Designed mosaic nanoparticles enhance cross-reactive immune responses in mice

Abstract

1Using computational methods, we designed 60-mer nanoparticles displaying SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs) by (i) creating RBD sequences with 6 mutations in the SARS-COV-2 WA1 RBD that were predicted to retain proper folding and abrogate antibody responses to variable epitopes (mosaic-2_COMs; mosaic-5_COM), and (ii) selecting 7 natural sarbecovirus RBDs (mosaic-7_COM). These antigens were compared with mosaic-8b, which elicits cross-reactive antibodies and protects from sarbecovirus challenges in animals. Immunizations in naïve and COVID-19 pre-vaccinated mice revealed that mosaic-7_COM elicited higher binding and neutralization titers than mosaic-8b and related antigens. Deep mutational scanning showed that mosaic-7_COM targeted conserved RBD epitopes. Mosaic-2_COMs and mosaic-5_COM elicited higher titers than homotypic SARS-CoV-2 Beta RBD-nanoparticles and increased potencies against some SARS-CoV-2 variants than mosaic-7_COM. However, mosaic-7_COM elicited more potent responses against zoonotic sarbecoviruses and highly mutated Omicrons. These results support using mosaic-7_COM to protect against highly mutated SARS-CoV-2 variants and zoonotic sarbecoviruses with spillover potential.

Keywords: RBD; SARS-CoV-2; antibody; computational methods; nanoparticle; protein design; sarbecovirus; vaccination.

Figure 1.. Overview of the design process.

(A) Structures of representative class 1 (C102, PDB 7K8M), class 2 (C144, PDB 7K90), class 3 (S309, PDB 7JMX), and class 4 (CR3022, PDB 6W41) antibodies bound to the WA1 SARS-CoV-2 RBD, and the structure of the WA1 RBD (PDB 6W41) colored based on conservation scores calculated using the ConSurf database. (B) Overview of mosaic-2_COM and mosaic-5_COM RBD-NP designs. Starting from the WA1 RBD, computational analysis and machine learning models were used to calculate properties of potential RBD immunogens based on expression, antibody binding, and solubility. A set of selected RBDs were further filtered based on expression and binding measurements and used to construct the mosaic-2_COM and mosaic-5_COM RBD-NPs. (C) Overview of designing mosaic-7_COM. A set of 8 RBDs were selected from naturally occurring zoonotic sarbecovirus RBDs to maximize (i) sequence diversity and (ii) binding to class 3 and 4 but not class 1 and 2 RBD epitopes (RBD epitopes defined as described. The 8 selected RBDs were further filtered based on experimentally determined properties (see text), and the 7 remaining RBDs were used for mosaic-7_COM.

Figure 2.. Overview of computational methods.

(A) Architecture of the neural network used to predict RBD expression. The input is an expression matrix, which is the element-wise product (multiplication of entries at the same positions) of the one-hot encoded sequence (each residue is represented as a 20-dimensional vector with entries of 1 for the matching amino acid and 0 for other amino acids) and the matrix of single-mutation expression changes. This is processed through a convolutional neural network to produce the predicted change in expression as an output. (B) ~800,000 possible RBD sequences are screened for predicted expression relative to the WA1 RBD using a threshold value of −0.2 logMFI. Rejected RBD pairs are in blue and selected pairs are in red. (C) ~100,000 RBD sequences that passed predicted expression screening and further screened for solubility based on a change in aggregation score relative to WA1 calculated using Aggrescan. Rejected RBD pairs are in blue and selected pairs are in red. (D) The distribution of total mutational entropy over sets of 10 RBDs, and the set selected for experimental testing is the one with maximum entropy indicated by the red line. (E) Mean escape against class 1 and 2 anti-RBD antibodies and the mean escape against class 3 and 4 anti-RBD antibodies for naturally occurring sarbecoviruses. Rejected RBDs are in blue and selected RBDs are in red.

Figure 3.. Designed SARS-CoV-2 RBDs and sarbecovirus RBDs exhibit desired properties.

(A, B) HiLoad 16/600 Superdex 200 SEC profiles of designed RBDs (A) and sarbecovirus RBDs (B). RBD3 and RBD8 exhibited sub-optimal expression, indicated by no signal for an RBD monomer (RBD3) or a peak in the void volume (RBD8). (C, D) Fold reduction of selected monoclonal anti-RBD antibodies (mAbs) or a human ACE2-Fc construct (hACE2) to designed SARS-CoV-2 RBDs (C) and sarbecovirus RBDs (D) compared with binding to WA1 RBD. (E) Superose 6 Increase 10/300 SEC profiles after SpyTagged RBDs were conjugated to SpyCatcher-mi3 showing peaks for RBD-NPs and free RBDs. (F) SDS-PAGE for each RBD-NP after pooling appropriate SEC fractions.

Figure 4.. Computationally designed mosaic RBD-NPs elicit cross-reactive antibody binding and neutralization responses in immunized mice.

