Glycosylated rbd and use thereof

Abstract

The present invention provides, as a means for efficiently producing anti-coronavirus antibodies having cross-reactivity, a polypeptide having an amino acid sequence that has at least 60% identity with an amino acid sequence of SEQ ID NO: 2 and having an amino acid sequence modified so that a sugar chain binds at positions corresponding to the 85 position, the 164 position, and the 172 position of an amino acid sequence of SEQ ID NO: 2.

Description

Glycosylated RBD and its use

[0001] Techniques relating to glycosylation of the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 are disclosed.

[0002] The COVID-19 pandemic caused by the β-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global health crisis. Coronavirus entry into host cells is mediated by the viral S protein, which forms trimeric spikes on the viral surface. The entry receptor for SARS-CoV-2 and SARS-CoV (referred to here as SARS-CoV-1) is human cell surface angiotensin-converting enzyme 2 (ACE2), and the receptor-binding domain (RBD) of the spikes of both these viruses binds to ACE2 with high affinity. Therefore, the RBD is thought to be a potential primary target for neutralizing antibodies.

[0003] However, given that previous coronavirus epidemics (e.g., SARS-CoV-1 and MERS-CoV) were caused by zoonotic coronaviruses that crossed the species barrier (Non-Patent Document 1), the possibility of the future emergence of similar viruses poses a significant threat to global public health, even with effective vaccines against current viruses. For example, WIV1-CoV, a pre-emergent bat coronavirus not yet seen in humans, shows high homology to SARS-CoV-1 and SARS-CoV-2 and has been shown to be capable of infecting human ACE2-expressing cells (Non-Patent Document 2). However, sera from SARS-CoV-2-infected individuals show very limited cross-neutralization with WIV1-CoV, except for rare individuals with low levels of neutralizing antibodies (Non-Patent Document 3).

[0004] Wacharapluesadee , S. , Tan , CW , Maneeorn , P. , Duengkae , P. , Zhu , F. , Joyjinda , Y. , Kaewpom , T. , Chia , WN , Ampoot , W. , Lim , BL , et al. (2021). Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia. Nat Commun 12, 972.Menachery, VD, BL Yount Jr, K Debbink, S Agnihothram, LE Gralinski, JA Plante, RL Graham, T Scobey, XY Ge, EF Donaldson, et al. 2015. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nat. Med. 21:1508-1513.Garcia-Beltran, WF, Lam, EC, Astudillo, MG, Yang, D, Miller, TE, Feldman, J, Hauser, BM, Caradonna, TM, Clayton, KL, Nitido, AD, et al. (2021). COVID-19-neutralizing antibodies predict disease severity and survival. Cell 184, 476–488–411.

[0005] One of the snowflakes is a slightly less expensive one.

[0006] The SARS-CoV-2 RBD is composed of two subdomains (Figure 1A). The core-RBD subdomain consists of a central β-sheet flanked by α-helices, providing a stable folding framework for the receptor-binding motif (RBM, residues 437-508) that encodes ACE2 binding and receptor specificity (Shang et al., 2020, Structural basis of receptor recognition by SARS-CoV-2. Nature 581, 221-224). (Here, the region encoded by the RBM is referred to as the head-RBD subdomain) (Shang et al., 2020, ibid.). Initial analysis of convalescent sera from SARS-CoV-2 infected individuals demonstrated that many neutralizing antibodies (class 1 and 2) recognize the head-RBD subdomain (Figure 1B). However, as mentioned above, cross-neutralization by known antibodies is quite limited. Therefore, the present inventors conducted extensive research and found that the core-RBD subdomain may be a more promising target in terms of broad-spectrum defense responses.

[0007] One potential obstacle to targeting the core-RBD subdomain is the immunorecessive nature of epitopes in this subdomain. Potential approaches to circumvent this problem include at least the following: i) altering the immunogen surface by targeted point mutations and deletions (Jardine et al., 2013, Rational HIV immunogen design to target specific germline B cell receptors. Science 340, 711-716; Impagliazzo et al., 2015, A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen. Science 349, 1301-1306; Valkenburg et al., 2016, Stalking influenza by vaccination with pre-fusion headless HA mini-stem. Sci Rep 6, 22666); ii) introducing N-linked glycans that may mask adjacent epitopes via NxS / T sequons (Duan et al., 2018, Glycan Masking Focuses Immune Responses to the HIV-1 CD4-Binding Site and Enhances Elicitation of VRC01-Class Precursor Antibodies. Immunity 49, 301-311 e305; Eggink et al., 2014, Guiding the immune response against influenza virus hemagglutinin toward the conserved stalk domain by hyperglycosylation of the globular head domain. J Virol 88, 699-704).The present inventors have demonstrated that introducing glycans into specific sites in the SARS-CoV-2 head-RBD subdomain to promote immune responses against the core-RBD subdomain promotes the induction of potent cross-neutralizing antibodies against SARS-CoV-1 and WIV1-CoV (clade 1 sarbecoviruses). Based on this finding, further studies and research have led to the provision of the following representative inventions.

[0008] Item 1. A polypeptide having an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2 and that has been modified so that sugar chains are attached at positions corresponding to positions 85, 164, and 172 of the amino acid sequence of SEQ ID NO: 2. Item 2. The polypeptide according to Item 1, wherein the modified amino acid sequence is further modified so that sugar chains are attached at positions corresponding to positions 118 and / or 147 of the amino acid sequence of SEQ ID NO: 2. Item 3. The polypeptide according to Item 1 or 2, wherein the sugar chain is an N-glycan. Item 4. The polypeptide according to any one of Item 1 to 3, wherein sugar chains are attached at positions corresponding to positions 85, 164, and 172 of the amino acid sequence of SEQ ID NO: 2. Item 5. The polypeptide according to Item 4, wherein sugar chains are further attached at positions 118 and / or 147. Item 6. The polypeptide according to Item 4 or 5, wherein the sugar chain is an N-glycan. Item 7. A polynucleotide having a nucleotide sequence encoding the polypeptide according to Item 1 or 2. Item 8. A composition comprising the polypeptide of any one of Items 1 to 6 or the polynucleotide of Item 7. Item 9. The composition of Item 8, which is a vaccine. Item 10. A method for producing an anti-coronavirus antibody, which comprises immunizing an animal with the polypeptide of any one of Items 1 to 6. Item 11. An antibody obtained by the method of Item 10.

[0009] A means for efficiently producing cross-reactive anti-coronavirus antibodies is provided.

