Coronavirus stem helix and fusion peptide nanoparticle vaccines

Abstract

Disclosed are coronavirus-based immunogens, including immunogens comprising spike protein and or domains thereof, comprised in immunogen displays (e.g., nanoparticles). Provided are also immunogenic compositions and methods of using these immunogens to induce immunogenic responses in a subject.

Description

CORONAVIRUS STEM HELIX AND FUSION PEPTIDENANOPARTICLE VACCINES

[0001] This International Patent Application claims the benefit of and priority to U.S. Application No. 63 / 590,120, filed October 13, 2023, entitled “Coronavirus Stem Helix and Fusion Peptide Nanoparticle Vaccines,” the content of which is hereby incorporated by reference in its entirety.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as know n to those skilled therein as of the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.GOVERNMENT INTERESTS

[0004] This invention w as made with government support from the NIH / NIAID / DMID, grant PO1-AI158571. The government has certain rights in the invention.FIELD

[0005] Disclosed are compositions and methods for eliciting broadly neutralizing antibodies against coronavirus.BACKGROUND

[0006] COVID-19 has been associated ith significant morbidity and mortality, representing the largest public health crisis of the present century. Current SARS-CoV-2 vaccines have been unable to keep pace with the rapid evolution of highly divergent viral variants, posing a challenge to mitigating the ongoing pandemic. There is a need to develop broadly protective pancoronavirus vaccines.SUMMARY

[0007] Disclosed herein are recombinant proteins that are coronavirus stem helix or fusion peptide sequences (defined here as the amino acids 816-833 of the SARS-CoV-2 spikeprotein) that are configured to be incorporated into an immunogen display (e g., nanoparticle). The coronavirus sequences can be selected by choosing a stem helix or fusion peptide epitope that is bound by a broadly -neutralizing antibody. The coronavirus sequences can include SEQ ID NOs: 7-9 or sequences with identity thereto. The recombinant proteins can include SEQ ID NOs: 4-6 or sequences with identity thereof.

[0008] Disclosed herein are immunogen displays that display on a surface, any of the coronavirus stem helix or fusion peptide sequences, or recombinant proteins containing the sequences. The immunogen displays can include a protein scaffold, nanoparticle (e g., selfassembling nanoparticle), or virus-like particle.

[0009] Disclosed are nucleic acids encoding the coronavirus stem helix or fusion peptide sequences, the recombinant proteins or the immunogen displays and host cells containing the nucleic acids.

[0010] Disclosed are immunogenic compositions containing the coronavirus stem helix or fusion peptide sequences, the recombinant proteins or the immunogen displays.

[0011] Disclosed are methods for inducing an immune response to a coronavirus in a subject by administering to a subject, containing the coronavirus stem helix or fusion peptide sequences, the recombinant proteins or the immunogen displays.BRIEF DESCRIPTION OF THE FIGURES

[0012] The patent or application file contains at least one drawing executed in color.Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] FIG. 1. Structural location of broadly neutralizing epitopes on SARS-CoV-2 Spike ectodomain. Each protomer of the Spike is colored a different shade of gray or black.Receptor binding domain, fusion peptide, and stem helix epitopes are colored. PDB:6RX8.

[0014] FIG. 2. High conservation of fusion peptide and stem helix sequences among SARS-CoV-2 variants of concern. Number of amino acid changes in each epitope is shown for the various isolates of SARS-CoV-2.

[0015] FIG. 3. Example vaccine Design Strategy for broadly protective CoV vaccine. In some embodiments, vaccine immunogens are nanoparticle scaffolds displaying sthe neutralizing antibody epitopes of interest.

[0016] FIG. 4. Structure-guided Stem-helix peptide design. The stem helix peptide bound to broadly neutralizing antibodies CC40.8 and S2P6 identified the section of the stem helix peptide.

[0017] FIG. 5. Example design of a stem-helix peptide 60-mer nanoparticle. Stem helix peptides of two different lengths were genetically fused to the N-terminus of mI3 nanoparticles.

[0018] FIG. 6. NSEM shows well-formed Stem-helix peptide nanoparticles.

[0019] FIG. 7. Stem-helix peptide nanoparticles can be antigenic for stem-helix neutralizing antibodies. Biolayer interferometry binding response shows the longer stem helix peptide exhibited better binding to stem helix antibody CC40.8 as predicted by the CC40.8 liganded structure.

[0020] FIG. 8A and 8B. Stem helix nanoparticles can react with each member of the stem helix neutralizing antibody lineage, including its unmutated common ancestor (UCA). A. The DH1057 phylogenetic tree. B. Biolayer interferometry binding of the different stem helix nanoparticles to the DH1057 antibody clone.

[0021] FIG. 9. Stem-helix peptide serum IgG can be a subdominant antibody response after SARS-CoV-2 infection or vaccination. Plasma IgG ELISA binding magnitudes for humans who were infected, vaccinated, or both for SARS-CoV-2 are shown. Uninfected / unvaccinated human serum was used as a negative control group. mI3 is the nanoparticle without any stem helix peptide.

[0022] FIG. 10A and 10B. Example design of SARS-CoV-2 fusion peptide nanoparticles (10A). Green indicates the fusion peptide on the spike and nanoparticle. An amino acid alignment comparing the fusion peptide sequences of different viruses shows high sequence conservation for this target epitope (10B).

[0023] FIG. 11A and 11B-C. Size and structure of example SARS-CoV-2 fusion peptide nanoparticles. A. Analytical size exclusion chromatography using a Superose 6 10 / 300 column is shown. Electron microscopy of nanoparticles includes, B. Shows negative staining. C. shows 2D class average of negative-stained electron microscopy images.

[0024] FIG. 12A, 12B and 12C. Reactivity of SARS-CoV-2 fusion peptide nanoparticles. Biolayer interferometry (BLI) binding response showing fusion peptide nanoparticles are antigenic for fusion peptide antibodies (12A). BLI binding response is shown for DH1294 antibody clonal lineage members binding to SARS-CoV-2 fusion peptide nanoparticles (FP- NP), Spike ectodomain (2P-Spike), or fusion peptide alone (FP) (12B). A heatmap indicating BLI binding response for a panel of fusion peptide antibodies binding to either SARS-CoV-2 FP-NP or the ferritin nanoparticle without the FP (Ferritin NP)(12C).

[0025] FIG. 13A and 13B. SARS-CoV-2 infection or vaccination and infection elicits human serum IgG that binds SARS-CoV-2 fusion peptide nanoparticles. Plasma IgG ELISAbinding magnitudes for humans who were infected, vaccinated, or both for SARS-CoV-2 are shown. Uninfected / unvaccinated human serum was used as a naive control group. Ferritin NP is the nanoparticle without any fusion peptide.

[0026] FIG. 14A, 14B, 14C and 14D. SARS-CoV-2 fusion peptide nanoparticles induce broad binding antibodies to different betacoronavirus spikes (specific virus indicated in lettering at top of each graph). ELISA measuring serum IgG binding as area-under-the-log- transformed-curve. Values are the group mean with error bars depicting the standard error of the mean. Arrows indicate immunization timepoints.

