Polypeptides, compositions, and their use for treating or limiting the onset of infectious diseases

Abstract

Disclosed herein are polypeptides comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-84, 138-146, and 167-184, nanoparticles thereof, related nanoparticle compositions, and their uses for treating or limiting the onset of infectious diseases.

Description

[Technical Field]

[0001] cross reference This application claims priority to U.S. Provisional Application No. 62 / 977,036 filed on 14 February 2020, No. 63 / 046,159 filed on 30 June 2020, and No. 63 / 064,235 filed on 11 August 2020, which are incorporated herein by reference in their entirety.

[0002] Description of funding provided by the federal government. This invention was made with government support under grant numbers HHSN272201700059C and R01GM120553, awarded by the National Institutes of Health (NIH). The government has certain rights to this invention.

[0003] Description regarding sequence listings A computer-readable sequence listing is submitted electronically with this application and is incorporated in its entirety by reference. The sequence listing is contained in a file named "20-1008-PCT_SeqList_ST25.txt" created on February 11, 2021, and has a size of 1077kb. [Background technology]

[0004] The emergence of a previously unknown virus in Wuhan, China, has led to the ongoing COVID-19 pandemic, which as of August 6, 2020, has caused more than 18.7 million infections and over 700,000 deaths (WHO). By January 2020, rapid virus isolation and sequencing revealed that the newly emerged zoonotic pathogen is a coronavirus closely related to SARS-CoV, and it was thus named SARS-CoV-2. SARS-CoV-2 is thought to be of bat origin based on the isolation of the closely related RaTG13 virus from Rhinolophus affinis and the identification of the RmYN02 genomic sequence by metagenomic analysis of Rhinolophus malayanus, both from Yunnan Province, China.

Summary of the Invention

[0005] In one aspect, the present disclosure provides a polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-84, 138-146, and 167-184, wherein X1 is absent or is an amino acid linker, the residues within parentheses are optional and may or may not be present, and some or all of the optional residues may be absent. In various specific embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and 142-151, an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, or an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or 5. In another embodiment, the present disclosure provides nanoparticles comprising a plurality of such polypeptides.

[0006] In another aspect, the present disclosure provides (a) a plurality of first assemblies, each first assembly comprising a plurality of identical first proteins, and (b) A plurality of second aggregates, each second aggregate comprising a plurality of second aggregates containing a plurality of second proteins, and a nanoparticle comprising The amino acid sequence of the first protein differs from that of the second protein, where multiple first aggregates interact non-covalently with multiple second aggregates to form nanoparticles, the nanoparticles presenting an immunogenic portion of the SARS-CoV-2 antigen or its variant or homolog present on the surface of at least one second protein. In one embodiment, the second protein comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 85-124 or 185-193, or SEQ ID NOs. 85-88, where X1 of at least one second protein comprises an immunogenic portion of the SARS-CoV-2 antigen or its variant or homolog, and X2 is absent or an amino acid linker, with the residues in parentheses being optional. In another embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of X1 of the second protein includes an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity, or a variant or homolog thereof, with respect to the SARS-CoV-2 derived spike (S) protein extracellular domain (ECD) amino acid sequence, S1 subunit amino acid sequence, S2 subunit amino acid sequence, S1 receptor-binding domain (RBD) amino acid sequence, and / or domain (NTD) amino acid sequence to the N-terminus. In a further embodiment, at least 20%, 30%, 40%, 50%, 60%, 65%, 70%, 80%, 90%, or 100% of X1 of the second protein contains at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to amino acid sequences selected from the group consisting of SEQ ID NOs. 125-137.In a further embodiment, the first protein comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequences selected from the group consisting of SEQ ID NOs: 152 - 159, where the residues in parentheses are optional, may be present, or some or all of any residues may not be present.

[0007] In various other aspects, the present disclosure provides compositions comprising a plurality of nanoparticles disclosed herein, nucleic acid molecules such as mRNA encoding the polypeptides disclosed herein, expression vectors comprising the nucleic acid molecules disclosed herein operably linked to appropriate control sequences, cells comprising the polypeptides, nanoparticles, compositions, nucleic acids, and / or expression vectors disclosed herein, and pharmaceutical compositions, kits, and vaccines comprising the polypeptides, nanoparticles, compositions, nucleic acids, expression vectors, and / or cells disclosed herein.

[0008] In another aspect, the present disclosure provides a method of restricting the treatment or onset of SARS-CoV-2 infection, comprising administering to a subject in need thereof an effective amount of a polypeptide, nanoparticle, composition, nucleic acid, pharmaceutical composition, or vaccine disclosed herein to restrict the treatment or onset of the infection.

Brief Description of the Drawings

[0009] [Figure 1A-H] It is a diagram of the design, in vitro assembly, and characterization of a SARS-CoV-2 RBD nanoparticle immunogen. [Figure 1A] It is a diagram representing the molecular surface of the prefusion conformation (PDB 6VYB) of the SARS-CoV-2 S-2P trimer. Each protomer is clearly colored, and the N-linked glycan is shown in dark blue (the glycan at position N343 was modeled based on PDB 6WPS, and the receptor-binding motif (RBM) was modeled from PDB 6M0J). A single open RBD is boxed. [Figure 1B]This figure shows the molecular surface of SARS-CoV-2S RBD, including N-linked glycans at positions 331 and 343. The ACE2 receptor binding site or RBM is indicated by a black outline. [Figure 1C] This is a structural model of the trimer RBD-I53-50A (light blue RBD and light gray I53-50A) and pentamer I53-50B (orange) components. When mixed in vitro, 20 trimer components and 12 pentamer components assemble to form an icosahedral symmetric nanoparticle immunogen. Each nanoparticle represents 60 copies of RBD. [Figure 1D] This is a structural model of the RBD-12GS-I53-50 nanoparticle immunogen. For simplicity, the RBD antigen and the 12-residue linker are shown in a unidirectional orientation, although these regions are expected to be flexible relative to the I53-50 nanoparticle scaffold. [Figure 1E] This shows the dynamic light scattering (DLS) of RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles compared to unmodified I53-50 nanoparticles. [Figure 1F] Representative electron micrographs of negatively stained RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles. Samples were imaged after one freeze / thaw cycle. Scale bar, 100 nm. [Figure 1G] Hydrogen / deuterium exchange mass spectrometry of monomer RBD and trimer RBD-8GS-I53-50A components, represented herein as a butterfly plot, confirms the conservation of the RBD stereochemistry, including the epitope recognized by known neutralization Abs. In the plot, each point along the horizontal sequence axis represents a peptide whose deuterium incorporation was monitored from 3 seconds to 20 hours. The error range shown in the butterfly plot indicates the standard deviation from the replicates of the two experiments. The difference plot below shows that monomer RBD and RBD-8GS-I53-50A are substantially identical in the local structural sequence of the entire RBD. [Figure 1H]This is a pie chart summarizing the glycan populations present at N-linked glycosylation sites N331 and N343 of five protein samples: monomer RBD, S-2P trimer, and RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50A trimer components. The majority of complex glycans at both sites are fucosylated, with a small population of afucosylated glycans indicated by dashed lines. Oligo and oligomannoses are also shown. [Figure 2A-B] This is a diagram showing the antigenic characterization of SARS-CoV-2 RBD-I53-50 nanoparticle immunogen. [Figure 2A] This is a biolayer interferometry image of immobilized mACE2-Fc, CR3022mAb, and S309mAb conjugated to RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles exhibiting RBD antigen at 50% or 100% valence. The monomer SARS-CoV-2RBD was included in each experiment for reference. [Figure 2B] The binding signals at 880 seconds, near the end of the association phase, are plotted for each experiment in panel (A), allowing for comparison of the binding signals obtained from each nanoparticle. [Figure 3A-E] This diagram shows the physical and antigenic stability of RBD nanoparticle immunogens and S-2P trimers. [Figure 3A] This figure shows chemical denaturation by guanidine hydrochloride. The tertiary structure of the protein was monitored using the intrinsic tryptophan fluorescence emission ratio at 350 / 320 nm. Major transitions are indicated by shaded regions. Representative data from one of three independent experiments is shown. [Figure 3B] This is a summary of SDS-PAGE and nsEM stability data over a four-week period. SDS-PAGE showed no detectable degradation in any sample. nsEM revealed substantial unfolding of the S-2P trimer at 2–8°C after a three-day incubation and at 22–27°C after four weeks. N / A: Not evaluated. [Figure 3C]This is a summary of antigenic data over a four-week period. Antigens were stored at various temperatures and then analyzed for mACE2-Fc (solid line) and CR3022mAb (dashed line) binding by biolayer interferometry. The plotted values ​​represent the signal amplitude near the end of the association phase, normalized for the corresponding <-70°C sample at each time point. [Figure 3D] This is a summary of UV / vis stability data over four weeks. The ratio of absorbance at 320 / 280 nm is plotted as a measure of particle scattering. Only the S-2P trimer and RBD-12GS-I53-50 nanoparticles showed increased scattering only at ambient temperature. [Figure 3E] DLS of RBD-12GS-I53-50 nanoparticles showed monodisperse species without detectable aggregates at all temperatures and time points. Data in panels B-E are from a single 4-week real-time stability test. [Figure 4A-D] RBD-I53-50 nanoparticle immunogen induces a potent antibody response in BALB / c and human immunorepertory mice. [Figure 4A] This is the anti-S binding titer of BALB / c mice after priming (week 2), as measured by ELISA. [Figure 4B] This chart shows the anti-S binding titer of BALB / c mice after boosting (week 5) as measured by ELISA. Each symbol represents an individual animal, and the geometric mean of each group is shown by the horizontal line. The dotted line represents the detection limit of the assay. Data sources: 8GS, RBD-8GS-I53-50; 12GS, RBD-12GS-I53-50; 16GS, RBD-16GS-I53-50; HCS, human convalescent serum. The inset shows the study timeline. The immunization experiment was repeated twice, and representative data are shown. [Figure 4C] The anti-S binding titer of Kymab Darwin™ mice after priming (week 2), which is the unreconstituted human antibody variable and transgenic constant-region germ cell repertoire, measured by ELISA and plotted as shown in (A). [Figure 4D]The anti-S binding titer of Kymab Darwin™ mice after boosting (week 5) is shown, which is the transgenicity of the unreconstituted human antibody variable and constant-region germ cell repertoire, measured by ELISA and plotted as shown in (A). The inset shows the study timeline. Immunization experiments were performed once. [Figure 5A-H] RBD-I53-50 nanoparticle immunogen induces a potent and protective neutralizing antibody response. [Figure 5A] This is the post-priming serum pseudotyped virus neutralizing titer from mice immunized with monomer RBD, S-2P trimer, or RBD-I53-50 nanoparticles. [Figure 5B] These are the boosted serum pseudotyped virus neutralizing titers from mice immunized with monomer RBD, S-2P trimer, or RBD-I53-50 nanoparticles. Each circle represents the inter-animal IC50. The geometric mean of each group is shown by the horizontal line. The detection limit is shown by the gray dotted line. Animal experiments were performed twice, and representative data from duplicate measurements are shown. [Figure 5C] This is the post-prime serum live virus neutralizing titer from mice immunized as described in (A). [Figure 5D] This is the boosted serum live virus neutralizing titer from immunized mice, as explained in (A). [Figure 5E] This is the serum pseudotype virus neutralizing titer from immunized, primed Kymab Darwin™ mice, as explained in (A). [Figure 5F] This is the serum pseudotyped virus neutralizing titer from immunized, boosted Kymab Darwin® mice, as described in (A). Animal experiments were performed once, and neutralization assays were performed at least twice. [Figure 5G] This figure shows the results after 7 weeks of boosting, with 8 BALB / c mice per group being challenged with SARS-CoV-2MA. [Figure 5H]This figure shows 8 BALB / c mice per group challenged with SARS-CoV-2MA 7 weeks after boosting. Two days after challenge, viral titers were evaluated in lung tissue (G) and nasal turbinates (H). The detection limit is indicated by the gray dotted line. [Figure 6A-J] RBD nanoparticle vaccines induce robust B-cell responses and antibodies targeting multiple epitopes in mice and non-human primates. [Figure 6A] The number of RBD+ B cells (B220+CD3-CD138-) was detected in each immunization group. [Figure 6B] The number of RBD+GC precursors and B cells (CD38+ / -GL7+) was detected in each immunization group. [Figure 6C] This refers to the frequency of RBD+GC precursors and B cells (CD38+ / -GL7+). [Figure 6D] The frequencies of IgD+, IgM+, or class-switched (IgM-IgD-;swIg+) RBD+GC precursors and B cells were determined. (A-D) N=6 in two experiments for each group. Statistical significance was determined by one-way ANOVA, and Tukey's multiple comparison test was performed for any group with a p-value less than 0.05. Significance is indicated by an asterisk: *p<0.05, ****p<0.0001. [Figure 6E] Figure 4D shows the ratio of S-2P ELISA binding titer (5D) to pseudotyped virus neutralizing titer (5F) after boosting (week 5) in Kymab Darwin™ mice. The ratio is [GMT(EC50) of 5 mice]:[GMT(IC50) of 5 mice], or EC50:IC50, for all HCSs tested. A lower value indicates a higher quality response. [Figure 6F] The S-2P ELISA binding titer after boosting (week 5) (Figure 4B) is shown as the ratio of the pseudotyped virus (Figure 5B) or live virus (Figure 5D) neutralizing titer in BALB / c mice. The ratio is [GMT (EC50) of 10 mice]:[GMT (IC50) of 10 mice] or EC50:IC50 for all HCS tested. [Figure 6G]This is a SARS-CoV-2 RBD conjugated with monomers ACE2, CR3022Fab, and S309Fab. [Figure 6H] This involves determining vaccine-induced Ab epitope specificity using competitive BLI. [Figure 6I] This involves determining vaccine-induced Ab epitope specificity using competitive BLI. [Figure 6J] This study determined vaccine-induced Ab epitope specificity using competitive BLI. A dilution series of polyclonal NHP Fab was pre-incubated with RBD using BLI chips. Polyclonal Fab concentrations were maintained by adding competition at each dilution point. The 1:3 dilution series of polyclonal Fab is represented by a range of colors from dark to light, with the dark gray line representing competition loaded into apo-RBD (no competition). Competition is with (H) 200 nM ACE2, (I) 400 nM CR3022, or (J) 20 nM S309. [Figure 7A-E] This involves determining additional properties of RBD nanoparticle immunogens. [Figure 7A] Superose® 6 Increase is a size exclusion chromatography method for RBD-I53-50 nanoparticles, unmodified I53-50 nanoparticles, and trimer RBD-I53-50A components at 10 / 300GL. [Figure 7B] This is SDS-PAGE of SEC-purified RBD-I53-50 nanoparticles under reducing and non-reducing conditions before and after a single freeze / thaw cycle. [Figure 7C] Dynamic light scattering of RBD-I53-50 nanoparticles before and after a single freeze / thaw cycle shows monodisperse nanoparticles without detectable aggregates in each sample. [Figure 7D] In this specification, hydrogen / deuterium exchange mass spectrometry, represented as a heatmap, reveals the structural accessibility and dynamics of RBD (PDB6W41). Color codes indicate deuterium uptake levels. The uptake patterns of monomer RBD and RBD-8GS-I53-50A are indistinguishable and are displayed in a single heatmap at each time point. [Figure 7E]The upper bar graph shows similar glycan profiles at N-linked glycosylation sites N331 and N343 for five protein samples: monomer RBD, the S-2P trimer, and the components of the RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50A trimer. Below is a comprehensive glycan profiling for other N-linked glycosylation sites other than N331 and N343 found in the S-2P trimer. The axes of each bar graph are scaled from 0 to 80%. Oligomannoses with 9 to 5 mannose residues, M9 to M5, are colored dark gray. Hybrid and FHybrid, with or without fucosylation, are gray. Subtypes of complex types, shown in light gray, are classified based on antenna number and fucosylation. [Figure 8A-B] This involves determining the hACE2 and CR3022Fab affinity using biolayer interferometry. [Figure 8A] This study analyzes the monomer hACE2 binding to immobilized monomer RBD and trimers RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50A. [Figure 8B] This study analyzes the binding of CR3022Fab to the immobilized monomer RBD and its trimers RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50A components. Affinity constants (Table 5) were determined by globally fitting kinetic data from six analyte concentrations to a 1:1 binding model. [Figure 9A-D] This involves characterizing partially valence RBD nanoparticles. [Figure 9A] Representative electron micrographs of negatively stained RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles displaying RBD at 50% valence. Samples were imaged after one freeze / thaw cycle. Scale bar, 100 nm. [Figure 9B]This is an SDS-PAGE of purified RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles displaying RBD at 50% valence. Both subunits with RBD and unmodified I53-50A subunits are displayed on the gel. [Figure 9C] Dynamic light scattering (DLS) of 50% valence RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles before and after freezing / thawing. No aggregates or unassembled components were observed. [Figure 9D] These are the UV / vis absorption spectra of RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles with a valence of 50%. As can be seen from the low absorbance at 320 nm, the turbidity of the sample is low. [Figure 10A-E] This is the stability data for day 28. [Figure 10A] SDS-PAGE of purified monomer RBD, S-2P trimer, RBD-I53-50A component, and RBD-12GS-I53-50 nanoparticles under reducing and non-reducing conditions. No immunogen degradation was observed after 4 weeks of incubation at any of the temperatures analyzed. [Figure 10B] This study analyzed the binding of mACE2-Fc and CR3022IgG to monomer RBD, RBD-I53-50A trimer components, and RBD-12GS-I53-50 nanoparticles by BLI after 4 weeks of incubation at three different temperatures. Monomer RBD was used as a reference standard for the nanoparticle components and nanoparticle BLI experiments. RBD-12GS-I53-50 nanoparticles lost minimal binding at high temperatures after 4 weeks. The remaining antigens did not lose mACE2-Fc or CR3022IgG binding during the course of the study. [Figure 10C]UV / vis spectroscopy showed minimal absorbance near UV, suggesting the absence of aggregation / particles after 4 weeks of incubation at three temperatures, with the exception of the S-2P trimer, which showed significant absorbance at approximately 320 nm at ambient temperature. RBD-12GS-I53-50 nanoparticle samples at 22–27°C showed a similar peak around 320 nm at several previous time points (see Supplementary Item 2). [Figure 10D] These are nsEM images of RBD-12GS-I53-50 nanoparticles (top) and S-2P trimers (bottom) after incubation at three different temperatures for four weeks. Undamaged monodisperse nanoparticles were observed at all temperatures, and no degradation or aggregation was observed. The S-2P trimers remained fully folded in samples incubated below -70°C and between 22 and 27°C, but were unfolded in samples incubated at 2 to 8°C. Scale bars: RBD-12GS-I53-50, 100nm; S-2P, 50nm. [Figure 10E] DLS of RBD-12GS-I53-50 nanoparticles after 4 weeks of incubation at three different temperatures. No aggregation was observed at any of the temperatures. [Figure 11] These are subclasses of vaccine-induced Ab and anti-scaffold antibody titers. In BALB / c mice, the images show (top) trimer I53-50A component, (center) pentamer I53-50B component, and (bottom) vaccine-induced IgG levels specific to assembled I53-50 nanoparticles, after priming (left) and after boosting (right). [Figure 12A-D] This concerns the B-cell gating strategy and persistence of the vaccine-induced immune response. [Figure 12A]This is a representative gating strategy for evaluating RBD-specific B cells, germinal center (GC) precursors and B cells (CD38+ / -GL7+), and B cell isotypes. The top row shows a gating strategy for measuring the number of viable non-doublet B cells. These cells were further analyzed as shown in the middle and bottom rows. The middle row shows representative data from mice immunized with monomer RBD formulated with AddaVax®. RBD+CD38+ / -GL7+ cells that did not bind to the decoy were counted as antigen-specific GC precursor cells and B cells. The bottom row shows representative data from mice immunized with RBD-12GS-I53-50 nanoparticles formulated with AddaVax®. GC precursors and B cells were further analyzed to characterize B cell receptor isotypes. [Figure 12B] This is the level of (B)S-specific IgG in serum, recovered after a 20 (RBD-16GS-I53-50) or 24 (monomer RBD, S-2P, RBD-8GS-I53-50, and RBD-12GS-I53-50) week boost. [Figure 12C] This refers to the level of pseudotyped virus neutralization in serum recovered after a 20 (RBD-16GS-I53-50) or 24 (monomer RBD, S-2P, RBD-8GS-I53-50, and RBD-12GS-I53-50) week boost. Serum was collected from two animals in each group that were not challenged with MA-SARS-CoV-2. [Figure 12D] This is the number of S-2P-specific Ab-secreting cells in the bone marrow of BALB / c mice immunized with S-2P trimers or RBD-16GS-I53-50 nanoparticles, as measured by ELISpot. Cells were harvested 17 weeks after boosting (see inset in panel B). Animal experiments were performed once. Statistical significance was determined by a two-sided, unpaired t-test *, p=0.02. [Modes for carrying out the invention]

