WO2022146484A1 - Stable coronavirus proteins and vaccine compositions thereof - Google Patents
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the field of the invention relates to methods and compositions for improving stability of protein-based vaccines.
- Coronaviruses maintain a prominent pandemic threat, with the 2019/2020 pandemic induced by the SARS-CoV-2 virus causing hundreds of thousands of deaths worldwide and a massive economic slowdown. It is likely that SARS-CoV-2 could maintain persistent epidemics even after the current pandemic is diminished. Effective vaccines against SARS-CoV-2 or other future emerging coronaviruses are therefore highly desirable.
- compositions and methods described herein are based, in part, on the discovery of single or paired amino acid mutations in the SARS-CoV-2 S “spike” protein amino acid sequence that enhance both yield and stability of the expressed protein (under the same or similar culture conditions).
- This enhanced spike protein also referred herein as a “spike protein-derived antigen” stability allows for the production of a vaccine having a longer shelf-life than vaccines based on the wild-type or native protein (under the same or similar storage conditions).
- 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 the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M;
- RBD coronavirus receptor binding domain
- the Blast-p program used is the National Center for Biotechnology Information (NCBI) online alignment tool.
- NCBI National Center for Biotechnology Information
- the Blast-p program can be downloaded onto a device and used locally.
- Protocol 1 For use with the BLASTp alignment online from the National Center for
- NCBI Biotechnology Information
- blastp protein-protein BLAST
- Protocol 2 For use with the protein BLASTp alignment tool downloaded onto a local computer or server.
- Another aspect provided herein comprises 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 the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M;
- RBD coronavirus receptor binding domain
- I358F/Y365W/L513M I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V;
- Another aspect provided herein comprises 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 the receptor binding domain of a second coronavirus as determined by a 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 the 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.
- the stability of the non-naturally occurring coronavirus receptor binding domain polypeptide and the stability of its corresponding wild-type polypeptide are assessed under the same conditions.
- the at least two mutations are at amino acids: 338 & 365; 365 & 513; 365 & 392; 338, 363, 365 & 377; 365 & 392; 365 & 395; 365, 392 & 395; 365, 513, & 515; 338, 363, & 365; 338, 358 & 365; 358, 365, & 513; 358, 365 & 392; 338, 358, 363, 365 & 377; 358, 365, & 392; 358, 365 & 395; 358, 365, 392, & 395; 358, 365, 513 & 515; and/or 338, 358, 363, & 365 of SEQ ID NO: 1, or at corresponding residues of the second coronavirus receptor binding domain as determined by a sequence alignment of SEQ ID NO: 1 with the sequence
- the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I;
- Y365F/F392W/V395I Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V;
- the receptor binding domain polypeptide further comprises additional amino acid residues outside of the RBD of SEQ ID NO: 1.
- the receptor binding domain polypeptide as described herein is comprised by a coronavirus spike protein polypeptide.
- the receptor binding domain polypeptide or the coronavirus spike protein polypeptide can include, for example, a fusion polypeptide.
- the receptor binding domain polypeptide or the coronavirus spike protein polypeptide can also include, for example, a leader sequence, e.g., for secretion. In various embodiments, a leader sequence and/or an amino terminal methionine can be present or, alternatively, can be removed, e.g., by proteolytic cleavage.
- the coronavirus receptor binding domain comprises at least 95% identity to residues 328-531 of SEQ ID NO: 1.
- the at least two mutations at amino acids 338 & 365; 365 & 513; 365 & 392; 338, 363, 365 & 377; 365 & 392; 365 & 395; 365, 392 & 395; 365, 513, & 515; 338, 363, & 365; 338, 358 & 365; 358, 365, & 513; 358, 365 & 392; 338, 358, 363, 365 & 377; 358, 365, & 392; 358, 365 & 395; 358, 365, 392, & 395; 358, 365, 513 & 515; and/or 338, 358, 363, & 365 of SEQ ID NO: 1, or at corresponding residues of the second coronavirus receptor binding domain are the only mutations in the receptor binding domain relative to wild type.
- the expression of the RBD polypeptide when expressed in a cell is increased as compared to expression of the wild-type RBD polypeptide lacking the at least two mutations (i.e., under the same or similar expression conditions or culture conditions).
- the RBD polypeptide binds to a coronavirus antibody or binds a coronavirus cognate receptor.
- the coronavirus antibody comprises a SARS-CoV-2 antibody.
- the receptor for the coronavirus corresponding to the polypeptide comprises an angiotensin converting enzyme (ACE) receptor.
- ACE angiotensin converting enzyme
- the ACE receptor is the ACE2 receptor.
- the second coronavirus comprises a sequence of a coronavirus selected from: Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2), Severe acute respiratory syndrome associated coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; HKU1, or a naturally occurring variant thereof.
- SARS-CoV-2 Severe acute respiratory syndrome associated coronavirus 2
- SARS-CoV Severe acute respiratory syndrome associated coronavirus
- MERS Middle East respiratory syndrome
- 229E NL63; OC43; HKU1, or a naturally occurring variant thereof.
- the receptor binding domain polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1.
- the RBD is fused to a second, heterologous polypeptide.
- the RBD is fused to a nanoparticle, nano-structure or heterologous protein scaffold.
- the heterologous protein scaffold comprises the 153-50 trimeric “A” component of SEQ ID NO: 3.
- the heterologous protein scaffold comprises a heterologous protein scaffold as described in Table 1 of US Patent No. 10,351,603, the contents of which are incorporated by reference in its entirety.
- polypeptide and/or the second polypeptide is an antigenic polypeptide.
- coronavirus spike protein comprising the polypeptide of claim 1.
- compositions comprising the polypeptide as described herein and a pharmaceutically acceptable carrier.
- the polypeptide is in an admixture with the pharmaceutically acceptable carrier.
- the polypeptide and the pharmaceutically acceptable carrier are provided as a suspension.
- the pharmaceutical composition further comprises an adjuvant.
- the shelf- life of the composition is longer than a composition comprising a wild-type RBD polypeptide lacking the at least two mutations.
- composition is formulated as a vaccine.
- 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 a second mutation.
- 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.
- the at least one cavity-filling mutation comprises mutation of a residue in a linoleic acid binding pocket of the coronavirus spike protein, subunit 1.
- the at least one cavity-filling mutation comprises mutation of a residue within residues 328-531 of SEQ ID NO: 1 or at corresponding residues of a second coronavirus spike protein, subunit 1 as determined by a sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus spike protein, subunit 1 using Blast-p (e.g., protocols 1 or 2 as described herein).
- the at least one cavity-filling mutation comprises mutation of a residue of SEQ ID NO: 1 between residues 335- 345; 355-375, or 378-395 or at corresponding residues of a second coronavirus spike protein, subunit 1 as determined by a sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus spike protein, subunit 1 using Blast-p (e.g., protocols 1 or 2 as described herein).
- the at least one cavity-filling mutation comprises mutation of a residue of SEQ ID NO: 1 at amino acid 336, 338, 341, 342, 358, 361, 363, 365, 368, 374, 377, 387, or 392 or of a corresponding residue of a second coronavirus spike protein, subunit 1 as determined by a sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus using Blast-p (e.g., protocols 1 or 2 as described herein).
- Blast-p e.g., protocols 1 or 2 as described herein.
- the at least one cavity-filling mutation and the at least one second mutation are at residues 338 & 365 ; 365 & 513 ; 365 & 392; 338, 363, 365 & 377; 365 & 392; 365 & 395; 365, 392 & 395; 365, 513, & 515; 338, 363, & 365; 338, 358 & 365; 358, 365, & 513; 358, 365 & 392; 338, 358, 363, 365 & 377; 358, 365, & 392; 358, 365 & 395; 358, 365, 392, & 395; 358, 365, 513 & 515; and/or 338, 358, 363, & 365 of SEQ ID NO: 1, or at corresponding residues of a second coronavirus spike protein, subunit 1 as determined
- the at least one cavity-filling mutation and the at least one second mutation are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M;
- Blast-p e.g., protocol 1 or 2 as described herein.
- the coronavirus spike protein, subunit 1 polypeptide comprises at least 95% identity to residues 328-531 of SEQ ID NO: 1 or a receptor binding domain sequence of a second coronavirus spike protein, subunit 1 as determined by a 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).
- the at least two mutations at amino acids 338 & 365; 365 & 513; 365 & 392; 338, 363, 365 & 377; 365 & 392; 365 & 395; 365, 392 & 395; 365, 513, & 515; 338, 363, & 365; 338, 358 & 365; 358, 365, & 513; 358, 365 & 392; 338, 358, 363, 365 & 377; 358, 365, & 392; 358, 365 & 395; 358, 365, 392, & 395; 358, 365, 513 & 515; and/or 338, 358, 363, & 365 of SEQ ID NO: 1, or 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, or at corresponding residues of the second coronavirus receptor binding
- the coronavirus polypeptide comprises at least 95% identity to SEQ ID NO: 1 or to a wild-type spike protein, subunit 1 amino acid sequence of a second coronavirus.
- the expression of the coronavirus polypeptide when expressed in a cell is increased as compared to expression of a wild-type polypeptide lacking the at least one cavity-fdling mutation and at least one second mutation under the same expression conditions.
- the coronavirus polypeptide binds to a coronavirus antibody or binds a cognate coronavirus receptor.
- the coronavirus antibody comprises a SARS-CoV-2 antibody.
- the cognate coronavirus receptor comprises an angiotensin converting enzyme (ACE) receptor.
- ACE angiotensin converting enzyme
- the ACE receptor is the ACE2 receptor.
- the coronavirus polypeptide is an engineered mutant polypeptide of a coronavirus selected from: Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2), Severe acute respiratory syndrome associated coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; or HKU1.