[0027] FIG. 15A, 15B and 15C. Combination of SARS-CoV-2 fusion peptide and stem helix nanoparticles induce broad binding IgG responses against different betacoronavirus spikes in BALB / c mice (specific virus indicated in lettering at top of each graph). ELISA measuring serum IgG binding as area-under-the-log-transformed-curve. Values are the group mean with error bars depicting the standard deviation. Arrows indicate immunization timepoints.DETAILED DESCRIPTION

[0028] Neutralizing antibodies generated during SARS-CoV-2 infection or vaccination predominantly target the spike receptor binding domain (reference 1 at end of Examples). Other neutralizing epitopes exist on the SARS-CoV-2 spike protein including the stem helix and fusion peptide epitopes (FIG. 1), but elicitation of antibodies against the stem helix and the fusion peptide epitopes can be low compared to the immune response elicited against the receptor binding domain (references 2 and 3 at end of Examples). The peptide that mediates fusion in the SARS-CoV-2 S is debated (Shi, Wei, et al. "Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane." Nature 619.7969 (2023): 403-409). thus fusion peptide is used here to describe the antibody epitope spanning positions 816-833 in SARS-CoV-2 S.The stem helix and fusion peptide are epitopes within the highly conserved S2 region of spike protein that can confer broad neutralization across Alphacoronaviruses andBetacoronaviruses (references 4 and 5 at end of examples). The stem helix and fusion peptide amino acid sequences have remained conserved from Alpha variant of concern through XBB variant of concern (FIG. 2). In some embodiments, compositions and methods for eliciting an immune response that targets the stem helix and / or fusion peptide region is disclosed. In some embodiments, compositions and methods for eliciting an immune response that targets stem helix and / or fusion peptide and the receptor binding domain is disclosed (FIG. 3).

[0029] In some embodiments, protein nanoparticles were designed and produced, inwhich the SARS-CoV-2 fusion peptide and / or the stem-helix peptide is incorporated as part of an immunogen display. In some embodiments, the fusion peptide and / or stem helix is fused to the N-terminus of a self-assembling protein nanoparticle. Disclosed are coronavirusbased immunogens, including immunogens comprising spike protein and or domains thereof, comprised in immunogen displays that can elicit broad immune responses against diverse members of the Coronaviridae family of viruses. Provided are also immunogenic compositions and methods of using these immunogens to induce broad immunogenic responses in a subject.Definitions

[0030] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms.Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0031] The singular forms “a”, '‘an’’ and ’‘the” include plural reference unless the context clearly dictates otherwise. The use of the word ‘’a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0032] Wherever any of the phrases “for example.” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.

[0033] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0034] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a. b and c.Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0035] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).Coronavirus

[0036] A “coronavirus” can refer to an enveloped virus with a positive-sense singlestranded RNA genome and a nucleocapsid of helical symmetry that constitutes the subfamily Orthocoronavirinae, in the family Coronaviridae. In some embodiments, the coronavirus can be an animal or human coronavirus. In some embodiments, the coronavirus can comprise a genus Alphacoronavirus or Betacoronavirus. In some embodiments, the genus Betacoronavirus can comprise a subgenus Sarbecovirus, Embecovirus and Merbecovirus. In some embodiments, the subgenus Sarbecovirus can comprise a species severe acute respiratory syndrome-related coronavirus (SARSr-CoV or SARS-CoV).^ SARSr-CoV or SARS-CoV species contains a strain of coronavirus called “SARS-CoV-2” which causes COVID-19 (coronavirus disease 2019), the respiratory illness responsible for the ongoing COVID- 19 pandemic.

[0037] In 2020, the World Health Organization (WHO) characterized the spread of COVID- 19, a highly contagious respiratory disease caused by the Betacoronavirus SARS- CoV-2 (SARS-2), as a pandemic. As the virus continues to spread, there is a need to mount effective defenses against emerging SARS-CoV-2 variants and other human-infecting coronaviruses that has prompted interest in developing broadly protective vaccines. In some embodiments, the immunogens disclosed herein can provide a way to focus the immune response to a site on the virus that may be rarely targeted by vaccination or infection.

[0038] Described herein are recombinant fusion proteins and multimeric protein complexes (nanoparticles) containing immunogens, or complete antigens composed of a macromolecular carrier and epitopes (determinants) that can induce immune response. The immunogens described herein can elicit immune responses to coronavirus epitopes that are highly conserved across diverse members of the Coronaviridae family. In some embodiments, the recombinant fusion proteins and multimeric protein complexes (nanoparticles) can elicit an immune response to Alphacoronavirus, Betacoronavirus, or acombination thereof. In some embodiments, the recombinant fusion proteins and multimeric protein complexes (nanoparticles) can elicit an immune response to Embecovirus, a Sarbecovirus, a Merbecovirus, other viruses, or a combination thereof.Spike Protein

[0039] The viral-host cell fusion (S) protein Spike is a target owing to its role in the virus lifecycle (FIG. 1). The Spike protein can be divided into two regions: the N-terminal SI domain and the S2 domain. Binding to host receptor via the Receptor Binding Domain (RBD) in SI is followed by proteolytic cleavage of the spike by host proteases. Large conformational changes in the S-protein result in SI shedding and exposure of the fusion machinery in S2. See Walls, A. C. et al. Structure. Function, and Antigenicity of the SARS- CoV-2 Spike Glycoprotein. Cell, 2020 Apr 16; 181(2):281-292); Wrapp. D. et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263, doi: 10. 1126 / science.abb2507 (2020).

[0040] The "‘Spike protein / ’ “S protein” or “S” is a membrane anchored class I fusion protein expressed on the coronavirus surface, which gives the characteristic shape to the coronavirus under the electronic microscope. Spike proteins can be targets of host immune responses elicited by infection or vaccination. Spike proteins are comprised of two distinct functional subunits: an N-terminal, membrane distal subunit designated SI, and a C-terminal, membrane proximal subunit designated S2.

[0041] The SI domain of the coronavirus spike contains the receptor binding domain (RBD). RBD binds to cell-surface receptors and attaches to cells prior to virus entry into cells. Current SARS-CoV-2 vaccines contain sequences from RBD. However, frequent mutations in RBD can result in reduced efficacy of antibodies produced by an immune response to a prior virus. In some examples, this can result in reduced efficacy of current SARS-CoV-2 vaccines.

[0042] For SARS-CoV-1 and SARS-CoV-2, the cellular receptor is “angiotensin converting enzyme-2” or “ACE2”. ACE2 is a transmembrane protein that has an extracellular domain, a transmembrane domain, and an intracellular domain. It can be expressed in cells of the nasal passages, lungs, arteries, heart, kidney and intestines, for example. The human version of the enzyme can be referred to as hACE2.

[0043] The S2 domain of the coronavirus spike contains the fusion machinery that undergoes structural rearrangements to mediate fusion of the host and viral membranes. The SI domain serves to stabilize the S2 subunit in the pre-fusion state and facilitates theattachment to ligands on host cells through the RBD. The S2 domain includes the fusion protein region (fusion protein) and S2 stem helix region (stem helix), conserved amino acid regions that are not subject to the variability of RBD. In some embodiments, because they are conserved, the fusion peptide and S2 stem helix regions can be the basis for broad vaccines that may stimulate protection against a variety of diverse coronaviruses and be more efficacious over time, due to their conservation (e g., virus mutations in these regions are less likely to occur).

[0044] In aspects of the invention, recombinant fusion proteins and / or immunogen displays that comprise the proteins can elicit immune responses (e.g., antibodies) to the S2 region of the human coronavirus spike protein, which can comprise a fusion peptide epitope, a S2 stem helix epitope, or a combination thereof. In some embodiments, the recombinant fusion proteins and / or immunogen displays can include immunogens that can elicit immune responses specific for an S2 region of a coronavirus spike protein. In some embodiments, the recombinant fusion proteins and / or immunogen displays can include immunogens that can elicit immune responses specific for a fusion peptide region (amino acids 816-833) of a coronavirus spike protein. The peptide that mediates fusion in the SARS-CoV-2 S is debated, thus fusion peptide is used here to describe the antibody epitope and refers to the amino acids spanning positions 816-833 in SARS-CoV-2 S. It is understood that this region may not mediate virus fusion (Shi, Wei, et al. "Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane." Nature 619.7969 (2023): 403-409). In some embodiments, the recombinant fusion proteins and immunogen displays can include immunogens that can elicit immune responses specific for a stem helix region of a coronavirus spike protein. In some embodiments, the recombinant fusion proteins and / or immunogen displays can include immunogens that can elicit immune responses specific for a stem helix region, a fusion peptide region, or to a combination thereof. In some embodiments, the multimeric protein complex can include at least one recombinant fusion protein that can elicit immune responses specific for a fusion peptide region of a coronavirus spike protein and at least one recombinant fusion protein that can elicit immune responses specific for a stem helix region. In some embodiments, immune responses against coronavirus receptor binding domain (RBD) can also be elicited.

