Sarbecovirus monoclonal antibodies and uses thereof

Abstract

The present disclosure relates to the field of monoclonal antibodies, as well as preparations and methods of their use in the treatment and / or prevention of disease. Described are monoclonal antibodies and antigen binding fragment thereof, compositions containing the same, and uses thereof for treating, preventing, reducing the risks of, or detecting coronavirus infections or H. pylori infection.

Description

SARBECOVIRUS MONOCLONAL ANTIBODIES AND USES THEREOFCross-Reference to Related Applications

[0001] This application claims priority to U.S. provisional application 63 / 529,104, filed July 26, 2023, the entire contents of which are incorporated herein by reference.Sequence Listing

[0002] This application includes a Sequence Listing submitted electronically, having file name “103783-0350_SL.xml”, with a creation date of July 23, 2024, and a size of 899 kB. The Sequence Listing constitutes part of the specification and is incorporated herein by reference in its entirety.Field of Invention

[0003] The present disclosure relates to monoclonal antibodies and uses thereof in the treatment and / or prevention of disease. In particular, described herein are monoclonal antibodies, pharmaceutical compositions comprising them, and uses thereof for treating or preventing coronavirus infections, including sarbecovirus infections.Government Support Clause

[0004] This invention was made with government support under W81XWH- 18-2-0040 awarded by the United States Army Medical Research and Development Command and 0130602D16 awarded by the Defense Health Agency. The Government has certain rights in the invention.Background

[0005] The following discussion is merely provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.

[0006] SARS-CoV-2 infections continue to cause significant morbidity and mortality worldwide (Zhang et al., 2022). While vaccination is a fundamental tool to prevent SARS- CoV-2 infections and limit the COVID-19 pandemic, the continuous emergence of SARS- CoV-2 variants with increased neutralization resistance has raised serious concerns about the efficacy of the first-generation of vaccines and antibody therapeutics (Abu-Raddad et al., 2021; Hansen et al., 2021; Madhi et al., 2021). Viral variants, such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.l), Delta (B.1.617.2), and Omicron (B.1.1.529) subvariants, characterized byincreased infectivity, pathogenicity, or immune escape, are defined as variants of concern (VOC) by the WHO (Chen et al., 2021b; Planas et al., 2021; Andrews et al., 2022; Pulliam et al., 2022; Saito et al., 2022). Specific population groups, such as age > 60 years, immunocompromised individuals, or individuals with certain pre-existing conditions are particularly prone to breakthrough infections that can develop into life-threatening disease (Beaudoin-Bussieres et al., 2020; Kustin et al., 2021; Long et al., 2020). Previous studies have demonstrated that neutralization antibody titers against Omicron subvariants were low or undetectable after two immunizations of the monovalent Pfizer / BioNTech CO VID-19 vaccine, while additional immunizations significantly boosted neutralizing antibodies against most Omicron subvariants (Lusvarghi et al., 2021; Rossler et al., 2022). Emergence of the highly mutated SARS-CoV-2 VOC Omicron, with even greater escape capability, has further advanced vaccine formulations towards bivalent versions, or towards annually updated vaccines, that elicit immune responses against the most-recent VOC (Cao et al., 2022; Hachmann et al., 2022;; Planas et al., 2022; Tuekprakhon et al., 2022; Willett et al., 2022; Zhang et al., 2022). However, even with subsequent boosting, breakthrough infections continue to occur (Assawakosri et al., 2022; Kuhlmann et al., 2022), reinforcing the ongoing need for development of next-generation vaccines.

[0007] Thus, there remains a need for treatments and prophylactics against sarbecovirus infection.Summary

[0008] Described herein are monoclonal antibodies, pharmaceutical compositions comprising them, and uses thereof for treating or preventing coronavirus infections, including sarbecovirus infections.

[0009] In one aspect, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain complementary determining region 3 (HCDR3) and a light chain complementary determining region 3 (CDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 6 (having HCDR3 of SEQ ID NOs 663-759 and LCDR3 of SEQ ID NOs 913-1009, respectively), optionally wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 7.

[0010] In some aspects, the monoclonal antibody comprises three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 6 (having HCDR1 of SEQ ID NOs 421 -517, HCDR2 of SEQ ID NOs 542-638, HCDR3 of SEQ ID NOs 663-759, LCDR1 of SEQ ID NOs 784-880, and LCDR3 of SEQ ID NOs 913-1009, respectively), optionally wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 7.

[0011] In some aspects, the monoclonal antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 6 (having HCDR1 of SEQ ID NOs 421-517, HCDR2 of SEQ ID NOs 542-638, HCDR3 of SEQ ID NOs 663-759, LCDR1 of SEQ ID NOs 784-880, and LCDR3 of SEQ ID NOs 913-1009, respectively), optionally wherein the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 7.

[0012] In some aspects, the monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 6, or of any one of the antibodies set forth in Table 7.

[0013] In some embodiments, the monoclonal antibody or antigen binding fragment thereof comprises a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 6, or of any one of the antibodies set forth in Table 7.

[0014] In some embodiments, the monoclonal antibody or antigen binding fragment thereof comprises the amino acid sequence of any one of the antibodies set forth in Table 6, of any one of the antibodies set forth in Table 7, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0015] In one aspect, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain complementary determining region 3 (HCDR3) and a light chain complementary determining region 3 (CDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 1 (having HCDR3 of SEQ ID NOs 764-779 and LCDR3 of SEQ ID NOs 1014-1029, respectively), optionally wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 2

[0016] In some aspects, the monoclonal antibody comprises three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 1 (having HCDR1 of SEQ ID NOs 522-537, HCDR2 of SEQ ID NOs 643-658, and HCDR3 of SEQ ID NOs 764-779, and LCDR1 of SEQ ID NOs 885-900, and LCDR3 of SEQ ID NOs 1014-1029, respectively), optionally wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 2.

[0017] In some aspectes, the monoclonal antibody or antigen binding fragment thereof comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 1 (having HCDR1 of SEQ ID NOs 522-537, HCDR2 of SEQ ID NOs 643-658, and HCDR3 of SEQ ID NOs 764-779, and LCDR1 of SEQ ID NOs 885-900, and LCDR3 of SEQ ID NOs 1014-1029, respectively), optionally wherein the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 2.

[0018] In some aspects, the monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 1, or of any one of the antibodies set forth in Table 2.

[0019] In some embodiments, the monoclonal antibody or antigen binding fragment thereof comprises a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 1, or of any one of the antibodies set forth in Table 2.

[0020] In some embodiments, the monoclonal antibody or antigen binding fragment thereof comprises the amino acid sequence of any one of the antibodies set forth in Table 1, or of any one of the antibodies set forth in Table 2, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

[0021] In some embodiments, the monoclonal antibody or antigen binding fragment thereof described herein is a coronaviruses reactive monoclonal antibody that binds to one or more epitopes on a region of a coronavirus spike protein of one or more coronaviruses selected from severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the monoclonal antibody or antigen binding fragment thereof neutralizes one or more coronaviruses selected from SARS-CoV-1, MERS-CoV, and SARS-CoV-2. In some embodiments, the monoclonal antibody or antigen binding fragment thereof binds to the Spike Ferritin nanoparticle (SpFN).

[0022] In another aspect, the present disclosure provides a pharmaceutical composition comprising a monoclonal antibody as disclosed herein, or antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In specific embodiments, the pharmaceutical composition comprises a cocktail of two or more monoclonal antibodies against SARS-CoV- 2 or other coronaviruses, or antigen binding fragments thereof, wherein at least one of the monoclonal antibodies or antigen binding fragments thereof is a coronaviruses reactive monoclonal antibody as described herein or an antigen binding fragment thereof.

[0023] In another aspect, the present disclosure provides a method of preventing, treating, or reducing the risks of a coronavirus infection, comprising administering a coronaviruses reactive monoclonal antibody as disclosed herein, or antigen binding fragment thereof, or a pharmaceutical composition comprising such a coronaviruses reactive monoclonal antibody as disclosed herein, or antigen binding fragment thereof, to a subject in need thereof, optionally wherein the coronavirus infection is selected from SARS-CoV-1, SARS-CoV-2, and MERS- CoV.

[0024] In another aspect, the present disclosure provides a coronaviruses reactive monoclonal antibody as disclosed herein, or antigen binding fragment thereof, or a pharmaceutical composition comprising such a coronaviruses reactive monoclonal antibody as disclosed herein, or antigen binding fragment thereof, for use in preventing, treating, or reducing the risks of a coronavirus infection in a subject in need thereof, optionally wherein the coronavirus infection is selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

[0025] In another aspect, there is provided an in vitro method for detecting a coronavirus infection in a subject or assessing a subject’s condition, comprising contacting a biological sample obtained from the subject with a coronaviruses reactive monoclonal antibody as disclosed herein, or antigen binding fragment thereof, and detecting any coronavirus antigen bound by the monoclonal antibody or antigen binding fragment thereof, optionally wherein the coronavirus infection is selected from SARS-CoV-1, SARS-CoV2, and MERS-CoV.

[0026] In another aspect, the present disclosure provides an in vitro method for detecting a coronavirus antigen in a sample, comprising contacting a sample with coronaviruses reactive monoclonal antibody as described herein, and detecting any coronavirus antigen bound by the monoclonal antibody or antigen binding fragment thereof, optionally wherein the sample is obtained from a composition comprising mRNA encoding a coronavirus antigen, further optionally wherein the sample is obtained from a composition comprising mRNA encoding SpFN.

[0027] In another aspect, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody is selected from: (A) a monoclonal antibody comprising a heavy chain complementary determining region 3 (HCDR3) and a light chain complementary determining region 3 (CDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 9 (having HCDR3 of SEQ ID NOs 760-763 and LCDR3 of SEQ ID NOs 1010-1013, respectively); (B) a monoclonal antibody comprising three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 9 (having HCDR1 of SEQ ID NOs 518-521, HCDR2 of SEQ ID NOs 639-642, and HCDR3 of SEQ ID NOs 760- 763 and LCDR1 of SEQ ID NOs 881-884, and LCDR3 of SEQ ID NOs 1010-1013,respectively); (C) a monoclonal antibody comprising a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 9 (SEQ ID NOs 413, 415, 417, and 419), optionally wherein the heavy chain variable region sequence comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 9; (D) a monoclonal antibody comprising a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 9 (SEQ ID NOs 414, 416, 418, and 420), optionally wherein the light chain variable region sequence comprises the light chain variable region sequence of any one of the antibodies set forth in Table 9; (E) a monoclonal antibody comprising a heavy chain variable region sequence and a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence and light chain variable region sequence, respectively, of any one of the antibodies set forth in Table 9, optionally wherein the heavy and light chain variable region sequences comprise the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 9; and (F) a monoclonal antibody comprising the amino acid sequence of any one of the antibodies set forth in Table 9, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, optionally wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 9.

[0028] In another aspect, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody is selected from: (A) a monoclonal antibody comprising a heavy chain complementary determining region 3 (HCDR3) and a light chain complementary determining region 3 (CDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 4 (having HCDR3 of SEQ ID NOs 780-783 and LCDR3 of SEQ ID NOs 1030-1033, respectively); (B) a monoclonal antibody comprising three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 4 (having HCDR1 of SEQ ID NOs 538-541, HCDR2 of SEQ ID NOs 659-662, and HCDR3 of SEQ ID NOs 780-783, and LCDR1 of SEQ ID NOs 901-904, and LCDR3 of SEQ ID NOs 1030-1033, respectively); (C) a monoclonal antibody comprising a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 4 (SEQ ID NOs 3, 13, 23, and 39), optionally wherein the heavy chain variable region sequence comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 4; (D) a monoclonal antibody comprising a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 4 (SEQ ID NOs. 4, 14, 24, and 40), optionally wherein the light chain variable region sequence comprises the light chain variable region sequence of any one of the antibodies set forth in Table 4; (E) a monoclonal antibody comprising a heavy chain variable region sequence and a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence and light chain variable region sequence, respectively, of any one of the antibodies set forth in Table 4, optionally wherein the heavy and light chain variable region sequences comprise the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 4; and (F) a monoclonal antibody comprising the amino acid sequence of any one of the antibodies set forth in Table 4, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, optionally wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 4.

[0029] In some embodiments, the monoclonal antibody is a ferritin reactive monoclonal antibody and the monoclonal antibody or antigen binding fragment thereof binds to H. pylori ferritin, optionally wherein the monoclonal antibody or antigen binding fragment thereof binds to empty H. pylori ferritin particles. In some embodiments, the ferritin reactive monoclonal antibody or antigen binding fragment thereof binds to a ferritin moiety of the Spike Ferritin nanoparticle (SpFN).

[0030] In other aspects, there is provided a method of preventing, treating, or reducing the risks of H. pylori infection, comprising administering a ferritin reactive monoclonal antibody or antigen binding fragment thereof to a subject in need thereof. In some aspects, there is provided a ferritin reactive monoclonal antibody or antigen binding fragment thereof for use in preventing, treating, or reducing the risks of H. pylori infection in a subject in need thereof. In some aspects, there is provided an in vitro method for detecting H. pylori infection in a subjector assessing a subject’s condition, comprising contacting a biological sample obtained from the subject with a ferritin reactive monoclonal antibody or antigen binding fragment thereof, and detecting any H. pylori bound by the monoclonal antibody or antigen binding fragment thereof. In some aspects, there is provided an in vitro method for detecting an H. pylori antigen in a sample, comprising contacting a sample with a ferritin reactive he monoclonal antibody or antigen binding fragment thereof, and detecting any H. pylori antigen bound by the monoclonal antibody or antigen binding fragment thereof, optionally wherein the sample is obtained from a composition comprising mRNA encoding an H. pylori antigen, further optionally wherein the sample is obtained from a composition comprise mRNA encoding H. pylori ferritin particles, further optionally wherein the sample is obtained from a composition comprising mRNA encoding SpFN.

[0031] In another aspect, the present disclosure provides a polynucleotide encoding a monoclonal antibody of the present disclosure, or antigen binding fragment thereof.

[0032] In another aspect, the present disclosure provides a vector comprising a polynucleotide encoding a monoclonal antibody of the present disclosure, or an antigen binding fragment thereof.

[0033] In another aspect, the present disclosure provides a host cell comprising a monoclonal antibody or antigen-binding fragment of, a polynucleotide, or a vector, of the present disclosure.

[0034] In another aspect, the present disclosure provides a method for detecting a coronavirus infection in a subject or assessing a subject’s condition, comprising contacting a biological sample obtained from the subject with a monoclonal antibody described herein, or antigen binding fragment thereof, and detecting any coronavirus antigen bound by the monoclonal antibody or antigen binding fragment thereof, optionally wherein the coronavirus infection is selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

[0035] The foregoing general description and following detailed description are exemplary and explanatory and are intended to provide further explanation of the disclosure. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the disclosure.Brief Description of the Drawings

[0036] FIGs. 1A-1H: Isolation of SARS-CoV-2 neutralizing antibodies elicited by SpFN- vaccinated rhesus macaques. FIG. 1A SpFN vaccination timeline. Animals received 50pg at week 0 and 4. Samples were taken at week 6 for B cell sorting and mAb isolation. FIG IB Reactivity of isolated WRAIR mAbs disclosed herein towards SpFN1(expressing SARS-CoV-1 S protein) and SpFN (expressing SARS-CoV-2 S protein) through flow cytometry. FIG. 1C Monoclonal antibody (mAb) binding to SARS-CoV-2 antigens or stabilized S protein (Hexapro) in a multiplexed bead-based assay. FIG. ID The proportion of isolated mAbs disclosed herein binding to H. pylori ferritin, subdomains of S protein or the stabilized S protein (Hexapro). FIG. IE Neutralization potency of isolated WRAIR mAbs disclosed herein segregated by subdomain binding specificity. Shown are the mean IC50 values (pg ml-1) from the SARS-CoV-2 (IL1 / 2020) pseudotyped assay, calculated from three independent experiments. FIG. IF Neutralization curves of RBD-directed and NTD-directed neutralizing mAbs disclosed herein against SARS-CoV-2. Shown in parentheses are the reciprocal IC50 values for each mAb. FIG. 1G Neutralization of SARS-CoV-1 as measured in pseudotyped viral inhibition assays, with the IC50 value for mAb WRAIR-5001 shown in parenthesis. Plotted are the mean ± s.e.m. from two independent experiments. FIG. 1H Assessment of RBD, NTD and S2-directed mAbs disclosed herein in Fc-mediated effector functions including antibody dependent cell surface S binding (opsonization), cellular phagocytosis (ADCP), cell membrane transfer (trogocytosis), and cellular cytotoxicity (ADCC). Black horizontal lines indicate the mean value and asterisks represent significance by two-tailed Mann-Whitney Z-test;** < 0.001, * <0.01. The dotted line indicates the positivity threshold as determined by negative antibody control. CR3022 is used as a positive control. Shown are representative data (n = 2) from a single experiment.

[0037] FIGs. 2A-2G: Epitope binning of NTD- and RBD-targeted WRAIR mAbs against variants of concern. FIGs. 2A-2B Epitope binning of NTD-directed (FIG. 2A) or RBD- directed (FIG. 2B) mAbs disclosed herein as measured through a BLI-based competition assay. Values are the percentage of residual binding of the indicated WRAIR second antibody after saturation of the antigen (NTD or RBD subdomain) with a representative first antibody (NTD: Group A: WRAIR-2025, Group B: WRAIR-2137, Group C: WRAIR-2054) (RBD: Group A: WRAIR-2125, Group B: WRAIR-2063, Group C: WRAIR-2151). Competition groups are indicated by boxes in shades of gray.). Closed S trimer (PDB 6ZGE) with epitopes of the NTD- targeted mAb competition groups indicated in light grey (Group A), medium grey (Group B),and dark grey (Group C). Each protomer of the S trimer is colored white, light grey, or dark grey. (Inset, left) The isolated NTD is shown in surface representation, with residues identified for competition Group A shown in light grey, and Group C in dark grey. The possible interface for Group B NTD-targeted mAbs is shaded in medium grey. Residue deletions observed in Omicron subvariants are indicated by *. FIG. 2C ACE2 inhibition by WRAIR RBD mAbs disclosed herein in a BLI-based assay. RBD mAbs were assessed for their ability to block ACE2 binding to SARS-CoV-2 RBD (top) or S-2P (bottom). The half-maximal effective concentration (EC50) in pg ml-lis indicated in parentheses. FIG. 2D Mapping of RBD A and B mAbs disclosed herein using an alanine mutagenesis across the spike glycoprotein (table). Residues of each group are shown in sphere representation on the SARS-CoV-2 RBD. Residues targeted by more than one antibody are bolded. FIG. 2E-2F Cross-reactivity of mAbs disclosed herein was assessed for SARS-CoV-2 VOCs and / or SARS-CoV-1, using BLI (FIG. 2E) or IC50 (FIG. 2F) values measured against pseudotyped virus. FIG. 2G Binding kinetics of WRAIR-5001 with SARS-CoV-2 WA-1, Delta, Omicron BA.1, Omicron BA.5, and SARS- CoV-1 RBDs, as measured by BLI.