- SARS-CoV-2 Severe acute respiratory syndrome associated coronavirus 2
- SARS-CoV Severe acute respiratory syndrome associated coronavirus
- MERS Middle East respiratory syndrome
- 229E Middle East respiratory syndrome
- NL63 OC43
- HKU1 Middle East respiratory syndrome
- the coronavirus spike protein, subunit 1 polypeptide comprises at least 90% sequence identity to SEQ ID NO: 1.
- the coronavirus polypeptide is fused to a second, heterologous polypeptide.
- the coronavirus polypeptide is fused to a nanoparticle, nano-structure or protein scaffold.
- the heterologous protein scaffold comprises the 153-50 trimeric “A” component of SEQ ID NO: 3.
- the heterologous protein scaffold comprises a heterologous protein scaffold as described in Table 1 of US Patent No. 10,351,603, the contents of which are incorporated by reference in its entirety.
- the coronavirus polypeptide or the second, heterologous polypeptide is an antigenic polypeptide.
- composition comprising a coronavirus polypeptide as described herein and a pharmaceutically acceptable carrier (e.g., in an admixture or forming a suspension).
- composition comprising a coronavirus polypeptide and a pharmaceutically acceptable carrier further comprises an adjuvant.
- the shelf- life of the composition is longer than a composition comprising a wild-type coronavirus polypeptide lacking the at least one cavity-fdling mutation and at least second mutation when stored under the same or similar storage conditions.
- composition comprising a coronavirus polypeptide and a pharmaceutically acceptable carrier is formulated as a vaccine.
- Another aspect provided herein relates to a cell expressing a receptor binding domain having at least two mutations as described herein, or a coronavirus polypeptide having at least two mutations as described herein.
- Another aspect provided herein relates to a nucleic acid sequence encoding a receptor binding domain having at least two mutations as described herein, or a coronavirus polypeptide having at least two mutations as described herein.
- Another aspect provided herein relates to a method of making 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.
- a fusion polypeptide composition comprising a coronavirus receptor binding domain (RBD) comprising a mutation selected from the group consisting of I358F, Y365F, Y365W, V367F and F392W, relative to the coronavirus polypeptide of SEQ ID NO: 1, fused to a heterologous protein scaffold.
- the heterologous protein scaffold comprises a polypeptide of SEQ ID NO: 3.
- each cysteine in the polypeptide of SEQ ID NO: 3 is mutated to alanine.
- the heterologous protein scaffold comprises a heterologous protein scaffold as described in Table 1 of US Patent No. 10,351,603, the contents of which are incorporated by reference in their entirety.
- the fusion polypeptide composition further comprises a pharmaceutically acceptable carrier.
- the fusion polypeptide composition further comprises an adjuvant.
- a vaccine composition comprising the fusion polypeptide composition.
- a cell expressing the fusion polypeptide is provided herein.
- composition comprising a nucleic acid encoding the fusion polypeptide.
- a method of vaccinating a subject against a coronavirus comprising administering a composition comprising a fusion polypeptide composition as described herein to the subject.
- a method of making 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.
- a polypeptide comprising a coronavirus receptor binding domain (RBD) comprising a mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L, relative to a coronavirus polypeptide of SEQ ID NO: 1.
- RBD coronavirus receptor binding domain
- the mutation is selected from the group consisting of I358F, Y365F, Y365W, V367F, and F392W.
- the polypeptide comprises a second mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L.
- the polypeptide comprises a third mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L.
- the polypeptide comprises the polypeptide sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
- the polypeptide comprises a heterologous protein scaffold.
- the heterologous protein scaffold has at least 90%, at least 95%, or at least 98% identity to a polypeptide sequence of SEQ ID NO: 3.
- the heterologous protein scaffold comprises a polypeptide of SEQ ID NO: 3.
- the polypeptide comprises the polypeptide sequence of SEQ ID NO: 6 or SEQ ID NO: 7.
- polypeptide complex comprising or consisting of a first component consisting of the polypeptide of any one of claims 59-62 and a second component that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 13-18.
- a vaccine composition comprising a composition or the polypeptide complex as described herein.
- the composition further comprises a pharmaceutically acceptable carrier.
- the vaccine composition further comprises an adjuvant.
- Another aspect provided herein relates to a cell expressing a polypeptide as described herein.
- Another aspect provided herein relates to a nucleic acid encoding a polypeptide as described herein.
- Another aspect provided herein relates to a method of vaccinating a subject against a coronavirus, the method comprising administering a polypeptide, a protein complex, or a vaccine composition as described herein to the subject.
- Another aspect provided herein relates to a method of making a vaccine, the method comprising combining a polypeptide as described herein with an adjuvant and a pharmaceutically acceptable carrier.
- Another aspect provided herein relates to a method of making a vaccine, the method comprising combining a first component consisting of a polypeptide as described herein; a second component that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 13-18; a pharmaceutically acceptable carrier; and optionally an adjuvant.
- 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 the at least one mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L, or at corresponding residues of a second coronavirus receptor binding domain as determined by a 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.
- the Blast-p program used is the National Center for Biotechnology Information (NCBI) online alignment tool.
- NCBI National Center for Biotechnology Information
- the Blast-p program can be downloaded onto a device and used locally.
- the mutation is selected from the group consisting of I358F, Y365F, Y365W, V367F, and F392W.
- the polypeptide comprises a second mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L.
- the polypeptide comprises a third mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L.
- 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 the at least two mutations are selected from the group consisting of:
- RBD coronavirus receptor binding domain
- F338L/I358F/A363L/Y365M or, a second coronavirus RBD comprising at least two mutations corresponding to F338L/Y365W;
- FIGs. 1A-1B Exemplary non-naturally occurring SARS-CoV-2 stabilized receptor binding domains having one or more mutations and show enhanced expression compared to the starting construct (i.e., native or wild-type SARS-CoV-2 spike protein).
- FIG. 1A Reducing and non-reducing SDS-PAGE analysis of supernatants used to express designed repacked RBD (“Rpk”) variants genetically fused to the I53-50A trimeric nanoparticle component.
- the wildtype control contains no mutations to the RBD, and the negative control uses a plasmid that does not encode for any secreted protein. Migration of monomeric and oxidized dimeric species are marked.
- FIG. IB List of mutations included in all constructs. Mutations listed in bold were validated alone in Starr et al. 2020.
- FIGs. 2A-2C Structural models showing the location of stabilizing mutations to the
- FIG. 2A Surface representation of the SARS-CoV-2 spike protein based on PDB- ID 6VYB (left) with the box highlighting the RBD, and a zoom-in of the RBD with a transparent surface representation, including a representation of N-glycans.
- FIG. 2B Zoom-in on the region of the SARS- CoV-2 receptor binding domain that contains most designed mutations.
- Fig. 2C Structural models of two representative designed sets of mutations, named Rpk4 and Rpk9, which stabilize the RBD.
- FIG. 3 Bio-layer interferometry (BLI) measuring binding of human ACE2 receptor and CR3022 antibody to supernatants used to express stabilized RBDs genetically fused to the I53-50A trimeric component.
- ACE2 and CR3022 were immobilized on sensors before exposing to supernatants.
- Data from (i) all designs, (ii) a construct containing a wildtype RBD, and (iii) negative control serum is shown in the graph.
- FIGs. 4A-4E Biochemical, biophysical and antigenic characterization of stabilized
- FIG. 4A Size- exclusion chromatography (SEC) purification of wild-type and stabilized RBDs after expression from equal volumes of HEK293F cultures followed by IMAC purification and concentration. Monomeric RBDs (left) were purified using a Superdex 75 Increase 10/300 GL while fusions to the I53-50A trimer (right) were purified using a Superdex 200 Increase 10/300 GL.
- SEC Size- exclusion chromatography
- FIG. 4B Thermal denaturation of wild-type and stabilized RBD monomers (left) and fusions to the I53-50A trimer (right), monitored by nanoDSF using intrinsic tryptophan fluorescence. Top panels show the barycentric mean (BCM) of each fluorescence emission spectrum as a function of temperature, while lower panels show smoothed first derivatives used to calculate melting temperatures.
- FIG. 4C Hydrogen/deuterium -exchange mass spectrometry (HDX-MS) of wild-type and stabilized RBDs fused to I53-50A trimers.
- HDX-MS Hydrogen/deuterium -exchange mass spectrometry
- Structural model (top, from PDB 6W41) shows panoramic difference uptake results of both Rpk4-I53-50A and Rpk9-I53-50A trimers compared to wild-type RBD-I53-50A trimer, with shading determined based on decreased uptake level in mutant trimers measured at 1 min.
- the box highlights the peptide segment from residues 392-399, with exchange for this peptide shown at multiple timepoints: 3 sec, 15 sec, 1 min, 30 min, and 20 h (bottom). Each point is an average of two measurements. Standard deviations are shown unless smaller than the points ploted.
- FIG. 4E Binding kinetics of immobilized CV30 and CR3022 monoclonal antibodies to monomeric wild-type and stabilized RBDs as assessed by BLI.
- Experimental data from five concentrations of RBDs in two-fold dilution series (grey traces) were fited (black lines) with binding equations describing a 1 : 1 interaction.
- Structural models (left) were generated by structural alignment of the SARS-CoV-2 bound to CV30 Fab (PDB 6XE1) and CR3022 Fab (PDB 6W41).
- FIGs. 5A-5E Stabilized RBDs presented on assembled 153-50 nanoparticles enhance solution stability compared to the wild-type RBD.
- FIG. 5A Schematic of assembly of 153-50 nanoparticle immunogens displaying RBD antigens (designated by addition "‘-153-50”).
- FIG. 5B Negative stain electron microscopy (nsEM) of wild-type RBD-I53-50, Rpk4-I53-50, and Rpk9-I53-50 (scale bar, 200 nm).
- FIG. 5C-5E show summarized quality control results for 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 The ratio of absorbance at 320 to 280 nm in UV-Vis spectra, an indicator of the presence of soluble aggregates.