[0010] All cited references are incorporated herein in their entirety by reference. Unless otherwise stated, the techniques used in this application can be found in any of several well-known documents. For example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, CA), ´´Guide to Protein Purification´´ in Methods in Enzymology (MP Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, CA), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (RIFreshney. 1987. Liss, Inc. New York, NY), Gene Transfer and Expression Protocols, pp. 109-128, ed. EJ Murray, The Humana Press. Inc., Clifton, NJ), and the Ambion 1998 Catalog (Ambion, Austin, TX).

[0011] As used herein, the singular forms "a," "an," and "the" refer to multiple objects unless the context explicitly indicates otherwise.

[0012] Where used herein, "approximately" means + / - 5% of the parameter stated.

[0013] As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0014] All embodiments of any aspect of this disclosure can be used in combination unless the context expressly indicates otherwise.

[0015] Unless otherwise clearly required by context, throughout the modes for carrying out the invention and the claims, words such as “comprise,” “comprising,” etc., should be interpreted in an inclusive or comprehensive sense, as opposed to an exclusive sense, that is, “including but not limited to.” Words used in the singular or plural also include the plural and singular, respectively. Furthermore, the words “herein,” “above,” and “below,” and words of similar meaning, when used in this application, refer to the application as a whole and not to any particular part of this application.

[0016] In a first aspect, the disclosure provides a polypeptide comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-84, 138-146, and 167-184, wherein X1 is absent or an amino acid linker, and the residues in parentheses are optional and may be present, or some or all of the optional residues may be absent.

[0017] As shown in the following examples, polypeptides of this embodiment can be used to generate self-assembling protein nanoparticle immunogens that induce a potent, protective antibody response against SARS-CoV-2. The nanoparticle vaccine induces neutralizing antibody titers approximately 10-fold higher than those of the pre-fusion-stabilized S-external domain trimer, even at one-fifth of a lower dose. Antibodies induced by the nanoparticle immunogen are not susceptible to easily evading mutations because they target multiple different epitopes and exhibit a significantly lower binding-to-neutralization ratio than convalescent human serum, suggesting that the risk of vaccine-related enhanced respiratory illness may be minimized.

[0018] The amino acid sequences of exemplary polypeptides of this embodiment of the present disclosure are provided below. [Table 1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 Table 1-22 Table 1-23 Table 1-24 Table 1-25 Table 1-26 Table 1-27 Table 1-28 Table 1-29 Table 1-30 Table 1-31 Table 1-32 Table 1-33 Table 1-34 Table 1-35

[0019] HexaPro-12GS-He-I5350A*-His:

[0020] >HexaPro-FO-12GS-He-I5350A*-His:

[0021] >HexaPro-delHR2-12GS-He-I5350A*-His:

[0022] >HexaPro-delHR2-FO-12GS-He-I5350A*-His:

[0023] RBD-noRpk-50A variant

[0024] >SARS-CoV-2 RBD_N501Y_16GS-he-I53-50A*-His(UK): (MGILPSPGMPALLSLVSLLSVLLMGCVAETGT)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSGGSGGSGSEKAAKAEEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHHH)(SEQ ID NO: 142)

[0025] >SARS-CoV-2 RBD_K417N_E484K_N501Y_16GS-he-I53-50A*-His(South Africa) (MGILPSPGMPALLSLVSLLSVLLMGCVAETGT)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSGGSGGSGSEKAAKAEEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHHH)(SEQ ID NO: 143)

[0026] >SARS-CoV-2_RBD-noRpk_16GS_I53-50A*_Brazil-ver_K417T_E484K_N501Y(Brazil): (MGILPSPGMPALLSLVSLLSVLLMGCVAETGT)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG TIADYNYKLPDDFTGCVIAWNSNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSG SGGSGGSGSKAAKAEEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEK GVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHHHH)

[0027] >SARS-CoV-2_RBD-noRpk_16GS_I53-50A*_E484K: (MGILPSPGMPALLSLVSLLSVLLMGCVAETGT)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSGGSGGSGSEKAAKAEEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHHH)(SEQ ID NO: 145)

[0028] >SARS-CoV-2_RBD-noRpk_16GS_I53-50A*_L452R: (MGILPSPGMPALLSLVSLLSVLLMGCVAETGT)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSGGSGGSGSEKAAKAEEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHHH)(SEQ ID NO: 146)

[0029] >SARS-CoV-2 RBD_N501Y_16GS-he-I53-50A*-His(UK): (MGILPSPGMPALLSLVSLLSVLLMGCVA)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSGGSGGSGSEKAAKAEEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHHH)(SEQ ID NO: 147)

[0030] >SARS-CoV-2 RBD_K417N_E484K_N501Y_16GS-he-I53-50A*-His(South Africa) (MGILPSPGMPALLSLVSLLSVLLMGCVA)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSG GSGGSGSEKAAKAAEAAR)KMEELFKKHKIVAVLRANSVEEAIKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHH)

[0031] >SARS-CoV-2_RBD-noRpk_16GS_I53-50A*_Brazil-ver_K417T_E484K_N501Y(Brazil): (MGILPSPGMPALLSLVSLLSVLLMGCVA)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSG GSGGSGSEKAAKAAEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHH)

[0032] >SARS-CoV-2_RBD-noRpk_16GS_I53-50A*_E484K: (MGILPSPGMPALLSLVSLLSVLLMGCVA)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSG GSGGSGSEKAAKAAEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHH)

[0033] >SARS-CoV-2_RBD-noRpk_16GS_I53-50A*_L452R: (MGILPSPGMPALLSLVSLLSVLLMGCVA)RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST(GGSGGSGSG GSGGSGSEKAAKAEEAAR)KMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(GGSHHHHHHH)(Sequence ID 151)

[0034] In various embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and 142-151. In various other embodiments, the polypeptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8, or the group consisting of SEQ ID NOs: 1-4, SEQ ID NOs: 5-8, or the group consisting of SEQ ID NOs: 1 and 5, and exemplary embodiments are provided in the following examples.

[0035] As used throughout this application, the term “polypeptide” is used in its broadest sense to refer to a sequence of subunit D or L amino acids, including regular and non-regular amino acids. Polypeptides described herein can be chemically synthesized or recombinantly expressed. Polypeptides can be conjugated to other compounds, such as by pegylation, HESization, PASization, glycosylation, etc., to promote an increase in half-life in vivo, or can be generated as Fc fusions or in deimmunized mutants. Such conjugations can be covalent or non-covalent, as will be understood by those skilled in the art.

[0036] In a second aspect, the Disclosure provides nanoparticles comprising multiple polypeptides in any embodiment or combination of embodiments of the First Aspect of the Disclosure. In this aspect, multiple (2, 3, 4, 5, 10, 20, 25, 50, 60, 100, or more) polypeptides of the First Aspect of the Disclosure are present in any suitable nanoparticle.

[0037] Nanoparticles of any embodiment or aspect of this disclosure may be of any size suitable for the intended application, including but not limited to a diameter of about 10 nm to about 100 nm.

[0038] In a third aspect, this disclosure is: (a) A plurality of first aggregates, each first aggregate comprising a plurality of identical first proteins, (b) A plurality of second assemblies, each second assembly comprising a plurality of second assemblies containing a plurality of second proteins, and providing nanoparticles comprising The amino acid sequence of the first protein is different from that of the second protein. Multiple first aggregates interact non-covalently with multiple second aggregates to form nanoparticles. The nanoparticles present, on their surface, the immunogenic portion of the SARS-CoV-2 antigen or its variant or homolog present in at least one second protein.

[0039] In this embodiment, the nanoparticles form a three-dimensional structure formed by non-covalent interactions between first and second assemblies. Multiple (2, 3, 4, 5, 6, or more) first polypeptides self-assemble to form a first assembly, and multiple (2, 3, 4, 5, 6, or more) second polypeptides self-assemble to form a second assembly. Non-covalent interactions of individual self-assembling proteins cause the first proteins to self-assemble into the first assembly and the second proteins to self-assemble into the second assembly. Then, multiple of these first and second assemblies self-assemble non-covalently across interfaces to produce nanoparticles. The number of first polypeptides in the first assembly may be the same as or different from the number of second polypeptides in the second assembly. The nanoparticles of this disclosure may have any shape and / or symmetry suitable for the intended application, including but not limited to tetrahedrons, octahedrons, icosahedrons, dodecahedrons, and their cleavage forms. In one exemplary embodiment, each first aggregate is a pentamer, and each second aggregate is a trimer.

[0040] The aggregation of the first and second assemblies onto the nanoparticles is not random, but is determined by various non-covalent interactions between the assemblies (e.g., hydrogen bonds, electrostatic, van der Waals, hydrophobic, etc.) (i.e., the cumulative effect of interactions between the first assemblies, between the second assemblies, and between the first and second assemblies). As a result, the nanoparticles of this disclosure contain symmetrically repeating, unnatural, non-covalent protein-protein interfaces that orient the first and second assemblies onto nanoparticles having a highly ordered structure. While the formation of the nanoparticles is due to the non-covalent interactions of the first and second assemblies, in some embodiments, once formed, the nanoparticles can be stabilized by covalent bonds between proteins in the first and second assemblies. Any suitable covalent bonds, including but not limited to disulfide bonds and isopeptide bonds, can be used.

[0041] The first and second proteins suitable for generating the aggregates of this disclosure may be of any length suitable for a given nanoparticle. The first and second proteins may have lengths of 30 to 250 amino acids.

