Trivalent SARS-CoV-2 S1 Subunit Protein Vaccination Induces Broad Humoral Responses in BALB/c Mice

This paper presents a novel approach for improving the efficacy of COVID-19 vaccines against emergent SARS-CoV-2 variants. We have evaluated the immunogenicity of unadjuvanted wild-type (WU S1-RS09cg) and variant-specific (Delta S1-RS09cg and OM S1-RS09cg) S1 subunit protein vaccines delivered either as a monovalent or a trivalent antigen in BALB/c mice. Our results show that a trivalent approach induced a broader humoral response with more coverage against antigenically distinct variants, especially when compared to monovalent Omicron-specific S1. This trivalent approach was also found to have increased or equivalent ACE2 binding inhibition, and increased S1 IgG endpoint titer at early timepoints, against SARS-CoV-2 spike variants when compared monovalent Wuhan, Delta, or Omicron S1. Our results demonstrate the utility of protein subunit vaccines against COVID-19 and provide insights into the impact of variant-specific COVID-19 vaccine approaches on the immune response in the current SARS-CoV-2 variant landscape. Particularly, our study provides insight into effects of further increasing valency of currently approved SARS-CoV-2 vaccines, a promising approach for improving protection to curtail emerging viral variants.

The spike (S) protein of SARS-CoV-2 has been the main target of currently approved COVID-19 vaccines and of most COVID-19 vaccines in development [13]. The S protein mediates virus binding and infection of susceptible cells through interaction with host receptor angiotensin-converting enzyme 2 (ACE2) [14]. The S protein is composed of two

Construction of Recombinant Protein Expressing Vectors
The coding sequence for SARS-CoV-2-S1 amino acids 1 to 661; having C-terminal tag known as 'C-tag', composed of the four amino acids (aa), glutamic acid-proline-glutamic acid-alanine (E-P-E-A) flanked with Sal I & Not I was codon-optimized using UpGene algorithm for optimal expression in mammalian cells [28,40]. The construct also contained a Kozak sequence (GCCACC) at the 5 end. The plasmid, pAd/SARS-CoV-2-S1 was then created by subcloning the codon-optimized SARS-CoV-2-S1 inserts into the shuttle vector, pAdlox (GenBank U62024), at Sal I/Not I sites. The plasmid constructs were confirmed by DNA sequencing.

Transient Production in Expi293 Cells
pAd/S1RS09cg proteins were amplified and purified using ZymoPURE II plasmid maxiprep kit (Zymo Research). For Expi293 cell transfection, we used ExpiFectamieTM 293 Transfection Kit (ThermoFisher, Waltham, MA, USA) and followed the manufacturer's instructions. Cells were seeded 3.0 × 10 6 cells/mL one day before transfection and grown Vaccines 2023, 11,314 3 of 14 to 4.5-5.5 × 10 6 cells/mL. 1 µg of DNA and ExpiFectamine mixtures per 1 mL culture were combined and incubated for 15 min before adding into 3.0 × 10 6 cells/mL culture. At 20 h post-transfection, enhancer mixture was added, and culture was shifted to 32 • C. The supernatants were harvested 5 days post transfection and clarified by centrifugation to remove cells, filtration through 0.8 µm, 0.45 µm, and 0.22 µm filters and either subjected to further purification or stored at 4 • C before purification.

Purification of Recombinant Proteins
The recombinant proteins were purified using a CaptureSelect TM C-tagXL Affinity Matrix prepacked column (ThermoFisher) and followed the manufacturer's guideline [41]. Briefly, The C-tagXL column was conditioned with 10 column volumes (CV) of equilibrate/wash buffer (20 mM Tris, pH 7.4) before sample application. Supernatant was adjusted to 20 mM Tris with 200 mM Tris (pH 7.4) before being loaded onto a 5-mL prepacked column per the manufacturer's instructions at 5 mL/min rate. The column was then washed by alternating with 10 CV of equilibrate/wash buffer, 10 CV of strong wash buffer (20 mM Tris, 1 M NaCl, 0.05% Tween-20, pH 7.4), and 5 CV of equilibrate/wash buffer. The recombinant proteins were eluted from the column by using elution buffer (20 mM Tris, 2 M MgCl 2 , pH 7.4). The eluted solution was concentrated and desalted with preservative buffer (PBS) in an Amicon Ultra centrifugal filter devices with a 50,000 molecular weight cutoff (Millipore, Burlington, MA, USA). The concentrations of the purified recombinant proteins were determined by the Bradford assay using bovine serum albumin (BSA) as a protein standard, aliquoted, and stored at −80 • C until use.