- FIG. 5D Dynamic Light Scattering (DLS) measurements, which monitor both proper nanoparticle assembly and formation of aggregates.
- FIG. 5E Fractional reactivity of 153-50 nanoparticle immunogens against immobilized hACE2-Fc receptor (top) and CR3022 (bottom). The pre-freeze and post-freeze data were separately normalized to the respective CHAPS-containing samples for each nanoparticle.
- DLS Dynamic Light Scattering
- FIGs. 6A-6C Potent immunogenicity of the parental wdld-type RBD-L53-50 nanoparticle immunogen is maintained with addition of Rpk mutations.
- FIG. 6A Female BALB/c mice (six per group) were immunized at weeks 0 and 3. Each group received equimolar amounts of RBD antigen adjuvanted with AddaVax, which in total antigen equates to 5 pg per dose for HexaPro-foldon and 0.88 pg per dose for all other immunogens. Serum collection was performed at wrecks 2 and 5 weeks. The RBD-I53-50 immunogen was prepared in two different buffer conditions, with one group including CHAPS as an excipient. FIG.
- FIG. 6B Binding titers against HexaPro-foldon at weeks 2 and 5, as assessed by AUC from ELISA measurements of serial dilutions of serum. Each circle represents the AUC measurement from an individual mouse and horizontal lines show the geometric mean of each group. One mouse with a near-zero AUC at week 2 for group four was not ploted but still included in the geometric mean calculation.
- FIG. 6C Autologous (D614G) pseudovirus neutralization using a lentivirus backbone. Each circle represents the neutralizing antibody titer at 50% inhibition (IC 50 ) for an individual mouse and horizontal lines show the geometric mean of each group. Statistical analysis was performed using one-sided nonparametric Kruskal-Wallis test with Dunn’s multiple comparisons. *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001.
- FIGs. 7A-7C Shelf-life stability of RBD-based nanoparticle immunogens is improved by Rpk mutations.
- FIG. 7A Summary of DLS measurements over four weeks. Hydrodynamic diameter remained consistent for all nanoparticles except wild -type RBD-I53-50 at 35-40°C, which showed signs of aggregation after 28 days of storage.
- FIG. 7B Binding against immobilized hACE2-Fc receptor (dashed lines) and CR3022 mAb (solid lines) by BLI, normalized to -80°C sample for each time point.
- FIG. 7C Summary of SDS-PAGE and nsEM over four weeks. No degradation was observed by SDS-PAGE. Partial aggregation was only observed by nsEM on day 28 for the WT nanoparticle stored at 35-40°C. Electron micrographs for day 28 after storage at 35 ⁇ 40°C are shown, with boxes indicating instances of aggregates (scale bar, 200 nm). All samples were formulated in TBS, 5% glycerol, 100 mM L-arginine.
- FIGs. 8A-8D Rpk9 mutations can be incorporated into full length SARS-CoV-2 S ectodomains containing HexaPro mutations.
- FIG. 8A SEC purification of wild-type (HexaPro-foldon) and Rpk9 (Rpk9 ⁇ HexaPro ⁇ foldon) prefusion-stabilized S ectodomains after expression from equal volumes of HEK293F cultures followed by IMAC purification and concentration. S ectodomains were purified using a Superose 6 Increase 10/300 GL.
- FIG. 8A SEC purification of wild-type (HexaPro-foldon) and Rpk9 (Rpk9 ⁇ HexaPro ⁇ foldon) prefusion-stabilized S ectodomains after expression from equal volumes of HEK293F cultures followed by IMAC purification and concentration. S ectodomains were purified using a Superose 6 Increase 10/300 GL.
- FIG. 8B Reducing and non-reducing SDS-PAGE of intermediates and final products during the purification of HexaPro-foldon and Rpk9-HexaPro-foldon.
- FIG. 8C Thermal denaturation of HexaPro-foldon and Rpk9 ⁇ HexaPro-foldon, monitored by nanoDSF using intrinsic tryptophan fluorescence. The barycentric mean (BCM) of the fluorescence emission spectra is plotted as a function of temperature.
- FIG. 8D nsEM of HexaPro-foldon and Rpk9-HexaPro- foldon (scale bar, 100 nm).
- FIG. 9 When added to the RBD of the B.1.351 variant, the Rpk9 mutations improved relative recovery of 153-50 nanoparticles displaying the RBD at the proper SEC elution volume in simpler buffer formulations, which shows that Rpk mutations improve the integrity of immunogens containing RBDs from different variants.
- FIG. 10 SDS-PAGE for nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100 mM L-arginine.
- DTT +/- reducing agent
- F/T pre- and post-freeze thaw
- FIG. 11 hACE2-Fc binding for nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100 mM L-arginine.
- FIG. 12 CR3022 binding for nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100 mM L-arginine.
- FIG. 13 Dynamic light scattering for nanoparticles in TBS, 5% glycerol, 0.75%
- FIG. 14 UV-Vis for nanoparticles in TBS, 5% glycerol, 0.75% CHAPS, 100 mM L-arginine. UV-Vis spectra (nm) for each sample in 50 mM Tris pH 7.4, 185 mM NaCl, 4.5% glycerol, 0.75% CHAPS, 100 mM L-arginine, plotted as normalized absorbance.
- FIG. 15 SDS-PAGE for nanoparticles in TBS, 5% glycerol, 100 mM L-arginine.
- DTT +/- reducing agent
- F/T pre- and post-freeze thaw
- FIG. 16 hACE2-Fc binding for nanoparticles in TBS, 5% glycerol, 100 mM L- arginine.
- FIG. 17 CR3022 binding for nanoparticles in TBS, 5% glycerol, 100 mM L- arginine.
- FIG. 18 Dynamic light scattering for nanoparticles in TBS, 5% glycerol, 100 mM
- FIG. 19 UV-Vis for nanoparticles in TBS, 5% glycerol, 100 mM L-arginine. UV- Vis spectra (nm) for each sample in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, 100 mM L-arginine, plotted as normalized absorbance.
- FIG. 20 SDS-PAGE for nanoparticles in TBS, 5% glycerol.
- FIG. 22 CR3022 binding for nanoparticles in TBS, 5% glycerol.
- FIG. 23 Dynamic light scattering for nanoparticles in TBS, 5% glycerol.
- FIG. 24 UV-Vis for nanoparticles in TBS, 5% glycerol. UV-Vis spectra (nm) for each sample in 50 mM Tris pH 8, 150 mM NaCl, 5% glycerol, plotted as normalized absorbance.
- FIG. 25 SDS-PAGE for nanoparticles in TBS.
- the integrity of samples in 50 mM Tris pH 8, 150 mM NaCl was analyzed by SDS-PAGE. Molecular weights of the standard are noted in kDa.
- DTT +/- reducing agent
- F/T pre-and post-freeze thaw
- FIG. 26 hACE2-Fc binding for nanoparticles in TBS.
- H ACE2-Fc binding of antigen in 50 mM Tris pH 8, 150 mM NaCl was analyzed by bio-layer interferometry (BLI).
- FIG. 27 CR3022 binding for nanoparticles in TBS.
- CR3022 IgG binding of antigen in 50 mM Tris pH 8, 150 mM NaCl was analyzed by bio-layer interferometry (BLI).
- FIG. 28 Dynamic light scattering for nanoparticles in TBS. Hydrodynamic diameter
- FIG. 29 UV-Vis for nanoparticles in TBS. UV-vis spectra (nm) for each sample in
- FIG. 30 SDS-PAGE for RBD-I53-50 nanoparticle.
- the integrity of samples after incubation at four temperatures over a 28 day (D) study was analyzed by SDS-PAGE. Molecular weights of the standard are noted in kDa. Each sample was analyzed +/-reducing agent (DTT).
- FIG. 31 hACE2-Fc binding for RBD-I53-50 nanoparticle.
- hACE2-Fc binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by bio-layer Interferometry (BLI).
- FIG. 32 CR3022 binding for RBD-I53-50 nanoparticle.
- CR3022 IgG binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by bio-layer Interferometry (BLI).
- FIG. 33 nsEM for RBD-I53-50 nanoparticle. Representative negative stain electron micrographs for each sample at days (D) 1 and 28 following incubation at four temperatures. Scale bar, 50 nm.
- FIG. 34 Dynamic light scattering for RBD-I53-50 nanoparticle. Hydrodynamic diameter (nm) for each sample over a 28 day (D) period, plotted as normalized intensity.
- FIG. 35 SDS-PAGE for Rpk4-I53-50 nanoparticle.
- the integrity of samples after incubation at four temperatures over a 28 day (D) study was analyzed by SDS-PAGE. Molecular weights of the standard are noted in kDa. Each sample was analyzed +/-reducing agent (DTT).
- FIG. 36 hACE2-Fc binding for Rpk4-I53-50 nanoparticle.
- hACE2-Fc binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by bio-layer Interferometry (BLI).
- FIG. 37 CR3022 binding for Rpk4-I53-50 nanoparticle.
- CR3022 IgG binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by bio-layer Interferometry (BLI).
- FIG. 38 nsEM for Rpk4-I53-50 nanoparticle. Representative negative stain electron micrographs for each sample at days (D) 1 and 28 following incubation at four temperatures. Scale bar, 50 nm.
- FIG. 39 Dynamic light scattering for Rpk4-I53-50 nanoparticle. Hydrodynamic diameter (nm) for each sample over a 28 day (D) period, plotted as normalized intensity.
- FIG. 40 SDS-PAGE for Rpk9-I53-50 nanoparticle.
- the integrity of samples after incubation at four temperatures over a 28 day (D) study was analyzed by SDS-PAGE. Molecular weights of the standard are noted in kDa. Each sample was analyzed +/-reducing agent (DTT).