[0045] Described herein are recombinant fusion proteins and immunogen displays (e.g., nanoparticles), containing immunogens that are directed to epitopes from the S2 region of the coronavirus spike protein. In some embodiments, these recombinant fusion proteins and immunogen displays can contain immunogens that can stimulate immune responses toepitopes that are conserved across diverse currently circulating and / or emerging coronaviruses. These recombinant fusion proteins and immunogen displays can induce high antibody titers against viral targets that have a low propensity to acquiring mutations.

[0046] In some aspects, the invention provides recombinant fusion proteins and immunogen displays (e.g., nanoparticles) containing immunogens that can be the basis for broad vaccines (e.g., vaccines that can elicit immune responses against multiple coronaviruses). In some embodiments, the recombinant fusion proteins and immunogen displays (e.g., nanoparticles) containing immunogens described herein can elicit protection against a variety of diverse coronaviruses and be more efficacious over time, due to their conserv ed immunogens. In some embodiments, the recombinant fusion proteins and immunogen displays can stimulate broad immune protection against diverse coronaviruses by eliciting at the same time an immune response against an Alphacoronavirus and a Betacoronavirus . In some embodiments, the recombinant fusion proteins and multimeric protein complexes can stimulate immune protection against Alphacoronavirus and Betacoronavirus . In some embodiments, the recombinant fusion proteins and multimeric protein complexes can stimulate a broad immune protection against Betacoronavirus by generating a cross-reactive immune response against Betacoronavirus subgenera lineages including lineages A (Embecovirus), B (Sarbecovirus), C (Merbecovirus), or a combination thereof.

[0047] Several broadly cross-reactive antibodies against epitopes in the fusion peptide and in the S2 stem helix region of the spike protein implicate these regions as highly conserved viral targets for inclusion in vaccines. However, antibodies against these targets can be weakly elicited by vaccines, possibly owing to the poor accessibility of the fusion peptide and stem helix on the native spike.

[0048] In some embodiments, disclosed herein are stem helix and fusion peptide regions from coronavirus. In some embodiments, the stem helix region can be an epitope that can be bound by a broadly neutralizing antibody, such as CC40.8 or S2P6. In some embodiments, the stem helix regions can be 14- and 22-amino acid sequences disclosed herein.

[0049] In some embodiments, the 14-amino acid sequence is part of the sequence in SEQ ID NO: 5. In some embodiments, the 14-amino acid sequence is DSFKEELDKYFKNH (SEQ ID NO: 7)

[0050] In some embodiments, the 22-amino acid sequence is part of the sequence in SEQ ID NO: 4. In some embodiments, the 22-amino acid sequence is YDPLQPELDSFKEELDKYFKNH (SEQ ID NO: 8).

[0051] In some embodiments, the fusion peptide region can be a 26-amino acid sequence from the S2 region of Spike that encompasses the fusion peptide. In some embodiments, the 26-amino acid sequence is part of the sequence in SEQ ID NO: 6. In some embodiments, the 26-amino acid sequence is DPSKPSKRSFIEDLLFNKVTLADAGF (SEQ ID NO: 9), where DPSKPSKR is an alpha helix in S2 that can interact with the fusion peptide and may stabilize the conformation of the fusion peptide. An originally reported fusion peptide sequence included SFIEDLLFNKVTLADAGF.Epitopes

[0052] Epitopes can include any protein determinant capable of specific binding to an immunoglobulin, a scFv, or a T-cell receptor. The variable region, of an antibody for example, allows the antibody to selectively recognize and specifically bind epitopes on antigens. For example, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. Epitopic determinants can have chemically active surface groupings of molecules such as amino acids or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- terminal or C-terminal peptides of a polypeptide. In some embodiments, the antigen-binding site is defined by three CDRs on each ofthe VH and VL chains (i.e. CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR- L2 and CDR-L3).

[0053] In embodiments, disclosed herein are an amino acid epitopes for antibody binding from a SARS-CoV-2 spike protein can stimulate immune responses against a fusion peptide amino acid epitope, a S2 stem helix amino acid epitope, or a combination thereof. The epitope can be any described herein related to the fusion peptide, or as described herein as related to the S2 stem helix.

[0054] In some aspects, the invention provides recombinant fusion protein sequences that can include, in order, a full-length coronavirus spike protein or a portion thereof, a peptide linker and a self-assembling protein, wherein non-limiting embodiments of sequences are shown in Table 2. In some embodiments, the recombinant fusion protein sequences described herein can be encoded by nucleotide sequences of a spike protein antigen fused via a linker / s pacer to a nucleotide sequence of H. Pylori ferritin (Table 1). In some embodiments, the nucleotide sequence of the spike protein includes a C-Terminal signal peptide, an N-terminal His purification tag, a twinStrepTagll purification tag, an HRV3C cleavage site, or a combination thereof. In some embodiments, a nucleic acid sequence corresponding to 26 amino acids of the fusion peptide is fused to a glycine-serine-glycine linker and to the fourth amino acid of H. pylori ferritin. In some embodiments, nucleic acid sequences corresponding to stem helix peptides of two different lengths (22 and 14 aa) are genetically fused to the N- terminus of mI3 nanoparticles (Table 1).

[0055] In some aspects, the invention provides modified coronavirus spike protein designs including but not limited to protein designs comprising spike protein and / or various spike portions / domains from SARS-CoV-2 (SARS-2), wherein in some embodiments these proteins are designed to form immunogen displays, including nanoparticles. The invention provides amino acid and nucleic acid sequences of recombinant coronavirus spike proteins or portions thereof, wherein in certain embodiments, these recombinant spike proteins or portions / domains can form nanoparticles that display the spike protein or portion thereof. The displayed spike protein or potion thereof can be used as an antigen to induce an immunogenic response.

[0056] In some embodiments the nanoparticle comprises any suitable portion from a spike protein. In non-limiting embodiments are portions of the spike protein which comprise epitopes conserved between different coronaviruses. In some embodiments the epitope comprises an FP domain from a spike protein. In some embodiments, the FP domain can include the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the nucleic acid sequence can include the nucleic acid sequence set forth in SEQ ID NO: 3.

[0057] In some embodiments the epitope comprises a stem helix domain from a spike protein. In some embodiments, the stem helix domain includes any of the amino acid sequences set forth in SEQ ID NO: 4 and SEQ ID NO: 5. In some embodiments, the nucleic sequence of the stem helix domain includes any one of the sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 2

[0058] In some embodiments, the nanoparticles described herein can include a recombinant protein complex including a fusion peptide domain from a coronavirus and another independent recombinant protein complex including a stem helix domain from a coronavirus. In some embodiments, recombinant protein complexes including a fusion peptide domain and recombinant protein complexes including a stem helix domain can both assemble together to form nanoparticles that include both recombinant protein complexes (stem-FP nanoparticles). The stem-FP nanoparticles can display both stem helix epitopes and fusion peptide epitopes.

[0059] The nanoparticles described herein can be composed of various portions of SARS- CoV-2 spike protein and a self-assembling ferritin protein. Any suitable ferritin could be used in the immunogens of the invention (see, for example, W 0 / 2018 / 005558 which is incorporated by reference herein for all purposes). In non-limiting embodiments, the ferritin can be derived from Helicobacter pylori. In non-limiting embodiments, the ferritin can be an insect ferritin. In non-limiting embodiments, each nanoparticle can display 24 copies of the spike protein on its surface.Epitopes that Stimulate Immune Responses to the Fusion Peptide Region

[0060] In some aspects, a “fusion peptide,’' “fusion protein,” and “FP” can be used. The fusion peptide described herein can refer to a region in the S2 subunit of the SARS-CoV-2 spike which shows high conservation across diverse coronaviruses.

