CN116761624A - Stable coronavirus proteins and vaccine compositions thereof - Google Patents

Abstract

Provided herein are compositions and methods comprising a mutant coronavirus "S" spike protein or receptor binding domain thereof that has increased expression levels, yields, and stability under the same expression, culture, or storage conditions as its corresponding native or wild-type coronavirus spike protein. These mutated spike proteins can be used to produce protein-based vaccines against one or more coronaviruses.

Description

Stable coronavirus protein and vaccine composition thereof

Government support

This invention was made with government support under Grant AI 141707 from the National Institutes of Health. The government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/132,863, filed on December 31, 2020, and U.S. Provisional Application No. 63/188,651, filed on May 14, 2021, under 35 U.S.C. §119(e), the contents of each of which are incorporated herein by reference in their entirety.

Technical Field

The field of the invention relates to methods and compositions for improving the stability of protein-based vaccines.

Background Art

Coronaviruses remain a prominent pandemic threat, with the 2019/2020 pandemic caused by the SARS-CoV-2 virus resulting in hundreds of thousands of deaths and a significant economic slowdown worldwide. SARS-CoV-2 is likely to remain a persistent epidemic even after the current pandemic abates. Therefore, effective vaccines against SARS-CoV-2 or other future emerging coronaviruses are highly desirable.

Summary of the invention

The compositions and methods described herein are based in part on the discovery of single or paired amino acid mutations in the amino acid sequence of the SARS-CoV-2S "spike" protein that increase both the yield and stability of the expressed protein (under the same or similar culture conditions). This enhanced spike protein (also referred to herein as "spike protein-derived antigen") stability allows the production of vaccines with longer shelf lives (under the same or similar storage conditions) than vaccines based on wild-type or native proteins.

Thus, in one aspect, a non-naturally occurring polypeptide is provided herein, comprising a first coronavirus receptor binding domain (RBD), wherein the first coronavirus RBD comprises at least 90% identity to residues 328-531 of SEQ ID NO: 1, and further comprising residues 328-531 of SEQ ID NO: 1. NO: at least two mutations of the RBD of 1, wherein the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/Y365W/F377V 92W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or by using Blast-p (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J.Mol.Biol.215:403-410) SEQ ID NO: 1 is aligned with the sequence of the second coronavirus receptor binding domain to determine the corresponding residues of the second coronavirus receptor binding domain. In one embodiment, the Blast-p program used is the National Center for Biotechnology Information (NCBI) online alignment tool. Alternatively, the Blast-p program can be downloaded to the device and used locally. Those skilled in the art will readily understand the use of the Blast-p alignment tool, however, for the avoidance of doubt, Protocols 1 and 2 are provided herein for use with online and downloaded alignment tools, respectively.

Protocol 1 : For use with online BLASTp alignments from the National Center for Biotechnology Information (NCBI) server.

1. Set up a BLAST alignment using the following settings:

Use the "Align two or more sequences" option

Enter the reference strain sequence of the relevant SARS-CoV-2 protein (i.e., SEQ ID NO: 1) into the "Enter Query Sequence" section

Enter any corresponding coronavirus spike protein sequence into the "Enter Subject Sequence" section

Algorithm: blastp (protein-protein BLAST)

Expected threshold: 0.1

Word length: 6

Maximum matches within query range: 0

Matrix: BLOSUM62

Vacancy cost:

Presence: 11

Extension: 1

Filter low complexity areas?: No

Mask:

For lookup table only? : No

Lowercase letters?: No.

2. Run the analysis by clicking the "BLAST" button.

3. Click the "Alignments" tab to display the alignment between the two sequences.

4. For each sequence position of interest, identify the number according to the "Query" sequence. Then identify the corresponding residue position in the "Sbjct" sequence that has been aligned with the position of the "Query" sequence.

Protocol 2 : For use with the protein BLASTp alignment tool downloaded to a local computer or server.

1. Install BLAST using the manufacturer's instructions for command line execution, or identify a computer or server where BLAST is installed.

2. Generate a FASTA formatted file containing the desired SARS-CoV-2 protein subtype-specific reference strain (i.e., SEQ ID NO: 1). In the following commands, this file will be named "query.fasta".

3. Generate a second file in FASTA format, which contains the corresponding protein sequences of different coronaviruses from the same subtype. In the following command, this file will be named "sbjct.fasta".

4. Execute the following command using a program such as Terminal, iTerm2, Windows Console, Linux console, or other similar terminal emulator. This will generate the results in a file called "results.txt". blastp -query query.fasta -subject sbjct.fasta -matrix BLOSUM62 -evalue 0.1 -

Word length 6-space open 11-space extension 1-output results.txt

(blastp-query query.fasta-subject sbjct.fasta-matrix BLOSUM62-evalue0.1-

word size 6-gapopen 11-gapextend 1-out results.txt).

5. Open results.txt and view the section showing the alignment of the two sequences. For each sequence position of interest, identify the number according to the "Query" sequence. Then identify the corresponding residue position in the "Sbjct" sequence that has been aligned with the position of the "Query" sequence.

It will be apparent to those skilled in the art that other protein alignment tools can also be used to identify sequence identity between a query sequence and a reference sequence (e.g., SEQ ID NO: 1). Given that the query sequence and the reference sequence share significant sequence identity, it is expected that other protein alignment tools will produce similar (if not identical) results to Blast-p using the protocol described herein. The protocol described herein has been shown to be accurate and effective for this purpose, and is provided herein to assist the skilled artisan in identifying amino acid residues to be mutated in the query sequence.

Another aspect provided herein includes a non-naturally occurring polypeptide comprising: a first coronavirus receptor binding domain (RBD), the first coronavirus RBD comprising at least 90% identity to residues 328-531 of SEQ ID NO: 1, and further comprising at least two mutations relative to the RBD of SEQ ID NO: 1, wherein the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/ Y365W/L513M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M; or a second coronavirus RBD comprising a fragment corresponding to SEQ ID NO:1's F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; ;F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/Y3 65W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; or at least two mutations of F338L/I358F/A363L/Y365M, wherein the corresponding positions are determined by sequence alignment of SEQ ID NO:1 with the spike protein sequence of the second coronavirus receptor binding domain using the Blast-p parameters of Protocol 1 or Protocol 2.

Another aspect provided herein includes a non-naturally occurring polypeptide, the non-naturally occurring polypeptide comprising: a first coronavirus receptor binding domain (RBD), the first coronavirus RBD comprising at least 90% identity with residues 328-531 of SEQ ID NO:1 or with the corresponding residues of the receptor binding domain of the second coronavirus determined by using Blast-p to align SEQ ID NO:1 with the sequence of the second coronavirus receptor binding domain, and further comprising at least two mutations relative to the corresponding residues in the RBD of SEQ ID NO:1 or the second coronavirus, wherein the at least two mutations enhance the stability of the polypeptide relative to the stability of the wild-type polypeptide lacking the at least two mutations. In certain embodiments, the stability of the non-naturally occurring coronavirus receptor binding domain polypeptide and the stability of its corresponding wild-type polypeptide are assessed under the same conditions.

In one embodiment of this aspect and all other aspects provided herein, the at least two mutations are in the range of SEQ ID NO:1 at the following amino acids: 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365, or by using Blast-p to convert SEQ ID NO: 1 is aligned with the sequence of the second coronavirus receptor binding domain to determine the corresponding residues of the second coronavirus receptor binding domain.

In another embodiment of this aspect and all other aspects provided herein, the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513I of SEQ ID NO: 1. 3M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or at the corresponding residues of a second coronavirus determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus receptor binding domain using Blast-p.

In another embodiment of this aspect and all other aspects provided herein, the receptor binding domain polypeptide further comprises additional amino acid residues outside the RBD of SEQ ID NO: 1. In another embodiment of this aspect and all other aspects provided herein, the receptor binding domain polypeptide as described herein is composed of a coronavirus spike protein polypeptide. In another embodiment of this aspect and all other aspects provided herein, the receptor binding domain polypeptide or coronavirus spike protein polypeptide may include, for example, a fusion polypeptide. The receptor binding domain polypeptide or coronavirus spike protein polypeptide may also include, for example, a leader sequence (e.g., for secretion). In various embodiments, the leader sequence and/or the amino-terminal methionine may be present or alternatively may be removed (e.g., by proteolytic cleavage).

In another embodiment of this aspect and all other aspects provided herein, the coronavirus receptor binding domain (RBD) comprises at least 95% identity to residues 328-531 of SEQ ID NO:1.

In another embodiment of this aspect and all other aspects provided herein, in the amino acids 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or at least two mutations at 338, 358, 363 and 365 or at corresponding residues in a second coronavirus receptor binding domain are unique mutations in the receptor binding domain relative to wild type.

In another embodiment of this aspect and all other aspects provided herein, when expressed in a cell, expression of the RBD polypeptide is increased compared to expression of a wild-type RBD polypeptide lacking the at least two mutations (i.e., under the same or similar expression or culture conditions).

In another embodiment of this aspect and all other aspects provided herein, the RBD polypeptide binds to a coronavirus antibody or binds to a coronavirus cognate receptor.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus antibody comprises a SARS-CoV-2 antibody.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus receptor corresponding to the polypeptide comprises angiotensin converting enzyme (ACE) receptor.

In another embodiment of this aspect and all other aspects provided herein, the ACE receptor is an ACE2 receptor.

In another embodiment of this aspect and all other aspects provided herein, the second coronavirus comprises a sequence of a coronavirus selected from: severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome-associated coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; HKU1, or a naturally occurring variant thereof.

In another embodiment of this aspect and all other aspects provided herein, the receptor binding domain polypeptide comprises at least 90% sequence identity to SEQ ID NO:1.

In another embodiment of this aspect and all other aspects provided herein, the RBD is fused to a second heterologous polypeptide.

In another embodiment of this aspect and all other aspects provided herein, the RBD is fused to a nanoparticle, nanostructure or a heterologous protein scaffold. In certain embodiments, the heterologous protein scaffold comprises the I53-50 trimer "A" component of SEQ ID NO: 3. In other embodiments, the heterologous protein scaffold comprises a heterologous protein scaffold as described in Table 1 of U.S. Patent No. 10,351,603, the contents of which are incorporated herein by reference in their entirety.

In another embodiment of this aspect and all other aspects provided herein, the polypeptide and/or the second polypeptide is an antigenic polypeptide.

Another aspect provided herein is a coronavirus spike protein comprising the polypeptide of claim 1.

Another aspect provided herein is a composition comprising a polypeptide as described herein and a pharmaceutically acceptable carrier. In one embodiment, the polypeptide is in the form of an admixture with a pharmaceutically acceptable carrier. In one embodiment, the polypeptide and the pharmaceutically acceptable carrier are provided as a suspension.

In one embodiment of this aspect and all other aspects provided herein, the pharmaceutical composition further comprises an adjuvant.

In another embodiment of this aspect and all other aspects provided herein, the composition has a longer shelf life than a composition comprising a wild-type RBD polypeptide lacking the at least two mutations.

In another embodiment of this aspect and all other aspects provided herein, the composition is formulated as a vaccine.

In another aspect, also provided herein is a non-naturally occurring coronavirus spike protein subunit 1 polypeptide comprising at least two mutations, wherein the at least two mutations include at least one cavity filling mutation and at least one second mutation.

In another embodiment of this aspect and all other aspects provided herein, the at least two mutations enhance the stability of the coronavirus polypeptide relative to the stability of a wild-type polypeptide lacking the at least one cavity-filling mutation and the at least second mutation.

In another embodiment of this aspect and all other aspects provided herein, the at least one cavity-filling mutation comprises a mutation of a residue in the linoleic acid binding pocket of subunit 1 of the coronavirus spike protein.

In another embodiment of this aspect and all other aspects provided herein, the at least one cavity-filling mutation comprises a mutation of a residue within residues 328-531 of SEQ ID NO: 1, or a mutation at the corresponding residue of the second coronavirus spike protein subunit 1 as determined by sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using Blast-p (e.g., Protocol 1 or Protocol 2 as described herein).

In another embodiment of this aspect and all other aspects provided herein, the at least one cavity-filling mutation comprises a mutation of a residue between residues 335-345; 355-375, or 378-395 of SEQ ID NO:1, or a mutation at the corresponding residue of the second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO:1 with the sequence of the second coronavirus spike protein subunit 1 using Blast-p (e.g., protocol 1 or 2 as described herein).

In another embodiment of this aspect and all other aspects provided herein, the at least one cavity-filling mutation comprises a mutation of a residue at amino acid 336, 338, 341, 342, 358, 361, 363, 365, 368, 374, 377, 387 or 392 of SEQ ID NO: 1, or a mutation at the corresponding residue of the spike protein subunit 1 of a second coronavirus as determined by aligning SEQ ID NO: 1 with the sequence of a second coronavirus using Blast-p (e.g., protocol 1 or 2 as described herein).

In another embodiment of this aspect and all other aspects provided herein, the at least one cavity filling mutation and the at least one second mutation are at residues 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 377 of SEQ ID NO: 1. 92; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365, or at the corresponding residues of the second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using Blast-p (e.g., Protocol 1 or 2 as described herein).

In another embodiment of this aspect and all other aspects provided herein, the at least one cavity filling mutation and the at least one second mutation are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/ Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or selected from the corresponding residues of the second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using Blast-p (e.g., protocol 1 or 2 as described herein).

In another embodiment of this aspect and all other aspects provided herein, the coronavirus spike protein subunit 1 polypeptide comprises at least 95% identity to residues 328-531 of SEQ ID NO: 1 or to the receptor binding domain sequence of a second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO: 1 with the sequence of a second coronavirus spike protein subunit 1 using Blast-p (e.g., protocol 1 or 2 as described herein).

In another embodiment of this aspect and all other aspects provided herein, in the amino acids 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 3 38, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or at least two mutations at 338, 358, 363 and 365 or at corresponding residues in the second coronavirus receptor binding domain are unique mutations in spike protein subunit 1 relative to SEQ ID NO: 1.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus polypeptide comprises at least 95% identity to SEQ ID NO: 1 or to the wild-type spike protein subunit 1 amino acid sequence of a second coronavirus.

In another embodiment of this aspect and all other aspects provided herein, when expressed in a cell, expression of the coronavirus polypeptide is increased compared to expression of a wild-type polypeptide lacking the at least one cavity-filling mutation and the at least one second mutation under the same expression conditions.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus polypeptide binds to a coronavirus antibody or binds to a cognate coronavirus receptor.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus antibody comprises a SARS-CoV-2 antibody.

In another embodiment of this aspect and all other aspects provided herein, the cognate coronavirus receptor comprises an angiotensin converting enzyme (ACE) receptor.

In another embodiment of this aspect and all other aspects provided herein, the ACE receptor is an ACE2 receptor.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus polypeptide is an engineered mutant polypeptide of a coronavirus selected from: severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome-associated coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; or HKU1.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus spike protein subunit 1 polypeptide comprises at least 90% sequence identity to SEQ ID NO:1.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus polypeptide is fused to a second heterologous polypeptide.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus polypeptide is fused to a nanoparticle, nanostructure or protein scaffold. In certain embodiments, the heterologous protein scaffold comprises the I53-50 trimer "A" component of SEQ ID NO: 3. In other embodiments, the heterologous protein scaffold comprises a heterologous protein scaffold as described in Table 1 of U.S. Patent No. 10,351,603, the contents of which are incorporated herein by reference in their entirety.

In another embodiment of this aspect and all other aspects provided herein, the coronavirus polypeptide or the second heterologous polypeptide is an antigenic polypeptide.

In another aspect, the present invention also provides a composition comprising a coronavirus polypeptide as described herein and a pharmaceutically acceptable carrier (eg, in admixture form or as a suspension).

In one embodiment of this aspect and all other aspects provided herein, the composition comprising a coronavirus polypeptide and a pharmaceutically acceptable carrier further comprises an adjuvant.

In another embodiment of this aspect and all other aspects provided herein, the composition has a longer shelf life than a composition comprising a wild-type coronavirus polypeptide lacking the at least one cavity-filling mutation and the at least second mutation when stored under the same or similar storage conditions.

In another embodiment of this aspect and all other aspects provided herein, the composition comprising a coronavirus polypeptide and a pharmaceutically acceptable carrier is formulated as a vaccine.

Another aspect provided herein relates to a cell expressing a receptor binding domain having at least two mutations as described herein, or a coronavirus polypeptide having at least two mutations as described herein.

Another aspect provided herein relates to a nucleic acid sequence encoding a receptor binding domain having at least two mutations as described herein, or a coronavirus polypeptide having at least two mutations as described herein.

In another aspect, also provided herein is a method for vaccinating a subject against a coronavirus, the method comprising administering to the subject a drug or vaccine composition as described herein.

Another aspect provided herein relates to a method of preparing a vaccine, comprising combining a composition comprising a receptor binding domain having at least two mutations as described herein or a coronavirus polypeptide having at least two mutations as described herein with an adjuvant and a pharmaceutically acceptable carrier.

Another aspect provided herein relates to a fusion polypeptide composition comprising a coronavirus receptor binding domain (RBD) fused to a heterologous protein scaffold, the coronavirus RBD comprising a mutation selected from the group consisting of I358F, Y365F, Y365W, V367F and F392W relative to the coronavirus polypeptide of SEQ ID NO: 1. In one embodiment, the heterologous protein scaffold comprises a polypeptide of SEQ ID NO: 3. In another embodiment, each cysteine in the polypeptide of SEQ ID NO: 3 is mutated to alanine.

In another embodiment, the heterologous protein scaffold comprises a heterologous protein scaffold as described in Table 1 of US Patent No. 10,351,603, the contents of which are incorporated herein by reference in their entirety.

In another embodiment, the fusion polypeptide composition further comprises a pharmaceutically acceptable carrier.

In another embodiment, the fusion polypeptide composition further comprises an adjuvant.

In another embodiment, provided herein is a vaccine composition comprising the fusion polypeptide composition.

In another embodiment, provided herein is a cell expressing the fusion polypeptide.

In another embodiment, provided herein is a composition comprising a nucleic acid encoding the fusion polypeptide.

In another embodiment, provided herein is a method of vaccinating a subject against a coronavirus, the method comprising administering to the subject a composition comprising a fusion polypeptide composition as described herein.

In another embodiment, provided herein is a method for preparing a vaccine, the method comprising combining a fusion polypeptide composition as described herein or a nucleic acid encoding such a fusion polypeptide composition with an adjuvant and a pharmaceutically acceptable carrier.

Another aspect provided herein relates to a polypeptide comprising a coronavirus receptor binding domain (RBD), wherein the coronavirus RBD comprises a mutation relative to the coronavirus polypeptide of SEQ ID NO: 1 selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L.

In one embodiment of this aspect and all other aspects provided herein, the mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367F and F392W.

In another embodiment of this aspect and all other aspects provided herein, the polypeptide comprises a second mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L.

In another embodiment of this aspect and all other aspects provided herein, the polypeptide comprises a third mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L.

In another embodiment of this aspect and all other aspects provided herein, the polypeptide comprises the polypeptide sequence of SEQ ID NO:4 or SEQ ID NO:5.

In another embodiment of this aspect and all other aspects provided herein, the polypeptide comprises a heterologous protein scaffold.

In another embodiment of this aspect and all other aspects provided herein, the heterologous protein scaffold is at least 90%, at least 95%, or at least 98% identical to the polypeptide sequence of SEQ ID NO:3.

In another embodiment of this aspect and all other aspects provided herein, the heterologous protein scaffold comprises the polypeptide of SEQ ID NO:3.

In another embodiment of this aspect and all other aspects provided herein, the polypeptide comprises the polypeptide sequence of SEQ ID NO:6 or SEQ ID NO:7.

Another aspect provided herein relates to a polypeptide complex comprising or consisting of: a first component consisting of a polypeptide according to any one of claims 59-62 and a second component having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with any one of SEQ ID NOs: 13-18.

In another aspect, also provided herein is a vaccine composition comprising the composition or polypeptide complex as described herein.

In one embodiment of this aspect and all other aspects provided herein, the composition further comprises a pharmaceutically acceptable carrier.

In another embodiment of this aspect and all other aspects provided herein, the vaccine composition further comprises an adjuvant.

Another aspect provided herein relates to a cell expressing a polypeptide as described herein.

Another aspect provided herein relates to a nucleic acid encoding a polypeptide as described herein.

Another aspect provided herein relates to a method of vaccinating a subject against a coronavirus, the method comprising administering to the subject a polypeptide, protein complex, or vaccine composition as described herein.

Another aspect provided herein relates to a method of preparing a vaccine, the method comprising combining a polypeptide described herein with an adjuvant and a pharmaceutically acceptable carrier.

Another aspect provided herein relates to a method for preparing a vaccine, comprising combining: a first component consisting of a polypeptide as described herein; a second component having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 13-18; a pharmaceutically acceptable carrier; and optionally an adjuvant.

Thus, in one aspect, a non-naturally occurring polypeptide is provided herein, comprising a first coronavirus receptor binding domain (RBD), wherein the first coronavirus RBD comprises at least 90% identity to residues 328-531 of SEQ ID NO: 1, and further comprising residues 328-531 of SEQ ID NO: 1. NO:1, wherein the at least one mutation is selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L, or at the corresponding residue of the second coronavirus receptor binding domain as determined by aligning SEQ ID NO:1 with the sequence of the second coronavirus receptor binding domain using Blast-p (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215: 403-410). In one embodiment, the Blast-p program used is the National Center for Biotechnology Information (NCBI) online alignment tool. Alternatively, the Blast-p program can be downloaded to the device and used locally.

In one embodiment of this aspect and all other aspects provided herein, the mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367F and F392W.

In another embodiment of this aspect and all other aspects provided herein, the polypeptide comprises a second mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L.

In another embodiment of this aspect and all other aspects provided herein, the polypeptide comprises a second mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L.

Another aspect described herein relates to a polypeptide comprising: a first coronavirus receptor binding domain (RBD), the first coronavirus RBD comprising at least 90% identity to residues 328-531 of SEQ ID NO: 1, and further comprising at least two mutations relative to the RBD of SEQ ID NO: 1, wherein the at least two mutations are selected from the group consisting of:

F338L/Y365W;

Y365W/L513M;

Y365W/F392W;

F338M/A363L/Y365F/F377V;

Y365F/F392W;

Y365F/V395I;

Y365F/F392W/V395I;

Y365W/L513I/F515L;

F338L/A363L/Y365M;

F338L/I358F/Y365W;

I358F/Y365W/L513M;

I358F/Y365W/F392W;

F338M/I358F/A363L/Y365F/F377V;

I358F/Y365F/F392W;

I358F/Y365F/V395I;

I358F/Y365F/F392W/V395I;

I358F/Y365W/L513I/F515L; and

F338L/I358F/A363L/Y365M;

Alternatively, a second coronavirus RBD, wherein the second coronavirus RBD comprises at least two mutations corresponding to: F338L/Y365W of SEQ ID NO: 1;

Y365W/L513M;

Y365W/F392W;

F338M/A363L/Y365F/F377V;

Y365F/F392W;

Y365F/V395I;

Y365F/F392W/V395I;

Y365W/L513I/F515L;

F338L/A363L/Y365M;

F338L/I358F/Y365W;

I358F/Y365W/L513M;

I358F/Y365W/F392W;

F338M/I358F/A363L/Y365F/F377V;

I358F/Y365F/F392W;

I358F/Y365F/V395I;

I358F/Y365F/F392W/V395I;

I358F/Y365W/L513I/F515L; and

F338L/I358F/A363L/Y365M, where the corresponding sites were determined by aligning SEQ ID NO:1 with the spike protein sequence of the second coronavirus receptor binding domain using the Blast-p parameters of protocol 1 or protocol 2.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A to Figure 1B. Exemplary non-naturally occurring SARS-CoV-2 stabilized receptor binding domains, which have one or more mutations and show enhanced expression compared to the starting construct (i.e., natural or wild-type SARS-CoV-2 spike protein). Figure 1A is a reduction and non-reduction SDS-PAGE analysis of the supernatant of the repackaged RBD ("Rpk") variants for expressing genetic fusions to the I53-50A trimer nanoparticle component. The wild-type control does not contain RBD mutations, and the negative control uses a plasmid that does not encode any secreted protein. The migration of monomers and oxidized dimer species is marked. Figure 1B is a list of mutations contained in all constructs. The mutations listed in bold have been verified separately in Starr et al., 2020.

FIG2A to FIG2C. Structural models showing the locations of stabilizing mutations of the SARS-CoV-2 RBD. FIG2A Surface representation of the SARS-CoV-2 spike protein based on PDB-ID 6VYB (left), with the box highlighting the RBD; and a zoomed-in view of the RBD with a transparent surface representation, including a representation of the N-glycans. FIG2B Zoomed-in view of the region of the SARS-CoV-2 receptor binding domain containing the majority of designed mutations. FIG2C Structural models of two sets of representative designed mutations, named Rpk4 and Rpk9, that stabilize the RBD.

Figure 3. Biolayer interferometry (BLI) measures the binding of human ACE2 receptors and CR3022 antibodies to supernatants for expressing stabilized RBD genetically fused to I53-50A trimer components. ACE2 and CR3022 are immobilized on the sensor before exposure to the supernatant. Data from (i) all designs, (ii) constructs containing wild-type RBD, and (iii) negative control serum are shown in the chart. Each mutant SARS-CoV-2 spike protein of Figure 1 was determined to be antigenically intact when tested for SARS-CoV-2 homologous receptors, angiotensin-converting enzyme 2 (ACE2) receptors, and CR3022 mAbs that recognize SARS-CoV-2 spike proteins. Measurements from the supernatant also confirmed that each of these mutants was expressed at a level much higher than the starting construct.

Figures 4A to 4E. Biochemical, biophysical and antigenic characterization of stabilized RBDs containing Rpk4 or Rpk9 mutations in monomeric form and when fused to the I53-50A trimer (indicated by the addition of "-I53-50A"). Expression, thermal stability and local structural order were improved while maintaining antigenicity similar to that of wild-type SARS-CoV-2 RBD. Figure 4A Size exclusion chromatography (SEC) purification of wild-type and stabilized RBDs after expression from equal volumes of HEK293F cultures followed by IMAC purification and concentration. Monomeric RBDs (left) were purified using Superdex 75Increase 10/300GL, while fusions with the I53-50A trimer (right) were purified using Superdex 200Increase 10/300GL. The cropped gel shows equivalently diluted SEC load samples. Fig. 4B uses the wild-type and stabilized RBD monomer (left) monitored by intrinsic tryptophan fluorescence by nanoDSF and the thermal denaturation of the fusion body (right) with I53-50A trimer.The top inset shows the center of gravity mean (BCM) of each fluorescence emission spectrum as a function of temperature, and the bottom inset shows the smoothing first-order derivative for calculating the melting temperature.The hydrogen/deuterium exchange mass spectrometry (HDX-MS) of the wild-type and stabilized RBD fused to the I53-50A trimer of Fig. 4C.The structural model (top, from PDB 6W41) shows the panoramic difference uptake results of Rpk4-I53-50A and Rpk9-I53-50A trimers compared with wild-type RBD-I53-50A trimers, wherein the shadow is determined by the reduction of the uptake level of the mutant trimer measured at 1min. The box highlights the peptide segment from residues 392-399, where the exchange of this peptide at multiple time points is shown: 3 seconds, 15 seconds, 1 minute, 30 minutes and 20 hours (bottom). Each point is the average of two measurements. Standard deviations are shown unless less than the plotted points. Fig. 4D shows the fluorescence of SYPRO Orange when mixed with equal concentrations of wild-type and stabilized RBD monomers, where larger signals indicate higher levels of exposed hydrophobicity. Fig. 4E shows the binding kinetics of immobilized CV30 and CR3022 monoclonal antibodies to monomeric wild-type and stabilized RBD as assessed by BLI. Experimental data of five concentrations of RBD in a two-fold dilution series (grey trace) are fitted (black line) using a binding equation describing a 1:1 interaction. The structural model (left) is generated by the structural alignment of SARS-CoV-2 bound to CV30 Fab (PDB 6XE1) and CR3022 Fab (PDB6W41).