- FIG. 41 hACE2-Fc binding for Rpk9-I53-50 nanoparticle.
- hACE2-Fc binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by bio-layer Interferometry (BLI).
- FIG. 42 CR3022 binding for Rpk9-I53-50 nanoparticle.
- CR3022 IgG binding of antigen incubated at four different temperatures for 28 days (D) was analyzed by bio-layer Interferometry (BLI).
- FIG. 43 nsEM for Rpk9-I53-50 nanoparticle. Representative negative stain electron micrographs for each sample at days (D) 1 and 28 following incubation at four temperatures. Scale bar, 50 nm.
- FIG. 44 Dynamic light scattering for Rpk9-I53-50 nanoparticle. Hydrodynamic diameter (nm) for each sample over a 28 day (D) period, plotted as normalized intensity.
- compositions and methods comprising coronavirus “S” spike proteins with at least one, two or more amino acid mutations that increase their expression level, yield and/or stability compared to a native or wild-type coronavirus spike protein (e.g., SARS-CoV-2 S protein) under the same expression, culture or storage conditions.
- SARS-CoV-2 S protein a native or wild-type coronavirus spike protein
- These mutated spike proteins can be used for generating a protein-based vaccine against SARS-CoV-2, a different coronavirus known to infect humans, or a pan-coronavirus vaccine that provides protection against multiple coronaviruses known to infect humans.
- the S protein comprises a single mutation that increases the expression level, yield and/or stability of a mutant coronavirus spike protein under certain expression, culture, or storage conditions compared to a native or wild-type coronavirus spike protein.
- the S protein comprises a plurality of mutations (e.g., 2, 3, 4, or 5).
- non-naturally occurring or “mutant” as used herein refer to a coronavirus polypeptide (e.g., stabilized coronavirus S protein or RBD polypeptide) that comprises at least one or at least two amino acid residue mutations and that preferably comprise enhanced stability and/or expression compared to its corresponding native or wild-type coronavirus sequence.
- the mutant polypeptides described herein are “substantially similar” to their native counterpart, for example, except for the at least two mutations.
- the native counterpart can include a naturally occurring coronavirus variant.
- coronavirus sequence e.g., SARS- Cov-2 variants: B.1.1.7; B.1.351; P.l; B.1.427; B.1.429; B.1.526; B.l.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 “mutant” coronavirus sequences, however such variants can be used as a reference coronavirus sequence as that term is used herein.
- SARS- Cov-2 variants e.g., SARS- Cov-2 variants: B.1.1.7; B.1.351; P.l; B.1.427; B.1.429; B.1.526; B.l.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 “mutant” coronavirus sequences, however
- non-naturally occurring coronavirus spike protein subunit 1 polypeptide refers to a polypeptide that comprises, at a minimum, the receptor binding domain sequence (residues 328-531 of SEQ ID NO: 1), residues that permit the structural formation of a linoleic acid binding pocket, and at least one or at least two amino acid mutations.
- one of the at least two mutations comprises a “cavity-filling mutation,” as that term is used herein.
- a molecule is said to be "substantially similar" to another molecule if both molecules have substantially similar structures (i.e., they are at least 90% similar in amino acid sequence as determined by Blast-p alignment set at default parameters) and are substantially similar in at least one relevant function (e.g., antigenic activity as determined by recognition of the polypeptide by an antibody that binds the native coronavirus counterpart). That is, a mutant polypeptide differs from the naturally occurring polypeptide or nucleic acid by one or more amino acid or nucleic acid deletions, additions, substitutions or side-chain modifications, yet retains one or more specific functions or biological activities of the naturally occurring molecule.
- Amino acid substitutions include alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Some substitutions can be classified as “conservative,” in which case an amino acid residue contained in a polypeptide is replaced with another naturally occurring amino acid of similar character either in relation to polarity, side chain functionality or size.
- substitutions encompassed by variants as described herein can also be “non-conservative,” in which an amino acid residue which is present in a peptide is substituted with an amino acid having different properties (e.g., substituting 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.
- mutant when used with reference to a polypeptide, are variations in primary, secondary, or tertiary structure, as compared to a reference polypeptide (e.g., as compared to a wild- type coronavirus polypeptide).
- Mutants can also include insertions, deletions or substitutions of amino acids, including insertions and substitutions of amino acids and other molecules) that do not normally occur in the peptide sequence that is the basis of the variant, including but not limited to insertion of ornithine which does not normally occur in human proteins.
- corresponding to or corresponding wild-type coronavirus refers to a wild-type coronavirus polypeptide sequence (or a naturally occurring variant thereof) from which the non-naturally occurring coronavirus polypeptide (e.g., spike polypeptide) or RBD polypeptide is produced.
- the wild-type coronavirus sequence or its naturally occurring variant is from the same strain as the non-naturally occurring coronavirus polypeptide.
- a mutated SARS-CoV-2 polypeptide as described herein will correspond to a wild-type coronavirus polypeptide for a SARS-CoV-2 sequence or a naturally occurring variant thereof (e.g., South Africa variant, Brazilian variant, Los Angeles variant etc).
- naturally occurring variant refers to a coronavirus sequence that spontaneously arises in a population of susceptible individuals.
- the term “increased stability” or “enhanced stability” refers to a mutated coronavirus protein sequence that degrades at a slower rate in a cell, solution or formulation than the corresponding native or wild-type coronavirus protein sequence (or naturally occurring variant thereof) under the same conditions and thus persists for at least 12 h longer than the corresponding native or wild-type coronavirus protein sequence (or naturally occurring variant thereof), for example, as assessed using a thermal melt assay as described in the working Examples herein.
- the mutated coronavirus protein sequence persists in a cell, solution or formulation for at least 24h, 36h, 48h, 72h, 7 days, 8 days, 9 days, 10 days, 2 weeks, one month, 2 months, 3 months, 4 months, 5 months, 6 months, 9 months, one year, two years or more compared to the persistence of the corresponding native or wild-type coronavirus protein sequence.
- higher expression levels can also be indicative of, or a result of, enhanced stability of a polypeptide.
- the term “increased yield” or “enhanced yield” refers to an increase of at least 10% in the amount of mutated coronavirus protein recovered from a cell system in which the protein is produced compared to the amount of native or wild-type protein (or a naturally occurring variant thereof) recovered from the same cell system under the same growth and isolation conditions.
- “enhanced yield” refers to an increase of 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 mutated coronavirus protein recovered from a cell system in which the protein is produced compared to the amount of native or wild-type protein recovered from the same cell system under the same growth conditions.
- the term “cavity fdling mutation” refers to the substitution of an amino acid residue in a wild-type coronavirus spike protein by an amino acid that is expected to “fill” an internal cavity of the mature coronavirus spike protein.
- the substituted amino acid is of an appropriate size or charge that it protrudes into the cavity and sterically reduces the cavity size and/or impairs a cognate ligand from binding within the cavity or pocket.
- adjuvant refers to a protein or chemical that, when administered with a vaccine antigen, enhances the immune response to the vaccine antigen.
- An adjuvant is distinguished from an antigenic moiety or carrier protein in that the adjuvant is not chemically coupled to the immunogen or the antigen.
- Adjuvants are well known in the art and include, for example, mineral oil emulsions such as Freund's complete or Freund's incomplete adjuvant (Freund, Adv. Tuberc. Res. 7: 130 (1956); Calbiochem, San Diego Calif.), aluminum salts, especially aluminum hydroxide or ALHYDROGELTM (approved for use in humans by the U.S.
- MDP muramyl dipeptide
- [Thrl]-MDP Boers and Allison, Vaccine 5:223 (1987)
- monophosphoryl lipid A Johnson et al., Rev. Infect. Dis. 9:S512 (1987)
- the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
- compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
- Coronaviruses are a family of hundreds of viruses that can cause fever, respiratory problems, and sometimes gastrointestinal symptoms.
- SARS-CoV-2 the virus that causes Coronavirus Disease 2019 (COVID-19) is one of seven members of this family known to infect humans, and the third in the past three decades to jump from animals to humans.
- Other coronaviruses known to infect humans include alphacoronaviruses 229E and NL63, and betacoronaviruses OC43, HKU 1, SARS-CoV (the coronavirus that causes severe acute respiratory syndrome, or SARS), and MERS-CoV (the coronavirus that causes Middle East Respiratory Syndrome, or MERS).
- 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 deltacoronavirus (PDCoV), bovine coronavirus (BCV), feline coronavirus (FCoV), canine coronavirus (CCoV), avian infectious bronchitis virus (IBV), and turkey coronavirus (TCV).
- the coronaviruses described herein include those that are currently known and those that are later discovered. Specifically contemplated herein are coronaviruses that are the cause of an ongoing or future epidemic or pandemic.
- coronavirus refers to an enveloped virus with a positive- sense single -stranded RNA genome and a helical symmetry.
- the genomic size of coronaviruses ranges from approximately 27 to 32 kilobases, which is the longest size for any known RNA virus.
- Large Spike (S) glycoproteins protrude from the virus particle giving coronaviruses a distinctive corona-like appearance when visualized by electron microscopy.
- Coronaviruses infect a wide variety of species, including canine, feline, porcine, murine, bovine, avian and human (Holmes, et al., 1996. Coronaviridae: the viruses and their replication, p. 1075-1094.
- Coronaviruses typically bind to target cells through Spike-receptor interactions and enter cells by receptor mediated endocytosis or fusion with the plasma membrane (Holmes, et al., 1996, supra).
- the Spike-receptor interaction is a strong determinant of species specificity as demonstrated for both group 1 and group 2 coronaviruses.
- the genome of SARS-CoV contains a single stranded (+)- sense RNA.