[0010] [Correction under Rule 91 06.07.2022] Design of SARS-CoV-2 RBD glycan masking mutants. (A) shows the structure of the RBD of CoV-2 S (PDB:6YZ5) that binds to the ACE-2 extracellular domain (PDB:6M0J). (B) shows the epitopes of representative anti-CoV-2 mAbs. (C) Left shows glycosylation sites in RBD WT, GM9, and GM14. (C) Right shows ribbon models of RBD GM9 and GM14. Spheres represent introduced N-glycan sites. (D) shows the glycan occupancy at N-linked glycosylation sites of RBD WT, GM9, and GM14 as measured by LC-MS. (E) ELISA binding of previously reported mAbs against SARS-CoV-2 RBD (CB6, C002, S309, CR3022, and EY6A) to WT, GM9, and GM14. The anti-C. albicans hIgG1 mAb 23B12 was used as a negative control. The heat map shows the percentage of binding (the area under the curve [AUC] value in ELISA for RBD WT is set to 100% for each mAb). Representative results from three independent experiments are shown. Figure 2: Design and Expression of CoV-2 RBD Glycan-Masking Mutants. (A) The structure of the RBD-ACE2 complex is shown. (B) The parent amino acid sequence and the introduced NXT sequon are shown. (C) SDS-PAGE of RBD WT, GM9, and GM14. The glycan mutants of the CoV-2 spike RBD immunogen effectively elicited CoV-1 S RBD-specific antibodies. (A) Schematic diagram of the experimental design. (B) CoV-2 RBD (left) or CoV-1 RBD (right)-specific serum IgG1 antibody levels were measured by ELISA in the serum of pre-immunized mice or mice immunized with CoV-2 RBD, GM9, GM14, or CoV-1 RBD WT. Samples from three independent experiments (three or four mice per group) were pooled. (C) Class 3 / 4 serum antibodies from CoV-2 RBD, GM9, GM14, or CoV-1 RBD WT immunized mice were measured by epitope-blocking ELISA using a mixture of CR3022 / EY6A / S309 human IgG1 mAbs (left).Affinity measurements were determined by ELISA and expressed as the binding ratio for low-density / high-density plate-bound CoV-1 RBD protein (right). (D) WIV1 RBD-specific serum IgG1 antibody levels (left) and affinity (right) were measured as in (B) and (C, right), respectively. Horizontal lines indicate mean values; each symbol represents one mouse. ＊ , p<0.05; ＊＊ , p<0.01; ＊＊＊ , p<0.001; unpaired Student's t-test. Immunization with nanoparticle antigens results in the effective induction of CoV-2 S RBD-specific antibodies. ELISA plots for CoV-2 RBD wt probe recognition in sera from mice immunized with biotin(+)RBD / St-avi nanoparticles, biotin(-)RBD / St-avi nanoparticles, or biotin(+)RBD alone 3 weeks after primary immunization. Horizontal lines indicate the mean values; each symbol represents one mouse. CR3022 mouse IgG1 monoclonal antibody (Invivogen) was used as a standard. ＊ , p<0.05; ＊＊ , p<0.01; ＊＊＊ , p<0.001; unpaired Student's test. These results show that glycan engineering of SARS-CoV-2 head-RBD elicited cross-neutralizing activity against SARS-CoV-1 and WIV-1-CoV. (A) BALB / c mice were prime-boost immunized with CoV-2 RBD WT, GM9, GM14, or CoV-1 RBD as shown in Figure 3. Serum was collected 7 days after the boost and preincubated with CoV-2 S, CoV-1 S, or WIV-1 CoV S-pseudotyped VSVΔG-luc for 1 hour. The mixture was incubated with VeroE6 TMPRESS2 cells overnight. The NT50 values ​​for mice in each group are shown on the graph. The NT50 values ​​for samples below the detection limit (1:20) were set at 20. ＊*P<0.05, Wilcoxon test. RBD WT (n=11); GM9 (n=11); GM14 (n=11); CoV-1 RBD (n=8). (B) Neutralizing activity of plasma samples from pre-pandemic healthy donors (HC, n=8) or convalescent COVID-19 patients (n=24) against CoV-2, CoV-1, and WIV-1 CoV S-pseudotyped VSVΔG-luc. The NT50 for samples below the detection limit (1:20) was set as 20. (C) Donor information. (D) ADE of infection with DENV1 single infectious particle (SRIP) by anti-DENV1 E mAb 3H12. DENV1 SRIP was administered with different concentrations of 3H12 or control mouse IgG. １ (E) Sera from immunized mice were serially diluted and mixed with CoV-2 S-pseudotyped VSVΔG-luc for 1 hour, then incubated with RAW264.7 cells. Luciferase activity was measured 24 hours after infection. (F) Representative results from two or three independent experiments are shown. Data are means ± SEM. The dotted line in the graphs (NT50 = 20) represents the lower limit of detection (A and B). ＊ , p<0.05; ＊＊ , p<0.01; ＊＊＊, p<0.001; unpaired Student's t-test. Figure 1 shows the cross-reactivity of serum and GC B cells in mice immunized with GM9 or GM14. (A) ELISA plot for CoV-2 RBD wt probe recognition in mouse serum after prime / boost with GM9 or GM14. Serum was collected 3 weeks after primary immunization. (B) Representative FACS plot of GC B cells derived from dLN after immunization. Cells were gated for antigen-binding IgG+ GC B cells (CD138-B220+IgG+GL7+Fas+). The percentage of cells positive for RBD wt binding among GM9- or GM14-binding cells is shown in the right graph (n=4 each). Immunization with CoV-2 GM antigen results in the activation of CoV-1 and CoV-2 cross-reactive GC B cells. (A) Representative flow cytometry plots analyzing GC B cells (CD138-B220+IgG+GL7+Fas+) derived from lymph nodes of mice 3 weeks after primary immunization with each antigen. Plots are representative of findings from two independent experiments (left). Quantification of absolute numbers of CD138-B220+IgG+GL7+Fas+ GC B cells from multiple mice in a single experiment (right). Results are representative of two independent experiments. Horizontal lines indicate mean values; each symbol represents one mouse. (B) Representative results of flow cytometric analysis of CoV-2 RBD-binding MLN GC B cells (CD138-B220+IgG+GL7+Fas+CoV-2 RBD(APC)+CoV-2 RBD). Plots are representative of findings from two independent experiments (left). Absolute numbers of GC B cells from multiple mice per experiment are shown (right). Results are representative of two independent experiments. (C) Flow cytometric analysis of CoV-2 RBD-binding MLN GC B cells (CD138-B220+IgG+GL7+Fas+CoV-2 RBD(APC)+CoV-2 RBD(PE-Cy7+) and CoV-1 Cross-reactivity with CoV-1 and CoV-2 RBD. Plots are representative of findings from two independent experiments (left). Frequency of CoV-1 and CoV-2 RBD cross-reactive GC B cells from multiple mice in a single experiment (right). Results are representative of two independent experiments.The horizontal line indicates the mean value; each symbol represents one mouse. ＊ , P < 0.05; ＊＊ , P < 0.01; ＊＊＊ , P<0.001; unpaired Student's t-test. Authentic virus neutralization assay. A mixture of 100 TCID50 virus and serially diluted heat-inactivated plasma samples was incubated for 1 hour and then placed on VeroE6-TMPRSS2 cells seeded in a 96-well plate. After 4 days of culture, the cells were fixed with formalin and stained with crystal violet solution. The cutoff dilution index for a cytopathic effect greater than 50% was presented as the microneutralization titer. The microneutralization titer of samples below the detection limit (1:40 dilution) was set as 20. ＊ , P < 0.05; ＊＊ , *P<0.01; unpaired Student's t-test. GC-derived mAbs exhibited broad RBD binding and neutralization against clade 1 SARS-associated CoVs. (A) Clonality, VH and VK mutations, and antibody class of CoV-1, WIV1-CoV, CoV-2, BatCoV Anlong-103 (AL-103), and BtCoV BM48-31, binding to the S RBD, and neutralizing activity against CoV-1, WIV1-CoV, and CoV-2 pseudoviruses of mAbs derived from GC B cells from mice immunized with GM9 (GM9-1 and GM9-2) or GM14 (GM14-1 and GM14-2). (B) Correlation of mAb binding to CoV-2 RBD with CoV-1 or WIV1-CoV RBD. Binding of mAbs to CoV-1, WIV1, and CoV-2 RBD was measured by bead-based flow cytometry assay. Binding signals (gMFI) to each RBD are plotted on a graph. Spearman's rank correlation coefficient and p-value are shown. (C) Binding affinity of mAbs to RBD proteins. Equilibrium dissociation constants (M) and IC of individual clones. ５０ (μg / ml) are shown. (D) Neutralizing activity (IC) of a representative cross-reactive mAb and a murine IgG2c recombinant antibody bearing the variable regions of the previously isolated S304 (class 3) and CR3022 (class 4) human anti-RBD antibodies. ５０[μg / ml]). Correlation between gMFI by bead-based flow cytometry assay and Rmax by Biolayer Interferometry (Octet) assay. gMFI of bivalent mAb-CoV-2 RBD interactions, as well as K Ｄ (M), RMax, k ｏｎ (1 / Ms) and k ｏｆｆ (1 / s) are shown. Spearman rank test. Generation of cross-reactive memory B cells and long-lived plasma cells in mice immunized with GM9 or GM14. (A) Schematic diagram of experimental design. (B) Cross-reactivity of CoV-2 RBD-specific memory B cells (CD138-B220+IgG1+CD38+GL7-CoV-2 RBD (APC)+CoV-2 RBD (PE-Cy7)+) with CoV-1 RBD derived from the spleens of mice 1 month after booster immunization with each antigen. Quantification of the frequency of CoV-1 and CoV-2 RBD cross-reactive memory B cells (top) and the absolute number of total CoV-2-specific memory B cells (bottom) from multiple mice in a single experiment. Results are representative of two independent experiments. (C) ELISPOT analysis of CoV-1 (left) or CoV-2 (right)-specific IgG1 ASC responses in pre-immune mice or 1 month after booster immunization with each antigen. Results are representative of two independent experiments. Horizontal lines indicate the mean; each symbol represents one mouse. ＊ , P < 0.05; ＊＊ , P < 0.01; ＊＊＊, P<0.001; unpaired Student's t-test. The characteristics of mAbs generated from GC B cells of mice immunized with GM9 or GM14 are shown. A comparison of the RBD sequences of representative β-coronavirus sarbecoviruses is shown. The boxed region is the receptor-binding motif (RBD head region). Asterisks indicate glycosylation insertion sites. The amino acid sequences of each RBD used for the alignment in Figure 13 are SEQ ID NOS: 2, 107, 108, 109, 110, 111, 112, 113, and 114, from top to bottom. A phylogenetic tree of the RBD sequences of representative β-coronavirus sarbecoviruses is shown. The numbers in parentheses indicate homology with the SARS-CoV-2 RBD. The amino acid sequence of the SARS-CoV-2 spike protein (SEQ ID NOS: 1) is shown. The region in bold is the receptor-binding domain (SEQ ID NOS: 2).

