Coronavirus vaccine compositions

Abstract

Described herein are compositions, e.g., vaccine compositions, comprising core envelope peptides and method of using such compositions in immunization and stimulation of immune responses.

Description

Attorney Docket No: 077734-000100WOPT CORONAVIRUS VACCINE COMPOSITIONS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 654,145 filed May 31, 2024, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 2, 2025, is named 077734-000100WOPT_SL.xml and is 21,784 bytes in size. TECHNICAL FIELD

[0003] The technology described herein relates to vaccines and methods of immunization and treatment relating to the vaccine compositions described herein. BACKGROUND

[0004] To date, the Spike (S) protein is the primary antigen in all currently licensed SARS-CoV-2 vaccines, but the increasing emergence of variants with spike mutations that confer resistance to vaccines, antibody therapeutics, and natural immunity represents a significant global public health threat. There is a need for vaccine compositions that provide broad protective effects against numerous coronaviruses, and which are not readily evaded by mutation of viral proteins. SUMMARY

[0005] The SARS-CoV-2 genome encodes 4 major structural proteins including the Spike (S), Nucleocapsid (N), Membrane (M), and Envelope (E) protein. The E protein is the smallest, sparsest, and least understood protein in the mature SARS-CoV-2 virion. The E protein of all Coronaviruses contains three distinct domains: a 7-12 amino acid hydrophilic N-terminus, a 25 amino acid hydrophobic transmembrane (TMB) domain, and a 38-43 amino acid hydrophilic carboxyl terminus–an overall structure that has led to the classification of the E protein as a viroporin As demonstrated herein, the inventors have surprisingly found that peptides derived from the transmembrane domain of the E protein can induce a robust and broad immune response that confers protection against many coronaviruses.

[0006] In one aspect of any of the embodiments, described herein is a a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide comprises the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto. 1 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0007] In one aspect of any of the embodiments, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto.

[0008] In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise SEQ ID NO: 16 or a sequence with at least 80% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise SEQ ID NO: 17 or a sequence with at least 80% sequence identity thereto.

[0009] In some embodiments of any of the aspects, the composition further comprises an adjuvant. In some embodiments of any of the aspects, the adjuvant is conjugated to a cysteine residue of the isolated core envelope peptide.

[0010] In some embodiments of any of the aspects, the adjuvant comprises mannose. In some embodiments of any of the aspects, the mannose is alpha-D-mannose.

[0011] In some embodiments of any of the aspects, the mannose is conjugated to a lysine residue of the isolated core envelope peptide. In some embodiments of any of the aspects, the mannose is conjugated to the isolated core envelope peptide via a methyl carboxylic acid linkage.

[0012] In some embodiments of any of the aspects, the adjuvant comprises KLH, BSA, or OVA. In some embodiments of any of the aspects, the adjuvant comprises KLH. In some embodiments of any of the aspects, the KLH, BSA, or OVA is conjugated to a cysteine residue of the isolated core envelope peptide.

[0013] In some embodiments of any of the aspects, the composition further comprises a pharmaceutically acceptable carrier.

[0014] In one aspect of any of the embodiments, described herein is a method of immunizing a subject, the method comprising administering to the subject a composition as described herein. In one aspect of any of the embodiments, described herein is a method of stimulating an immune response of a subject, the method comprising administering to the subject a composition described herein.

[0015] In some embodiments of any of the aspects, the administration is by injection, subcutaneous injection, or mucosal administration. In some embodiments of any of the aspects, the injection is an intramuscular injection. In some embodiments of any of the aspects, the composition is administered twice. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 2 months apart. In some embodiments of any of the aspects, the 2 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least two weeks apart and no more than four weeks apart. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Fig.1 depicts a graph demonstrating that sera from mice immunized with two doses of KLH- E vaccine exhibited stronger inhibitory activities than those immunized with one dose of KLH-E vaccine. Indeed, the sera's inhibitory activities from different animal groups correlated with the sera's titers against the KLH-E vaccine and the E peptide.

[0017] Fig.2. Amino acid sequence alignment of human Coronavirus Envelope (E) proteins. The amino acids of the proteins are labelled corresponding to their relative location: virion surface (gray), transmembrane (yellow), intravirion (white). Amino acid sequences are listed from left to right corresponding to the N-terminus and C-terminus, respectively. Figure discloses SEQ ID NOS 18-24, respectively, in order of appearance.

[0018] Fig.3A-3C. Serum IgG antibody titers of individual mice immunized with KLH-E or aluminum control measured against KLH-E conjugate, KLH, and E-peptide. Serum from individual mice immunized with the KLH-E conjugate (squares) or aluminum control (circles) measured for IgG antibodies against the vaccine immunogen KLH-E (Fig.3A) and each subunit KLH (Fig.3B) and E- peptide (Fig.3C). Mice were given one or two doses of vaccine in 30 day intervals. Each symbol represents the reciprocal antibody titer of individual animals in each vaccine group, with each data point representing the average antibody titer enumerated in duplicate, in two independent assays. Mice with no detectable IgG to the KLH-E, KLH, or E-peptide were assigned a titer of one half lower limit of the assay (i.e. a titer of <1:8 was assigned as 4). Numbers above each group represent the geometric mean IgG antibody titer for all animals in the respective vaccine group. Differences between vaccine groups were assessed using a two-tailed students t test of the log-transformed reciprocal titers. Differences with a probability of <0.5 (two-tailed) were considered significant.

[0019] Fig.4A-4C. T-cell responses of KLH-E vaccinated mice measured against KLH-E, KLH, and Epeptide. The number of spleenocytes tested for each antigen was fixed at 750,000 cells. Concentrations of antigens for each T-cell activation was 2µg (KLH-E), 100µg (KLH), and 1µg (SARS-CoV-2 E- peptide). Each bar represents a representative animal from each vaccine group at different time points. Spleenocytes from each animal were measured against each antigen in duplicate and the number of IL-2 secreting foci were enumerated in duplicate by two independent observers for accuracy. Error bars represent + / - standard deviation. 3 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0020] Fig.5. Anti-E IgG ELISA of pooled sera from mice immunized with KLH-E or aluminum control vaccines measured against hybrid alphavirus SARS-CoV-2 pseudovirions (HA-CoV-2). Pooled serum from groups of mice immunized with two doses of KLH-E (left, n=8 mice) or Aluminum control (right, n=5 mice) at 30 days post-immunization were measured for anti-E IgG antibodies against hybrid alphavirus HA-CoV-2 pseudovirions. The HA-CoV-2 pseudovirions all expressed the four SARS-CoV-2 structural proteins (Spike (S), Matrix (M), Nucleocapsid (N), Envelope (E)).

[0021] Fig.6A-6B. Anti-E Antibody and T-cell reactivity of KLH-E vaccinated mice measured against HCoV-229E infected or uninfected human MRC5 cell lysates. Anti-E IgG ELISA of pooled sera of mice immunized with KLH-E or aluminum control vaccines measured against HCoV-229E infected, or uninfected human MRC5 cell lysates (Fig.6A). Pooled serum from groups of mice immunized with the aluminum control vaccine were combined in equal parts using serum from each vaccine group and measured for IgG antibodies to the native E protein expressed in uninfected (top) or HCoV-229E infected MRC5 cell lysates (bottom). The serum pools were from mice immunized with the control aluminum vaccine collected at 30 days postdose 1 (n= 5 mice), 60 days post-dose 1 (n=4 mice), or 30 days post-dose 2 (n=5 mice). The serum pools were from mice immunized with the KLH-E vaccine collected at 30 days post-dose 1 (n= 8 mice), 60 days post-dose 1 (n=8 mice), or 30 days post-dose 2 (n=8 mice). (Fig.6B) Antigen specific T-cell responses of KLH-E vaccinated mice measured against HCoV-229E infected, or uninfected human MRC5 cell lysates. The number of spleenocytes tested for each antigen was fixed at 750,000 cells. Concentrations of antigens for each T-cell activation was 200µg of uninfected or HCoV- 229E infected human MRC5 cell lysate. As an additional control, spleenocytes from both KLH-E and Aluminum vaccinated mice were stimulated with Concanavalin A to ensure that mouse T-cells retained functional activity after cryopreservation, irrespective of vaccine dose or formulation.

[0022] Figs.7A-7C. Neutralization activity induced by KLH-E Vaccination. (Fig.7A) Neutralization of HA-CoV-2 Pseudovirions expressing Spike proteins from SARS-CoV-2 variants of concern. The horizontal axis lists the pooled serum, monoclonal antibody, or virus and cell only compared to the % infection on the vertical axis. The figure depicts the quantification of neutralizing antibodies in pooled sera at 30 days post-immunization from mice vaccinated with two doses of KLH-E or aluminum control vaccine. Each bar represents an individual HA-CoV-2 pseudovirion expressing a S protein sequence from a SARS-CoV-2 variant of concern (Beta, Delta, Omicron, XBB). The % infection was defined as the change (∆) in relative luciferase units of pseudovirions incubated in test serum or control monoclonal antibody (mAb) compared to the luciferase signal of pseudovirions and cells only. The background luciferase signal of uninfected cells was also measured but was negligible (data not shown). The control antibody (27VB1) used in the assay is a broadly neutralizing rabbit anti-RBD S antibody (Virongy 4 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT Biosciences). Each pooled serum was tested at a 1:2 dilution, and the control mAb was tested at a concentration of 5μg. All samples were tested in independent replicates and error bars represent the + / - standard deviation. (Fig.7B) Neutralization of SARS-CoV-2-ΔORF3-E. Virus samples were incubated with different sera samples and assayed for their infectivity by infection of Vero cells, following the experimental procedures described previously and in Materials and Methods. (Fig.7C) Neutralization of human Coronavirus 229E (HCoV-229E). Virus samples were incubated with different sera samples and assayed for their infectivity by infection of human foreskin fibroblasts, following the experimental procedures described previously and in Materials and Methods of Example 2. In Fig.7B and Fig.7C, the horizontal axis lists the pooled serum from mice in each vaccine group against the vertical axis which lists the level of viral infection measured (%), compared to that of viral infection with PBS in the absence of sera. Each pooled serum was tested at a 1:2 dilution by combining diluted serum and virus as described in Materials and Methods of Example 2. Experiments were conducted in duplicates and repeated three times.

[0023] Fig.8A-8B. In vivo challenge of SCID mice with HCoV-229E after passive immunization with pooled KLH-E sera (Fig.8A) and in-vivo challenge of BALB / c mice with HCoV-229E after KLHE vaccination (Fig.8B). In (Fig.8A), groups of SCID mice were first intravenously administered with PBS, sera samples from aluminum-DMSO injected animals, or sera samples from KLH-E immunized animals and intranasally challenged with HCoV-229E (1x105 pfu / mice) at 24 hours postadministration. Animals were sacrificed and lungs were collected at 10 days post-challenge. In (Fig.8B), groups of BALB / c mice were first immunized with two doses of PBS, the aluminum-DMSO control vaccine, or the KLH-E vaccines for two doses at 30 days intervals, and intranasally challenged with HCoV-229E (1x105 pfu / mice) at 30 days post dose two treatment. At 7 days post-challenge, mice were sacrificed, and the lungs were collected. Viral load in the lung tissues were determined as described in Materials and Methods of Example 2. Experiments for assaying viral loads were conducted in duplicated and repeated three times. DETAILED DESCRIPTION

[0024] It is demonstrated herein that the transmembrane domain of the coronavirus envelope protein, surprisingly, can induce a protective immune response. This is unexpected as the transmembrane domain would typically be expected to be inaccessible when present in a viral envelope. The naturally-occuring envelope protein does not have the activity of inducing such an immune response, but when the isolated core envelope proteins described herein are used to induce an immune response, that immune response can then recognize the naturally-occuring envelope protein. Additionally, this domain can induce a broadly (e.g., pan-coronavirus) immune response, which cannot be accomplished with the spike proteins 5 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT used in current vaccines. Further surprisingly, this effect is observed even when the sequence identity of the transmembrane domain is not highly conserved.

[0025] The envelope protein transmembrane domain identified herein as an antigen is referred to herein as the “core envelope peptide.” As used herein, “envelope protein” or “E protein” refers to one of the four major structural proteins of a coronavirus viron. The envelope protein is a transmembrane protein with a short hydrophilic N-terminal region, a transmembrane domain, and a C-terminal region. As used herein “core envelope peptide” refers to a peptide having the sequence of one or more of SEQ ID NOs: 1-15 provided herein.

[0026] Compositions described herein can relate to an isolated core envelope peptide. As used herein, “isolated” means artificially produced and / or removed from a naturally occurring environment. As used herein with respect to proteins or peptides, the term "isolated" refers to a protein or peptide that has been isolated from its natural environment or artificially produced (e.g., by chemical synthesis, by recombinant DNA technology, etc.). In some embodiments of any of the aspects, an isolated peptide or polypeptide is not embedded in a membrane, e.g., a lipid bilayer. Isolated peptides not embedded in or present in a membrane can induce a protective immune response (e.g., immunize a subject), a property not possessed by peptides of the same sequence which are embedded in a membrane.

[0027] Provided herein are the relevant transmembrane domain sequences from a number of coronaviruses, and a consensus core envelope peptide sequence. Accordingly, in one aspect of any of the embodiments, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto.

[0028] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 85% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 1 or a sequence with at least 85% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 1 or a sequence with at least 85% sequence identity thereto. 6 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0029] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 1 or a sequence with at least 90% sequence identity thereto.

[0030] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 95% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 1 or a sequence with at least 95% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 1 or a sequence with at least 95% sequence identity thereto.

[0031] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 98% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 1 or a sequence with at least 98% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 1 or a sequence with at least 98% sequence identity thereto.

[0032] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 1. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 1.

[0033] Accordingly, in one aspect of any of the embodiments, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L;or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16 is A or T; and X17 is I, F, or Lor a sequence with at least 80% sequence identity thereto. In one embodiment of any of the aspects, the 7 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT composition comprises an isolated core envelope peptide consisting essentially of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13is V, F, or L; X14is T, C, V, or A; X15is L, M, V, or I; X16is A or T; and X17is I, F, or L or a sequence with at least 80% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2 is L,I, or F; X3 is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8is V, A, L, or I; X9is V, I, L, or F; X10is F, T, C, or I; X11is L, T, or F; X12is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16 is A or T; and X17 is I, F, or Lor a sequence with at least 80% sequence identity thereto.

[0034] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2 is L,I, or F; X3 is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6is A, V, I, S, C, or V; X7is F, C, M, or L; X8is V, A, L, or I; X9is V, I, L, or F; X10is F, T, C, or I; X11is L, T, or F; X12is L, I, or V; X13is V, F, or L; X14is T, C, V, or A; X15is L, M, V, or I; X16 is A or T; and X17 is I, F, or L or a sequence with at least 85% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L; X6is A, V, I, S, C, or V; X7is F, C, M, or L; X8is V, A, L, or I; X9is V, I, L, or F; X10is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16is A or T; and X17is I, F, or L or a sequence with at least 85% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1is V,F,I, or L; X2 is L,I, or F; X3 is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11is L, T, or F; X12is L, I, or V; X13is V, F, or L; X14is T, C, V, or A; X15is L, M, V, or I; X16is A or T; and X17 is I, F, or L or a sequence with at least 85% sequence identity thereto.