SDS-PAGE, Silver Staining, and Western Blot
The purified proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Silver Staining, and Western blot. Briefly, after the supernatants were boiled in Laemmli sample buffer containing 2% SDS with beta-mercaptoethanol (β-ME), the proteins were separated by Tris-Glycine SDS-PAGE gels and transferred to nitrocellulose membrane. After blocking for 1 h at room temperature (RT) with 5% non-fat milk in TBS-T, rabbit anti-SARS-CoV spike polyclonal antibody (1:3000) (Sino Biological), or rabbit anti-SARS-CoV nucleoprotein (1:3000) (Sino Biological) was added and incubated overnight at 4 • C as primary antibody, and horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (1:10,000) (Jackson immuno research) was added and incubated at RT for 1 hs as secondary antibody. After washing, the signals were visualized using ECL Western blot substrate reagents and iBright 1500 (Thermo Fisher).

Animals and Immunization
At week 0 female BALB/c mice (n = 5 animals per group) were bled from retro-orbital vein and primed with 45 µg of either WU S1-RS09cg, Delta S1-RS09cg, OM S1-RS09cg, or trivalent WU/Delta/OM S1-RS09cg. Mice were bled on week 3 and received a homologous booster of 45 µg. Mice were bled on week 5, 7, 9, 12, 16, and 20. Mice were maintained under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were conducted in accordance with animal use guidelines and protocols approved by the University of Pittsburgh's Institutional Animal Care and Use (IACUC) Committee.

ELISA
Sera from all mice were collected prior to immunization (week 0) and at weeks indicated after immunization and evaluated for SARS-CoV-2-S1-specific IgG, IgG1, and IgG2a antibodies using ELISA [28]. Briefly, ELISA plates were coated with 200 ng of recombinant SARS-CoV-2-S1 protein per well overnight at 4 • C in carbonate coating buffer (pH 9.5) and then blocked with PBS-T and 2% bovine serum albumin (BSA) for one hour. For ELISA coating antigens, Wuhan S1 was purchased from Sino Biological, Delta S1cg was produced by our lab, and Omicron S1-RS09cg was used to elucidate Omicron (BA.1) specific response. Mouse sera were serially diluted in PBS-T with 1% BSA and Vaccines 2023, 11, 314 4 of 14 incubated overnight. After the plates were washed, anti-mouse IgG-horseradish peroxidase (HRP) (1:10,000, SantaCruz, Dallas, Texas, USA) was added to each well and incubated for 60 min. The plates were washed three times, developed with 3,3 5,5 -tetramethylbenzidine, and the reaction was stopped. Next, absorbance was determined at 450 nm using a plate reader. For IgG1 and IgG2a ELISAs, mouse sera were diluted in PBS-T with 1% BSA and incubated overnight. After the plates were washed, biotin-conjugated IgG1 and IgG2a (1:1000, eBioscience, San Diego, CA, USA) and biotin horseradish peroxidase (Av-HRP) (1:50,000, Vector Laboratories, Newark, CA, USA) were added to each well and incubated for 1 h. The plates were washed three times and developed with 3,3 5,5tetramethylbenzidine, the reaction was stopped, and absorbance at 450 nm was determined using a plate reader. ELISA data graphed is relative to preimmunization sera, using week 0 sera as the standardized cutoff. were detected with a V-PLEX SARS-CoV-2 Panel X (ACE2) Kit (Meso Scale Discovery (MSD)) according to the manufacturer's instructions. The assay plate was blocked for 30 min and washed. Serum samples were diluted (1:25, 1:100 or 1:400) and 25 µL were transferred to each well. The plate was then incubated at room temperature for 60 min with shaking at 700 rpm, followed by the addition of SULFO-TAG conjugated ACE2, and continued incubation with shaking for 60 min. The plate was washed, 150 µL MSD GOLD Read Buffer B was added to each well, and the plate was read using the QuickPlex SQ 120 Imager. Electrochemiluminescent values (ECL) were generated for each sample. Results were calculated as % inhibition compared to the negative control for the ACE2 inhibition assay, and % inhibition is calculated as follows: % neutralization = 100 × (1 − (sample signal/negative control signal)).