[0038] FIGs. 3A-3G: Mapping and in vivo efficacy of select RBD-targeted mAbs. FIG. 3A Relative distribution of RBD antigenic sites targeted by SpFN vaccination-induced serum antibodies, determined by SPR competition assays against previously defined control mAbs as outlined below. FIG. 3B Groups 1 / 2 (medium grey) and Groups 3 / 4 (dark grey) antigenic sites shown in surface representation on the RBD (light grey). Residues identified in the viral escape assays indicated by stick representation and indicated with * to indicate 2E1,#to indicate Omicron BA.1.1 andAto indicate Omicron BA.2, as shown in the table of FIG. 3C. FIG. 3D In vivo protection study plan and design of prophylactic administration of RBD mAbs. RBD mAbs WRAIR-5001 and WRAIR-5021 were given at single dose of lOmg / kg intravenously in K18 mice, followed by challenge with SARS-CoV-2 Delta (B.1.617.2) intranasally 24 hours later (n=13 / group). 5 mice from each group were sacrificed on day 2 for plaque reduction neutralization test (PRNT) assay, and surviving mice were measured for body weight changes and survival out to day 14. RBD mAbWRAIR-2125 was used a positive control, and the negative control was an IgG isotype Zika-specific mAb, MZ4. FIG. 3E Particle forming units (PFU) measured in the lungs or BAL of mice on study day 2 (n=5 mice / group). FIG. 3F Percent loss of body weight out to study day 10 and FIG. 3G survival curves out to study day 14 (study end date). (**** <0.0001 using ordinary one-way ANOVA compared to IgG isotype control, MZ4).

[0039] FIGs. 4A-4G: Structure and epitope analysis of mAb WRAIR-5021. FIG. 4A Left: crystal structure of WRAIR-5021, in complex with SARS-CoV-2 RBD (white) shown in cartoon representation. Heavy and light chains of antibody are colored dark and light grey, respectively. Right: structure is shown at 90° rotation. FIG. 4B Left: overlay of SARS-CoV-2 RBD bound ACE2 structure (PDB: 6m0j) onto the WRAIR-5021 -RBD complex structure. ACE2 is shown in white surface. Right: epitope footprint of WRAIR- 5021 shown on the surface of the RBD and colored based on the antibody heavy and light chain colors. ACE2 epitope is drawn as light grey line. FIG. 4C Buried surface area (BSA) for the heavy and light chain CDR loops are shown as a bar diagram. FIG. 4D Left: key antibody contacting residues of RBD are shown as sticks, with residues reported in VOCs indicated by a *. Right: important heavy and light chain contacting residues of contributing CDRs are shown as thin sticks and labeled as per antibody scheme. RBD residues reported in VOCs are indicated with#. The ACE2 binding ridge (RBD Tip) in FIG. 4D is indicated by (J). The ACE2 binding ridge (RBD Tip) is indicated by (J). FIG. 4E Structure of WRAIR-5001-RBD complex is overlaid on previously reported antibodies in complex with SARS-CoV-2 RBD (representing frequently observed SARS-CoV-2 epitopes) (Rappazo et al., 2021). FIG. 4F Left: Omicron mutations are highlighted as dark gray spheres on the surface of SARS-CoV-2 RBD. The WRAIR-5021 epitope is shown in tubular representation and medium gray. Omicron mutations that fall in the mAb epitope are dark-gray spheres and labeled. Right: sequence alignment of WRAIR-5021 epitope on RBDs from WAI and major VOCs (SEQ ID NOs 241-246). Mutated residues in the VOCs are bolded. Buried surface area (BSA) for epitope residues are shown as bar graphs at the bottom. FIG. 4G Structural superimposition of the WRAIR-5021 -RBD complex with closed (all RBD down conformation, PDB code: 6ZGE) and open (1-RBD-up, PDB: 6X2B) conformations of SARS-CoV-2 S-2P. WRAIR- 5021 -RBD is overlaid onto the RBD (dark gray surface) from one protomer. Side and top views are shown.FIGs. 5A-5J: Structure and epitope analysis of mAb WRAIR-5001. FIG. 5A Left: crystal structure of mAb WRAIR-5001 in complex with SARS-CoV-2 RBD (white) shown in cartoon representation. Heavy and light chains of antibody are colored dark and light grey, respectively. Right: structure is shown at 180 rotation. FIG. 5B SARS-CoV-2 RBD is shown in surface representation with WRAIR-5001 heavy and light chain epitopes highlighted in dark and light grey, respectively. FIG. 5C Overlay of SARS-CoV-2 RBD bound ACE2 structure (PDB: 6m0j) onto the WRAIR-5001 -RBD complex structure. ACE2 is shown in cartoon representation and light gray a helices and 0 sheets. FIG. 5D Buried surface area (BSA) forthe WRAIR-5001 heavy and light chain CDR loops are shown as a bar diagram. FIG. 5E Left: key antibody contacting residues of RBD are shown as sticks. WRAIR- 5001 epitope is shaded as per heavy and light chain shading scheme. Right: important heavy and light chain contacting residues of contributing CDRs are shown as thin sticks and labeled as per antibody scheme. FIG. 5F Structure of WRAIR-5001 -RBD complex is overlaid on previously reported antibodies in complex with SARS-CoV-2 RBD (representing frequently observed SARS-CoV- 2 epitopes) (Rappazo et al., 2021). FIG. 5GLeft / Middle: crystal structures of S309 (light gray)- RBD and WRAIR-2057 (black)-RBD complexes are overlaid on the WRAIR-5001 (medium gray)-RBD structure. Antibodies S309 and WRAIR-2057 are shown in ribbon representation. Right: the WRAIR-5001 epitope is colored gray (dark for heavy chain and light for light chain) on the surface of SARS-CoV-2 RBD while S309 and WRAIR-2057 epitopes are drawn as lines and labeled accordingly. The ACE2 binding ridge (RBD Tip) is indicated by (J). FIG. 5H Omicron mutations are highlighted as dark gray spheres on the surface of SARS-CoV-2 RBD. The WRAIR-5001 epitope is shown in tubular representation and colored dark gray. FIGs. 51-5 J Structural superimposition of the WRAIR-5001- (a helices and b sheets) RBD complex with closed (all RBD down conformation, PDB code: 6ZGE) and open (1-RBD-up, PDB: 6X2B) conformations of SARS-CoV-2 S-2P. WRAIR-5001 -RBD is overlaid onto the RBD (dark gray) from one protomer. Side and top views are shown.

[0040] FIGs. 6A-6D: Epitope conservation and mAb cross-reactivity. FIG. 6A-6B Structural and sequence analysis of the WRAIR-5021 and WRAIR-5001 footprints across sarbecoviruses. FIG. 6A The RBD structure is shown in surface representation and depicts mutations between SARS-CoV-1 and SARS-CoV-2 in dark gray; the WRAIR-5021 and WRAIR-5001 epitopes are outlined and labeled. FIG. 6B Sequence alignment of mAb epitopes across sarbecoviruses (SEQ ID NOs 247-372). The epitope residues are numbered according to the Wuhan reference; the strength of the interaction between the mAb and the RBD is indicated by the height and color of the histogram bars above the sequence alignment. Sequences are ordered based on their phylogenetic relationships based on a maximum likelihood phylogenetic tree derived from RBD protein sequences. FIG. 6C The binding of mAbs was measured with a set of sarbecovirus RBDs using BLI to assess cross-reactivity. Heat-map represents the relative binding strengths for WRAIR mAbs disclosed herein. Shading legend indicating the relative binding strength is shown on the right. FIG. 6D ACE2 blocking activity of WRAIR RBD-targeting mAbs in a BLLbased assay. mAbs were assessed for their ability to block human ACE2 binding to selected sarbecovirus clade lb or la RBDs.

[0041] FIGs. 7A-7H: Plasma neutralizing and binding antibody activity in rhesus macaques. FIG. 7A Serum antibody binding to Spike trimer in rhesus macaques receiving two doses of SpFN or mRNA-1273. FIG. 7B Competition of serum antibody binding with a panel of monoclonal antibodies to Spike subdomains to map serum antibody specificity in macaques receiving SpFN or mRNA-1273. FIG. 7C Reciprocal plasma ID50 values across study weeks 0-8 using pseudotyped SARS-CoV-2 (WA-1) neutralization assays from animals vaccinated with PBS (grey) or 2x with 50pg SpFN (black). Non-human primate (NHP).O2 (Hsl606375) was chosen for mAb isolation (light grey overlayed on black). FIG. 7D Reciprocal plasma ID50 values from authentic virus neutralization assays across SARS-CoV-2 VOCs and SARS- CoV-1 at the peak neutralization timepoint (week 6). FIGs. 7E-7F Plasma IgG binding antibody activity across SARS-CoV-2 (FIG. 7E) and SARS-CoV-1 (FIG. 7F) S protein subdomains at study week 6 and 9 / 10. FIGs. 7G-7H Plasma IgM binding antibody activity across SARS-CoV-2 (FIG. 7G) and SARS-CoV-1 (FIG. 7H) S protein subdomains at study week 6 and 9 / 10. In FIGs. 7C-H light grey denotes NHP.02.

[0042] FIGs. 8A-8D: Sort strategy and frequency of SARS-CoV-binding B cells. FIG. 8A Gating strategy used for the isolation of SARS-CoV+ B cells from the PBMCs at the peak neutralization time point (week 6) from NHP.02, after two doses of 50pg of SpFN. B cells were identified as lymphocytes, singlets, dump channel / viability-, CD19+, and IgG+ and IgM+IgG+. FIG. 8B Schematic of the novel sorting strategy used to identify SARS-CoV specific B cells (left). SpFNs presenting either SARS-1 spike (SpFN1) or SARS-CoV-2 spike (SpFN) were incubated with PBMCs and sequential stained to identify SARS-CoV specific B cells. Flow cytometry panel used to stain PBMCs from the peak neutralization time point from NHP.02 (right) FIG. 8C SARS-CoV specific B cells were identified to both SARS-1 and SARS-CoV-2 in SpFN-vaccinated macaque (NHP.02) compared to a naive animal FIG. 8D Quantification of the frequency of SpFN+, SpFNJ+ B cells using fluorochrome conjugated antigenic tetramers or the SpFN and SpFN1particles as bait.

[0043] FIGs. 9A-9E: Screening mAbs for SARS CoV reactivity and identification of ferritin reactive mAbs. FIGs. 9A-9C Purified mAbs were tested for binding in an enzyme immunoassay (EIA) to the SpFN particle expressing SARS-CoV-2 spike (Fig. 9A), SARS- CoV- 1 spike (Fig. 9B), or empty ferritin particles not expressing spike proteins (empty) (Fig. 9C). The dotted lines represent background level of binding of isotype control mAbrhMZ134 when used at either 2pg / mL (black) or 0.02pg / mL (gray). FIG. 9D mAbs positive in the EIA for binding to the empty ferritin particle (WRAIR-5002, WRAIR-5008, WRAIR-5014, andWRAIR-5023) were titrated for binding against the SpFN, the stabilized spike protein alone (S2P), ferritin derived from H. pylori, or the human ferritin light chain (FTL) or heavy chain (FTH). BSA and the IgG isotype control (MZ134) were used as negative controls. WRAIR- 5021 was used as the positive control mAb with known binding to SpFN and S-2P. FIG. 9E Summary of binding of ferritin reactive mAbs and control mAbs at a single concentration of 0.4 g / mL. Ferritin reactive mAbs WRAIR-5002, WRAIR-5008, WRAIR-5014, and WRAIR- 5023 were found to bind exclusively to H. pylori- nNQ ferritin and not human-derived ferritin.

[0044] FIGs. 10A-10F: Monoclonal antibody binding, neutralization, and ACE-2 competition. FIGs. 10A-10C Binding activity of isolated WRAIR mAbs to the S protein subdomains of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and four seasonal hCoVs as measured in a multiplexed bead-based Luminex assay. Antibodies fell into distinct patterns of domain recognition, with mAbs identified as targeting the NTD (Fig. 10A), RBD (Fig. 10B), or S2 (Fig. 10C). SARS-CoV-1 subdomains are boxed, and WRAIR mAbs that demonstrated ability to cross-bind SARS-CoV-1 subdomains are marked with *. FIG. 10D IC50 and IC80 values of NTD and S2 directed mAbs as measured through a PSV neutralization assay against SARS-CoV-2 VOCs and SARS-CoV-1. FIGs. 10E-10F ACE-2 binding inhibition curves of isolated NTD (FIG. 10E) and S2 (FIG. 10F) mAbs for their ability to inhibit binding of the stabilized S protein (S2P) to hACE2 as measured by BLI.

[0045] FIGs. 11A-11D: SpFN with ALFQ vaccination elicits cross-sarbecovirus neutralizing IgG FIG. 11A SpFN + AEFQ vaccination timeline including the time points where mAbs were isolated post-vaccination from a single donor, Participant C. FIG. 11B Neutralizing IgG titers against pseudotyped SARS-CoV-2 (D416G) measured throughout the course of the Phase I clinical trial, with the donor used for mAb isolation highlighted in yellow. FIGs. 11C-11D Neutralizing IgG titers against pseudotyped SARS-CoV-2 variants of concern (VOCs) at the peak neutralization time points SD43 (post 2nddose) (Fig. 11C) and SD95 (post 3rddose) (Fig. 11D), with the donor used for mAb isolation highlighted in yellow.

[0046] FIGs. 12A-12E: SpFN with ALFQ vaccination elicits cross-sarbecovirus binding plasma IgG FIG. 12A SpFN + ALFQ vaccination timeline. FIGs. 12B-12D Median signaknoise binding over negative control as measured through a bead-based binding assay (Luminex) on the IgG fraction of the plasmas to antigenic domains of the Spike protein against SARS-CoV-2 (Fig. 12B), SARS-CoV-1 (Fig. 12C), MERS-CoV (Fig. 12D) and in donors enrolled in a Phase I clinical trial that were given 2 or 3 doses of SpFN + ALFQ. FIG. 12ESignal:noise binding of the IgG fraction of the plasmas of all donors given SpFN + ALFQ across all human coronavirus antigenic measured, including the Spike domains of seasonal coronaviruses, at the peak neutralization time point study day (SD) 43.

[0047] FIGs. 13A-13C: Mabs isolated from a SpFN + ALFQ vaccinated donor cross-bind and neutralize SARS-CoV-2 VOCs and SARS-CoV-1. FIG. 13A Sorting strategy used to isolate sarbecovirus reactive B cells using SARS-CoV-2 Spike domains NTD and RBD, as well as the SpFN molecules expressing SARS-CoV-2 and SARS-CoV-1 (SpFN1) as bait for BCR binding. FIG. 13B The percentage of mAbs reactive to unique domains of the SARS- CoV-2 Spike protein out of 136 total mAbs isolated and purified, with 93 mAbs found to be reactive to either the ferritin molecule or a domain of SARS-CoV-2 or stabilized Spike (S) trimer. FIG. 13C IC50 heatmap of neutralizing, RBD, and NTD directed WRAIR mAbs isolated post 2ndand post 3rdSpFN vaccination (left y-axis) and broken into their epitope binning groups (right y-axis) across SARS-CoV-2 pseudotyped variants of concern (VOCs) and SARS-CoV-1.

[0048] FIGs. 14A-14B: NTD-directed mAbs generated from the BCR sequences from a single SpFN vaccinated donor bin into unique epitopes shared across previously described and unique locations on the NTD. FIG. 14A NTD-directed mAbs were tested for binding to the isolated NTD in the presence of each other and previously published control of known epitope on the NTD. Several NTD mAbs bound to virus neutralization sites known as the site of vulnerability and the NTD supersite but were binned into epitope groups of unknown location on the NTD. FIG. 14B IC50 values calculated using pseudotyped virus of NTD- directed mAbs are shown grouped from the results of the epitope binning, with previously defined sites of neutralization are indicated on the left, and SARS-CoV-2 variants as well as SARS-CoV-2 as indicated below.

[0049] FIGs. 15A-15C: RBD-directed mAbs generated from the BCR sequences from a single SpFN vaccinated donor bin into known and unique RBD epitope classes. FIG. 15A RBD-directed mAbs were tested for binding to the isolated RBD in the presence of each other and previously published control mAbs of known epitope. The majority of the RBD- directed mAbs bin into previously identified RBD classes, with a majority clustering into the more conserved RBD class IV and V. FIG. 15B, IC50 values calculated using pseudotyped virus of RBD-directed mAbs are shown grouped from the results of the epitope binning, with previously defined RBD classes indicated on the left, and SARS-CoV-2 variants as well as SARS-CoV-2 as indicated below. FIG. 15C Binding breadth across sarbecovirus spike RBDsas measured by Bio-Layer Interferometry (BLI). Darker color indicates strong binding. Class IV and V mAbs such as WRAIR-4045, WRAIR-4066, WRAIR-4112, and WRAIR-4150 demonstrate wide breadth of binding across the RBDs of different sarbecovirus clades.

[0050] FIGs. 16A-16B: Screening mAbs for reactivity to the SpFN expressing SARS- CoV-2 Spike protein or empty ferritin scaffold particle. Purified mAbs that previously showed no reactivity to a component of a sarbecovirus Spike protein via a multiplexed beadbased assay (Luminex) were tested for binding in an enzyme immunoassay (EIA) to the SpFN particle expressing SARS-CoV-2 spike (FIG. 16A) or empty ferritin particles not expressing spike proteins (empty) (FIG. 16B). The dotted lines represent background level of binding of isotype control mAb MZ4 when used at either 2pg / mL (black) or 0.2pg / mL (gray).Detailed DescriptionDefinitions:

[0051] As used herein, “a”, “an” and “the” signify one or more (singular or plural), unless the context clearly indicates otherwise.

[0052] As used herein, a phrase in the form “A / B” or in the form “A and / or B” means (A), (B), or (A and B); a phrase in the form “at least one of A, B, or C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B, and C).

[0053] As used herein, the meaning of the term “about” will be understood by persons of ordinary skill in the art and may vary to some extent depending upon the context in which it is used. If further definition is required to be clear to persons of ordinary skill in the art in the context in which it is used, “about” will mean up to plus or minus 10% of the particular value modified.

[0054] As used herein, the terms “comprising”, “comprise”, “comprises”, “includes”, and “including” are open-ended and signify that the described substance or method includes the stated element(s) or step(s), and may include other unspecified element(s) or step(s).

[0055] As used herein, “subject” denotes any mammal, including humans.

[0056] Described herein are monoclonal antibodies (mAbs) elicited following immunization of rhesus macaques or humans with a ferritin nanoparticle coronavirus vaccine (SpFN) described in more detail below, compositions comprising the mAbs, and diagnostic, therapeutic, prophylactic, and research methods using the mABs.

[0057] In accordance with some aspects, the present disclosure describes mAbs that bind coronavirus antigens, and are useful for diagnosing, treating, reducing the risks of, or preventing coronavirus infections and coronavirus infectious diseases, and related infections and diseases caused by sarbecoviruses and merbecoviruses, or for research purposes, e.g., to detect coronavirus antigens or expression of mRNA encoding coronavirus antigens. In accordance with other aspects, the present disclosure describes mAbs that bind ferritin derived from H. pylori (e.g., ferritin particles of ferritin from H. pylori) and are useful for diagnosing, treating, reducing the risks of, or preventing H. pylori infection and related infections and diseases caused by H. pylori, or for research purposes, e.g., to detect ferritin or ferritin particles or expression of mRNA encoding ferritin or ferritin particles, such as SpFN.

