CN116761624A

CN116761624A - Stabilized coronavirus proteins and vaccine compositions thereof

Info

Publication number: CN116761624A
Application number: CN202180088941.3A
Authority: CN
Inventors: D·埃利斯; N·金; J·布鲁姆; T·思达; A·格雷尼
Original assignee: University of Washington; Fred Hutchinson Cancer Research Center
Current assignee: University of Washington; Fred Hutchinson Cancer Center
Priority date: 2020-12-31
Filing date: 2021-06-15
Publication date: 2023-09-15

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

Stabilized coronavirus proteins and vaccine compositions thereof

Government support

The present application was developed with government support under AI141707 awarded by the national institutes of health (National Institutes of Health). The government has certain rights in this application.

Cross Reference to Related Applications

The present application claims the benefit of U.S. c. ≡119 (e) from U.S. provisional application No. 63/132,863 filed on 12/31/2020 and U.S. provisional application No. 63/188,651 filed on 14/2021, each of which is incorporated herein by reference in its entirety.

Technical Field

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

Background

Coronaviruses remain a prominent pandemic threat, with the 2019/2020 pandemic induced by the SARS-CoV-2 virus leading to hundreds of thousands of deaths worldwide and a great economic delay. SARS-CoV-2 may remain continuously epidemic even after the current pandemic has diminished. Thus, there is a great need for effective vaccines against SARS-CoV-2 or other coronaviruses that occur in the future.

Disclosure of Invention

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

Accordingly, in one aspect, provided herein is 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 at least two mutations relative to the RBD of SEQ ID NO:1, wherein said 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 at the corresponding residue 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 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 a National Center for Biotechnology Information (NCBI) online alignment tool. Alternatively, the Blast-p program may be downloaded to the device and used locally. The use of Blast-p alignment tools will be readily understood by those skilled in the art, however for the avoidance of doubt, protocol 1 and protocol 2 are provided herein for alignment tools for online and download, respectively.

Protocol 1: for use with an online BLASTp alignment from a National Center for Biotechnology Information (NCBI) server.

1. BLAST alignment was set using the following settings:

using the "Align two or more sequences (aligned two or more sequences)" option

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

Inputting any corresponding coronavirus spike protein sequence into the "Enter Subject Sequence (input subject sequence)" segment

Algorithm: blastp (protein-protein BLAST)

Expected threshold: 0.1

Word length: 6

Maximum match within query range: 0

Matrix: BLOSUM62

Vacancy cost:

the presence is: 11

Extension: 1

Filtering low complexity regions? : whether or not

Masking:

is only for look-up tables? : whether or not

Lower case letters? : and (3) if not.

2. The analysis is run by clicking on the "BLAST" button.

3. Click on the "alignment" mark to show the alignment between the two sequences.

4. For each sequence position of interest, a number is identified according to the "Query" sequence. The corresponding residue positions in the "Sbjct" sequence that have been aligned with the position of the "Query" sequence are then identified.

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

1. The BLAST is installed to execute the command line using the manufacturer's instructions, or a computer or server to which the BLAST has been installed is determined.

2. A FASTA format file was generated 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.

3. A second file in FASTA format is generated, the second file comprising corresponding protein sequences from different coronaviruses of the same subtype. In the following commands, this file will be named "sbjct.

4. The following commands are executed using a program such as Terminal, iTerm2, windows Console, linux Console or other similar terminal emulator. This will generate a result in a file named "results. blastp-query. Fasta-topic sbjct. Fasta-matrix BLOSUM 62-evaluation 0.1-

Word length 6-vacancy open 11-vacancy extend 1-output results. Txt

(blastp-query query.fasta-subject sbjct.fasta-matrix BLOSUM62-evalue 0.1-

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

5. The results. Txt is opened and the aligned segments showing the two sequences are viewed. For each sequence position of interest, a number is identified according to the "Query" sequence. The corresponding residue positions in the "Sbjct" sequence that have been aligned with the position of the "Query" sequence are then identified.

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 query and reference sequences share significant sequence identity, it is expected that other protein alignment tools will produce similar (if not identical) results as Blast-p using the protocols described herein. The protocols described herein have proven to be accurate and effective for this purpose, and are provided herein to assist the skilled artisan in identifying amino acid residues to be mutated in a query sequence.

Another aspect provided herein includes 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 at least two mutations relative to the RBD of SEQ ID NO:1, wherein said 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 F338L/Y365W corresponding to 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; or at least two mutations of F338L/I358F/A363L/Y365M, wherein the corresponding sites 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 comprising: a first coronavirus Receptor Binding Domain (RBD) comprising at least 90% identity to residues 328-531 of SEQ ID No. 1 or to corresponding residues of a receptor binding domain of a second coronavirus determined by sequence alignment of SEQ ID No. 1 with the sequence of the second coronavirus receptor binding domain using Blast-p, and further comprising at least two mutations relative to the RBD of SEQ ID No. 1 or to corresponding residues in the second coronavirus, wherein the at least two mutations enhance the stability of the polypeptide relative to the stability of a wild-type polypeptide lacking the at least two mutations. In certain embodiments, the stability of a 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 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 Blast-p.

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 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 at the corresponding residue of the second coronavirus determined by sequence alignment of SEQ ID NO:1 with the sequence of the second coronavirus receptor binding domain using Blast-p.

In this aspect and in another embodiment of all other aspects provided herein, the receptor binding domain polypeptide further comprises additional amino acid residues beyond 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 consists 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 comprise, for example, a fusion polypeptide. The receptor binding domain polypeptide or coronavirus spike protein polypeptide may further comprise, for example, a leader sequence (e.g., for secretion). In various embodiments, the leader sequence and/or amino terminal methionine may be present or alternatively may be removed (e.g., by proteolytic cleavage).

In this aspect and in another embodiment of 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 this aspect and in another embodiment of all other aspects provided herein, amino acids 338 and 365 in SEQ ID NO. 1; 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 of the second coronavirus receptor binding domain are the only mutations in the receptor binding domain relative to wild type.

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

In another embodiment of this aspect and all other aspects provided herein, the RBD polypeptide binds to a coronavirus antibody or 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 and all other aspects provided herein, the coronavirus receptor corresponding to the polypeptide comprises an Angiotensin Converting Enzyme (ACE) receptor.

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

In this aspect and all other aspects provided herein, 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; HKU1, or a naturally occurring variant thereof.

In this aspect and all other aspects provided herein in another embodiment, 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 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 a blend 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 and all other aspects provided herein, the shelf-life of the composition is longer 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, provided herein is 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.

In this aspect and in another embodiment of 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 at least a second mutation.

In another embodiment of this 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 coronavirus spike-protein subunit 1.

In this aspect and in another embodiment of 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 a 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 this aspect and in another embodiment of all other aspects provided herein, the at least one cavity filling mutation comprises residues 335-345 of SEQ ID No. 1; 355-375, or 378-395, or 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 2 as described herein).

In this aspect and in another embodiment of all other aspects provided herein, the at least one cavity filling mutation comprises a mutation at the residue at amino acids 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 second coronavirus spike protein subunit 1 as determined by sequence alignment of SEQ ID No. 1 with the sequence of the second coronavirus using Blast-p (e.g., protocol 1 or 2 as described herein).

In this aspect and all other aspects provided herein in another embodiment, the at least one cavity filling mutation and the at least one second mutation are at residues 338 and 365 of SEQ ID No. 1; 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 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 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 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 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 Blast-p (e.g., protocol 1 or 2 as described herein).

In this aspect and in another embodiment of 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 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 2 as described herein).

In this aspect and in another embodiment of all other aspects provided herein, amino acids 338 and 365 in SEQ ID NO. 1; 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 of the second coronavirus receptor binding domain are the only mutations in spike protein subunit 1 relative to SEQ ID No. 1.

In this aspect and in another embodiment of 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 and all other aspects provided herein, the expression of the coronavirus polypeptide is increased when expressed in the cell as compared to the 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 to a cognate coronavirus receptor.

In another embodiment of this 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 coronavirus polypeptide is an engineered mutant polypeptide 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.

In this aspect and in another embodiment of 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, provided herein is also a composition comprising a coronavirus polypeptide as described herein and a pharmaceutically acceptable carrier (e.g., in admixture or forming 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 and all other aspects provided herein, 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 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 that expresses 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, provided herein is also a method of vaccinating a subject against coronavirus, the method comprising administering to the subject a pharmaceutical or vaccine composition as described herein.

Another aspect provided herein relates to a method of preparing a vaccine, the method 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, said coronavirus RBD comprising a mutation of a coronavirus polypeptide relative to SEQ ID NO:1 selected from the group consisting of: I358F, Y365F, Y365W, V367F and F392W. 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 an alanine.

In another embodiment, 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, 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 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 of preparing a vaccine 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) comprising a mutation relative to the coronavirus polypeptide of SEQ ID No. 1 selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N F, 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, N F, 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, N F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L.

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

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

In this aspect and in another embodiment of 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 the polypeptide of 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 to any one of SEQ ID NOs 13-18.

In another aspect, provided herein is also a vaccine composition comprising a 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 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 comprising combining a polypeptide described herein with an adjuvant and a pharmaceutically acceptable carrier.

Another aspect provided herein relates to a method of preparing a vaccine, the method 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; optionally an adjuvant.

Accordingly, in one aspect, provided herein is 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 at least one mutation relative to the RBD of SEQ ID NO:1, wherein said at least one mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367F, F W, G502D, N F, N501T, Q498Y, F338L, F338 39324 363L, Y365M, F377 4639I, L513I, L M and F515L, or at the corresponding residues of the second coronavirus receptor binding domain as determined by sequence alignment of SEQ ID NO:1 with the sequences 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 a National Center for Biotechnology Information (NCBI) online alignment tool. Alternatively, the Blast-p program may be downloaded to the device and used locally.

Another aspect described herein relates to a 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 at least two mutations relative to the RBD of SEQ ID NO:1, wherein said 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 comprising 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, wherein the corresponding site is determined by sequence alignment of SEQ ID NO:1 with the spike protein sequence of the second coronavirus receptor binding domain using Blast-p parameters of protocol 1 or protocol 2.

Drawings

FIGS. 1A-1B illustrate an exemplary non-naturally occurring SARS-CoV-2 stabilizing receptor binding domain that has one or more mutations and that exhibits enhanced expression compared to the starting construct (i.e., the natural or wild-type SARS-CoV-2 spike protein). FIG. 1A reducing and non-reducing SDS-PAGE analysis of supernatants from designed repackaged RBD ("Rpk") variants for expression of genetic fusion to I53-50A trimeric nanoparticle components. The wild type control did not contain RBD mutations and the negative control used a plasmid that did not encode any secreted proteins. Migration of monomer and oxidized dimer species is marked. FIG. 1B is a list of mutations contained in all constructs. Mutations listed in bold have been validated alone in Starr et al 2020.

FIGS. 2A-2C show structural models of the location of the stabilizing mutations of SARS-CoV-2 RBD. FIG. 2A is a surface representation of SARS-CoV-2 spike protein based on PDB-ID 6VYB (left), wherein the boxes highlight RBD; and an enlarged view of the RBD with a transparent surface representation, including a representation of N-glycans. FIG. 2B is an enlargement of the region containing most of the designed mutated SARS-CoV-2 receptor binding domain. FIG. 2C is a structured model of two representative sets of design mutations, designated Rpk4 and Rpk9, which stabilize RBD.

FIG. 3 Biological Layer Interferometry (BLI) measures binding of human ACE2 receptor and CR3022 antibodies to supernatants used to express stabilized RBDs genetically fused to I53-50A trimer components. ACE2 and CR3022 were immobilized on the sensor prior to exposure to the supernatant. Data from (i) all designs, (ii) constructs comprising wild-type RBD, and (iii) negative control serum are shown in the graph. Each mutated SARS-CoV-2 spike protein of figure 1 was determined to be antigenically intact when tested against the SARS-CoV-2 cognate receptor, the angiotensin converting enzyme 2 (ACE 2) receptor, and the CR3022 mAb that recognizes the SARS-CoV-2 spike protein. Measurements from the supernatant also confirm that each of these mutants is expressed at a much higher level than the starting construct.

FIGS. 4A-4E. Biochemical, biophysical and antigenic characterization of stabilized RBDs containing monomeric forms of Rpk4 or Rpk9 mutations and when fused to I53-50A trimer (indicated by the addition of "-I53-50A"). Expression, thermostability and local structural order are improved while maintaining antigenicity similar to wild-type SARS-CoV-2 RBD. Figure 4A Size Exclusion Chromatography (SEC) purification of wild-type and stabilized RBD after expression from an equal volume of HEK293F culture followed by IMAC purification and concentration. Monomer RBD (left) was purified using Superdex 75Increate 10/300GL, while fusion with I53-50A trimer (right) was purified using Superdex 200Increate 10/300 GL. The cut gel shows an equivalently diluted SEC-loaded sample. FIG. 4B thermal denaturation of wild-type and stabilized RBD monomers (left) and fusions with I53-50A trimer (right) monitored by nanoDSF using intrinsic tryptophan fluorescence. The top plot shows the center of gravity average (BCM) of each fluorescence emission spectrum as a function of temperature, while the bottom plot shows the smoothed first derivative used to calculate the melting temperature. FIG. 4C hydrogen/deuterium exchange mass spectrum (HDX-MS) of wild-type and stabilized RBD fused with I53-50A trimer. The structured model (top, from PDB 6W 41) shows the panoramic differential uptake results of both Rpk4-I53-50A and Rpk9-I53-50A trimers compared to wild-type RBD-I53-50A trimers, where shading is determined based on the reduced uptake level of mutant trimers measured at 1 min. This box highlights the peptide segment from residues 392-399, showing the exchange of this peptide at various time points: 3 seconds, 15 seconds, 1 minute, 30 minutes and 20 hours (bottom). Each point is the average of the two measurements. Standard deviations are shown unless less than the plotted points. Fig. 4D fluorescence of SYPRO Orange when mixed with equal concentrations of wild-type and stabilized RBD monomers, with a larger signal indicating a higher level of exposed hydrophobicity. FIG. 4E binding kinetics of immobilized CV30 and CR3022 monoclonal antibodies to monomeric wild type and stabilized RBD as assessed by BLI. Experimental data for RBD at five concentrations in a two-fold dilution series (gray trace) were fitted (black line) using a binding equation describing 1:1 interactions. The structured model (left) was generated by structural alignment of SARS-CoV-2 binding to CV30 Fab (PDB 6XE 1) and CR3022 Fab (PDB 6W 41).

FIGS. 5A-5E. Stabilized RBDs presented on assembled I53-50 nanoparticles enhance solution stability compared to wild-type RBDs. FIG. 5A shows a schematic representation of the assembly of the I53-50 nanoparticle immunogen (labeled by addition of "-I53-50") of RBD antigen. FIG. 5B negative staining electron microscopes (nsEM) (scale bar, 200 nm) for wild type RBD-I53-50, rpk4-I53-50 and Rpk 9-I53-50. FIGS. 5C-5E show summarized quality control results 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. Fig. 5D Dynamic Light Scattering (DLS) measurement, which monitors both proper nanoparticle assembly and aggregate formation. FIG. 5E I53-50 nanoparticle immunogen was partially reactive to immobilized hACE2-Fc receptor (top) and CR3022 (bottom). The pre-and post-freezing data were individually normalized to the corresponding CHAPS-containing samples for each nanoparticle.

FIGS. 6A-6℃ Strong immunogenicity of the parental wild-type RBD-I53-50 nanoparticle immunogens was maintained by the addition of Rpk mutations. FIG. 6A female BALB/c mice (6 per group) were immunized at weeks 0 and 3. Each group received equimolar amounts of RBD antigen supplemented with adavax, which corresponded to 5 μg per dose for HexaPro-foldon and 0.88 μg per dose for all other immunogens, on a total antigen basis. Serum collection was performed at weeks 2 and 5. The RBD-I53-50 immunogen was prepared in two different buffer conditions, one of the groups comprising CHAPS as excipient. FIG. 6B binding titers to HexaPro-foldon at weeks 2 and 5 as assessed by ELISA measurements of serial dilutions of AUC from serum. Each circle represents AUC measurements from individual mice, and the horizontal line shows the geometric mean of each group. One mouse of the fourth group with AUC close to zero at week 2 was not drawn but was still included in the geometric mean calculation. FIG. 6C self (D614G) pseudovirus neutralization using lentiviral scaffolds. Each circle represents neutralizing antibody titers (IC) at 50% inhibition in individual mice ₅₀ ) And the horizontal line shows the geometric mean of each group. Statistical analysis was performed using a one-sided nonparametric Kruskal-Wallis test and multiple comparisons of Dunn. * P, p<0.05；**，p<0.01；***，p<0.001。

Fig. 7A-7℃ Shelf life stability of RBD-based nanoparticle immunogens was improved by Rpk mutation. Fig. 7A is a summary of DLS measurements over four weeks. The hydrodynamic diameters of all nanoparticles remained consistent at 35-40℃except for wild-type RBD-I53-50, which showed signs of aggregation after 28 days of storage. FIG. 7B BLI binding to immobilized hACE2-Fc receptor (dotted line) and CR3022 mAb (solid line), normalized to-80℃samples at each time point. The antigen integrity of the stabilized nanoparticle immunogens remained consistent, while the binding signal of wild-type RBD-I53-50 incubated at 35-40℃was reduced by 60% (hACE 2-Fc) and 30% (CR 3022). FIG. 7C is a 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 ℃ was observed by nsEM on day 28. Electron micrographs at day 28 after storage at 35-40 ℃ are shown, with boxes indicating examples of aggregates (scale bar, 200 nm). All samples were formulated in TBS, 5% glycerol, 100mM L-arginine.