[0011] 1. Definitions In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0012] As used herein, "identity" of amino acid sequences refers to the degree of amino acid sequence match between two or more comparable amino acid sequences. Therefore, the greater the identity between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul. A program called BLASTX based on this BLAST algorithm has been developed. Specific techniques for these analysis methods are known; see the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0013] As used herein, "corresponding positions" with respect to amino acid sequences and nucleotide sequences can be determined by aligning a subject sequence with a reference sequence (e.g., the amino acid sequence of SEQ ID NO: 2) so as to maximize homology among residues present in each sequence. Alignment can be performed using known algorithms, and procedures for doing so are known to those skilled in the art. For example, alignment can be performed using the Clustal W multiple alignment program (Nucleic Acids Res, 1994, 22:4673-4680) with default settings. Alternatively, revised versions of Clustal W, such as Clustal W2 and Clustal omega, can also be used. Clustal W, Clustal W2, and Clustal omega are available, for example, on the websites of the European Bioinformatics Institute (EBI [www.ebi.ac.uk / index.html]) and the DNA Data Bank of Japan (DDBJ [www.ddbj.nig.ac.jp / Welcome-j.html]), operated by the National Institute of Genetics.

[0014] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0015] 2. Polypeptide The polypeptide preferably has a certain level of identity to the amino acid sequence of SEQ ID NO: 2. "A certain level or higher" means, for example, 60% or higher, 65% or higher, 70% or higher, 75% or higher, 80% or higher to 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, or 99% or higher.

[0016] SEQ ID NO: 2 is the amino acid sequence of the RBD consisting of 199 amino acid residues from positions 331 to 529 of the amino acid sequence of the S protein of SARS-CoV-2 (SEQ ID NO: 1). In one embodiment, the polypeptide may be shorter or longer than 199 amino acid residues. In one embodiment, the polypeptide is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues longer than 199 residues, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues shorter than 199 residues. When the polypeptide is longer than 199 residues, it is preferable that the sequence of the longer portion has a certain level of identity to the corresponding amino acid sequence of SEQ ID NO: 1. Here, "certain level or higher" is as described above. In one embodiment, the polypeptide may include a tag sequence, for example, at the C-terminus or N-terminus.

[0017] In one embodiment, if the amino acid sequence of the polypeptide does not completely match the amino acid sequence of SEQ ID NO: 1 or 2, the mismatched amino acid residues preferably represent conservative amino acid substitutions for the corresponding amino acid residues in the amino acid sequence of SEQ ID NO: 1 or 2.

[0018] The polypeptide preferably has an amino acid sequence modified so that sugar chains are attached at positions corresponding to positions 85, 164, and 172 of the amino acid sequence of SEQ ID NO: 2. The type of sugar chain is not limited, and may be either an N-type sugar chain or an O-type sugar chain. In one embodiment, the sugar chain is preferably an N-type sugar chain.

[0019] Techniques for attaching a sugar chain to a specific position in a polypeptide are known. For example, in order for an N-glycan to be attached to the position corresponding to position 85 in the amino acid sequence of SEQ ID NO: 2, it is preferable that the amino acid residue corresponding to position 85 in the amino acid sequence of SEQ ID NO: 2 is an aspartic acid residue, and the amino acid residue corresponding to position 87 in the amino acid sequence of SEQ ID NO: 2 is a serine or threonine residue (preferably a threonine residue). Similarly, in order for an N-glycan to be attached to the position corresponding to position 164 in the amino acid sequence of SEQ ID NO: 2, it is preferable that the amino acid residue corresponding to position 164 in the amino acid sequence of SEQ ID NO: 2 is an aspartic acid residue, and the amino acid residue corresponding to position 166 in the amino acid sequence of SEQ ID NO: 2 is a serine or threonine residue (preferably a threonine residue). Similarly, in order for an N-glycan to be attached to the position corresponding to position 172 in the amino acid sequence of SEQ ID NO: 2, it is preferable that the amino acid residue corresponding to position 172 in the amino acid sequence of SEQ ID NO: 2 is an aspartic acid residue, and the amino acid residue corresponding to position 174 is a serine or threonine residue.

[0020] In one embodiment, the polypeptide preferably has an amino acid sequence modified so that sugar chains are attached at positions corresponding to positions 85, 164, and 172 in the amino acid sequence of SEQ ID NO: 2, as well as positions 118 and / or 147. In one embodiment, the polypeptide preferably has an amino acid sequence modified so that sugar chains are attached at positions corresponding to positions 85, 164, and 172 in the amino acid sequence of SEQ ID NO: 2, as well as positions 118 and 147. In one embodiment, the polypeptide preferably has an amino acid sequence modified so that sugar chains are attached at positions corresponding to positions 85, 164, and 172 in the amino acid sequence of SEQ ID NO: 2, but not modified so that sugar chains are attached at positions corresponding to positions 118 and / or 147 in the amino acid sequence of SEQ ID NO: 2. In one embodiment, from the viewpoint of stable expression, the polypeptide preferably has a polypeptide that has not been modified so that sugar chains are attached at at least one position selected from the group consisting of positions 87, 126, 141, 152, 154, and 157 in the amino acid sequence of SEQ ID NO: 2. Here, "at least one" may be at least two, at least three, at least four, at least five, or six. The type of sugar chain is not limited, and may be either an N-type sugar chain or an O-type sugar chain. In one embodiment, the sugar chain is preferably an N-type sugar chain.

[0021] In order for an N-glycan to be bound to a position corresponding to position 118 in the amino acid sequence of SEQ ID NO: 2, it is preferred that the amino acid residue corresponding to position 118 in the amino acid sequence of SEQ ID NO: 2 is an aspartic acid residue, and the amino acid residue corresponding to position 120 is a serine or threonine residue (preferably a threonine residue). Similarly, in order for an N-glycan to be bound to a position corresponding to position 147 in the amino acid sequence of SEQ ID NO: 2, it is preferred that the amino acid residue corresponding to position 147 in the amino acid sequence of SEQ ID NO: 2 is an aspartic acid residue, and the amino acid residue corresponding to position 149 is a serine or threonine residue (preferably a threonine residue).

[0022] In one embodiment, the polypeptide preferably has the amino acid residues at positions 85, 164, and 172 substituted with aspartic acid residues and the amino acid residues at positions 87, 166, and 174 substituted with serine or threonine residues (preferably threonine residues) in the amino acid sequence of SEQ ID NO: 2. In one embodiment, the polypeptide preferably has the amino acid residues at positions 85, 118, 147, 164, and 172 substituted with aspartic acid residues and the amino acid residues at positions 87, 120, 149, 166, and 174 substituted with serine or threonine residues (preferably threonine residues) in the amino acid sequence of SEQ ID NO: 2.

[0023] In one embodiment, the polypeptide preferably has a glycan occupancy rate of at least a certain level at positions corresponding to one or more positions selected from the group consisting of positions 85, 118, 147, 164, and 172 of the amino acid sequence of SEQ ID NO: 2. The glycan occupancy rate can be measured by the method described in the Examples below. An example of a glycan occupancy rate of at least a certain level is, for example, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more. In one embodiment, "one or more" means two or more, three or more, or four or more.

[0024] The polypeptide may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0025] The polypeptide may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0026] The polypeptide can be easily produced according to its amino acid sequence using known genetic engineering techniques, such as PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, and recombinant protein production techniques. In one embodiment, the polypeptide can be obtained by expressing a polynucleotide encoding it in a suitable host.

[0027] The polypeptide may be purified after synthesis. For example, the polypeptide can be obtained by extracting the cells collected from the culture by centrifugation, filtration, or the like. Specifically, the disruption of the cells in the first extraction step can be achieved by enzymatic digestion, osmotic disruption, rapid pressure / pressure / vacuum application, ultrasound, various homogenizers, and the like. The disrupted cells can then be fractionated using physical means such as low-speed centrifugation, ultracentrifugation, filtration, molecular sieving, and membrane concentration; chemical means such as precipitants, solubilizers, adsorbents, and dispersants; and physicochemical means such as electrophoresis, column chromatography, supports, dialysis, and salting out, in combination with other means. Furthermore, when applying these means, physicochemical conditions such as temperature, pressure, pH, and ionic strength can be appropriately set.

[0028] The polypeptide is useful for efficiently producing cross-reactive anti-coronavirus antibodies. In one embodiment, cross-reactivity refers to reactivity with SARS-CoV-2 and coronaviruses other than SARS-CoV-2. In one embodiment, the coronavirus other than SARS-CoV-2 belongs to the β-coronavirus genus, preferably the Sarbecovirus subgenus of the β-coronavirus genus. In one embodiment, the coronavirus other than SARS-CoV-2 is a SARS-associated coronavirus. In one embodiment, examples of coronaviruses other than SARS-CoV-2 include SARS-CoV-1, WIV1-CoV, MERS-CoV, and Pangolin-CoV. In one embodiment, the cross-reactive anti-coronavirus antibody is preferably a neutralizing antibody, and more preferably a broadly neutralizing antibody. Such antibodies are useful for preventing, treating, or alleviating diseases or symptoms caused by coronaviruses. Therefore, the polypeptide can be used as a vaccine or therapeutic agent for these purposes.

[0029] 3. Polynucleotide The polynucleotide preferably has a base sequence encoding the above-described polypeptide. In one embodiment, the polynucleotide is preferably in the form of an expression cassette for the above-described polypeptide. The expression cassette is not particularly limited as long as it is a polynucleotide capable of expressing the polypeptide in cells. Typical examples of expression cassettes include a promoter and a polynucleotide having a base sequence encoding the polypeptide under the control of the promoter.

[0030] The promoter contained in the expression cassette is not particularly limited and can be appropriately selected depending on the type of cell into which the expression cassette is introduced. For example, Pol II promoters, specifically, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β-actin promoter, CAG promoter, etc., can be mentioned. Other examples include tryptophan promoter, lac promoter, T7 promoter, T5 promoter, T3 promoter, SP6 promoter, arabinose-inducible promoter, cold shock promoter, tetracycline-inducible promoter, etc.

[0031] The type (origin) of cells used to introduce a polynucleotide and produce a polypeptide is not particularly limited. Examples include animal cells, plant cells, and fungi such as yeast. Examples of animals include various mammals, such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cattle, sheep, goats, and deer, as well as non-mammalian vertebrates and invertebrates. In one embodiment, mammalian cells are preferred. The type of cells is not particularly limited, and examples include cells derived from various tissues or with various properties, such as blood cells, hematopoietic stem and progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, neurons, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, and cancer cells.

[0032] The expression cassette may contain other elements as needed (for example, a multicloning site (MCS), a drug resistance gene, a replication origin, an enhancer sequence, a repressor sequence, an insulator sequence, a reporter protein (for example, a fluorescent protein) coding sequence, a drug resistance gene coding sequence, etc.). The MCS is not particularly limited as long as it contains multiple (for example, 2 to 50, preferably 2 to 20, more preferably 2 to 10) restriction enzyme sites.

[0033] Examples of drug resistance genes include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, and a puromycin resistance gene.

[0034] The reporter protein is not particularly limited as long as it is a luminescent (color-producing) protein that emits light (color) upon reaction with a specific substrate, or a fluorescent protein that emits fluorescence upon excitation light. Examples of luminescent (color-producing) proteins include luciferase, β-galactosidase, chloramphenicol acetyltransferase, and β-glucuronidase. Examples of fluorescent proteins include GFP, Azami-Green, ZsGreen, GFP2, HyperPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, JRed, KillerRed, Cherry, HcRed, and mPlum.

[0035] The expression cassette, together with other sequences as necessary, can constitute an expression vector. The type of vector is not particularly limited, and examples thereof include plasmid vectors such as animal cell expression plasmids, and viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, and Sendai viruses.

[0036] Polynucleotides can be easily prepared using known genetic engineering techniques, such as PCR, restriction enzyme digestion, and DNA ligation techniques.

[0037] The polynucleotides allow for the production of the above-mentioned polypeptides, and thus may be used for the same purposes as the above-mentioned polypeptides, such as vaccines against diseases or conditions caused by coronaviruses.

[0038] 4. Compositions Preferably, compositions comprise the above-described polypeptides or polynucleotides. Compositions comprising the above-described polypeptides or polynucleotides are useful as vaccines or pharmaceutical compositions.

[0039] The composition may contain other components depending on the purpose. Examples of other components include stabilizers that enhance the heat resistance of the vaccine and adjuvants that serve as auxiliary agents to enhance immunogenicity. Examples of stabilizers include sugars and amino acids. Examples of adjuvants include aluminum compounds (e.g., aluminum hydroxide gel, etc.), CpG oligodeoxynucleotides, mineral oil, vegetable oil, alum, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, etc. The adjuvant may be a single type or a combination of two or more types.

[0040] The composition may contain, as necessary, a base, carrier, solvent, dispersant, emulsifier, buffer, stabilizer, excipient, binder, disintegrant, lubricant, thickener, moisturizer, colorant, flavor, chelating agent, etc.

[0041] The dosage form of the vaccine or pharmaceutical composition is not particularly limited, and examples include injectable preparations such as aqueous injectable preparations, non-aqueous injectable preparations, suspension injectable preparations, and solid injectable preparations; oral preparations such as tablets, capsules, granules, powders, fine granules, syrups, enteric-coated preparations, sustained-release capsules, chewable tablets, drops, pills, oral liquid preparations, confectionery tablets, sustained-release preparations, and sustained-release granules; and external preparations such as nasal drops, inhalants, rectal suppositories, inserts, enemas, and jellies.

[0042] The content of the active ingredient in the composition can be determined based on the subject of administration, the administration route, the dosage form, the condition of the patient, the judgment of the physician, etc. For example, the content of the polypeptide or polynucleotide in the composition can be 0.0001 to 95% by weight, preferably 0.001 to 50% by weight.

[0043] The amount of vaccine or pharmaceutical composition used can be determined taking into consideration various factors such as the route of administration, the subject's health condition, the subject's age, sex, body weight, pharmacological knowledge such as pharmacokinetics and toxicological characteristics, whether a drug delivery system is used, whether the vaccine or pharmaceutical composition is administered as part of a combination of other drugs, and whether the vaccine or pharmaceutical composition is intended to provide an immune or boosting effect. For example, the active ingredient can be administered in a single dose of 1 μg to 100 mg / kg (body weight), or 50 μg to 10 mg / kg (body weight). The interval and frequency of administration are not particularly limited, but it is preferable to administer the vaccine or pharmaceutical composition at intervals of, for example, about 1 to 30 weeks.

[0044] 5. Antibodies and methods for producing the same By using the above-mentioned polypeptides as antigens, cross-reactive anti-coronavirus antibodies can be produced.

[0045] Types of antibodies include polyclonal antibodies, monoclonal antibodies, human chimeric antibodies, humanized antibodies, fully human antibodies, single-chain antibodies, and antigen-binding fragments (Fab, F(ab')2, minibody, scFv-Fc, Fv, scFv, diabody, triabody, and tetrabody). A human chimeric antibody is an antibody whose variable regions have sequences derived from an animal other than human (e.g., mouse, cow, etc.) and whose constant regions have sequences derived from human.

[0046] Methods for producing these antibodies are known and can be produced according to standard methods (Current Protocols in Molecular Biology, Chapters 11.12-11.13 (2000)). Specifically, polyclonal antibodies can be obtained by immunizing a non-human animal such as a rabbit with an antigen and extracting the antibody from the serum of the immunized animal according to standard methods. Monoclonal antibodies can be obtained from hybridoma cells prepared by immunizing a non-human animal such as a mouse or cow with the antigen and fusing the resulting spleen cells with myeloma cells (Current Protocols in Molecular Biology, ed., Ausubel et al. (1987) Publish. John Wiley and Sons. Sections 11.4-11.11). Immunization of an animal can be achieved by administering the above-described polypeptide to the animal or by administering the above-described polynucleotide to the animal and allowing it to be expressed in the animal's body.

[0047] In producing antibodies, various adjuvants can be used depending on the type of host to enhance the immunological response, including Freund's adjuvant, mineral gels such as aluminum hydroxide, lysolecithin, pluronic polyol, polyanions, peptides, oil emulsions, surfactants such as keyhole limpet hemocyanin and dinitrophenol, and human adjuvants such as Bacillus Calmette-Guerin (BCG) and Corynebacterium parvum.

[0048] In one embodiment, the cross-reactivity of an antibody refers to reactivity with SARS-CoV-2 and coronaviruses other than SARS-CoV-2. In one embodiment, the coronavirus other than SARS-CoV-2 belongs to the β-coronavirus genus, preferably the Sarbecovirus subgenus of the β-coronavirus genus. In one embodiment, the coronavirus other than SARS-CoV-2 is a SARS-associated coronavirus. In one embodiment, examples of coronaviruses other than SARS-CoV-2 include SARS-CoV-1, WIV1-CoV, MERS-CoV, and Pangolin-CoV. In one embodiment, the antibody preferably recognizes the core-RBD subdomain of the S protein of SARS-CoV-2. In one embodiment, the cross-reactive anti-coronavirus antibody is preferably a neutralizing antibody, and more preferably a broadly neutralizing antibody. Such antibodies are useful for preventing, treating, or alleviating diseases or symptoms caused by coronaviruses.

[0049] Materials and Test Methods Mice BALB / c mice were purchased from Japan SLC Co., Ltd. and maintained under specific pathogen-free conditions. Gender-matched mice aged 7 to 8 weeks were used in all experiments. All animal experiments were conducted in accordance with the guidelines for animal experiments of Osaka University.

[0050] Human blood samples were collected at Tokyo Shinagawa Hospital, and plasma was isolated using Vacutainer CPT Tubes (Becton, Dickinson). The study protocol was approved by the Ethics Committee for Medical Research Involving Human Subjects of the National Institute of Infectious Diseases, the Ethics Committee of Tokyo Shinagawa Hospital, and Osaka University. All participants provided written informed consent in accordance with the Declaration of Helsinki.

[0051] [Correction under Rule 91 06.07.2022] Vaccine Design: The mammalian expression construct of the parental rRBD (pCAGGS-CoV-2 RBD-His-Avi) encodes aa 331-530 of SARS CoV-2 S and contains an N-terminal signal peptide from the MERS S protein (MIHSVFLLMFLLTPTESYVD) and a C-terminal 6xHis-Avi tag (HHHHHHGLNDIFEAQKIEWHE). For glycan masking, NxT sequons were introduced at the RBD surface residues shown in Figure 2. Mutations predicted to disrupt the RBD structure or with a low glycosylation score (<0.5) according to NetNGlyc (http: / / www.cbs.dtu.dk / services / NetNGlyc / ) were avoided. Candidate constructs were transfected into Expi293F cells to test whether additional glycosylation interferes with RBD expression. The antigenicity of the GM mutants was assessed by ELISA using a known anti-RBD antibody.

[0052] Recombinant protein expression and purification. The recombinant SARS-CoV RBD and GM mutant plasmids were transiently transfected into Expi293F cells (Thermo Fisher Scientific) using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. After 3 to 4 days, the supernatant was collected and passed through a 0.45-micrometer filter. The recombinant protein was purified from the supernatant using Talon metal affinity resin (Takara Bio Inc.) according to the manufacturer's protocol. The eluate from the resin was concentrated using an Amicon Ultra 4 10,000 NWML, with the elution buffer exchanged for PBS. For nanoparticle antigens and probes, post-translational biotinylation of the rRBD and GM mutants was performed in culture by co-expression of the BirA enzyme via co-transfection of the BirA-Flag expression vector (Addgene). Cells were maintained in culture with 100 μM biotin (Sigma-Aldrich) after transfection as described above.

[0053] For preparation of ELISA or ELISPOT antigens, mammalian expression constructs of CoV-2 RBD WT, GM9, GM14, and SARS-associated CoV RBD containing a thrombin cleavage site (LVPRGS) before the C-terminal 6xHis-Avi tag were transiently transfected into Expi293F cells. The purified proteins were treated with thrombin using a Thrombin kit (Merck) according to the manufacturer's protocol. The cleaved 6xHis-Avi tag and thrombin were removed using Talon metal affinity resin (Takara Bio Inc.) and benzamidine Sepharose (GE), respectively. The purity and biotinylation efficiency of the recombinant proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0054] Anti-CoV-2 S RBD Monoclonal Antibodies DNA fragments encoding the heavy and κ or λ chain variable regions of known anti-CoV-2 mAbs (Pinto et al., 2020, Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583, 290-295; Yuan et al., 2020, Structural basis of a shared antibody response to SARS-CoV-2. Science 369, 1119-1123; Zhou et al., 2020, Structural basis for the neutralization of SARS-CoV-2 by an antibody from a convalescent patient. Nat Struct Mol Biol 27, 950-958) were synthesized (Eurofins) and cloned into human IgG1, Igκ, and Igλ expression vectors, respectively. The heavy and light chain expression vectors were co-transfected into Expi293F. Each mAb was purified using Protein G Sepharose (GE) according to the manufacturer's protocol.

[0055] N-linked glycan occupancy analysis by liquid chromatography / mass spectrometry (LC / MS) was performed according to Tajiri-Tsukada et al., 2020 (Establishment of a highly precise multi-attribute method for the characterization and quality control of therapeutic monoclonal antibodies. Bioengineered 11, 984-1000). Sample protein (10 μg) was treated with MPEX PTS Reagents (GL Sciences). The reduced and carboxymethylated protein was digested with 2 μg of chymotrypsin (1 μg / ml, Promega) at 37°C for 3 days. The resulting peptides were desalted using an Oasis HLB μElution plate (Waters), dried, and dissolved in 50 μl of 0.1% FA solution. The sample solution was analyzed by liquid chromatography / mass spectrometry (LC / MS) using high-energy collisional dissociation (HCD) and electron transfer / high-energy collisional dissociation (EThcD) in parallel acquisition mode. The EThcD-MS / MS conditions were as follows: auxiliary activation collision energy 23%, isolation window m / z 2, and maximum injection time 100 ms. Peptide identification was performed by database search using BioPharma Finder™ 3.1 (Thermo Fisher Scientific). Search parameters were as follows: mass tolerance ±5 ppm, confidence score >80, and ID type MS2. Carboxymethylation (+58.005 Da) was set as a static modification of Cys residues. The human glycan database stored in the software was used for glycopeptide identification.The integrated peak areas of multiple precursor ions derived from each glycopeptide were calculated, and the relative peak area and glycan occupancy (%) were calculated using the following formulas: Relative peak area = (peak area of ​​glycopeptide) / (total peak area of ​​glycopeptide and non-glycosylated peptide) Glycan occupancy (%) = (total relative peak area of ​​glycopeptide) / (total relative peak area of ​​glycopeptide and non-glycosylated peptide) × 100.

[0056] Nanoparticle coating: Streptavidin-coated 0.11 μm nanoparticles (Bangs Laboratories) were washed twice with PBS, and 5.5 μg of nanoparticles were incubated with 50 μg of biotinylated protein in 80 μl of PBS per mouse for 5 hours at 4°C. Coating efficiency was measured by flow cytometry.

[0057] Immunization: Mice were vaccinated with each antigen group by prime immunization at 7 to 8 weeks of age and boosted 3 weeks later with a second vaccination. Prior to inoculation, the immunogen suspension was gently mixed with AddaVax adjuvant (Invivogen) at a 2:1 (volume / volume) ratio to achieve a final concentration of 0.4 mg / mL antigen. Mice were intramuscularly injected with 60 μL of immunogen per injection site (total 120 μL) under isoflurane anesthesia using a 29G × 1 / 2 needle syringe (Terumo Corporation). Antigens were used as nanoparticles for prime immunization and as monomeric proteins for boost immunization.

[0058] Flow cytometry. Single-cell suspensions were prepared from inguinal and iliac lymph nodes or spleens. Inguinal and iliac lymph nodes were harvested and pooled from individual mice. Detection of CoV-1 or CoV-2 RBD-specific B cells was performed using biotinylated rRBD prelabeled with fluorophore-conjugated streptavidin. Samples were prestained with decoy probes to exclude induced 6xHis, Avi tag, biotin, and streptavidin-specific B cells. Cell samples were analyzed using an Attune NxT flow cytometer (Thermo Fisher Scientific) and sorted using a FACS Aria II cell sorter (BD Bioscience). Data were analyzed using FlowJo software V10 (Tree Star).

[0059] BCR cloning and antibody expression. CoV-1 and CoV-2-S RBD-specific B cells were single-cell sorted from lymph nodes of immunized mice 7 days after the boost as described above. Antibody cloning and expression were performed using published methods (von Boehmer et al., 2016, Sequencing and cloning of antigen-specific antibodies from mouse memory B cells. Nat Protoc 11, 1908-1923; Inoue et al., 2021, Exit from the germinal center to become quiescent memory B cells depends on metabolic reprogramming and provision of a survival signal. J Exp Med 218). Monoclonal antibodies were expressed using the Expi293 Expression System (Thermo Fisher Scientific) and purified from the culture supernatant of Expi293F cells using Protein G Sepharose (GE).

[0060] Flow cytometry analysis of mAb binding to antigen. Anti-mouse Igκ microparticles and negative control particles (BD CompBeads) were mixed at a 1:1 ratio. The beads were then mixed with 250 ng of purified mAb (mIgG2c / mIgκ) for 20 minutes, washed with PBS containing 2% FBS, and subsequently labeled with PE-conjugated antigen for 20 minutes. The binding ability of the mAb to the antigen was assessed by flow cytometry (BD FACSCanto II and FACS Aria II).

[0061] ELISA Nunc Maxisorp immunoplates (Thermo Fisher Scientific) were coated with each rRBD (100 ng / 50 μl). Blocking was performed using BlockingOne solution (Nacalai Tesque). The plates were sequentially incubated with serially diluted serum samples or monoclonal antibodies cloned from single GC B cells and IgG1-HRP detection antibodies (Southern Biotech). Detection was performed using KPL SureBlue TMB Microwell Peroxidase Substrate (SeraCare). CR3022 mouse IgG1e3 monoclonal antibody (Invivogen) was used as a standard.

[0062] For affinity ELISA, serially diluted sera were incubated on plates coated with 1 μg / ml CoV-1 RBD protein (low density) or 8 μg / ml CoV-1 RBD (high density). The affinity of CoV-1 RBD-specific IgG1 was expressed as the ratio of binding to low-density:high-density CoV-1 RBD-coated plates as described by Wang et al., 2015 (Anti-HA Glycoforms Drive B Cell Affinity Selection and Determine Influenza Vaccine Efficacy. Cell 162, 160-169).

[0063] For MAb-based epitope blocking ELISA, CoV-1 RBD- or CoV-2 RBD-coated plates were prepared for serum samples or cloned monoclonal antibodies, respectively. After blocking, a mixture of CR3022 (class 4), EY6A (class 4), and / or S309 (class 3) human IgG1 mAbs (150 ng / 50 μl each) or a single antibody was added. After 3 hours of incubation at room temperature, the plates were washed with PBST, and then serum samples or cloned monoclonal antibodies were added and incubated at 37°C for 1 hour. After three washes, HRP-conjugated goat anti-mouse IgG1 or IgG2c, respectively, was added and incubated at room temperature for 1 hour. The share of class 3 / 4 antibodies in the total anti-CoV-1 RBD antibodies was calculated by subtracting the titers measured by epitope blocking ELISA and conventional serum titer ELISA, and then expressed as a percentage.

[0064] ELISPOT assay: Plates with cellulose membrane bottoms were coated with CoV-1 RBD or CoV-2 RBD (100 ng / 50 μl). Bone marrow cells were added to the wells and incubated in RPMI 1640 medium supplemented with 10% FBS, 50 μM 2-ME, and 2 mM sodium pyruvate at 5% CO. ２ The plates were incubated at 37°C for 5 hours under reduced pressure. After washing with PBS containing 0.05% Tween 20, goat anti-mouse IgG1 Ab was added to the wells, followed by alkaline phosphatase-labeled anti-goat IgG Ab. Spots were visualized with BCIP / NBT substrate (Promega) and counted.

[0065] Pseudovirus assay: SARS-CoV S protein-pseudotyped VSVΔG-luc was generated as described by Tani et al., 2010 (Involvement of ceramide in the propagation of Japanese encephalitis virus. J Virol 84, 2798-2807) and Yoshida et al., 2021 (SARS-CoV-2-induced humoral immunity through B cell epitope analysis in COVID-19 infected individuals. Sci Rep 11, 5934). HEK293T cells were transfected with expression plasmids for each CoV S protein (SARS-CoV-2 Wuhan strain S, SARS-CoV-1 S, and SARS-associated CoV WIV1 S) using TransIT-LT1 (Mirus) according to the manufacturer's instructions. Twenty-four hours after transfection, the transfectants were inoculated with VSVΔG-luc virus (MOI = 0.1). After 2 hours of incubation, the cells were washed with DMEM and further cultured for 24 to 48 hours. Cell-free supernatants were collected and used for neutralization assays as described by Nie et al., 2020 (Quantification of SARS-CoV-2 neutralizing antibodies by a pseudotyped virus-based assay. Nat Protoc 15, 3699-3715). Mouse serum and human plasma samples were incubated at 56°C for 30 minutes and then serially diluted from 1 / 20 in culture medium. SARS-CoV S-pseudotyped VSVΔG-luc was incubated with different dilutions of mouse serum, human plasma, or recombinant antibodies for 1 hour at 4°C and then inoculated onto monolayer cultures of VeroE6-TMPRSS2 cells in a 96-well plate. Sixteen hours after seeding, the cells were washed with PBS and then lysed with luciferase cell culture lysis reagent (Promega).After centrifugation, the clarified cell lysates were incubated with firefly luciferase assay substrate (Promega) in a 96-well white polystyrene plate (Corning). Luciferase activity was measured using a GloMax Discover luminometer (Promega). NT50 or IC. ５０ was calculated by Prism software (GraphPad).

[0066] Antibody-dependent enhancement (ADE) assay DENV1 single-round infectious particles (SRIPs) were prepared as described by Matsuda et al., 2018 (High-throughput neutralization assay for multiple flaviviruses based on single-round infectious particles using dengue virus type 1 reporter replicon. Sci Rep 8, 16624). To evaluate the ADE assay system using RAW264.7 cells, the anti-E mouse monoclonal antibody 3H12 (Yamanaka et al., 2013, A mouse monoclonal antibody against dengue virus type 1 Mochizuki strain targeting envelope protein domain II and displaying strongly neutralizing but not enhancing activity. J Virol 87, 12828-12837) was preincubated with DENV1 SRIP (multiplicity of infection [MOI] = 0.1) for 1 hour at 4°C and then inoculated into RAW264.7 cells in a 96-well plate. After 2 hours, the cells were washed with PBS and cultured in fresh medium for 3 days. Infectivity was measured using a NanoLuc assay kit (Promega). To test whether the antisera had an ADE of CoV-2 infection, SARS-CoV-2 S-pseudotyped VSVΔG-luc was incubated with sera from immunized mice for 1 hour at 4°C and then inoculated into RAW264.7 cells in a 96-well plate. After 2 hours, the cells were washed with PBS and cultured in fresh medium for 16 to 20 hours. Luciferase activity in infected cells was measured as described above.

[0067] Virus neutralization assay: A mixture of 100 TCID50 virus and serially diluted heat-inactivated plasma samples (two-fold serial dilutions starting from a 1:40 dilution) was incubated at 37°C for 1 hour and then placed on VeroE6-TMPRSS2 cells seeded in a 96-well flat-bottom plate (TPP). VeroE6-TMPRSS2 cells were cultured in low-glucose DMEM (Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10% heat-inactivated fetal bovine serum, 1 mg / mL geneticin (Thermo Fisher Scientific), and 100 U / mL penicillin / streptomycin (Thermo Fisher Scientific) at 5% CO. ２ The cells were maintained at 37°C while receiving 1000 mg of ...

[0068] Biolayer Interferometry Assay: The kinetics of monoclonal antibody binding to antigens were determined using an OctetRED96e system (ForteBio) with shaking at 1000 rpm at 30°C. Biotinylated RBD proteins from CoV-1, CoV-2, WIV1-CoV, bat SARS-associated AL-103, and BM48-31 were loaded onto a streptavidin biosensor (ForteBio) at 6 μg / ml in 1× kinetic buffer (0.1% BSA, 0.02% Tween-20 in PBS) for 900 seconds and incubated with serially diluted Fab antibodies (100 nM, 33.3 nM, 11.1 nM, and 0 nM) for 120 seconds, followed by a 300-second dissociation time in 1× kinetic buffer. Binding curves were fitted to a 1:1 binding model and dissociation constant (KD) values ​​were calculated by Octet data analysis software (ForteBio).

[0069] Phylogenetic analysis of antibody clones. For each cell, V, D, J gene and CDR3 assignments were performed against the full-length BCR sequence using IgBlast and the IMGT mouse reference. Clones were defined separately for each experiment based on the heavy chain alone using the DefineClones function in the Change-O package, and 38 / 38 cells from GM14#1 and 26 / 31 cells from GM14#2 were considered to be the same clone. Clone tree reconstruction was performed for these two primary clones using Alakazam (both Change-O and Alakazam are part of the Immcantation analysis framework) (Gupta et al., 2015, Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire sequencing data. Bioinformatics 31, 3356-3358). The reconstructed clone tree was finally plotted using Cytoscape.

[0070] Statistics Statistical analysis was performed by unpaired and paired two-tailed Student's t-test using GraphPad Prism software. A p-value of <0.05 was considered significant. Error bars represent ±SD.

[0071] [Correction under Rule 91 06.07.2022] Design of SARS-CoV-2 RBD Glycan Mutants To engineer the SARS-CoV-2 head-RBD subdomain (Figure 1A), we introduced an N-linked glycosylation site (NxT motif) into this subdomain, which contains residues essential for ACE-2 binding (Barnes et al., 2020, Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies. Cell 182, 828-842 e816) (Figures 1A and 1C). Established monoclonal antibodies (mAbs) that recognize the SARS-CoV-2 RBD are classified into four types; as shown in Figure 1B, classes 1 / 2 and 3 / 4 recognize epitopes within the head-RBD and core-RBD subdomains, respectively. Five potential sites for introduction of the NxT sequon (GM14) were identified based on the following criteria: (1) a surface residue on a class 1 or 2 epitope; (2) a residue outside of the class 3 and 4 epitopes within the core-RBD subdomain (Barnes et al., 2020, supra) (Figure 1C); (3) a residue within or conformationally adjacent to a non-conserved patch of the head-RBD subdomain (Figure 2A); (4) the NxT mutation is not expected to disrupt the overall structure of the RBD; and (5) it is not expected to reduce RBD protein expression or stability (Starr et al., 2020, Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell 182, 1295-1310 e1220).Although the N415 site is somewhat distal to the head-RBD subdomain (Figure 1C), this site has previously been reported to be part of the epitope recognized by the class 1 mAb CB6 (Shi et al., 2020, "A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2." Nature 584, 120-124). Two glycan mutants, GM9 and GM14, were designed with three and five additional glycosylation sites, respectively. Structural modeling predicted that these additional glycans would interfere with antigen recognition by class 1 and class 2 antibodies (Barnes et al., 2020, ibid.; Yuan et al., 2020, ibid.).

[0072] [Rule 91 Correction 06.07.2022] Results: The glycan mutants were C-terminally tagged with a 6xHis-Avi tag and expressed in mammalian Expi293 cells. They exhibited higher molecular weights than the wild-type RBD on SDS-PAGE (Figure 2B). The glycan occupancy of each introduced glycosylation site was determined by liquid chromatography-mass spectrometry (LC-MS). All introduced glycosylation sites were over 75% occupied, except for the N494 and N502 glycans of GM9, which exhibited 70% and 57%, respectively (Figure 1D). The process of N-glycosylation in the endoplasmic reticulum (ER) is subject to conformational constraints (Breitling and Aebi, 2013, N-linked protein glycosylation in the endoplasmic reticulum. Cold Spring Harb Perspect Biol 5, a013359). The somewhat lower glycan occupancy at N494 and N502 may be due to limited accessibility of oligosaccharyltransferase to these asparagine residues, which are located in close proximity to each other.

[0073] We next investigated whether glycan modifications interfere with class 1 / 2 antibody binding while preserving antigenic epitopes for class 3 / 4 antibodies. In ELISA, all tested antibodies were potent binders of the wild-type RBD (Figure 1E). mAb CB6, a typical class 1 neutralizing antibody, was completely unable to bind to either of the glycan mutants (GM9 and GM14). Binding of the class 2 antibody mAb C002 (Robbiani et al., 2020, Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature 584, 437-442) to the RBD was abolished by GM14, but this mAb still weakly bound to GM9, indicating that five mutations are required for complete inhibition of class 2 mAb C002. In contrast, mAb S309 (Class 3), mAb CR3022 (Class 4), and mAb EY6A (Class 4) targeting the core-RBD subdomain (Pinto et al., 2020, ibid.; Yuan et al., 2020, ibid.; Zhou et al., 2020, ibid.) bound to wild-type RBD, GM9, or GM14 at similar levels, indicating that the mutations introduced for additional glycosylation do not affect the overall structure of the core-RBD subdomain (Figure 1E).

[0074] [Correction under Rule 91 06.07.2022] Glycan engineering of the SARS-CoV-2 head-RBD generated more antibodies against the core-RBD with higher affinity. To overcome the limited immunogenicity of the relatively small SARS-CoV-2 RBD, it was multivalently displayed on streptavidin polystyrene nanoparticles. Consistent with the description by Walls et al., 2020 (Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2. Cell 183, 1367-1382 e1317), particulate RBD, in contrast to soluble monomeric RBD, elicited a more robust primary antibody response (Figure 4). These particulate antigens (wild-type RBD, GM9, or GM14) were injected intramuscularly into BALB / c mice with Addavax adjuvant. Three weeks after priming, mice were boosted with each non-particulate antigen (Figure 3A). Serum collected 7 days after boosting was tested for reactivity against the SARS-CoV-2 RBD. Like Alum, AddaVax predominantly induces IgG1-type antibodies in mice (Sato et al., 2020, Repurposing the psoriasis drug Oxarol to an ointment adjuvant for the influenza vaccine. Int Immunol 32, 499-507). Therefore, IgG1 responses were measured. To address the possibility that mice might have mounted an immune response against neoepitopes generated by glycan engineering that were not detected by the parent SARS-CoV-2 RBD probe, we first tested serum and GC B cell responses following each immunization protocol using immunogen-adapted glycosylation mutants and the parent probe. ELISA assay analysis showed similar RBD-specific serum titers among the probe sets; flow cytometry analysis showed that approximately 10% or 15% of GC B cells bound only the immunogen-matched glycosylation mutant probes (Fig. 6A and B).Therefore, GM9 or GM14 immunization likely results in the emergence of some neoepitope-recognizing B cells. Nevertheless, the parental SARS-CoV-2 RBD probe was generally used in this assay, as over 85% of responding cells were reactive to the parental CoV-2 RBD probe.

[0075] [Correction under Rule 91 06.07.2022] IgG1 from mice immunized with the glycan mutants showed similar reactivity to CoV-2 as wild-type RBD immunization, as assessed by ELISA (Figure 3B, left). Regarding reactivity to CoV-1, mice immunized with GM9 or GM14 showed approximately 8-fold higher reactivity than wild-type RBD immunization (Figure 3B, right). Given the sequence conservation of the RBD-core subdomain between SARS-CoV-2 and CoV-1 (Figure 2A), the enhanced cross-reactivity to CoV-1 by GM9 or GM14 immunization is most likely due to the generation of more IgG1 against the conserved RBD-core subdomain of CoV-2. Supporting this possibility, CoV-1-reactive IgG1 elicited by GM9 or GM14 immunization, similar to that elicited by wild-type RBD immunization, was inhibited by approximately 90% or 85%, respectively, by pretreatment with a mixture of validated class 3 / 4 anti-CoV-2 core-RBD antibodies (mAb S309, mAb CR3022, and mAb EY6A) (Fig. 3C, left). Next, we characterized the affinity of the cross-reactive IgG1 elicited by GM9 or GM14 immunization. The affinity of IgG1 for CoV-1 RBD was measured using a modified nitrophenyl-based affinity ELISA assay; this assay measures the ratio of high-affinity binding IgG1 to total affinity binding IgG1. As shown in Figure 3C (right), cross-reactive IgG1 elicited by GM9 or GM14 immunization had significantly higher affinity than wild-type RBD immunization. When WIV1-CoV was used instead of the SARS-CoV-1 RBD probe, both glycan mutants induced approximately eightfold higher serum titers (Figure 3D (left)). Regarding affinity, two of 11 wild-type RBD-immunized mice showed a high ratio of high-affinity binding to total affinity binding, whereas sera from the remaining nine mice had relatively lower affinity compared to GM9- or GM14-immunized mice (Figure 3D (right)). Collectively, glycan engineering of the SARS-CoV-2 head-RBD subdomain facilitates the generation of core-RBD subdomain-specific antibodies with higher affinity.

[0076] [Rule 91 Correction 06.07.2022] Glycan engineering of SARS-CoV-2 head-RBD elicited cross-neutralizing antibodies against SARS-CoV-1 and other related viruses. To determine neutralizing activity, we primarily used a vesicular stomatitis virus (VSV)-based pseudovirus method (Nie et al., 2020, ibid.) and calculated half-maximal neutralizing titers (NT50). Immunization with CoV-2 wild-type RBD, GM9, or GM14 produced similar neutralizing activity against SARS-CoV-2. GM9 or GM14 immunization showed some increase or decrease in activity, respectively, compared to CoV-2 wild-type RBD immunization (Figure 5A). This trend with GM9 or GM14 immunization was also observed using a virus neutralization assay (Figure 8).

[0077] To measure neutralizing activity against CoV-2 variants, we used a VSV-based virus containing the K417N / E484K / N501Y mutations from the B.1.351 variant. This demonstrated that even immunization with the wild-type CoV-2 RBD elicited somewhat higher neutralizing activity than wild-type CoV-2. Given that human sera from mRNA vaccines showed low neutralizing activity against viruses containing the E484K mutation (Chen et al., 2021, Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies. Nat. Med. 27:717-726), our immunization approach may generate more resistant sera.