[0058] Since 2002, three human coronaviruses (hCoVs), which had previously only caused common cold-like symptoms in humans, have escaped from animal reservoirs, and entered the human population. Beginning with SARS-CoV-1, then MERS-CoV in 2012, and finally with the emergence global pandemic-causing SARS-CoV-2 in late 2019, these highly pathogenic hCoVs have been responsible for the deaths of millions of people in the span of less than a quarter of a century. Towards that end, as sarbecoviruses have, to date, been responsible for the majority of deaths surrounding hCoV infection, understanding cross-sarbecovirus neutralizing epitopes elicited by SARS-CoV-2 vaccine strategies may not only inform future vaccine design, but also have potential for utilization in clinical settings both prophylactically and therapeutically to prevent disease or death.

[0059] Considering the continued changing landscape of SARS-CoV-2 viral evolution, nextgeneration vaccines are needed for protection against Covid-19. Novel nanoparticle-based SARS-CoV-2 and pan-CoV vaccines in development show promising elicitation of potent and broad sarbecovirus neutralization responses (Cohen et al., 2022; Li et al., 2022; Walls et al., 2021), including protection against highly divergent VOC including Beta, Delta and Omicron (Yu et al., 2022b). The design and development of a SARS-CoV-2 ferritin nanoparticle-based vaccine candidate, SpFN (Spike Ferritin Nanoparticle) has been previously reported. Administration with the adjuvant Army Liposomal Formulation containing QS21 (ALFQ) induced robust and broad immune responses in multiple preclinical animal models and protection against viral challenge in mice, Syrian golden hamsters, cynomolgus macaques, and rhesus macaques (Johnston et al., 2022; Joyce et al., 2021; Joyce et al., 2022; Wuertz et al., 2021; Yu et al., 2022b) and is being evaluated in a Phase I clinical trial (NCT04784767). The SpFN molecule displays eight SARS-CoV-2 (WA-1) Spike glycoprotein trimers on a self-assembling ferritin nanoparticle backbone and is administered together with the ALFQ adjuvant, which is a cholesterol-dense, liposomal adjuvant that includes QS21 saponin. Serum samples from SpFN-immunized rhesus macaques potently neutralized several SARS-CoV-2 variants of concern (VOC) including the highly transmissible and pathogenic Delta and Omicron variants (Johnston et al., 2022; Joyce et al., 2022; Yu et al., 2022b) . Neutralizing antibody activity after two doses of SpFN was an order of magnitude higher than that of convalescent serum samples (Joyce et al., 2022) . In addition, potent neutralization titers were observed for another sarbecovirus, SARS-CoV-1, in both authentic and pseudotyped viruses (Johnston et al., 2022; Joyce et al., 2022). However, the targets of cross-neutralizing antibodies after SpFN vaccination are unknown. Without being bound by theory, the neutralization potency elicited by SpFN can likely be attributed to multiple aspects, including the repetitive array of the viral SARS-CoV-2 Spike glycoprotein on the ferritin nanoparticle (Darricarrere et al., 2021; Kanekiyo et al., 2013; Kelly et al., 2020; Nelson et al., 2022; Yassine et al., 2015), underlining the importance of multiplicity of antigen display to the immune system to generate robust and highly cross-reactive immune responses (Yu et al., 2022a). Determination of the targets of SARS-CoV-2 neutralizing antibodies induced by novel vaccine strategies may be key to understanding their mechanism of protection.

[0060] Described herein are mAbs elicited following immunization of rhesus macaques or human donors enrolled in a Phase I clinical trial with SpFN-ALFQ. SpFN, which comprises an H. pylori- QnNQ ferritin scaffold particle displaying eight SARS-CoV-2 Spike trimers, elicited cross-sarbecovirus neutralizing and binding antibody responses in the serum of vaccinated rhesus macaques and human donors enrolled in a Phase I clinical trial. Participants and animals with higher neutralizing titers were selected for the isolation of Spike-reactive B cells, and BCRs were sequenced from sarbecovirus reactive sorted B cells and constructed as rhesus (rh) or human (hu) IgGl. The mAbs described herein were identified from Spike-or SpFN-reactive B cells post-SpFN-ALFQ vaccination and comprise novel variable regions that facilitate sarbecovirus binding or bind to the H. pylori- Qvw' rQ ferritin particle itself. Many harbor the ability to neutralize sarbecovirus viral entry using pseudotyped virus, and target sites on the SARS-CoV-2 or SARS-CoV-1 Spike protein such as the receptor binding domain (RBD), N- terminal domain (NTD), and the cryptic S2 site. The coronavirus reactive mAbs described herein may be prophylactically or therapeutically protective against SARS-CoV-2 or other coronavirus infection or pathogenesis. The coronavirus reactive mAbs may be cross- sarbecovirus neutralizing and may cross-protect against existing and future coronaviruspandemics. The ferritin reactive mAbs described herein may be prophylactically or therapeutically protective against H. pylori infection or pathogenesis. The mAbs described herein may be used in diagnostic applications, e.g., for diagnosing coronavirus or H. pylori infection, respectively. The mAbs described herein may be used (diagnostically, prophylactically, or therapeutically) individually or in cocktails with each other or other mAbs. The mAbs described herein may be used as research tools in research applications, e.g., to detect coronavirus or ferritin antigens or expression of mRNA encoding coronavirus or ferritin antigens.

[0061] The polyclonal humoral response in the serum of SpFN-vaccinated macaques and donors reported herein targets overlapping, but distinct, epitopes of the Spike glycoprotein (S) when compared to a first-generation COVID-19 mRNA-LNP vaccine in macaques. The isolated mAbs described herein target multiple regions of the SpFN immunogen, including the ferritin nanoparticle, and Spike N-terminal domain (NTD), receptor-binding domain (RBD) and S2 subunit, and have advantageous activity and cross-reactivity profiles. The coronavirus reactive mAbs induced by SpFN vaccination included both neutralizing and non-neutralizing molecules which displayed high, yet differential, activity in multiple antibody effector assays dependent on the antigenic target, suggesting a role for the CDR3 in facilitating both neutralization and Fc effector functions because all isolated mAbs were cloned into the same rh-IgGl backbone.

[0062] One aim of the non-human primate (NHP) study described herein was to define the epitopes targeted by antibodies at the molecular level induced by SpFN vaccination in non- human primates. To achieve this aim, a novel sorting strategy was used, using both SARS- CoV-2 SpFN and SARS-CoV-1 SpFN from the Urbani strain (SpFN1) as probes to sort Spikespecific B cells and isolated mAbs directed at NTD, RBD, and S2. Neutralizing antibodies only targeted NTD or RBD. Using alanine mutagenesis epitope mapping and hACE2 competition, it was found that the RBD-targeting mAbs largely fell into two competition groups, one group that blocked hACE2 binding (deemed Group A), and another that minimally inhibited hACE2 binding but mainly targeted a cryptic, but conserved, epitope spanning across class III and V epitopes (deemed Group B). X-ray crystal structures were determined for SARS-CoV-2 RBD in complex with representative neutralizing mAbs from Groups A and B, termed the Walter Reed Army Institute of Research (WRAIR)-5021 and WRAIR-5001 mAbs, respectively. These NHP antibodies were tested for in vivo protection, structural analysis and binding and neutralization across diverse sarbecovirus and VOCs to further elucidate the molecularmechanism of broad antibody binding following SpFN vaccination, as discussed in more detail below.

[0063] One of the principal components of vaccine-elicited SARS-CoV-2 humoral immunity are neutralizing RBD-targeting antibodies (Walsh et al., 2020; Wang et al., 2022b). As noted above, the case of SpFN-elicited RBD-targeting NHP mAbs described herein could be divided into two broad epitope categories targeting either (i) the hACE2 -binding site (Group A RBD mAbs), or (ii) a conserved cryptic site (Group B RBD mAbs). The structural characterization of WRAIR-5021 and WRAIR-5001 showed that the WRAIR-5021 epitope overlaps considerably and competes with hACE2, a characteristic of the Group A RBD-directed mAbs. WRAIR-5021 also showed robust binding to RBDs from variants of concern, including Delta, Omicron BA.l, and Omicron BA.4 / 5, with low nanomolar affinity. However, potent neutralization was absent for most of the Omicron variants. Matching with the neutralization data, WRAIR-5021 showed robust protection in the K18 mouse model challenge using the Delta variant. The WRAIR-5001 mAb, while having more modest neutralization potency, targeted a highly conserved epitope located between the class III and class V epitopes. This epitope is cryptic and only seen in the RBD-up conformation of Spike and has high structural and sequence conservation. WRAIR-5001 showed robust binding to a panel of clade la and lb sarbecoviruses RBD molecules with sub-nanomolar affinity and neutralized Delta, Omicron BA. l, Omicron BA.2, and SARS-CoV-1.

[0064] Also described herein are molecular details on the NHP antibodies elicited following SpFN vaccination that may explain broad neutralizing and protective immune responses. The use of mAb-competition based characterization of the serum response highlighted differences between SpFN-immune responses compared to typical mRNA-LNP based vaccination. Analysis using mAb-blocking assays with DH1041 and DH1047 showed other nanoparticlebased vaccines elicited immune responses that are focused on epitopes consistent with the WRAIR-5001 and WRAIR-5021 mAbs (Saunders et al., 2021). The studies described herein link vaccine-elicited rhesus macaque antibodies that recapitulate the properties and epitopetargeting of broadly neutralizing and potent mAbs found in humans.

[0065] As previously noted for nanoparticle-based vaccines, without being bound by theory, the difference in immune responses when compared to single-valence vaccines is likely due to multiple reasons including the repetitive nature of a displayed antigen, improved T-cell help, and increased antigen duration in lymph nodes. In numerous cases, this has been shown to generate improved quality of immune responses against multiple viral pathogens (Boyoglu-Bamum et al., 2021; Brouwer et al., 2022; Brouwer et al., 2021; Carmen et al., 2021; Cohen et al., 2021; Cohen et al., 2022; Kanekiyo et al., 2019; Kanekiyo et al., 2013; Kelly et al., 2020; Zhang et al., 2020). Adjuvant selection likely also enhances the overall adaptative immune responses observed after SpFN vaccination, as ALFQ has been shown to also elicit robust T cell stimulation (Johnston et al., 2022; Joyce et al., 2021; Joyce et al., 2022). Thus, vaccination with SpFN and ALFQ in combination leads to strong engagement of the both the humoral and cellular components of the immune system, including the ability of AFLQ to stimulate CD4 T cell help leading to the development of mature, vaccine-responsive B cells. The further development of these SARS-CoV-2 nanoparticle vaccines and characterization in humans is important, with the Phase I clinical evaluation of SpFN currently underway, with clinical and immunological data in preparation and recently published (Ober Shepherd et al., 2024) [NCT04784767], Preliminary data from this trial indicates the SpFN and ALFQ vaccination recapitulates findings from the pre-clinical animal model studies and stimulates robust CD4 T cell mediated immunity concurrently with peak neutralizing titers. Understanding the context of SpFN-vaccine elicited responses in context of prior coronavirus exposure in humans will yield insight into the effectiveness of these responses to protect against current VOCs. In addition, the data will further speak to the ability of these vaccines to generate immune coverage against SARS-CoV-2 emerging VOCs and potential spillover events from distinct sarbecoviruses.

[0066] The mAbs identified herein represent the types of antibodies that may be present in SpFN-immunized macaque and human serum, and are useful to define the antibody molecular recognition of neutralization and cross-recognition of sarbecoviruses elicited by SpFN vaccination. Efforts to isolate and identify the targets of neutralization elicited in the ongoing Phase I clinical trial of SpFN and ALFQ will confirm and expand upon these findings. Understanding antibody epitope specificities and functional characteristics of cross-reactive antibodies following vaccination will further aid the development of antiviral countermeasures for SARS-CoV-2 and potential future CoV pandemic pathogens.

[0067] Isolation and characterization of next-generation SARS-CoV-2 neutralizing mAbs as described herein are important contributions, because the efficacy of previously identified therapeutic mAbs has severely diminished against VOCs, particularly the Omicron subvariants. Multiple antibody therapies received initial Emergency Use Authorization (EUA) in the U.S., but the emergence of resistant viral VOCs ultimately led to their withdrawal due to ineffectiveness. The mAbs disclosed herein could be combined with other mAbs targeting theNTD to generate immunotherapeutic agents with improved breadth in either prophylactic or therapeutic settings. Combinations with complementary nanobody-based antibodies (Chen et al., 2023; Wrapp et al., 2020a; Xu et al., 2021), bispecific modalities (Weidenbacher et al., 2022), or novel delivery platforms could further enhance the effectiveness of antibody-based therapies as we advance into new phases of the COVID-19 pandemic (Vanover et al., 2022). Methodologies described herein may allow the identification of further mAbs with high development potential.Monoclonal Antibodies

[0068] Table 1 below sets forth the variable region amino acid sequences of coronavirus reactive mAbs constructed from the BCR sequences of sorted B cells following immunization of rhesus macaques with SpFN, with the CDRs underlined. Table 2 below sets forth the variable region amino acid sequences of a selected subset of the NHP mAbs of Table 1. Table 3 below sets forth the CDR amino acid sequences of the NHP-derived mAbs of Table 1. Table 4 below sets forth the variable region amino acid sequences of H. pylori ferritin reactive mAbs constructed from the BCR sequences of sorted B cells following immunization of rhesus macaques with SpFN, with the CDRs underlined. Table 5 below sets forth the CDR sequences of the ferritin reactive NHP mAbs of Table 4.

[0069] Table 6 below sets forth the variable region amino acid sequences of coronavirus reactive mAbs constructed from the BCR sequences of sorted B cells following immunization of humans with SpFN-ALFQ, with the CDRs underlined. Table 7 below sets forth the variable region amino acid sequences of a selected subset of the human-derived mAbs of Table 6. Table 8 below sets forth the CDR amino acid sequences of the human mAbs of Table 6. Table 9 below sets forth the variable region amino acid sequences of H. pylori ferritin reactive mAbs elicited following immunization of humans with SpFN-ALFQ, with the CDRs underlined. Table 10 below sets forth the CDR sequences of the ferritin reactive human mAbs of Table 9.

[0070] In one aspect, the present disclosure provides a monoclonal antibody comprising a heavy chain complementary determining region 3 (HCDR3) and light chain complementary determining region 3 (LCDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the antibodies set forth in Table 1 (NHP) and Table 6 (human) below, or an antigen binding fragment thereof. In specific aspects, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein themonoclonal antibody comprises a HCDR3 and LCDR3, wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the antibodies set forth in Table 2 (NHP) and Table 7 (human) below.

[0071] In another aspect, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the antibodies set forth in Table 1 (NHP) and Table 6 (human). In specific aspects, the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the antibodies set forth in Table 2 (NHP) and Table 7 (human).

[0072] In some embodiments, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 1 (NHP) and Table 6 (human). In some embodiments, the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 2 (NHP) and Table 7 (human), or antigen binding fragment thereof.

[0073] In some embodiments, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 1 and Table 6. In some embodiments, the monoclonal antibody comprises a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 2 and Table 7. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 1 and Table 6. In some embodiments, the monoclonal antibody thereof comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 2 and Table 7, or antigen binding fragment thereof.

[0074] In some embodiments, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 1 and Table 6. In some embodiments, the monoclonal antibody comprises a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 2 and Table 7. In some embodiments, the monoclonal antibody comprises the light chain variable region sequence of any one of the antibodies set forth in Table 1 and Table 6. In some embodiments, the monoclonal antibody comprises the light chain variable region sequence of any one of the antibodies set forth in Table 2 and Table 7, or an antigen binding fragment thereof.

[0075] In some embodiments, the present disclosure provides a monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 1 and Table 6, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto. In some embodiments, the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 2 and Table 7, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, or antigen binding fragment thereof.

[0076] In accordance with other aspects, the present disclosure provides a monoclonal antibody or antigen binding fragment thereof, wherein the monoclonal antibody comprises a HCDR3 and LCDR3, wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the antibodies set forth in Table 4 (NHP) and Table 9 (human) below. In some embodiments, the monoclonal antibody comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the antibodies set forth in Table 4 (NHP) and Table 9 (human). In some embodiments, the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 4 (NHP) and Table 9 (human). In some embodiments, the monoclonal antibody comprises a heavy chain variable regionsequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 4 and Table 9, or antigen binding fragment thereof. In some embodiments, the monoclonal antibody comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 4 and Table 9, or antigen binding fragment thereof. In some embodiments, the monoclonal antibody comprises a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 4 and Table 9, or antigen binding fragment thereof. In some embodiments, the monoclonal antibody comprises the light chain variable region sequence of any one of the antibodies set forth in Table 4 and Table 9, or antigen binding fragment thereof. In some embodiments, the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 4 and Table 9, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, or antigen binding fragment thereof.

[0077] The tables below set forth amino acid sequences of monoclonal antibodies disclosed herein, which were elicited following immunization of rhesus macaques (Tables 1-5, NHP- derived mAbs) or humans (Tables 6-10, human-derived mAbs) with SpFN-ALFQ, and isolated and characterized as described in more detail below.The following abbreviations are used in Tables 1 and 6:RBD = SARS-CoV-2 (WA-1) Spike receptor binding domainNTD = SARS-CoV-2 (WA-1) Spike N-terminal domain S2 = SARS-CoV-2 (WA-1) Spike S2 subunitHexapro = Stabilized SARS-CoV-2 (WA-1) Spike trimerOC43 S2 = OC43 Spike S2 subunitSARS1 S2 = SARS-CoV-1 (Urbani) Spike S2 subunitTable 1 - Amino Acid Sequences of Coronavirus Reactive NHP mAb Variable Regions(CDRs are underlined)Table 2 - Selected Coronavirus Reactive NHP mAbs (CDRs are underlined)Table 3 - CDRs of SARS-CoV Reactive NHP mAb Variable RegionsTable 4 - Amino Acid Sequences of H. pylori Ferritin Reactive NHP mAb Variable Regions(CDRs are underlined)Table 5 - CDRs of H. pylori Ferritin Reactive NHP mAb Variable RegionsTable 6- Amino Acid Sequences of Coronavirus Reactive Human mAb Variable Regions (CDRs are underlined)* Two different lots of these antibodies were usedTable 7 - Selected Coronavirus Reactive Human mAbs (CDRs are underlined)Table 8 - CDRs of Coronavirus Reactive Human mAb Variable RegionsTable 9 - Amino Acid Sequences of H. pylori Ferritin Reactive Human mAb Variable Regions (CDRs are underlined)Table 10 - CDRs of H. pylori Ferritin Reactive Human mAb Variable Regions

[0078] As used herein, a “coronavirus reactive mAb” of the present disclosures includes (i) the mAbs disclosed in Tables 1-2 (NHP) and Table 6-7 (human), as well as (ii) mAbs havingHCDR3 and LCDR3 amino acid sequences at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the HCDR3 and LCDR3 sequences of an mAb disclosed in Tables 1-2 and 6-7; (iii) mAbs having HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences of an mAb disclosed in Tables 1-2 and 6-7; and (iv) mAbs having amino acid sequences at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of an mAb disclosed in Tables 1-2 and 6- 7. (As noted above, the amino acid sequences of the CDRs of the antibodies set forth in Tables 1-2 and Tables 6-7 are set forth in Table 3 (NHP) and Table 8 (human), respectively.)

