S protein was prepared, extracted, and purified according to our previous report (56). Purified mRNA was formulated with lipids (ALC-3015, ALC-0159, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol) using the ethanol-drop process to obtain RNA-lipid nanoparticles (LNP@mRNA) according to Pfizer’s report (32). The molar ratios of the ALC-3015:ALC-0159:cholesterol:DSPC used are 46.3:1.6:42.7:9.4, and each dose of LNP@mRNA vaccine consists of 0.43 mg of ALC-3015, 0.05 mg of ALC-0159, 0.09 mg of DSPC, 0.19 mg of cholesterol, and 30 μg of mRNA according to the released document from Pfizer (34).

With respect to World Organization for Animal Health recommendation, CD1 mice are strain for the vaccine potency assay in mouse (57), which have been used for evaluating the immunogenicity of various vaccines including RB51 vaccine, Zika vaccine, PCV15 vaccine, etc. Accordingly, female CD1 mice [Crl:CD1(ICR)] that are 6 to 8 weeks old and purchased from Charles River Laboratory (MA, USA) were used for assessing the immunogenicity of Flu-RBD vaccine. All animal studies were compliant with the Institutional Animal Care and Use Committee (IACUC) at North Carolina State University (protocol # 19-806-B). RBD-RhB and Flu-RBD-RhB were administered into the right hint limb of mice via i.m. injection. After 4 or 24 hours postinjection, the right, left, and cervical draining LNs as well as major organs were harvested and imaged via the Xenogen Live Imager (PerkinElmer, MA, USA).

PBS, Flu (10¹⁰/kg), RBD (0.71 μg/kg), Flu-RBD (10¹⁰/kg), and the mixture of Flu and RBD (Flu-M-RBD) vaccinations were i.m. injected with two doses 1 week apart. Seven days later, blood, spleen, BALF, and LNs were collected for further analysis. To evaluate the immune responses of booster, CD1 mice administrated with two doses of Flu-RBD (prime) were received a homologous booster dose 4 weeks later. Seven days later, the blood of mice was collected for further analysis.

One hundred microliters of RBD or influenza A H1N1 HA recombinant protein (4 μg/ml; A42579, Thermo Fisher Scientific) was added into micro titer plates (Nunc Cell Culture, Thermo Fisher Scientific) for overnight at 4°C. After that, 200 μl of 5% (w/v) bovine serum albumin (B6717, Sigma-Aldrich) in phosphate-buffered saline with 0.1% Tween 20 (PBS-T) buffer was added to each well to block nonspecific binding and incubated at 37°C for 1 hour. After three times of wash with PBS-T, serum samples with serial dilutions (1:100, 1:1000, 1:3000, 1:5000, 1:10,000, 1:50,000, 1:100,000, and 1:200,000) and control serum samples (1:100 dilution) were added to the wells and incubated at 37°C for 1.5 hours. After washing intensively, 100 μl of horseradish peroxidase–labeled anti-mouse IgG secondary antibody with 1:10,000 dilution was added for 1 hour of incubation at 37°C. After three times of wash, 100 μl of trimethylboron was added into each well and incubated at room temperature for 30 min. Fifty microliters of 2 M H₂SO₄ (Sigma-Aldrich) was used for stopping the reaction, and the optical absorption was determined at 450 nm via a plate reader. The endpoint IgG titer was determined as the highest reciprocal dilution of serum that exhibits an optical density greater than twofold of the mean control group.

Splenocytes from each vaccinated mouse were seeded in an ELISpot plate (EL485, R&D Systems) (10⁶ cells per well) and stimulated with RBD peptide pool (PP002-A, SinoBiological) and H1N1 HA peptide pool (130-097-285, Miltenyi Biotec). Antigen-specific cells secreting IFN-γ were detected according to the protocol of the manufacturer. The formed SFUs were imaged by an anatomical microscope (Nikon, Minato City, Tokyo, Japan) and counted by ImageJ software. Furthermore, the collected splenocytes were also plated in six-well plates (5 × 10⁶ per well) and restimulated with RBD peptide pool and H1N1 HA peptide pool. After 48 hours of incubation, the culture supernatant was collected. The Mouse IL-6 Kit (RAB0308, Sigma-Aldrich) and the Tumor Necrosis Factor α Kit (RAB0477, Sigma-Aldrich) were used to detect IL-6 and TNF-α levels from collected supernatant.

At day 7 postprime, mouse splenocytes were collected and seeded to the plate (1 × 10⁶ per well). The RBD peptide pool or influenza A HA peptide pool was added to stimulate splenocytes for 12 hours at 37°C, respectively. Brefeldin A solution was added into the plate with a final concentration of 1 μg/ml and incubated for another 4 hours. Following that, the splenocytes were collected and stained with CD4–fluorescein isothiocyanate (FITC) (100406, BioLegend) antibody or CD8-FITC (ab22504, Abcam) antibody at a dilution of 1:100. After washing with magnetic-activated cell sorting flow buffer, splenocytes were fixed with 4% paraformaldehyde (PFA) and permeabilized using saponin and incubated with IFN-γ–phycoerythrin (PE) (507806, BioLegend), IL-4–PE (504103, BioLegend), or IL-17a–APC (17-7177-81, Invitrogen) Abs for 1 hour. The stained splenocytes were analyzed by a CytoFLEX flow cytometer (Beckman Coulter). Data analyses were carried out by FCS Express V6.

