Neutralization of SARS-CoV-2 and zoonotic coronavirus threats by mosaic nanoparticle vaccination

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.

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.
SpyTag003-RBDs were coupled to SpyCatcher003-mi3 (60 potential conjugation sites) (36,43) to make homotypic and mosaic nanoparticles (Fig 2A). Particles were purified by size exclusion chromatography (SEC) and analyzed by SDS-PAGE, revealing monodisperse SEC profiles and . CC-BY-NC-ND 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint nearly 100% conjugation (Fig. 2B,C). Representative RBDs were conjugated to SpyCatcher003-mi3 with similar or identical efficiencies ( fig. S2), suggesting that mosaic particles contained approximately equimolar mixtures of different RBDs.
. 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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint To address whether RBD-nanoparticles elicited antibodies against totally mismatched strains, we evaluated sera for binding to SARS, Yun11, BM-4831, and BtKY72 RBDs (Fig. 3E,F) and neutralization of SARS (Fig. 3E). Against these RBDs, we found significantly higher and more cross-reactive antibody responses for mosaic immunizations compared with homotypic SARS-2 immunizations: e.g., mosaic-8-primed and boosted animals showed significantly higher titers against SARS RBD than sera from homotypic SARS-2-immunized mice (Fig. 3E). After the prime, sera from the homotypic SARS-2-immunized animals did not neutralize SARS, whereas the mosaic-4b and mosaic-8 sera were neutralizing (Fig. 3E), perhaps facilitated by these nanoparticles including WIV1 RBD, which is related by 95% amino acid identity to SARS RBD ( Fig. 1D). After boosting, SARS-2 and mosaic-4a sera were also neutralizing, although titers were ~4-fold lower than for mosaic-8-immunized animals (Fig. 3E). ELISA titers against other mismatched RBDs (Yun11, BM-4831, BtKY72) were significantly higher for sera collected after mosaic-8 priming compared to sera from homotypic SARS-2 priming, and heightened binding was retained after boosting (Fig. 3F). 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.
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).
. 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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint 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 (44) (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 In conclusion, we confirmed that multimerization of RBDs on nanoparticles enhances immunogenicity compared with soluble antigen (33,45) 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 . 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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint 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 RBD-nanoparticles 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. . 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 December 1, 2020. ; 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 December 1, 2020. ; 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 December 1, 2020. ; 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 December 1, 2020. ; 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 December 1, 2020. ; 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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint

Phylogenetic tree. A sequence alignment of coronavirus RBD domains was made using Clustal
Omega (48). A phylogenetic tree was calculated from this amino acid alignment using PhyML 3.0 (49), and a figure of this tree was made using PRESTO (http://www. atgc-montpellier.fr/presto).  (44). 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 (50) was expressed and purified as described (24). To prepare fluorochrome-. 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
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).
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 . 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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint 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 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).
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 . 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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint 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. S3C,D). AUC calculations were used as they better capture changes in maximum binding (52). 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 (18,53) using genes encoding S protein sequences lacking C-terminal residues in the cytoplasmic tail: 21 amino acid deletions for SARS-CoV-2, WIV1, and SHC014 and a 19 amino acid deletion for SARS-CoV. For neutralization assays, four-fold serially diluted sera from immunized mice were incubated with a pseudotyped virus for 1 hour at 37˚C. After incubation with 293TACE2 target cells for 48 hours at 37˚C, cells were washed twice with phosphate-buffered saline (PBS) and lysed with Luciferase Cell Culture Lysis 5x reagent (Promega). NanoLuc Luciferase activity in lysates was measured using the Nano-Glo Luciferase Assay System (Promega). Relative luminescence units (RLUs) were normalized to values derived from cells infected with pseudotyped virus in the absence of serum. Half-maximal inhibitory dilutions (ID50 values) were determined using 4-parameter nonlinear regression in AntibodyDatabase (54). Statistical significance of titer differences between groups were calculated using Tukey's multiple comparison test of ID50s converted to log 10 scale using Graphpad Prism 8.3.
. 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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint Statistical Analysis. Comparisons between groups for ELISAs and neutralization assays were calculated with one-way analysis of variance (ANOVA) using Tukey's post hoc test in Prism 9.0 (Graphpad). For correlation analysis between ELISA and neutralization titers, significance (p), Spearman coefficients (rs), and linear plots were calculated using Prism 9.0 (Graphpad).
Flow cytometry. B-cell analysis using flow cytometry was carried out as described (51). 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     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 (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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint 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.  represents the Rs4081 RBD + / SARS-2 RBD + population, Q3 represents the SARS-2 RBD + population, and Q4 represents the RBDpopulation. (C-E) Percent single-positive (SP) and double-positive (DP) cells for the indicated groups. Significant differences between groups linked . 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 December 1, 2020. . 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 December 1, 2020. ; 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 December 1, 2020. ; 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 December 1, 2020. ; 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 December 1, 2020. ; 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 December 1, 2020. ; 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 December 1, 2020. ; https://doi.org/10.1101/2020.11.17.387092 doi: bioRxiv preprint