SARS-COV-2 receptor binding domain nanoparticles and methods of use thereof

Abstract

Disclosed herein are receptor binding domain (RBD) SARS-CoV-2 immunogens, engineered nanoparticle vaccines comprising receptor binding domain (RBD) SARS-CoV-2 immunogens and methods of use thereof for SARS- CoV-2 vaccines.

Description

SARS-COV-2 RECEPTOR BINDING DOMAIN NANOPARTICLES AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 701,255, filed September 30, 2024, which is hereby incorporated by reference herein in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an XML Document formatted sequence listing with a file name “206193-0I46-00WO_Sequence_Listing.xml,” creation date of September 30, 2025, and having a size of 37,014 bytes. The sequence listing submitted via EFS-Web is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] New seasonal outbreaks fueled by emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to highlight the urgent need for next-generation vaccines that are resilient to antigenic drift. The World Health Organization (WHO) designates variants under monitoring (VUM), of interest (VOI), and of concern (VOC) to identify the viruses that pose the greatest present threats to public health (World Health Organization, 2023b). Variants can arise from errors during replication, recombination (Goldstein et al., 2022, Genome Biol Evol, 14; Turakhia et al., 2022, Nature, 609, 994-997), replication in immunocompromised individuals (Weigang et al., 2021, Nat Commun, 12, 6405; Choi et al., 2020, N Engl J Med, 383, 2291-2293; Scherer et al., 2022, NEngl J Med, 386, 2436-2438), and spillback events (Oude Munnink et al., 2021, Science, 371, 172-177; Kuchipudi et al., 2022, Proc Natl Acad Sci USA, 119:e2121644119; Yen et al., 2022, Lancet, 399, 1070-1078), posing an ongoing threat via immune evasion to vaccines and monoclonal antibody therapies (Wang et aL, 2021, Nature, 592, 616-622; Clark et al., 2021, Cell, 184, 2605- 2617 e!8; Cao et al., 2022, Nature, 602, 657-663; Liu et al., 2022, Nature, 602, 676-681).In May 2021, VOC B.1.617.2 (Delta) emerged (World Health Organization, 2023a) with 8 mutations as well as deletions in the spike relative to ancestral, US A-WA 1 / 2020 (Hodcroft, 2024, covariants.org / variants). In November 2021, VOC B.1.1.529 (Omicron) emerged (World Health Organization, 2023a) and was the most dramatic antigenic drift yet, with over 30 substitutions in addition to deletions and insertions in the spike relative to USA-WA1 / 2020 (Hodcroft, 2024, covariants.org / variants). Due to these events, vaccine efficacy against symptomatic Omicron infection dropped by nearly 30% relative to ancestral efficacy (Zou et al., 2022, Front Public Health, 10, 940956). Strategies to mitigate the immune evasion of future viruses remains an important goal to protect from severe disease.

[0004] Early in the pandemic, receptor binding domain (RBD)-directed antibodies were determined to account for 90% of the neutralizing response (Piccoli et al., 2020, Cell, 183, 1024-1042 e21) and have been a key focus of vaccine design. RBD-directed antibodies target four major epitopes, denoted classes 1-4 (Barnes et al., 2020, Nature, 588, 682-687). Classes 1 and 2 footprints partially overlap and target the receptor binding site (RBS), while classes 3 and 4 footprints recognize the RBD core (Barnes et al., 2020, Nature, 588, 682-687). Mutations in variants located in these four major epitopes contribute to the immune evasion.

[0005] Accordingly, a need remains in the art for the development of a safe and effective vaccine for the treatment of SARS-CoV-2 infection or the treatment or prevention of a disease or disorder associated with SARS-CoV-2 infection.BRIEF SUMMARY OF THE INVENTION

[0006] Disclosed herein are receptor binding domain (RBD) SARS-CoV-2 immunogens, engineered nanoparticle vaccines comprising receptor binding domain (RBD) SARS-CoV-2 immunogens and methods of use thereof for SARS-CoV-2 vaccines.

[0007] In some embodiments, the invention relates to an antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the RBD comprises a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 10, SEQ IDNO: 11, or SEQ ID NO: 12. In some embodiments, the RBD comprises a polypeptide comprising an amino acid sequence comprising at least 70% of the length of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the RBD comprises a polypeptide comprising an amino acid sequence at least 90% identical to and comprising at least 70% of the length of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0008] In some embodiments, the polypeptide further comprises a nanoparticle scaffold.

[0009] In some embodiments, the antigenic polypeptide comprises an amino acid sequence of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO 9, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO:21. In some embodiments, the antigenic polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 15, SEQ ID NO:18 or SEQ ID NO:21. In some embodiments, the antigenic polypeptide comprises an amino acid sequence comprising at least 70% of the length of SEQ ID NON, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO:21. In some embodiments, the antigenic polypeptide comprises an amino acid sequence at least 90% identical to and comprising at least 70% of the length of SEQ ID NON, SEQ ID NON, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO 18 or SEQ ID NONE

[0010] In some embodiments, the antigenic polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NON, SEQ ID NO:5, SEQ ID NON, SEQ ID NON, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:20. In some embodiments, the antigenic polypeptide is encoded by a nucleotide sequence at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the antigenic polypeptide is encoded by a nucleotide sequence comprising at least 70% of the length of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:20. In some embodiments, the antigenic polypeptide is encoded by a nucleotide sequence at least 90% identical to and comprising at least 70% of the length of SEQ ID NO: 1, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:20.

[0011] In some embodiments, the invention relates to an immunogenic composition comprising an antigenic polypeptide comprising a receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0012] In some embodiments, the invention relates to a nucleic acid molecule encoding an immunogenic composition comprising an antigenic polypeptide comprising a receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: !, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO 8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:20. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:20. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence comprising at least 70% of the length of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 19 or SEQ ID NO:20. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence at least 90% identical to and comprising at least 70% of the length of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO: 20.

[0013] In some embodiments, the invention relates to an immunogenic composition comprising a nucleic acid molecule encoding an immunogenic composition comprising an antigenic polypeptide comprising a receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0014] In some embodiments, the immunogenic composition comprises a nanoparticle.

[0015] In some embodiments, the invention relates to amethod of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the methodcomprising administering to the subject an antigenic polypeptide comprising a receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO:11 or SEQ ID NO: 12. In some embodiments, the subject is a human. In some embodiments, the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

[0016] In some embodiments, the invention relates to a method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising administering to the subject an immunogenic composition comprising an antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the subject is a human. In some embodiments, the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

[0017] In some embodiments, the invention relates to a method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule encoding an antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the subject is a human. In some embodiments, the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

[0018] In some embodiments, the invention relates to a method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising administering to the subject an immunogenic composition comprising a nucleic acid molecule encoding the antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the subject is a human. In some embodiments, the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

[0019] In some embodiments, the invention relates to a method of treating orpreventing infection by more than one human coronavirus in a subject in need thereof, the method comprising administering to the subject an antigenic polypeptide comprising a receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the subject is a human. In some embodiments, the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

[0020] In some embodiments, the invention relates to a method treating or preventing infection by more than one human coronavirus in a subject in need thereof, the method comprising administering to the subject an immunogenic composition comprising an antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the subject is a human. In some embodiments, the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

[0021] In some embodiments, the invention relates to a method of treating or preventing infection by more than one human coronavirus in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule encoding an antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the subject is a human. In some embodiments, the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

[0022] In some embodiments, the invention relates to a method of treating or preventing infection by more than one human coronavirus in a subject in need thereof, the method comprising administering to the subject an immunogenic composition comprising a nucleic acid molecule encoding the antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises a polypeptide comprising an amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12. In some embodiments, the subject is a human. In some embodiments, the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A through Figure 1H depict data demonstrating that the USA- WA1 / 2020 based RBD g5.1 24mer design induces cross-reactive responses through early SARS-CoV-2 variants and protects B.1.617.2 variant. Figure 1A) Model of RBD g5.1 24mer nanoparticle where the surfaces for RBD, RBS, and ferritin scaffold are shown by surfaced colored dark blue, yellow, and gray respectively. Glycans are in cyan spheres. Figure IB) Pseudovirus endpoint titers to variant viruses of sera and Figure 1C) live virus neutralization of sera from mice after single 2 pg immunization (n=5 mice / group pooled sera). Figure ID) Immunization scheme for in vivo lethal B.1.617.2 (Delta) variant challenge, with single immunization at lOug or 20ug, followed 64 days after by IxlO5PFU intranasal B.1.617.2 challenge for data in Figure IE - Figure 1H (n=10 mice / group). Figure IE) Weight loss curves from B.1.617.2 challenge. Individual mice represented by dashed lie and solid line is the average. Figure IF) Survival was monitored post-challenge. Survival was compared by Mantel-Cox log-rank test. Figure 1G) Viral loads in lung tissue four days post-challenge and differences analyzed by Kruskal -Wallis test followed by post hoc Dunn’s analysis. Figure 1H) Pathology scoring of H&E stained lung in challenged mice in subset of mice at day 4 post-challenge and compared by Kruskal-Wallis test followed by a post hoc Dunn’s analysis * p < 0.05, ** p < 0.01. LOD is limit of detection. Data in Figure IB and Figure 1C were generated using pooled samples, so no statistical tests were run.

[0024] Figure 2A through Figure 2G depict data demonstrating the bioinformatic analysis of the RBS epitope in Omicron variants. Figure 2A) CDC variant proportion database entire U.S. dataset was sorted by variant over time. Variants that had at least a reported proportion of 4 for at least one reported week (above the line) are plotted. For Figure 2B - Figure 2E, top view of the RBD (top row) shows where ACE2 binds and side view of the RBD (bottom row). RBD surface colored to represent the most common positions contacted by Figure 2B) class 1 and Figure 2C) class 2 neutralizing antibodies. The surface is colored where red positions are most contacted by antibodies of the given class and blue is the least contacted by antibodies of the given class. Figure 2D) Spheres represent the positions of mutations occurring in significant variants. Sphere size representshow many variants contain defining mutations (mutations that occur in >50% of sequenced viruses) at that position and red spheres are positions of mutations that are within 4A of ACE2. Positions that have been mutated in variants but to multiple amino acid identities are not indicated with spheres. The four labeled mutations were used in immunogen design. Figure 2E) Regions where deletions have been identified in non-human mammal (non- SARS-CoV-1 or -2) sarbecovirus RBDs are shown in yellow. Figure 2F) RED 4mut g5.1 immunogen has selective BA. 1 mutations (red patches) and a set of glycans (sticks) to mask non-RBS epitopes. RBD 4mut g5.1 was stabilized in the RBD core to make the RBD 4mut g5.2. The numbers next to glycans indicate the position of the asparagine. Figure 2G) Plasmid construction of immune focused monomer (top), epitope resurfaced modification (middle), and cavity filled-glycan occupancy modified (bottom).

[0025] Figure 3 A through Figure 3F depict data demonstrating the in vitro characterization of redesigned immunogens redesigned for cross-reactivity to ancestral and Omicron strains. Figure 3A) Mass spectrometry analysis of glycan occupancy and species at each of the designed glycan positions. Figure 3B) Heatmap of AUCs determined by ELISA for class 1 and 2 antibodies to immunogens and SARS-CoV-2 RBD antigens. Figure 3C) Sensograms of most cross-reactive antibodies. KDs are corrected for glycan mass and occupancy and are indicated in the top right of each sensogram. Figure 3D) Model of RBD 4mut g5.1 24mer nanoparticle where the surfaces for RBD, 4 mutations of BA.1, and ferritin scaffold are shown by surfaced colored dark blue, red, and gray respectively. Glycans are in cyan spheres. Figure 3E) Size exclusion chromatography trace of RBD 4mut g5.2 24mer. Figure 3F) On the left, a negative stain electron micrograph of RBD 4mut g5.2 24mer with a zoomed in view of nanoparticles. A representative 2D class average of the nanoparticle is to the bottom right.

[0026] Figure 4A through Figure 4G depict data demonstrating the comparison of immunogenicity of next generation Omicron RBD nanoparticle designs in vivo. Figure 4A) Binding endpoint and Figure 4B) pseudovirus neutralization titers 4 weeks after lOug immunization of BALB / c mice with RBD 4mut g5.1 24mer or RBD g5.1. Figure 4C) Binding endpoint and Figure 4D) pseudovirus neutralization titers 64 weeks after 10 ug immunization of RBD 4mut g5.1 24mer. For A-D, n=5 mice / group. Figure 4E) Binding endpoint and Figure 4F) pseudovirus neutralization titers 16 weeks after 10 ug immunization of RBD 4mut g5.1 24mer (optimized codons) or after 10 ug immunization ofRBD 4mut g5.2 24mer. Figure 4G) Pseudovirus neutralization titers over time following 10 pg immunization of RBD 4mut g5.1 24mer (optimized codons) vs. RBD 4mut g5.2 24mer. For Figure 4E - Figure 4G, n=10 mice / group. For Figure 4A - Figure 4B and Figure 4E - Figure 4F, one-tailed t tests were performed to compare responses between vaccines for each variant. For Figure 4G, differences between vaccines were assessed by Sidak multiple comparisons. * p < 0.05, ** p < 0.01. LOD is limit of detection.

[0027] Figure 5 A through Figure 5F depict data demonstrating that RBD 4mut g5. 124mer protects mice in BA.2 memory challenge model. Figure 5A) Immunization scheme of K18 hACE2 mice with two 10 pg doses followed 109 days after boost with intranasal infection with BA.2 at IxlO3PFU (n=10 mice / group). Figure 5B) Pseudovirus neutralization from pre-challenge sera. Figure 5C) Weight loss curves from BA.2 challenge. Figure 5D) TCIDso of lungs and nasal turbinates from BA.2 challenge 2 and 4 days after challenge. Figure 5E - Figure 5F) Lung pathology scoring at day 4 postchallenge. * p < 0.05, ** p < 0.01. LOD is limit of detection. For Figure 5B, one-tailed t tests were performed to compare responses between vaccines for each variant. For Figure 5D - Figure 5F, metrics of viral burden were compared by Kruskal-Wallis tests followed by a post hoc Dunn’s analysis.

