Mosaic nanoparticles elicit cross-reactive immune responses to zoonotic coronaviruses in mice

Fighting zoonotic coronaviruses In the past 20 years, three betacoronaviruses thought to have originated in bats have caused devastating disease in humans. The global pandemic caused by the latest such virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need to protect against other strains that could present a threat to humans. Cohen et al. constructed nanoparticles displaying the protein domain that binds the host cell receptor (receptor-binding domain or RBD), either a homotypic SARS-CoV-2 particle or mosaic particles displaying RBDs from four or eight different betacoronaviruses. In mice, antibodies to the SARS-CoV-2 RBD were elicited just as well by mosaic particles as by homotypic nanoparticles. The mosaic nanoparticles elicited antibodies that, beyond recognizing the strains displayed, also recognized mismatched strains. Science, this issue p. 735

involve a nanoparticle that enables antigen multimerization. Many nanoparticles and coupling strategies have been explored for vaccine design (29), with "plug and display" strategies being especially useful (30,31). In one such approach, multiple copies of an engineered protein domain called SpyCatcher fused to subunits of a virus-like particle form spontaneous isopeptide bonds to purified antigens tagged with a 13-residue SpyTag (29)(30)(31)(32). The SpyCatcher-SpyTag system was used to prepare multimerized SARS-CoV-2 RBD or S trimer that elicited high titers of neutralizing antibodies (33,34). Although promising for protection against SARS-CoV-2, coronavirus reservoirs in bats suggest future cross-species transmission (6,7,35), necessitating a vaccine that protects against emerging coronaviruses as well as SARS-CoV-2. Here we prepared SpyCatcher003-mi3 nanoparticles (31,36) simultaneously displaying SpyTagged RBDs from human and animal coronaviruses to evaluate whether mosaic particles can elicit cross-reactive antibody responses, as previously demonstrated for influenza head domain mosaic particles (37).
We show that mice immunized with homotypic or mosaic nanoparticles produced broad binding and neutralizing responses, in contrast to plasma antibodies elicited in humans by SARS-CoV-2 infection. Moreover, mosaic nanoparticles showed enhanced heterologous binding and neutralization properties against human and bat SARS-like betacoronaviruses (sarbecoviruses) compared with homotypic SARS-CoV-2 nanoparticles.
We immunized mice with either soluble SARS-CoV-2 spike trimer (SARS-2 S), nanoparticles displaying only SARS-2 RBD (homotypic SARS-2), nanoparticles co-displaying RBDs (mosaic-4a, mosaic-4b, mosaic-8), or unconjugated nanoparticles (mi3). IgG responses were evaluated . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint after prime or boost immunizations (Fig. 3A) by ELISA against SARS-2 S (Fig. 3B) or a panel of RBDs (Fig. 3C-F; fig. S3). Sera from unconjugated nanoparticle-immunized animals (black in Fig. 3, fig. S3) showed no responses above background. Anti-SARS-2 S trimer and anti-SARS-2 RBD serum responses were similar (Fig. 3B,C), demonstrating that antibodies elicited against RBDs can access their epitopes on SARS-2 S trimer. We also conducted in vitro neutralization assays using a pseudotyped virus assay that quantitatively correlates with authentic virus neutralization (44) (27,28). Homotypic SARS-2 nanoparticles, but not soluble SARS-2 trimer, induced heterologous responses to zoonotic RBDs and neutralization of heterologous coronaviruses (Fig. 3D-F). To address whether co-display of SARS-2 RBD along with other RBDs on mosaic-4a and mosaic-8 versus homotypic display of SARS-2 RBD (homotypic SARS-2) diminished anti-SARS-2 responses, we compared SARS-2-specific ELISA and neutralization titers for mosaic versus homotypic immunizations (Fig. 3C): there were no significant differences in IgG anti-SARS-2 titers for animals immunized with homotypic (red in . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint Fig. 3C) versus mosaic nanoparticles (green and blue in Fig. 3C). Thus there was no advantage of immunization with a homotypic RBD-nanoparticle versus a mosaic-nanoparticle that included SARS-2 RBD in terms of the magnitude of immune responses against SARS-2.