[0079] In some embodiments, a coronavirus reactive mAb or antigen binding fragment thereof as disclosed herein is reactive with a coronavirus antigen. As used herein, “reactive with a coronavirus antigen” can be determined by a signal to noise value that is three times over the binding of an isotype control mAb in a multiplexed, bead based assay (as illustrated in the examples below) to an isolated coronavirus domain, e.g., to an isolated domain of SARS-CoV- 1, SARS-CoV-2, or MERS-CoV (as illustrated in FIGs. 10A-C, and FIG. 12E). It should be understood that an mAb as described herein may dominantly bind one domain (e.g., SARS- CoV-2 RBD), and also may bind another domain (e.g., another domain of SARS-CoV-2 or another sarbecovirus), and that such cross-binding may be advantageous.

[0080] Thus, in some embodiments, a coronavirus reactive mAb or antigen binding fragment thereof of the present disclosure binds to one or more epitopes on a region of a coronavirus spike protein of one or more coronaviruses selected from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS- CoV), and severe acute respiratory syndrome coronavirus 2 (SARS CoV-2). In some embodiments, a coronavirus reactive mAb is reactive with SARS-CoV, e.g., the mAb binds to an antigenic domain of a SARS-CoV Spike protein, such as an antigenic domain of a SARS- CoV-2 Spike protein or an antigenic domain of a SARS-CoV- 1 Spike protein. In some embodiments, the region of the spike protein bound by a coronavirus reactive mAb or antigen binding fragment thereof is one or more selected from the RBD region, the NTD region, and the S2 region. In some embodiments, a coronavirus reactive mAb or antigen binding fragment thereof neutralizes one or more coronaviruses selected from SARS-CoV- 1, MERS-CoV, and SARS-CO-V2.

[0081] Additionally or alternatively, in some embodiments, a coronavirus reactive mAb or antigen binding fragment thereof is reactive to SpFN, e.g., binds to an epitope on SpFN. Additionally or alternatively, in some embodiments, a coronavirus reactive mAb is not reactive with H. pylori ferritin.

[0082] Also provided are pharmaceutical compositions comprising a coronavirus reactive mAb as disclosed herein, or antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a cocktail of two or more monoclonal antibodies against SARS-CoV-2 or other coronaviruses, or antigen binding fragments thereof, wherein at least one of the monoclonal antibodies or antigen binding fragments thereof is a coronavirus reactive mAb as described herein or an antigen binding fragment thereof.

[0083] The coronavirus reactive mAbs disclosed herein, or antigen binding fragment thereof, or pharmaceutical compositions comprising one or more thereof, can be used in methods of preventing, treating, or reducing the risks of a coronavirus infection, comprising administering a coronavirus reactive mAb as described herein, or antigen binding fragment thereof, or a pharmaceutical composition comprising one or more thereof, to a subject in need thereof. In some embodiments, the coronavirus infection is selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV. In some embodiments, the subject is at risk of infection by one or more coronaviruses selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV. In some embodiments, the subject is infected by one or more coronaviruses selected from SARS-CoV- 1, SARS-CoV-2, and MERS-CoV.

[0084] The coronavirus reactive mAbs described herein, or antigen-binding fragments thereof, may be used in methods for detecting a coronavirus infection in a subject or assessing a subject’s condition, comprising contacting a biological sample obtained from the subject with a coronavirus reactive mAb as described herein, or antigen binding fragment thereof, and detecting any coronavirus antigen bound by the mAb or antigen binding fragment thereof. In some embodiments, the coronavirus infection is selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV. In some embodiments, the subject has been exposed to one or more coronaviruses selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV. In some embodiments, the subject is infected by one or more coronaviruses selected from SARS-CoV- 1, SARS-CoV-2, and MERS-CoV.

[0085] As used herein, a "ferritin reactive mAb” of the present disclosures includes (i) the mAbs disclosed in Tables 4 and 9, as well as (ii) mAbs having HCDR3 and LCDR3 amino acid sequences at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the HCDR3 and LCDR3 sequences of a monoclonal antibody disclosed in Tables 4 and 9; (iii) mAbs having HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences of a mAb disclosed in Tables 4 and 9; and (iv) mAbs having amino acid sequences at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of an antibody disclosed in Tables 4 and 9. (The amino acid sequences of the CDRs of the antibodies set forth in Tables 4 and 9 are set forth in Table 5 (NHP) and Table 10 (human), respectively.)

[0086] In some embodiments, a ferritin reactive mAb or antigen binding fragment thereof is reactive with H. pylori ferritin. As used herein, “reactive with a H. pylori antigen” can be determined by a signal to noise value that is three times over the binding of a control mAb in an EIA assay (as illustrated in the examples below) to an isolated H. pylori ferritin antigen (as illustrated in FIGs. 9A-B, and FIG. 16A-B). In some embodiments, a ferritin reactive mAb or antigen binding fragment thereof is reactive with empty H. pylori ferritin particles. Additionally or alternatively, in some embodiments, a ferritin reactive mAb or antigen binding fragment is reactive with a ferritin moiety of SpFN. Additionally or alternatively, in some embodiments, a ferritin reactive mAb or antigen binding fragment thereof neutralizes H. pylori. Additionally or alternatively, in some embodiments, a ferritin reactive mAb or antigen binding fragment thereof is not reactive with coronavirus antigens, e.g., does not specifically bind coronavirus antigens.

[0087] Thus, also provided are pharmaceutical compositions comprising a ferritin reactive mAb as disclosed herein, or antigen binding fragment thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises a cocktail of two or more ferritin reactive mAbs, or antigen binding fragments thereof, wherein at least one of the mAbs or antigen binding fragments thereof is a ferritin reactive mAb as described herein or an antigen binding fragment thereof. The ferritin reactive mAbs disclosed herein, or antigen binding fragment thereof, or pharmaceutical compositions comprising one or more thereof, can be used in methods of preventing, treating, or reducing the risks of H. pylori infection, comprising administering a ferritin reactive mAb as described herein, or antigenbinding fragment thereof, or a pharmaceutical composition comprising one or more thereof, to a subject in need thereof. The ferritin reactive mAbs described herein, or antigen-binding fragments thereof, may be used in methods for detecting H. pylori infection in a subject or assessing a subject’s condition, comprising contacting a biological sample obtained from the subject with a ferritin reactive mAb as described herein, or antigen binding fragment thereof, and detecting any ferritin antigen bound by the mAb or antigen binding fragment thereof.

[0088] As noted above, the coronavirus reactive and ferritin reactive mAbs described herein may be used to detect coronavirus or ferritin antigens, respectively, or expression of mRNA encoding coronavirus or ferritin antigens, , respectively, including to detect SpFN or expression of mRNA encoding SpFN. 27. Thus, provided are methods for detecting a coronavirus antigen or an H. pylori antigen in a sample, comprising contacting a sample with a mAb as disclosed herein, or antigen binding fragment thereof, and detecting any antigen bound by the mAb or antigen binding fragment thereof. In some embodiments, the sample is obtained from a composition comprising mRNA encoding a coronavirus or H. pylori antigen. In some embodiments, the sample is obtained from a composition comprising mRNA encoding H. pylori ferritin particles. In some embodiments, the sample is obtained from a composition comprising mRNA encoding SpFN.

[0089] Also provided are polynucleotides encoding a mAb as described herein, or antigen binding fragment thereof. Also provided are vectors comprising a polynucleotide encoding a mAb as described herein, or an antigen binding fragment thereof. Also provided are host cells comprising a mAb as described herein, or antigen-binding fragment thereof, or a polynucleotide as described herein, or a vector as described herein.

[0090] The following examples are provided for illustration, and are not limiting of the present disclosure. It should be understood that the invention is not limited to the specific conditions or details described in these examples.ExamplesMethodsSorting of SARS-CoV-2-positive B cells.

[0091] Cryopreserved PBMCs from naive or SpFN vaccinated rhesus macaques or human Phase I SpFN-vaccines were thawed in warm media containing benzonase, then washed with PBS and stained for viability using the Aqua Live / Dead stain (ThermoFisher) and Fc receptorsblocked using normal mouse IgG (Invitrogen #02-6502). After centrifuging, cells were then stained for 30 minutes at room temperature (RT) for SARS-CoV-2 and SARS-CoV-1 reactive B cells by incubating with SpFN and SpFN1particles, followed by a wash with staining buffer (lx PBS containing with FCS and NaNs). Cells were then incubated at RT for 30 minutes with fluorochrome conjugated secondary antibodies specific to the S protein of either SARS-CoV- 2 (WRAIR-2054-APC) or SARS-CoV-1 (mmO2-PE, SinoBiological #40150-MM02). Finally, after another wash, cells were incubated at RT for 30 min with a cocktail of phenotyping antibodies including CD3 BV510 (BD Biosciences Cat# 740187, Lot# 0261796), CD4 BV510 (BD Biosciences Cat# 562970, Lot# 0177290), CD14 BV510 (BioLegend Cat# 301842, Lot# B264335), and CD16 BV510 (Biosciences Cat# 563830, Lot# 1307468) as dump channel markers, and CD 19 PE-Cy5 (Beckman Coulter Cat# IM2643U Lot# 200100), CD20 AlexaFluor700 (BD Biosciences Cat# 560631 Cat# 134775), IgG PE-Cy7 (BioLegend Cat# 410721 Lot# M1310G05), IgM BV650 (Biolegned Cat# 314525 Lot# B294373). SARS-CoV- 2 RBD, and NTD (ThermoFisher) that had been biotinylated, tetramerized and conjugated to streptavidin-PE were also included in this step. Viable, dump channel -, IgG+, IgM-, CD19+B cells that were antigen positive to either a SpFN particle or tetramerized subdomain, or a combination, were single-cell sorted into PCR plates containing lysis buffer composed of murine RNAse inhibitor (New England Biolabs), dithiothreitol (DTT), SuperScript III First Strand Buffer (ThermoFisher), Igepal (Sigma), and carrier RNA (Qiagen) at one cell per well using a FACS ARIA (Becton Dickinson) and stored at -80°C until subsequent reverse transcription. Analysis was performed using FlowJo 10 (BD Bioscience).Antibody sequencing and production.

[0092] RNA from single B cells was reverse-transcribed using random hexamers and the SuperScriptlll kit (ThermoFisher). Antibody V (D) J genes were amplified from the cDNA by nested PCR, using the HotStar Taq DNA Polymerase kit (Qiagen) using a combination of primer sets and methods described previously (Dussupt et al., 2020). V(D)J gene assignment, somatic hypermutation and CDR3 determinations were performed using IgBlast28. Sequences for the antibody variable regions for the monoclonal antibodies from the rhesus macaques (Tables 1, 2, and 4) and human donors (Table 6, 7, and 9) are shown below. Antibody variable regions were synthesized and cloned (Genscript) into CMVR expression vectors (NIH AIDS reagent program) between a murine Ig leader (GenBank DQ407610) and the constant regions of human IgGl (GenBank AAA02914), IgK (GenBank AKL91145) or IgZ. (GenBank AAA02915). Antibodies were first expressed using a 24-well plate format by co-transfectingplasmids encoding paired heavy and light chains into Expi293F cells (ThermoFisher) according to the manufacturer’s instructions. After 5 days, clarified cell culture supernatants were screened for neutralization in the pSV assay and binding to SARS-CoV-2 antigens using a bead-based multiplex assay (see above). Positive hits from the supernatant screen for neutralization activity and / or binding were scaled up by transfecting 30 ml cultures of Expi293F cells as indicated above. Monoclonal antibodies were purified 4 to 5 days posttransfection using AmMag Protein A magnetic beads and the AmMagSA purification system (Genscript), according to the manufacturer’s recommendations. Purified mAbs were buffer exchanged into Phosphate-Buffered Saline (PBS). The purity and stability of monoclonal antibodies was assessed by SDS-PAGE and Coomassie staining in both reducing and nonreducing conditions. Control antibodies were all expressed as human IgGl and purified from Expi293F cells, as described above.

[0093] In some circumstances, gene usage, CDR3 length, and % somatic hypermutation may be used to identify similar antibodies. These characteristics of the NHP mAbs are listed in Table 11. Table 12 indicates how tightly these mABs bind to their respective epitope on the spike protein. Tables 13-15 provide crystal structure characteristics. This information supports the uniqueness, specificity, and function of these mAbs.Table 11. Gene usage of coronavirus reactive NHP mAbs isolated from SpFN vaccinated rhesus macaques.

[0094] Annotation of nucleotide sequences was performed using IGMT / VQuest. Table 11 shows the mAh ID; reactivity of the mAh to either a domain of the SARS-CoV-2 Spike protein or the H. pylori ferritin molecule (Ferr); the variable heavy (VH) chain germline gene annotation; VH percent somatic hypermutation (%SHM) difference from the germline sequence; length of the heavy chain CDR3 (CDRH3) region; amino acid sequence of the variable (V), diversity (D), and joining (J) junction; the variable light (VL) chain germline gene usage; the VL chain percent somatic hypermutation (%SHM) difference from the germline sequence, the length of the light chain CDR3 (CDRL3), and the amino acid sequence of the variable (V) joining (J) of the light chain. The sequences and pairing of the VDJ and VJ junctions of the heavy and light chain, respectively, are unique for each mAb and are the most variable region of their respective chains. These regions contribute significantly to the specificity of each mAb. The percent SHM and CDR3 lengths further characterize these mAbs, as a higher SHM rate and shorter CDR3 may indicate a high degree of antigen stimulation, leading to affinity maturation of a B cell. Taken together, these data detail the uniqueness of each antibody at the most variable regions of both the heavy and light chain.Table 12. Binding affinity constants of WRAIR RBD NHP mAbs measured against selected RBDs, using BLI.

[0095] Table 12 shows the binding affinity of each mAb to the receptor binding domain (RBD) of the Spike protein of SARS-CoV-2 against wild type WA-1, Delta (B.1.617.2), Omicron (BA. l or BA.4 / 5), and SARS-CoV-1. Low equilibrium dissociation constants (KD) values, measured as a ratio of the dissociation rate constant in min'n(KOff) over the association rate constant, in units of M'1min'1(Kon), suggesting high affinity, were observed for all mAbs to WA-1. KD values rose dramatically against VoCs, especially Omicron, suggesting a weaker affinity of many of the mAbs across VoCs compared to WA-1. While weak affinity was observed for WRAIR-5004, WRAIR-5005, and WRAIR-5011 to SARS-CoV-1, WRAIR-5001had the smallest KD value and thus highest affinity to SARS-CoV-1. WRAIR-5020 and WRAIR-5021 showed no binding data (NBD) to SARS-CoV-1.Table 13. X-ray crystallographic data collection and refinement statistics of WRAIR-5001 in complex with SARS-CoV-2 RBD, and WRAIR-5021 in complex with SARS- CoV-2 RBD.

[0096] Table 13 shows the X-ray diffraction data and subsequent structure model building and refinement statistics for WRAIR-5001 in complex with SARS-CoV-2 RBD, and for WRAIR- 5021 in complex with SARS-CoV-2 RBD. Data completeness was 99.6% and 97.8% for the two independent structures, respectively. Structure quality as measured by Ramachandran plot analysis showed no amino acid outliers for either structure. R-free values of 29.8% and 25.6% indicate suitable structure modeling, with root mean square deviations for bond lengths and bond angles at low and acceptable levels of 0.003 and 0.63 for both structures. Values in parentheses pertain to highest-resolution shells. *Rfree was calculated using ~5% randomly selected reflections.Table 14A and 14B. WRAIR-5021 mAb detailed contacts with SARS-CoV-2 receptorbinding domain defined by high-resolution crystal structure.Table 14ATable 14B

[0097] Table 14A shows amino acids that form hydrogen bonds or salt bridge bonds between WRAIR-5021 and SARS-CoV-2 RBD indicative of important interacting residues between the mAb and RBD. Table 14B shows accessible and buried surface area of WRAIR-5021 heavy and light chain amino acids and the SARS-CoV-2 RBD. Residues with greater buried surface area have greater contribution to the binding energy of the complex.Table 15A and 15B. WRAIR-5001 mAb detailed contacts with SARS-CoV-2 receptorbinding domain defined by high-resolution crystal structure.Table 15ATable 15B

[0098] Table 15A shows amino acids that form hydrogen bonds or salt bridge bonds between WRAIR-5001 and SARS-CoV-2 RBD indicative of important interacting residues between the mAb and RBD. Table 15B shows accessible and buried surface area of WRAIR-5001 heavy and light chain amino acids and the SARS-CoV-2 RBD. Residues with greater buried surface area have greater contribution to the binding energy of the complex.Fab production.

[0099] Freshly purified IgGs in PBS buffer (pH 7.4) were mixed with Lys C protease (New England Biolabs) at 1 :2000 (w:w) ratio. Reaction was allowed to proceed for 2-3 hours in a water bath incubator at 37 °C. Digestion was assessed by SDS-PAGE and upon completion, the reaction mixture was passed through Protein-A beads (Cytiva) three times and the final flow through was assessed by SDS-PAGE for purity.Production of recombinant proteins.

[0100] Recombinant SARS-CoV-2 proteins RBD (318-514), NTD (1-290) and SI (1-665) were made from a synthesized full-length Spike sequence (Genscript) from strain USA / IL1 / 2020 (GenBank # MN988713) and were cloned with C-terminal AviTag and polyhistidine tags into the CMVR vector under the bovine prolactin leader sequence. The coding sequence for the SARS-CoV-2 (Genbank # MN908947) stabilized trimer (S2P) was a generous gift from Jason McLellan (Wrapp et al., 2020b). The S-2P sequence was subcloned into the pCMVR vector with C-terminal AviTag and poly-histidine tags. Four additional stabilizing mutations were added using the Quickchange multi site-directed mutagenesis kit (Agilent) to make the HexaPro variant with improved stability (Hsieh et al., 2020), referred to as stabilized S trimer throughout the manuscript. SARS-CoV-2 RBD constructs (331 - 527) were also modified to incorporate a N-terminal hexa-histidine tag were derived from the Wuhan-Hu- 1 strain genome sequence (GenBank # MN9089473). Subsequent RBD VOC with point mutations were generated using a modified QuikChange site-directed mutagenesis protocol (Agilent). A S-2P construct derived from SARS-CoV-1 was generated as previously described (Kirchdoerfer et al., 2018). Spike probes were expressed and biotinylated as previously described (Zhou et al., 2020), with mutations for B.1.1.7, B.1.351, P.1 and other variants added by QuikChange site-directed mutagenesis. Mutated residues were as follows: B. l.1.7 (69- 70del, Y144del, N501Y, A570D, D614G, P681H, T718I, S982A, D1118H), B.1.351 (L18F, D80A, D215G, 241-243del, R246I, K417N, E484K, N501Y, D614G, A701V, E1195Q), P.l (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y,T1027I), B.1.427 / 429 (W152C, L452R, D614G), B.1.526a (T95I, D253G, S477N, D614G, A701V) and B.1.526b (T95I, D253G, E484K, D614G, A701V). hACE2-Ig, a fusion protein made by connecting the human hACE2 (Q9BYF1) extracellular domain (residues 19-611) to the constant domain of a human IgGl was expressed and purified as described above for antibodies. All proteins were produced transiently from Expi293F or FreeStyle 293F (stabilized trimer) cells (both ThermoFisher) and purified from cell culture supernatants using Ni-NTA (Qiagen) affinity. The stabilized trimer was further purified by gel filtration on an ENrich SEC 650 column (Bio-Rad).Multiplex antibody binding assay.