[0028] Figure 6A through Figure 6J depict data demonstrating that immune focused nanoparticles effectively boost vaccine responses against variants in antigen experienced mice. Figure 6A) Schematic of boosting DNA spike antigen experienced BALB / c mice for Figure 6B - Figure 6D (n=5 mice / group). Figure 6B) Binding, Figure 6C) pseudovirus neutralization, and Figure 6D) live virus neutralization of DNA spike antigen experienced BALB / c mice. Labels denote boosting group after DNA Spike prime. Figure 6E) Schematic of boosting Comirnaty mRNA antigen experienced BALB / c mice for Figure 6F - Figure 6G (n=5 mice / group). Figure 6F) Binding and Figure 6G) pseudovirus neutralization of Comirnaty mRNA antigen experienced BALB / c mice. Labels denote boosting group after Comirnaty mRNA prime. H) Schematic of boosting DNA spike antigen experienced human antibody repertoire mice for Figure 61 - Figure 6J (n=3 mice). Figure 61) Binding and Figure 6J) pseudovirus neutralization of DNA spike antigen experienced human antibody repertoire mice. LOD is limit of detection. For Figure 6B - Figure 6D and Figure 6F - Figure 6G, differences between vaccination regiments were assessed by Kruskal-Wallis tests followed by a post hoc Dunn’s analysis.

[0029] Figure 7A through Figure 7F depict data demonstrating vaccine induced antibodies targeting the RBD. Figure 7 A) Representative Fabs for class 1-C105 blue (EMDB 22128) and class 2-C121 orange (EMDB 22735) on a gray spike. Week 6 terminal sera pooled from BALB / c mice was tested against USA-WA1 / 2020 spike following 10 ug DNA immunization of Figure 7B) spike Figure 7C) RBD g5.1 24mer Figure 7D) RBD g5.1 4mut 24mer (n=10 mice / group). Representative Fabs recognizing BA.l spike following immunization with Figure 7E) RBD g5.1 4mut 24mer. In Figure 7A - Figure 7E, class 1- directed Fabs are colored blue, class 2-directed Fabs are orange / yellow, USA-WA1 / 2020 spike is light gray, and BA.l spike is dark gray; side and top views of the spike-Fab complexes are shown. Figure 7F) Summary of class 1, 2, and XI -2 epitopes on the 4mut RBD.

[0030] Figure 8A through Figure 8E depict data demonstrating the additional immunogenicity characterization of USA-WA1 / 2020 based nanoparticle vaccine construct. Figure 8A) Binding titers to series of ancestral variants following single, 2 pg immunization of DNA encoding RBD g5.1 24mer. Figure 8B) Scoring of pathology of IHC stained lung in a subset of B.1.617.2 challenged mice at day 4 post-challenge (n=4 mice / group). Figure 8C) Representative lung images of K18-hACE mice that were B.1.617.2 challenged mice at day 4 post-challenge. Figure 8D) Binding endpoint titers of RBD g5.1 24mer or RBD monomer vs USA-WA 1 / 2020 and BA.l RBD antigens after 2 pg immunization (n=5 mice / group). Figure 8E) Pseudovirus neutralization of RBD g5.1 24mer vs USA / WA1 / 2020 and BA.1 (n=5 mice / group). For Figure 8A, data was generated using pooled samples, so no statistical tests were run. For Figure 8B, differences between pathology was assessed by Kruskal-Wallis tests followed by a post hoc Dunn’s analysis. For Figure 8D and Figure 8E, differences were assessed by Sidak multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0031] Figure 9 A through Figure 9C depict data demonstrating an analysis of sarbecovirus RBDs. Non-human mammalian non-classified sarbecovirus and SARS-related sarbecovirus RBDs were extracted from spike sequences from the Bacterial and Viral Bioinformatics Resource Center database. Complete and non-redundant RBD sequences were aligned to the SARS-CoV-2 RBD for analysis of insertions and deletions. Figure 9A) The deletions are labeled relative to the USA-WA1 / 2020 numbering (PDB ID: 6M0J). A total of 102 sequences were used for this analysis and excluded any SARS-CoV-2sequences. AlphaFold models of sarbecovirus RBDs were generated and aligned to the SARS-CoV-2 RBD structure. Examples of models of sarbecovirus RBDs containing deletions in the Figure 9B) 444-448 and Figure 9C) 472-490 loops are indicated by colored cartoons compared to SARS-CoV-2 RBD in gray.

[0032] Figure 10 depicts data demonstrating an additional in vitro characterization. Mass spectrometry analysis of glycan occupancy and species at each of the designed glycan positions.

[0033] Figure 11 A and Figure 1 IB depict data demonstrating an additional immune characterization. Pooled Figure 11 A) pseudovirus and Figure 1 IB) live virus neutralization of RBD 4mut g5.1 24mer vs. RBD g5.1 24mer following 10 pg immunization. Data were generated using pooled samples (n=5 mice / group), so no statistical tests were run.

[0034] Figure 12A and Figure 12B depict data demonstrating a comparison of inhouse batch to optimized commercial construct. Figure 12 A) Binding and Figure 12B) pseudovirus neutralization comparison to variants panel of week 4 sera after lOug immunization of BALB / c mice (n=10 mice / group).

[0035] Figure 13 A and Figure 13D depict data demonstrating Omicron breadth in spike antigen experienced BALB / c mice. For Figure 13A - Figure 13B, immunization scheme described in Figure 5A; in brief, mice are experienced with DNA encoded D614G spike (n=5 mice / group). Sera 3 weeks post boost was assessed for Figure 13 A) binding to a panel of Omicron RBDs, and Figure 13B) pseudovirus neutralization of XBB. Labels denote boosting group after DNA spike prime. LOD is limit of detection. For Figure 13C - Figure 13D, immunization scheme described in Figure 5E; in brief, mice are mRNA-LNP spike experience (n=5 mice / group). Sera 3 weeks post boost was assessed for Figure 13C) binding to a panel of Omicron RBDs, and Figure 13D) pseudovirus neutralization of XBB. Labels denote boosting group after mRNA-LNP spike prime. LOD is limit of detection. For Figure 13 A - Figure 13D, differences in vaccine responses were assessed by Kruskal- Wallis tests followed by a post hoc Dunn’s analysis.

[0036] Figure 14 depicts data demonstrating the prevalence of mutation and variants. CDC variant proportion database entire U.S. dataset was sorted by variant over time. Variants with a reported proportion of 4 for at least one reported week were analyzed for mutations in each variant. In the case of identical RBD sequences, only the variant with the higher maximum variant burden is included. The percent of sequenced variantscontaining mutations are shown as a percent according to Outbreak Info database. Mutations in the majority of viruses of a given variant are colored red and indicated as mutation relative to USA-WA1 / 2020 virus. The relative maximum burden of each variant according to US-wide data from the CDC is shown as the maximum proportion and colored on gray scale where darker shades indicate greater burden of the variant. Residues are categorized by epitope based on which residues are within 5A of a representative Fabs for classl-B38 (7BZ5), class2- C002 (7K8S), class 3-S309 (6WPT), class 4-CR3022 (6Y0R). Mutations highlighted in yellow are incorporated in 4mut design. ND=not detected in sequenced viruses of the variant.DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention relates to the development of receptor binding domain (RBD) SARS-CoV-2 immunogens and the engineering of variants to increase cross- reactive protection and neutralization. In some embodiments, the immunogen is suitable for vaccination strategies to stimulate an immune response (e.g., SARS-CoV-2 immune response) in a subject. In one embodiment, the immunogen is able to elicit detectable antibody responses and heterologous neutralizing antibodies against BA.1 (Omicron). In some embodiment, the immunogen of the invention elicits neutralizing antibodies with cross-reactive properties.

[0038] In one embodiment, the composition comprises a peptide comprising an amino acid sequence capable of expressing one or more SARS-CoV-2 antigens in the subject and a pharmaceutically acceptable excipient.

[0039] In one embodiment, the composition comprises nucleotide sequences capable of expressing a SARS-CoV-2 antigen in the subject and a pharmaceutically acceptable excipient. In one embodiment, the nucleic acid molecule comprises a promoter operably linked to a coding sequence that encodes a SARS-CoV-2 antigen. In some embodiments, the composition comprises nucleotide sequences capable of expressing a self-assembling nanoparticle decorated with a SARS-CoV-2 antigen. In some embodiments, the self-assembling nanoparticle comprises a receptor binding domain (RBD) and variants thereof.Definitions

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0041] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0042] “Adjuvant” as used herein means any molecule added to the vaccine described herein to enhance the immunogenicity of the antigen.

[0043] “Antibody” as used herein means an antibody of classes IgG, IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof. The antibody can be an antibody isolated from the serum sample of mammal, a polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.

[0044] “Coding sequence” or “encoding nucleic acid” as used herein means the nucleic acids (RNA or DNA molecule) that comprise a nucleotide sequence which encodes a protein. The coding sequence can further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0045] “Complement” or “complementary” as used herein means Watson-Crick (e.g., A-T / U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules.

[0046] “Consensus” or “Consensus Sequence” as used herein may mean a synthetic nucleic acid sequence, or corresponding polypeptide sequence, constructed based on analysis of an alignment of multiple subtypes of a particular antigen. The sequence may be used to induce broad immunity against multiple subtypes, serotypes, or strains of a particular antigen. Synthetic antigens, such as fusion proteins, may be manipulated to generate consensus sequences (or consensus antigens).

[0047] “Electroporation,” “electro-permeabilization,” or “electro-kinetic enhancement” (“EP”) as used interchangeably herein means the use of a transmembrane electric field pulse to induce microscopic pathways (pores) in a bio-membrane; their presence allows biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water to pass from one side of the cellular membrane to the other.

[0048] “Fragment” as used herein means a nucleic acid sequence or a portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal. The fragments can be DNA fragments selected from at least one of the various nucleotide sequences that encode protein fragments set forth below.

[0049] “Fragment” or “immunogenic fragment” with respect to polypeptide sequences means a polypeptide capable of eliciting an immune response in a mammal that cross reacts with a full length wild type strain SARS-CoV-2 antigen. Fragments of consensus proteins can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of a consensus protein. In some embodiments, fragments of consensus proteins can comprise at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids or more, at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more, at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more of a consensus protein.

[0050] As used herein, the term “genetic construct” refers to the DNA or RNA molecules that comprise a nucleotide sequence which encodes a protein. The codingsequence includes initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of the individual to whom the nucleic acid molecule is administered. As used herein, the term “expressible form” refers to gene constructs that contain the necessary regulatory elements operable linked to a coding sequence that encodes a protein such that when present in the cell of the individual, the coding sequence will be expressed.

[0051] “Identical” or “identity” as used herein in the context of two or more nucleic acids or polypeptide sequences, means that the sequences have a specified percentage of residues that are the same over a specified region. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0052] “Immune response” as used herein means the activation of a host’s immune system, e.g., that of a mammal, in response to the introduction of antigen. The immune response can be in the form of a cellular or humoral response, or both.

[0053] “Nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0054] Nucleic acids can be single stranded or double stranded, or can contain portions of both double stranded and single stranded sequence. The nucleic acid can be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.

[0055] Nucleic acids can be obtained by chemical synthesis methods or by recombinant methods. “Operably linked” as used herein means that expression of a gene is under the control of a promoter with which it is spatially connected. A promoter can be positioned 5' (upstream) or 3' (downstream) of a gene under its control. The distance between the promoter and a gene can be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance can be accommodated without loss of promoter function.

[0056] A “peptide,” “protein,” or “polypeptide” as used herein can mean a linked sequence of amino acids and can be natural, synthetic, or a modification or combination of natural and synthetic.

[0057] “Promoter” as used herein means a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell. A promoter can comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter can also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A promoter can be derived from sources including viral, bacterial, fungal, plants, insects, and animals. A promoter can regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 late promoter and the CMV IE promoter.

[0058] “Signal peptide” and “leader sequence” are used interchangeably herein and refer to an amino acid sequence that can be linked at the amino terminus of a SARS-CoV-2 protein set forth herein. Signal peptides / leader sequences typically direct localization of a protein. Signal peptides / leader sequences used herein preferably facilitate secretion of the protein from the cell in which it is produced. Signal peptides / leader sequences are often cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell. Signal peptides / leader sequences are linked at the N terminus of the protein.

[0059] “Subject” as used herein can mean a mammal that wants to or is in need of being immunized with the herein described vaccine. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

[0060] “Substantially identical” as used herein can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more amino acids. Substantially identical can also mean that a first nucleic acid sequence and a second nucleic acid sequence are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or more nucleotides.

[0061] “Treatment” or “treating,” as used herein can mean protecting of an animal from a disease through means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a vaccine of the present invention to an animal prior to onset of the disease. Suppressing the disease involves administering a vaccine of the present invention to an animal after induction of the disease but before its clinical appearance. Repressing the disease involves administering a vaccine of the present invention to an animal after clinical appearance of the disease.

[0062] “Variant” used herein with respect to a nucleic acid means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to areferenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.

[0063] Variant can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of “biological activity” include the ability to be bound by a specific antibody or to promote an immune response.

[0064] Variant can also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substituted and still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0065] A variant may be a nucleic acid sequence that is substantially identical overthe full length of the full gene sequence or a fragment thereof. The nucleic acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant may be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0066] “Vector” as used herein means a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self- replicating extrachromosomal vector, and preferably, is a DNA plasmid.