Fig. 5A to Fig. 5E. Compared with wild-type RBD, the stabilized RBD presented on the assembled I53-50 nanoparticles enhances solution stability. Fig. 5A shows the schematic diagram of the assembly of the I53-50 nanoparticle immunogen (marked by adding "-I53-50") of the RBD antigen. Fig. 5B Negative staining electron microscopy (nsEM) of wild-type RBD-I53-50, Rpk4-I53-50 and Rpk9-I53-50 (scale bar, 200nm). Fig. 5C to Fig. 5E show the quality control results of the summary of wild-type RBD-I53-50, Rpk4-I53-50 and Rpk9-I53-50 before and after a single freeze/thaw cycle in four different buffers. Fig. 5C Absorbance ratio at 320nm to 280nm in UV-Vis spectrum, which is an indicator of the presence of soluble aggregates. FIG5D Dynamic light scattering (DLS) measurements, which monitor both proper nanoparticle assembly and aggregate formation. FIG5E Partial reactivity of I53-50 nanoparticle immunogen to immobilized hACE2-Fc receptor (top) and CR3022 (bottom). Pre-freezing and post-freezing data were normalized to the corresponding CHAPS-containing sample for each nanoparticle individually.

Figure 6A to Figure 6C. The strong immunogenicity of the parent wild-type RBD-I53-50 nanoparticle immunogen is maintained by adding Rpk mutations. Figure 6A Female BALB/c mice (6 per group) were immunized at weeks 0 and 3. Each group received an equimolar amount of RBD antigen supplemented with AddaVax, which was equivalent to 5 μg per dose for HexaPro-foldon in terms of total antigen, and 0.88 μg per dose for all other immunogens. Serum collection was performed at weeks 2 and 5. The RBD-I53-50 immunogen was prepared under two different buffer conditions, one of which contained CHAPS as an excipient. Figure 6B shows the binding titer for HexaPro-foldon at weeks 2 and 5, as assessed by ELISA measurements of serial dilutions of serum from AUC. Each circle represents the AUC measurement from a single mouse, and the horizontal line shows the geometric mean of each group. One mouse in the fourth group with an AUC close to zero at week 2 was not drawn, but was still included in the geometric mean calculation. Figure 6C uses autologous (D614G) pseudovirus neutralization of the lentiviral backbone. Each circle represents the neutralizing antibody titer ( _IC50 ) at 50% inhibition in a single mouse, and the horizontal line shows the geometric mean of each group. Statistical analysis was performed using a one-sided nonparametric Kruskal-Wallis test and Dunn's multiple comparisons. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 7A to Figure 7C. Improved shelf life stability of RBD-based nanoparticle immunogens by Rpk mutations. Figure 7A Summary of DLS measurements over four weeks. The hydrodynamic diameters of all nanoparticles remained consistent at 35-40°C except for wild-type RBD-I53-50, which showed signs of aggregation after 28 days of storage. Figure 7B BLI binding to immobilized hACE2-Fc receptor (dashed line) and CR3022 mAb (solid line), which was normalized to -80°C samples at each time point. The antigenic integrity of the stabilized nanoparticle immunogens remained consistent, while the binding signal of wild-type RBD-I53-50 incubated at 35-40°C was reduced by 60% (hACE2-Fc) and 30% (CR3022). Figure 7C Summary of SDS-PAGE and nsEM over four weeks. No degradation was observed by SDS-PAGE. Only partial aggregation of WT nanoparticles stored at 35-40°C was observed by nsEM at day 28. Electron micrographs at day 28 after storage at 35-40°C are shown, where boxes indicate examples of aggregates (scale bar, 200 nm). All samples were formulated in TBS, 5% glycerol, 100 mM L-arginine.

Figures 8A to 8D. Rpk9 mutations can be incorporated into the full-length SARS-CoV-2 S extracellular domain containing HexaPro mutations. Figure 8A SEC purification of the pre-fused stabilized S extracellular domain of wild type (HexaPro-foldon) and Rpk9 (Rpk9-HexaPro-foldon) from an equal volume of HEK293F culture expression followed by IMAC purification and concentration. The S extracellular domain was purified using Superose 6Increase 10/300GL. Figure 8B Reducing and non-reducing SDS-PAGE of intermediates and final products during purification of HexaPro-foldon and Rpk9-HexaPro-foldon. Figure 8C Thermal denaturation of HexaPro-foldon and Rpk9-HexaPro-foldon monitored by nanoDSF using intrinsic tryptophan fluorescence. The center of gravity mean (BCM) of the fluorescence emission spectrum is plotted as a function of temperature. Figure 8D nsEM of HexaPro-foldon and Rpk9-HexaPro-foldon (scale bar, 100 nm).

Figure 9. When added to the RBD of the B.1.351 variant, the Rpk9 mutation improves the relative recovery of I53-50 nanoparticles displaying the RBD in a simpler buffer formulation with an appropriate SEC elution volume, indicating that the Rpk mutation improves the integrity of the immunogen containing RBDs from different variants. For I53-50 nanoparticles displaying the Wuhan-1 RBD (without the Rpk mutation), the B.1.351 RBD without the Rpk9 mutation, and the B.1.351 RBD with the Rpk9 mutation, assembly and SEC were performed in 50mM Tris pH7.4, 185mM NaCl, 100mM L-arginine, 0.75% CHAPS, 4.5% glycerol or 50mM Tris pH 8, 150mM NaCl, 100mM L-arginine, 5% glycerol. While the Rpk9 mutation increased yield and other measures of RBD stability under either buffer condition compared to equivalent samples without the Rpk9 mutation, nanoparticles displaying the B.1.351 RBD with the Rpk9 mutation better maintained relative yield and SEC mobility in the absence of CHAPS detergent.

FIG. 10 SDS-PAGE of nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100 mM L-arginine. The integrity of the samples in 50 mM Tris pH 7.4, 185 mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100 mM L-arginine was analyzed by SDS-PAGE. The molecular weight of the standards is expressed in kDa. Each sample was analyzed +/- reducing agent (DTT), before freeze-thaw, and after freeze-thaw (F/T).

Figure 11 hACE2-Fc binding of nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100mM L-arginine. hACE2-Fc binding of antigens in 50mM Tris pH 7.4, 185mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100mM L-arginine was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with hACE2-Fc were incubated with immunogens (association, x = 590-889s) and then with buffer (dissociation, x = 890-1190s).

Figure 12 CR3022 binding of nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100 mM L-arginine. CR3022 IgG binding of antigen in 50 mM Tris pH 7.4, 185 mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100 mM L-arginine was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with CR3022 IgG were incubated with immunogen (association, x = 590-889 s) and then with buffer (dissociation, x = 890-1190 s).

Figure 13 Dynamic light scattering of nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100 mM L-arginine. Hydrodynamic diameter (nm) of each sample in 50 mM Tris pH 7.4, 185 mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100 mM L-arginine, plotted as normalized intensity.

Figure 14 UV-Vis of nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100 mM L-arginine. UV-Vis spectra (nm) of each sample in 50 mM Tris pH 7.4, 185 mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100 mM L-arginine, plotted as normalized absorbance.

FIG. 15 SDS-PAGE of nanoparticles in TBS, 5% glycerol, 100 mM L-arginine. The integrity of the samples in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 100 mM L-arginine was analyzed by SDS-PAGE. The molecular weight of the standards is expressed in kDa. Each sample was analyzed +/- reducing agent (DTT), before freeze-thaw, and after freeze-thaw (F/T).

Figure 16 hACE2-Fc binding of nanoparticles in TBS, 5% glycerol, 100mM L-arginine. hACE2-Fc binding of antigens in 50mM Tris pH 8, 150mM NaCl, 5% glycerol, 100mM L-arginine was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with hACE2-Fc were incubated with immunogens (association, x = 590-889s) and then with buffer (dissociation, x = 890-1190s).

FIG. 17 CR3022 binding of nanoparticles in TBS, 5% glycerol, 100 mM L-arginine. CR3022 IgG binding of antigen in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 100 mM L-arginine was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with CR3022 IgG were incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

Figure 18 Dynamic light scattering of nanoparticles in TBS, 5% glycerol, 100 mM L-arginine. Hydrodynamic diameter (nm) of each sample in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 100 mM L-arginine, plotted as normalized intensity.

Figure 19 UV-Vis of nanoparticles in TBS, 5% glycerol, 100 mM L-arginine. UV-Vis spectra (nm) of each sample in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 100 mM L-arginine, plotted as normalized absorbance.

Figure 20 SDS-PAGE of nanoparticles in TBS, 5% glycerol. The integrity of samples in 50mM Tris pH 8, 150mM NaCl, 5% glycerol was analyzed by SDS-PAGE. The molecular weight of the standards is expressed in kDa. Each sample was analyzed +/- reducing agent (DTT), before freeze-thaw and after freeze-thaw (F/T).

Figure 21 hACE2-Fc binding of nanoparticles in TBS, 5% glycerol. ACE2-Fc binding of antigens in 50mM Tris pH 8, 150mM NaCl, 5% glycerol was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with hACE2-Fc were incubated with immunogens (association, x = 590-889s) and then with buffer (dissociation, x = 890-1190s).

Figure 22 CR3022 binding of nanoparticles in TBS, 5% glycerol. CR3022 IgG binding of antigen in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with CR3022 IgG were incubated with immunogen (association, x = 590-889 s) and then with buffer (dissociation, x = 890-1190 s).

Figure 23 Dynamic light scattering of nanoparticles in TBS, 5% glycerol. Hydrodynamic diameter (nm) of each sample in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, plotted as normalized intensity.

Figure 24 UV-Vis of nanoparticles in TBS, 5% glycerol. UV-Vis spectra (nm) of each sample in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, plotted as normalized absorbance.

Figure 25 SDS-PAGE of nanoparticles in TBS. The integrity of samples in 50mM Tris pH 8, 150mM NaCl was analyzed by SDS-PAGE. The molecular weight of the standards is expressed in kDa. Each sample was analyzed +/- reducing agent (DTT), before freeze-thaw and after freeze-thaw (F/T).

Figure 26 hACE2-Fc binding of nanoparticles in TBS. HACE2-Fc binding of antigens in 50mMTris pH 8, 150mM NaCl was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with hACE2-Fc were incubated with immunogens (association, x=590-889s) and then with buffer (dissociation, x=890-1190s).

Figure 27 CR3022 binding of nanoparticles in TBS. CR3022 IgG binding of antigen in 50 mM Tris pH 8, 150 mM NaCl was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with CR3022 IgG were incubated with immunogen (association, x = 590-889 s) and then with buffer (dissociation, x = 890-1190 s).

Figure 28 Dynamic light scattering of nanoparticles in TBS. Hydrodynamic diameter (nm) of each sample in 50 mM Tris pH 8, 150 mM NaCl, plotted as normalized intensity.

Figure 29 UV-Vis of nanoparticles in TBS. UV-Vis spectra (nm) of each sample in 50 mM Tris pH 8, 150 mM NaCl, plotted as normalized absorbance.

Figure 30 SDS-PAGE of RBD-I53-50 nanoparticles. The integrity of the samples after incubation at four temperatures in the 28-day (D) study was analyzed by SDS-PAGE. The molecular weight of the standards is expressed in kDa. Each sample was analyzed with +/- reducing agent (DTT).

Figure 31 hACE2-Fc binding of RBD-I53-50 nanoparticles. hACE2-Fc binding of antigens incubated for 28 days (D) at four different temperatures was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with hACE2-Fc were incubated with immunogens (association, x = 590-889s) and then with buffer (dissociation, x = 890-1190s).

Figure 32 CR3022 binding of RBD-I53-50 nanoparticles. CR3022 IgG binding of antigens incubated for 28 days (D) at four different temperatures was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with CR3022 were incubated with immunogens (association, x=590-889s) and then with buffer (dissociation, x=890-1190s).

Figure 33 nsEM of RBD-I53-50 nanoparticles. Representative negative staining electron micrographs of each sample at day 1 (D) and day 28 after incubation at four temperatures. Scale bar, 50 nm.

Figure 34 Dynamic light scattering of RBD-I53-50 nanoparticles. The hydrodynamic diameter (nm) of each sample over a period of 28 days (D), plotted as normalized intensity.

Figure 35 SDS-PAGE of Rpk4-I53-50 nanoparticles. The integrity of the samples after incubation at four temperatures in the 28-day (D) study was analyzed by SDS-PAGE. The molecular weight of the standards is expressed in kDa. Each sample was analyzed with +/- reducing agent (DTT).

Figure 36 hACE2-Fc binding of Rpk4-I53-50 nanoparticles. hACE2-Fc binding of antigens incubated for 28 days (D) at four different temperatures was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with hACE2-Fc were incubated with immunogens (association, x = 590-889s) and then with buffer (dissociation, x = 890-1190s).

Figure 37 CR3022 binding of Rpk4-I53-50 nanoparticles. CR3022 IgG binding of antigens incubated for 28 days (D) at four different temperatures was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with CR3022 were incubated with immunogen (association, x = 590-889s) and then with buffer (dissociation, x = 890-1190s).

Figure 38 nsEM of Rpk4-I53-50 nanoparticles. Representative negative staining electron micrographs of each sample at day 1 (D) and day 28 after incubation at four temperatures. Scale bar, 50 nm.

Figure 39 Dynamic light scattering of Rpk4-I53-50 nanoparticles. The hydrodynamic diameter (nm) of each sample over a 28 day (D) period, plotted as normalized intensity.

Figure 40 SDS-PAGE of Rpk9-I53-50 nanoparticles. The integrity of the samples after incubation at four temperatures in the 28-day (D) study was analyzed by SDS-PAGE. The molecular weight of the standards is expressed in kDa. Each sample was analyzed with +/- reducing agent (DTT).

Figure 41 hACE2-Fc binding of Rpk9-I53-50 nanoparticles. hACE2-Fc binding of antigens incubated for 28 days (D) at four different temperatures was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with hACE2-Fc were incubated with immunogens (association, x = 590-889s) and then with buffer (dissociation, x = 890-1190s).

Figure 42 CR3022 binding of Rpk9-I53-50 nanoparticles. CR3022 IgG binding of antigens incubated for 28 days (D) at four different temperatures was analyzed by biolayer interferometry (BLI). Protein A biosensors loaded with CR3022 were incubated with immunogen (association, x = 590-889s) and then with buffer (dissociation, x = 890-1190s).

Figure 43 nsEM of Rpk9-I53-50 nanoparticles. Representative negative staining electron micrographs of each sample at day 1 (D) and day 28 after incubation at four temperatures. Scale bar, 50 nm.

Figure 44 Dynamic light scattering of Rpk9-I53-50 nanoparticles. The hydrodynamic diameter (nm) of each sample over a 28 day (D) period, plotted as normalized intensity.

DETAILED DESCRIPTION

Provided herein are compositions and methods comprising coronavirus "S" spike proteins having at least one, two or more amino acid mutations, which have increased expression levels, yields and/or stability compared to native or wild-type coronavirus spike proteins (e.g., SARS-CoV-2 S proteins) under the same expression, culture or storage conditions. These mutant spike proteins can be used to generate protein-based vaccines against SARS-CoV-2 (a different coronavirus known to infect humans), or pan-coronavirus vaccines that can provide protection against multiple coronaviruses known to infect humans.

In one embodiment, the S protein comprises a single mutation that increases the expression level, yield and/or stability of the mutant coronavirus spike protein under specific expression, culture, or storage conditions compared to the native or wild-type coronavirus spike protein. In alternative embodiments, the S protein comprises multiple mutations (e.g., 2, 3, 4, or 5).

definition

As used herein, the term "non-naturally occurring" or "mutant" refers to a coronavirus polypeptide (e.g., a stabilized coronavirus S protein or RBD polypeptide) that comprises at least one or at least two amino acid residue mutations and preferably comprises enhanced stability and/or expression compared to its corresponding natural or wild-type coronavirus sequence. In some embodiments, for example, in addition to the at least two mutations, the mutant polypeptides described herein are "substantially similar" to their natural counterparts. In some embodiments, the natural counterparts may include naturally occurring coronavirus variants.

For the avoidance of doubt, naturally occurring variants of coronavirus sequences (e.g., SARS-Cov-2 variants: B.1.1.7; B.1.351; P.1; B.1.427; B.1.429; B.1.526; B.1.526.1; B.1.525; P.2; B.1.617; B.1.617.1; B.1.617.2; and B.1.617.3) are not considered "non-naturally occurring" or "mutated" coronavirus sequences, however such variants may be used as reference coronavirus sequences as that term is used herein.

As used herein, the term "non-naturally occurring coronavirus spike protein subunit 1 polypeptide" refers to a polypeptide that comprises at least the receptor binding domain sequence residues that allow for the structural formation of the linoleic acid binding pocket (residues 328-531 of SEQ ID NO: 1), and at least one or at least two amino acid mutations. In one embodiment, one of the at least two mutations comprises a "cavity filling mutation," as the term is used herein.

If one molecule and another molecule have a substantially similar structure (i.e., as determined by a Blast-p comparison set under default parameters, they are at least 90% similar in amino acid sequence) and are substantially similar in at least one relevant function (e.g., antigenic activity determined by recognition of the polypeptide by antibodies binding to natural coronavirus counterparts), then the two molecules are said to be "substantially similar". That is, the mutant polypeptide differs from the naturally occurring polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side chain modifications, but retains one or more specific functions or biological activities of the naturally occurring molecule. Amino acid substitutions include changes in which amino acids are replaced by different naturally occurring or unconventional amino acid residues. Some substitutions can be classified as "conservative", in which case the amino acid residues contained in the polypeptide are replaced by another naturally occurring amino acid having similar properties in terms of polarity, side chain functionality or size. Substitutions covered by variants as described herein can also be "non-conservative", in which amino acid residues present in the peptide are replaced by amino acids having different properties (e.g., replacing charged or hydrophobic amino acids with uncharged or hydrophilic amino acids), or alternatively, in which naturally occurring amino acids are replaced by unconventional amino acids. When the term "mutant" is used in relation to a polypeptide, the term "mutant" also encompasses changes in primary structure, secondary structure, or tertiary structure as compared to a reference polypeptide (e.g., as compared to a wild-type coronavirus polypeptide). Mutants may also include insertions, deletions, or substitutions of amino acids that do not normally appear in the peptide sequence underlying the variant (including insertions and substitutions of amino acids and other molecules), including but not limited to ornithine insertions that do not normally appear in human proteins.

As used herein, the term "corresponding to" or "corresponding wild-type coronavirus" refers to a wild-type coronavirus polypeptide sequence (or a naturally occurring variant thereof) that produces a non-naturally occurring coronavirus polypeptide (e.g., a spike polypeptide) or an RBD polypeptide. Typically, the wild-type coronavirus sequence (or a naturally occurring variant thereof) is from the same strain as the non-naturally occurring coronavirus polypeptide. For example, the mutant SARS-CoV-2 polypeptides described herein will correspond to a wild-type coronavirus polypeptide or a naturally occurring variant thereof (e.g., a South African variant, a Brazilian variant, a Los Angeles variant, etc.) of a SARS-CoV-2 sequence.

As used herein, the term "naturally occurring variant" refers to a coronavirus sequence that arises spontaneously within a population of susceptible individuals.

As used herein, the term "increased stability" or "enhanced stability" refers to that the mutant coronavirus protein sequence is degraded at a slower rate than the corresponding natural or wild-type coronavirus protein sequence (or its naturally occurring variant) under the same conditions in a cell, solution or preparation and therefore persists for at least 12h longer than the corresponding natural or wild-type coronavirus protein sequence (or its naturally occurring variant), for example, as described in the working examples herein, the hot melt assay is used to assess. In some examples, compared with the persistence of the corresponding natural or wild-type coronavirus protein sequence, the mutant coronavirus protein sequence persists in a cell, solution or preparation for at least 24h, 36h, 48h, 72h, 7 days, 8 days, 9 days, 10 days, 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, one year, two years or longer. In some embodiments, higher expression levels can also indicate that the stability of the polypeptide is enhanced, or the result of the enhanced stability of the polypeptide.

As used herein, the term "yield increase" or "yield enhancement" refers to an increase of at least 10% in the amount of a mutant coronavirus protein recovered from a cell system producing a protein compared to the amount of a natural or wild-type protein (or its naturally occurring variant) recovered from the same cell system under the same growth and separation conditions. In certain embodiments, "yield enhancement" refers to an increase of at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, at least 1 times, at least 2 times, at least 5 times, at least 10 times, at least 100 times or more in the amount of a mutant coronavirus protein recovered from a cell system producing a protein compared to the amount of a natural or wild-type protein recovered from the same cell system under the same growth conditions.

As used herein, the term "cavity-filling mutation" refers to the substitution of an amino acid residue in a wild-type coronavirus spike protein with an amino acid that is expected to "fill" the internal cavity of the mature coronavirus spike protein. For example, the substituted amino acid has an appropriate size or charge such that the substituted amino acid protrudes into the cavity and sterically reduces the size of the cavity and/or weakens the binding of a cognate ligand within the cavity or pocket.

As used herein, the term "adjuvant" refers to a protein or chemical substance that enhances the immune response to a vaccine antigen when administered with the vaccine antigen. The difference between an adjuvant and an antigenic moiety or a carrier protein is that the adjuvant is not chemically coupled to an immunogen or antigen. Adjuvants are well known in the art and include, for example, mineral oil emulsions, such as Freund's complete adjuvant or Freund's incomplete adjuvant (Freund, Adv. Tuberc. Res. 7: 130 (1956); Calbiochem, San Diego Calif.); aluminum salts, especially aluminum hydroxide or ALHYDROGEL ^TM (approved for use in humans by the U.S. Food and Drug Administration (US Food and Drug Administration)); muramyl dipeptide (MDP) and its analogs, such as [Thr1]-MDP (Byers and Allison, Vaccine 5: 223 (1987)), monophosphoryl lipid A (Johnson et al., Rev. Infect. Dis. 9: S512 (1987)), etc.

As used herein, the term "comprising" means that there may be other elements in addition to the defined elements given. The use of "comprising" indicates inclusion rather than limitation.

As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiments of the invention.

The term "consisting of" means that the compositions, methods, and corresponding components thereof, as described herein, do not include any elements not recited in the description of the embodiment.

Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” When used in conjunction with a percentage, the term “about” may mean ±1%.

Coronavirus

Coronaviruses are a family of hundreds of viruses that can cause fever, respiratory problems, and sometimes gastrointestinal symptoms. SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is one of seven members of this family known to infect humans and the third to jump from animals to humans in the past three decades. Other coronaviruses known to infect humans include alphacoronaviruses 229E and NL63, and betacoronaviruses OC43, HKU1, SARS-CoV (the coronavirus that causes severe acute respiratory syndrome, or SARS), and MERS-CoV (the coronavirus that causes Middle East respiratory syndrome, or MERS).

Although the methods and compositions described herein are discussed in the context of coronaviruses that infect humans, the methods and compositions described herein can also be used to produce stable coronavirus proteins from viruses that infect other mammals, including pets or livestock (e.g., pigs, cattle, dogs, etc.). Such viruses include, but are not limited to, porcine transmissible gastroenteritis virus, porcine respiratory coronavirus, porcine epidemic diarrhea virus (PEDV), porcine hemagglutinating encephalomyelitis virus, porcine delta coronavirus (PDCoV), bovine coronavirus (BCV), feline coronavirus (FCoV), canine coronavirus (CCoV), avian infectious bronchitis virus (IBV), and turkey coronavirus (TCV). In addition, the coronaviruses described herein include those currently known and later discovered coronaviruses. Specifically contemplated herein are coronaviruses that are the cause of ongoing or future epidemics or pandemics.

As used herein, the term "coronavirus" refers to an enveloped virus with a positive single-stranded RNA genome and helical symmetry. The genome size of coronavirus ranges from about 27 kilobases to 32 kilobases, which is the longest size of any known RNA virus. The large spike (S) glycoprotein protrudes from the virion, giving the coronavirus a unique crown appearance when visualized by an electron microscope. Coronavirus infects a variety of species, including canines, felines, swine, murines, bovines, birds and humans (Holmes et al., 1996. Coronaviridae: the viruses and their replication, pp. 1075-1094. In D.M.K.a.P.M.H.B.N.Fields (ed.), Fields Virology.Lippincott-Raven, Philadelphia, Pa.). However, the natural host range of each coronavirus strain is very narrow, usually consisting of a single species.

