Pan-beta-coronavirus subunit vaccine prevents SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV challenge
ABSTRACT
Three highly pathogenic coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, belonging to the genus beta-CoV, have caused outbreaks or pandemics. SARS-CoV-2 has evolved into many variants with increased resistance to the current vaccines. Spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are important vaccine targets; however, the RBD of the SARS-CoV-2 Omicron variant is highly mutated, rending neutralizing antibodies elicited by ancestral-based vaccines targeting this region ineffective, emphasizing the need for effective vaccines with broad-spectrum efficacy against SARS-CoV-2 variants and other CoVs with pandemic potential. This study describes a pan-beta-CoV subunit vaccine, Om-S-MERS-RBD, by fusing the conserved and highly potent RBD of MERS-CoV into an RBD-truncated SARS-CoV-2 Omicron S protein, and evaluates its neutralizing immunogenicity and protective efficacy in mouse models. Om-S-MERS-RBD formed a conformational structure, maintained effective functionality and antigenicity, and bind efficiently to MERS-CoV receptor, human dipeptidyl peptidase 4, and MERS-CoV RBD or SARS-CoV-2 S-specific antibodies. Immunization of mice with Om-S-MERS-RBD and adjuvants (Alum plus monophosphoryl lipid A) induced broadly neutralizing antibodies against pseudotyped MERS-CoV, SARS-CoV, and SARS-CoV-2 original strain, as well as T-cell responses specific to RBD-truncated Omicron S protein. Moreover, the neutralizing activity against SARS-CoV-2 Omicron subvariants was effectively improved after priming with an Omicron-S-RBD protein. Adjuvanted Om-S-MERS-RBD protein protected mice against challenge with SARS-CoV-2 Omicron variant, MERS-CoV, and SARS-CoV, significantly reducing viral titers in the lungs. Overall, these findings indicated that Om-S-MERS-RBD protein could develop as an effective universal subunit vaccine to prevent infections with MERS-CoV, SARS-CoV, SARS-CoV-2, and its variants.
IMPORTANCE
Coronaviruses (CoVs), SARS-CoV-2, SARS-CoV, and MERS-CoV, the respective causative agents of coronavirus disease 2019, SARS, and MERS, continually threaten human health. The spike (S) protein and its receptor-binding domain (RBD) fragment of these CoVs are critical vaccine targets. Nevertheless, the highly mutated RBD of SARS-CoV-2 variants, especially Omicron, significantly reduces the efficacy of current vaccines against SARS-CoV-2 variants. Here a protein-based pan-beta-CoV subunit vaccine is designed by fusing the potent and conserved RBD of MERS-CoV into an RBD-truncated Omicron S protein. The resulting vaccine maintained effective functionality and antigenicity, induced broadly neutralizing antibodies against all of these highly pathogenic human CoVs, and elicited Omicron S-specific cellular immune responses, protecting immunized mice from SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV infections. Taken together, this study rationally designed a pan-beta-CoV subunit vaccine with broad-spectrum efficacy, which has the potential for development as an effective universal vaccine against SARS-CoV-2 variants and other CoVs with pandemic potential.
INTRODUCTION
Three highly pathogenic beta-coronaviruses (CoVs), including severe acute respiratory syndrome (SARS)-CoV, Middle East respiratory syndrome (MERS)-CoV, and SARS-CoV-2, which were first identified in 2002, 2012, and 2019, respectively, caused global outbreaks or pandemics (1–3). Infections with SARS-CoV and MERS-CoV resulted in mortality rates of 10% and ~35%, respectively (2, 4). According to the World Health Organization, as of 28 April 2024, SARS-CoV-2 has infected more than 775.3 million persons worldwide, with at least 7.04 million persons dying of coronavirus disease 2019 (COVID-19), the disease caused by SARS-CoV-2 (5). SARS-CoV-2 has been found to undergo constant mutations, with these mutations resulting in significant resistance to vaccines and therapeutic antibodies against the original or earlier variants (6–8). In addition, MERS-CoV continues to infect humans, and some SARS-like or MERS-like CoVs from bats use the same receptors as SARS-CoV-2 or MERS-CoV for viral entry, having pandemic potential (9–11). Effective vaccines with broad-spectrum protective efficacy against these CoVs are therefore urgently needed to prevent infection with MERS-CoV, SARS-CoV-2 variants, or other CoVs with pandemic potential.