Six to 8 weeks old of 20 male and female Syrian golden hamsters (Envigo) were randomly assigned to four groups and housed at Bioqual. Hamsters were i.m. administered with two doses of PBS, Flu (10¹⁰/kg) or Flu-M-RBD, Flu-RBD (10¹⁰/kg) at week 0 and week 1 (n = 5 per group, 2 females/3 males). At week 2, the hamsters were challenged with 100 μl of 2 × 10⁴ 50% tissue culture infective dose SARS-CoV-2 via intranasal routes (50 μl in each nare). BAL, OS, and blood were harvested at the indicated time. All hamster experiments were conducted in accordance with all relevant local, state, and federal regulations that were approved by the Bioqual IACUC (20-091P).

Following procedures previously described by Duke Human Vaccine Institute (58), SARS-CoV-2 RNA copies per milliliter were tested by qRT-PCR assay. Briefly, viral RNA was extracted using the QIAsymphony SP sample preparation platform with the DSP Virus/Pathogen Midi Kits (Qiagen). A primer specific to SARS-CoV-2 envelope gene was annealed to extracted RNA, which was reverse-transcribed into cDNA using SuperScript III Reverse Transcriptase and RNaseOut. The cDNA was treated with ribonuclease and then added to the custom 4× TaqMan Gene Expression Master Mix including primers and a fluorescently labeled hydrolysis probe. The qRT-PCR reaction was performed on a QuantStudio 3 Real-Time PCR system. SARS-CoV-2 RNA copies per reaction were interpolated via quantification cycle data and a serial dilution of a highly characterized custom DNA plasmid with SARS-CoV-2 envelope gene sequence. The limit of quantification for the assay was approximately 62 RNA copies/ml of sample.

SARS-CoV-2 Delta pseudoviruses were constructed by cotransfecting HEK293T cells with the plasmids of plv-spike-v8 (InvivoGen), pLenti-EF1pluciferase-PGK-RFP-T2A-PURO lentiviral reporter (LR252, ALSTEM), and pspax2 (64586, addgene) via Lipo3000 (L3000015, Thermo Fisher Scientific). After 48 to 72 hours, Delta pseudoviruses were harvested from culture medium through centrifugation (3000 rpm, 10 min), aliquoted, and stored at −80°C before use.

Seven-week-old female CD1 mice were vaccinated with two doses of PBS, RBD, Flu, Flu-M-RBD, or Flu-RBD once a week. Seven days later, mice were inoculated with SARS-CoV-2 Delta pseudovirus carrying both RFP and luciferase reporters. Lung organs were harvested and imaged at 24 hours postchallenge by the Xenogen Live Imager. To study the distribution of Delta pseudovirus in the lung, the collected lung tissues were fixed in 4% PFA, dehydrated using 30% sucrose solution, and then frozen in O.C.T. compound (Tissue-Tek). Cryo-sections were permeabilized and blocked with Dako containing 0.1% saponin solution for 1 hour and then stained with phalloidin antibody (ab176753, Abcam). The ProLong Gold Antifade reagent with DAPI was used to counterstain nuclei and prevent the fade of the fluorophore. Imaging was performed via Olympus FLUOVIEW CLSM.

The sera samples from vaccinated mice were collected to assess their neutralization activity against SARS-CoV-2 Delta pseudovirus. A549 cells expressing human ACE2 and transmembrane serine protease 2 (TMPRSS2) were obtained from InvivoGen (a549-hace2tpsa). Sera samples with the indicated dilutions were incubated with SARS-CoV-2 Delta pseudovirus for 1 hour at 37°C. After that, the mixture was added in the A549 cells and incubated for another 24 hours. The luciferase signals from infected cells were determined by the Dual-Glo Luciferase Assay System (Promega). Furthermore, the A549 cells infected with the mixture of SARS-CoV-2 Delta with serum at a ratio of 1:1 were collected for flow cytometry analysis via the RFP reporter.

Wild-type influenza A H1N1 (VNV-019) inactivated virus was purchased from Creative Biogene and labeled with 1,1-Dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DID) dye (V22887, Thermo Fisher Scientific). Seven-week-old female CD1 mice were i.m. administered with PBS, Flu-RBD, LNP@mRNA, or LNP@mRNA plus Fluz in two doses once a week. Seven days after two vaccinations, every mouse was challenged with H1N1 inactivated virus with red fluorescence. Lung organs were harvested and imaged at 24 hours postadministration by the Xenogen Live Imager. Immunofluorescence imaging of lung tissues was performed by an Olympus FLUOVIEW CLSM.

All experiments were performed at least three times independently. Results are shown as means ± SD. Comparisons between any two groups were performed using the two-tailed, unpaired Student’s t test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by the post hoc Bonferroni test. Group data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Single, double, triple, and four asterisks represent P < 0.05, 0.01, 0.001, and 0.0001, respectively; P < 0.05 was considered statistically significant.