[0042] In one embodiment, the second protein comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-124 or 185-193 (Table 2), wherein X1 of at least one second protein comprises an immunogenic portion of the SARS-CoV-2 antigen or its variant or homolog, and X2 is absent or an amino acid linker, with the residues in parentheses being optional. Optional residues may be present, or some (i.e., 1, 2, 3, 4, 5, 6, or more) or all of the optional residues may be absent. [Table 2] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0043] In various embodiments of this third aspect, the second protein comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 85-88, or the group consisting of SEQ ID NOs. 85-86, or SEQ ID NO. 85, and exemplary embodiments are provided in the following examples.

[0044] The nanoparticles of this third embodiment exhibit an immunogenic moiety of the SARS-CoV-2 antigen or its variant or homolog present on at least one second protein on their surface. In one embodiment, the immunogenic moiety of the SARS-CoV-2 antigen or its variant or homolog exists as a fusion protein with at least one second protein, which may be present on a single second protein in the nanoparticle (present as a single copy on the nanoparticle) or on multiple second proteins present in the nanoparticle. In various embodiments, the SARS-CoV-2 antigen or its variant or homolog is present in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second protein in the nanoparticle.

[0045] In these fusion proteins, the second protein can be directly bound to the SARS-CoV-2 antigen or its variant or homolog, or the second protein and the SARS-CoV-2 antigen or its variant or homolog can be linked using a linker. As used throughout this disclosure, the linker is a short (e.g., 2-30) amino acid sequence used to covalently bond two polypeptides. Any suitable linker sequence, including but not limited to those disclosed herein, can be used.

[0046] Any suitable SARS-CoV-2 antigen or its variant or homolog may be used. In one embodiment of this third aspect, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of X1 of the second protein comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity, or a variant or homolog thereof, with respect to the SARS-CoV-2 derived spike (S) protein extracellular domain (ECD) amino acid sequence, S1 subunit amino acid sequence, S2 subunit amino acid sequence, S1 receptor-binding domain (RBD) amino acid sequence, and / or N-terminal identity domain (NTD) amino acid sequence.

[0047] In various further embodiments, at least 20%, 30%, 40%, 50%, 60%, 65%, 70%, 80%, 90%, or 100% of X1 of the second protein contains at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical amino acid sequences to amino acid sequences selected from the group consisting of SEQ ID NOs. 125-137.

[0048] RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST (RBD)(Sequence ID 125)

[0049] ETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST(RBD)(Sequence No. 126)

[0050]

[0051]

[0052] ETGT It remains as a remnant after the cleavage of the signal peptide.

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] In a particular embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of X1 of the second protein contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 125, and SARS-CoV-2 RBD is provided as an exemplary embodiment in the following example. In various embodiments, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of X1 of the second protein contains mutations at 1, 2, 3, 4, 5, 6, 7, or 8 positions in sequence number 125, selected from the group consisting of K90N, K90T, G119S, Y126F, T151I, E157K, E157A, S167P, N174Y, and L125R, including the following naturally occurring mutations or combinations of mutations: N174Y (UK variant); K90N / E157K / N174Y (South African variant); K90N or T / E157K / N174Y (Brazilian variant); or This includes, but is not limited to, mutations containing one of the L125R (LA variants).

[0062] The numbering of amino acid residues in these naturally occurring variants is based on their positions in Sequence ID No. 125, but is generally described based on the residue numbers of the spike protein (i.e., K417 of spike = K90 of RBD; G446 of spike = G119 of RBD; L452 of spike = L125 of RBD; Y453 of spike = Y126 of RBD; T478 of spike = T151 of RBD; E484 of spike = E157 of RBD; S494 of spike = S167 of RBD; N501 of spike = N174 of RBD).

[0063] In various further embodiments, X1 in at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the second protein includes mutations at 1, 2, 3, 4, 5, 6, 7, or 8 positions for SEQ ID NO: 130 selected from the group consisting of L18F, T20N, P26S, deletion of residues 69-70, D80A, D138Y, R190S, D215G, K417N, K417T, G446S, L452R, Y453F, T478I, E484K, S494P, N501Y, A570D, D614G, H655Y, P681H, A701V, and T716L, including the following naturally occurring mutations or combinations of mutations: In some cases, N501Y (UK variant) may further include one, two, three, four, or five deletions of one or both of residues 69-70, A570D, D614G, P681H, and / or T716L; K417N / E484K / N501Y (South African variant) optionally further including one, two, three, four, or five L18F, D80A, D215G, D614G, and / or A701V; K417N or T / E484K / N501Y, which optionally further comprises one, two, three, four, or five of L18F, T20N, P26S, D138Y, R190S, D614G, and / or H655Y (Brazilian variant); or This includes, but is not limited to, mutations containing one of the L452R (LA variants).

[0064] As will be understood by those skilled in the art, X1 is identical to the amino acid sequence of SEQ ID NO: 125 (or any other disclosed antigen) by at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its amino acid sequence, and it may contain additional amino acids at its amino or carboxyl terminus. Therefore, for example, if X1 is identical to the amino acid sequence of SEQ ID NO: 125 by at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of its amino acid sequence, X1 may contain the amino acid sequence of SEQ ID NO: 126, and the amino acid sequence of SEQ ID NO: 126 contains additional amino acids at its N terminus compared to SEQ ID NO: 125.

[0065] In a further embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of X1 of the second protein contains all one, two, three, or four mutations of SEQ ID NO: 125, selected from the group consisting of K90N, K90T, E157K, and N174Y.

[0066] The multiple second assemblies may collectively contain a single SARS-CoV-2 antigen or two or more different SARS-CoV-2 antigens. In one embodiment, the multiple second assemblies collectively contain 2, 3, 4, 5, 6, 7, 8, or more different SARS-CoV-2 antigens. In one exemplary such embodiment, the multiple second assemblies collectively contain 2, 3, 4, 5, 6, 7, 8, or more polypeptides, each containing one amino acid sequence from SEQ ID NOs: 1 to 84.

[0067] In one embodiment, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of X1 in the second protein contains the amino acid sequence of SEQ ID NO: 125. In another embodiment, X1 in 100% of the second protein contains the amino acid sequence of SEQ ID NO: 125, and all of the second protein is identical.

[0068] In a further embodiment, all second assemblies include at least one second protein containing one of the amino acid sequences of SEQ ID NOs: 1 to 84. In another embodiment, all second proteins contain one of the amino acid sequences of SEQ ID NOs: 1 to 84.

[0069] The nanoparticles contain multiple identical first proteins. In one embodiment, the first protein contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 152-159, where the residues in parentheses are optional, may be present, or any part (i.e., 1, 2, 3, 4, 5, 6, or more) or all of any residues may be absent. In a particular embodiment, the first protein contains an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO. 155. [Table 3]

[0070] I53-50-v4 Pentamer Components (MGSSHHHHHHSSGLVPRGSEQKLISEEDLGS)NQHSQKDQETVRIAVVRARWHAFIVDACVSAFEAAMRKIGGERFAVDVFDVPGAYEIPLHARTLAKTGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLDTGVPVLSAVLTPHNYDKSNAKTLLFLALFAVKGMEAARACVEILAAREKIAA(GSLEGS)(Sequence ID 156)

[0071] I53-50-v1 pentameric component B (M)NQHSHKDHETVRIAVVRARWHAEIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLDTGVPVLSAVLTPHNYDKSKAHTLLFLALFAVKGMEAARACVEILAAREKIAA(GS)(Sequence ID 157)

[0072] I53-50-v2 pentameric component B (M)NQHSHKDHETVRIAVVRARWHAFIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLDTGVPVLSAVLTPHNYDKSNAKTLLFLALFAVKGMEAARACVEILAAREKIAA(GS)(Sequence ID 158)

[0073] I53-50-v3 pentameric component B (M)NQHSHKDHETVRIAVVRARWHAFIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHARTLAETGRYGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLDTGVPVLSAVLTPHNYDKSNAKTLLFLALFAVKGMEAARACVEILAAREKIAA(GS)(Sequence ID 159)

[0074] In exemplary embodiments, the first protein comprises the amino acid sequence of SEQ ID NO: 155. In various further such embodiments, at least one or more of the second assemblies (20%, 33%, 40%, 50%, 75%, etc.) comprises at least one second protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-88, or all of the second assemblies comprise at least one second protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 85-88.

[0075] In one particular embodiment, (a) The first protein contains the amino acid sequence of SEQ ID NO: 155, (b) All second proteins contain the amino acid sequence of SEQ ID NO: 85, and at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of X1 of the second protein contain 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 125.

[0076] In certain other embodiments, (a) The first protein contains the amino acid sequence of SEQ ID NO: 155, (b) All second proteins contain the amino acid sequence of SEQ ID NO: 85, and at least 50%, 60%, 70%, 80%, 90%, or 100% of X1 in the second protein contain an amino acid sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with respect to the amino acid sequence of SEQ ID NO: 125.

[0077] In a further specific embodiment, (a) The first protein contains the amino acid sequence of SEQ ID NO: 155, (b) All second proteins contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-8.

[0078] In one particular embodiment, (a) The first protein contains the amino acid sequence of SEQ ID NO: 155, (b) All second proteins contain the amino acid sequence of SEQ ID NO: 1 or 5.

[0079] This disclosure further provides compositions comprising a plurality of nanoparticles of any embodiment or combination of embodiments of this disclosure. In one embodiment, the composition comprises a plurality of nanoparticles of the particular embodiment disclosed above.

[0080] In a fourth aspect, the Disclosure provides nucleic acids encoding polypeptides or fusion proteins of the Disclosure. Nucleic acid sequences may include RNA (e.g., mRNA) or DNA. Such nucleic acid sequences may include, but are not limited to, poly(A) sequences, modified Kosack sequences, and sequences encoding epitope tags, transport signals, and secretion signals, nuclear localization signals, and cell membrane localization signals, as well as additional sequences useful for promoting the expression and / or purification of the encoded protein. Based on the teachings herein, it will be apparent to those skilled in the art which nucleic acid sequences encode the proteins of the present invention.

[0081] In a fifth aspect, the Disclosure provides an expression vector comprising an isolated nucleic acid of any embodiment or combination of embodiments of the Disclosure functionally ligated to a preferred regulatory sequence. An “expression vector” includes a vector that functionally ligates a nucleic acid coding region or gene to any regulatory sequence capable of resulting in the expression of a gene product. A “regulatory sequence” operably ligated to a nucleic acid sequence of the Disclosure is a nucleic acid sequence capable of resulting in the expression of a nucleic acid molecule. The regulatory sequences do not need to be adjacent to the nucleic acid sequence insofar as they function to direct its expression. For example, an intervening, untranslatable but transcribed sequence may exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence may still be considered “operably ligated” to the coding sequence. Other such regulatory sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors may be any type known in the Art, including, but are not limited to, plasmid and virus-based expression vectors. Regulatory sequences used to promote the expression of disclosed nucleic acids in mammalian systems may be constitutive (promoted by any of a variety of promoters, including CMV, SV40, RSV, actin, and EF) or inductive (promoted by any of a number of inductive promoters, including tetracycline, ecdysone, and steroid-responsive).

[0082] In a sixth aspect, the Disclosure provides cells comprising polypeptides, nanoparticles, compositions, nucleic acids, and / or expression vectors of any embodiment or combination of embodiments of the Disclosure, the cells of which may be prokaryotic or eukaryotic cells, such as mammalian cells. In one embodiment, cells may be transiently or stably transfected with nucleic acids or expression vectors of the Disclosure. Such transfection of expression vectors into prokaryotic and eukaryotic cells can be achieved by any technique known in the Art. A method for producing polypeptides according to the present invention is an additional part of the Invention. The method comprises (a) culturing a host under conditions that promote polypeptide expression according to this aspect of the Invention, and (b) optionally recovering the expressed polypeptide.

[0083] In the seventh aspect, this disclosure is (a) Polypeptides, nanoparticles, compositions, nucleic acids, expression vectors, and / or cells of embodiments or combinations of embodiments thereof as specified herein, (b) Provide a pharmaceutical composition / vaccine comprising a pharmaceutically acceptable carrier.

[0084] As illustrated in the following example, nanoparticle immunogens induce a potent, protective antibody response against SARS-CoV-2. The nanoparticle vaccines of this disclosure induce neutralizing antibody titers approximately 10-fold higher than those of pre-fusion-stabilized S-external domain trimers, even at doses less than one-fifth of the original dose. Because antibodies induced by nanoparticle immunogens target multiple distinct epitopes, they are not susceptible to easily evading mutations and exhibit significantly lower binding-to-neutralization ratios than those of convalescent human serum, suggesting that the risk of vaccine-related enhanced respiratory illness may be minimized.

[0085] The pharmaceutical composition / vaccine may further comprise (a) a lyophilization protectant, (b) a surfactant, (c) a bulking agent, (d) a tonicity modifier, (e) a stabilizer, (f) a preservative, and / or (g) a buffering agent. In some embodiments, the buffering agent in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer, or an acetate buffer. The composition may also comprise a lyophilization protectant, such as sucrose, sorbitol, or trehalose. In certain embodiments, the composition comprises preservatives, such as benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercury nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the composition comprises a bulking agent such as glycine. In yet another embodiment, the composition includes surfactants, such as polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80, polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or combinations thereof. The composition may also include tonicity modifiers, such as compounds that make the formulation substantially isotonic or isostomal to human blood. Exemplary tonicity modifiers include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine, and arginine hydrochloride. In other embodiments, the composition further comprises stabilizers, such as molecules that substantially prevent or reduce the chemical and / or physical instability of the nanostructure in freeze-dried or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.

[0086] The nanoparticles may be the sole activator in the composition, or the composition may further contain one or more other agents suitable for the intended use, including but not limited to adjuvants for generally stimulating the immune system and improving the overall immune response. Any suitable adjuvant may be used. The term “adjuvant” refers to a compound or mixture that enhances the immune response to an antigen. Exemplary adjuvants include Adju-Phos®, Adjumer®, albumin-heparin nanoparticles, Algal Glucan, Algammulin, alum, antigen preparations, AS-2 adjuvant, autologous dendritic cells, autologous PBMCs, Avridine®, B7-2, BAK, BAY R1005, bupivacaine, bupivacaine-HCl, BWZL, calcitriol, calcium phosphate gel, CCR5 peptide, CFA, cholera holotoxin (CT) and cholera toxin B subunit (CTB), cholera toxin A1 subunit-protein A D-fragment fusion protein, CpG, CRL1005, cytokine-containing liposomes, D-mura palmitin, DDA, DHEA, diphtheria toxoid, DL-PGL, DMPC, DMPG, DOC / alum complex, fowlpox, Freund's complete adjuvant, gamma inulin, gelb adjuvant, GM-CSF, GMDP, hGM-CSF, hIL-12(N222L), hTNF-α, IFA, IFN-γ of pcDNA3, IL-12DNA, IL-12 plus Mido, IL-12 / GMCSF plasmid (Sykes), IL-2 in pcDNA3, IL-2 / Ig plasmid, IL-2 / Ig protein, IL-4, IL-4 in pcDNA3, Imiquimod (trademark), ImmTher (trademark), immunoliposomes containing antibodies against costimulatory molecules, interferon-γ, interleukin-1β, interleukin-12, interleukin-2, interleukin-7, ISCOM(s) (trademark), Iscoprep 7.0.3 (Trademark), Keyhole Limpet Hemocyanin, Lipid-based adjuvant, Liposome, Loxoribine, LT(R192G), LT-OA or LT Oral adjuvant, LT-R192G, LTK63, LTK72, MF59, MONTANIDE ISA51, MONTANIDE ISA720, MPL.TM, MPL-SE, MTP-PE, MTP-PE liposomes, Muramethide, Murapalmitin, NAGO, nCT natural cholera toxin, non-ionic surfactant vesicles, non-toxic variant E112K of cholera toxin mCT-E112K, p-hydroxybenzoate methyl ester, pCIL-10, pCIL12, pCMVmCAT1, pCMVN, peptomer-NP, Pleuran, PLG, PLGA, PGA, and PLA, Pluronic L121, PMMA, PODDS (trademark), PolyrA:PolyrU, Polysorbate 80, Ta This includes, but is not limited to, protein cocreates, QS-21, QuadriA saponin, Quil-A, hydrogel HPA, hydrogel LV, RIBI, Rivirike adjuvant systems (MPL, TMD, CWS), S-28463, SAF-1, Sclavo peptide, Sendai proteoliposomes, Sendai lipid matrix, Span 85, Specol, squalane 1, squalene 2, stearyl tyrosine, tetanus toxoid (TT), Theramide®, threonyl muramyl dipeptide (TMDP), Ty particles, and Walter Reed liposomes. The choice of adjuvant depends on the target being treated. Preferably, a pharmaceutically acceptable adjuvant is used.