Demonstrating advantages of mosaic versus homotypic SARS-2 nanoparticles, sera from mosaic-8-immunized mice bound SHC014 and WIV1 RBDs significantly better after the prime than sera from homotypic SARS-2-immunized mice and retained better binding to SHC014 RBD after boosting (Fig. 3D). Thus the potential increased avidity of the homotypic SARS-2 nanoparticle displaying only one type of RBD over the mosaic-8 nanoparticles did not confer increased breadth. Moreover, mosaic-8-immunized and boosted sera were 7-44-fold more potent than sera from homotypic SARS-2-immunized animals in neutralizing SHC014 and WIV1 (Fig.   3D). Neutralization of the SHC014 and WIV1 pseudoviruses by mosaic-8 sera suggests that . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint combining RBDs on a mosaic nanoparticle does not diminish the immune response against a particular RBD, also suggested by ELISA binding of sera to Rs4081 and RaTG13 ( fig. S3A,B).
Thus mosaic nanoparticles, particularly mosaic-8, induce higher antibody titers against mismatched RBDs than homotypic SARS-2 nanoparticles, again favoring the co-display approach for inducing broader anti-coronavirus responses, especially after a single prime.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint We investigated the potential for cross-reactive recognition using flow cytometry to ask whether B-cell receptors on IgG+ splenic B-cells from RBD-nanoparticle-boosted animals could simultaneously recognize RBDs from SARS-2 and Rs4081 (related by 70% sequence identity) ( Fig. 1D; fig. S5). Whereas control animals were negative, all other groups showed B-cells that recognized SARS-2 and Rs4081 RBDs simultaneously, suggesting the existence of antibodies that cross-react with both RBDs (fig. S5E).
To compare antibodies elicited by RBD-nanoparticle immunization to antibodies elicited by SARS-CoV-2 infection, we repeated ELISAs against the RBD panel using IgGs from COVID-19 plasma donors (47) (Fig. 4). Most of the convalescent plasmas showed detectable binding to SARS-2 RBD (Fig. 4A). However, binding to other sarbecovirus RBDs (RaTG13, SHC014, WIV1, Rs4081 and BM-4831) was significantly weaker than binding to SARS 2 RBD, with many human plasma IgGs showing no binding above background ( Fig. 4B-G). In addition, although convalescent plasma IgGs neutralized SARS-CoV-2 pseudoviruses, they showed weak or no neutralization of SARS, SHC014, or WIV1 pseudoviruses (Fig. 4H). These results are consistent with little to no cross-reactive recognition of RBDs from zoonotic coronavirus strains resulting from SARS-CoV-2 infection in humans.
In conclusion, we confirmed that multimerization of RBDs on nanoparticles enhances immunogenicity compared with soluble antigen (33,48) and further showed that homotypic SARS-2 nanoparticle immunization produced IgG responses that bound zoonotic RBDs and neutralized heterologous coronaviruses after boosting. By contrast, soluble SARS-2 S . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint immunization and natural infection with SARS-CoV-2 resulted in weak or no heterologous responses in plasmas. Co-display of SARS-2 RBD along with diverse RBDs on mosaic nanoparticles showed no disadvantages for eliciting neutralizing antibodies against SARS-CoV-2 compared with homotypic SARS-2 nanoparticles, suggesting mosaic nanoparticles as a candidate vaccine to protect against COVID-19. Furthermore, compared with homotypic SARS-2 RBD particles, the mosaic co-display strategy demonstrated advantages for eliciting neutralizing antibodies against zoonotic sarbecoviruses, thus potentially also providing protection against emerging coronaviruses with human spillover potential., Neutralization of matched and mismatched strains was observed after mosaic priming, suggesting a single injection of a mosaic-RBD nanoparticle might be sufficient in a vaccine. Since COVID-19 convalescent plasmas showed little to no recognition of coronavirus RBDs other than SARS-CoV-2, COVD-19induced immunity in humans may not protect against another emergent coronavirus. However, the mosaic nanoparticles described here could be used as described or easily adapted to present RBDs from newly-discovered zoonotic coronaviruses.

29.
K. D. Brune . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021.  . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint

Materials and Methods
Phylogenetic tree. A sequence alignment of coronavirus RBD domains was made using Clustal Omega (51). A phylogenetic tree was calculated from this amino acid alignment using PhyML 3.0 (52), and a figure of this tree was made using PRESTO (http://www. atgcmontpellier.fr/presto).