The surface spike (S) protein of CoVs binds to its respective receptor on host cells, initiating viral entry and the process of infection. The S proteins of CoVs consist of S1 and S2 subunits. The receptor-binding domains (RBDs) of SARS-CoV-2 and SARS-CoV bind to their cellular receptor, angiotensin-converting enzyme 2 (ACE2), whereas the RBD of MERS-CoV binds dipeptidyl peptidase 4 (DPP4) for efficient entry into host cells (9, 12–15). Thus, the CoV S proteins and the RBDs are important vaccine targets. The RBDs in the S1 subunit of SARS-CoV and MERS-CoV contain critical neutralizing epitopes and induce potent neutralizing antibodies against infection by different strains of these viruses (2, 16, 17). The RBD of SARS-CoV-2, however, has been found to mutate rapidly, resulting in at least five variants of concern to date (Alpha, Beta, Gamma, Delta, and Omicron) (18, 19). Currently available SARS-CoV-2 vaccines are relatively ineffective against emerging Omicron subvariants, with the latter found to significantly escape neutralizing immunity or protective efficacy of vaccines targeting the wild-type (WT) strain or earlier variants (20, 21).
The present study describes the development of a pan-beta-CoV subunit vaccine, called Om-S-MERS-RBD, by fusing the highly neutralizing RBD region of MERS-CoV into an RBD-truncated S protein of SARS-CoV-2 Omicron variant. Its conformational structure, antigenicity, as shown by its ability to bind antibodies against MERS-CoV RBD and SARS-CoV-2 S, and functionality, as shown by its ability to bind the MERS-CoV receptor, human DPP4, were investigated. This protein was tested for its ability to neutralize multiple SARS-CoV-2 strains, SARS-CoV, and MERS-CoV. Moreover, its protective efficacy against the three CoVs (i.e., SARS-CoV-2 Omicron, SARS-CoV, and MERS-CoV) was evaluated in mouse models.
RESULTS
Construction and characterization of the pan-beta-CoV subunit vaccine
The S protein of SARS-CoV-2 contains S1 and S2 subunits, with the RBD being located on the S1 (Fig. 1A). The pan-beta-CoV subunit vaccine was designed to contain an RBD-truncated extracellular domain (with a HexaPro sequence) of S protein of SARS-CoV-2 Omicron variant (Om-S) and a RBD of MERS-CoV (Om-S-MERS-RBD) (Fig. 1B). Om-S-SARS-RBD and Om-S-BA5-RBD proteins, which contained the above RBD-truncated Om-S sequence and the RBD of SARS-CoV or the RBD of Omicron-BA.5 variant, were constructed as controls (Fig. 1B). Each construct had a N-terminal tissue plasminogen activator (tPA) signal peptide sequence, as well as a C-terminal foldon trimeric sequence and His6 tag (Fig. 1B); the respective protein was purified from transfected HEK293F cell culture supernatant.
Fig 1
Cryo-EM analysis demonstrated that Om-S-MERS-RBD protein formed a trimeric open conformational structure consisting of three truncated Om-S molecules, with two RBDs of MERS-CoV in a standing-up position and one RBD of MERS-CoV in a lying-down position (Fig. 2A and B; Fig. S1). Om-S-MERS-RBD bound efficiently to polyclonal antibodies (pAb) specific to the SARS-CoV-2 S protein and MERS-CoV RBD, respectively (Fig. 2C). As expected, Om-S-MERS-RBD did not bind to a SARS-CoV-2 RBD-specific nanobody (Nb) or a SARS-CoV RBD-specific monoclonal antibody (mAb), as it lacked the RBD sequences bound by these antibodies (Fig. 2D). By contrast, Om-S-SARS-RBD bound to anti-SARS-CoV-2-S pAb and anti-SARS-CoV-RBD mAb (Fig. 2E and F), whereas Om-S-BA5-RBD only bound to anti-SARS-CoV-2-S pAb, but not to the other antibodies tested (Fig. 2G and H). In contrast to Om-S-SARS-RBD and Om-S-BA5-RBD proteins, which bind to the SARS-CoV and SARS-CoV-2 receptor, human ACE2, Om-S-MERS-RBD bound effectively to the MERS-CoV receptor, human DPP4 protein, but not binding to human ACE2 (Fig. 3A and B). These data indicated that Om-S-MERS-RBD formed a conformational structure and maintained functionality and antigenicity.