[0101] A high-throughput bead-based antibody binding assay was performed as previously described (Brown et al., 2012; Tomaras et al., 2008) with modifications to adapt to sarbecovirus antigens. Briefly, heat-inactivated plasma from rhesus macaques that were vaccinated twicewith 50 pg of SpFN or PBS, or purified monoclonal antibodies, were diluted and loaded into 384-well assay plates by use of a Biomek NXP® automated liquid handler (Beckman Coulter). A cocktail of 11 sarbecovirus antigens and 1 control proteins (HIV-1 antigens), obtained commercially (SinoBiological) or internally produced (see below), spanning Spike SI and S2 domains for SARS-CoV-2 or SARS-CoV-1 were covalently coupled to uniquely coded magnetic microspheres (Luminex) per manufacturer’ s protocol and added to the plate in a final volume of 50 pl / well. Following a 2 hour incubation with vigorous shaking, microspheres were washed using a magnetic 384-well automated plate washer (Bio-Tek) to remove unbound sample. Microspheres were then resuspended with 0.5 pg mF1mouse anti-human IgG-PE (Southern Biotech), vortexed for 1 minute with a microplate vortex at 3,000 rpm, sonicated for 1 minute and then incubated with vigorous shaking for 1 hour. A final wash removed unbound detection reagent, and microspheres were resuspended in 40 pl sheath fluid (Luminex). Data was collected on a Bio-Plex®3D Suspension Array system (Bio-Rad) running xPONENT® v.4.2 (Luminex). Signal to Noise (S / N) ratio were calculated by the dividing the MFI for each sample by either Ig-depleted healthy plasma or a negative control antibody (MZ4) according to the type of sample analyzed.SARS-CoV-2 pseudovirus neutralization assay.

[0102] SARS-CoV-2 pseudovirions (pSV) were produced by co-transfection of HEK293T / 17 cells with a pcDNA3.1 encoding SARS-CoV-2 S and an HIV-1 NL4-3 luciferase reporter plasmid (pNL4-3.Luc.R-E-, NIH AIDS Reagent Program). The S expression plasmid sequence was derived from the Wuhan Hu-1 strain or other indicated strain (Genscript) (GenBank # NC_045512), which is also identical to the IL1 / 2020 and WA1 / 2020 strains. The S expression plasmid sequence was also codon optimized and modified to remove the last 18 amino acids of the cytoplasmic tail to improve S incorporation into the pseudovirions and thereby enhance infectivity. S expression plasmids for current SARS-CoV-2 VOC were similarly codon optimized, modified and included the following mutations compared to WA-1: Beta / B.1.351 (= D80A, D215G, del 241-243, K417N, E484K, N501Y, D614G, A701V,), Delta / B.1.617.2 (T19R, E156G, del 157-158, L452R, T478K, D614G, P681R, D950N), Omicron BA. l (A67V, A69 / 70, T95I, G142D, del 143-145, N211I, del 212R214, insG339D S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), Omicron BA.2 (T19I, L24-, P25-, P26-, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R,N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), and Omicron BA.5 (T19I, del L24, del P25, del P26, A27S, del H69, del V70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K) from GenscriptA D614G variant was also made from the Wuhan Hu-1 construct using the Q5 site-directed mutagenesis kit (NEB). In addition, a codon-optimized S expression plasmid encoding SARS-CoV-1 (Sino 1-11, GenBank # AY485277) was generated that incorporated a 28 amino acid C-terminal deletion to improve infectivity (Moore et al., 2004). Virions pseudotyped with the vesicular stomatitis virus (VSV) G protein were used as control. Infectivity and neutralization titers were determined using hACE2-expressing HEK293 target cells (Integral Molecular) in a semi-automated assay format using robotic liquid handling (Biomek NXp Beckman Coulter). Samples were diluted 1 :40 in growth medium and serially diluted, then 25 pl / well was added to a white 96-well plate. Purified mAbs started at a concentration of 1 mg ml'1. An equal volume of diluted SARS-CoV-2 pSV was added to each well and plates were incubated for 1 hour at 37 °C. Target cells were added to each well (40,000 cells / well) and plates were incubated for an additional 48 h. RLUs were measured with the EnVision Multimode Plate Reader (Perkin Elmer) using the Bright-Glo Luciferase Assay System (Promega). Neutralization dose-response curves were fitted by nonlinear regression using the LabKey server, and the final titers are reported as the reciprocal of the dilution of plasma necessary to achieve 50% neutralization (ID50, 50% inhibitory dose or IC50, 50% inhibitory concentration) and 80% neutralization (ID80, 80% inhibitory dose or IC80, 80% inhibitory concentration). Assay equivalency was verified by participation in the SARS-CoV-2 Neutralizing Assay Concordance Survey (SNACS) run by the Virology Quality Assurance Program and External Quality Assurance Program Oversite Laboratory (EQAPOL) at the Duke Human Vaccine Institute, sponsored through programs supported by the National Institute of Allergy and Infectious Diseases, Division of AIDS.Authentic SARS-CoV-2 variant and SARS-CoV-1 neutralization assay.

[0103] Assays were performed as previously described (Joyce et al., 2022) (34914540), with SARS-CoV-2 viruses USA-WA1 / 2020 (WAI), USA / CA_CDC_5574 / 2020 (BL 1.7), hCoV- 19 / South Africa / KRISP-EC-K005321 / 2020 (B.1.351), hCoV-19 / Japan / TY7-503 / 2021, and hCoV-19 / USA / PHC658 / 2021 (B.1.617.2) obtained from BEI Resources (National Institute of Allergy and Infectious Diseases, NIH) and propagated for one passage using Vero clone E6 cells. Virus infectious titer was determined by an endpoint dilution and cytopathic effect (CPE)assay on Vero-E6 cells. An endpoint dilution microplate neutralization assay was performed to measure the neutralization activity of macaque serum samples. In brief, serum samples were heat-inactivated and subjected to successive threefold dilutions starting from 1 :50. Triplicates of each dilution were incubated with SARS-CoV-2 at a multiplicity of infection of 0.1 in Eagle’s minimum essential medium with 7.5% inactivated fetal calf serum for 1 hour at 37 °C. After incubation, the virus-antibody mixture was transferred onto a monolayer of Vero-E6 cells grown overnight. The cells were incubated with the mixture for about 70 hours. CPE of viral infection was visually scored for each well in a blinded fashion by two independent observers. The results were then reported as the percentage of neutralization at a given sample dilution. A SARS-CoV-1 authentic plaque reduction virus neutralization assay was performed similarly to previously described (Joyce et al., 2022), with the following modifications. The starting dilution of serum was 1 :5, and about 100 plaque-forming units of virus were used for virus and serum incubation. The overlay used after virus adsorption was Dulbecco’s modified Eagle’s medium (Gibco) containing 2% FBS and 20% methylcellulose. Plates were then incubated for 5 days, and after crystal violet staining, the washing step used water. Plaques were graded as follows: about 25 plaques / 25% monolayer damage (MD; ±); about 50 plaques / 50% MD (+); about 75 plaques / 75% MD (++); and about 100 plaques / 100% MD (+++). All negative control wells were solid monolayers.Measurements of antibody Fc effector functions using recombinant proteins.

[0104] ADCP. ADCP was measured as previously described (Ackerman et al., 2011). Briefly, biotinylated SARS-CoV-2 S stabilized trimer was incubated with red streptavidin-fluorescent beads (Molecular Probes) for 2 hours at 37 °C. Ten pl of a 100-fold dilution of beads-protein mixture was incubated for 2 hours at 37 °C with 100 pl of monoclonal antibodies diluted at 5 pg mF1before addition of THP-1 cells (20,000 cells per well; Millipore). After 19 hours incubation at 37 °C, the cells were fixed with 2% formaldehyde solution and fluorescence was evaluated on a LSRII flow cytometer (BD Bioscience). The phagocytic score was calculated by multiplying the percentage of bead-positive cells by the geometric mean fluorescence intensity (MFI) of the bead -positive cells and dividing by 104.Measurements of antibody Fc effector functions using cell surface-expressed Spikes. Opsonization. SARS-CoV-2 S-expressing FreeStyle 293F cells were generated by transfection with linearized plasmid encoding a codon-optimized full-length SARS-CoV-2 S protein matching the amino acid sequence of the IL 1 / 2020 isolate (GenBank # MN988713). Stable transfectants were single-cell sorted and selected to obtain a high-level Spike surfaceexpressing clone (293F-Spike-S2A). 293F-Spike-S2A cells were incubated with 100 pl of monoclonal antibodies diluted at 5 pg ml’1for 30 minutes at 37 °C. Cells were washed twice and stained with anti-human IgG PE (Southern Biotech). Cells were then fixed with 4% formaldehyde solution and fluorescence was evaluated on a LSRII (BD Bioscience).

[0105] Trogocytosis. Trogocytosis was measured using a previously described assay (Alrubayyi et al., 2018). Briefly, SARS-CoV-2 Spike-expressing Expi293F cells were stained with PKH26 (Sigma- Aldrich). Cells were then washed with and resuspended in R10 media. Cells were then incubated with monoclonal antibodies diluted at 5 pg ml’1for 30 minutes at 37 °C. Effector peripheral blood mononuclear cells were next added to the R10 media at an effector to target (E:T) cell ratio of 50: 1 and then incubated for 5 hours at 37 °C. After the incubation, cells were washed, stained with live / dead aqua fixable cell stain (Life Technologies) and CD 14 APC-Cy7 (clone M(|)P9) for 15 minutes at RT, washed again, and fixed with 4% formaldehyde (Tousimis) for 15 minutes at RT. Fluorescence was evaluated on an LSRII flow cytometer (BD Biosciences). Trogocytosis was evaluated by measuring the PKH26 mean fluorescence intensity of the live CD14+cells.

[0106] CD 16 reporter assay (ADCC). WT Spike-CEM cells were plated at 100,000 per well in round bottom 96-well plates and incubated with 5 pg ml’1of mAbs for 30 minutes at 4 °C. Cells were washed and 200,000 lurkat-Lucia NFAT-CD16 cells (Invivogen) were added to each well in 100 pl of IMDM 10% FBS. The cells were then centrifuge for 1 minute at low speed and co-cultured for 24 hours at 37 °C. Fifty pl of Quanti-Luc was added to 20 pl of coculture supernatant and luminescence was measured immediately on a luminometer (2104 Multilabel reader, PerkinElmer).Epitope binding.

[0107] Epitopes of the NTD and RBD mAbs were first mapped by binding competition against a set of characterized control antibodies (RBD) using Biolayer interferometry (BLI) on an Octet RED96 instrument (ForteBio) similar to what was reported previously (Dussupt et al., 2021). Avi-tagged recombinant NTD and RBD proteins, biotinylated with the BirA biotinylation kit (Avidity), were diluted to 2.5 and 1 pg ml-1, respectively, in kinetic buffer (0.1% [w / v] bovine serum albumin [BSA], 0.02% [v / v] Tween-20 in PBS; ForteBio) and loaded onto Streptavidin (SA) sensors (ForteBio) for 250 s, to reach -50% of the sensor maximum binding capacity. Loaded biosensors were immersed into wells containing the first competing antibody at 100 nM for 900 s to saturate all binding sites. Next, biosensors were dipped into wells containingthe second antibody, in presence of the first competing antibody (all at 100 nM), and binding was measured after 900 s of association. Residual binding signal of the second antibody was expressed as a percentage of the maximum binding signal obtained in absence of the first competing antibody, ran in parallel. Residual binding signal was further corrected for any increase in signal obtained with the first competing antibody alone. Antibodies were defined as competing when binding signal of the second antibody was reduced to less than 25% of its maximum binding capacity and non-competing when binding was greater than 50%. Intermediate competition was defined by binding levels of 25-50%. Control antibodies representing RBD-A, RBD-B and RBD-C, as described previously (Dussupt et al., 2021), were WRAIR-2125, WRAIR-2063 and WRAIR-2151, respectively. Control antibodies representing NTD-A, NTD-B and NTD-C, as described previously (Dussupt et al., 2021), were WRAIR- 2025, WRAIR-2137neut and WRAIR-2054, respectively. The same approach was used to assess binding competition between NTD and RBD antibodies within the stabilized S trimer. hACE2-Ig was used like an antibody to assess the ability of NTD and RBD antibodies to block hACE2 binding to the S trimer. Epitope bins were also generated via high-throughput SPR epitope binning. High throughput SPR classical epitope binning experiments were performed on a Carterra LSA system, using an CMDP sensor chip in a 384-ligand array format. Chip surface was primed with lOmM MES, pH5.5 (Carterra) run buffer, and conditioned using 50mMNaOH (Carterra), IM NaCl (Teknova), and lOmM Glycine pH2 (Carterra). Antibodies were coupled to the chip surface using a solution of 400 nM l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) +100 mM N-hydroxy sulfosuccinimide (Sulfo- NHS) in lOmM MES pH5.5 running buffer for 7 minutes, followed by IM ethanolamine quenching of the chip surface. A non-regenerative kinetics experiment is preformed to determine the antibody surface using either CoV-RBD or CoV-NTD in a four-fold dilution series (0-400nM) in lx HBST+0.5mg / mL BSA run buffer (Carterra). In order, to determine the regenerative ability of the chip surface, regeneration scouting by super saturating the chip surface with the antigen of interest, followed by two, thirty second pulses of lOmM Glycine pH2 with subsequent washes of lx HBST+0.5mg / mL BSA run buffer. The epitope binning experiment was performed by injecting lOOnM of either CoV-RBD or CoV-NTD for five minutes (Capture Antigen Phase), then by 20 mg / mL of of mAb for five minutes (Capture mAb Phase), followed by a HBST+BSA running buffer injection for 1 minute. Two regeneration pulses of thirty seconds were performed after each anitgen-mAb reactions and buffer runs were performed after every eleven antigen-mAb reactions. Data were analyzed using the Carterra Epitope Software to determine mAb groupings to either the CoV-RBD or CoV-NTD.Sensogram thresholds were normalized to the antigen-only binding signal to determine which mAbs could be either blocking or sandwiching interactions with the mAb coupled surface (y- axis). Antibodies that showed poor regeneration or did not self-block were excluded from the binning analysis. Antibodies that fell into sensogram thresholds and not excluded were clustered together to form a heatmap, community plot, or dendrogram where antibodies with identical blocking / sandwiching interactions can be grouped into a bin. Data analysis from each epitope binning experiment were visualized as a dendrogram.Affinity binding assays.

[0108] Real-time interactions between purified SARS-CoV-2 proteins and antibodies were monitored on an Octet RED96 instrument (ForteBio). For affinity measurement, mAbs were immobilized onto Anti-human IgG Fc capture (AHC) biosensors (Sartorius). Baseline was established in PBS. Loaded biosensors were dipped into wells containing serial dilutions of RBD (starting from 500 nM) for 180 s. Complexes were then allowed to dissociate in PBS for 300 s. After reference subtraction, apparent binding kinetic constants were determined, from at least 4 concentrations of RBD, by fitting the curves to a 1 : 1 binding model using the Data analysis software 12.0 (ForteBio). To assess binding to a panel of RBD mutants, HIS IK biosensors (ForteBio) were equilibrated in assay buffer (PBS) for 15 s before loading of His- tagged SARS-CoV-2 RBD, VOC RBDs, or SARS-CoV-1 RBD (30 pg ml’1diluted in PBS) for 100 s. Immobilized RBD proteins were then dipped in antibodies (30 pg ml’1diluted in PBS) for 180 s followed by dissociation for 60 s. To assess binding to the panel of S mutants, biotinylated probes were loaded on SA biosensors (ForteBio) and subsequently dipped into antibodies (30 pg ml’1diluted in IX kinetic buffer) for 450 s followed by a 120 s dissociation step. Binding responses were measured at the end of the association step using the Data analysis software 10.0 (ForteBio). hACE2-RBD competition assays were carried out as follows: SARS- CoV-2 RBD (30 pg ml’1diluted in PBS) was immobilized on HIS1K biosensors (ForteBio) for 220 s. Test antibodies were allowed to bind for 200 s, followed by baseline equilibration (30 s), and then incubation with hACE2 protein (30 pg ml’1) for 120 s. Percent inhibition (PI) of RBD binding to hACE2 by antibodies was determined using the equation: PI = [(hACE2 binding following RBD-antibody incubation)) / (hACE2 binding)] x 100. Antibody concentration was titrated from 100 pg ml’1by serial two-fold dilutions. All assays were performed at 30 °C with agitation set at 1,000 rpm.Epitope mapping of antibodies by alanine scanning.

[0109] Epitope mapping was performed essentially as described previously(Davidson and Doranz, 2014) using SARS-CoV-2 (strain Wuhan-Hu-1) S protein RBD shotgun mutagenesis mutation libraries, made using a full-length expression construct for S protein. 184 residues of the RBD (between S residues 335 and 526) were mutated individually to alanine, and alanine residues to serine. Mutations were confirmed by DNA sequencing, and clones arrayed in 384- well plates, one mutant per well. Binding of mAbs to each mutant clone in the alanine scanning library was determined, in duplicate, by high-throughput flow cytometry. Each S protein mutant was transfected into HEK-293T cells and allowed to express for 22 h. Cells were fixed in 4% (v / v) paraformaldehyde (Electron Microscopy Sciences), and permeabilized with 0.1% (w / v) saponin (Sigma- Aldrich) in PBS plus calcium and magnesium (PBS++) before incubation with mAbs diluted in PBS++, 10% normal goat serum (Sigma), and 0.1% saponin. MAb screening concentrations were determined using an independent immunofluorescence titration curve against cells expressing wild-type S protein to ensure that signals were within the linear range of detection. Antibodies were detected using 3.75 pg ml’1of AlexaFluor488- conjugated secondary antibody (Jackson ImmunoResearch Laboratories) in 10% normal goat serum with 0.1% saponin. Cells were washed three times with PBS++ / 0.1% saponin followed by two washes in PBS and mean cellular fluorescence was detected using a high-throughput Intellicyte iQue flow cytometer (Sartorius). Antibody reactivity against each mutant S protein clone was calculated relative to wild-type S protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type S-transfected controls. Mutations within clones were identified as critical to the mAb epitope if they did not support reactivity of the test mAb, but supported reactivity of other SARS-CoV-2 antibodies. This counter- screen strategy facilitates the exclusion of S mutants that are locally misfolded or have an expression defect.Serum antibody epitope mapping.