[0078] [Correction under Rule 91 06.07.2022] Antisera elicited by GM9 or GM14 immunization possessed neutralizing activity against SARS-CoV-1, as assessed by a VSV-based pseudovirus assay, with approximately 15-fold and 10-fold higher potency, respectively, than CoV-2 wild-type RBD immunization (Figure 5A). Similarly, these sera exhibited substantial neutralizing activity against WIV1-CoV, SHC014, and PaGX (Figure 5A). Importantly, the neutralizing levels against SARS-CoV-1 and WIV1-CoV observed after GM9 or GM14 immunization were similar to the neutralizing activity against SARS-CoV-2 in convalescent-phase plasma samples from SARS-CoV-2-infected individuals analyzed by the same laboratory using the same assay format (Figures 5B and 5C). Given that the major epitopes of CoV-1-reactive antibodies elicited by GM9 or GM14 immunization are located within the core-RBD subdomain (Fig. 3C, left), these anti-core-RBD antibodies most likely contribute to the cross-neutralization of SARS-CoV-1, WIV1-CoV, SHC014, and PaGX.

[0079] [Correction under Rule 91 06.07.2022] Previous studies of SARS-CoV-1 and other viruses have suggested that antibody-dependent enhancement of infection (ADE) can occur after vaccination (Smatti et al., 2018, Viral-Induced Enhanced Disease Illness. Front Microbiol 9, 2991). To examine this possibility, VSV-based pseudoviruses were incubated with serially diluted sera from immunized mice and then inoculated into Raji, a human B lymphoma cell line frequently used to assess ADE activity of SARS-CoV (Verma et al., 2009, Analysis of the Fc gamma receptor-dependent component of neutralization measured by anthrax toxin neutralization assays. Clin Vaccine Immunol 16, 1405-1412). As a positive control for this assay, the infectivity of CoV-2 S pseudoviruses was significantly enhanced by the addition of anti-CoV-2 SRBD MW05 chimeric human / mouse IgG1 antibody (Fig. 5D, center). In this experimental setting, sera from mice immunized with GM9 or GM14, as well as sera from mice immunized with the SARS-CoV-2 wild-type RBD, did not exhibit significant ADE activity (Fig. 5D, right).

[0080] [Correction under Rule 91 06.07.2022] Reactivity Profile of Cross-Reactive GC B Cells To complement the serological characterization described above, the reactivity of GC B cells isolated after priming was assessed. As demonstrated by flow analysis, wild-type RBD, GM9, or GM14 immunization resulted in similar levels of overall GC B cells (Figure 7A). ＋ Among the cells, the frequency of GC B cells positive for both CoV-2 and CoV-1 probes was increased by immunization with GM9 or GM14 compared with wild-type RBD immunization (Fig. 7B and C).

[0081] [Amendment under Rule 91 06.07.2022] Each CoV-2＋ / CoV-1 ＋ To examine the cross-reactivity profile for GC B cells, we characterized mAbs derived from single-cell-sorted GC B cells from four individual mice (GM9-1, GM9-2, GM14-1, or GM14-2). Sequence analysis revealed that in each mouse, >85% of the isolated mAbs were members of expanded clonal lineages. Specifically, in GM14-1 or GM14-2 mice, nearly all mAbs were encoded by the same VH / VK combinations, with distinct DH and JK segments: GM14-1 (VH14-3-DH3-2-JH4 / VK4-111-JK2) and GM14-2 (VH14-3-DH2-3-JH4 / VK4-111-JK1) (Figure 12).

[0082] [Correction under Rule 91 06.07.2022] Of all the mAbs analyzed, most showed blocking of binding to CoV-2 RBD by authentic class 4 mAbs, except for the mAb derived from the VH14-1 / VK8-27 gene pair, which was blocked by authentic class 3 mAbs (Figure 12). To assess the breadth of recognition of the sarbecovirus RBD by these mAbs, we assigned a relative binding index to each antibody using flow cytometry-based measurements (Figure 9A) (Bajic et al., 2019, Influenza Antigen Engineering Focuses Immune Responses to a Subdominant but Broadly Protective Viral Epitope. Cell Host Microbe 25, 827-835 e826; Leach et al., 2019, Requirement for memory B-cell activation in protection from heterologous influenza virus reinfection. Int Immunol 31, 771-779); the relative binding index was in good agreement with the RMax of bivalent IgG antibodies measured by Octet kinetic assay (Figure 10). In addition to clade 1 (SARS-CoV-1 and WIV1-CoV as clade 1a, and SARS-CoV-2 and PaGX as clade 1b), we selected RBDs from the distantly related clades 2 (Anlong-103 [AL-103]) and 3 (BM48-31) (Lau et al., 2020, Possible Bat Origin of Severe Acute Respiratory Syndrome Coronavirus 2. Emerg Infect Dis 26, 1542-1547) (Letko et al., 2020, Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat Microbiol 5, 562-569).As expected, nearly all mAbs isolated from GM9-1, GM9-2, GM14-1, or GM14-2 mice showed binding reactivity to clade 1a and clade 1b RBDs. The relative binding indices correlated well with CoV-1 / WIV1 / SHC014 / PaGX and CoV-2 in GM9- or GM14-immunized mice (Fig. 9B). For clade 2 and 3 RBDs, several mAbs isolated from GM9-1 or GM9-2 mice showed significant binding reactivity, whereas only a small proportion showed binding reactivity in GM14-1 mice (Fig. 9A).

[0083] [Correction under Rule 91 06.07.2022] The neutralizing activity of each mAb against CoV-1, WIV1, SHC014, or PaGX or CoV-2 was then evaluated by the VSV-based pseudotype assay described above (Figures 9A and 12). Due to the large number of antibodies, screening was performed using the following two criteria, focusing on nine mAbs (Figure 9C). First, neutralizing activity was determined to be greater than 10 μg / ml IC against at least two of the three virus types. ５０ The highest efficacy against these viruses was less than 3 μg / ml IC ５０ As shown in Figure 9D, similar to the authentic mAbs S309 or CR3022, these mAbs have higher IC against CoV-2 than that observed for CoV-1 or WIV1. ５０ In the VH14-3-DH3-2-JH4 / VK4-111-JK2 (GM14-1) or VH14-3-DH2-3-JH4 / VK4-111-JK1 (GM14-2) series, the mAb with the highest binding index for CoV-2 did not have the highest neutralizing potency against CoV-2 (Figure 9C). Thus, antibody neutralizing potency may be at least partially governed by factors other than binding affinity.

[0084] [Rule 91 Amendment 06.07.2022] Elicitation of Cross-Reactive Long-Lived Plasma Cells and Memory B Cells Most successful vaccine approaches rely on the generation of memory B cells and long-lived plasma cells (LLPCs). As shown in Figure 11A, analysis was performed at week 7. At this time point, class-switched IgG1 memory B cells were similarly generated by wild-type RBD, GM9, or GM14 immunization, although GM9 and GM14 were significantly more potent at eliciting CoV-1 ＋ CoV-2 ＋ Furthermore, GM9 or GM14 immunization resulted in more SARS-CoV-1 specific antibody-secreting cells (ASCs) in the bone marrow. ＋ ASCs were induced (Fig. 11C), collectively demonstrating that both cross-reactive memory compartments were generated more efficiently by this immunization protocol with GM9 or GM14 compared to wild-type RBD immunization.

Claims

1. A polypeptide having an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:2, and having an amino acid sequence modified so that a glycan is attached to one or more of positions corresponding to positions 85, 118, 147, 164, and 172 of the amino acid sequence of SEQ ID NO:

2.

2. A polypeptide described in claim 1 having at least 95% identity to the amino acid sequence of SEQ ID NO:

2.

3. A polypeptide described in claim 1, having at least 97% identity to the amino acid sequence of SEQ ID NO:

2.

4. A polypeptide described in claim 1 having at least 98% identity to the amino acid sequence of SEQ ID NO:

2.

5. The polypeptide according to claim 1 , 2 , 3 or 4 , wherein the glycan is an N-glycan.

6. 5. The polypeptide according to claim 1, 2, 3 or 4, wherein a sugar chain is bound to each of the positions corresponding to positions 85, 164, and 172 of the amino acid sequence of SEQ ID NO:

2.

7. The polypeptide according to claim 6 , further comprising a sugar chain attached at a position corresponding to position 118 and / or position 147.

8. A polynucleotide having a base sequence encoding the polypeptide according to claim 1, 2, 3 or 4.

9. 5. A vaccine or pharmaceutical composition comprising a polypeptide according to claim 1, 2, 3 or 4.

10. A vaccine or pharmaceutical composition comprising the polynucleotide described in claim 8.

11. A method for producing an anti-coronavirus antibody, comprising immunizing an animal with a polypeptide according to claim 1, 2, 3 or 4.

12. A method for producing an anti-coronavirus antibody, comprising immunizing an animal with the polynucleotide described in claim 8.

13. An antibody obtained by the method of claim 11.