[0087] In the eighth aspect, the Disclosure provides a method for treating or limiting the onset of SARS-CoV-2 infection, comprising administering a polypeptide, nanoparticles, composition, nucleic acid, pharmaceutical composition, or vaccine (referred to as “immunogenic composition”) of any embodiment herein to a subject in need of such a substance, in an amount effective for treating or limiting the onset of the infection. The subject may include, but is not limited to, any suitable mammalian subject.

[0088] Where the method involves limiting SARS-CoV-2 infection, the immunogenic composition is administered prophylactically to subjects who are not known to be infected but may be at risk of exposure to SARS-CoV-2. As used herein, “limiting the onset” includes, but is not limited to, achieving one or more of the following: (a) generating an immune response (antibodies and / or cell-based) to SARS-CoV-2 in the subject; (b) generating neutralizing antibodies against SARS-CoV-2 in the subject; (c) limiting the accumulation of SARS-CoV-2 titers in the subject after exposure to SARS-CoV-2; and / or (d) limiting or preventing the onset of SARS-CoV-2 symptoms after infection. Exemplary symptoms of SARS-CoV-2 infection include, but are not limited to, fever, fatigue, cough, shortness of breath, chest tightness and / or pain, loss or reduction of smell, loss or reduction of taste, pneumonia, and respiratory problems including bronchitis, severe acute respiratory syndrome (SARS), and upper and lower respiratory tract infections.

[0089] In one embodiment, the method generates an immune response in subjects not known to be infected with SARS-CoV-2, and the immune response helps limit infection and the development of symptoms of SARS-CoV-2 infection. In one embodiment, the immune response includes the production of neutralizing antibodies against SARS-CoV-2. In an exemplary embodiment, the immune response is at least 1 x 10⁻⁶ 5 This includes the generation of a SARS-CoV-2 spike protein antibody-specific response having an average geometric titer. In further embodiments, the immune response includes the generation of antibodies against multiple antigenic epitopes.

[0090] As used herein, “therapeutic dose” refers to the amount of immunogen composition effective in treating and / or limiting SARS-CoV-2 infection. Polypeptides, nanoparticles, compositions, nucleic acids, pharmaceutical compositions, or vaccines of any embodiment herein are typically formulated as pharmaceutical compositions such as those disclosed above and can be administered via any suitable route, including oral, parenteral, inhalation spray, rectal, or topical, including a conventional pharmaceutically acceptable dosage unit formulation comprising a carrier, adjuvant, and vehicle. As used herein, the term parenteral includes subcutaneous, intravenous, intra-arterial, intramuscular, intrasternal, intratendinous, intraspinal, intracranial, intrathoracic, infusion techniques, or intraperitoneal. Polypeptide compositions may also be administered via microspheres, liposomes, immunostimulatory complexes (ISCOMs), or other microparticle delivery systems, or sustained-release formulations introduced into suitable tissues (such as blood). Dosage regimens may be modified to provide the optimal desired response (e.g., a therapeutic or prophylactic response). An appropriate dose range may be, for example, 0.1 μg to 100 mg of polypeptide or its nanoparticles per kg of body weight. The composition may be delivered in a single bolus or administered two or more times (e.g., two, three, four, five or more times) as determined by the healthcare professional.

[0091] In one embodiment, administration comprises administering a first dose and a second dose of the immunogenic composition, the second dose being administered about 2 to 12 weeks, or about 4 to 12 weeks, after the first dose was administered. In various further embodiments, the second dose is administered about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the first dose. In another embodiment, three doses may be administered, the second dose being administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the first dose, and the third dose being administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after the second dose.

[0092] In various other embodiments of prime-boost administration, the administration is, (a) Administering a prime dose of a DNA, mRNA, or adenovirus vector vaccine, wherein the DNA, mRNA, or adenovirus vector vaccine encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NOs. 125-137, and administering a prime dose of the vaccine. (b) administering any embodiment or combination disclosed herein to a target of a boost dose of a polypeptide, nanoparticles, composition, nucleic acid, pharmaceutical composition, or vaccine.

[0093] In another embodiment, administration is, (a) To administer the prime dose of any embodiment or combination disclosed herein, (b) Administering a boost dose of a DNA, mRNA, or adenovirus vector vaccine to a target, wherein the boost dose encodes an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of SEQ ID NOs. 125-137.

[0094] In any of these embodiments, any suitable DNA, mRNA, or adenovirus vector vaccine may be used in combination with the immunogenic composition of the Disclosure, including, but not limited to, vaccines under development, as well as vaccines available from Moderna, Pfizer / BioNTech, Johnson & Johnson, and others.

[0095] In another embodiment of the method, the subject is infected with a severe acute respiratory (SARS) virus, including but not limited to SARS-CoV-2, and the administration includes inducing an immune response to the SARS virus in the subject and treating the SARS subject's viral infection. If the method includes the treatment of a SARS-CoV-2 infection, the immunogenic composition is administered to a subject that is already infected with SARS-CoV-2 and / or a subject suffering from symptoms (such as those described above) that suggest the subject is likely to have been infected with SARS-CoV-2.

[0096] As used herein, “to treat” or “to treat” includes, but is not limited to, achieving one or more of the following: (a) reducing the SARS-CoV-2 titer in a control; (b) limiting the increase in the SARS-CoV-2 titer in a subject; (c) reducing the severity of SARS-CoV-2 symptoms; (d) limiting or preventing the onset of SARS-CoV-2 symptoms after infection; (e) suppressing the exacerbation of SARS-CoV-2 symptoms; (f) limiting or preventing the recurrence of SARS-CoV-2 symptoms in a subject who has previously had symptoms of SARS-CoV-2 infection; and / or (e) survival.

[0097] This disclosure further provides kits that may be used to prepare the nanoparticles and compositions of this disclosure. In one embodiment, the kit is: (a) polypeptides of any embodiment or combination of embodiments disclosed herein, (b) A first protein comprising an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 152-159, wherein the residues in parentheses are optional and may or may not be present.

[0098] In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 5, and the first protein comprises the amino acid sequence of SEQ ID NO: 155.

[0099] In another embodiment, the kit is (a) A nucleic acid encoding a polypeptide of any embodiment or combination of embodiments disclosed herein, such as the first embodiment, (b) A nucleic acid encoding a first protein having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 152-159, wherein the residues in parentheses are optional and may or may not be present.

[0100] In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 5, and the first protein comprises the amino acid sequence of SEQ ID NO: 155.

[0101] In further embodiments, the kit includes: (a) An expression vector comprising nucleic acids encoding a polypeptide of any embodiment or combination of embodiments disclosed herein, such as the first embodiment, operably linked to a suitable control sequence, (b) An expression vector comprising a nucleic acid encoding a first protein having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 152-159, wherein the residues in parentheses are optional and may or may not be present, and the nucleic acid is operably linked to appropriate regulatory sequences.

[0102] In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 5, and the first protein comprises the amino acid sequence of SEQ ID NO: 155.

[0103] In another embodiment, the kit is (a) A cell comprising an expression vector, wherein the expression vector comprises a nucleic acid encoding a polypeptide of any embodiment or combination of embodiments disclosed herein, such as the first embodiment, operably linked to a suitable control sequence, (b) A kit comprising cells containing an expression vector, wherein the expression vector comprises a nucleic acid encoding a first protein having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 152-159, where the residues in parentheses are optional and may or may not be present, and the nucleic acid is operably linked to appropriate regulatory sequences.

[0104] In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 5, and the first protein comprises the amino acid sequence of SEQ ID NO: 155.

[0105] Examples Induction of a potent neutralizing antibody response by a protein nanoparticle vaccine designed for SARS-CoV-2. overview To halt the ongoing SARS-CoV-2 global pandemic, a safe, effective, and scalable vaccine is urgently needed. Here, we describe a structure-based design of a self-assembling protein nanoparticle immunogen that induces a potent, protective antibody response against SARS-CoV-2 in mice. The nanoparticle vaccine displays 60 copies of the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) in a highly immunogenic sequence, inducing a neutralizing antibody titer approximately 10-fold higher than that of the pre-fusion-stabilized S ectodomain trimer at one-fifth a dose. Because the antibodies induced by the nanoparticle immunogen target multiple distinct epitopes on the RBD, they are not susceptible to easily evading mutations and exhibit a significantly lower binding-to-neutralization ratio than convalescent human serum, suggesting that the risk of vaccine-related enhanced respiratory illness may be minimized. In particular, the high yield and stability of the protein components and assembled nanoparticles compared to the SARS-CoV-2 pre-fusion-stabilized S trimer indicate that the production of the nanoparticle vaccine is highly scalable.

[0106] Design, in vitro assembly, and characterization of SARS-CoV-2 RBD nanoparticle immunogens. To design a vaccine candidate that induces a potent neutralizing Ab response, we focused on the RBD of the SARS-CoV-2S glycoprotein (Figure 1A-B). To overcome the limited immunogenicity of this small monomeric antigen, we polyvalently displayed the RBD on the outer surface of two-component protein nanoparticles I53-50. I53-50 is a computer-designed 28 nm, 120-subunit complex with icosahedral symmetry, constructed from trimer (I53-50A) and pentamer (I53-50B) components (all amino acid sequences are listed in Table 3). The nanoparticles can be assembled in vitro by simply mixing independently expressed and purified I53-50A and I53-50B. RBD (residues 328-531) is genetically fused to I53-50A using a linker containing 8, 12, or 16 glycine and serine residues (hereinafter referred to as RBD-8GS-, RBD-12GS-, or RBD-16GS-I53-50A), enabling flexible presentation of antigens extending from the nanoparticle surface (Figure 1C). All RBD-I53-50A constructs were recombinantly expressed using mammalian (Expi293F) cells to ensure proper folding and glycosylation of the viral antigen. The initial yield of purified RBD-I53-50A protein (approximately 30 mg of purified protein per liter of Expi293F cells) was about 20 times higher than that of the S-2P trimer stabilized before fusion (Kirchdoerfer et al., 2018; Pallesen et al., 2017; Walls et al., 2020; Wrapp et al., 2020) (~1.5 mg / L), and increased to approximately 60 mg / L after promoter optimization. RBD-I53-50A protein was mixed with the pentamer I53-50B purified from E. coli in a 1:1 molar ratio (subunit:subunit) to initiate nanoparticle aggregation (Figure 1D). [Table 4] [Table 4-2] [Table 4-3] [Table 4-4]

[0107] Size exclusion chromatography (SEC) of SARS-CoV-2 RBD-I53-50 nanoparticles revealed a main peak corresponding to the icosahedral aggregate of the target, and smaller peaks containing the remaining RBD-I53-50A components that were not aggregated (Figures 7A and 7B). Dynamic light scattering (DLS) and negative staining electron microscopy (nsEM) confirmed the homogeneity and monodispersity of various RBD-I53-50 nanoparticles before and after freezing / thawing (Figures 1E, 1F, and 7C). The mean hydrodynamic diameter and percent polydispersity of RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50, measured by DLS before freezing / thawing, were 38.5 (27%), 37 (21%), and 41 (27%) nm, respectively, compared to 30 (22%) nm for unmodified I53-50 nanoparticles. Hydrogen / deuterium exchange mass spectrometry confirmed that the RBD display on the trimer RBD-8GS-I53-50A component preserves the conformation of the antigen and the structural order of several different antibody epitopes (Figure 1G and Figure 7D). Finally, using glycoproteomics, we showed that all three RBD-I53-50A components are N-glycosylated at the N331 and N343 positions, similar to the SARS-CoV-2S-2P external domain trimer (Watanabe et al., 2020), again suggesting that the displayed antigen retains its native antigenic properties (Figure 1H and Figure 7E). [Table 5]

[0108] Each experiment was performed at least twice, and the presented values ​​and fitting errors are representative of the experiments. The corresponding joint curves and fits are shown in Figure 8.

[0109] Antigen Characterization of SARS-CoV-2 RBD-I53-50 Nanoparticle Components and Immunogens The antigenicity of RBD when fused to I53-50A, as well as the accessibility of multiple RBD epitopes in the context of assembled nanoparticle immunogens, were characterized using recombinant human ACE2 ectodomains and two S-specific mAbs (CR3022 and S309). Both CR3022 and S309 were isolated from individuals infected with SARS-CoV and cross-react with SARS-CoV-2 RBD. CR3022 is a weakly neutralizing AB that becomes accessible when RBD opens, but binds to a conserved, potential epitope of RBD that is different from the receptor-binding motif (RBM), which is the surface of RBD that interacts with ACE2 (Huo et al., 2020; ter Meulen et al., 2006; Yuan et al., 2020). S309 neutralizes both SARS-CoV and SARS-CoV-2 by binding to a glycan-containing epitope that is conserved among salvecoviruses and accessible in both open and closed prefusion S conformations (Pinto et al., 2020).

[0110] The binding affinity of CR3022Fab to the human ACE2 (hACE2) external domain of the monomer and to the RBD monomer was confirmed using biolayer interferometry (BLI). The equilibrium dissociation constant (K) of these reagents to the immobilized RBD-I53-50A fusion protein was determined. D The values ​​obtained were in close agreement with those obtained with monomer RBD (Table 4 and Figure 8). These data further support the idea that the RBD-I53-50A fusion protein represents RBD in its original three-dimensional structure.