- SARS coronavirus Urbani GenBank accession # AY278741
- SARS coronavirus Tor2 GenBank accession # AY274119
- SARS coronavirus CUHK-W1 GenBank accession # AY278554
- SARS-CoV Shanghai LY GenBank accession # H012999; GenBank accession # AY322205; GenBank accession # AY322206
- SARS-CoV Shanghai QXC GenBank accession # AH013000; GenBank accession # AY322208; GenBank accession # AY322197; GenBank accession # AY322199
- SARS-CoV ZJ-HZ01 GenBank accession # AY322206
- the S (spike) protein may form non-covalently linked homotrimers (oligomers), which may mediate receptor binding and virus infectivity. Homotrimers of S proteins are likely necessary for presenting the correct native conformation of receptor binding domains and for eliciting a neutralizing antibody response.
- intracellular processing of S protein is associated with significant post- translation oligosaccharide modification.
- the post-translation oligosaccharide modification (glycosylation) expected by N-glycan motif analysis indicates that the S protein has as many as 23 sites for such modification.
- C-terminal cysteine residues may also participate in protein folding and preserving the native (functional) S protein conformation.
- the S protein of some coronaviruses can be proteolytically processed near the center of the S protein by a trypsin-like protease in the Golgi apparatus or by extracellularly localized enzymes into to a linked polypeptide, containing an N-terminal SI and a C-terminal S2 polypeptide.
- Some members of the type II group of coronaviruses and group I viruses may not be so processed.
- a subject e.g., a human, is diagnosed as having a coronavirus infection based on diagnostic test results.
- a subject can be suspected of having a coronavirus infection (e.g., COVID- 19, SARS, MERS etc.) based on one or more presenting symptoms such as, fever, chills, cough, shortness of breath/difficulty breathing, fatigue, muscle/body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea, vomiting, or diarrhea.
- a coronavirus infection e.g., COVID- 19, SARS, MERS etc.
- an asymptomatic infection e.g., SARS-CoV-2 infection
- an active coronavirus infection can be confirmed using a method known in the art that detects one or more of viral antigens and viral nucleic acid in a sample taken from a subject. Examples include using a reverse -transcriptase polymerase chain reaction (RT-PCR) diagnostic assay from a nasopharyngeal swab or sputum to detect viral RNA.
- RT-PCR reverse -transcriptase polymerase chain reaction
- nucleic acid amplification methods e.g., any of a number of isothermal amplification methods, can also be used, and have sensitivity close to, if not equal to RT-PCR.
- Isothermal amplification methods have the advantage of not requiring a thermal cycler to generate an amplified product, and can more rapidly provide results in a highly sensitive manner.
- an active coronavirus infection can be determined by detecting one or more coronavirus polypeptides (such as an antigen) in a biological sample obtained from the subject. Lateral flow assays for viral antigens can provide qualitative, and sometimes quantitative diagnostic results. Viral polypeptides can also be detected by other methods known in the art, such as Western blot.
- Assessing the presence of a coronavirus antibody can be used to determine if a subject has been exposed to a coronavirus in the past or alternatively as a means to monitor the effectiveness of a vaccine (i.e., ability of a mutated spike protein to raise an immune response).
- Methods for assessing the presence of a coronavirus antibody are known in the art and are not discussed in detail herein.
- coronavirus virions can be determined directly or indirectly by using, for example, electron microscopy.
- Protein Sequence The amino acid sequence of the native or wild-type SARS-CoV-2 S protein, subunit 1 is:
- SEQ ID NO: 1 is used as a ‘base’ or ‘reference’ sequence to which other coronavirus amino acid sequences can be aligned using an alignment program known in the art (e.g., Blast-p).
- An alignment of a different (or second) coronavirus sequence’s spike protein 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 a given amino acid mutation or mutations to achieve stabilization as described herein.
- the different coronavirus sequence is aligned against 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).
- the Blast-p program used is the National Center for Biotechnology Information (NCBI) online alignment tool.
- NCBI National Center for Biotechnology Information
- the Blast-p program can be downloaded onto a device and used locally.
- Protocol 1 For use with the BLASTp alignment online from the National Center for
- NCBI Biotechnology Information
- Protocol 2 For use with the protein BLASTp alignment tool downloaded onto a local computer or server.
- Clustalw or Clustal-omega can also be used to identify sequence identity between a query sequence and a reference sequence (e.g., SEQ ID NO: 1). Given that the query sequence and the reference sequence share significant sequence identity, it is expected that other protein alignment tools will produce similar, if not identical results to Blast-p using the protocols described herein.
- the protocols described herein have been shown to be accurate and effective for this purpose and are provided herein to aid the skilled artisan in identifying amino acid residues to be mutated in the query sequence.
- a virus-surface “spike” protein mediates the entry of coronavirus into host cells.
- the spike proteins of SARS-CoV and SARS-CoV-2 each contain a receptor binding domain that specifically recognizes angiotensin converting enzyme-2 (ACE2) as its receptor. Given the importance of this domain in viral uptake and function, the receptor binding domain is relatively well conserved among the coronaviruses that are known to infect humans.
- the sequence of the receptor binding domain for the spike protein of SARS-CoV-2 is:
- SEQ ID NO: 2 is used as a ‘base’ or ‘reference’ receptor binding domain sequence to which other coronavirus RBD sequences can be aligned using an alignment program known in the art (e.g., Blast-p, ClustalW etc.).
- An alignment of at least a second coronavirus RBD sequence with SEQ ID NO: 2 can be used to determine the corresponding site in the second coronavirus RBD sequence for a given amino acid mutation in SARS-CoV-2.
- Blast-p can be used to align the coronavirus RBD query sequence with SEQ ID NO: 2 using protocol 1 or protocol 2 as described herein.
- the receptor binding domain polypeptides comprise at least two mutations within SEQ ID NO: 2 (or equivalent for a different coronavirus).
- the receptor binding domain polypeptides can comprise an additional mutation. This additional mutation can also be in the RBD region or can occur outside of the RBD region.
- the at least one, two or more amino acid mutations do not include those mutations that are found in naturally occurring variants of a given coronavirus sequence. For example, L452R and E484K, which are present in naturally occurring variants of SARS-CoV-2 are not counted as the at least one, two or more amino acid mutations as described herein.
- the receptor binding domain polypeptides described herein are used in the generation of a protein vaccine against one or more coronaviruses.
- the mutated coronavirus protein need not necessarily retain receptor binding properties to its cognate receptor; thus the at least one amino acid mutation or at least two amino acid mutations do not need to be designed to retain the function of the RBD.
- coronavirus proteins having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2 are specifically contemplated herein (e.g., at least 95%, at least 99% identity to SEQ ID NO: 1 or 2) provided that they retain the ability to act as a coronavirus antigen (i.e., stimulate the production of coronavirus-binding antibodies in a subject or bind a coronavirus antibody directed against the corresponding wild-type coronavirus).
- the receptor binding domain polypeptides described herein are “immunogenic,” i.e., immunization of a subject with a receptor binding domain polypeptide (optionally bound to an appropriate support (such as a protein, a lipid or a polypeptide)), induces an immune response (of the B cell type and/or of the T cell type), directed against the RBD polypeptide.
- an appropriate support such as a protein, a lipid or a polypeptide
- epitope refers to an antigenic determinant in a molecule such as an antigen, i.e., to a part in or fragment of the molecule that is recognized by the immune system, for example, that is recognized by a T cell or B cell, in particular when presented in the context of MHC molecules.
- An epitope of a protein antigen can comprise a continuous or discontinuous portion of said protein and can be between 5 and 100, between 5 and 50, between 8 and 30, between 10 and 25 amino acids in length, for example, the epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
- the ability of a protein to raise an immune response can be attributed, in part, to its secondary structure and the conformation in which the protein is folded. In some embodiments, a certain conformation is preferred for generating an antigenic response and/or for augmenting stability of the protein.
- the secondary structure of SARS-CoV-2 bound to the ACE2 receptor is described in Shang et al. “Structural Basis of Receptor Recognition by SARS-CoV-2” Nature 581:221-224 (2020), the contents of which are incorporated herein by reference in their entirety. With knowledge of the crystal structure of SARS-CoV-2, one of skill in the art can use educated reasoning or computational software to determine whether a given receptor binding domain polypeptide is likely to comprise a shape or secondary structure that would induce an immune response in a subject.
- At least two amino acids are mutated in a coronavirus receptor binding domain, which enhance the yield of the protein in a cell system and/or enhance the stability of the coronavirus protein in a cell, solution, or formulation as compared to its corresponding wild-type protein.
- at least one amino acid is mutated in a coronavirus receptor binding domain, which enhances the yield of the protein in a cell system and/or enhances the stability of the coronavirus protein in a cell, solution, or formulation as compared to its corresponding wild-type protein.
- the at least two mutations comprise at least two mutations are introduced within the RBD region of the spike protein (SEQ ID NO: 2 or residues 328-531 of SEQ ID NO: 1). In some embodiments, the at least one amino acid mutation is introduced within the RBD region of the spike protein.
- the at least two mutations are at amino acids residues: 338 &
- the at least one amino acid mutation is at amino acid residue:
- the at least one amino acid mutation is: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, or F515L.
- the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I; Y365W/L513I/F515L; F338L/A363L/Y365M; F338L/I358F/Y365W; I358F/Y365W/L513M; I358F/Y365W/F392W;
- the receptor binding domain polypeptide comprises a fusion protein.
- SARS-CoV-2 “S” (spike) protein has been shown to comprise a “pocket” or
- mutations within the linoleic acid binding pocket or sub-domains of the linoleic acid binding pocket can be used to mimic the effects of linoleic acid and/or to stabilize the closed conformation of the S protein.
- an amino acid mutation in this region is a ‘cavity-filling’ mutation.
- the cavities in a native coronavirus spike protein can be identified by methods known in the art, such as by visual inspection of a crystal structure representation of the spike protein of, e.g., SARS-CoV-2 (see e.g., Shang et al.