[0035] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; 8 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT wherein X1is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16is A or T; and X17is I, F, or L or a sequence with at least 90% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2 is L,I, or F; X3 is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11is L, T, or F; X12is L, I, or V; X13is V, F, or L; X14is T, C, V, or A; X15is L, M, V, or I; X16is A or T; and X17is I, F, or L or a sequence with at least 90% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L; X6is A, V, I, S, C, or V; X7is F, C, M, or L; X8is V, A, L, or I; X9is V, I, L, or F; X10is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16 is A or T; and X17is I, F, or L or a sequence with at least 90% sequence identity thereto.

[0036] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16is A or T; and X17is I, F, or L or a sequence with at least 95% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2 is L,I, or F; X3 is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11is L, T, or F; X12is L, I, or V; X13is V, F, or L; X14is T, C, V, or A; X15is L, M, V, or I; X16 is A or T; and X17 is I, F, or L or a sequence with at least 95% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L; X6is A, 9 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT V, I, S, C, or V; X7is F, C, M, or L; X8is V, A, L, or I; X9is V, I, L, or F; X10is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16 is A or T; and X17 is I, F, or L or a sequence with at least 95% sequence identity thereto.

[0037] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16is A or T; and X17is I, F, or L or a sequence with at least 98% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2 is L,I, or F; X3 is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11is L, T, or F; X12is L, I, or V; X13is V, F, or L; X14is T, C, V, or A; X15is L, M, V, or I; X16is A or T; and X17is I, F, or L or a sequence with at least 98% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L; X6is A, V, I, S, C, or V; X7is F, C, M, or L; X8is V, A, L, or I; X9is V, I, L, or F; X10is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16 is A or T; and X17 is I, F, or L or a sequence with at least 98% sequence identity thereto.

[0038] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide comprising the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1is V,F,I, or L; X2is L,I, or F; X3is L,F,I, or W; X4is F, T, V, L, or C; X5is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8 is V, A, L, or I; X9 is V, I, L, or F; X10 is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16is A or T; and X17is I, F, or L. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2 is L,I, or F; X3 is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6 is A, V, I, S, C, or V; X7 is F, C, M, or L; X8is V, A, L, or I; X9is V, I, L, or F; X10is F, T, C, or I; X11is L, T, or F; X12is L, I, or V; 10 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT X13is V, F, or L; X14is T, C, V, or A; X15is L, M, V, or I; X16is A or T; and X17is I, F, or L. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18; wherein X1 is V,F,I, or L; X2 is L,I, or F; X3 is L,F,I, or W; X4 is F, T, V, L, or C; X5 is L, V, A, or L; X6 is A, V, I, S, C, or V; X7is F, C, M, or L; X8is V, A, L, or I; X9is V, I, L, or F; X10is F, T, C, or I; X11 is L, T, or F; X12 is L, I, or V; X13 is V, F, or L; X14 is T, C, V, or A; X15 is L, M, V, or I; X16 is A or T; and X17 is I, F, or L.

[0039] In one aspect of any of the embodiments, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto.

[0040] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto.

[0041] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 85% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 85% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 85% sequence identity thereto.

[0042] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 90% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 90% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 90% sequence identity thereto.

[0043] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with 11 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT at least 95% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 95% sequence identity thereto. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 95% sequence identity thereto.

[0044] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of one of SEQ ID NOs: 2-15. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of one of SEQ ID NOs: 2-15.

[0045] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 2. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 2. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 2.

[0046] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 3. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 3. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 3.

[0047] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 4. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 4. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 4.

[0048] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 5. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 5. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 5.

[0049] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 6. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the 12 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT sequence of SEQ ID NO: 6. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 6.

[0050] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 7. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 7. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 7.

[0051] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 8. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 8. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 8.

[0052] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 9. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 9. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 9.

[0053] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 10. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 10. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 10.

[0054] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 11. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 11. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 11.

[0055] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 12. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 12. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 12. 13 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0056] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 13. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 13. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 13.

[0057] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 14. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 14. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 14.

[0058] In one embodiment of any of the aspects, described herein is a composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 15. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting essentially of the sequence of SEQ ID NO: 15. In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide consisting of the sequence of SEQ ID NO: 15.

[0059] Table 1 SEQ Description Sequence ID NO 1 Consensus Core X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18Envelope Peptide X1is V,F,I, or L X2is L,I, or F X3is L,F,I, or W X4is F, T, V, L, or C X5is L, V, A, or L X6is A, V, I, S, C, or V X7is F, C, M, or L X8is V, A, L, or I X9is V, I, L, or F X10is F, T, C, or I X11is L, T, or F X12is L, I, or V X13is V, F, or L 14 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT X14is T, C, V, or A X15is L, M, V, or I X16is A or T X17is I, F, or L 2 TLIVNSVLLFLAFVVFLLVTLAILTALRLC 3 GTLIVNSVLLFLAFVVFLLVTLAILTA 4 LIVNSVLLFLAFVVFLLVTLAIL 5 GTLIVNSVLLFLAFVVFLLVTLAIL 6 LIVNSVLLFLAFVVFLLVTLAILTA 7 TLIVNSVLLFLAFVVFLLVTLAILTALR 8 LIVNSVLLFLAFVVFLLVTLAIL 9 SARS CoV2 Core VNSVLLFLAFVVFLLVTLAIL Envelope Peptide 10 SARS CoV1 Core VLLFLAFVVFLLVTLAILTAL Envelope Peptide 11 MERS Core Envelope FIFTVVCAITLLVCMAFLTAT Peptide 12 OC43 Core Envelope IFIVAICLLCTIVVVAFLATF Peptide 13 HKU1 Core Envelope FFLVLSCVIFLIFVVALLATI Peptide 14 NL63 Core Envelope ILWLLCMIFFFVLAMTFIKLI Peptide 15 229E Core Envelope LLWCVVLIVILLVCITIIKLI 15 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT Peptide 16 N-terminus of SARS MYSFVSEET CoV2 Envelope Protein 17 C-terminus of SARS LCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV CoV2 Envelope Protein

[0060] In some embodiments of any of the aspects, the isolated core envelope peptides described herein are not naturally-occuring. In some embodiments of any of the aspects, the isolated core envelope peptides described herein are engineered. In some embodiments of any of the aspects, the isolated core envelope peptides described herein do not comprise envelope protein sequences other than the isolated core envelope peptide sequence.

[0061] The isolated core envelope peptides described herein do not comprise the N-terminus and / or C-terminus of the envelope protein. Accordingly, in some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 16 or a sequence with at least 80% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 17 or a sequence with at least 80% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of either of SEQ ID NO: 16 and 17, or a sequence with at least 80% sequence identity thereto.

[0062] In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 16 or a sequence with at least 85% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 17 or a sequence with at least 85% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of either of SEQ ID NO: 16 and 17, or a sequence with at least 85% sequence identity thereto.

[0063] In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 16 or a sequence with at least 90% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 17 or a sequence with at least 90% sequence identity thereto. In some 16 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of either of SEQ ID NO: 16 and 17, or a sequence with at least 90% sequence identity thereto.

[0064] In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 16 or a sequence with at least 95% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 17 or a sequence with at least 95% sequence identity thereto. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of either of SEQ ID NO: 16 and 17, or a sequence with at least 95% sequence identity thereto.

[0065] In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 16. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of SEQ ID NO: 17. In some embodiments of any of the aspects, the isolated core envelope peptide does not comprise the sequence of either of SEQ ID NO: 16 and 17.

[0066] In some embodiments of any of the aspects, the composition described herein further comprises an adjuvant. As used herein in the context of immunization, immune response and vaccination, the term “adjuvant” refers to any substance than when used in combination with a specific antigen that produces a more robust immune response than the antigen alone. When incorporated into a vaccine formulation, an adjuvant acts generally to accelerate, prolong, or enhance the quality of specific immune responses to the vaccine antigen(s).

[0067] Adjuvants typically promote the accumulation and / or activation of accessory cells or factors to enhance antigen-specific immune responses and thereby enhance the efficacy of vaccines, i.e., antigen- containing or encoding compositions used to induce protective immunity against the antigen.

[0068] Adjuvants, in general, include adjuvants that create a depot effect, immune-stimulating adjuvants, and adjuvants that create a depot effect and stimulate the immune system. An adjuvant that creates a depot effect is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen. This class of adjuvants includes but is not limited to alum (e.g., aluminum hydroxide, aluminum phosphate); emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion, oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.); and PROVAX™ (an oil-in-water emulsion containing a stabilizing detergent and a micelle- forming agent; IDEC Pharmaceuticals Corporation, San Diego, Calif.). 17 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0069] An immune-stimulating adjuvant is an adjuvant that causes activation of a cell of the immune system. It may, for instance, cause an immune cell to produce and secrete cytokines and interferons. This class of adjuvants includes but is not limited to saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.). This class of adjuvants also includes CpG DNA.

[0070] Adjuvants that create a depot effect and stimulate the immune system are those compounds which have both of the above-identified functions. This class of adjuvants includes but is not limited to ISCOMS (immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is an oil-in-water emulsion containing MPL and QS21: SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxypropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, an oil-in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

[0071] In one embodiment of any of the aspects, the composition comprises an isolated core envelope peptide as described herein and at least one adjuvant. In one embodiment of any of the aspects, the composition consists essentially of an isolated core envelope peptide as described herein and at least one adjuvant. In one embodiment of any of the aspects, the composition consists of an isolated core envelope peptide as described herein and at least one adjuvant.

[0072] In one embodiment of any of the aspects, the active ingredients of a composition described herein comprises an isolated core envelope peptide as described herein and at least one adjuvant. In one embodiment of any of the aspects, the active ingredients of a composition described herein consists essentially of an isolated core envelope peptide as described herein and at least one adjuvant. In one embodiment of any of the aspects, the active ingredients of a composition described herein consists of an isolated core envelope peptide as described herein and at least one adjuvant.

[0073] In some embodiments of any of the aspects, the isolated core envelope peptide and the adjuvant are in an admixture. In some embodiments of any of the aspects, the isolated core envelope 18 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT peptide and the adjuvant are in a single formulation. In some embodiments of any of the aspects, the isolated core envelope peptide and the adjuvant are provided in separate formulations of a single kit.

[0074] In some embodiments of any of the aspects, the adjuvant is conjugated to the isolated core envelope peptide. The term “conjugated” refers to the attachment of at least two entities to form one entity. The joining of the two entities can be direct (e.g., via covalent or non-covalent bonds) or indirect (e.g., via linkers etc.). Thus, conjugation can be by means of linkers, chemical modification, peptide linkers, chemical linkers, covalent or non-covalent bonds, or protein fusion or by any means known to one skilled in the art. The joining or conjugation can be permanent or reversible. In some embodiments of any of the aspects, the conjugation is by a covalent bond.

[0075] In some embodiments of any of the aspects, the adjuvant is conjugated to a cysteine residue of the isolated core envelope peptide. In some embodiments of any of the aspects, the adjuvant is conjugated to a lysine residue of the isolated core envelope peptide. In some embodiments of any of the aspects, the adjuvant is conjugated to a cysteine residue of the isolated core envelope peptide. In some embodiments of any of the aspects, the adjuvant is conjugated to a cysteine residue or lysine residue of the isolated core envelope peptide. In some embodiments of any of the aspects, the adjuvant is conjugated to the isolated core envelope peptide via a methyl carboxylic acid linkage.

[0076] In some embodiments of any of the aspects, the adjuvant comprises mannose. In some embodiments of any of the aspects, the mannose is alpha-D-mannose. In some embodiments of any of the aspects, the mannose is conjugated to a lysine residue of the isolated core envelope peptide. In some embodiments of any of the aspects, the mannose is conjugated to the isolated core envelope peptide via a methyl carboxylic acid linkage.

[0077] In some embodiments of any of the aspects, the adjuvant comprises Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), or Ovalbumin (OVA). The sequences of such proteins are known for a variety of species, e.g., seqences can be found in the NCBI database for KLH under GI Accessions 1859630622, 1859630623, 568786454, 568786453, 568786452, and 568786451; for BSA under Gene ID 280717; and for OVA under Gene ID 396058, e.g., as of May 31, 2024. These proteins are readily obtained from commercial sources.

[0078] In some embodiments of any of the aspects, the adjuvant comprises KLH.

[0079] In some embodiments of any of the aspects, the KLH, BSA, or OVA is conjugated to a cysteine residue of the isolated core envelope peptide. In some embodiments of any of the aspects, the KLH is conjugated to a cysteine residue of the isolated core envelope peptide.

[0080] In some embodiments, a peptide, can be modified, e.g. by addition of a moiety to one or more of the amino acids that together comprise the peptide. In some embodiments, a peptide as described 19 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT herein can comprise one or more moiety molecules, e.g.1 or more moiety molecules per polypeptide, 2 or more moiety molecules per polypeptide, 5 or more moiety molecules per polypeptide, 10 or more moiety molecules per polypeptide or more moiety molecules per polypeptide. In some embodiments, a peptide as described herein can comprise one more types of modifications and / or moieties, e.g.1 type of modification, 2 types of modifications, 3 types of modifications or more types of modifications. Non- limiting examples of modifications and / or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end-capping modifications; cyano groups; phosphorylation; albumin, and cyclization. In some embodiments, an end-capping modification can comprise acetylation at the N-terminus, N-terminal acylation, and N-terminal formylation. In some embodiments, an end-capping modification can comprise amidation at the C-terminus, introduction of C- terminal alcohol, aldehyde, ester, and thioester moieties. The half-life of a polypeptide can be increased by the addition of moieties, e.g. PEG, albumin, or other fusion partners (e.g. Fc fragment of an immunoglobin).

[0081] In some embodiments, a peptide can comprise one or more substitutions or modifications. In some embodiments, the substitutions and / or modifications can prevent or reduce proteolytic degradation and / or prolong half-life of the peptide in a subject. In some embodiments, a peptide can be modified by conjugating or fusing it to other polypeptide or polypeptide domains such as, by way of non-limiting example, transferrin (WO06096515A2), albumin (Yeh et al., 1992), growth hormone (US2003104578AA); cellulose (Levy and Shoseyov, 2002); and / or Fc fragments (Ashkenazi and Chamow, 1997). The references in the foregoing paragraph are incorporated by reference herein in their entireties.

[0082] In some embodiments of any of the aspects, the compositions of the disclosure--comprising a peptide as taught herein--exhibit markedly different characteristics / properties compared to their closest naturally occurring counterpart. That is, the compositions of the disclosure exhibit markedly different functional and / or structural characteristics / properties, as compared to their closest naturally occurring counterpart. For instance, the peptide of the disclosure are structurally different from an envelope protein as it naturally exists in a virus and / or membrane for at least the following reasons: said peptide can be isolated and purified, such that it is not found in the milieu of the virus and / or membrane, said peptide can be present at concentrations that do not occur for the wild-type envelope protein, said peptide can be associated with acceptable carriers and / or adjuvants that do not occur with the wild-type envelope protein, said peptide can be formulated to be shelf-stable and exist outside the viral / cellular environment, and said peptide can be combined with other polypeptides or drugs at concentrations that do not exist in nature. Further, the peptides of the disclosure are functionally different from an envelope proteinas it naturally 20 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT exists, for at least the following reasons: said peptide when applied in an isolated and purified form can lead to an inducation of immune response not observed in nature, said peptide can be formulated to be shelf-stable and able to exist outside the cellular environment, such that the peptide now has a new utility as a therapeutic capable of administration to a subject, wherein the envelope protein could not have such a utility in its natural state, as the envelope protein would be embedded in the membrane and / or inaccessible to the immune system for the purposes of raising an effective immune response without the intervention of the hand of man to formulate the peptide into a state described herein and impart this new utility that has the aforementioned functional characteristics not possessed by the envelope proteins in its natural state of existence.