[0110] Serum antibody epitope mapping competition assays were performed as previously described (Corbett et al., 2021), using the Biacore 8K+ surface plasmon resonance system (Cytiva). Briefly, anti-histidine antibody was immobilized on Series S Sensor Chip CM5 (Cytiva) via primary amine coupling using a His capture kit (Cytiva). His-tagged SARS-CoV- 2 S protein containing 2 proline stabilization mutations (S-2P) was then captured on active sensor surface. Human IgG monoclonal antibodies (mAbs) used for these analyses include: S2- specific mAbs S652-112, and S2P6; NTD-specific mAbs 4-8, S652-118, 5-7, and N3C; Sl- specific mAb A20-36.1; and RBD-specific mAbs Bl-182, CB6, A20-29.1, A19-46.1, LY-COV555, A19-61.1, S309, A23-97.1, A19-30.1, 5001, 5011, A23-80.1, and CR3022. Negative control antibody or competitor mAb was injected over both active and reference surfaces, followed by subsequent injection of non-human primate sera (diluted 1 : 100). Active and reference sensor surfaces were regenerated following each analysis cycle. Prior to analysis, sensorgrams were aligned to Y (Response Units) = 0, beginning at the serum association phase, using Biacore 8K Insights Evaluation Software (Cytiva). Relative “analyte binding late” report points (RU) were collected and used to calculate percent competition (% C) using the following formula: % C = [1 - (100 * ( (RU in presence of competitor mAb) / (RU in presence of negative control mAb) )]. Absolute competition (ARUs) was calculated with the following formula: ARUs = [(RUs in presence of negative control mAb) - (RUs in presence of competitor mAb)]. Results are reported as percent competition or absolute competed RUs and statistical analysis was performed using unpaired, two-tailed t-test (GraphPad Prism v.8.3.1). Assays were performed in duplicate, with average data point represented on corresponding graphs.In vivo protection studies in K18-hACE2 transgenic mice.[OHl] All research in this study involving animals was conducted in compliance with the Animal Welfare Act, and other federal statutes and regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 1996 edition. The research protocol was approved by the Institutional Animal Care and Use Committee of the Trudeau Institute. KI 8- hACE2 transgenic mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in the animal facility of the Trudeau Institute and cared for in accordance with local, state, federal, and institutional policies in a National Institutes of Health American Association for Accreditation of Laboratory Animal Care-accredited facility. For the prophylactic protection studies, on day -1, groups of 13 K18-hACE2 mice (8-10 weeks of age) were injected intravenously with the purified antibodies at the indicated dose of 10 mg ml'1. On study day 0, all mice were inoculated with 1.25>< 104PFU of SARS-CoV-2 Delta B.1.618.2 via intranasal instillation, a challenge dose determined from a previous study (Joyce et al., 2021). All mice were monitored from study day 0 to study day 1, with body weight measurements taken every day, twice daily, every 12 h, out to day 10. Mice were euthanized if they displayed any signs of pain or distress as indicated by the failure to move after stimulated or inappetence, or if mice have greater than 25% weight loss compared to their study day 0 body weight. From each group, a subset (5) of mice, were sacrificed 2 days after challenge fordetermination of infectious virus titers in lower respiratory tract (from bronchoalveolar lavage and lung tissue) using a PRNT assay.Evaluation of escape and selection of virus variants.

[0112] For the evaluation of antibody escape ability, and generation of putative antibody escape S variants, a previously described chimeric recombinant VSV derivative (rVSV / SARS- CoV-2 / GFP2El) that encodes a SARS-CoV-2 S protein in place of VSV-G, recapitulating the neutralization properties of authentic SARS-CoV-2 (Schmidt et al., 2020), was prepared and passaged to generate diversity. Then, rVSV / SARS-CoV-2 / GFP2El populations containing 106infectious units were incubated with individual antibodies (at 1.25, 2.5, 5 or 10 pg mF1final concentration) or 1 : 1 mixtures of two antibodies (5 pg ml’1of each antibody) for 1 hour at 37 °C. Then, the virus-antibody mixtures were incubated with 5 * 105293T / hACE2cl.22 cells in 6-well plates. Two days later, supernatants were harvested from these passage 1 cultures, a 100 pl aliquot of the cleared supernatant was incubated with the same concentration of antibodies and then used to infect 5 x io5293T / hACE2cl.22 cells in 6-well plates, as before. After a second passage, infectious rVSV / SARS-CoV-2 / GFP2El titers were measured in the passage 2 supernatants to indicate escape or lack thereof from the neutralizing antibodies. Titers were measured by inoculating 293T / hACE2cl.22 cells in 96-well plates with serially diluted supernatant, and determining the number of infected cells by FACS, 16 hours later.

[0113] For passage 2 cultures in which clear escape was observed, as evidenced by the appearance of numerous GFP-positive cells, RNA was isolated from aliquots of supernatant containing selected viral populations using NucleoSpin 96 Virus Core Kit (Macherey -Nagel). The purified RNA was subjected to reverse transcription using random hexamer primers and SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). The cDNA was amplified using KOD Xtreme Hot Start DNA 396 Polymerase (Millipore Sigma) flanking the S encoding sequences. The PCR products were gel-purified and subjected to bulk Sanger-sequencing.

[0114] Identification of ferritin reactive antibodies by enzyme linked immunosorbent assay. 96-well Immulon “U” Bottom plates were coated with 1 pg ml’1of ferritin proteins in Dulbecco’s Phosphate buffered saline, pH 7.4 (PBS). Plates were incubated at 4 °C overnight. After 30 minutes of blocking with blocking buffer (Dulbecco’s PBS containing 0.2% bovine serum albumin, pH 7.4), at RT, the plates were washed 3x with wash buffer (Dulbecco’s PBS containing 0.05% Tween 20, pH 7.4). Antibodies were serially diluted 5-fold in sample buffer (Dulbecco’s PBS containing 0.2% bovine serum albumin and 0.05% Tween 20, pH 7.4), or ata single concentration as indicated, and added to duplicate wells. The plates were incubated for 1 hour at RT. The plates were then washed 4 times with wash buffer. Horseradish peroxidase (HRP)-conjugated goat anti-human IgG, gamma chain specific antibody (Sigma) was added and incubated at RT for 30 min, followed by 4 washes with wash buffer and one wash with PBS. For development, the substrate mixture from TMB Substrate set (Biolegend) was added and incubated for 10 min, before the addition of the Stop Solution for TMB Substrate (Biolegend). Absorbance (A) was measured at 450 nm or 650 nm as indicated, using an ELISA reader iD3 (Molecular Devices, San Jose, CA).

[0115] X-ray crystallography and structure analysis. WRAIR-5001-RBD (10 mg mF1) and WRAIR-5021-RBD (9 mg ml’1) complexes were screened for crystallization conditions using an Art Robbins Gryphon crystallization robot, 0.2 pl drops, and a set of 1200 conditions. Crystal drops were observed daily using a Jan Scientific UVEX-PS with automated UV and brightfield drop imaging. Initial crystallization conditions were optimized manually by mixing protein and reservoir solutions in 1 : 1 (v:v) ratios. Crystals used for data collection grew in the following crystallization conditions: WRAIR-5001-RBD complex: 0.2 M Sodium chloride, 0.1 M Phosphate-citrate pH 4.5, 20% PEG 8,000 and WRAIR-5021-RBD complex: 0.2 M Sodium malonate pH 7.0, 20% w / v Polyethylene glycol 3,350.

[0116] Diffraction data for the WRAIR-5001-RBD and WRAIR-5021-RBD complexes were collected at Advanced Photon Source (APS), Argonne National Laboratory beamline 24-ID-E and measured using a Dectris Eiger 16M PIXEL detector to a final resolution of 4.3 A and 2.5 A, respectively. Diffraction data indexing, integration, and scaling were carried out using the XDS-GUI(Diederichs). Data collection statistics are reported in Table 12.

[0117] All the crystal structures described in this study were solved by molecular replacement using PHASER, and iterative model building, and refinement were performed in COOT and Phenix (Adams et al., 2009; Emsley and Cowtan, 2004; McCoy et al., 2007). Phenix xtriage was used to analyze all the scaled diffraction data output from HKL2000 and XDS. Primarily, data was analyzed for measurement value significance, completeness, asymmetric unit volume, and possible twinning and / or pseudotranslational pathologies. To determine the structure of the Fab-SARS-CoV-2 RBD structures, the previously reported crystal structure of SARS-CoV-2 RBD was used. The heavy chain variable domain (VH) of A17 mAb (PDB code: 3ZL4) and light chain variable domain (VL) of 20350 mAb (PDB code: 5CZV) were used as the search models for WRAIR-5021. The heavy chain of antibody CH235UCA (PDB code: 6UDA) and light chain of 059-152-Fv (PDB code: 5XWD) were combined and used as the search modelfor WRAIR-5001 mAb. Search models for Fabs, were divided into Fv and Fc domains to carry out the molecular replacement searches. This approach was critical in finding solutions for all complexes. All structures were refined using Phenix refine with positional, global isotropic B- factor refinement and defined TLS groups. Manual model building was performed in COOT. The Ramachandran plot as determined by MOLPROBITY showed > 96% of all residues in favored regions and ~4% of all residues in the allowed regions for the WRAIR-5001-RBD complex. For the WRAIR-5021-RBD complex >96% residues were located in the favored and allowed regions. Data collection and refinement statistics are reported in Table 12. Interactive surfaces were analyzed using PISA (www.ebi.ac.uk / pdbe / pisa / ) and are provided in Table 14- 15 Structure figures were prepared using PyMOL (The PyMOL Molecular Graphics System, Version 2.1 Schrodinger, LLC). Software used in this work was curated by SBGrid (Morin et al., 2013).Statistical analysis.

[0118] Neutralization is the geometric mean of the IC50 values calculated using 5-parameter logistic regression from at least two-independent experiments performed in triplicates (R package nplr). Non-parametric Spearman correlations were used to assess relationship between neutralization and binding or neutralization and effector function data as well as between neutralization data obtained from the pseudotyped and authentic SARS-CoV-2 neutralization assays. Two-tailed Mann-Whitney t-tests were used to verify the existence of significant differences between NTD and RBD mAbs in several binding and functional assays. In the animal studies, one-way ANOVA with Dunnett’s multiple comparisons tests were used to assess significance in weight changes and viral loads across groups compared to the isotype control antibody-treated animals. Survival curves were compared individually to the isotype control antibody using a Mantel-Cox log-rank test. Fold change in binding to mutant proteins was calculated relative to the wild-type WA1 / 2020 Spike or RBD proteins. In absence of binding, a background binding value (0.05 nm in BLI assays) was attributed. Fold change in neutralization to VOC was calculated relative to the IL 1 / 2020 virus. Non-neutralizing mAbs were assigned the IC50 of 25 pg ml’1antibody, the mAb starting concentration in the assay. All tests, except for the 5-parameter logistic regression performed in R (version 3.6.3) and R studio (1.2.1355), were performed in Prism (version 9, GraphPad Software). Data were graphed using Prism software (version 9, GraphPad Software).Example 1 - Rhesus macaque plasma neutralizing and binding antibody activity

[0119] As previously reported, rhesus macaques were vaccinated twice, 4 weeks apart, with 50 pg of SpFN adjuvanted with the adjuvant Army Liposomal Formulation containing saponin QS-21 (ALFQ) (King et al., 2021). The magnitude of serum binding antibodies from SpFN- vaccinated macaques were comparable to the magnitude of serum binding antibodies elicited by macaques vaccinated with two doses of mRNA-1273 (FIG. 7A) (Baden et al., 2021). However, distinct mapping of the polyclonal antibodies was found between vaccination regimens using a panel of SARS-CoV-2 mAbs to compete with plasma antibody binding to the Spike trimer (FIG. 7B). Broad coverage of Spike antigenic sites for both vaccine regimens was observed, however SpFN-derived plasma showed strong binding to RBD antigen site G (described in FIG. 3A), a site with weak binding measured in mRNA-1273 -derived plasma (Cerutti et al., 2021a; Liu et al., 2020; Pinto et al., 2021). Plasma from the SpFN-vaccinated macaques were tested for neutralization using SARS-CoV-2 WA-1 pseudotyped virus and authentic virus assays. Peak neutralization of SARS-CoV-2 was observed 2 weeks following the second dose (week 6) (FIG. 7C), with cross-neutralizing activity against SARS-CoV-2 VOCs and SARS-CoV-1 in authentic virus assays (FIG. 7D). Robust levels of plasma IgG binding were detected to both SARS-CoV-1 SI and RBD, and SARS-CoV-2 SI, Hexapro, RBD and NTD at the peak time point (week 6) and the necropsy timepoint week 9 / 10 (FIGs. 7E-F), whereas minimal IgM binding was detected across domains (FIGs. 7G-H).Example 2 - Isolation of SARS-CoV-2 neutralizing antibodies elicited in SpFN- vaccinated rhesus macaques.

[0120] To understand the molecular targeting of broadly reactive antibodies elicited by SpFN vaccination, monoclonal antibodies were isolated from SpFN vaccinated rhesus macaques 2 weeks following the second SpFN immunization (FIG. 1A). Non-human primate (NHP).O2 (animal Hsl606375, as previously published (Joyce et al., 2022)) was selected based upon representative binding and neutralization profiles as shown in FIG. 7. B cells from NHP.02 from the peak neutralization time point (week 6) were single cell sorted using a novel sequential sorting strategy (FIG. 8). SpFN molecules containing either the S protein for SARS-CoV-2 (WA-1) (SpFN) or SARS-CoV-1 Urbani (SpFN1), in addition to fluorochrome-conjugated streptavidin tetramerized subdomains of the S protein (NTD and RBD), were used as probes to identify SARS-CoV-2 and / or SARS-CoV-1 reactive B cells from animal PBMCs (FIG. IB, FIGs. 8A-B). SpFN was used to mimic the SARS-CoV-2 virus with the goal to isolate mAbs targeting potential conformational or quaternary epitopes. The majority of SARS-CoV antigen positive B cells reacted to either one or both SpFN molecules (FIGs. 8C-D). As a control, Bcells from a naive animal showed minimal reactivity to both SpFN molecules and tetramerized S protein subdomain baits (FIG. 8C).

[0121] In aggregate, 85 antibody heavy and light chain pairs were recovered and sequenced from single-cell Spike-reactive B cells. Antibodies were produced as human IgGl in Expi293T cells and screened as cell culture supernatants for binding and neutralization. A total of 25 mAbs were subsequently purified and tested for binding to SARS-CoV-2 subdomains and for neutralization using a S pseudotyped lentivirus (pSV) neutralization assay. Based on reactivity to the SpFN and SpFN1molecules in an enzyme immunoassay (EIA), 20 mAbs were down- selected for further characterization (FIGs. 9A-B; Table 1). Four of the isolated mAbs, Walter Reed Army Institute of Research (WRAIR)-5002, -5008, -5014, and -5023 were found to react to the empty ferritin particle (FIG. ID, FIG. 9C). However, this reactivity was limited to ferritin derived from H. pylori, with no cross-reactivity to ferritin derived from humans (FIGs. 9D-E). The remaining 16 mAbs were tested for binding to a panel of 28 antigens spanning SARS- CoV-2, SARS-CoV-1, MERS-CoV, and the 4 seasonal coronaviruses 229E, HKU1, NL63, and OC43, using a multiplex bead-based Luminex assay (FIGs. 10A-C). Eleven of the mAbs were directed towards either the RBD or NTD, and three reacted to S2 (FIGs. 1C-D). While most mAbs directed towards the NTD or RBD could cross-bind stabilized S protein (HexaPro), two of the mAbs were HexaPro-specific (FIGs. 1C-D). More than half also cross-bound to the same domains of SARS-CoV-1 (FIGs. 10A-C). Potent neutralization activity, as measured with pseudotyped virus (WA-1), was observed only for RBD and NTD mAbs, ranging from subnanomolar to micromolar concentrations (FIGs. 1E-F). All RBD-specific mAbs neutralized, while 60% of NTD-specific mAbs, and none of the S2- or Hexapro-specific mAbs harbored neutralization activity against SARS-CoV-2 WA-1 (FIGs. 1E-F). Neutralization plateaued around 75% for WRAIR mAbs targeting NTD, as previously described (FIG. IF) (Dussupt et al., 2021).

[0122] One RBD-directed mAb, WRAIR-5001, demonstrated neutralization activity against pseudotyped SARS-CoV-1 at 0.20 pg / ml (FIG. 1G).

[0123] While the ability to facilitate cell surface S protein binding (opsonization) did not vary based on epitope targeted, RBD and NTD-directed mAbs mediated higher levels of antibodydependent cellular phagocytosis (ADCP) compared to S2-directed mAbs (FIG. 1H). In contrast, S2-directed mAbs had higher functionality in antibody-dependent cellular cytotoxicity assays (ADCC), compared to the RBD and NTD-directed mAbs. Together, this data indicates that two doses of SpFN induced IgG antibodies that targeted a wide range ofneutralizing epitopes, with cross-sarbecovirus neutralizing activity, while also facilitating Fc effector functions.Example 3 - SpFN vaccination of rhesus macaques elicits antibodies directed towards multiple epitopes

[0124] To define the epitope specificity of the isolated mAbs the percent residual binding to either the NTD or RBD Spike domain in the presence of a control mAb with previously defined epitope was measured. The NTD-directed mAbs fell into three previously defined competition groups using a set of competing mAbs (Dussupt et al., 2021) (FIG. 2A). Of the NTD-directed mAbs, the NTD Group B mAbs were the only mAbs that neutralized, with no crossneutralization to other SARS-CoV-2 VOCs or SARS-CoV-1 (FIG. IF and FIG. 10D). Modest hACE2 inhibition activity was observed (FIG. 10E). One S2 directed mAb was tested for neutralization and hACE2 -blocking but had minimal activity in either assay (FIGs. 10D-F). The epitopes of all NTD-targeted mAbs appear to lie near or overlap with the NTD supersite (FIG. 2A, inset). Most mutations found in the VOC are found on the NTD face distal to the rest of the Spike, explaining the limited neutralization activity of these mAbs against VOCs.

[0125] The RBD-directed WRAIR mAbs fell into RBD antigen competition Groups A and B (FIG. 2B). The WRAIR RBD-A mAbs (WRAIR-5005, -5021, and -5020) facilitated potent neutralization and completely blocked hACE2 binding to both RBD and the Spike trimer S-2P (FIG. 2C). Shotgun alanine mutagenesis epitope mapping revealed that RBD residues critical for mAb interaction were conserved within Group A and overlapped with previously identified class I RBD epitopes (Barnes et al., 2020) (FIG. 2D). Macaque RBD-directed mAb breadth and neutralization potency against SARS-CoV-2 VOC was assessed. WRAIR RBD-A mAbs demonstrated binding across earlier pandemic VOC RBDs (FIG. 2E). However, interaction with Omicron VOC or SARS-CoV-1 RBDs was severely impacted or completely abrogated (FIG. 2E). Similarly, potent neutralization was observed across WA-1, Beta (B.1.351) and Delta (B.1.617.2) VOCs, but a lack of neutralization was found to Omicron subvariants and SARS-CoV-1 (FIG. 2F, Table 12) for Group A RBD WRAIR mAbs.