- amino acids to be replaced for cavity-filling mutations can include small aliphatic amino acids
- a charged amino acid can replace or be replaced by a non-charged amino acid, thereby altering the secondary structure of the protein and the cavity.
- residues for replacement can also include amino acids that are buried in a given protein conformation, but exposed to solvent in a second conformation.
- At least one of the at least two mutations in the SARS-CoV-2 spike protein is at a residue involved in the linoleic binding pocket.
- Such mutations can stabilize a particular conformation of the protein and/or decrease the rate of degradation of the protein compared to the corresponding wild-type coronavirus.
- the residues involved in linoleic acid binding are separated herein into three sub- domains. These sub-domains are based simply on the close proximity of several residues involved in linoleic acid binding.
- Non-naturally occurring coronavirus spike protein subunit 1 polypeptides can comprise at least one cavity filling mutation or mutation in a residue in the linoleic acid binding pocket and at least one additional mutation that together enhance the stability and/or yield of the polypeptides as those terms are used herein.
- the cavity-filling mutation comprises mutation of a residue of
- SEQ ID NO: 1 at amino acid 336, 338, 341, 342, 358, 361, 363, 365, 368, 374, 377, 387, or 392 or of a corresponding residue of a second coronavirus spike protein, subunit 1 as determined by a sequence alignment of SEQ ID NO: 1 (or a naturally occurring variant thereof) with the sequence of the second coronavirus (or a naturally occurring variant thereof) using Blast-p.
- the cavity- filling mutation and the at least one second mutation are at residues 338 & 365; 365 & 513; 365 & 392; 338, 363, 365 & 377; 365 & 392; 365 & 395; 365, 392 & 395; 365, 513, & 515; 338, 363, & 365; 338, 358 & 365; 358, 365, & 513; 358, 365 & 392; 338, 358, 363, 365 & 377; 358, 365, & 392; 358, 365 & 395; 358, 365, 392, & 395; 358, 365, 513 & 515; and/or 338, 358, 363, & 365 of SEQ ID NO: 1, or at corresponding residues of a second coronavirus spike protein, subunit 1 as determined by a sequence alignment of SEQ ID NO: 1 with the sequence of the second
- the cavity-filling mutation and the at least one second mutation are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I; Y365F/F392W/V395I;
- I358F/Y365W/F392W F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W;
- I358F/Y365F/V395I I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and
- Coronavirus spike proteins having a cavity-filling mutation as described herein and further having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2 are specifically contemplated herein (e.g., at least 95%, at least 99% identity to SEQ ID NO: 1 or 2) provided that they retain the ability to act as an antigen for the coronavirus (i.e., stimulate the production of coronavirus antibodies in a subject).
- the non-naturally-occurring coronavirus spike protein polypeptides described herein are “immunogenic,” i.e., immunization of a subject with the polypeptide (optionally bound to an appropriate support (such as a protein, a lipid or a polypeptide)), induces an immune response (of the B cell type and/or of the T cell type) directed against the polypeptide.
- an appropriate support such as a protein, a lipid or a polypeptide
- the RBD polypeptides or spike polypeptides as described herein may have one of more amino acid substitutions from known variants of the coronavirus.
- the polypeptides may comprise 1, 2, 3, 4, 5, 6, 7, or all 8 positions relative to SEQ ID NO: 1 selected from the group consisting of L18F, T20N, P26S, deletion of residues 69-70, D80A, D138Y, R190S, D215G, K417N, K417T, G446S, L452R, Y453F, T478I, E484K, S494P, N501Y, A570D, D614G, H655Y, P681H, A701V, and T716L.
- the polypeptides may comprise one of the following naturally occurring mutations or combinations of mutations:
- N501Y optionally further including 1, 2, 3, 4, or 5 of deletion of one or both of residues
- K417N/E484K/N501Y optionally further including 1, 2, 3, 4, or 5 of L18F, D80A,
- D215G, D614G, and/or A701V South African variant
- K417N or T/E484K/N501Y optionally further including 1, 2, 3, 4, or 5 of L18F, T20N,
- polypeptides as disclosed herein comprise the polypeptide sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
- mutated coronavirus S proteins or receptor binding domains thereof having at least two amino acid mutations or substitutions that confer enhanced stability of the mutated protein as compared to its corresponding native or wild-type coronavirus S protein.
- mutated SARS-CoV-2 S proteins or receptor binding domains thereof Exemplified in this specification are mutated SARS-CoV-2 S proteins or receptor binding domains thereof, however the methods and compositions described herein can be applied to any coronavirus S protein, including both coronaviruses that infect humans and those that infect other mammals (i.e., bats, bovines, porcines etc).
- SARS-CoV-2 One of skill in the art can readily identify corresponding residues to those outlined in the specification for SARS-CoV-2 by aligning the amino acid sequence for another coronavirus with that of SARS-CoV-2 (i.e., SEQ ID NO: 1 or 2).
- Alignment can provide guidance regarding residues likely to be necessary for function, whether the function is direct contact of a given residue or residues with receptor, or, for example, residues involved in maintaining a conformation that permits other residues to make such contact; non- limiting examples of the latter include those residues likely to line the linoelic acid binding pocket or aid in maintaining a given conformation of the spike protein. Where, for example, an alignment shows two identical or similar amino acids at corresponding positions, it is more likely that that site is important functionally (i.e., linoleic acid binding or receptor binding).
- the variant amino acid sequence can 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 and a mutant sequence can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose and freely available on the world wide web.
- the variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to the sequence from which it is derived (referred to herein as an “original,” “native,” or “wild-type” sequence).
- the degree of similarity (percent similarity) between an original and a mutant sequence can be determined, for example, by using a similarity matrix. Similarity matrices are well known in the art and a number of tools for comparing two sequences using similarity matrices are freely available online, e.g. BLASTp (available on the world wide web at http://blast.ncbi.nlm.nih.gov), with default parameters set or using protocol 1 or protocol 2 as described herein.
- a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn), preferably where a smaller residue is substituted by a larger one that sterically “fills” the cavity or is altered in charge to induce changes in the cavity size and/or structure.
- substitutions e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known and can conserve function.
- Polypeptides comprising desired amino acid substitutions can be tested in any one of the assays described herein to confirm that (i) a desired conformation is maintained such that the antigenic activity of a native or reference polypeptide is substantially retained, or (ii) the stability of the protein is enhanced.
- the amino acid substitutions can comprise a conservative amino acid substitution.
- conservative amino acid substitution is well known in the art, and relates to substitution of a particular amino acid by one having a similar characteristic (e.g., similar charge or hydrophobicity).
- Conservative mutations as described herein can include substitution of amino acid residues with e.g., similar charge or hydrophobicity but differing in size or bulkiness (e.g., to provide a cavity-filling function).
- a list of exemplary conservative amino acid substitutions is given in the table below.
- Non-conservative amino acid substitution may be preferred, for example, when eradication of a flexible portion of the native coronavirus S protein secondary structure is desired, for example, by adding a cysteine residue (or vice versa).
- Non-conservative substitution refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala with Asp, Asn, Glu, or Gin.
- non- conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
- a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine
- a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.
- a mutant coronavirus polypeptide e.g., a RBD polypeptide or a stabilized coronavirus S polypeptide
- a mutant coronavirus polypeptide as described herein can have a mixture of conservative and non-conservative amino acid substitutions in any desired configuration.
- the polypeptides described herein can be tested for antigenic activity, receptor binding domain activity or conformation using methods known in the art or described in the Examples.
- Cysteine residues can be important for protein secondary structure or conformation.
- cysteine residues are contemplated herein provided that the secondary structure of the mutated protein is functional and/or antigenic as determined, for example, by assessing binding to its cognate receptor (e.g., ACE2 receptor), assessing secondary structure using crystallography or EM, or confirming binding to an antibody against the native or wild-type protein.
- Cysteine residues not involved in maintaining the proper conformation of the polypeptide also can be substituted, for example, with serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
- cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.
- a stabilized coronavirus S protein or RBD polypeptide can comprise naturally occurring amino acids commonly found in polypeptides and/or proteins produced by living organisms, e.g. 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).
- the stabilized coronavirus S protein or RBD polypeptide as described herein can comprise alternative amino acids.
- Non-limiting examples of alternative amino acids include, D-amino acids; beta-amino acids; homocysteine, phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2- carboxylic acid, statine, l,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine (3-mercapto- D-valine), ornithine, citruline, alpha-methyl-alanine, para-benzoylphenylalanine, para-amino phenylalanine, p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, and tert- butylglycine), diaminobutyric acid, 7 -hydroxy -tetrahydroisoquinoline carboxylic acid, naphth
- a polypeptide e.g. a mutant coronavirus polypeptide
- modifications and/or moieties include PEGylation; glycosylation; HESylation; ELPylation; lipidation; acetylation; amidation; end-capping modifications; cyano groups; phosphorylation; albumin conjugation, and cyclization. Modifications or moieties that improve solubilization in a given solution (i.e., aqueous) solution are specifically contemplated herein.
- Alterations of the original amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced at the nucleic acid level, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required.
- a polypeptide as described herein can be chemically synthesized and mutations can be incorporated as part of the chemical synthesis process.
- mutated spike proteins or RBC polypeptides described herein can be synthesized using well known methods including recombinant methods and chemical synthesis.
- Recombinant methods of producing a polypeptide through the introduction of a vector including 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, 2d Ed, Vols 1 to 8, Cold Spring Harbor, NY (1989); M.W. Pennington and B.M. Dunn, Methods in Molecular Biology: Peptide Synthesis Protocols, Vol 35, Humana Press, Totawa, NJ (1994), contents of both of which are herein incorporated by reference.