[0111] In the context of high-density multivalent antigen arrays, to assess the potential for the magnitude and quality of nanoparticle immunogen-induced Ab responses to be modulated by the accessibility of specific epitopes, we measured the binding of nanoparticle immunogens to immobilized dimeric macaq ACE2 (mACE2-Fc) and CR3022 and S309 mAb, the latter broadly mimicking the B cell receptor (BCR)-antigen interaction central to B cell activation. This approach is considered due to the multivalent nature of the interaction. D While this does not allow for the calculation of values, it enables a quantitative comparison of epitope accessibility across different nanoparticle constructs. Leveraging the versatility of an in vitro assembly for preparing nanoparticle immunogens displaying RBD antigens at 50% valence (approximately 30 RBDs per nanoparticle), we compared 60 RBD-displaying full-valence nanoparticles with a low-density antigen array (Figure 9). This was achieved by adding pentamer I53-50B to an equimolar mixture of RBD-I53-50A and unmodified I53-50A lacking fusion antigens. We found that all RBD nanoparticles bound well to immobilized mACE2-Fc, CR3022, and S309 (Figure 2A) (Figure 2A). There was no consistent trend between 50% and 100% valence RBD-8GS- and RBD-12GS-I53-50 nanoparticles, but 100% valence RBD-16GS-I53-50 nanoparticles yielded the best binding signal for all three binders (Figure 2B). While longer linkers in RBD-16GS-I53-50 nanoparticles may improve access to the target epitopes of ACE2, CR3022, and S309, our data do not rule out other possible explanations. We conclude that multiple different epitopes targeted by the neutralizing antibody are exposed and can be accessed for binding in the context of the RBD antigen array presented outside the nanoparticle.

[0112] Physical and antigenic stability of RBD nanoparticle immunogen and S-2P trimer First, the stability of the RBD-I53-50A fusion protein and RBD-12GS-I53-50 nanoparticle immunogens was compared with that of recombinant monomer RBD and S-2P external domain trimers using chemical denaturation with guanidine hydrochloride (GdnHCl) (Figure 3A). Fluorescence emission spectra from samples incubated with 0–6.5 M GdnHCl revealed that all three RBD-I53-50A fusion proteins and RBD-12GS-I53-50 nanoparticles transitioned between 4–5 M GdnHCl, indicating at least partial unfolding, while the S-2P trimer showed transition at lower GdnHCl levels between 2–4 M. Monomer RBD showed less cooperative unfolding transitions to 0–5 M GdnHCl. Next, physical and antigenic stability was monitored at three temperatures over four weeks post-purification using a series of analytical assays. <-70°C, 2-8°C, and 22-27°C (Figures 3B-E). Consistent with previous reports, the monomer RBD was very stable, showing little change in appearance on SDS-PAGE (Figure 10A), mACE2-Fc and CR3022 binding (Figure 10B), or on the ratio of UV / vis absorption at 320 / 280 nm and the particle scattering scale (Figure 10C). The S-2P trimer was unstable at 2-8°C, showing clear signs of unfolding by nsEM even at the initial stage (Figure 9D). Once unfolding was evident by nsEM and some aggregation was shown by UV / vis (Figure 10C), the structure was fairly well maintained at 22-27°C until the latest time (28 days). All three RBD-I53-50A components were very stable, with no substantial changes in readings at any point (data not shown). Finally, the RBD-12GS-I53-50 nanoparticles were also very stable over the 4-week study, showing only changes in UV / vis absorbance, with a peak appearing around 320 nm after 7 days at 22–27°C (data not shown). Electron micrographs and DLS of the RBD-12GS-I53-50 nanoparticle samples consistently showed monodisperse, well-formed nanoparticles at all temperatures over the 4-week period (Figures 10D, 10E).In summary, these data indicate that the RBD-I53-50A component and RBD-12GS-I53-50 nanoparticles possess superior physical and antigenic stability compared to the S-2P external domain trimer.

[0113] RBD-I53-50 nanoparticle immunogen induces a potent neutralizing antibody response in BALB / c and human immunorepertory mice. The immunogenicity of three RBD-I53-50 nanoparticles, each exhibiting RBD at 50% or 100% valence, was compared to that of S-2P external domain trimers and monomeric RBDs in BALB / c mice. Groups of 10 mice were intramuscularly immunized at weeks 0 and 3 with AddaVax® adjuvant formulations containing 0.9 or 5 μg of soluble or particulate SARS-CoV-2 antigen. Three weeks after priming, all RBD nanoparticles exhibited 8x10⁻¹⁶ immunogenicity. 2 ~1x10 4 A potent S-specific Ab response was induced at geometric mean reciprocal half-maximal effective concentrations in the range (Figure 4A). In contrast, monomeric RBDs and low-dose S-2P trimers did not induce detectable levels of S-specific Ab, while high-dose S-2P trimers induced a weak response. After a second immunization, an enhancement of S-specific Ab titer was observed in all RBD nanoparticle groups, with a geometric mean titer (GMT) of 1 x 10⁻⁶. 5 ~2x10 6 The levels were within the range shown (Figure 4B). These S-specific Ab levels were consistent with or exceeded those of most samples from the Washington State 29 COVID-19 human convalescent serum (HCS) panel and the NIBSC benchmark 20 / 130 COVID-19 plasma (Figures 4A-B, Table 5). Double immunization with 5 μg of S-2P trimer induced an S-specific Ab response that was 1-2 orders of magnitude weaker than that of RBD nanoparticles, and monomeric RBDs did not induce detectable antigen-specific Abs after double immunization. As expected, Ab responses to I53-50 scaffolds were also detected, and these were of a consistent magnitude across all RBD nanoparticle groups (Figure 11). These data demonstrate that polyvalent representation of RBDs on self-assembling nanoparticle scaffolds dramatically improves their immunogenicity.

Table 6

Table 6-2

[0114] Using Kymab's proprietary IntelliSelect™ transgenic mouse platform (known as "Darwin"), which is transgenic for the non-reconstituted human antibody variable and constant region germline repertoires, a prototype of the potential human antibody response to the RBD nanoparticle immunogen was generated. In contrast to previous mice with the described chimeric antibody loci (Lee et al., 2014), the mice in this study differed in that they were engineered to express fully human kappa light chain Abs. A group of five Darwin mice was immunized intramuscularly with S-2P trimer, 100% RBD-12GS-, or 100% RBD-16GS-I53-50 nanoparticles at an antigen dose of 0.9 μg (nanoparticles only) or 5 μg (Figure 4C). All groups immunized with RBD nanoparticles elicited an S-directed Ab response after priming (EC 50 2x10 3 -1x10 4 ), which was significantly boosted by the second immunization at week 3 (EC 50 was in the range of 4x10 5 ~8x10 5 )(Figure 4C and 4D). In this animal model, the S-2P trimer elicited S-specific Abs at levels comparable to those of RBD nanoparticles after each immunization.

[0115] Next, neutralization assays using both pseudotyped virus and live virus were used to evaluate the neutralizing activity induced by each immunogen. In BALB / c mice, all RBD nanoparticle immunogens elicited serum neutralizing Abs after a single immunization, and the half-maximal inhibitory dilution reciprocal (IC 50 ) was 1x10 2 ~5x10 2 (GMT) for pseudotyped virus and 3x10 3 ~7x10 3(Figures 5A and 5C). Consistent with S-specific Ab data, no significant difference was observed in the neutralization of pseudotyped or live viruses at 50% (pseudotyped virus neutralization only) or 100% titer between low-dose or high-dose RBD-8GS-, RBD-12GS-, or RBD-16GS-I53-50 nanoparticles. The GMT of all three 100% valence RBD nanoparticle groups was consistent with or exceeded the GMT of the panel of 29 HCS tested in the pseudotyped virus neutralization assay (Figure 5A). Immunization with monomeric RBD or S-2P trimer did not induce neutralizing Abs after a single immunization (Figures 5A and 5C). Similar to BALB / c mice, both high and low doses of RBD-I53-50 nanoparticles in Darwin mice did not induce HCS (IC) after a single immunization. 50 1x10 2 ) equivalent pseudovirus neutralizing Ab titer (IC) 50 8x10 1 ~2.5x10 2 ) induced total S-specific absorption, while 5 μg of S-2P trimer did not induce a detectable level of neutralizing absorption, although it did induce a similar level of total S-specific absorption (Figure 5E).

[0116] In both mouse models, a second immunization with RBD-I53-50 nanoparticles significantly increased neutralizing Ab titers. In BALB / c mice, pseudotyped viral neutralizing GMT increased by 2x10⁻¹⁰. 3 ~3x10 4 It reached a level exceeding that of HCS by 1-2 orders of magnitude, and the live virus neutralizing titer was 2x10 4 ~3x10 4 This was achieved (Figures 5B and 5D). A second immunization with 5 μg of S-2P trimer also strongly enhanced neutralizing activity, but neutralization of pseudotyped and live viruses (3 x 10⁻¹⁰ each) was also reduced. 2 and 6x10 3The GMT of the titer (of the titer) remained lower than that of the serum of animals immunized with RBD nanoparticles. The increase between the S-2P trimer and RBD nanoparticles ranged from 7–90-fold and 4–9-fold, respectively, in pseudotyped virus and live virus neutralization assays. Neither the 0.9 μg dose of the S-2P trimer nor both doses of monomeric RBD elicited detectable neutralization after two immunizations. Similar increases in pseudotyped virus neutralization were observed after a second immunization in Darwin mice, but titers were generally lower than in BALB / c mice (Figure 5F).

[0117] Several conclusions can be drawn from these data. First, RBD nanoparticles induce a potent neutralizing Ab response in two mouse models, exceeding that induced by pre-fusion-stabilized S-2P trimers and by human infection after two doses. Second, while linker length and antigen valency do not substantially affect the overall immunogenicity of RBD nanoparticles, RBD-16GS-I53-50 tends to suggest that it may be more immunogenic than nanoparticles with shorter linkers. These observations are consistent with the antigenicity and accessibility data shown in Table 4 and Figure 2, indicating that multiple epitopes are intact and accessible in all RBD nanoparticle immunogens. Finally, the induction of equivalent neutralizing Ab titers at both 0.9 μg and 5 μg doses of each nanoparticle immunogen suggests that RBD presentation with I53-50 nanoparticles allows for dose savings, which is an important consideration in vaccine manufacturing and distribution.

[0118] Eight mice immunized with monomeric RBD, S-2P trimer, or RBD-8GS- or RBD-12GS-I53-50 nanoparticles using AddaVax® alone were challenged with mouse-adapted SARS-CoV-2 virus (SARS-CoV-2 MA) after a 7-week boost to determine whether these immunogens provide protection from viral replication. RBD-8GS and RBD-12GS-I53-50 nanoparticles provided complete protection from detectable SARS-CoV-2 MA replication in the lungs and nasal turbinates of mice (Figure 5G-H). Immunization with monomeric RBD, 0.9 μg of S-2P trimer, and adjuvant controls did not provide protection from SARS-CoV-2 MA replication. These results reflect pseudotyped and live virus neutralization data, indicating that RBD nanoparticles induce a potent anti-SARS-CoV-2 Ab response at either dose or valence.

[0119] RBD nanoparticle vaccines induce robust B-cell responses and antibodies targeting multiple epitopes in mice and non-human primates. The germinal center (GC) response is a crucial process in the formation of durable B cell memory, resulting in the development of affinity-mature class-switch memory B cells and long-lived plasma cells. Therefore, we evaluated antigen-specific GC B cell responses in mice immunized with monomer RBD, S-2P trimers, and RBD-8GS-, RBD-12GS-, or RBD-16GS-I53-50 nanoparticles. The quantity and phenotype of RBD-specific B cells were assessed 11 days after immunization, and GC precursors and B cells (B220) were evaluated. + CD3 - CD138 - CD38 - GL7 +The level of ) was determined (Figure 12). Immunization with RBD nanoparticles resulted in an expansion of RBD-specific B cells and GC precursors and B cells (Figures 6A-C). The S-2P trimer resulted in a detectable but low number and frequency of RBD-specific B cells and GC precursors and B cells compared to RBD nanoparticles, while the monomer RBD construct did not induce a sensible B cell response. Consistent with these findings, immunization with the three RBD nanoparticles and the trimer S-2P resulted in an expansion of CD38 + / - GL7 + IgM + and class switch (swIg + RBD-specific B cells appear, exhibiting functional GC precursors and GC B cells (Figure 6D). Robust GC B cell response and IgM in mice immunized with RBD-nanoparticles and, to a lesser extent, S-2P constructs. + and swIg + The increased proportion of RBD-specific B cells is consistent with the ongoing GC response, at which point memory B cells and long-lived plasma cells should be formed. To assess the persistence of the humoral response induced by the RBD nanoparticle vaccine, serum Ab responses were analyzed 20–24 weeks after boost. The magnitudes of both binding and neutralizing titers were similar to those at 2 weeks after boost for all nanoparticle groups (Figure 12B, C), indicating that the designed immunogen is not only potent but also induces durable neutralizing Abs. This may be partly due to improved induction of long-lived plasma cells by the nanoparticle vaccine, as the number of S-2P-specific Ab-secreting cells in the bone marrow was approximately three times higher in mice immunized with RBD-16GS-I53-50 nanoparticles compared to S-2P trimers (Figure 12D).

[0120] The binding rates to neutralizing antibodies induced by S-2P, RBD-8GS-, RBD-12GS-, and RBD-16GS-I53-50 nanoparticles and HCS were compared as a measure of the quality of the Ab response induced by the nanoparticle immunogens. In Kymab Darwin® mice, the nanoparticle vaccines showed lower (better) rates than S-2P immunized mice, but higher rates than HCS (Figure 6E). In BALB / c mice, the binding rates to pseudotyped viral neutralizing titers induced by RBD-12GS- and RBD-16GS-I53-50 were significantly reduced compared to S-2P and HCS (Figure 6F). This pattern was consistent when the rates were calculated using live viral neutralizing titers, but the magnitude of the differences between groups was smaller due to the higher values ​​obtained in the live viral neutralization assay. These results suggest that the Ab responses induced by RBD-12GS and RBD-16GS-I53-50 nanoparticle immunogens are of higher quality than those obtained from immunization with S-2P trimers or acquired during natural infection, likely because they focus on the RBD epitopes, which are targets for most neutralizing Abs.

[0121] We embarked on identifying epitopes recognized by Abs induced by immunization with nanoparticle immunogens in non-human primate models whose immune response to vaccination is more similar to that of humans. Macacuils were immunized with 250 μg of RBD-12GS-I53-50 (88 μg of RBD antigen) at weeks 0 and 4, and serum collected at week 8 showed high levels of S-specific antibody (EC). 50 Approximately 1x10 6We found that the polyclonal Fab possesses the following properties. The polyclonal Fab was generated and purified for use in competitive BLI with hACE2, CR3022, and S309, and recognizes three different sites targeted by neutralizing Abs on the SARS-CoV-2 RBD (Figure 6G). Polyclonal serum dose-dependently inhibited the binding of hACE2, CR3022Fab, and S309Fab at concentrations exceeding their respective dissociation constants (Figures 6H-J). These data indicate that immunization with 12GS-RBD-I53-50 induced Abs targeting several non-overlapping epitopes, and we anticipate that the possibility of escape mutation emergence and selection is limited, especially since coronaviruses do not mutate rapidly compared to viruses such as influenza or human immunodeficiency virus (Li et al., 2020; Smith et al., 2014).

[0122] Consideration Here, we demonstrate that a two-component self-assembling SARS-CoV-2 RBD nanoparticle vaccine candidate induces a potent neutralizing Ab response targeting multiple distinct RBD epitopes. The greater neutralizing Ab response induced by RBD nanoparticles is highly promising compared to the pre-fusion-stabilized external domain trimer. Our data show that RBD-12GS-I53-50 and RBD-16GS-I53-50 induce nearly 10-fold higher levels of S-specific Abs, and more importantly, approximately 10-fold higher levels of neutralizing activity compared to the S-2P external domain trimer. This enhanced efficacy is maintained at an antigen dose one-fifth lower by mass, suggesting that presentation to nanoparticles also has a dose-saving effect. Both enhanced efficacy and dose-saving may be crucial in addressing the need to produce unprecedented numbers of vaccines to respond to a SARS-CoV-2 global pandemic.