[0083] In one aspect of any of the embodiments, described herein is a nucleic acid encoding an isolated core envelope peptide and / or composition as described herein. In one aspect of any of the embodiments, described herein is a vector encoding an isolated core envelope peptide and / or composition as described herein. In some embodiments of any of the aspects, described herein is a nucleic acid molecule comprising a sequence encoding promoter operably linked to a sequence encoding an isolated core envelope peptide as described herein. In some embodiments of any of the aspects, a nucleic acid sequence described herein can be in a vector, or a nucleic acid molecule described herein can be a vector. In some embodiments of any of the aspects, a nucleic acid sequence described herein can be in an expression vector, or a nucleic acid molecule described herein can be an expression vector. In some embodiments of any of the aspects, the nucleic acid sequence is optimized for mammalian expression.

[0084] The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.

[0085] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like). 21 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0086] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence, but will be transcribed and / or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native / wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon- optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and / or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.

[0087] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.

[0088] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non- essential viral genes. The vector and / or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0089] In one aspect of any of the embodiments, described herein is a composition comprising an isolated core envelope peptide as described herein and further comprising a pharmaceutically acceptable carrier.

[0090] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising an isolated core envelope peptide as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprises an isolated core envelope peptide as described herein. In some embodiments, the 22 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT active ingredients of the pharmaceutical composition consist essentially of an isolated core envelope peptide as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of an isolated core envelope peptide as described herein. In some embodiments, the active ingredients of the pharmaceutical composition comprises an isolated core envelope peptide as described herein and at least one adjuvant. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of an isolated core envelope peptide as described herein and at least one adjuvant. In some embodiments, the active ingredients of the pharmaceutical composition consist of an isolated core envelope peptide as described herein and at least one adjuvant.

[0091] Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. an isolated core envelope peptide as described herein.

[0092] In some embodiments, the pharmaceutical composition comprising an isolated core envelope peptide as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be 23 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.

[0093] Suitable vehicles that can be used to provide parenteral dosage forms of an isolated core envelope peptide as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a composition as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

[0094] Pharmaceutical compositions comprising an isolated core envelope peptide as described herein can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).

[0095] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the 24 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT toxicity level for the drug. In some embodiments, the isolated core envelope peptide can be administered in a sustained release formulation.

[0096] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled- release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0097] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0098] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS®(Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.

[0099] In some embodiments of any of the aspects, the pharmaceutical composition described herein is a vaccine. The term "vaccine" used herein is defined as a composition used to elicit an immune response against an antigen (e.g., the isolated core envelope peptide described herein) within the composition in 25 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT order to protect or treat an organism against disease. The terms "vaccine composition" and “vaccine” are used interchangeably. The term “vaccinate” refers to the act of administering a vaccine to a subject.

[0100] In one aspect of any of the embodiments, described herein is a method of stimulating an immune response of a subject, the method comprising administering an isolated core envelope peptide or composition or pharmaceutical composition as described herein to the subject. In one aspect of any of the embodiments, described herein is a method of immunizing a subject, the method comprising administering an isolated core envelope peptide, or composition, or pharmaceutical composition described herein to the subject.

[0101] In one aspect of any of the embodiments, described herein is an isolated core envelope peptide, or composition, or pharmaceutical composition as described herein for use in a method of stimulating an immune response of a subject. Optionally, in some embodiments, the method of the foregoing aspect is not a method for treatment of the human or animal body by surgery or therapy practised on the human or animal body.

[0102] In one aspect of any of the embodiments, described herein is an isolated core envelope peptide, or composition, or pharmaceutical composition as described herein for use in a method of immunizing a subject. Optionally, in some embodiments, the method of the foregoing aspect is not a method for treatment of the human or animal body by surgery or therapy practised on the human or animal body.

[0103] In some embodiments of any of the aspects the subject may have or be at risk of having a coronavirus infection, which may be Middle East Respiratory Syndrome, SARS-CoV-1, SARS-CoV-2, or SARS-CoV-2 any variants of concern. In some such embodiments, the disease in the subject may be a coronavirus infection. In some embodiments of any of the aspects, the method or use may be for inducing an immune response against coronavirus, optionally to prevent coronavirus infection. In some embodiments of any of the aspects, the method or use may be for prevention or treatment of a coronavirus infection. In some embodiments of any of the aspects, the method or use may be for immunizing a subject against coronavirus, optionally to prevent coronavirus infection.

[0104] The term “immunize” as used herein is defined as elicit an immune response, e.g., either a cellular (T-cell) or humoral (B-cell or antibody) response, or both, as measured by standard assays known to one skilled in the art.

[0105] As used herein, an “immune response” refers to a response by a cell of the immune system, such as a B cell, T cell (CD4 or CD8), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus (e.g., to an antigen and / or adjuvant). In some embodiments of the aspects described herein, the response is specific for a particular antigen (an "antigen-specific response") and refers to a response by a CD4 T cell, CD8 T cell, 26 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT or B cell via their antigen-specific receptor. In some embodiments of the aspects described herein, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response. Stimulation of an immune response refers to an induction or increase of the immune response.

[0106] An immune response to an antigen and / or adjuvant can be the development in a subject of a humoral and / or a cell-mediated immune response to molecules present in the antigen or vaccine composition of interest. For purposes of the present invention, a "humoral immune response" is an antibody-mediated immune response and involves the induction and generation of antibodies that recognize and bind with some affinity for the antigen in the immunogenic composition of the invention, while a "cell-mediated immune response" is one mediated by T-cells and / or other white blood cells. A "cell-mediated immune response" is elicited by the presentation of antigenic epitopes in association with Class I or Class II molecules of the major histocompatibility complex (MHC), CD1 or other non-classical MHC-like molecules. This activates antigen-specific CD4+ T helper cells or CD8+ cytotoxic lymphocyte cells ("CTLs"). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by classical or non-classical MHCs and expressed on the surfaces of cells. CTLs help induce and promote the intracellular destruction of intracellular microbes, or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide or other antigens in association with classical or non-classical MHC molecules on their surface. A "cell-mediated immune response" also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and / or other white blood cells, including those derived from CD4+ and CD8+ T-cells. The ability of a particular antigen or composition to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, by assaying for T- lymphocytes specific for the antigen in a sensitized subject, or by measurement of cytokine production by T cells in response to re-stimulation with antigen. Such assays are well known in the art. See, e.g., Erickson et al. (1993) J. Immunol.151:4189-4199; and Doe et al. (1994) Eur. J. Immunol.24:2369-2376.

[0107] In some embodiments of any of the aspects, the immune response comprises an increase in Ig levels in the subject. Humans have four Ig subclasses; IgG1, IgG2, IgG3, and IgG4. IgG2 and IgG3 are most indicative of inflammatory and desired vaccine-induced responses, respectively. In some embodiments of any of the aspects, the Ig is IgG2, IgG3, or IgG2a. In some embodiments of any of the aspects, the Ig is IgG2. In some embodiments of any of the aspects, the Ig is IgG3. In some embodiments 27 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT of any of the aspects, the Ig is IgG2a. In some embodiments of any of the aspects, the Ig is an Ig that specifically binds the isolated core envelope peptide and / or the coronavirus envelope protein. In some embodiments of any of the aspects, the Ig is an IgG2a that specifically binds the binds the isolated core envelope peptide and / or the coronavirus envelope protein.

[0108] In some embodiments of any of the aspects, the immune response comprises a CD4+ T cell response in the subject. In some embodiments of any of the aspects, an immune response can be cytokine production by CD4+ T cells. In some embodiments of any of the aspects, cytokine production by a CD4+ T cell can comprise production of one or more of IL-2 (proliferation); IL-2, IFN-γ, TNF, TNF-β (Th1); IL-4, IL-5, IL-9 and IL-13 (Th2); IL-1-β, IL-17A, IL-17E, IL-17F, IL-21, IL-22, IL-23 (Th17); IL-6, IL21, (Tfh); TGF-β, IL-10, IL-35 (multiple and Tregs). In some embodiments of any of the aspects, an immune response can be an increase in the level of CD4+ T cells, e.g., antigen-specific CD4+ cells.

[0109] In some embodiments of any of the aspects, the immune response comprises a CD8+ T cell response in the subject. In some embodiments of any of the aspects, an immune response can be cytokine production by CD8+ T cells. In some embodiments of any of the aspects, cytokine production by a CD8+ T cell can comprise production of one or more of IL-2, IFN-γ, TNF, and IL-10. In some embodiments of any of the aspects, an immune response can be the release of perforin and / or granzymes by CD8+ T cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of CD8+ T cells.

[0110] In some embodiments of any of the aspects, the immune response comprises a Th1 cell response in the subject. In some embodiments of any of the aspects, an immune response can be cytokine production by Th1 cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of Th1 cells, e.g., antigen-specific Th1 cells.

[0111] In some embodiments of any of the aspects, the immune response comprises a NK cell response in the subject. In some embodiments of any of the aspects, an NK cell response comprises the production of one or more of IFN-γ and TNF. In some embodiments of any of the aspects, an immune response can be the release of perforin and / or granzymes by NK cells. In some embodiments of any of the aspects, an immune response can be an increase in the level of NK cells.

[0112] In some embodiments of any of the aspects, the immune response stimulates or is an increase of the production of an interferon gamma (IFNγ) response from T cells in the subject, e.g., an increase in IFNγ levels.

[0113] In some embodiments of any of the aspects, the immune response initiates or comprises an increase in phagocytosis via the Fc region of each IgG subclass via improved affinity for phagocyte membrane Fc-gamma-receptors (FcγR). 28 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0114] In some embodiments of any of the aspects, the immune response comprises immunization of the subject against the antigen (e.g., binds the isolated core envelope peptide) or an organism comprising the antigen or a portion of the antigen.

[0115] In some embodiments of any of the aspects, the administration is by injection, subcutaneous injection, or mucosal administration. In some embodiments of any of the aspects, the administration is by injection. In some embodiments of any of the aspects, the administration is by intramuscular injection.

[0116] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy.

[0117] The term "treatment" (including variations thereof, e.g., "treat" or "treated") as used herein means any one or more of the following: (i) the prevention of infection or re-infection, as in a traditional vaccine, (ii) the reduction in the severity of, or, in the elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen or disorder in question. Hence, treatment may be effected prophylactically (prior to infection) or therapeutically (following infection). In some embodiments of any of the aspects, the treatment is prophylactic. In some embodiments of any of the aspects, compositions and methods are provided that treat, including prophylactically and / or therapeutically immunize, a host animal against an infection (e.g., a virus). The methods disclosed herein are useful for conferring prophylactic and / or therapeutic immunity to a subject. The methods disclosed herein can also be practiced on subjects for biomedical research applications.

[0118] The compositions and methods described herein can be administered to a subject in need of vaccination, immunization, and / or stimulation of an immune response. In some embodiments of any of the aspects, the methods described herein comprise administering an effective amount of compositions described herein, e.g. to a subject in order to stimulate an immune response or provide protection against the relevant pathogen the antigen was derived from. Providing protection against the relevant pathogen is stimulating the immune system such that later exposure to the antigen (e.g., on or in a pathogen) triggers a more effective immune response than if the subject was naïve to the antigen. Protection can include faster clearance of the pathogen, reduced severity and / or time of symptoms, and / or lack of development of disease or symptoms. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, injection, or topical, administration. Administration can be local or systemic. In some embodiments of any of the aspects, the administration can be intramuscular or subcutaneous. 29 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0119] The term “effective amount" as used herein refers to the amount of an isolated core envelope peptide or composition described herein needed to stimulate the immune system or to provide a protective effect against subsequent infections and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of the isolated core envelope peptide or composition described herein (and optionally, an adjuvant) that is sufficient to provide a particular immune stimulatory effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slowing the progression of a symptom of the disease), or prevent a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0120] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a composition which achieves a half-maximal inhibition of symptoms or induction of desired responses) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for antibody titers, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0121] In some embodiments of any of the aspects, an effective dose of a composition comprising an isolated core envelope peptide or composition as described herein can be administered to a patient once. In some embodiments of any of the aspects, an effective dose of the isolated core envelope peptide or composition as described herein can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of the composition, such as, e.g., 0.01 mg / kg, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, or more. 30 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0122] In some embodiments of any of the aspects, the isolated core envelope peptide or composition described herein is administered once. In some embodiments of any of the aspects, the isolated core envelope peptide or composition described herein is administered twice. In some embodiments of any of the aspects, the isolated core envelope peptide or composition described herein is administered at least twice. In some embodiments of any of the aspects, the isolated core envelope peptide or composition described herein is administered repeatedly. In some embodiments of any of the aspects, the isolated core envelope peptide or composition described herein is administered once and then subsequent booster(s) are administered.

[0123] In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least two days apart. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least two weeks apart. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least three weeks apart. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least four weeks apart.

[0124] In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 6 months apart. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 3 months apart. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 2 months apart.

[0125] In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart and no more than 2 months apart. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart and no more than 6 months apart. In some embodiments of any of the aspects, the method comprises a first administration 31 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT and at least a second administration, wherein the first administration and the at least a second administration are at least two weeks apart and no more than four weeks apart.

[0126] In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration comprises a first isolated core envelope peptide, and the second administration comprises a second isolated core envelope peptide that does not have a sequence identical to the first isolated core envelope peptide. In some embodiments of any of the aspects, the method comprises a first administration and at least a second administration, wherein the first administration comprises a first adjuvant, and the second administration comprises a second adjuvant that is not identical to the first adjuvant.

[0127] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the isolated core envelope peptide or composition as described herein. The desired dose or amount of a composition can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments of any of the aspects, administration can be chronic, e.g., one or more doses over a period of weeks or months.

[0128] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the minimal effective dose and / or maximal tolerated dose. The dosage can vary depending upon the dosage form employed and the route of administration utilized. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a dosage range between the minimal effective dose and the maximal tolerated dose. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for immune response among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0129] The dosage ranges for the administration of an isolated core envelope peptide or composition as described herein depend upon, for example, the form of the isolated core envelope peptide or composition, its potency, and the extent to which symptoms, markers, or indicators of a response described herein are desired to be induced, for example the percentage induction desired for an immune response. The dosage 32 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT should not be so large as to cause adverse side effects, such as inflammatory responses. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0130] The efficacy of an isolated core envelope peptide or composition as described herein in, e.g. to induce a response as described herein (e.g. an immune response or immunization) can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted signs or symptoms are improved, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate. Immune responses can be detected by a variety of methods known to those skilled in the art, including but not limited to, antibody production, cytotoxicity assay, proliferation assay and cytokine release assays. For example, samples of blood can be drawn from the immunized mammal and analyzed for the presence of antibodies against the antigen administered in the respective vaccine and the titer of these antibodies can be determined by methods known in the art.

[0131] Efficacy of an agent can be determined by assessing physical indicators of a desired response, (e.g., immune response, cytokine production, antibody titers, etc.). It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example immunization of mice or monkeys. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.

[0132] In vitro and animal model assays are provided herein which allow the assessment of a given dose of an adjuvant and / or antigen. By way of non-limiting example, the effects of a dose of adjuvant can be assessed by measuring the antibody titers.