Statistical Analysis
Statistical analyses were performed using GraphPad Prism v9 (San Diego, CA, USA). Antibody endpoint titers and neutralization data were analyzed by Kruskal-Wallis test, followed by Dunn's multiple comparisons. Significant differences are indicated by * p < 0.05. Comparisons with non-significant differences are not indicated.
Against Wuhan S1, all vaccinated groups had significantly higher geometric mean Wuhan S1 IgG EPT at week 5 when compared to week 3, illustrating the superior immunogenicity conferred by boost immunization (Figure 2, p < 0.05, Kruskal-Wallis test, followed by Dunn's multiple comparisons). Interestingly, WU S1-RS09cg vaccinated mice achieved lower geometric mean Wuhan S1 IgG EPT by week 9 when compared to the other immunization groups (Figure 2). Indeed, trivalent WU/Delta/OM S1-RS09cg had increased Wuhan S1 IgG EPT when compared to monovalent OM S1-RS09cg at weeks 3, 5, and 7 ( Figure 2). However, as waning of the immune response occurred, the trivalent WU/Delta/OM S1-RS09cg vaccinated mice reached similar geometric mean Wuhan S1 IgG EPT as monovalent OM S1-RS09cg vaccinated mice at week 9 with waning continuing to occur through week 20 ( Figure 2).
To assess whether the IgG antibody response was Th1-or Th2-specific, serum samples were collected at week 5 and serially diluted to determine Wuhan and BA.1-specific S1, IgG1 (indicating a Th2 bias) and IgG2a (indicating a Th1 bias) endpoint titers for each immunization group ( Figure 5A-D). Interestingly, against Wuhan S1 all vaccinated mice groups achieved similar IgG1 and IgG2a geometric mean S1 IgG1 and IgG2a EPT, with no significant differences between groups ( Figure 5A,B). Differences between vaccine groups were illuminated against BA.1 S1 ( Figure 5C,D). Both OM-S1RS09cg and trivalent WU/Delta/OM S1-RS09cg vaccinated mice had the greatest geometric mean Omicron-S1 IgG1 EPT than WU S1-RS09cg and Delta S1-RS09cg vaccinated mice ( Figure 5C,D). OM S1-RS09cg vaccinated mice achieved the highest BA.1 S1 IgG2a geometric mean EPT ( Figure 5C,D). As expected for unadjuvanted protein subunit vaccine in BALB/c mouse, all vaccinated groups had a trend to a IgG1 dominant IgG response, indicating a Th2 bias.
To combine the data on Wuhan, Delta, and Omicron (BA.1) S binding IgG EPT and ACE2-percent binding inhibition we plotted the respective mean values at week 5 against each other ( Figure 7A-C). In the context of Wuhan S, Delta S1-RS09cg and trivalent WU/Delta/OM S1-RS09cg vaccinated mice grouped together with the highest mean S1 IgG EPT and mean ACE2 binding inhibition ( Figure 7A). Against Delta S, Delta S1-RS09cg and trivalent WU/Delta/OM S1-RS09cg vaccinated mice group together with the highest mean S1 IgG EPT and mean ACE2 binding inhibition ( Figure 7B). For BA.1 S, OM S1-RS09cg and trivalent WU/Delta/OM S1-RS09cg vaccinated mice grouped together with the highest mean S1 IgG EPT and mean ACE2 binding inhibition ( Figure 7C). Taken together, a prime and boost of non-adjuvanted recombinant S1 protein subunit vaccine induced a robust humoral antibody response against SARS-CoV-2 in BALB/c mice. Particularly, trivalent WU/Delta/OM vaccinated mice induced a broad and crossreactive neutralizing antibodies against SARS-CoV-2 variants with increased breadth when compared to monovalent WU S1-RS09cg, Delta S1-RS09cg, and OM S1-RS09cg vaccinated mice.

