Improved coronavirus vaccine

Abstract

To provide improved immunogens, vaccines, and methods for better prevention and treatment of the emerging coronavirus infections.SOLUTION: The present disclosure provides a glycoengineered SARS-CoV-2 spike protein which is capable of eliciting an enhanced immune response relative to a native spike protein of SARS-CoV-2 and its variants. The glycoengineered spike protein exposes the glycosylation sites and at the same time preserves the tertiary structure of the spike protein.SELECTED DRAWING: Figure 1-1

Description

[Technical field]

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 173,752, filed April 12, 2021, and U.S. Provisional Application No. 63 / 63 / 190,199, filed May 18, 2021, the contents of which are incorporated by reference in their entireties herein. [Background technology]

[0002] The Covid-19 outbreak has resulted in more than 3 million deaths worldwide since the first cases were reported in December 2019. Currently, several COVID-19 vaccines against SARS-CoV-2 infection are in clinical trial development, and some vaccines have been approved for human use. Although many licensed vaccines have shown high efficacy, genetic variants of SARS-CoV-2 have emerged and are circulating worldwide throughout the COVID-19 pandemic.

[0003] Therefore, improved COVID-19 vaccines are urgently needed to better prevent infection and / or reduce the severity of life-threatening coronavirus infections. Summary of the Invention [Problem to be solved by the invention]

[0004] Summary of the Disclosure The spike protein of SARS-CoV-2 is extensively glycosylated. The present disclosure is based on the recognition that immunization with modified SARS-CoV-2 spike proteins that lack or have a reduced glycan shield compared to the native spike protein of SARS-CoV-2 or its variants induces an enhanced immune response against SARS-CoV-2 and variants of concern (e.g., alpha, gamma, delta, and omicron). Twelve highly conserved epitopes (SEQ ID NOs: 1-12) located in the receptor binding domain (RBD) and subunit 2 (S2) containing the heptad repeat 2 (HR2) domain were identified based on an alignment of over 6 million S protein sequences from GISAID. Removal of the glycan shield by N-glycan trimming to better expose these highly conserved epitopes provides an effective approach to develop broadly protective vaccines against SARS-CoV-2 and variants. N-glycan trimming of spike protein can be achieved by in vitro glycoengineering, whereby the glycoengineered spike protein exposes highly conserved epitopes shielded by glycans while retaining the tertiary structure of the spike protein. Thus, the present disclosure provides improved immunogens, vaccines, and methods for better preventing and treating novel coronavirus (e.g., SARS-CoV-2) infections. [Means for solving the problem]

[0005] Thus, the present disclosure provides immunogens comprising a glycoengineered coronavirus spike protein comprising a plurality of truncated N-glycans and unmodified O-glycans (e.g., O-linked oligosaccharides). In some embodiments, the plurality of truncated N-glycans are located in the receptor binding domain (RBD), thereby exposing a plurality of highly conserved epitopes having the amino acid sequences of TESIVRFPNITNL (SEQ ID NO: 41), NITNLCPFGEVFNATR (SEQ ID NO: 42), LYNSASFSTFK (SEQ ID NO: 43), LDSKVGGNYN (SEQ ID NO: 44), KSNLKPFERDIST (SEQ ID NO: 45), KPFERDISTEIYQAG (SEQ ID NO: 46), and / or GPKKSTNLVKNKC (SEQ ID NO: 47). In some embodiments, the multiple truncated N-glycans are located in the heptad repeat 2 (HR2) domain, thereby exposing multiple highly conserved epitopes having the amino acid sequences of NCDVVIGIVNNTVY (SEQ ID NO: 48), PELDSFKEELDKYFKNHTS (SEQ ID NO: 49), VNIQKEIDRLNEVA (SEQ ID NO: 50), NLNESLIDLQ (SEQ ID NO: 51) and / or LGKYEQYIKWP (SEQ ID NO: 52).

[0006] In some embodiments, the multiple truncated N-glycans are located in the receptor binding domain (RBD) and heptad repeat 2 (HR2) domain, thereby exposing multiple highly conserved epitopes having the amino acid sequences of TESIVRFPNITNL (SEQ ID NO: 41), NITNLCPFGEVFNATR (SEQ ID NO: 42), LYNSASFSTFK (SEQ ID NO: 43), LDSKVGGNYN (SEQ ID NO: 44), KSNLKPFERDIST (SEQ ID NO: 45), KPFERDISTEIYQAG (SEQ ID NO: 46), GPKKSTNLVKNKC (SEQ ID NO: 47), NCDVVIGIVNNTVY (SEQ ID NO: 48), PELDSFKEELDKYFKNHTS (SEQ ID NO: 49), VNIQKEIDRLNEVA (SEQ ID NO: 50), NLNESLIDLQ (SEQ ID NO: 51), and / or LGKYEQYIKWP (SEQ ID NO: 52).

[0007] The glycoengineered coronavirus spike protein described herein comprises the amino acid sequence of SEQ ID NO:1, or a variant thereof having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1, or an immunologically active fragment of the amino acid sequence or variant.

[0008] In some embodiments, the glycoengineered coronavirus spike protein comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, wherein the polypeptide consists of 22 truncated N-glycans, each having a GlcNAc moiety.

[0009] In some embodiments, the glycoengineered coronavirus spike protein comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, wherein the polypeptide consists of 21 truncated N-glycans, each having a GlcNAc moiety.

[0010] In some embodiments, the truncated N-glycan is a monosaccharide, a disaccharide, or a trisaccharide. In some embodiments, the truncated N-glycan is a monosaccharide. In a preferred embodiment, the monosaccharide is N-acetylglucosamine (GlcNAc).

[0011] In a preferred embodiment, the truncated N-glycans described herein are substantially homogeneous. The term "homogeneous" is intended to mean a glycosylation pattern represented by one desired glycan species. The term "substantially homogeneous" as used herein is intended to mean that at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the glycoproteins present in the composition are represented by one desired glycoform (e.g., GlcNAc modification), with trace amounts of undesired glycoforms present in the composition. By "trace amounts" is intended that any given undesired glycoform present in the glycoprotein composition is present at less than 5%, preferably less than 4%, less than 3%, less than 2%, less than 1%, or even less than 0.5%, or even less than 0.1% of the total glycoprotein.

[0012] As described herein, the terms "spike protein" and "spike glycoprotein" and "coronavirus spike protein" are used interchangeably. The glycoengineered spike proteins described herein can be generated from native coronavirus spike proteins by glycoengineering (e.g., in vitro or in vivo glycoengineering). In some embodiments, the glycoengineered spike proteins are generated using one or more chemical or enzymatic methods. In some embodiments, the glycoengineered spike proteins are generated using Endoglycosidase H (Endo H).

[0013] In some embodiments, the native coronavirus spike protein described herein is the spike protein of severe acute respiratory syndrome coronavirus 2 (SAR-CoV-2) or a variant thereof. The SARS-CoV-2 described herein is the Wuhan strain of SARS-CoV-2 (hCoV / Wuhan / WH01 / 2019). Variants of SARS-CoV-2 described herein include, but are not limited to, D614G, alpha (B.1.1.7 and Q lineages), beta (B.1.351 and descendant lineages), gamma (P.1 and descendant lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), ​​kappa (B.1.617.1), 1.617.3, mu (B.1.621, B.1.621.1), zeta (P.2), delta (B.1.617.2 and AY lineages), and omicron (B.1.1.529 and BA lineages). In some embodiments, the native coronavirus spike protein is the spike protein of bat coronavirus RaTG13 or a variant thereof.

[0014] As used herein, the terms "native coronavirus spike protein," "native coronavirus spike glycoprotein," "native spike glycoprotein," and "native spike protein" are interchangeable.

[0015] In some embodiments, the glycoengineered spike proteins described herein exist as trimers (e.g., trimers in solution). The glycoengineered spike proteins described herein may retain the same tertiary structure as their native coronavirus spike proteins.

[0016] As described herein, a glycoengineered spike protein is capable of inducing an enhanced immune response compared to the native coronavirus spike protein, the enhanced immune response being an increased IgG titer, an increased IgM titer, an increased CD4 T cell response, an increased CD8 T cell response, an increased neutralization titer, or a combination thereof.

[0017] In another aspect, the present invention provides an immunogenic composition comprising (a) an immunogen of the present disclosure, and (b) optionally an adjuvant.

[0018] As described herein, adjuvants include, but are not limited to, aluminum hydroxide, aluminum phosphate, Freund's incomplete adjuvant (IFA), squalene, alum, Alhydrogel, MF59, QS-21, CpG 1018, AS03, AS37, Matrix-M, or combinations thereof.

[0019] The coronaviruses described herein may include SARS-CoV-2 and variants thereof, and may include bat coronavirus RaTG13 or variants thereof. In a preferred embodiment, the coronavirus infection is caused by SARS-CoV-2 and variants thereof.

[0020] As described herein, the immunogenic compositions can elicit an enhanced immune response compared to vaccines that use the native SAR-CoV-2 spike protein, thereby serving as improved COVID-19 vaccines against coronavirus infection caused by SAR-CoV-2 or its variants.

[0021] In another aspect, the present invention provides a method for inducing an immune response against SAR-CoV-2 or a variant in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic composition of the present invention.

[0022] In another aspect, the present invention provides a method for protecting a subject in need thereof from infection by SAR-CoV-2 or a variant, comprising administering to the subject an effective amount of an immunogenic composition of the present invention.

[0023] In another aspect, the present invention provides a method of preventing a subject in need thereof from contracting COVID-19 disease, comprising administering to the subject an effective amount of the immunogenic composition of the present invention.

[0024] In another aspect, the present invention provides a use of the immunogenic composition of the present invention for inducing an immune response against SARS-CoV-2 in a subject in need thereof.

[0025] In another aspect, the present invention provides a use of the immunogenic composition of the present invention for protecting a subject in need thereof from infection by SARS-CoV-2.

[0026] In another aspect, the present invention provides a use of the immunogenic composition of the present invention for preventing a subject in need thereof from contracting COVID-19 disease.

[0027] These and other aspects will become apparent from the following description of the preferred embodiments taken in conjunction with the following drawings, although variations and modifications may be effected without departing from the spirit and scope of the novel concepts of the present disclosure.