[0133] In one aspect, described herein is method of producing an antibody, the method comprising the steps of: a) injecting a mammal with an isolated core envelope peptide or composition as described herein, and b) isolating an antibody specific for the isolated core envelope peptide or producing a monoclonal antibody specific for the isolated core envelope peptide from at least one cell of the mammal. In some embodiments of any of the aspects, the mammal is a mouse. In some embodiments of any of the aspects, the mammal is a rabbit. In some embodiments of any of the aspects, the mammal comprises a humanized immune system. Methods of isolating antibodies and / or antibody-producing cells are known in the art, and can include, by way of non-limiting example, producing a monoclonal antibody via, e.g., the 33 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT production of hybridomas or phage display. See, e.g., Little et al. Immunology Today 200021:364-370; Pasqualini et al. PNAS 2004101:257-259; Reichert et al. Nature Reviews Drug Discovery 20076:349- 356; and Wang et al. Antibody Technology Journal 20111:1-4; each of which is incorporated by reference herein in its entirety.

[0134] A kit is any manufacture (e.g., a package or container) comprising at least one reagent, e.g., an isolated core envelope peptide, a composition, and / or an adjuvant, the manufacture being promoted, distributed, or sold as a unit for performing the methods described herein. The kits described herein can optionally comprise additional components useful for performing the methods described herein. By way of example, the kit can comprise fluids and compositions (e.g., buffers, needles, syringes etc.) suitable for performing one or more of the administrations according to the methods described herein, an instructional material which describes performance of a method as described herein, and the like. Additionally, the kit may comprise an instruction leaflet.

[0135] In one respect, the present invention relates to the herein described compositions, methods, and respective component(s) thereof, as essential to the technology, yet open to the inclusion of unspecified elements, essential or not ("comprising). In some embodiments of any of the aspects, other elements to be included in the description of the composition, method or respective component thereof are limited to those that do not materially affect the basic and novel characteristic(s) of the technology (e.g., the composition, method, or respective component thereof “consists essentially of” the elements described herein). This applies equally to steps within a described method as well as compositions and components therein. In other embodiments of any of the aspects, the compositions, methods, and respective components thereof, described herein are intended to be exclusive of any element not deemed an essential element to the component, composition or method (e.g., the composition, method, or respective component thereof “consists of” the elements described herein). This applies equally to steps within a described method as well as compositions and components therein.

[0136] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail. 34 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0137] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

[0138] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0139] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0140] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0141] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans 35 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT can be advantageously used as subjects that represent animal models of infection or immunization. A subject can be male or female.

[0142] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. an infection) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.

[0143] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0144] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha- amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, of the reference protein, polypeptide, or peptide. Conservative substitution variants that maintain the activity of the reference or wild-type protein, polypeptide, or peptide will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with homologs or paralogs from other species. Amino acids that are identical between homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and / or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of immunization as described herein. 36 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0145] In some embodiments, a polypeptide, e.g., an isolated core envelop peptide, can be a variant of a sequence described herein. In some embodiments, the variant is a conservative substitution variant. Variants can be obtained by mutations of native nucleotide sequences, for example. A “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains the relevant biological activity relative to the reference protein, e.g., can induce an immune response at least 50% as well as the reference or wild-type. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage, (i.e.5% or fewer, e.g.4% or fewer, or 3% or fewer, or 1% or fewer) of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. It is contemplated that some changes can potentially improve the relevant activity, such that a variant, whether conservative or not, has more than 100% of the activity of the reference or wildtype, e.g.110%, 125%, 150%, 175%, 200%, 500%, 1000% or more.

[0146] One method of identifying amino acid residues which can be substituted is to align, for example, an isolated core envelope peptide to a homolog from one or more species. Alignment can provide guidance regarding not only residues likely to be necessary for function but also, conversely, those residues likely to tolerate change. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally. Where, conversely, alignment shows residues in corresponding positions to differ significantly in size, charge, hydrophobicity, etc., it is more likely that that site can tolerate variation in a functional polypeptide. The variant amino acid or DNA sequence can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original” sequence). The degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools 37 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp or BLASTn (available on the world wide web at blast.ncbi.nlm.nih.gov), with default parameters set.

[0147] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0148] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. immune inducing activity and specificity of a native or reference polypeptide is retained.

[0149] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity of a native or reference polypeptide is retained. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0150] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non- polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, 38 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Val, into Ile or into Leu. Typically conservative substitutions for one another also include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).

[0151] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0152] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide- encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0153] In some embodiments, a polypeptide, e.g., an isolated core envelope peptide can comprise one or more amino acid substitutions or modifications. In some embodiments, the substitutions and / or modifications can prevent or reduce proteolytic degradation and / or prolong half-life of the polypeptide in a subject. In some embodiments, a polypeptide can be modified by conjugating or fusing it to other polypeptide or polypeptide domains such as, by way of non-limiting example, transferrin (WO06096515A2), albumin (Yeh et al., 1992), growth hormone (US2003104578AA); cellulose (Levy 39 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT and Shoseyov, 2002); and / or Fc fragments (Ashkenazi and Chamow, 1997). The references in the foregoing paragraph are incorporated by reference herein in their entireties.

[0154] In some embodiments, a polypeptide, e.g., an isolated core envelope peptide, as described herein can comprise at least one peptide bond replacement. A peptide as described herein can comprise one type of peptide bond replacement or multiple types of peptide bond replacements, e.g.2 types, 3 types, 4 types, 5 types, or more types of peptide bond replacements. Non-limiting examples of peptide bond replacements include urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine, ortho- (aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylacetic acid, meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronic ester, olefinic group, and derivatives thereof.

[0155] In some embodiments, a polypeptide, e.g., an isolated core envelope peptide, as described herein can comprise naturally occurring amino acids commonly found in polypeptides and / or proteins produced by living organisms, e.g. Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, a polypeptide as described herein can comprise alternative amino acids. Non-limiting examples of alternative amino acids include, D-amino acids; beta-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma- carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, 1,2,3,4,- tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citruline, alpha- methyl-alanine, para-benzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine), diaminobutyric acid, 7-hydroxy- tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, amino- isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-l- cyclopentanecarboxylic acid, 1-amino-l-cyclohexanecarboxylic acid, amino-benzoic acid, amino- naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine, nipecotic acid, alpha-amino butyric acid, thienyl-alanine, t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs; azide- modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and derivatives thereof.

[0156] In some embodiments, a polypeptide, e.g. an isolated core envelope peptide, can be modified, e.g. by addition of a moiety to one or more of the amino acids that together comprise the peptide. In some embodiments, a polypeptide as described herein can comprise one or more moiety molecules, e.g.1 or more moiety molecules per polypeptide, 2 or more moiety molecules per polypeptide, 5 or more moiety molecules per polypeptide, 10 or more moiety molecules per polypeptide or more moiety molecules per 40 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT polypeptide. In some embodiments, a polypeptide as described herein can comprise one more types of modifications and / or moieties, e.g.1 type of modification, 2 types of modifications, 3 types of modifications or more types of modifications. Non-limiting examples of modifications and / or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end- capping modifications; cyano groups; phosphorylation; albumin, and cyclization. In some embodiments, an end-capping modification can comprise acetylation at the N-terminus, N-terminal acylation, and N- terminal formylation. In some embodiments, an end-capping modification can comprise amidation at the C-terminus, introduction of C-terminal alcohol, aldehyde, ester, and thioester moieties. The half-life of a polypeptide can be increased by the addition of moieties, e.g. PEG, albumin, or other fusion partners (e.g. Fc fragment of an immunoglobin).

[0157] Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0158] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide- directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established. Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Khudyakov et al. “Artificial DNA: Methods and Applications” CRC Press, 2002; Braman “In Vitro Mutagenesis Protocols” Springer, 2004; and Rapley “The Nucleic Acid Protocols Handbook” Springer 2000; which are herein incorporated by reference in their entireties. In some embodiments, a polypeptide 41 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

[0159] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0160] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and / or to the translation of mRNA into a polypeptide.

[0161] In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is / are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is / are global. In some embodiments, the expression of a biomarker(s), target(s), or gene / polypeptide described herein is systemic.

[0162] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g.5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0163] In some embodiments of any of the aspects, a polypeptide of nucleic acid as described herein can be engineered. In some embodiments of any of the aspects, a polypeptide or nucleic acid as described herein is not a naturally-occurring polypeptide or nucleic acid. In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be modified. As used herein, “modified" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “modified" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. 42 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0164] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0165] In some embodiments of any of the aspects, a polypeptide or nucleic acid as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature.

[0166] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. an infection. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with an infection. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0167] In some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein 43 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT can be prophylactic relative to infection. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.

[0168] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.

[0169] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.

[0170] As used herein, the term “antibody” refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab’, a F(ab’)2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope-binding portion thereof, and / or bifunctional hybrid antibodies.

[0171] As used herein, the term “antigen” refers to a substance or substances alone or in combination that when introduced into a subject, e.g., a mammalian subject, induces production of antibodies that bind to at least a fraction of the antigen molecules. An antigen will comprise an epitope.

[0172] As used herein, an “epitope” can be formed on a polypeptide both from contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from 44 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. An “epitope” includes the unit of structure conventionally bound by an immunoglobulin VH / VL pair. Epitopes define the minimum binding site for an antibody, and thus represent the target of specificity of an antibody. In the case of a single domain antibody, an epitope represents the unit of structure bound by a variable domain in isolation. The terms “antigenic determinant” and “epitope” can also be used interchangeably herein. In certain embodiments, epitope determinants include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and / or specific charge characteristics.

[0173] “Avidity” is the measure of the strength of binding between an antibody and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding molecule, and the number of pertinent binding sites present on the antigen-binding molecule. Typically, antibodies will bind to their cognate or specific antigen with a dissociation constant (KD of 10−5to 10−12moles / liter or less, such as 10−7to 10−12moles / liter or less, or 10−8to 10−12moles / liter (i.e., with an association constant (KA) of 105to 1012liter / moles or more, such as 107to 1012liter / moles or 108to 1012liter / moles). Any KD value greater than 10−4mol / liter (or any KA value lower than 104M−1) is generally considered to indicate non-specific binding. The KD for biological interactions which are considered meaningful (e.g., specific) are typically in the range of 10−10M (0.1 nM) to 10−5M (10000 nM). The stronger an interaction, the lower is its KD. For example, a binding site on an antibody will bind to the desired antigen with an affinity less than 500 nM, such as less than 200 nM, or less than 10 nM, such as less than 500 pM. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as other techniques as mentioned herein.

[0174] Accordingly, as used herein, “selectively binds” or “specifically binds” refers to the ability of a first entity (e.g., an antibody or portion thereof) to bind to a second entity, such as an antigen, with a KD 10−5M (10000 nM) or less, e.g., 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, 10−12M, or less. Specific binding can be influenced by, for example, the affinity and avidity of the first entity and the concentration of the first entity. The person of ordinary skill in the art can determine appropriate conditions under which a first entity (e.g., an antibody described herein) selectively binds a second entity 45 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT (e.g., an antigen) using any suitable methods, such as titration of an antibody in a suitable binding assay. A first entity specifically bound to a second entity (e.g., an antigen) is not displaced by a non-similar competitor. In certain embodiments, a first entity is said to specifically bind a second entity when it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, specific binding does not refer to covalent bonding.

[0175] As used herein, the term "administering," refers to the placement of a compound or composition as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the peptides or compositions disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0176] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and / or decanting; and / or manipulation of a delivery device or machine.

[0177] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0178] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0179] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0180] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0181] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0182] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates 46 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0183] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0184] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978- 0911910421); Bruce Alberts et al., Molecular Biology of the Cell, published by W.W. Norton & Company, 2022 (ISBN 0393884821, 978-0393884821); John M. Lackie eat al. (eds.), The Dictionary of Cell and Molecular Biology, 5thEdition, published by Academic Press, 2013 (ISBN 0123849314, 978- 0123849311); Nalini Chandar et al., Lippincott Illustrated Reviews: Cell and Molecular Biology, 3rdEdition, published by LWW, 2023 (ISBN 1975180895, 978-1975180898); Teresa Atwood et al., Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006; Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); Johnathan Law et al., (eds.), A Dictionary of Chemistry, 8thEdition, published by Oxford University Press, 2020 (ISBN 9780198841227, 9780191876783); Robert C. King et al. (eds.), A Dictionary of Genetics, 8thEdition, published by Oxford University Press, 2013 (ISBN 9780199766444, 9780199376865); Richard Cammack et al. (eds.), Oxford Dictionary of Biochemistry and Molecular Biology, 2ndEdition, published by Oxford University Press, 2006 (ISBN 9780198529170, 9780191727641); John Lackie et al. (eds.), A Dictionary of Biomedicine, 2ndEdition, published by Oxford University Press, 2019 (ISBN 9780191829116); Lodish et al., Molecular Cell Biology, 8thEdition, published by W.H. Freeman, 2016 47 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT (ISBN 1464183392, 978-1464183393); Abul K. Abbas et al., Cellular and Molecular Immunology, 10thEdition, published by Elsevier, 2021 (ISBN 0323757480, 978-0323757485); Kenneth M. Murphy et al., Janeway's Immunobiology, 10thEdition, published by W. W. Norton & Company, 2022 (ISBN 0393884899, 978-0393884890); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 0444569464); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, 1987-2010 (ISBN 047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0185] In all embodiments where a sample is obtained or has been obtained or provided, the sample can be sample taken, obtained, or provided via minimally invasive methods and / or involves only a minor intervention. In some embodiments of any of the aspects, a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and / or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc), fecal sampling, semen / seminal fluid sampling, or clippings (e.g., of hair or nails). In some emodiments of any of the aspects, the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and / or hair or nail clippings.

[0186] Other terms are defined herein within the description of the various aspects of the invention.

[0187] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to 48 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0188] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0189] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0190] In some embodiments, the present technology may be defined in any of the following numbered paragraphs: 1. A composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto. 2. The composition of paragraph 1, wherein the isolated core envelope peptide comprises the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto. 3. A composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto. 4. The composition of any one of the preceding paragraphs, wherein the isolated core envelope peptide does not comprise SEQ ID NO: 16 or a sequence with at least 80% sequence identity thereto. 49 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT 5. The composition of any one of the preceding paragraphs, wherein the isolated core envelope peptide does not comprise SEQ ID NO: 17 or a sequence with at least 80% sequence identity thereto. 6. The composition of any one of the preceding paragraphs, further comprising an adjuvant. 7. The composition of paragraph 6, wherein the adjuvant is conjugated to a cysteine residue of the isolated core envelope peptide. 8. The composition of any one of paragraphs 6-7, wherein the adjuvant comprises mannose. 9. The composition of paragraph 8, wherein the mannose is alpha-D-mannose. 10. The composition of any one of paragraphs 8-9, wherein the mannose is conjugated to a lysine residue of the isolated core envelope peptide. 11. The composition of any one of paragraphs 8-9, wherein the mannose is conjugated to the isolated core envelope peptide via a methyl carboxylic acid linkage. 12. The composition of any one of paragraphs 6-7, wherein the adjuvant comprises KLH, BSA, or OVA. 13. The composition of paragraph 12, wherein the adjuvant comprises KLH. 14. The composition of any one of paragraphs 12-13, wherein the KLH, BSA, or OVA is conjugated to a cysteine residue of the isolated core envelope peptide. 15. The composition of any one of the preceding paragraphs, further comprising a pharmaceutically acceptable carrier. 16. A method of immunizing a subject, the method comprising administering to the subject a composition of any one of paragraphs 1-15. 17. A method of stimulating an immune response of a subject, the method comprising administering to the subject a composition of any one of paragraphs 1-15. 18. The method of any of the preceding paragraphs, wherein the administration is by injection, subcutaneous injection, or mucosal administration. 19. The method of any of the preceding paragraphs, wherein the injection is an intramuscular injection. 20. The method of any of the preceding paragraphs, wherein the composition is administered twice. 21. The method of any of the preceding paragraphs, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart. 50 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT 22. The method of any of the preceding paragraphs, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 2 months apart. 23. The method of any of the preceding paragraphs, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least two weeks apart and no more than four weeks apart.