The mean of mean titers is compared in panels B and C by Tukey’s multiple comparison test with the Geisser-Greenhouse correction calculated using GraphPad Prism, with pairings by viral strain. Significant differences between immunized groups linked by horizontal lines are indicated by asterisks: p<0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****. (A) Left: Schematic of immunization regimen. Middle: numbers and colors used for sarbecovirus strains within clades throughout the figure. Right: Colors and symbols (squares) used to identify immunizations (colors) and matched (filled in) versus mismatched (not filled in) viral strains. (B) Left: ELISA binding titers at day 56 for serum IgG binding to RBDs, represented as mean ED₅₀ values. Middle left: Means of ELISA binding titers for each immunization. Middle right: Means of ELISA binding titers for each immunization against only SARS-CoV-2 variant RBDs. Right: Means of ELISA binding titers for each immunization against zoonotic sarbecovirus RBDs. Each circle represents the mean serum IgG binding titer against matched (solid circles) and mismatched (open circles) RBDs. (C) Left: ELISA binding titers at day 84 for serum IgG binding to RBDs, represented as mean ED₅₀ values. Middle left: Means of ELISA binding titers for each immunization. Middle right: Means of ELISA binding titers for each immunization against only SARS-CoV-2 variant RBDs. Right: Means of ELISA binding titers for each immunization against zoonotic sarbecovirus RBDs. Each circle represents the mean serum IgG binding titer against matched (solid circles) and mismatched (open circles) RBDs. (D) Left: Neutralization titers at day 84 for serum IgG neutralization of pseudoviruses derived from the virus strains in panel A, represented as mean ID₅₀ values. Middle left: Means of all neutralization titers for each immunization. Each circle represents the mean neutralization titer against matched (Khosta-2 for mosaic-7_COM; solid circle) and mismatched (open circles) pseudoviruses. Middle right and right: Neutralization titers against XBB.1.5 and Khosta-2. Each circle represents a neutralization titer from an individual mouse serum sample.

Figure 5.. Differences in epitope targeting of antibodies elicited in mice immunized with mosaic and homotypic RBD-NPs.

(A) DMS line plots for analyses of sera from mice that were immunized as shown in Figure 4A. DMS was conducted using a SARS-CoV-2 Beta RBD library. The x-axis shows RBD residue positions, and the y-axis shows the total sum of Ab escape for all mutations at a given site, with larger values indicating greater Ab escape. Each faint line represents a single antiserum with heavy lines indicating the average of n=4 sera for each group. Lines are colored differently based on RBD epitopes from the 4 major classes (color definitions are shown in the legend below this panel; gray for residues not assigned to an epitope). (B) Mean site-total antibody escape for a SARS-CoV-2 Beta RBD library determined using sera from mice immunized with the indicated immunogens mapped to the surface of the WA1 RBD (PDB 6M0J). White indicates no escape and dark pink indicates sites with the most escape (residue numbers are denoted with epitope-specific colors as denoted by the legend between panels A and B).

Figure 6.. Mosaic-7_COM immunization in pre-vaccinated mice elicits superior cross-reactive antibody responses.

The mean of mean titers is compared in panels C and E by Tukey’s multiple comparison test with the Geisser-Greenhouse correction calculated using GraphPad Prism, with pairings by viral strain. Significant differences between immunized groups linked by horizontal lines are indicated by asterisks: p<0.05 = *, p<0.01 = **, p<0.001 = ***, p<0.0001 = ****. Binding responses at day 0 (before NP or other vaccine immunizations) showed significant differences across cohorts in titers elicited by the pre-vaccinations. To account for different mean responses at day 0 between cohorts, we applied baseline corrections (see Methods). Uncorrected binding data for panels B and C are shown in Figure S4B-C. (A) Left: Schematic of vaccination regimen. Mice were pre-vaccinated with mRNA-LNP encoding WA1 spike and bivalent WA1/BA.5 prior to prime and boost immunizations with RBD-NPs at day 0 and day 28 or an additional WA1/BA.5 mRNA-LNP immunization at day 0. Middle: Colors and symbols (squares) used to identify immunizations (colors) and matched (filled in), mismatched (not filled in), or matched to pre-vaccination (half-filled in) viral strains (squares). Right: numbers and colors used for sarbecovirus strains within clades throughout the figure. (B) Log₁₀ mean fold change in ELISA ED₅₀ binding titers from day 0 at the indicated days after priming with the indicated immunogens against spike or RBD proteins from the indicated sarbecovirus strains (numbers and color coding as in panel A). (C) Log₁₀ means of fold change in ELISA titers for each type of immunization at the indicated days. Each circle represents the log₁₀ mean fold change in ED₅₀ titers from mice against a single viral strain of sera from mice that were immunized with the specified immunogen (solid circles=matched; open circles=mismatched; colors for different strains defined in panel A). (D) Mean change in neutralization ID₅₀ titers from day 0 at the indicated days against the indicated sarbecovirus strains (numbers and color coding as in panel A). (E) Means of all neutralization titers for each type of immunization at the indicated days. Each circle represents the mean neutralization IC₅₀ titer against a single viral strain of sera from mice that were immunized with the specified immunogen (solid circles=matched; open circles=mismatched; colors for different strains defined in panel A).