Expression of RBD and S proteins. Mammalian expression vectors encoding the RBDs of SARS-
CoV-2 (GenBank MN985325.1; S protein residues 319-539) and SARS-CoV S (GenBank AAP13441.1; residues 318-510) with an N-terminal human IL-2 or Mu phosphatase signal peptide were previously described (47). Expression vectors were constructed similarly for RBDs (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint ER-directed BirA enzyme (kind gift of Michael Anaya, Caltech). RBD proteins were purified from transiently-transfected Expi293F cell (Gibco) supernatants by nickel affinity and size-exclusion chromatography (47). Peak fractions corresponding to RBDs were identified by SDS-PAGE and then pooled and stored at 4˚C. A trimeric SARS-CoV-2 ectodomain with 6P stabilizing mutations (53) was expressed and purified as described (24). Correct folding of the soluble SARS-CoV-2 S trimer was verified by a 3.3 Å cryo-EM structure of a neutralizing antibody complexed with the trimer preparation used for immunizations (24). To prepare fluorochrome-conjugated streptavidin-tetramerized RBDs, biotinylated SARS-2 and Rs4081 RBDs were incubated with streptavidin-APC (eBioscience TM ) and streptavidin-PE (ThermoFisher), respectively, overnight at 4 o C at a 1:1 molar ratio of RBD to streptavidin subunit.
Preparation of human plasma IgGs. Plasma samples collected from COVID-19 convalescent and healthy donors are described in (18). Human IgGs were isolated from heat-inactivated plasma samples using 5-mL HiTrap MabSelect SuRe columns (GE Healthcare Life Sciences) as described (24).
Lysates were spun at 21,000xg for 30 min, filtered with a 0.2 µm filter, and mi3 particles were isolated by Ni-NTA chromatography using a pre-packed HisTrap TM HP column (GE Healthcare).
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint Eluted particles were concentrated using an Amicon Ultra 15 mL 30K concentrator (MilliporeSigma) and purified by SEC using a HiLoad® 16/600 Superdex® 200 (GE Healthcare) column equilibrated with 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.02% NaN3 (TBS). SpyCatcher-mi3 particles were stored at 4°C and used for conjugations for up to 1 month after filtering with a 0.2 µm filter or spinning at 21,000xg for 10 min.
Purified SpyCatcher003-mi3 was incubated with a 3-fold molar excess (RBD to mi3 subunit) of purified SpyTagged RBD (either a single RBD for making homotypic SARS-CoV-2 RBD particles or an equimolar mixture of four or eight RBDs for making mosaic particles) overnight at room temperature in TBS. Conjugated mi3 particle were separated from free RBDs by SEC on a Superose 6 10/300 column (GE Healthcare) equilibrated with PBS (20 mM sodium phosphate pH 7.5, 150 mM NaCl). Fractions corresponding to conjugated mi3 particles were collected and analyzed by SDS-PAGE. Concentrations of conjugated mi3 particles were determined using a Bio-Rad Protein Assay.
Immunizations. Animal procedures and experiments were performed according to protocols approved by the IACUC. Experiments were done using 4-6 week old female Balb/c mice (Charles River Laboratories), with 5 animals each for cohorts immunized with soluble SARS-CoV-2 S or SpyCatcher003-mi3, and 10 animals each for remaining cohorts (Fig 3A). Immunizations were carried out with intraperitoneal (ip) injections of either 5 µg of conjugated RBD (calculated as the mass of the RBD, assuming 100% efficiency of conjugation to SpyCatcher003-mi3), 5 µg of soluble SARS-CoV-2 S, or 6 µg of unconjugated SpyCatcher003-mi3, in 100 µL of 50% v/v . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint AddaVax TM adjuvant (Invivogen). Animals were boosted 4 weeks after the prime with the same quantity of antigen in adjuvant. Animals were bled every 2 weeks via tail veins, and then euthanized 8 weeks after the prime (Day 56, 57) and bled through cardiac puncture. Blood samples were allowed to clot at room temperature in MiniCollect® Serum and Plasma Tubes (Greiner), and serum was harvested, preserved in liquid nitrogen, and stored at -80˚C until use.
ELISAs. 10 µg/ml of a purified RBD (not SpyTagged) in 0.1 M NaHCO3 pH 9.8 was coated onto Nunc® MaxiSorp™ 384-well plates (Sigma) and stored overnight at 4 o C. Plates were washed with Tris-buffered saline with 0.1% Tween 20 (TBS-T) after blocking with 3% bovine serum albumin (BSA) in TBS-T for 1 hr at room temperature. Mouse serum was diluted 1:100 and then serially diluted by 4-fold with TBS-T/3% BSA and added to plates for 3 hr at room temperature.
A 1:50,000 dilution of secondary HRP-conjugated goat anti-mouse IgG (Abcam) was added after washing for 1 hr at room temperature. Plates were developed using SuperSignal™ ELISA Femto Maximum Sensitivity Substrate (ThermoFisher) and read at 425 nm. Curves were plotted and integrated to obtain the area under the curve (AUC) using Graphpad Prism 8.3 assuming a onesite binding model with a Hill coefficient ( Fig. 3; fig. S3). We also calculated EC50s and endpoint titers, which were determined using the dilution that was at or below the mean + 2 x the standard deviation of the plate control (no primary serum added) for ELISA binding data ( fig.   S3E,F). AUC calculations were used as they better capture changes in maximum binding (55).