[0067] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.Description

[0068] The invention is based on the development of an immunogen that recapitulates the antigenicity of a SARS-CoV-2 antigen. In some embodiments, the immunogen is suitable for vaccination strategies to stimulate an immune response (e.g., SARS-CoV-2 immune response) in a subject. In some embodiments, the immunogen is able to elicit detectable antibody responses and heterologous neutralizing antibodies against BA.1 (Omicron). In some embodiments, the immunogen of the invention elicits neutralizing antibodies with cross-reactive properties.

[0069] In some embodiments, the composition comprises a peptide comprising an amino acid sequence capable of expressing one or more SARS-CoV-2 antigens in the subject and a pharmaceutically acceptable excipient.

[0070] In some embodiments, the composition comprises nucleotide sequences capable of expressing a SARS-CoV-2 antigen in the subject and a pharmaceuticallyacceptable excipient. In some embodiments, the nucleic acid molecule comprises a promoter operably linked to a coding sequence that encodes a SARS-CoV-2 antigen. In some embodiments, the composition comprises nucleotide sequences capable of expressing a self-assembling nanoparticle decorated with a SARS-CoV-2 antigen. In some embodiments, the self-assembling nanoparticle comprises a receptor binding domain (RBD) or a fragment or variants thereof.

[0071] In some embodiments, the RBD comprises a polypeptide having a sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, or a fragment thereof. In some embodiments, the SARS- CoV-2 antigen can be an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity over an entire length of the amino acid sequence set forth in SEQ ID NON, SEQ ID NO:6, SEQ ID NON, SEQ ID NO:15, SEQ ID NO: 18 or SEQ ID NO:21, or a fragment thereof. In some embodiments, the fragment of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NON, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO:21 comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO: 15, SEQ ID NO: 18 or SEQ ID NO:21.

[0072] In some embodiments, the immunogen is encoded by a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, SEQ ID NON, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NON, SEQ ID NO:8, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:20, or a fragment thereof. In some embodiments, the fragment of SEQ ID NO: 1, SEQ ID NON, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NON, SEQ ID NON, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NO:20 comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the full length of SEQ ID NO: 1, SEQ ID NON, SEQ ID NO:4, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 or SEQ ID NONO.

[0073] Compositions that comprise one or more nucleotide sequence that encode a SARS-CoV-2 antigen may be on a single plasmid. In one embodiment, a composition comprises a single plasmid that encodes a SARS-CoV-2 antigen under a single promoter.

[0074] In one embodiment, a SARS-CoV-2 antigen is operably linked to one or more regulatory elements. In one embodiment, a regulatory element is a leader sequence. In one embodiment, a regulatory element is a start codon. In one embodiment, a regulatory element is at least one stop codon.

[0075] When taken up by a cell, the DNA plasmids can remain in the cell as separate genetic material. Alternatively, RNA may be administered to the cell. It is also contemplated to provide a genetic construct as a linear minichromosome including a centromere, telomeres and an origin of replication. Genetic constructs include regulatory elements necessary for gene expression of a nucleic acid molecule. The elements include: a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for gene expression of the sequence that encodes the target protein or the immunomodulating protein. It is necessary that these elements be operable linked to the sequence that encodes the desired proteins and that the regulatory elements are operably in the individual to whom they are administered. Such genetic constructs may be therefore be recombinant nucleic acid molecules.

[0076] The recombinant nucleic acid molecule can include one or more recombinant nucleotide sequence constructs. The recombinant nucleotide sequence construct can include a heterologous nucleotide sequence that encodes a viral antigen, a fragment thereof, a variant thereof, or a combination thereof.

[0077] The recombinant nucleotide sequence construct can include one or more leader sequences. The leader sequence can encode a signal peptide. The signal peptide can be an immunoglobulin (Ig) signal peptide, for example, but not limited to, an IgG signal peptide and a IgE signal peptide. In some embodiments, nucleic acid constructs may be provided in which the coding sequences for the proteins described herein are linked to IgE leader peptide, or such IgE leader is removed. In some embodiments, proteins described herein are linked to IgE signal peptide, or such IgE leader is removed.

[0078] The one or more vectors can be a plasmid. The plasmid may be useful for transfecting cells with the recombinant nucleotide sequence construct. The plasmid may be useful for introducing the recombinant nucleotide sequence construct into the subject. Theplasmid may also comprise a regulatory sequence, which may be well suited for gene expression in a cell into which the plasmid is administered.Vaccines and Immunogenic Compositions

[0079] Immunogenic compositions, such as vaccines, are provided comprising an optimized consensus sequence, an optimized consensus-encoded antigen, a fragment thereof, a variant thereof, or a combination thereof. The immunogenic composition can significantly induce an immune response of a subject administered with the immunogenic composition against the SARS-CoV-2 antigen. The vaccine may comprise a plurality of the nucleic acid molecules, or combinations thereof. The vaccine may be provided to induce a therapeutic or prophylactic immune response.

[0080] The immunogenic composition can be a DNA vaccine, an RNA vaccine, a peptide vaccine, or a combination vaccine. The vaccine can include an optimized consensus nucleotide sequence encoding an antigen. The nucleotide sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleotide sequence can also include additional sequences that encode linker, leader, or tag sequences that are linked to the antigen by a peptide bond. The peptide vaccine can include an antigen, a variant thereof, a fragment thereof, or a combination thereof. The combination DNA and peptide vaccine can include the above described optimized consensus nucleotide sequence and the encoded antigen.

[0081] The vaccine can be a DNA vaccine. DNA vaccines are disclosed in US Patent Nos. 5,593,972, 5,739,118, 5,817,637, 5,830,876, 5,962,428, 5,981,505, 5,580,859, 5,703,055, and 5,676,594, which are incorporated herein fully by reference. The DNA vaccine can further comprise elements or reagents that inhibit it from integrating into the chromosome.

[0082] The vaccine can be an RNA of the one or more SARS-CoV-2 antigens. The RNA vaccine can be introduced into the cell.

[0083] The vaccine can use recombinant vectors to deliver antigen, subunit vaccines, and glycoprotein vaccines, for example, but not limited, the vaccines described in U.S. Patent Nos.: 4,510,245; 4,797,368; 4,722,848; 4,790,987; 4,920,209; 5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424; 5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668; 5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,3 64;5,462,734; 5,470,734; 5,474,935; 5,482,713; 5,591,439; 5,643,579; 5,650,309; 5,698,202; 5,955,088; 6,034,298; 6,042,836; 6,156,319 and 6,589,529, which are each incorporated herein by reference.

[0084] The vaccine of the present invention can have features required of effective vaccines such as being safe so that the vaccine itself does not cause illness or death; being protective against illness; inducing protective T cell responses; and providing ease of administration, few side effects, biological stability, and low cost per dose.

[0085] Provided herein is an immunogenic composition capable of generating in a mammal an immune response against SARS-CoV-2. The immunogenic composition may comprise each plasmid as discussed above. The immunogenic composition may comprise a plurality of the plasmids, or combinations thereof. The immunogenic composition may be provided to induce a therapeutic or prophylactic immune response. In one embodiment, the composition of the invention can induce immunization after one immunization.

[0086] Immunogenic compositions may be used to deliver nucleic acid molecules that encode one or more consensus SARS-CoV-2 antigen. Immunogenic compositions are preferably compositions comprising plasmids.

[0087] The antigen can be a nucleic acid sequence, an amino acid sequence, a polysaccharide or a combination thereof. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The amino acid sequence can be a protein, a peptide, a variant thereof, a fragment thereof, or a combination thereof. The polysaccharide can be a nucleic acid encoded polysaccharide.

[0088] In one embodiment, the nucleic acid molecule comprises an optimized nucleic acid sequence. The optimized sequence can comprise a consensus sequence and / or modification(s) for improved expression. Modification can include codon optimization, RNA optimization, addition of a kozak sequence for increased translation initiation, and / or the addition of an immunoglobulin leader sequence to increase immunogenicity. The SARS-CoV-2 antigen encoded by the optimized sequence can comprise a signal peptide such as an immunoglobulin signal peptide, for example, but not limited to, an immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. The SARS-CoV-2 antigen encoded by the optimized sequence can be designed to elicit stronger cellular and / or humoral immune responses than a corresponding native antigen.

[0089] The immunogenic composition can induce an immune response in thesubject administered the composition. The induced immune response can be specific for at least one SARS-CoV-2 antigen.

[0090] The immunogenic composition can induce a humoral immune response in the subject administered the immunogenic composition. The induced humoral immune response can be specific for at least one SARS-CoV-2 antigen. The induced humoral immune response can be reactive with at least one SARS-CoV-2 antigen related to an administered optimized encoded antigen. The humoral immune response can be induced in the subject administered the immunogenic composition by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3 -fold to about 10-fold. The humoral immune response can be induced in the subject administered the immunogenic composition by at least about 1.5-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0- fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5-fold, or at least about 16.0- fold as compared to a subject not administered the immunogenic composition of the invention.

[0091] The humoral immune response induced by the immunogenic composition can include an increased level of one or more of IgG, IgM, IgA, and the likes associated with the subject administered the immunogenic composition as compared to a subject not administered the immunogenic composition. These IgG antibodies can be specific for at least one SARS-CoV-2 antigen genetically related to an administered optimized encoded antigen. These IgG antibodies can be reactive with at least one SARS-CoV-2 antigen genetically related to an administered optimized encoded antigen. The level of IgG antibody associated with the subject administered the immunogenic composition can be increased by about 1.5-fold to about 16-fold, about 2-fold to about 12-fold, or about 3-fold to about 10-fold as compared to the subject not administered the immunogenic composition. The level of IgG antibody associated with the subject administered the immunogenic composition can be increased by at least about 1.5-fold, at least about 2.0- fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about4.0-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 5.5-fold, at least about6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about10.0-fold, at least about 10.5-fold, at least about 1 l.O-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5- fold, or at least about 16.0-fold as compared to a subject not administered the immunogenic composition.

[0092] The immunogenic composition can induce a cellular immune response in the subject administered the immunogenic composition. The induced cellular immune response can be specific for at least one SARS-CoV-2 antigen genetically related to an administered optimized encoded antigen. The induced cellular immune response can be reactive to at least one SARS-CoV-2 antigen genetically related to an administered optimized consensus-encoded antigen. The induced cellular immune response can include eliciting a CD8+ T cell response. The elicited CD8+ T cell response can be reactive with at least one SARS-CoV-2 antigen genetically related to an administered optimized encoded antigen. The elicited CD8+ T cell response can be polyfunctional. The induced cellular immune response can include eliciting a CD8+ T cell response, in which the CD8+ T cells produce interferon-gamma (IFN-y), tumor necrosis factor alpha (TNF-a), interleukin-2 (IL- 2), or a combination of IFN-y and TNF-a.

[0093] The induced cellular immune response can include an increased CD8+ T cell response associated with the subject administered the immunogenic composition as compared to the subject not administered the immunogenic composition. The CD8+ T cell response associated with the subject administered the immunogenic composition can be increased by about 2-fold to about 30-fold, about 3-fold to about 25-fold, or about 4-fold to about 20-fold as compared to the subject not administered the immunogenic composition. The CD8+ T cell response associated with the subject administered the immunogenic composition can be increased by at least about 1.5-fold, at least about 2.0-fold, at least about 3.0-fold, at least about 4.0-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 6.5-fold, at least about 7.0-fold, at least about 7.5-fold, at least about 8.0-fold, at least about 8.5-fold, at least about 9.0-fold, at least about 9.5-fold, at least about 10.0-fold, at least about 10.5-fold, at least about 11.0-fold, at least about 11.5-fold, at least about 12.0-fold, at least about 12.5-fold, at least about 13.0-fold, at least about 13.5-fold, at least about 14.0-fold, at least about 14.5-fold, at least about 15.0-fold, at least about 16.0-fold, at least about 17.0-fold, at least about 18.0-fold, at least about 19.0-fold, at least about 20.0-fold, at least about 21.0-fold, at least about 22.0-fold, at least about 23.0-fold, at least about 24.0- fold, at least about 25.0-fold, at least about 26.0-fold, at least about 27.0-fold, at least about 28.0-fold, at least about 29.0-fold, or at least about 30.0-fold as compared to a subject not administered the immunogenic composition.

[0094] The induced cellular immune response can include an increased frequency of CD107a / IFNy / T-bet triple-positive CD8 T cells that are reactive against the native antigen. The frequency of CD107a / IFNy / T-bet triple-positive CD8 T cells associated with the subject administered the immunogenic composition can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13- fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to a subject not administered the immunogenic composition.

[0095] The induced cellular immune response can include an increased frequency of CD107a / IFNy double-positive CD8 T cells that are reactive against the native antigen. The frequency of CD107a / IFNy double-positive CD8 T cells associated with the subject administered the immunogenic composition can be increased by at least about 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14- fold as compared to a subject not administered the immunogenic composition.

[0096] The cellular immune response induced by the immunogenic composition can include eliciting a CD4+ T cell response. The elicited CD4+ T cell response can be reactive with the native antigen genetically related to the optimized consensus antigen. The elicited CD4+ T cell response can be polyfunctional. The induced cellular immune response can include eliciting a CD4+ T cell response, in which the CD4+ T cells produce IFN-y, TNF-a, IL-2, or a combination of IFN-y and TNF-a.

[0097] The induced cellular immune response can include an increased frequency of CD4+ T cells that produce IFN-y. The frequency of CD4+IFN-y+ T cells associated with the subject administered the immunogenic composition can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13- fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold as compared to a subject not administered the immunogenic composition.

[0098] The induced cellular immune response can include an increased frequency of CD4+ T cells that produce TNF-a. The frequency of CD4+TNF-a+ T cells associated with the subject administered the immunogenic composition can be increased by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, or 22-fold as compared to a subject not administered the immunogenic composition.