[0061] In some aspects, a recombinant fusion protein sequence comprising, in order, a full-length coronavirus spike protein or a portion thereof, a peptide linker and a selfassembling protein (or a peptide, polypeptide or protein that provides for linking the epitope to an immunogen display), wherein the coronavirus spike protein comprises a fusion peptide region or a portion thereof of a coronavirus is described. In embodiments, the recombinant fusion protein can be assembled into an immunogen display that displays the FP epitopes described herein. In some embodiments, the immunogen display that displays the FP can stimulate immune responses to, and / or can bind antibodies that bind to. fusion peptide epitopes of coronavirus. In some embodiments, the fusion peptide epitopes can include a 26 amino acid peptide that can be bound by antibodies that recognize the FP region of the coronavirus Spike protein.

[0062] In some embodiments, the fusion peptide region or a portion thereof of the coronavirus can include a sequence corresponding to a fusion peptide region epitope of coronavirus. In some embodiments, the fusion peptide region has the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%. at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 6. In some embodiments, the fusion peptide region has the nucleic acid sequence set forth in SEQ ID NO: 3, or a nucleic acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%. at least at or about 93%. at least at or about 94%, at least at or about 95%, at leastat or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity’ to SEQ ID NO: 3.

[0063] In some embodiments, the fusion peptide nanoparticles described herein can bind and / or react with serum and / or antibodies from serum of humans that were infected, vaccinated, or both for SARS-CoV-2. In some embodiments, the fusion peptide nanoparticles described herein can bind and / or react with serum and / or antibodies from serum of humans that were infected with SARS-CoV-2. In some embodiments, the fusion peptide nanoparticles described herein can bind and / or react with serum and / or antibodies from serum of humans that were vaccinated for SARS-CoV-2. In some embodiments, the fusion peptide nanoparticles described herein can bind and / or react with serum and / or antibodies from serum of humans that were infected and vaccinated for SARS-CoV-2. In some embodiments, the fusion peptide nanoparticles described herein can be used to elicit an immune reaction in a subject.

[0064] In some embodiments, an effective amount of any one of the recombinant fusion proteins and / or nanoparticles displaying the fusion peptide described herein can be administered to a subject to induce an immune response. In some embodiments, the effective amount of the recombinant fusion proteins and / or nanoparticles displaying the fusion peptide can be administered to a subject that was previously vaccinated. In some embodiments, the effective amount of the recombinant fusion proteins and / or nanoparticles displaying the fusion peptide can be administered to a subject as a boost. In some embodiments, the effective amount of the of the recombinant fusion proteins and / or nanoparticles displaying the fusion peptide can be administered to engage naive or pre-existing memory’ B cells.

[0065] In embodiments, the fusion peptide nanoparticles described herein can induce a broad immune response including broad binding antibodies to different subgenera of the Betacoronaviruses . In some embodiments, the antibody stimulated by, and / or that react with, the coronavirus fusion peptide described herein, can cross-react with other coronavirus disclosed herein. In some embodiments, the antibodies that react to coronavirus FP epitopes can also bind / react with, and / or cross-react, with Betacoronaviruses . In some embodiments, the antibodies that react to coronavirus FP epitopes can also bind / react with, and / or cross- react, with Embecovirus (lineage A). In some embodiments, the antibodies that react to coronavirus FP epitopes can also bind / react with, and / or cross-react, with Sarbecovirus (lineage B). In some embodiments, the antibodies that react with coronavirus FP epitopes can also bind / react with, and / or cross-react. vnX Merbecovirus (lineage C). In some embodiments, the antibodies that react to coronavirus FP epitopes can also bind / reactwith, and / or cross-react, with Embecovirus, Sarbecovrius, Merceovirus, or a combination thereof. Non-limiting examples of antibodies that can bind to the FP epitopes can include DH1058, DH1294. Non-limiting examples of antibodies that cannot bind to the FP epitopes can include DH1047 (an antibody specific to RBD) and VRC34.1 (an antibody specific to the FP).Epitopes that Stimulate Immune Responses to the S2 Stem Helix

[0066] In some aspects, “S2 stem helix” can refer to a region in the S2 subunit of the SARS-CoV-2 spike which shows high conservation across diverse coronaviruses.

[0067] In some aspects, a recombinant stem helix sequence comprising in order a full- length coronavirus spike protein or a portion thereof, a peptide linker and a self-assembling protein, wherein the coronavirus spike protein comprises a stem helix region or a portion thereof of a coronavirus is described. In some embodiments, the recombinant fusion protein can be assembled into an immunogen display that displays the stem helix region / epitope described herein.

[0068] In some embodiments, the immunogen display that displays the stem helix epitope can stimulate immune responses to, and / or can bind antibodies that bind to, stem helix epitopes of coronaviruses. In some embodiments, the stem helix region includes a 14 amino acid peptide (SEQ ID NO: 5) that can be recognized by the anti-SARS-CoV-2 broadly neutralizing antibodies CC40.8 and S2P6. In some embodiments, the stem helix region can include a 22 amino acid peptide (SEQ ID NO: 4) that can be recognized by the anti-SARS- CoV-2 broadly neutralizing antibodies CC40.8 and S2P6.

[0069] In some embodiments, the stem helix region or a portion thereof of the coronavirus can include a sequence corresponding to a stem helix epitope of coronavirus. In some embodiments, the stem helix region can have the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 4. In some embodiments, the stem helix region can have the amino acid sequence set forth in SEQ ID NO: 5, or an amino acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%. at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 5.

[0070] In some embodiments, the stem helix region can have the nucleic acid sequence set forth in SEQ ID NO: 2, or an nucleic acid sequence having at least at or about 90%, at least at or about 91%, at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 2. In some embodiments, the stem helix region has the nucleic acid sequence set forth in SEQ ID NO: 1, or an nucleic acid sequence having at least at or about 90%. at least at or about 91%. at least at or about 92%, at least at or about 93%, at least at or about 94%, at least at or about 95%, at least at or about 96%, at least at or about 97%, at least at or about 98%, or at least at or about 99% sequence identity to SEQ ID NO: 1.

[0071] In some embodiments, the nanoparticles displaying the stem helix described herein can bind and / or react with serum and / or antibodies from serum of humans that were infected, vaccinated, or both for SARS-CoV-2. In some embodiments, the nanoparticles displaying the stem helix can bind and / or react with serum and / or antibodies from serum of humans that were infected with SARS-CoV-2. In some embodiments, the nanoparticles displaying the stem helix can bind and / or react with serum and / or antibodies from serum of humans that were vaccinated for SARS-CoV-2. In some embodiments, nanoparticles displaying the stem helix can bind and / or react with serum and / or antibodies from serum of humans that were infected and vaccinated for SARS-CoV-2.

[0072] In embodiments, the nanoparticles displaying the stem helix describe herein can induce a broad immune response including broad binding antibodies to different subgenera of the Betcicoronaviruses . In some embodiments, the antibodies stimulated by, and / or that react with, the nanoparticles displaying the stem helix describe herein, can cross-react with other members of the coronavirus family disclosed herein. In some embodiments, the antibodies that react to coronavirus FP epitopes can also bind / react with, and / or cross-react, with Betacoronaviruses . In some embodiments, the antibodies that react to the nanoparticles displaying the stem helix describe herein can also bind / react with, and / or cross-react, with Embecovirus (lineage A). In some embodiments, the antibodies that react to the nanoparticles displaying the stem helix describe herein can also bind / react with, and / or cross-react. with Sarhecovirus (lineage B). In some embodiments, the antibodies that react to the nanoparticles displaying the stem helix describe herein can also bind / react with, and / or cross-react, with Merbecovirus (lineage C). In some embodiments, the antibodies that react to the nanoparticles displaying the stem helix describe herein can also bind / react with, and / or cross- react, with Embecovirus, Sarbecovrius, Merceovirus, or a combination thereof. Non-limitingexamples of antibodies that can bind to the nanoparticles displaying the stem helix describe herein include CC40.8, DH1057.1, and S2P6. Other non-limiting examples of antibodies that can bind to the nanoparticles displaying the stem helix described herein can include antibodies belonging to the stem helix neutralizing antibody lineage including unmutated common ancestor (UCA) including DH1057 UCA, DH1057.1, DH1057. i l, DH1057.12, DH1057.2, and DH1057.3. Non-limiting examples of antibodies that cannot bind to the stem helix epitopes can include DH1047, Sx259 and JC57-14 (antibodies specific to RBD) and VRC34. 1 (an antibody specific to the FP).