[0126] The WRAIR RBD Group B mAbs (WRAIR-5001, -5004, -5011) demonstrated potent neutralization to WA-1 that plateaued around 75% and had incomplete hACE2 blocking activity against RBD and the Spike trimer S-2P (FIG. IF, FIG. 2C). Shotgun alanine mutagenesis epitope mapping revealed that RBD residues critical for mAb interaction were conserved within Group B, and overlapped with epitopes spanning class III and IV RBDepitopes that were previously identified (Barnes et al., 2020) (FIG. 2D). WRAIR RBD-B mAbs demonstrated significant breadth across SARS-CoV-2 VOCs, where none of the Group B mAbs were affected by any of the variant mutations including Omicron subvariants (FIG. 2E). Modest neutralization was observed across Beta (B.1.351) Delta (B.1.617.2) and Omicron (BA. l, BA.2), but a lack of neutralization was found to Omicron BA.4 / 5 (FIG. 2f, Table 12). Of note, one RBD-B mAb, WRAIR-5001, demonstrated both cross-binding activity to SARS- CoV-1 RBD and neutralization to SARS-CoV-1 (FIGs. 2E-G). Interestingly, while WRAIR- 5001 was able to bind with high affinity to SARS-CoV-2 Omicron subvariants BA. l and BA.4 / 5 (FIG. 2G), these mAbs had diminished neutralization activity (FIG. 2F).

[0127] To determine if these mAb epitopes recapitulated the plasma polyclonal antibody targets, surface plasmon resonance (SPR) plasma-mAb competition assays were used to map the specificity of the plasma polyclonal responses. Evaluation of the plasma antibodies revealed that SpFN elicited polyclonal responses targeted towards known protective RBD epitopes (FIGs. 3A-B), which overlapped with epitopes found by alanine scanning (FIG. 2D). Using viral escape assays, residues that could contribute to escape from two representative RBD group A mAbs (WRAIR-5001, and -5011) and one mAb from RBD group B (WRAIR-5021) were determined (FIG. 3C), which confirmed the targeting of these mAbs overlapped with the targeting of the polyclonal antibodies in the plasma (FIG. 3B). Combined, these data map the antibody specificity following SpFN vaccination and demonstrate that cross-neutralizing epitopes are targeted towards the RBD.Example 4 - in vivo efficacy of NHP mAbs elicited after SpFN vaccination

[0128] Previous studies showed that SpFN-vaccinated rhesus macaques were protected against viral challenge25. To determine if protection from challenge was due to targeting these defined epitopes, one representative mAb from each RBD competition group (Group A, WRAIR-5021 and Group B, WRAIR-5001) were further tested for in vivo protection against lethal SARS- CoV-2 Delta (B.1.617.2) challenge in the K18-hACE2 transgenic mouse model. Prophylactic in vivo protection was assessed by intravenous administration of WRAIR-5001 or WRAIR- 5021 at a dose equivalent to 10 mg / kg, side-by-side with control mAb WRAIR-2125, (a SARS- CoV-2 neutralizing mAb isolated from a convalescent donor that previously showed protective efficacy in vivo (Dussupt et al., 2021)), and an IgG isotype control (MZ4), 24 hours prior to administration of SARS-CoV-2 Delta (B.1.617.2) (FIG. 3D).

[0129] At 48 hours post-challenge, 5 animals from each group were sacrificed for viral replication analysis. Group A RBD WRAIR-5021 showed reduced viral replication in the lung and bronchoalveolar lavage (BAL) of challenged mice at a level akin to the WRAIR- 2125positive control, and significantly better than the IgG isotype control (P<0.0001) (FIG. 3E). However, there was no significant difference in the viral replication in the lung or BAL between Group B WRAIR-5001 and the IgG isotype control (FIG. 3E.) The remaining animals (n=8) in each group were monitored for loss of body weight out to 10 days and survival out to 14 days. WRAIR-5021 also conferred in vivo protection against body weight loss and lethal challenge (P<0.0001) (FIGs. 3F-G). WRAIR-5001 was able to extend the median survival by two days compared to the IgG isotype control but was unable to completely protect against weight loss and death. The lack of protection by Group B WRAIR-5001 to Delta (B.1.617.2) aligned with modest neutralization of WRAIR-5001 to Delta (IC50=1.581 pg / ml, FIG. 2F), whereas Group A WRAIR-5021 was able to potently neutralize Delta (IC50=0.002pg / ml) and therefore offered complete protection (FIG. 3G).Example 5 - Crystal structure of RBD-A mAb, WRAIR-5021, in complex with SARS- CoV-2 RBD

[0130] To further define the molecular recognition of vaccine-elicited RBD mAbs elicited by SpFN vaccination, structural studies of Group A RBD mAb WRAIR-5021 and Group B RBD mAb WRAIR-5001 were performed. Binding competition assay -based epitope mapping experiments, using previously reported antibodies, indicated the hACE2 binding site as the target for RBD-A antibodies (FIGs. 2B-C). To understand the structural basis of RBD recognition by these mAbs, WRAIR-5021 Fab was crystallized in complex with the SARS- CoV-2 RBD and the structure was analyzed at a final resolution of 2.3 A (FIG. 4A and Table 13). WRAIR-5021 exhibited low levels of somatic hyper mutations (SHM) with heavy and light chain V-genes consisting of 4 and 6 changes, respectively. Of these mutations, 2 residues of the heavy chain, Thr53 and Thr54, are found at the interface with the RBD, with the Thr54 hydroxyl group hydrogen bonded to the carboxyl side chain of RBD Glu484. From the light chain, the 2 nitrogens of the Arg53 guanidino group form hydrogen bonds with the carboxyl side chains of RBD residues Asp420 and Asn460, and Phe32 and Asp50 are -54% and -28% buried at the interface. Structure analysis confirmed that WRAIR-5021, which potently neutralizes SARS-CoV-2 (FIG. IF) targets the hACE2 binding site with a distinct epitope centered on the hACE2 binding ridge loop (sites 475-478 and 484-490) (FIG. 4B). A careful structure superimposition of WRAIR-5021 -RBD complex structure onto the hACE2 boundRBD structure (PDB: 6M0J) revealed nineteen of the twenty-five hACE2 binding residues are part of WRAIR-5021 epitope, demonstrating 76% epitope-binding site overlap. WRAIR-5021 forms extensive interactions across the entire length of the receptor binding motif, with a BSA of 1221.6 A2with heavy and light chains contributing 61.9% and 38.1% of the total BSA, respectively (FIG. 4C). Heavy and light chain interactions form a total of 13 and 9 hydrogen bonds, respectively, with the light chain forming three additional salt-bridge interactions. WRAIR-5021 heavy chain contacts are mediated by all the CDR loops with CDR Hl contributing the most, covering >300 A2of the RBD interface (FIG. 4C). Major heavy chain contacts are formed by a set of Tyr residues in the CDR Hl and H2 (residues Tyr27, Tyr33, Tyr34, and Tyr51). Light chain contacts are primarily mediated by CDR L2 and L3, with limited contributions from CDR LI. Major light chain contacts are mediated by a set of polar and charged residues in the CDRL1-L3 (Ser30, Asp50, R53, Ser56, Asp92, Ser93and Asp94). (FIG. 4D and Table 14). Next, structural superimposition of WRAIR-5021 was performed with representative antibodies from previously defined classes (Rappazzo et al., 2021). Based on this analysis, WRAIR-5021 can be classified as a class I mAb (FIG. 4E). With respect to VOCs, except Delta variant, all the RBD mutations in the Alpha, Beta and Gamma VOCs are within the epitope site of WRAIR-5021 mAb. However, based on the structure modeling, none of these mutations, including those found in the Delta variant, would offer enough steric / electrostatic clashing to prevent RBD binding by WRAIR-5021 (FIG. 4F). Assessment of WRAIR-5021 binding by BLI, against a panel of RBD molecules showed high-affinity nanomolar binding to WA-1, Delta, and Omicron subvariants BA.l and BA.4 / 5 (Table 12). In the case of the Omicron BA. l variant, 9 of the 15 RBD mutations overlap with the WRAIR- 5021 epitope, with many of the mutations altering the epitope structure, and were expected to negatively impact Omicron VOC binding (FIG. 4F). However, significant changes in affinity was not observed indicating some plasticity within the epitope, similar to that seen for hACE2- binding.

[0131] To further understand the WRAIR-5021 epitope in the context of full-length Spike, WRAIR-5021 was modeled to the closed conformation (all RBD down; PDB code: 6VXX), and 1- or 2-RBD in the up conformations (PDB codes: 7DWZ and 6X2B) (FIG. 4G) (Henderson et al., 2020; Walls et al., 2020; Yan et al., 2021). Structure superimposition demonstrated that despite a few minor clashes, the binding of WRAIR-5021 is compatible with both the up and down conformations of S protein, indicating accessibility of the epitope on theSpike trimer. Antibodies similar to WRAIR-5021 may arise from the precursor germline VH4- 99*01 in rhesus macaques (SEQ ID NO: 237).Example 6 - Crystal structure of RBD-B mAb, WRAIR-5001, in complex with SARS- CoV-2 RBD

[0132] A set of previously described RBD neutralizing antibodies (WRAIR-2057, WRAIR- 2063 and WRAIR-2134) isolated from a human subject were of particular interest because of their novel epitope on the SARS-CoV-2 RBD (Dussupt et al., 2021). Binding competition, and alanine scanning experiments indicated that the WRAIR-5001 mAb of the present disclosure targets a similar epitope (FIG. 2B). WRAIR-5001 exhibited low levels of somatic hyper mutations (SHM) with heavy and light chain V-genes consisting of 7 and 6 changes, respectively. Of these SHMs, 2 residues of the heavy chain, Val56 and Val58, are -46% and 67% buried at the RBD interface. The carboxamide of Asn32 of the light chain is hydrogen bonded to the backbone amine and carboxyl oxygen of RBD Arg357; Asn66 and Ala29 are 34% and 100% buried, respectively, at the RBD interface. To further our knowledge of this epitope, the crystal structure of WRAIR-5001 Fab was determined in complex with SARS- CoV-2 RBD. The crystal structure of the WRAIR-5001 -RBD complex was determined to a final resolution of 4.3A and refined to an Rwork / Rfree of -0.25 / 0.30 (Table 13). WRAIR-5001 binds to a less typical, cryptic epitope, located on the “side” of the RBD, distal from the hACE2 binding site (FIGs. 5A-C). Overall, the WRAIR-5001 epitope covers a total BSA of 1087.3 A2with heavy and light chains contributing 26.6% and 73.4% of total BSA, respectively (FIG. 5D and Tables 15A and 15B). WRAIR-5001 recognition of SARS-CoV-2 RBD is primarily based on CDR H2, H3, and CDR L1-L3 loops. Heavy and light chain interactions form a total of 2 and 9 hydrogen bonds, respectively, with the light chain forming three additional salt-bridge interactions. WRAIR-5001 heavy chain contacts are mediated by CDR H2 and H3 with CDR H3 contributing the most, covering -200 A2of the RBD interface (FIG. 5D). Major heavy chain contacts are formed by a set of hydrophobic residues (Trp47, Trp50, Val57, Val59, Leul03, Vall04 and Vall05). Leul03 inserts into a hydrophobic pocket on the RBD (Trp353, Arg355 and Phe464) and contributes 127.1 A2of the binding interface area. Light chain contacts are primarily mediated by CDR LI and L3, with limited contributions from CDR L2. Major CDR LI contacts are mediated by a set of polar and charged residues (Asp25, Asn26, Ala28, Ser29, Lys30 and Asn31). Asn26 inserts in a shallow pocket lined by RBD residues Glu340-Thr345 and buries 73.3 A2of the surface area (FIG. 5E and Tables 15A and 15B). Major CDR L3 contacts are mediated by a set of hydrophobic residues (Trp90, Tyr92-93, andHis96) with Tyr93 and His96 contributing the most. Tyr93 inserts into a deep pocket lined by RBD residues Val341, Ala344, Phe347 and Ser399, and buries 143.4 A2of the surface area. While His96 inserts into a deep hydrophobic pocket lined by RBD residues Ala351, Tyr352, Arg466 and Ile468, and buries 105.7 A2of the surface area (FIG. 5E and Tables 15A and 15B).

[0133] Structural superimposition of WRAIR-5001 with representative antibodies from previously defined classes indicated that the WRAIR-5001 epitope exists between class III and class V epitopes (FIGs. 5F-G). Further analysis revealed that light chain of the WRAIR-5001 overlaps with S309 epitope, a class-III mAb, and the heavy chain epitope extends along the WRAIR-2057 epitope (FIG. 5G). Structural mapping of the variant mutations on the RBD demonstrated that none of the mutant residues in the Alpha, Beta, Gamma, Delta and Omicron BA.1 and BA.2 variants are within the epitope site of WRAIR-5001 mAb and therefore were not expected to impact the WRAIR-5001 binding and neutralization (FIG. 5H). This finding was verified by affinity assessment of WRAIR-5001 for select RBDs, all of which bound with low nanomolar or high picomolar affinity (FIG. 2G, Table 12). Our analysis revealed that even in the context of highly mutated variants, i.e. Delta or Omicron, only a single mutation overlaps with the WRAIR-5001 epitope (G339D). As binding affinity between WA-1 and Omicron variants BA. l and BA.5 were comparable (all at high picomolar range), this reinforces the value of this class of mAbs for protection against highly mutated variants.

[0134] To further analyze the WRAIR-5001 epitope in the context of the SARS-CoV-2 prefusion stabilized Spike trimer, WRAIR-5001 was modeled onto the closed (all RBD down; PDB code: 6VXX), and 1- or 2-RBD up conformations (PDB codes: 7DWZ and 6X2B) (FIGs. 5I-J) (Henderson et al., 2020; Walls et al., 2020; Yan et al., 2021). Structure superimposition demonstrated that the epitope for WRAIR-5001 is occluded by the adjacent S protomers when the RBD is in the “down” conformation while more accessible when RBD is in the open conformation, indicating the cryptic nature of this epitope in line with other mAbs in class III or class V, which may also originate from the precursor germline in Rhesus macaques, VH1200*01 (SEQ ID NO: 239).Example 7 - Epitope conservation analysis of NHP mAbs

[0135] Structural and sequence analysis revealed that the WRAIR-5001 epitope is highly conserved between SARS-CoV-2 and SARS-CoV-1 RBDs with >85% sequence identity (FIG. 6A-B). Given this high sequence identity, the conservation of WRAIR-5001 epitope among SARS-CoV-2 VOC and other representative sarbecoviruses was studied (FIG. 6A). Sequencealignment and phylogenetic analysis demonstrated that the epitope for WRAIR-5001, is considerably conserved amongst sarbecoviruses; in contrast, significant variations for the WRAIR-5021 epitope were observed (FIG. 6B), consistent with other mAbs that target the hACE2 epitope.

[0136] Intrigued by the sequence conservation of the WRAIR-5001 epitope, the antigenic cross-reactivity of all the macaque RBD-directed mAbs against purified VOC and sarbecovirus RBDs was tested. Using BLI, we further tested the breadth of cross-reactivity of RBD mAbs with a panel of sarbecovirus RBDs (FIG. 6C). Overall, the RBD-B mAbs (WRAIR-5001, WRAIR-5004, and WRAIR-5011) exhibited greater breadth than RBD-A mAbs (WRAIR- 5005, WRAIR-5020, and WRAIR-5021), cross-reacting with diverse RBDs from sarbecovirus clades lb and la, which utilize hACE2 for cell entry. Amongst RBD-A group mAbs, modest cross-reactivity was only observed against SARS-CoV-1 from clade la. The ability of the RBD-A and RBD-B mAbs to block hACE2 binding in the presence of RBDs from diverse sarbecoviruses was assessed (FIG. 6D). RBD-A mAbs only blocked hACE2 binding to BANAL-20-103 RBD, another clade lb sarbecovirus, with equivalent activity to WA-1. Interaction of WRAIR-5005 and -5021 with RaTG13 was minimal, limiting hACE2 inhibition. RBD-B mAbs displayed modest inhibition potential to WA-1, but this was consistently maintained in the hACE2 -blocking assays using BANAL-20-103, RaTG13, and WIV1 RBD molecules. Combined, these data indicate that SpFN elicited antibodies that cross-recognize clade la and clade lb sarbecoviruses.Example 8- SpFN vaccination elicits cross-sarbecovirus neutralizing IgG titers in human donors after two or three doses.

[0137] One donor from the SpFN+ALFQ human clinical trial was chosen for monoclonal antibody (mAb) isolation at the peak neutralizing time points of SD42 (post 2nddose) and SD195 (post 3rddose) (FIG. 11 A). Donor down selection was performed to increase the likelihood of identifying B cells that exhibit potent and broad sarbecovirus neutralization post vaccination. Participant C was selected as ideal for this purpose as the IgG fraction of the plasma exhibited high viral titers to SARS-CoV-2 WA-1, the Spike trimer displayed on the surface of SpFN (FIG. 1 IB). Furthermore, at the peak neutralization time points of SD43 (post 2nddose) and SD195 (post 3rddose), Participant C displayed a strong ability to neutralize across SARS-CoV-2 variants of concern, including against pseudotyped virus of the Omicron variants (FIGs. 11C-D). Selection of this donor and the maintained ability to neutralize against Omicron, including the recently circulating BQ.1.1 variant, enhanced the change of identifyingthe target of any cross-sarbecovirus neutralizing epitopes elicited post SpFN+ALFQ vaccination.Example 9- SpFN with ALFQ vaccination in human donors elicits cross-sarbecovirus binding human plasma IgG

[0138] Donors (n=20) were enrolled in a Phase I human clinical trial and received 25 pg of the SARS-CoV-2 vaccine strategy Spike Ferritin Nanoparticle (SpFN) together with the adjuvant Army Liposomal Formulation containing QS21 (ALFQ) (FIG. 12A). Donors that received SpFN+ALFQ were given 2 doses, 4 weeks apart, with a subset (n=9) also receiving a third dose at least 20 weeks later. SpFN + ALFQ vaccination elicited plasma IgG capable of binding multiple domains commonly associated with sarbecovirus neutralization to SARS-CoV-2, SARS-CoV-1 and MERS spike protein (FIGs. 12B-D), including but not limited to the receptor binding domain (RBD), the N-terminal domain (NTD), and the more cryptic but highly conserved S2 domain. IgG capable of binding across Spike domains to the seasonal coronaviruses was also measured, however measurable amounts above baseline were not observed post SpFN vaccination.Example 10-mAbs isolated from a SpFN + ALFQ vaccinated human donor cross-bind and neutralize SARS-CoV-2 VOCs and SARS-CoV-1.

[0139] Cryopreserved PBMC from donor Participant C were stained to identify sarbecovirus- reactive B cells using fluorochrome conjugated tetramers to Spike domains (NTD and RBD) or SpFN molecule displaying the Spike trimer of SARS-CoV-2 (SpFN2) or SARS-CoV-1 (SpFN1) together with a fluorochrome conjugated secondary antibody as bait (FIG. 13 A, Table 6). In total 136 mAbs were isolated and sequenced, with 96 showing reactivity on a bead-based assay (Luminex) to an antigenic domain of the SARS-CoV-2 Spike protein, stabilized Spike (S) trimer, or the ferritin molecule used by SpFN, itself (FIG. 13B). Antibodies were further characterized by measuring their neutralization activity against pseudotyped virus, with the RBD directed mAbs display more potent activity against SARS-CoV-2 WA-1 compared to the NTD, S2, and S-trimer specific mAbs (FIG. 14C).Example 11 - NTD-directed mAbs generated from the BCR sequences from a single SpFN vaccinated donor bin into unique epitopes shared across previously described and unique locations on the NTD.