FIGS. 8A-8 D.Rpk9 mutations can be incorporated into the full length SARS-CoV-2S ectodomain containing the HexaPro mutation. FIG. 8A SEC purification of the S ectodomain pre-fusion stabilized for wild-type (HexaPro-foldon) and Rpk9 (Rpk 9-HexaPro-foldon) after expression from an equal volume of HEK293F culture followed by IMAC purification and concentration. The S ectodomain was purified using Superose 6increase 10/300 GL. FIG. 8B reducing and non-reducing SDS-PAGE of intermediates and end products during purification of HexaPro-foldon and Rpk 9-HexaPro-foldon. FIG. 8C thermal denaturation of HexaPro-foldon and Rpk9-HexaPro-foldon monitored by nanoDSF using intrinsic tryptophan fluorescence. The center of gravity average (BCM) of the fluorescence emission spectrum is plotted as a function of temperature. FIG. 8D HexaPro-foldon and Rpk 9-HexaPro-foldon's nsEM (scale bar, 100 nm).

FIG. 9. Rpk9 mutation when added to RBD of B.1.351 variant improved the relative recovery of I53-50 nanoparticles displaying the RBD at the appropriate SEC elution volume in simpler buffer formulation, indicating that Rpk mutation improved the integrity of immunogen containing RBD from different variants. For I53-50 nanoparticles displaying Wuhan-1 RBD (no Rpk mutation), no Rpk9 mutation, and B.1.351RBD with Rpk9 mutation, assembly and SEC were performed in 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. While the Rpk9 mutation increased yield and other measures of RBD stability under either buffer condition compared to an equivalent sample without the Rpk9 mutation, nanoparticles exhibiting b.1.351rbd with the Rpk9 mutation better maintained relative yield and SEC migration without CHAPS detergent.

FIG. 10 SDS-PAGE of nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100mM L-arginine. The integrity of the samples was analyzed by SDS-PAGE in 50mM Tris pH 7.4, 185mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100mM L-arginine. Molecular weights of the standards are expressed in kDa. Each sample was subjected to +/-reductant (DTT), pre-freeze-thaw and post-freeze-thaw (F/T) analysis.

FIG. 11 hACE2-Fc binding of nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100mM L-arginine. hACE2-Fc binding of antigen in 50mM Tris pH 7.4, 185mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100mM L-arginine was analyzed by Biological Layer Interferometry (BLI). The hACE2-Fc loaded protein a biosensor was incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

FIG. 12 CR3022 binding of nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100mM L-arginine. Antigen binding by CR3022 IgG was analyzed by Biological Layer Interferometry (BLI) in 50mM Tris pH 7.4, 185mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100mM L-arginine. 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).

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

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

FIG. 15 SDS-PAGE of nanoparticles in TBS, 5% glycerol, 100mM L-arginine. The integrity of the samples was analyzed by SDS-PAGE in 50mM Tris pH 8, 150mM NaCl, 5% glycerol, 100mM L-arginine. Molecular weights of the standards are expressed in kDa. Each sample was subjected to +/-reductant (DTT), pre-freeze-thaw and post-freeze-thaw (F/T) analysis.

FIG. 16 hACE2-Fc binding of nanoparticles in TBS, 5% glycerol, 100mM L-arginine. hACE2-Fc binding of antigen was analyzed by Biological Layer Interferometry (BLI) in 50mM Tris pH 8, 150mM NaCl, 5% glycerol, 100mM L-arginine. The hACE2-Fc loaded protein a biosensor was incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

FIG. 17 CR3022 binding of nanoparticles in TBS, 5% glycerol, 100mM L-arginine. CR3022 IgG binding to antigen was analyzed by Biological Layer Interferometry (BLI) in 50mM Tris pH 8, 150mM NaCl, 5% glycerol, 100mM L-arginine. 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).

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

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

FIG. 20 SDS-PAGE of nanoparticles in TBS, 5% glycerol. The integrity of the samples was analyzed by SDS-PAGE in 50mM Tris pH 8, 150mM NaCl, 5% glycerol. Molecular weights of the standards are expressed in kDa. Each sample was subjected to +/-reductant (DTT), pre-freeze-thaw and post-freeze-thaw (F/T) analysis.

FIG. 21 hACE2-Fc binding of nanoparticles in TBS, 5% glycerol. ACE2-Fc binding of antigen in 50mM Tris pH 8, 150mM NaCl, 5% glycerol was analyzed by Biol Layer Interferometry (BLI). The hACE2-Fc loaded protein a biosensor was incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

Figure 22 CR3022 binding of nanoparticles in TBS, 5% glycerol. Antigen binding by CR3022 IgG was analyzed by Biological Layer Interferometry (BLI) in 50mM Tris pH 8, 150mM NaCl, 5% glycerol. 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 at 50mM Tris pH 8, 150mM NaCl, 5% glycerol, plotted as normalized intensity.

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

FIG. 25 SDS-PAGE of nanoparticles in TBS. The integrity of the samples was analyzed by SDS-PAGE in 50mM Tris pH 8, 150mM NaCl. Molecular weights of the standards are expressed in kDa. Each sample was subjected to +/-reductant (DTT), pre-freeze-thaw and post-freeze-thaw (F/T) analysis.

FIG. 26 hACE2-Fc binding of nanoparticles in TBS. HACE2-Fc binding of antigen was analyzed by Biological Layer Interferometry (BLI) in 50mM Tris pH 8, 150mM NaCl. The hACE2-Fc loaded protein a biosensor was incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

Figure 27 CR3022 binding of nanoparticles in TBS. Antigen binding by CR3022IgG was analyzed by Biological Layer Interferometry (BLI) in 50mM Tris pH 8, 150mM NaCl. Protein a biosensors loaded with CR3022IgG 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 at 50mM Tris pH 8, 150mM NaCl, plotted as normalized intensity.

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

FIG. 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. Molecular weights of the standards are expressed in kDa. Each sample was analyzed with +/-reductant (DTT).

FIG. 31 hACE2-Fc binding of RBD-I53-50 nanoparticles. hACE2-Fc binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by Biological Layer Interferometry (BLI). The hACE2-Fc loaded protein a biosensor was incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

FIG. 32 CR3022 binding of RBD-I53-50 nanoparticles. Antigen CR3022 IgG binding to antigen incubated at four different temperatures for 28 days (D) was analyzed by Biol Layer Interferometry (BLI). Protein a biosensors loaded with CR3022 were incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

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

FIG. 34 dynamic light scattering of RBD-I53-50 nanoparticles. Hydrodynamic diameter (nm) of each sample over a 28 day (D) period, plotted as normalized intensity.

FIG. 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. Molecular weights of the standards are expressed in kDa. Each sample was analyzed with +/-reductant (DTT).

FIG. 36 hACE2-Fc binding of Rpk4-I53-50 nanoparticles. hACE2-Fc binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by Biological Layer Interferometry (BLI). The hACE2-Fc loaded protein a biosensor was incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

FIG. 37 CR3022 binding of Rpk4-I53-50 nanoparticles. Antigen CR3022 IgG binding to antigen incubated at four different temperatures for 28 days (D) was analyzed by Biol Layer Interferometry (BLI). Protein a biosensors loaded with CR3022 were incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

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

FIG. 39Rpk4-I53-50 dynamic light scattering of nanoparticles. Hydrodynamic diameter (nm) of each sample over a 28 day (D) period, plotted as normalized intensity.

FIG. 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. Molecular weights of the standards are expressed in kDa. Each sample was analyzed with +/-reductant (DTT).

FIG. 41Rpk9-I53-50 hACE2-Fc binding of nanoparticles. hACE2-Fc binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by Biological Layer Interferometry (BLI). The hACE2-Fc loaded protein a biosensor was incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

FIG. 42 CR3022 binding of Rpk9-I53-50 nanoparticles. Antigen CR3022 IgG binding to antigen incubated at four different temperatures for 28 days (D) was analyzed by Biol Layer Interferometry (BLI). Protein a biosensors loaded with CR3022 were incubated with immunogen (association, x=590-889 s) and then with buffer (dissociation, x=890-1190 s).

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

FIG. 44Rpk9-I53-50 dynamic light scattering of nanoparticles. 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 protein having at least one, two, or more amino acid mutations that increase its expression level, yield, and/or stability as compared to a native or wild-type coronavirus spike protein (e.g., SARS-CoV-2S protein) under the same expression, culture, or storage conditions. These mutated spike proteins can be used to generate protein-based vaccines against SARS-CoV-2, a different coronavirus known to infect humans, or ubiquitin coronavirus vaccines that provide protection against a variety of 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 as 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 of the definition

The term "non-naturally occurring" or "mutant" as used herein 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 native or wild-type coronavirus sequence. In some embodiments, for example, a mutant polypeptide described herein is "substantially similar" to its natural counterpart, except for the at least two mutations. In some embodiments, the natural counterpart may include naturally occurring coronavirus variants.

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 comprising at least the receptor binding domain sequence residues (residues 328-531 of SEQ ID NO: 1) and at least one or at least two amino acid mutations that allow the structural formation of a linoleic acid binding pocket. In one embodiment, one mutation of the at least two mutations comprises a "cavity filling mutation", as that term is used herein.

Two molecules are said to be "substantially similar" if they have substantially similar structures (i.e., they are at least 90% similar in amino acid sequence as determined by Blast-p alignment set under default parameters) and are substantially similar in at least one relevant function (e.g., antigen activity determined by recognition of a polypeptide by an antibody that binds to a native coronavirus counterpart). That is, a mutant polypeptide differs from a 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 alterations in which the amino acid is replaced by a different naturally occurring or non-conventional amino acid residue. Some substitutions may be classified as "conservative", in which case the amino acid residue contained in the polypeptide is replaced with another naturally occurring amino acid having similar properties in terms of polarity, side chain functionality, or size. Substitutions encompassed by variants as described herein may also be "non-conservative" in which an amino acid residue present in the peptide is substituted with an amino acid having a different property (e.g., substitution of a charged or hydrophobic amino acid with an uncharged or hydrophilic amino acid), or alternatively, in which a naturally occurring amino acid is substituted with a non-conventional amino acid. When the term "mutant" is used in reference to a polypeptide, the term "mutant" also encompasses a change in primary, secondary, 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 (including insertions and substitutions of amino acids and other molecules) that are not normally present in the peptide sequence underlying the variant, including, but not limited to, ornithine insertions that are not normally present in human proteins.

As used herein, the term "corresponding to" or "corresponding wild-type coronavirus" refers to wild-type coronavirus polypeptide sequences (or naturally occurring variants thereof) that produce non-naturally occurring coronavirus polypeptides (e.g., spike polypeptides) or RBD polypeptides. Typically, the wild-type coronavirus sequence (or naturally occurring variant thereof) is from the same strain as the non-naturally occurring coronavirus polypeptide. For example, a mutant SARS-CoV-2 polypeptide described herein will correspond to a wild-type coronavirus polypeptide of the SARS-CoV-2 sequence or a naturally occurring variant thereof (e.g., a south africa variant, a brazil variant, a los angeles variant, etc.).

As used herein, the term "naturally occurring variant" refers to coronavirus sequences that spontaneously occur in a population of susceptible individuals.

As used herein, the term "increased stability" or "enhanced stability" refers to a mutant coronavirus protein sequence that degrades at a slower rate in a cell, solution, or formulation under the same conditions than a corresponding native or wild-type coronavirus protein sequence (or naturally occurring variant thereof) and thus persists for a longer period of at least 12 hours than the corresponding native or wild-type coronavirus protein sequence (or naturally occurring variant thereof), e.g., as assessed using a hot melt assay as described in working examples herein. In certain examples, the mutant coronavirus protein sequence is persisted in the cell, solution, or formulation for at least 24 hours, 36 hours, 48 hours, 72 hours, 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 more, as compared to the persisting of the corresponding native or wild-type coronavirus protein sequence. In some embodiments, higher expression levels may also indicate increased stability of the polypeptide, or the result of increased stability of the polypeptide.

As used herein, the term "increased yield" or "increased yield" refers to an increase in the amount of mutant coronavirus protein recovered from a protein-producing cell system by at least 10% as compared to the amount of the native or wild-type protein (or naturally occurring variant thereof) recovered from the same cell system under the same growth and isolation conditions. In certain embodiments, "yield enhancement" refers to an increase in the amount of mutant coronavirus protein recovered from a protein-producing cell system by at least 20%, at least 30%, at least 50%, at least 75%, at least 90%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or more as compared to the amount of native 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 the 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 cavity size and/or impairs the binding of cognate ligands within the cavity or pocket.

As used herein, the term "adjuvant" refers to a protein or chemical that enhances an immune response to a vaccine antigen when administered with the vaccine antigen. Adjuvants differ from antigen moieties or carrier proteins in that they are 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. Tubec. Res.7:130 (1956); calbiochem, san Diego Calif.); aluminium salts, especially aluminium hydroxide or ALHYDROGEL ^TM (approved for use in humans by the U.S. food and drug administration (U.S. food and Drug Administration); muramyl Dipeptide (MDP) and analogs thereof, such as [ Thr1 ]]MDP (Byers and Allison, vaccine 5:223 (1987)), monophosphoryl lipid A (Johnson et al, rev. Effect. Dis.9:S512 (1987)), and the like.

As used herein, the term "comprising" means that other elements may be present in addition to the given definition elements. The use of "including" is meant to be inclusive, and not limiting.

As used herein, the term "consisting essentially of. The term allows for 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 as described herein do not include any elements not listed in the description of the embodiments.

Furthermore, unless the context requires otherwise, singular terms shall include the plural 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 being modified in all instances by the term "about". The term "about" when used in conjunction with a percentage 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 is a virus that causes 2019 coronavirus disease (COVID-19), one of seven members of this family known to infect humans, and is the third virus from animals to infect humans in the past three decades. Other coronaviruses known to infect humans include coronaviruses A229E and NL63, and coronaviruses B OC43, HKU1, SARS-CoV (coronavirus leading to Severe acute respiratory syndrome or SARS), and MERS-CoV (coronavirus leading to 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., swine, 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 butyl coronavirus (PDCoV), bovine Coronavirus (BCV), feline coronavirus (FCoV), canine coronavirus (CCoV), avian Infectious Bronchitis Virus (IBV), and Turkey Coronavirus (TCV). In addition, coronaviruses described herein include those currently known and those later discovered. Particularly contemplated herein are coronaviruses that are the cause of an ongoing or future epidemic or pandemic.

As used herein, the term "coronavirus" refers to an enveloped virus having a positive-sense single-stranded RNA genome and helical symmetry. Coronaviruses range in genome size from about 27 kilobases to 32 kilobases, the longest size in any known RNA virus. The large spike (S) glycoprotein protrudes from the virion, giving the coronavirus a unique coronal appearance when visualized by electron microscopy. Coronaviruses infect a variety of species including canine, feline, porcine, murine, bovine, avian and human (Holmes et al, 1996.Coronaviridae:the viruses and their replication, pages 1075-1094. In d.m.k.a.p.m.b.n.fields (editions), fields virology.lippincott-Raven, philiadelphia, pa.). However, the natural host range of each coronavirus strain is very narrow, typically consisting of a single species.

Coronaviruses typically bind to target cells via spike receptor interactions and enter the 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 important determinants of species specificity. The genome of SARS-CoV comprises single stranded (+) sense RNA. Several SARS coronavirus isolates have been reported for complete and partial genomic sequences, including SARS coronavirus Urbani (GenBank accession number AY 278741), SARS coronavirus Tor2 (GenBank accession number AY 274119), SARS coronavirus CUHK-W1 (GenBank accession number AY 278554), SARS-CoV Shanghai LY (GenBank accession number H012999; genBank accession number AY322205; genBank accession number AY 322206), SARS-CoV Shanghai QXC (GenBank accession number AH013000; the part of the part (part (part.

The S (spike) protein may form a non-covalently linked homotrimer (oligomer) that may mediate receptor binding and viral infectivity. Homotrimers of the S protein may be necessary to assume the correct native conformation of the receptor binding domain and elicit a neutralizing antibody response. Furthermore, intracellular processing of S proteins is associated with significant post-translational oligosaccharide modifications. Post-translational oligosaccharide modification (glycosylation) expected from N-glycan motif analysis suggests that S proteins have up to 23 such modification sites. Furthermore, the C-terminal cysteine residues may also be involved in protein folding and maintain the native (functional) S protein conformation. The S proteins of some coronaviruses (e.g., some strains of group II and III viruses) can be proteolytically processed near the center of the S protein into linked polypeptides, including N-terminal S1 polypeptides and C-terminal S2 polypeptides, by trypsin-like proteases in the golgi apparatus or by extracellular localized enzymes. Some members of group II coronaviruses and group III virus types may not do so.

Diagnosis: based on the diagnostic test results, the subject (e.g., human) is diagnosed as having a coronavirus infection. Based on one or more symptoms exhibited, such as fever, chills, cough, shortness of breath/dyspnea, fatigue, muscle/body pain, headache, emerging 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., covd-19, SARS, MERS, etc.). However, certain subjects may exhibit asymptomatic infections (e.g., SARS-CoV-2 infection), and thus may be suspected of having a coronavirus infection when contacted with a subject having a coronavirus infection. In both cases, active coronavirus infection may be confirmed using methods known in the art for detecting one or more of viral antigen and viral nucleic acid in a sample taken from a subject. Examples include the detection of viral RNA using reverse transcriptase polymerase chain reaction (RT-PCR) diagnostic assays from nasopharyngeal swabs or sputum. Other nucleic acid amplification methods (e.g., any of a number of isothermal amplification methods) may also be used and have a sensitivity that approximates, if not is equal to, RT-PCR. Isothermal amplification methods have the advantage of not requiring a thermal cycler to produce amplified products and can provide results more rapidly in a highly sensitive manner. In another embodiment, an active coronavirus infection may be determined by detecting one or more coronavirus polypeptides (such as antigens) in a biological sample obtained from a subject. Lateral flow assays of viral antigens can provide qualitative, and sometimes quantitative, diagnostic results. Viral polypeptides may 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 enhance immune responses). Methods of 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, an electron microscope.