[0191] In some embodiments, the present technology may be defined in any of the following numbered paragraphs: 1. A composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto. 2. The composition of paragraph 1, wherein the isolated core envelope peptide comprises the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto. 3. A composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto. 4. The composition of any one of the preceding paragraphs, wherein the isolated core envelope peptide does not comprise SEQ ID NO: 16 or a sequence with at least 80% sequence identity thereto. 5. The composition of any one of the preceding paragraphs, wherein the isolated core envelope peptide does not comprise SEQ ID NO: 17 or a sequence with at least 80% sequence identity thereto. 6. The composition of any one of the preceding paragraphs, further comprising an adjuvant. 7. The composition of paragraph 6, wherein the adjuvant is conjugated to a cysteine residue of the isolated core envelope peptide. 8. The composition of any one of paragraphs 6-7, wherein the adjuvant comprises mannose. 9. The composition of paragraph 8, wherein the mannose is alpha-D-mannose. 10. The composition of any one of paragraphs 8-9, wherein the mannose is conjugated to a lysine residue of the isolated core envelope peptide. 11. The composition of any one of paragraphs 8-9, wherein the mannose is conjugated to the isolated core envelope peptide via a methyl carboxylic acid linkage. 12. The composition of any one of paragraphs 6-7, wherein the adjuvant comprises KLH, BSA, or OVA. 13. The composition of paragraph 12, wherein the adjuvant comprises KLH. 51 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT 14. The composition of any one of paragraphs 12-13, wherein the KLH, BSA, or OVA is conjugated to a cysteine residue of the isolated core envelope peptide. 15. The composition of any one of the preceding paragraphs, further comprising a pharmaceutically acceptable carrier. 16. A method of immunizing a subject, the method comprising administering to the subject a composition of any one of paragraphs 1-15. 17. A method of stimulating an immune response of a subject, the method comprising administering to the subject a composition of any one of paragraphs 1-15. 18. The method of any of the preceding paragraphs, wherein the administration is by injection, subcutaneous injection, or mucosal administration. 19. The method of any of the preceding paragraphs, wherein the injection is an intramuscular injection. 20. The method of any of the preceding paragraphs, wherein the composition is administered twice. 21. The method of any of the preceding paragraphs, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart. 22. The method of any of the preceding paragraphs, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 2 months apart. 23. The method of any of the preceding paragraphs, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least two weeks apart and no more than four weeks apart. 24. The composition of any one of paragraphs 1-15 for use in a method of immunizing a subject. 25. The composition of any one of paragraphs 1-15 for use in a method of stimulating an immune response of a subject. 26. The composition of any one of paragraphs 24-25, wherein the composition is administered by injection, subcutaneous injection, or mucosal administration. 27. The composition of paragraph 26, wherein the injection is an intramuscular injection. 28. The composition of any one of paragraphs 24-27, wherein the composition is administered twice. 29. The composition of any one of paragraphs 24-28, wherein the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart. 52 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT 30. The composition of any one of paragraphs 24-28, wherein the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 2 months apart. 31. The composition of any one of paragraphs 24-28, wherein the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least two weeks apart and no more than four weeks apart.

[0192] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. EXAMPLES Example 1

[0193] The increasing emergence of SARS-CoV-2 variants with mutations that confer resistance to vaccines, monoclonal antibody therapeutics, and natural immunity represents a public health emergency of global concern. Currently, there is a significant unmet medical need to investigate additional antigens for possible inclusion in the next generation of Coronavirus vaccines. The inventors investigated the vaccine potential of a highly conserved 30 amino acid transmembrane domain epitope from the SARS- CoV-2 Envelope (E) protein in mice. To increase immunogenicity, the E protein epitope was conjugated to Keyhole Limpet Hemocyanin (KLH) and mice were immunized with one or two vaccine doses, administered intramuscularly in thirty-day intervals. The KLH-E conjugate vaccine elicited serum IgG antibodies and antigen-specific T-cells in mice against both the E transmembrane domain epitope, as well as the KLH carrier. A single dose of KLH-E vaccine was sufficient to elicit E-specific humoral and cellular responses, though a decline at 60 days post-immunization was observed.

[0194] A second dose of KLH-E vaccine increased E-specific T-cells and restored but did not fully increase the overall level of anti-E IgG antibody compared to a single dose. Pooled sera from KLH-E vaccinated mice had similar neutralization activity against four different hybrid alphavirus (HA-CoV-2) pseudovirions expressing spike (S) proteins from SARS-CoV-2 variants of concern (Beta, Delta, Omicron, XBB) and neutralized SARS-CoV-2 and HCoV-229E, which indicates that antibodies directed at the transmembrane domain of the SARS-CoV-2 E protein can confer broad immunity against emerging SARS-CoV-2 variants and other genetically diverse human Coronaviruses.

[0195] E-peptide-induced antibodies inhibit SARS-CoV-2 intracellularly

[0196] The E protein is found in the envelope of mature infectious virions. It is also expressed in the cellular secretory pathways / compartments (e.g., endoplasmic reticulum (ER), trans-Golgi networks, etc.), where virus particle assembly and envelope maturation occur. The E protein has been shown to be 53 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT essential for viral infectivity / entry and virus assembly, two very different steps of the virus replication cycle. However, the molecule mechanism of how E protein facilitates virus assembly has yet to be extensively studied. Specifically, little is known about the domains and sequences of the E protein that are important for virus assembly and envelope maturation. Equally elusive is whether the E protein engages various conformations with the exposure of different regions, including the E peptide, during virus assembly and envelope maturation.

[0197] The E peptide used as the immunogen herein represents the transmembrane domain of the E protein. Thus, this peptide sequence is buried in the viral membrane and is not exposed to the surface of the viral envelope. This raises the possibility that a part of the observed neutralizing activity of the antibodies may be due to its action inside the cells. In this case, the antibodies potentially enter the cells via endocytosis and interact with the E protein, inhibiting virus assembly and neutralizing viral replication and infection.

[0198] To determine if this is the case during HCoV-229E infection, human foreskin fibroblasts were first infected with HCoV-229E, washed extensively to remove all extracellular virus at 24 hours post-infection, and then treated with pools of sera obtained from different groups of immunized mice. Cell and culture media were collected at different time points of infection and assayed for the level of virus titers and replication. The salient features of the results are as follows (Fig.1):

[0199] All the sera collected from mice immunized with the KLH-E peptide exhibited inhibitory activities against human coronavirus 229E.

[0200] Sera from mice immunized with two doses of KLH-E vaccine exhibited stronger inhibitory activities than those immunized with one dose of KLH-E vaccine. Indeed, the sera's inhibitory activities from different animal groups correlated with the sera's titers against the KLH-E vaccine and the E peptide.

[0201] These exciting and surprising results indicate that (i) the antibodies induced from the E peptide work intracellularly to block HCoV-229E infection and replication, and (ii) the antibodies can be used as therapeutic antivirals since they effectively inhibit pre-existing HCoV infection.

[0202] The E-peptide is essential for SARS-CoV-2 replication and assembly

[0203] While the E protein is essential for HCoV infection, little is known about the domains and sequences of the E protein that are important for virus assembly. For example, it is currently unknown if the E peptide sequence is required for HCoV maturation and replication.

[0204] To address this issue, the inventors utilized the trans-complementation system of SARS-CoV- 2 infection and replication. See Zhang et al. Cell 184:2229-2238 (2021); which is incorporated by reference herein in its entirety. In this system, Vero-ORF3-E cells express full length ORF3 and E 54 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT proteins and support the replication of defective ΔORF3-E mNG SARS-CoV-2 mutant virus. Using CRISPR-Cas genome editing methods, ΔE-peptide cells were generated by deleting the E peptide coding sequence from Vero-ORF3-E cells. Further experiments showed no complementation of defective ΔORF3-E mNG SARS-CoV-2 mutant virus in these cells. These results indicate that (a) the constructed cells without E-peptide expression do not support ΔORF3-E mNG SARS-CoV-2 replication and (b) the E peptide sequence is essential for HCoV infection and assembly.

[0205] Summary

[0206] The results described herein are exciting and surprising. This is because the antisera immunized against the E-peptide of SARS-CoV-2 exhibits therapeutic activity intracellularly against genetically diverse human coronaviruses such as the 229E strain. Antibodies are known to work extracellularly, and little has been reported about their intracellular activity. This is potentially a paradigm-shifting concept. Several sets of experiments can be carried out to understand how these antibodies achieve their intracellular antiviral therapeutic activity. First, the different steps (e.g. gene expression, genome replication, and particle assembly) of the replication cycle of HCoV-229E in the absence and presence of the antibodies will be studied to understand which step(s) (e.g. assembly and envelope maturation) is blocked by the antibodies. Second, the localization of the antibodies will be imaged to confirm which cellular compartments these antibodies are in. Third, additional experiments will be performed to determine if the antisera immunized against the E-peptide also exhibits therapeutic activity intracellularly against SARS-CoV-2 with the trans-complementation system. Fourth, the mutated E-peptide sequence will be generated using mutagenesis or CRISP-CAS genome editing procedures. Cells expressing these E protein mutants containing the mutated E-peptide sequences will be constructed and tested to determine if they can support defective ΔORF3-E mNG SARS-CoV-2 mutant virus and are resistant to the inhibitory activities of the antibodies due to the mutations of the E-peptide that disrupt the antibody binding. These studies will elucidate the mechanism of how these antibodies achieve antiviral activity. Example 2: A Highly Conserved SARS-CoV-2 Envelope Protein Transmembrane Domain Induces Humoral and Cellular Responses in Mice and Elicits Broad Coronavirus Immunity

[0207] The increasing emergence of SARS-CoV-2 variants with mutations that confer resistance to vaccines, monoclonal antibody therapeutics, and natural immunity represents a public health emergency of global concern. Currently, there is a significant unmet medical need to investigate additional antigens for possible inclusion in the next generation of Coronavirus vaccines. The inventors studied the vaccine potential of a highly conserved 30 amino acid transmembrane domain epitope from the SARS-CoV-2 55 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT Envelope (E) protein in mice. To increase immunogenicity, the E protein epitope was conjugated to Keyhole Limpet Hemocyanin (KLH) and mice were immunized with one or two doses of vaccine, administered intramuscularly at thirty-day intervals. The KLH-E conjugate vaccine elicited serum IgG antibodies and antigen-specific T-cells in mice against both the E transmembrane domain epitope and the KLH carrier. A single dose of KLH-E vaccine was sufficient to elicit E-specific humoral and cellular responses, though we observed a decline at 60 days post-immunization. A second dose of KLH-E vaccine increased E-specific T-cells and restored the antibody response but did not increase the overall level of anti-E IgG antibody compared to a single dose. Sera from KLH-E vaccinated mice exhibited strong neutralization activity against four different hybrid alphavirus (HA-CoV-2) pseudovirions expressing spike (S) proteins from SARS-CoV-2 variants of concern (Beta, Delta, Omicron, XBB) and neutralized SARS-CoV-2 and HCoV-229E. These results indicate that the induced anti-E specific antibodies confer broad immunity against emerging SARS-CoV-2 variants and other genetically diverse human Coronaviruses. Passive immunization of SCID mice with sera from KLH-E vaccinated mice reduced the titers of HCoV-229E in lungs after challenge. Furthermore, immunization of BALB / c mice with the KLH-E vaccine inhibited the infection after challenge with HCoV-229E. These results demonstrate the potential of using the KLH-E conjugated vaccine against different human Coronaviruses, including SARS-CoV2 and HCoV-229E.

[0208] Introduction

[0209] The SARS-CoV-2 genome encodes 16 nonstructural proteins and 4 major structural proteins including the Spike (S), Nucleocapsid (N), Membrane (M), and Envelope (E) protein [1]. The E protein is the smallest, sparsest, and least understood protein in the mature SARS-CoV-2 virion. It is implicated to be involved in assembly, budding, and envelope formation based on protein-protein interactions with other structural and accessory viral proteins [2] and high (95%) sequence homology to the SARS-CoV-1 E protein, which has been more extensively studied [3]. During infection, the E protein is highly expressed in the endoplasmic reticulum (ER), Golgi apparatus, and the ER-Golgi intermediate compartment (ERGIC), which serves as the Coronavirus envelopment site. Yet only a small fraction of E protein is incorporated into assembled virions [3].

[0210] The E protein of all Coronaviruses contains three distinct domains: a 7-12 amino acid hydrophilic N-terminus, a 25 amino acid hydrophobic transmembrane (TMB) domain, and a 38-43 amino acid hydrophilic carboxyl terminus–an overall structure that has led to the classification of the E protein as a viroporin [4]. Viroporins are a broad class of virally encoded small proteins with distinct hydrophobic transmembrane domains that interact with membrane surfaces to produce a pore with selective ion and small molecule specificity. Viroporins have been identified in many highly pathogenic 56 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT human viruses and are capable of modulating ionic gradients, pH, membrane vascularization, and cell permeability to influence diverse cellular processes, including protein trafficking, signal transduction, and apoptosis to produce an optimal cellular environment for viral replication[5] [6] [7] [8] [9]. The viroporin activity of the E protein has been documented in many human Coronaviruses including SARS-CoV-1

[0010]

[0011]

[0012]

[0013] , MERS-CoV

[0014] , SARS-CoV-2

[0015] , and HCoV-229E

[0016] . In all Coronaviruses, the transmembrane domain of the E protein is required for functional viroporin activity and strongly correlated with virulence

[0017] .

[0211] To date, the Spike (S) protein is the primary antigen in all currently licensed SARS-CoV-2 vaccines, but the increasing emergence of variants with spike mutations that confer resistance to vaccines, antibody therapeutics, and natural immunity represents a significant global public health threat. It is contemplated herein that since the E protein is moderately conserved across various human Coronaviruses, the induction of humoral and cellular immune responses to this conserved antigen has the potential to elicit pan-Coronavirus immunity.

[0212] The objective of the current study is to (1) test an unusual and novel vaccine concept with a highly hydrophobic transmembrane peptide as the immunogen and (2) develop a new vaccine against SARS-CoV-2, the causative agent of COVID-19. The antigen used in the present study is a 30-amino acid long peptide corresponding to the transmembrane region of the E protein from SARS-CoV-2. The immunogenicity of the 30 amino acid domain was investigated in eliciting effective immune responses in mice against SARS-CoV-2 and other related human Coronaviruses to determine if the host immune system can recognize and mount an effective immune response to a completely occluded transmembrane peptide. The present results provide the first direct evidence that the highly conserved SARS-CoV-2 E protein transmembrane domain epitope induces humoral and cellular responses in mice and elicits broad Coronavirus immunity.