[0028] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0029] [Figure 1-1] (a)-(b). Design and characterization of glycoengineered spike protein with mono-GlcNAc-decorated (SMG) protein vaccine. (a) Schematic diagram of recombinant SARS-CoV-2 spike glycoprotein construct. Protein domains are depicted as N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), central helix (CH), and linking domain (CD). The C-terminus of the soluble spike protein is joined to a sequence fold and His tag (His6). The furin cleavage site is replaced with a GSAG (SEQ ID NO:9) residue, and two proline mutations (K986P and V987P) lock the spike protein in the prefusion state. The positions of N-linked glycosylation sequons (NXS / T, where X≠P) are shown as branches (N, Asn; X, any residue; S, Ser; T, Thr; P, Pro). The pound sign sites indicate thrombin cleavage sites. (b) Schematic of mono-GlcNAc decorated spike vaccine production. SHM, spike containing high mannose type N-glycans. SMG, spike with GlcNAc at the N-glycosylation site. [Figure 1-2] (c) Design and characterization of glycoengineered spike protein from mono-GlcNAc-decorated (SMG) protein vaccine. (c) Size-exclusion chromatography of purified SMG. The black curve represents SMG and the grey curve represents protein molecular weight markers. [Figure 1-3] (d)-(e). Design and characterization of glycoengineered spike proteins from mono-GlcNAc-decorated (SMG) protein vaccines. (d) SDS-PAGE analysis of SFG (spike containing typical complex N-glycans), SHM and SMG. (e) Structural model of SMG. The model was generated with Protein Data Bank (PDB) ID code 7CN9 by adding glycans using Wincootmand displayed in the program ChimeraX. [Figure 1-4](f) Design and characterization of glycoengineered spike proteins from mono-GlcNAc-decorated (SMG) protein vaccines. (f) Mass spectrometry analysis of the N-glycan composition of SFG and SMG. [Figure 2-1] (A)-(C). SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (A) Immunization schedule for Syrian hamsters: SFG (blue), SMG (red), and control (gray). (B) Body weight change was measured in Syrian hamsters after WT SARS-CoV-2 antigen challenge. (C) Lung viral titers in antigen-challenged hamsters. The dashed line indicates the lower limit of detection. [Figure 2-2] (D). SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (D) Representative images show histopathology, immunohistochemistry, and immunofluorescence (3 dpi) of lungs of infected hamsters. 1st row: hematoxylin and eosin (H&E) staining; scale bar, 50 μm. 2nd row: immunohistochemistry (IHC) staining; scale bar, 50 μm. 3rd row: immunofluorescence (IF) staining; scale bar, 100 μm. A SARS-CoV-2 N-specific polyclonal antibody was used for virus detection as brown dots in IHC and green dots in IF staining. Blue: 4,6-diamidino-2-phenylindole (DAPI). [Figure 2-3] (E). SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (E) Immunization schedule for CAG-hACE2 or K18-hACE2 transgenic mice. [Figure 2-4] (F)-(I). SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (F)-(I) Anti-S IgG titers (F), SARS-CoV-2 WT microneutralization titers (G), and subtype IgG analysis including IgG1, IgG2c (H), and IgG2c:IgG1 ratio (I) are shown for serum samples collected from immunized CAG-hACE2 transgenic mice (n=7). [Figure 2-5](J). SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (J) Representative histopathology, immunohistochemistry, and immunofluorescence of lungs from infected mice (7 dpi) are shown. Scale bars are the same as in (D). [Figure 2-6] (K)-(M). (K)-(M) SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (K) Lung viral titers in infected CAG-hACE2 mice (n=3). The dashed line indicates the lower limit of detection. (L)-(M) Body weight change (L) and survival analysis (M) are shown for CAG-hACE2 transgenic mice (n=4) challenged with WT-SARS-CoV-2. [Diagram 2-7] (N)-(O). SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (N)-(O) Body weight change (N) and survival analysis (O) are shown for CAG-hACE2 transgenic mice (n=5) challenged with SARS-CoV-2 alpha variants. [Figure 2-8] (P)-(Q). SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (P)-(Q) Body weight change (P) and survival analysis (Q) are shown for CAG-hACE2 transgenic mice (n=5) challenged with SARS-CoV-2 gamma variants. [Figure 2-9] (R)-(S). SMG vaccination provides enhanced protection against SARS-CoV-2 infection in vivo. (R)-(S) Body weight change (R) and survival analysis (S) are shown for K18-hACE2 transgenic mice (n=4) challenged with SARS-CoV-2 delta variants. Data are presented as mean ± SEM and analyzed by two-tailed Mann-Whitney U test to compare the two experimental groups. ns, not significant; *P<0.05. [Figure 3-1] (a). SMG vaccination provides better protection against lethal SARS-CoV-2 infection in transgenic hACE2 mice. (a) Immunization and antigen challenge schedule for transgenic mice. [Figure 3-2] (b)-(c). SMG vaccination provides better protection against lethal SARS-CoV-2 infection in transgenic hACE2 mice. (b) Sera collected 28 and 42 days after first vaccination were tested for anti-spike-specific IgG. (c) Sera collected 42 days after first vaccination were tested for neutralizing antibody titers by CPE assay. [Figure 3-3] (d)-(e). SMG vaccination provides better protection against lethal SARS-CoV-2 infection in transgenic hACE2 mice. (d) Body weight change of transgenic mice after antigen challenge (n=3). (e) Survival rate of transgenic mice after antigen challenge (n=3). Data are mean ± SEM (standard error of the mean). Comparisons were performed by Student's t-test (unpaired, two-tailed). Binding and fold neutralization increases of antibodies elicited by SMG against SFG vaccine are shown by numbers above the bars. Blue squares, transgenic mice immunized with SFG; red triangles, transgenic mice immunized with SMG; grey dots, control animals (alum only group). [Figure 4-1] (a). Antibodies induced by SMG vaccine in mice to enhance breadth and potency of binding and neutralization against SARS-CoV-2 variants. (a) Schematic diagram of SARS-CoV-2 spike protein structure and the mutational landscape of the variants used in this study. RBD, receptor binding domain. In the mutation map, a dot (.) indicates an amino acid at that position that is the same as the wild type, and a dash (-) indicates a deletion. [Figure 4-2] (b). Antibodies induced by SMG vaccine in mice to enhance breadth and potency of binding and neutralization against SARS-CoV-2 variants. (b) SARS-CoV-2-specific IgG antibody titers were measured by ELISA. The fold increase in antibody binding induced by SMG against SFG vaccine is indicated by numbers above the bars. [Figure 4-3](c) Antibodies induced by SMG vaccine in mice to enhance breadth and potency of binding and neutralization against SARS-CoV-2 variants. (c) SARS-CoV-2 variant pseudovirus neutralization curves. [Figure 4-4] (d). Antibodies induced by SMG vaccine in mice to enhance breadth and potency of binding and neutralization against SARS-CoV-2 variants. (d) Titers to achieve 50% pseudovirus neutralization (pNT50) of SARS-CoV-2 variants are plotted. Fold neutralization increase of antibodies induced by SMG against SFG vaccine is indicated by numbers above the bars. [Figure 4-5] (e) Antibodies induced by SMG vaccines in mice to enhance breadth and potency of binding and neutralization against SARS-CoV-2 variants. (e) Neutralization curves of infectious SARS-CoV-2 variants as determined by plaque reduction neutralization test (PRNT). [Figure 4-6] (f). Antibodies induced by SMG vaccine in mice to enhance the breadth and potency of binding and neutralization against SARS-CoV-2 variants. (f) Titers to achieve 50% neutralization (PRNT50) of SARS-CoV-2 variants are plotted. Data are mean ± SEM (standard error of the mean). Curves were fitted by nonlinear regression using GraphPrism 9.0 and comparisons were performed by multiple t-tests (unpaired, two-tailed). Titer comparisons were performed by Student's t-test (unpaired, two-tailed). Fold-neutralization increase of antibodies induced by SMG against SFG vaccine is shown as numbers on the bars. Blue squares, transgenic mice immunized with SFG; red triangles, transgenic mice immunized with SMG; grey dots, control animals (PBS group). [Figure 5-1](A)-(B). S protein glycosylation influences ACE2 receptor binding and SARS-CoV-2 infection. (A)-(B) ACE2 binding activity was measured for differently glycosylated S protein ectodomains from BEAS-2B (A), HEK293T (B), and HEK293S (GnTI-) cells with or without Endo H digestion (C) (SFG; original fully glycosylated, blue; deS, non-sialylated, light blue; SHM, high mannose, yellow; SMG, mono-GlcNAc modified, red). Data from three technical replicates are shown as mean ± SD and curves are fitted by nonlinear regression of EC50 values. [Figure 5-2] (C)-(D). S protein glycosylation affects ACE2 receptor binding and SARS-CoV-2 infection. ACE2 binding activity was measured for differently glycosylated S protein ectodomains from BEAS-2B (A), HEK293T (B), and HEK293S (GnTI-) cells with or without Endo H digestion (C) (SFG; original fully glycosylated, blue; deS, non-sialylated, light blue; SHM, high mannose, yellow; SMG, mono-GlcNAc modified, red). Data from three technical replicates are shown as mean ± SD and curves are fitted by nonlinear regression of EC50 values. (D) Viral infectivity was measured for pseudoviruses with different glycosylated S proteins at the same input amount (0.3 μg / ml of p24 equivalent) colored as in (C). RLU, relative light units. Data from six technical replicates are presented as mean ± SD and analyzed by ordinary one-way ANOVA test followed by Tukey's multiple comparison test. ns, not significant; ****P<0.0001. [Figure 5-3](E). S protein glycosylation affects ACE2 receptor binding and SARS-CoV-2 infection. (E) Schematic diagram of SARS-CoV-2 S protein [wild type (WT)] colored by domains including the N-terminal domain (NTD; 14–306; orange), receptor binding domain (RBD; 319–541; red), two subdomains (SD1 / 2; 542–685; yellow), fusion peptide proximal region (FPPR; 816–856; green), heptad repeat 1 (HR1; 912–984; cyan), linking domain (CD; 1063–1162; blue), heptad repeat 2 (HR2; 1163–1211; purple), and transmembrane domain (TM; 1214–1234; white). The sites of N-glycans (depicted as branches) and O-glycans (circles) are numbered with residue numbers. The S1 and S2 domains are shown at the top. [Figure 5-4] (F)-(G). S protein glycosylation affects ACE2 receptor binding and SARS-CoV-2 infection. (F) Viral titers are shown for pseudoviruses carrying WT S protein or mutants with deleted glycans at each indicated glycosite, normalized by p24 quantification and colored according to (E). (G) Infectivity of the same panel of pseudoviruses as in (F) tested in five hACE2-expressing cell lines. Values ​​in (F) and (G) are normalized to the WT value (defined as 100%, colored dark grey) by the mean ± SD of three independent experiments. [Figure 6-1](A). S protein glycan profiles show differences in the two cell lines and correlate with sequence conservation. (A) Comparison of N-glycosylation profiles of recombinant S protein expressed from BEAS-2B lung epithelial cells. Glycans are grouped and colored according to: complex-S (sialylated complex type; dark blue), complex (non-sialylated complex type; light blue), hybrid-S (sialylated hybrid type; dark yellow), and hybrid (non-sialylated hybrid type; light yellow), high mannose (green), and unoccupied (gray). The percentage of each group for each glycosite is shown in a pie chart, and the proportion of each glycoform (numbered 1-27) is shown in a bar chart. Bar charts show the mean ± SD of three biological replicates. Hybrid indicates fucosylated hybrid type glycans. [Figure 6-2] (B). S protein glycan profiles show differences in the two cell lines and correlate with sequence conservation. (B) Comparison of N-glycosylation profiles of recombinant S protein expressed from HEK293T kidney epithelial cells. Glycans are grouped and colored according to: complex-S (sialylated complex type; dark blue), complex (non-sialylated complex type; light blue), hybrid-S (sialylated hybrid type; dark yellow), and hybrid (non-sialylated hybrid type; light yellow), high mannose (green), and unoccupied (gray). The percentage of each group for each glycosite is shown in a pie chart, and the proportion of each glycoform (numbered 1-27) is shown in a bar chart. Bar charts show the mean ± SD of three biological replicates. Hybrid indicates fucosylated hybrid type glycans. [Figure 6-3](C). S protein glycan profiles show differences in the two cell lines and correlate with sequence conservation. Glycan profiles from (A) and (B) were mapped onto the 3D structure of the S ectodomain (modeled from 6VSB). Glycans are colored by the most abundant group (complex, blue; hybrid, yellow; high mannose, green) for BEAS-2B (left) or HEK293T (right) data, as indicated. Non-complex N-glycosites are indicated by residue number. [Figure 6-4] (D)-(E). S protein glycan profiles show differences in the two cell lines, correlating with sequence conservation. (D) Mapping of the relative surface accessibility (RSA) of the modeled S structure protein, with buried residues colored dark yellow, shielded glycans colored yellow, and exposed ones colored light yellow. (E) Mapping of sequence variants of the modeled S protein structure, colored in a heatmap, with dark red indicating high mutation rates. Several highly conserved glycan shield regions are highlighted. Further details regarding (D) and (E) can be found in Figure S9 and Data File S1. [Figure 7-1] (A)-(B). SMG vaccination induces stronger humoral and cellular immune responses than SFG in BALB / c mice. (A) Structural models of SFG and SMG protein vaccines are shown (according to Fig. 2C). Blue: glycans; grey: protein. SFG was expressed by HEK293E without further modification. SMG was obtained by enzymatic digestion of HEK293S GnTI-expressed SHM to cleave all N-glycans to a single GlcNAc, whereas O-glycans were unmodified. (B) Immunization schedule using proteins as in (A) as immunogens in BALB / c mice (n=5 in each experiment). SFG (blue), SHM (yellow), SMG (red), and control (grey). Alum, aluminum hydroxide. [Figure 7-2](C)-(G). SMG vaccination induces stronger humoral and cellular immune responses than SFG in BALB / c mice. (C) Anti-S protein IgG titers of serum samples were analyzed by ELISA. (D) Neutralization titers of serum samples were measured using a pseudovirus containing the WT S protein. (E)-(G) IgG subtype analysis of serum, including IgG1 (E), IgG2a (F), and IgG2a:IgG1 ratio (G). [Figure 7-3] (H)-(N). SMG vaccination induces stronger humoral and cellular immune responses in BALB / c mice than SFG. (H)-(K) Percentage of Tfh among activated non-regulatory CD4 T cells (H) and percentage of IFN-γ (I), IL-4 (J), and IL-21 (K) expressing Tfh cells (CD4+CD19-CD44hiFoxp3-PD-1+CXCR5+) in lymph nodes (LN) of BALB / c mice by flow cytometry. (L) Percentage of granzyme B producing CD8+ T cells (CD3+B220-CD8+CD49b-) in LN of BALB / c mice analyzed by flow cytometry. (M) The percentage of S protein-specific B cells (CD3-CD19+S protein+) normalized to the fluorescence minus one (FMO) control staining (stained without S protein) in the spleen. (N) Kappa and lambda light chain usage is shown. [Figure 7-4] (O)-(P). SMG vaccination induces stronger humoral and cellular immune responses than SFG in BALB / c mice. (O)-(P) Heavy chain (O) and kappa chain (P) distribution from B cell repertoire analysis. Usage less than 5% is shown in white. [Figure 7-5](Q)-(S). SMG vaccination induces stronger humoral and cellular immune responses in BALB / c mice than SFG. (Q)-(S) Anti-S protein IgG titers (Q), pseudovirus neutralization titers (R), and authentic virus neutralization titers (S) are shown for sera isolated from BALB / c mice after three doses of the presented vaccines against SARS-CoV-2 WT (or D614G) and variants (numbers above each bar indicate the fold increase in SMG compared to the SFG group). pNT50 indicates the reciprocal dilution achieving 50% neutralization. The dotted line in the bar graph indicates the lower limit of detection. Data are presented as mean ± SEM and analyzed by two-tailed Mann-Whitney U test to compare the two experimental groups, except in (N), where five samples were pooled together and chi-square test was used. P values ​​are shown above each bar. *P<0.05; **P<0.01. [Figure 8-1] (A)-(C). Functional, prophylactic, and structural characterization of antibody m31A7 elicited by SMG vaccination suggests cross-neutralizing capacity. (A) ELISA binding of m31A7 to the entire S1, S2, RBD, or S ectodomain. (B) Flow cytometry analysis of m31A7 binding to HEK293T cells expressing SARS-CoV-2 WT and variant S proteins. (C) Neutralizing activity of m31A7 against pseudoviruses with WT or variant S proteins. Data from three technical replicates for (A), (B), and (C) are shown as mean ± SD and curves are fitted by nonlinear regression of EC50 values. [Figure 8-2] (D)-(F). Functional, prophylactic, and structural characterization of antibody m31A7 induced by SMG vaccination suggests cross-neutralizing capacity. (D) Antibody injection and antigen challenge schedule for K18-hACE2 transgenic mice (n=3). (E)-(F) Body weight change (E) and temperature change (F) for mice treated with m31A7 or PBS. Data are presented as mean ± SEM. [Figure 8-3](G). Functional, protective, and structural characterization of antibody m31A7 induced by SMG vaccination suggests cross-neutralizing capacity. (G) Binding kinetics of m31A7 IgG and Fab to S protein are shown, with dissociation constants (Kd) shown at the top. [Figure 8-4] (H). Functional, preventive, and structural characterization of antibody m31A7 induced by SMG vaccination suggests cross-neutralizing capabilities. (H) Time course epitope mapping of m31A7 by HDX-MS reveals >10% ΔHDX at 15 s for two peptide candidates 419–433 and 471–482. Data are presented as mean ± SD and analyzed by multiple t-test at each time point. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001. [Figure 8-5] (I)-(J). Functional, prophylactic, and structural characterization of antibody m31A7 induced by SMG vaccination suggests cross-neutralizing capabilities. (I) Cryo-EM maps fitted to the m31A7-Fab / S protein complex structure are shown. Heavy chain, dark green; light chain, light green; RBD, red; NTD, orange; remainder of S1, light grey; S2, dark grey; N-glycan, blue. (J) A close-up of the RBD-m31A7 interface is shown. The asterisk marks the vicinity between the m31A7 light chain and the N165-glycan. [Figure 8-6] (K)-(L). Functional, prophylactic, and structural characterization of antibody m31A7 induced by SMG vaccination suggests cross-neutralizing capabilities. (K) Superposition of previously reported mAbs S2E12 (magenta), COV253 (pink), and B1-182.1 (light blue) (PDB 7BEN, 7K4N, and 7MLZ) onto the m31A7-bound RBD (gray). The receptor binding motif and RBD tip are highlighted. (L) Footprint comparison of COV253 (pink) and m31A7 (green) on the RBD (gray) shows similarities, with VOC residues depicted as red spheres. [Figure 9-1](A)-(B). Delta-SMG vaccination elicited more neutralizing antibodies against SARS-CoV-2 variant pseudoviruses. (A) Scheme of vaccination. BALB / c mice (n=5) were immunized twice at weeks 0 and 2 with delta-SFG or SMG vaccines. Serum was collected at week 6 and subsequently tested for neutralizing capacity using pseudovirus assay. (B) Reciprocal neutralization titers (pNT50) of sera against SARS-CoV-2 variant pseudoviruses including wild type (WH01), and variants of concern (VOCs) including alpha, beta, gamma, delta, and omicron. Data are presented as mean (shown above each bar) ± SEM and analyzed by two-tailed Mann-Whitney U test to compare the two experimental groups. *P<0.05. [Figure 9-2] (C). Delta SMG vaccination induced more neutralizing antibodies against SARS-CoV-2 variant pseudoviruses. (C) Inhibition (%) of SARS-CoV-2 variant pseudovirus cell infection provided by different dilutions of serum. Data are shown as mean ± SEM and curves were fitted by nonlinear regression using Graph Prism 9.0. [Figure 10] (A)-(B). Delta or WT SMG vaccination induced more neutralizing antibodies against SARS-CoV-2 omicron variant pseudovirus. (A) Scheme of vaccination. BALB / c mice (n=5) were immunized twice with Delta / WT SFG or SMG vaccine at weeks 0 and 2. Serum was collected at week 6 and subsequently tested for neutralizing capacity using pseudovirus assay. (B) Inhibition (%) of SARS-CoV-2 variant pseudovirus cell infection provided by different dilutions of serum. Data are shown as mean ± SEM and curves were fitted by nonlinear regression using Graph Prism 9.0. [Figure 11-1](A)-(B). Delta SMG protein can be stored at room temperature in solution or powder. (A)-(B) Delta SMG protein in phosphate buffered saline additive containing two amino acids (pbs-aa), 50 mM L-arginine and 50 mM L-glutamic acid was filtered using a 0.22 μm filter and stored at room temperature (RT) or 4°C. Protein was harvested at various time points including 3 days, 7 days, 14 days, 21 days, and 3 months. The harvested samples were mixed with 5X SDS-PAGE Loading Dye, heated at 100°C for 5 minutes, and stored at 4°C until gel running. [Figure 11-2] (C). Delta SMG protein can be stored at room temperature in solution or powder. (C) Storage study of delta SMG protein in different buffers and lyophilization conditions. PBS with or without 50 mM L-arginine and 50 mM L-glutamic acid was tested for storage at 4°C of delta SHM or SMG (first three lanes). Delta SMG was lyophilized with or without excipients and stored at RT for more than 2 weeks (last two lanes). [Figure 12] Schematic diagram of highly conserved epitopes (E1, E2, E3, E4, E5, E6 and E7) present in the receptor binding domain (RBD) of SARS-CoV-2 spike protein, including the N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptapeptide repeat sequence 1 (HR1), heptapeptide repeat sequence 2 (HR2), transmembrane domain (TM), cytoplasmic domain (CD), and S2 subunit (SEQ ID NO: 41-47). [Figure 13] Schematic diagram of highly conserved epitopes (E8, E9, E10, E11, E12) present in heptapeptide repeat sequence 2 (HR2) of SARS-CoV-2 spike protein (SEQ ID NOs: 48-52). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In the following detailed description of the embodiments of the present disclosure, reference is made to the accompanying drawings, in which like references indicate like elements and which show, by way of example, specific embodiments in which the present disclosure may be practiced. These embodiments have been described in sufficient detail to enable those skilled in the art to practice the present disclosure, and it is to be understood that other embodiments may be utilized, and that logical, structural, functional, and other changes may be made without departing from the scope of the present disclosure. Therefore, the following detailed description should not be taken in a limiting sense.

[0031] All technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise defined below. References to technology used herein are intended to refer to technology commonly understood in the art, including variations of those technologies that would be expected to be obvious to those skilled in the art, or equivalent or alternatives to later developed technologies. In order to further more clearly and concisely describe the subject matter that is the present invention, the following definitions are provided for certain terms used in this specification and the appended claims.

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "protein" can refer to one protein or a mixture of such proteins, and reference to a "method" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0033] As used herein, the term "mutation" refers to a single change in the genome (genetic code) of a virus. Mutations occur frequently, but only occasionally change the characteristics of the virus.

[0034] As used herein, the term "lineage" refers to a group of closely related viruses that share a common ancestor. SARS-CoV-2 has many lineages; all cause COVID-19.

[0035] As used herein, the term "variant" refers to a viral genome (genetic code) that may contain one or more mutations. In some cases, a group of variants with similar genetic changes, such as a lineage or group of lineages, may be designated by public health agencies as variants of concern (VOC) or variants of interest (VOI) due to common attributes and characteristics that may require public health action.

[0036] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, enhances or alters or modifies the immune response elicited against the immunogen. Modification of the immune response can include enhancing or broadening the specificity of either or both the antibody and cellular immune responses.

[0037] As used herein, the term "about" or "approximately" preceding a numerical value indicates a range of plus or minus 10% from that numerical value. For example, "about 100" includes 90 and 110.

[0038] As used herein, an "immunogenic composition" is a composition comprising an antigen that, upon administration of the composition to a subject, develops in a subject a humoral and / or a cellular immune response against the antigen.

[0039] As described herein, the terms "spike protein" and "spike glycoprotein" and "coronavirus spike protein" are used interchangeably.

[0040] The term "substantially homogeneous" as used herein is intended to mean that at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the glycoproteins present in the composition are represented by one desired glycoform (e.g., mono-GlcNAc-modified), with trace amounts of undesired glycoforms being present in the composition. By "trace amounts" is intended that any given undesired glycoform present in the glycoprotein composition is present at less than 5%, preferably less than 4%, less than 3%, less than 2%, less than 1%, or even less than 0.5% or even less than 0.1% of the total glycoprotein.

[0041] The terms "treat," "treatment," and "treating," as used herein, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. For purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the onset or progression of an infection or disease; ameliorating the symptoms of, or reducing the onset of, an infection or disease; or a combination thereof.

[0042] The terms "prevent" and "prevention", as used herein, are used interchangeably with "prophylaxis" and can mean the complete prevention of an infection or the prevention of the onset of symptoms of the infection; a delay in the onset of an infection or its symptoms; or a reduction in the severity of an infection or its symptoms that subsequently occurs.

[0043] As used herein, an "effective amount" refers to an amount of an immunogen sufficient to induce an immune response that alleviates at least one symptom of a pathogen infection. An effective dose or amount can be determined, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), or microneutralization assay.

[0044] As used herein, the term "vaccine" refers to an immunogenic composition, such as a coronavirus-derived immunogen (with or without an adjuvant), that is used to induce an immune response against a coronavirus that provides protective immunity (e.g., immunity that protects a subject from infection with a coronavirus and / or immunity that reduces the severity of a condition caused by infection with a coronavirus). A protective immune response can include the formation of antibodies and / or a cellular response. Depending on the context, the term "vaccine" can also refer to a suspension or solution of immunogens administered to a subject to generate protective immunity.

[0045] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, a teenager, a child (ages 2-14), an infant (ages birth-2), or a newborn (up to 2 months). In certain embodiments, the subject is 4 months or younger, or 6 months or younger. In some embodiments, the adult is about 65 years or older, or about 60 years or older. In some embodiments, the subject is a pregnant woman or a woman who wishes to become pregnant. In other embodiments, the subject is not a human. For example, the subject is a non-human primate, such as a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject may be a pet, such as a dog or cat.

[0046] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the U.S. Federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other pharmacopoeias generally recognized for use in mammals, more particularly humans. These compositions may be useful as vaccine and / or antigen compositions for inducing a protective immune response in vertebrates.

[0047] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped, positive-sense, single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). Viral particles contain RNA genetic material and structural proteins required for entry into host cells. Once inside the cell, the infectious RNA is used to encode the structural proteins that make up the viral particle, the nonstructural proteins that direct viral assembly, transcription, replication, and host control, and accessory proteins whose functions have yet to be determined. The structural proteins of SARS-CoV-2 include the envelope protein (E), the spike or surface glycoprotein (S), the membrane protein (M), and the nucleocapsid protein (N). The spike glycoprotein has been identified on the outside of the viral particle and gives the coronavirus virus its crown-like appearance. This glycoprotein mediates attachment of the viral particle and entry into the host cell.

[0048] The S protein produced by lung epithelial cells has a glycoform that is associated with increased infectivity. Compared to the fully glycosylated S protein, the mono-GlcNAc decorated state (S MG Immunization with S protein bearing N-glycans trimmed to 100% glycan induced stronger immune responses and better protection of human angiotensin-converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOCs). Furthermore, broadly neutralizing monoclonal antibodies (BBAs) were identified that could neutralize wild-type SARS-CoV-2 and VOCs with subpicomolar potency. MG Identified from immunized mice.

[0049] Glycoengineered spike proteins of the present disclosure include a polypeptide having the amino acid sequence of SEQ ID NO: 1, or a variant having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, or an immunologically active fragment of that amino acid sequence or variant. The amino acid sequence of SEQ ID NO: 1 is shown below.

[0050] [ka]

[0051] In one embodiment, a glycoengineered spike protein of the disclosure comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 below. SEQ ID NO:2

[0052] [ka]

[0053] As described herein, glycoengineered spike protein of SARS-CoV-2 or variants thereof can include those that comprise an amino acid sequence that (i) is substantially identical to the amino acid sequence set forth in SEQ ID NO:1 (e.g., at least 90%, 95% or 97% identical to SEQ ID NO:1, such as SEQ ID NO:2); and (ii) is capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding a spike protein described herein, or is encoded by a nucleic acid sequence that is capable of hybridizing under at least moderately stringent conditions to any nucleic acid sequence encoding a spike protein described herein due to the use of synonymous codons (e.g., codons that do not have the same nucleotide sequence but encode the same amino acid).

[0054] Analysis of cell-specific glycoform distribution, sequence conservation, glycan shields, and their correlations revealed that essentially all glycan shields were removed from S MGThis led to the design of a vaccine. Glycosylation of the SARS-CoV-2 S protein has a profound effect on viral infection, protein integrity, and immune responses. S protein from lung epithelial cells contains more sialylated complex-type glycans to facilitate receptor binding, and glycosites N801 and N1194 were shown to be essential for S protein folding and viral infection. This results in better exposure of conserved epitopes to the immune system, and more effective and broadly protective B- and T-cell responses can be elicited against the virus and variants.

[0055] The disclosed glycoengineered colonovirus spike protein targets the entire S protein ectodomain, specifically the conserved domain shielded by glycans, and stimulates CD8 T cell responses that are important for eliciting both RBD and non-RBD neutralizing antibodies and cross-protection.

[0056] In certain aspects, the disclosure provides a vaccine or pharmaceutical composition comprising an immunogen described herein. The disclosure also provides a method of treating or preventing a coronavirus infection comprising administering to a subject (e.g., a mammal) in need thereof an effective amount of an immunogen, pharmaceutical composition, or vaccine described herein.

[0057] In one embodiment, the vaccine may include an adjuvant. Exemplary adjuvants include, but are not limited to, aluminum hydroxide, aluminum phosphate, Freund's incomplete adjuvant (IFA), squalene, alum, alhydrogel, MF59, QS-21, CpG 1018, AS03, AS37, Matrix-M, or combinations thereof.

[0058] The vaccine or pharmaceutical composition may be formulated using any suitable method. Formulation using standard pharma- ceutically acceptable carriers and / or excipients may be carried out using conventional methods in the pharmaceutical art. The exact nature of the formulation will depend on several factors, including the vaccine to be administered and the desired route of administration. Suitable types of formulations are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

[0059] The vaccines or pharmaceutical compositions described herein can be administered by any route. Such methods include, for example, parenteral application, such as application by any injection route, e.g., intramuscular, intravenous, intraperitoneal, intradermal, mucosal, submucosal, or subcutaneous, into or through the skin. They can also be applied by topical application as drops, sprays, gels, or ointments to the mucosal epithelium of the eyes, nose, mouth, anus, or vagina, or to the epidermis of the integument of any part of the body. Another possible route of application is by application of a spray, aerosol, or powder by inhalation via the respiratory tract. Alternatively, application can be via the gastrointestinal route. The effective amount of the vaccine composition can depend on any number of variables, including, but not limited to, the species, breed, size, height, weight, age, overall health of the patient, type of formulation, or mode or method of administration. An appropriate effective amount can be routinely determined by one of skill in the art using routine optimization techniques and the skilled and informed judgment of the skilled artisan, as well as other factors that will be apparent to one of skill in the art.

[0060] In one embodiment, the composition can include an additional therapeutic agent, such as an antiviral agent. The provided pharmaceutical compositions are useful for treating coronavirus infections. Examples of additional antiviral agents include, but are not limited to, ribavirin, penciclovir, nitazoxanide, nafamostat, chloroquine, remdesivir (GS-5734) and favipiravir (T-705), interferon, adefovir, tenofovir, acyclovir, brivudine, cidofovir, fomivirsen, foscarnet, ganciclovir, amantadine, rimantadine, zanamivir, remdesivir, molnupiravir, and paxlobid.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, and suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, shall govern. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting. EXAMPLES

[0062] [Example 1] Gene construct The SARS-CoV-2 spike sequence was designed based on gene sequences downloaded from the GISAID database. A total of 1,117,474 S protein sequences of all available SARS-CoV-2 strains were extracted from the GISAID (Global Initiative on Sharing Avian Influenza Data) database (version: April 18, 2021).

[0063] DNA sequences of spike proteins from SARS-CoV-2 Wuhan / WH01 / 2019 and delta variants were synthesized with codons optimized for human cell expression. The furin cleavage site was replaced with GSAG (SEQ ID NO: 9) and 2P substitutions were designed to maintain the protein in the prefusion state. The transmembrane domain was replaced with a thrombin cleavage site, a foldon, and a histidine tag at the C-terminus of the spike. The modified HA sequence was cloned into the pTT vector and the protein was expressed and purified.

[0064] Example 2: Expression and purification of SARS-CoV-2 S protein A plasmid encoding the secreted SARS-CoV-2 spike was transfected into HEK293EBNA (ATCC no. CRL-10852) or HEK293S GnTI cells by using a transfection reagent (either polyethylenimine or FectoPRO). - The cells were transfected into either human embryonic kidney cell line or human ES cell line and cultured in Freestyle 293 expression medium (Invitrogen) supplemented with 0.5% bovine serum. Supernatants were harvested 5 days after transfection and clarified by centrifugation. The spike protein was then purified by nickel chelate chromatography, and the eluted fraction was concentrated using a Millipore Amicon Ultrafilter (100 kDa) and loaded onto a Superpose™ 6 gel filtration column (10 / 300 GL; GE) pre-equilibrated with a Tris-based buffer (20 mM Tris / HCl, 20 mM NaCl, 50 mM glutamic acid, 50 mM arginine), and the corresponding trimer fraction was collected. The purified S HM was treated with Endo H (NEB) overnight at room temperature to obtain spike protein, spike mg To remove Endo H, S was extracted by buffer exchange using a Millipore-Amicon ultrafilter (100 kDa). MG The expression and purification methods were modified from existing work in our lab published in PNAS.

[0065] Generation of glycoengineered pseudoviruses in (Figure 1D) followed previous studies (Y. Watanabe, J.D. Allen, D. Wrapp, J.S. McLellan, M. Crispin, Site-specific glycan analysis of the SARS-CoV-2 spike. Science 369, pp. 330-333 (2020); Q. Yang, T.A. Hughes, A. Kelkar, X. Yu, K. Cheng, S. Park, W.-C. Huang, J.F. Lovell, S. Neelamegham, Inhibition of SARS-CoV-2 viral entry upon blocking N-and O-glycan elaboration. eLife 9, e61552 (2020)). To generate glycosite-specific S mutant pseudoviruses ( Fig. 1F ), HEK293T cells were transiently transfected with pVax-nCoV-SΔ19 constructs carrying mutations at each glycosite and a luciferase-expressing HIV-1 genome plasmid (pNL4-3.luc.RE).

[0066] [Example 3] S MG Protein characterization S MG To characterize the proteins, size-exclusion chromatography was performed using an ENrich™ SEC 650 (10×300 column; Biorad) pre-equilibrated with a Tris-based buffer (20 mM Tris / HCl, 20 mM NaCl, 50 mM glutamic acid, 50 mM arginine). To confirm the purity of the protein samples, the samples were then combined with loading buffer and separated on 7.5% SDS-PAGE and stained with Coomassie Brilliant Blue-Plus (EBL).

[0067] [Example 4] Glycopeptide analysis by mass spectrometry Two 20 μg aliquots of SARS-CoV-2 spike protein from two biological replicates were denatured for 1 h at 55 °C in 50 mM triethylammonium bicarbonate buffer containing 10 mM tris(2-carboxyethyl)phosphine. The spike protein was then reduced and alkylated by adding 18 mM iodoacetamide (IAA) and incubated in the dark for 30 min. The alkylated Env protein was digested separately using different combinations of chymotrypsin or alpha lytic protease in a ratio of 1:10 (w / w), or trypsin in a ratio of 1:20 (w / w). (Mass Spectrometry Grade, Promega). After overnight digestion, the samples were dried in a SpeedVac concentrator and processed for LC-MS / MS measurement. Glycan classification followed previous studies based on the composition detected and visualized by Graphpad Prism 9.0.0.

[0068] [Example 5] Vaccination experiment and antigen challenge experiment Female 6- to 7-week-old Golden Syrian hamsters (n=5) were treated with 25 μg of purified S mixed with 250 μg of aluminum hydroxide. FG Or S MG Hamsters were immunized intramuscularly with the protein on days 0 and 14. Blood was collected 28 and 42 days after the first immunization, and serum samples were collected from each hamster. ... 4 Hamsters were challenged intranasally with PFU of SARS-CoV-2 TCDC#4 (hCoV-19 / Taiwan / 4 / 2020, GISAID registration ID: EPI_ISL_411927) in a volume of 100 μL per hamster. The weight of each hamster was recorded daily after infection. On day 3 after challenge, the hamsters were euthanized by carbon dioxide. The right lung was examined for viral load determination (TCID 50The left lung was fixed in 4% paraformaldehyde for histopathological examination. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Academia Sinica.

[0069] Alternatively, for mouse vaccination with a two-dose schedule, female 6- to 8-week-old BALB / c mice (n = 5) were vaccinated with 10 μg of purified S mixed with aluminum hydroxide (50 μg). FG , S HM , or S MG Mice were immunized intramuscularly with 100 mg / kg / day on days 0 and 14. Sera were collected 28 days after the first vaccination to assess anti-S IgG levels, IgG subtypes, and neutralization titers (as described in Supplementary Materials and Methods). For T cell response analysis (as described in Supplementary Materials and Methods), S FG Or S MG Lymph nodes of immunized mice were harvested 21 days after the first vaccination. For B cell repertoire analysis and serum titers to variants, female 6-8 week old BALB / c mice (n=5) were immunized with 20 μg of purified S mixed with aluminum hydroxide (20 μg). FG Or S MG on days 0, 14, and 56. Mice were euthanized on day 84 and whole blood was collected for assessment of anti-S IgG and neutralizing titers, and spleens were harvested for sorting of S protein-specific B cells (as described in Supplementary Materials and Methods).

[0070] To study hamster vaccination and viral antigen challenge, male 6- to 7-week-old golden Syrian hamsters (n=5) were vaccinated with 25 μg of purified S. cerevisiae mixed with aluminum hydroxide (250 μg). FG Or S MG Hamsters were immunized intramuscularly with 1 × 10 IgG in 100 μl of PBS on days 0 and 14. Four weeks after the second immunization, each hamster was immunized intramuscularly with 1 × 10 IgG in 100 μl of PBS. 4 TCID 50Hamsters were challenged intranasally with SARS-CoV-2 (hCoV-19 / Taiwan / 4 / 2020). Body weights were recorded daily after infection. On day 3 after challenge, the hamsters were euthanized by carbon dioxide. The upper lobe of the left lung was fixed in 10% paraformaldehyde for histopathological examination, and the remaining lung was analyzed for viral load measurements (TCID 50 Assay).

[0071] For vaccination and viral antigen challenge studies of transgenic mice, male 6- to 8-week-old CAG-hACE2 transgenic mice or male 12-week-old K18-hACE2 transgenic mice (purchased from Jackson Laboratory) were immunized with 10 μg of purified S mixed with aluminum hydroxide (50 μg). FG Or S MG on days 0 and 14. CAG-hACE2 transgenic mice were immunized intramuscularly with 1 × 10 3 TCID 50 Mice were challenged intranasally with WT SARS-CoV-2. In the first study (n=3), all mice were euthanized at 7 dpi for histopathological examination of the upper lobe of the left lung. In the second study (n=7), three mice were euthanized at 4 dpi for lung viral titers and four mice were kept until 14 dpi for survival analysis. Serum was collected 1 day before viral challenge.

[0072] For antigen challenge studies using VOCs, CAG-hACE2 mice were challenged with 1 × 10 of either the alpha variant (hCoV-19 / Taiwan / 792 / 2020) (n=5) or the gamma variant (hCoV-19 / Taiwan / 906 / 2021) of SARS-CoV-2 in 50 μl of PBS per mouse. 3 TCID 50 Additionally, K18-hACE2 mice were challenged with 1 × 10 delta SARS-CoV-2 (hCoV-19 / Taiwan / 1144 / 2021) (n=4) in 50 μl PBS per mouse. 4 TCID 50and intranasally challenged 4 weeks after the second immunization. For all SARS-CoV-2 variant challenge models, the body weight of each mouse was recorded daily up to 14 dpi.

[0073] For antibody prophylactic protection studies, male 8-week-old K18-hACE2 transgenic mice (n=3) were intraperitoneally injected with m31A7 (15mg / kg) or PBS, followed by 1×10 3 TCID 50 Mice were challenged intranasally with WT SARS-CoV-2 (hCoV-19 / Taiwan / 4 / 2020). Body weight and temperature were recorded daily up to 5 dpi. All animal experiments were evaluated and approved by the Animal Care and Use Committee of Academia Sinica (approval numbers 21-10-1716, 18-12-1272, and 20-10-1522).

[0074] [Example 6] Histology and immunohistochemistry (IHC) staining Hamster lungs at 3 dpi were immediately harvested and placed in 10% neutral buffered formalin fixation for 24 h, followed by transfer to 70% ethanol for 72 h. Paraffin-embedded lung tissues were cut at a thickness of 5 mm. For histological staining, tissues were stained with hematoxylin and eosin (H&E). For immunohistochemistry (IHC) staining, sectioned tissues were deparaffinized in xylene and rehydrated in an ethanol gradient. Antigen retrieval was performed by heating slides in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven at 95°C for 10 min. After cooling at room temperature and washing with PBS, 3% H2O2 was applied to remove endogenous peroxidase activity. Sectioned tissues were blocked with 5% normal goat serum and 1% BSA in 1X PBST for 1 hour, followed by incubation with rabbit anti-N and anti-S primary antibodies (anti-SARS-CoV-2 polyclonal antibodies) at a dilution of 1:50 overnight at 4° C. Tissues were then incubated with goat anti-rabbit HRP secondary antibody at a dilution of 1:500 for 1 hour and visualized by incubation with 3,3-diaminobenzidine (DAB) substrate and counterstained with hematoxylin.

[0075] [Example 7] Immunofluorescence (IF) staining For immunofluorescence staining, after the antigen retrieval step, tissues were permeabilized with Triton X-100 in PBS. Sectioned tissues were blocked with 5% normal goat serum and 1% BSA in 1X PBST for 1 h. Then, they were incubated with autofluorescence quencher for 5 min. Subsequently, samples were incubated with rabbit anti-N and anti-S primary antibodies (anti-SARS-CoV-2 polyclonal antibodies) at 1:50 dilution overnight at 4 °C, incubated with secondary antibody Alexa Fluor-488 (1:500, Thermo Fisher) for 1 h at room temperature, and incubated with the nuclear dye 4,6-diamidino-2-phenylindole (DAPI) for 3 min at room temperature. Coverslips were mounted on microscope slides and imaged with a Leica TCS SP8X confocal microscope equipped with a HC PL APO CS210x / 1.40 lens (Leica, Wetzlar, Germany).

[0076] [Example 8] Vaccination experiment and antigen challenge experiment of transgenic mice Male 8-week-old CAG-hACE2 transgenic mice (n = 3) were treated with purified S mixed with 50 μg of aluminum hydroxide. FG Or S MG The transgenic mice were immunized intramuscularly with 10 μg of protein on days 0 and 14. Blood was collected on days 28 and 42 after the first immunization, and serum samples were taken from each transgenic mouse. Hamsters were immunized with 1×10 3 Mice were challenged intranasally with PFU of SARS-CoV-2 TCDC#4 (hCoV-19 / Taiwan / 4 / 2020, GISAID accession ID: EPI_ISL_411927) at a volume of 100 μL per mouse 6 weeks after the second vaccination. Body weight and survival rate were recorded for each transgenic mouse daily after infection. On day 7 after challenge, all transgenic mice were euthanized with carbon dioxide. Lungs were fixed in 4% paraformaldehyde for histopathological examination. All animal experiments were evaluated and approved by the Animal Care and Use Committee of Academia Sinica.

[0077] [Example 9] Quantification of viral titers in lung tissue by cell culture infection assay (TCID 50 ) Hamster mid, lower, and posterior lung lobes were homogenized in 600 μl of DMEM containing 2% FBS and 1% penicillin / streptomycin using a homogenizer. Tissue homogenates were centrifuged at 15,000 rpm for 5 min, and the supernatants were collected for live virus titration. Briefly, 10-fold serial dilutions of each sample were added in quadruplicate to Vero E6 cell monolayers and incubated for 4 days. Cells were then fixed with 10% formaldehyde and stained with 0.5% crystal violet for 20 min. Plates were washed with tap water and scored for infection. 50% tissue culture infectious dose (TCID 50 ) / mL was calculated by the Reed and Muench method.

[0078] Example 10: Mouse vaccination study Female 6- to 8-week-old BALB / c mice (n = 5) were administered purified S mixed with 20 μg of aluminum hydroxide. FG Or S MG They were immunized intramuscularly with 20 μg of protein on days 0, 14, and 56. Blood was collected on day 14 after the third immunization, and serum samples were collected from each mouse. All animal experiments were evaluated and approved by the Animal Care and Use Committee of Academia Sinica.

[0079] [Example 11] Evaluation of serum antibody titers Anti-S ELISA was used to determine serum IgG titers. Plates were blocked with 5% skim milk, and mouse polyclonal anti-S primary antibody and HRP-conjugated secondary antibody were added sequentially. Peroxidase substrate solution (TMB) and 1M H2SO4 stop solution were used, and absorbance (OD450nm) was read by a microplate reader. Strains tested included SARS-CoV-2 (wild type, variants B.1.1.7 and B.1.135), RnGT13, and SARS-CoV-1.

[0080] The m31A7 antibody was isolated by single B cell screening assay and then characterized. Primers were designed based on a previous publication (T. Tiller, CE Busse, H. Wardemann, Cloning and expression of murine Ig genes from single B cells. J. Immunol. Methods 350, pp. 183-193 (2009)). Polymerase chain reaction (PCR) was performed at 50°C for 30 min, 95°C for 15 min, followed by 40 cycles of incubation at 94°C for 30 s, 50°C for 30 s, 72°C for 1 min, and a final extension at 72°C for 10 min. Seminested second-round PCR was performed with 1 μl of unpurified first-round PCR product using KOD One PCR Master Mix (TOYOBO) at 98°C for 2 min, followed by 45 cycles of incubation at 98°C for 10 s, 55°C for 10 s, 68°C for 10 s, and a final extension at 68°C for 1 min. PCR products were then analyzed by electrophoresis and sequencing. Ig V and L genes were identified by the international ImMunoGeneTics information system (http: / / imgt.org / IMGT_vquest / input). Genes were then amplified from the second-round PCR products using single gene-specific V and L gene primers containing restriction sites for cloning into vectors containing human IgH or IgL expression backbones. Chimeric IgH and IgL expression constructs were co-transfected into Expi293 for antibody production. Following isolation of m31A7, the antibody was subsequently evaluated for S protein binding by ELISA and fluorescence-activated cell sorting, pseudovirus neutralization potency, binding kinetics, epitope mapping, and structure determination.

[0081] [Example 12] Pseudovirus neutralization assay To determine the infectious units of pseudotyped lentiviral vectors, we seeded 293T-ACE2 cells at appropriate density in 96-well (100 μL per well) tissue culture plates one day before infection. After overnight incubation (37° C., 5% CO2), 100 mL of three premixed pseudoviral supernatants and four-fold serial dilutions of immunized mouse serum were added to the seeded cells. The cells were incubated for 48 hours at 37° C. / 5% CO2 to allow expression of the nanoluciferase reporter gene. Luciferase activity was measured by an ELISA reader. Percent inhibition was calculated according to the following formula: 100*[1-(RLU) 試料 / RLU 偽性-処置 Data was analyzed using Graphpad Prism and pNT50 values ​​were calculated by taking the 50% inhibitory concentration values ​​for all samples.

[0082] [Example 13] Plaque reduction assay Vero E6 cells were seeded in DMEM containing 10% FBS and antibiotics in 24-well culture plates 1 day before infection. SARS-CoV-2 was incubated with antibodies for 1 h at 37 °C and then added to the cell monolayer for another 1 h. The virus-antibody mixture was then removed, and the cell monolayer was washed once with PBS and then covered with 1% methylcellulose-containing medium for 5–7 days. The cells were fixed overnight with 10% formaldehyde. After removing the overlay medium, the cells were stained with 0.7% crystal violet and plaques were counted. The percentage of inhibition was calculated as [1-(VD / VC)] × 100%, where VD and VC refer to the virus titer in the presence and absence of serum, respectively.

[0083] For the CPE-based neutralization assay, Vero E6 cells were plated at 2 × 105 cells / well in 6-well plates to 90% confluence overnight. Serum and virus were mixed and then added to the monolayer for an additional hour. Plates were allowed to solidify at room temperature for 30 minutes and then incubated at 37 °C until cytopathic effect (CPE) was observed.

[0084] Statistical analysis: All data are expressed as the mean ± standard error of the mean. In all analyses, P values ​​were obtained from Student's t-test (unpaired, two-tailed) except for curve comparisons, where Student's t-test (paired, two-tailed) was used. All graphs were generated with GraphPad Prism version 9.0.0 software.

[0085] [Example 14] Glycosylated mono-GlcNAc-modified spike (S MG ) Protein vaccine design and characterization Recombinant native with sequence (amino acids 14-1209) from the original SARS-CoV-2 Wuhan strain (hCoV / Wuhan / WH01 / 2019) is codon optimized for human cell expression, has a GSAG (SEQ ID NO: 9) residue replacing the original furin cleavage site and two proline mutations locking spike native in the prefusion state, has a foldon trimerization sequence and a His tag appended to its C-terminus, and is expressed in human HEK293S cells (Figure 1). Intermediate high mannose type spike recombinant native (S), in which all N-glycans are Man5 N-glycans, as indicated by branch symbols (Figure 1a), is expressed in human HEK293S cells (Figure 1b). HM ) was first obtained and purified. Then, endoglycosylase EndoH was used to remove excess glycans, generating the final product, monoglycosylated spike-native (Figure 1b). MG It is a highly pure trimer with an apparent molecular weight of about 520 kDa in solution (Fig. 1c and 1d). The size reduction by removal of N-glycans is comparable to that of normal S FG and intermediate S HM and analyzed by SDS-PAGE (Fig. 1d). MG Mass spectrometry analysis of showed that most N-glycosylation sites contain nearly 100% monosaccharide, N-acetylglucosamine (GlcNAc), except for N603 and N1194, which are more than 90% GlcNAc (Fig. 1f). In contrast, the original fully glycosylated spike native has heterologous N-glycans at all its N-glycosylation sites, including complex, hybrid, etc.

[0086] [Example 15] S MG Vaccine provides excellent protection against SARS-CoV-2 and variants in vivo S against SARS-CoV-2 MG To evaluate the in vivo protective efficacy of the vaccine, we first MG Or S FG WT SARS-CoV-2 antigen challenge was performed in Syrian hamsters vaccinated with S. MG Hamsters vaccinated with S. FG Although a smaller weight loss was observed in the rats treated with PBS compared with the rats treated with PBS (Fig. 2B), the S FG and S MGSimilar reductions in viral titers were observed in the lungs of both vaccinated hamsters (Figure 2C). Furthermore, histopathological staining and anti-nucleocapsid (N) protein immunostaining data showed fewer lesions in the lungs of immunized hamsters (Figure 2D). Because hamsters showed only mild to moderate pathology upon SARS-CoV-2 infection, we then tested highly susceptible CAG-hACE2 (C.-Y. Tsai, C.-Y. Chen, J.-T. Jan, Y.-C. Chou, M.-L. Chang, LA Lu, P.-Y. Huang, MFC Chul, T.-T. Hsu, Y.-P. Hsueh, Sex-biased response to and brain cell infection by SARS-CoV-2 in a highly susceptible human ACE2 transgenic model. bioRxiv, 2021) or K18-hACE2 (ES Winkler, AL Bailey, NM Kafai, S. Nair, BT McCune, J. Yu, JM Fox, RE Chen, JT Earnest, SP Keeler, JH Ritter, L.-I. Kang, S. Dort, A. Robichaud, R. Head, MJ Holtzman, MS Diamond, SARS-CoV-2 infection of human ACE2-transgenic mice causes severe lung inflammation and impaired function. Nat. Immunol.21, pp. 1327-1335 (2020) transgenic mice were used (Figure 2E). Analysis of anti-S IgG binding titers, neutralization titers, anti-S subtype IgG, and IgG2c:IgG1 ratios in CAG-hACE2 mice (Figure 2, F-I) all showed similar results to BALB / c mice (Figure 6, C-G). After intranasal challenge with WT SARS-CoV-2, the virus was detected by anti-N staining at 7 days post-infection (dpi) (Figure 2J) or median tissue culture infectious dose (TCID 50) assay (Fig. K), S FG and S MG Not detected in both lungs of vaccinated CAG-hACE2 mice (n=3), but at TCID 50 Viral titers of more than 1,000 were observed in the control group (Fig. 2K). MG Group (n=4) was treated with S at 14 dpi. FG The survival rate of CAG-hACE2 mice (75%) was better than that of alpha variants (50%) (Fig. 2, L and M). We then performed a quantitative analysis of the survival rate of CAG-hACE2 mice (n=5) against antigen challenge with alpha variants. MG The degree of protection conferred by vaccination was evaluated. MG We confirmed that vaccination resulted in a 100% survival rate up to 14 dpi (Fig. 2, N and O). MG Vaccinated mice showed a 60% survival rate upon gamma variant antigen challenge in CAG-hACE2 mice (n = 5) (Fig. S2P and Q) and a 75% survival rate upon delta variant antigen challenge in K18-hACE2 mice (n = 4) (Fig. S4R and S), but less than 50% survival rate upon S challenge. FG Vaccinated mice survived up to 14 dpi with gamma and delta variant antigen challenges (Figure 2, Q–S). MG The improvement in in vivo protection conferred by vaccination provides further evidence that removal of the glycan shield from immunogens is an advantageous strategy to elicit superior immune responses.

[0087] [Example 16] S MG Vaccination provides better protection against lethal SARS-CoV-2 infection in transgenic hACE2 mice CAG-hACE2 transgenic mice can develop severe disease and die when infected with the SARS-CoV-2 virus. FG , S MG The vaccine or adjuvant alone was administered intramuscularly twice on days 0 and 14, and serum was collected on days 28 and 42. 3 TCID 50Each mouse was intranasally infected with SARS-CoV-2 (Figure 3a). MG Spike-specific antibody IgG titers in immunized mice were FG 1.9 times higher than the S group (40,000 vs. 20,000). MG The neutralization titer of S. FG Surprisingly, in this severe disease model, S. cerevisiae was significantly more aggressive than S. cerevisiae, as evidenced by the rapid loss of body weight and death of all models by day 7 after viral infection. FG Vaccination did not protect CAG-hACE2 transgenic mice from severe disease. FG Disease progression in the vaccinated group was slightly better than in the adjuvant-only group (Fig. 3d and Fig. 3e). MG The vaccinated group showed minimal weight loss and all survived SARS-CoV-2 infection. This result supports the conclusion that SARS-CoV-2 severe disease in a transgenic mouse model of S. MG This shows the advantages of vaccination.

[0088] Example 17: S in mice to enhance breadth and potency of binding and neutralization against SARS-CoV-2 variants MG Vaccine-induced antibodies We then compared S. cerevisiae, which has the spike native sequence from the Wuhan strain, for its ability to neutralize newly emerged SARS-CoV-2 variants of concern. FG Or S MG We analyzed whether there were differences in the antibody responses induced by vaccination with either of the two vaccines (Figure 4). FG Compared to S MG Immunization elicited superior spike-specific antibody IgG that bound not only to spike native from the WT (Wuhan strain) but also to variants D614G, B.1.1.7, B.1.351, bat CoV RnGT13, and SARS-CoV-1 (Figure 4b). Pseudoviruses of SARS-CoV-2 variants were used to measure the ability to neutralize infection from vaccine-immunized sera. Again, S MGSera from the immunization had excellent neutralizing ability against D614G, B.1.1.7, and B.1.351 pseudoviruses, respectively, S FG 1.4-fold, 2.7-fold, and 1.5-fold superior to the induced sera (Figures 4c and 4d). Finally, SARS-CoV-2 real viruses D614G and B.1.1.7 were used in plaque reduction neutralization assays (Figures 4e and 4f). MG Vaccination is S. FG Compared to vaccination, it induced better neutralizing antibody responses against the D614G and B.1.1.7 variants, 2.0-fold and 1.4-fold better, respectively.

[0089] Example 18: Glycosylation influences pseudovirus S protein interaction with ACE2 on several cell types To understand the importance of glycosylation, we expressed S protein from lung epithelial cells, the primary cells for infection, and found that sialylation of S protein was required for high avidity to the receptor (Figure 5A). A similar pattern was observed for S protein produced in HEK293T cells (Figure 5B), and avidity was also reduced in the case of S protein containing only high-mannose glycans or in the form of glycoforms in which all N-glycans were trimmed to a single N-acetylglucosamine (GlcNAc) (Figure 5C). The effect of glycosylation was further tested by pseudovirus infection in HEK293T cells expressing human angiotensin-converting enzyme 2 (hACE2), and a consistent trend was observed when the same amount of virus was applied (Figure 5D). From this, it could be concluded that complex glycans and sialylation are functionally important for S protein-mediated infectivity. A full panel of 24 lentivirus-based pseudovirus variants (containing 22 N-glycosides and 2 O-glycosides) was also generated to evaluate the viral entry efficiency in five hACE2-expressing cell lines, including HEK293T, Vero-E6, and three human lung cell lines, A549, Calu-1, and Calu-3 (Figure 5, E-G). These pseudoviruses were based on the S construct with a C-terminal 19 amino acid deletion and showed the highest viral titers. Pseudovirus production was quantified by p24 immunoassay, and the results were normalized to the titer of each mutant (Figure 5F). All asparagines (Asn) in the N-glycosides were replaced with glutamines (Gln) to minimize the impact on the structure due to their chemical similarity, and each O-glycoside threonine (Thr) or serine (Srn) was replaced with alanine (Ala). As amino acids were changed by mutagenesis, the resulting changes in infectivity arise from multiple factors, including glycosylation-associated structural shifts that affect receptor binding, as well as surface abundance of the S protein, which is affected by protein expression, folding, and trafficking. Results demonstrated that disruption of S protein glycosylation reduced infectivity.Substantial reductions were observed with two mutations in the receptor binding domain (RBD), N331Q and N343Q, as well as two O-glycosite mutations, T323A and S325A, despite the lower occupancy of the latter (Figure 5G). Furthermore, loss of N122 glycosylation in the N-terminal domain (NTD) reduced infectivity and reduced protein expression (Figure 5G). Two NTD mutations, N149Q and N165Q, increased infectivity in Vero-E6 and Calu-1 cells, respectively, whereas reduced infectivity was observed in other cells (Figure 5G). It is noted that the glycan attached to this N165 residue is structurally proximal to the adjacent RBD in the trimeric S protein, and the mutation reduces ACE2 binding, likely due to a structural shift of the RBD to the "down" state. We identified two mutants, N801Q and N1194Q, that universally abolished virus infectivity in all five cell lines (Figure 5F). Glycosite N801 is located near the fusion peptide proximal region (FPPR), and N1194 is near the center of the heptad repeat 2 (HR2) helix and is the last N-glycosite preceding the transmembrane domain (Figure 5E). Both of these mutants caused low-yield expression. The N801Q mutant was more prone to degradation, and the N1194Q mutant inhibited trimerization of the S protein, which may be one of the factors explaining the reduced infectivity by pseudoviruses carrying these mutations.

[0090] Example 19: S protein from lung epithelial cells contains more sialylated complex-type glycans, and highly conserved epitopes in S protein are largely shielded by glycans Glycan profile analysis of S protein showed that S protein produced more complex glycans (78%) and less hybrid glycans (<1%) in human lung epithelial cell line BEAS-2B (Fig. 6A) compared with S protein produced in human kidney epithelial cell line HEK293T (61% and 23%, respectively) (Fig. 6B). Among high-mannose glycans, N-linked mannose-5 glycan (man5) was the predominant type found throughout HEK293T-expressed S protein, but was found only at N61 site in BEAS-2B cells. Furthermore, complex glycans at N74, N149, N282, and N1194 sites in BEAS-2B cells were more diversely processed (multiple antennary, galactosylated, fucosylated, or sialylated) than those in HEK293T cells. In contrast, glycans at sites N122, N331, N1098, and N1134 were less diverse. Furthermore, N149 and N17 did not have a core fucose in BEAS-2B. We observed that sialylation at all 22 N-glycosites in BEAS-2B (53%) was generally higher than that in HEK293T (35%), HEK293E (26%), or previously reported HEK293F cells (15%) (Y. Watanabe, J. D. Allen, D. Wrapp, JS McLellan, M. Crispin, Site-specific glycan analysis of the SARS-CoV-2 spike. Science 369, pp. 330-333 (2020)). In particular, two N-glycosites in the RBD (N331 and N343) are more sialylated in BEAS-2B (99% and 39%) than in HEK293T (49% and 15%). Despite the differences, the S proteins of all cell types contain a noncomplex glycan belt located near the central part of the S2 domain (Fig. 6C), where N-glycosite N801 is important for infection (Fig. 5G), N1074 contains diverse glycans, and N717 is essential for S protein expression.

[0091] From the modeled SARS-CoV-2 S protein structure and glycan profile from BEAS-2B cells, we performed a structural analysis of the glycan coverage on the protein surface region and superimposed the multiple alignment results using 1,117,474 S protein sequences (S. Elbe, G. Buckland-Merrett, Data, disease and diplomacy: GISAID's innovative contribution to global health. Global Chall.1, pp. 33-46 (2017)). Several regions were revealed that were highly conserved but shielded by glycans, including the lower flank of the RBD, the S2 stem region containing the non-complex glycan belt, and the C-terminal part of S2 involving the connecting domain (CD) and HR2 (Figure 6, D and E). At the primary sequence level, these regions were shown as conserved epitopes. Sequence conservation analysis also showed that most glycosite regions were highly conserved, and the most conserved (mutation rate <0.02%) included those in the NTD (N61, N122, N165, and N234), RBD (S325, N331, and N343), subdomain 1 / 2 (SD1 / 2) (N603 and N657), stem region of subunit 2 (S2) (N709, N1098, N1134, N1158, N1173, and N1194), and N801 near the FPPR. Most of these regions contained approximately 20–40% conserved surface residues, among which a certain percentage of residues were shielded by glycans, 36% in the RBD and approximately 50% in the other regions. The HR1 region, which does not have N-glycosides, had 69% conserved surface residues covered by glycans derived from adjacent domains. These results highlight the importance of S protein glycosylation both structurally and evolutionarily, and raise the idea that exposure of conserved regions shielded by glycans may elicit immune responses against conserved epitopes. (More importantly, it is important to account for the 12 conserved epitopes as vaccine targets. Ten of the 12 epitopes are shielded by glycans.)Removal of the shielded glycans exposes conserved epitopes and elicits a broad-based protective response.

[0092] [Example 20] S MG was developed as a vaccine, and S MG The vaccine used different antibody subclasses to induce a better immune response Our initial attempt to mutate multiple glycosites led to a significant reduction in expression of the S protein. However, when we expressed it from GnTI HEK293S cells, the high mannose glycoform of the S protein (S HM ) in good yield and purity. We then used endoglycosidase H (Endo H) to trim the glycans to a single GlcNAc at each N-glycosite, which we then used to produce S MG We generated a soluble trimeric mono-GlcNAc-modified S protein, designated S (Figure 7A). MG confirmed by mass spectrometry that all N-glycosites were nearly occupied by a single GlcNAc, and the occupancy of unprocessed O-glycans was too low to be detected. MG , S HM , and the original fully glycosylated S protein (S FG ) was mixed with aluminum hydroxide (alum) as an adjuvant and then used to immunize BALB / c mice (n=5) by intramuscular injection (Figure 7B). FGwas expressed by HEK293E cells and contained diverse glycans. This is consistent with the insect cell-expressed S protein vaccines from Sanofi and Novavax (PJ Klasse, DF Nixon, JP Moore, Immunogenicity of clinically relevant SARS-CoV-2 vaccines in nonhuman primates and humans. Sci. Adv.7, eabe8065(2021)) and Medigen's Chinese hamster ovary (CHO) cell-expressed recombinant S vaccine (T.-Y. Kuo, M.-Y. Lin, RL Coffman, JD Campbell, P. Traquina, Y.-J. Lin, LT-C. Liu, J. Cheng, Y.-C. Wu, C.-C. Wu, W.-H. Tang, C.-G. Huang, K.-C. Tsao, C. Chen, Development of CpG-adjuvanted stable prefusion SARS-CoV-2 spike antigen as a subunit vaccine against COVID-19. Sci. Rep.10, 20085 (2020)), the adenovirus-based vaccines from AstraZeneca and Johnson & Johnson, and the mRNA vaccines from Pfizer-BioNTech and Moderna (PJ Klasse, DF Nixon, JP Moore, Immunogenicity of clinically relevant SARS-CoV-2 vaccines in nonhuman primates and humans. Sci. Adv.7, eabe8065 (2021)). FG Protein and S MG Both proteins show essentially the same trimeric structure in solution by negative staining analysis.

[0093] S MG Mice immunized with S.FG Compared with the control group, the immunoglobulin G (IgG) titer against the S protein was 1.44-fold higher (endpoint titer: S FG , 39,408±1,619;S MG , 56,957 ± 5,091; P = 0.0079) (Figure 7C), and a 3.6-fold stronger antibody neutralization potency based on inhibition of SARS-CoV-2 pseudovirus infection (reciprocal half-maximal neutralization titer, pNT 50 :S FG , 1346±285;S MG , 4791 ± 767; P = 0.0159) (Fig. 6D), which induced superior humoral immune responses after the second immunization, whereas S HM The immune group was S FG showed similar anti-S IgG titers (39,086 ± 11,654) compared with the pNT group. 50 Analysis of IgG subtype titers and interferon-γ (IFN-γ) or interleukin-4 (IL-4) production by follicular helper T (Tfh) cells showed that S MG The vaccine is S. FG and S HM Compared with the vaccinated group, T helper 1 cells (T H 1) Induce more IgG2a, a lymphocyte marker, for a more balanced T H 1 / T H It was confirmed that the S. cerevisiae induced a 2-fold increase in the IFN-γ-expressing Tfh cells and a higher number of IFN-γ-expressing Tfh cells (Figure 7, E–J). MG The vaccine is designed to deliver a higher frequency of IL-21 + Induce Tfh cells (Figure 7K) and granzyme B-producing CD8 + These data suggest that the S FG Compared to those induced by S. MG It was suggested that the inflammatory response was induced by S. FG Or S MG S protein-specific B cells (CD3 - CD19 + S+ ) frequency was examined (Fig. 6A). MG It was confirmed that mice immunized with S generated more S protein-specific B cells (Figure 6M). FG Mice immunized with and S MG Analysis of the B cell repertoire from mice immunized with S FG Compared with those in the group, S MG It was shown that more lambda light chain genes were used in the S FG , 1.92%;S MG , 9.68%) (Figure 7N). Furthermore, antibodies derived from several specific loci of Ig heavy chain variable region (IGHV) (Figure 7O) and Ig kappa chain variable region (IGKV) genes (Figure 7P), especially IGHV1-18 genes (Figure 7O), were found to be S FG S rather than the group MG This finding suggests that B cell epitopes may be processed differently in these two groups, and whether and why this difference is immunologically beneficial requires further investigation. MG Vaccination with the wild-type (WT) S protein induced higher endpoint titers of IgG than two vaccinations with the wild-type (WT) S protein (Endpoint titers: S FG , 208,911 ± 50,092;S MG We found that the S protein was significantly higher in serum IgG binding curves measured by enzyme-linked immunosorbent assay (ELISA) against S proteins from SARS-CoV-2 VOCs, including alpha (B.1.1.7; P = 0.0488), beta (B.1.351; P = 0.0010), gamma (B.1; P = 0.0068), and delta (B.1.617.2; P = 0.0068). MG Groups and S FGAlthough we observed differences between groups (PR Krause, TR Fleming, IM Longini, R. Peto, S. Briand, DL Heymann, V. Beral, MD Snape, H. Rees, A.-M. Ropero, RD Balicer, JP Cramer, C. Munoz-Fontela, M. Gruber, R. Gaspar, JA Singh, K. Subbarao, MD Van Kerkhove, S. Swaminathan, MJ Ryan, A.-M. Henao-Restrepo, SARS-CoV-2 variants and vaccines. N. Engl. J. Med 385, 179-186 (2021)), there were no statistical differences in the endpoint titer analysis (Figure 7Q). Differences in neutralizing antibody responses to VOCs were also identified by pseudovirus neutralization curves, including alpha (P = 0.0156), beta (P = 0.0156), and delta (P = 0.0078), but no significant differences in pNTs for neutralization of pseudoviruses or authentic viruses. 50 The strength values ​​(Figure 7, R and S) are FG No differences were observed compared with

[0094] [Example 21] Broadly neutralizing antibodies against S MG isolated from B cells of mice immunized with S MG Selection of S protein-specific B cells from mice immunized with S MG We identified a monoclonal antibody (mAb) m31A7 from an IGHV1-18 amplified clone that is uniquely enriched in the B cell repertoire immunized with SARS-CoV-2 (Figure 8O). This mAb interacts with the full-length S protein, S1, and RBD, but not S2 (Figure 8A), and binds to HEK293T cells expressing S proteins from different SARS-CoV-2 variants (Figure 8B). Moreover, m31A7 exhibits sub-pM half maximal inhibitory concentrations (ICs) up to 1000-fold higher than the reported human mAb EY6A. 50) and was shown to neutralize various pseudoviral variants (WT, D614G, alpha, beta, and delta) (Figure 8C). Prophylactic studies also demonstrated good in vivo efficacy of m31A7 in K18-hACE2 mice (n=3) challenged with WT SARS-CoV-2 (Figure 8D). Prophylactically treated mice maintained both body weight and temperature (Figure 8, E and F). Using biolayer interferometry (BLI) analysis, the dissociation constants of m31A7 and its Fab binding to S protein were measured at 70.pM and 4.66nM, respectively (Figure 8G). Epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed its potential binding region on the RBD (Figure 8H), which overlapped with the epitope observed in the crystal structure of RBD in complex with m31A7-Fab. The cryo-electron microscopy (EM) structure further revealed that m31A7 binds only to the RBD in the "up" state (Figure 8I), and that the N165-glycan from the adjacent NTD is close to the RBD-m31A7 interface (Figure 8J). The footprint of m31A7 on the RBD resembles that of human VH1-58 class mAbs (Figure 8K), but approaches the RBD from a different angle, shifting the local contact area on the apical loop and bypassing most of the key mutated residues of VOCs, such as E484 and K417, but not T478 (Figure 8L). The detailed RBD-m31A7 interface and this S MG The inhibitory mechanism of the triggered mAbs is currently being further investigated.