[0073] In some embodiments, an effective amount of any one of the recombinant fusion proteins and / or nanoparticles displaying the stem helix epitope described herein can be administered to a subject to induce an immune response. In some embodiments, the effective amount of the recombinant fusion proteins and / or nanoparticles displaying the stem helix epitope can be administered to a subject that was previously vaccinated. In some embodiments, the effective amount of the recombinant fusion proteins and / or nanoparticles displaying the fusion peptide can be administered to a subject as a boost. In some embodiments, the effective amount of the of the recombinant fusion proteins and / or nanoparticles displaying the fusion peptide can be administered to engage naive or preexisting memory7B cells.

[0074] In some aspects, a nanoparticle displaying an epitope corresponding to the stem helix region of a coronavirus can be used in combination with another nanoparticle displaying an epitope corresponding to the FP region of a coronavirus to elicit an immune response. In some embodiments, the fusion peptide nanoparticles described herein can induce a broad immune response including broad binding antibodies to different subgenera of the Alphacoronaviruses and Betacoronaviruses. In some embodiments, the antibodies stimulated by, and / or that react with, the combination stem helix and FP epitopes described herein, can cross-react with other coronavirus disclosed herein. In some embodiments, the antibodies stimulated by, and / or that react with, the combination stem helix and FP epitopes described herein can cross react with other members of subfamilies and genus of the Coronaviridae family of viruses. In some embodiments, the antibodies stimulated by, and / or that react with, the combination stem helix and FP epitopes described herein can cross react with members of the Betacoronaviruses including Embecovirus (lineage A), Sarbecovirus (lineage B) and Merbecovirus (lineage C), or a combination thereof. In some aspects of the invention, a nanoparticle can be a combination (stem-FP) nanoparticle that displays stem helix epitopes and FP epitopes on the surface of the same nanoparticle. In some embodiments, the stem-FPnanoparticles described herein can induce a broad immune response including broad binding antibodies to different members of the Coronaviridae family. In some embodiments, the stem-FP nanoparticles described herein can induce a broad immune response including broad binding antibodies to different members of the Alphacoronaviruses and Betacoronaviruses, or a combination thereof. In some embodiments, the stem-FP nanoparticles described herein can cross-react with another coronavirus disclosed herein. In some embodiments, the antibodies stimulated by, and / or that react with, the stem-FP nanoparticles described herein can cross react with members of the Betacoronaviruses including Embecovirus (lineage A), Sarbecovirus (lineage B) and Merbecovirus (lineage C), or a combination thereof.TABLE 1. DNA SequencesTABLE 2. Amino Acid SequencesImmunogen Displays

[0075] In certain embodiments, the recombinant proteins described herein (e.g.. fusion peptide and / or stem helix) are designed to form an immunogen display. The immunogen displays are generally platforms (e.g., molecules, particles, and the like) that can “display” the fusion peptide / stem helix peptide sequences or combinations of sequences to the immune system in a way that an immune response is produced that is better than an immune response produced by the fusion peptide / stem helix sequences or combinations alone. In some embodiments, the immunogen displays can include protein scaffolds, nanoparticles, virus-like particles, liposomes and the like. In some embodiments, the immunogen display can include a self-assembling protein nanoparticle. In some embodiments, the immunogen display can augment the immunogenicity of a fusion peptide / stem helix by arraying multiple fusioncopies on the surface of the scaffolds, protein nanoparticles and the like (e.g., mi3 nanoparticles, ferritin nanoparticles and the like). In some embodiments, the immunogen display can augment the immunogenicity of displayed peptides / polypeptides / proteins by carrying inside it, T cell helper epitopes. In some embodiments, the immunogen display can be modified to mask its epitopes by pegylation or by adding de novo glycosylation sites, for example.

[0076] In some instances, the nucleotide sequence of an antigen can be fused via a linker / spacer to a gene of a protein which can self-assemble. Upon translation, a fusion protein is made that can self-assemble into a multimeric complex or an immunogen display (e.g., nanoparticle), displaying multiple copies of the antigen. In other instances, the protein antigen could be conjugated to the self-assembling protein or immunogen display via an enzymatic or other reaction, thereby forming a nanoparticle displaying multiple copies of the antigen.

[0077] In non-limiting embodiments, immunogen displays (e.g., nanoparticles) can comprise a ferritin sequence. In non-limiting embodiments, the recombinant proteins disclosed herein can include a ferritin sequence (e.g., a self-assembling protein) that can facilitate assembly of the recombinant protein into a nanoparticle with display of the coronovirus sequences on the surface of the nanoparticle. In non-limiting embodiments, the immunogen displays can comprise encapsulin. These immunogen displays (e.g., multimeric complexes) can be nanoparticles.

[0078] Any suitable ferritin sequence can be used. In non-limiting embodiments, ferritin sequences are disclosed in WO / 2018 / 005558, which is incorporated herein by reference for all purposes. In some embodiments, ferritin protein can self assemble into a nanoparticle with three-fold axis of symmetry. At these axes the protein is fused. Therefore, the assembly of the three-fold axis also clusters three proteins together. Each ferritin particle has 8 axes formed by three ferritin subunits which equates to 24 proteins being displayed per particle (Sliepen et al.). Ferritin nanoparticles can display the fusion peptide epitope attached to its N-terminus and / or C-terminus separated or not by a glycine-serine linker. The ferritin nanoparticle can be derived from different species. For example, Trichoplusia ni ferritin sequence can be used to encode the same or different fusion peptides on the heavy and light chains of the ferritin. Other non-limiting examples of ferritins that can be used include H. Pylori ferritin, and encapsulins from various bacterial species.

[0079] Trichoplusia ni (T. ni) ferritin can be alternative sequence to the H. pylori ferritin. T. ni has two ferritin chains whose structures are different from Helicobacter pylori ferritin.In contrast to H. pylori ferritin chains, the two chains of T. ni ferritin have long flexible N- terminal regions. The limited intramolecular interactions of these N-terminus with other ferritin chains allows it to be easily fused to a heterologous protein without disrupting the overall ferritin chain structure or assembly of the particle.

[0080] In some emboidments, the nanoparticles can include encapsulins. Encapsulin shell protomer proteins self-assemble to form icosahedral structures of various diameters (24-42 nm). Inside of nanocompartments shells, one or several cargo proteins, diverse in their functions, can be encapsulated.

[0081] Encapsulin nanoparticles can have 60 or more subunits to which heterologous proteins can attach. This provides an increase of 36 copies of a heterologous antigen of choice compared to ferritin nanoparticles. Encapsulins also have the advantage of being able to encapsulate other proteins inside the nanoparticle. Structures of Thermotoga maritima. Myxococcus xanthus, and Pyrococcus fiiriosus encapsulins have been solved, which allows for determination of the sites of attachment. The structure of Thermotoga maritima encapsulin showed that it had 60 repeat subunits that comprised one nanoparticle. Some encapsulin nanoparticles such as Pdu nanoparticles are composed of three or more subunits making it more complicated to express and form as an intact nanoparticle. The N-terminus of T. maritima encapsulin points into the lumen of the particle whereas the C-terminus points out away from the nanoparticle. Thus, the C-terminus of Thermotoga maritima encapsulin is the optimal attachment site for a heterologous protein so that the folding of the encapsulin subunit or the assembly of the nanoparticle in not disrupted. The C-terminus is also an optimal site for attachment since it lacks secondary structure and is just a flexible loop pointing away from the nanoparticle. Encapsulin nanoparticles displaying Epstein-Bar virus glycoproteins have been successfully achieved validating their use as a nanoparticle scaffold.