Discussion
As SARS-CoV-2 variants continue to emerge more vaccination platforms against SARS-CoV-2, which induce a broader immune response covering multiple variants, will be necessary [8,9,11,12,43,47]. Further, as COVID-19 booster doses are distributed, it will be critical to ensure that global vaccine equity is met [48,49]. Protein subunit vaccines are ideal for worldwide distribution due to their excellent safety, low cost, scalability, and thermostability [23,24,50]. Protein subunit vaccine platforms can be further improved through use of alternative vaccine delivery methods such as intranasal or intradermal vaccination, with microneedle arrays [28,51]. The versatility of protein subunit vaccines lends to their utility for mass distribution and vaccination.
In this study, we demonstrate the robust antibody response elicited by our unadjuvanted S1 protein subunit vaccine in BALB/c mice. Wuhan S1-RS09cg, Delta S1-RS09cg, OM-S1RS09cg, and trivalent WU/Delta/OM S1-RS09cg vaccinated mice all elicited a robust IgG binding antibody response against Wuhan S1, Delta S1, and Omicron (BA.1) S1. Particularly, trivalent WU/Delta/OM S1-RS09cg vaccinated mice mounted cross-reactive ACE2 binding inhibiting antibodies against SARS-CoV-2 variants with increased breadth when compared to monovalent WU S1-RS09cg, Delta S1-RS09cg, and OM S1-RS09cg vaccinated mice. We believe that this gives credence to investigating SARS-CoV-2 vaccines that are multivalent to expand variant specific immune responses. Our data also suggests that increasing valency of SARS-CoV-2 vaccines may not reduce magnitude of the individual variant immune response, a key added piece of information for development of next-generation SARS-CoV-2 vaccines. A particularly unexpected result of our study is the low immunogenicity of our WU S1-RS09cg vaccine against Wuhan S1, and other VOCs, when compared to Delta and OM S1-RS09cg. Indeed, Delta and Omicron (BA.1) mutations in S have been shown to increase pathogenicity and S fusogenicity, along with increased ACE2 binding to S, when compared to wild-type Wuhan SARS-CoV-2 [47,[52][53][54][55]. We hypothesize that this increased ACE2 binding by Delta and Omicron S may Taken together, a prime and boost of non-adjuvanted recombinant S1 protein subunit vaccine induced a robust humoral antibody response against SARS-CoV-2 in BALB/c mice. Particularly, trivalent WU/Delta/OM vaccinated mice induced a broad and cross-reactive neutralizing antibodies against SARS-CoV-2 variants with increased breadth when compared to monovalent WU S1-RS09cg, Delta S1-RS09cg, and OM S1-RS09cg vaccinated mice.