[0123] Although RBD is not sufficiently immunogenic as a monomer, our data demonstrate that when presented polyvalently in our design, it can form the basis for a highly immunogenic vaccine. The very low binding:neutralization ratio induced by immunization with RBD nanoparticles suggests that the presentation of RBD on I53-50 focuses on a humoral response to epitopes recognized by neutralizing Abs. This metric could be an important indicator of vaccine safety, as Abs that bind at high levels but do not neutralize, or only weakly neutralize, may contribute to the enhancement of vaccine-related respiratory illness. Our data further show that RBD-12GS-I53-50 induced Ab responses targeting several non-overlapping epitopes recognized by neutralizing Abs identified by RBD. Such polyclonal responses targeting multiple different epitopes may explain the magnitude of neutralization observed and should minimize the risk of escape mutation selection or emergence. Finally, the high production yield of the RBD-I53-50A components and the robust stability of the antigen-containing RBD nanoparticles make them suitable for large-scale production.

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[0125] Method

Table 7

Table 7-2

Table 7-3

[0126] Cell line HEK293F is a female human fetal kidney cell line transformed and adapted for growth in suspension (Life Technologies). HEK293F cells were grown in 293FreeStyle® expression medium (Life Technologies), cultured at 37°C and 8% CO2, and shaken at 130 rpm. Expi293F® cells are derived from the HEK293F cell line (Life Technologies). Expi293F® cells were grown in Expi293® Expression Medium (Life Technologies), cultured at 36.5°C and 8% CO2, and shaken at 150 rpm. VeroE6 is a female kidney epithelial cell line derived from African green monkeys. HEK293T / 17 is a female human fetal kidney cell line (ATCC). HEK-ACE2 adherent cell lines were obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK-293T) Expressioning Human Angiotensin-Converting Enzyme 2, HEK-293T-hACE2 Cell Line, NR-52511. All adherent cells were cultured at 37°C in 8% CO2 in flasks containing DMEM + 10% FBS (Hyclone) + 1% penicillin-streptomycin. Cell lines other than Expi293F have not been tested for or certified for mycoplasma contamination.

[0127] mouse Four-week-old female BALB / c mice were obtained from Jackson Laboratory, Bar Harbor, Maine. Animal procedures were performed under the approval of the Institutional Animal Care and Use Committee of the University of Washington, Seattle, WA and the University of North Carolina, Chapel Hill, NC. Kymab's proprietary IntelliSelect® transgenic mouse platform, known as Darwin®, possesses a complete human antibody locus with unreconstituted human antibody variables and a distinct germline repertoire. Therefore, the antibodies produced by these mice are entirely human.

[0128] Pigtail macaques In this study, two adult male pig-tailed macaques (Macaca nemestrina) were immunized. As previously mentioned, all animals were housed at the Washington National Primate Research Center (WaNPRC), an American Association for the Accreditation of Laboratory Animal Care International (AAALAC) accredited facility (Erasmus et al., 2020). All procedures performed on the animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Washington.

[0129] Convalescent human serum Samples collected between 1 and 60 days post-infection from 31 individuals who tested positive for SARS-CoV-2 by PCR were profiled for anti-SARS-CoV-2 S antibody response, and 29 individuals with anti-S Ab response were maintained in the cohort (Figures 4 and 5). Individuals were enrolled as part of the HAARVI study at the University of Washington in Seattle, Washington. Baseline sociodemographic and clinical data of these individuals are summarized in Table 5. This study was approved by the Institutional Review Board of the Human Subjects Division at the University of Washington (STUDY00000959 and STUDY00003376). All experiments were performed with at least two technical copies and two biological copies (for ELISA and pseudotyped virus neutralization assays). One sample was NIBSC 20 / 130 COVID-19 plasma.

[0130] Plasmid construction The SARS-CoV-2 RBD (BEI NR-52422) construct was synthesized by GenScript into pcDNA3.1- having an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag (GHHHHHHHH) (SEQ ID NO: 164). The construct boundary is N- 328 RFPN 331 and 528 KKST 531 -C (Walls et al., 2020). The SARS-CoV-2S-2P external domain trimer (GenBank: YP_009724390.1, BEI NR-52420) was synthesized by GenScript into pCMV (SEQ ID NO: 165) with an N-terminal mu-phosphatase signaling peptide and a C-terminal TEV cleavage site (GSGRENLYFQG), T4 fibrintinfoldon (GGGSGYIPEAPRDGQAYVRKDGEWVLLSTFL) (SEQ ID NO: 166), and octa-histidine tag (GHHHHHHHH) (SEQ ID NO: 164) (Walls et al., 2020). Constructs were found to have 2P mutations (proline substitution at residues 986 and 987; (Pallesen et al., 2017)) and fulin cleavage sites. 682 SGAG 685Substitutions were included. SARS-CoV-2 RBD was genetically fused to the N-terminus of the trimer I53-50A nanoparticle component using linkers of 8, 12, or 16 glycine and serine residues. RBD-8GS- and RBD-12GS-I53-50A fusions were synthesized and cloned to pCMV by GenScript. The RBD-16GS-I53-50A fusion was cloned to pCMV / R using the Xba1 and AvrII restriction sites and the Gibson assembly (Gibson et al., 2009). All RBD-supported components contained an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag. Macac or human ACE2 external domains were genetically fused at the C-terminus to a thrombin cleavage site and a sequence encoding a human Fc fragment. hACE2-Fc was synthesized and cloned with the BM40 signal peptide by GenScript. To obtain plasmids for transient transfection into Expi293F cells, the plasmids were transformed into E. coli (New England Biolabs) NEB5-α strain for subsequent DNA extraction from bacterial cultures (NucleoBond Xtra Midi® kit). The amino acid sequences of all novel proteins used in this study are listed in Table 3.

[0131] Transient transfection SARS-CoV-2 S and ACE2-Fc proteins were generated in Expi293F cells grown in suspension using Expi293F expression medium (Life Technologies) at 33°C, 70% humidity, 8% CO2, and 150 rpm. The cultures were transfected using PEI-MAX® (Polyscience), and cells were grown to a density of 3 million cells per mL and cultured for 3 days. The supernatant was clarified by centrifugation (4000 rcf for 5 minutes), addition of PDADMAC solution (Sigma Aldrich, #409014) to a final concentration of 0.0375%, and a second spin (4000 rcf for 5 minutes).

[0132] The genes encoding the CR3022 heavy and light chains were ordered from GenScript and cloned into pCMV / R. Antibodies were expressed by transient co-transfection of both the heavy and light chain plasmids in Expi293F cells using PEI MAX® (Polyscience) transfection reagent. As described above, the cell supernatant was collected and clarified after 3 or 6 days.

[0133] Protein purification Proteins containing the His tag were purified from the clarified supernatant by a batch fusion method, in which each clarified supernatant was supplemented with 1 M Tris-HCl, pH 8.0 to a final concentration of 45 mM, and then with 5 M NaCl to a final concentration of approximately 310 mM. The supernatants treated with Talon cobalt affinity resin (Takara) were added and incubated for 15 minutes with gentle shaking. The resin was collected using vacuum filtration with a 0.2 μm filter and transferred to a gravity column. The resin was washed with 20 mM Tris pH 8.0 and 300 mM NaCl, and the proteins were eluted using three column volumes of 20 mM Tris pH 8.0, 300 mM NaCl, and 300 mM imidazole. The batch fusion process was then repeated, and the first and second elutions were combined. Purity was assessed using SDS-PAGE. The RBD-I53-50A fusion protein IMAC eluate was concentrated to >1 mg / mL and dialyzed three times using a Thermo Scientific 10K molecular weight cutoff dialysis cassette to 50 mM Tris, pH 7, 185 mM NaCl, 100 mM arginine, 4.5% glycerol, and 0.75% w / v 3-[(3-collamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The S-2P IMAC eluate fraction was concentrated to approximately 1 mg / mL and dialyzed three times using a Thermo Scientific 10K molecular weight cutoff dialysis cassette to 50 mM Tris, pH 8, 150 mM NaCl, and 0.25% L-histidine. Due to its inherent instability, the S-2P trimer was immediately flash-frozen and stored at -80°C.

[0134] The clarified supernatants of monoclonal antibodies and cells expressing human or macaque ACE2-Fc were purified using an AKTA Avant150FPLC (Cytiva) with a MabSelect PrismA™ 2.6x5cm column (Cytiva). The conjugated antibodies were washed with 5 columns of 20 mM NaPO4, 150 mM NaCl, pH 7.2, followed by 5 columns of 20 mM NaPO4, 1 M NaCl, pH 7.4, and eluted with 3 columns of 100 mM glycine at pH 3.0. The eluate was neutralized with 2 M trisma base to a final concentration of 50 mM. Purity was evaluated using SDS-PAGE.

[0135] Recombinant S309 was expressed as Fab in expiCHO cells transiently co-transfected with plasmids expressing the heavy and light chains, as described above (see transient transfection) (Stettler et al., 2016). The protein was affinity-purified using a HiTrap® Protein A Mab select Xtra® column (Cytiva), followed by desalting with a HiTrap® Fast desalting column (Cytiva) in 20 mM NaPO4, 150 mM NaCl, pH 7.2. The protein was sterilized through a 0.22 μm filter and stored at 4°C until use.

[0136] Bacterial protein expression and purification The I53-50A and I53-50B.4.PT1 proteins were expressed in Lemo21(DE3)(NEB) LB (10g tryptone, 5g yeast extract, 10g NaCl) grown in a 2L baffled shaking flask or a 10L BioFlo320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 of approximately 0.8, and then induced with 1mM IPTG. The expression temperature was lowered to 18°C, and the cells were shaken for approximately 16 hours. Cells were harvested and lysed by microfluidics using Microfluidics M110P in 50mM Tris, 500mM NaCl, 30mM imidazole, 1mM PMSF, and 0.75% CHAPS at 18,000 psi. The lysate was clarified by centrifugation at 24,000 g for 30 minutes and applied to a 2.6 x 10 cm Ni Sepharose® 6FF column (Cytiva) for IMAC purification on an AKTA Avant150 FPLC system (Cytiva). The target protein was eluted with a linear gradient of 30 mM to 500 mM imidazole against a background of 50 mM Tris, pH 8, 500 mM NaCl, and 0.75% CHAPS buffer. The peak fractions were pooled, concentrated through a 10K MWCO centrifuge filter (Millipore), and sterile filtered (0.22 μm). The elutions were then applied to Superdex® 200 Increase 10 / 300 or HiLoad® S200pg GL SEC columns (Cytiva) using 50 mM Tris, pH 8, 500 mM NaCl, and 0.75% CHAPS buffer. I53-50A eluted approximately 0.6 column volume (CV). I53-50B.4PT1 eluted approximately 0.45 CV. After sizing, bacterial components were tested to confirm low endotoxin levels before use in nanoparticle aggregates.

[0137] In vitro nanoparticle aggregate The total protein concentration of the purified individual nanoparticle components was determined by measuring the absorbance at 280 nm using a UV / vis spectrophotometer (Agilent Cary8454) and calculating the extinction coefficient (Gasteiger et al., 2005). The assembly process was carried out at room temperature, with the following steps added in order: RBD-I53-50A trimer fusion protein, then additional buffer to achieve the desired final concentration as needed, and finally I53-50B.4PT1 pentamer component (50 mM Tris, pH 8, 500 mM NaCl, 0.75% w / v CHAPS), with a molar ratio of RBD-I53-50A:I53-B.4PT1 of 1.1:1. To produce partially valence RBD-I53-50 nanoparticles (50% RBD-I53-50), both RBD-I53-50A and unmodified I53-50A trimers (50 mM Tris, pH 8, 500 mM NaCl, 0.75% w / v CHAPS) were added to I53-50B.4PT1 in a slight molar excess (1.1x). All RBD-I53-50 in vitro assemblies were incubated at 2–8°C with gentle shaking for at least 30 minutes before subsequent purification by SEC to remove any remaining aggregated and unassembled components. Various columns were used depending on the purpose. Superose® 6Increase 10 / 300GL columns were used analytically to estimate nanoparticle size, Superdex® 200Increase 10 / 300GL columns were used for small pilot assemblies, and HiLoad® 26 / 600Superdex® 200pg columns were used for nanoparticle generation. The assembled particles eluted in approximately 11 mL of void volume using Superose® 6 and Superdex® 200 columns. The assembled nanoparticles were aseptically filtered (0.22 μm) immediately before column application and after fractionation pooling.

[0138] HACE2-Fc and CR3022 digestion hACE2-Fc was digested with thrombin protease (Sigma Aldrich) in the presence of 2.5 mM CaCl2 at a thrombin:protein ratio of 1:300 w / w. The reaction mixture was incubated at ambient temperature for 16–18 hours with gentle shaking. After incubation, the reaction mixture was concentrated using an Ultracel® 10K centrifugation filter (Millipore Amicon Ultra) and filtered by sterile filtration (0.22 μM). The cleaved hACE2 monomers were separated from the uncleaved hACE2-Fc and cleaved Fc regions using protein A purification (see protein purification above) on a HiScreen MabSelect SuRe® column (Cytiva) with an AKTA avant25FPLC (Cytiva). The cleaved hACE2 monomers were collected by flow-through, filtered by sterile filtration (0.22 μm), and quantified by UV / vis.

[0139] LysC (New England BioLabs) was diluted to 10 ng / μL in 10 mM Tri pH 8 and added to CR3022IgG in a 1:2000 w / w LysC:IgG ratio. The mixture was then incubated at 37°C for 18 hours with orbital shaking at 230 rpm. The cleavage reaction product was concentrated using an Ultracel® 10K centrifuge filter (Millipore Amicon Ultra) and filtered sterile (0.22 μM). The cleaved CR3022 mAb was separated from the Fc portion of the uncleaved CR3022IgG and the cleaved IgG using protein A purification as described above. The cleaved CR3022 was collected by flow-through, filtered sterile (0.22 μm), and quantified by UV / vis.

[0140] Biolayer interference (antigenic) Antigenicity assays were performed and analyzed using BLI with an Octet® Red 96 System (Pall Forte Bio / Sartorius) at ambient temperature with shaking at 1000 rpm. The RBD-I53-50A trimer component and monomer RBD were diluted to 40 μg / mL in Kinetics buffer (1x HEPES-EP+ (Pall Forte Bio), 0.05% skim milk, 0.02% sodium azide). The monomers hACE2 and CR3022Fab were diluted to 750 nM in Kinetics buffer and serially diluted three-fold to a final concentration of 3.1 nM. The reagents were applied to a Black 96-well Greiner Bio-one microplate at a rate of 200 μL per well, as described below. The RBD-I53-50A component or monomer RBD was immobilized on an Anti-Penta-HIS (HIS1K) biosensor according to the manufacturer's instructions (Forte Bio), except for using the following sensor incubation times. The HIS1K biosensor was hydrated in water for 10 minutes, then equilibrated in Kinetics buffer for 60 seconds. Diluted trimer RBD-I53-50A component or monomer RBD was loaded onto the HIS1K tip for 150 seconds and washed in Kinetics buffer for 300 seconds. The association step was performed by immersing the immunogen-immobilized HIS1K biosensor in diluted hACE2 monomer or CR3022Fab for 600 seconds, and then measuring dissociation by inserting the biosensor into Kinetics buffer for 600 seconds. Pall® ForteBio / Sartorius analysis software (version 12.0) was used to subtract baselines from the data and fit the plots. The plot in Figure 8 shows the processes of association and dissociation.