Protein sequence: the amino acid sequence of the natural or wild SARS-CoV-2S protein subunit 1 is: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAA (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 procedures known in the art (e.g., blast-p). Alignment of the spike protein sequence of the 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 site in the different (or second) coronavirus at which to introduce one or more given amino acid mutations to achieve stabilization as described herein. In one embodiment, different coronavirus sequences are aligned with SEQ ID NO. 1 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 a National Center for Biotechnology Information (NCBI) online alignment tool. Alternatively, the Blast-p program may be downloaded to the device and used locally. The use of Blast-p alignment tools will be readily understood by those skilled in the art, however for the avoidance of doubt, protocol 1 and protocol 2 are provided herein for alignment tools for online and download, respectively.

1. BLAST alignment was set using the following settings:

using the "Align two or more sequences (aligned two or more sequences)" option

Algorithm: blastp (protein-protein BLAST)

Expected threshold: 0.1

Word length: 6

Maximum match within query range: 0

Matrix: BLOSUM62

Vacancy cost:

the presence is: 11

Extension: 1

Filtering low complexity regions? : whether or not

Masking:

is only for look-up tables? : whether or not

Lower case letters? : and (3) if not.

2. The analysis is run by clicking on the "BLAST" button.

4. The following commands are executed using a program such as Terminal, iTerm2, windows Console, linux Console or other similar terminal emulator. This will generate a result in a file named "results.

blastp-query query.fasta-subject sbjct.fasta-matrix BLOSUM62-evalue 0.1-

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

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

Receptor Binding Domain (RBD) polypeptides

The viral surface "spike" proteins mediate the entry of coronaviruses into host cells. The spike proteins of both SARS-CoV and SARS-CoV-2 comprise a receptor binding domain that specifically recognizes angiotensin converting enzyme 2 (ACE 2) 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 receptor binding domain of the spike protein of SARS-CoV-2 has the sequence:

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 aligned with other coronavirus RBD sequences using alignment procedures known in the art (e.g., blast-p, clustalW, etc.). At least the alignment of the 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 align 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 an 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 a naturally occurring variant 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 a protein vaccine against one or more coronaviruses. As will be appreciated by those skilled in the art and particularly when used in vaccine settings, the mutant coronavirus proteins do not have to retain the receptor binding properties of their cognate receptors; thus, there is no need to design at least one amino acid mutation or at least two amino acid mutations to preserve the function of the RBD. Whereas maintenance of coronavirus protein function is not necessary, coronavirus proteins having at least 90% identity (e.g., at least 95%, at least 99% identity) to SEQ ID No. 1 or SEQ ID No. 2 are specifically contemplated herein, provided that they retain the ability to act as coronavirus antigens (i.e., to stimulate production of coronavirus binding antibodies or binding to coronavirus antibodies against the corresponding wild-type coronavirus) in a subject. That is, the receptor binding domain polypeptides described herein are "immunogenic," i.e., immunization of a subject with the receptor binding domain polypeptide (optionally bound to a suitable carrier such as a protein, lipid, or polypeptide) induces 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 T cells or B cells), particularly in the context of MHC molecules. Epitopes of a protein antigen may comprise contiguous or non-contiguous portions of the protein and may be between 5 and 100 amino acids in length, between 5 and 50 amino acids, between 8 and 30 amino acids, between 10 and 25 amino acids, for example, the length of 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.

The ability of a protein to enhance an immune response may be due in part to its secondary structure and conformation of the protein fold. In some embodiments, a conformation is preferred for generating an antigenic response and/or increasing the stability of the protein. The secondary structure of SARS-CoV-2 binding to 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 knowing the crystal structure of SARS-CoV-2, one skilled in the art can use inferential or computational software to determine whether a given receptor binding domain polypeptide is likely to contain a shape or secondary structure that would 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 protein in the cellular system and/or enhances the stability of the coronavirus protein in a cell, solution or formulation 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 protein in the cellular system and/or enhances the stability of the coronavirus protein in a cell, solution or formulation compared to its corresponding wild-type protein.

In some embodiments, the at least two mutations comprise at least two mutations introduced within the RBD region of the spike protein (residues 328-531 of SEQ ID NO:2, or 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 at the following amino acid 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 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 Blast-p.

In certain embodiments, the at least one amino acid mutation is at an amino acid residue of: 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, N F, 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 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 at the corresponding residue of the second coronavirus determined by sequence alignment of 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 combined bag

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-2spike protein" (2020)). Residues in the linoleic acid binding pocket were conserved among all 7 human-infected coronaviruses (Toelzer, supra), indicating that this cavity was functionally conserved. Toelzer et al also show that binding of linoleic acid to SARS-CoV-2S protein stabilizes the closed conformation of the S protein. It is contemplated herein that linoleic acid binding pockets or mutations within the 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 mutation in this region is a 'cavity filling' mutation.

In one embodiment, the "cavity filling mutation," as that term is used herein, fills a site within the linoleic acid binding pocket (e.g., protrudes spatially into the cavity). Cavities in native coronavirus spike proteins can be identified by methods known in the art, such as by visual inspection of the crystal structure representation of spike proteins, e.g., 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 use of 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,) and the like). 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.

Amino acids to be substituted 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 substituted with similar amino acids that are sterically larger and capable of "filling" 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 be substituted for uncharged amino acids or by uncharged amino acids, thereby altering the secondary structure of proteins and cavities. Such residues for substitution may also include amino acids that are hidden in a given protein conformation but exposed to 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 that is 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 may stabilize a particular conformation of the protein and/or reduce the rate of degradation of the protein compared to the corresponding wild-type coronavirus. For ease of reference, residues involved in linoleic acid binding are divided into three subdomains herein.

These subdomains are based on the close proximity of only 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 mutation of a residue in a linoleic acid binding pocket and at least one additional mutation, which together enhance the stability and/or yield of the polypeptide, as those terms are used herein.

In some embodiments, the cavity filling mutations include mutations at residues at amino acids 336, 338, 341, 342, 358, 361, 363, 365, 368, 374, 377, 387, or 392 of SEQ ID No. 1, or mutations of corresponding residues of the determined second coronavirus spike protein subunit 1 by sequence alignment of SEQ ID No. 1 (or naturally occurring variant thereof) with the sequence of the second coronavirus (or naturally occurring variant thereof) using Blast-p. In other embodiments, the cavity filling mutation and the at least one second mutation are at residues 338 and 365 of SEQ ID NO. 1; 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 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 2 as described herein).

In some embodiments, the cavity filling abrupt change and the at least one second abrupt change 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 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 Blast-p (e.g., protocol 1 or 2 as described herein).

Coronavirus spike proteins having cavity filling mutations as described herein and further having at least 90% identity (e.g., at least 95%, at least 99% identity to SEQ ID NO:1 or SEQ ID NO: 2) to SEQ ID NO:1 or SEQ ID NO:2, provided that they retain the ability to act as coronavirus antigens (i.e., to stimulate production of coronavirus antibodies in a subject) are specifically contemplated herein. That is, the non-naturally occurring coronavirus spike protein polypeptides described herein are "immunogenic," i.e., immunization of a subject with the polypeptide, optionally conjugated to a suitable 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.

An RBD polypeptide or spike polypeptide as described herein may have one or more amino acid substitutions from a known variant of a coronavirus. For example, but not by way of limitation, a 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, P S, deletion of residues 69-70, D80A, D138Y, R S, 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 comprises 1, 2, 3, 4 or 5 of: 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: L18F, D80A, D215G, D614G and/or a701V (south africa variant);

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

L452R (los angeles variant).

In some embodiments, the polypeptides as disclosed herein comprise 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 are mutant coronavirus S proteins or receptor binding domains thereof having at least two amino acid mutations or substitutions that confer enhanced stability to the mutant proteins as compared to their corresponding native or wild-type coronavirus S proteins. The mutated SARS-CoV-2S protein or receptor binding domain thereof is exemplified in this specification, however, 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, cows, pigs, etc.). By aligning 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), one skilled in the art can readily identify residues corresponding to the residues of SARS-CoV-2 listed in the present specification.

Alignment may provide guidance regarding residues that may be necessary for a function, whether the function is direct contact of one or more given residues with the receptor, or, for example, residues that participate in maintaining a conformation that allows other residues to make such contact; non-limiting examples of the latter include those residues that may be arranged in the linoleic acid binding pocket or that help maintain a given conformation of the spike protein. For example, where an alignment shows two identical or similar amino acids at corresponding positions, the site is more likely to be functionally important (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 a native or reference sequence (e.g., SEQ ID NO: 1). The degree of homology (percent identity) between a native sequence and a mutated sequence can be determined, for example, by comparing the two sequences using computer programs commonly used for this purpose and available on the world wide web for free. A variant amino acid or DNA sequence may 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 the "original", "natural" or "wild-type" sequence). The degree of similarity (percent similarity) between the original sequence and the mutant sequence may be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and many tools for comparing two sequences using similarity matrices are available on-line at no charge, such as BLASTp (available on the world wide web at http:// blast. Ncbi. Lm. Nih. Gov), with default parameters set or using protocol 1 or protocol 2 as described herein.

When cavity filling mutations are desired, a given amino acid may be replaced by a residue having similar physiochemical characteristics, e.g., substitution of one aliphatic residue for another (such as Ile, val, leu or Ala for each other), or substitution of one polar residue for another (such as between Lys and Arg; between Glu and Asp; or between gin and Asn), preferably wherein smaller residues are replaced by larger residues that "fill" the cavity in space or change in charge to induce a change in cavity size and/or structure. Other such substitutions, for example substitutions of the entire region 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 involves substitution of a particular amino acid by an amino acid having similar properties (e.g., similar charge or hydrophobicity). Conservative mutations described herein may include substitutions of amino acid residues that have similar charge or hydrophobicity, but differ in size or volume (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 eradication of a flexible portion of the native coronavirus S protein secondary structure is desired. "non-conservative substitution" refers to the substitution of one type of amino acid for another; for example, substitution of Asp, asn, glu or Gln for Ala. Additional non-limiting examples of non-conservative substitutions include the substitution of a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine with a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine, and/or the substitution of a non-polar residue with a polar residue.

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

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

In some embodiments, a stabilized coronavirus S protein or RBD polypeptide as described herein can comprise naturally occurring amino acids common 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), gin (Q), asp (D), glu (E), lys (K), arg (R), and His (H). In some embodiments, a stabilized coronavirus S protein or RBD polypeptide as described herein can comprise a surrogate amino acid. Non-limiting examples of substitute amino acids include D-amino acids; a beta-amino acid; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, and gamma-carboxyglutamic acid; hippuric acid, octahydroindole-2-carboxylic acid, statin, 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-tetrahydroisoquinoline carboxylic acid, naphthylalanine, biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline, norleucine, t-leucine, tetrahydroisoquinoline carboxylic acid, pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine, dehydroleucine, 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, t-butylglycine, trifluorovaline; hexafluoroleucine; fluorinated analogs; an azide-modified amino acid; alkyne-modified amino acids; cyano-modified amino acids; and their derivatives.

In some embodiments, the polypeptide, e.g., mutant coronavirus polypeptide, may be modified, e.g., 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; HES conversion; ELP-ing; lipidation; acetylation; amidation; end capping modification; cyano group; phosphorylation; albumin conjugation and cyclization. Modifications or moieties that improve solubility in a given solution (i.e., aqueous solution) are particularly contemplated herein.

The alteration of the original amino acid sequence may be accomplished by any of a number of techniques known to those skilled in the art. Mutations can be introduced at the nucleic acid level, for example, by synthesizing oligonucleotides containing a mutant sequence flanked by restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertions, substitutions or deletions. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide altered nucleotide sequences with specific codons altered according to the desired substitution, deletion or insertion. Techniques for making such changes include those described by Khudyakov et al, "Artificial DNA: methods and Applications" CRC Press,2002; braman "In Vitro Mutagenesis Protocols" Springer,2004; and the technology disclosed by 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 may be chemically synthesized and mutations may be incorporated as part of the chemical synthesis process.

The mutant spike proteins or RBC polypeptides described herein can be synthesized using well known methods, including recombinant methods and chemical synthesis. Recombinant methods for producing a polypeptide by introducing a vector comprising a nucleic acid encoding the polypeptide into a suitable host cell are well known in the art, e.g., as described in Sambrook et al, molecular Cloning: A Laboratory Manual, 2 nd edition, volumes 1 to 8, cold Spring Harbor, NY (1989); described in M.W. Pennington and B.M. Dunn, methods in Molecular Biology: peptide Synthesis Protocols, vol.35, humana Press, totawa, N.J. (1994), the contents of both of which are incorporated herein by reference. Peptides can also be chemically synthesized using methods well known in the art. See, e.g., merrifield et al, J.am.chem.Soc.85:2149 (1964); bodanszky, M. Principles of Peptide Synthesis, springer-Verlag, new York, N.Y. (1984); kimmerlin, T.and Seebach, D.J.Pept.Res.65:229-260 (2005); nilsson et al, annu.Rev.Biophys.Biomol.Structure. (2005) 34:91-118; W.C. Chan and P.D. white (eds.) Fmoc Solid Phase Peptide Synthesis: A Practical Approach, oxford University Press, cary, NC (2000); benoiton, chemistry of Peptide Synthesis, CRC Press, boca Raton, FL (2005); jones, amino Acid and Peptide Synthesis, 2 nd edition, oxford University Press, cary, NC (2002); and P.Lloyd-Williams, F.Alberidio 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 may also be as described in U.S. patent No. 4,612,302;4,853,371; and 4,684,620, and as described in U.S. patent application publication No. 2009/0263843, the contents of each of which are incorporated herein by reference.

Production and purification of RBD polypeptides or mutant coronavirus S proteins

RBD polypeptides or mutant coronavirus S proteins (as those terms are used herein) can be produced chemically, e.g., by solution or solid phase peptide synthesis, or semisynthesis 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 may be used in the methods and compositions described herein include a variety of microorganisms and cells that are well known in the recombinant arts and therefore not described in detail herein. These include, for example, various strains of E.coli (E.coli), bacillus (Bacillus), streptomyces (Streptomyces) and Saccharomyces (Saccharomyces), as well as mammalian, yeast and insect cells. If desired, polypeptides as described herein may be produced as peptides or fusion proteins. Suitable vectors for producing peptides and polypeptides are known and available from private and public laboratory and preservation institutions and from commercial suppliers. Then transfected with a recipient cell capable of expressing the gene product. The transfected recipient cells are cultured under conditions that allow expression of the recombinant gene product, which is recovered from the culture. Host mammalian cells, such as chinese hamster ovary Cells (CHO) or COS-1 cells, may be used. These hosts may be used in combination with poxvirus expression vectors (e.g., vaccinia or suipoxviruses). Suitable non-pathogenic viral expression vectors that can be engineered to carry synthetic genes into host cells include poxviral expression vectors, such as vaccinia virus, adenovirus, retrovirus, and the like. Many such non-pathogenic viral expression vectors are commonly used in human gene therapy and as vectors for other vaccine agents and are known and selectable by those of skill in the art. Selection of other suitable host cells and methods for transformation, culture, amplification, screening, and product production and purification can be performed by one of skill in the art with reference to known techniques.

In some embodiments, as described herein, it may be desirable to isolate and/or purify a synthetic 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 homogenizing and coarsely separating cells, tissues or organs at one level into polypeptide and non-polypeptide fractions. Mutant coronavirus spike proteins or receptor binding domain polypeptides may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods which are particularly suitable for the preparation of pure peptides or polypeptides are ion exchange chromatography, gel exclusion chromatography, polyacrylamide gel electrophoresis, affinity chromatography, immunoaffinity chromatography and isoelectric focusing. A particularly effective method of purifying peptides/polypeptides is fast high performance liquid chromatography (FPLC) or even High Performance Liquid Chromatography (HPLC).

"purified polypeptide" is intended to mean a composition that is separable from other components in which the mutant coronavirus S polypeptide or receptor binding domain thereof is purified to any degree relative to the organism producing the recombinant protein or to a naturally available state thereof. Thus, an isolated or purified polypeptide also refers to a polypeptide isolated from an environment in which the polypeptide may naturally occur. In one embodiment, "purified" will refer to a polypeptide composition that has been fractionated to remove various other components, and which substantially retains the ability to bind to coronavirus antibodies that bind to native or wild-type coronavirus S proteins. Where the term "substantially purified" is used, this designation will refer to a composition in which the coronavirus polypeptide forms the major component of the composition, such as a protein comprising about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition.

The general requirements for the polypeptides described herein are not provided 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 may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it will be appreciated that cation exchange column chromatography performed using an HPLC apparatus will typically result in greater "fold" purification than the same technique using a low pressure chromatography system. Methods that exhibit a lower degree of relative purification may have advantages in terms of overall recovery of the protein product or in terms of maintaining the activity of the expressed protein.

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

Coronavirus fusion proteins and scaffolds

In some embodiments, the receptor binding domain polypeptides or mutant coronavirus S proteins described herein comprise fusion proteins. In some embodiments, the RBD polypeptides or mutant coronavirus S proteins described herein are fused to a scaffold, nanoparticle, heterologous protein scaffold, or 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 polypeptides described herein are provided as fusion proteins. Such fusion proteins may comprise, for example, an antigenic moiety to enhance the resulting immune response. Such antigenic moieties may include exogenous molecules, such as carrier proteins that are foreign to the individual to be vaccinated with the fusion proteins described herein. Foreign proteins that activate an immune response and that can be conjugated to a fusion protein 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, more preferably at least about 60,000 daltons. Carrier proteins useful in this context 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, and the like.