Coronaviruses typically bind to target cells via spike receptor interactions and enter cells by receptor-mediated endocytosis or fusion with the plasma membrane (Holmes et al., 1996, supra). As demonstrated for both group 1 and group 2 coronaviruses, spike receptor interactions are an important determinant of species specificity. The genome of SARS-CoV contains single-stranded (+) sense RNA. The complete and partial genome sequences of several SARS coronavirus isolates have been reported, including SARS coronavirus Urbani (GenBank accession number AY278741), SARS coronavirus Tor2 (GenBank accession number AY274119), SARS coronavirus CUHK-W1 (GenBank accession number AY278554), SARS-CoV Shanghai LY (GenBank accession number H012999; GenBank accession number AY322205; GenBank accession number AY322206), SARS-CoV Shanghai QXC (GenBank accession number AH013000; GenBank accession number AY322208; GenBank accession number AY322197; GenBank accession number AY322199), and SARS-CoV Shanghai LY (GenBank accession number H012999; GenBank accession number AY322205; GenBank accession number AY322206). ZJ-HZ01 (GenBank accession number AY322206), gi|31416292|gb|AY278487.3|SARS coronavirus BJ02, gi|30248028|gb|AY274119.3|SARS coronavirus TOR2, gi|30698326|gb|AY291451.1|SARS coronavirus TW1, gi|33115118|gb|AY323977.2|SARS coronavirus HSR 1. gi|35396382|gb|AY394850.1|SARS coronavirus WHU, gi|33411459|dbj|AP006561.1|SARS coronavirus TWY, gi|33411444|dbj|AP006560.1|SARS coronavirus TWS, gi|33411429|dbj|AP006559.1|SARS coronavirus TWK, gi|33411414|dbj|AP006558.1|SARS coronavirus TWJ, gi|33411399|dbj|AP006557.1|SARS coronavirus TWH, gi|30023963|gb|AY278 491.2|SARS coronavirus HKU-39849、gi|33578015|gb|AY310120.1|SARS coronavirus FRA、gi|33518725|gb|AY362699.1|SARS coronavirus TWC3、gi|33518724|gb|AY362698.1|SARS coronavirus TWC2、gi|30027617|gb|AY278741.1|SARS coronavirus Urbani、gi|31873092|gb|AY321118.1|SARS coronavirus TWC、gi|33304219|gb|AY351680.1|SARS coronavirus ZMY 1. gi|31416305|gb|AY278490.3|SARS coronavirus BJ03, gi|30910859|gb|AY297028.1|SARS coronavirus ZJ01, gi|30421451|gb|AY282752.1|SARS coronavirus CUHK-Su10, gi|34482146|gb|AY304495.1|SARS coronavirus GZ50, gi|344821 39|gb|AY304488.1|SARS coronavirus SZ16、gi|34482137|gb|AY304486.1|SARS coronavirus SZ3、gi|30027610|gb|AY278554.2|SARS coronavirus CUHK-W1、gi|31416306|gb|AY279354.2|SARS coronavirus BJ04、gi|37576845|gb|AY427439 .1|SARS coronavirus AS, gi|37361915|gb|AY283798.2|SARS coronavirus Sin2774, gi|31416290|gb|AY278489.2|SARS coronavirus GD01, gi|30468042|gb|AY283794.1|SARS coronavirus Sin2500, gi|30468043|gb|AY283795.1|SARS coronavirus S in2677, gi|30468044|gb|AY283796.1|SARS coronavirus Sin2679, gi|30468045|gb|AY283797.1|SARS coronavirus Sin2748, gi|31982987|gb|AY286320.2|SARS coronavirus isolate ZJ-HZ01, gi|30275666|gb|AY278488.2|SARS coronavirus BJ0 1.

S (spike) protein can form non-covalently linked homotrimers (oligomers), which can mediate receptor binding and viral infectivity. The homotrimers of S protein may be necessary for presenting the correct native conformation of the receptor binding domain and eliciting neutralizing antibody reactions. In addition, the intracellular processing of S protein is associated with significant post-translational oligosaccharide modifications. The post-translational oligosaccharide modifications (glycosylation) expected by N-glycan motif analysis indicate that S protein has up to 23 such modification sites. In addition, the C-terminal cysteine residue may also be involved in protein folding and maintain the native (functional) S protein conformation. The S protein of some coronaviruses (e.g., some strains of Group II and Group III viruses) can be processed into connected polypeptides by trypsin-like proteases in the Golgi apparatus or by extracellularly located enzyme proteases near the center of the S protein, including N-terminal S1 polypeptides and C-terminal S2 polypeptides. Some members of Group II coronavirus and Group III virus types may not be processed in this way.

Diagnosis: Based on the diagnostic test results, the subject (e.g., a person) is diagnosed with a coronavirus infection. Based on one or more symptoms presented, such as fever, chills, cough, shortness of breath/dyspnea, fatigue, muscle/body pain, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea, vomiting, or diarrhea, the subject may be suspected of having a coronavirus infection (e.g., COVID-19, SARS, MERS, etc.). However, some subjects may exhibit asymptomatic infection (e.g., SARS-CoV-2 infection), and therefore may be suspected of having a coronavirus infection when in contact with a subject with a coronavirus infection. In both cases, active coronavirus infection can be confirmed using methods known in the art to detect one or more of viral antigens and viral nucleic acids in samples taken from the subject. Examples include the use of reverse transcriptase polymerase chain reaction (RT-PCR) diagnostic assays from nasopharyngeal swabs or sputum to detect viral RNA. Other nucleic acid amplification methods (e.g., any of many isothermal amplification methods) can also be used, and have a sensitivity close to that of RT-PCR, if not equal to RT-PCR. Isothermal amplification methods have the advantage of not needing a thermal cycler to produce an amplified product, and can provide results more quickly in a highly sensitive manner. In another embodiment, active coronavirus infection can be determined by detecting one or more coronavirus polypeptides (such as antigens) in a biological sample obtained from a subject. The lateral flow assay of viral antigens can provide qualitative and sometimes quantitative diagnostic results. Viral polypeptides can also be detected by other methods known in the art (such as Western blotting).

Assessing the presence of coronavirus antibodies can be used to determine whether a subject has been exposed to coronavirus in the past, or alternatively as a means of monitoring vaccine effectiveness (i.e., the ability of mutant spike proteins to increase immune response). Methods for assessing the presence of coronavirus antibodies are known in the art and are not discussed in detail herein.

Alternatively, the presence or production of coronavirus virions can be determined directly or indirectly by using, for example, electron microscopy.

Protein sequence: The amino acid sequence of the native or wild-type SARS-CoV-2 S protein subunit 1 is: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVD LPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLD SKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPL QSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENS VAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQY GSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVV NQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAA (SEQ ID NO: 1).

Throughout this specification, SEQ ID NO: 1 is used as a 'base' or 'reference' sequence that can be aligned with other coronavirus amino acid sequences using alignment programs known in the art (e.g., Blast-p). The alignment of the spike protein sequence of a different (or second) coronavirus sequence with the SARS-CoV-2 spike protein sequence of SEQ ID NO: 1 can be used to determine the corresponding sites in the different (or second) coronavirus to introduce one or more given amino acid mutations to achieve stabilization as described herein. In one embodiment, Blast-p (Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J.Mol.Biol.215: 403-410) is used to align different coronavirus sequences with SEQ ID NO: 1. In one embodiment, the Blast-p program used is the National Center for Biotechnology Information (NCBI) online alignment tool. Alternatively, the Blast-p program can be downloaded to the device and used locally. The use of the Blast-p alignment tool will be readily understood by those skilled in the art, however for the avoidance of doubt, Protocols 1 and 2 are provided herein for use with the online and downloaded alignment tools respectively.

Protocol 1 : For use with online BLASTp alignments from the National Center for Biotechnology Information (NCBI) server.

1. Set up a BLAST alignment using the following settings:

Use the "Align two or more sequences" option

Enter the reference strain sequence of the relevant SARS-CoV-2 protein (i.e., SEQ ID NO: 1) into the "Enter Query Sequence" section

Enter any corresponding coronavirus spike protein sequence into the "Enter Subject Sequence" section

Algorithm: blastp (protein-protein BLAST)

Expected threshold: 0.1

Word length: 6

Maximum matches within query range: 0

Matrix: BLOSUM62

Vacancy cost:

Presence: 11

Extension: 1

Filter low complexity areas?: No

Mask:

For lookup table only? : No

Lowercase letters?: No.

2. Run the analysis by clicking the "BLAST" button.

3. Click the "Alignments" tab to display the alignment between the two sequences.

4. For each sequence position of interest, identify the number according to the "Query" sequence. Then identify the corresponding residue position in the "Sbjct" sequence that has been aligned with the position of the "Query" sequence.

Protocol 2 : For use with the protein BLASTp alignment tool downloaded to a local computer or server.

1. Install BLAST using the manufacturer's instructions for command line execution, or identify a computer or server where BLAST is installed.

2. Generate a FASTA formatted file containing the desired SARS-CoV-2 protein subtype-specific reference strain (i.e., SEQ ID NO: 1). In the following commands, this file will be named "query.fasta".

3. Generate a second file in FASTA format, which contains the corresponding protein sequences of different coronaviruses from the same subtype. In the following command, this file will be named "sbjct.fasta".

4. Execute the following command using a program such as Terminal, iTerm2, Windows Console, Linux console, or other similar terminal emulator. This will generate the results in a file called "results.txt".

blastp-query query.fasta-subject sbjct.fasta-matrix BLOSUM62-evalue0.1-

word size 6-gapopen 11-gapextend 1-out results.txt

5. Open results.txt and view the section showing the alignment of the two sequences. For each sequence position of interest, identify the number according to the "Query" sequence. Then identify the corresponding residue position in the "Sbjct" sequence that has been aligned with the position of the "Query" sequence.

It will be apparent to those skilled in the art that other protein alignment tools (e.g., Clustalw or Clustal-omega) can also be used to identify sequence identity between a query sequence and a reference sequence (e.g., SEQ ID NO: 1). Given that the query sequence and the reference sequence share significant sequence identity, it is expected that other protein alignment tools will produce similar (if not identical) results to Blast-p using the protocol described herein. The protocol described herein has been shown to be accurate and effective for this purpose, and is provided herein to assist the skilled artisan in identifying amino acid residues to be mutated in the query sequence.

Receptor Binding Domain (RBD) Peptides

The viral surface "spike" protein mediates coronavirus entry into host cells. The spike proteins of both SARS-CoV and SARS-CoV-2 contain a receptor binding domain that specifically recognizes angiotensin-converting enzyme 2 (ACE2) as its receptor. Given the importance of this domain in viral uptake and function, the receptor binding domain is relatively well conserved among coronaviruses known to infect humans. The sequence of the receptor binding domain of the spike protein of SARS-CoV-2 is:

RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCY

GVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGC

VIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKK (SEQ ID NO: 2).

Throughout this specification, SEQ ID NO: 2 is used as a 'base' or 'reference' receptor binding domain sequence that can be compared with other coronavirus RBD sequences using alignment programs known in the art (e.g., Blast-p, ClustalW, etc.). The alignment of at least a second coronavirus RBD sequence with SEQ ID NO: 2 can be used to determine the corresponding site in the second coronavirus RBD sequence for a given amino acid mutation in SARS-CoV-2. In such embodiments, Blast-p can be used to compare the coronavirus RBD query sequence with SEQ ID NO: 2 using Protocol 1 or Protocol 2 described herein. The receptor binding domain polypeptide comprises at least two mutations within SEQ ID NO: 2 (or the equivalent of a different coronavirus). In some embodiments, the receptor binding domain polypeptide may comprise additional mutations. Such additional mutations may also be in the RBD region, or may occur outside the RBD region. Typically, at least one, two or more amino acid mutations do not include those found in naturally occurring variants of a given coronavirus sequence. For example, L452R and E484K present in naturally occurring variants of SARS-CoV-2 are not counted as at least one, two or more amino acid mutations as described herein.

In some embodiments, the receptor binding domain polypeptides described herein are used to produce protein vaccines against one or more coronaviruses. As will be understood by those skilled in the art and particularly when used in the setting of a vaccine, the mutant coronavirus protein does not have to retain the receptor binding properties of its cognate receptor; therefore, it is not necessary to design at least one amino acid mutation or at least two amino acid mutations to retain the function of the RBD. In view of the fact that the maintenance of the function of the coronavirus protein is not necessary, coronavirus proteins having at least 90% identity with SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., at least 95%, at least 99% identity with SEQ ID NO: 1 or SEQ ID NO: 2) are particularly contemplated herein, provided that they retain the ability to act as coronavirus antigens (i.e., stimulate the production of coronavirus binding antibodies in a subject or bind to coronavirus antibodies against the corresponding wild-type coronavirus). That is, the receptor binding domain polypeptides described herein are "immunogenic", i.e., the subject is immunized with a receptor binding domain polypeptide (optionally bound to a suitable carrier (such as a protein, lipid or polypeptide)) to induce an immune response (of the B cell type and/or T cell type) against the RBD polypeptide.

The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., a portion or fragment of the molecule that is recognized by the immune system (e.g., by a T cell or B cell), particularly when presented in the context of an MHC molecule. An epitope of a protein antigen may comprise a continuous or discontinuous portion of the protein and may be between 5 and 100 amino acids, between 5 and 50 amino acids, between 8 and 30 amino acids, between 10 and 25 amino acids in length, for example, an epitope may preferably be 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, or 25 amino acids in length.

The ability of a protein to improve an immune response can be attributed in part to its secondary structure and the conformation of protein folding. In some embodiments, a certain conformation is preferred for producing an antigenic response and/or increasing the stability of the protein. The secondary structure of SARS-CoV-2 bound to the ACE2 receptor is described in Shang et al., "Structural Basis of Receptor Recognition by SARS-CoV-2" Nature 581:221-224 (2020), the contents of which are incorporated herein by reference in their entirety. By understanding the crystal structure of SARS-CoV-2, those skilled in the art can use well-founded reasoning or computational software to determine whether a given receptor binding domain polypeptide is likely to contain a shape or secondary structure that will induce an immune response in a subject.

In certain embodiments, at least two amino acids are mutated in the coronavirus receptor binding domain, which increases the yield of the protein in the cell system and/or enhances the stability of the coronavirus protein in cells, solutions or formulations compared to its corresponding wild-type protein. In other embodiments, at least one amino acid is mutated in the coronavirus receptor binding domain, which increases the yield of the protein in the cell system and/or enhances the stability of the coronavirus protein in cells, solutions or formulations compared to its corresponding wild-type protein.

In some embodiments, the at least two mutations include at least two mutations introduced within the RBD region of the spike protein (SEQ ID NO: 2, or residues 328-531 of SEQ ID NO: 1). In some embodiments, at least one amino acid mutation is introduced within the RBD region of the spike protein.

In certain embodiments, the at least two mutations are in SEQ ID NO:1 at the following amino acid residues: 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365, or by using Blast-p to convert SEQ ID NO: 1 is aligned with the sequence of the second coronavirus receptor binding domain to determine the corresponding residues of the second coronavirus receptor binding domain.

In certain embodiments, the at least one amino acid mutation is at the following amino acid residues: 338, 358, 363, 365, 367, 377, 392, 395; 498, 501, 502, 513 or 515. In other embodiments, the at least one amino acid mutation is: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, or F515L.

In certain embodiments, the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513I of SEQ ID NO: 1. 3M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or at the corresponding residues of a second coronavirus determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus receptor binding domain using Blast-p.

In one embodiment, the receptor binding domain polypeptide comprises a fusion protein.

Linoleic acid binding pocket

The SARS-CoV-2 "S" (spike) protein has been shown to contain a "pocket" or "cavity" that has recently been identified as a linoleic acid binding pocket (Toelzer et al. Science "Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein" (2020)). The residues in the linoleic acid binding pocket are conserved in all seven coronaviruses that infect humans (Toelzer, supra), suggesting that this cavity is functionally conserved. Toelzer et al. also showed that binding of linoleic acid to the SARS-CoV-2 S protein stabilizes the closed conformation of the S protein. It is contemplated herein that mutations within the linoleic acid binding pocket or a subdomain of the linoleic acid binding pocket can be used to mimic the effects of linoleic acid and/or stabilize the closed conformation of the S protein. In some embodiments, the amino acid mutations in this region are 'cavity filling' mutations.

In one embodiment, a "cavity-filling mutation," as that term is used herein, fills a site within the linoleic acid binding pocket (e.g., spatially protrudes into the cavity). Cavities in natural coronavirus spike proteins can be identified by methods known in the art, such as by visual inspection of crystal structure representations of, for example, spike proteins of SARS-CoV-2 (see, e.g., Shang et al., "Structural Basis of Receptor Recognition by SARS-CoV-2" Nature 581: 221-224 (2020)), or by using computational protein design software (such as BioLuminate ^TM (BioLuminate, Schrodinger LLC, New York), Discovery Studio ^TM (Discovery Studio Modeling Environment, Accelrys, San Diego), MOE ^TM (Molecular Operating Environment, Chemical Computing Group Inc., Montreal), and Rosetta ^TM (Rosetta, University of Washington, Seattle, etc.). Such models allow one skilled in the art to design cavity-filling mutations that are expected to enhance the stability of a given coronavirus spike protein.

The amino acids to be replaced for cavity-filling mutations may include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr) that are replaced by similar amino acids that are sterically larger and able to "fill" the cavity (e.g., large aliphatic amino acids (Ile, Leu, and Met) or large aromatic amino acids (His, Phe, Tyr, and Trp)). In other embodiments, charged amino acids may replace uncharged amino acids or be replaced by uncharged amino acids, thereby changing the secondary structure of the protein and the cavity. Such residues for replacement may also include amino acids that are hidden in a given protein conformation but exposed to the solvent in a second conformation.

In certain embodiments, at least one of the at least two mutations in the SARS-CoV-2 spike protein is at a residue involved in the linoleic acid binding pocket. In some embodiments, it is preferred that the mutation "fills" the linoleic acid binding pocket (e.g., using larger amino acids with similar charge or hydrophobicity). Such mutations can stabilize a specific conformation of the protein and/or reduce the degradation rate of the protein compared to the corresponding wild-type coronavirus. For ease of reference, the residues involved in linoleic acid binding are divided into three subdomains herein.

These subdomains are based solely on the close proximity of a few residues involved in linoleic acid binding.

The non-naturally occurring coronavirus spike protein subunit 1 polypeptide may comprise at least one cavity filling mutation or a mutation of a residue in the linoleic acid binding pocket and at least one additional mutation that together enhance the stability and/or productivity of the polypeptide, as those terms are used herein.

In some embodiments, the cavity filling mutation comprises a mutation of a residue at amino acid 336, 338, 341, 342, 358, 361, 363, 365, 368, 374, 377, 387, or 392 of SEQ ID NO: 1, or a mutation of a corresponding residue of the spike protein subunit 1 of a second coronavirus as determined by aligning SEQ ID NO: 1 (or a naturally occurring variant thereof) with the sequence of a second coronavirus (or a naturally occurring variant thereof) using Blast-p. In other embodiments, the cavity filling mutation and the at least one second mutation are in the range of SEQ ID NO: 1 to 336, 338, 341, 342, 358, 361, 363, 365, 368, 374, 377, 387, or 392 of SEQ ID NO: 1. Residues 338 and 365 of NO:1; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 358, 365 and 3 92; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365, or at the corresponding residues of the second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using Blast-p (e.g., Protocol 1 or 2 as described herein).

In some embodiments, the cavity filling mutation and at least one second mutation are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/ Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or selected from the corresponding residues of the second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using Blast-p (e.g., protocol 1 or 2 as described herein).

Specifically contemplated herein are coronavirus spike proteins having cavity-filling mutations as described herein and further having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., at least 95%, at least 99% identity to SEQ ID NO: 1 or SEQ ID NO: 2), provided that they retain the ability to act as coronavirus antigens (i.e., stimulate the production of coronavirus antibodies in a subject). That is, the non-naturally occurring coronavirus spike protein polypeptides described herein are "immunogenic", i.e., immunizing a subject with the polypeptide (optionally bound to an appropriate carrier (such as a protein, lipid or polypeptide)) induces an immune response (of the B cell type and/or T cell type) against the polypeptide.

RBD polypeptides or spike polypeptides as described herein may have one or more amino acid substitutions from known variants of coronaviruses. For example, but not limitation, the polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, or all 8 positions relative to SEQ ID NO: 1 selected from the group consisting of: L18F, T20N, P26S, deletion of residues 69-70, D80A, D138Y, R190S, D215G, K417N, K417T, G446S, L452R, Y453F, T478I, E484K, S494P, N501Y, A570D, D614G, H655Y, P681H, A701V, and T716L. The polypeptide may comprise one of the following naturally occurring mutations or combinations of mutations:

N501Y, optionally further comprising 1, 2, 3, 4 or 5 of the following: a deletion of one or both of residues 69-70, A570D, D614G, P681H and/or T716L (UK variant);

K417N/E484K/N501Y, optionally further comprising 1, 2, 3, 4 or 5 of the following: L18F, D80A, D215G, D614G and/or A701V (South African variant);

K417N or T/E484K/N501Y, optionally further comprising 1, 2, 3, 4 or 5 of: L18F, T20N, P26S, D138Y, R190S, D614G and/or H655Y (Brazilian variant); or

L452R (Los Angeles variant).

In some embodiments, a polypeptide as disclosed herein comprises the polypeptide sequence of SEQ ID NO:4 or SEQ ID NO:5.

RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADFSVLYNSASFSTFKCYG

VSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI

AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKK(SEQ ID NO:4)

RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADFSVLYNSASFSTFKCYG

VSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVI

AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKK(SEQ ID NO:5)

Amino acid mutation/substitution

Provided herein is a mutant coronavirus S protein or its receptor binding domain, wherein the mutant coronavirus S protein or its receptor binding domain has at least two amino acid mutations or substitutions that confer enhanced stability to the mutant protein compared to its corresponding natural or wild-type coronavirus S protein. The mutated SARS-CoV-2 S protein or its receptor binding domain is exemplified in this specification, but the methods and compositions described herein can be applied to any coronavirus S protein, including coronaviruses that infect humans and coronaviruses that infect other mammals (i.e., bats, cattle, pigs, etc. ......). By comparing the amino acid sequence of another coronavirus with the amino acid sequence of SARS-CoV-2 (i.e., SEQ ID NO: 1 or SEQ ID NO: 2), those skilled in the art can easily identify the residues corresponding to the residues of SARS-CoV-2 listed in this specification.

Alignment can provide guidance about residues that may be essential for function, whether the function is direct contact of one or more given residues with the receptor, or, for example, residues involved in maintaining a conformation that allows other residues to make such contacts; non-limiting examples of the latter include those residues that may be arranged in the linoleic acid binding pocket or help maintain a given conformation of the spike protein. For example, where the alignment shows that there are two identical or similar amino acids at corresponding positions, it is more likely that the site is important in function (i.e., linoleic acid binding or receptor binding). The variant amino acid sequence may be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the native or reference sequence (e.g., SEQ ID NO: 1). The degree of homology (percent identity) between the native sequence and the mutant sequence can be determined, for example, by comparing the two sequences using a computer program commonly used for this purpose and freely available on the World Wide Web. The variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more similar to the sequence from which the variant amino acid or DNA sequence was derived (referred to herein as "original," "native" or "wild-type" sequence). The degree of similarity (similarity percentage) between the original sequence and the mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and many tools that use similarity matrices to compare two sequences are available online for free, such as BLASTp (available on the World Wide Web at http://blast.ncbi.nlm.nih.gov), with default parameters set or using Protocol 1 or Protocol 2 as described herein.

When a cavity-filling mutation is desired, a given amino acid may be substituted with a residue having similar physiochemical characteristics, for example, one aliphatic residue for another (such as substitution of Ile, Val, Leu or Ala for each other), or one polar residue for another (such as between Lys and Arg; between Glu and Asp; or between Gln and Asn), preferably where a smaller residue is substituted with a larger residue that "fills" the cavity spatially or changes in charge to induce a change in the size and/or structure of the cavity. Other such substitutions, such as substitutions of entire regions with similar hydrophobic properties, are well known and may retain function. Polypeptides comprising the desired amino acid substitutions may be tested in any of the assays described herein to confirm that (i) the desired conformation is maintained, such that the antigenic activity of the native or reference polypeptide is substantially retained, or (ii) the stability of the protein is enhanced.

In some embodiments, amino acid substitutions may include conservative amino acid substitutions. The term "conservative amino acid substitution" is well known in the art and relates to the replacement of a particular amino acid by an amino acid having similar properties (e.g., similar charge or hydrophobicity). Conservative mutations described herein may include replacing amino acid residues having similar charge or hydrophobicity, but different sizes or volumes, (e.g., to provide a cavity-filling function).

A list of exemplary conservative amino acid substitutions is given in the table below.

Alternatively, non-conservative amino acid substitutions may be preferred, for example, by adding cysteine residues (or vice versa), for example, when it is desired to eradicate the flexible portion of the secondary structure of the native coronavirus S protein. "Non-conservative substitutions" refer to substitutions of one class of amino acids for another class of amino acids; for example, substitutions of Ala with Asp, Asn, Glu or Gln. Additional non-limiting examples of non-conservative substitutions include substitutions of polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid or lysine with non-polar (hydrophobic) amino acid residues such as isoleucine, valine, leucine, alanine, methionine and/or substitutions of non-polar residues with polar residues.

As will be appreciated by those skilled in the art, mutant coronavirus polypeptides as described herein (e.g., RBD polypeptides or stabilized coronavirus S polypeptides) can have a mixture of conservative and non-conservative amino acid substitutions of any desired configuration. Antigenic activity, receptor binding domain activity or conformation of polypeptides described herein can be tested using methods known in the art or described in the Examples.

Cysteine residues may be important for protein secondary structure or conformation. Mutations of cysteine residues are contemplated herein, provided that the secondary structure of the mutant protein is functional and/or antigenic, such as, for example, by assessing the binding to its cognate receptor (e.g., ACE2 receptor), using crystallography or EM to assess the secondary structure, or confirming the binding to antibodies against natural or wild-type proteins. Cysteine residues that are not involved in maintaining the appropriate conformation of a polypeptide may also be replaced by, for example, serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. On the contrary, cysteine bonds may be added to a polypeptide to improve the stability of the polypeptide or to promote oligomerization.

In some embodiments, the stabilized coronavirus S protein or RBD polypeptide as described herein may comprise naturally occurring amino acids commonly found in polypeptides and/or proteins produced by living organisms, such as Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M), Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q), Asp (D), Glu (E), Lys (K), Arg (R), and His (H). In some embodiments, the stabilized coronavirus S protein or RBD polypeptide as described herein may comprise alternative amino acids. Non-limiting examples of alternative amino acids include D-amino acids; β-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, γ-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statins, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto-D-valine), ornithine, citrulline, α-methyl-alanine, p-benzoylphenylalanine, p-aminophenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine and t-butylglycine), diaminobutyric acid, 7-hydroxy-tetrahydroisoquinolinecarboxylic acid, naphthylalanine, biphenylalanine , cyclohexylalanine, amino-isobutyric acid, norvaline, norleucine, tert-leucine, tetrahydroisoquinolinecarboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, amino-benzoic acid, amino-naphthoic acid, γ-aminobutyric acid, difluorophenylalanine, hexahydronicotinic acid, α-aminobutyric acid, thienyl-alanine, tert-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs; azide-modified amino acids; alkyne-modified amino acids; cyano-modified amino acids; and their derivatives.

In some embodiments, the polypeptide can be modified, such as a mutant coronavirus polypeptide, for example, by adding a moiety to one or more of the amino acids that together make up the peptide. Non-limiting examples of modifications and/or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end-capping modification; cyano; phosphorylation; albumin conjugation and cyclization. Modifications or moieties that improve solubility in a given solution (i.e., aqueous solution) are particularly contemplated herein.

The change of the original amino acid sequence can be achieved by any of many techniques known to those skilled in the art. Mutations can be introduced at the nucleic acid level, for example, by synthesizing oligonucleotides containing mutant sequences flanked by restriction sites that allow connection to fragments of the native sequence to introduce mutations at specific sites. After connection, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis program can be used to provide a changed nucleotide sequence with a specific codon changed according to the desired substitution, deletion or insertion. Techniques for making such changes include those disclosed by Khudyakov et al., "Artificial DNA: Methods and Applications" CRC Press, 2002; Braman "In Vitro Mutagenesis Protocols" Springer, 2004; and Rapley "The Nucleic Acid Protocols Handbook" Springer 2000; the entire contents of these documents are incorporated herein by reference. In some embodiments, polypeptides as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.