- Peptides can also be chemically synthesized using methods well known in the art. See for example, Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964); Bodanszky, M., Principles of Peptide Synthesis, Springer-Verlag, New York, NY (1984); Kimmerlin, T. and Seebach, D. J. Pept. Res. 65:229-260 (2005); Nilsson et al., Annu. Rev. Biophys. Biomol. Struct. (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); N.L.
- a RBD polypeptide or mutated coronavirus S protein can be produced chemically by e.g., solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods, as described by Dugas et al (1981). However, it is generally preferred to synthesize the polypeptides described herein using recombinant methods.
- Systems for cloning and expressing polypeptides useful with the methods and compositions described herein include various microorganisms and cells that are well known in recombinant technology and thus are not described in detail herein. These include, for example, various strains of E. coli, Bacillus, Streptomyces, and Saccharomyces, as well as mammalian, yeast and insect cells.
- a polypeptide as described herein can be produced as a peptide or fusion protein, if so desired.
- Suitable vectors for producing peptides and polypeptides are known and available from private and public laboratories and depositories and from commercial vendors. Recipient cells capable of expressing the gene product are then transfected.
- the transfected recipient cells are cultured under conditions that permit expression of the recombinant gene products, which are recovered from the culture.
- Host mammalian cells such as Chinese Hamster ovary cells (CHO) or COS-1 cells, can be used. These hosts can be used in connection with poxvirus expression vectors, such as vaccinia or swinepox.
- Suitable non-pathogenic viral expression vectors that can be engineered to carry the synthetic gene into the cells of the host include poxvirus expression vectors, such as vaccinia, adenovirus, retroviruses and the like. A number of such non-pathogenic viral expression vectors are commonly used for human gene therapy, and as carrier for other vaccine agents, and are known and selectable by one of skill in the art.
- the 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 by reference to known techniques.
- Protein purification techniques are well known to those of skill in the art and as such are not described in detail herein. These techniques can involve, at one level, the homogenization and crude fractionation of the cells, tissue or organ to polypeptide and non-polypeptide fractions.
- the mutant coronavirus spike protein or receptor binding domain polypeptide can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity).
- a “purified polypeptide” is intended to refer to a composition, isolatable from other components, wherein the mutant coronavirus S polypeptide or receptor binding domain thereof is purified to any degree relative to the organism producing recombinant protein or in its naturally- obtainable state.
- an isolated or purified polypeptide therefore, also refers to a /polypeptide free from the environment in which it may naturally occur.
- purified will refer to a polypeptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains the ability to bind to a coronavirus antibody that binds the native or wild-type coronavirus S protein.
- substantially purified will refer to a composition in which the coronavirus polypeptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.
- polypeptides described herein are provided in the most purified state. Indeed, it is contemplated that less purified products will have utility in certain embodiments. Partial purification can be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
- the receptor binding domain polypeptides or the mutated coronavirus S proteins described herein comprise a fusion protein.
- the RBD polypeptides or mutated coronavirus S proteins described herein are fused to a scaffold, a nanoparticle, a heterologous protein scaffold, or a polymer.
- the heterologous protein scaffold comprises the 153-50 trimeric “A” component of SEQ ID NO: 3.
- the heterologous protein scaffold comprises a heterologous protein scaffold as described in Table 1 of US Patent No. 10,351,603, the contents of which are incorporated by reference in its entirety.
- the mutant coronavirus polypeptides described herein are provided as a fusion protein.
- fusion proteins can comprise, for example, an antigenic moiety to enhance the resulting immune response.
- antigenic moieties can include a foreign molecule such as a carrier protein that is foreign to the individual to be vaccinated using the fusion proteins described herein.
- Foreign proteins that activate the immune response and can be conjugated to a fusion protein as described herein include proteins or other molecules with molecular weights of at least about 20,000 Daltons, preferably at least about 40,000 Daltons and more preferably at least about 60,000 Daltons.
- Carrier proteins useful in this context include, for example, GST, hemocyanins such as from the keyhole limpet, serum albumin or cationized serum albumin, thyroglobulin, ovalbumin, various toxoid proteins such a tetanus toxoid or diphtheria toxoid, immunoglobulins, heat shock proteins, and the like.
- Methods to chemically couple one protein e.g., a RBD polypeptide or mutated coronavirus S protein
- another protein e.g., carrier or antigenic moiety
- conjugation by a water soluble carbodiimide such as l-ethyl-3- (3dimethylaminopropyl)carbodiimide hydrochloride
- conjugation by a homobifunctional cross-linker having, for example, NHS ester groups or sulfo-NHS ester analogs
- conjugation by a heterobifunctional cross-linker having, for example, and NHS ester and a maleimide group such as sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane -1 -carboxylate and, conjugation with glutaraldehyde
- the disclosure further provides protein-based Virus-Like Particles (VLPs).
- VLPs may comprise a receptor binding domain polypeptide or mutated coronavirus S proteins described, comprising a fusion protein, wherein the fusion protein comprises a multimerization domain, such as a “first component” as described herein.
- the VLPs for use with the methods and compositions described herein can comprise multimeric protein assemblies adapted for display of the ectodomain of the RBD or an ectodomain of the coronavirus spike protein, or an antigenic fragment thereof.
- the VLPs for use with the methods and compositions described herein can comprise at least a first plurality of polypeptides.
- the first plurality of polypeptides (also referred to a “first component”) can be derived from a naturally -occurring protein sequence by substitution of at least one amino acid residue or by additional at the N- or C-terminus of one or more residues.
- the first component comprises a protein sequence determined by computational methods.
- This first component can form the entire core of the VLP; or the core of the VLP can comprise one or more additional polypeptides (also referred to a “second component” or third, fourth, fifth component and so on), such that the VLP comprises two, three, four, five, six, seven, or more pluralities of polypeptides.
- the first plurality will form trimers related by 3 -fold rotational symmetry and the second plurality will form pentamers related by 5-fold rotational symmetry.
- the VLP forms an “icosahedral particle” having 153 symmetry. Together these one or more pluralities of polypeptides can be arranged such that the members of each plurality of polypeptides are related to one another by symmetry operators.
- the “core” of the VUP is used herein to describe the central portion of the VUP that links together the several copies of the RBD or coronavirus S protein ectodomain, or antigenic fragments thereof, displayed by the VUP.
- the first component comprises a first polypeptide comprising an RBD, a linker, and a first polypeptide comprising a multimerization domain.
- FIG. 6A depicts an RBD genetically fused to a component of the VUP, which optionally is expressed recombinantly in a host cell (e.g. , 293F cells); along with a pentameric protein assembly, which is optionally expressed recombinantly in the same or a different host cell (e.g. , E. coli cells), these two pluralities of polypeptides self-assembling into a VUP displaying 20 antigen trimers around an icosahedral core.
- a host cell e.g. 293F cells
- a pentameric protein assembly which is optionally expressed recombinantly in the same or a different host cell
- the VUP is adapted to display the RBD or spike protein from two or more diverse strains of coronavirus.
- the same VUP displays mixed populations of protein antigens or mixed heterotrimers of protein antigens from different strains of coronavirus.
- VUPs for use with the methods and compositions described herein display antigenic proteins in various ways including as gene fusion or by other means disclosed herein.
- “linked to” or “attached to” denotes any means known in the art for causing two polypeptides to associate.
- the association can be direct or indirect, reversible or irreversible, weak or strong, covalent or non-covalent, and selective or nonselective.
- attachment is achieved by genetic engineering to create an N- or C- terminus fusion of an antigen to one of the pluralities of polypeptides composing the VUP.
- the VUP can consist of, or consist essentially of, one, two, three, four, five, six, seven, eight, nine, or ten pluralities of polypeptides displaying one, two, three, four, five, six, seven, eight, nine, or ten pluralities of antigens, where at least one of the pluralities of antigens is genetically fused to at least one of the plurality of polypeptides.
- the VUP consists essentially of one plurality of polypeptides capable of self-assembly and comprising the plurality of antigenic proteins genetically fused thereto. In some cases, the VUP consists essentially of a first plurality of polypeptides comprising a plurality of antigens; and a second plurality of polypeptides capable of co-assembling into a two-component VUP, one plurality of polypeptides linking the antigenic protein to the VLP and the other plurality of polypeptides promoting self-assembly of the VLP.
- attachment is achieved by post-translational covalent attachment between one or more pluralities of polypeptides and one or more pluralities of antigenic protein.
- chemical cross-linking is used to non-specifically attach the antigen to a VLP polypeptide.
- chemical cross-linking is used to specifically attach the antigenic protein to a VLP polypeptide (e.g. to the first polypeptide or the second polypeptide).
- Various specific and non-specific cross-linking chemistries are known in the art, such as Click chemistry and other methods. In general, any cross-linking chemistry used to link two proteins can be adapted for use with the presently disclosed VLPs.
- chemistries used in creation of immunoconjugates or antibody drug conjugates may be used.
- an VLP is created using a cleavable or non-cleavable linker.
- the components of the VLP of the present disclosure can have any of various amino acids sequences.
- U.S. Patent Pub No. US 2015/0356240 Al (the contents of which are incorporated herein by reference in their entirety) describes various methods for designing protein assemblies.
- the polypeptides were designed for their ability to self-assemble in pairs to form VLPs, such as icosahedral particles.
- the design involved design of suitable interface residues for each member of the polypeptide pair that can be assembled to form the VLP.
- the VLPs so formed include symmetrically repeated, non-natural, non-covalent polypeptide-polypeptide interfaces that orient a first assembly and a second assembly into a VLP, such as one with an icosahedral symmetry.
- the RBD or coronavirus S protein ectodomain, or antigenic fragments thereof are expressed as a fusion protein with the first multimerization domain.
- the first multimerization domain and RBD or coronavirus S protein ectodomain are joined by a linker sequence.
- the linker sequence comprises a foldon, wherein the foldon sequence is EKAAKAEEAARK (SEQ ID NO: 8).