Methods of chemically coupling one protein (e.g., RBD polypeptide or mutant coronavirus S protein) to another protein (e.g., carrier or antigenic moiety) are well known in the art and include, for example, conjugation by water-soluble carbodiimides such as 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide hydrochloride, conjugation by homobifunctional crosslinkers having, for example, NHS ester groups or sulfo-NHS ester analogs, conjugation by heterobifunctional crosslinkers 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). The pbVLP may comprise the receptor binding domain polypeptide or a mutant coronavirus S protein, including fusion proteins, wherein the fusion protein comprises a multimerization domain, such as a "first component" as described herein.

VLPs for use with the methods and compositions described herein may comprise multimeric protein assemblies suitable for displaying the extracellular domain of RBD or the extracellular domain of coronavirus spike protein, or antigenic fragments 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 a "first component") can be derived from a naturally occurring protein sequence by substitution of at least one amino acid residue or by addition of an amino acid residue 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 "second component" or 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 set will form trimers related by 3-fold rotational symmetry and the second set will form pentamers related by 5-fold rotational symmetry. In this case, VLPs form "icosahedral particles" with I53 symmetry. These one or more polypeptide groups may be arranged together such that the members of each polypeptide group are related to each other by a symmetry operator. A general computational approach for designing self-assembled protein materials is disclosed in U.S. patent publication No. 2015/0356240A1, the disclosure of which is incorporated herein by reference in its entirety, involving symmetrical docking of protein building blocks in a target symmetrical architecture.

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 comprises a first polypeptide comprising an RBD, a linker, and a first polypeptide comprising a multimerization domain.

Non-limiting embodiments are shown in fig. 6A, which depicts RBDs genetically fused with components of VLPs, optionally recombinantly expressed in host cells (e.g., 293F cells); together with a pentameric protein assembly, which is optionally recombinantly expressed in the same or different host cells (e.g. e.coli cells), the two polypeptide groups self-assemble into VLPs displaying 20 antigen trimers around the icosahedral core.

In some cases, the VLP is 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 mixed heterotrimers of protein antigens from different coronavirus strains.

VLPs for use with the methods and compositions described herein display antigen proteins in a variety of 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 association of two polypeptides. The association may be direct or indirect, reversible or irreversible, weak or strong, covalent or non-covalent, and selective or non-selective.

In some embodiments, attachment is achieved by genetic engineering to produce N-terminal or C-terminal fusion of the antigen to one polypeptide in the set of polypeptides comprising the VLP. Thus, a VLP may consist of, or consist essentially of, one, two, three, four, five, six, seven, eight, nine, or ten sets of polypeptides displaying one, two, three, four, five, six, seven, eight, nine, or ten antigen groups, wherein at least one antigen of said antigen groups is genetically fused to at least one polypeptide of said polypeptide groups. In some cases, VLPs consist essentially of one polypeptide set that is capable of self-assembly and comprises an antigenic protein set genetically fused thereto. In some cases, the VLP consists essentially of: a first set of polypeptides comprising a set of antigens; and a second polypeptide group capable of co-assembling into a bi-component VLP, one polypeptide group linking the antigenic protein to the VLP, and other polypeptide groups facilitating self-assembly of the VLP.

In some embodiments, the attachment is by post-translational covalent attachment between the one or more sets of polypeptides and the one or more sets of antigenic proteins. In some cases, chemical cross-linking is used to non-specifically attach the antigen to the VLP polypeptide. In some cases, chemical cross-linking is used to specifically attach an antigenic protein to a VLP polypeptide (e.g., to a first polypeptide or a 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 linking two proteins may be suitable for use with the presently disclosed VLPs. In particular, chemical methods for producing immunoconjugates or antibody drug conjugates can be used. In some cases, VLPs are created using cleavable or non-cleavable linkers. The process and method for conjugating an antigen to a carrier is provided, for example, by U.S. patent publication No. US 2008/0145373 A1, the disclosure of which is incorporated herein by reference in its entirety.

Components of VLPs of the present disclosure may have any of a variety of amino acid sequences. U.S. patent publication No. US 2015/0356240 A1 (the contents of which are incorporated herein by reference in their entirety) describes various methods for designing protein assemblies. As described in U.S. patent publication No. US 2016/012392 A1 and international patent publication No. WO 2014/124301 A1, the polypeptides are designed to have the ability to self-assemble in pairs to form VLPs, such as icosahedral particles. The design involves designing the appropriate interface residues for each member of the polypeptide pair that can be assembled to form the VLP. The VLPs so formed include symmetrically repetitive, non-native, non-covalent polypeptide-polypeptide interfaces that orient the first assembly and the second assembly into a VLP, such as a VLP having icosahedral symmetry.

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

Non-limiting examples of designed protein complexes that can be used in the protein-based VLPs of the present disclosure include those described in U.S. patent No. 9,630,994; international patent publication No. WO2018187325A1; U.S. patent publication No. 2018/01374234 A1; those disclosed in 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 Table 3

In some embodiments, the VLP comprises: a fusion protein 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 9 to 13 and comprising RBD or coronavirus spike protein as disclosed herein; and 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 to 18.

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

In some embodiments, the polypeptides as disclosed herein comprise the polypeptide sequence of SEQ ID NO. 6 or SEQ ID NO. 7.

RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADFSVLYNSASFSTFKCYG

VSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVI

AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF

NCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGGSGGSG

SGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFA

GGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFI

VSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQ

FVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(SEQ ID NO:6)

RFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADFSVLYNSASFSTFKCYG

VSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVI

AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGF

NCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGGSGGSG

SGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRANSVEEAIEKAVAVFA

GGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFI

VSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQ

FVKAMKGPFPNVKFVPTGGVNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATE(SEQ ID NO:7)。

Protein stability

Most biological products are susceptible to degradation, such as thermal, photochemical or oxidative degradation. Because biological products such as vaccines and insulin need to be distributed worldwide, and because the ambient temperatures vary widely from region to region, vaccines (or stabilized proteins therein) with an extended shelf life compared to rapidly degrading proteins/compositions are preferred. Increased intracellular stability may also provide higher recombinant protein yields. The mutant coronavirus spike proteins described herein, or receptor binding domains thereof, have increased stability compared to their wild-type counterparts. 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 tracing, bleach tracing, cyclohexylamide tracing, circular dichroism spectroscopy, and fluorescence-based activity assays.

Given the stability of a composition, also referred to herein as the "shelf life" of the composition when applied to an isolated preparation or vaccine preparation, depends on the storage conditions of the composition as well as the formulation of the composition (e.g., adding chemical components) or the physical state at which the composition is provided (e.g., lyophilization, drying, freezing, etc.).

The term "lyophilization" or "freeze-dried" as used herein refers to a dehydration process for preserving or making more convenient the storage and transportation of a composition as described herein, commonly referred to as "freeze-drying". Lyophilization works by: the composition is frozen and then the ambient pressure is reduced to sublimate the chilled water in the composition directly from the solid phase to the gas phase. In some embodiments, lyophilization may be used to preserve the vaccine compositions as described herein, allowing the vaccine compositions to be portable and stored at room temperature without refrigeration.

In addition to the increased antigen stability provided by the mutations described herein, the addition of antioxidants or other agents intended to extend the shelf life of vaccine compositions is contemplated herein. In some embodiments, it is contemplated that the compositions described herein are stored at room temperature and do not require refrigeration.

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

Formulations comprising the mutant coronavirus proteins or peptides described herein are stable because their properties change little over a given period of time at a given temperature. In general, a formulation as described herein may be stable for at least about one month. In some embodiments, the formulation is stable 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 formulations are generally stable at temperatures below about 30 ℃. In some embodiments, the stability of the formulation is with respect to a temperature of less than about 25 ℃, about 20 ℃, about 15 ℃, about 10 ℃, about 8 ℃, about 5 ℃, about 4 ℃, or about 2 ℃. Thus, in some embodiments, the temperature is in the range of about 25 ℃ to about 2 ℃, about 20 ℃ to about 2 ℃, about 15 ℃ to about 2 ℃, about 10 ℃ to about 2 ℃, about 8 ℃ to about 2 ℃, or about 5 ℃ to about 2 ℃. In other embodiments, the temperature is in the range of about 25 ℃ to about 5 ℃, about 20 ℃ to about 5 ℃, about 15 ℃ to about 5 ℃, about 10 ℃ to about 5 ℃, or about 8 ℃ to about 5 ℃. In other embodiments, the temperature is in the range of about 25 ℃ to about 8 ℃, about 20 ℃ to about 8 ℃, about 15 ℃ to about 8 ℃, or about 10 ℃ to about 8 ℃. In other embodiments, the temperature is in the range of about 25 ℃ to about 10 ℃, about 20 ℃ to about 10 ℃, about 15 ℃ to about 10 ℃, about 25 ℃ to about 15 ℃, about 20 ℃ to about 15 ℃, or about 25 ℃ to about 20 ℃. In some embodiments, the composition may be stored at 4 ℃ or-20 ℃ to achieve longer term storage.

Vaccine composition

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

In some embodiments, the immunity raised against the coronavirus antigens described herein is durable (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 cycle of the subject). Alternatively, in some embodiments, it is contemplated herein that vaccine formulations described herein are administered on an annual basis, tailored for a epidemic or predicted epidemic strain of a target virus, similar to immunization with an influenza vaccine, and may 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 coronavirus vaccine formulations are not required to have 100% efficacy in conferring community-based or group immunity against one or more coronaviruses. Thus, in some embodiments, the vaccine formulations described herein are at least 40% effective in a vaccinated population of individuals, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% effective in a vaccinated population of individuals.

In some embodiments, to reduce community transmission and prevent/control coronavirus infection and transmission, the vaccines described herein may be administered as a universal vaccination for healthy children and individuals. Children may play an important role in the spread of coronaviruses in schools, households and communities, particularly because they tend to have lighter symptoms and are not necessarily diagnosed as having a coronavirus infection. Influenza vaccine studies have shown that vaccination of about 80% of school-age children in the community has reduced respiratory disease in adults and excessive death in the elderly (Reichert et al, 2001). This concept is known as community immunity or "group immunity" and is believed to play an important role in protecting communities from diseases. Since vaccinated persons have antibodies that neutralize a particular virus, they are much less likely to transmit the virus to others. This concept can also be applied to coronavirus infections. Thus, even non-vaccinated persons (as well as persons whose vaccination has been reduced or whose vaccine is not fully effective) can often be protected by group immunity, since the vaccinated persons around them will not be ill or spread the virus. As the percentage of vaccinated people increases, the population immunity will be more effective. It is believed that about 60% (but preferably more nearly 90-95%) of the people in the community must be protected by the vaccine to achieve group immunity. Non-immunized people can increase the chances that they and others will become infected with the disease.

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

The vaccine formulations described herein may prevent at least one of the symptoms associated with a coronavirus infection, or may completely prevent the appearance of any symptoms. Common symptoms of coronavirus infection include, but are not limited to, fever, chills, cough, shortness of breath/dyspnea, fatigue, muscle/body pain, headache, emerging loss of taste or smell, sore throat, congestion or runny nose, nausea, vomiting or diarrhea. The reduction of symptoms may be determined subjectively or objectively, e.g., by a subject's own assessment, by a clinician, or by making appropriate determinations or measurements (e.g., body temperature, degree of pneumonia infection, lung scarring, etc.), including, e.g., quality of life assessment, slowing of progression of coronavirus infection or additional symptoms, reduction in severity of symptoms of coronavirus-related disease, or suitable determinations (e.g., antibody titer and/or T cell activation assays).

Preferably, the vaccine formulations described herein will reduce the transmission of active coronaviruses from a vaccinated individual to other individuals within the community of individuals 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 the vaccinated individual and two or more individuals).

In some embodiments, the vaccine formulations described herein comprise one or more adjuvants. Non-limiting examples of adjuvants include Freund's complete adjuvant (a nonspecific stimulator of immune response containing killed Mycobacterium tuberculosis (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). Also contemplated is a RIBI containing three components MPL, trehalose Dimycolate (TDM), and Cell Wall Skeleton (CWS) extracted from bacteria in a 2% squalene/Tween 80 emulsion. MF-59 may also be used,MHC antigens.

In one aspect, the adjuvant effect is achieved by using an agent (such as alum) that is used in the form of a phosphate buffered saline solution of about 0.05% to about 0.1%. Alternatively, a vaccine as described herein may be formulated with a synthetic sugar polymerThe blend is used in about 0.25% solution. Some adjuvants, such as certain organic molecules obtained from bacteria, act on the host without acting on itIs an antigen. An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)), which is a bacterial peptidoglycan. In other embodiments, hemocyanin and erythrosine may also be used with the vaccine formulations described herein. Hemocyanin from Keyhole Limpet (KLH) may be used in certain embodiments, but other molluscs and arthropod hemocyanins and hematoxylin may be used in alternative embodiments.

Various polysaccharide adjuvants may also be used. For example, the effects of various pneumococcal polysaccharide adjuvants on mouse antibody responses have been described (Yin et al, 1989). Dosages that produce optimal response or no inhibition should be used as indicated (Yin et al, 1989). Polyamine-based polysaccharides are particularly preferred, such as chitin and chitosan, including chitosan. In another embodiment, lipophilic disaccharide-tripeptide derivatives of muramyl dipeptide may be used, described as being used in artificial liposomes formed from phosphatidylcholine and phosphatidylglycerol.

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

In addition, the purified detoxified endotoxin may be used alone or in a multi-adjuvant formulation to produce an adjuvant response in vertebrates. For example, combinations of detoxified endotoxins with trehalose dimycolate or combinations of detoxified endotoxins with trehalose dimycolate and endotoxin glycolipids are contemplated herein. Alternatively, combinations of detoxified endotoxin with Cell Wall Skeletons (CWS) or CWS and trehalose dimycolate, or combinations of CWS and trehalose dimycolate alone without detoxified endotoxin are also contemplated.

Those skilled in the art will appreciate different kinds of adjuvants that can be conjugated or blended with the vaccines as described herein, and these include Alkyl Lysophospholipids (ALP); BCG; and biotin (including biotinylated derivatives), and the like. Some adjuvants that are particularly contemplated for use are teichoic acid from gram-negative bacterial cells. These adjuvants include lipoteichoic acid (LTA), ribitol Teichoic Acid (RTA), and glycerophospholipid acid (GTA). Active forms of their synthetic counterparts may also be used.

Various adjuvants, even those not commonly used in humans, are contemplated for use in vaccines for other vertebrates.

Vaccine formulations may comprise a coronavirus polypeptide or fusion protein thereof as described herein, microencapsulated or macroencapsulated into an undergarment capsid protein of, for example, bovine rotavirus (Redmond et al, mol. Immunol.28:269 (1991)), into an immunostimulatory molecule (ISCOMS) composed of saponins such as Quil A (Morein et al, nature 308:457 (1984)), morein et al, in Immunological Adjuvants and Vaccines (G. Gregordidis et al) pages 153-162, plenum Press, NY (1987)), or into controlled release biodegradable microspheres composed of, for example, lactide-glycolide copolymers (O 'Hagan et al, immunol 73:239 (1991); O' Hagan et al, vaccine 11:149 (1993)), using methods well known in the art (e.g., liposomes (see, e.g., garcon and Six, J. Immunol.146:3697 (1991)).

The stabilized coronavirus polypeptides described herein, or fusion proteins thereof, may also be adsorbed onto 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 editions), pages 431-449, butterworth-Hinemann, stoneman N.Y. (1992)).

The vaccine formulations described herein comprise an "effective amount" or "therapeutically effective amount" of a stabilized coronavirus polypeptide as described herein or a fusion protein thereof. As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to a dose sufficient to provide a concentration high enough to generate (or contribute to the generation of) an immune response in its recipient. The specific effective dosage level of any particular subject will depend upon a variety of factors including the condition being treated, the severity of the condition, the activity of the particular compound, the route of administration, the rate of clearance of the administered agent, the duration of treatment, the drugs used in conjunction or concurrently with the administered agent, the age, weight, sex, diet and general health of the subject, and like factors well known in the medical arts and sciences. Various general considerations that are considered in determining a "therapeutically effective amount" are known to those skilled in the art and are described, for example, in Gilman et al, editors Goodman and Gilman's The Pharmacological Bases of Therapeutics, 8 th edition, pergamon Press,1990; and Remington's Pharmaceutical Sciences, 17 th edition, mack Publishing co., easton, pa., 1990.

The pH of the formulations may also vary. Typically, the pH of the formulation 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, the pH may also be within certain values. Thus, 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, the formulation may comprise one or more salts, such as sodium chloride, sodium phosphate, or a combination thereof. Typically, each salt is present in the formulation at about 10mM to about 200 mM. Thus, in some embodiments, any salt present is present at 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 200 mM. In other embodiments, any salt present is present at about 10mM to about 175mM, about 20mM to about 175mM, about 25mM to about 175mM, about 30mM to about 175mM, about 40mM to about 175mM, about 50mM to about 175mM, about 75mM to about 175mM, about 100mM to about 175mM, about 125mM to about 175mM, or about 150mM 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 150 mM. 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 125 mM. 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 100 mM. In yet 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 75 mM. In still 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 50 mM. 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 30mM, about 25mM to about 30mM, about 10mM to about 25mM, about 20mM to about 25mM, or about 10mM to about 20 mM. In one embodiment, sodium chloride is present in the formulation at about 100 mM. In one embodiment, sodium phosphate is present in the formulation at about 25 mM.

Formulations comprising the mutant coronavirus proteins described herein may further comprise a solubilizing agent, such as a nonionic detergent. Such detergents include, but are not limited to polysorbate 80%80 Triton x100 and polysorbate 20).