The mutant spike protein or RBC polypeptide described herein can be synthesized using well-known methods, including recombinant methods and chemical synthesis. Recombinant methods for producing polypeptides by introducing a vector containing a nucleic acid encoding a polypeptide into a suitable host cell are well known in the art, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Vol. 1 to Vol. 8, Cold Spring Harbor, NY (1989); M. W. Pennington and B. M. Dunn, Methods in Molecular Biology: Peptide Synthesis Protocols, Vol. 35, Humana Press, Totawa, NJ (1994), the contents of which are incorporated herein by reference. Peptides can also be chemically synthesized using methods well known in the art. See, for example, Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964); Bodanszky, M., Principles of Peptide Synthesis, Springer-Verlag, New York, NY (1984); Kimmerlin, T. and Seebach, D.J. Pept. Res. 65:229-260 (2005); Nilsson et al., Annu. Rev. Biophy s.Biomol.Struct.(2005)34:91-118; W.C.Chan and P.D.White (editors) Fmoc Solid Phase Peptide Synthesis: A Practical Approach, Oxford University Press, Cary, NC (2000); N.L.Benoiton, Chemistry of PeptideSynthesis, CRC Press, Boca Raton, FL (2005); J.Jones, Amino Acid and Peptide Synthesis, 2nd Edition, Oxford University Press, Cary, NC (2002); and P. Lloyd-Williams, F. Albericio and E. Giralt, Chemical Approaches to the synthesis of peptides and proteins, CRC Press, Boca Raton, FL (1997), the contents of all of which are incorporated herein by reference. Peptide derivatives can also be prepared as described in U.S. Patent Nos. 4,612,302; 4,853,371; and 4,684,620, and U.S. Patent Application Publication No. 2009/0263843, the contents of which are incorporated herein by reference.

Production and purification of RBD peptides or mutant coronavirus S proteins

RBD polypeptides or mutant coronavirus S proteins (as those terms are used herein) can be produced chemically by, for example, solution or solid phase peptide synthesis, or semi-synthesis in solution starting from protein fragments coupled by conventional solution methods, as described by Dugas et al. (1981). However, it is generally preferred to use recombinant methods to synthesize the polypeptides described herein.

Systems for cloning and expressing polypeptides that can be used for the methods and compositions described herein include various microorganisms and cells that are well known in recombinant technology and therefore not described in detail herein. These include various strains of, for example, E. coli, Bacillus, Streptomyces, and Saccharomyces, as well as mammals, yeast, and insect cells. If desired, polypeptides as described herein can be produced as peptides or fusion proteins. Suitable vectors for producing peptides and polypeptides are known and available from private and public laboratories and depository institutions and from commercial suppliers. Receptor cells capable of expressing gene products are then transfected. The transfected receptor cells are cultured under conditions that allow expression of the recombinant gene product, and the recombinant gene product is recovered from the culture. Host mammalian cells, such as Chinese hamster ovary cells (CHO) or COS-1 cells can be used. These hosts can be used in combination with poxvirus expression vectors (e.g., vaccinia or swine poxvirus). Suitable non-pathogenic viral expression vectors that can be engineered to carry synthetic genes into host cells include poxvirus expression vectors, such as vaccinia virus, adenovirus, retrovirus, etc. Many such non-pathogenic viral expression vectors are commonly used in human gene therapy and as carriers for other vaccine agents, and are known and selectable by those skilled in the art. Other suitable host cells and methods for transformation, culture, amplification, screening, and product production and purification can be selected by those skilled in the art with reference to known techniques.

In some embodiments, as described herein, it may be necessary to separate and/or purify the synthesized mutant polypeptide. Protein purification techniques are well known to those skilled in the art and are therefore not described in detail herein. These techniques may involve homogenization and coarse fractionation of cells, tissues or organs into polypeptide and non-polypeptide fractions at one level. Mutated coronavirus spike protein or receptor binding domain polypeptides can be further purified using chromatography and electrophoresis techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for preparing pure peptides or polypeptides are ion exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. Particularly effective methods for purifying peptides/polypeptides are fast high performance liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC).

"Purified polypeptide" is intended to refer to a composition separable from other components, wherein the mutant coronavirus S polypeptide or its receptor binding domain is purified to any degree relative to the organism producing the recombinant protein or its naturally obtainable state. Therefore, an isolated or purified polypeptide also refers to a polypeptide separated from an environment in which the polypeptide can naturally exist. In one embodiment, "purified" will refer to a polypeptide composition that has been fractionated to remove various other components, and the composition substantially retains the ability to bind to coronavirus antibodies that bind to natural or wild-type coronavirus S proteins. In the case of using the term "substantially purified", this name will refer to a composition in which the coronavirus polypeptide forms the main component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the protein in the composition.

There is no general requirement to provide the polypeptides described herein in the most purified state. In fact, it is expected that products with a lower degree of purification will be useful in certain embodiments. Partial purification can be accomplished by combining fewer purification steps, or by utilizing different forms of the same general purification scheme. For example, it should be understood that the cation exchange column chromatography performed using an HPLC device will generally result in a greater "fold" of purification than the same technology utilizing a low pressure chromatography system. Methods that exhibit a lower degree of relative purification may have advantages in terms of the total recovery of the protein product or in terms of maintaining the activity of the expressed protein.

Various methods for quantifying the extent of purification of a given polypeptide are known to those of skill in the art and include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptide within a fraction by SDS/PAGE analysis.

Coronavirus fusion proteins and scaffolds

In some embodiments, the receptor binding domain polypeptide or mutant coronavirus S protein described herein comprises a fusion protein. In some embodiments, the RBD polypeptide or mutant coronavirus S protein described herein is fused to a scaffold, a nanoparticle, a heterologous protein scaffold, or a polymer. In certain embodiments, the heterologous protein scaffold comprises the I53-50 trimer "A" component of SEQ ID NO:3. In other embodiments, the heterologous protein scaffold comprises a heterologous protein scaffold as described in Table 1 of U.S. Patent No. 10,351,603, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the mutant coronavirus polypeptide described herein is provided as a fusion protein. Such fusion proteins may include, for example, antigenic portions to enhance the resulting immune response. Such antigenic portions may include exogenous molecules, such as carrier proteins that are foreign to individuals to be vaccinated using the fusion proteins described herein. Exogenous proteins that activate immune responses and can be conjugated to fusion proteins as described herein include proteins or other molecules having a molecular weight of at least about 20,000 daltons, preferably at least about 40,000 daltons, and more preferably at least about 60,000 daltons. In this case, useful carrier proteins include, for example, GST, hemocyanin (such as hemocyanin from keyhole limpet), serum albumin or cationized serum albumin, thyroglobulin, ovalbumin, various toxoid proteins (such as tetanus toxoid or diphtheria toxoid), immunoglobulins, heat shock proteins, etc.

Methods for chemically coupling one protein (e.g., an RBD polypeptide or a mutant coronavirus S protein) to another protein (e.g., a carrier or an antigenic portion) are well known in the art and include, for example, conjugation via water-soluble carbodiimides (such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), conjugation via homobifunctional cross-linkers having, for example, NHS ester groups or sulfo-NHS ester analogs, conjugation via heterobifunctional cross-linkers having, for example, NHS ester and maleimide groups (such as sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), and conjugation with glutaraldehyde.

Protein-based virus-like particles

The present disclosure further provides protein-based virus-like particles (VLPs). pbVLPs may comprise the receptor binding domain polypeptide or mutant coronavirus S protein, including fusion proteins, wherein the fusion proteins comprise a multimerization domain, such as the "first component" as described herein.

VLPs for use with the methods and compositions described herein may comprise a multimeric protein assembly suitable for displaying the extracellular domain of the RBD or the extracellular domain of the coronavirus spike protein, or an antigenic fragment thereof. VLPs for use with the methods and compositions described herein may comprise at least a first polypeptide group (plurality of polypeptides). The first polypeptide group (also referred to as the "first component") may be derived from a naturally occurring protein sequence by replacing at least one amino acid residue or by appending amino acid residues at the N-terminus or C-terminus of one or more residues. In some cases, the first component comprises a protein sequence determined by a computational method. This first component may form the entire core of the VLP; or the core of the VLP may comprise one or more additional polypeptides (also referred to as the "second component" or the third, fourth, fifth component, etc.), such that the VLP comprises two, three, four, five, six, seven or more polypeptide groups. In some cases, the first group will form trimers related by 3-fold rotational symmetry and the second group will form pentamers related by 5-fold rotational symmetry. In this case, the VLP forms an "icosahedral particle" with I53 symmetry. These one or more polypeptide groups can be arranged together so that the members of each polypeptide group are related to each other by a symmetry operator. A general computational method for designing self-assembling protein materials is disclosed in U.S. Patent Publication No. 2015/0356240A1, which is incorporated herein by reference in its entirety, involving symmetric docking of protein building blocks in a target symmetric framework.

The "core" of a VLP is used herein to describe the central portion of the VLP that links together several copies of the RBD or coronavirus S protein ectodomain displayed by the VLP, or antigenic fragments thereof. In embodiments, the first component includes a first polypeptide containing an RBD, a linker, and a first polypeptide containing a multimerization domain.

A non-limiting embodiment is shown in Figure 6A, which depicts an RBD genetically fused to a component of a VLP, optionally recombinantly expressed in a host cell (e.g., a 293F cell); together with a pentameric protein assembly, optionally recombinantly expressed in the same or different host cell (e.g., an E. coli cell), the two polypeptide groups self-assemble into a VLP displaying 20 antigen trimers around an icosahedral core.

In some cases, VLPs are adapted to display RBD or spike proteins from two or more different coronavirus strains. In a non-limiting example, the same VLP displays a mixed population of protein antigens or a mixed heterotrimer of protein antigens from different coronavirus strains.

VLPs for use with the methods and compositions described herein display antigenic proteins in various ways, including as gene fusions or by other means disclosed herein. As used herein, "connected to" or "attached to" means any means known in the art for causing the association of two polypeptides. The association can be direct or indirect, reversible or irreversible, weak or strong, covalent or non-covalent, and selective or non-selective.

In some embodiments, realize attachment to produce antigen and the N-terminal or C-terminal fusion of a kind of polypeptide in the polypeptide group constituting VLP by genetic engineering.Therefore, VLP can be by or basically by displaying one, two, three, four, five, six, seven, eight, nine or ten kinds of one, two, three, four, five, six, seven, eight, nine or ten kinds of polypeptide groups of one, two, three, four, five, six, seven, eight, nine or ten kinds of antigen groups, wherein at least one antigen in the antigen group is genetically fused with at least one polypeptide in the polypeptide group.In some cases, VLP is basically composed of a kind of polypeptide group that can self-assemble and comprise the antigenic protein group that is genetically fused with it.In some cases, VLP is basically composed of the following: the first polypeptide group comprising antigen group; And the second polypeptide group that can be assembled into two-component VLP altogether, antigenic protein is connected to a kind of polypeptide group of VLP, and other polypeptide groups that promote the self-assembly of VLP.

In some embodiments, attachment is achieved by post-translational covalent attachment between one or more polypeptide groups and one or more antigenic protein groups. In some cases, chemical crosslinking is used for non-specific attachment of antigen to VLP polypeptide. In some cases, chemical crosslinking is used for antigenic protein specific attachment to VLP polypeptide (for example, attached to the first polypeptide or the second polypeptide). Various specific and non-specific crosslinking chemistries are known in the art, such as click chemistry and other methods. In general, any crosslinking chemistry for connecting two proteins can be applicable to use with currently disclosed VLP. In particular, the chemical method for producing immunoconjugates or antibody drug conjugates can be used. In some cases, VLP is created using a cleavable or non-cleavable joint. The process and method for conjugating antigen to a carrier are provided by, for example, U.S. Patent Publication No. US 2008/0145373 A1, and the content disclosed in this U.S. Patent is incorporated herein by reference in its entirety.

The component of VLP of the present disclosure can have any amino acid sequence in multiple amino acid sequences. U.S. Patent Publication No. US 2015/0356240 A1 (its content is incorporated herein by reference in its entirety) describes a variety of methods for designing protein assemblies. As described in U.S. Patent Publication No. US 2016/0122392 A1 and International Patent Publication No. WO 2014/124301 A1, the polypeptide is designed to have the ability of self-assembly in pairs to form VLP, such as icosahedral particles. The design relates to suitable interface residues for each member design of the polypeptide pair that can be assembled to form VLP. The VLP formed in this way comprises a symmetrically repeated, non-natural, non-covalent polypeptide-polypeptide interface, and the first assembly and the second assembly are directed into VLP by the interface, such as VLP with icosahedral symmetry.

In some embodiments, the RBD or coronavirus S protein extracellular domain or its antigenic fragment is expressed as a fusion protein with a first multimerization domain. In some embodiments, the first multimerization domain and the RBD or coronavirus S protein extracellular domain are joined by a linker sequence. In some embodiments, the linker sequence comprises a foldon, wherein the foldon sequence is EKAAKAEEAARK (SEQ ID NO: 8).

Non-limiting examples of designed protein complexes that can be used for protein-based VLPs of the present disclosure include those disclosed in U.S. Pat. No. 9,630,994; International Patent Publication No. WO2018187325A1; U.S. Patent Publication No. 2018/0137234A1; U.S. Patent Publication No. 2019/0155988A2, each of which is incorporated herein by reference in its entirety. Illustrative sequences are provided in Table 3.

Table 3

In some embodiments, the VLP comprises: a fusion protein that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NO:9 to SEQ ID NO:13 and comprises an RBD or a coronavirus spike protein as disclosed herein; and a second component that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NO:13 to SEQ ID NO:18.

In some embodiments, the VLP comprises: a fusion protein that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 19 and comprises an RBD or a coronavirus spike protein as disclosed herein; and a second component that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 20.

In some embodiments, a polypeptide as disclosed herein comprises the polypeptide sequence of SEQ ID NO:6 or SEQ ID NO:7.

RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADFSVLYNSASFSTFKCYG

VSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI

AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF

NCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGGSGGSG

SGGSGGSGSEKAAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFA

GGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFI

VSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQ

FVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(SEQ ID NO:6)

RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADFSVLYNSASFSTFKCYG

VSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVI

AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGF

NCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGGSGGSG

SGGSGGSGSEKAAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFA

GGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFI

VSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQ

FVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE (SEQ ID NO: 7).

Protein stability

Most biological products are susceptible to degradation, such as thermal degradation, photochemical degradation or oxidative degradation. Because biological products such as vaccines and insulin need to be distributed worldwide, and because the ambient temperature in different regions varies greatly, vaccines (or stabilized proteins therein) with extended shelf life are preferred compared to rapidly degradable proteins/compositions. Increased intracellular stability can also provide a higher recombinant protein yield. The mutant coronavirus spike protein or its receptor binding domain described herein has increased stability compared to its wild-type counterpart. The stability of these proteins can be determined using one or more assays known in the art or using the methods described in the working examples. Exemplary protein stability assays include, but are not limited to, differential scanning calorimetry, pulse tracer method, bleach tracer method, cyclohexamide tracer method, circular dichroism spectroscopy, and fluorescence-based activity assays.

The stability of a given composition, also referred to herein as the "shelf life" of the composition when applied to an isolated preparation or a vaccine preparation, depends on the storage conditions of the composition as well as the formulation of the composition (e.g., addition of chemical components) or the physical state in which the composition is provided (e.g., lyophilized, dried, frozen, etc.).

The term "lyophilization" or "freeze-dried" as used herein refers to a dehydration process, commonly referred to as "freeze drying," used to preserve a composition as described herein or to make the composition more convenient for storage and transport. Freeze drying works by freezing the composition and then reducing the surrounding pressure to allow the frozen water in the composition to sublime directly from the solid phase to the gas phase. In some embodiments, lyophilization can be used to preserve a vaccine composition as described herein, thereby allowing the vaccine composition to be portable and stored at room temperature without the need for refrigeration.

In addition to the increased antigenic stability provided by the mutations described herein, the addition of antioxidants or other agents intended to extend the shelf life of the vaccine compositions is also contemplated herein. In some embodiments, it is contemplated that the compositions described herein are stored at room temperature and without refrigeration.

Regardless of the storage method, both the protein stability and the shelf life of the composition of the mutant coronavirus spike protein or its RBD are increased compared to the protein stability and the shelf life of the composition of the corresponding wild-type coronavirus spike protein or its RBD.

The preparation comprising mutant coronavirus protein or peptide as described herein is stable, because their characteristics in a given time period at a specified temperature are almost unchanged. Generally speaking, preparations as described herein can be stabilized for at least about one month. In some embodiments, preparations are stabilized for at least about 6 weeks, at least about 2 months, at least about 4 months, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months (1 year), at least about 14 months, at least about 16 months, at least about 18 months (1.5 years), at least about 20 months, at least about 22 months, at least about 24 months (2 years), at least about 26 months, at least about 28 months, at least about 30 months, at least about 32 months, at least about 34 months, at least about 36 months (3 years), at least about 38 months, at least about 40 months, at least about 42 months, at least about 44 months, at least about 46 months or at least about 48 months (4 years).

The formulation is generally stable at temperatures below about 30°C. In some embodiments, the stability of the formulation is about temperatures below about 25°C, about 20°C, about 15°C, about 10°C, about 8°C, about 5°C, about 4°C, or about 2°C. Thus, in some embodiments, the temperature is in the range of about 25°C to about 2°C, about 20°C to about 2°C, about 15°C to about 2°C, about 10°C to about 2°C, about 8°C to about 2°C, or about 5°C to about 2°C. In other embodiments, the temperature is in the range of about 25°C to about 5°C, about 20°C to about 5°C, about 15°C to about 5°C, about 10°C to about 5°C, or about 8°C to about 5°C. In other embodiments, the temperature is in the range of about 25°C to about 8°C, about 20°C to about 8°C, about 15°C to about 8°C, or about 10°C to about 8°C. In other embodiments, the temperature is in the range of about 25° C. to about 10° C., about 20° C. to about 10° C., about 15° C. to about 10° C., about 25° C. to about 15° C., about 20° C. to about 15° C., or about 25° C. to about 20° C. In some embodiments, the composition can be stored at 4° C. or -20° C. for longer term storage.

Vaccine composition

RBD polypeptides, mutant coronavirus S proteins, or fusion proteins comprising them as described herein can be used to produce vaccine preparations. Such vaccine preparations can provide protection for each coronavirus in the seven coronaviruses known to infect humans individually, or can provide protection for at least 2, at least 3, at least 4, at least 5, or at least 6 coronaviruses in the seven coronaviruses known to infect humans. In one embodiment, the vaccine preparations as described herein can provide protection for all 7 coronaviruses known to infect humans. It is also particularly contemplated herein that the vaccine preparations described herein can provide protection for coronaviruses expected to be transferred from animal species (e.g., bats) to humans in the future. In other embodiments, the vaccine preparations described herein can provide protection for the animal against one or more coronaviruses to which the animal is susceptible. RNA and/or DNA vaccine preparations encoding mutant spike protein polypeptides described herein are also particularly contemplated herein.

In some embodiments, immunity generated against coronavirus antigens described herein is long-lasting (e.g., at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, or even the entire life span of the subject). Alternatively, in some embodiments, it is contemplated herein that the vaccine formulations described herein are administered on an annual basis, customized for the prevalence or predicted prevalence of the target virus, similar to immunization with influenza vaccines, and can provide protection for at least 3 months, at least 6 months, at least 8 months, at least one year, at least 1.5 years, or at least two years from the last administration.

Those skilled in the art will recognize that a coronavirus vaccine formulation need not be 100% effective to confer community-based immunity or herd immunity against one or more coronaviruses. Thus, in some embodiments, the vaccine formulations described herein are at least 40% effective in a population of vaccinated individuals, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% effective in a population of vaccinated individuals.

In some embodiments, to reduce community transmission and prevent/control coronavirus infection and transmission, the vaccines described herein can be administered as universal vaccinations for healthy children and individuals. Children can play an important role in the spread of coronaviruses in schools, families, and communities, especially because they tend to have milder symptoms and are not necessarily diagnosed with coronavirus infection. Flu vaccine studies have shown that vaccinating about 80% of school-age children in the community has reduced respiratory diseases in adults and excess deaths in the elderly (Reichert et al., 2001). This concept is called community immunity or "herd immunity" and is believed to play an important role in protecting communities from disease. Since vaccinated people have antibodies that neutralize specific viruses, they are much less likely to spread the virus to others. This concept can also be applied to coronavirus infections. Therefore, even people who have not been vaccinated (and those whose vaccinations have weakened or the vaccines are not completely effective) can often be protected by herd immunity because the vaccinated people around them will not get sick or spread the virus. As the percentage of vaccinated people increases, herd immunity will be more effective. It is believed that about 60% (but preferably closer to 90-95%) of people in the community must be protected by vaccines to achieve herd immunity. People who are not immunized increase their chances of contracting the disease, as well as others.

Therefore, in another aspect, a method is provided herein for inducing sufficient protective immunity for coronavirus infection to a group or community by administering a vaccine formulation as described herein to a group in a community, to reduce the incidence of coronavirus infection in an immunocompromised individual or an unvaccinated individual. In one embodiment, most school-age children are immunized against coronavirus infection by administering a vaccine as described herein (for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more school-age children have been vaccinated). In another embodiment, most healthy individuals in the community (for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more) are immunized against a given coronavirus or a group of coronaviruses by administering a vaccine as described herein. In another embodiment, vaccines as described herein can be used as part of a "dynamic vaccination" strategy. Dynamic vaccination is the stable production of inefficient vaccines associated with emerging or existing pandemic strains, but due to antigenic drift, it may not be possible to provide complete protection in mammals (see Germann et al., 2006). Due to the uncertainty of the future characteristics of pandemic strains, it is almost impossible to store well-matched pandemic strains. However, vaccination with a poorly matched but potentially effective vaccine may slow the spread of the pandemic virus and/or reduce the severity of the symptoms of pandemic coronavirus strains. In one embodiment, the vaccine as described herein is directed against one or more SARS-CoV-2 strains that caused the 2019/2020 COVID-19 pandemic.

Vaccine formulations as described herein can prevent at least one symptom in the symptoms associated with coronavirus infection, or can completely prevent the appearance of any symptom. Common symptoms of coronavirus infection include, but are not limited to, fever, chills, cough, shortness of breath/dyspnea, fatigue, muscle/body pain, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea, vomiting or diarrhea. The alleviation of symptoms can be determined subjectively or objectively, such as by subject self-assessment, by clinician assessment or by performing appropriate determination or measurement (such as body temperature, pneumonia infection degree, lung scarring, etc.), including, for example, quality of life assessment, coronavirus infection or the progression of additional symptoms slows down, the severity of coronavirus-related disease symptoms is reduced or suitable determination (such as antibody titer and/or T cell activation determination).

Preferably, the vaccine formulations described herein will reduce the transmission of active coronavirus from a vaccinated individual to other individuals within the individual's community or between two different individuals by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or even 100% (i.e., no transmission can be detected between a vaccinated individual and two or more individuals).

In some embodiments, the vaccine formulations described herein include one or more adjuvants. Non-limiting examples of adjuvants include Freund's complete adjuvant (a non-specific stimulator of the immune response, containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, aluminum hydroxide, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI is also considered, which contains three components extracted from bacteria in a 2% squalene/Tween 80 emulsion: MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS). MF-59, MHC antigens.

In one aspect, the adjuvant effect is achieved by using an agent such as alum in the form of about 0.05% to about 0.1% phosphate buffered saline solution. Alternatively, the vaccines described herein can be formulated with synthetic sugar polymers. of a mixture which is used in about a 0.25% solution. Some adjuvants, such as certain organic molecules obtained from bacteria, act on the host rather than the antigen. An example is muramyl dipeptide (N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP)), which is a bacterial peptidoglycan. In other embodiments, hemocyanin and hemoerythrin may also be used with the vaccine formulations described herein. Hemocyanin from keyhole limpet (KLH) may be used in certain embodiments, but other mollusc and arthropod hemocyanins and hemoerythrin may be used in alternative embodiments.

Various polysaccharide adjuvants can also be used. For example, the effects of various pneumococcal polysaccharide adjuvants on mouse antibody responses have been described (Yin et al., 1989). The dosage that produces the best response or does not produce inhibition should be adopted as indicated (Yin et al., 1989). Polyamine polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin. In another embodiment, lipophilic disaccharide-tripeptide derivatives of muramyl dipeptide can be used, which are described as being used for artificial liposomes formed by phosphatidylcholine and phosphatidylglycerol.

Amphiphilic and surfactants, such as saponins and derivatives, such as QS21 (Cambridge Biotech), also form another group of adjuvants for vaccine formulations as described herein. Nonionic block copolymer surfactants (Rabinovich et al., 1994) may also be used. Oligonucleotide adjuvants (Yamamoto et al., 1988), Quil A and Lentinan are other adjuvants that may be used in certain embodiments.

In addition, refined detoxified endotoxins can be used alone or in the form of multiple adjuvant preparations to produce auxiliary reactions in vertebrates. For example, the combination of detoxified endotoxins and trehalose dimycolate or the combination of detoxified endotoxins and trehalose dimycolate and endotoxin glycolipids is contemplated herein. Alternatively, the combination of detoxified endotoxins and cell wall skeletons (CWS) or CWS and trehalose dimycolate, or only CWS and trehalose dimycolate without the combination of detoxified endotoxins is also contemplated.

Those skilled in the art will appreciate the different types of adjuvants that can be conjugated or admixed with vaccines as described herein, and these include alkyl lysophospholipids (ALP); BCG; and biotin (including biotinylated derivatives), among others. Certain adjuvants specifically contemplated for use are teichoic acids from gram-negative bacterial cells. These adjuvants include lipoteichoic acid (LTA), ribitol teichoic acid (RTA), and glyceroteichoic acid (GTA). Active forms of their synthetic counterparts may also be used.

A variety of adjuvants, even those not commonly used in humans, are anticipated to be useful in vaccines for other vertebrates.

Vaccine formulations can include a coronavirus polypeptide or fusion protein thereof as described herein, microencapsulated or macroencapsulated, for example, into the inner capsid protein of bovine rotavirus (Redmond et al., Mol. Immunol. 28:269 (1991)), into immunostimulatory molecules (ISCOMS) composed of saponins such as Quil A (Morein et al., Nature 308:457 (1984)); Morein et al., in Immunological Adjuvants and Vaccines (G. Gregoriadis et al., eds.) pp. 153-162, Plenum Press, NY (1987)), or into controlled-release biodegradable microspheres composed of, for example, lactide-glycolide copolymers (O'Hagan et al., Immunology 73:239 (1991); O'Hagan et al., Vaccine 11:149 (1993)).

The stabilized coronavirus polypeptides or fusion proteins thereof described herein can also be adsorbed to the surface of lipid microspheres containing squalene or squalane emulsions prepared using PLURONIC block copolymers (such as L-121) and stabilized with detergents (such as TWEEN 80) (see Allison and Byers, Vaccines: New Approaches to Immunological Problems (R. Ellis, ed.) pp. 431-449, Butterworth-Hinemann, Stoneman N.Y. (1992)).