[0099] The induced cellular immune response can include an increased frequency of CD4+ T cells that produce both IFN-y and TNF-a. The frequency of CD4+IFN-Y+TNF- a+ associated with the subject administered the immunogenic composition can be increased by at least about 2-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0-fold, 4.5-fold, 5.0-fold, 5.5-fold, 6.0-fold, 6.5-fold, 7.0-fold, 7.5-fold, 8.0-fold, 8.5-fold, 9.0-fold, 9.5-fold, 10.0-fold, 10.5- fold, 11.0-fold, 11.5-fold, 12.0-fold, 12.5-fold, 13.0-fold, 13.5-fold, 14.0-fold, 14.5-fold, 15.0-fold, 15.5-fold, 16.0-fold, 16.5-fold, 17.0-fold, 17.5-fold, 18.0-fold, 18.5-fold, 19.0- fold, 19.5-fold, 20.0-fold, 21-fold, 22-fold, 23-fold 24-fold, 25-fold, 26-fold, 27-fold, 28- fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, or 35-fold as compared to a subject not administered the immunogenic composition.

[0100] In one embodiment, the immunogenic composition can be a DNA vaccine, an RNA vaccine, a peptide vaccine, or a combination thereof. The immunogenic composition can include a nucleic acid molecule comprising a sequence encoding the SARS-CoV-2 antigen in the form of a self-assembling nanoparticle. The nucleic acid sequence can be DNA, RNA, cDNA, a variant thereof, a fragment thereof, or a combination thereof. The nucleic acid sequence can also include additional sequences that encode linker, leader, or tag sequences that are linked to the SARS-CoV-2 antigen by a peptide bond.

[0101] In one embodiment, the immunogen can be used as a priming immunogen. In another embodiment, the immunogen can be used as the second, third, fourth, fifth, sixth immunogen of a series.Other Components of the Composition

[0102] In some embodiments, the immunogenic composition of the invention further includes a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient can include such functional molecules as vehicles, adjuvants, carriers or diluents,which are known and readily available to the public. Preferably, the pharmaceutically acceptable excipient is an adjuvant or transfection facilitating agent. In some embodiments, the nucleic acid molecule, or DNA plasmid, is delivered to the cells in conjunction with administration of a polynucleotide function enhancer or a genetic vaccine facilitator agent (or transfection facilitating agent). Polynucleotide function enhancers are described in U.S. Serial Number 5,593,972, 5,962,428 and International Application Serial Number PCT / US94 / 00899 fded January 26, 1994, which are each incorporated herein by reference. Genetic vaccine facilitator agents are described in US. Serial Number 021,579 fded April 1, 1994, which is incorporated herein by reference. The transfection facilitating agent can be administered in conjunction with nucleic acid molecules as a mixture with the nucleic acid molecule or administered separately simultaneously, before or after administration of nucleic acid molecules. Examples of transfection facilitating agents includes surface active agents such as immune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the genetic construct. In some embodiments, the DNA plasmid vaccines may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. Preferably, the transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid.

[0103] In some embodiments of the present invention, the immunogenic compositions can further include an adjuvant. In some embodiments, the adjuvant is selected from the group consisting of: alpha-interferon, gamma-interferon, platelet derived growth factor (PDGF), TNFa, TNF , GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL- 15 having the signal sequence deleted and optionally including the signal peptide from IgE. Other genes which may be useful adjuvants include those encoding: MCP-1, MIP-l-alpha, MIP-lp, IL-8, RANEES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2,ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, 0x40, 0x40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAPI, TAP2 and functional fragments thereof. In some preferred embodiments, the adjuvant is selected from IL-12, IL-15, CTACK, TECK, or MEC.

[0104] The immunogenic compositions according to the present invention are formulated according to the mode of administration to be used. In cases where DNA plasmid vaccines are injectable compositions, they are sterile, and / or pyrogen free and / or particulate free. An isotonic formulation is preferably used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. In some embodiments, a stabilizing agent that allows the formulation to be stable at room or ambient temperature for extended periods of time, such as LGS or other polycations or polyanions is added to the formulation.

[0105] The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be functional molecules such as vehicles, carriers, or diluents. The pharmaceutically acceptable excipient can be a transfection facilitating agent, which can include surface active agents, such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents.

[0106] The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. The transfection facilitating agent is poly-L-glutamate, and the poly-L-glutamate may be present in the composition at a concentration less than 6 mg / ml. The transfection facilitating agent may also include surface active agents such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs and vesicles such as squalene and squalene, and hyaluronic acid may also be used administered in conjunction with the composition. The composition may also include a transfection facilitating agent such as lipids, liposomes, including lecithin liposomes or other liposomes known in the art, as a DNA-liposome mixture (see for example W09324640), calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent is a polyanion, polycation, including poly-L-glutamate (LGS), or lipid. Concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, less than 0.100 mg / ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

[0107] The composition can be formulated according to the mode of administration to be used. An injectable pharmaceutical composition can be sterile, pyrogen free and particulate free. An isotonic formulation or solution can be used. Additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The composition can comprise a vasoconstriction agent. The isotonic solutions can include phosphate buffered saline. The composition can further comprise stabilizers including gelatin and albumin. The stabilizers can allow the formulation to be stable at room or ambient temperature for extended periods of time, including LGS or polycations or polyanions.Methods of Delivery of the Composition

[0108] The present invention also relates to methods of delivering the composition to the subject in need thereof. The method of delivery can include, administering the composition to the subject. The mammal receiving delivery of the composition may be human, primate, non-human primate, cow, cattle, sheep, goat, antelope, bison, water buffalo, bison, bovids, deer, hedgehogs, elephants, llama, alpaca, mice, rats, and chicken.

[0109] The composition may be administered by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intranasal intrathecal, and intraarticular or combinations thereof. For veterinary use, the composition may be administered as a suitably acceptableformulation in accordance with normal veterinary practice. The veterinarian can readily determine the dosing regimen and route of administration that is most appropriate for a particular animal. The composition may be administered by traditional syringes, needleless injection devices, “microprojectile bombardment gone guns”, or other physical methods such as electroporation (“EP”), “hydrodynamic method”, or ultrasound.Method of Treatment

[0110] Also provided herein is a method of treating, protecting against, and / or preventing disease in a subject in need thereof by inducing an immune response against a viral antigen in the subject. In certain embodiments, the invention provides a method of treating, protecting against, and / or preventing at least one of an SARS-CoV-2 virus infection or an SARS-CoV-2 associated pathology in a subject.[Oi l 1] The method can include administering an immunogenic composition of the invention to the subject. Administration of the composition to the subject can be done using the method of delivery described above.

[0112] The composition dose can be between 1 pg to 10 mg active component / kg body weight / time, and can be 20 pg to 10 mg component / kg body weight / time. The composition can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of composition doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0113] The vaccine can be administered prophylactically or therapeutically. In prophylactic administration, the vaccines can be administered in an amount sufficient to induce an immune response. In therapeutic applications, the vaccines are administered to a subject in need thereof in an amount sufficient to elicit a therapeutic effect. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition of the vaccine regimen administered, the manner of administration, the stage and severity of the disease, the general state of health of the patient, and the judgment of the prescribing physician.

[0114] The vaccine can be administered by methods well known in the art as described in Donnelly et al. (Ann. Rev. Immunol. 15:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580,859, issued Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30, 1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21, 1997), the contents of allof which are incorporated herein by reference in their entirety. The DNA of the vaccine can be complexed to particles or beads that can be administered to an individual, for example, using a vaccine gun. One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the expression vector.

[0115] The vaccine can be delivered via a variety of routes. Typical delivery routes include parenteral administration, e.g., intradermal, intramuscular or subcutaneous delivery. Other routes include oral administration, intranasal, and intravaginal routes. For the DNA of the vaccine in particular, the vaccine can be delivered to the interstitial spaces of tissues of an individual (Feigner et al., U.S. Pat. Nos. 5,580,859 and 5,703,055, the contents of all of which are incorporated herein by reference in their entirety). The vaccine can also be administered to muscle, or can be administered via intradermal or subcutaneous injections, or transdermally, such as by iontophoresis. Epidermal administration of the vaccine can also be employed. Epidermal administration can involve mechanically or chemically irritating the outermost layer of epidermis to stimulate an immune response to the irritant (Carson et al., U.S. Pat. No. 5,679,647, the contents of which are incorporated herein by reference in its entirety).

[0116] The vaccine can also be formulated for administration via the nasal passages. Formulations suitable for nasal administration, wherein the carrier is a solid, can include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. The formulation can be a nasal spray, nasal drops, or by aerosol administration by nebulizer. The formulation can include aqueous or oily solutions of the vaccine.

[0117] The vaccine can be a liquid preparation such as a suspension, syrup or elixir. The vaccine can also be a preparation for parenteral, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration), such as a sterile suspension or emulsion.Kit

[0118] Provided herein is a kit, which can be used for treating a subject using the method of vaccination described above. In one embodiment, the kit can comprise thevaccine. In one embodiment, the kit can comprise a nucleic acid molecule encoding a modified SARS-CoV-2 envelope immunogen of the invention.

[0119] The kit can also comprise instructions for carrying out the vaccination method described above and / or how to use the kit. Instructions included in the kit can be affixed to packaging material or can be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, links to websites, QR codes, electronic storage media (e.g., magnetic discs, tapes, cartridges), optical media (e g., CD ROM), and the like.EXPERIMENTAL EXAMPLES

[0120] The present invention has multiple aspects, illustrated by the following non-limiting examples.Example 1 : Immune-Focused RBD Nanoparticles Induce Cross-Reactive, RBS-Directed Responses Capable Of Variant-Proof SARS-CoV-2 Neutralization

[0121] New SARS-CoV-2 variants pose an ongoing threat due to persistent immune escape of natural and vaccine-induced immunity. The emergence of BA.l (Omicron) produced a large antigenic shift in the spike protein, rendering many antibodies ineffective with concomitant loss of Emergency Use Authorization (EUA) status. In this study, an engineered self-assembling nanoparticle displaying RBD 4mut g5. 1, an immunogen developed using structure-guided design was used to focus antibody responses to the receptor binding site (RBS) epitope and promote cross-reactivity by inclusion of four rationally selected BA. l mutations in the RBS. Unlike multi-component RBD approaches, these results demonstrate a single, rationally designed component is sufficient for generating broad immunity. These results demonstrate that in both naive and antigen- experienced mice, RBD 4mut g5.1 nanoparticle induced cross-reactive and durable B cells with antibodies capable of potent neutralization of ancestral SARS-CoV-2 and many Omicron variants. RBD 4mut g5.1 provided heterologous protection at a memory timepoint. By showcasing how subtle changes in an epitope can trigger a diversifiedantibody response, this study offers a promising new avenue for developing vaccines that can effectively tackle the ever-evolving threat of immune escape, not only in SARS-CoV-2 but potentially in a wide range of pathogens.

[0122] Omicron viruses have continued to diversify since the emergence of B.1.1.529 (Hodcroft, 2024, covariants.org / variants; Skowronski et al., 2024, Euro Surveill, 29, 2400076). In fact, VOC B.1.1.529 (Omicron) has sixteen mutations in the RBD including eight mutations in class 1 and 2 epitopes. Thus, updated seasonal vaccines with new strains are constantly being developed. One study of a VOC XBB 1.5 vaccine reported a 71% vaccine effectiveness at preventing COVID-19 hospitalization in those over 60 years old, suggesting updating the strains included in vaccines can protect vulnerable populations (van Werkhoven et al., 2024, Euro Surveill, 29, 2300703). Current Omicron vaccines can induce protective responses in part through boosting neutralizing antibodies from memory against conserved epitopes (Jain et al., 2024, bioRxiv [Preprint] 2024.02.03.578771; Cao et al., 2022, Nature, 608, 593-602; Park et al., 2022, Science, 378, 619-627; Yisimayi et al., 2024, Nature, 625, 148-156). However, the development of vaccines that can induce broader immunity could circumvent the need to take seasonal VOC boosters. Significant efforts have been undertaken to induce broad SARS-CoV-2 responses such as through focusing antibody responses to conserved class 3 or class 4 epitopes (Cohen et al., 2021, Science, 371, 735-741; Fan et al., 2022, Immunity, 55, 2419- 2435 elO; Wang et al., 2024, Cell, 188, 1036-1050; Zhang et al., 2023, Adv Sci (Weinh), 10, e2301034; Brinkkemper et al., 2022, iScience, 25, 105649). While less effort has been dedicated to broadening class 1 and class 2 immune responses, there are cross-reactive class 1 and class 2 monoclonals that have been discovered (Huang et al., 2022, Immunity, 55, 1501-1514 e3; Yisimayi et al., 2024, Nature, 625, 148-156) suggesting class 1 and 2 epitopes are also a promising target for broad SARS-CoV-2 immunity.