Vaccine administration and efficacy

The 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 detrimental to the vertebrate receiving the composition, and that can be administered without undue toxicity. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia, european pharmacopeia, or other generally recognized pharmacopeia for use in vertebrates, and more particularly in humans. These compositions are useful as vaccine and/or antigen compositions to induce protective immune responses in vertebrates.

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

The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. 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 contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

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

While it is preferred to administer a single dose for substantial immune stimulation, additional doses may be administered by the same or different routes to achieve the desired effect. For example, in newborns and infants, multiple administrations may be required to generate adequate levels of immunity. If necessary, administration may be continued at intervals throughout childhood to maintain a sufficient level of protection against coronavirus infection. Also, adults particularly susceptible to severe disease or recurrent infections (such as, for example, health care workers, day care workers, family members of young children, elderly people, and people with impaired cardiopulmonary function) may require multiple vaccinations 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 secreted and serum antibodies, and adjusting the dose or repeat vaccination as needed to elicit and maintain the desired level of protection.

The prophylactic vaccine formulation may be administered systemically, for example by subcutaneous or intramuscular injection using a needle and syringe or needleless 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. Although any of the above delivery routes may result in an immune response, intranasal administration may bring additional benefits of eliciting mucosal immunity at one of the entry sites for coronaviruses.

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, the vaccine composition as described herein is administered intramuscularly, intravenously, subcutaneously, transdermally, or intradermally. The compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, bladder, and intestinal mucosa, etc.), and may be administered with other bioactive agents. In some embodiments, intranasal or other mucosal routes of administration of vaccine compositions as described herein can induce significantly higher antibodies or other immune responses than other routes of administration. In another embodiment, the intranasal or other mucosal route of administration of the vaccine compositions as described herein may induce antibodies or other immune responses that will induce cross-protection against other coronavirus strains. Administration may be systemic or local. In some embodiments, the vaccine formulation is administered in a manner that targets mucosal tissue in order to elicit an immune response at the immune site. For example, immunization can be targeted to mucosal tissue, such as intestinal associated lymphoid tissue (GALT), by oral administration using a composition containing an adjuvant with specific mucosal targeting properties. Other mucosal tissues, such as nasopharyngeal lymphoid tissue (NALT) and Bronchi Associated Lymphoid Tissue (BALT), may also be targeted.

Vaccine formulations as described herein may also be administered in a dosage regimen, such as initial administration and subsequent booster administration of the vaccine composition. In certain embodiments, the second dose of the composition is administered at any time from two weeks to one year, preferably about 1 month, about 2 months, about 3 months, about 4 months, about 5 months to about 6 months, after the initial administration. Further, the third dose may be administered after the second dose, and about three months to about two years, or even longer, preferably about 4 months, about 5 months, or about 6 months, or about 7 months to about one year, after the initial administration. A third dose may optionally be administered when no or low levels of specific immunoglobulins are detected in the serum and/or urine or mucosal secretions of the subject following the second dose. In a preferred embodiment, the second dose is administered about one month after the first administration and the third dose is administered about six months after the first administration. In another embodiment, the second dose is administered about six months after the first administration.

The dosage of the pharmaceutical formulation can be readily determined by the skilled person, for example by first determining the dosage effective to elicit a prophylactic or therapeutic immune response, for example by measuring the serum potency of a virus-specific immunoglobulin or by measuring the inhibition of antibodies in a serum sample, urine sample or mucosal secretion. The dose may be determined from animal studies. A non-limiting list of animals used to study coronaviruses includes guinea pigs, syrian hamsters, chinchiles, hedgehog, chickens, rats, mice, pigs, cattle, bats, and ferrets. Bats are particularly considered to be the natural host of coronaviruses and are particularly useful in the study or testing of vaccines. However, any of the above animals may be dosed with a vaccine formulation as described herein to partially characterize the immune response induced, and/or to determine whether any neutralizing antibodies have been raised.

Furthermore, human clinical studies may be performed by the skilled person to determine a preferred effective dose to the human. Such clinical studies are routine and well known in the art. The exact dosage employed will also depend on the route of administration. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal test systems.

Another method of inducing or enhancing an immune response may be accomplished by: vaccine compositions as described herein are prepared to include one or more immunostimulants, such as one or more cytokines, lymphokines or chemokines (e.g., interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13) with immunostimulatory, immunopotentiating, and/or pro-inflammatory activity), growth factors (e.g., granulocyte-macrophage (GM) -Colony Stimulating Factor (CSF)), and other immunostimulatory molecules, such as macrophage inflammatory factors, flt3 ligands, B7.1, B7.2, and the like. Such immunostimulatory molecules may be administered in the same formulation as the coronavirus vaccine formulation, or may be administered separately.

The RBD polypeptides or stabilized coronavirus spike proteins described herein can be used in vaccine formulations that, when administered to a vertebrate, can induce substantial immunity in the vertebrate (e.g., a human). The substantial immunity results from an immune response against the RBD polypeptide or the stabilized coronavirus spike protein and prevents or ameliorates coronavirus infection or at least alleviates symptoms of coronavirus infection in the vertebrate. In some cases, the vaccinated subject that is subsequently infected will be asymptomatic. However, this reaction may not be a fully protective reaction, and partial immune reactions are also contemplated herein. For example, a partially protected subject subsequently infected with coronavirus will experience a reduced severity of symptoms or a shorter duration of symptoms than an unimmunized vertebrate. For example, a subject vaccinated with a formulation comprising SARS-CoV-2 stabilized spike protein may not need a long hospital stay or use of a ventilator to treat COVID-19.

In one embodiment, provided herein is a method of 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 or stabilized coronavirus S protein as described herein. In another embodiment, the induction of substantial immunity shortens the duration of symptoms of a coronavirus-related disease (e.g., SARS, COVID-19).

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

Kit for detecting a substance in a sample

Also provided herein are kits for vaccination of an individual or animal comprising one or more containers filled with one or more of the components of the vaccine formulations as described herein. In one embodiment, the kit comprises two containers, one container containing the stabilized coronavirus polypeptide or fusion protein thereof and the other container containing the adjuvant. Associated with such containers may be a notice in the form prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The vaccine formulation is packaged in a hermetically sealed container, such as an ampoule or pouch that notes the amount of the composition. In one embodiment, the vaccine composition is supplied as a liquid, in another embodiment as a dry sterile lyophilized powder or anhydrous concentrate in a hermetically sealed container, and may be reconstituted to an appropriate concentration, for example with water or saline, for administration to a subject. In some embodiments, the vaccine composition is supplied as a dry sterile lyophilized powder in a hermetically sealed container in 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 vaccine composition may be administered in a unit dose of 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 polypeptide 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, vaccine compositions as described herein are supplied in liquid form in hermetically sealed containers that indicate the amount and concentration of the composition. Preferably, the liquid form of the vaccine composition is supplied in a hermetically 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 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 at least two mutations relative to the RBD of SEQ ID NO:1, wherein said 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 at 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 Blast-p parameters of protocol 1 or protocol 2.

2. 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 or to corresponding residues of a second coronavirus receptor binding domain determined by sequence alignment of SEQ ID NO:1 with the sequence of the second coronavirus receptor binding domain using 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 to corresponding residues in the 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 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 corresponding residues of said second coronavirus receptor binding domain as determined by sequence alignment of SEQ ID NO:1 with said sequence of said second coronavirus receptor binding domain using Blast-p parameters of protocol 1 or protocol 2.

4. The polypeptide of 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; 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 residue of the second coronavirus determined by sequence alignment of SEQ ID NO:1 with the sequence of the second coronavirus receptor binding domain using Blast-p parameters of protocol 1 or protocol 2.

5. The polypeptide of any one of paragraphs 2-4, further comprising an additional amino acid residue beyond the RBD of SEQ ID NO. 1.

6. The polypeptide of any of paragraphs 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 paragraphs 2-6, wherein amino acids 338 and 365 in SEQ ID NO. 1; 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 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 paragraphs 2-7, wherein expression of the polypeptide is increased when expressed in a cell as compared to expression of the wild-type RBD polypeptide lacking the at least two mutations.

9. The polypeptide of any one of paragraphs 2-8, wherein the polypeptide binds to a coronavirus antibody or 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 paragraphs 9 or 10, wherein the receptor for the coronavirus corresponding to the polypeptide comprises an Angiotensin Converting Enzyme (ACE) receptor.

12. The polypeptide of paragraph 11 wherein the ACE receptor is an ACE2 receptor.

13. The polypeptide of 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 one 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 of any one of paragraphs 1-17 and a pharmaceutically acceptable carrier.

19. The composition of paragraph 18 further comprising an adjuvant.

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

21. The composition of any one 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 comprise at least one cavity-filling mutation and at least one second mutation.

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

25. The coronavirus polypeptide according to any one of paragraphs 22-24, wherein the at least one cavity filling mutation comprises a mutation at a residue within residues 328-531 of SEQ ID NO:1, or a mutation at a 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 parameters of protocol 1 or protocol 2.

26. The coronavirus polypeptide according to any one of paragraphs 22-25, wherein the at least one cavity filling mutation comprises a mutation of the 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 sequence alignment of SEQ ID No. 1 with the sequence of the second coronavirus spike protein subunit 1 using Blast-p parameters of protocol 1 or protocol 2.

27. The coronavirus polypeptide of any one 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 a corresponding residue of second coronavirus spike protein subunit 1 as determined by sequence alignment of SEQ ID No. 1 with the sequence of the second coronavirus using Blast-p parameters of protocol 1 or protocol 2.

28. The coronavirus polypeptide of any one of paragraphs 22-27, wherein the at least one cavity filling mutation and the at least one second mutation are at residues 338 and 365 of SEQ ID No. 1; 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 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 parameters of protocol 1 or protocol 2.

29. The 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 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 a corresponding residue selected from 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 parameters of protocol 1 or protocol 2.

30. The coronavirus polypeptide according to any one 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 to the receptor binding domain sequence 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 parameters of protocol 1 or protocol 2.

31. The coronavirus spike protein subunit 1 polypeptide of any one of paragraphs 22-30, wherein amino acids 338 and 365 in SEQ ID No. 1; 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 of the second coronavirus receptor binding domain are the only 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 expression of the coronavirus polypeptide is increased when expressed in a cell as 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. The coronavirus polypeptide of any one of paragraphs 22-33, wherein the coronavirus polypeptide binds to a coronavirus antibody or binds to a cognate coronavirus receptor.

35. The coronavirus polypeptide of paragraph 34 wherein the coronavirus antibody comprises a SARS-CoV-2 antibody.

36. The coronavirus polypeptide of paragraph 35 wherein the homologous coronavirus receptor comprises an Angiotensin Converting Enzyme (ACE) receptor.

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

38. The coronavirus polypeptide of any one of paragraphs 22-37, wherein the coronavirus polypeptide is an engineered mutant polypeptide 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.

39. The coronavirus polypeptide according to 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. A coronavirus polypeptide according to paragraph 40, wherein said coronavirus polypeptide or said 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. The composition of paragraphs 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 one of paragraphs 43-45, wherein the composition is formulated as a vaccine.

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

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

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

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

51. A coronavirus spike protein comprising the polypeptide of 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 comprise:

algorithm: blastp (protein-protein BLAST)

Expected threshold: 0.1

Word length: 6

Maximum match within query range: 0

Matrix: BLOSUM62

Vacancy cost:

the presence is: 11

Extension: 1

Filtering low complexity regions? : whether or not

Masking:

is only for look-up tables? : whether or not

Lower case letters? : and (3) if not.

53. The method or composition of any of the preceding paragraphs, wherein the Blast-p parameters of protocol 2 comprise:

blastp-query, fasta-topic sbjct, fasta-matrix BLOSUM 62-value 0.1-word length 6-gap open 11-gap extend 1-output results, txt

(blastp-query query.fasta-subject sbjct.fasta-matrix BLOSUM62-evalue 0.1-

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

54. A polypeptide comprising a coronavirus Receptor Binding Domain (RBD) comprising a mutation in the coronavirus polypeptide or variant thereof relative to SEQ ID No. 1 selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M and F515L.

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, N F, 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, N F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L.

58. A 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 one of paragraphs 54-58, wherein the polypeptide comprises a heterologous protein scaffold.

60. The polypeptide of paragraph 58, wherein the heterologous protein scaffold has at least 90%, at least 95%, or at least 98% identity 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. A 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 the polypeptide of 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 to any one of SEQ ID NOs 13-18.

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

65. The vaccine composition of 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 one of paragraphs 54-62.

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

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

70. A method of preparing a vaccine, the method comprising combining the polypeptide of any one 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 the polypeptide of 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; optionally an 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 at least one mutation relative to the RBD of SEQ ID NO:1, wherein said at least one mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367 of SEQ ID NO 1 with respect to its use in a Blast-p parameter of protocol 1 or protocol 2 the corresponding sites in the second coronavirus reference sequence 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 parameter of protocol 1 or protocol 2, with D, N501F, N501W, G502D, N501 with respect to its use in a Blast-binding domain of SEQ ID NO 1 with respect to its use in a method of making a pharmaceutical composition for the treatment of a viral infection.

73. The polypeptide of 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/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.

Examples

Example 1

An ideal protein-based coronavirus vaccine must be both efficient and scalable to manufacture, with the latter property being largely affected by vaccine yield and stability. Genetic vaccines using RNA or DNA immunization have also been more frequently explored for SARS-CoV-2, the efficacy of which is affected by the level of target antigen expression. Many coronaviruses, particularly SARS-CoV-2 and related sarbecoviruses (subgenera of coronaviruses), have been considered very valuable domain-based vaccine targets due to the isolation of many strongly neutralized RBD-directed antibodies. While RBD is suitable for production in a variety of forms, limitations in its yield and stability may prevent scalable manufacturing and distribution of RBD-based protein vaccines.

Exemplary mutations in coronavirus Receptor Binding Domain (RBD) polypeptides designed to increase the yield and stability of immunogens containing such coronavirus RBDs are provided herein. Immunogens containing receptor binding domains are generated using a stabilized collection of mutations that exhibit highly improved expression and/or yield in addition to improved stability in solution compared to equivalent immunogens having native (i.e., wild-type) RBD sequences. As will be appreciated by those skilled in the art, increased expression of a given protein may be due in part to increased stability of the protein. The designed immunogen was antigenically intact as verified by SARS-CoV-2 directed antibodies and ACE2 receptor. Overall, these sets of mutations allow for an improved ability to extendably make vaccines against multiple coronaviruses, which may also contribute to the performance of genetic vaccines against RBD.

Base sequence and number of mutations: 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 SARS-CoV-2 sequence is shown in bold underlined text (SEQ ID NO: 2): the following sequences (or receptor binding domains thereof; SEQ ID NO: 2) may be aligned with at least a second coronavirus sequence.

An exemplary list of mutations that can enhance both yield and stability of coronavirus proteins is provided in the following list (all amino acid residue numbers are based on the "base sequence" described above (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.I 356F/Y365W, and

20.I358F/F392W。

materials and methods

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

The sequences of the components I53-50 trimer "A" are:

MKMEELFKKHKIVAVLRANSVEEAIEKAVAVFAGGVHLIEITFTVP

DADTVIKALSVLKEKGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLD

EEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQ

FVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVK

GTPDEVREKAKAFVEKIRGCTE(SEQ ID NO:3)

all sequences were preceded by a "MGILPSPGMPALLSLVSLLSVLLMGCVAETGT" secretion signal and labeled 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 a helix sequence "EKAAKAEEAAR" to increase antigen accessibility. All cysteines in the I53-50 trimer component were mutated to alanine. The human ACE2 ectodomain is genetically fused to a sequence encoding a thrombin cleavage site and a human Fc fragment at the C-terminus. hACE2-Fc was synthesized and cloned by GenScript using the BM40 signal peptide. Genes encoding the CR3022 heavy and light chains were purchased from GenScript and cloned into pCMV/R.

All proteins were expressed in an Expi293F expression medium (Life Technologies) at 33 ℃, 70% humidity, 8% CO ₂ And spun at 150rpm, generated in the Expi293F cells grown in suspension. Transfection of cell cultures with PEI-MAX (Polyscience), cell productionUp to a density of 300 tens of thousands of cells/mL and cultured for 3 days. Supernatant was clarified by centrifugation at 4000rcf, adding PDADMAC to a final concentration of 0.0375% (Sigma Aldrich), and spinning a second time at 4000 rcf.

His-tagged proteins were purified from clarified supernatants via batch-binding, where 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 with 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 eluted with 3 column volumes of 20mM Tris pH 8.0, 300mM NaCl, 300mM imidazole. Clear supernatants of monoclonal antibody and human ACE2-Fc expressing cells were purified on AKTA Avant150 FPLC (Cytiva) using a MabSelect prism A2.6X15 cm column (Cytiva). Bound antibody was washed with five column volumes of 20mM NaPO4, 150mM NaCl pH 7.2, then with five column volumes of 20mM NaPO4, 1M NaCl pH 7.4, and eluted with three column volumes of 100mM glycine pH 3.0. The eluate was neutralized to a final concentration of 50mM with 2M Trizma base.