[0141] Biolayer Interferometry (Accessibility) The binding of mACE2-Fc, CR3022IgG, and S309IgG to RBD, RBD-I53-50A trimers, and RBD-I53-50 nanoparticles was analyzed for accessibility experiments and real-time stability studies using the Octet® Red96 system (Pall® ForteBio / Sartorius) with shaking at 1000 rpm at ambient temperature. Protein samples were diluted to 100 nM with Kinetics buffer. The buffer, immunogen, and analyte were then applied to a Black 96-well Greine Bio-one microplate at 200 μL per well. Protein A biosensors (ForteBio / Sartorius) were first hydrated in Kinetics buffer for 10 minutes, and then immersed in either mACE2-Fc, CR3022, or S309IgG diluted to 10 μg / mL in Kinetics buffer in the immobilization step. After 500 seconds, the chip was transferred to Kinetics buffer for 60 seconds to reach baseline. The association step was performed by immersing the loaded biosensor in immunogen for 300 seconds, and subsequent dissociation was performed by immersing the biosensor in Kinetics buffer for another 300 seconds. The data were subtracted from baseline before plotting using ForteBio analysis software (version 12.0). The plot in Figure 2 shows the association and dissociation over 600 seconds.

[0142] Negative staining electron microscope RBD-I53-50 nanoparticles were first diluted to 75 μg / mL with 50 mM Tris, pH 7, 185 mM NaCl, 100 mM arginine, 4.5% v / v glycerol, and 0.75% w / v CHAPS. The S-2P protein was diluted to 0.03 mg / mL with 50 mM Tris, pH 8, 150 mM NaCl, and 0.25% L-histidine. 3 μL of each sample was applied to a 300-mesh copper grid that had just been glow-discharged. After incubating the sample on the grid for 1 minute, the grid was immersed in 50 μL of water droplets, and excess liquid was absorbed with filter paper (Whatman). Next, the grid was immersed in 6 μL of 0.75% w / v uranyl formate staining solution. The staining solution was absorbed with filter paper, and then the grid was immersed in another 6 μL of staining solution and incubated for approximately 70 seconds. Finally, the staining solution was absorbed and the grid was dried for 1 minute. The prepared grids were imaged at 45,000x (nanoparticles) or 92,000x (S-2P) using a Talos model L120C electron microscope.

[0143] Dynamic light scattering The hydrodynamic diameter (Dh) and % polydispersity (%Pd) of RBD-I53-50 nanoparticle samples were measured using dynamic light scattering (DLS) at UNcle Nano-DSF (UNchained Laboratories). Samples were applied to 8.8 μL quartz capillary cassettes (UNi, UNchained Laboratories), and measurements were taken using 10 acquisitions of 5 seconds each, with automatic laser decay. The increase in viscosity due to 4.5% v / v glycerol in the RBD nanoparticle buffer was explained by UNcle® Client software in the Dh measurement.

[0144] Guanidine HCl denaturation Monomer RBD, RBD-I53-50A fusion protein, and RBD-I53-50 nanoparticle immunogen were diluted to 2.5 μM with 50 mM Tris, pH 7.0, 185 mM NaCl, 100 mM arginine, 4.5% v / v glycerol, and 0.75% w / v CHAPS, and prepared in triplicates with guanidine chloride [GdnHCl] ranging from 0 M to 6.5 M (increasing by 0.25 M). The S-2P trimer was also diluted to 2.5 μM using 50 mM Tris, pH 8, 150 mM NaCl, 0.25% L-histidine, and the same GuHCl concentration range. The dilutions were mixed 10-fold by pipetting. The samples were then incubated at ambient temperature for 18–19 hours. Using Nano-DSF (UNcle®, UNchained Laboratories) and an 8.8 μL quartz capillary cassette (UNi®, UNchained Laboratories), fluorescence spectra were collected in triplicate, excited at 266 nm, and emission from 200 nm to 750 nm was measured at 25°C.

[0145] Endotoxin measurement Endotoxin levels in protein samples were measured using the EndoSafe® Nexgen-MCS System (Charles River). Samples were diluted 1:50 or 1:100 with endotoxin-free LAL reagent water and applied to the wells of the EndoSafe® LAL reagent cartridge. Endotoxin content was analyzed and the dilution factor was automatically calculated using Charles River EndoScan®-V software. Endotoxin values ​​were reported as EU / mL and then converted to EU / mg based on UV / vis measurements. The threshold for samples suitable for immunization was <50 EU / mg.

[0146] UV / vis Ultraviolet-visible spectrophotometry (UV / vis) was performed using an Agilent Technologies Cary® 8454 instrument. Samples were applied to 10 mm, 50 μL quartz cells (Starna Cells, Inc.), and absorbance was measured from 180 to 1000 nm. Protein concentration was obtained using the net absorbance at 280 nm, obtained from the measurement and baseline subtraction of a single reference wavelength, along with the calculated extinction coefficient and molecular weight. The relative aggregation level of real-time stability study samples was determined using the absorbance ratio at 320 / 280 nm. Samples were diluted with their respective purification / instrument blanking buffers to obtain absorbances from 0.1 to 1.0. All data generated from the UV / vis instrument were processed with 845x UV / visible system software.

[0147] Glycan profiling A bottom-up mass spectrometry (MS) approach was used to identify site-specific glycosylation profiles, including the determination of glycoform distribution and occupancy. Fixed amounts of 1 mg / mL monomer, 8GS, 12GS, and 16GS RBD protein were prepared to evaluate the glycosylation profiles of four RBD variants at N331 and N343. Comprehensive glycosphing of the stabilized spike outer domain (S-2P) was performed in parallel using 1.5 mg / mL SARS-CoV-2 S-2P protein. All samples were decomposed at 90°C for 30 minutes in a solution containing 25 mM Tris (pH 8.0), 7 M guanidinium chloride (GdnHCl), and 50 mM dithiothreitol (DTT). Reduced cysteine ​​was alkylated by adding fresh iodoacetamide (IAA) to 100 mM and incubating in the dark at room temperature for 1 hour. Next, the remaining IAA was quenched by adding 50 mM excess DTT. The GndHCl concentration was reduced to 0.6 M by diluting the sample 11-fold with 10 mM Tris (pH 8.0) and 2 mM calcium chloride solution. Then, each sample was divided in half. One half (275 μL) was mixed with 10 units of recombinant peptide N-glycanase F (GST-PNGase F) (Krenkova et al., 2013) and incubated at 37°C for 1 hour to convert glycosylated Asn to deglycosylated Asp.

[0148] Protease digestion was performed as follows: All RBD samples and one S-2P sample were digested at 37°C for 4 hours in a ratio of 1:40 (w / w) for RBD and 1:30 (w / w) for S-2P, followed by overnight Glu-C digestion under the same ratios and conditions. The other three S-2P samples were digested overnight at 37°C using trypsin, chymotrypsin, and α-soluble protease, each in a ratio of 1:30 (w / w). All digestive proteases used were MS grade (Promega). The digestion reaction was quenched the following day with 0.02% formic acid (FA, Optima®, Fisher).

[0149] Glycoform analysis of four S-2P samples was performed by nanoLC-MS using an Orbitrap Fusion® mass spectrometer (Thermo Fisher). Digested samples were desalted using a Sep-Pak C18 cartridge (Waters) according to the manufacturer's recommended protocol. 2cm trap and 35cm analytical columns were freshly prepared with fused silica (100μm inner diameter) containing 5μM ReproSil-Pur® C18AQ beads (Dr.Maisch). 8μL of sample was injected and analyzed by a linear gradient of 2% to 30% acetonitrile in 0.1% FA for 60 minutes, followed by analysis in 80% acetonitrile for 10 minutes. The EThcD method was optimized as follows: Ion source: 2.1kV in positive mode, ion transfer tube temperature: 350°C, resolution: MS 1 =120000, MS 2 =30000, AGC target: MS 1 =2e 5 MS 2 =1e 5 , and injection time: MS 1 = 50 milliseconds, MS 2 = 60ms.

[0150] Glycopeptide data were visualized and processed using Byonic® and Byologic® (version 3.8, Protein Metrics Inc.) with precursor mass tolerances of 6 ppm and fragment mass tolerances of 10 ppm. Glycopeptides were searched using the Protein Metrics PMI-Suite N-glycan 309 mammalian database and scored based on the correct assignment of c- and z-fragment ions. True positive entities were further verified by the presence of glycan oxonium ions m / z in 204 (HexNAc ions) and 366 (HexNAcHex ions), and the absence of corresponding spectra in deglycosylated samples. The relative abundance of each glycoform was determined by peak area analyzed with Byologic®. Glycoforms were classified into oligo(oligomannose), hybrid, complex, and complex subtypes, as described in previous studies (Watanabe et al., 2020). HexNAc(2)Hex(9-5) are M(annose)9-M5, HexNAc(3)Hex(5-6) are classified as hybrids, HexNAc(3)Hex(3-4)X are A1 subtype, HexNAc(4)X are A2 / A1B, HexNAc(5)X are A3 / A2B, and HexNAc(6)X are A4 / A3B subtypes. Hybrids and complexes with fucosylation are described separately as FHybrid and FComplex (e.g., FA1), respectively.

[0151] Glycan occupation analysis and glycoform determination of the four RBD variants were performed by LC-MS on a SynaptG2-Si® TOF mass spectrometer coupled to an Acquity® ULC system (Waters). Samples were separated over 30 minutes on a Waters CSH C18 1x100 mm 1.7 μm column with a linear gradient from 3% to 40% B (A: 98% water, 2% acetonitrile, 0.1% FA; B: 100% acetonitrile, 0.1% FA). Data-dependent acquisition (DDA) methods were used with a precursor mass range of 300–2000, MS / MS mass range of 50–2000, and collision energies of a gradient of 70–100 V. MassLynx® (Waters) was used to determine and integrate the most numerous, non-overlapping isotope peaks. Unless otherwise specified, all water and organic solvents used were MS grade (Optima®, Fisher). The glycan occupancy rate at each site was measured using the peak area ratio of unglycosylated (Asn) glycopeptides to deglycosylated (Asp) glycopeptides.

[0152] Hydrogen / deuterium exchange mass spectrometry 3 μg of monomer RBD and RBD-8GS-I53-50A were incubated in deuterated buffer (pH*7.6, 85% D2O, Cambridge Isotope Laboratories, Inc.) for 3, 60, 1800, and 72000 seconds at 23°C, followed by HDX with H / D exchange. Subsequently, the samples were mixed 1:1 with ice-cold quench buffer (200 mM tris(2-chloroethyl) phosphate (TCEP), 8 M urea, 0.2% formic acid) to a final pH of 2.5 and immediately flash-frozen in liquid nitrogen. As previously described, the samples were digested inline with pepsin and analyzed by LC-MS-IMS using a Synapt G2-Si™ TOF mass spectrometer (Waters) (Verkerke et al., 2016) with an 18-minute gradient applied. A complete deuterated control was prepared by collecting pepsin digestate eluate from non-deuterated sample LC-MS runs, drying it in a speedvac, incubating it in deuterated buffer at 85°C for 1 hour, and quenching it in the same manner as all other HDX samples. Internal exchange standards (Pro-Pro-Pro-Ile [PPPI] and Pro-Pro-Pro-Phe [PPPF]) were added to each sample to ensure consistent labeling conditions across all samples (Zhang et al., 2012). Pepsin digests from non-deuterated samples were also analyzed by nanoLC-MS using an Orbitrap Fusion® mass spectrometer (Thermo Fisher) with the above settings for sugar profiling. The data were then processed with Byonic® to obtain a peptide reference list. Peptides were manually validated using DriftScope® (Waters) and identified by orthogonal retention time (rt) and drift time (dt) coordinates. Deuterium uptake analysis was performed using HX-Express v2 (Guttman et al., 2013; Weis et al., 2006). Binary fitting was applied to identify peaks from the peptide spectra. Deuterium uptake levels were normalized against a fully deuterated standard.

[0153] Mouse immunization and challenges Female BALB / c (stock: 000651) mice were purchased at 4 weeks of age from The Jackson Laboratory, Bar Harbor, Maine, and maintained at the Comparative Medicine Facility at the University of Washington, Seattle, WA, which is accredited by the American Association for the Accreditation of Laboratory Animal Care International (AAALAC). At 6 weeks of age, 10 mice per treatment group were primed, and a second booster dose was administered 3 weeks later. Prior to inoculation, the immunogen suspension was gently mixed with AddaVax® adjuvant (Invivogen, San Diego, CA) at a 1:1 vol / vol ratio to reach a final antigen concentration of 0.009 or 0.05 mg / mL. Under isoflurane anesthesia, 50 μL of immunogen (100 μL total) was intramuscularly injected into the gastrocnemius muscle of each hind limb using a 27-gauge needle (BD, San Diego, CA) at each injection site. To obtain serum, blood was collected from all mice two weeks after prime and boost. Blood was collected by subchinicular venipuncture and allowed to coagulate in 1.5 mL plastic Eppendorf tubes at room temperature for 30 minutes. Serum was separated from hematocrit by centrifugation at 2000 g for 10 minutes. Complement factors and pathogens in the separated serum were inactivated by heat incubating the serum at 56°C for 60 minutes. The serum was stored at 4°C or -80°C until use. Six weeks after boost, the mice were exported from the Comparative Medicine Facility at the University of Washington, Seattle, WA to the AAALAC accredited Animal Biosafety Level 3 (ABSL3) Laboratory in Chapel Hill, North Carolina. After a 7-day acclimatization period, the mice were anesthetized with a ketamine / xylazine mixture and given 10 doses of the SARS-CoV-2MA strain adapted to the mice to evaluate the efficacy of the vaccine. 5Plaque-forming units (pfus) were used to challenge the nasal cavity (IACUC protocol 20-114.0). Post-infection lung and nasal turbinate tissues were collected and viral load was assessed by plaque assay. Body weight was monitored daily until the end of the study on day 2 post-infection. All experiments were conducted at the University of Washington, Seattle, WA and the University of North Carolina, Chapel Hill, NC, in accordance with approved Institutional Animal Care and Use Committee protocols.

[0154] Immunized (Kymab Darwin® mouse) Kymab Darwin® mice (mixed males and females, 10 weeks old) were primed at a rate of 5 mice per treatment group, with a second vaccination administered 3 weeks later. Prior to inoculation, the immunogen suspension was gently mixed with AddaVax® adjuvant (Invivogen) at a 1:1 vol / vol ratio to reach a final antigen concentration of 0.009 or 0.05 mg / mL. Under isoflurane anesthesia, mice were intramuscularly injected into the tibialis muscle of each hind limb using a 30-gauge needle (BD) with 20 μL of immunogen per injection site (40 μL total). The final boost was administered intravenously (50 μL) without adjuvant at 7 weeks. Mice were sacrificed 5 days later according to UK Home Office Schedule 1 (increased CO2 concentration), and the spleen, lymph nodes, and bone marrow were cryopreserved. Whole blood (0.1 ml) was collected 2 weeks after each administration (final blood collection at weeks 0, 2, 5, and 8). Serum was separated from hematocrit by centrifugation at 2000g for 10 minutes. The serum was stored at -20°C and used for titer monitoring by ELISA. All mice were maintained, and all procedures were carried out under UK Home Office license 70 / 8718, with the approval of the Wellcome Trust Sanger Institute Animal Welfare and Ethical Review Body.