[0082] In some embodiments, the encapsulin can include Thermotoga maritima, Myxococcus xanthus, and Pyrococcus fiiriosus . Any suitable encapsulin described in the art can be used in this invention, including any of the encapulins described in Chmelyuk N., Biochemistry (Mose), 2023; 88(1): 35-49 and Matthias Ktinzle.

[0083] In some embodiments, the nanoparticle can include mi3 nanoparticles (Bruun. Theodora UJ, et al. "Engineering a rugged nanoscaffold to enhance plug-and-display vaccination." ACS nano 12.9 (2018): 8855-8866). These nanoparticles can use an engineered, thermostable protein from Thermotoga maritima to form a dodecahedron that contains 60 copies of the protein. Each of the 60 copies of the protein can have a “SpyCatcher’ molecule. In a mixture, the SpyCatcher molecules can bind a “SpyTag”molecule. When the SpyTag molecules are fused, herein with the coronavirus fusion peptide or stem helix sequences, nanoparticle immunogen displays can result that display the fusion peptide / stem helix epitopes on the nanoparticle surface.

[0084] Other suitable protein-based self-assembling nanoparticle platforms that can be used for the presentation of the fusion peptides described herein include lumazine synthase, dihydrolipoly acetyltransferase (E2p), a computationally designed hyperstable 60-mer (13-01) and ferritin.

[0085] The fusion peptides described herein can be included in a any biological nanoparticle including, but not limited to, virus like particles (VLPs), outer membrane vesicles (OMVs), protein cages (e.g., ferriting protein cages), gold nanocages, microparticles, polymer networks, liposomes, and the like, as described in Curley SM and Putnam D (2022), Biological Nanoparticles in Vaccine Development. Front. Bioeng. Biotechnol. 10:867119. doi: 10.3389 / fbioe.2022.867119, which is incorporate herein by reference for all purposes.

[0086] In some embodiments, the immunogen displays disclosed herein can be produced by contacting / reacting the coronavirus sequences disclosed herein with an assembled immunogen display. In some embodiments, when the immunogen display is a nanoparticle, the coronavirus sequences can be reacted with the nanoparticle in the presence of an enzy me in a conjugation reaction. In some embodiments, the enzyme can be a sortase.Nucleic Acids, Vectors and Cells Expressing Recombinant Proteins or Immunogen Displays

[0087] Also disclosed are nucleic acids encoding all or part of the molecules described herein. Also disclosed are various vectors (e.g., plasmids, viral, and the like) that include the nucleic acids. Also disclosed are various cells (e.g., prokaryotic, eukaryotic) that contain nucleic acids or vectors and can express the fusion proteins.

[0088] In certain aspects, the invention discloses using immunogenic compositions wherein immunogens are delivered as recombinant proteins. Various methods for production and purification of recombinant proteins, including fusion peptide ferritin fusion recombinant proteins as described herein, and suitable for use in immunization are known in the art.

[0089] DNA sequences encoding the disclosed recombinant proteins can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokary otic or eukaryotic.

[0090] Host systems for recombinant production can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Non-limiting examples of suitable hostcells include bacteria, archea, insect, fungi (for example, yeast), plant, and animal cells (for example, mammalian cells, such as human). Exemplary cells of use include Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Salmonella typhimurium, SF9 cells, C 129 cells, 293 cells, Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.

[0091] Techniques for the propagation of mammalian cells in culture are well-known (see, e.g., Helgason and Miller (Eds.). 2012, Basic Cell Culture Protocols (Methods in Molecular Biology), 4.sup.th Ed.. Humana Press). Examples of mammalian host cell lines are VERO and HeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although cell lines may be used, such as cells designed to provide higher expression, desirable glycosylation patterns, or other features. In some embodiments, the host cells include HEK293 cells or derivatives thereof, such as GnTI.sup.- / - cells , or HEK-293F cells.

[0092] In some embodiments, the disclosed recombinant proteins can be expressed in cells under conditions where the recombinant protein can self-assemble and / or are secreted from the cells into the cell media. In such embodiments, each recombinant protein can contain a leader sequence (signal peptide) that causes the protein to enter the secretory system, where the signal peptide is cleaved, and the protomers form a trimer, before being secreted in the cell media. The medium can be centrifuged, and recombinant protein can be purified from the supernatant.Compositions and Antibodies

[0093] In some aspects, this invention describes recombinant fusion protein sequences and immunogen displays (e.g., nanoparticles) displaying coronavirus immunogens.

[0094] In one aspect this disclosure provides compositions including any one of the recombinant proteins, immunogen displays, nucleic acids, multimeric protein complexes, virus-like particles described herein, or a combination thereof, and a pharmaceutically acceptable carrier and an adjuvant.

[0095] The composition describe herein can be used for a prime boost immunization regimen. In certain embodiments the composition for a prime boost immunization regimen comprises one or more stem-helix or fusion peptide regions or portions thereof described herein. In some embodiments, the effective amount of the of the recombinant fusion proteins and / or nanoparticles displaying the fusion peptide can be administered to engage naive or pre-existing memory B cells.

[0096] In non-limiting embodiments, compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for immunization. Insome embodiments, the compositions comprise a suitable carrier. Any suitable carrier can be used.

[0097] In certain aspects the compositions can comprise a suitable adjuvant. Any suitable adjuvant can be used. In certain embodiments, TLR agonists are used as adjuvants. In certain embodiments, TLR agonists are TLR7 / 8 agonist. Non-limiting examples of TLR 7 / 8 are described in Evans et al. ACS Omega 2019, 4, 13, 15665-15677; Patinote et al. Eur J Med Chem. 2020 May 1; 193: 112238, Miller et al. in Front. Immunol., 10 March 2020 | / / doi.org / 10.3389 / fimmu.2020.00406. In certain embodiments, adjuvants are TLR 4 agonists. In some embodiments, the compositions include the TLR4 agonist GLA-SE as the adjuvant. In some embodiments, the composition is the TLR4 agonist GLA-SE as the adjuvant.

[0098] The compositions can include adjuvants, such as. for example but not limited to alum, 3M052, poly IC, MF-59 or another squalene-based oil-in-water emulsion adjuvant, AS01B, or other liposomal based adjuvant suitable for protein. In non-limiting embodiments, LNPs are used as adjuvants for immunogenic formulations comprising proteins, including multimeric protein complexes and nanoparticles. In certain embodiments, the adjuvant is GSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shown by GSK to be as potent as the similar adjuvant AS01B but to be less reactogenic. In certain embodiments, AS04, a combination of alum and 3-0-desacyl-4'-monophosphoryl lipid A (MPL) developed by GSK. In certain embodiments the adjuvant is AS03, an oil-in water emulsion combination adjuvant developed by GSK. Non-limiting embodiments of adjuvants are described in the NIH 2018 Strategic Plan for Research on Vaccine Adjuvants, www.niaid.nih.gov / sites / default / files / NIAIDStrategicPlanVaccineAdjuvants2018.pdf, which is incorporated by reference herein for all purposes.

[0099] In certain embodiments the compositions described herein can be used to elicit an immune response including autologous and / or heterologous neutralizing antibodies against coronavirus. Various assays that analyze whether an immunogenic composition induces an immune response, and the type of antibodies induced are know n in the art and are also described herein.Methods for Inducing an Immune Response

[0100] Aspects of the invention include method for inducing an immune response to a coronavirus in a subject, the method comprising administering to the subject and effective amount of the recombinant fusion protein, the immunogen display, the nucleic acids, or theimmunogenic compositions describe herein, or combinations thereof, in an amount sufficient to induce an immune response in a subject.