Biological layer interferometry: to measure the secretion levels of proteins in the supernatant, all supernatants from protein expression were diluted 1:10 into KB1 (25 mM 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 forte Bio/Sartorius) using an 8-channel Octet system (Pall forte Bio/Sartorius) for 300 seconds. After 60 seconds of baseline collection 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 KB 1. For affinity measurements, all proteins and antibodies were diluted at 200 μl/well into phosphate buffered saline containing 0.5% bovine serum albumin and 0.01% tween-20 (KB 2) in black 96-well Greiner Bio-one microwell plates, with separate buffers used for the baseline and dissociation steps. CV30 or CR3022IgG at 10 μg/mL was loaded onto a pre-hydrated protein A biosensor (Pall forte Bio/Sartorius) for 150s followed by a baseline of 60 s. The biosensor was then transferred to a step associated with one of five serial two-fold dilutions of wild-type or stabilized monomeric RBD for 120s, where 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 CR 3022. After association, the biosensor was transferred to buffer for 300s of dissociation. The baseline was subtracted from the data from the association and dissociation steps and kinetic measurements were calculated globally across all five serial dilutions of RBD using a 1:1 binding model (fortebio analysis software, version 12.0). For the fractionated antigenicity measurement of nanoparticle immunogens, hACE2-Fc (dimeric receptor) and CR3022IgG were assayed for binding to monomeric RBD and RBD-I53-50 nanoparticles at ambient temperature with shaking at 1000 rpm. Protein samples were diluted to 100nM in kinetic buffer (Pall forttiebio/Sartorius). The buffer, antibodies, receptors and immunogens were then applied to a black 96-well Greiner Bio-one microplate at 200 μl/well. Protein A biosensors were first hydrated in kinetic buffer for 10min and then immersed in hACE2-Fc or CR3022 diluted to 10. Mu.g/mL in kinetic buffer in a immobilization step. After 500s, tips were transferred to Kinetics buffer for 90s to reach baseline. The association step is performed by immersing the loaded biosensor in the immunogen for 300s, and the subsequent dissociation step is performed by immersing the biosensor back in the hydrodynamic buffer for an additional 300 s. The baseline was subtracted from the data before mapping using the forte Bio analysis software (version 12.0).

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

SYPRO orange fluorescence: 5000 XSYPRO orange protein gel stain (Thermo Fisher) was diluted to 25mM Tris pH 8.0, 150mM NaCl, 5% glycerol and further added to monomeric RBD prepared in the same buffer, where the final concentration of RBD was 1.0mg/mL and the final concentration of SYPRO orange was 20X. Samples were loaded into unclle nano DSF (UNChained Laboratories) and 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 the purified individual nanoparticle component was determined by measuring absorbance at 280nm using a UV/vis spectrophotometer (Agilent Cary 8454) and calculating the extinction coefficient. The assembly step is performed at room temperature, with the following additions: wild-type or stabilized RBD-I53-50A trimeric fusion protein, followed by the addition of an appropriate amount of 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, wherein the molar ratio of RBD-I53-50A: I53-50B.4PT1 is 1.1:1. An appropriate amount of buffer contains 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 2-8deg.C for at least 30min, followed by subsequent purification by SEC to remove residual unassembled components. Nanoparticle production was performed using a Superose 6 Increate10/300 GL column. The assembled particles were eluted at about 11mL on a Superose 6 column. The assembled nanoparticles were sterile filtered (0.22 μm) immediately before column application and after SEC fractions were combined.

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

Dynamic light scattering: dynamic Light Scattering (DLS) was used to measure hydrodynamic diameter (Dh) and polydispersity (% Pd) of RBD-I53-50 nanoparticle samples at UNcle (UNchained Laboratories). Samples were applied to 8.8 μl quartz capillary cartridges (UNi, UNchained Laboratories) and 10 acquisition measurements were made using laser automated decay for 5s each. Viscosity increase due to 5% v/v glycerol in RBD nanoparticle buffer was explained by unclle Client software in Dh measurement.

Hydrogen/deuterium exchange mass spectrometry: 3mg of RBD-I53-50A, rpk4-I53-50A and Rpk9-I53-50A trimer were buffered in deuteration buffer (pH 7.5, 85% D ₂ O, cambridge Isotope Laboratories, inc.) H/D exchange (HDX) was performed at 22 ℃ for 3 seconds, 15 seconds, 60 seconds, 1800 seconds and 72000 seconds, respectively. The exchanged samples were then mixed at 1:1 with ice-cold quench buffer (200 mM tris (2-chloroethyl) phosphate (TCEP), 8M 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 G2-Si mass spectrometer using a loading system that maintained all columns, loops, valves and lines at 0 ℃. Frozen samples were thawed on ice and loaded onto an immobilized pepsin column (2.1×50 mm) with a flow of 200mL/min of 0.1% trifluoroacetic acid (TFA) and 2% acetonitrile. Peptides were captured on a Waters CSH C18 capture column (2.1X105 mm) and then resolved on a Waters CSH C18 1X 100mm 1.7 μm column with a linear gradient from 3% to 40% B (A: 98% water, 2% acetonitrile, 0.1% FA, 0.025% TFA; B:100% acetonitrile, 0.1% FA, flow rate 40 mL/min) over 18 min. A series of washes was performed between sample runs to minimize carryover. Unless otherwise specified, all water and organic solvents used were of MS grade (Optima ^TM Fisher). Non-deuterated sample by digestion from pepsinLC eluate, speed vac dried, incubated in deuteration buffer at 85 ℃ for 1 hour and quenched identically to all other HDX samples, complete deuteration control was performed for each sample series. Internal exchange standards (Pro-Pro-Pro-Ile [ PPPI ] were added to each sample]And Pro-Pro-Pro-Phe [ PPPF]) To ensure consistent labeling conditions for all samples. Peptide use of DriftScope ^TM (Waters) manual verification and authentication using orthogonal retention time and drift time coordinates. Deuterium uptake analysis was performed using HX-Express v 2. Peaks were identified from the peptide spectra based on the peptide m/z values and a binomial fit was applied. Deuterium uptake levels were normalized to the fully deuterated control.

Immunization of mice: female BALB/c (stock number: 000651) mice were purchased from The Jackson Laboratory, bar Harbor, maine at 4 weeks of age and maintained in Comparative Medicine Facility approved by the International Association of laboratory animal Care (AAALAC) of Washington university of Seattle, washington. At 6 weeks of age, 6 mice per dosing group were vaccinated with a primary vaccine and three weeks later mice received a second vaccination boost. Prior to vaccination, the immunogen suspension was gently mixed with AddaVax adjuvant (Invivogen, san Diego, calif.) at 1:1 volume/volume to achieve a final concentration of either 0.009mg/mL or 0.05mg/mL antigen. Under isoflurane anesthesia, mice were injected intramuscularly into the gastrocnemius muscle of each hindleg using a 27 gauge needle (BD, san Diego, CA), with 50 μl (100 μl total) of immunogen injected per injection site. To obtain serum, all mice were bled two weeks after primary and booster immunization. Blood was collected via a subchin venipuncture and allowed to stand in a 1.5mL plastic eppendorf tube at room temperature for 30min to effect clotting. Serum was separated from hematocrit by centrifugation at 2,000g for 10 min. Complement factors and pathogens in the isolated serum were heat inactivated via incubation at 56 ℃ for 60 min. Serum was stored at 4 ℃ or-80 ℃ prior to use. All experiments were conducted at the university of washington, seattle, washington, according to the approved institutional animal care and use committee (Institutional Animal Care and Use Committee) protocol.

ELISA: binding of mouse serum to the delivered antigen was determined using an enzyme-linked immunosorbent assay (ELISA). Briefly, maxisorp (Nunc) ELISA plates were coated overnight at 4℃with 0.08. Mu.g/mL protein of interest per well in 0.1M sodium bicarbonate buffer (pH 9.4). Plates were then blocked with dry milk powder (BioRad) in 4% (w/v) solution in TBS with 0.05% (v/v) Tween 20 (TBST) for 1 hour at room temperature. Serial dilutions of serum were added to the plates and after washing, antibody binding was revealed using catalase-conjugated equine anti-mouse IgG antibodies. Plates were then thoroughly washed in TBST, colorimetric substrates (TMB, thermo Fisher) were added and absorbance at 450nm was read. Area Under Curve (AUC) calculations were generated by adding the trapezoidal area generated between adjacent absorbance measurement pairs and baseline.

Pseudovirus neutralization assay: spike pseudotyped lentivirus neutralization assay uses spike HDM_spike δ21_D614G available from Addgene (# 158762) or BEI (NR-53765), where the complete sequence can be obtained over the world Wide Web at www.addgene.org/158762 for errors-! The hyperlink references are not valid. ). Briefly, 293T-ACE2 cells (BEI NR-52511) were isolated at 1.25X10 ⁴ Individual cells/well were inoculated in 50ul d10 growth medium (containing 10% heat-inactivated FBS, 2mM L-glutamine, 100U/mL penicillin and 100 μg/mL streptomycin) in poly L-lysine coated black wall clear bottom 96 well plates (Greiner 655930). The following day, the mouse serum samples were heat-inactivated at 56 ℃ for 30min, then serially diluted in D10 growth medium. The spike pseudotyped lentiviruses were diluted 1:50 to yield about 200,000RLU per well and incubated with serum dilutions for 1 hour at 37 ℃. mu.L of the virus-serum mixture was then added to the cells and luciferase activity was measured after about 52 hours using the Bright-Glo luciferase assay system (Promega, E2610). The neutralization assay for each batch 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 as compared to two "serum-free" control wells in the same row of the plate. The "neutecurve" package (available on the world wide web at jbloom lab. Gitub. Io/neutecurve, version 0.5.2) was used to calculate 50% inhibition concentration (IC ₅₀ ) And 50% neutralization potency (NT ₅₀ ) For a pair ofFor each serum sample, it was simply 1/IC by fitting Hill curve with bottom fixed at 0 and top fixed at 1 ₅₀ 。

Quantification and statistical analysis: multiple sets of comparisons were performed using the nonparametric Kruskal-Wallis test in GraphPad Prism 8 and Dunn post hoc analysis. When the P value is less than 0.05, the difference is considered significant.

Example 2

Materials and methods

Cell lines

The Expi293F cells were derived from the HEK293F cell line, a female human embryonic kidney cell line transformed and adapted to suspension growth (Life Technologies). The Expi293F cells were grown in Expi293 expression medium (Life Technologies) at 36.5℃and 8% CO ₂ And culturing with shaking at 150 rpm. VeroE6 is a female kidney epithelial cell from african green monkey. HEK-ACE2 adherent cell lines were obtained by BEI Resources, NIAID, NIH: human embryonic kidney cells (HEK 293T), HEK293T-hACE2 cell lines, NR-52511 expressing human angiotensin converting enzyme 2. All adherent cells were treated with 8% CO at 37℃ ₂ Cultures were carried out in flasks with dmem+10% FBS (Hyclone) +1% penicillin-streptomycin. Cell lines other than Expi293F were not tested for mycoplasma contamination nor validated.

A mouse

Female BALB/c mice (four weeks old) were obtained from Jackson Laboratory, bar Harbor, maine. Animal surgery is performed under the approval of the institutional animal care and use committee at the university of washington, seattle.

Design of stabilizing mutations

All calculations in Rosetta were performed using version v2020.22-dev 61287. All design traces assess RBD in a closed symmetrical trimer conformation observed in the low temperature EM structure of spike (PDB 6 VXX) and in the context of the crystal structure of RBD (PDB 6YZ 5). Triple symmetry axis of PDB entry 6VX is aligned with [0, 1 ]]Aligned and individual protomers were saved in the. Pdb format. RBD monomers from PDB 6YZ5 are structurally superimposed and similarly conserved with the original PDB 6 VXX. Writing design protocols using Rosettascripts (58, 59)The design protocol takes as input aligned precursors and custom resiles, which specify the side chain properties and conformation that are sampled during design. The protocol applies a two-round design to the symmetric model based on the input profile, with side-chain minimization applied after each design step. This protocol allows skeleton minimization to be performed simultaneously with side chain minimization and trajectories to be performed with or without skeleton minimization. Allowing both the design step and the minimization step to repackage or minimize all of the mutated or packagable residues listed in the resfileResidues within. Residue positions were manually picked based on spike and RBD structures to include positions 358, 365, 392 and surrounding residues, and the 'PIKAA' option was used to assign possible residue characteristics for each position in the profile. The resfile inputs are diversified to include various combinations of I358F, Y365F, Y365W and/or F392W, while also limiting or allowing mutation to surrounding residues. Further resfile inputs are similarly set, but do not limit positions 358, 365, and 392 to particular characteristics. The design model and score were manually examined to identify interactions that seemed to be favorable in structure. If the mutation buries polar groups that are naturally exposed to solvents or participate in hydrogen bonding, the mutation is discarded. Mutations to surface exposed residues are not frequently considered in order to prevent accidental alteration of antigenicity. The advantageous set of mutations is retested iteratively from the optimized profiles and manually refined to complete a different set of designs.

Plasmid construction

Wild-type and stabilizing sequences of SARS-CoV-2RBD were genetically fused to the N-terminus of trimeric I53-50A nanoparticle component using a linker having 16 glycine and serine residues. Each I53-50A fusion protein also carries a C-terminal octahistidine tag, and the monomer sequence comprises both Avi and octahistidine 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 using the BM40 signal peptide. HexaPro-fold sub-constructs for immunization studies were produced as described (Hsieh et al Science 369:1501-05 (2020)) and placed in octahistidine-tagged pCMV/R. The HexaPro-folding sub construct used for expression and stability comparison with and without Rpk9 mutation contained BM40 signal peptide and was placed in pCMV/R. The plasmid was transformed into NEB 5 a strain of escherichia coli (New England Biolabs) for subsequent DNA extraction from the bacterial culture (NucleoBond Xtra Midi kit) to obtain a 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 through SEQ ID NO. 47.

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

SEQ ID NO:21

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRF

ASVYAWNRKRISNCVADY

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG

KIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk1-I53-50A trimer; SEQ ID NO. 22

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk2-I53-50A trimer; SEQ ID NO. 23

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPLGEVFNATRFASVYAWNRKRISNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

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

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk5-I53-50A trimer; SEQ ID NO. 26

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk6-I53-50A trimer; SEQ ID NO. 27

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPMGEVFNATRFASVYAWNRKRISNCVLDF

SVLYNSASFSTVKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk7-I53-50A trimer; SEQ ID NO. 28

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk8-I53-50A trimer; SEQ ID NO. 29

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCFTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk9-I53-50A trimer; SEQ ID NO. 30

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

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

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

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

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

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

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk16-I53-50A trimer; SEQ ID NO. 37

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRFSNCVADW

SVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGGSHHHHHHHH

Rpk17-I53-50A trimer; SEQ ID NO. 38

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRFASVYAWNRKRFSNCVADF

SVLYNSASFSTFKCYGVSPTKLNDLCWTNIYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGGSGGSGSGGSGGSGSEKAAKAEEAARKMEELFKKHKIVAVLRA

NSVEEAIEKAVAVFAGGVHLIEITFTVPDADTVIKALSVLKEKGAIIGAGTV

TSVEQARKAVESGAEFIV

SPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGP

QFVKAMKGPFPNVKFVPTGG

VNLDNVAEWFKAGVLAVGVGSALVKGTPDEVREKAKAFVEKIRGATEGG

SHHHHHHHH

RBD monomers (with Avi and hexahistidine tags); SEQ ID NO. 39

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRF

ASVYAWNRKRISNCVADY

SVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTG

KIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG

FNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGLNDIFEAQKIEWHEHHHHHHHH > Rpk4 monomer (with Avi and hexahistidine tag); SEQ ID NO. 40

MGILPSPGMPALLSLVSLLSVLLMGCVAETGTRFPNITNLCPFGEVFNATRF

ASVYAWNRKRISNCVADY

SVLYNSASFSTFKCYGVSPTKLNDLCWTNVYADSFVIRGDEVRQIAPGQT

GKIADYNYKLPDDFTGCVIA

WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEG

FNCYFPLQSYGFQPTNGVGY

QPYRVVVLSFELLHAPATVCGPKKSTGLNDIFEAQKIEWHEHHHHHHHH > Rpk9 monomer (with Avi and hexahistidine tag); 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-foldlon 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-fold for expression and stability comparison with Rpk 9-Hexapro-fold (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

Rpk 9-Hexapro-fold (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

LNEVAKNLNESLIDLQELGKYEQGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGRSLEVLFQGPGHHHHHH

HH

>hACE2-FC；SEQ ID NO:47

MARAWIFFLLCLAGRALASTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKWS

AFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQE

CLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQLRPLYEEYVVLKNE

MARANHYEDYGDYWRGDYEVN

GVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGC

LPAHLLGDMWGRFWTNLYS

LTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWE

NSMLTDPGNVQKAVCHPTAWD

LGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYAAQPFLLRNGANEG

FHEAVGEIMSLSAATPKHLKS

IGLLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKD

QWMKKWWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTL

YQFQFQEALCQAAKHEGPLHKCDISNSTEAGQKLFNMLRL

GKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQNKNSFVGWST

DWSPYADPLVPRGSGGGGDPEP

KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH

EDPEVKFNWYVDGVEVHNA

KTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

KAKGQPREPQVYTLPPSRDE

LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

KLTVDKSRWQQG

Transient transfection

In the use of an Expi293F expression cultureNutrient medium (Life Technologies), at 33deg.C, 70% humidity, 8% CO ₂ SARS-CoV-2S and ACE2-Fc proteins were produced in the suspension grown Expi293F cells by rotation at 150 rpm. Cells were grown to a density of 300 ten thousand cells/mL using PEI-MAX (Polyscience) transfection cultures 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.

Genes encoding the CV30 and CR3022 heavy and light chains were purchased from GenScript and cloned into pCMV/R. Antibodies were expressed by transiently co-transfecting both heavy and light chain plasmids in Expi293F cells using PEI MAX (Polyscience) transfection reagents. Cell supernatants were harvested and clarified as described above after 3 or 6 days.