[0123] This study used structure-guided design to selectively incorporate Omicron mutations in an immunogen as an approach to improve cross-reactive, RBS-directed responses that broadly neutralize both ancestral and contemporary Omicron viruses. The RBS surface was carefully examined, and three datasets were mapped onto the class 1 and class 2 epitopes: structures of neutralizing antibodies (nAbs), frequency of viral mutations and viral indels. These RBS-focused nanoparticle designs demonstrated cross-reactive protection in a heterologous B.1.617.2 (Delta) challenge and by incorporating just 4Omicron mutations can provide near-sterilizing immunity against BA.2 (Omicron) challenge. These results demonstrated that the antibody response by negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) discovered a new class of antibody, ‘Xl-2’, targeting the RBS. These results showed that structure-guided design can be employed to rationally resurface epitopes on an immune-focused RBD nanoparticle as powerful strategy for eliciting broad and durable SARS-CoV-2 immunity, potentially reducing the need for frequent vaccine reformulations.The results of the experiments are now described.Ancestral -based RBD g5.1 24mer exhibits cross-reactivity and protects against B, 1,617, 2 variant lethal challenge

[0124] Previously, RBD g5.1 24mer was developed by incorporating potential N- linked glycosylation sites (PNGS) into the ancestral USA-WA1 / 2020 RBD to focus responses toward the RBS epitope which is recognized by potently neutralizing class 1 and 2 antibodies (Konrath et al., 2022, Cell Rep, 38, 110318) (Figure 1 A). Upon the emergence of early VOCs, it was observed in human sera that there was a reduction in neutralization relative to the USA-WA1 / 2020 ancestral virus against B.1.351 (Beta), B.l.1.7 (Alpha), P. l (Gamma), B.1.617.2 (Delta) (Wang et al., 2021, Nature, 592, 616-622; Hoffmann et al., 2021, Cell, 184, 2384-2393 el2; Zhou et al., 2021, Cell, 184, 2348-2361 e6; Wang et al., 2021, Nature, 593, 130-135) which had 1, 3, 3, and 2 mutations respectively in the RBS epitope (Hodcroft, 2024, covariants.org / variants). It was previously demonstrated that a single 5 pg dose of RBD g5.1 24mer induced cross-reactive responses to all these variants and were durable in mice for at least 6 months (Konrath et al., 2022, Cell Rep, 38, 110318). To further assess the potency of the observed cross-reactivity, BALB / c mice were immunized with a single 2 pg dose of DNA encoding RBD g5.1 24mer or USA-WA1 / 2020 RBD monomer. These results showed that RBD g5.1 24mer elicited high neutralizing titers against B.1.351 (Beta), and B.1.617.2 (Delta) and autologous USA-WA1 / 2020 ancestral isolate (Figure IB). Similar trends were observed for binding titers (Figure 8A). RBD monomer at the same dose induced weaker binding and minimal-to-no neutralizing titers to USA-WA1 / 2020, B.1.351 (Beta), P. l (Gamma), and B.1.617.2 (Delta) (Figure IB, Figure 8A). Sera from RBD g5.1 24mer animals showed higher live virus neutralizationthan the RBD monomer vaccine sera of USA-WA1 / 2020 (ID50 >250 vs below Limit of Detection) and B.1.617.2 (ID50 474 vs 91) (Figure 1C).

[0125] To assess the functional protection against infection, 40 K18-hACE2 mice (n=10 per group) were immunized with 20 pg of empty pVAX vector, 10 pg of RBD monomer, 10 pg of RBD g5.1 24mer, or 20 pg of RBD g5.1 24mer, followed by a heterologous, lethal B.1.617.2 challenge 64 days post-immunization (Figure ID). Six of ten mice per group were monitored 21 days post-challenge for morbidity and mortality and the other 4 mice per group were necropsied at 4 days post-challenge for viral burden analysis (Figure ID). The antigen-naive, pVAX immunized mice succumbed to infection and rapidly lost weight in the first week post-challenge (Figure IE). RBD monomer immunized mice succumbed to infection and rapidly lost weight within the first week, but two mice recovered by the end of the challenge (Figure IE). In the RBD g5.1 24mer groups, only one mouse in the lower dose exhibited weight loss but recovered by the end of the challenge (Figure IE). RBD monomer provided 33% protection, but remarkably both doses of RBD g5.1 24mer achieved 100% protection with just a single immunization, a statistically significant improvement over RBD monomer (Figure IF). In the mice necropsied at day 4 post challenge, these results showed that lower viral loads within lungs in the RBD g5.1 24mer groups, with the high dose group exhibiting viral burdens near the limit of detection which was a significant reduction compared to pVAX group (Figure 1G). RBD g5.1 24mer at 20 pg dose significantly reduced the lung pathology compared to the other groups (Figure 1H, Figure 8B-C). The potent protection observed in heterologous B.1.617.2 challenge following RBD g5.1 24mer immunization demonstrate that immune- focused nanoparticles can maintain potency even against viruses that have some mutations in the epitope being targeted by antibodies (Figure 14).Immunogen is minimally resurfaced to improve Omicron variant-reactivity

[0126] Despite the powerful cross-reactivity observed through B.1.617.2, RBD g5.1 24mer induced immunity exhibited decreased binding and significant loss of neutralization against BA.l (Omicron) pseudovirus compared to USA-WA1 / 2020 (Figure 8D-E). Approved vaccines based on the ancestral isolates displayed similar losses in crossreactivity (Braeye et al., 2023, Vaccine, 41, 3292-3300; Muik et al., 2022, Science, 375, 678-680; Planas et al., 2022, Nature, 602, 671-675; Cao et al., 2023, Nature, 614, 521-529). BA.1 and subsequent Omicron variants contain considerably more mutations (up to 27) within the RBD than previous variants (Figure 14). Eight of the sixteen RBD mutations in BA.l strain were in class 1 and 2 epitopes and explains the loss of neutralization by RBD g5. 1 24mer vaccine sera since the RBS was being targeted by antibodies (Figure 14). New Omicron viruses have continued to emerge since the end of 2021 and contribute heavy proportions of disease in the U.S (Figure 2A).

[0127] To understand the potential impact of Omicron lineage mutations on class 1 and 2 antibodies, a structural analysis was performed to identify the most common positions contacted by class 1 and class 2 neutralizing antibodies. Class 1 and 2 antibodies contact slightly overlapping surfaces near the RBS where ACE2 binds (Figure 2B-C). To understand the relative frequency of mutations in the most impactful Omicron viruses, viruses with the heaviest burden on the U.S. using the U.S. Centers for Disease Control and Prevention (CDC) virus proportions database were identified (Ma et al., 2023, MMWR Morb Mortal WklyRep, 72, 651-656; Lambrou et al., 2022, MMWR Morb Mortal Wkly Rep, 71, 206-211; Paul et al., 2021, MMWR Morb Mortal Wkly Rep, 70, 846-850) (Figure 2A) and analyzed the major mutations for each variant (Figure 14). There were nine mutations in major Omicron variants (B.1.1.529-JN.1) that are within 4A of ACE2 (Figure 2D). Four mutations that are frequently occurring in Omicron strains cluster together in one patch: Q493R, Q498R, N501Y, and Y505H (Figure 2D). In non-human, mammalian sarbecoviruses (excluding SARS-CoV-1 and -2), there are deletions in three portions of the RBD: 424, 444-448, 472-490 (Figure 2E). Deletions of 5 or more amino acids near the RBS occur in 444-448 and 472-490 loops (USA-WA1 / 2020 numbering) in >80% of sarbecoviruses (Figure 9A), resulting in important changes to the RBS backbone (Figure 9B-C). 4 common mutations of Omicron were identified — Q493R, Q498R, N501Y, and Y505H — that occur within the class 1 or 2 epitopes and cluster away from the less conserved 444-448 and 472-490 RBS loops that may be susceptible to deletion in future VOCs (Figure 2E-F). The four identified mutations can perturb monoclonal and polyclonal antibody binding confirming the validity of the structural analysis in identifying mutations that can antigenically resurface epitopes (Willett et al., 2022, Nat Microbiol, 7, 1161-1179; Wang et al., 2021, Nature, 592, 616-622; Li et al., 2021, Cell, 184, 2362-2371 e9).

[0128] These four Omicron mutations were incorporated into an USA-WA1 / 2020- based RBD with the same PNGS used to immune focus in RBD g5.1 24mer construct tocreate a monomer, RBD 4mut g5.1 (Figure 2F). To further refine of the design for improved expression, folding, and immune focusing capacity, the cavity was mutated to stabilize the RBD and amino acids near designed PNGS to promote glycan occupancy in a design called RBD 4mut g5.2 (Figure 2F-G). Stabilizing mutations were selected from deep mutational scanning (dms) hits that improved stability and expression (Starr et al., 2020, Cell, 182, 1295-1310 e20; Ellis et al., 2021, Front Immunol, 12, 710263). Three of the dms hits were used in the RBD 4mut g5.2 design — I358F, Y365W, F392W — to improve folding through Van der Waals forces in the core of the RBD (Figure 2F). Previous work identified that PNGS at 383 and 460 were unoccupied in the RBD g5.1 24mer (Konrath et al., 2022, Cell Rep, 38, 110318), perhaps due to prolines near the PNGS sequons (Petrescu et al., 2004, Glycobiology, 14, 103-14; Taguchi et al., 2021, Commun Biol, 4, 941). Prolines at the i+1 position of the 383 PNGS and an i+4 position of the 460 PNGS were both mutated to glycine to promote occupancy in the RBD 4mut g5.2 design (Figure 2G).

[0129] RBD 4mut g5.1 and RBD 4mut g5.2 were expressed and purified to compare the designs biochemically and biophysically. The glycan occupancy and species of the two designs were analyzed. The occupancy of the RBD 4mut g5.1 design was consistent with RBD g5.1 as expected with the 383 and 460 positions being almost entirely unoccupied (Konrath et al., 2022) (Figure 3A). The RBD 4mut g5.2 had improved occupancy at 383 and 460 suggesting that the prolines near the PNGS were limiting occupancy and that g5.2 may improve immune focusing (Figure 3 A).

[0130] The binding of a panel of class 1 and 2 monoclonal antibodies were compared against USA-WA1 / 2020 RBD, 4mut immunogens, and a panel of variant RBDs (Wang et al., 2021, Science, 373, eabhl766; Du et al., 2020, Cell, 183, 1013-1023 el3; Park et al., 2022, Science, 375, 449-454; Guo et al., 2023, Nat Commun, 14, 3537; Fenwick et al., 2022, Nat Microbiol, 7, 1376-1389; Gruell et al., 2022, C ell Host Microbe, 30, 1231- 1241 e6; Vanshylla et al., 2022, Cell Host Microbe, 30, 69-82 elO; Chen et al., 2022, Cell Rep, 41, 111528; Yin et al., 2022, Science, 375, 1048-1053; Huang et al., 2022, Immunity, 55, 1501-1514 e3; Seydoux et al., 2020, Immunity, 53, 98-105 e5; Hansen et al., 2020, Science, 369, 1010-1014; Rogers et al., 2020, Science, 369, 956-963; Ju et al., 2020, Nature, 584, 115-119; Liu et al., 2020, Nature, 584, 450-456; Jones et al., 2021, Sci Transl Med, 13:eabfl906). The level of cross-reactivity of the antibodies against USA-WA1 / 2020through JN.1 occurred on a spectrum for both class 1 and 2 antibodies, but the binding was completely lost for all monoclonals tested against HK.3 and JN.1 (Figure 3B) and was consistent with the continued antibody escape observed in human sera and with monoclonal antibodies (Li et al., 2023, PLoS Pathog, 19, elOl 1856; Huang et al., 2022, Immunity, 55, 1501-1514 e3). Three of the least broad class 1 and class 2 antibodies in the set (CC12.1, 2-4, C121, and LY-CoV555) had minimal interaction with RBD 4mut g5.1 and RBD 4mut g5.2 which is suggestive that these antibodies are particularly sensitive to the four Omicron mutations selected in the design (Figure 3B) and is consistent with the reported restricted reactivity of these antibodies (Huang et al., 2022, Immunity, 55, 1501- 1514 e3). RBD 4mut g5.1 and RBD 4mut g5.2 bound well to the majority of class 1 and 2 antibodies in the panel displaying intermediate binding between USA-WA1 / 2020 and BA.l (Figure 3B). Interestingly, RBD 4mut g5.1 and g5.2 displayed strong binding to the most cross-reactive antibodies for class 1 (TH281, TH132) and class 2 (R200-1F9, JMB2002). Affinities were measured using SPR for class 1 and 2 antibodies for both the RBD 4mut g5.1 and RBD 4mut g5.2 (Figure 3C). The most cross-reactive antibodies showed nanomolar or higher affinity for RBD 4mut g5.1 and RBD 4mut g5.2 (Figure 3C).

[0131] To enhance the potency, breadth, and durability of the immunogens, nanoparticles were engineered to display RBD 4mut g5.1 and g5.2 (Figure 3D). The transgene contained an IgE leader to promote expression and secretion followed by the LS- 3 CD4 helper epitope (Xu et al., 2020, iScience, 23, 101399) with a glycine-serine linker to the RBD immunogen which was connected to ferritin (24mer) scaffold. RBD 4mut g5.1 24mer and RBD 4mut g5.2 24mer nanoparticle designs were transfected, harvested supernatant, and purified supernatant by lectin affinity chromatography then further refined by size exclusion chromatography (SEC). A representative SEC trace for RBD 4mut g5.2 24mer showed a homogenous peak eluting at the expected size (Figure 3E). The nanoparticle was assessed by negative stain electron microscopy and results showed a central ring-like scaffold decorated by small densities, consistent with the design of ferritin displaying multiple RBDs (Joyce et al., 2021, Cell Rep, 31, 110143) (Figure 3F).Resurfaced immunogen achieves potent, cross-reactive immunogenicity

[0132] To compare differences in immunogenicity to the original RBD g5.1 24mer design, BALB / c mice were immunized with a single 10 pg dose of a DNA plasmidencoding RBD g5.1 24mer or RBD 4mut g5.1 24mer and binding and neutralization were analyzed at week 4. Compared to RBD g5.1 24mer, RBD 4mut g5.1 24mer induced comparable binding titers to USA-WA1 / 2020 RBD as RBD g5.1 24mer and high, albeit slightly lower binding titers against B.1.617.2 (Figure 4A). Interestingly, RBD 4mut g5.1 24mer induced equivalent or greater binding titers to subsequent Omicron sublineages through EG.5.1 as compared to RBD g5.1 24mer (Figure 4A). The neutralizing response is significantly higher for mice immunized with RBD 4mut g5. 1 24mer than mice immunized with RBD g5.1 24mer across all the Omicron variants that were measured (Figure 4B, Figure 11 A-B). Notably, RBD 4mut g5.1 24mer immunized-mice maintained breadth and neutralization against USA-WA1 / 2020, BA.l, BA.2, and BA.4 through at least week 64 post-immunization, indicating durable and potent antibody responses (Figure 4C-D).