Fig 2
Fig 3
The pan-beta-CoV subunit vaccine induced broadly neutralizing antibodies against multiple CoVs and durable T-cell responses
To evaluate the broadly neutralizing immunogenicity of the Om-S-MERS-RBD vaccine, BALB/c mice were immunized with each of the aforementioned proteins, or sequentially with two proteins, or PBS control, in the presence of aluminum hydroxide gel (Alum for short) and monophosphoryl lipid A (MPL) adjuvants and the ability of induced neutralizing antibodies (nAbs) in their sera against infection with multiple CoVs was compared (Fig. 1C). Om-S-MERS-RBD protein elicited potent nAbs against pseudotyped MERS-CoV, and effective nAbs against pseudotyped SARS-CoV, as well as against the original WT strain and some variants of SARS-CoV-2 (Fig. 4A through H). By contrast, the nAb titers against SARS-CoV and SARS-CoV-2 (original and variant strains) were significantly lower, or lower, than the titers induced by immunization with Om-S-SARS-RBD or Om-S-BA5-RBD protein (Fig. 4B through H). Compared with Om-S-BA5-RBD protein, Om-S-SARS-RBD protein induced significantly higher, or higher, titers of nAbs against the SARS-CoV and SARS-CoV-2-WT, but significantly lower, or lower, titers of nAbs against the SARS-CoV-2 variants tested (Fig. 4B through H). Immunization with Om-S-MERS-RBD or Om-S-SARS-RBD protein following Om-S-BA5-RBD protein priming efficiently improved nAb titers to neutralize these SARS-CoV-2 variants (Fig. 4D through H). Neither Om-S-SARS-RBD nor Om-S-BA5-RBD protein, nor the priming-boosting regimen, however, induced nAbs against MERS-CoV (Fig. 4A). By contrast, PBS plus adjuvant control only induced a background level of nAbs (Fig. 4).
Fig 4
To evaluate whether the Om-S-MERS-RBD vaccine elicited durable T-cell responses, BALB/c mice were immunized as described above and collected for peripheral blood mononuclear cells (PBMCs) 5 months after the last immunization for measurement of cytokine secretion after stimulation with an RBD-truncated Omicron-S protein. Compared with the PBS plus adjuvant control, all groups vaccinated with the respective protein(s) elicited a significantly high level of T-cell responses represented by secretion of IL-4 cytokine, and immunization with Om-S-MERS-RBD following Om-S-BA5-RBD priming further significantly increased the cytokine production than Om-S-SARS-RBD (Fig. S2).
The above findings indicate that the Om-S-MERS-RBD subunit vaccine was sufficiently immunogenic to induce effective and broadly nAbs against MERS-CoV, SARS-CoV, and SARS-CoV-2, whereas Om-S-SARS-RBD protein elicited nAbs against SARS-CoV and SARS-CoV-2. Moreover, the ability of Om-S-MERS-RBD to neutralize SARS-CoV-2 variants could be improved by priming with Om-S-BA5-RBD. All proteins tested induced durable T-cell responses specific to the Omicron-S protein of SARS-CoV-2.
The pan-beta-CoV subunit vaccine protected against SARS-CoV-2 Omicron variant
BALB/c mice are susceptible to SARS-CoV-2 Omicron-BA.5 variant (22). To evaluate the protective efficacy of the designed subunit vaccines against SARS-CoV-2 infection, BALB/c mice were immunized with each protein or sequentially with the two proteins, or PBS control, in the presence of adjuvants described above, then challenged with SARS-CoV-2 (Omicron-BA.5 variant) 3 weeks after the last vaccination, and viral titers and viral replication in the lungs were measured (Fig. 5A). Viral titers and viral replication in the lungs were lower in all vaccinated mice than in control mice injected with PBS plus adjuvants (Fig. 5B and C). Viral titers in the lungs were relatively lower in challenged Om-S-MERS-RBD protein-immunized mice, similar to titers in mice immunized with the Om-S-SARS-RBD protein than in mice immunized with the Om-S-BA5-RBD protein (Fig. 5B). Compared with the respective protein alone, viral replication was further decreased by >95% when priming mice with Om-S-BA5-RBD followed by boosting with the Om-S-MERS-RBD or Om-S-SARS-RBD (Fig. 5C). These findings indicated that Om-S-MERS-RBD or Om-S-SARS-RBD protein may be used as an effective subunit vaccine to protect immunized mice against SARS-CoV-2 Omicron-BA.5 variant infection and replication.