Vaccine formulations described herein include stabilized coronavirus polypeptides or fusion proteins thereof as described herein of an "effective amount" or "therapeutically effective amount". As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to provide a concentration high enough to produce (or contribute to the production of) an immune response in its recipient. The specific effective dose level of any particular subject will depend on a variety of factors, including the illness being treated, the severity of the illness, the activity of a particular compound, the route of administration, the clearance rate of the administered agent, the duration of treatment, the drug used in combination with the administered agent or used simultaneously, the age, weight, sex, diet and overall health of the subject, and similar factors well known in medical art and science. The various general considerations considered in determining a "therapeutically effective amount" are known to those skilled in the art and are described in, for example, Gilman et al., Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th edition, Pergamon Press, 1990; and Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Easton, Pa., 1990.

The pH of preparation may also be different. Typically, the pH of preparation is between about pH 6.2 and about pH 8.0. In some embodiments, the pH is about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8 or about 8.0. Of course, pH can also be in the range of certain values. Therefore, in some embodiments, the pH is between about 6.2 and about 8.0, between about 6.2 and 7.8, between about 6.2 and 7.6, between about 6.2 and 7.4, between about 6.2 and 7.2, between about 6.2 and 7.0, between about 6.2 and 6.8, between about 6.2 and about 6.6 or between about 6.2 and 6.4. In other embodiments, the pH is between 6.4 and about 8.0, between about 6.4 and 7.8, between about 6.4 and 7.6, between about 6.4 and 7.4, between about 6.4 and 7.2, between about 6.4 and 7.0, between about 6.4 and 6.8, or between about 6.4 and about 6.6. In still other embodiments, the pH is between about 6.6 and about 8.0, between about 6.6 and 7.8, between about 6.6 and 7.6, between about 6.6 and 7.4, between about 6.6 and 7.2, between about 6.6 and 7.0, or between about 6.6 and 6.8. In other embodiments, it is between about 6.8 and about 8.0, between about 6.8 and 7.8, between about 6.8 and 7.6, between about 6.8 and 7.4, between about 6.8 and 7.2, or between about 6.8 and 7.0. In other embodiments, it is between about 7.0 and about 8.0, between about 7.0 and 7.8, between about 7.0 and 7.6, between about 7.0 and 7.4, between about 7.0 and 7.2, between about 7.2 and 8.0, between about 7.2 and 7.8, between about 7.2 and about 7.6, between about 7.2 and 7.4, between about 7.4 and about 8.0, between about 7.4 and about 7.6, or between about 7.6 and about 8.0.

In some embodiments, preparation can comprise one or more salts, such as sodium chloride, sodium phosphate or their combination.Usually, every kind of salt is present in preparation with about 10mM to about 200mM.Therefore, in some embodiments, any salt present is present with about 10mM to about 200mM, about 20mM to about 200mM, about 25mM to about 200mM, about 30mM to about 200mM, about 40mM to about 200mM, about 50mM to about 200mM, about 75mM to about 200mM, about 100mM to about 200mM, about 125mM to about 200mM, about 150mM to about 200mM or about 175mM to about 200mM. In other embodiments, any salt present is present at about 10 mM to about 175 mM, about 20 mM to about 175 mM, about 25 mM to about 175 mM, about 30 mM to about 175 mM, about 40 mM to about 175 mM, about 50 mM to about 175 mM, about 75 mM to about 175 mM, about 100 mM to about 175 mM, about 125 mM to about 175 mM, or about 150 mM to about 175 mM. In other embodiments, any salt present is present at about 10mM to about 150mM, about 20mM to about 150mM, about 25mM to about 150mM, about 30mM to about 150mM, about 40mM to about 150mM, about 50mM to about 150mM, about 75mM to about 150mM, about 100mM to about 150mM, or about 125mM to about 150mM. In other embodiments, any salt present is present at about 10mM to about 125mM, about 20mM to about 125mM, about 25mM to about 125mM, about 30mM to about 125mM, about 40mM to about 125mM, about 50mM to about 125mM, about 75mM to about 125mM, or about 100mM to about 125mM. In some embodiments, any salt present is present at about 10mM to about 100mM, about 20mM to about 100mM, about 25mM to about 100mM, about 30mM to about 100mM, about 40mM to about 100mM, about 50mM to about 100mM, or about 75mM to about 100mM. In other embodiments, any salt present is present at about 10mM to about 75mM, about 20mM to about 75mM, about 25mM to about 75mM, about 30mM to about 75mM, about 40mM to about 75mM, or about 50mM to about 75mM. In more other embodiments, any salt present is present at about 10mM to about 50mM, about 20mM to about 50mM, about 25mM to about 50mM, about 30mM to about 50mM, or about 40mM to about 50mM. In other embodiments, any salt present is present at about 10mM to about 40mM, about 20mM to about 40mM, about 25mM to about 40mM, about 30mM to about 40mM, about 10mM to about 30mM, about 20mM to about 30, about 25mM to about 30mM, about 10mM to about 25mM, about 20mM to about 25mM, or about 10mM to about 20mM. In one embodiment, sodium chloride is present in the formulation at about 100mM. In one embodiment, sodium phosphate is present in the formulation at about 25mM.

The formulations comprising the mutant coronavirus proteins described herein may further comprise a solubilizing agent, such as a non-ionic detergent. Such detergents include, but are not limited to, polysorbate 80 ( 80), TritonX100 and polysorbate 20.

Vaccine administration and efficacy

Vaccine formulations as described herein may further comprise a pharmaceutically acceptable carrier, including any suitable diluent or excipient, including any agent that does not itself induce an immune response harmful to the vertebrate receiving the composition and can be administered without excessive toxicity. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the U.S. Pharmacopoeia, European Pharmacopoeia or other recognized pharmacopoeia for use in vertebrates, more specifically in humans. These compositions can be used as vaccines and/or antigenic compositions to induce a protective immune response in vertebrates.

Pharmaceutically acceptable carriers or excipients include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof. Pharmaceutically acceptable carriers, diluents, and other excipients are discussed comprehensively in Remington's Pharmaceutical Sciences (Mack Pub. Co. NJ current edition). The formulation should be suitable for the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans, preferably sterile, particulate-free, and/or non-pyrogenic.

If necessary, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. The composition may be in solid form, such as a lyophilized powder, liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation or powder suitable for reconstitution. Oral formulations may include standard carriers, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc.

Typically, the coronavirus vaccines described herein are administered in an effective amount or an amount sufficient to stimulate an immune response to one or more coronavirus strains. Preferably, the administration of the vaccine formulation causes substantial immunity to at least one coronavirus. Typically, the dosage can be adjusted based on, for example, age, physical condition, weight, sex, diet, administration time, and other clinical factors.

Although it is preferred to carry out substantial immunostimulation with a single dose, additional doses can be applied by the same or different routes to achieve the desired effect. For example, in newborns and infants, multiple administrations may be required to produce sufficient immunity levels. If necessary, administration can be continued at intervals throughout childhood to maintain sufficient protection levels for coronavirus infection. Similarly, adults who are particularly susceptible to severe diseases or repeated infections (such as, for example, health care workers, day care workers, family members of young children, the elderly, and people with impaired cardiopulmonary function) may need multiple immunizations to establish and/or maintain a protective immune response. The level of induced immunity can be monitored, for example, by measuring the amount of neutralizing secretory antibodies and serum antibodies, and the dosage can be adjusted as needed or repeated vaccinations can be used to induce and maintain the desired protection level.

Prophylactic vaccine formulations can be administered systemically, for example, by subcutaneous or intramuscular injection using a needle and syringe or a needle-free injection device. Alternatively, the vaccine formulation is administered intranasally by drops, large particle aerosols (greater than about 10 microns), or sprays into the upper respiratory tract. While any of the above delivery routes may result in an immune response, intranasal administration may have the added benefit of eliciting mucosal immunity at one of the coronavirus's sites of entry.

Non-limiting methods of administering vaccine formulations as described herein include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and oral or pulmonary routes or by suppositories). In specific embodiments, vaccine compositions as described herein are administered intramuscularly, intravenously, subcutaneously, transdermally, or intradermally. The composition can be administered by any convenient route, such as by infusion or bolus injection, by absorption via epithelial or mucocutaneous linings (e.g., oral mucosa, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, bladder, and intestinal mucosa, etc.), and can be administered together with other bioactive agents. In some embodiments, the intranasal or other mucosal administration routes of vaccine compositions as described herein can induce antibodies or other immune responses that are significantly higher than other administration routes. In another embodiment, the intranasal or other mucosal administration routes of vaccine compositions as described herein can induce antibodies or other immune responses that will induce cross-protection against other coronavirus strains. Administration can be systemic or local. In some embodiments, vaccine formulations are administered in a manner that targets mucosal tissues so as to induce an immune response at the immunization site. For example, by oral administration of a composition containing an adjuvant with specific mucosal targeting properties, mucosal tissues such as gut-associated lymphoid tissue (GALT) can be targeted for immunization. Other mucosal tissues such as nasopharyngeal lymphoid tissue (NALT) and bronchus-associated lymphoid tissue (BALT) can also be targeted.

Vaccine formulations as described herein can also be administered in a dosage regimen, such as the initial administration of vaccine compositions and subsequent booster administration. In a specific embodiment, the composition of the second dose is administered at any time from two weeks to one year after the initial administration, preferably from about 1 month, about 2 months, about 3 months, about 4 months, about 5 months to about 6 months. In addition, the 3rd dose can be administered after the second dose, and from about three months to about two years after the initial administration, or even longer, preferably from about 4 months, about 5 months, or about 6 months, or about 7 months to about one year. When low-level specific immunoglobulin is not detected or detected in the serum and/or urine or mucosal secretions of the subject after the second dose, the 3rd dose can be optionally administered. In a preferred embodiment, the second dose is administered for about one month after the first administration, and the 3rd dose is administered for about six months after the first administration. In another embodiment, the second dose is administered for about six months after the first administration.

The dosage of the pharmaceutical preparation can be easily determined by a technician, for example, by first determining the dosage that effectively triggers a preventive or therapeutic immune response, for example, by measuring the serum titer of virus-specific immunoglobulins or by measuring the inhibition rate of antibodies in serum samples, urine samples or mucosal secretions. The dosage can be determined from animal studies. A non-limiting list of animals used to study coronaviruses includes guinea pigs, Syrian hamsters, chinchillas, hedgehogs, chickens, rats, mice, pigs, cattle, bats and ferrets. Bats are particularly considered to be natural hosts of coronaviruses and are particularly useful in studying or testing vaccines. However, any of the above animals can be dosed with a vaccine formulation as described herein to partially characterize the induced immune response, and/or to determine whether any neutralizing antibodies have been produced.

In addition, human clinical studies can be performed by technicians to determine the preferred effective dose for humans. Such clinical studies are routine and well known in the art. The exact dose used will also depend on the route of administration. The effective dose can be extrapolated from a dose-response curve derived from an in vitro or animal test system.

Another method of inducing or enhancing an immune response can be achieved by preparing a vaccine composition as described herein to include one or more immunostimulants, such as one or more cytokines, lymphokines or chemokines with immunostimulatory, immunoenhancing and/or proinflammatory activity (e.g., interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factors, Flt3 ligand, B7.1; B7.2, etc.). Such immunostimulatory molecules can be administered in the same formulation as the coronavirus vaccine formulation, or can be administered separately.

The RBD polypeptide or stabilized coronavirus spike protein described herein can be used in a vaccine formulation that, when administered to a vertebrate, can induce substantial immunity in a vertebrate (e.g., a human). The substantial immunity is produced by an immune response to the RBD polypeptide or stabilized coronavirus spike protein, and prevents or improves coronavirus infection or at least alleviates the symptoms of coronavirus virus infection in the vertebrate. In some cases, the subsequently infected vaccinated subject will be asymptomatic. However, the response may not be a completely protective response, and partial immune responses are also contemplated herein. For example, compared to unimmunized vertebrates, partially protected subjects who are subsequently infected with coronavirus will experience reduced symptom severity or a shorter duration of symptoms. For example, subjects vaccinated with a preparation containing a SARS-CoV-2 stabilized spike protein may not need to be hospitalized for a long time or use a ventilator to treat COVID-19.

In one embodiment, provided herein is a method for inducing substantial immunity to one or more coronavirus infections or at least one symptom thereof in a subject, the method comprising administering at least one effective dose of a vaccine formulation comprising an RBD polypeptide as described herein or a stabilized coronavirus S protein. In another embodiment, the induction of the substantial immunity shortens the duration of coronavirus-related disease symptoms (e.g., SARS, COVID-19).

In one embodiment, the vaccine formulations as described herein can elicit an immune response that will provide protection against more than one coronavirus strain. This cross-protection of vertebrates using stabilized coronavirus S proteins or RBD polypeptides constructed from specific strains of specific subgroups can induce cross-protection against coronaviruses of different strains and/or subgroups.

Reagent test kit

Also provided herein is a kit for individual or animal vaccination, the kit comprising one or more containers filled with one or more components of the components of the vaccine formulation as described herein. In one embodiment, the kit comprises two containers, one container containing stabilized coronavirus polypeptides or their fusion proteins, and the other container containing adjuvants. Associated with such containers may be a notification in the form of a government agency regulating the manufacture, use or sale of a drug or biological product, which reflects the agency's approval of manufacture, use or sale for human administration.

The vaccine preparation is packaged in an airtight sealed container, such as an ampoule or a pouch indicating the amount of the composition. In one embodiment, the vaccine composition is supplied as a liquid, in another embodiment, as a dry sterilized lyophilized powder or anhydrous concentrate in an airtight sealed container, and can be reconstituted to an appropriate concentration for administration to a subject, for example, with water or saline. In some embodiments, the vaccine composition is supplied as a dry sterile lyophilized powder in an airtight sealed container with a unit dose of preferably about 1 μg, about 5 μg, about 10 μg, about 20 μg, about 25 μg, about 30 μg, about 50 μg, about 100 μg, about 125 μg, about 150 μg, or about 200 μg. Alternatively, the unit dose of the vaccine composition is less than about 1 μg (e.g., about 0.08 μg, about 0.04 μg; about 0.2 μg, about 0.4 μg, about 0.8 μg, about 0.5 μg or less, about 0.25 μg or less, or about 0.1 μg or less), or greater than about 125 μg (e.g., about 150 μg or more, about 250 μg or more, or about 500 μg or more). These doses can be measured as total coronavirus polypeptides or as μg HA. The vaccine composition should be administered within about 12 hours, preferably within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour after reconstitution from the lyophilized powder.

In alternative embodiments, the vaccine composition as described herein is supplied in a liquid form in an airtight sealed container indicating the amount and concentration of the composition. Preferably, the liquid form of the vaccine composition is supplied in an airtight sealed container at least about 50 μg/ml, more preferably at least about 100 μg/ml, at least about 200 μg/ml, at least 500 μg/ml, or at least 1 mg/ml.

The invention may be as described in any of the following numbered paragraphs.

1. A non-naturally occurring polypeptide, the non-naturally occurring polypeptide comprising a first coronavirus receptor binding domain (RBD), the first coronavirus RBD comprising at least 90% identity to residues 328-531 of SEQ ID NO:1, and further comprising at least two mutations relative to the RBD of SEQ ID NO:1, wherein the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F /Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or at the corresponding residues of a second coronavirus receptor binding domain as determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus receptor binding domain using the Blast-p parameters of Protocol 1 or Protocol 2.

2. A non-naturally occurring polypeptide, the non-naturally occurring polypeptide comprising: a first coronavirus receptor binding domain (RBD), the first coronavirus RBD comprising at least 90% identity with residues 328-531 of SEQ ID NO:1 or with the corresponding residues of the receptor binding domain of a second coronavirus determined by sequence alignment of SEQ ID NO:1 with the sequence of a second coronavirus receptor binding domain using the Blast-p parameters of protocol 1 or protocol 2, and further comprising at least two mutations relative to the RBD of SEQ ID NO:1 or the corresponding residues in the second coronavirus, wherein relative to the stability of a wild-type polypeptide lacking the at least two mutations, the at least two mutations enhance the stability of the polypeptide.

3. The polypeptide according to paragraph 2, wherein the at least two mutations are at the following amino acids of SEQ ID NO: 1: 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358 , 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365, or at the corresponding residues of the second coronavirus receptor binding domain as determined by sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus receptor binding domain using the Blast-p parameters of Protocol 1 or Protocol 2.

4. The polypeptide according to paragraph 2 or 3, wherein the at least two mutations are selected from the group consisting of: F338L/Y365W of SEQ ID NO: 1; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I377V/A363L/Y365F 58F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or at the corresponding residues of a second coronavirus determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus receptor binding domain using the Blast-p parameters of Protocol 1 or Protocol 2.

5. The polypeptide according to any one of paragraphs 2-4, further comprising additional amino acid residues outside the RBD of SEQ ID NO: 1.

6. A polypeptide according to any one of paragraphs 2-5, wherein the coronavirus receptor binding domain (RBD) comprises at least 95% identity with residues 328-531 of SEQ ID NO:1.

7. A polypeptide according to any of paragraphs 2-6, wherein at amino acids 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or at least two mutations at 338, 358, 363 and 365 or at corresponding residues in a second coronavirus receptor binding domain are unique mutations in the receptor binding domain relative to wild type.

8. A polypeptide according to any one of paragraphs 2-7, wherein when expressed in a cell, expression of the polypeptide is increased compared to expression of the wild-type RBD polypeptide lacking the at least two mutations.

9. A polypeptide according to any one of paragraphs 2-8, wherein the polypeptide binds to a coronavirus antibody or binds to a coronavirus cognate receptor.

10. The polypeptide of paragraph 9, wherein the coronavirus antibody comprises a SARS-CoV-2 antibody.

11. The polypeptide of paragraph 9 or 10, wherein the receptor for the coronavirus corresponding to the polypeptide comprises angiotensin converting enzyme (ACE) receptor.

12. The polypeptide according to paragraph 11, wherein the ACE receptor is an ACE2 receptor.

13. A polypeptide according to any one of paragraphs 2-12, wherein the second coronavirus comprises a sequence of a coronavirus selected from the group consisting of severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome-associated coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; or HKU1.

14. The polypeptide of any of paragraphs 2-13, wherein the polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1.

15. The polypeptide of any one of paragraphs 2-14, wherein the RBD is fused to a second heterologous polypeptide.

16. The polypeptide of paragraph 15, wherein the RBD is fused to a nanoparticle, nanostructure or heterologous protein scaffold.

17. The polypeptide of any one of paragraphs 2-16, wherein the RBD polypeptide and/or the second polypeptide is an antigenic polypeptide.

18. A composition comprising the polypeptide according to any one of paragraphs 1-17 and a pharmaceutically acceptable carrier.

19. The composition according to paragraph 18, further comprising an adjuvant.

20. The composition of paragraph 18 or 19, wherein the composition has a longer shelf life than a composition comprising a wild-type RBD polypeptide lacking the at least two mutations.

21. The composition of any of paragraphs 18-20, wherein the composition is formulated as a vaccine.

22. A non-naturally occurring coronavirus spike protein subunit 1 polypeptide comprising at least two mutations, wherein the at least two mutations include at least one cavity filling mutation and at least one second mutation.

23. A coronavirus polypeptide according to paragraph 22, wherein the at least two mutations enhance the stability of the coronavirus polypeptide relative to the stability of a wild-type polypeptide lacking the at least one cavity-filling mutation and the at least second mutation.

24. A coronavirus polypeptide according to paragraph 22 or 23, wherein the at least one cavity-filling mutation comprises a mutation of a residue in the linoleic acid binding pocket of subunit 1 of the coronavirus spike protein.

25. A coronavirus polypeptide according to any of paragraphs 22-24, wherein the at least one cavity-filling mutation comprises a mutation of a residue within residues 328-531 of SEQ ID NO: 1, or a mutation at the corresponding residue of the second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using the Blast-p parameters of Protocol 1 or Protocol 2.

26. A coronavirus polypeptide according to any of paragraphs 22-25, wherein the at least one cavity-filling mutation comprises a mutation of a residue between residues 335-345, 355-375, or 378-395 of SEQ ID NO: 1, or a mutation at the corresponding residue of the second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using the Blast-p parameters of Protocol 1 or Protocol 2.

27. A coronavirus polypeptide according to any of paragraphs 22-26, wherein the at least one cavity-filling mutation comprises a mutation of a residue at amino acid 336, 338, 341, 342, 358, 361, 363, 365, 368, 374, 377, 387 or 392 of SEQ ID NO: 1, or a mutation of the corresponding residue of the second coronavirus spike protein subunit 1 as determined by sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus using the Blast-p parameters of Protocol 1 or Protocol 2.

28. A coronavirus polypeptide according to any one of paragraphs 22-27, wherein the at least one cavity filling mutation and the at least one second mutation are in SEQ ID 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365 of NO:1, or by using the Blast-p parameters of Protocol 1 or Protocol 2 to convert SEQ ID NO: NO: 1 is compared with the sequence of the second coronavirus spike protein subunit 1 and determined by comparing the sequence of the second coronavirus spike protein subunit 1 with the corresponding residues of the second coronavirus spike protein subunit 1.

29. A coronavirus polypeptide according to any one of paragraphs 22-28, wherein the at least one cavity filling mutation and the at least one second mutation are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358 8F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or selected from the corresponding residues of the second coronavirus spike protein subunit 1 as determined by sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using the Blast-p parameters of Protocol 1 or Protocol 2.

30. A coronavirus polypeptide according to any of paragraphs 22-29, wherein the coronavirus spike protein subunit 1 polypeptide comprises at least 95% identity to residues 328-531 of SEQ ID NO: 1 or the receptor binding domain sequence of the second coronavirus spike protein subunit 1 as determined by aligning SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using the Blast-p parameters of Protocol 1 or Protocol 2.

31. The coronavirus spike protein subunit 1 polypeptide of any of paragraphs 22-30, wherein at amino acids 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392 of SEQ ID NO: 1; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or at least two mutations at 338, 358, 363 and 365 or at corresponding residues in the second coronavirus receptor binding domain are unique mutations in spike protein subunit 1 relative to SEQ ID NO: 1.

32. The coronavirus polypeptide of any one of paragraphs 22-31, wherein the coronavirus polypeptide comprises at least 95% identity to SEQ ID NO: 1 or to the wild-type spike protein subunit 1 amino acid sequence of a second coronavirus.

33. The coronavirus polypeptide of any one of paragraphs 22-32, wherein when expressed in a cell, expression of the coronavirus polypeptide is increased compared to expression of a wild-type polypeptide lacking the at least one cavity-filling mutation and the at least one second mutation under the same expression conditions.

34. A coronavirus polypeptide according to any one of paragraphs 22-33, wherein the coronavirus polypeptide binds to a coronavirus antibody or binds to a cognate coronavirus receptor.

35. A coronavirus polypeptide according to paragraph 34, wherein the coronavirus antibody comprises a SARS-CoV-2 antibody.

36. The coronavirus polypeptide of paragraph 35, wherein the cognate coronavirus receptor comprises angiotensin converting enzyme (ACE) receptor.

37. The coronavirus polypeptide of paragraph 36, wherein the ACE receptor is an ACE2 receptor.

38. A coronavirus polypeptide according to any one of paragraphs 22-37, wherein the coronavirus polypeptide is an engineered mutant polypeptide of the following coronaviruses: severe acute respiratory syndrome-associated coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome-associated coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; or HKU1.

39. The coronavirus polypeptide of any one of paragraphs 22-38, wherein the coronavirus spike protein subunit 1 polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1.

40. The coronavirus polypeptide of any one of paragraphs 22-39, wherein the coronavirus polypeptide is fused to a second heterologous polypeptide.

41. The coronavirus polypeptide of any one of paragraphs 22-40, wherein the coronavirus polypeptide is fused to a nanoparticle, nanostructure or protein scaffold.

42. The coronavirus polypeptide of paragraph 40, wherein the coronavirus polypeptide or the second heterologous polypeptide is an antigenic polypeptide.

43. A composition comprising a coronavirus polypeptide according to any one of paragraphs 22-42 and a pharmaceutically acceptable carrier.

44. The composition of paragraph 43, further comprising an adjuvant.

45. A composition according to paragraph 43 or 44, wherein the shelf life of the composition is longer than a composition comprising a wild-type coronavirus polypeptide lacking the at least one cavity filling mutation and the at least second mutation when stored under the same shelf conditions.

46. The composition of any of paragraphs 43-45, wherein the composition is formulated as a vaccine.

47. A cell expressing the receptor binding domain of any one of paragraphs 1-15 or the coronavirus polypeptide of any one of paragraphs 22-42.

48. A nucleic acid sequence encoding a receptor binding domain according to any one of paragraphs 1-15 or a coronavirus polypeptide according to any one of paragraphs 22-42.

49. A method of vaccinating a subject against a coronavirus, the method comprising administering to the subject a composition according to paragraph 21 or paragraph 46.

50. A method of preparing a vaccine, the method comprising combining the composition of any of paragraphs 1-15 or 22-42 with an adjuvant and a pharmaceutically acceptable carrier.

51. A coronavirus spike protein comprising a polypeptide according to any one of paragraphs 1-25.

52. The method or composition of any of the preceding paragraphs, wherein the Blast-p parameters of Protocol 1 include:

Algorithm: blastp (protein-protein BLAST)

Expected threshold: 0.1

Word length: 6

Maximum matches within query range: 0

Matrix: BLOSUM62

Vacancy cost:

Presence: 11

Extension: 1

Filter low complexity areas?: No

Mask:

For lookup table only? : No

Lowercase letters?: No.

53. A method or composition according to any of the preceding paragraphs, wherein the Blast-p parameters of Protocol 2 include:

blastp -query query.fasta -subject sbjct.fasta -matrix BLOSUM62 -evalue 0.1 -wordlength 6 -gap opening 11 -gap extension 1 -output results.txt

(blastp-query query.fasta-subject sbjct.fasta-matrix BLOSUM62-evalue0.1-

word size 6-gapopen 11-gapextend 1-out results.txt).

54. A polypeptide comprising a coronavirus receptor binding domain (RBD), wherein the coronavirus RBD comprises a mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L relative to a coronavirus polypeptide or a variant thereof of SEQ ID NO:1.

55. The polypeptide of paragraph 54, wherein the mutation is selected from the group consisting of I358F, Y365F, Y365W, V367F and F392W.

56. The polypeptide of paragraph 54 or paragraph 55, wherein the polypeptide comprises a second mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L.

57. The polypeptide of paragraph 58, wherein the polypeptide comprises a third mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L.

58. The polypeptide according to paragraph 57, wherein the polypeptide comprises the polypeptide sequence of SEQ ID NO:4 or SEQ ID NO:5.

59. The polypeptide of any of paragraphs 54-58, wherein the polypeptide comprises a heterologous protein scaffold.

60. The polypeptide of paragraph 58, wherein the heterologous protein scaffold is at least 90%, at least 95%, or at least 98% identical to the polypeptide sequence of SEQ ID NO:3.

61. The polypeptide of paragraph 59, wherein the heterologous protein scaffold comprises the polypeptide of SEQ ID NO:3.

62. The polypeptide according to paragraph 61, wherein the polypeptide comprises the polypeptide sequence of SEQ ID NO: 6 or SEQ ID NO: 7.