Purification of glycoproteins

His-tagged proteins were purified from clarified supernatants via batch-binding, where 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 with 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 10 column volumes of 20mM Tris pH 8.0, 300mM NaCl and the binding protein eluted with 3 column volumes of 20mM Tris pH 8.0, 300mM NaCl, 300mM imidazole. The batch binding process was then repeated for the same supernatant sample and the first and second eluents were combined. Purity was assessed using SDS-PAGE. To quantify the yield of RBD-based constructs, IMAC eluate extracted from comparable cell culture conditions and volumes was supplemented with 100mM L-arginine and 5% glycerol and concentrated to 1.5mL. The concentrated samples were then loaded into a 1mL loop and applied to either a Superdex 75 Increate 10/300GL column (for monomeric RBD) or a Superdex 200 Increate 10/300GL column (for fusion of RBD with I53-50A trimer) pre-equilibrated with 50mM Tris pH8, 150mM NaCl, 100mM L-arginine, 5% glycerol. To quantify the yield of the HexaPro-folding sub-construct with and without Rpk9 mutation, IMAC eluate extracted from comparable cell culture conditions and volumes was supplemented with 5% glycerol and concentrated to 1.5mL, which was then loaded into a 1mL loop and applied to a Superose 6increase 10/300GL column pre-equilibrated with 50mM Tris pH 8.0, 150mM NaCl, 0.25% w/v L-histidine, 5% glycerol. HexaPro-foldon for immunization studies was purified by IMAC and dialyzed against 50mM Tris pH 8.0, 150mM NaCl, 0.25% w/v L-histidine, 5% glycerol three times at room temperature for four hours.

Thermal denaturation (nanometer DSF)

RBD-based samples were prepared in buffer containing 50mM Tris pH8, 150mM NaCl, 100mM L-arginine, 5% glycerol, while HexaPro-foldon-based samples were prepared in buffer containing 50mM Tris pH8.0, 150mM NaCl,0.25%w/v L-histidine, 5% glycerol. The non-equilibrium melting temperature is that of UNcle ^TM (UNchained Labs) was determined based on the barycentric mean of intrinsic tryptophan fluorescence emission spectra collected from 20-95 ℃ using a thermal ramp of 1 ℃/min. The melting temperature is defined as the maximum point of the first derivative of the melting curve, where the first derivative is calculated using GraphPad Prism software after smoothing the four neighboring points using a second order polynomial set-up.

SYPRO orange fluorescence

5000 XSYPRO ^TM Orange protein gel stain (Thermo Fisher) was mixed diluted to 25mM Tris pH8.0, 150mM NaCl, 5% glycerol and further added to monomeric RBD prepared in the same buffer, wherein the final concentration of RBD was 1.0mg/mL and SYPRO ^TM The final concentration of orange was 20×. Loading a sample into a unclle ^TM Nano DSF (UNChained Laboratories), and adding SYPRO to the sample ^TM Fluorescence emission spectra were collected for all samples after orange for 5 min.

Microbial protein 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. 2016/012392 (the disclosure of which is incorporated herein by reference in its entirety), and the same protocol was used for purification of the 2OBX unassembled control pentamer.

In vitro nanoparticle assembly

The total protein concentration of the purified individual nanoparticle component was determined by measuring absorbance at 280nm using a UV/vis spectrophotometer (Agilent Cary 8454) and calculating the extinction coefficient. The assembly step is performed at room temperature, with the following additions: wild-type or stabilized RBD-I53-50A trimeric fusion protein, followed by the addition of an appropriate amount of buffer as needed to achieve the desired final concentration, and finally the I53-50B.4PT1 pentamer component (SEQ ID NO: 17) (in 50mM Tris pH 8, 500mM NaCl, 0.75% w/v CHAPS, wherein the molar ratio of RBD-I53-50A: I53-50B.4PT1 is 1.1:1. The appropriate amount of buffer contains 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 2-8deg.C for at least 30min, followed by subsequent purification by SEC to remove residual unassembled components. Using Superose ^TM Nanoparticle production was performed on a 6 incoease 10/300GL column. The assembled particles were subjected to Superose ^TM The column was eluted at about 11mL on 6 columns. The assembled nanoparticles were sterile filtered (0.22 μm) immediately before column application and after SEC fractions were combined.

Biological layer interferometry for kinetic analysis of monomeric RBD

Kinetic measurement using Octet ^TM Red 96 system (Pall forte Bio +.) Performed at 25℃with shaking at 1000 rpm. All proteins and antibodies were diluted at 200 μl/well into Phosphate Buffered Saline (PBS) containing 0.5% bovine serum albumin and 0.01% TWEEN-20 in black 96-well Greiner Bio-one microwell plates, with separate buffers used for the baseline and dissociation steps. CV30 or CR3022 IgG at 10. Mu.g/mL was loaded onto a pre-hydrated protein A biosensor (Pall forte Bio/. About.>) Last 150s, then a baseline of 60 s. The biosensor was then transferred to a step associated with one of five serial two-fold dilutions of wild-type or stabilized monomeric RBD for 120s, where 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 CR 3022. After association, the biosensor was transferred to buffer for 300s of dissociation. Baseline was subtracted from the data from association and dissociation steps and a 1:1 binding model (+. >Analysis software, version 12.0) calculated kinetic measurements globally across all five serial dilutions of RBD.

Biological layer interferometry for hierarchical antigenicity of RBD nanoparticles

Using Octet ^TM The Red 96 system was run at 1000rpm at ambient temperature to analyze the binding of hACE2-Fc (dimerization receptor) and CR3022 IgG to monomeric RBD and RBD-I53-50 nanoparticles for real-time stability studies. In kinetic buffer (Pall forte Bio +.) Protein samples were diluted to 100nM. The buffer, antibodies, receptors and immunogens were then applied to a black 96-well Greiner Bio-one microplate at 200 μl/well. Protein A biosensors were first hydrated in kinetic buffer for 10min and then immersed in hACE2-Fc or CR3022 diluted to 10. Mu.g/mL in kinetic buffer in a immobilization step. After 500s, the tips were transferred to Kinetics buffer for 90s to reach baseline. The association step is performed by immersing the loaded biosensor in the immunogen for 300s, and the subsequent dissociation step is performed by immersing the biosensor back in the dynamic buffer for an additional 300 s. In use +.>The baseline was subtracted from the data prior to drawing by the analysis software (version 12.0).

Negative staining electron microscopy

Wild-type RBD-I53-50 nanoparticles and Rpk-I53-50 nanoparticles were first diluted to 75. Mu.g/mL in 50mM Tris pH8, 150mM NaCl, 100mM L-arginine, 5% v/v glycerol, and then 3. Mu.L of the sample was applied to a freshly glow-discharged 300 mesh copper grid. The sample was incubated on the grid for 1 minute, then the grid was immersed in a 50 μl drop of water and excess liquid was aspirated with filter paper. The mesh was then immersed in 3. Mu.L of 0.75% w/v uranyl formate stain. The stain was sucked off with filter paper and then the grid was immersed in another 6 μl of stain and incubated for about 90 seconds. Finally, the stain was blotted dry and the grid dried for 1 minute and then stored or imaged. The prepared grid is positioned in Talos ^TM Gatan used in L120C type transmission electron microscope ^TM The camera images at 57,000 x.

Dynamic light scattering

Dynamic Light Scattering (DLS) for use in unclle ^TM The hydrodynamic diameter (Dh) and polydispersity (% Pd) of the RBD-I53-50 nanoparticle samples were measured on (UNchained Laboratories). The sample was applied to an 8.8 μl quartz capillary cartridge (UNi ^TM UNchained Laboratories) and 10 acquisition measurements were made using laser auto-decay, 5s each. From UNcle in Dh measurement ^TM The Client software accounts for the viscosity increase due to 5% v/v glycerol in RBD nanoparticle buffer.

Endotoxin measurement

Using EndoSafe ^TM The Nexgen-MCS system (Charles River) measures endotoxin levels in protein samples. Samples were diluted 1:50 or 1:100 in LAL reagent water without endotoxin and applied to EndoSafe ^TM The LAL reagent cartridge is in the well. Using Charles River EndoScan ^TM V software analyses endotoxin content, which software automatically back calculates dilution factors. Endotoxin values are reported as EU/mL, which are then converted to EU/mg based on UV-Vis measurements. Our sample threshold suitable for immunization is<100EU/mg。

UV-Vis

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

Hydrogen/deuterium exchange mass spectrometry

3mg of RBD-I53-50A, rpk4-I53-50A and Rpk9-I53-50A trimer were buffered in deuteration buffer (pH 7.5, 85% D ₂ O, cambridge Isotope Laboratories, inc.) H/D exchange (HDX) was performed at 22 ℃ for 3 seconds, 15 seconds, 60 seconds, 1800 seconds and 72000 seconds, respectively. The exchanged samples were then mixed at 1:1 with ice-cold quench buffer (200 mM tris (2-chloroethyl) phosphate (TCEP), 8M urea, 0.2% Formic Acid (FA)) to a final pH of 2.5 and immediately flash frozen in liquid nitrogen. In Syntat ^TM Samples were analyzed by LC-MS on a G2-Si mass spectrometer using a custom-built loading system that maintained all columns, loops, valves and lines at 0 ℃. Frozen samples were thawed on ice and loaded onto custom-packed immobilized pepsin columns (2.1x50 mm) with 200mL/min flow of 0.1% trifluoroacetic acid (TFA) and 2% acetonitrile. Peptides were captured on a Waters CSH C18 capture column (2.1X105 mm) and then resolved on a Waters CSH C18 1X 100mm 1.7 μm column with a linear gradient from 3% to 40% B (A: 98% water, 2% acetonitrile, 0.1% FA, 0.025% TFA; B:100% acetonitrile, 0.1% FA, flow rate 40 mL/min) over 18 min. A series of washes was performed between sample runs to minimize carryover. Unless otherwise specified, all water and organic solvents used were of MS grade (Optima ^TM Fisher). Complete deuteration control was performed on each sample series by collecting LC eluate from pepsin digested non-deuterated samples, performing speedvac drying, incubating in deuteration buffer at 85 ℃ 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]) To ensure consistent labeling conditions for all samples.

The peptide reference list was updated from the wild-type RBD peptide list, with the addition of new peptides encompassing the mutation. These peptides were used with DriftScope ^TM Manual verification is performed and authentication is performed using orthogonal retention time and drift time coordinates. Deuterium uptake analysis was performed using HX-Express v 2. Peaks were identified from the peptide spectra based on the peptide m/z values and a binomial fit was applied. Deuterium uptake levels were normalized to the fully deuterated control.

Immunization of mice

Female BALB/c (stock number: 000651) mice were purchased at four weeks of age. At 6 weeks of age, 6 mice per dosing group were vaccinated with a primary vaccine and three weeks later mice received a second vaccination boost. Prior to vaccination, the immunogen suspension was combined with adavax ^TM The adjuvant was gently mixed at 1:1 volume/volume to achieve a final concentration of 0.009mg/mL or 0.05mg/mL antigen. Under isoflurane anesthesia, mice were injected intramuscularly into the gastrocnemius muscle of each hind leg using a 27 gauge needle, with 50 μl (100 μl total) of immunogen injected per injection site. To obtain serum, all mice were bled two weeks after primary and booster immunization. Blood was collected via a subchin venipuncture and allowed to stand in a 1.5mL plastic eppendorf tube at room temperature for 30min to effect clotting. Serum was separated from hematocrit by centrifugation at 2,000g for 10 min. Complement factors and pathogens in the isolated serum were heat inactivated via incubation at 56 ℃ for 60 min. Serum was stored at 4 ℃ or-80 ℃ prior to use.

ELISA

Binding of mouse serum to the delivered antigen was determined using an enzyme-linked immunosorbent assay (ELISA). Briefly, maxisorp ^TM ELISA plates were coated overnight at 4℃with 0.08. Mu.g/mL protein of interest per well in 0.1M sodium bicarbonate buffer (pH 9.4). Plates were then blocked with dry milk powder in a 4% (w/v) solution of TBS containing 0.05% (v/v) Tween 20 (TBST) for 1 hour at room temperature. Continuous serum preparationDilutions were added to the plates and after washing, antibody binding was revealed using catalase-conjugated equine anti-mouse IgG antibodies. Plates were then thoroughly washed in TBST, colorimetric substrates (TMB, thermo Fisher) were added and absorbance at 450nm was read. Area Under Curve (AUC) calculations were generated by adding the trapezoidal area generated between adjacent absorbance measurement pairs and baseline. Mid-point potency calculations (EC) were generated based on fitting four-point logistic equations using the SciPy library in Python ₅₀ ) Wherein EC is ₅₀ Is the serum dilution at which the curve reaches 50% of its maximum.

Lentivirus-based pseudovirus neutralization assay

Spike pseudotyped lentivirus neutralization assay was performed essentially as described in (67). The protocol of this study was modified to use a cytoplasmic tail truncated SARS-CoV-2 spike with 21 amino acids (which increases spike pseudotyped lentiviral titers) and a D614G mutation (which is now dominant in human SARS-CoV-2). Plasmid HDMSpikedelta 21_D614G encoding this spike is available from Addgene (accession number 158762) or BEI (NR-53765), and the complete sequence is available on the world Wide Web at Addgene. Org/158762).

Briefly, 293T-ACE2 cells (BEI NR-52511) were isolated at 1.25X10 ⁴ Individual cells/well were inoculated in 50ul d10 growth medium (containing 10% heat-inactivated FBS, 2mM L-glutamine, 100U/mL penicillin and 100 μg/mL streptomycin) in poly L-lysine coated black wall clear bottom 96 well plates (Greiner 655930). The following day, the mouse serum samples were heat-inactivated at 56 ℃ for 30min, then serially diluted in D10 growth medium. The spike pseudotyped lentiviruses were diluted 1:50 to yield about 200,000RLU per well and incubated with serum dilutions for 1 hour at 37 ℃. mu.L of the virus-serum mixture was then added to the cells and after about 52 hours Bright-Glo was used ^TM Luciferase assay System [ ]E2610 Luciferase activity was measured. The neutralization assay for each batch included human serum negative control samples collected in 2017-2018 and known neutralizing antibodies to ensure batchesConsistency between them. The infection rate score for each well was calculated as compared to two "serum-free" control wells in the same row of the plate. The "neutecurve" package (available on the world wide web at jbloom. Gitub. Io/neutecurve, version 0.5.2) was used to calculate 50% inhibition concentration (IC ₅₀ ) And 50% neutralization titers (NT 50), for each serum sample, by fitting a Hill curve with bottom fixed at 0 and top fixed at 1, which is simply 1/IC ₅₀ . All neutralization assay data is available on the world wide web at gitsub.

MLV-based pseudovirus neutralization assay

Preparation of MLV-based SARS-CoV-2S pseudotyped 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, lipofectamine was used according to the manufacturer's instructions ^TM 2000 (Life Technologies) HEK293T cells were co-transfected with a plasmid encoding SARS-CoV-2S, an MLV Gag-Pol packaging construct and an MLV transfer vector encoding a luciferase reporter gene. Cells were washed 3 times with Opti-MEM and incubated with transfection medium for 5h at 37 ℃. DMEM containing 10% FBS was added to culture for 60 hours. The supernatant was harvested by spin-harvesting at 2,500g, filtered through a 0.45 μm filter, concentrated with a 100kDa membrane at 2,500g for 10min, then aliquoted and stored at-80 ℃.

For neutralization assays, HEK-hACE2 cells were incubated with 8% CO in a 37℃incubator (ThermoFisher) ₂ Cultured in DMEM containing 10% FBS (Hyclone) and 1% PenStrep. One day or more prior to infection, 40 μl polylysine (Sigma) was placed in 96-well plates and incubated for 5min with spin. Polylysine was removed, the plates were dried for 5min and then washed 1 time with water before plating the cells HEK-hACE2 cells. The following day, the cells were examined to 80% confluence. Serial dilutions of serum were prepared in DMEM at a final volume of 22 μl in half-area 96-well plates. Then 22. Mu.L of pseudovirus was added to the serial dilutions and inIncubating for 30-60min at room temperature. The mixture was added to the cells and after 2 hours 44 μl DMEM supplemented with 20% FBS and 2% PenStrep was added and the cells were incubated for 48 hours. After 48h, 40. Mu.L/well of One-Glo-EX was taken ^TM Substrate (Promega) was added to cells and incubated in the dark for 5-10min, then in BioTek ^TM Reading on a microplate reader. Determination of curve-fitted IC using nonlinear regression of log (inhibitor) versus normalized response ₅₀ Values.

Quantification and statistical analysis

Multiple sets of comparisons were performed using the nonparametric Kruskal-Wallis test in GraphPad Prism 8 and Dunn post hoc analysis. When the P value is less than 0.05, the difference is considered significant. Statistical methods and P-value ranges can be found in the graphs and legends.

Results

The following five mutations of SARS-CoV-2S RBD are considered as starting points for the design of stabilized RBD antigens: I358F, Y365F, Y365W, V367F and F392W. Frozen EM structure using pre-fusion S ectodomain trimer (PDB ID 6 VX) for use in PyMol ^TM And Rosetta ^TM Five mutations were analyzed. Only the V367F mutation was found to be exposed to the solvent and was therefore not considered for inclusion in the stabilized RBD design to avoid the risk of adversely altering antigenicity. The other four mutations were observed to be near or within the recently identified Linoleic Acid (LA) binding pocket, with Y365 being identified as the key gating residue for this interaction (fig. 2A-2B). Increased expression and stability was observed by DMS for several mutations in the LA binding pocket, indicating that this region of RBD is structurally suboptimal.

It is next examined whether combinations of these mutations can further enhance these and other properties of RBD. The computing protocol is in Rosetta ^TM Modeling one or more of I358F, Y365F, Y W and/or F392W, while also allowing mutation of nearby residues (fig. 2C). Also performed is a design trajectory that does not force any of these four validated mutations to be contained in the LA binding pocket, rather than allowing Rosetta ^TM The novel set of stabilizing mutations is designed in the same region. All designsThe traces were all performed in the context of the intact S ectodomain (PDB ID 6 VXX) and the crystal structure of RBD monomers (PDB ID 6YZ 5) that showed subtly different skeletal conformations in the region surrounding the LA binding pocket. Seventeen repackaging designs (abbreviated as "Rpk") with mutations that fill cavities and/or remove buried polar groups were selected for experimental analysis, which also included 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 reported DMS-identified mutations or Rosetta-identified mutations.