Fig 5
The pan-beta-CoV subunit vaccine protected against MERS-CoV infection
Wild-type mice, including BALB/c, can be infected with MERS-CoV after the mice are transduced with adenovirus-5 (Ad5)-hDPP4 (23). To evaluate the protective efficacy of the designed subunit vaccines against MERS-CoV infection, BALB/c mice, which were immunized with each protein or sequentially with the two proteins, or PBS control, in the presence of adjuvants described above, were transduced with Ad5-hDPP4 3 weeks after the last vaccination, followed by the challenge with MERS-CoV, and measurement of viral titers in the lungs (Fig. 6A). Viral titers were lowest in mice immunized with Om-S-MERS-RBD protein, with these titers being significantly lower than those in the other vaccinated mice (Fig. 6B). Notably, the sera of mice immunized with Om-S-SARS-RBD, or primed with the Om-S-BA5-RBD protein and boosted with either Om-S-SARS-RBD or Om-S-MERS-RBD protein, effectively blocked viral infection with these mice having significantly lower, or lower, levels of viral titers than mice immunized with Om-S-BA5-RBD protein (Fig. 6B). Viral titers were significantly lower in most of the immunized mice than in control mice injected with PBS plus adjuvants (Fig. 6B). These findings indicated that Om-S-MERS-RBD protein is an effective subunit vaccine that can protect immunized mice against MERS-CoV infection.
Fig 6
The pan-beta-CoV subunit vaccine provided durable protection against SARS-CoV infection
BALB/c mice are susceptible to mouse-adapted SARS-CoV strain (MA15) (24). Therefore, to evaluate the durable protective efficacy of the designed subunit vaccines against SARS-CoV infection, BALB/c mice immunized with each protein or sequentially with the two proteins, or PBS control, in the presence of adjuvants described above, were challenged with the mouse-adapted SARS-CoV (MA15) 5 months after the last vaccination (Fig. 6C). Viral titers in the lungs were significantly lower in mice immunized with Om-S-MERS-RBD protein, or in mice primed with Om-S-BA5-RBD protein and boosted with Om-S-MERS-RBD protein, than in control mice injected with PBS plus adjuvants, although these viral titers were significantly higher than those of the mice immunized with Om-S-SARS2-RBD protein, or Om-S-BA5-RBD protein prime and Om-S-SARS2-RBD protein boost (Fig. 6D). These results demonstrated the ability of Om-S-MERS-RBD or other proteins to induce broad and long-term protection against SARS-CoV infection.
DISCUSSION
A variety of COVID-19 vaccines have been developed, but many of those targeting the S protein of the original strain of SARS-CoV-2 and subsequent variants have lower or no neutralizing activity or protection against the newly emerging SARS-CoV-2 Omicron subvariants (20, 25). Thus, variant-specific COVID-19 vaccines have to be constantly designed to prevent individuals from infection with these new Omicron subvariants or other future variants with pandemic potential. Developing new vaccines, however, normally requires additional time and costs. Moreover, COVID-19-specific vaccines are not expected to show neutralizing activity or significant protection against pathogenic Merbecovirus, such as MERS-CoV, due to lower homology among their sequences of S and other proteins. These findings indicate a need to develop pan-beta-CoV vaccines, without the need for constant change of vaccine antigens based on newly emerging viral strains. These pan-beta-CoV vaccines should not only protect against infection with newly emerging SARS-CoV-2 variants but should also protect against MERS-CoV and other beta-CoVs, which may infect human hosts and have future pandemic potential.
Several pan-CoV vaccines have been designed based on the nanoparticles or mRNAs, displaying a mosaic RBD or S protein of SARS-CoV-2 or other beta-CoVs on their surfaces (26–29). For example, a chimeric mRNA vaccine expressing S protein (with different NTD, RBD, and S2 regions) of SARS-CoV-2 or SARS-related CoVs was found to induce neutralizing antibodies against several SARS-CoV-2 variants and to protect mice from infection with SARS-CoV-2 (WT strain and B.1.351 variant), SARS-CoV, Bt-CoV RsSHC014, and Bt-CoV WIV-1 (26). In addition, a mosaic-8 nanoparticle vaccine, which presents the RBD of SARS-CoV-2 Beta variant and seven animal Sarbecoviruses, was found to elicit neutralizing antibodies against SARS-CoV-2 Omicron subvariants (BA.1, BA.2, BA.2.12.1, or BA.4/5), protecting animals from challenge with SARS-CoV and the Beta and Delta variants of SARS-CoV-2 (27).
In this study, a different approach was applied to design the pan-beta-CoV subunit vaccines. Since the RBD of SARS-CoV-2 Omicron is highly variable, it was removed during the vaccine design, and replaced by the RBD of MERS-CoV or SARS-CoV, which shows low variations among different strains but potent neutralizing activity against multiple viral infections (2, 16, 30). The RBD of SARS-CoV-2 is divergently mutated, but the other region of the S protein has high homology among all SARS-CoV-2 strains, including the Omicron variant (31–33). Therefore, the RBD-truncated region was used to fuse with the RBD of MERS-CoV or SARS-CoV during the vaccine design.
Om-S-MERS-RBD protein (which expresses the MERS-CoV RBD and lacks the SARS-CoV-2 RBD) formed a conformational structure, allowing it to strongly bind MERS-CoV receptor DPP4, and antibodies specific to SARS-CoV-2 S protein and MERS-CoV RBD, respectively. This subunit vaccine elicited potent or effective neutralizing antibodies against MERS-CoV, SARS-CoV, and SARS-CoV-2 WT strain. Its ability to neutralize SARS-CoV-2 Omicron subvariants was significantly improved by priming with Om-S-BA5-RBD protein. Further studies will be conducted to evaluate the neutralizing activities and protective efficacy of this vaccine after priming with a SARS-CoV-2 S protein from the ancestral strain, or a variant of current or future emerging strains, and compared for the improved neutralizing antibodies and protection against infection of different SARS-CoV-2 strains and other CoVs. Of note, vaccination with Om-S-MERS-RBD simultaneously protected animals from infection with SARS-CoV-2 (Omicron-BA.5), SARS-CoV, and MERS-CoV. In addition, Om-S-SARS-RBD (which expresses the SARS-CoV RBD and lacks the SARS-CoV-2 RBD) induced favorable neutralizing antibodies and protection against SARS-CoV-2 and SARS-CoV, rather than MERS-CoV, potentially due to the lack of homologous sequences between the MERS-CoV and SARS-CoV RBDs.
Om-S-MERS-RBD protein elicited low-titer neutralizing antibodies against the pseudotyped Omicron-BA.5 variant, but it induced high-level RBD-truncated S-specific IL-4 cytokine-associated T-cell responses in the immunized mice. Therefore, although vaccine-induced neutralizing antibodies may play a critical role in the ability of Om-S-MERS-RBD to protect against SARS-CoV and MERS-CoV infections, its ability to protect against infection with the SARS-CoV-2 Omicron variant would likely be due to the vaccine-induced T-cell responses. Notably, vaccination with Om-S-MERS-RBD or other proteins did not elicit a significant level of S-specific Th1 cytokines, such as TNF-α, and IFN-γ, partially due to the use of Alum adjuvant, which is a strong Th2 inducer (34, 35). Future studies will be performed to optimize the ratio of Alum and MPL (Th1 inducer) adjuvant combination or use other adjuvants, such as SMQ or a combination of CpG and MF59, which tend to induce Th1 cytokines or a balanced T-cell response (34–36).
Overall, the present study demonstrated that a pan-beta-CoV subunit vaccine with protective efficacy against three highly pathogenic CoVs (i.e., SARS-CoV-2, SARS-CoV, and MERS-CoV), and a pan-sarbecovirus subunit vaccine with protective efficacy against SARS-CoV-2 and SARS-CoV could be designed. These vaccines were characterized, and their ability to protect against viral infections was confirmed. Further development of these vaccines may protect against highly pathogenic CoVs and other beta-CoVs with pandemic potential.
MATERIALS AND METHODS
Cell lines
HEK293F, 293T, 293T expressing human ACE2 receptor (hACE2/293T), Huh-7, Vero, Vero E6, and Vero E81 cells were cultured at 37°C in the presence of 5% CO2. The 293F cells were cultured in an ESF serum-free medium (Expression Systems). The other cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (R&D Systems) and 1% Gibco Penicillin-Streptomycin (Thermo Fisher Scientific).
Plasmids and viruses
DNA sequences encoding S protein of SARS-CoV-2 WT strain (GenBank accession number QHR63250.2), Omicron BA.1 variant (GISAID accession number EPI_ISL_6795835), SARS-CoV Tor2 strain (GenBank accession number AY274119), or MERS-CoV EMC2012 strain (GenBank accession number JX869059.2) were inserted into pcDNA3.1/V5-His-TOPO vector to construct the respective recombinant plasmids. SARS-CoV-2 BA.2 (GISAID accession number EPI_ISL_12030355), BA.5 (GISAID accession number EPI_ISL_12043290), BQ.1.1 (GISAID accession number EPI_ISL_15370776), and XBB (GISAID accession number EPI_ISL_15341139) recombinant plasmids containing the respective RBD mutant residues were constructed based on the above BA.1-S recombinant plasmid using multi-site-directed mutagenesis kit. Live SARS-CoV-2 Omicron-BA.5 variant, MERS-CoV (EMC2012 strain, GenBank accession number JX869059.2), and SARS-CoV (MA15 strain) were used in this study.
Vaccine preparation
This was prepared as described below (37). Briefly, the codon-optimized RBD of MERS-CoV (GenBank accession number JX869059.2) or SARS-CoV (GenBank accession number AY274119) was fused with the RBD-truncated, codon-optimized S extracellular domain sequence of SARS-CoV-2 Omicron variant (GISAID accession number EPI_ISL_6795835), and inserted into a pLenti expression vector using ClonExpress MultiS One Step Cloning kit (Cellagen Technology). Each recombinant plasmid contains an N-terminal tPA signal sequence, HexaPro sequence, a C-terminal foldon trimerization motif, and a His6 tag. The sequence-confirmed recombinant plasmids were respectively transfected into HEK293F cells, and the culture supernatant was collected for purification of the respective proteins using Ni-NTA Superflow (Qiagen).
Cryo-EM grid preparation and data acquisition
The isolated spike (4 µL at 0.96 µM) was applied to freshly glow-discharged Quantifoil R1.2/1.3 300-mesh copper grids (EM Sciences), and then blotted for 4 s at 22°C under 100% chamber humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI). Cryo-EM data were collected using EPU (Thermo Fisher Scientific) on a Titan Krios electron microscope (Thermo Fisher Scientific) equipped with a K3 direct electron detector with a Biocontinuum energy filter (Gatan) in CDS mode at the Hormel Institute, University of Minnesota. The movies were collected at a nominal magnification of 81,000× (corresponding to 1.1 Å per pixel), a 20 eV slit width, a dose rate of 25 e– per Å2 per second, and a total dose of 50 e−/Å2. The statistics of cryo-EM data collection are summarized in Table S1.
Image processing
Cryo-EM data were processed using cryoSPARC v4.0.3 (38), and the data processing procedures are outlined in Fig. S1. Dose-fractionated movies were first subjected to Patch motion correction and Patch CTF estimation with MotionCor2 (39) and CTFFIND-4.1.13 (40), respectively. Images with defocus values outside of −1.0 to −2.0 µm or the CTF fit resolutions worse than 6 Å were excluded from the further steps. Particles were picked using both Blob picker and Template picker accompanied by removing duplicate particles. Three rounds of 2D classifications were applied to remove junk particles and particles (213,507) extracted from the good 2D classes were used for Ab-initio Reconstruction of four maps and then for the heterogeneous refinements. The good 3D class (192,374 particles) was finally subjected to further homogeneous, non-uniform, and CTF refinements to generate a 3.45 Å resolution final map. Map resolution was determined by gold-standard Fourier shell correlation (FSC) at 0.143 between the two half-maps. Local resolution variation was estimated from the two half-maps in cryoSPARC v4.0.3.
Model building and refinement
Initial model building of the Om-S-MERS-RBD was performed in Coot-0.8.9 using PDB 7TGW as the starting model (41). Several rounds of refinement in Phenix-1.16 (42) and manual building in Coot-0.8.9 were performed until the final reliable models were obtained. The final model has good stereochemistry by evaluation in MolProbity (43). The statistics of 3D reconstruction and model refinement are shown in Table S1. Figures were generated using UCSF Chimera X v0.93 (44).
Enzyme-linked immunoassay
Enzyme-linked immunoassay (ELISA) was performed to test the binding of proteins to CoV S/RBD-specific antibodies (45, 46). Briefly, ELISA plates were coated with each protein (1 µg/mL) at 4°C overnight and blocked with a blocking buffer [2% non-fat milk in PBS containing 0.05% Tween-20 (PBST)] at 37°C for 2 h. After washing five times with PBST, the plates were incubated with serially diluted SARS-CoV-2 S or MERS-CoV RBD protein-immunized mouse sera (pAb) (17, 37, 47), SARS-CoV-2 RBD-targeting nanobody (Nb) (46), or SARS-CoV RBD-targeting mouse monoclonal antibody (mAb, 33G4) (45), which was followed by incubation with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Fab-specific 1:10,000, Sigma; for mouse sera or mAb) or anti-Camelid VHH antibody (1:10,000, GenScript; for Nb) antibody at 37°C for 1 h. The plates were washed and then incubated sequentially with substrate TMB (3,3′,5,5′-Tetramethylbenzidine) (Sigma), and H2SO4 (1 N). Absorbance at 450 nm (A450 value) was measured using Cytation 7 Microplate Multi-Mode Reader (BioTek Instruments).
