Mosaic RBD nanoparticles elicit neutralizing antibodies against SARS-CoV-2 and zoonotic coronaviruses

Protection against SARS-CoV-2 and SARS-related zoonotic coronaviruses with pandemic potential is urgently needed. To evaluate immunization strategies, we made nanoparticles displaying the receptor-binding domain (RBD) of only SARS-CoV-2 (homotypic nanoparticles) or co-displaying the SARS-CoV-2 RBD along with RBDs from animal betacoronaviruses that represent threats to humans (mosaic nanoparticles; 4-8 distinct RBDs). Mice immunized with RBD-nanoparticles, but not soluble antigen, elicited cross-reactive antibody binding and neutralization responses, confirming increased immunogenicity from multimerization. Mosaic-RBD-nanoparticles elicited antibodies with superior cross-reactive recognition of heterologous RBDs compared to sera from immunizations with homotypic SARS-CoV-2–RBD-nanoparticles or antibodies from COVID-19 convalescent human plasmas. Moreover, sera from mosaic-RBD– immunized mice neutralized heterologous pseudotyped coronaviruses equivalently or better after priming than sera from homotypic SARS-CoV-2–RBD-nanoparticle immunizations, demonstrating no loss of immunogenicity against any particular RBD resulting from co-display. Thus, a single immunization with mosaic-RBD-nanoparticles provides a potential strategy to simultaneously protect against SARS-CoV-2 and emerging zoonotic coronaviruses.

pandemics. In particular, the WIV1 and SHC014 bat strains are thought to represent an ongoing threat to humans (6,7).
Multivalent display of antigen enhances B-cell responses and can provide longer-lasting immunity than monovalent antigens (27,28), thus protein-based vaccine candidates often involve a nanoparticle that enables antigen multimerization. Many nanoparticles and coupling strategies have been explored for vaccine design (29), with a "plug and display" strategy being especially useful (30,31). In this approach, a virus-like particle fused to a SpyCatcher protein is covalently conjugated to a purified antigen 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 a SARS-CoV-2 vaccine, large coronavirus reservoirs in bats suggest future cross-species transmission (6,7,35), necessitating a vaccine that could protect against emerging coronaviruses as well as SARS-CoV-2. Here we used plug and display to prepare SpyCatcher003-mi3 nanoparticles (31) simultaneously displaying SpyTagged RBDs from human and animal coronaviruses to evaluate whether mosaic particles can elicit cross-reactive antibody responses. 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 sarbecoviruses compared with homotypic SARS-CoV-2 nanoparticles.
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) 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 (42) (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 (33,43) 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 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. Importantly, 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, greatly simplifying large-scale immunizations. Since COVID-19 convalescent plasmas showed little to no recognition of coronavirus RBDs other than SARS-CoV-2, COVD-19-induced immunity in humans may not protect against another emergent coronavirus. However, the mosaic nanoparticles described here could be used as described and/or easily adapted to present RBDs from newly-discovered zoonotic coronaviruses. Since these types of RBDnanoparticles retain immunogenicity after lyophilization (33), they could be easily stored for widespread use. Thus this modular vaccine platform could provide protection from SARS-CoV-2 and potential future coronavirus pandemics resulting from emergent zoonotic sarbecoviruses.        ectodomain with 6P stabilizing mutations (44) was expressed and purified as described (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). Sera for ELISAs were collected at Day 14 (Prime) and Day 42 (Boost). Sera for neutralization assays were collected at Day 28 (Prime) and Day 56 (Boost) (Fig. 3, fig. S3). one-site 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.   S3C,D). AUC calculations were used as they better capture changes in maximum binding (46).

ELISAs
Statistical significance of titer differences between groups were calculated using Tukey's multiple comparison test using Graphpad Prism 8.3.
Neutralization assays. SARS-CoV-2, SARS, WIV1, and SHC014 pseudoviruses based on HIV lentiviral particles were prepared as described (18,47)  Flow cytometry. B-cell analysis using flow cytometry was carried out as described (45). 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 was enriched for IgG+ B-cells using the negative selection protocol in a mouse memory B-cell   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