[0213] Results

[0214] Vaccine design and immunization. Using a bioinformatics approach, the amino acid sequences of the full length E proteins from all seven coronaviruses known to infect humans were aligned and the highest degree of sequence conservation and amino acid homology observed was concentrated in the transmembrane domain of the E protein

[0019] as illustrated in Fig.2. In particular, the E transmembrane domain in SARS-CoV-2 is 100% and 32% conserved with those of SARS-CoV-1 and HCoV-229E, respectively. Previous immunoinformatic studies predicted the presence of multiple B and T-cell epitopes and possible MHC binding sites on the SARS-CoV-2 E protein

[0020] . It was determined herein whether a synthetic peptide alone corresponding to the hydrophobic 30 amino acid (TLIVNSVLLFLAFVVFLLVTLAILTALRLC) (SEQ ID NO: 2) SARS-CoV-2 transmembrane domain would 57 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT be capable of inducing an immune response. To enhance immunogenicity, the SARS-CoV-2 E protein transmembrane domain sequencewas synthesized and conjugated to the immunogenic carrier protein Keyhole Limpet Hemocyanin (KLH)

[0021] , producing an antigen herein referred to as KLH-E. KLH is a very large, copper-containing protein derived from the hemolymph of the mollusk Megathura crenulata and is highly immunogenic to the mammalian immune system. KLH is also a T-cell dependent antigen that is non-toxic and elicits robust humoral and cellular immune responses

[0021] . Groups of inbred mice were immunized with one or two doses (50µg per dose) of KLH-E or control vaccine (Aluminum-DMSO) intramuscularly at 30-day intervals. After the end of the vaccination study, mice were terminally bled and their serum and splenocytes were collected and analyzed to assess the immunogenicity of the KHL-E vaccine.

[0215] Antibody response to KLH-E vaccine in mice. As illustrated in Fig.3A against the KLH-E antigen, mice immunized with a single dose of the KLH-E vaccine had an IgG antibody geometric mean titer of 220 (range 159-358) at 30 days post-immunization. A small, but significant decrease in KLH-E IgG titer was observed in mice immunized with a single dose of the KLH-E vaccine at 60 days post-immunization (GMT: 220 vs.132, 30 days vs.60 days post-dose 1; *P=0.0194). The 0.6-fold reduction in geometric mean titers at 30 days compared to 60 days post-immunization was not surprising, as it is widely understood that antibody levels induced by vaccination ultimately decrease over time, irrespective of the vaccine, especially after a single dose. Despite the waning antibody titers, a second dose of KLH-E vaccine elicited a robust 7.2- fold significant increase in geometric mean titer in mice at 30 days post-immunization (GMT: 220 vs.1588, one dose vs. two doses at 30 days; **P=0.0060)(Fig.3A). In contrast to the robust IgG antibody responses observed in KLH-E vaccinated mice, mice immunized with one or two doses of the control aluminum vaccine had no detectable IgG antibodies to KLH-E as expected.

[0216] Since the IgG antibody responses measured against the KLH-E vaccine antigen represent a mixture of antibodies to KLH as well as to the SARS-CoV-2 E peptide transmembrane domain, the IgG antibody responses of mice to individual components of the vaccine were measured to delineate the relative contribution of each in the overall humoral response elicited by the KLH-E vaccine. Fig.3B depicts the KLH carrier IgG antibody responses of individual mice induced by the KLH-E vaccine. The geometric mean KLH IgG titer of mice immunized with a single dose of KLH-E was 226 at 30 days post-immunization. A similarly small but significant decrease was observed in the KLH specific IgG titer of mice vaccinated with a single dose of KLH-E vaccine at 60 days post-immunization (GMT: 226 vs.137, 30 days vs.60 days post-dose 1; *P=0.0227). A second dose of KLH-E vaccine elicited a 7.2-fold increase in KLH specific geometric mean IgG antibody titers at one month post-immunization (GMT: 226 vs.1481, 1 dose vs.2 doses at 30 days; **P=0.0047)(Fig.3B). No KLH IgG responses were detected in mice immunized with the control aluminum vaccine. The highly similar trends in the geometric means and individual IgG antibody titers to KLH-E and 58 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT KLH in vaccinated mice led us to infer that most of the antibodies induced by vaccination were directed at the immunogenic carrier KLH. These results are unsurprising, considering that the SARS-CoV-2 E peptide is only 30 amino acids in length with an approximate molecular weight of 3.29 kD compared to the 3,414 amino acid KLH carrier with a molecular weight of 370 kD

[0021] .

[0217] Since the SARS-CoV-2 E peptide is the primary antigenic target of the present immunogenicity study, the anti-E specific IgG antibody responses of individual mice immunized with the KLH-E vaccine were quantified. As illustrated in Fig.3C, the geometric mean anti-E IgG titer of mice vaccinated with a single dose of KLH-E was 87 at 30 days post-immunization. As with the antibodies directed at KLH, a similar decrease in the anti-E specific IgG antibody titers of mice immunized with a single dose of KLH-E vaccine was observed at 60 days post-immunization, though this decrease was not significant (GMT: 87 vs.46, 30 days vs.60 days post-dose 1; P=0.1325, ns). A second dose of KLH-E vaccine elicited an increase in anti-E specific antibody, but there was no significant difference in the geometric mean anti-E antibody titers at 30 days post- immunization when comparing mice that received one or two doses of vaccine (GMT: 87 vs.91, 1 vs.2 doses at 30 days; P=0.5201, ns). These results indicate that the KLH-E vaccine induces anti-E specific antibody response.

[0218] T-cell response to KLH-E vaccine in mice. To investigate the antigen-specific T-cell response induced by KLH-E vaccination, a single representative mouse was selected from each of the following vaccine groups: KLH-E (30 and 60 days post-dose 1 and 30 days post-dose 2) or control aluminum vaccine (30 days post-dose 2). A single dose of KLH-E vaccine elicited similar proportions of T-cells at 30 and 60 days post-immunization (44 vs.37 IL-2 secreting foci; 30 days vs.60 days) when measured against the KLH-E conjugate that was used as the vaccine as illustrated in Fig.4A. There was a slight decrease in KLH-E specific T-cells at 60 days vs.30 days post-immunization with a single dose, but this decrease was not statistically significant. A second dose of KLH-E vaccine increased KLH-E specific T- cells at 30 days post-immunization, compared to a single dose at either time point (104 vs.44 or 37 IL-2 secreting foci; 2 doses vs. a single dose at 30 or 60 days post-immunization). Since the cellular responses measured using the KLH-E antigen for stimulation represent a mixture of T-cells specific to KLH as well as the SARS-CoV-2 E peptide transmembrane domain, the T-cell responses of the same mice to individual components of the vaccine were measured to delineate the relative contribution of each in the overall T-cell response induced by KLH-E vaccination.

[0219] As depicted in Fig.4B, a single dose of KLH-E vaccine elicited similar proportions of T-cells at 30 and 60 days post-immunization (141 vs.127 IL-2 secreting foci, respectively) to the KLH carrier. There was a slight decrease in KLH-specific T-cells at 60 days vs.30 days post-immunization with a single dose, but this decrease was not statistically significant (P=0.068). A second dose of KLH-E vaccine elicited an increase 59 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT in KLH-specific T-cells compared to a single dose at either timepoint (199 vs.141 [**P=0.016] or 199 vs. 127 [***P=0.008] IL-2 secreting foci; 2 doses vs.1 dose at 30 or 60 days post-immunization). In contrast to the robust T-cell responses of KLH-E vaccinated mice, control aluminum vaccinated mice had no KLH carrier specific T-cells.

[0220] A single dose of KLH-E vaccine elicited substantial proportions of T-cells at 30 days and 60 days post-immunization (12 vs.5 IL-2 secreting foci; 30 days vs.60 days) when measured against a 30 amino acid synthetic peptide corresponding to the SARS-CoV-2 E peptide transmembrane domain as illustrated in Fig.4C. There was a significant decrease in the E-peptide specific T-cells at 60 days vs.30 days post- immunization with a single dose (**P=0.038). A second dose of KLH-E vaccine induced a three to six- fold increase in E-peptide specific T-cells compared to a single dose at either timepoint (29 vs.12, or 29 vs.5, IL-2 secreting foci; 2 doses vs. a single dose at 30 or 60 days post-immunization). The induction of elevated anti-E specific T-cells after a second dose of vaccine was statistically significant when compared to a single dose at 30 days post-immunization (**P=0.033) or 60 days post-immunization (**P=0.018).

[0221] Cross-reactivity of antibodies induced by KLH-E vaccination. Since the E protein transmembrane domain is highly conserved across emerging SARS-CoV-2 variants of concern and moderately conserved across different human coronaviruses (Fig.2), the cross-reactivity of antibodies induced by KLH-E vaccination were investigated. First, the ability of anti-E antibodies induced by KLH- E vaccination to recognize SARS-CoV-2 E protein expressed on the surface of a hybrid alphavirus SARS-CoV-2 pseudovirions (HA-CoV-2) were measured by ELISA. Ha-CoV-2 is a non-replicating SARS-CoV-2 virus-like particle composed of four SARS-CoV-2 structural proteins (S, M, N, and E)

[0022] . The Ha-SARS-CoV-2 pseudovirions have been used for rapid and accurate quantification of antiviral drugs, SARS-CoV-2 variants, and their responses to neutralizing antibodies. Each of the four pseudovirions tested expressed a different S protein sequence from a SARS-CoV-2 variant of concern (Beta, Delta, Omicron, XBB)

[0022] . Thus, As illustrated in Fig.5, at 30 days post-immunization, pooled sera from mice vaccinated with two doses of KLH-E, but not the control aluminum vaccine, contained anti-E antibodies that recognize the E protein on the surface of HA-CoV-2 pseudovirions. As expected, pooled sera from KLH-E vaccinated mice reacted similarly by ELISA, irrespective of the S protein sequence expressed by the HA-CoV-2 pseudovirions (Fig.5). As an additional assay control, the ELISA reactivity of a control rabbit monoclonal antibody (mAb 27VB1) that is broadly neutralizing and recognizes the receptor binding domain (RBD) of the SARS-CoV-2 S protein

[0022] was also measured. The mAb bound to all four SARS-CoV-2 pseudovirions tested to varying degrees (data not shown), which is consistent with both in vitro and in vivo studies that report SARS-CoV-2 variants of concern 60 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT (particularly omicron and XBB) have divergent S protein sequences that result in impaired antibody binding

[0023] .

[0222] The same serum pools were also tested by ELISA against HCoV-229E infected human MRC5 whole cell lysates to determine whether anti-E antibodies induced by KLH-E vaccination could recognize native full length E protein from a genetically divergent human coronavirus (Fig.6). As depicted in Fig.6A (left), pooled serum from mice immunized with the control aluminum vaccine had no reactivity to both uninfected and HCoV-229E infected MRC5 cells irrespective of the number of doses or timepoint as expected. Likewise, as illustrated in Fig.6A (right), pooled serum from mice immunized with KLH-E had no reactivity to uninfected MRC5 cell lysates, irrespective of the number of vaccine doses or time points. Surprisingly, serum pools from mice immunized with the KLH-E vaccine contained antibodies that were reactive to HCoV-229E infected MRC5 cell lysates with serum pools from mice immunized with two doses of KLH-E vaccine yielded higher antibody reactivity compared to pools from mice immunized with a single dose of vaccine (Fig.6A). Moreover, a decline in antibody reactivity in pooled sera from mice immunized with a single dose of KLH-E vaccine at 60 days post-immunization was observed compared to 30 days post-immunization. Using the same mouse serum pools, these findings share similar trends with the previously observed antibody reactivity measured against the 30-amino acid E-peptide corresponding to the SARS-CoV-2 E protein transmembrane domain. One important distinction, however, was that the same dilution of pooled KLH-E mouse sera produced approximately half the amount of signal when tested against the HCoV-229E infected cell lysate compared to the 30- amino E-peptide, which may be due to the low sequence homology (32%) between the E protein transmembrane domains of SARS-CoV-2 and HCoV-229E. Conceivably, differences in the concentration of uninfected vs. HCoV-229E infected cell lysates could result in differences in antibody reactivity that could confound interpretation. To eliminate this possibility, the results were controlled for differences in coating antigen concentration in each ELISA plate by first measuring the total protein concentration of each cell lysate preparation as described in the materials and methods. As an additional control, to show that the uninfected and HCoV-229E infected MRC5 cell lysate preparations were equivalent in total protein concentration and overall protein composition (excluding the E protein), the level of β-actin in each MRC5 cell lysate preparation were measured for uninfected and HCoV-229E infected cells. As expected, no significant difference was observed in the levels of β-actin in each of the MRC5 cell lysate preparations (data not shown). This observation further supports that the reactivity observed in pooled KLH-E immune mouse sera measured against HCoV-229E infected cell lysates was specific for anti-E antibodies and that both plates were coated with equivalent total protein concentrations. 61 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0223] Cross-reactivity of T-cells induced by KLH-E vaccination. After demonstrating that KLH- E vaccination induced anti-E antibodies with cross-reactivity against a genetically divergent human coronavirus, it was determined whether the KLH-E vaccine also induced similar cross-reactive T-cells. To address this question, antigen-specific T-cell responses were measured as before but used HCoV-229E infected and uninfected human MRC5 cell lysates for stimulation. Fig.6B depicts the antigen-specific T- cell responses of mice immunized with two doses of KLH-E or aluminum control vaccine at 30 days post- immunization stimulated with uninfected and HCoV-229E infected human MRC5 cell lysates. As expected, no significant T-cell response was observed in cells stimulated with the uninfected human MRC5 cell lysates irrespective of vaccination (1 vs.1 IL-2 secreting foci, P=1). In contrast, when the same cells were treated with HCoV-229E infected cell lysates, a 14-fold increase in IL-2 secreting foci was observed in mice receiving two doses of KLH-E vaccine compared to mice immunized with the same number of doses of aluminum vaccine (14 vs.1 IL-2 secreting foci; **P=0.036). The differences we observed were due to antigenic-specificity of the T-cells and not defects in the ability to become activated, as noted by the similar proportions of IL-2 secreting foci when T-cells from both KLH-E and aluminum vaccinated mice were stimulated with Concanavalin A (Fig.6B). Taken together, these results indicate that KLH-E vaccination induces not only cross-reactive antibodies, but also cross-reactive T-cells that recognize a conserved epitope in the E protein, even when the overall sequence homology is low.

[0224] Serum neutralization activity of KLH-E vaccinated mice against SARS-CoV-2 and HCoV-229E. To date, the most common immunological metric used to infer SARS-CoV-2 vaccine efficacy is the induction of neutralizing antibodies. To investigate the functional activity of anti-E transmembrane antibodies induced by KLH-E vaccination, the neutralization activity of pooled sera from mice receiving two doses of KLH-E or aluminum control vaccine was measured at 30 days post- immunization against the four HA-CoV-2 pseudovirions

[0022] used in previous experiments. As illustrated in Fig. 7A, pooled KLH-E immune serum had similar neutralization activity (% infection: range; 0 - 4%) against all four HA-CoV-2 pseudovirions expressing different spike protein sequences from SARS-CoV- 2 variants of concern (Beta, Delta, Omicron, XBB). As expected, pooled serum from control aluminum vaccinated mice had negligible or no neutralization activity (% infection; range: 97-100%), indicating that the neutralization of HA-CoV-2 pseudovirions was mediated by specific antibody, and not due to a global serum sensitivity. As a control, a broadly neutralizing monoclonal antibody directed at the receptor binding domain (RBD) of the SARS-CoV-2 S protein was tested and disparate levels of neutralization activity were observed

[0022] . Specifically, HA-CoV-2 Omicron and XBB pseudovirions had greater resistance to neutralization, consistent with both in-vivo and in-vitro reports that these highly mutated S 62 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT protein sequences confer resistance to vaccine induced and natural immunity mediated by anti-S antibody against SARS-CoV-2 variants of concern.