[0101] The immune response against the compositions of the invention can be generated by one or more inoculations of a subject with an immunogenic composition of the invention. A first inoculation can be termed a “primary inoculation” and subsequent immunizations can be termed “booster inoculations”. Booster inoculations can enhance the immune response, and immunization regimens including at least one booster inoculation can be used. Inoculations can also occur as escalating dose regimens where increasing amounts of the vaccine are given as a series of injections over a small number of days (Tam, Hok Hei, et al. "Sustained antigen availability during germinal center initiation enhances antibody responses to vaccination." Proceedings of the National Academy of Sciences 113.43 (2016): E6639- E6648). Any composition of the invention may be used for a primary or booster immunization. The adequacy of the vaccination parameters chosen, e.g., formulation, dose, regimen and the like, can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program. Alternatively. T cell populations can by monitored by conventional methods. The clinical condition of a subject can be monitored for the desired effect, e g., limiting SARS-CoV-2 infection, improvement in disease state (e.g., reduction in viral load), etc. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response. Thus, for example, the dose of the polypeptide, immunogen display, or composition, and / or adjuvant, can be increased or the route of administration can be changed.

[0102] In some embodiments, the immunogens described herein can be administered to engage naive or pre-existing memory B cells. For example, in instances where there can be serum antibodies reactive against stem helix and / or fusion peptide sequences, but the levels of these antibodies are low (i.e., there is some antibody), the immunogens described herein can be used to boost the antibody levels.

[0103] In some embodiments, one or more of the following effects can result from the administration of “sufficient amount,” or “effective amount” of any of the immune inducing compositions described herein: (i) the reduction or amelioration of the severity of a viral infection and / or a symptom associated therewith; (ii) the reduction in the duration of a viral infection and / or a symptom associated therewith; (iii) the regression of a viral infection and / or a symptom associated therewith; (iv) the reduction of the titer of a virus; (v) the reduction in organ reduced function or failure associated with a viral infection; (vi) thereduction in hospitalization of a subject; (vii) the reduction in hospitalization length; (viii) the increase in the survival of a subject; (ix) the elimination of a virus infection; (x) the inhibition of the progression of a viral infection and / or a symptom associated therewith; (xi) the prevention of the spread of a virus from a cell, tissue or subject to another cell, tissue or subject; and / or (xii) the enhancement or improvement the therapeutic effect of another therapy.

[0104] In embodiments, a therapeutically effective amount can comprise less than about 0.1 mg / kg, about 0.1 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg. about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 135 mg / kg, about 150 mg / kg, about 175 mg / kg, about 200 mg / kg, about 225 mg / kg, about 250 mg / kg, about 275 mg / kg, about 300 mg / kg, about 325 mg / kg, about 350 mg / kg, about 375 mg / kg, about 400 mg / kg, about 425 mg / kg, about 450 mg / kg, about 475 mg / kg, about 500 mg / kg, about 525 mg / kg. about 550 mg / kg, about 575 mg / kg, about 600 mg / kg, about 625 mg / kg, about 650 mg / kg. about 675 mg / kg, about 700 mg / kg. about 725 mg / kg, about 750 mg / kg, about 775 mg / kg, about 800 mg / kg, about 825 mg / kg, about 850 mg / kg, about 875 mg / kg, about 900 mg / kg, about 1.0 g / kg, about 1.5 g / kg, about 2.0 g / kg, about 2.5 g / kg, about 5 g / kg, about 10 g / kg, about 25 g / kg, about 50 g / kg, or more than 50 g / kg of compound per body weight of a subject.

[0105] In embodiments, the therapeutically effective amount comprises less than about 0.1 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg. about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, about 135 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg. about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 1.0 g, about 1.5 g, about 2.0 g, about 2.5 g, about 5 g, about 10 g, about 25 g, about 50 g, or more than 50 g.

[0106] Aspects of the invention can comprise administering to a subject an immunogenic composition comprising an epitope from an RBD region of a human coronavirus spike protein.

[0107] In embodiments, administering can refer to providing a therapeutically effective amount of the composition to a subject. The formulation or pharmaceutical compound can be administered alone, but can be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. In some embodiments, an immune response can be stimulated in an individual by administering to the individual the immunogenic composition described herein or the amino acid epitope grafted into the scaffold protein described herein. In embodiments, the method is for eliciting an immune response to a fusion peptide epitope and / or an S2 stem helix of human coronavirus spike protein.

[0108] Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.

[0109] In embodiments, the compound can be administered alone, or can be administered as a pharmaceutical composition together with other compounds, excipients, carriers, diluents, fillers, binders, or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.

[0110] Embodiments can be administered to a subject in one or more doses. The dose level can vary as a function of the specific composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by a variety of means. For example, dosages can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be employed to help identify optimal dosage ranges. The precise dose to be employed can also depend on the route of administration and can be decided according to the judgment of the practitioner and each patient's circumstances.

[0111] In some embodiments, multiple doses of the composition can be administered. The frequency of administration and the duration of administration of the composition can vary depending on any of a variety' of factors, e.g., subject response, severity of thesymptoms, and the like. For example, in an embodiment, the pharmaceutical composition can be administered once per month, twice per month, three times per month, every’ other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily7(ad), twice a day7(qid), three times a day (tid), or four times a day. In an embodiment, the pharmaceutical composition can be administered 1 to 4 times a day over a period of time, such as 1 to 10-day time period, or longer than a 10-day period of time.

[0112] In embodiments, the composition can be administered in combination with one or more additional active agents. For example, a first agent (e.g., a prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour. 2 hours, 4 hours. 6 hours, 12 hours, 24 hours, 48 hours, 72 hours. 96 hours. 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second agent (e.g.. a prophylactic or therapeutic agent) to a subject with a disease or disorder or a symptom thereof.EXAMPLES

[0113] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.EXAMPLE 1: Generation of nanoparticles that display stem helix epitopes

[0114] For the stem helix, the structures of broadly neutralizing antibodies CC40.8 and S2P6 were used to determine regions of the stem helix to display on the nanoparticle (3, 5). CC40.8 has a long groove in its paratope that contacts the N-terminus and C-terminus of the stem helix (FIG. 4). S2P6 has a small pocket into which a shorter fragment of stem helix peptide inserts (FIG. 4). Based on these structures, a 22 amino acid stem helix and a 14 amino acid stem helix sequence were designed, and each sequence was genetically fused to the N-terminus of mI3 nanoparticles (FIG. 5). Negative stain electron microscopy (NSEM) of the stem-helix nanoparticles showed well-formed nanoparticles (FIG. 6). In biolayer interferometry experiments, the stem helix nanoparticles bound to all 3 stem helix neutralizing antibodies tested, with the 22 amino acid peptide binding to a higher magnitude to CC40.8 than the 14 amino acid peptide (FIG. 7).EXAMPLE 2: Nanoparticles that display stem helix epitopes stimulate antibodies

[0115] To assess whether either version of the stem helix nanoparticle (22 or 14 amino acid version) could initiate stem helix antibody responses, the reactivity of the inferred unmutated common ancestor (UCA) and the inferred intermediate antibodies of the DH1057 antibody clone were evaluated (FIG. 8A). Both stem helix nanoparticles bound to the inferred precursor of DH1057 and maintained binding throughout the lineage (FIG. 8B). Plasma IgG binding responses were assessed in samples from humans who were vaccinated against SARS-CoV-2, infected with SARS-COV-2, or both. Uninfected / unvaccinated individuals were assessed as negative controls. In these studies, there were few people that had binding to the stem helix nanoparticles that was specific to the stem helix peptide (FIG. 9). Most binding IgG responses appeared in vaccinated individuals (FIG. 9). Thus, in someembodiments, vaccinated people could be a population to target for boosting with the stem helix nanoparticle or for engaging naive or pre-existing memory B cells.EXAMPLE 3: Nanoparticles that display fusion protein epitopes

[0116] A similar approach was taken for designing fusion peptide nanoparticles. The 26 amino acids of the fusion peptide were fused to a glycine-serine-glycine linker and then fused to the fourth amino acid of H. pylori ferritin (FIG. 10A and 10B). Fusion peptide nanoparticles were expressed by 293F cells. Analytical size exclusion (FIG. 11A) and negative stain electron microscopy (FIG. 11B-C) showed well-formed nanoparticles.Biolayer interferometry experiments, showed the fusion peptide nanoparticle was antigenic for multiple neutralizing SARS-CoV-2 fusion peptide antibodies (FIG. 12A, 12B and 12C). Since antibodies evolve over time to bind with higher affinity to their corresponding antigen, we tested binding of the fusion peptide nanoparticle to DH1294 antibodies at various stages of its evolutionary pathway. The fusion peptide nanoparticle bound to members of the DH1294 phylogeny that had undergone different levels of evolution (FIG. 12B).