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint Statistical significance of titer differences between groups were calculated using Tukey's multiple comparison test using Graphpad Prism 8.3.
Flow cytometry. B-cell analysis using flow cytometry was carried out as described (54). Briefly, single-cell suspensions were prepared from mouse spleens using mechanical dissociation, and red blood cells were removed using ACK lysing buffer (Gibco). The white blood cell preparation . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint , and BtKY72 (KY352407). SARS-2 RBD residues that interact directly with ACE2 (58) are indicated by an asterisk. We note that antibody neutralization by direct binding of ACE2-binding residues does not represent the only mechanism of neutralization for ACE2-tropic viruses. This has been shown for monoclonal human antibodies derived from COVID-19 patients: some neutralizing antibodies do not directly interact with the ACE2-binding site on RBD (for example, class 3 anti-SARS-CoV-2 neutralizing antibodies (24)). Red shading indicates strains known to use ACE2 as a receptor.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint Fig. S3. Day 14 serum IgG responses to RBDs evaluated by ELISA shown as area under the curve (AUC) from mice immunized with soluble SARS-CoV-2 S trimers (SARS-2 S) or RBDs on nanoparticles (homotypic SARS-2, mosaic-4a, mosaic-4b, mosaic-8, or unconjugated SpyCatcher003-mi3 (mi3)). Each dot represents serum from one animal, with means and standard deviations represented by rectangles (mean) and horizontal lines (SD). RBDs from strains that were not present on an immunized particle or were present on an immunized particle are indicated by red and gray rectangles, respectively, below the ELISA data. Significant differences between groups linked by horizontal lines are indicated by asterisks and p-values. NS=not significant. (A,B) Binding of serum IgGs to (A) Rs4081 and (B) RaTG13 RBDs. (C) Binding of serum IgGs to SARS-2 RBD (left), a triple RBD mutant in a mouse-adapted SARS-CoV-2 (46) that includes substitutions adjacent to the N501Y RBD mutation in an emergent UK SARS-CoV-2 lineage (https://virological.org/t/preliminary-genomic-characterisation-of-an-emergent-sarscov-2-lineage-in-the-uk-defined-by-a-novel-set-of-spike-mutations/563) (middle), and Y453F, the "Danish mink variant" (45) (right). (D) Binding of serum IgGs to RBD from MERS-CoV (a non-ACE2-binding merbecovirus, representing a different subgenus from sarbecoviruses). (E,F) Comparison of ELISA data for serum binding to selected RBDs presented as AUC, endpoint titers, midpoint titers, or binding curves. Day 14 serum IgG responses to (E) SARS-2 or (F) SARS RBDs evaluated by ELISA shown as AUC (left), endpoint titers (middle left), midpoint (EC50) titers (middle right), or binding curves (right). For AUC, each dot represents serum from one animal, with means and standard deviations represented by rectangles (mean) and horizontal lines (SD). For endpoint and midpoint titers, each dot represents serum from one animal, with geometric means and geometric standard deviations represented by rectangles (mean) and horizontal lines (SD). Binding curves are shown with data points representing the mean and SD of duplicate measurements fit to a binding model (see Methods) for animals immunized with mosaic-8 and homotypic SARS-2.
. CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint Fig S5. Antigen-specific IgG + B-cell analysis of splenocytes isolated from animals immunized with mosaic-RBD nanoparticles. (A) Flow cytometry gating strategy for characterizing RBDspecific IgG + B-cells isolated from splenocytes. Anti-CD4, anti-CD8, anti-F4/80, anti-Ly6G, and anti-IgM were used in the dump to remove T-cells, macrophages, and IgM + B-cells. Antigenspecific IgG + B-cells were isolated using labeled anti-CD19 and anti-IgG antibodies, and probed for binding RBD with a pair of fluorophore-conjugated RBD tetramers (SARS-2 RBD and Rs4081 RBD). (B) Complete flow cytometry analysis for antigen-specific IgG + splenocytes isolated from animals immunized with mosaic-RBD particles. The 4-way gate shown for each animal separates each population of RBD single-positive and double-positive cells and was used for the % antigen-specific populations shown in panels C-E. Q1 represents the Rs4081 RBD + population, Q2 represents the Rs4081 RBD + / SARS-2 RBD + population, Q3 represents the SARS-2 RBD + population, and Q4 represents the RBDpopulation. . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted January 5, 2021. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint