RU2816182C2 - Advanced coronavirus vaccine - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: immunogen containing a glycoengineered coronavirus spike protein containing a plurality of truncated N-glycans and one or more unmodified O-glycans is described, wherein the glycoengineered coronavirus spike protein consists of the amino acid sequence SEQ ID NO: 1 or a variant thereof having at least 96% identity with the amino acid sequence of SEQ ID NO: 1, or an immunologically active fragment thereof. The following is proposed: an immunogenic composition for inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof in a subject in need thereof, containing an effective amount of the immunogen described. The following is disclosed: a method of inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof in a subject in need thereof, comprising administering to the subject an effective amount of the proposed immunogenic composition.

EFFECT: invention provides improved immunogens, vaccines and methods of more effectively preventing and treating novel coronavirus infections.

20 cl, 13 dwg, 22 ex

Description

Cross reference to related applications

This application has the benefit and benefit of U.S. Provisional Patent Application No. 63/173,752, filed April 12, 2021, and U.S. Provisional Patent Application No. 63/63/190,199, filed May 18, 2021, the contents of which are hereby incorporated by reference in their entirety. completeness.

Background to this Disclosure

The Covid-19 outbreak has killed more than three million people worldwide since the first case was reported in December 2019. Currently, several COVID-19 vaccines for the prevention of SARS-CoV-2 are in clinical trials, and some vaccines have been approved for medical use. Although a number of approved vaccines have demonstrated high efficacy, genetic variants of SARS-CoV-2 have been emerging and circulating around the world throughout the COVID-19 pandemic.

Thus, there is an urgent need for improved COVID-19 vaccines to better prevent novel coronavirus infections and/or to reduce the severity of life-threatening coronavirus infections.

Brief Description of the Subject of the Present Disclosure

The spike protein of SARS-CoV-2 is highly glycosylated. The present invention is based on the recognition that immunization with a modified SARS-CoV-2 spike protein, without or less glycan-shielded, produced an enhanced immune response against SARS-CoV-2 and its variants of concern (e.g., alpha, gamma, delta and omicron) compared to the native SARS-CoV-2 spike protein or its variant. 12 highly conserved epitopes (SEQ ID: 1-12) located in the receptor binding domain (RBD) and subunit 2 (S2), including the heptad repeat 2 (HR2) domain, were identified based on the alignment of over 6 million S protein sequences from GISAID. Removing glycan screens by removing N-glycans to better expose these highly conserved epitopes offers an effective approach to developing protective vaccines against SARS-CoV-2 and its broad-spectrum variants. Removal of spike protein N-glycans can be accomplished by in vitro glycoengineering techniques. Thus, the glycoengineered spike protein exposes highly conserved epitopes hidden by glycans while maintaining the tertiary structure of the spike protein. Thus, the present disclosure provides improved immunogens, vaccines, and methods for more effectively preventing and treating novel coronavirus (eg, SARS-CoV-2) infections.

Accordingly, the present invention provides an immunogen comprising a glycoengineered coronavirus spike protein containing a variety of truncated N-glycans and unmodified O-glycans (eg, O-linked oligosaccharides). In some embodiments, a plurality of truncated N-glycans are localized in the receptor binding domain (RBD), thereby exposing a large number of highly conserved epitopes having the amino acid sequences 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 accordance with some embodiments, multiple truncated N-glycans are localized in the heptad repeat 2 (HR2) domain, thereby exposing a large number of highly conserved epitopes having the amino acid sequences 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).

In accordance with some embodiments, a plurality of truncated N-glycans are localized in the receptor binding domain (RBD) and heptad repeat 2 (HR2) domain, thereby exposing a large number of highly conserved epitopes having amino acid sequences) TESIVRFPNITNL (SEQ ID NO.: 41) , NITNLCPF GEVFNATR (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: 51) and/or LGKYEQYIKWP (SEQ ID NO: 52 ).

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

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 of which contains a GlcNAc moiety.

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 of which contains a GlcNAc moiety.

In accordance with some embodiments, the truncated N-glycans are monosaccharides, disaccharides, or trisaccharides. In accordance with some embodiments, the truncated N-glycans are monosaccharides. According to preferred embodiments, the monosaccharides are N-acetylglucosamines (GlcNAc).

In accordance with preferred embodiments, the truncated N-glycans described herein are substantially homogeneous. The term “homogeneous” is intended to denote a glycosylation profile represented by a single desired glycan species. As used herein, "substantially homogeneous" means that at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, or at least 99% of the glycoprotein present in the composition is represented by one desired glycoform (eg, GlcNAc-decorated) with trace amounts of undesired glycoforms in the composition. By "trace amounts" is meant that any given undesired glycoform that is present in the glycoprotein composition is present in an amount of less than 5%, preferably less than 4%, less than 3%, less than 2%, less than 1% and even less than 0.5% or even less than 0.1% of the total glycoprotein.

As described herein, the terms “spike protein,” “spike glycoprotein,” and “coronavirus spike protein” are used interchangeably. The glycoengineered spike protein described herein can be produced from the native coronavirus spike protein through glycoengineering (e.g., in vitro or in vivo glycoengineering). In accordance with some embodiments, the glycoengineered spike protein is produced using one or more chemical or enzymatic methods. In some embodiments, the glycoengineered spike protein is created using endoglycosidase H (Endo H).

In some embodiments, the native coronavirus spike protein described herein is the severe acute respiratory syndrome coronavirus 2 (SAR-CoV-2) spike protein or variants thereof. The SARS-CoV-2 described herein is the Wuhan strain of SARS-CoV-2 (hCoV/Wuhan/WHO 1/2019). SARS-CoV-2 variants described herein include, but are not limited to, D614G, alpha (B.1.1.7 and Q lineages), beta (B.1.351 lineages and descendants), gamma (P.1 and descendants 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 (lines B. 1.617.2 and AY) and omicron (lines B. 1.1.529 and BA). In some embodiments, the native coronavirus spike protein is the bat coronavirus spike protein RaTG13 or variants thereof.

As described herein, the terms “native coronavirus spike protein,” “native coronavirus spike glycoprotein,” “native spike glycoprotein,” and “native spike protein” are used interchangeably.

In accordance with some embodiments, the glycoengineered spike protein described herein is present as a trimer (eg, a trimer in solution). The glycoengineered spike protein described herein may retain the same tertiary structure as its native coronavirus spike protein.

As described herein, the glycoengineered spike protein is capable of inducing an enhanced immune response compared to its native coronavirus spike protein. The enhanced immune response is an increased IgG titer, an increased IgM titer, an increased CD4 T cell-mediated cellular response, an increased CD8 T cell-mediated cellular response, an increased neutralization titer, or a combination thereof.

In accordance with another aspect, the present invention provides an immunogenic composition comprising: (a) an immunogen according to the present disclosure and (b) optionally an adjuvant.

As described herein, the adjuvant may include, but is not limited to, aluminum hydroxide, aluminum phosphate, incomplete Freund's adjuvant (IFA), squalene, alum, alhydrogel, MF59, QS-21, CpG 1018, AS03, AS37, Matrix-M, or their combination.

The coronavirus described herein may include SARS-CoV-2 and its variants, and may also include bat coronavirus RaTG13 or its variants. According to preferred embodiments, coronavirus infection is caused by SARS-CoV-2 and variants thereof.

As described herein, the immunogenic composition is capable of inducing an enhanced immune response compared to a vaccine using the native SAR-CoV-2 spike protein, thereby serving as an improved COVID-19 vaccine for the prevention of coronavirus infections caused by SAR-CoV-2 or a variant thereof.

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

In accordance with another aspect, the present invention provides a method of protecting a subject in need thereof from infection with SAR-CoV-2 or variants thereof, comprising administering to the subject an effective amount of an immunogenic composition of the present invention.

In accordance with another aspect, the present invention provides a method of preventing infection with COVID-19 in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic composition of the present invention.

In accordance with another aspect, the present invention provides the use of an immunogenic composition of the present invention to induce an immune response against SARS-CoV-2 in a subject in need thereof.

In accordance with another aspect, the present invention provides the use of an immunogenic composition of the present invention to protect a subject in need thereof from SARS-CoV-2 infection.

In accordance with another aspect, the present invention provides the use of an immunogenic composition of the present invention to prevent infection of a subject in need thereof with COVID-19.

These and other aspects will become apparent from the following description of preferred embodiments when used in conjunction with the following drawings, although changes and modifications are possible without departing from the spirit and scope of the new concepts of the invention.

Brief description of drawings

The following drawings form a part of this specification and are included to further demonstrate certain aspects of the present invention, the invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

Fig. 1 (a)-(f). Design and characterization of a glycoengineered spike protein mono-GlcNAc (S _MG ) decorated protein vaccine. (a) Schematic representation of the recombinant SARS-CoV-2 spike glycoprotein construct. Protein domains are shown as N-terminal domain (NTD), receptor binding domain (RBD), fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), central helix (CH) and junction domain (CD ). The C-terminus of the soluble spike protein is associated with a sequence fold and a His tag (His ₆ ). The furin cleavage site has been replaced by GSAG residues, and two proline mutations (K986P and V987P) fix the spike protein in a prefusion state. The positions of N-linked glycosylation sequences (NXS/T, where X ≠ P) are represented as branches (N, Asn; X, any residue; S, Ser; T, Thr; P, Pro). The recessed site represents the thrombin cleavage site, (b) Schematic overview of mono-GlcNAc spike protein vaccine production; _SHM , spike with high-mannose type N-glycans; S _MG , spike with GlcNAc at N-glycosylation sites, (c) Size exclusion chromatography of purified S _MG . The black curve represents S _MG and the gray curve shows protein molecular weight markers, (d) SDS-PAGE analysis of S _FG (a peak with common complex type N-glycans), S _HM and S _MG , (e) structural model of S _MG . Models were generated from Protein Data Bank (PDB) ID 7CN9 by glycan addition using Wincootmand and displayed using the ChimeraX program. (f) Mass spectrometric analysis of N-glycan compositions S _FG and S _MG .

Fig. 2 (A)-(S). S _MG vaccination provides improved protection against SARS-CoV-2 infection in vivo. (A) Shown is the immunization schedule for Syrian hamsters. S _FG (blue), S _MG (red) and control (gray). (B) Body weight change was measured in Syrian hamsters after challenge with WT SARS-CoV-2. (C) Lung virus titers from infected hamsters are shown. The dotted line indicates the lower limit of detection. (D) Representative images showing histopathology, immunohistochemistry, and immunofluorescence of infected hamster lungs (3 dpi). First row: hematoxylin and eosin (H&E) staining; scale bar, 50 µm. Second row: immunohistochemical (IHC) staining; scale bar, 50 µm. Third row: immunofluorescence (IF) staining; scale bar, 100 µm. N-specific polyclonal antibodies to SARS-CoV-2 were used to detect the virus, as shown by brown dots in IHC and green dots in IF staining. Blue color: 4,6-diamidino-2-phenylindole (DAPI). (E) Immunization schedule for CAG-hACE2 or K18-hACE2 transgenic mice is shown. (F - I) Anti-S IgG titers (F), SARS-CoV-2 WT microneutralization titers (G), and IgG subtype analysis including IgG1, IgG2c (H), and IgG2c:IgGl ratio (I) for serum samples are shown. collected from immunized CAG-hACE2 transgenic mice (n = 7). (J) Representative histopathology, immunohistochemistry, and immunofluorescence of lungs from infected mice are shown (7 dpi). Scale bars are the same as in (D). (K) Virus titers in the lungs of infected CAG-hACE2 mice (n = 3). The dotted line represents the lower limit of detection. (L and M) Body weight change (L) and survival analysis (M) are presented for CAG-hACE2 transgenic mice infected with WT-SARS-CoV-2 (n = 4). (N and O) Body weight change (N) and survival analysis (O) are presented for CAG-hACE2 transgenic mice infected with the alpha variant of SARS-CoV-2 (n = 5). (P and Q) Body weight change (P) and survival analysis (Q) are presented for CAG-hACE2 transgenic mice infected with the gamma variant of SARS-CoV-2 (n = 5). (R and S) Body weight change (R) and survival analysis (S) are presented for K18-hACE2 transgenic mice infected with the SARS-CoV-2 delta variant (n = 4). Data are presented as mean ± SEM and analyzed using two-tailed Mann-Whitney U tests for comparisons between two experimental groups, ns, not significant; *P<0.05.

Fig. 3(a)-(e). S _MG vaccination provides better protection against lethal dose of SARS-CoV-2 infection in hACE2 transgenic mice. (a) Immunization and challenge schedule for transgenic mice, (b) Sera collected 28 days and 42 days after initial vaccination were examined for the presence of specific IgG against the spike protein. (c) Sera collected 42 days after initial vaccination were tested for neutralizing antibody titers using the CPE assay. (d) Change in body weight of transgenic mice after infection. (n = 3) (e) Survival rate of transgenic mice after infection. (n = 3). Data represent mean ± SEM (standard error of the mean). Comparisons were made using Student's t test (unpaired, two-tailed). The increase in the fold of binding and neutralization of antibodies induced by S _MG vaccines compared to S _FG vaccines is indicated by the numbers above the bars. Blue squares are transgenic mice immunized with S _FG ; red triangles - transgenic mice immunized with S _MG ; gray dots, animals of the control group (the group that received only alum).

Fig. 4 (a) - (f). Antibodies induced by S _MG vaccine in mice to enhance binding and neutralization width and efficacy against SARS-CoV-2 variants. (a) Schematic representations of the SARS-CoV-2 spike protein structure and mutation landscape of variants used in this study are illustrated. RBD, receptor binding domain. On the mutation map, a dot (.) denotes the same amino acid at that position as the wild type, and a dash (-) denotes a deletion. (b) SARS-CoV-2-specific IgG antibody titer was determined by ELISA. The multiplicity of binding of antibodies induced by S _MG vaccines compared to S _FG vaccines is indicated by the numbers above the bars. (c) Pseudovirus neutralization curves of SARS-CoV-2 variants are shown. (d) The graph shows titers that achieve 50% pseudovirus neutralization (pNT50) of SARS-CoV-2 variants. The multiplicity of neutralization of antibodies induced by S _MG vaccines compared to S _FG vaccines is indicated by the numbers above the bars. (e) Neutralization curves of infectious SARS-CoV-2 variants determined by the plaque reduction neutralization test (PRNT) are shown. (f) Titers achieving 50% neutralization (PRNT50) of SARS-CoV-2 variants are plotted. Data are means ± SEM (standard error of the mean). Curves were fitted using nonlinear regression using GraphPrism 9.0, and comparisons were made using multiple t tests (paired, two-tailed). Comparison of titers was performed using Student's t test (unpaired, two-tailed). The increase in the fold neutralization of antibodies induced by S _MG vaccines compared to S _FG vaccines is indicated by the numbers above the bars. Blue squares are transgenic mice immunized with S _FG ; red triangles - transgenic mice immunized with S _MG ; gray dots, animals of the control group (group that received phosphate-buffered saline solution, PBS group).

Fig. 5 (A)-(G). S protein glycosylation influences ACE2 receptor binding and SARS-CoV-2 infection. (A-C) ACE2 binding avidity was measured for differentially glycosylated S protein ectodomains (S _FG ; native fully glycosylated, blue; deS , unsialylated, light blue; S _HM , high-mannose, yellow; and S _MG , mono-GlcNAc-decorated, red) from BEAS-2B (A), HEK293T (B), and HEK293S (GnTI) cells without or with Endo H cleavage (C). (WITH). Data from three technical replicates are shown as mean ± SD and curves fit nonlinear regression for EC50 values. (D) Viral infectivity was measured for pseudoviruses carrying differently glycosylated S protein with the same input amount (0.3 μg/ml p24 equivalent), colored accordingly as in (C). RLU, relative luminescence unit. Data from six technical replicates are shown as mean ± SD and analyzed using a conventional one-way ANOVA test followed by Tukey's multiple comparisons test, ns, not significant. **** P < 0.0001. (E) Schematic representation of the SARS-CoV-2 S protein [wild type (WT)] shown by the colored domain, 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; turquoise), junction domain (CD; 1063- 1162; blue), heptad repeat 2 (HR2; 1163-1211; purple), and transmembrane domain (TM; 1214-1234; white). N-glycan (indicated by branches) and O-glycan (circles) sites are indicated by residue numbers. The SI and S2 domains are shown above. (F) Virus titers are shown for pseudoviruses carrying the WT S protein or glycan-deleted mutants at each glycosylation site shown, normalized by p24 quantitation and colored accordingly as in (E). (G) Infectivity of the same panel of pseudoviruses as in (F) was tested in five hACE2-expressing cell lines. Values in (F) and (G) are normalized to WT values (defined as 100%, colored dark gray) with the mean ± SD of three independent experiments.

Fig. 6 (A)-(E). S protein glycan profiles show differences in the two cell lines and correlate with the degree of sequence conservation. (A and B) Shown is a comparison of the N-glycosylation profile of recombinant S protein expressed by BEAS-2B lung epithelial cells (A) and HEK293T kidney epithelial cells (B). Glycans are grouped and colored accordingly: 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 the pie chart, and the percentage of each glycoform (#1-27) is shown in the histogram. Histograms represent the mean ± SD of three biological replicates. F-hybrid means fucosylated hybrid type glycans. (C) Glycan profiles from (A) and (B) were compared to the 3D structure of the S ectodomain (modeled from 6VSB). Glycans are colored by the group with the highest abundance for BEAS-2B (left) or HEK293T (right) data as labeled (complex type - blue; hybrid type - yellow; and high-mannose type - green). Non-complex type N-glycosites are labeled by residue number. (D) Shown is a relative surface accessibility (RSA) mapping on a simulated S-structure protein, where closed residues are colored dark yellow, glycans are colored yellow, and open regions are colored light yellow. (E) Shown is a mapping of sequence variation onto a simulated S protein structure, colored in a heatmap fashion, with deeper dark red color indicating higher mutation rates. Several highly conserved regions shielded by glycans have been identified. Additional information for (D) and (E) can be found in FIG. S9 and in data file SI.

Fig. 7 (A)-(S). Vaccination with S _MG induces stronger humoral and cellular immune responses than S _FG in BALB/c mice. (A) Structural models of the SFG and SMG protein vaccines are shown (according to Fig. 2C). Blue: glycans; grey: protein. S _FG was expressed by HEK293E without further modification. S _MG was produced by enzymatic digestion to truncate all N-glycans of S _HM expressed by HEK293S GnTI 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 per experiment). S _FG (blue), S _HM (yellow), S _MG (red) and control (gray). Alum, aluminum hydroxide. (C) S protein IgG titers in serum samples were analyzed by ELISA. (D) Neutralization titers of serum samples were measured using wild-type S protein pseudovirus. (EG) Serum analysis for IgG subtype, including IgG1 (E), IgG2a (F), and IgG2a:IgG1 ratio (G). (H - K) Percentage of Tfh in 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 ^- CD44 ^hi Pohr3 ^- PD-1 ⁺ CXCB5 ⁺ ) in the lymph nodes (LN) of BALB/c mice by flow cytometry. (L) Percentage of granzyme B-producing CD8 ⁺ T cells (CD3 ⁺ B220 ⁻ CD8 ⁺ CD49b ⁻ ) in the LN of BALB/c mice analyzed by flow cytometry. (M) Shown is the ratio of S protein-specific B cells (CD3 ^- CD19 ⁺ S ^{protein +} ) (percentage) normalized to fluorescence minus one (FMO) control staining (stained without S protein) (percentage). in the spleen. (N) Use of kappa and lambda light chains is indicated. (O and P) Heavy (O) and kappa (P) chain distributions of B cell repertoire analysis. Less than 5% usage is highlighted in white. (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 administration of three doses of the indicated SARS-CoV- vaccines 2 WT (or D614G) and variants (the numbers above each bar indicate the fold increase in S _MG compared to the S _FG group). pNT ₅₀ represents the reverse dilution, achieving 50% neutralization. The dotted line in the histogram represents the lower limit of detection. Data are presented as mean ± SEM and analyzed using a two-tailed Mann-Whitney U test to compare two experimental groups, except in (N) where five samples were pooled together and the chi-square test was used. P values are shown above each bar. *P <0.05; **P < 0.01.

Fig. 8 (A)-(L). The functional, prophylactic and structural characteristics of the m31A7 antibody obtained by vaccination with S _MG indicate the ability to cross-neutralize. (A) Enzyme-linked immunosorbent assay (ELISA) of m31A7 binding to SI, S2, RBD, or the entire S ectodomain. (B) Flow cytometry analysis of m31A7 binding to HEK293T cells expressing SARS-CoV-2 WT S protein and variants. (C) Neutralizing activity of m31A7 against pseudoviruses carrying WT or S variant proteins. Data from three technical replicates for (A), (B), and (C) are shown as mean ± standard deviation (SD), and curves are fit to nonlinear regression for values EC ₅₀ . (D) Shown is the antibody injection and challenge schedule for K18-hACE2 transgenic mice (n = 3). (E and F) Change in body weight (E) and change in body temperature (F) are shown for mice treated with m31A7 or PBS. Data are presented as mean ± standard error of the mean (SEM). (G) The binding kinetics of m31A7 IgG and Fab to protein S are shown with the dissociation constants (Kd) shown above. (H) HDX-MS epitope mapping of m31A7 is shown as a function of time, revealing two candidate peptides, 419-433 and 471-482, with more than 10% AHDX at 15 s. Data are presented as mean ± SD and analyzed using multiple t tests at each time point. *P <0.05; **P <0.01; ***P <0.001; ****P < 0.0001. (I) Shown is a cryo-electron microscopy (cryo-EM) map corresponding to the structure of the m31A7-Fab/S protein complex. Heavy chain, dark green; light chain, light green; RED, red; NTD, orange; the remaining SI are light gray; S2, dark gray; and N-glycans, blue. (J) An enlarged view of the RBD-m31A7 interface is shown. The asterisk marks the proximity between the m31A7 light chain and the N165 glycan. (K) Overlay of previously reported mAbs S2E12 (magenta), COV253 (pink), and Bl-182.1 (cyan) (PDB 7BEN, 7K4N, and 7MLZ) on the RBD bound to m31A7 (gray). The receptor binding motif and RBD tip are highlighted. (L) Comparison of images of COV253 (pink) and m31A7 (green) on RBD (gray) shows similarity, VOC residues labeled and drawn as red spheres.

Fig. 9 (A)-(C). Delta S _MG vaccination revealed more neutralizing antibodies against SARS-CoV-2 variant pseudovirus. (A) Vaccination schedule. BALB/c mice (n=5) were immunized twice at week 0 and week 2 with Delta S _FG or S _MG vaccine. Sera were collected at week 6 and subsequently determined for neutralization capacity using a pseudovirus assay. (B) 50% reverse neutralization titer (pNT50) of serum against SARS-CoV-2 pseudovirus variant, including wild type (WH01) and variants of concern (VOC), including alpha, beta, gamma, delta, and omicron. Data were shown as mean (indicated above each bar) ± SEM and analyzed using a two-tailed Mann-Whitney U test to compare two experimental groups. *P < 0.05. (C) Inhibition (%) of SARS-CoV-2 variant pseudovirus infection of cells provided by differentially diluted serum. Data are presented as mean ± SEM and curves were fitted by nonlinear regression using Graph Prism 9.0.

Fig. 10 (A) and (B). Vaccination with Delta or WT S _MG induced more neutralizing antibodies against the SAR.S-CoV-2 Omicron variant pseudovirus. (A) Vaccination schedule. BALB/c mice (n=5) were immunized twice at week 0 and week 2 with Delta/WT S _FG or S _MG vaccine. Sera were collected at week 6 and subsequently tested for neutralization capacity using a pseudovirus assay. (B) Inhibition (%) of SARS-CoV-2 variant pseudovirus cell infection provided by differentially diluted serum. Data are presented as mean ± SEM and curves were fitted by nonlinear regression using Graph Prism 9.0.

Fig. 11 (A)-(C). Delta S _MG protein can be stored at room temperature in solution or in powder form. (A and B) Delta S _MG protein in phosphate buffered saline supplemented with two amino acids (pbs-aa), 50 mM L-arginine, and 50 mM L-glutamate was filtered through a 0.22 μm filter and stored at room temperature (RT). or at 4°C. Proteins were collected at different time points including 3, 7, 14, 21 days and 3 months. Collected samples were mixed with 5X SDS-PAGE Loading Dye, heated at 100°C for 5 minutes and stored at 4°C until a gel appeared. (C) Storage test of Delta S _MG protein in different buffers and lyophilization conditions. PBS with or without 50 mM L-arginine and 50 mM L-glutamate was tested for storage of delta S _HM or S _MG (first 3 lanes) at 4°C. Delta-SMG was tested for lyophilization with or without excipient and stored at room temperature for more than 2 weeks (last 2 lanes).

In fig. 12 shows a schematic representation of the highly conserved epitopes (E1, E2, E3, E4, E5, E6 and E7) (SEQ ID NO: 41-47) present in the receptor binding domain (RBD) of the SARS-CoV-2 spike protein containing 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 subunit S2.

In fig. 13 shows a schematic representation of highly conserved epitopes (E8, E9, E10, E11, E12) (SEQ ID NO: 48-52) present in the heptapeptide repeat 2 (HR2) sequence of the SARS-CoV-2 spike protein.

Detailed description

In the following detailed description of embodiments of the present invention, reference is made to the accompanying drawings, wherein like references indicate like elements and wherein by way of illustration specific embodiments are shown in which the present invention may be practiced. These embodiments have been described in sufficient detail to enable those skilled in the art to practice the present invention, and it is understood that other embodiments may be used and that logical, structural, functional and other changes may be made without departing from the scope of the present invention. Therefore, the following detailed description should not be construed in a limiting sense.

All technical and scientific terms used herein, unless otherwise defined below, have the same meaning as commonly understood by one skilled in the art. References to techniques used herein are intended to refer to techniques as they are generally understood in the art, including variations of these techniques or replacements for equivalent or later developed techniques that would be apparent to one skilled in the art. In addition, to provide a clearer and more concise description of the subject matter, certain terms that are used in the specification and appended claims are defined as follows.

As used herein and in the accompanying claims, the singular number includes references to the plural unless the context clearly indicates otherwise. Thus, for example, a reference to “protein” may refer to a single protein or a mixture of such proteins, and a reference to “method” includes reference to equivalent steps and/or methods known to those skilled in the art, etc.

As used herein, the term “mutation” refers to a single change in the viral genome (genetic code). Mutations occur frequently, but only sometimes change the characteristics of the virus.

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

As used herein, the term “variant” refers to a viral genome (genetic code) that may contain one or more mutations. Sometimes a group of variants with similar genetic changes, such as a lineage or group of lineages, may be flagged as a variant of concern (VOC) or variant of interest (VOI) by public health organizations due to common features and characteristics that may warrant action public health.

As used herein, the term “adjuvant” refers to a compound that, when used in combination with an immunogen, enhances or otherwise alters or modifies the immune response induced against the immunogen. Modification of the immune response may involve enhancing or expanding the specificity of one or both antibodies and the cellular immune response.

As used herein, the term “about” or “approximately” before a numerical value means a value plus or minus in the range of 10%. For example, "about 100" includes 90 and 110.

As used herein, the term “immunogenic composition” is a composition containing an antigen, wherein administration of the composition to a subject results in the subject developing a humoral and/or cellular immune response to the antigen.

As described herein, the terms “spike protein,” “spike glycoprotein,” and “coronavirus spike protein” are used interchangeably.

As used herein, the term "substantially homogeneous" means that at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%, of the glycoprotein present in the composition is represented by one desired glycoform (eg, mono-GlcNAc-decorated) with a trace amount of undesired glycoforms present in the composition. By "trace amount" is meant that any given undesired glycoform that is present in the glycoprotein composition is present in an amount of less than 5%, preferably less than 4%, less than 3%, less than 2%, less than 1% and even less than 0.5%, or even less than 0.1% of the total glycoprotein.

As used herein, the terms “treat,” “treat,” and “treat” mean an approach that produces useful or desired results, such as clinical results. For purposes of the present invention, beneficial or desired results may include inhibiting or suppressing the onset or progression of an infection or disease; relief or reduction in the development of symptoms of infection or disease; or a combination of the above.

Here, the terms "prevention" and "avoidance" are used interchangeably with the term "prevention" and can mean preventing an infection entirely or preventing the development of symptoms of that infection; delay in onset of infection or symptoms; or reduction in the severity of subsequent infection or its symptoms

As used herein, the term “effective amount” means an amount of immunogen sufficient to induce an immune response that reduces at least one symptom of a pathogenic infection. An effective dose or effective 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.

As used herein, the term “vaccine” means an immunogenic composition (with or without an adjuvant), such as an immunogen derived from a coronavirus, that is used to induce an immune response against the coronavirus that provides protective immunity (e.g., immunity that protects the subject from infection with the coronavirus and/or or reduces the severity of the condition caused by coronavirus infection). The protective immune response may include antibody production and/or a cell-mediated response. Depending on the context, the term "vaccine" may also refer to a suspension or solution of an immunogen that is administered to a subject to produce protective immunity.

As used herein, the term “subject” includes humans and other animals. Typically, the subject is a person. For example, the subject may be an adult, an adolescent, a child (2 to 14 years), an infant (birth to 2 years), or a newborn (up to 2 months). According to specific aspects, the age of the subject is up to 4 months or up to 6 months. In some aspects, the adults are senior citizens, about 65 years of age or older, or about 60 years of age or older. In certain aspects, the subject is a pregnant woman or a woman intending to become pregnant. According to other aspects, the subject is not human; for example, it could be a non-human primate; for example, baboon, chimpanzee, gorilla or macaque. In some aspects, the subject may be a pet such as a dog or cat.

As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the United States federal or state government or listed in the United States Pharmacopoeia, European Pharmacopoeia, or other generally accepted pharmacopoeia for use in mammals and, more specifically, in humans. These compositions can be used as vaccines and/or antigen compositions to induce a protective immune response in vertebrates.

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 include RNA genetic material and structural proteins necessary for invasion of host cells. Once inside the cell, the infecting RNA is used to encode the structural proteins that make up the virus particles, nonstructural proteins that control virus assembly, transcription, replication, and host control, as well as accessory proteins whose function is unknown. 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 is found on the outside of the virus particle and gives coronavirus viruses their crown-like appearance. This glycoprotein ensures the attachment of the viral particle and its penetration into the host cell.

S protein generated by lung epithelial cells has glycoforms associated with increased infectivity. Compared with fully glycosylated S protein, immunization of S protein coated with N-glycans truncated to mono-GlcNAc (S _MG ) elicited stronger immune responses and better protection of human angiotensin converting enzyme 2 (hACE2) transgenic mice against variants of concern (VOC). In addition, a broadly neutralizing monoclonal antibody was identified in mice immunized with S _MG that could neutralize wild-type SARS-CoV-2 and VOC at subpicomolar potency.

The glycoengineered spike protein of the present disclosure comprises a polypeptide having the amino acid sequence of SEQ ID NO: 1, or a variant thereof, the sequence of which is at least 90% identical to the amino acid sequence of SEQ ID NO: 1, or an immunologically active fragment of the amino acid sequence or variant . The amino acid sequence of SEQ ID NO: 1 is shown below.

SEQ ID NO: 1

QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNR KRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDIS TEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCG PKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLE ILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGS NVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTM SLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLL QYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSK PSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDE MIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIAN QFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILS RLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKR VDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFV SNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELD KYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQY

In accordance with one embodiment, the glycoengineered spike protein of the present disclosure comprises a polypeptide comprising the amino acids set forth in SEQ ID NO: 2 below.

SEQ ID NO: 2

QCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNATNWIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKI YSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIA PGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGV NCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSRGSAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLF NKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLA ATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLG DISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ

As described herein, the glycoengineered SARS-CoV-2 spike protein or variant thereof may comprise proteins containing 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) are encoded by a nucleic acid sequence capable of hybridizing under at least moderately stringent conditions with any nucleic acid sequence encoding the spike protein specified herein, or capable of hybridizing under at least moderately stringent conditions with any nucleic acid sequence encoding spike protein as defined herein, but using synonymous codons (eg, codons that do not have an identical nucleotide sequence but encode an identical amino acid).

Analysis of cell-specific glycoform distribution, sequence conservation, glycan shielding, and their cross-correlations led to the development of the S _MG vaccine, in which virtually all glycan shields are removed. Glycosylation of the SARS-CoV-2 S protein has major effects on viral infection, protein integrity, and immune response. Protein S from lung epithelial cells contains more complex sialylated glycans to facilitate receptor binding, and glycosites N801 and N1194 have been shown to be required for Protein S folding and viral infection. This made conserved epitopes more susceptible to the immune system so that more effective and broadly protective B and T cell responses could be generated against the virus and its variants.

The glycoengineered coronavirus spike protein of the present invention targets the entire ectodomain of the S protein, in particular the conserved glycan-shielded domains, stimulating the production of both RBD and non-RBD neutralizing antibodies and CD8 T cell responses that have critical for cross protection.

In accordance with some aspects, the present invention provides a vaccine or pharmaceutical composition containing an immunogen described herein. The present invention also provides a method of treating or preventing a coronavirus infection, comprising administering to a subject (eg, a mammal) in need thereof an effective amount of an immunogen, pharmaceutical composition or vaccine as described herein.

In accordance with one embodiment, the vaccine may include an adjuvant. Examples of adjuvants include, but are not limited to, 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.

The vaccine or pharmaceutical composition can be prepared by any suitable method. Mixing with standard pharmaceutically acceptable carriers and/or excipients can be accomplished using conventional pharmaceutical techniques. The exact nature of the drug will depend on several factors, including the vaccine being administered and the desired route of administration. Suitable drug types are fully described in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Company, Eastern Pennsylvania, USA.

The vaccine or pharmaceutical composition as described herein can be administered by any route. Such methods include administration, for example, parenterally, for example, by all routes of administration into or through the skin: i.e. intramuscularly, intravenously, intraperitoneally, intradermally, mucosally, submucosally or subcutaneously. In addition, they can be applied topically in the form of drops, spray, gel or ointment to the epithelium of the mucous membrane of the eyes, nose, mouth, anus or vagina or to the epidermis of the outer skin of any part of the body. Other possible routes of administration are spray, aerosol or powder application by inhalation through the respiratory tract. Alternatively, administration can be done with food. The effective amount of a vaccine composition may depend on any number of variables, including, without limitation, species, breed, size, height, body weight, age, general health of the patient, type of formulation, or mode or route of administration. The appropriate effective amount can be routinely determined by those skilled in the art using standard optimization techniques, as well as the qualified and informed judgment of a practitioner and other factors apparent to those skilled in the art.

In accordance with one embodiment, the composition may contain an additional therapeutic agent, such as an antiviral agent. The proposed pharmaceutical composition is useful for the treatment of coronavirus infection. Examples of an additional antiviral agent 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, foskamet, ganciclovir, amantadine , rimantadine, zanamivir, remdesivir, molnupiravir and paxlovid.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art. Although methods and materials similar or equivalent to those described herein may be used in the practice or study of the present invention, 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 the event of a conflict, this specification, including definitions, will control. In addition, the materials, methods, and examples are for illustrative purposes only and are not intended to be limiting.

Examples

Example 1. Gene constructs

SARS-CoV-2 spike protein sequences were constructed 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 for Sharing Avian Influenza Data) database (version: 18 April 2021).

The DNA sequences of the SARS-CoV-2 spike protein Wuhan/WHOl/2019 and the delta variant were synthesized with codons optimized for expression in human cells. The furin cleavage site was replaced with GSAG (SEQ ID NO: 9) and 2P substituents were designed to keep the protein in its prefusion state. The transmembrane domain was replaced by 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 for protein expression and purification.

Example 2 Expression and Purification of SARS-CoV-2 S Protein

The plasmid that encodes the secreted SARS-CoV-2 spike was transfected into human embryonic kidney cell lines HEK293EBNA (ATCC number CRL-10852) or HEK293S GnTI cells using a transfection reagent (either polyethylenimine or FectoPRO) and cultured in Freestyle 293 expression medium (Invitrogen) with the addition of 0.5% calf serum. The supernatant was collected 5 days after transfection and purified by centrifugation. Spike proteins were then purified by nickel chelation chromatography and eluted fractions were concentrated using a Millipore Amicon Ultra Filter (100 kDa) and loaded onto a superpose™ gel filtration column (10/300 GL; GE) pre-equilibrated in Tris -buffer. (20 mM Tris/HCl, 20 mM NaCl, 50 mM glutamate, 50 mM arginine) and the corresponding trimer fractions were collected. Purified _SHM was treated with Endo H (NEB) overnight at room temperature to produce spike protein with one GlcNAc at the glycosylation sites, Spike _mg . To remove EndoH S _{, MG} was further purified by buffer exchange using a Millipore Amicon Ultra Filter (100 kDa). The expression and purification method was modified from a previous laboratory study published in PNAS.

Production of glycoengineered pseudoviruses (Figure 1D) is based on 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, 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, Life 9, e61552 (2020)). To produce glycosylation site mutant pseudoviruses (Fig. IF), HEK293T cells were transiently transfected with the pVax-nCoV-SA19 construct containing mutations at each glycosylation site and an HIV-1 luciferase-expressing genomic plasmid (pNL4-3.luc.RE).

Example 3: Characterization of S _MG Protein

To characterize the S _MG protein, size exclusion chromatography was performed using an ENrich™ SEC 650 (10 x 300 column; Biorad) pre-equilibrated with Tris buffer (20 mM Tris/HCl, 20 mM NaCl, 50 mM glutamate, 50 mM arginine) . To check protein sample purity, samples were then combined with loading buffer, separated by 7.5% SDS-PAGE, and stained with Coomassie Brilliant Blue-plus (EBL).

Example 4. Analysis of glycopeptides by mass spectrometry

Two 20-μg aliquots of SARS-CoV-2 spike protein from two biological replicates were denatured at 55°C for 1 h 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 incubating for 30 min in the dark. Alkylated envelope proteins were digested separately using various combinations of 1:10 (w/w) chymotrypsin or alpha-lytic protease or 1:20 (w/w) trypsin (Mass Spectrometry Grade, Promega). After overnight enzymatic digestion, samples were dried in a SpeedVac and processed for LC-MS/MS determination. The classification of glycans was followed by a preliminary study according to the composition detected and visualized using Graphpad Prism 9.0.0.

Example 5: Vaccination and challenge experiments

6- to 7-week-old female golden Syrian hamsters (n = 5) were immunized intramuscularly with 25 μg of purified S _FG or S _MG proteins mixed with 250 μg of aluminum hydroxide on day 0 and day 14. Blood was collected 28 and 42 days after first immunization, and serum samples were collected from each hamster. 4 weeks after the second vaccination, hamsters were infected with 1 x 10 ⁴ PFU of SARS-CoV-2 TCDC#4 virus (hCoV-19/Taiwan/4/2020, GISAID ID: EPI ISL 411927) intranasally in a volume of 100 μl per hamster. The body weight of each hamster was recorded daily after infection. On the 3rd day after infection, the hamsters were killed with carbon dioxide. The right lung was collected for viral load determination (TCID50 assay). The left lung was fixed in 4% paraformaldehyde for histological examination. All animal experiments were evaluated and approved by the Academia Sinica Laboratory Animal Care and Use Committee.

Alternatively, for a two-dose vaccination schedule, 6- to 8-week-old female BALB/c mice (n=5) were immunized intramuscularly with 10 μg of purified S _FG , S _HM , or S _MG mixed with aluminum hydroxide (50 μg) on days 0 and 14. Serum was collected on day 28 after the first vaccination to assess anti-S IgG content, IgG subtype, and neutralizing titers (described in Supplementary Materials and Methods). Lymph nodes from mice immunized with S _FG or S _MG were collected on day 21 after the first vaccination to analyze T cell responses (described in Supplementary Materials and Methods). To analyze the B cell repertoire and serum titers against variants, female BALB/c mice aged 6 to 8 weeks (n=5) were immunized intramuscularly with 20 μg of purified S _FG or S _MG mixed with aluminum hydroxide (20 μg) on days 0, 14 and 56; mice were sacrificed on day 84 to collect whole blood for anti-S IgG and neutralizing titer assessment, as well as spleens for S protein-specific B cell sorting (described in Supplementary Materials and Methods).

For the hamster vaccination and virus challenge study, 6- to 7-week-old male Golden Syrian hamsters (n = 5) were immunized intramuscularly with 25 μg of purified S _FG or S _MG mixed with aluminum hydroxide (250 μg) on days 0 and 14. Four days later weeks after the second immunization, each hamster was intranasally injected with 1 x 10 ⁴ TCID ₅₀ SARS-CoV-2 (hCoV-19/Taiwan/4/2020) in 100 μl PBS. Body weight was recorded daily after infection. On the 3rd day after infection, the hamsters were killed with carbon dioxide. The upper lobe of the left lung was fixed in 10% paraformaldehyde for histopathological examination, and the rest of the lung was collected for viral load determination (TCID ₅₀ assay).

To vaccinate transgenic mice and study virus challenge, 6- to 8-week-old male CAG-hACE2 transgenic mice or 12-week-old male K18-hACE2 transgenic mice (purchased from The Jackson Laboratory) were immunized intramuscularly with 10 μg of purified S _FG or S _MG mixed with aluminum hydroxide (50 μg) on days 0 and 14. CAG-hACE2 transgenic mice were challenged intranasally 4 weeks after the second immunization with 1 x 10 ₃ TCID ₅₀ WT SARS-CoV-2. In the first trial (n=3), all mice were sacrificed at day 7 postinfection (dpi) for histopathological examination of the left upper lobe; in the second trial (n = 7), three mice were sacrificed at 4 dpi to determine lung virus titer, and four mice were kept until 14 dpi for survival analysis. Serum was collected 1 day before virus infection.

For VOC challenge studies, CAG-hACE2 mice were injected with 1 x 10 ³ TCID ₅₀ alpha variant (hCoV-19/Taiwan/792/2Q20) (n = 5) or gamma variant (hCoV-19/Taiwan/906/2021 ) SARS-CoV-2 in 50 µl PBS per mouse. In addition, K18-hACE2 mice were infected intranasally 4 weeks after the second immunization with 1 x 10 ⁴ TCID ₅₀ delta-SARS-CoV-2 (hCoV-19/Taiwan/1144/2021) (n = 4) in 50 μl PBS per mouse . For all SARS-CoV-2 variant infection models, the body weight of each mouse was recorded daily until 14 dpi.

To test prophylactic antibody protection, 8-week-old male K18-hACE2 transgenic mice (n = 3) were intraperitoneally injected with m31A7 (15 mg/kg) or PBS 1 day before intranasal challenge with 1 x 10 ³ TCID ₅₀ wild-type SARS-CoV-2 (WT) (hCoV-19/Taiwan/4/2020). Body weight and temperature were recorded daily until 5 dpi. All animal experiments were evaluated and approved by the Academia Sinica Laboratory Animal Care and Use Committee (approvals no. 21-10-1716, 18-12-1272, and 20-10-1522).

Example 6: Histological and Immunohistochemical (IHC) Staining

Hamster lungs at 3 dpi were collected promptly and fixed in 10% neutral buffered formalin for 24 hours, then transferred to 70% ethanol for 72 hours. Paraffin-embedded lung tissue was trimmed to a thickness of 5 mm. For histological staining, tissue was stained with hematoxylin and eosin (H&E). For immunohistochemical (IHC) staining, tissue sections were deparaffinized with xylene and rehydrated with an ethanol gradient. Antigen retrieval was performed by heating the slides to 95°C for 10 minutes in 10 mM sodium citrate buffer (pH 6.0) in a microwave oven. After cooling at room temperature and washing with PBS, 3% H ₂ O ₂ is applied to eliminate endogenous peroxidase activity. Tissue sections were blocked with 5% normal goat serum and 1% BSA in PBST IX for 1 hour, followed by incubation with rabbit anti-N and anti-S primary antibodies at a dilution of 1:50 (polyclonal anti-SARS-CoV-2 antibodies) in overnight at 4°C. The tissue was then incubated with goat anti-rabbit anti-horseradish peroxidase (HRP) secondary antibody at a dilution of 1:500 for 1 hour and visualized by incubation with 3,3-diaminobenzidine (DAB) substrate and counterstaining with hematoxylin.

Example 7. Immunofluorescence (IF) staining

For immunofluorescence staining, after antigen retrieval steps, tissue was permeabilized with Triton X-100 in PBS. Tissue sections were blocked with 5% normal goat serum and 1% BSA in PBST IX for 1 hour. Then incubated with autofluorescence quencher for 5 minutes. After this, the samples were incubated with rabbit anti-N and anti-S primary antibodies at a dilution of 1:50 (polyclonal antibodies against SARS-CoV-2) overnight at 4°C, secondary antibodies Alexa Fluor-488 (1:500, Thermo Fisher) for 1 hour at room temperature and 4,6-diamidino-2-phenylindole (DAPI), a nuclear dye, for 3 minutes at room temperature. Coverslips were mounted on microscope slides and imaged under a Leica TCS SP8X confocal microscope with an HC PL APO CS2 10x/1.40 objective (Leica AG, Wetzlar, Germany).

Example 8. Experiments on vaccination and infection of transgenic mice

Male 8-week-old CAG-hACE2 transgenic mice (n=3) were immunized intramuscularly with 10 μg of purified S _FG or S _MG protein mixed with 50 μg of aluminum hydroxide on day 0 and day 14. ¹⁴ Blood was collected at 28 days and 42 days after first immunization, and serum samples were collected from each transgenic mouse. 6 weeks after the second vaccination, hamsters were challenged with 1 x 10 ³ PFU of SARS-CoV-2 TCDC#4 (hCoV-19/Taiwan/4/2020, GISAID ID: EPI_ISL 411927) intranasally in a volume of 100 μl per mouse. Body weight and survival of each transgenic mouse were recorded daily after infection. On day 7 postinfection, all transgenic mice were sacrificed with carbon dioxide. The lung was fixed in 4% paraformaldehyde for histopathological examination. All animal experiments were evaluated and approved by the Academia Sinica Laboratory Animal Care and Use Committee.

Example 9 Quantitative determination of virus titer in lung tissue using cell culture infectious assay (TCID ₅₀ )

The middle, lower and postcaval lobes of hamster lungs were homogenized in 600 μl of Dulbecco's modified Eagle's minimal essential medium (DMEM) with 2% fetal bovine serum (FBS) and 1% penicillin/streptomycin using a homogenizer. The tissue homogenate was centrifuged at 15,000 rpm for 5 minutes, and the supernatant was collected for titration of live viruses. Briefly, 10-fold serial dilutions of each sample were added to a monolayer of Vero E6 cells in quadruplicate and incubated for 4 days. Cells were then fixed with 10% formaldehyde and stained with 0.5% crystal violet for 20 minutes. The plates were washed with tap water and assessed for infection. The 50% tissue culture infectious dose (TCID ₅₀ )/ml was calculated using the method of Reed and Muench.

Example 10 Mice Vaccination Studies

6- to 8-week-old female BALB/c mice (n = 5) were immunized intramuscularly with 20 μg of purified S _FG or S _MG proteins mixed with 20 μg of aluminum hydroxide on day 0, day 14, and day 56. Blood was collected at 14 days after the third immunization, and serum samples were collected from each mouse. All animal experiments were evaluated and approved by the Academia Sinica Laboratory Animal Care and Use Committee.

Example 11. Evaluation of antibody titer in serum

Anti-S ELISA was used to determine serum IgG titer. The plates were blocked with 5% skim milk and mouse polyclonal anti-S primary antibody and HRP-conjugated secondary antibody were added sequentially. Peroxidase substrate (TMB) solution and 1 M H ₂ SO ₄ stop solution were used and the absorbance (OD 450 nm) was read using a microplate reader. The strain tested included SARS-CoV-2 (wild type, variants B.1.1.7 and B.1.135), RnGT13 and SARS-CoV-1.

The m31A7 antibody was isolated by single B cell screening and then characterized. The primers were designed based on a previous publication (Tiller T., Busse E. E., Wardemann H. Cloning and expression of murine Ig genes from single B cells./ Immunol. Methods 350, 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, and 72°C for 1 min, with a final extension at 72°C for 10 min. Semi-nested second-round PCR was performed using KOD One PCR mix (TQYQRO) along with 1 μl of crude first-round PCR product at 98°C for 2 min followed by 45 cycles of incubation at 98°C for 10 s, 55°C for 10 s and 68°C for 10 s, with a final extension at 68°C for 1 min. The PCR products were then analyzed by electrophoresis and sequencing. The Ig V and L genes were identified in the international information system ImMunoGeneTics (http://imgt.org/IMGT_vquest/input). Genes were then amplified from the second-round PCR product with single gene-specific primers for the V and L genes containing restriction sites for cloning into vectors containing a human IgH or IgL expression framework. Chimeric IgH and IgL expression constructs were cotransfected into Expi293 to produce antibodies. After m31A7 was isolated, the antibody was subsequently assessed for S protein binding by ELISA and fluorescence-activated cell sorting, pseudovirus neutralization activity, binding kinetics and epitope mapping, and structure determinations.

Example 12: Pseudovirus Neutralization Assay

To determine infectious units of pseudotyped lentiviral vectors, we plated 293T-ACE2 cells at appropriate densities in 96-well (100 μl per well) tissue culture plates 1 day before infection. After overnight incubation (37°C, 5% CO ₂ ), 100 ml of three premixed pseudovirus-containing supernatants and four-fold serial dilutions of serum from immunized mice were added to seeded cells. Cells were incubated for 48 hours at 37°C/5% CO ₂ to allow expression of the nano-luciferase reporter gene. Luciferase activity was measured using an ELISA reader. The percentage of inhibition was calculated using the following equation 100*[1-(RLU _Sample /RLU _{mock-treatment} )] (RLU _Sample is the relative units of luminescence emitted by the sample, RLU _{mock-treatment} is the relative units emitted by the mock treatment). Data were analyzed using Graphpad Prism and pNT50 values were calculated using the 50% inhibitory concentration value for all samples.

Example 13 Plaque Reduction Assays

Vero E6 cells were seeded in 24-well culture plates in DMEM with 10% FBS and antibiotics 1 day before infection. SARS-CoV-2 was incubated with antibodies for 1 hour at 37°C, after which it was added to the cell monolayer for another hour. Virus-antibody mixtures were then removed, and the cell monolayer was washed once with PBS before covering with medium containing 1% methylcellulose for 5-7 days. Cells were fixed with 10% formaldehyde overnight. After removal of the covering medium, cells were stained with 0.7% crystal violet and plaques were counted. Percent inhibition was calculated as [1 - (VD/VC)] x 100%, where VD and VC refer to the virus titer in the presence and absence of serum, respectively.

For the CPE neutralization assay, Vero E6 cells were seeded onto a 6-well plate at 2×10 ⁵ cells/well overnight to achieve 90% monolayer confluence. Serum and viruses were mixed before adding to the monolayer for another hour. The plates are left to solidify at room temperature for 30 minutes, then incubated at 37°C until cytopathic effects (CPE) appear.

Statistical analysis: All data are expressed as mean ± standard error of the mean. For all analyses, P values were obtained from Student's t test (unpaired, two-tailed), except for comparisons of curves using Student's t test (paired, two-tailed). All graphs were generated using GraphPad Prism software version 9.0.0.

Example 14: Design and Characterization of a Glycoengineered S Protein Vaccine (S _MG ) Coated with Mono-GlcNAc

The recombinant native protein with the sequence (amino acids 14-1209) from the original SARS-CoV-2 Wuhan strain (hCoV/Wuhan/WHO 1/2019) was codon optimized for expression in human cells with GSAG residues (SEQ ID NO: 9) for replacement the original furin cleavage site and 2 proline mutations to fix the native spike in its prefusion state, and a foldon trimerization sequence and a His tag were added to its C-terminus and expressed in human HEK293S cells (Fig. 1). First, a high-mannose recombinant native spike intermediate was produced and purified, where all N-glycans indicated by the branch sign (Fig. 1a) are Man5 N-glycans. Excess glycans were then removed using EndoH endoglycosylase to obtain the final product, monoglycosylated native spike (Figure 1b). Purified S _MG is a trimer with an apparent molecular mass of ~520 kDa in solution and a high degree of purity (Fig. 1c and 1d). The reduction in size due to the removal of N-glycans was analyzed by SDS-PAGE compared to conventional S _FG and intermediate S _HM (Fig. 1d). Mass spectrometry analysis of S _MG revealed that most N-glycosylation sites contain almost 100% of the single sugar, N-acetylglucosamine (GlcNAc), with the exception of N603 and N1194, where GlcNAc constitutes >90% (Fig. 1f). In contrast, the original fully glycosylated native spike has heterogeneous N-glycans at all N-glycosylation sites, containing complex type, hybrid type, and others.

Example 15: S _MG Vaccine Provided Best Protection Against SARS-COV-2 and Variants in Vivo

To evaluate the in vivo protective efficacy of the S _MG vaccine against SARS-CoV-2, we first challenged SMG- or SFG-vaccinated Syrian hamsters with wild-type SARS-CoV-2 ( Fig. 2A ). Hamsters vaccinated with S _MG (n=5) showed less reduction in body weight compared to the S _FG and phosphate-buffered saline (PBS) groups (Fig. 2B), while a similar reduction in virus titer was observed in the lungs of both S _FG - and S _MG -vaccinated hamsters (Fig. 2C). In addition, according to histopathological and antinucleocapsid (N) protein immunostaining, fewer lesions were observed in the lungs of immunized hamsters (Fig. 2D). Because hamsters exhibited only mild to moderate disease upon SARS-CoV-2 infection, we then used severe disease models highly sensitive to CAG-hACE2 (C.-Y. Tsai, C.-Y. Chen, J.- T. Jan, Y.-C. Chou, M.-L. Chang, L. A. Lu, P.-Y. Huang, M. F. C. Chu, 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 (E.S. Winkler, A.L. Bailey, N.M. Kafai, S. Nair, V.T. McCune, J. Yu , J. M. Fox, R. E. Chen, J. T. Earnest, S. P. Keeler, J. H. Ritter, L.-I. Kang, S. Dort, A. Robichaud, R. Head, M. J. Holtzman, MS Diamond, SARS-CoV-2 infection of human ACE2 -transgenic mice causes severe lung inflammation and impaired function. Nat. Immunol. 21, 1327-1335 (2020)) transgenic mice (Fig. 2E). Analysis of anti-S IgG binding titers, neutralizing antibody titers, anti-S IgG subtype, and IgG2c:IgG1 ratio (Figure 2, F to I) in CAG-hACE2 mice showed results similar to BALB/c mice (Figure 6, F to I). CG). Following intranasal challenge with WT SARS-CoV-2, virus was undetectable in the lungs of both S _FG - and S _MG -vaccinated CAG-hACE2 mice (n = 3) by anti-N staining at 7 days postinfection (dpi) (Fig. 2J) or median tissue culture infectious dose (TCID ₅₀ ) at 4 dpi (Figure 2K), whereas the control group had a viral titer greater than 1000 TCID ₅₀ (Figure 2K). The S _MG group (n = 4) showed better (75%) survival than the S _FG (50%) at 14 dpi ( Fig. 2, L and M ). We next assessed the degree of protection provided by S _MG vaccination against alpha variant challenge in CAG-hACE2 mice (n = 5). We found that SMG vaccination resulted in 100% survival up to 14 dpi ( Fig. 2, N and O ). Mice vaccinated with S _MG also showed 60% survival against gamma variant challenge in CAG-hACE2 mice (n=5) (Fig. 22 P and Q) and 75% survival against delta variant challenge in K18-hACE2 mice. (n = 4) (Fig. 4, R and S), whereas less than 50% of mice vaccinated with S _FG survived to 14 dpi when challenged with gamma and delta variants (Fig. 2, QS). The improved in vivo protection afforded by S _MG vaccination provides further evidence that removing glycan shields from an immunogen is an advantageous strategy for eliciting a superior immune response.

Example 16 S _MG Vaccination Provides Better Protection Against Lethal Dose of SARS-CoV-2 Infection in hACE2 Transgenic Mice

CAG-hACE2 transgenic mice can become severely ill and die when infected with the SARS-CoV-2 virus. Two doses of vaccines S _FG , S _MG or adjuvant alone were administered intramuscularly on days 0 and 14, serum was collected on days 28 and 42, and 1x10 ³ TCID ₅₀ SARS-CoV-2 was used for intranasal infection of each mouse (Fig. 3a). Spike-specific IgG antibody titers in mice immunized with S _MG are 1.9 times higher than in the S _FG group (40,000 vs. 20,000), and neutralization titers of S _MG are 2.8 times higher than in the S _FG group (Figs. 3b and 3c). Surprisingly, in this model of severe disease, S _FG vaccination failed to protect CAG-hACE2 transgenic mice from severe disease as observed by rapid loss of body weight, and all mice died before day 7 postvirus infection. Disease progression in the S _FG -vaccinated group was only slightly better than in the adjuvant-only group (Figs. 3d and 3e). In contrast, the group vaccinated with S _MG showed little weight loss, and all mice in the group survived SARS-CoV-2 infection. This result demonstrates the superiority of S _MG vaccination in a transgenic mouse model of severe SARS-CoV-2 disease.

Example 17: Antibodies induced by S _MG vaccine in mice to enhance binding and neutralization and efficacy against SARS-CoV-2 variants

We next analyzed whether there were differences in antibody responses induced by vaccination with S _FG or S _MG , which have the native spike sequence from the Wuhan strain, with respect to their ability to neutralize recently emerged SARS-CoV-2 variants of concern (Figure 4). Compared with S _FG , immunization with S _MG caused better spike-specific IgG binding not only to native WT spikes (Wuhan strain), but also to variants D614G, Bl1.7, B.1.351, bat CoV RnGT13 and SARS- CoV-1 (Fig. 4b). Pseudotyped SARS-CoV-2 variant viruses were used to evaluate the ability to neutralize infections from vaccine-immunized sera. Again, sera from S _MG immunization have superior neutralizing ability against pseudoviruses D614G, B.1.1.7, and B.1.351, and they are 1.4-, 2.7-, and 1.5-fold better than sera induced by S _FG , respectively (Fig. 4c and 4d). Finally, real SARS-CoV-2 virus D614G and B.1.1.7 were used in the plaque reduction neutralization assay (Figures 4e and 4f). S _MG vaccination induced better neutralizing antibody responses to the D614G and B.1.1.7 variants compared to S _FG vaccination, being 2.0- and 1.4-fold better, respectively.

Example 18: Glycosylation affects pseudovirus S protein by interacting with ACE2 on several cell types

To understand the importance of glycosylation, we expressed S protein in lung epithelial cells, the cells first susceptible to infection, and found that S protein sialylation is required for higher receptor avidity ( Fig. 5A ). A similar pattern was also observed in S protein generated by HEK293T cells (Figure 5B), and avidity was also reduced in S protein with only high-mannose glycans or in a glycoform with all N-glycans truncated to a single N-glucosamine ( GlcNAc) (Fig. 5C). The effect of its glycosylation was further studied by pseudoviral infection in human angiotensin converting enzyme 2 (hACE2)-expressing HEK293T cells, revealing a consistent trend when the same amount of virus was applied ( Fig. 5D ). This led to the conclusion that complex glycans and sialylation are functionally important for S protein-mediated infectivity. A complete panel of 24 lentivirus-based pseudovirus variants (containing 22 N- and 2 O-glycosylation sites) was also generated to evaluate the efficiency of viral entry into five hACE2-expressing cell lines, including HEK293T, Vero-E6, and three human lung cell lines , A549, Calu-1, and Calu-3 (Fig. 5, E-G). These pseudoviruses were based on the S construct with a 19 amino acid deletion at the C terminus, which produced the highest virus titer. Pseudovirus production was quantified using a p24 immunoassay, and the results were normalized to the titer of each mutant strain (Fig. 5F). Each N-site glycosylation asparagine (Asn) was replaced by a glutamine (Gln) to minimize structural impact due to their chemical similarity, and each G-site glycosylation threonine (Thr) or serine (Ser) was replaced by an alanine (Ala). . Since mutagenesis did change the amino acids, the resulting change in infectivity would be due to collective factors including conformational shifts associated with glycosylation, which influence receptor engagement, and S protein surface content, which is affected by protein expression, folding, and trafficking. The results showed that disruption of S protein glycosylation reduces infectivity. Significant reductions were observed for two mutations in the receptor binding domain (RBD), N33 IQ and N343Q, as well as for mutations in two O-glycosylation sites (T323A and S325A), despite the low occupancy of the latter (Figure 5G). In addition, deletion of N122 glycosylation in the N-terminal domain (NTD) resulted in decreased infectivity and low protein expression ( Fig. 5G ). Two NTD mutations, N149Q and N165Q, increased infectivity in Vero-E6 and Calu-1 cells, respectively, although a decrease in infections was observed in other cells ( Fig. 5G ). Note that the glycans attached to this N165 residue are structurally proximal to the adjacent RBD in the trimeric S protein, and its mutation reduces ACE2 binding, likely due to a conformational shift of the RBD toward a “down” state. We identified two mutants, N801Q and N1194Q ( Fig. 5F ), that universally abolished virus infectivity in all five cells. The glycosylation site N801 is located near the fusion peptide proximal region (FPPR), and N1194 is located near the center of heptad repeat 2 (HR2) helix and is the last N-glycosylation site preceding the transmembrane domain (Fig. 5E). Both of these mutations caused low-yield expression. The N80IQ mutant was more prone to degradation, and the N1194Q mutant impaired S protein trimerization, which may partly explain the reduced infectivity of pseudoviruses carrying these mutants.

Example 19. S protein from lung epithelial cells contains more sialylated glycans of a complex type, and highly conserved epitopes in the S protein are more shielded by glycans

Analysis of the S protein glycan profile revealed a higher content of complex type glycans (78%) and a lower number of hybrid type glycans (less than 1%) for S protein in the human lung epithelial cell line BEAS-2B (Fig. 6A) compared to S -protein produced in the human kidney epithelial cell line, HEK293T (61 and 23%, respectively) (Fig. 6B). Among the high-mannose glycans, N-linked glycan mannose-5 (man5) was the predominant type found in HEK293T-expressed S protein, although it is only observed at the N61 site of S protein from BEAS-2B cells. In addition, complex type glycans at sites N74, N149, N282, and N1194 in BEAS-2B cells were subject to more diverse processing (multiple antennas, galactosylation, fucosylation, or sialylation) than glycans in HEK293T cells. In contrast, glycans at sites N122, N331, N1098, and N1134 were less diverse. Moreover, N149 and N17 did not contain core fucose in BEAS-2B. We observed an overall higher degree of sialylation of all 22 N-glycosylation sites in BEAS-2B (53%) than in HEK293T (35%), HEK293E (26%) or previously described HEK293F cells (15%) (Y. Watanabe , J. D. Allen, D. Wrapp, J. S. McLellan, M. Crispin, Site-specific glycan analysis of the SARS-CoV-2 spike. Science 369, 330-333 (2020)). In particular, the two N-glycosylation sites (N331 and N343) of the RBD are more sialylated in BEAS-2B (99 and 39%) than in HEK293T (49 and 15%). Despite the differences, the S protein from all cell types contains a non-complex type glycan belt located around the middle part of the S2 domain (Fig. 6C), where the N-glycosylation site N801 is critical for infection (Fig. 5G), N1074 contains various glycans, and N717 is required for S protein expression.

Based on the modeled structure of the SARS-CoV-2 S protein and the glycan profile from BEAS-2B cells, we performed a structural analysis of glycan coverage of protein surface areas and overlaid 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, 33-46 (2017). Several regions were identified that were highly conserved but shielded glycans, including the downstream edge of the RBD, the stem region of S2 with a noncomplex-type glycan belt, and the C-terminal portion of S2 including the junction domain (CD) and HR2 ( Figure 6, D and E ). . At the primary sequence level, these regions were shown to be conserved epitopes. Sequence conservation analysis also showed that most of the glycosylation site regions were highly conserved, and the most conserved (mutation rate below 0.02%) were the NTD (N61, N122, N165 and N234), RBD (S325, N331 and N343), subdomain 1 regions /2 (SD1/2) (N603 and N657), stem region subunit 2 (S2) (N709, N1098, N1134, N1158, N1173, N1194) and N801 near FPPR. Most of these regions contain between 20 and 40% conserved surface residues, and among them, a certain percentage of the residues were screened by glycans: 36% in the RBD and about 50% in other regions. Despite the absence of N-glycosylation sites, in the HR1 region, 69% of the conserved surface residues were covered by glycans derived from neighboring domains. These results highlighted the importance of S protein glycosylation both structurally and evolutionarily, leading to the idea that exposure of conserved glycan-shielded regions may induce immune responses against conserved epitopes. (It is more important to describe the 12 conserved epitopes as targets for vaccine design. 10 of the 12 epitopes are shielded by glycans, and removal of the shielded glycans exposes the conserved epitopes, causing a variety of protective responses.)

Example 20: S _MG was developed as a vaccine, and the S _MG vaccine produced the best immune response using different antibody subclasses

An initial attempt to mutate several glycosylation sites resulted in a marked reduction in S protein expression. However, when the authors expressed it in GnTI HEK293S cells, they were able to obtain high mannose ( _SHM ) S protein glycoforms in good yield and purity. The glycans were then trimmed by endoglycosidase H (Endo H) to one GlcNAe at each N-glycosylation site, resulting in a soluble mono-GlcNAc-coated S protein truncation product called S _MG (Fig. 7A). In S _MG , according to mass spectrometry data, all N-glycosylation sites are mainly occupied by single GlcNAc, and the loading of raw O-glycans is too low to be detected. This modified S _MG and S _HM as well as the original fully glycosylated S protein (S _FG ) were mixed with aluminum hydroxide (alum) as an adjuvant and then used to immunize BALB/c mice (n = 5) by intramuscular injection (Fig. 7B ). For comparison, the S _FG in this study was expressed by HEK293E cells and contained a variety of glycans, similar to the immunogens used in many currently available COVID-I9 vaccines that are either approved or in clinical trials, including insect cell-expressed vaccines with S -protein from Sanofi and Novax (PJ Klasse, DF Nixon, JP Moore, Immunogemeity of clinically relevant SARS-CoV-2 vaccines in nonhuman primates and humans. Sci. Adv. 7, eabe8065 (2021)), a recombinant S-vaccine from cells Chinese Hamster Ovary (CHO) from Medigen (T.-Y, Kuo. M.-Y. Lin, R.L. Coffman, J.D. Campbell, P. Traquina, Y.-J. Lin, L.T.-C. Lin, J. Cheng, Y'.-C. Wu, C.-C. Wu, W.-H. Tang, C.-G. Hoang, K.-C. Tsao, C. Chen, Development of CpG-adjuvanted stable prefusion of SARS-CoV -2 spike antigen as a subunit vaccine against COVID-19. Sci. Rep. 10, 20085 (2020)), adenovirus-based vaccines from AstraZeneca and Johnson & Johnson, as well as mRNA vaccines from Pfizer-BioNTech and Moderna (PJ Klasse , D. F. Nixon. JP Moore, Immuhogenicity of clinically relevant SARS-CoV-2 vaccines in nonhuman primates and humans. Sci. Adv. 7, eabe8065 (2021)). The S _FG and S _MG proteins exhibit essentially the same three-dimensional structure in solution, as shown by negative staining analysis.

Mice immunized with S _MG induced a higher humoral immune response after the second immunization compared to S _FG , with a significantly higher (1.44-fold) titer of immunoglobulin G (IgG) against S protein (endpoint titer: S _FG , 39 408 ± 1619; S _MG , 56,957 ± 5091; P = 0.0079) (Figure 7C) and 3.6-fold higher antibody neutralization efficiency, as shown by inhibition of SARS-CoV-2 pseudoviral infection (reverse half-maximal neutralization titer _pNT50 : S _FG , 1346 ± 285; S _MG , 4791 ± 767; P = 0.0159) (Fig. 6D), whereas the S _HM immunized group showed similar anti-S IgG titers (39,086 ± 11 654) and no difference in pNT ₅₀ titer compared to the S _FG group. Analysis of IgG subtype titer and production of interferon-γ (IFN-γ) or interleukin-4 (IL-4) by T-follicular helper (Tfh) cells showed that the S _MG vaccine induces more IgG2a, which is a marker for T-helper 1- cells (T _H 1) lymphocytes in BALB/c mice, a more balanced TH1/T _H 2 response and a greater number of Tfh cells expressing IFN-γ compared with the S _FG and S _HM vaccinated groups (Fig. 7, E - J). Moreover, the S _MG vaccine induced a higher frequency of IL-21 ⁺ Tfh cells (Fig. 7K) and an increased frequency of granzyme B-producing CD8 ⁺ T cells (Fig. 7L). These data indicate that S _MG induces a stronger humoral and cellular adaptive immune response compared to the response induced by S _FG . We then examined the frequency of S protein-specific B cells (CD3 ⁻ CD19 ⁺ S ⁺ ) in the spleen of mice immunized after the third dose of S _FG or S _MG (Fig. 6A) and found that mice immunized with S _MG produced more S protein-specific B cells (Fig. 6M). Analysis of the B cell repertoire in mice immunized with S _FG and S _MG (n = 5) showed that the S _MG group used more lambda light chain genes compared to the S _FG group (S _FG , 1.92%; S _MG , 9.68%) (Fig. 7N). In addition, antibodies derived from several specific loci of the Ig heavy chain variable region (IGHV) (Fig. 7O) and Ig kappa chain variable region (IGHV) (Fig. 7P) gene loci were more represented in the S _MG group than in group S _FG , especially the IGHV1-18 gene (Fig. 7O). This finding suggests that B cell epitopes may be processed differently in the two groups, and whether and why this difference is immunologically beneficial remains to be investigated. In addition, three-dose S _MG vaccination induced a higher final IgG titer than two-dose wild-type (WT) S protein vaccination (final titer: S _FG , 208,911 ± 50,092; S _MG , 376,410 ± 80,873). We observed differences between the S _MG and S _FG groups in serum IgG binding curves measured using an enzyme-linked immunosorbent assay (ELISA) against S protein from the SARS-CoV-2 VOC (PR Kraisse, TR Fleming, IM Longing R. Peto, S. Briand, D. L. Heymann, V. Beral, M. D. Snape, H. Rees, A.-M, Ropero, R. D. Ralicer, J. P. Cramer, C. Munoz-Fonteia, M. Gruber, R. Gaspar, J. A. Singh , K. Suhbarao, MD Van Kerkhove, S. Swatninathan, M. J. Ryan, A.-M. Henao-Restrepo, SARS-CoV-2 variants and vaccines. N. Engl. J Med 385, 179-186 (2021)), including alpha (B.1.1.7; P = 0.0488), beta (B.1.351; P = 0.0010), gamma (P.1; P = 0.0068), and delta (B.1.617.2; P = 0.0068) but found no statistical difference in the endpoint titer analysis (Figure 7Q). There were also differences in anti-VOC neutralizing antibody responses across the pseudovirus neutralization curve, including alpha (P = 0.0156), beta (P = 0.0156), and delta (P = 0.0078), but no differences were observed in titer values pNT ₅₀ neutralization of pseudovirus or authentic virus (Fig. 7, R and S) compared with S _FG .

Example 21 A broadly neutralizing antibody was isolated from B cells of mice immunized with S _MG

Sorting of S protein-specific B cells from S _MG -immunized mice led to the identification of the monoclonal antibody (mAb) m31A7 from amplified IGHV1-18 clones, a subset that is uniquely abundant in the SMG-immunized B cell repertoire (Fig. 8O). This mAb interacts with full-length S protein, SI and RBD, but not S2 (Figure 8A), and binds to HEK293T cells that express S protein from different SARS-CoV-2 variants (Figure 8B). In addition, m31A7 was shown to neutralize various pseudovirus variants (WT, D614G, alpha, beta and delta) at a sub-picomolar half-maximal inhibitory concentration ( _IC50 ), up to 1000-fold higher than that reported for human mAb EY6A (Fig. 8C). The prophylaxis study also demonstrated good in vivo efficacy of m31A7 in K18-hACE2 mice (n = 3) infected with WT SARS-CoV-2 (Figure 8D). Mice receiving prophylactic treatment maintained both body weight and temperature ( Fig. 8, E and F ). Biolayer interferometric assay (BLI) was used to measure the dissociation constants of m31A7 and its Fab binding to protein S at 70.9 pM and 4.66 nM, respectively ( Figure 8G ). Epitope mapping by hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed regions of its potential binding in the RBD (Figure 8H) that overlap with the observed epitope in the crystal structure of the RBD complexed with m31A7-Fab. The structure obtained by cryoelectron microscopy (EM) further clarified the binding of m31A7 only to the RBD in the “up” state (Figure 8I), with the N165 glycan from the adjacent NTD near the RBD-m31A7 interface (Figure 8J). The pattern of m31A7 in the RBD is similar to that of the human VH1-58 class mAb (Figure 8K), but it approaches the RBD from a different angle, with offset local contact regions at the tip of the loop, bypassing most of the key mutated VOC residues such as E484 and K417, but not T478 (Fig. 8F). The detailed interface of RBD-m31A7 and the inhibitory mechanism of this S _MG- induced mAb are under further investigation.

Example 22: Delta S _MG Vaccination Induced More Neutralizing Antibodies Against SARS-CoV-2 Pseudovirus Variant

Expression construct of the S _MG protein of the delta variant of SARS-CoV-2:

Signal peptide from A/Brisbane/59/2007(HlNl)): MKVKLLVLLCTFTATYAGT (SEQ ID NO: 4)

His Mark: HHHHHHJSEQ ID NO: 8)

BALB/c mice (n=5) were immunized twice at week 0 and week 2 with Delta S _FG or S _MG vaccine. Serum was collected at week 6 and subsequently examined for neutralizing capacity using a pseudovirus assay (Fig. 9A). Pseudotyped SARS-CoV-2 delta variant viruses were used to evaluate the ability to neutralize infections from vaccine-immunized sera. Again, serum from S _MG immunization has superior ability to neutralize WT, alpha, beta, gamma, delta and omicron pseudoviruses. Their respective delta S _FG titers versus S _MG (pNT50, higher is better) are 1018 versus 3336 (against WT pseudovirus), 1396 versus 2680 (against alpha pseudovirus), 337 versus 2282 (versus beta -pseudovirus), 814 versus 4424 (against gamma pseudovirus), 1138 versus 6680 (against delta pseudovirus), and 144 versus 2628 (against omicron pseudovirus), respectively.

Example 23. Vaccination with delta or WT S _MG induced more neutralizing antibodies against the SARS-CoV-2 omicron variant pseudovirus

BALB/c mice (n=5) were immunized twice at week 0 and week 2 with delta/WT S _FG or S _MG vaccine. Serum was collected at week 6 and subsequently tested for neutralizing capacity using a pseudovirus assay (Fig. 10A). Pseudotyped SARS-CoV-2 Omicron variant viruses were used to evaluate the ability to neutralize infections from vaccine-immunized sera from either WT S _FG /S _MG or delta S _FG /S _MG . Vaccination with WT S _FG does not produce neutralization against Omicron pseudovirus, while WT S _MG provides little protection. Similarly, vaccination with delta S _FG produces little neutralization of Omicron pseudovirus infection, but delta S _MG provides excellent neutralization.

Example 24 Delta S _MG Protein can be stored at room temperature in solution or powder form

Delta SMG protein in phosphate-buffered saline supplemented with two amino acids (pbs-aa), 50 mM L-arginine and 50 mM L-glutamate, was filtered through a 0.22-μm filter and stored at room temperature (RT) or 4°C. Proteins were collected at different time points including 3, 7, 14, 21 days and 3 months. The collected samples were mixed with 5X SDS-PAGE Loading Dye, heated at 100°C for 5 minutes and stored at 4°C until a gel appeared (Fig. 11 A and B). PBS with or without 50 mM L-arginine and 50 mM L-glutamate was tested for retention of delta S _HM or S _MG (first 3 lanes) at 4°C. Delta-S _MG was tested for lyophilization with or without excipient and stored at room temperature for over 2 weeks (last 2 lanes) (Fig. 11C). Stability testing shows that Delta S _MG protein can be stored in solution and is stable at both room temperature and 4 degrees C for at least 21 days. Moreover, delta S _MG can be lyophilized and stored at room temperature, remaining stable for at least 3 months.

Sequences

SEQ ID NO: 1

QCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG NFKNLREFVFKNIDGY FKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDE VRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ DVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLL FNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDWNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRAS ANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELD KYFKNHTSPDVDLGDISGINASWNIQKEIDRLNEVAKNLNESLIDLQELGKYEQY

SEQ ID NO: 2

QCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNATNWIKVCEFQFCNDPFLDVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGY FKI YSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAA YYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVR FPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVR QIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLF RKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVWLSFELLHA PATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEI LDITPCSFGGVSVITPGTNTSNQVAV LYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAG CLIGAEHVNNSYECDIPIGAGICASYQTQTNSRGSAGSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSK RSFIEDLLFNKVTLADAGFIKQYGDC LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAY RFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNWNQNAQALNTLVKQLSS NFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLI RAAEIRASANLAATKMSEC VLGQSKRVDFCGKGYHLMSFPQSAPHGWFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFV SNGTHWFVTQRNFYEPQIITTDNTFVSGNCDWIGIVNNTVYDPLQPELDSFKEELDKYFKNHT SPDVDLGDIS GINASWNIQKEIDRLNEVAKNLNE S LIDLQELGKY EQ

Expression construct of the S _MG protein of the delta variant of SARS-CoV-2

SEQ ID NO:3

Signal peptide modified from A/Brisbane/59/2007(HlNl)

MKVKLLLVLLCTFTATYAGT (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 label

HHHHHH (SEQ ID NO: 8)

Residues replacing the original furin cleavage site

GSAG (SEQ ID NO: 9)

Highly conserved epitopes

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)

N CD V VIGIVNNT V Y (SEQ ID NO: 48)

PELDSFKEELDKYFKNHTS (SEQ ID NO: 49)

VNIQKEIDRLNEVA (SEQ ID NO: 50)

NLNESLIDLQ (SEQ ID NO: 51)

LGKYEQYIKWP (SEQ ID NO: 52)

--->

RЎРџРРЎРћРљ RџРћРЎР›Р•Р”ОВАТЕЛЬНОСТЕЙ

<110> АКАДЕМРРђ РЎРРќРРљРђ

МА, Чэ

R'RћRќ, R§Рё-РҐСѓСЌР№

РҐРЈРђРќ, Хань-Р

<120> RЈРЎРћР'ЕРRЁР•РќРЎРўR'RћR'RђRќРќРђРЇ RљRћR RћRќRђR'RR RЈРЎРќРђРЇ R'РђРљР¦РРќРђ

<130>G4590-16000PCT

<140> PCT/US2022/071682

<141> 2022-04-12

<150> US 63/173,752

<151> 2021-04-12

<150> US 63/190,199

<151> 2021-05-18

<160> 52

<170> Patent In version 3.5

<210> 1

<211> 1196

<212>PRT

<213> SARS-CoV-2

<400> 1

Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr

1 5 10 15

Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser

20 25 30

Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn

35 40 45

Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys

50 55 60

Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala

65 70 75 80

Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr

85 90 95

Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn

100 105 110

Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu

115 120 125

Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe

130 135 140

Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln

145 150 155 160

Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu

165 170 175

Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser

180 185 190

Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser

195 200 205

Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg

210 215 220

Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp

225 230 235 240

Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr

245 250 255

Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile

260 265 270

Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys

275 280 285

Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn

290 295 300

Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr

305 310 315 320

Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser

325 330 335

Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr

340 345 350

Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly

355 360 365

Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala

370 375 380

Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly

385 390 395 400

Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe

405 410 415

Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val

420 425 430

Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu

435 440 445

Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser

450 455 460

Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln

465 470 475 480

Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg

485 490 495

Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys

500 505 510

Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

515 520 525

Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys

530 535 540

Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr

545 550 555 560

Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro

565 570 575

Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser

580 585 590

Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro

595 600 605

Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser

610 615 620

Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala

625 630 635 640

Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly

645 650 655

Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg

660 665 670

Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala

675 680 685

Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn

690 695 700

Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys

705 710 715 720

Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys

725 730 735

Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg

740 745 750

Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val

755 760 765

Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe

770 775 780

Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser

785 790 795 800

Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala

805 810 815

Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala

820 825 830

Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu

835 840 845

Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu

850 855 860

Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala

865 870 875 880

Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile

885 890 895

Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn

900 905 910

Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr

915 920 925

Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln

930 935 940

Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile

945 950 955 960

Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala

965 970 975

Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln

980 985 990

Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser

995 1000 1005

Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln

1010 1015 1020

Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser

1025 1030 1035

Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr

1040 1045 1050

Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile

1055 1060 1065

Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val

1070 1075 1080

Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu

1085 1090 1095

Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys

1100 1105 1110

Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu

1115 1120 1125

Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe

1130 1135 1140

Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly

1145 1150 1155

Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu

1160 1165 1170

Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln

1175 1180 1185

Glu Leu Gly Lys Tyr Glu Gln Tyr

1190 1195

<210> 2

<211> 1193

<212>PRT

<213> SARS-CoV-2

<400> 2

Gln Cys Val Asn Leu Arg Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr

1 5 10 15

Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser

20 25 30

Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn

35 40 45

Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys

50 55 60

Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala

65 70 75 80

Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr

85 90 95

Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn

100 105 110

Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu

115 120 125

Asp Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Gly Val

130 135 140

Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe

145 150 155 160

Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu

165 170 175

Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His

180 185 190

Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu

195 200 205

Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln

210 215 220

Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser

225 230 235 240

Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln

245 250 255

Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp

260 265 270

Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu

275 280 285

Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg

290 295 300

Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

305 310 315 320

Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr

325 330 335

Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val

340 345 350

Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser

355 360 365

Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser

370 375 380

Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr

385 390 395 400

Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly

405 410 415

Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly

420 425 430

Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

435 440 445

Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro

450 455 460

Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr

465 470 475 480

Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val

485 490 495

Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro

500 505 510

Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe

515 520 525

Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe

530 535 540

Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala

545 550 555 560

Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser

565 570 575

Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln

580 585 590

Val Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala

595 600 605

Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly

610 615 620

Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His

625 630 635 640

Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys

645 650 655

Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Gly Ser Ala Gly Ser Val

660 665 670

Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn

675 680 685

Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr

690 695 700

Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser

705 710 715 720

Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn

725 730 735

Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu

740 745 750

Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala

755 760 765

Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly

770 775 780

Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg

785 790 795 800

Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala

805 810 815

Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg

820 825 830

Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro

835 840 845

Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala

850 855 860

Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln

865 870 875 880

Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val

885 890 895

Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe

900 905 910

Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser

915 920 925

Ala Leu Gly Lys Leu Gln Asn Val Val Asn Gln Asn Ala Gln Ala Leu

930 935 940

Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser

945 950 955 960

Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val

965 970 975

Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr

980 985 990

Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn

995 1000 1005

Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys

1010 1015 1020

Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1025 1030 1035

Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val

1040 1045 1050

Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His

1055 1060 1065

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn

1070 1075 1080

Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln

1085 1090 1095

Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val

1100 1105 1110

Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro

1115 1120 1125

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1130 1135 1140

His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn

1145 1150 1155

Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1160 1165 1170

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu

1175 1180 1185

Gly Lys Tyr Glu Gln

1190

<210> 3

<211> 1271

<212>PRT

<213> SARS-CoV-2 (Delta strain)

<400> 3

Met Lys Val Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr

1 5 10 15

Ala Gly Thr Gln Cys Val Asn Leu Arg Thr Arg Thr Gln Leu Pro Pro

20 25 30

Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val

35 40 45

Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe

50 55 60

Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn

65 70 75 80

Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val

85 90 95

Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe

100 105 110

Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn

115 120 125

Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp

130 135 140

Pro Phe Leu Asp Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu

145 150 155 160

Ser Gly Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser

165 170 175

Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn

180 185 190

Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr

195 200 205

Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe

210 215 220

Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr

225 230 235 240

Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly

245 250 255

Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly

260 265 270

Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr

275 280 285

Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys

290 295 300

Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser

305 310 315 320

Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile

325 330 335

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

340 345 350

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

355 360 365

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

370 375 380

Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr

385 390 395 400

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

405 410 415

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

420 425 430

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

435 440 445

Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn

450 455 460

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

465 470 475 480

Ser Lys Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

485 490 495

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

500 505 510

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

515 520 525

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

530 535 540

Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn

545 550 555 560

Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr

565 570 575

Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr

580 585 590

Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr

595 600 605

Ser Asn Gln Val Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val

610 615 620

Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr

625 630 635 640

Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly

645 650 655

Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala

660 665 670

Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Arg Gly Ser Ala

675 680 685

Gly Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly

690 695 700

Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr

705 710 715 720

Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr

725 730 735

Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu

740 745 750

Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn

755 760 765

Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu

770 775 780

Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp

785 790 795 800

Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro

805 810 815

Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu

820 825 830

Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile

835 840 845

Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val

850 855 860

Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala

865 870 875 880

Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala

885 890 895

Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly

900 905 910

Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala

915 920 925

Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser

930 935 940

Thr Ala Ser Ala Leu Gly Lys Leu Gln Asn Val Val Asn Gln Asn Ala

945 950 955 960

Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala

965 970 975

Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Pro Pro Glu

980 985 990

Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu

995 1000 1005

Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg

1010 1015 1020

Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu

1025 1030 1035

Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu

1040 1045 1050

Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu His

1055 1060 1065

Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro

1070 1075 1080

Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val

1085 1090 1095

Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe

1100 1105 1110

Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly

1115 1120 1125

Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp

1130 1135 1140

Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys

1145 1150 1155

Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile

1160 1165 1170

Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp

1175 1180 1185

Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp

1190 1195 1200

Leu Gln Glu Leu Gly Lys Tyr Glu Gln Asp Ile Arg Ser Leu Val

1205 1210 1215

Pro Arg Gly Ser Pro Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg

1220 1225 1230

Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu

1235 1240 1245

Ser Thr Phe Leu Gly Gly Ser Gly Ser Leu Glu Val Leu Phe Gln

1250 1255 1260

Gly Pro His His His His His

1265 1270

<210> 4

<211> 19

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 4

Met Lys Val Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr

1 5 10 15

Ala Gly Thr

<210> 5

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 5

Leu Val Pro Arg Gly Ser

15

<210> 6

<211> 31

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 6

Pro Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr

1 5 10 15

Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly

20 25 30

<210> 7

<211> 12

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 7

Gly Ser Gly Ser Leu Glu Val Leu Phe Gln Gly Pro

1 5 10

<210> 8

<211> 6

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 8

His His His His His

15

<210> 9

<211> 4

<212>PRT

<213> SARS-CoV-2

<400> 9

Gly Ser Ala Gly

1

<210> 10

<400> 10

000

<210> 11

<400> 11

000

<210> 12

<400> 12

000

<210> 13

<400> 13

000

<210> 14

<400> 14

000

<210> 15

<400> 15

000

<210> 16

<400> 16

000

<210> 17

<400> 17

000

<210> 18

<400> 18

000

<210> 19

<400> 19

000

<210> 20

<400> 20

000

<210> 21

<400> 21

000

<210> 22

<400> 22

000

<210> 23

<400> 23

000

<210> 24

<400> 24

000

<210> 25

<400> 25

000

<210> 26

<400> 26

000

<210> 27

<400> 27

000

<210> 28

<400> 28

000

<210> 29

<400> 29

000

<210> 30

<400> 30

000

<210> 31

<400> 31

000

<210> 32

<400> 32

000

<210> 33

<400> 33

000

<210> 34

<400> 34

000

<210> 35

<400> 35

000

<210> 36

<400> 36

000

<210> 37

<400> 37

000

<210> 38

<400> 38

000

<210> 39

<400> 39

000

<210> 40

<400> 40

000

<210> 41

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 41

Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

1 5 10

<210> 42

<211> 16

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 42

Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg

1 5 10 15

<210> 43

<211> 11

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 43

Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys

1 5 10

<210> 44

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 44

Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn

1 5 10

<210> 45

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 45

Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr

1 5 10

<210> 46

<211> 15

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 46

Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

1 5 10 15

<210> 47

<211> 13

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 47

Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys

1 5 10

<210> 48

<211> 14

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 48

Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr

1 5 10

<210> 49

<211> 19

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 49

Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1 5 10 15

His Thr Ser

<210> 50

<211> 14

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 50

Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala

1 5 10

<210> 51

<211> 10

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 51

Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln

1 5 10

<210> 52

<211> 11

<212>PRT

<213> Artificial Sequence

<220>

<223>Polypeptide

<400> 52

Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro

1 5 10

<---

Claims (20)

1. An immunogen comprising a glycoengineered coronavirus spike protein comprising a plurality of truncated N-glycans and one or more unmodified O-glycans, wherein the glycoengineered coronavirus spike protein consists of the amino acid sequence of SEQ ID NO: 1 or a variant thereof characterized by at least 96% identity with the amino acid sequence of SEQ ID NO: 1, or an immunologically active fragment thereof. 2. The immunogen according to claim 1, wherein the plurality of truncated N-glycans are monosaccharides. 3. The immunogen according to claim 2, where the monosaccharides are N-acetylglucosamines (GlcNAc). 4. The immunogen of claim 1, wherein a plurality of truncated N-glycans are located in the receptor binding domain (RBD), thereby exposing a large number of highly conserved epitopes having the amino acid sequences 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) . 5. The immunogen of claim 1, wherein a plurality of truncated N-glycans are localized in the heptad repeat 2 (HR2) domain, thereby revealing a large number of highly conserved epitopes having the amino acid sequences 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. The immunogen of claim 1, wherein the glycoengineered coronavirus spike protein is derived from a native coronavirus spike protein by in vitro glycoengineering, wherein the native coronavirus spike protein is a severe acute respiratory syndrome coronavirus 2 (SAR-CoV-2) spike protein, or its options. 7. The immunogen according to claim 6, where the option is selected from the group consisting of D614G, alpha (lines B.1.1.7 and Q), beta (lines B.1.351 and descendants), gamma (lines P.1 and descendants), 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 (lines B. 1.617.2 and AY) and omicron (lines B. 1.1.529 and BA). 8. The immunogen according to claim 1, where the coronavirus spike protein constructed using glycoengineering is present in the form of a trimer. 9. The immunogen according to claim 6, wherein the glycoengineered coronavirus spike protein retains the same tertiary structure as the native coronavirus spike protein. 10. The immunogen according to claim 4, wherein the glycoengineered coronavirus spike protein is capable of inducing an enhanced immune response compared to the native coronavirus spike protein. 11. The immunogen of claim 10, wherein the enhanced immune response is an increased IgG titer, an increased IgM titer, an increased neutralization titer, an increased CD4 T cell-mediated cellular response, an increased CD8 T cell-mediated cellular response, or a combination thereof. 12. The immunogen of claim 6, wherein the glycoengineered spike protein is produced using one or more chemical or enzymatic methods. 13. The immunogen according to claim 12, wherein the glycoengineered spike protein is produced using endoglycosidase H (Endo H). 14. The immunogen according to claim 1, wherein the glycoengineered coronavirus spike protein contains a polypeptide consisting of the amino acid sequence SEQ ID NO: 1, where the polypeptide consists of 22 truncated N-glycans, each of which contains a GlcNAc fragment. 15. The immunogen according to claim 1, wherein the glycoengineered coronavirus spike protein contains a polypeptide consisting of the amino acid sequence SEQ ID NO: 2, where the polypeptide consists of 21 truncated N-glycans, each of which contains a GlcNAc fragment. 16. An immunogenic composition for inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof in a subject in need thereof, comprising an effective amount of an immunogen according to any one of claims. 1-15. 17. The immunogenic composition according to claim 16, further containing an adjuvant, and said adjuvant is selected from aluminum hydroxide, aluminum phosphate, incomplete Freund's adjuvant (IFA), squalene, alum, alhydrogel, MF59, QS-21, CpG 1018, AS03, AS37, Matrix-M and their combinations. 18. The immunogenic composition of claim 16, wherein the immunogenic composition is an improved coronavirus vaccine capable of inducing an enhanced immune response compared to a vaccine using the native coronavirus spike protein, where the coronavirus is SAR-CoV-2 or a variant thereof. 19. A method of inducing an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or a variant thereof in a subject in need thereof, comprising administering to the subject an effective amount of an immunogenic composition according to claim 16 or 17. 20. The method of claim 19, wherein the immune response comprises the production of neutralizing antibodies against SARS-CoV-2 or a variant thereof, wherein the variant is selected from the group consisting of D614G, alpha (lines B.1.1.7 and Q), beta (lines B.1.351 and descendants), gamma (P.1 lineages and descendants), 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 (lines B. 1.617.2 and AY) and omicron (lines B. 1.1.529 and BA).