Discussion
As SARS-CoV-2 variants continue to emerge more vaccination platforms against SARS-CoV-2, which induce a broader immune response covering multiple variants, will be necessary [8,9,11,12,43,47]. Further, as COVID-19 booster doses are distributed, it will be critical to ensure that global vaccine equity is met [48,49]. Protein subunit vaccines are ideal for worldwide distribution due to their excellent safety, low cost, scalability, and thermostability [23,24,50]. Protein subunit vaccine platforms can be further improved through use of alternative vaccine delivery methods such as intranasal or intradermal vaccination, with microneedle arrays [28,51]. The versatility of protein subunit vaccines lends to their utility for mass distribution and vaccination.
In this study, we demonstrate the robust antibody response elicited by our unadjuvanted S1 protein subunit vaccine in BALB/c mice. Wuhan S1-RS09cg, Delta S1-RS09cg, OM-S1RS09cg, and trivalent WU/Delta/OM S1-RS09cg vaccinated mice all elicited a robust IgG binding antibody response against Wuhan S1, Delta S1, and Omicron (BA.1) S1. Particularly, trivalent WU/Delta/OM S1-RS09cg vaccinated mice mounted cross-reactive ACE2 binding inhibiting antibodies against SARS-CoV-2 variants with increased breadth when compared to monovalent WU S1-RS09cg, Delta S1-RS09cg, and OM S1-RS09cg vaccinated mice. We believe that this gives credence to investigating SARS-CoV-2 vaccines that are multivalent to expand variant specific immune responses. Our data also suggests that increasing valency of SARS-CoV-2 vaccines may not reduce magnitude of the individual variant immune response, a key added piece of information for development of next-generation SARS-CoV-2 vaccines. A particularly unexpected result of our study is the low immunogenicity of our WU S1-RS09cg vaccine against Wuhan S1, and other VOCs, when compared to Delta and OM S1-RS09cg. Indeed, Delta and Omicron (BA.1) mutations in S have been shown to increase pathogenicity and S fusogenicity, along with increased ACE2 binding to S, when compared to wild-type Wuhan SARS-CoV-2 [47,[52][53][54][55]. We hypothesize that this increased ACE2 binding by Delta and Omicron S may explain the increased immunogenicity exhibited by Delta and OM S1-RS09cg when compared to Wuhan S1-RS09cg, however, this will need to be explored further. Furthermore, Omicron BA.1 spike G446S mutation has been shown to potentiate antiviral T-cell recognition which may further explain the increased immunogenicity demonstrated by our OM S1-RS09cg and trivalent vaccine candidates [56].
The IgG isotype of the induced IgG antibodies skew to be IgG1 dominant, indicating a Th2-type bias. Indeed, BALB/c mice are the prototypical Th2-type mouse strain which necessitates the investigation of this protein subunit vaccine in additional animal models to examine the risk of vaccine-associate enhanced respiratory disease (VAERD) [57]. Our previous research has suggested that a booster of unadjuvanted subunit vaccine after an Adenoviral prime vaccine might avoid Th2-based immune response and the occurrence of VAERD [30]. Indeed, there have been numerous Adenoviral vector vaccine platforms used in the SARS-CoV-2 pandemic and constitute a large population necessitating variantspecific boosting [7,58]. Further so, the Th1-and Th2-type immune response may be further augmented using an adjuvant. In the context of SARS-CoV-2 vaccines there have been numerous adjuvants that have shown beneficial effects on immunogenicity [59][60][61][62]. Interestingly, a AS01-like adjuvanted SARS-CoV-2 subunit vaccine enhanced Th1-type IgG2a isotype and IFN-γ secreting T cell immune responses in BALB/c mice when compared to unadjuvanted control [63].
An important limitation regarding our study is the lack of T-cell immunity investigation and SARS-CoV-2 challenge, which were not performed to assess the protection ability of our vaccine constructs. S-specific binding antibodies were positively correlated with S-specific T-cell responses indicating induction of T cell immune response by our vaccine constructs [64]. We chose to focus on the induction of antibodies because they are the hypothesized correlate of protection against severe COVID-19 [20]. Furthermore, prior studies have shown the positive correlation and high concordance between binding antibodies and traditional virus-based microneutralization tests [30]. Our past work has also shown the positive correlation between the MSD ACE2 binding inhibition and virusbased microneutralization tests [30]. As a conventional and multiplex test, measurement of competitive immunoassay for quantifying inhibition of the spike-ACE2 interaction can serve as a surrogate for traditional virus-based microneutralization tests with high levels of correlation [43,45,46]. Our future studies will probe the protection ability elicited by our monovalent and trivalent vaccines through challenge studies using BALB/c mice and K18-hACE2 mice. The BALB/c mouse model of SARS-CoV-2 infection only supports infection of SARS-CoV-2 variants that carry the N501Y variant, necessitating the use of hACE2-transgenic mice to evaluate protection efficiency against other variants [65].
Overall, this study illustrates the potential of subunit protein vaccine targeting SARS-CoV-2-S1 as it induces significant induction of humoral immune responses against SARS-CoV-2 even without adjuvant. Particularly, immunizing with trivalent WU/Delta/OM S1-RS09cg increased binding antibodies and ACE2-binding inhibiting antibodies against SARS-CoV-2 variant spikes versus monovalent approaches. Furthermore, combining our protein subunit protein vaccine targeting SARS-CoV-2-S1 with an immunostimulatory adjuvant should provide even higher levels of immunogenicity when compared to the unadjuvanted studies presented here. Our findings support the use of trivalent Wuhan, Delta, and Omicron targeting COVID-19 vaccines to increase variant antigenic coverage. Funding: AG is funded by NIH grants (UM1-AI106701, R01DK119936-S1 and U01-CA233085) and UPMC Enterprises IPA 25565. These funding institutions had no role in the study design, data collection, data analysis, and interpretation of this publication.
Institutional Review Board Statement: Mice were maintained under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were conducted in accordance with animal use guidelines and protocols approved by the University of Pittsburgh's Institutional Animal Care and Use (IACUC) Committee. Experiments with animals were performed in accordance local and national legal requirements such as AAALAC International and the United States Department of Agriculture.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data that support the finding of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.