ELISA was also performed to test the binding of proteins to CoV receptors (17, 37, 47). Briefly, ELISA plates were coated with each protein, and blocked as described above, followed by the addition of diluted human DPP4 (MERS-CoV receptor) or human ACE2 (SARS-CoV-2 or SARS-CoV receptor) protein (Laboratory stock) for incubation at 37°C for 1 h. After washes, the plates were sequentially incubated with goat anti-hDPP4 or anti-hACE2 antibody (1:1,000, R&D Systems) and HRP-conjugated rabbit-anti-goat IgG antibody (1:5,000, Abcam). Other steps were the same as described above.
CoV pseudovirus generation and neutralization assay
The pseudoviruses were prepared as described below (48–50). Briefly, each recombinant plasmid DNA encoding the respective S protein of SARS-CoV-2, SARS-CoV, or MERS-CoV was transfected, in the presence of PS-PAX2 and pLenti-CMV-luciferase plasmids, into 293T cells using the PEI transfection reagent. The culture supernatant containing each pseudovirus was collected 72 h post-transfection, and incubated with immunized mouse sera at 37°C for 1 h. The mixture of virus and sera was incubated in 293T cells expressing SARS-CoV-2 or SARS-CoV receptor, human ACE2 (hACE2/293T) (for SARS-CoV-2 or SARS-CoV pseudovirus), or Huh-7 cells expressing MERS-CoV receptor human DPP4 (for MERS-CoV pseudovirus), for 24 h. The cells were further cultured for 48 h after the addition of fresh medium. The lysed cell supernatant was incubated with luciferase substrate (Promega), and relative luciferase activity was measured using Cytation 7 Microplate Multi-Mode Reader and Gen5 software. Neutralizing antibody activity against pseudovirus infection was reported as 50% neutralizing antibody titer (NT50).
Multiplex immunoassay
Immunized mice were evaluated for specific T-cell responses by Multiplex immunoassay. Specifically, PBMCs were isolated by gradient centrifugation using Histopaque-1083 solution (Sigma-Aldrich). Residual red blood cells were removed using Red Blood Cell Lysis Buffer (Sigma-Aldrich). The isolated PBMCs were resuspended in RPMI 1640 cell culture medium containing 10% FBS, penicillin (100 U/mL), streptomycin (100 µg/mL), β-mercaptoethanol (55 µM), non-essential amino acids, sodium pyruvate (1 mM), and mouse IL-2 (1 ng/mL), and stimulated with RBD-truncated SARS-CoV-2 Omicron-S protein (5 µg/mL). 48 hours later, the cells were re-stimulated with the same protein (5 µg/mL) for an additional 24 hours. The supernatant was collected by centrifugation, and the cytokines in the supernatant were measured using a Bio-Plex Pro Mouse Cytokine Th1/Th2 Assay kit (Bio-Rad). The results were analyzed using the Bio-Plex 200 System (Bio-Rad).
Mouse immunization and sample collection
Female BALB/c mice (6- to 8-week-old) were used in the study based on the preliminary studies, and they were randomly assigned to the indicated vaccination groups. Three separate immunizations were performed as described below (37, 51). The mice were intramuscularly (i.m.) immunized with the following proteins (10 µg/mouse) for three doses at a 3-week interval: (i) Om-S-BA5-RBD, for three doses; (ii) Om-S-SARS-RBD, for three doses; (iii) Om-S-MERS-RBD, for three doses; (iv) Om-S-BA5-RBD for the 1st dose and Om-S-SARS-RBD for the 2nd and 3rd doses; and (v) Om-S-BA5-RBD for the 1st dose and Om-S-MERS-RBD for the 2nd and 3rd doses. An optimal combination of Alum (500 µg/mouse) and MPL (10 µg/mouse) (InvivoGen) adjuvants was thoroughly mixed with the respective proteins before use. PBS plus the above adjuvants were included as control. Sera were collected 10 days after the last dose to test neutralizing antibodies against pseudotyped CoV infection, and PBMCs were isolated 5 months post-last immunization to detect T-cell responses, as described above. The immunized mice were proceeded to the subsequent challenge studies.
Challenge of mice with SARS-CoV-2 Omicron variant
This was performed as described below (37, 51). The immunized mice described above were intranasally (i.n.) challenged with an Omicron-BA.5 variant of SARS-CoV-2 at an optimal dose of 105 plaque-forming unit (PFU)/mouse (50 µL/mouse) 3 weeks after the last vaccination. The lungs of challenged mice were collected 2 days after the challenge, and tested for viral titers and viral replication by plaque assay and qPCR method, respectively, as described below.