[0095] [Example 22] Delta S MG Vaccination elicited more neutralizing antibodies against SARS-CoV-2 variant pseudoviruses SARS-CoV-2 delta variant S MG Protein expression constructs

[0096] SEQ ID NO:3

[0097] [ka]

[0098] Signal peptide from A / Brisbane / 59 / 2007 (H1N1): MKVKLLVLLCTFTATYAGT (SEQ ID NO: 4) Thrombin cleavage site: LVPRGS (SEQ ID NO:5) T4 foldon: PGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG (SEQ ID NO:6) HRV3C cleavage site: GSGSLEVLFQGP (SEQ ID NO:7) His Tag: HHHHHH (SEQ ID NO:8)

[0099] BALB / c mice (n=5) were cultured with Delta S FG Or S MG Subjects were immunized twice with the vaccine at weeks 0 and 2. Sera were collected at week 6 and subsequently tested for neutralization capacity using a pseudovirus assay (Figure 9A). A pseudovirus of the SARS-CoV-2 delta variant was used to measure the ability of vaccine-immunized sera to neutralize infection. Again, S MG Immune sera have excellent neutralizing capacity against WT, alpha, beta, gamma, delta, and omicron pseudoviruses. Delta S FG vs. S MG Their respective titers (pNT 50 The correlation coefficients (higher is better) were 1018 vs. 3336 (for WT pseudovirus), 1396 vs. 2680 (for alpha pseudovirus), 337 vs. 2282 (for beta pseudovirus), 814 vs. 4424 (for gamma pseudovirus), 1138 vs. 6680 (for delta pseudovirus), and 144 vs. 2628 (for omicron pseudovirus).

[0100] [Example 23] Delta or WT S MG Vaccination induced more neutralizing antibodies against SARS CoV 2 omicron variant pseudoviruses BALB / c mice (n=5) were cultured with delta / WT S FG Or S MGVaccine was administered twice at weeks 0 and 2. Sera were collected at week 6 and subsequently tested for neutralization capacity using a pseudovirus assay (Figure 10A). Pseudoviruses of the SARS-CoV-2 omicron variant were used to neutralize WT S FG / S MG or Delta S FG / S MG The ability of vaccine-immune sera from either WT or S FG Vaccination does not induce neutralization against Omicron pseudovirus, but WT S MG provides little protection. Similarly, Delta S FG Vaccination induces little neutralization against Omicron pseudovirus infection, but not against Delta S. MG provides excellent neutralization.

[0101] [Example 24] Delta S MG The protein can be stored at room temperature in solution or as a powder. Delta S in phosphate buffered saline supplement (pbs-aa) containing two amino acids, 50 mM L-arginine and 50 mM L-glutamate MG Proteins were filtered through a 0.22 μm filter and stored at room temperature (RT) or 4° C. Proteins were harvested at various time points including 3 days, 7 days, 14 days, 21 days, and 3 months. Harvested samples were mixed with 5× SDS-PAGE Loading Dye, heated at 100° C. for 5 minutes, and stored at 4° C. until gel electrophoresis (FIGS. 11A and B). PBS with or without 50 mM L-arginine and 50 mM L-glutamic acid was added to Delta S. HM Or S MG (first three lanes) were tested for storage at 4°C. Delta S MG The stability studies were performed using lyophilization with and without excipients and stored at RT for more than 2 weeks (last two lanes) (Figure 11C). MG This indicates that the protein can be stored in solution and is stable for at least 21 days at room temperature or 4°C. Furthermore, the delta S MGIt can be lyophilized and stored at RT and will remain stable for at least 3 months.

[0102] array SEQ ID NO:1

[0103] [ka]

[0104] SEQ ID NO:2

[0105] [ka]

[0106] SARS-CoV-2 delta variant S MG Protein expression constructs

[0107] SEQ ID NO:3

[0108] [ka]

[0109] Modified signal peptide from A / Brisbane / 59 / 2007(H1N1) MKVKLLVLLCTFTATYAGT (SEQ ID NO: 4)

[0110] Thrombin cleavage site LVPRGS (SEQ ID NO:5)

[0111] T4 foldon PGSGYIPEAPRDGQAYVRKDGEWVLLSTFLG (SEQ ID NO: 6)

[0112] HRV3C cleavage site GSGSLEVLFQGP (SEQ ID NO: 7)

[0113] His Tag HHHHHH (SEQ ID NO: 8)

[0114] Residues replacing the original furin cleavage site GSAG (SEQ ID NO: 9)

[0115] Highly conserved epitopes

[0116] [ka]

Claims

1. 1. An immunogen comprising a glycomodified coronavirus spike protein comprising a plurality of truncated N-glycans and one or more unmodified O-glycans, wherein the glycomodified coronavirus spike protein consists of the amino acid sequence of SEQ ID NO:1, or a variant thereof having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:1, or an immunologically active fragment thereof.

2. 2. The immunogen of claim 1, wherein the plurality of truncated N-glycans are monosaccharides.

3. The immunogen of claim 2, wherein the monosaccharide is N-acetylglucosamine (GlcNAc).

4. 2. The immunogen of claim 1, wherein multiple truncated N-glycans are located in the receptor binding domain (RBD) thereby exposing multiple highly conserved epitopes having the amino acid sequences of TESIVRFPNITNL (SEQ ID NO: 41), NITNLCPFGEVFNATR (SEQ ID NO: 42), LYNSASFSTFK (SEQ ID NO: 43), LDSKVGNYN (SEQ ID NO: 44), KSNLKPFERDIST (SEQ ID NO: 45), KPFERDISTEIYQAG (SEQ ID NO: 46), and / or GPKKSTNLVKNKC (SEQ ID NO: 47).

5. 2. The immunogen of claim 1, wherein multiple truncated N-glycans are located in the heptad repeat 2 (HR2) domain, thereby exposing multiple highly conserved epitopes having the amino acid sequences of NCDVVIGIVNNTVY (SEQ ID NO: 48), PELDSFKEELDKYFKNHTS (SEQ ID NO: 49), VNIQKEIDRLNEVA (SEQ ID NO: 50), NLNESLIDLQ (SEQ ID NO: 51) and / or LGKYEQYIKWP (SEQ ID NO: 52).

6. 2. The immunogen of claim 1, wherein the glycoengineered coronavirus spike protein is derived from a native coronavirus spike protein by in vitro glycoengineering.

7. 7. The immunogen of claim 6, wherein the native coronavirus spike protein is the spike protein of severe acute respiratory syndrome coronavirus 2 (SAR-CoV-2) or a variant thereof.

8. 8. The immunogen of claim 7, wherein the variant is selected from the group consisting of D614G, alpha (B.1.1.7 and Q lineages), beta (B.1.351 and descendant lineages), gamma (P.1 and descendant lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), ​​kappa (B.1.617.1), 1.617.3, mu (B.1.621, B.1.621.1), zeta (P.2), delta (B.1.617.2 and AY lineage), and omicron (B.1.1.529 and BA lineage).

9. The immunogen of claim 1 , wherein the glycoengineered coronavirus spike protein exists as a trimer.

10. The immunogen of claim 6 , wherein the glycosylated coronavirus spike protein retains the same tertiary structure as the native coronavirus spike protein.

11. The immunogen of claim 4, wherein the glycoengineered coronavirus spike protein is capable of eliciting an enhanced immune response compared to the native coronavirus spike protein.

12. 12. The immunogen of claim 11, wherein the enhanced immune response is an increase in IgG titer, an increase in IgM titer, an increase in neutralization titer, an increase in CD4 T cell response, an increase in CD8 T cell response, or a combination thereof.

13. 7. The immunogen of claim 6, wherein the glycoengineered spike protein is produced using one or more of chemical or enzymatic methods.

14. 14. The immunogen of claim 13, wherein the glycoengineered spike protein is produced using Endoglycosidase H (Endo H).

15. 2. The immunogen of claim 1, wherein the glycoengineered coronavirus spike protein comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, the polypeptide consisting of 22 truncated N-glycans each having a GlcNAc moiety.

16. 2. The immunogen of claim 1, wherein the glycoengineered coronavirus spike protein comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, the polypeptide consisting of 21 truncated N-glycans each having a GlcNAc moiety.

17. An immunogenic composition comprising: (a) an immunogen according to any one of claims 1 to 16; and (b) optionally an adjuvant.

18. 18. The immunogenic composition of claim 17, wherein the adjuvant is aluminum hydroxide, aluminum phosphate, incomplete Freund's adjuvant (IFA), squalene, alum, Alhydrogel, MF59, QS-21, CpG 1018, AS03, AS37, Matrix-M, or a combination thereof.

19. 20. The immunogenic composition of claim 17, which is an improved coronavirus vaccine capable of eliciting an enhanced immune response compared to a vaccine that uses a native coronavirus spike protein.

20. The immunogenic composition of claim 19, wherein the coronavirus is SAR-CoV-2 or a variant thereof.

21. 19. A method of inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a subject in need thereof, comprising administering to the subject an effective amount of the immunogenic composition of claim 17 or 18.

22. 22. The method of claim 21, wherein the immune response comprises the production of neutralizing antibodies against SARS-CoV-2 or a variant thereof.

23. 23. The method of claim 22, wherein the variant is selected from the group consisting of D614G, alpha (B.1.1.7 and Q lineages), beta (B.1.351 and progeny lineages), gamma (P.1 and progeny lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), ​​kappa (B.1.617.1), 1.617.3, mu (B.1.621, B.1.621.1), zeta (P.2), delta (B.1.617.2 and AY lineages), and omicron (B.1.1.529 and BA lineages).

24. 19. Use of the immunogenic composition of claim 17 or 18 for inducing an immune response against SARS-CoV-2 or a variant thereof in a subject in need thereof, comprising administering an effective amount of the immunogenic composition to a subject in need thereof.

25. 25. The use of claim 24, wherein the immune response comprises the production of neutralizing antibodies against SARS-CoV-2 or a variant thereof.

26. 26. The use of claim 25, wherein the variant is selected from the group consisting of D614G, alpha (B.1.1.7 and Q lineages), beta (B.1.351 and descendant lineages), gamma (P.1 and descendant lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), ​​kappa (B.1.617.1), 1.617.3, mu (B.1.621, B.1.621.1), zeta (P.2), delta (B.1.617.2 and AY lineages), and omicron (B.1.1.529 and BA lineage).

27. 19. Use of the immunogenic composition of claim 17 or 18 for protecting a subject in need of protection from infection by SARS-CoV-2 or a variant thereof, comprising administering an effective amount of the immunogenic composition to a subject in need of protection from infection by SARS-CoV-2 or a variant thereof.

28. 20. Use of the immunogenic composition of claim 17 or 18 for preventing COVID-19 disease in a subject in need thereof, comprising administering an effective amount of the immunogenic composition to a subject in need thereof.

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