[0133] Next, experiments were focused on comparing the RBD 4mut g5.1 24mer to RBD 4mut g5.2 24mer by DNA immunization. To remove the impact of codon optimization, which can affect expression and downstream immunogenicity, RBD 4mut g5.1 24mer were re-codon optimized to match the RBD 4mut g5.2 24mer in a design called RBD 4mut g5.1 24mer (opt). RBD 4mut g5.1 24mer and RBD 4mut g5.1 24mer (opt) induced equivalent binding titers and pseudovirus neutralization against USA-WA1 / 2020 ancestral strain and panel of Omicron variants (Figure 12). These results supported that these two constructs were immunologically equivalent.

[0134] Next, the immunogenicity of RBD 4mut g5.1 (opt) and RBD 4mut g5.2 24mer were compared. To explore the difference in immunogenicity due to RBD stability and glycan occupancy, BALB / c mice were immunized with a single 10 pg dose of DNA encoding RBD 4mut g5.1 24mer (opt) or RBD 4mut g5.224mer and analyzed week 16 serology for breadth. Strong but equivalent binding titers to all Omicron RBDs was observed after both vaccines with only a significant improvement against BA.4 / BA.5 in the RBD 4mut g5.2 24mer group, suggesting overall comparable humoral responses (Figure 4E). Neutralization titers to variants were similarly equivalent at all time points measured up to week 16 (Figure 4F-G). The robust humoral responses in SARS-CoV-2 antigen- inexperienced mice following RBD 4mut g5.1 or g5.2 24mer immunization suggests that this immunogen would effectively induce broad, variant-resistant immunity in naive individuals. The data here supports that minimal resurfacing of an epitope can simultaneously induce both ancestral and contemporary SARS-CoV-2 immunity.RBD 4mut g5.1 24mer protects in heterologous BA.2 memory challenge

[0135] To evaluate the protection afforded from long-term immune memory, K18 hACE2 mice were immunized twice with 10 pg of pVAX, RBD g5.1 24mer, or RBD 4mut g5.1 24mer and performed a non-lethal challenge with BA.2 virus >100 days after boost (Figure 5 A). In pre-challenge sera, RBD g5.1 24mer induced higher pseudovirus neutralization titers than RBD 4mut g5.1 24mer against USA-WA1 / 2020, but RBD 4mut g5.1 24mer induced higher neutralization against BA.4 and significantly higher BA.1 neutralization than RBD g5.1 24mer (Figure 5B). After infection, pVAX and RBD g5.1 24mer mice exhibited mild weight loss while RBD 4mut g5.1 24mer ameliorated this (Figure 5C). Relative to pVAX, RBD g5.1 24mer slightly reduced viral loads in the lungs and nasal turbinates and RBD 4mut g5.1 24mer significantly reduced viral loads (Figure 5D). Notably, RBD 4mut g5.1 24mer reduced viral loads within nasal turbinates below the limit of detection by day 2 post-infection and in both nasal turbinates and lungs by day 4 post-infection. Like the B.1.617.2 challenge, lung pathology was scored in a subset of mice at day 4 post-BA.2 challenge. Overall lung pathology scores were reduced in the RBD 4mut g5.1 24mer group compared to RBD g5.1 24mer (Figure 5E-F). The BA.2 memory challenge demonstrated that RBD 4mut g5.1 24mer immunity protects from disease burden of heterologous virus, even at a memory timepoint.RBD 4mut g5.1 24mer boosts cross-reactive responses after COVID-19 immunization

[0136] To evaluate the performance of RBD 4mut g5.1 24mer as a booster in mice with pre-existing SARS-CoV-2 spike immunity, mice were primed with 10 pg of plasmid DNA encoding D614G SARS-CoV-2 spike and boosted 4 weeks later with 10 pg of DNA plasmid encoding D614G SARS-CoV-2 spike or RBD 4mut g5.1 24mer. A group of control mice were primed and boosted with an empty pVAX vector (Figure 6A). Results showed that boosting with RBD 4mut g5.1 24mer induced significantly greater antibody responses compared to naive mice in terms of binding, pseudovirus neutralization, and live virus neutralization against ancestral USA-WA1 / 2020 RBD (Figure 6B-D). Interestingly, D614G spike immunized mice produced binding antibodies to BA.l RBD (Figure 6B), but those antibodies did not neutralization BA.l pseudovirus (Figure 6C). On the other hand,RBD 4mut g5.1 24mer immunized mice elicited BA.1 binding and neutralizing antibodies (Figure 6B-D). RBD 4mut g5.1 24mer vaccine also induced stronger binding and neutralization across Omicron sublineages through EG.5.1 compared to D614G spike boosted mice (Figure 13A-B).

[0137] To simulate immunity in the mRNA-LNP -immunized population, BALB / c mice were primed with a low-dose 0.5 pg of Comirnaty SARS-CoV-2 vaccine (similar dosing to previous studies (Ying et al., 2022, Cell, 185, 1572-1587 el 1)) and heterologously boosted with 10 pg of pVAX, D614G spike, or RBD 4mut g5.1 24mer. A control group was boosted with 0.5 pg of Comirnaty (Figure 6E). Boosting with RBD 4mut g5.1 24mer induced stronger binding and significantly stronger neutralization titers against both USA-WA1 / 2020 and B.1.1.529 compared to pVAX boosting (Figure 6F-G). Relative to Comirnaty boosting, RBD 4mut g5.1 24mer improved binding titers against both USA- WA1 / 2020 and BA.l as well as neutralization against BA.l and almost significantly improved USA-WA1 / 2020 neutralization (Figure 6F-G). Results showed that binding titer improvements of the RBD 4mut g5.1 24mer booster compared to all the other booster vaccines including Comirnaty against a set of Omicron subvariants (Figure 13C-D).

[0138] Finally, RBD 4mut g5.1 24mer was assessed as a booster to mice with existing immunity from vaccination with D614G spike in the Omni mouse model which has humanized immunoglobulin loci with human V, D, and J gene segments (Geurts et al., 2009, Science, 325, 433) as this might better recapitulate the existing antibody lineages in humans. OmniMice were primed with 25 pg of DNA encoding the full D614G spike and boosted 31 weeks later with 25 pg of DNA encoding RBD 4mut g5.1 24mer (Figure 6H). Prior to boost, antibody binding and neutralization were poorly cross-reactive to BA.1 and had waned against matched USA-WA1 / 2020 since peak responses (Figure 6I-J). After RBD 4mut g5.1 24mer boost, responses to ancestral USA-WA1 / 2020 were boosted and novel cross-reactive responses to BA.l were conferred. This shows that human VDJ antibody configurations can be elicited by RBD 4 mut g5.1 24mer that are cross-reactive between ancestral and the BA.1 variant.

[0139] Robust humoral responses were observed among SARS-CoV-2 antigen- experienced BALB / c mice following RBD 4mut g5.1 24mer immunization regardless of the initial COVID-19 vaccine delivery platform (mRNA-LNP or DNA). The data in the antigen-experienced experiments support that RBD 4mut g5.1 24mer would effectivelyinduce broad, variant resistant immunity in antigen-experienced individuals. Assessing the differences a memory timepoint rather than an acute one may better differentiate the response between boosting with USA-WA1 / 2020 spike and boosting with nanoparticle. The data in the waning immunity experiment in human antibody repertoire mice supports that RBD 4mut g5.1 24mer can quickly recall USA-WA1 / 2020 antibodies from memory and facilitate their development into cross-reactive responses.Antibody diversity confers improved breadth

[0140] To compare antibody specificity as a potential mechanism for breadth, ten BALB / c mice were immunized once with 10 pg of DNA encoding spike, RBD g5.1 24mer, or RBD 4mut g5.1 24mer and collected a terminal bleed after six weeks to visualize antibody specificity with electron microscopy polyclonal epitope mapping (EMPEM). The EMPEM technique isolates antibodies from sera samples, digests antibodies into Fabs, and then complexes Fabs with antigen for electron microscopy image collection and processing to reconstruct 3D structures of Fab-antigen complexes (Bianchi et al., 2018, Immunity, 49, 288-300 e8; Turner et al., 2023, STAR Protoc, 4, 102476). Representative low-resolution densities were created using class 1 (C105) and 2 (C121) Fab structures in complex with spike to assign putative class 1 and 2 binding to Fabs found in the mouse sera (Figure 7A) (Barnes et al., 2020, Nature, 588, 682-687).

[0141] First, Fabs were complexed from three different immunization groups with an ancestral (USA-WA1 / 2020) spike protein. In the spike-immunized group, eight unique Fab densities against the RBS were observed (Figure 7B), consistent with observations that the RBS is immunodominant in vaccine sera following DNA full spike immunization (Smith et al., 2020, Nat Commun, 11, 2601). Four Fabs bound to the class 1 epitope and four Fabs bound to class 2 epitope (Figure 7B). Results showed that the five RBD g5.1 24mer vaccine elicited antibodies that bound to ancestral spike at both the class 1 and class 2 epitopes, but were found to be more restrictive in the angle of approach relative to D614G Spike elicited antibodies (Figure 7B, 7C). In the RBD 4mut g5.1 24mer group, six unique densities were observed against the RBS, with four class 1 Fabs and two class 2 Fabs were observed (Figure 7D). The angles of approach for class 1 Fabs are more restricted than those observed in D614G immunizations, however they are slightly less confined than the RBD g5.1 24mer antibodies.

[0142] Next, complexes of RBD 4mut g5.1 24mer Fabs were purified with a BA.l variant spike to compare the BA.1 and ancestral antibody specificities. Results showed four class 1 Fabs and two class 2 Fabs (Figure 7E). Interestingly, results showed two Fabs with unique angles of approach in complex with the BA.1 spike that approached at opposing angles across the RBS in a novel way across the class 1 and 2 epitopes (denoted Xl-2 Fabs) (Figure 7F). One of the Xl-2 Fabs appeared to straddle the two sides of the RBS from at an angle from the exterior of the spike (dark green Figure 7F). The other Xl-2 Fab approached at a southwest angle from the center part of the spike more parallel with the top of the trimer than observed in the other samples (light green Figure 7F). These novel Xl-2 antibodies may not be cross-reactive to USA-WA1 / 2020 (Figure 7D) as they do not appear in the USA-WA1 / 2020 complex (Figure 7D). Further investigation of Xl-2 antibodies could be done to understand their contribution to the observed breadth across Omicron sublineages. EMPEM with BA.l spike was not performed for the spike and RBD g5.1 24mer groups given that the groups had minimal neutralization and the affinity was likely too weak to robustly purify complexes (Huang et al., 2022, Immunity, 55, 1501-1514 e3). Higher resolution structures and monoclonal isolation could provide additional insights on the mechanism of antibody lineages achieving breadth.

[0143] SARS-CoV-2 vaccine campaigns prevented an estimated 18 million deaths from COVID-19 in the first year of immunizations alone (Watson et al., 2022, Lancet Infect Dis, 22, 1293-1302) and have continued to reduce hospitalization risks against variants (Albreiki et al., 2023, Front Immunol, 14, 1049393), yet waves of infection continue. Omicron sublineages limit cross-reactivity of previously induced antibodies through the introduction of 8 mutations within the class 1 and 2 epitopes relative to the ancestral USA-WA1 / 2020 sequence (Chen et al., 2022, Du et al., 2020, Cell, 183, 1013- 1023 el3; Fenwick et al., 2022, Nat Microbiol, 7, 1376-1389; Gruell et al., 2022, Cell Host Microbe, 30, 1231-1241 e6; Guo et al., 2023, Nat Commun, 14, 3537; Huang et al., 2022, Park et al., 2022, Science, 378, 619-627; Vanshylla et al., 2022, Cell Host Microbe, 30, 69- 82 elO; Wang et al., 2021, Science, 373, eabhl766; Yin et al., 2022, Science, 375, 1048- 1053). A new analysis was developed by collating the structural footprints of nAbs with mutations most frequently observed in SARS-CoV-2 variants. This analysis led us to 4 RBS mutations — Q493R, Q498R, N501Y, and Y505H — that occur frequently in Omicron sublineages, are commonly located in the footprints of class 1 and 2 antibodies and form asurface patch that could stimulate cross-reactive antibodies. Given the observed reversion mutations from Omicron back to ancestral strains, and the possibility of zoonotic spillover reintroducing ancestral-like viruses (Oude Munnink et al., 2021, Science, 371, 172-177; Kuchipudi et al., 2022, Proc Natl Acad Sci USA, 119:e2121644119; Yen et al., 2022, Lancet, 399, 1070-1078), RBD 4mut g5.1 / g5.2 24mer was developed using the ancestral RBD sequence with the 4 RBS mutations engineered to broaden SARS-CoV-2 responses to include immunity to both ancestral and Omicron strains.

[0144] The antigenic and immunologic impacts of these mutations on an immune focused ancestral RBD are highly significant. RBD 4mut g5.1 bound with high affinity to many neutralizing antibodies that bind ancestral variants and to the most cross-reactive monoclonal antibodies. RBD 4mut g5.1 24mer induced strong titers to USA-WA1 / 2020 and cross-reactive binding and neutralization against all Omicron viruses tested. Antibodies induced by RBD 4mut g5.1 24mer were highly durable as results showed both potent heterologous BA.2 protection at an immunological memory timepoint of 100 days post-immunization and high neutralization titers against BA.1 for over a year after a single immunization. RBD 4mut g5.1 was able to induce cross-neutralizing antibodies in antigen- experienced animal, such as those primed with the Comimaty mRNA-LNP vaccine. Further, RBD 4mut g5.1 was able to induce cross-reactive immunity in human antibody repertoire mice demonstrating the vaccine can elicit human antibodies that neutralize both USA-WA1 / 2020 and BA.l. EMPEM analysis of RBD 4mut g5.1 24mer immunized mice identified class 1 and 2 antibodies with similar angles of approach against both USA- WA1 / 2020 and BA.l antigens suggesting that some cross-reactive clones was elicited. RBD 4mut induced unique XI -2 antibodies that cross over the RBS at angles not described by any previous vaccine. Further exploration of XI -2 antibodies to understand their contribution to polyclonal Omicron breadth is needed to understand their immunological importance.