63. A polypeptide complex comprising or consisting of: a first component consisting of a polypeptide according to any one of paragraphs 59-62 and a second component having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with any one of SEQ ID NOs: 13-18.

64. A vaccine composition comprising the composition according to any one of paragraphs 54-62 or the polypeptide complex according to paragraph 63.

65. The vaccine composition according to paragraph 64, further comprising a pharmaceutically acceptable carrier.

66. The vaccine composition of paragraph 64 or paragraph 65, further comprising an adjuvant.

67. A cell expressing the polypeptide of any of paragraphs 54-62.

68. A nucleic acid encoding the polypeptide of any of paragraphs 54-62.

69. A method of vaccinating a subject against a coronavirus, the method comprising administering to the subject a polypeptide according to any of paragraphs 54-62, a protein complex according to paragraph 63, or a vaccine composition according to any of paragraphs 64-68.

70. A method of preparing a vaccine, the method comprising combining the polypeptide of any of paragraphs 54-62 with an adjuvant and a pharmaceutically acceptable carrier.

71. A method of preparing a vaccine, the method comprising combining: a first component consisting of a polypeptide according to any one of paragraphs 59-62; a second component having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 13-18; a pharmaceutically acceptable carrier; and optionally an adjuvant.

72. A non-naturally occurring polypeptide comprising a first coronavirus receptor binding domain (RBD), the first coronavirus RBD comprising at least 90% identity to residues 328-531 of SEQ ID NO:1, and further comprising at least one mutation relative to the RBD of SEQ ID NO:1, wherein the at least one mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L of SEQ ID NO:1, or a second coronavirus reference sequence, wherein the corresponding sites in the second coronavirus reference sequence are obtained by converting SEQ ID NO:1 to a residue 328-531 of SEQ ID NO:1 using the Blast-p parameters of protocol 1 or protocol 2. NO:1 was determined by sequence alignment with the spike protein sequence of the second coronavirus receptor binding domain.

73. A polypeptide according to paragraph 72, wherein the polypeptide comprises two or more mutations selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I35 8F/Y365W; I358F/Y365W/L513M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M.

Example

Example 1

An ideal protein-based coronavirus vaccine must be both highly efficient and scalable to manufacture, with the latter property being largely influenced by vaccine yield and stability. Genetic vaccines using RNA or DNA immunization have also been more frequently explored against SARS-CoV-2, with their efficacy being influenced by the level of target antigen expression. The receptor binding domain (RBD) of many coronaviruses, especially SARS-CoV-2 and related sarbecoviruses (coronavirus subgenus), is considered to be a very valuable domain-based vaccine target due to the isolation of many strongly neutralizing RBD-directed antibodies. Although the RBD is amenable to production in a variety of formats, its yield and stability limitations may hinder the scalable manufacturing and distribution of RBD-based protein vaccines.

Provided herein are exemplary mutations in coronavirus receptor binding domain (RBD) polypeptides, which are designed to improve the yield and stability of immunogens containing such coronavirus RBDs. Immunogens containing receptor binding domains are produced with stabilizing mutation sets, and in addition to having improved stability in solution compared to equivalent immunogens with natural (i.e., wild-type) RBD sequences, also show highly improved expression and/or yield. As will be appreciated by those skilled in the art, the increased expression of a given protein may be due in part to the improved stability of the protein. As verified by SARS-CoV-2 directed antibodies and ACE2 receptors, the designed immunogens are complete in antigenicity. In general, these mutation sets allow for improved ability to scalably manufacture vaccines for a variety of coronaviruses, which can also contribute to the performance of genetic vaccines for RBD.

Base sequence and mutation numbers: The SARS-CoV-2 sequence (SEQ ID NO: 1) is used throughout this specification as the basis for mutation numbering in other coronaviruses. The receptor binding domain of the SARS-CoV-2 sequence is shown below in bold underlined text (SEQ ID NO: 2): The following sequence (or its receptor binding domain; SEQ ID NO: 2) can be aligned with at least a second coronavirus sequence.

An exemplary list of mutations that can enhance both the yield and stability of coronavirus proteins is provided in the following list (all amino acid residue numbers are based on the above-mentioned "base sequence" (SEQ ID NO: 1)):

1.F338L/Y365W,

2.Y365W/L513M,

3.Y365W/F392W,

4.F338M/A363L/Y365F/F377V,

5.Y365F/F392W,

6.Y365F/V395I,

7.Y365F/F392W/V395I,

8.Y365W/L513I/F515L,

9.F338L/A363L/Y365M,

10.F338L/I358F/Y365W,

11.I358F/Y365W/L513M,

12.I358F/Y365W/F392W,

13.F338M/I358F/A363L/Y365F/F377V,

14.I358F/Y365F/F392W,

15.I358F/Y365F/V395I,

16.I358F/Y365F/F392W/V395I,

17.I358F/Y365W/L513I/F515L,

18.F338L/I358F/A363L/Y365M,

19.I358F/Y365W, and

20.I358F/F392W.

Materials and methods

Expression and protein purification: The gene encoding the mutant receptor binding domain (native receptor binding domain residues 328-531 of SEQ ID NO: 1, or the equivalent sequence from a variant (e.g., the B.1.351 variant)) genetically fused to the "A" component of the I53-50 trimer with a 16-residue linker was cloned in the pCMV/R vector using the XbaI and AvrII restriction sites.

The sequence of the "A" component of the I53-50 trimer is:

MKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVP

DADTVIKALSVLKEKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLD

EEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQ

FVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVK

GTPDEVREKAKAFVEKIRGCTE(SEQ ID NO:3)

All sequences are preceded by a "MGILPSPGMPALLSLVSLLSVLLMGCVAETGT" secretion signal and tagged at the C-terminus with a "GGSHHHHHHHH" sequence to allow purification. In some embodiments, the N-terminal methionine of the I53-50 trimer component is replaced with the helical sequence "EKAAKAEEAAR" to improve antigen accessibility. All cysteines in the I53-50 trimer component are mutated to alanine. The human ACE2 extracellular domain is genetically fused to sequences encoding the thrombin cleavage site and the human Fc fragment at the C-terminus. hACE2-Fc was synthesized and cloned by GenScript with the BM40 signal peptide. Genes encoding the CR3022 heavy and light chains were ordered from GenScript and cloned into pCMV/R.

All proteins were produced in Expi293F cells grown in suspension using Expi293F expression medium (Life Technologies) at 33°C, 70% humidity, 8% _CO2 at 150 rpm. Cell cultures were transfected with PEI-MAX (Polyscience) and grown to a density of 3 million cells/mL and cultured for 3 days. The supernatant was clarified by centrifugation at 4000 rcf, addition of PDADMAC to a final concentration of 0.0375% (Sigma Aldrich), and a second spin at 4000 rcf.

The protein containing the His tag was purified from the clarified supernatant via a batch binding method, wherein each clarified supernatant was supplemented with 1M Tris-HCl pH 8.0 to a final concentration of 45mM and 5M NaCl to a final concentration of about 310mM. Talon cobalt affinity resin (Takara) was added to the treated supernatant and incubated for 15 minutes under gentle shaking. The resin was collected using vacuum filtration with a 0.2 μm filter and transferred to a gravity column. The resin was washed with 20mM Tris pH 8.0, 300mM NaCl, and the protein was eluted with 3 column volumes of 20mM Tris pH 8.0, 300mM NaCl, 300mM imidazole. The clarified supernatant of cells expressing monoclonal antibodies and human ACE2-Fc was purified using MabSelect PrismA 2.6×5cm columns (Cytiva) on AKTA Avant150 FPLC (Cytiva). The bound antibody was washed with five column volumes of 20 mM NaPO4, 150 mM NaCl pH 7.2, then five column volumes of 20 mM NaPO4, 1 M NaCl pH 7.4, and eluted with three column volumes of 100 mM glycine, pH 3.0. The eluate was neutralized with 2 M Trizma base to a final concentration of 50 mM.

Biolayer interferometry: To measure secretion levels of proteins in the supernatant, all supernatants from protein expression were diluted 1:10 into KB1 (25mM Tris pH 8.0, 150mM NaCl, 0.5% bovine serum albumin, and 0.01% TWEEN-20). Purified ACE2-Fc or CR3022 antibodies were diluted into KB1 at 0.02mg/mL and immobilized on an AHC tip (Pall FortéBio/Sartorius) for 300 seconds using an 8-channel Octet system (Pall FortéBio/Sartorius). After collecting a 60-second baseline in KB1, the sensor was exposed to each of the diluted ACE2-Fc or CR3022 solutions for 300 seconds, followed by a 300-second dissociation step in KB1. For affinity measurements, all proteins and antibodies were diluted at 200 μL/well into black 96-well Greiner Bio-one microplates in phosphate-buffered saline containing 0.5% bovine serum albumin and 0.01% TWEEN-20 (KB2), with separate buffers used for baseline and dissociation steps. 10 μg/mL of CV30 or CR3022 IgG was loaded onto prehydrated protein A biosensors (Pall FortéBio/Sartorius) for 150 s, followed by a 60 s baseline. The biosensor was then transferred to a step of association with one of five serial two-fold dilutions of wild-type or stabilized monomeric RBD for 120 s, wherein the RBD concentrations were 125 μM, 62.5 μM, 31.3 μM, 15.6 μM and 7.8 μM for CV30 and 31.3 μM, 15.6 μM, 7.8 μM, 3.9 μM and 2.0 μM for CR3022. After association, the biosensor was transferred to the buffer for 300 s of dissociation. The data from the association and dissociation steps were subtracted from the baseline, and the kinetic measurements were calculated globally across all five serial dilutions of RBD using a 1:1 binding model (FortéBio analysis software, version 12.0). For the hierarchical antigenicity measurement of nanoparticle immunogens, the binding of hACE2-Fc (dimeric receptor) and CR3022 IgG to monomeric RBD and RBD-I53-50 nanoparticles was analyzed at ambient temperature with 1000 rpm oscillation. Protein samples were diluted to 100 nM in kinetic buffer (Pall FortéBio/Sartorius). Buffer, antibody, receptor and immunogen were then applied to black 96-well Greiner Bio-one microplates at 200 μL/well. Protein A biosensor was first hydrated in kinetic buffer for 10 min, then immersed in hACE2-Fc or CR3022 diluted to 10 μg/mL in kinetic buffer in the fixed step. After 500 s, the tip was transferred to Kinetics buffer for 90 s to reach baseline. The association step was performed by immersing the loaded biosensor in the immunogen for 300 s, and the subsequent dissociation step was performed by immersing the biosensor back in the kinetic buffer for an additional 300 s. Before drawing using FortéBio analysis software (version 12.0), the data was subtracted from the baseline.

Thermal melting: RBD-based samples were prepared in a buffer containing 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-arginine, 5% glycerol, while HexaPro-foldon-based samples were prepared in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.25% w/v L-histidine, 5% glycerol. The non-equilibrium melting temperature was determined using UNcle ^™ (UNchained Labs) based on the centroid mean of the intrinsic tryptophan fluorescence emission spectra collected from 20-95°C using a 1°C/min thermal ramp. The melting temperature was defined as the maximum point of the first derivative of the melting curve, where the first derivative was calculated using GraphPad Prism software after smoothing four adjacent points using a second-order polynomial setting.

SYPRO Orange fluorescence: 5000× SYPRO Orange protein gel stain (Thermo Fisher) was diluted into 25 mM Tris pH 8.0, 150 mM NaCl, 5% glycerol and further added to monomeric RBD prepared in the same buffer, where the final concentration of RBD was 1.0 mg/mL and the final concentration of SYPRO Orange was 20×. The samples were loaded into UNcle nano DSF (UNChained Laboratories) and the fluorescence emission spectra of all samples were collected after adding SYPRO Orange to the samples for 5 min.

In vitro nanoparticle assembly: The total protein concentration of purified individual nanoparticle components was determined by measuring the absorbance at 280 nm using a UV/vis spectrophotometer (Agilent Cary 8454) and calculating the extinction coefficient. The assembly steps were performed at room temperature, where the following order was added: wild-type or stabilized RBD-I53-50A trimeric fusion protein, followed by appropriate buffer as needed to achieve the desired final concentration, and finally the I53-50B.4PT1 pentamer component (in 50mM Tris pH 8, 500mM NaCl, 0.75% w/v CHAPS, with a molar ratio of RBD-I53-50A:I53-50B.4PT1 of 1.1:1. The appropriate buffer contained 50mM Tris pH 7.4, 185mM NaCl, 100mM L-arginine, 0.75% CHAPS, 4.5% glycerol, or 50mM Tris pH 8, 150mM NaCl, 100mM L-arginine, 5% glycerol). All RBD-I53-50 in vitro assemblies were incubated at least 30 min at 2-8 ° C, and then subjected to subsequent purification by SEC to remove residual unassembled components. Nanoparticle production was carried out using Superose 6 Increase 10/300GL posts. The assembled particles were eluted with approximately 11 mL on Superose 6 posts. The assembled nanoparticles were sterile filtered (0.22 μm) immediately before post application and after SEC fractions were merged.

Negative stain electron microscopy: Wild-type RBD-I53-50 nanoparticles and Rpk-I53-50 nanoparticles were first diluted to 75 μg/mL in 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-arginine, 5% v/v glycerol, and 3 μL of the sample was then applied to a freshly glow-discharged 300 mesh copper grid. The sample was incubated on the grid for 1 minute, and then the grid was immersed in a 50 μL drop of water and the excess liquid was blotted off with filter paper (Whatman). The grid was then immersed in 3 μL of 0.75% w/v uranyl formate stain. The stain was blotted off with filter paper, and the grid was then immersed in another 6 μL of stain and incubated for approximately 90 seconds. Finally, the stain was blotted and the grid was dried for 1 minute before storage or imaging. The prepared grid was imaged at 57,000× using a Gatan camera in a Talos L120C model transmission electron microscope.

Dynamic light scattering: Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter (Dh) and polydispersity (%Pd) of RBD-I53-50 nanoparticle samples at UNcle (UNchained Laboratories). The samples were applied to 8.8 μL quartz capillary cartridges (UNi, UNchained Laboratories) and 10 acquisition measurements were performed with 5 s each using laser auto-attenuation. The viscosity increase due to 5% v/v glycerol in the RBD nanoparticle buffer was accounted for by the UNcle Client software in the Dh measurement.

Hydrogen/deuterium exchange mass spectrometry: 3 mg of RBD-I53-50A, Rpk4-I53-50A and Rpk9-I53-50A trimers were subjected to H/D exchange (HDX) at 22°C in deuterated buffer (pH*7.5, 85% D ₂ O, Cambridge Isotope Laboratories, Inc.) for 3 seconds, 15 seconds, 60 seconds, 1800 seconds and 72000 seconds, respectively. The exchanged samples were then mixed 1:1 with ice-cold quenching buffer (200 mM tris(2-chloroethyl)phosphate (TCEP), 8 M urea, 0.2% formic acid (FA)) to a final pH of 2.5 and immediately flash frozen in liquid nitrogen. The samples were analyzed by LC-MS on a Synapt G2-Si mass spectrometer using a loading system that maintained all chromatographic columns, loops, valves and lines at 0°C. Frozen samples were thawed on ice and loaded onto an immobilized pepsin column (2.1×50 mm) with 0.1% trifluoroacetic acid (TFA) and 2% acetonitrile at a flow rate of 200 mL/min. Peptides were trapped on a Waters CSH C18 trap column (2.1×5 mm) and then resolved on a Waters CSH C18 1×100 mm 1.7 μm column with a linear gradient from 3% to 40% B in 18 min (A: 98% water, 2% acetonitrile, 0.1% FA, 0.025% TFA; B: 100% acetonitrile, 0.1% FA, flow rate 40 mL/min). A series of washes were performed between sample runs to minimize carryover. All water and organic solvents used were MS grade (Optima ^™ , Fisher) unless otherwise specified. A full deuteration control was performed for each sample series by collecting LC eluate from a pepsin-digested undeuterated sample, speedvac drying, incubation in deuterated buffer at 85°C for 1 hour, and quenching identically to all other HDX samples. Internal exchange standards (Pro-Pro-Pro-Ile [PPPI] and Pro-Pro-Pro-Phe [PPPF]) were added to each sample to ensure consistent labeling conditions for all samples. Peptides were manually verified using DriftScope ^TM (Waters) and identified using orthogonal retention time and drift time coordinates. Deuterium uptake analysis was performed using HX-Express v2. Peaks were identified from peptide spectra based on peptide m/z values and a binomial fit was applied. Deuterium uptake levels were normalized relative to the full deuteration control.

Mouse immunization: Female BALB/c (stock number: 000651) mice were purchased from The Jackson Laboratory, Bar Harbor, Maine at 4 weeks of age and maintained in the Comparative Medicine Facility accredited by the American Association for Laboratory Animal Care International Accreditation (AAALAC) at the University of Washington in Seattle, Washington. At 6 weeks of age, 6 mice in each dosing group were vaccinated with a primary vaccination, and mice received a second vaccination booster three weeks later. Before vaccination, the immunogen suspension was gently mixed with AddaVax adjuvant (Invivogen, San Diego, CA) at 1:1 volume/volume to achieve a final concentration of 0.009 mg/mL or 0.05 mg/mL antigen. Under isoflurane anesthesia, a 27-gauge needle (BD, San Diego, CA) was used to inject intramuscularly into the gastrocnemius muscle of each hind leg of the mouse, with 50 μL (100 μL in total) of the immunogen injected at each injection site. In order to obtain serum, all mice were bled two weeks after the primary and booster immunizations. Blood was collected via submental venous puncture and allowed to stand for 30 min at room temperature in a 1.5 mL plastic Eppendorf tube for coagulation. Serum was separated from hematocrit by centrifugation at 2,000 g for 10 min. Complement factors and pathogens in the separated serum were heat inactivated by incubation at 56° C. for 60 min. Serum was stored at 4° C. or −80° C. before use. All experiments were performed at the University of Washington, Seattle, WA, according to approved Institutional Animal Care and Use Committee protocols.

ELISA: Enzyme-linked immunosorbent assay (ELISA) was used to determine the binding of mouse sera to the delivered antigen. Briefly, Maxisorp (Nunc) ELISA plates were coated overnight at 4°C with 0.08 μg/mL of the protein of interest per well in 0.1 M sodium bicarbonate buffer (pH 9.4). The plates were then blocked for 1 hour at room temperature with a 4% (w/v) solution of dry milk powder (BioRad) in TBS containing 0.05% (v/v) Tween 20 (TBST). Serial dilutions of sera were added to the plates, and after washing, antibody binding was revealed using a catalase-conjugated horse anti-mouse IgG antibody. The plates were then washed thoroughly in TBST, a colorimetric substrate (TMB, Thermo Fisher) was added, and the absorbance at 450 nm was read. The area under the curve (AUC) calculation was generated by adding the trapezoidal area generated between adjacent pairs of absorbance measurements and the baseline.

Pseudovirus neutralization assay: Spike pseudotyped lentivirus neutralization assays used spike HDM_Spikeδ21_D614G available from Addgene (#158762) or BEI (NR-53765), where the complete sequence is available on the World Wide Web at www.addgene.org/158762 Error! Hyperlink reference is not valid. ). Briefly, 293T-ACE2 cells (BEI NR-52511) were seeded at 1.25×10 ⁴ cells/well in 50uL D10 growth medium (containing 10% heat-inactivated FBS, 2mM L-glutamine, 100U/mL penicillin, and 100μg/mL streptomycin) in a black-walled, clear-bottomed 96-well plate (Greiner 655930) coated with poly-L-lysine. The next day, mouse serum samples were heat-inactivated at 56°C for 30min and then serially diluted in D10 growth medium. The spike pseudotyped lentivirus was diluted 1:50 to produce approximately 200,000 RLU per well and incubated with serum diluents at 37°C for 1 hour. 100 μL of the virus-serum mixture was then added to the cells, and luciferase activity was measured approximately 52 hours later using the Bright-Glo Luciferase Assay System (Promega, E2610). Each batch of neutralization assays included human serum negative control samples collected in 2017-2018 and known neutralizing antibodies to ensure consistency between batches. The infection rate score for each well was calculated compared to the two "serum-free" control wells in the same row of the plate. The "neutcurve" package (available on the World Wide Web at jbloomlab.github.io/neutcurve, version 0.5.2) was used to calculate the 50% inhibitory concentration ( _IC50 ) and 50% neutralizing titer ( _NT50 ) for each serum sample by fitting a Hill curve with the bottom fixed to 0 and the top fixed to 1, which is simply 1/ _IC50 .

Quantification and statistical analysis: Multiple group comparisons were performed using the nonparametric Kruskal-Wallis test with Dunn's post hoc analysis in GraphPad Prism 8. Differences were considered significant when the P value was less than 0.05.

Example 2

Materials and methods

Cell lines

Expi293F cells are derived from the HEK293F cell line, a female human embryonic kidney cell line (Life Technologies) that has been transformed and adapted to growth in suspension. Expi293F cells were grown in Expi293 expression medium (Life Technologies) and cultured at 36.5°C and 8% CO ₂ and 150rpm shaking. VeroE6 is a female renal epithelial cell from an African green monkey. The HEK-ACE2 adherent cell line was obtained through BEI Resources, NIAID, and NIH: human embryonic kidney cells (HEK293T) expressing human angiotensin-converting enzyme 2, HEK293T-hACE2 cell line, NR-52511. All adherent cells were cultured in flasks with DMEM+10% FBS (Hyclone)+1% penicillin-streptomycin at 37°C with 8% CO _2. Cell lines other than Expi293F were not tested for mycoplasma contamination and were not verified.

Mouse

Female BALB/c mice (four weeks old) were obtained from the Jackson Laboratory, Bar Harbor, Maine. Animal procedures were performed with the approval of the Institutional Animal Care and Use Committee at the University of Washington, Seattle, WA.

Design of stabilizing mutations

All calculations in Rosetta were performed using version v2020.22-dev61287. All design trajectories evaluated the RBD in the closed symmetric trimer conformation observed in the cryo-EM structure of the spike (PDB 6VXX) and in the context of the crystal structure of the RBD (PDB 6YZ5). The three-fold symmetry axis of the PDB entry 6VXX was aligned to [0,0,1] and the individual protomers were saved in .pdb format. The RBD monomer from PDB 6YZ5 was structurally superimposed with the protomer from PDB 6VXX and saved similarly. Design protocols were written using RosettaScripts (58,59) that take as input the aligned protomers and a custom resfile, where the resfile specifies the side chain properties and conformations sampled during design. The protocol applies two rounds of design to the symmetric model based on the input resfile, with side chain minimization applied after each design step. The protocol allows backbone minimization to be performed simultaneously with side chain minimization, and trajectories are executed with or without backbone minimization allowed. Allows both the design step and the minimization step to repack or minimize all mutable or packable residues listed in the resfile Residues within. Residue positions were manually picked based on the spike and RBD structures to include positions 358, 365, 392 and surrounding residues, and possible residue properties were specified for each position in the resfiles using the 'PIKAA' option. The resfile input was diversified to include various combinations of I358F, Y365F, Y365W and/or F392W, while also restricting or allowing mutations to surrounding residues. Further resfile inputs were set similarly, but positions 358, 365 and 392 were not restricted to specific properties. The design models and scores were manually checked to identify interactions that appeared structurally favorable. If the mutation buried a polar group that was naturally exposed to the solvent or involved in hydrogen bonding, the mutation was discarded. In order to prevent unexpected changes in antigenicity, mutations to surface exposed residues were not frequently considered. Favorable sets of mutations were iteratively retested from the optimized resfiles and manually refined to complete different design sets.

Plasmid construction

The wild-type and stabilized sequences of SARS-CoV-2 RBD were genetically fused to the N-terminus of the trimeric I53-50A nanoparticle component using a linker with 16 glycine and serine residues. Each I53-50A fusion protein also carries a C-terminal eight-histidine tag, and the monomer sequence contains both Avi and eight-histidine tags. All sequences were cloned into pCMV/R using Xba1 and AvrII restriction sites and Gibson assembly. All RBD-bearing components contain an N-terminal μ-phosphatase signal peptide. hACE2-Fc was synthesized and cloned by GenScript with a BM40 signal peptide. The HexaPro-foldon construct for immune studies was produced as described (Hsieh et al. Science 369: 1501-05 (2020)) and placed in pCMV/R with an eight-histidine tag. The HexaPro-foldon construct for expression and stability comparison with and without Rpk9 mutations contained the BM40 signal peptide and was placed in pCMV/R. The plasmid was transformed into the NEB 5ɑ strain of Escherichia coli (New England Biolabs) and then DNA was extracted from the bacterial culture (NucleoBond Xtra Midi Kit) to obtain the plasmid for transient transfection into Expi293F cells. The amino acid sequences of all novel proteins used in this study are provided as SEQ ID NO: 21 to SEQ ID NO: 47.