* n/a represents inapplicability

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

The stabilized design was secreted from HEK293F cells, as well as wild-type RBD ("RBD") fused to I53-50A trimer and negative control plasmid. Reduced SDS-PAGE of cell culture supernatants showed increased expression for all designs compared to wild type (fig. 1A). Furthermore, non-reducing SDS-PAGE showed significant differences in the number of disulfide-linked dimers formed for each design (fig. 1A). Designs involving the F392W mutation produced significantly lower levels of disulfide-linked dimers. In addition to F392W partially filling the LA binding pocket cavity, the proximity of this mutation to disulfide between C391 and C525 suggests that it is detrimental to off-target intermolecular disulfide formation involving these cysteines.

The following two designs were chosen for more detailed analysis as both monomer and trimer: rpk4, featuring F392W only; and Rpk9, which combines F392W with DMS-identified Y365F to remove buried side chain hydroxyl groups and V395I identified Rosetta to refill the resulting cavity with hydrophobic filler (data not shown). The scaled-up expression from HEK293F cells and purification by Immobilized Metal Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC) demonstrated an increase in the yields of Rpk4 and Rpk9 both as monomers and as fusions with the I53-50A trimer, wherein Rpk9 showed a distinct advantage for the I53-50A trimer (fig. 4A and 10A). All constructs feature low levels of off-target disulfide-linked dimer formation highlighting the importance of including F392W in the stabilized RBD design. Measurement of melting temperature (T) of both monomer and trimer by nano-differential scanning fluorescence (nano DSF) _m ) Intrinsic tryptophan fluorescence was monitored, which showed an increase in Rpk4 protein of 1.9-2.4 ℃ and an increase in Rpk9 protein of 3.8-5.3 ℃ (fig. 4B) compared to the wild-type counterpart. All monomeric RBDs were indistinguishable by circular dichroism and appeared to refold after denaturation at 95 ℃ (fig. 10B). Furthermore, hydrogen/deuterium exchange mass spectrometry (HDX-MS) of stabilized RBDs fused with the I53-50A trimer showed reduced deuterium uptake in two different peptide segments in the LA binding pocket compared to the wild-type RBD (fig. 4C, fig. 11), indicating improved local ordering in the stabilization design. Peptide segments remote from the LA binding pocket (including those in the ACE2 binding motif) showed a retained structural order compared to the wild type. To further evaluate the structural order, all three monomeric RBDs were mixed alone with a syno orange dye to measure exposure of hydrophobic groups (fig. 4D). Both Rpk4 and Rpk9 showed reduced signal compared to wild-type RBD, with least fluorescence generated by Rpk9, indicating that improved local order of LA binding pockets in the stabilized RBD results in reduced hydrophobic exposure. Consistent with HDX-MS data, neither stabilizing set of mutations affected the antigenicity of the ACE2 binding motif as assessed by binding of antibody CV30 recognizing antigenic site Ia (Hur lbut et al Nature Communications 11:5413 (2020)) (FIG. 4E). The affinity for non-neutralizing antibody CR3022 for site IIc and closer to the LA binding pocket (Yuan et al Science 386:630-644 (2020)) was slightly reduced [ ]<3.5 times). In summary, both stabilizing sets of mutations enhance antigen expression, thermostability and structural order while minimizing impact on antigenicity, with Rpk9 showing excellent improvement in all categories for both monomeric RBD and its genetic fusion to the I53-50A trimer.

Although stabilizing mutations were designed to take into account isolated RBDs, such mutations were also evaluated in the context of the intact S ectodomain. The total yield of pre-fusion stabilized HexaPro antigen fused to the T4 fibrin folder was measured using the Rpk9 mutation (Rpk 9-HexaPro-folder) and compared to the wild-type version (HexaPro-folder). The term "HexaPro" refers to spike proteins with four beneficial proline substitutions (F817P, A892P, A899P, A942P) and two proline substitutions in S-2P (proline at 986 and 987). See Hsieh et al, science 369:1501-05 (2020); a slight increase in yield was seen with the Rpk9 mutation, however a slightly earlier SEC elution volume was observed, which may indicate a decrease in stability against the S ectodomain background. The Rpk 9-HexaPro-fold showed nano DSF characteristics similar to the HexaPro-fold, but the intrinsic fluorescence change at above 60 ℃ was slightly accelerated with the Rpk 9-HexaPro-fold (fig. 8C). Negative staining electron microscopy (nsEM) showed that the typical pre-fusion spike morphology remained largely identical to the mutation (fig. 8D). These data indicate that although mutations in the LA binding pocket can be incorporated into pre-fusion spike trimers, the stabilizing effect of the Rpk9 mutation appears to be unique to isolated RBDs.

Next it was investigated whether RBD stabilizing mutations would improve nanoparticle stability in simpler buffers lacking adjuvants such as glycerol, L-arginine and the detergent 3- [ (3-cholamidopropyl) dimethylamino ] -1-propanesulfonate (CHAPS), which would otherwise be useful for stabilizing preparations of nanoparticle immunogens. Wild-type and stabilized RBD-I53-50A trimers were assembled into nanoparticles (RBD-I53-50, rpk4-I53-50 and Rpk 9-I53-50) by adding the complementary I53-50B.4PT1 pentamer component (SEQ ID NO: 17) (FIG. 5A). The excess residual components were removed by SEC using a mobile phase comprising Tris Buffered Saline (TBS) containing glycerol, L-arginine and CHAPS, and the formation of highly monodisperse nanoparticles was confirmed by negative dye electron microscopy (nsEM) (fig. 5B). The purified nanoparticles were then dialyzed into a buffer solution with less adjuvant to assess the solution stability before and after a single freeze/thaw cycle (fig. 5C-5E). In TBS supplemented with glycerol and L-arginine, wild-type RBD-I53-50 showed slight signs of aggregation by UV-Vis spectroscopy (FIG. 5C) and Dynamic Light Scattering (DLS) (FIG. 5D), whereas no signs of aggregation were observed for Rpk4-I53-50 and Rpk 9-I53-50. The difference in solution stability was further compared after dialysis into TBS containing only glycerol: rpk4-I53-50 and Rpk9-I53-50 were both more resistant to aggregation than RBD-I53-50 and better maintained binding to immobilized human ACE2 (hACE 2-Fc) and CR3022 (FIG. 5E). Dialysis into TBS alone showed clear evidence of aggregation and loss of antigenicity for all samples, with Rpk9-I53-50 retaining slightly better antigenicity than RBD-I53-50 and Rpk4-I53-50. The improved solution stability observed for stabilized RBD appears to be consistent with its enhanced thermal stability and structural order, and provides subtle but important improvements in formulation stability that are highly relevant to vaccine manufacture.

The immunogenicity of the stabilized RBD was then assessed in a mouse immunization study. Immunogens comprising wild-type and stabilized RBD were prepared in two forms: the I53-50 nanoparticles of each antigen, as well as a non-assembly control of nearly identical proteins, were displayed in which trimeric fusions with I53-50A were mixed with slightly modified pentameric scaffolds lacking hydrophobic interfaces driving nanoparticle assembly ("2 OBX"; SEQ ID NO:43; FIG. 6A). In addition to allowing the immunogenicity of different RBDs in the form of trimers and nanoparticles to be assessed, this comparison also directly controls the effect 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 buffers further comprising CHAPS to enable direct comparison with other immunogenicity studies. HexaPro-fold (characterized by wild-type RBD) was included as a comparison. Female BALB/c mice were immunized twice every three weeks with each immunogen and serum was collected two weeks after each immunization (fig. 6A). All doses were administered with equimolar amounts of RBD and contained adavax adjuvant.

Binding titers against HexaPro-folates were measured using an enzyme-linked immunosorbent assay (ELISA) and analyzed by measuring the area under the curve (AUC) (fig. 6B) and the midpoint titers (fig. 12A). Serum from all nanoparticle groups showed antigen specific antibody levels slightly above the HexaPro-folder after priming and significantly above the non-assembled control. Binding signals increased in all groups after the second immunization, with less gap between them. Pseudovirus neutralization using lentiviral scaffolds showed a similar trend after primary immunization, with all nanoparticle groups showing significantly higher neutralization activity than the non-assembled control and more effective neutralization by nearly two orders of magnitude than the HexaPro-fold (fig. 6C). After the second immunization, neutralization was greatly increased for all groups, with nanoparticles and HexaPro-fold exhibiting the highest level of neutralization activity. At each time point, there was no significant difference in neutralization activity between the various nanoparticle groups or between the various non-assembled control groups. Comparable results were obtained with different pseudovirus assays using Murine Leukemia Virus (MLV) frameworks (fig. 12B). These data indicate that stabilized RBD has similar immunogenicity to wild-type RBD when presented in trimeric or microparticle form, with nanoparticle presentation significantly enhancing RBD immunogenicity and being most significant after a single immunization.

The improved shelf life stability of SARS-CoV-2 vaccine makes it possible to directly enhance global vaccination efforts by simplifying manufacturing and distribution. The stability of the two stabilized RBD nanoparticle immunogens was compared to wild-type RBD-I53-50 by DLS (FIG. 7A), BLI (FIG. 7B), SDS-PAGE and NSEM (FIG. 7C) during 28 days storage at-80 ℃, 2-8 ℃, 22-27 ℃ and 35-40 ℃. No significant deviation from baseline was observed for any of the immunogens at-80 ℃, 2-8 ℃, or 22-27 ℃ during the study. However, storage of wild-type RBD-I53-50 at 35-40℃for 28 days resulted in a significant reduction in aggregation and antigenicity detectable by DLS and nsEM. In contrast, after 28 days of storage at 35-40 ℃, both the particle stability and antigenicity of Rpk4-I53-50 and Rpk9-I53-50 remained unchanged. These results indicate that the stabilizing mutations identified in RBD can improve manufacturability and stability of RBD-based nanoparticle immunogens without compromising their potent immunogenicity.

Experimental information to reduce potential design space to particularly valuable regions and mutations can greatly facilitate structure-based protein design. Here, the utility of DMS data in directing viral glycoprotein stabilization is demonstrated by characterizing the LA binding pocket of SARS-CoV-2S RBD as a structurally suboptimal region and providing the characteristics of potential stabilizing mutations. Under the guidance of these data, rosetta ^TM The structural modeling of (a) determines additional stabilizing mutations and promising combinations of mutations. All designs screened by experiments successfully improved expression of wild-type RBD, which is an exceptionally high efficiency compared to many purely structure-based design experiments.

This in-depth biochemical and biophysical characterization of RBD variants enabled the inventors to choose to enhance expression; minimizing off-target disulfides; improving the local structural order; and improving thermal stability, solution stability and shelf life stability; while maintaining the design of strong immunogenicity of the wild-type RBD displayed on the I53-50 nanoparticles. In the two mutants studied in detail, rpk4 (F392W) is more conserved, characterized by only a single amino acid change, but is less stable than Rpk9 (Y365F, F392W, V I) comprising additional mutations that specifically improve expression and thermostability. 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 due to the improvement in local structural order and the reduction in hydrophobic surface area exposure, as indicated by HDX-MS and SYPRO orange fluorescence. More generally, these results suggest the following possibilities: other RBD antigens may take on dynamic conformations not observed in the S ectodomain or existing structures of isolated RBDs, such as transitions between the 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 main focus of the neutralization reaction towards RBD, and the fact that stabilizing mutations are not exposed on the antigen surface. These immunogenicity data also clearly indicate that the higher priced RBD nanoparticle immunogens are far more immunogenic than the trimeric form of RBD, especially after a single immunization. Trimer spikes (HexaPro-folder) trigger a higher level of neutralizing activity than trimer RBD. This result suggests that removal of RBD from the spike environment while maintaining the spike oligomerization state is not an inherent advantage for enhancing antibody responses against RBD and underscores the importance of nanoparticle presentation in RBD-based vaccines.

The observed improvements in manufacturability, stability and solution properties may have a significant impact on the manufacture and distribution of protein-based vaccines against SARS-CoV-2. Since SARS-CoV-2 vaccines have been updated in response to antigen drift, such improvements may be important to maximize the scale and speed of vaccine production and to buffer unexpected changes in the stability or solution properties of antigens from novel SARS-CoV-2 strains. Furthermore, improving denaturation resistance and shelf life stability at various temperatures may have a particular impact on reliable distribution in less developed areas of the world where cold chain infrastructure is lacking. Finally, since knowledge of the pre-fusion stabilizing "2P" mutation prior to the emergence of SARS-CoV-2 proved critical to work with pandemic responses, the ability to use stabilizing mutations on RBDs to reliably improve vaccine manufacturability could be an important tool for optimizing vaccine design against other coronaviruses transmitted in animal infectious disease hosts that are threatening to infect humans.

Claims

1. 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 at least two mutations relative to the RBD of SEQ ID NO:1, wherein said 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 at the corresponding residue 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 Blast-p parameters of protocol 1 or protocol 2.

2. 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 or to corresponding residues of said receptor binding domain of said second coronavirus as determined by sequence alignment of SEQ ID NO:1 with the sequence of a second coronavirus receptor binding domain by using Blast-p parameters of protocol 1 or protocol 2, and

further comprising at least two mutations relative to said RBD of SEQ ID NO. 1 or said corresponding residue in said second coronavirus,

Wherein the at least two mutations enhance the stability of the polypeptide relative to the stability of a wild-type polypeptide lacking the 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,

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 at the corresponding residue of the second coronavirus as determined by sequence alignment of SEQ ID NO. 1 with said sequence of said second coronavirus receptor binding domain using Blast-p parameters of protocol 1 or protocol 2.

5. The polypeptide of any one of claims 2-4, further comprising additional amino acid residues outside 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 expression of the RBD polypeptide is increased when expressed in a cell as compared to expression of the wild-type RBD polypeptide lacking the at least two mutations.

9. The polypeptide of any one of claims 2-8, wherein the RBD polypeptide binds to a coronavirus antibody or to 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 comprises 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 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 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 shelf life that is longer 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.

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

25. The coronavirus polypeptide according to 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 a mutation at a 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 parameters of protocol 1 or protocol 2.

26. The coronavirus polypeptide according to any one of claims 22-25, wherein the at least one cavity filling mutation comprises a mutation of residues 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 sequence alignment of SEQ ID No. 1 with the sequence of the second coronavirus spike protein subunit 1 using Blast-p parameters of protocol 1 or protocol 2.

27. The coronavirus polypeptide according to any one of claims 22-26, wherein the at least one cavity filling mutation comprises a mutation of the 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 Blast-p parameters of protocol 1 or protocol 2.

28. The coronavirus polypeptide according to 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 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 parameters of protocol 1 or protocol 2.

29. The coronavirus polypeptide according to 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/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 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 Blast-p parameters of protocol 1 or protocol 2.

30. The coronavirus polypeptide according to any one of claims 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 to the receptor binding domain sequence 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 parameters of protocol 1 or protocol 2.

31. The coronavirus spike protein subunit 1 polypeptide of 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 95% identity with SEQ ID No. 1 or with the wild-type spike protein subunit 1 amino acid sequence of a second coronavirus.

33. The coronavirus polypeptide according to any one of claims 22-31, wherein the expression of the coronavirus polypeptide is increased when expressed in a cell compared to the 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. The coronavirus polypeptide according to any one of claims 22-33, wherein the coronavirus polypeptide binds to a coronavirus antibody or to 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, wherein the homologous coronavirus receptor comprises 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 according to 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-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 according to 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 of 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 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 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, the method 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 of 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 of any one of claims 1-25.

52. The method or composition of any of the preceding claims, wherein the Blast-p parameter of protocol 1 comprises:

algorithm: blastp (protein-protein BLAST)

Expected threshold: 0.1

Word length: 6

Maximum match within query range: 0

Matrix: BLOSUM62

Vacancy cost:

the presence is: 11

Extension: 1

Filtering low complexity regions? : whether or not

Masking:

is only for look-up tables? : whether or not

Lower case letters? : and (3) if not.

53. The method or composition of any of the preceding claims, wherein the Blast-p parameter of protocol 2 comprises:

blastp-query. Fasta-topic sbjct. Fasta-matrix BLOSUM 62-value 0.1-word length 6-gap open 11-gap extend 1-output results. Txt.

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, N F, 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, N F, 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 has at least 90%, at least 95%, or at least 98% identity to the polypeptide sequence of SEQ ID NO. 3.

61. The polypeptide of claim 59, wherein the heterologous protein scaffold comprises a 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 of 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 to any one of SEQ ID NOs 13-18.

64. A vaccine composition comprising the composition of any one of claims 54-62 or the polypeptide complex of 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 the polypeptide of any one of claims 54-62, the protein complex of claim 63, or the vaccine composition of any one of claims 64-68.

70. A method of preparing a vaccine, the method comprising combining the polypeptide of 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 a polypeptide according to any one of claims 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; optionally an 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 at least one mutation of the RBD relative to SEQ ID NO:1, wherein said at least one mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367 of SEQ ID NO 1, F, N501W, G502D, N501F, N501T, Q498Y, F338M, A363L, Y365M, F377V, V395I, L513I, L M and F515L, or a second coronavirus reference sequence, wherein the corresponding site in said second coronavirus reference sequence is determined by sequence alignment of SEQ ID NO 1 with the spike protein sequence of said second coronavirus receptor binding domain using Blast-p parameters of protocol 1 or protocol 2.

73. The polypeptide of claim 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/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.