Challenge of mice with MERS-CoV
This was performed as described below (23, 49). Three weeks after the last immunization, the vaccinated mice described above were i.n. transduced with an optimal dose (2.5 × 108 focus-forming unit: FFU) of Ad5-human DPP4 (Ad5-hDPP4: Ad5CMV/hDPP4-myc-flag; UI Viral Vector Core Web) suspended in 75 µL/DMEM culture medium, and then i.n. challenged with an optimal dose of MERS-CoV (EMC2012 strain; 105 PFU/mouse, 50 µL/mouse) 5 days after transduction. Lungs were collected 3 days after challenge, and measured for viral titers using plaque assay as described below.
Challenge of mice with SARS-CoV
This was performed as described below (52). Briefly, 5 months after the last immunization, the vaccinated mice described above were i.n. challenged with an optimal dose of SARS-CoV (MA15 strain; 200 PFU/mouse, 50 µL/mouse), and lungs were collected 2 days after the challenge to measure viral titers using plaque assay as described below.
Plaque assay
This was performed as described below (16, 53). Lungs from mice challenged with SARS-CoV-2 Omicron-BA.5, SARS-CoV, or MERS-CoV were homogenized in PBS. The tissue homogenate supernatant was serially diluted in a DMEM cell culture medium. Vero E6 cells (for SARS-CoV and SARS-CoV-2 wild-type), Vero in the presence of ACE2 and TMPRSS2 (for SARS-CoV-2 Omicron subvariants), or Vero E81 (for MERS-CoV) cells were plated in 12-well plates and cultured at 37°C for 1 h in 5% CO2 with gentle rocking every 15 min. The medium was removed 1 h later, and the plates were overlaid with 0.6% agarose, which were removed after 3 days. The plaques were visualized by staining with 0.1% crystal violet. Viral titers were quantified as PFU/mL of lung tissues.
qRT-PCR
This was performed as described below (54). Lungs from mice challenged with the SARS-CoV-2 Omicron-BA.5 variant were homogenized in Trizol buffer by Invitrogen, and RNA was extracted according to the manufacturer’s protocol (Thermo Fisher Scientific). Total RNA (1 µg) was used as a template for the first strand of cDNA, which was subjected to amplification of selected genes by real-time quantitative PCR (qRT-PCR) using Power SYBR Green PCR Master Mix (Applied Biosystems). The nucleocapsid (N) gene of Omicron-BA.5 was detected using nCOV_N1 primer (IDT, Cat# 10007031). The expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by the following threshold cycle (CT) equation: ΔCT = CT of the gene of interest − CT of GAPDH. All results are shown as a ratio to GAPDH calculated as 2−ΔCT.
Statistical analysis
Statistical software (GraphPad Prism 9) was used to determine statistical significance among different groups. Ordinary one-way ANOVA Tukey’s multiple comparison test was applied for statistical analysis. *, **, ***, and **** indicate P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively.
ACKNOWLEDGMENTS
This work was supported by National Institutes of Health grants (R01AI157975, R01AI139092, R01AI137472, and R01AI110700).
L.D. conceived the study. G.W. constructed the vaccines and prepared pseudoviruses. G.W. and X.G. characterized the vaccines and tested immune responses. G.W., A.K.V., X.G., and A.E.O immunized animals and performed challenge studies. F.B., F.L., and B.L. analyzed and supervised the cryo-EM structures. S.P. and L.D. supervised the study, wrote, and revised the paper with input from all authors.
SUPPLEMENTAL MATERIAL
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Information & Contributors
Information
Published In
Journal of Virology
Volume 98 • Number 9 • 17 September 2024
eLocator: e00376-24
Editor: Kanta Subbarao, Universite Laval, Quebec City, Quebec, Canada
PubMed: 39189731
Copyright
© 2024 American Society for Microbiology. All Rights Reserved.
History
Received: 24 February 2024
Accepted: 10 July 2024
Published online: 27 August 2024
Keywords
Data Availability
Special constructs and materials related to this study are available from the lead contact with a signed Material Transfer Agreement. All data generated in this study are available in the main text or the supplemental material. No special code was used in this study. The cryo-EM density map from this study has been deposited in the Electron Microscopy Data Bank (EMDB) with the entry ID EMD-42456 and the PDB ID 8UPX.
Contributors
Editor
Kanta Subbarao
Editor
Universite Laval, Quebec City, Quebec, Canada
Ethics Approval
The animal protocols were approved by the Institutional Animal Care and Use Committees (IACUC) of Georgia State University and University of Iowa. All animal-related studies were performed according to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and our approved protocols.
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Citation
2024.
Pan-beta-coronavirus subunit vaccine prevents SARS-CoV-2 Omicron,
SARS-CoV, and MERS-CoV challenge. J Virol
98:e00376-24.
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