[0145] The prevailing strategy of using RBD-based vaccines to achieve broad SARS-CoV-2 immunity is highly limited by the need to encode multiple RBDs. Mosaic RBD nanoparticles that co-present multiple diverse clades of sarbecoviruses or variants of SARS-CoV-2 on the same nanoparticle have been explored for their ability to engage B cells that bivalently bind conserved epitopes on diverse neighboring RBDs and improve breath (Cohen et al., 2021, Science, 371, 735-741; Fan et al., 2022, Immunity, 55, 2419-2435 elO; Wang et al., 2024, Cell, 188, 1036-1050). Despite these impressive results, multi-component vaccines face significant challenges that can limit their development and widespread use including complexities with formulation, manufacturing costs, regulatory hurdles, immunological competition between the antigens and less control of resulting immune response. Here, similar robust was achieved, cross-reactive immunity yet using a simplified system of a single nanoparticle harboring one epitope-resurfaced and immune- focused RBD.

[0146] In summary, broadly reactive was achieved, variant-resistant SARS-CoV-2 immunity with an immune-focused, epitope-resurfaced nanoparticle vaccine. This was possible through a newly developed immunogen engineering approach consisting of antibody-antigen structures, viral sequencing and nucleic acid vaccine delivery. It was also demonstrate that electron microscopy can be employed to validate immunity in mice driven by engineered RBD vaccines. These findings provide a blueprint for developing broadly protective vaccines against other pathogens using structural footprints of nAbs, frequency analysis of mutational variants and immune validation by electron microscopy.The materials and methods used in these experiments are now described.Animals

[0147] BALB / c, C57BL / 6, and K18-hACE2 mice were obtained from Jackson Laboratories. All studies were performed at Wistar Institute animal facilities in accordance with approved protocols under Institutional Animal Care and Use Committees. Animals were housed in ventilated cages and given free access to food and water. For DNA immunizations, plasmids were formulated in water and administered intramuscularly into the tibialis anterior muscle. Electroporation was then performed using CELLECTRA-EP delivery system consisting of two pulse sets at 0.2A of 52ms pulses and 198ms delay with 3 second interval. For RNA immunizations, Pfizer-BioNTech mRNA COVID-19 vaccine was obtained from pharmacy vial remnants, formulated in water, and administered intramuscularly into the tibialis anterior muscle. Blood was collected via submandibular bleed at specified time points for serology.ELISA

[0148] For analysis of mouse serum samples, high binding 96-well flat-bottom half-area microplates (Corning) were coated overnight at 4°C with I g / mL of SARS-CoV2 RBD of variants (Sino Biological). Plates were then blocked for 2 hours at ambient temperature in PBS with 5% Milk, 0.2% Tween-20. Serial dilutions of sera were made and applied to the plates, followed by 2 hours incubation at 37°C. Plates were then incubated for 1 hour at ambient temperature. For BALBc and K18-hACE2 mouse studies, secondary antibody goat anti-mouse IgG h+1 HRP -tagged antibody (Bethyl Laboratories) was used at a 1 :20,000 dilution. For OmniMouse® studies, goat anti-human K+L light chain HRP- tagged antibody was used at a 1 : 10,000 dilution. All dilutions were made in PBS with 1% NCS, 0.2% Tween-20, and plates were washed in between steps in PBS with 0.05% Tween-20 with a 405LS automated plate washer (Biotek Instruments). Plates were then developed with 1-step Ultra TMB substrate (Thermofisher) for 5 minutes and quenched with IN H2SO4 solution. Plates were read on a Synergy 2 plate reader (Biotek Instruments) at 450 and 570 nm absorbances.Surface Plasmon Resonance

[0149] RBD-antibody kinetics experiments were performed with a Series S Sensor Protein A capture chip (Cytiva) on a Biacore 8k instrument (GE). HBS-EP+ running buffer was used (Teknova). Each experiment began with two start up cycles with 60 s of contact time and a flow rate of 50 mL / min. For analysis methods, approximately 150-250 RUs of IgG antibodies was captured on each flow cell at a flow rate of 10 mL / min for 60 seconds. RBD or glycan variants samples were 6x serial diluted from 200 nM (based on theoretical mass of the RBD without glycans) in running buffer and flowed across the chip after capture at a 50 mL / min rate. The experiment had a 120 second contact time phase and a 600 second dissociation phase. Regeneration was performed with 10 mM glycine at pH=1.5 at a flow rate of 50 mL / min for 30 seconds after each cycle. Results were analyzed with 1 : 1 kinetic fitting in the Biacore Insight Evaluation software version 5.0.18.22102 (Cytiva).

[0150] For KD calculations, molecular weights of proteins were adjusted to include the mass contributed by glycans. The molecular weight of glycans in the RBD g5.1 monomer design was previously determined by SEC MAES as 12.27 kDa (Konrath et al., 2022). The occupancy determined here for RBD 4mut g5.1 monomer was an average of3.75 glycans and assumed to be approximately equivalent to the RBD g5.1 design. Based on SEC MALS data, this was approximately 3.27 kDa per glycan. The number of average glycans on the RBD as determined by mass spectrometry analysis for RBD 4mut g5.1 or RBD 4mut g5.2 and this 3.27 kDa mass per glycan was used to determine the molecular weight of the RBD and concentration range used in the experiment. Average occupancy of native glycans for positions 331 and 343 for the RBD 4mut g5.1 and RBD 4mut g5.2 designs were used to estimate the molar mass of variant RBDs.Pseudovirus generation and neutralization assay

[0151] HERK293T cells were obtained from ATCC. CHO-hACE2 cells were obtained from Creative Biolabs. Cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (P / S) antibiotic at 37°C under 5% CO2. To generate SARS-CoV2 spike containing pseudoviruses for luciferase-based virus neutralization assays, HEK293T cells were seeded at 5 million cells in T75 flasks and grown overnight. Cells were then treated with 48pL GeneJammer (Agilent), 6pg S IgE deltaCterm 19 pseudovirus plasmid (Genscript), and 6pg pNL4- 3.1uc.R-E- backbone (Aldevron). For variants, cells were treated similarly but instead with variant pseudovirus plasmids. Transfection supernatants were collected 72 hours later and supplemented with 12% FBS. Pseudovirus solutions were stored at -80°C until further use. Pseudovirus solutions were titered to determine dilutions for working solutions such that they yielded >15-fold greater relative luminescence compared to cells alone control. For pseudovirus assays, CHO-hACE2 cells were seeded in 96-well plates at 10,000 cells / well and incubated overnight. Sera from vaccinated mice were heat inactivated for 15 min at 56°C, then diluted in DMEM with 10% FBS and 1% P / S. Virus solutions at the determined working titer were then introduce to the dilutions and incubated for 90 min at ambient temperature. This media containing diluted sera and pseudovirus was then applied to CHO-hACE2 cells and incubated for 72 hrs. Plates were developed using the BriteLite plus luminescence reporter system (Perkin Elmer) and read on a plate reader. Percent neutralization was calculated based on virus only positive control signal with background subtraction of cells only negative control. ID50S were calculated using Graphpad Prism v8.1 using nonlinear curve fitting with a Hill Slope constraint of < 0.Live virus neutralization

[0152] Isolates of SARS-CoV2 USA-WA1 / 2020, B.l.617.2, or BA.l were obtained through BEI Resources, NIAID, NIH and handled in the BSL3 facility at the Wistar Institute. To grow virus stocks, Vero cells were inoculated with 0.01 MOI virus in DMEM. Supernatant was then collected 3 days post infection. To titer virus, Vero cells were seeded in DMEM with 1% FBS at 20,000 cells / well overnight. Virus stocks were serially diluted and transferred to wells. Five days post infection, wells were scored + / - for presence of cytopathic effect (CPE) by visual examination; virus titer was calculated using Reed- Munch method (Lei et al., 2021, Virologica Sinica, 36, 141-144). For neutralization assay, Vero cells were seeded at 20,000 cells / well overnight. Sera were heat inactivated for 30 min at 56°C. Sera were then serially diluted in DMEM with 1% FBS and 1% P / S and incubated for 1 hr at room temperature with 300 TCIDso / mL virus. This solution was then transferred to the Vero cells and scored five days post-infection for CPE. Neutralization titers were calculated using the Reed-Munch method.SARS-CoV-2 challenge

[0153] K18-hACE2 mice were immunized and shipped to the Public Health Agency of Canada (PHAC) for infection with Delta and BA.2 variants. Weight loss threshold for euthanasia post-infection was 20%. A subset of mice in each group were euthanized at specified time points post-infection for analyses of lung pathology and viral load.

[0154] For pathology, tissues were fixed in 10% neutral phosphate buffered formalin, routinely processed, sectioned at 5 um and stained with hematoxylin and eosin (HE) for histopathologic examination. Paraffin tissue sections were quenched for 10 rain in aqueous 3% hydrogen peroxide. Epitope retrieval was performed using an in-house glycan retrieval solution in a Biocare Medical Decloaking Chamber (Biocare Medical, Pacheco, CA, USA). The primary antibody applied to the sections was SARS-CoV-2 (2019-nCoV) Nucleocapsid, Rabbit MAb (&40143-R019, Sino Biological Inc., Beijing, China) used at a 1 : 6000 dilution for thirty minutes. They were then visualized using a horse radish peroxidase labelled polymer, Envision® + system (anti-rabbit) (Dako, Santa Clara, CA, USA) and reacted with the chromogen diaminobenzidine (DAB). The sections were then counter stained with Gill’s hematoxylin.

[0155] Semi-quantitative lesion scoring was performed at day 4 post-challenge as follows: The percentage affected of each section examined was scored as 0 = noPlasmid immunogen design

[0156] RBD nanoparticle constructs were encoded in the pVAX vector as follows: IgE secretion tag followed by the LS-3 CD4+T cell helper epitope, glycine-serine linker, SARS-CoV-2 spike positions 331-527 modified with glycans and select omicron positions, glycine serine linker, anti Helicobacter pylori ferritin (PDB ID: 3BVE) positions 2-167 construct modified at N19Q to remove a PNGS (Kanekiyo et al., 2013, Nature, 499, 102- 6). RBD monomer constructs were encoded in the pVAX vector with an IgE secretion tag, SARS-CoV-2 spike positions 331-527, and a C-terminal 6x His tag. Monoclonal antibodies were synthesized as human IgGl antibodies with heavy and light chains were encoded in separate pVAX plasmids. Plasmids were codon optimized for human and mouse by Genscript.Epitope surface and diversity analysis

[0157] To determine the most common residues that antibodies against class 1 and 2 antibodies make contact with, Pymol was used to identify RBD residues that are within 5 A of the heavy or light chains for 39 total antibodies, for the following class 1 structures were used: B38 (PDB ID: 7BZ5), CC12.1 (PDB ID: 6XC2), CC12.3 (PDB ID: 6XC4), C105 (PDB ID: 6XCN), REGN10933 (PDB ID: 6XDG), CV30 (PDB ID: 6XE1), CV07- 250 (PDB ID: 6XKQ), CB6 (PDB ID: 7C01), P2C-1F11 (PDB ID: 7CDI), BD-604 (PDB ID: 7CH4), BD-236 (PDB ID: 7CHB), COVA2-04 (PDB ID: 7JMO), C102(PDB ID: 7K8M), C1A-B12 (PDB ID: 7KFV), S728-1157 (PDB ID: 8D0Z), TH132 (PDB ID: 7YVH), TH281 (PDB ID: 7YVM), R40-1G8 (PDB ID: 7 SCI), S2K146 (PDB ID: 7TAS),A23-58.1(PDB ID: 7LRS), P5C3 (PDB ID: 7P40). The following class 2 structures were used: C002 (PDB ID: 7K8S), 2-4 (PDB ID: 6XEY), CV07-270 (PDB ID: 6XKP), P2C- 1A3 (PDB ID: 7CDJ), BD-368-2 (PDB ID: 7CHH), COVA2-39 (PDB ID: 7JMP), C104 (PDB ID: 7K8U), Cl 19 (PDB ID: 7K8W), C121 (PDB ID: 7K8Y), S2M11 (PDB ID: 7K43), Cl 44 (PDB ID: 7K90), NE12 (PDB ID: 7U9O), LY-CoV555 (PDB ID: 7KMG), BD23 (PDB ID: 7BYR), 5A6 (PDB ID: 7KQB), S2H13 (PDB ID: 7JV4), Cl 10 (PDB ID: 7K8V), BG7-20 (PDB ID: 7M6H). The positions mutated in the variant RBDs that are within 5 A of class 1 and 2 Fabs were used to generate Figure 2D. For the coloring of the RBD by B factor to indicate the most common residues contacted by antibodies, the insert- bfactor Python script from GitHub written by Axel Fischer github.com / axelfischer / insert- bfactor was used to make figures in Pymol.

[0158] For the SARS-CoV-2 variant analysis, the SARS-CoV-2 Variant Proportions data was downloaded from the Center for Disease Control and Prevention data.cdc.gov / Laboratory-Surveillance / SARS-CoV-2-Variant-Proportions / jr58- 6ysp / about_data. The proportion of each of the variants over time was determined for data at the national level. Maximums of the proportions was determined. Variants that had a burden of at least 4 was defined in this paper as a variant of “high burden.” For each of the high burden variants, the Outbreak.Info database outbreak.info / compare-lineages was used for the prevalence of mutations in the variants. Defining mutations for each variant was defined as mutations that occur in >50% of sequenced viruses. For the B-factor sphere scaling, the number of variants with a mutation was categorized by frequency where the B factor was set to a value.