[0140] To elucidate the epitope target on the Spike protein elicited by SpFN vaccination, SARS-CoV-2 N-terminal domain (NTD)-directed mAbs isolated from the BCR sequences ofa SpFN vaccinated donor were subjected to epitope binning technology using a Carterra LSA to test for competitive binding to the isolated NTD binding against each other, and mAbs with a previously defined epitope on the SARS-CoV-2 Spike protein (Table 16). Experimentation also included other WRAIR-derived mAbs isolated from the BCR sequences of SARS-CoV-2 convalescent donors (Dussupt et al., 2021) and SpFN vaccinated rhesus macaques (Sankhala et al., 2024) as controls. As shown in FIG. 14A using mAbs were clustered together based on identical blocking / sandwi ching interactions and visualized as a dendrogram, with the y-axis representing a similar index. Several SpFN vaccinated donor-derived WRAIR mAbs were binned into epitope groups that target the neutralization site coined the NTD-supersite supersite (Cerutti et al., 2021b) (WRAIR-4140 and -4005), and the site of vulnerability (WRAIR-4034 and -4014) (Cerutti et al., 2021a). However, some mAbs were binned into epitope groups representing previously undefined sites of neutralization, such as the epitope groups for WRAIR-4107 and -4019, and WRAIR-4023. One mAb, WRAIR-4032, demonstrates modest neutralization activity against SARS-CoV-2 but is grouped into an epitope bin that includes DH1052, a mAb that facilitates Fc effector functions that provides in vivo protection from SARS-CoV-2 challenge in animals models in the absence of neutralization activity (Pierre et al., 2024). Interestingly, the epitope target of vaccination with SpFN to the NTD of SARS- CoV-2 appears to target the NTD in sites that do not facilitate viral neutralization (Fig. 14B), with the vast majority of SpFN vaccinated donor derived mAbs that bind the NTD harboring no neutralization activity and binding in sites outside of the commonly found NTD supersite and site of vulnerability.Example 12 - The majority of RBD-directed mAbs generated from the BCR sequences from a single SpFN vaccinated human donor bin into epitope classes that bind to the broadly-sarbecovirus conserved Class IV and V epitopes.

[0141] Like the NTD-directed mAbs, the molecular target of the RBD-directed mAbs isolated from the BCR sequences of SpFN vaccinated human donors were subjected to epitope binning technology using a Carterra LSA to test for competitive binding to the isolated RBD binding against each other, and mAbs with a previously defined epitope (Table 16), and binned based on identical blocking / sandwi ching interactions and displayed as a dendrogram with the y-axis representing a similar index (FIG. 15 A). Of the RBD SpFN vaccinated donor-derived mAbs, ten were grouped into the Class IV, with eight harboring neutralization activity against SARS- CoV-2, and four also able to cross-neutralization SARS-CoV-1 (WRAIR-4066, -4112, -4098, and -4108) (Fig. 15B). 15 mAbs were grouped as Class V, 11 of which demonstratedneutralization activity against SARS-Cov-2 and variants, with 7 harboring SARS-CoV-1 crossneutralization activity (WRAIR-4150, -4043, -4087, -4045, -4018, -4015, -4036). mAbs in these epitope bins also displayed broad breadth of binding using BLI to the RBDs of SARS- CoV-2 variants (Table 17) and the RBDs of other sarbecovirus clades, with WRAIR-4066, - 4045, -4112, and -4150 showing the widest breadth of binding (Fig. 15C). SpFN-vaccination also elicited mAbs binned into the hACE2 binding site-occupying Class I and Class II epitope groups. While mAbs that target these epitope groups are generally less broadly binding and neutralizing across sarbecovirsues and SARS-CoV-2 variants (FIG. 14B), the mAb WRAIR- 4095 of the present disclosure is an exception with broad SARS-CoV-2 neutralization out to the Omicron XBB.1.5 variant, and potent neutralization of SARS-CoV-2 (WA-1). WRAIR- 4095, akin to other Class I RBD mAbs, also displayed strong binding to SARS-CoV-2, with low KD values to WT SARS-CoV-2 and diminishing potency of binding across variants (Table 17). Three mAbs were binned into the Class III epitope group, and WRAIR-4075 of the present disclosure shows neutralizing activity that targets an epitope unique to the five classically defined RBD epitope classes. Altogether, SpFN vaccination targets a wide range of RBD epitope groups, with the majority of mAbs generated from the BCR sequences post SpFN vaccination targeting conserved sites away from the hACE2 binding site such as the Class IV and V epitopes.Table 16. Control mAbs used for epitope binning of NTD- and RBD-directed mAbs derived from the BCR sequences from a single SpFN vaccinated human donor.Table 17. Binding affinity constants of SpFN human donor WRAIR RBD mAbs measured against selected VoC RBDs, using BLI.Example 13 - SpFN vaccination of human donors elicits antibodies directed towards H pylori ferritin

[0142] To test for humoral responses elicited by components of the SpFN molecule other than the SARS-CoV-2 Spike protein it displays, purified mAbs generated from the BCR sequences of a single donor vaccinated with two and three doses of SpFN that demonstrated no reactivity to any component of a sarbecovirus Spike protein via multiplexed bead-based assay (Luminex), were tested for binding to the whole SpFN particle (FIG. 16 A) or the empty H. pylori ierritin scaffold protein (FIG. 16B) via EIA. Eight mAbs demonstrated binding above background to the SpFN molecule at both concentrations tested (2pg / mL and 0.2pg / mL), WRAIR-4001, - 4057, -4060, -4063, -4082, -4088, -4101, and -4124. Of these, four had reactivity to the scaffold protein comprised of the H. pylori ferritin molecule alone, WRAIR-4001, -4057, -4063, -4101, demonstrating that SpFN vaccination also elicits immune responses against portions of the vaccine that do not target the Spike protein of SARS-CoV-2. H. pylori ferritin reactive mAbs harbor not only research-based potential to aid in the design and implementation of futureferritin nanoparticle-based vaccines towards fighting SARS-CoV-2 and other diseases, but could also, in theory, be used as a therapeutic for H. pylori infections, in vivo.

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Claims

What is claimed is:

1. A monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain complementary determining region 3 (HCDR3) and a light chain complementary determining region 3 (CDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 6 (having HCDR3 of SEQ ID NOs 663-759 and LCDR3 of SEQ ID NOs 913- 1009, respectively), optionally wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 7.

2. The monoclonal antibody or antigen binding fragment thereof of claim 1, wherein the monoclonal antibody comprises three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 6 (having HCDR1 of SEQ ID NOs 421-517, HCDR2 of SEQ ID NOs 542-638, HCDR3 of SEQ ID NOs 663-759, LCDR1 of SEQ ID NOs 784-880, and LCDR3 of SEQ ID NOs 913-1009, respectively), optionally wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 7.

3. The monoclonal antibody or antigen binding fragment thereof of claim 1 or claim 2, wherein the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 6 (having HCDR1 of SEQ ID NOs 421-517, HCDR2 of SEQ ID NOs 542-638, HCDR3 of SEQ ID NOs 663-759, LCDR1 of SEQ ID NOs 784-880, and LCDR3 of SEQ ID NOs 913-1009, respectively), optionally wherein the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 7.

4. The monoclonal antibody or antigen binding fragment thereof of any one of claims 1- 3, wherein the monoclonal antibody comprises:(A) a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 6 (SEQ ID NOs 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223,225, 227, 229, 231, 233, and 235), optionally wherein the heavy chain variable region sequence is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 7 (SEQ ID NOs. 85, 143, 111, 161, 59, 231, 171, 75, 121, 51, 131, 73, 57, 145, and 41), further optionally wherein the heavy chain variable region sequence comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 6, further optionally wherein the heavy chain variable region sequence comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 7; or(B) a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 6 (SEQ ID NOs 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 ,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 ,100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, and 236), optionally wherein light chain variable region sequence is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 7 (SEQ ID NOs 86, 144, 112, 162, 60, 232, 172, 76, 122, 52, 132, 74, 58, 146, and 42), further optionally wherein the light chain variable region sequence comprises the light chain variable region sequence of any one of the antibodies set forth in Table 6, further optionally wherein the light chain variable region sequence comprises the light chain variable region sequence of any one of the antibodies set forth in Table 7; or(C) a heavy chain variable region sequence and a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to theheavy chain variable region sequence and light chain variable region sequence, respectively, of any one of the antibodies set forth in Table 6, optionally wherein heavy and light chain variable region sequences are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 7, further optionally wherein the heavy and light chain variable region sequences comprise the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 6, further optionally wherein the heavy and light chain variable region sequences comprises the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 7.

5. The monoclonal antibody or antigen binding fragment thereof of any one of claims 1- 4, wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 6, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, optionally wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 7 or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

6. A monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody comprises a heavy chain complementary determining region 3 (HCDR3) and a light chain complementary determining region 3 (CDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 1 (having HCDR3 of SEQ ID NOs 764-779 and LCDR3 of SEQ ID NOs 1014- 1029, respectively), optionally wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 2.

7. The monoclonal antibody or antigen binding fragment thereof of claim 6, wherein the monoclonal antibody comprises three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 1 (having HCDR1 of SEQ ID NOs 522-537, HCDR2 of SEQ ID NOs 643-658, and HCDR3 of SEQ ID NOs 764-779, and LCDR1 of SEQ ID NOs 885-900, and LCDR3 of SEQ ID NOs 1014-1029, respectively), optionally wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 2.

8. The monoclonal antibody or antigen binding fragment thereof of claim 6 or claim 7, wherein the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 1 (having HCDR1 of SEQ ID NOs 522-537, HCDR2 of SEQ ID NOs 643-658, and HCDR3 of SEQ ID NOs 764- 779, and LCDR1 of SEQ ID NOs 885-900, and LCDR3 of SEQ ID NOs 1014-1029, respectively), optionally wherein the monoclonal antibody comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of any one of the antibodies set forth in Table 2.

9. The monoclonal antibody or antigen binding fragment thereof of any one of claims 6- 8, wherein the monoclonal antibody comprises:(A) a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 1 (SEQ ID NOs 1, 5, 7, 9, 11, 15, 17, 19, 21, 25, 27, 29, 31, 33, 35, and 37), optionally wherein heavy chain variable region sequence is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 2 (SEQ ID NOs 1 and 35), further optionally wherein the heavy chain variable region sequence comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 1, further optionally wherein the heavy chain variable region sequence comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 2; or(B) a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 1 (SEQ ID NOs 2, 6, 8, 10, 12, 16, 18, 20, 22, 26, 28, 30, 32, 34, 36, and 38), optionally wherein the light chain variable region sequence is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 2 (SEQ ID NOs 2 and36), further optionally wherein the light chain variable region sequence comprises the light chain variable region sequence of any one of the antibodies set forth in Table 1, further optionally wherein the light chain variable region sequence comprises the light chain variable region sequence of any one of the antibodies set forth in Table 2; or(C) a heavy chain variable region sequence and a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence and light chain variable region sequence, respectively, of any one of the antibodies set forth in Table 1, optionally wherein heavy and light chain variable region sequences are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 2, further optionally wherein the heavy and light chain variable region sequences comprise the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 1, further optionally wherein the heavy and light chain variable region sequences comprises the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 2.

10. The monoclonal antibody or antigen binding fragment thereof of any one of claims 6- 9, wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 1, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, optionally wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 2 or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto.

11. The monoclonal antibody or antigen binding fragment thereof of any one of claims 1- 10, wherein the monoclonal antibody or antigen binding fragment thereof binds to one or more epitopes on a region of a coronavirus spike protein of one or more coronaviruses selected from severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), optionally wherein the region of the spike protein bound by the antibody or antigen binding fragment thereof is one or more selected from the RBD region, the NTD region, and the S2 region.

12. The monoclonal antibody or antigen binding fragment thereof of claim 11, wherein the monoclonal antibody or antigen binding fragment thereof neutralizes one or more coronaviruses selected from SARS-CoV-1, MERS-CoV, and SARS-CoV-2.

13. The monoclonal antibody or antigen binding fragment thereof of any one of claims 1- 10, wherein monoclonal antibody or antigen binding fragment thereof binds to the Spike Ferritin nanoparticle (SpFN).

14. A pharmaceutical composition comprising the monoclonal antibody or antigen binding fragment thereof of any of claims 1-13 and a pharmaceutically acceptable carrier.

15. The composition of claim 14, further comprising one or more additional monoclonal antibodies against SARS-CoV-2 or other coronaviruses, or antigen binding fragments thereof.

16. A method of preventing, treating, or reducing the risks of a coronavirus infection, comprising administering the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-13 or the pharmaceutical composition of claim 14 or claim 15, to a subject in need thereof, optionally wherein the coronavirus infection is selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

17. The monoclonal antibody or antigen binding fragment thereof of any one of claims 1- 13 or the pharmaceutical composition of claim 14 or claim 15, for use in preventing, treating, or reducing the risks of a coronavirus infection in a subject in need thereof, optionally wherein the coronavirus infection is selected from SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

18. An in vitro method for detecting a coronavirus infection in a subject or assessing a subject’s condition, comprising contacting a biological sample obtained from the subject with the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-13, and detecting any coronavirus antigen bound by the monoclonal antibody or antigen binding fragment thereof, optionally wherein the coronavirus infection is selected from SARS-CoV-1, SARS-CoV2, and MERS-CoV.

19. An in vitro method for detecting a coronavirus antigen in a sample, comprising contacting a sample with the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-13, and detecting any coronavirus antigen bound by the monoclonal antibody or antigen binding fragment thereof, optionally wherein the sample is obtained from acomposition comprising mRNA encoding a coronavirus antigen, further optionally wherein the sample is obtained from a composition comprising mRNA encoding SpFN.

20. A monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody is selected from:(A) a monoclonal antibody comprising a heavy chain complementary determining region 3 (HCDR3) and a light chain complementary determining region 3 (CDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 9 (having HCDR3 of SEQ ID NOs 760-763 and LCDR3 of SEQ ID NOs 1010-1013, respectively);(B) a monoclonal antibody comprising three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 9 (having HCDR1 of SEQ ID NOs 518-521, HCDR2 of SEQ ID NOs 639-642, and HCDR3 of SEQ ID NOs 760-763 and LCDR1 of SEQ ID NOs 881-884, and LCDR3 of SEQ ID NOs 1010-1013, respectively);(C) a monoclonal antibody comprising a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 9 (SEQ ID NOs 413, 415, 417, and 419), optionally wherein the heavy chain variable region sequence comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 9;(D) a monoclonal antibody comprising a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 9 (SEQ ID NOs 414, 416, 418, and 420), optionally wherein the light chain variable region sequence comprises the light chain variable region sequence of any one of the antibodies set forth in Table 9;(E) a monoclonal antibody comprising a heavy chain variable region sequence and a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence and light chain variable region sequence, respectively, of any one of the antibodies set forth in Table 9, optionally wherein the heavy and light chain variable region sequences comprise the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 9; and(F) a monoclonal antibody comprising the amino acid sequence of any one of the antibodies set forth in Table 9, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, optionally wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 9.

21. A monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody is selected from:(A) a monoclonal antibody comprising a heavy chain complementary determining region 3 (HCDR3) and a light chain complementary determining region 3 (CDR3), wherein the HCDR3 and LCDR3 are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR3 and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 4 (having HCDR3 of SEQ ID NOs 780-783 and LCDR3 of SEQ ID NOs 1030-1033, respectively);(B) a monoclonal antibody comprising three heavy chain complementary determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) and three light chain CDRs (LCDR1, LCDR2, and LCDR3), wherein the CDRs are at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of any one of the monoclonal antibodies set forth in Table 4 (having HCDR1 of SEQ ID NOs 538-541, HCDR2 of SEQ ID NOs 659-662, and HCDR3 of SEQ ID NOs 780-783, and LCDR1 of SEQ ID NOs 901-904, and LCDR3 of SEQ ID NOs 1030-1033, respectively);(C) a monoclonal antibody comprising a heavy chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence of any one of the antibodies set forth in Table 4 (SEQ ID NOs 3, 13, 23, and 39), optionally wherein the heavy chain variable region sequence comprises the heavy chain variable region sequence of any one of the antibodies set forth in Table 4;(D) a monoclonal antibody comprising a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the light chain variable region sequence of any one of the antibodies set forth in Table 4 (SEQ ID NOs. 4, 14, 24, and 40), optionally wherein the light chain variable region sequence comprises the light chain variable region sequence of any one of the antibodies set forth in Table 4;(E) a monoclonal antibody comprising a heavy chain variable region sequence and a light chain variable region sequence at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the heavy chain variable region sequence and light chain variable region sequence, respectively, of any one of the antibodies set forth in Table 4, optionally wherein the heavy and light chain variable region sequences comprise the heavy and light chain variable region sequences of any one of the antibodies set forth in Table 4; and(F) a monoclonal antibody comprising the amino acid sequence of any one of the antibodies set forth in Table 4, or an amino acid sequence at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto, optionally wherein the monoclonal antibody comprises the amino acid sequence of any one of the antibodies set forth in Table 4.

22. The monoclonal antibody or antigen binding fragment thereof of claim 20 or claim 21, wherein the monoclonal antibody or antigen binding fragment thereof binds to H. pylori ferritin, optionally wherein the monoclonal antibody or antigen binding fragment thereof binds to empty H. pylori ferritin particles.

23. The monoclonal antibody or antigen binding fragment thereof of claim 20 or claim 21, wherein the monoclonal antibody or antigen binding fragment thereof binds to a ferritin moiety of the Spike Ferritin nanoparticle (SpFN).

24. A method of preventing, treating, or reducing the risks of H. pylori infection, comprising administering the monoclonal antibody or antigen binding fragment thereof of any one of claims 20-23 to a subject in need thereof.

25. The monoclonal antibody or antigen binding fragment thereof of any one of claims 20- 23 for use in preventing, treating, or reducing the risks of H. pylori infection in a subject in need thereof.

26. An in vitro method for detecting H. pylori infection in a subj ect or assessing a subj ect’ s condition, comprising contacting a biological sample obtained from the subject with the monoclonal antibody or antigen binding fragment thereof of any one of claims 20-23, and detecting any H. pylori bound by the monoclonal antibody or antigen binding fragment thereof.

27. An in vitro method for detecting an H. pylori antigen in a sample, comprising contacting a sample with the monoclonal antibody or antigen binding fragment thereof of any one of claims 20-23, and detecting any H. pylori antigen bound by the monoclonal antibody or antigen binding fragment thereof, optionally wherein the sample is obtained from a composition comprising mRNA encoding an H. pylori antigen, further optionally wherein the sample is obtained from a composition comprise mRNA encoding H. pylori ferritin particles, further optionally wherein the sample is obtained from a composition comprising mRNA encoding SpFN.

28. A polynucleotide encoding the monoclonal antibody or antigen binding fragment thereof of any one of claims 1-13 or 20-23.

29. A vector comprising the polynucleotide of claim 28.

30. A host cell comprising the antibody or antigen-binding fragment of any one of claims 1-13 or 20-23, the polynucleotide of claim 28, or the vector of claim 29.

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