[0155] ELISA For anti-S-2P ELISA, 25 μL of 2 μg / mL of S-2P was plated into a 384-well Nunc Maxisorp® (ThermoFisher) plate in PBS and sealed overnight at 4°C. The following day, the plate was washed four times with Tris Buffered Saline Tween (TBST) using a plate washer (BioTek) and blocked with 2% BSA in TBST at 37°C for 1 hour. The plate was washed four times with TBST, and 1:5 serial dilutions of mouse, NHP, or human serum were added in 25 μL of TBST, starting at 1:25 or 1:50, and incubated at 37°C for 1 hour. The plate was washed four times with TBST, and 25 μL of anti-mouse (Invitrogen) or anti-human (Invitrogen) horseradish peroxidase-conjugated antibody, diluted 1:5,000, was added to each well and incubated at 37°C for 1 hour. The plate was washed four times with TBST, and 25 μL of TMB (SeraCare) was added to each well at room temperature for 5 minutes. The reaction was quenched by adding 25 μL of 1N HCl. The plate was immediately read at 450 nm with a VarioSkanLux™ plate reader (ThermoFisher), the data were plotted, and the nonlinear regression sigmoid was fitted using 4PL (GraphPad), and the EC curve was fitted from the curve. 50 To determine the value, X is log(concentration).

[0156] Generation of pseudoviruses MLV-based SARS-CoV-2S, SARS-CoV S, and WIV-1 pseudotypes were prepared as described above (Millet and Whittaker, 2016; Walls et al., 2020). Briefly, HEK293T cells were cotransfected with Lipofectamine® 2000 (Life Technologies) along with a plasmid encoding S, an MLV Gag-Pol packaging construct, and an MLV transfer vector encoding a luciferase reporter, according to the manufacturer's instructions. The cells were washed three times with Opti-MEM and incubated in transfection medium at 37°C for 5 hours. DMEM containing 10% FBS was added for 60 hours. The supernatant was collected by 2,500 g spinning, filtered through a 0.45 μm filter, concentrated at 2,500 g for 10 minutes on a 100 kDa membrane, then reduced to a constant volume and stored at -80°C.

[0157] Pseudovirus Invasion and Serum Neutralization Assay HEK-hACE2 cells were cultured in DMEM with 10% FBS (Hyclone) and 1% PenStrep in a 37°C incubator (ThermoFisher) with 8% CO2. One day before infection, 40 μL of polylysine (Sigma) was added to a 96-well plate and incubated for 5 minutes with rotation. The polylysine was removed, the plate was dried for 5 minutes, and then washed once with DMEM before plating the cells. The following day, the cells were confirmed to be 80% confluent. In half of the 96-well plate, serum was serially diluted 1:3 times in DMEM to initiate initial dilutions of 1:3 to 1:66 with a final volume of 22 μL. Then, 22 μL of pseudotyped virus was added to the serial dilutions and incubated at room temperature for 30–60 minutes. The HEK-hACE2 plate medium was removed, 40 μL of serum / virus mixture was added to the cells, and incubated at 37°C with 8% CO2 for 2 hours. After incubation, 40 μL of DMEM containing 20% ​​FBS and 2% PenStrep was added to the cells for 48 hours. After 48 hours of infection, One-Glo-EX® (Promega) was added to the cells at half the culture volume (40 μL), incubated in the dark for 5 minutes, and then read on a Varioskan® LUX plate reader (ThermoFisher). Measurements were performed in at least two sequences for all 10 mouse serum samples from each group. Relative luciferase units were plotted and normalized using Prism® (GraphPad) with zero values ​​for cells only and 100% values ​​for 1:2 virus only. IC was calculated from curve fitting using nonlinear regression of log (inhibitor) vs. normalized response. 50 The values ​​were determined. The Mann-Whitney test was used to compare the two groups and determine whether they were statistically different.

[0158] Generation of live viruses SARS-CoV-2-nanoLuc virus (WA1 strain) in which ORF7 was replaced with the nanoluciferase gene (nanoLuc), and mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA) (Dinnon et al., 2020) were generated using the previously described coronavirus reverse genetics system (Hou et al., 2020). Recombinant viruses were generated in E6 cells (ATCC-CRL1586) grown in DMEM high-glucose medium (Gibco#11995065) supplemented with 10% Hyclone® Fetal Clone II (GE#SH3006603HI), 1% non-essential amino acids, and 1% Pen / Strep, in a 37°C + 5% CO2 incubator. To generate recombinant SARS-CoV-2, seven DNA fragments encoding the entire full-length SARS-CoV-2 genome, each containing a 5'T7 promoter and a 3' poly-A tail, were ligated in vitro. The transcribed RNA was electroporated into Vero E6 cells to generate a P0 virus stock. Seed viruses were amplified twice in Vero E6 cells at low MOI for 48 hours to create working stocks, whose titers were measured by plaque assay (Hou et al., 2020). All live virus experiments, including the ligation and electroporation steps, were performed under biosafety level 3 (BSL-3) conditions, under negative pressure, by operators in Tyvek suits wearing personal powered air purifiers.

[0159] Luciferase-based serum neutralization assay, SARS-CoV-2-nanoLuc 24 hours before the assay, Vero E6 cells were divided into 2x10⁶ cells. 4Cells were seeded in 96-well plates at a rate of cells / well. 100 pfu of SARS-CoV-2-nanoLuc virus (Hou et al., 2020) was mixed with serum in a 1:1 ratio and incubated at 37°C for 1 hour. For each sample, an 8-point 3-fold dilution curve was created at an initial concentration of 1:20 (standard) or 1:2000 (high-neutralizing agent). The virus and serum mixture was added to each well and incubated at 37°C + 5% CO2 for 48 hours. Luciferase activity was measured by the Nano-Glo® luciferase assay system (Promega, WI) using a SpectraMax® M3 illuminometer (Molecular Device) according to the manufacturer's protocol. Percent inhibition and 50% inhibitory concentrations (IC50) were calculated by the following formula: [1 - (RLU containing sample / RLU containing mock treatment)] x 100%. 50% inhibitory titer (IC50) 50 The values ​​were calculated in GraphPad Prism® 8.3.0 by fitting data points using a sigmoid dose-response (variable gradient) curve.

[0160] Formation of tetramers Recombinant SARS-CoV-2 S-2P trimers were biotinylated using the EZ-Link® Sulfo-NHS-LC biotinylation kit (ThermoFisher) and tetramerized with streptavidin-APC (Agilent) as described above (Krishnamurty et al., 2016; Taylor et al., 2012). The RBD domain of SARS-CoV-2 S was biotinylated and tetramerized with streptavidin-APC (Agilent). The APC decoy reagent was generated by conjugating SA-APC to Dylight® 755 using the DyLight755 antibody labeling kit (ThermoFisher), washing and removing the unbound DyLight755, and incubating in excess unrelated biotinylated His-tagged protein. PE decoys were generated in the same manner by conjugating SA-PE to Alexa Fluor647 using an AF647 antibody labeling kit (ThermoFisher).

[0161] Mouse immunization, cell enrichment, and flow cytometry To investigate the B cell phenotype, 6-week-old female BALB / c mice (3 mice per treatment group) were immunized intramuscularly on day 0 with 50 μL of a vaccine formulation per injection site containing 5 μg of SARS-CoV-2 antigen (S-2P trimer or RBD, but excluding the mass from I53-50 nanoparticles) mixed with AddaVax® adjuvant in a 1:1 vol / vol ratio. All experimental mice were euthanized on day 11 for inguinal and popliteal lymph node collection. The experiment was repeated twice. Popliteal and inguinal lymph nodes were collected and pooled for individual mice. Cell suspensions were prepared by grinding the lymph nodes and filtering through a 100 μM Nitex® mesh. Cells were resuspended in PBS containing 2% FBS and Fc block (2.4G2) and incubated with a 10 nM decoy tetramer at room temperature for 20 minutes. RBD-PE tetramer and Spike-APC tetramer were added at a concentration of 10 nM and incubated on ice for 20 minutes. Cells were washed and incubated with anti-PE and anti-APC magnetic beads on ice for 30 minutes, then passed through a magnetized LS column (Miltenyi Biotec). Binding B cells were stained with anti-mouse B220 (BUV737), CD3 (PerCP-Cy5.5), CD138 (BV650), CD38 (Alexa Fluor® 700), GL7 (eFluor® 450), IgM (BV786), IgD (BUV395), CD73 (PE-Cy7), and CD80 (BV605) on ice for 20 minutes. Cells were run on Cytek Aurora® and analyzed using FlowJo® software (Treestar). Cell counts were determined using Accucheck® cell counting beads.

[0162] NHP immunization Pig-tailed macaques were immunized with 250 μg of RBD-12GS-I53-50 nanoparticles (88 μg of RBD antigen) on days 0 and 28. Blood was collected on days 0, 10, 14, 28, 42, and 56 postpriming. Serum and plasma were collected as described above (Erasmus et al., 2020). Before vaccination or blood collection, animals were sedated with intramuscular injection of ketamine (Ketaset®; Henry Schein) (10 mg / kg). Before inoculation, the immunogen suspension was gently mixed with AddaVax® adjuvant (Invivogen, San Diego, CA) at a 1:1 vol / vol ratio to reach a final antigen concentration of 0.250 mg / mL. The vaccine was delivered intramuscularly to both quadriceps muscles at a dose of 1 mL per injection site on days 0 and 28. All injection sites were shaved before injection, and signs of local reaction were monitored post-injection. As previously mentioned, a complete physical examination and general health assessment were performed on the animals at each study time point (Erasmus et al., 2020), and no adverse events were observed.

[0163] Competitive Biolayer Interferometry The purification of Fab from NHP serum was adopted from Boyoglu-Barnum et al., 2020. Briefly, 1 mL of serum from day 56 was diluted to 10 mL with PBS and incubated overnight at 37°C with 1 mL of 3x PBS-washed protein A beads (GenScript) while stirring. The following day, the beads were thoroughly washed with PBS using a gravity flow column, and the conjugated antibody was eluted with 0.1 M glycine pH 3.5 in 1 M Tris-HCl (pH 8.0) to a final concentration of 100 mM. The serum and initial washing solution that passed through were re-conjugated to the beads overnight, and the elution process was repeated. IgG was concentrated (Amicon 30 kDa), and the buffer was changed to PBS. 2x digestion buffer (40 mM sodium phosphate pH 6.5, 20 mM EDTA, 40 mM cysteine) was added to the concentrated and pooled IgG. 500 μL of resuspended and immobilized papain resin (ThermoFisher Scientific), freshly washed with 1x digestion buffer (20 mM sodium phosphate, 10 mM EDTA, 20 mM cysteine, pH 6.5), was added to purified IgG in 2x digestion buffer, and the sample was stirred at 37°C for 5 hours. The supernatant was separated from the resin, the resin washing solution was collected, and pooled together with the resin flow-through. The pooled supernatant was sterile filtered through 0.22 μm and applied six times to PBS-washed protein A beads in a gravity flow column. The column was eluted as described above, and the papain procedure was repeated overnight with undigested IgG to increase yield. The protein A flow-through was pooled and concentrated (Amicon 10 kDa), and the buffer was replaced with PBS. Purity was confirmed by SDS-PAGE.

[0164] Epitope competition was performed and analyzed using BLI with the Octet® Red 96 System (Pall® Forte Bio / Sartorius) under shaking at 1000 rpm at 30°C. NTA biosensors (Pall® Forte Bio / Sartorius) were hydrated in water for at least 10 minutes and equilibrated in 10x Kinetics buffer (KB) (Pall® Forte Bio / Sartorius) for 60 seconds. After loading 10 ng / μL monomer RBD in 10x KB for 100 seconds, baseline was acquired in 10x KB for 300 seconds. The tips were then immersed in polyclonal Fab diluted in 10x KB for 2000 seconds, starting at 5000 nM and serially diluting in a 1:3 ratio, or maintained in 10x KB. The tips were bound at varying levels depending on the polyclonal Fab concentration. Next, the chips were immersed in either 200 nM hACE2, 400 nM CR3022, or 20 nM S309 of the same concentration and incubated for 300–2000 seconds. Using Pall® Forte Bio / Sartorius analysis software (version 12.0), the data were baseline subtracted, the polyclonal Fab was preloaded and aligned, and plotted in PRISM®.

Claims

1. (a) A plurality of first aggregates, each first aggregate comprising a plurality of identical first proteins, (b) A nanoparticle comprising a plurality of second aggregates, each second aggregate containing a plurality of second proteins, Each of the first proteins comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 152 to 159, and each of the second proteins comprises a polypeptide selected from the group consisting of SEQ ID NOs: 2, 14, and 22. The plurality of first aggregates interact non-covalently with the plurality of second aggregates to form the nanoparticles. The nanoparticles present, on their surface, an immunogenic portion of the SARS-CoV-2 antigen or its variant or homolog present in the one second protein.

2. The nanoparticles according to claim 1, wherein the plurality of second aggregates comprise a total of 2, 3, 4, 5, 6, 7, 8 or more polypeptides, each containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 14, and 22.

3. The nanoparticle according to claim 1, wherein all second aggregates comprise a second protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 14, and 22.

4. The nanoparticles according to claim 1, wherein all second proteins contain amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 14, and 22.

5. Each of the first proteins comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 152 to 155, Sequence ID 152 is a sequence ID 152 with residue 1 deleted. Sequence ID 153 is obtained by deleting residue 1 of sequence ID 153, Sequence ID 154 is missing residue 1 of sequence ID 154, or The nanoparticle according to claim 1, wherein sequence number 155 is missing residue 1 of sequence number 155.

6. The nanoparticle according to claim 1, wherein the first protein comprises the amino acid sequence of SEQ ID NO:

155.

7. The nanoparticle according to claim 1, wherein each first aggregate is a pentamer and each second aggregate is a trimer.

8. A composition comprising a plurality of nanoparticles according to any one of claims 1 to 7.

9. A nucleic acid molecule encoding a polypeptide selected from the group consisting of Sequence ID Nos. 2, 14, and 22.

10. The nucleic acid molecule according to claim 9, wherein the nucleic acid molecule is mRNA.

11. An expression vector comprising a nucleic acid molecule according to claim 9 or 10, operably linked to a suitable control sequence.

12. A cell comprising the nanoparticles described in any one of claims 1 to 7.

13. A cell comprising the expression vector described in Claim 11.

14. A pharmaceutical composition, (a) Nanoparticles according to any one of claims 1 to 7, (b) A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

15. The pharmaceutical composition according to claim 14, wherein the pharmaceutical composition further comprises an adjuvant.

16. A vaccine comprising nanoparticles according to any one of claims 1 to 7.

17. Nanoparticles according to any one of claims 1 to 7, for use in treating or limiting the onset of SARS-CoV-2 infection in a subject.

18. The nanoparticles according to claim 17, wherein the subject is not infected with SARS-CoV-2.

19. The nanoparticles according to claim 17, wherein the subject is infected with a severe acute respiratory (SARS) virus, including but not limited to SARS-CoV-2.

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