[0159] For the analysis of sarbecovirus RBD diversity, unique, complete RBD sequences extracted from non-human, mammal sequences for sarbecovirus spikes from the Bacterial and Viral Bioinformatics Resource Center. Sequence with the name SARS-CoV- 1 and -2 in their names were excluded from the analysis. An amino acid multisequence alignment with reference to SARS-CoV-2 RBD used to identify the percentage of viruses with deletions and the positions of these deletions on the SARS-CoV-2 spike to make figures in Pymol. Models of the Rssp7924 Yunnan (NCBI: MH615953) and Yunnan RP JCC9 2020 (NCBI: UUX91064) were created using the Google Collab AlphaFold2. Models were aligned to the SARS-CoV-2 RBD in Pymol to generate figures.Nanoparticle models

[0160] Models of each the RBD nanoparticles were created using three components: ferritin nanoparticle scaffold (PDB ID: 3BVE), GS linker, and USA- WA1 / 2020 SARS-CoV-2 RBD (PDB ID: 6M0J). The RBD structure was modified with asparagine and man9 sugars at all the sequons that were modified in designs using Rosetta’s Glycan Tree Modeler. The GS linker was generated using Google Collab AlphaFold2 (Mirdita et al., 2022, Nat Methods, 19, 679-682). A single subunit of the relevant combinations of antigen GS linker, and then copied to the biological assembly of ferritin to create 24-mers. The nanoparticle scaffold and RBDs are shown as surface filled models and GS linkers are shown in cartoon representation. Glycans are shown by spheres.In vitro production and purification

[0161] Nanoparticles, antigens, and antibodies were produced in the Expi293F transfection system (Thermo Fisher Scientific). Briefly, Expi293F cells were maintained in Expi293F media on a shaker at 37°C and 5% CO2. Expi293F cells transfected using ExpiFectamine (Gibco) according to manufacturer’s protocol. A 1 :1 ratio of heavy chain to light chain plasmid were co-transfected according to the ExpiFectamine manufacturer’s protocol for monoclonal antibody production. Supernatant was harvested following immunogen or monoclonal antibody plasmid transfection 7 days post-transfection.

[0162] Nanoparticle supernatant was run via an Akta Pure system over an in-house column packed with Galnthus Nivalis Lectin Beads (Vector Labs). Following lectin purification, nanoparticle elution fractions were then pooled and concentrated, before size exclusion chromatography purification on an Superose 6 Increase column (Cytiva) on the AKTA Pure system under the flow of PBS+ 0.02% sodium azide. Monomeric antigen supernatant was run over a HisTrap HP column (Cytiva) using an Akta Pure system. Following nickel purification, monomeric antigen elution fractions were then pooled and concentrated, before size exclusion chromatography purification on an Superose 200 Increase 10 / 300 column (Cytiva) on the AKTA Pure system under the flow of PBS+ 0.02% sodium azide. Monoclonal antibodies transfection supernatant was purified by HiTrap Mab Select on the Akta Pure system. Fractions were pooled and concentrated then buffer exchanged into PBS.Serum IgG isolation and fab digestion

[0163] Serum samples were heat inactivated at 56°C for 1 hour. To isolate polyclonal IgG, sera was incubated by individual mouse on Nab Protein A / G columns (Thermo Scientific) end over end at 4°C for at least 40 hours. Columns were washed and IgG eluted according to kit protocol. An incubation of 10 minutes of end over end mixing of wash buffer and elution buffer was done between each centrifugation step for elution. Antibodies were concentrated and buffer exchanged into phosphate buffered saline (PBS) in a 30 kDa Amicon concentrator.

[0164] To digest antibodies, papain from papaya latex (Sigma Aldrich) was activated at 37°C for 15 minutes in lOOmM Tris pH=8, 2mM EDTA, 10 mM L-cysteine. Activated papain was added to IgG for 5 hours at 37°C. The digest was quenched with 0.03M iodoacetamide then concentrated and buffer exchanged into tris-buffered saline (TBS) in a lOkDa Amicon concentrator. Approximately 200-400 ug of this antibody digest mixture was incubated with 15 ug of hexapro spike trimer with disulfides introduced via V705C and T883C (Bangaru et al., 2022, Sci Adv, 8, eabn2911). Complexes were incubated for -90 minutes at ambient temperature. Complexes were then purified on an Akta Pure system (GE Healthcare) on a Superose 6 Increase column under flow of TBS and detected with UV absorbance at 215nm. Fractions corresponding to complexes were concentrated to -100 uL in a 10 kDa Amicon concentrator and immediately used for making nsEM grids.Negative Stain Electron Microscopy

[0165] A total of 3 pL of purified proteins or EMPEM complex at -0.025 mg / mL were adsorbed onto glow discharged carbon-coated Cu300 EM grids (Electron Microscopy Sciences). Grids were then washed 3 times with 6 pL of TBS buffer, if protein was not already in TBS. The grids were then stained with 3 pL of 2% w / v uranyl formate, blotted, and stained for 90 seconds with 3 pL of the stain followed by a final blot. Micrographs were collected manually on a FEI Tecnai T12 microscope equipped with Oneview Gatan camera at lOOkV and —1.6 pm defocus. EMPEM datasets were collected at 52,000x magnification and 2.356 A / pixel.

[0166] Relion v5.0 was used for data processing. Laplacian of Guassian particle picking was performed to generate stacks of -250,000-400,000 particles per complex.Particles were extracted and classified in 2D classification, 3D reconstruction, and 3D autorefine. The reference for 3D classification and auto-refine was EMDB 25711. A subset of 3D classes with good Fab densities that were auto-refined were used for making composite figures on a spike model (EMDB 25711) using UCSF Chimera.Glycan occupancy and species analysis

[0167] Proteins (60 pg) were denatured with 9 M urea in 100 mM ammonium acetate, 20 mM glycine (pH 6) followed by reduction with 10 mM dithiothreitol (DTT) at 37°C for 1 h, and alkylation with 50 mM iodoacetamide (IAM) for 45 min at 37°C in the dark. Reduced and alkylated samples were buffer exchanged into 100 mM ammonium bicarbonate (pH 8), using 10 kDa centrifugal filters, and samples were divided into six equal (10 pg) aliquots for proteolytic digestions. Three out of the six aliquots were digested with a modified triple digestion method (Arg-C / trypsin, elastase, and subtilisin) and combined into a single sample as previously described (Cao et al., 2017, Nat Commun, 8, 14954). The fourth and fifth aliquots were digested with Arg-C / Trypsin and chymotrypsin, as previously described (Cao et al., 2017, Nat Commun, 8, 14954). The sixth aliquot was digested with a-Lytic Protease (New England Biolabs) at 1 :20 (w / w) enzyme / substrate ratio in ammonium bicarbonate (pH 8) at 37°C for 16 h. After incubations, each sample was heat inactivated for 5 min at 100°C followed by drying in a Speed-Vac centrifuge. Samples were then redissolved in ammonium acetate, pH 5.5.The digested samples were de-glycosyl ated with Endo-H (New England Biolabs) at a concentration of 250 units per 10 pg with incubation at 37°C for 1 h, followed by drying in a Speed-Vac. Samples were re-dissolved in 100 mM ammonium bicarbonate (pH 8) prepared in O18-H20 (Sigma). PNGase-F (lyophilized, Bulldog Bio), dissolved in O18-H20, was added at 500 units per 10 pg and incubated at 37°C for 1 h followed by heat inactivation for 5 min at 100°C.

[0168] Digests (1 pg) were analyzed on an Orbitrap Astral (Thermo Fisher Scientific) in-line with a Vanquish Neo UHPLC system (Thermo Fisher Scientific). Peptides were injected onto an Acclaim PepMapTM 100 trap column (75 pm i.d. x 2 cm packed with 3 pm C18 resin; ThermoFisher Scientific) and separated by reversed phase HPLC on a BEH C18 nanocapillary analytical column (75 pm i.d. x 25 cm, 1.7 pm particle size; Waters) using a 175 min gradient formed by 0.1% formic acid in water (mobile phaseA) and acetonitrile (mobile phase B). Full MS spectra were acquired in the orbitrap at 60,000 resolution with a scan range of 300-1800 m / z, automatic gain control (AGC) target of 3e6, and maximum injection time (max IT) of 50 ms. Data-dependent MS2 spectra were acquired in the orbitrap for the most abundant ions (intensity threshold of 2e4) over 3 sec. at 15,000 resolution with an isolation width of 1.5 m / z, standard AGC, and max IT of 50ms. Unassigned and singly charged ions were rejected.

[0169] MS data were analyzed using Fragpipe, v 22.0 (Kong et al., 2017, Nat Methods, 14, 513-520; Teo et al., 2021, J Proteome Res, 20, 498-505; Yu et al., 2021, Mol Cell Proteomics, 20, 100077). Tandem MS spectra were converted to mzML format using MSConvert from ProteoWizard, v 3.023045, and searched against the UniProt human sequence database (August 21, 2023) to which the RBD design sequences and common contaminants had been added. A target / decoy database containing reversed sequences appended to the target database was used to determine peptide probabilities and false discovery rate (FDR). For combined digest searches, no enzyme specificity was selected and a fixed carbamidom ethylation modification on cysteine (+57.02146) was used. In addition, oxidation (+15.9994, M), deamidation (+2.9883, N), GlcNAc (+203.0794, N), and N-terminal pyroglutamate formation (-17.0265, N) were considered as variable modifications. A cutoff of 1% FDR was used for peptide and protein identifications and a minimum glycosylation site localization probability of >0.75 was used (da Veiga Leprevost et al., 2020, Nat Methods, 17, 869-870; Keller et al., 2002, Anal Chem, 74, 5383-92).

[0170] Peptide abundances of RBD designs were summed for each of the four samples (1 triple protease digest+ 3 individual digests) at each glycosite (i.e. peptide containing the consensus motif: N-X-S / T). Specifically, the intensities of peptides containing high mannose (N+203.0794) and complex -type (N+2.9883) modifications were summed for each glycosite, and the unoccupied proportion was determined by summing the intensities of unmodified peptides that had an identical glycosylated peptide sequence, ng each glycosylation state (high-mannose, complex, and unoccupied) across the four digest samples for each site. The proportion of each glycosylation state was calculated as the total abundance of each glycosylation state divided by the total abundance for each glycosite.Statistical analyses

[0171] Statistical analyses were performed in Graphpad Prism vlO.O. For comparison between individual groups, unpaired two-tailed Student t-tests were performed. For comparisons between groups over time, two-way ANOVAs were performed. For comparisons of survival curves, Mantel-Cox tests were performed.Example 2: RBD SEQUENCESAnnotation Key:Leader sequenceNanoparticle scaffoldLS3 helper epitopeRBDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINN IVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVK GIAKSRKS**

[0172] The above sequences include an IgE leader sequence (bold). The invention also encompasses these sequences without an IgE leader sequence. SEQ ID NOs: 13-21 lack the N-terminal IgE leader sequence comparison to SEQ ID NOs: 1-9.

[0173] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.

[0174] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.

Claims

1. CLAIMSWhat is claimed is:

1. An antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2, wherein the RBD comprises one or more selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; b) a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12; c) a polypeptide comprising an amino acid sequence comprising at least 70% of the length of an amino acid sequence selected from the group consisting of: SEQ ID NO:10, SEQ ID NO: 11, and SEQ ID NO:12; and d) a polypeptide comprising an amino acid sequence at least 90% identical to and comprising at least 70% of the length of an amino acid sequence selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

2. The antigenic polypeptide of claim 1, wherein the polypeptide further comprises a nanoparticle scaffold.

3. The antigenic polypeptide of claim 1, wherein the antigenic polypeptide comprises one or more selected from the group consisting of: a) an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NON, SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO:21; b) a polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NON, SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO:21 ; c) a polypeptide comprising an amino acid sequence comprising at least 70% of the length of an amino acid sequence selected from the group consisting of:SEQ ID NON, SEQ ID N0:6, SEQ ID N0:9, SEQ ID NO: 15, SEQ ID NO: 18 and SEQ ID NO:21; and d) a polypeptide comprising an amino acid sequence at least 90% identical to and comprising at least 70% of the length of an amino acid sequence selected from the group consisting of: SEQ ID NON, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NON5, SEQ ID NO: 18 and SEQ ID NO:21.

4. The antigenic polypeptide of claim 1, wherein the polypeptide is encoded by a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence selected from the group consisting of: SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NON3, SEQ ID NON4, SEQ ID NO: 16, SEQ ID NO:17, SEQ ID NO: 19 and SEQ ID NO:20; b) a nucleotide sequence at least 90% identical to a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NON4, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 19 and SEQ ID NO:20; c) a nucleotide sequence comprising at least 70% of the length of a nucleotide sequence selected from the group consisting of: SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO: 13, SEQ ID NON4, SEQ ID NON6, SEQ ID NO:17, SEQ ID NO:19 and SEQ ID NO:20; and d) a nucleotide sequence at least 90% identical to and comprising at least 70% of the length of a nucleotide sequence selected from the group consisting of: SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NON, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NON9 and SEQ ID NO: 20.

5. An immunogenic composition comprising the antigenic polypeptide of any one of claims 1 to 4.

6. A nucleic acid molecule encoding the antigenic polypeptide of any one of claims 1 to 4.

7. An immunogenic composition comprising the nucleic acid molecule of claim 6.

8. The immunogenic composition of claim 7 comprising a nanoparticle.

9. A method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising administering to the subject the antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2 of any one of claims 1-4, the immunogenic composition of claim 5, the nucleic acid molecule of claim 6 or the immunogenic composition of any one of claims 7 or 8.

10. The method of claim 9, wherein said subject is a human.11 . The method of claim 9, wherein the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

12. A method of treating or preventing infection by more than one human coronavirus in a subject, comprising administering to the subject the antigenic polypeptide comprising an receptor binding domain (RBD) of SARS-CoV-2 of any one of claims 1-4, the immunogenic composition of claim 5, the nucleic acid molecule of claim 6 or the immunogenic composition of any one of claims 7 or 8.

13. The method of claim 12, wherein said subject is a human.

14. The method of claim 12, wherein the subject is infected with one or more human coronavirus or at risk of becoming infected with one or more human coronavirus.

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