>RBD-I53-50A trimer (16-GS linker, using wild-type RBD from Wuhan-Hu-1)

SEQ ID NO:21

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRF

ASVYAWNRKRISNCVADY

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG

KIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk1-I53-50A trimer; SEQ ID NO: 22

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk2-I53-50A trimer; SEQ ID NO: 23

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPLGEVFNATRFASVYAWNRKRISNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk3-I53-50A trimer; SEQ ID NO: 24

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVMSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk4-I53-50A; SEQ ID NO:25 trimer

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISN

CVADYSVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTG

CVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk5-I53-50A trimer; SEQ ID NO: 26

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk6-I53-50A trimer; SEQ ID NO: 27

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPMGEVFNATRFASVYAWNRKRISNCVLDF

SVLYNSASFSTVKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk7-I53-50A trimer; SEQ ID NO: 28

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk8-I53-50A trimer; SEQ ID NO: 29

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCFTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk9-I53-50A trimer; SEQ ID NO: 30

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk10-I53-50A trimer; SEQ ID NO: 31

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVISLELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk11-I53-50A trimer; SEQ ID NO: 32

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPLGEVFNATRFASVYAWNRKRISNCVLDM

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk12-I53-50A trimer; SEQ ID NO: 33

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRFSNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk13-I53-50A trimer; SEQ ID NO: 34

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPLGEVFNATRFASVYAWNRKRFSNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk14-I53-50A trimer; SEQ ID NO: 35

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRFSNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVMSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk15-I53-50A trimer; SEQ ID NO: 36

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRFSNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCFTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk16-I53-50A trimer; SEQ ID NO: 37

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRFSNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

>Rpk17-I53-50A trimer; SEQ ID NO: 38

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRFSNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTV

TSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGP

QFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGG

SHHHHHHHH

>RBD monomer (with Avi and hexahistidine tags); SEQ ID NO: 39

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRF

ASVYAWNRKRISNCVADY

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG

KIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG

FNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGLNDIFEAQKIEWHEHHHHHHHH>Rpk4 monomer (with Avi and hexa-histidine tags); SEQ ID NO: 40

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRF

ASVYAWNRKRISNCVADY

SVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQT

GKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG

FNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGLNDIFEAQKIEWHEHHHHHHHH>Rpk9 monomer (with Avi and hexahistidine tags); SEQ ID NO: 41

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRF

ASVYAWNRKRISNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTG

KIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG

FNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGLNDIFEAQKIEWHEHHHHHHHH

>I53-50B.4PT1 pentamer; SEQ ID NO:42

MNQHSHKDHETVRIAVVRARWHAEIVDACVSAFEAAMRDIGGDRFAVDVFDVPGAYEIPLHARTLAETGR

YGAVLGTAFVVNGGIYRHEFVASAVINGMMNVQLNTGVPVLSAVLTPHNYDKSKAHTLLFLALFAVKGME AARACVEILAAREKIAAGSLEHHHHHH

>2OBX pentamer; SEQ ID NO:43

MNQHSHKDYETVRIAVVRARWHADIVDQCVSAFEAEMADIGGDRFAVDVFDVPGAYEIPLHARTLAETGR

YGAVLGTAFVVNGGIYRHEFVASAVIDGMMNVQLSTGVPVLSAVLTPHNYHDSAEHHRFFFEHFTVKGKE AARACVEILAAREKIAAGSLEHHHHHH

>Hexapro-fold, for immunization (Wuhan-Hu-1); SEQ ID NO: 44

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHV

SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPF

LGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPI

NLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYN

ENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASV

YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD

YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYF

PLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFL

PFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLT

PTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSP

GSASSVASQSIIAYTMSLG

AENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLL

LQYGSFCTQLNRALTGI

AVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSPIEDLL

FNKVTLADAGFIKQYGDC

LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAG

PALQIPFPMQMAYRFNGIG

VTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALN

TLVKQLSSNFGAISSVLNDI

LSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSE

CVLGQSKRVDFCGKGYHLM

SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGT

HWFVTQRNFYEPQIITTDNT

FVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISG

INASVVNIQKEIDRLNEVA

KNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHHHSAW SHPQFEKGGGSGGGGSGGSAWSHPQFEK>Hexapro-foldon, used for expression and stability comparison with Rpk9-Hexapro-foldon (Wuhan-Hu-1); SEQ ID NO: 45

MARAWIFFLLCLAGRALAQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS

SVLHSTQDLFLPFFSNVTWF

HAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS

LLIVNNATNVVIKVCEFQF

CNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG

NFKNLREFVFKNIDGYFKIYS

KHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGW

TAGAAAYYVGYLQPRTF

LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIV

RFPNITNLCPFGEVFNAT

RFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNV

YADSFVIRGDEVRQIAPGQT

GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPF

ERDISTEIYQAGSTPCNGVEG

FNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVK

NKCVNFNFNGLTGTGVLTES

NKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQ

VAVLYQDVNCTEVPVAIH

ADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQT

QTNSPGSASSVASQSIIAY

TMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDST

ECSNLLLQYGSFCTQLNR

ALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSPI

EDLLFNKVTLADAGFIK

QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGW

TFGAGPALQIPFPMQMAYR

FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNA

QALNTLVKQLSSNFGAISS

VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAAT

KMSECVLGQSKRVDFCGK

GYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVF

VSNGTHWFVTQRNFYEPQII

TTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVD

LGDISGINASVVNIQKEIDR

LNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLST

FLGRSLEVLFQGPGHHHHHH

HH

>Rpk9-Hexapro-foldon (Wuhan-Hu-1); SEQ ID NO: 46

MARAWIFFLLCLAGRALAQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRS

SVLHSTQDLFLPFFSNVTWF

HAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQS

LLIVNNATNVVIKVCEFQF

CNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQG

NFKNLREFVFKNIDGYFKIYS

KHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGW

TAGAAAYYVGYLQPRTF

LLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNAT

RFASVYAWNRKRISNCVADFSVLYNSASFSTFKCYGVSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQT

GKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG

FNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTES

NKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIH

ADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPGSASSVASQSIIAY

TMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNR

ALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGFIK

QYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGPALQIPFPMQMAYR

FNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTPSALGKLQDVVNQNAQALNTLVKQLSSNFGAISS

VLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGK

GYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQII

TTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDR

LNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHHH

HH

>hACE2-FC; SEQ ID NO:47

MARAWIFFLLCLAGRALASTIEEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWS

AFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQE

CLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE

MARANHYEDYGDYWRGDYEVN

GVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGC

LPAHLLGDMWGRFWTNLYS

LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWE

NSMLTDPGNVQKAVCHPTAWD

LGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEG

FHEAVGEIMSLSAATPKHLKS

IGLLSPDFQEDNETEINFLLKQALTIVGTLPPFTYMLEKWRWMVFKGEIPKD

QWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTL

YQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRL

GKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWST

DWSPYADPLVPRGSGGGGDPEP

KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

EDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

KAKGQPREPQVYTLPPSRDE

LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

KLTVDKSRWQQG

Transient transfection

SARS-CoV-2S and ACE2-Fc proteins were produced in Expi293F cells grown in suspension using Expi293F expression medium (Life Technologies) at 33 ° C, 70% humidity, 8% CO ₂ at 150 rpm. Using PEI-MAX (Polyscience) transfection culture, cells grew to a density of 3 million cells/mL and cultured for 3 days. The supernatant was clarified by centrifugation (5 min at 4000 rcf), PDADMAC solution was added to a final concentration of 0.0375% (Sigma Aldrich, No. 409014), and a second separation step (5 min at 4000 rcf) was performed.

The genes encoding CV30 and CR3022 heavy and light chains were ordered from GenScript and cloned into pCMV/R. Antibodies were expressed by transient co-transfection of both heavy and light chain plasmids in Expi293F cells using PEI MAX (Polyscience) transfection reagent. Cell supernatants were harvested and clarified as described above after 3 or 6 days.

Glycoprotein purification

The protein containing the His tag was purified from the clarified supernatant via a batch binding method, wherein each clarified supernatant was supplemented with 1M Tris-HCl pH 8.0 to a final concentration of 45mM and 5M NaCl to a final concentration of about 313mM. Talon cobalt affinity resin (Takara) was added to the treated supernatant and incubated for 15min under gentle shaking. The resin was collected using vacuum filtration with a 0.45 μm filter and transferred to a gravity column. The resin was washed with 20mMTris pH 8.0, 300mM NaCl for 10 column volumes, and the bound protein was eluted with 20mM Tris pH 8.0, 300mM NaCl, 300mM imidazole for 3 column volumes. The batch binding process was then repeated for the same supernatant sample, and the first and second eluents were merged. Purity was assessed using SDS-PAGE. To quantify the yield of RBD-based constructs, IMAC eluates extracted from comparable cell culture conditions and volumes were supplemented with 100 mM L-arginine and 5% glycerol and concentrated to 1.5 mL. The concentrated sample was then loaded into a 1 mL loop and applied to a Superdex 75 Increase 10/300 GL column (for monomeric RBD) or a Superdex 200 Increase 10/300 GL column (for fusions of RBD and I53-50A trimers), which was pre-equilibrated with 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-arginine, and 5% glycerol. To quantify the yield of HexaPro-foldon constructs with and without Rpk9 mutations, IMAC eluates extracted from comparable cell culture conditions and volumes were supplemented with 5% glycerol and concentrated to 1.5 mL, which were then loaded into a 1 mL loop and applied to a Superose6Increase 10/300GL column pre-equilibrated with 50 mM Tris pH 8.0, 150 mM NaCl, 0.25% w/v L-histidine, 5% glycerol. HexaPro-foldon for immunological studies was purified by IMAC and dialyzed three times for four hours at room temperature against 50 mM Tris pH 8.0, 150 mM NaCl, 0.25% w/v L-histidine, 5% glycerol.

Thermal denaturation (Nano DSF)

RBD-based samples were prepared in a buffer containing 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-arginine, 5% glycerol, while HexaPro-foldon-based samples were prepared in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.25% w/v L-histidine, 5% glycerol. The non-equilibrium melting temperature was determined using UNcle ^™ (UNchainedLabs) based on the centroid mean of the intrinsic tryptophan fluorescence emission spectra collected from 20-95°C using a 1°C/min thermal ramp. The melting temperature was defined as the maximum point of the first derivative of the melting curve, where the first derivative was calculated using GraphPad Prism software after smoothing four adjacent points using a second-order polynomial setting.

SYPRO Orange Fluorescent

5000× SYPRO ^TM Orange protein gel stain (Thermo Fisher) was mixed and diluted into 25 mM Tris pH 8.0, 150 mM NaCl, 5% glycerol and further added to monomeric RBD prepared in the same buffer, where the final concentration of RBD was 1.0 mg/mL and the final concentration of SYPRO ^TM Orange was 20×. The samples were loaded into UNcle ^TM NanoDSF (UNChained Laboratories) and the fluorescence emission spectra of all samples were collected 5 min after adding SYPRO ^TM Orange to the samples.

Microprotein expression and purification of I53-50B.4PT1

Pentameric nanoparticle components complementary to RBD-I53-50A, I53-50B.4PT1 (SEQ ID NO: 17) were produced as described in U.S. Patent Publication No. 2016/0122392, the contents of which are incorporated herein by reference in their entirety, and the same protocol was used for the purification of the 2OBX non-assembled control pentamer.

In vitro nanoparticle assembly

The total protein concentration of purified individual nanoparticle fractions was determined by measuring the absorbance at 280 nm using a UV/vis spectrophotometer (Agilent Cary 8454) and calculating the extinction coefficient. The assembly steps were performed at room temperature, where the following order was added: wild-type or stabilized RBD-I53-50A trimeric fusion protein, followed by appropriate buffer as needed to achieve the desired final concentration, and finally the I53-50B.4PT1 pentamer component (SEQ ID NO:17) (in 50mMTris pH 8, 500mM NaCl, 0.75% w/vCHAPS, with a molar ratio of RBD-I53-50A:I53-50B.4PT1 of 1.1:1. The appropriate buffer contained 50mM Tris pH 7.4, 185mM NaCl, 100mM L-arginine, 0.75% CHAPS, 4.5% glycerol (for solution stability studies), or 50mM Tris pH 8, 150mM NaCl, 100mM L-arginine, 5% glycerol). All RBD-I53-50 in vitro assemblies were incubated at least 30 min at 2-8 ° C and then subjected to subsequent purification by SEC to remove residual unassembled components. Nanoparticle production was performed using Superose ^™ 6 Increase 10/300GL columns. The assembled particles were eluted with approximately 11 mL on Superose ^™ 6 columns. The assembled nanoparticles were sterile filtered (0.22 μm) immediately before column application and after SEC fractions were combined.

Biolayer interferometry for kinetic analysis of monomeric RBD

Kinetic measurements were performed using the Octet ^™ Red 96 system (Pall ForteBio/ ) was performed at 25°C with shaking at 1000 rpm. All proteins and antibodies were diluted into black 96-well Greiner Bio-one microplates at 200 μL/well in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and 0.01% TWEEN-20, with separate buffers used for baseline and dissociation steps. 10 μg/mL of CV30 or CR3022 IgG was loaded onto prehydrated protein A biosensors (Pall FortéBio/ ) for 150 s, followed by a 60 s baseline. The biosensor was then transferred to an association step with one of five serial two-fold dilutions of wild-type or stabilized monomeric RBD for 120 s, where the RBD concentrations were 125 μM, 62.5 μM, 31.3 μM, 15.6 μM, and 7.8 μM for CV30 and 31.3 μM, 15.6 μM, 7.8 μM, 3.9 μM, and 2.0 μM for CR3022. After association, the biosensor was transferred to buffer for 300 s of dissociation. The data from the association and dissociation steps were baseline subtracted and analyzed using a 1:1 binding model ( Analysis software, version 12.0) calculated kinetic measurements globally across all five serial dilutions of RBD.

Biolayer interferometry for hierarchical antigenicity of RBD nanoparticles

Binding of hACE2-Fc (dimeric receptor) and CR3022 IgG to monomeric RBD and RBD-I53-50 nanoparticles was analyzed using the Octet ^™ Red 96 system at ambient temperature with shaking at 1000 rpm for real-time stability studies. ) was diluted to 100 nM in . The buffer, antibody, receptor, and immunogen were then applied to a black 96-well Greiner Bio-one microplate at 200 μL/well. The Protein A biosensor was first hydrated in kinetic buffer for 10 min and then immersed in hACE2-Fc or CR3022 diluted to 10 μg/mL in kinetic buffer during the fixation step. After 500 s, the tip was transferred to Kinetics buffer for 90 s to reach baseline. The association step was performed by immersing the loaded biosensor in the immunogen for 300 s, and the subsequent dissociation step was performed by immersing the biosensor back in the kinetic buffer for 300 s. In use The data were baseline subtracted before plotting using the ANALYSIS software (version 12.0).

Negative stain electron microscopy

Wild-type RBD-I53-50 nanoparticles and Rpk-I53-50 nanoparticles were first diluted to 75 μg/mL in 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-arginine, 5% v/v glycerol, and 3 μL of the sample was then applied to a freshly glow-discharged 300-mesh copper grid. The sample was incubated on the grid for 1 minute, and then the grid was immersed in a 50 μL drop of water and the excess liquid was blotted off with filter paper. The grid was then immersed in 3 μL of 0.75% w/v uranyl formate stain. The stain was blotted off with filter paper, and the grid was then immersed in another 6 μL of stain and incubated for approximately 90 seconds. Finally, the stain was blotted off and the grid was dried for 1 minute before storage or imaging. The prepared grid was imaged at 57,000× in a Talos ^TM L120C model transmission electron microscope using a Gatan ^TM camera.

Dynamic Light Scattering

Dynamic light scattering (DLS) was used to measure the hydrodynamic diameter (Dh) and polydispersity (%Pd) of RBD-I53-50 nanoparticle samples on UNcle ^™ (UNchained Laboratories). The samples were applied to 8.8 μL quartz capillary cartridges (UNi ^™ , UNchained Laboratories) and 10 acquisition measurements were performed using laser auto-attenuation, each for 5 s. The viscosity increase due to 5% v/v glycerol in the RBD nanoparticle buffer was accounted for by the UNcle ^™ Client software in the Dh measurement.

Endotoxin measurement

Endotoxin levels in protein samples were measured using the EndoSafe ^™ Nexgen-MCS system (Charles River). Samples were diluted 1:50 or 1:100 in endotoxin-free LAL reagent water and applied to the wells of the EndoSafe ^™ LAL reagent cartridge. Endotoxin content was analyzed using Charles River EndoScan ^™ -V software, which automatically back-calculates the dilution factor. Endotoxin values are reported as EU/mL and then converted to EU/mg based on UV-Vis measurements. Our threshold for samples suitable for immunostaining is <100EU/mg.

UV-Vis

Agilent Technologies Cary 8454 was used to carry out ultraviolet-visible spectrophotometry (UV-Vis) measurement. Samples were applied to 10 mm, 50 μL quartz cells (Starna Cells, Inc.), and absorbance was measured at 180 nm to 1000 nm. The net absorbance at 280 nm obtained from measurement and single reference wavelength baseline subtraction was used together with calculated extinction coefficient and molecular weight to obtain protein concentration. The absorbance ratio at 320/280 nm was used to determine the relative aggregation level in the real-time stability study sample. Samples were diluted with corresponding blank buffer to obtain an absorbance between 0.1 and 1.0. All data generated from UV/vis instruments were processed in 845x UV/visible light system software.

Hydrogen/deuterium exchange mass spectrometry

3 mg of RBD-I53-50A, Rpk4-I53-50A and Rpk9-I53-50A trimers were subjected to H/D exchange (HDX) at 22°C for 3 seconds, 15 seconds, 60 seconds, 1800 seconds and 72000 seconds, respectively, in deuterated buffer (pH*7.5, 85% D ₂ O, Cambridge Isotope Laboratories, Inc.). The exchanged samples were then mixed 1:1 with ice-cold quenching buffer (200 mM tris(2-chloroethyl)phosphate (TCEP), 8 M urea, 0.2% formic acid (FA)) to a final pH of 2.5 and immediately flash frozen in liquid nitrogen. Samples were analyzed by LC-MS on a Synapt ^TM G2-Si mass spectrometer using a custom-built loading system that maintained all chromatographic columns, loops, valves and lines at 0°C. Frozen samples were thawed on ice and loaded onto a custom packed immobilized pepsin column (2.1×50 mm) with 0.1% trifluoroacetic acid (TFA) and 2% acetonitrile at a flow rate of 200 mL/min. Peptides were trapped on a Waters CSH C18 trap column (2.1×5 mm) and then resolved on a Waters CSH C18 1×100 mm 1.7 μm column with a linear gradient from 3% to 40% B in 18 min (A: 98% water, 2% acetonitrile, 0.1% FA, 0.025% TFA; B: 100% acetonitrile, 0.1% FA, flow rate 40 mL/min). A series of washes were performed between sample runs to minimize carryover. Unless otherwise specified, all water and organic solvents used were MS grade (Optima ^™ , Fisher). Full deuteration controls were performed for each sample series by collecting LC eluate from a pepsin-digested, nondeuterated sample, speedvac drying, incubating in deuterated buffer at 85°C for 1 hour, and quenching identically to all other HDX samples. Internal exchange standards (Pro-Pro-Pro-Ile [PPPI] and Pro-Pro-Pro-Phe [PPPF]) were added to each sample to ensure consistent labeling conditions for all samples.

The peptide reference list was updated from the wild-type RBD peptide list, with new peptides added covering mutations. The peptides were manually verified using DriftScope ^TM and identified using orthogonal retention time and drift time coordinates. Deuterium uptake analysis was performed using HX-Express v2. Peaks were identified from peptide spectra based on peptide m/z values, and a binomial fit was applied. Deuterium uptake levels were normalized relative to a fully deuterated control.

Mouse immunization

Female BALB/c (stock number: 000651) mice were purchased at four weeks of age. At 6 weeks of age, 6 mice in each dosing group were vaccinated with the primary vaccine, and mice received a second vaccination booster three weeks later. Before vaccination, the immunogen suspension was gently mixed with AddaVax ^TM adjuvant at 1:1 volume/volume to reach a final concentration of 0.009mg/mL or 0.05mg/mL antigen. Under isoflurane anesthesia, a 27-gauge needle was used to inject intramuscularly into the gastrocnemius muscle of each hind leg of the mouse, with 50μL (a total of 100μL) of immunogen injected at each injection site. In order to obtain serum, all mice were bled two weeks after the primary and booster immunizations. Blood was collected via submental venous puncture and allowed to stand for 30min at room temperature in a 1.5mL plastic Eppendorf tube for coagulation. Serum was separated from hematocrit by centrifugation at 2,000g for 10min. Complement factors and pathogens in the separated serum were heat-inactivated by incubation for 60 min at 56° C. Serum was stored at 4° C. or −80° C. before use.

ELISA

Binding of mouse sera to the delivered antigen was determined using an enzyme-linked immunosorbent assay (ELISA ⁾ . ELISA plates were coated overnight at 4°C with 0.08 μg/mL of the protein of interest per well in 0.1 M sodium bicarbonate buffer (pH 9.4). The plates were then blocked for 1 hour at room temperature with a 4% (w/v) solution of dry milk powder in TBS containing 0.05% (v/v) Tween 20 (TBST). Serial dilutions of serum were added to the plates, and after washing, antibody binding was revealed using a catalase-coupled horse anti-mouse IgG antibody. The plates were then washed thoroughly in TBST, a colorimetric substrate (TMB, Thermo Fisher) was added, and the absorbance at 450 nm was read. The area under the curve (AUC) calculation was generated by adding the trapezoidal area generated between adjacent absorbance measurement pairs and the baseline. The midpoint potency calculation (EC ₅₀ ) was generated based on fitting a four-point logistic equation using the SciPy library in Python, where EC ₅₀ is the serum dilution at which the curve reaches 50% of its maximum value.

Lentivirus-based pseudovirus neutralization assay

Spike-pseudotyped lentivirus neutralization assays were performed essentially as described in (67). The protocol for this study was modified to use a SARS-CoV-2 spike with a 21-amino acid cytoplasmic tail truncation (which increases spike-pseudotyped lentivirus titers) and the D614G mutation (which is now predominant in human SARS-CoV-2). The plasmid HDM_Spikedelta21_D614G encoding this spike is available from Addgene (no. 158762) or BEI (NR-53765), and the full sequence is available on the World Wide Web at addgene.org/158762).

Briefly, 293T-ACE2 cells (BEI NR-52511) were seeded at 1.25×10 ⁴ cells/well in 50uL D10 growth medium (containing 10% heat-inactivated FBS, 2mM L-glutamine, 100U/mL penicillin, and 100μg/mL streptomycin) in a poly-L-lysine-coated black-walled clear-bottom 96-well plate (Greiner 655930). The next day, mouse serum samples were heat-inactivated at 56°C for 30min and then serially diluted in D10 growth medium. The spike pseudotyped lentivirus was diluted 1:50 to produce approximately 200,000 RLU per well and incubated with serum dilutions at 37°C for 1 hour. 100μL of the virus-serum mixture was then added to the cells and the Bright-Glo ^™ luciferase assay system ( E2610) to measure luciferase activity. Each batch of neutralization assays includes human serum negative control samples and known neutralizing antibodies collected in 2017-2018 to ensure consistency between batches. Compared with the two "serum-free" control wells in the same row of the plate, the infection rate score of each well is calculated. The "neutcurve" package (available on the World Wide Web at jbloomlab.github.io/neutcurve, version 0.5.2) is used to calculate 50% inhibition concentration (IC ₅₀ ) and 50% neutralization titer (NT50), for each serum sample, by fitting the Hill curve with the bottom fixed to 0 and the top fixed to 1, which is simply 1/IC _50. All neutralization assay data are available on the World Wide Web at github.com/jbloomlab/RBD_nanoparticle_vaccine.

MLV-based pseudovirus neutralization assay

The MLV-based SARS-CoV-2S pseudotype was prepared as described (AC Walls et al., Cell 181, 281-292.e6 (2020); AC Walls et al., Cell 183, 1367-1382.e17 (2020); AC Walls et al., Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines, dx.doi.org/10.1101/2021.03.15.435528; JK Millet & GR Whittaker. Bio Protoc 6 (2016)). Briefly, HEK293T cells were co-transfected with a plasmid encoding SARS-CoV-2S, an MLVGag-Pol packaging construct, and an MLV transfer vector encoding a luciferase reporter gene using Lipofectamine ^™ 2000 (Life Technologies) according to the manufacturer's instructions. The cells were washed three times with Opti-MEM and incubated with transfection medium at 37°C for 5 h. DMEM containing 10% FBS was added for 60 h. The supernatant was harvested by spinning at 2,500 g, filtered through a 0.45 μm filter, concentrated with a 100 kDa membrane at 2,500 g for 10 min, and then aliquoted and stored at -80°C.

For neutralization assays, HEK-hACE2 cells were cultured in DMEM containing 10% FBS (Hyclone) and 1% PenStrep in a 37°C incubator (ThermoFisher) with 8% CO _2. One day or more before infection, 40 μL of polylysine (Sigma) was placed in a 96-well plate and incubated with rotation for 5 min. Polylysine was removed, the plate was dried for 5 min, and then washed with water once before plating the cells HEK-hACE2 cells. The next day, the cells were checked to reach 80% confluence. In a half-area 96-well plate, a 1:3 serial dilution of serum was prepared in DMEM with a final volume of 22 μL. Then 22 μL of pseudovirus was added to the serial dilutions and incubated at room temperature for 30-60 min. The mixture was added to the cells, and 44 μL of DMEM supplemented with 20% FBS and 2% PenStrep was added 2 hours later, and the cells were incubated for 48 hours. After 48 h, 40 μL/well of One-Glo-EX ^™ substrate (Promega) was added to the cells and incubated in the dark for 5-10 min before reading on a BioTek ^™ microplate reader. _IC50 values were determined from curve fits using nonlinear regression of log(inhibitor) versus normalized response.

Quantification and statistical analysis

Multiple group comparisons were performed using the nonparametric Kruskal-Wallis test and Dunn post hoc analysis in GraphPad Prism 8. Differences were considered significant when P values were less than 0.05. Statistical methods and P value ranges can be found in the figures and legends.

result

The following five mutations of SARS-CoV-2S RBD are considered to be the starting points for designing stabilized RBD antigens: I358F, Y365F, Y365W, V367F and F392W. Five mutations were analyzed in PyMol ^TM and Rosetta ^TM using the cryoEM structure of the S extracellular domain trimer (PDB ID 6VXX) before fusion. Only the V367F mutation was found to be exposed to the solvent, and therefore it was not considered to be included in the stabilized RBD design to avoid the risk of adversely changing antigenicity. It was observed that the other four mutations were close to or located in the recently identified linoleic acid (LA) binding pocket, where Y365 was identified as the key gating residue (Fig. 2 A to Fig. 2 B) for this interaction. The expression and stability of improvement were observed by DMS for several mutations in the LA binding pocket, indicating that this region of RBD is structurally suboptimal.

Next, we explored whether the combination of these mutations can further improve these and other properties of RBD. The computational protocol was developed in Rosetta ^TM , which models one or more of I358F, Y365F, Y365W and/or F392W, while also allowing nearby residues to mutate (Fig. 2C). Design trajectories that do not force any of these four validated mutations to be included in the LA binding pocket were also performed, rather than allowing Rosetta ^TM to design novel stabilizing mutation sets in the same region. All design trajectories were performed in the context of the crystal structure (PDB ID 6YZ5) of the RBD monomer that shows a subtle difference in backbone conformation in the region around the LA binding pocket. Seventeen repackaging designs (abbreviated as "Rpk") with mutations that fill the cavity and/or remove buried polar groups were selected for experimental analysis, including some of the individual mutations identified by DMS for comparison (Table 1).

Table 1: Mutations included in each stabilized RBD design. Mutations are classified as either reported DMS-identified mutations or mutations identified by Rosetta.

*n/a means not applicable

These designs were screened in the context of genetic fusion of the Wuhan-Hu-1 RBD to the I53-50A nanoparticle trimer, which could be incorporated into icosahedral I53-50 nanoparticles to enable their evaluation as candidate vaccines displaying 60 copies of the RBD. Thus, the stabilized RBD amino acid sequence was cloned into a vector for mammalian expression, in which the I53-50A sequence was fused C-terminally to the antigen and the two domains were joined by a 16-residue flexible Gly-Ser linker.

Stabilized designs, as well as wild-type RBD ("RBD") fused to the I53-50A trimer and negative control plasmids, were secreted from HEK293F cells. Reducing SDS-PAGE of cell culture supernatants showed increased expression of all designs compared to wild-type (Figure 1A). In addition, non-reducing SDS-PAGE showed significant differences in the number of disulfide-linked dimers formed by each design (Figure 1A). The design that included the F392W mutation produced significantly lower levels of disulfide-linked dimers. In addition to the partial filling of the LA binding pocket cavity by F392W, the proximity of this mutation to the disulfide between C391 and C525 suggests that it is not conducive to off-target intermolecular disulfide formation involving these cysteines.