[0225] To determine if the sera obtained from the immunized mice exhibit activity to neutralize infectious SARS-CoV2 virus, different sera were mixed with infectious particles of SARS-CoV-2 ΔORF3-E, a viral mutant with the deletion of the ORF3 and E genes. SARS-CoV-2 ΔORF3-E, which is defective in growth in normal human cells, can be generated in complementing cells expressing ORF3 and E proteins

[0024] . Thus, with the SARS-CoV-2 ΔORF3-E and complementing cell line, this trans- complementing system can produce single-round infectious SARS-CoV-2 that recapitulates authentic viral infection and replication. This system has been shown to be used safely as biosafety level 2 (BSL-2) for high-throughput antiviral testing, including successful screening of neutralizing antibodies against SARS-CoV2

[0024] .

[0226] As depicted in Fig.7B, there was no neutralization activity in pooled sera from mice immunized with one or two doses of aluminum vaccine as expected. In contrast, there was a significant reduction in viral infection when HCoV-229E virions were incubated with pooled sera from mice receiving one or two doses of KLH-E at 30 days post-immunization. The magnitude of neutralization against HCoV-229E virions was proportional to the number of doses of KLH-E vaccine in pooled serum, with two doses of KLH-E yielding a significant reduction in viral infection compared to a single dose of aluminum vaccine at either time point.

[0227] To further elucidate the breadth of immunity conferred by KLH-E vaccination, the serum neutralization activity of pooled sera from mice receiving one or two doses of KLH-E or control aluminum vaccine was measured against HCoV-229E, a genetically diverse human coronavirus with highly divergent E protein sequence to that of the vaccine antigen. As depicted in Fig.7C, there was no neutralization activity in pooled sera from mice immunized with one or two doses of aluminum vaccine, as expected. In contrast, there was a significant reduction in viral infection when HCoV-229E virions were incubated with pooled sera from mice receiving one or two doses of KLH-E at 30 days post- immunization. The magnitude of neutralization against HCoV-229E virions was proportional to the number of doses of KLH-E vaccine in pooled serum, with two doses of KLH-E yielding a significant reduction in viral infection compared to a single dose of aluminum vaccine at either time point.

[0228] Inhibition of virus growth in SCID mice induced by KLH-E passive immunization and challenge with HCoV-229E. In the absence of a defined immune correlate of protection that predicts immunity to coronavirus infection, passive immunization and challenge in a susceptible animal model can serve as a surrogate for inferring vaccine protection. To investigate whether antibodies induced by KLH- E vaccination can confer protection in vivo, we passively immunized immunodeficient SCID mice with 63 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT pooled sera from mice immunized with two doses of KLH-E or aluminum control vaccine and intranasally infected SCID mice with HCoV-229E. At 10 days post challenge, SCID mice were sacrificed, and HCoV-229E viral titers were enumerated in homogenized whole lung tissue samples. As illustrated in Fig.8A, viral titers in SCID mice treated with pooled KLH-E sera were about 4,000 folds lower than those treated with PBS without any sera. In contrast, there were no substantial differences in viral titers among SCID mice treated with pooled aluminum sera and those treated with PBS without any sera. The absence of a significant reduction in HCoV-229E titer in SCID mice passively immunized with control aluminum sera underscores that the HCoV-229E virions used for infection retained their virulence as they were not neutralized by the contributions of various innate immune molecules or cells in the immunodeficient mouse model. Moreover, the significant reduction in HCoV-229E viral titer in KLH-E vs. aluminum control treated SCID mice supports the conclusion that protection was specifically mediated by antibodies induced by vaccination.

[0229] Reduced virus infection in BALB / c mice immunized with the KLH-E vaccine and challenged with HCoV-229E. To determine if the E-peptide can serve as a vaccine and protect immunized mice against challenges with coronavirus, BALB / c mice were immunized with two doses of E-KLH vaccine at 30 days intervals and then challenged animals with HCoV-229E (Fig.8B). At 7 days post-challenge, lungs were harvested and viral titers were determined. The viral titers in the lungs of BALB / c mice immunized with the KLH-E vaccine were 2,000 folds lower than those in mice injected with PBS (Fig.8B). In contrast, there were no substantial differences in viral titers among BALB / c mice immunized with control aluminum vaccine and those injected with PBS. These results indicate that KLH-E immunization inhibits viral infection and reduces viral replication and growth in immunized mice.

[0230] Discussion

[0231] Described herein is the first investigation of the immunogenicity of the SARS-CoV-2 E protein transmembrane domain, using the immunogenic carrier KLH to enhance the immune response to the hapten. The humoral and cellular immune responses of mice induced by a novel KLH-E vaccine were characterized and it is shown herein that the sera obtained from the immunized mice neutralized SARS- CoV-2 and HCoV-229E in vitro. Moreover, provided herein is in vivo evidence that anti-E transmembrane domain antibodies can confer protection in a SCID mouse model against challenge with HCoV-229E and that KLH-E immunization protects BALB / c mice against challenge with HCoV-229E. These results are significant because they demonstrate for the first time, cross-protection against Coronaviruses from different subfamilies mediated by antibodies against a non-Spike antigen with extremely low sequence homology (32%). Without wishing to be bound by theory, these results indicate 64 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT that the SARS-CoV-2 E protein transmembrane domain contains epitopes capable of inducing broadly cross-reactive antibodies and T-cells with the ability to confer broad pan-Coronavirus immunity.

[0232] These results serve as proof of principle that humoral and cellular immunity can be induced against the SARS-CoV-2 E protein transmembrane domain and provides direct experimental evidence that the transmembrane domain contains important protective epitopes inducing pan-coronavirus immunity.

[0233] Whether due to size or differences in relative immunogenicity, a large number of antibodies induced by KLH-E vaccination were directed at the KLH carrier. Anti-E IgG was inducedafter a single dose of the KLH-E vaccine, a second dose did not elicit an overall increase in anti-E IgG. The KLH-E vaccine induced robust anti-KLH IgG after a single dose and significantly increased following subsequent immunization. These results suggest a skewed antibody repertoire directed at the KLH carrier rather than the SARS-CoV-2 E protein transmembrane domain. It is contemplated herein that primary immunization with KLH-E and boosting with a different immunogenic carrier E conjugate could potentially overcome the skewed antibody repertoire.

[0234] Neutralization of viruses by antibody generally occurs via four distinct mechanisms: (1) inhibition of viral-host receptor engagement by antibody, (2) antibody-dependent complement mediated neutralization, (3) antibody-dependent cellular cytotoxicity of virally infected cells or (4) opsonization of antibody coated viruses by phagocytosis. It is demonstrated herein that vaccination with KLH-E can induce specific and functional anti-E transmembrane antibodies. The neutralization activity of the anti-E antibodies against HA-CoV-2 pseudovirions, SARS-CoV-2 ΔORF3-E, and HCoV-229E was measured in vitro by neutralization assay but the molecular mechanisms responsible for the neutralization observed were not further determined. Without wishing to be bound by theory, it is contemplated that anti-E transmembrane antibodies could mediate protection by one or more mechanisms and may depend on the in vitro assay conditions. For example, anti-E transmembrane antibodies could function in an opsonophagocytic dependent manner mediated by specific Fc receptors expressed on cells used for in vitro neutralization assays

[0033]

[0034] . FcRn binds to IgG and has the greatest ligand binding affinity in cellular endosomes, as would be the case if anti-E antibodies neutralized coronaviruses in an opsonophagocytic manner. Taken together, it is futher contemplated herein that "non-neutralizing" antibodies could indeed neutralize SARS-CoV-2 or other Coronaviruses via unorthodox mechanisms such as opsonization

[0033]

[0034] .

[0235] Unlike the highly mutable S protein, largescale genomic studies of publicly available global SARS-CoV-2 genomes have demonstrated that the E protein, particularly the transmembrane domain, is highly conserved and accumulates mutations more slowly than in other structural proteins, suggesting an 65 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT evolutionary pressure to maintain E protein transmembrane domain amino acid residues

[0018]

[0035] . In view of the data presented in the present study, the SARS-CoV-2 E protein transmembrane domain represents an attractive vaccine antigen that warrants serious consideration as it has the potential to confer broad immunity against emerging SARS-CoV-2 variants and potentially other genetically diverse human coronaviruses.

[0236] Traditional vaccine approaches target highly abundant and surface exposed epitopes, therefore the concept that antibodies can be induced to a transmembrane domain of a protein and have neutralizing activity is surprising. Conventionally, it would be assumed that even if antibodies could be induced to a transmembrane domain, they would be unable to exert their effector functions if the epitope was inaccessible on the surface of the microorganism. To date, only the transmembrane domain of the SARS-CoV-2 E protein has been determined by nuclear magnetic resonance (NMR) spectroscopy data

[0036] , while the N and C termini of the protein remain incompletely understood. Overall, the SARS-CoV-2 E protein appears to be conformationally flexible, with some residues possibly accessible under different temperature and membrane conditions. Taken together, and without wishing to be bound by theory, it cannot be entirely excludes that the transmembrane domain of the SARS-CoV-2 E protein may be exposed or more accessible than is currently assumed. The present study serves as proof of principle that both humoral and cellular immunity can indeed be induced to a transmembrane region of a protein, using the SARS-CoV-2 E protein as a model. Furthermore, the present study demonstrates that antibodies directed at the SARS-CoV-2 E protein transmembrane domain can have functional activity.

[0237] Materials and Methods

[0238] SARS-CoV-2 E peptide. The 30 amino acid sequence (H- TLIVNSVLLFLAFVVFLLVTLAILTALRLC-OH) (SEQ ID NO: 2) corresponding to the full length transmembrane domain of the SARS-CoV-2 Envelope protein was synthetically produced (Pepscan, Netherlands) as either a purified peptide (77.1% Purity, UPLC / UV215) or conjugated to the Keyhole Limpet Hemocyanin carrier protein (77.1% Purity, UPLC / UV215). The extreme hydrophobicity of the transmembrane domain sequence proved challenging for synthesis and is reflected in the purity of each custom peptide.

[0239] Mouse immunization. The protocol was approved by the Institutional Animal Care and Use Committees (ACUC) at the University of California, Berkeley. At approximately six weeks of age, groups of male inbred BALB / c and SCID mice were purchased from Jackson Laboratories and housed at the University of California, Berkeley animal facility for one week before beginning the immunogenicity study. At the time of the first dose of vaccine, the ages of the BALB / c mice were eight weeks, and mice 66 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT were assigned to different vaccine groups. The mice were immunized with 50 µg of KLH-E per dose intramuscularly with 0.5mg Aluminum hydroxide salt in DMSO.

[0240] Viral strains and cell lines. Human coronavirus 229E (HuCoV-229E), MRC5 cells, and Vero cells were supplied by American Type Culture Collection (ATCC). Human primary foreskin Fibroblast (HFF) was obtained from Clonetics (San Diego). SARS-CoV-2 ΔORF3-E and VERO-ORF3-E propagation was carried out described previously

[0024] . Cells were maintained in minimal essential media (MEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Bio Whittaker, Walkersville, Maryland) and incubated at 37ºC and 5% CO2. Cells were passaged twice weekly using trypsin (0.25%)-EDTA. Viral stocks were prepared by infecting cells at a multiplicity of infection of 0.01 for 4 to 7 days until significant cytopathic effect (CPE) was observed. Infected cells were subjected to 3 freeze / thaw cycles, and infected-cell lysate was stored at -80ºC.

[0241] Recombinant protein ELISA. The antigens included Keyhole Limpet Hemocyanin (KLH) (EDM Millipore, CAT# 374817-50MG), KLH-E (Pepscan, Netherlands), and the purified 30 amino acids (TLIVNSVLLFLAFVVFLLVTLAILTALRLC) (SEQ ID NO: 2) E-peptide conjugate (Pepscan, Netherlands).96-well Medisorp ELISA plates (Thermo Fisher) were coated with 2µg of antigen (KLH, KLH-E, or E peptide) in ELISA coating buffer (50mM carbonate-bicarbonate buffer, 1.59g Na2CO3 + 2.93g NaHCO3 in 1L diH20, pH 9.4) and sensitized overnight at 4ºC. Next, plates were extensively washed by adding 200µl of ELISA wash buffer (0.05M Tris, 0.138M NaCl, 0.0027 M KCl, 0.05% Tween-20 pH 8.0 at 25ºC) to each well and aspirated, with each wash repeated in triplicate. After washing, the wells of the ELISA plate were blocked with 200µl per well of 5% non-fat powdered milk in PBS at room temperature for two hours. After blocking, the plate was washed as described previously, and 100µl of serum diluted in ELISA dilution buffer (5% Bovine Serum Albumin, BSA; VWR ) was added to the wells of the plate and incubated for one hour at room temperature on an orbital shaker (Roto mix, Thermolyne). After the primary antibody incubation, the plate was once again washed as previously described, and 100µl of goat anti-mouse IgG (H+L) (Cell Signaling Technologies, Massachusetts, USA) at a 1:2,000 dilution and incubated for one hour at room temperature on an orbital shaker. Next, the plate was again washed, and 100µl of chemiluminescent TMB substrate (BioLegend, California, USA) was added to each well, and optical density measured at OD=640nm in a plate reader (Spectramax ® M2, Molecular Devices).

[0242] Infected cell lysate ELISA. Serum anti-E IgG antibody reactivity was measured against HCoV-229E infected human MRC5 cell lysates or uninfected control cell lysates.

[0243] Cell lysates were prepared by growing MRC5 cells in Eagles Minimum Essential Medium (EMEM) (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Bio Whittaker, 67 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT Walkersville, Maryland) and incubated at 37ºC and 5% CO2.Once the cells reached 80% confluency, they were infected with HCoV-229E at an MOI= 1 or mock-infected with 1X PBS. After 7 days, cytopathic effect (CPE) was observed. Both infected and mock-infected MRC5 cells were subjected to 3 freeze / thaw cycles, quantified by measuring absorbance at 280nm in a spectrophotometer, and stored at -80ºC.96- well Medisorp ELISA plates (Thermo Fisher) were coated with 50µg / mL of cell lysate diluted in coating buffer (50mM carbonate-bicarbonate buffer, 1.59g Na2CO3 + 2.93g NaHCO3 in 1L diH20, pH 9.4) and sensitized overnight at 4ºC. Next, plates were extensively washed by adding 200µl of ELISA wash buffer (0.05M Tris, 0.138M NaCl, 0.0027 M KCl, 0.05% Tween-20 pH 8.0 at 25ºC) to each well and aspirated, with each wash repeated in triplicate. After washing, the wells of the ELISA plate were blocked with 200µl per well of 5% non-fat powdered milk in PBS at room temperature for two hours. After blocking, the plate was washed as described previously, and 100µl of serum diluted in ELISA dilution buffer (5% Bovine Serum Albumin, BSA; VWR) was added to the wells of the plate and incubated for one hour at room temperature on an orbital shaker (Roto mix, Thermolyne). After the primary antibody incubation, the plate was once again washed as previously described, and 100µl of goat anti-mouse IgG (H+L) AP secondary antibody (Cell Signaling Technologies, Massachusetts, USA) was added at a 1:2,000 dilution and incubated for one hour at room temperature on an orbital shaker. Next, the plate was once again washed as previously described, and 100µl of chemiluminescent TMB substrate (BioLegend, California, USA) was added to each well, and optical density measured at OD=640nm in a plate reader (Spectramax ® M2, Molecular Devices).