[0117] To investigate whether SARS-CoV-2 vaccinated or infected humans had antibodies capable of binding the fusion peptide displayed on the nanoparticle surface, plasma IgG reactivity' was assessed by ELISA. Binding by infected or infected and vaccinated individuals to the SARS-CoV-2 fusion peptide nanoparticle was observed (FIG.13A and 13B).

[0118] Immunogenicity of the fusion peptide nanoparticle was assessed in BALB / c mice. Mice were immunized with bolus injections of 10 mcg or with escalating doses that totaled 10 mcg. Binding IgG responses were high in both groups for the fusion peptide immunogen. The SARS-CoV-2 spike binding IgG tended to be higher for the escalating dose group. The breadth of the binding IgG was notable, with binding IgG detectable against Sarbecoviruses, Merbecoviruses, and Embecoviruses (FIG. 14A, 14B, 14C and 14D).EXAMPLE 4: Combinations of nanoparticles

[0119] Because a vaccine where Fusion peptide and stem helix nanoparticles are combined was envisioned, a group of BALB / c mice was immunized three times with a mixture of stem helix nanoparticles and fusion peptide nanoparticles. Serum was collected over time and binding IgG was measured by ELISA. The mice generated broad binding serum IgG responses against recombinant spikes from Sarbecoviruses, Merbecoviruses, and Embecoviruses genera of betacoronaviruses (FIG. 15A, 15B and 15C). Serum IgG bindingwas highest for betacoronaviruses such as OC43 compared to alphacoronaviruses (229E and NL63) (FIG. 15A, 15B and 15C-C). A single immunization was sufficient to detect binding antibody, but a subsequent boost six weeks later moderately increased binding magnitude (FIG. 15A, 15B and 15C)REFERENCES

[0120] 1. Piccoli L, Park YJ. Tortorici MA, Czudnochowski N. Walls AC, BeltramelloM, et al. Mapping Neutralizing and Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-Guided High-Resolution Serology. Cell. 2020; 183(4): 1024-42 e21.

[0121] 2. Dacon C, Tucker C, Peng L. Lee CD, Lin TH, Yuan M, et al. Broadly neutralizing antibodies target the coronavirus fusion peptide. Science. 2022;377(6607):728- 35.

[0122] 3. Zhou P, Song G, Liu H, Yuan M, He WT, Beutler N, et al. Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease. Immunity. 2023;56(3):669-86 e7.

[0123] 4. Low JS, Jerak J, Tortorici MA, McCallum M, Pinto D, Cassotta A, et al. ACE2- binding exposes the SARS-CoV-2 fusion peptide to broadly neutralizing coronavirus antibodies. Science. 2022;377(6607):735-42.

[0124] 5. Pinto D, Sauer MM. Czudnochowski N. Low JS, Tortorici MA, Housley MP, et al. Broad betacoronavirus neutralization by a stem helix-specific human antibody. Science.2021;373(6559):1109-16.EQUIVALENTS

[0125] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

CLAIMSWhat is claimed:

1. A recombinant protein configured for incorporation into an immunogen display, comprising a coronavirus stem helix sequence or fusion peptide sequence.

2. The recombinant protein of claim 1, wherein the stem helix or fusion peptide sequence comprises an epitope for a broadly -neutralizing antibody.

3. The recombinant protein of claim 1 or 2, wherein the stem helix sequence comprises SEQ ID NO: 7 or 8, or a sequence 90, 92, 94, 96 or 98% identical thereto.

4. The recombinant protein of claim 3, comprising SEQ ID NO: 4 or 5, or a sequence 90, 92, 94, 96 or 98% identical thereto.

5. The recombinant protein of claim 1 or 2, wherein the fusion peptide sequence comprises SEQ ID NO: 9, or a sequence 90, 92, 94, 96 or 98% identical thereto.

6. The recombinant protein of claim 5, comprising SEQ ID NO: 6, or a sequence 90, 92, 94. 96 or 98% identical thereto.

7. The recombinant protein of claim 1 or 2, wherein the immunogen display comprises a protein scaffold, nanoparticle, or virus-like particle.

8. The recombinant protein of claims 1 or 2, wherein the immunogen display comprises a self-assembling nanoparticle.

9. The recombinant protein of claims lor 2, wherein the recombinant protein is configured to be added to a surface of an existing immunogen display (nanoparticle).

10. The recombinant protein of claims 1 or 2, wherein the recombinant protein is incorporated into the immunogen display during assembly of the immunogen display.

11. A recombinant fusion protein sequence comprising in order, a coronavirus spike protein stem helix or fusion protein region, a peptide linker and a self-assembling protein, wherein non-limiting embodiments of sequences are shown in Table 2.

12. The recombinant fusion protein of claim 11, wherein the self-assembling protein comprises a ferritin.

13. The recombinant fusion protein of claim 1 1, wherein the self-assembling protein comprises an encapsulin that can form an encapsulin nanoparticle.

14. An immunogen display (nanoparticle), comprising the recombinant protein of any one of claims 3-4 (stem helix) and / or the recombinant protein sequence of any one of claims 5-6 (fusion peptide).

15. A nucleic acid encoding the recombinant protein of any one of claims 1-10 or the recombinant fusion protein of any one of claims 1 1-13.

16. A host ceil comprising a nucleic acid molecule encoding the recombinant protein of any one of claims 1-10 or the recombinant fusion protein of any one of claims 11-13.

17. An immunogenic composition for stimulating an immune response to a coronavirus, comprising the recombinant protein of any one of claims 1-10, the recombinant fusion protein of any one of claims 11-13, the immunogen display of claim 14, or the nucleic acid of claim 15.

18. The immunogenetic composition of claim 17, wherein the coronavirus comprises an Alphacoronavirus, a Betacoronavirus, or a combination thereof.

19. The immunogenetic composition of claim 18, wherein the Betacoronavirus comprises an Embecovirus, a Sarbecovirus, a Merbecovirus, or a combination thereof.

20. A method for inducing an immune response to a coronavirus in a subject, the method comprising administering to the subject an effective amount of the recombinant protein of any one of claims 1-10, the recombinant fusion protein of any one of claims 11-13, theimmunogen display of claim 14, or the nucleic acid of claim 15, or combinations thereof, in an amount sufficient to induce an immune response.

21. The method of claim 20, wherein the effective amount is administered to a subject that was previously vaccinated or infected.

22. The method of claim 21. wherein the effective amount is administered as a boost.

23. The method of claim 21, wherein the effective amount is administered to engage naive or pre-existing memory' B cells.

24. The method of claim 21, additionally comprising administering to the subject an effective amount of a receptor binding domain (RBD) region of a coronavirus.

25. An immunogen display, comprising a stem helix sequence, a fusion peptide sequence, or a stem helix sequence and a fusion peptide sequence from a coronavirus.

26. The immunogen display of claim 25, comprising a ferritin-based nanoparticle or a mi3 nanoparticle.

Images