The following two designs were selected for more detailed analysis as both monomers and trimers: Rpk4, which is characterized by only F392W; and Rpk9, which combines F392W with Y365F identified by DMS to remove buried side chain hydroxyls and with V395I identified by Rosetta to refill the resulting cavity with hydrophobic fillers (data not shown). Proportional expression from HEK293F cells and purification by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) confirmed that Rpk4 and Rpk9 increased the yield of both monomers and fusions with I53-50A trimers, wherein Rpk9 showed a clear advantage for I53-50A trimers (Fig. 4A and Fig. 10A). All constructs are characterized by low levels of off-target disulfide bonded dimer formation, highlighting the importance of including F392W in the stabilized RBD design. The melting temperatures (T _m ) of both monomers and trimers were measured by nano differential scanning fluorimetry (nano DSF), monitoring intrinsic tryptophan fluorescence, which showed that the Rpk4 protein increased by 1.9-2.4 ° C and the Rpk9 protein increased by 3.8-5.3 ° C ( FIG. 4B ) compared to the wild-type counterpart. All monomeric RBDs could not be distinguished by circular dichroism and appeared to refold after denaturation at 95 ° C ( FIG. 10B ). In addition, hydrogen/deuterium exchange mass spectrometry (HDX-MS) of the stabilized RBD fused to the I53-50A trimer showed that deuterium uptake in two different peptide segments in the LA binding pocket was reduced compared to the wild-type RBD ( FIG. 4C , FIG. 11 ), indicating that local ordering was improved in the stabilization design. Compared to the wild type, peptide segments away from the LA binding pocket (including those in the ACE2 binding motif) showed a retained structural order. To further evaluate the structural order, all three monomeric RBDs were mixed with SYPRO orange dye individually to measure the exposure of hydrophobic groups (Figure 4D). Compared with wild-type RBD, both Rpk4 and Rpk9 showed reduced signals, with Rpk9 producing the least fluorescence, indicating that the improved local order of the LA binding pocket in the stabilized RBD resulted in reduced hydrophobic exposure. Consistent with the HDX-MS data, as assessed by the binding of antibody CV30 that recognizes antigenic site Ia, neither of the two stabilizing mutation sets affected the antigenicity of the ACE2 binding motif (Hurlbut et al. Nature Communications 11: 5413 (2020)) (Figure 4E). The affinity for non-neutralizing antibody CR3022 (Yuan et al. Science 386: 630-644 (2020)) for site IIc and closer to the LA binding pocket was slightly reduced (<3.5 times). In summary, both stabilizing mutation sets enhanced antigen expression, thermal stability, and structural order with minimal impact on antigenicity, with Rpk9 showing superior improvements in all categories for both the monomeric RBD and its genetic fusion to the I53-50A trimer.

Although stabilizing mutations were designed with isolated RBDs in mind, such mutations were also evaluated in the context of the complete S extracellular domain. The total yield of pre-fused stabilized HexaPro antigen fused to the T4 fibrin foldon was measured using Rpk9 mutations (Rpk9-HexaPro-foldon) and compared with the wild-type version (HexaPro-foldon). The term "HexaPro" refers to a spike protein with four beneficial proline substitutions (F817P, A892P, A899P, A942P) and two proline substitutions in S-2P (proline is located at 986 and 987). See Hsieh et al., Science 369: 1501-05 (2020); a slight increase in yield was seen when using Rpk9 mutations, but a slightly earlier SEC elution volume was observed, which may indicate reduced stability in the context of the S extracellular domain. Rpk9-HexaPro-foldon showed similar nanoDSF features as HexaPro-foldon, but the intrinsic fluorescence change occurring above 60°C was slightly accelerated when using Rpk9-HexaPro-foldon (Figure 8C). Negative staining electron microscopy (nsEM) showed that the typical prefusion spike morphology was mainly consistent with the mutation (Figure 8D). These data suggest that although mutations in the LA binding pocket can be incorporated into the prefusion spike trimer, the stabilizing effect of the Rpk9 mutation appears to be unique to the isolated RBD.

Next, it was investigated whether the RBD stabilizing mutations would improve the stability of the nanoparticles in simpler buffers lacking excipients such as glycerol, L-arginine, and the detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), which otherwise could be used to stabilize preparations of nanoparticle immunogens. Wild-type and stabilized RBD-I53-50A trimers were assembled into nanoparticles (RBD-I53-50, Rpk4-I53-50, and Rpk9-I53-50) ( FIG. 5A ) by adding the complementary I53-50B.4PT1 pentamer component (SEQ ID NO: 17). Using a mobile phase comprising Tris-buffered saline (TBS) containing glycerol, L-arginine, and CHAPS, excess residual components were removed by SEC, and the formation of highly monodisperse nanoparticles was confirmed by negative staining electron microscopy (nsEM) ( FIG. 5B ). The purified nanoparticles were then dialyzed into a buffer solution with less excipients to assess solution stability before and after a single freeze/thaw cycle (Figure 5C to Figure 5E). In TBS supplemented with glycerol and L-arginine, wild-type RBD-I53-50 showed slight signs of aggregation by UV-Vis spectroscopy (Figure 5C) and dynamic light scattering (DLS) (Figure 5D), while no signs of aggregation were observed for Rpk4-I53-50 and Rpk9-I53-50. The differences in solution stability were further compared after dialysis into TBS containing only glycerol: both Rpk4-I53-50 and Rpk9-I53-50 were more resistant to aggregation than RBD-I53-50 and better maintained binding to immobilized human ACE2 (hACE2-Fc) and CR3022 (Figure 5E). Dialysis into TBS alone showed clear evidence of aggregation and loss of antigenicity for all samples, with Rpk9-I53-50 retaining antigenicity slightly better than RBD-I53-50 and Rpk4-I53-50. The improved solution stability observed for the stabilized RBD appears consistent with its enhanced thermal stability and structural order, and provides a subtle but important improvement in formulation stability, which is highly relevant to vaccine manufacturing.

The immunogenicity of stabilized RBD was subsequently evaluated in mouse immunization studies. Immunogens containing wild-type and stabilized RBD were prepared in two forms: I53-50 nanoparticles displaying each antigen, and non-assembly controls of nearly equivalent proteins, in which the trimeric fusion with I53-50A was mixed with a slightly modified pentamer scaffold lacking a hydrophobic interface that drives nanoparticle assembly ("2OBX"; SEQ ID NO:43; Figure 6A). In addition to allowing the evaluation of the immunogenicity of different RBDs in trimer and nanoparticle form, this comparison also directly controls the impact of nanoparticle assembly. All nanoparticle immunogens were prepared in Tris-buffered saline (TBS) supplemented with glycerol and L-arginine, while wild-type RBD-I53-50 nanoparticles were also prepared in a buffer further comprising CHAPS to enable direct comparison with other immunogenicity studies. HexaPro-fold (characterized by wild-type RBD) was included as a comparator. Female BALB/c mice were immunized twice with each immunogen at intervals of three weeks, and sera were collected two weeks after each immunization (Figure 6A). All doses were administered with an equimolar amount of RBD and contained AddaVax adjuvant.

Binding titers to HexaPro-foldon were measured using an enzyme-linked immunosorbent assay (ELISA) and analyzed by measuring the area under the curve (AUC) (Figure 6B) and midpoint titers (Figure 12A). Sera from all nanoparticle groups showed slightly higher levels of antigen-specific antibodies than HexaPro-foldon and significantly higher than non-assembly controls after the initial immunization. The binding signals of all groups increased after the second immunization, with fewer differences between them. Neutralization of pseudoviruses using a lentiviral backbone showed similar trends after the initial immunization, with all nanoparticle groups showing significantly higher neutralizing activity than non-assembly controls and more effective neutralization than HexaPro-foldon by nearly two orders of magnitude (Figure 6C). After the second immunization, the neutralization of all groups was greatly increased, with nanoparticles and HexaPro-foldon showing the highest levels of neutralizing activity. At each time point, there was no significant difference in neutralizing activity between various nanoparticle groups or between various non-assembly control groups. Comparable results were obtained using different pseudovirus assays using a murine leukemia virus (MLV) backbone (Figure 12B). These data suggest that stabilized RBD has similar immunogenicity to wild-type RBD when presented as trimers or microparticles, with nanoparticle presentation significantly enhancing RBD immunogenicity, most significantly after a single immunization.

Improved shelf-life stability of SARS-CoV-2 vaccines has the potential to directly enhance global vaccination efforts by streamlining manufacturing and distribution. The stability of two stabilized RBD nanoparticle immunogens was compared with wild-type RBD-I53-50 during 28 days of storage at -80°C, 2-8°C, 22-27°C, and 35-40°C by DLS (Figure 7A), BLI (Figure 7B), SDS-PAGE, and nsEM (Figure 7C). No significant deviations from baseline were observed for any immunogen at -80°C, 2-8°C, or 22-27°C during the study. However, storage of wild-type RBD-I53-50 at 35-40°C for 28 days resulted in a significant reduction in aggregation and antigenicity that was detectable by DLS and nsEM. In contrast, particle stability and antigenicity of both Rpk4-I53-50 and Rpk9-I53-50 were maintained after 28 days of storage at 35-40°C. These results suggest that the stabilizing mutations identified in the RBD can improve the manufacturability and stability of RBD-based nanoparticle immunogens without compromising their potent immunogenicity.

Narrowing the potential design space to particularly promising regions and experimental information on mutations can greatly facilitate structure-based protein design. Here, the utility of DMS data in guiding viral glycoprotein stabilization is demonstrated by characterizing the LA-binding pocket of the SARS-CoV-2S RBD as a structurally suboptimal region and providing properties of potential stabilizing mutations. Guided by these data, structural modeling in Rosetta ^TM identified additional stabilizing mutations as well as promising mutation combinations. All designs that passed the experimental screen successfully improved expression of the wild-type RBD, an unusually high efficiency compared to many purely structure-based design experiments.

This in-depth biochemical and biophysical characterization of RBD variants enables the inventors to select enhanced expression; minimize off-target disulfides; improve local structural order; and improve thermal stability, solution stability and shelf life stability; while maintaining the design of strong immunogenicity of wild-type RBD displayed on I53-50 nanoparticles. Of the two mutants studied in detail, Rpk4 (F392W) is more conservative, characterized by only a single amino acid change, but is more unstable than Rpk9 (Y365F, F392W, V395I) containing additional mutations that specifically improve expression and thermal stability. Without wishing to be bound by theory, the inventors speculate that the improved solution properties of Rpk4-I53-50 and Rpk9-I53-50 are most likely derived from improvements in local structural order and reductions in hydrophobic surface area exposure, as indicated by HDX-MS and SYPRO orange fluorescence. More generally, these results raise the possibility that other RBD antigens may adopt dynamic conformations not observed in existing structures of the S ectodomain or isolated RBD, such as transitions between open and closed states of the LA-binding pocket.

The similarly strong immunogenicity of Rpk4-I53-50 and Rpk9-I53-50 compared to wild-type RBD-I53-50 nanoparticles is consistent with the natural-like immunogenicity of the ACE2 binding motif, the primary focus of the neutralization response against RBD, and the fact that the stabilizing mutations are not exposed on the antigenic surface. These immunogenicity data also clearly demonstrate that the high-valent RBD nanoparticle immunogens are far more immunogenic than the trimeric form of RBD, especially after a single immunization. The trimeric spike (HexaPro-foldon) elicited higher levels of neutralizing activity than the trimeric RBD. This result suggests that removing the RBD from the spike environment while maintaining the oligomeric state of the spike is not an inherent advantage for enhancing antibody responses against RBD and highlights the importance of nanoparticle presentation in RBD-based vaccines.

The observed improvements in manufacturability, stability, and solution properties may have significant implications for the manufacture and distribution of protein-based vaccines against SARS-CoV-2. As SARS-CoV-2 vaccines are already being updated in response to antigenic drift, such improvements may be important for maximizing the scale and speed of vaccine production and for buffering against unexpected changes in stability or solution properties of antigens from novel SARS-CoV-2 strains. Furthermore, improved denaturation resistance and shelf-life stability across temperatures may be particularly impactful for reliable distribution in less developed regions of the world that lack cold chain infrastructure. Finally, as knowledge of the prefusion stabilizing “2P” mutation prior to the emergence of SARS-CoV-2 proved critical to pandemic response efforts, the ability to reliably improve vaccine manufacturability using stabilizing mutations to the RBD may be an important tool for optimizing vaccine design against other coronaviruses that circulate in zoonotic reservoirs and threaten to jump to humans.

Claims (73)

1. A non-naturally occurring polypeptide comprising a first coronavirus receptor binding domain (RBD), the first coronavirus RBD comprising residues 328-1 of SEQ ID NO:1 531 is at least 90% identical, and further comprises at least two mutations relative to the RBD of SEQ ID NO: 1, wherein the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M or in said second coronavirus receptor binding structure as determined by sequence alignment of SEQ ID NO: 1 with the sequence of a second coronavirus receptor binding domain using the Blast-p parameters of Protocol 1 or Protocol 2 at the corresponding residues of the domain. 2. A non-naturally occurring polypeptide comprising: A first coronavirus receptor binding domain (RBD) comprising residues 328-531 corresponding to SEQ ID NO: 1 or corresponding to SEQ. ID NO:1 is at least 90% identical to the corresponding residues of the receptor binding domain of the second coronavirus as determined by a sequence alignment with the sequence of the receptor binding domain of the second coronavirus, and further comprising at least two mutations relative to said RBD of SEQ ID NO: 1 or said corresponding residues in said second coronavirus, wherein said at least two mutations enhance the stability of said polypeptide relative to the stability of a wild-type polypeptide lacking said at least two mutations. 3. The polypeptide of claim 2, wherein the at least two mutations are at the following amino acids of SEQ ID NO: 1: 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365, or in said second coronavirus as determined by sequence alignment of SEQ ID NO: 1 with said sequence of said second coronavirus receptor binding domain using the Blast-p parameters of Protocol 1 or Protocol 2 corresponding residues in the receptor binding domain. 4. The polypeptide of claim 2 or 3, wherein the at least two mutations are selected from the group consisting of: F338L/Y365W of SEQ ID NO: 1; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or correspond to a second coronavirus as determined by sequence alignment of SEQ ID NO: 1 with said sequence of said second coronavirus receptor binding domain using the Blast-p parameters of Protocol 1 or Protocol 2 at the residue. 5. The polypeptide of any one of claims 2-4, further comprising additional amino acid residues other than the RBD of SEQ ID NO:1. 6. The polypeptide of any one of claims 2-5, wherein the coronavirus receptor binding domain (RBD) comprises at least 95% identity to residues 328-531 of SEQ ID NO:1. 7. The polypeptide of any one of claims 2-6, wherein at the following amino acids of SEQ ID NO:1 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365, Or the at least two mutations at corresponding residues of the second coronavirus receptor binding domain are the only mutations in the receptor binding domain relative to wild type. 8. The polypeptide of any one of claims 2-7, wherein when expressed in a cell, the expression of the RBD polypeptide is compared to the expression of the wild-type RBD polypeptide lacking the at least two mutations. increase. 9. The polypeptide according to any one of claims 2-8, wherein the RBD polypeptide binds a coronavirus antibody or binds a coronavirus cognate receptor. 10. The polypeptide of claim 9, wherein the coronavirus antibody comprises a SARS-CoV-2 antibody. 11. The polypeptide of claim 9, wherein the receptor for the coronavirus corresponding to the polypeptide includes an angiotensin-converting enzyme (ACE) receptor. 12. The polypeptide of claim 11, wherein the ACE receptor is an ACE2 receptor. 13. The polypeptide of any one of claims 2-12, wherein the second coronavirus comprises a sequence selected from a coronavirus: severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) , severe acute respiratory syndrome-related coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; or HKU1. 14. The polypeptide of any one of claims 2-13, wherein the polypeptide comprises at least 90% sequence identity to SEQ ID NO:1. 15. The polypeptide of any one of claims 2-14, wherein the RBD is fused to a second heterologous polypeptide. 16. The polypeptide of claim 15, wherein the RBD is fused to a nanoparticle, nanostructure or heterologous protein scaffold. 17. The polypeptide of any one of claims 2-16, wherein the RBD polypeptide and/or the second polypeptide is an antigenic polypeptide. 18. A composition comprising the polypeptide of any one of claims 1-17 and a pharmaceutically acceptable carrier. 19. The composition of claim 18, further comprising an adjuvant. 20. The composition of claim 18 or 19, wherein the composition has a longer shelf life than a composition comprising a wild-type RBD polypeptide lacking the at least two mutations. 21. The composition of any one of claims 18-20, wherein the composition is formulated as a vaccine. 22. A non-naturally occurring coronavirus spike protein subunit 1 polypeptide comprising at least two mutations, wherein the at least two mutations comprise at least one cavity-filling mutation and at least one second mutation. 23. The coronavirus polypeptide of claim 22, wherein said at least two mutations enhance said stability relative to a wild-type polypeptide lacking said at least one cavity-filling mutation and said at least a second mutation. Stability of coronavirus peptides. 24. The coronavirus polypeptide of claim 22 or 23, wherein the at least one cavity-filling mutation comprises a mutation of a residue in the linoleic acid binding pocket of the coronavirus spike protein subunit 1. 25. The coronavirus polypeptide of any one of claims 22-24, wherein the at least one cavity-filling mutation comprises a mutation of a residue within residues 328-531 of SEQ ID NO:1, or within The second coronavirus spike protein subunit as determined by sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus spike protein subunit 1 using the Blast-p parameters of Protocol 1 or Protocol 2 Mutation at the corresponding residue of 1. 26. The coronavirus polypeptide of any one of claims 22-25, wherein the at least one cavity-filling mutation comprises SEQ ID NO: 1 at residues 335-345, 355-375, or 378-395 Mutation of residues between, or as determined by sequence alignment of SEQ ID NO:1 with the sequence of the second coronavirus spike protein subunit 1 using the Blast-p parameters of Protocol 1 or Protocol 2 Mutations at the corresponding residues of the second coronavirus spike protein subunit 1. 27. The coronavirus polypeptide of any one of claims 22-26, wherein the at least one cavity-filling mutation comprises SEQ ID NO: 1 at amino acids 336, 338, 341, 342, 358, 361, 363 , 365, 368, 374, 377, 387 or 392, or as by using the Blast-p parameters of Protocol 1 or Protocol 2 to compare SEQ ID NO: 1 with the sequence of the second coronavirus Mutation of the corresponding residues of the second coronavirus spike protein subunit 1 determined by sequence alignment was performed. 28. The coronavirus polypeptide of any one of claims 22-27, wherein the at least one cavity-filling mutation and the at least one second mutation are at the following residues of SEQ ID NO: 1 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365, or in said second coronavirus spike protein as determined by sequence alignment of SEQ ID NO: 1 with the sequence of second coronavirus spike protein subunit 1 using the Blast-p parameters of Protocol 1 or Protocol 2 at the corresponding residue of subunit 1. 29. The coronavirus polypeptide of any one of claims 22-28, wherein the at least one cavity-filling mutation and the at least one second mutation are selected from the group consisting of: F338L of SEQ ID NO: 1 /Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M, or selected from said second coronavirus spike protein as determined by sequence alignment of SEQ ID NO: 1 with the sequence of second coronavirus spike protein subunit 1 using the Blast-p parameters of Protocol 1 or Protocol 2 Corresponding residues of subunit 1. 30. The coronavirus polypeptide of any one of claims 22-29, wherein the coronavirus spike protein subunit 1 polypeptide comprises residues 328-531 of SEQ ID NO: 1 or as described by using Protocol 1 Or the Blast-p parameter of Protocol 2 is used to determine the receptor binding of the second coronavirus spike protein subunit 1 by sequence alignment of SEQ ID NO:1 with the sequence of the second coronavirus spike protein subunit 1. At least 95% identity of domain sequences. 31. The coronavirus spike protein subunit 1 polypeptide according to any one of claims 22-30, wherein at the following amino acids of SEQ ID NO: 1 338 and 365; 365 and 513; 365 and 392; 338, 363, 365 and 377; 365 and 392; 365 and 395; 365, 392 and 395; 365, 513 and 515; 338, 363 and 365; 338, 358 and 365; 358, 365 and 513; 358, 365 and 392; 338, 358, 363, 365 and 377; 358, 365 and 392; 358, 365 and 395; 358, 365, 392 and 395; 358, 365, 513 and 515; and/or 338, 358, 363 and 365 Or the at least two mutations at corresponding residues of the second coronavirus receptor binding domain are the only mutations in the spike protein subunit 1 relative to SEQ ID NO: 1. 32. The coronavirus polypeptide according to any one of claims 22-31, wherein the coronavirus polypeptide comprises at least the same amino acid sequence as SEQ ID NO: 1 or the wild-type spike protein subunit 1 of a second coronavirus. 95% identical. 33. The coronavirus polypeptide of any one of claims 22-31, wherein when expressed in a cell, expression of the coronavirus polypeptide is consistent with the absence of the at least one cavity-filling mutation and the at least one first The expression of the double-mutated wild-type polypeptide increased under the same expression conditions. 34. The coronavirus polypeptide of any one of claims 22-33, wherein the coronavirus polypeptide binds a coronavirus antibody or binds a cognate coronavirus receptor. 35. The coronavirus polypeptide of claim 34, wherein the coronavirus antibody comprises a SARS-CoV-2 antibody. 36. The coronavirus polypeptide of claim 35, said cognate coronavirus receptor comprising an angiotensin-converting enzyme (ACE) receptor. 37. The coronavirus polypeptide of claim 36, wherein the ACE receptor is an ACE2 receptor. 38. The coronavirus polypeptide of any one of claims 22-37, wherein the coronavirus polypeptide is an engineered mutant polypeptide of a coronavirus selected from the group consisting of severe acute respiratory syndrome-related coronavirus 2 ( SARS-CoV-2), severe acute respiratory syndrome-related coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; or HKU1. 39. The coronavirus polypeptide of any one of claims 22-38, wherein the coronavirus spike protein subunit 1 polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1. 40. The coronavirus polypeptide of any one of claims 22-39, wherein the coronavirus polypeptide is fused to a second heterologous polypeptide. 41. The coronavirus polypeptide of any one of claims 22-40, wherein the coronavirus polypeptide is fused to a nanoparticle, nanostructure or protein scaffold. 42. The coronavirus polypeptide of claim 40, wherein the coronavirus polypeptide or the second heterologous polypeptide is an antigenic polypeptide. 43. A composition comprising the coronavirus polypeptide according to any one of claims 22-42 and a pharmaceutically acceptable carrier. 44. The composition of claim 43, further comprising an adjuvant. 45. The composition of claim 43 or 44, wherein when stored under identical shelf conditions, the composition has a shelf life greater than that of a composition lacking said at least one cavity-filling mutation and said at least a second mutation. The composition of wild-type coronavirus peptides is longer. 46. The composition of any one of claims 43-45, wherein the composition is formulated as a vaccine. 47. A cell expressing the receptor binding domain according to any one of claims 1-15 or the coronavirus polypeptide according to any one of claims 22-42. 48. A nucleic acid sequence encoding the receptor binding domain according to any one of claims 1-15 or the coronavirus polypeptide according to any one of claims 22-42. 49. A method of vaccinating a subject against coronavirus, comprising administering to the subject a composition according to claim 21 or claim 46. 50. A method of preparing a vaccine, the method comprising combining the composition according to any one of claims 1-15 or 22-42 with an adjuvant and a pharmaceutically acceptable carrier. 51. A coronavirus spike protein comprising the polypeptide according to any one of claims 1-25. 52. The method or composition of any one of the preceding claims, wherein the Blast-p parameters of Protocol 1 include: Algorithm: blastp (protein-protein BLAST) Expected threshold: 0.1 Word length: 6 Maximum match in query range: 0 Matrix: BLOSUM62 Space cost: Presence: 11 Extension: 1 Filter low complexity regions? :no Mask: Only for lookup tables? :no Lower case letters? :no. 53. The method or composition of any one of the preceding claims, wherein the Blast-p parameters of Protocol 2 include: blastp-query query.fasta-topic sbjct.fasta-matrix BLOSUM62-evalue 0.1-word length 6-gaps open 11-gaps extended 1-output results.txt. 54. A polypeptide comprising a coronavirus receptor binding domain (RBD), the coronavirus RBD comprising a mutation selected from the group consisting of I358F, or a variant thereof relative to the coronavirus polypeptide of SEQ ID NO: 1 or a variant thereof. Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L. 55. The polypeptide of claim 54, wherein the mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367F and F392W. 56. The polypeptide of claim 54 or claim 55, wherein the polypeptide comprises a second mutation selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L , F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L. 57. The polypeptide of any one of claims 54-56, wherein the polypeptide comprises a third mutation selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y , F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L. 58. The polypeptide of claim 57, wherein the polypeptide comprises the polypeptide sequence of SEQ ID NO:4 or SEQ ID NO:5. 59. The polypeptide of any one of claims 54-58, wherein the polypeptide comprises a heterologous protein scaffold. 60. The polypeptide of claim 59, wherein the heterologous protein scaffold is at least 90%, at least 95%, or at least 98% identical to the polypeptide sequence of SEQ ID NO:3. 61. The polypeptide of claim 59, wherein the heterologous protein scaffold comprises the polypeptide of SEQ ID NO:3. 62. The polypeptide of claim 61, wherein the polypeptide comprises the polypeptide sequence of SEQ ID NO:6 or SEQ ID NO:7. 63. A polypeptide complex comprising or consisting of: a first component consisting of the polypeptide according to any one of claims 59-62 and SEQ ID NO: 13-SEQ ID Any of NO: 18 has a second component that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical. 64. A vaccine composition comprising the composition according to any one of claims 54-62 or the polypeptide complex according to claim 63. 65. The vaccine composition of claim 64, further comprising a pharmaceutically acceptable carrier. 66. The vaccine composition of claim 64 or claim 65, further comprising an adjuvant. 67. A cell expressing the polypeptide of any one of claims 54-62. 68. A nucleic acid encoding the polypeptide of any one of claims 54-62. 69. A method of vaccinating a subject against coronavirus, the method comprising administering to the subject a polypeptide according to any one of claims 54-62, a polypeptide according to claim 63 Protein complex or vaccine composition according to any one of claims 64-68. 70. A method of preparing a vaccine, the method comprising combining the polypeptide according to any one of claims 54-62 with an adjuvant and a pharmaceutically acceptable carrier. 71. A method of preparing a vaccine, the method comprising combining: a first component consisting of the polypeptide according to any one of claims 59-62; and SEQ ID NO: 13-18 A second component that either has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity; a pharmaceutically acceptable carrier; and an optional adjuvant. 72. A non-naturally occurring polypeptide comprising: A first coronavirus receptor binding domain (RBD) comprising at least 90% identity to residues 328-531 of SEQ ID NO: 1 and further comprising a protein relative to SEQ ID NO: 1 At least one mutation of the RBD, wherein the at least one mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, of SEQ ID NO: 1 Y365M, F377V, V395I, L513I, L513M and F515L, or a second coronavirus reference sequence, wherein the corresponding site in the second coronavirus reference sequence is changed to SEQ ID NO. : 1 is determined by sequence comparison with the spike protein sequence of the second coronavirus receptor binding domain. 73. The polypeptide of claim 72, wherein the polypeptide comprises two or more mutations selected from: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W; I358F/Y365F/V395I; I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and F338L/I358F/A363L/Y365M.