[0244] Pseudovirion ELISA. Serum anti-E IgG antibody reactivity was measured against four commercially available hybrid alphavirus pseudovirions, which expressed all four SARS-CoV-2 structural proteins (Spike (S), Membrane (M), Nucleocapsid (N), Envelope (E)) (Virongy Biosciences; Manassas, VA, USA)

[0022] . The four pseudovirions tested differed only in the Spike protein sequences they expressed and corresponded to sequences from SARS-CoV-2 variants of concern: Beta (B1.351), Delta (B1.617), Omicron (B1.1.529), XBB (XBB.1). The HA-CoV-2 pseudovirions have been previously described in detail and validated for rapid quantification of neutralizing antibodies

[0022] . To measure anti- E IgG antibody reactivity, 96-well Medisorp ELISA plates (Thermo Fisher) were coated with 2000 PFU / mL of HA-CoV-2 Pseudovirions (Virongy Biosciences, Manassas, VA, USA) diluted in coating buffer (50mM carbonate-bicarbonate buffer, 1.59g Na2CO3 + 2.93g NaHCO3 in 1L diH20, pH 9.4) and sensitized overnight at 4ºC

[0022] . Next, plates were extensively washed by adding 200µl of ELISA wash buffer (0.05M Tris, 0.138M NaCl, 0.0027 M KCl, 0.05% Tween-20 pH 8.0 at 25ºC) to each well and aspirated, with each wash repeated in triplicate. After washing, the wells of the ELISA plate were blocked with 200µl per well of 5% non-fat powdered milk in PBS at room temperature for two hours. After 68 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT blocking, the plate was washed as described previously, and 100µl of serum diluted in ELISA dilution buffer (5% Bovine Serum Albumin, BSA; VWR ) was added to the wells of the plate and incubated for two hours at room temperature on an orbital shaker (Roto mix, Thermolyne)

[0022] . After the primary antibody incubation, the plate was once again washed as previously described, and 100µl of goat anti- mouse IgG (H+L) AP secondary antibody (Cell Signaling Technologies, Massachusetts, USA) was added at a 1:2,000 dilution and incubated for one hour at room temperature on an orbital shaker. Next, the plate was once again washed as previously described, and 100µl of chemiluminescent TMB substrate (BioLegend, California, USA) was added to each well, and optical density measured at OD=640nm in a plate reader (Spectramax ® M2, Molecular Devices)

[0022] .

[0245] Isolation of mouse splenocytes. Individual mouse splenocytes were isolated from freshly sacrificed animals and placed in a small sterile Petri dish with 5mL of Hanks Balanced Salt Solution (HBSS) (Sigma). In a sterile biosafety hood, the spleen was carefully minced into small pieces (~0.2cm) with a sterile razor blade before transferring the material to a 70µm cell strainer over a 50mL falcon conical tube. Using the plunger end of a sterile syringe, the material was homogenized through the cell strainer, and 10mL of sterile 1X PBS (Gibco) was used to wash the cell strainer. After collecting the homogenized spleen tissue, the suspension was centrifuged for 5 minutes (400-600 x g) at 4ºC. After centrifugation, the supernatant was discarded, and the cell pellet was resuspended in 5mL of sterile 1X Red Blood Cell (RBC) Lysis Buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) and incubated for 5 minutes on ice. After incubation, the cell suspension was centrifuged for 5 minutes (400-600 x g) at 4ºC and the supernatant was discarded. The cell pellet was then washed with 10mL of cold, sterile 1X PBS and centrifuged again. After the final centrifugation, the supernatant was discarded, and the splenocyte cell pellet was resuspended in 2mL of freezing buffer (90% heat-inactivated Fetal Bovine Serum (∆FBS, Bio Whittaker; Walkersville, Maryland) and 10% DMSO). The 2mL volume was separated into two 1mL aliquots, flash frozen, and stored at -80ºC.

[0246] Antigen-specific T-cell assay. Interleukin 2 (IL-2) is an important cytokine with various immunological functions. IL-2 is highly expressed by T-cells following activation and can, therefore, serve as an indirect surrogate of T-cell activation. To measure the activation of splenocytes from vaccinated mice, we adapted a commercially available IL-2 ELISPOTPLUSassay (Mabtech, catalog #3441-4APW-2). In brief, a 96-well plate precoated with a mouse anti-IL2 monoclonal antibody was washed four times with sterile 1X PBS (200µl / well). After washing, the wells of the plate were conditioned with 200µl of RPMI media (Gibco; Montana, USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (∆FBS, Bio Whittaker; Walkersville, Maryland) and incubated for 30 minutes at room temperature. 69 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT

[0247] A hemocytometer (Reichert, USA) and 4% Trypan Blue stain (Sigma) were used to quantify the number of freshly thawed splenocytes and assess the overall viability of cells to be added to each well. After the total number of splenocytes in each sample was enumerated and diluted in RMPI media, the medium from the 96-well plate was removed, and fixed concentrations of splenocytes (2,500-750,000 cells per well) from immunized mice were added in each well with a fixed concentration of antigen (KLH=100µg, KLH-E=2µg, SARS-CoV-2 E peptide=1µg) or infected cell lysate (uninfected MRC5 cell lysate=200µg or HCoV-229E infected MRC5 cell lysate=200µg) for stimulation. A total volume of 200µl of the splenocyte / antigen / RPMI mixture was added to each well of the covered 96-well plate, stored in a 37ºC humidified incubator with 5% CO2,and incubated for 48 hours.

[0248] As additional assay controls, some wells contained only splenocytes (no exogenous antigen) or splenocytes treated with a 1:500 dilution of 500x Concanavalin A (Thermo Fisher). To detect IL-2 secreting foci after the 48 hour incubation, the plate was removed and washed five times with PBS (200µl / well). The detection antibody (5HA-biotin) was diluted to 1µg / mL in PBS containing 0.5% FBS and 100µl was added to each well and incubated for two hours at room temperature. After the primary antibody incubation, the plate was washed as previously described, and 100µl / well of the streptavidin- ALP secondary antibody (1:1,000 dilution in PBS containing 0.5% FBS) was added and incubated for one hour at room temperature followed by another wash as previously described. Lastly, 100µl of BCIP / NTB- plus substrate was added to each well of the 96-well plate and incubated until distinct foci were visible. After color development, the plate was washed extensively with 200µl / well of distilled water and allowed to dry. To enumerate foci, the spots in each well were counted manually using a dissection microscope. All T-cell activation experiments were performed in duplicate at each concentration and for each antigen tested. All enumeration of foci were confirmed by two independent observers for accuracy.

[0249] Serum neutralization of HA-CoV-2 pseudovirions. HEK293T (ACE2 / TMRPSS2) cells were obtained from Virongy Biosciences (Manassas, VA, USA) and maintained in DMEM+10%∆FBS (Bio Whittaker, Walkersville, Maryland)

[0022] . Cells were plated at a density of 2.5x104cells / well in 100µl volume of DMEM+10%∆FBS in a sterile 96-well tissue culture treated plate and allowed to adhere for 4 hours before infection. In a separate sterile 96-well plate, a 75µl reaction mixture was prepared of diluted test sera (15µl of diluted serum + 25µl pseudovirion + 35µl media), broadly neutralizing rabbit anti-RBD Spike antibody control 27VB1 (Virongy Biosciences) (1µl mAb + 25µl pseudovirion + 49µl media), or virus only (25µl pseudovirion + 50µl media) and incubated for 1 hour at 37ºC

[0022] .

[0250] After incubation, the media from the adherent cells was removed and replaced with the neutralization mixture and allowed to infect for 24 hours in a 37ºC incubator. After infection, the liquid in each well was removed with a multichannel pipette and discarded, followed by carefully washing each 70 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT well with 200µl of sterile 1X PBS to wash away excess assay components

[0022] . After washing, 20µl per well of 1X Promega Lysis buffer was added to each well and allowed to lyse for 5 minutes at room temperature. After lysis, 100µl of Luciferase assay reagent was added to each well of the 96-well plate and read using a plate reader (Spectramax ® M2, Molecular Devices). Neutralization activity was calculated by comparing the reduction in relative luciferase units (∆RLU) in wells containing test serum or neutralizing monoclonal antibody to wells containing pseudovirion and cells in the absence of antibody

[0022] .

[0251] Serum neutralization against SARS-CoV2-ΔORF3-E and human Coronavirus 229E. In a 24-well tissue culture plate (Genesee Scientific), 5x104cells were plated in 1mL of DMEM media (Gibco) supplemented with 10% FBS (Bio Whittaker, Walkersville, Maryland) and incubated at 37ºC for 3 days or until the cells in each well reached 90% confluency

[0024] . In a sterile PCR tube, 20µl of viral stock (1x105PFU / mL) or 2,000 PFU of virus were incubated with 20µl of test serum or media only as a negative control. The mixture of virus and serum was mixed and centrifuged for 30 seconds and incubated at 37ºC for 1 hour in a PCR thermal cycler (Peltier Thermal Cycler PTC-200, MJ Research). After incubation, the 40µl serum and virus mixture was added to each well of the 24-well tissue culture plate containing 960µl of fresh DMEM media supplemented with 10% FBS and incubated at 37ºC in a CO2incubator overnight

[0024] . After 24 hours of infection, the media in each well was replaced with 1mL of fresh DMEM media and cultured at 37ºC in a CO2incubator. The neutralizing activity and inhibition of viral infection by the sera were determined as described previously

[0024] .

[0252] Passive immunization and HCoV-229E challenge of SCID mice. SCID mice were passively immunized with 400µl of serum by tail vein injection. Five SCID mice were transfused with pooled sera from BALB / c mice receiving two doses of KLH-E vaccine and collected at 30 days post- immunization. As a controls, five SCID mice were transfused with either PBS alone or with pooled sera from BALB / c mice receiving two doses of aluminum vaccine and collected at 30 days post- immunization. Twenty-fout hours after transfusion, the SCID mice were intranasally infected with 1 X 105PFU / dose of HCoV-229E. Ten days after HCoV-229E challenge, animals were sacrificed and lung tissues were collected to measure viral titers, following the procedures as described previously

[0037] .

[0253] KLH-E vaccination and HCoV-229E challenge of BALB / c mice. Ten BALB / c mice were immunized with 50 µg of KLH-E per dose intramuscularly with 0.5mg Aluminum hydroxide salt in DMSO for two doses at 30-day intervals. As controls, ten BALC / c mice were administered intramuscularly with PBS alone or with 0.5mg Aluminum hydroxide salt in DMSO for two doses at 30- day intervals. At 30 days post second dose immunization, animals were intranasally infected with 1 X 10571 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT PFU / dose of HCoV-229E. Seven days after HCoV-229E challenge, animals were sacrificed and lung tissues were collected to measure viral titers, following the procedures as described previously

[0037] .

[0254] Statistical methods in ELIZA. The proportions of mice responding to the vaccine with serum antibody titers ≥1:8 in post-immunization sera were compared by Fishers exact test. For the calculation of geometric mean titers, titers below the lower limit of detection were assigned a value half of the lower limit (i.e., a titer of <1:8 was assigned a titer of 1:4), and the reciprocal titers were log10transformed. To determine whether the geometric mean serum antibody titers between the two independent groups of mice were different, we used a two-tailed Student’s t test of the log-transformed reciprocal titers. Differences with a probability of <0.05 (two-tailed) were considered significant.

[0255] Statistical Analysis. Experiments were conducted in triplicate and repeated for three independent assays. The analysis of variance (ANOVA) was applied to analyze the data, and p-values (< 0.05) were thought to be statistically significant.

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Claims

Attorney Docket No: 077734-000100WOPT What is claimed herein is:

1. A composition comprising an isolated core envelope peptide comprising the sequence of SEQ ID NO: 1 or a sequence with at least 80% sequence identity thereto.

2. The composition of claim 1, wherein the isolated core envelope peptide comprises the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto.

3. A composition comprising an isolated core envelope peptide comprising the sequence of one of SEQ ID NOs: 2-15 or a sequence with at least 80% sequence identity thereto.

4. The composition of any one of the preceding claims, wherein the isolated core envelope peptide does not comprise SEQ ID NO: 16 or a sequence with at least 80% sequence identity thereto.

5. The composition of any one of the preceding claims, wherein the isolated core envelope peptide does not comprise SEQ ID NO: 17 or a sequence with at least 80% sequence identity thereto.

6. The composition of any one of the preceding claims, further comprising an adjuvant.

7. The composition of claim 6, wherein the adjuvant is conjugated to a cysteine residue of the isolated core envelope peptide.

8. The composition of any one of claims 6-7, wherein the adjuvant comprises mannose.

9. The composition of claim 8, wherein the mannose is alpha-D-mannose.

10. The composition of any one of claims 8-9, wherein the mannose is conjugated to a lysine residue of the isolated core envelope peptide.

11. The composition of any one of claims 8-9, wherein the mannose is conjugated to the isolated core envelope peptide via a methyl carboxylic acid linkage.

12. The composition of any one of claims 6-7, wherein the adjuvant comprises KLH, BSA, or OVA.

13. The composition of claim 12, wherein the adjuvant comprises KLH.

14. The composition of any one of claims 12-13, wherein the KLH, BSA, or OVA is conjugated to a cysteine residue of the isolated core envelope peptide.

15. The composition of any one of the preceding claims, further comprising a pharmaceutically acceptable carrier.

16. A method of immunizing a subject, the method comprising administering to the subject a composition of any one of claims 1-15.

17. A method of stimulating an immune response of a subject, the method comprising administering to the subject a composition of any one of claims 1-15.

18. The method of any of the preceding claims, wherein the administration is by injection, subcutaneous injection, or mucosal administration. 77 4928-8667-5759.1 077734-000100WOPTAttorney Docket No: 077734-000100WOPT 19. The method of any of the preceding claims, wherein the injection is an intramuscular injection.

20. The method of any of the preceding claims, wherein the composition is administered twice.

21. The method of any of the preceding claims, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart.

22. The method of any of the preceding claims, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 2 months apart.

23. The method of any of the preceding claims, comprising a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least two weeks apart and no more than four weeks apart.

24. The composition of any one of claims 1-15 for use in a method of immunizing a subject.

25. The composition of any one of claims 1-15 for use in a method of stimulating an immune response of a subject.

26. The composition of any one of claims 24-25, wherein the composition is administered by injection, subcutaneous injection, or mucosal administration.

27. The composition of claim 26, wherein the injection is an intramuscular injection.

28. The composition of any one of claims 24-27, wherein the composition is administered twice.

29. The composition of any one of claims 24-28, wherein the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least one week apart.

30. The composition of any one of claims 24-28, wherein the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are no more than 2 months apart.

31. The composition of any one of claims 24-28, wherein the method comprises a first administration and at least a second administration, wherein the first administration and the at least a second administration are at least two weeks apart and no more than four weeks apart. 78 4928-8667-5759.1 077734-000100WOPT

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