- Non-limiting examples of designed protein complexes useful in protein-based VLPs of the present disclosure include those disclosed in U.S. Patent No. 9,630,994; Int’l Pat. Pub No. WO2018187325 Al; U.S. Pat. Pub. No. 2018/0137234 Al; U.S. Pat. Pub. No. 2019/0155988 A2, each of which is incorporated by reference herein in its entirety. Illustrative sequences are provided in Table 3.
- the VLP comprises a fusion protein that has at least 95%, at least
- SEQ ID NOs: 9-13 comprises an RBD or coronavirus spike protein as disclosed herein; and a second component that has 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: 9-13 and comprises an RBD or coronavirus spike protein as disclosed herein; and a second component that has 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: 9-13 and comprises an RBD or coronavirus spike protein as disclosed herein; and a second component that has 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: 9-13 and comprises an RBD or coronavirus spike protein as disclosed herein; and a second component that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one
- the VLP comprises a fusion protein that has at least 95%, at least
- SEQ ID NO: 19 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 19 and comprises an
- RBD or coronavirus spike protein as disclosed herein and a second component that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 20.
- polypeptides as disclosed herein comprise the polypeptide sequence of SEQ ID NO: 6 or SEQ ID NO: 7.
- Exemplary protein stability assays include, but are not limited to, differential scanning calorimetry, pulse-chase method, bleach chase method, cyclohexamide -chase method, circular dichroism spectroscopy, and fluorescence -based activity assays.
- shelf-life of a given composition when applied to an isolated preparation or a vaccine preparation, depends on the conditions in which the composition is stored as well as the formulation of the composition (e.g., addition of chemical components) or the physical state in which the composition is provided (e.g., lyophilized, dried, frozen, etc).
- lyophilization refers to a dehydration process, commonly referred to as “freeze drying” used to preserve a composition as described herein or make the composition more convenient for storage and transport. Freeze-drying works by freezing the composition and then reducing the surrounding pressure to allow the frozen water in the composition to sublimate directly from the solid phase to the gas phase. In some embodiments, lyophilization can be used to preserve vaccine compositions as described herein, thereby allowing the vaccine compositions to be portable and to be stored at room temperature without the need of refrigeration.
- compositions described herein are contemplated to be stored at room temperature and avoid the need for refrigeration.
- the mutated coronavirus spike proteins or RBDs thereof will have both an increase in the protein stability and composition shelf-life over the protein stability and composition shelf-life for the corresponding wild-type coronavirus spike protein or RBD thereof.
- Formulations comprising the mutated coronavirus proteins or peptides described herein are stable in that their characteristics change little over a given period of time at a defined temperature. In general, formulations as described herein are stable for at least about a month.
- the formulations are 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 temperatures over which a formulation is stable are generally below about 30° C.
- the formulation's stability is in reference to a temperature below about 25° C, about 20° C, about 15° C, about 10° C, about 8° C, about 5° C, about 4° C, or about 2° C.
- the temperature is in the range of about 25° C to about 2° C, about 20° C. to about 2° C, about 15° C to about 2° C, about 10° C to about 2° C, about 8° C to about 2° C, or about 5° C to about 2° C.
- the temperature is in the range of about 25° C to about 5° C, about 20° C to about 5° C, about 15° C to about 5° C, about 10° C to about 5° C, or about 8° C to about 5° C. In still other embodiments, the temperature is in the range of about 25° C to about 8° C, about 20° C to about 8° C, about 15° C to about 8° C, or about 10° C to about 8° C. In yet other embodiments, the temperature is in the range of about 25° C to about 10° C, about 20° C to about 10° C, about 15° C to about 10° C, about 25° C to about 15° C, about 20° C to about 15° C, or about 25° C to about 20° C. In some embodiments, the composition can be stored at 4° C or -20 ° C for longer term storage.
- the RBD polypeptides, mutated coronavirus S proteins or fusion proteins comprising the same as described herein can be used in the production of a vaccine formulation.
- Such vaccine formulations can provide protection against each of the seven coronaviruses known to infect humans individually, or can 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.
- a vaccine formulation as described herein can provide protection against all 7 of the coronaviruses known to infect humans. It is also specifically contemplated herein that the vaccine formulations described herein can provide protection against coronaviruses that are expected to move from an animal species (e.g., bats) to humans in the future.
- the vaccine formulations described herein can provide protection of an animal against one or more coronaviruses to which they are susceptible .
- RNA and/or DNA vaccine formulations encoding the mutated spike protein polypeptides described herein are also specifically contemplated herein.
- the immunity generated against the coronavirus antigens described herein is long lasting (e.g., at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years, or even for the entire lifespan of the subject).
- the vaccine formulations as described herein are administered on an annual basis, tailored to prevalent or predicted prevalent strains of the target virus, analogous to immunization with the influenza vaccine, and can provide protection for at least 3 months from the last administration, at least 6 months, at least 8 months, at least one year, at least 1.5 years, or at least two years.
- the vaccine formulations described herein are at least 40% effective in a population of vaccinated 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 population of vaccinated individuals.
- the vaccines described herein can be administered as a universal vaccination to healthy children and individuals. Children can play an important role in the transmission of coronavirus within schools, families and communities, particularly because they tend to have milder symptoms and are not necessarily diagnosed as having a coronavirus infection. Studies with influenza vaccines have shown that vaccination of approximately 80% of schoolchildren in a community has decreased respiratory illnesses in adults and excess deaths in the elderly (Reichert et al., 2001). This concept is known as community immunity or “herd immunity” and is thought to play an important part of protecting the community against disease.
- vaccinated people have antibodies that neutralize a particular virus, they are much less likely to transmit the virus to other people. This concept can be applied to coronavirus infections as well. Thus, even people who have not been vaccinated (and those whose vaccinations have become weakened or whose vaccines are not fully effective) often can be shielded by the herd immunity because vaccinated people around them are not getting sick or transmitting the virus. Herd immunity is more effective as the percentage of people vaccinated increases. It is thought that approximately 60% (but preferably closer to 90-95%) of the people in the community must be protected by a vaccine to achieve herd immunity. People who are not immunized increase the chance that they and others will get the disease.
- most school-aged children are immunized against a coronavirus infection by administering 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-aged children are immunized).
- most healthy individuals in a community are immunized against a given coronavirus or group of coronaviruses by administering a vaccine as described herein.
- the vaccines described herein can be used as part of a “dynamic vaccination” strategy. Dynamic vaccination is the steady production of a low-efficacy vaccine that is related to an emerging or existing pandemic strain, but due to an antigenic drift may not provide complete protection in a mammal (see Germann et al., 2006).
- a vaccine as described herein is directed against one or more strains of SARS-CoV-2 that are responsible for the 2019/2020 COVID-19 pandemic.
- the vaccine formulations described herein can prevent at least one of the symptoms associated with the coronavirus infection or can completely prevent presentation of any symptom.
- Common symptoms of a coronavirus infection include, but are not limited to, fever, chills, cough, shortness of breath/difficulty breathing, fatigue, muscle/body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea, vomiting, or diarrhea.
- a reduction in a symptom may be determined subjectively or objectively, e.g., self-assessment by a subject, by a clinician's assessment or by conducting an appropriate assay or measurement (e.g.
- body temperature body temperature, degree of pneumonia infection, lung scarring etc.
- a quality of life assessment including, e.g., a quality of life assessment, a slowed progression of a coronavirus infection or additional symptoms, a reduced severity of coronavirus- associated disease symptoms or a suitable assays (e.g. antibody titer and/or T-cell activation assay).
- a suitable assays e.g. antibody titer and/or T-cell activation assay.
- the vaccine formulations described herein will reduce transmission of active coronavirus from a vaccinated individual to others within a 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 detectable transmission among a vaccinated individual and two or more individuals).
- the vaccine formulations described herein comprise one or more adjuvants.
- adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
- Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL).
- RIBI which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion also is contemplated.
- MPL trehalose dimycolate
- CWS cell wall skeleton
- MF-59, Novasomes®, MHC antigens may also be used.
- an adjuvant effect is achieved by use of an agent, such as alum, used in about 0.05 to about 0.1% solution in phosphate buffered saline.
- an agent such as alum
- vaccines as described herein can be made as an admixture with synthetic polymers of sugars (Carbopol®) used as an about 0.25% solution.
- Some adjuvants for example, certain organic molecules obtained from bacteria, act on the host rather than on the antigen.
- An example is muramyl dipeptide (N-acetylmuramyl-L-alanyl-D- isoglutamine (MDP)), a bacterial peptidoglycan.
- hemocyanins and hemoerythrins may also be used with the vaccine formulations as described herein.
- Hemocyanin from keyhole limpet (KLH) can be used in certain embodiments, although other molluscan and arthropod hemocyanins and hemoerythrins may be used in alternative embodiments.
- polysaccharide adjuvants can also be used.
- various pneumococcal polysaccharide adjuvants on the antibody responses of mice has been described (Yin et al., 1989).
- the doses that produce optimal responses, or that otherwise do not produce suppression, should be employed as indicated (Yin et al., 1989).
- Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.
- Amphipathic and surface active agents e.g., saponin and derivatives such as QS21
- Nonionic block copolymer surfactants Roshanovich et al., 1994
- Oligonucleotide adjuvants Y amamoto et al., 1988
- Quil A and lentinen are other adjuvants that can be used in certain embodiments.
- refined detoxified endotoxins can be used to produce an adjuvant response in vertebrates, either alone or in a multi-adjuvant formulation.
- a combination of detoxified endotoxins with trehalose dimycolate or a combination of detoxified endotoxins with trehalose dimycolate and endotoxic glycolipids are contemplated herein.
- a combination of detoxified endotoxins with cell wall skeleton (CWS) or CWS and trehalose dimycolate or combinations of just CWS and trehalose dimycolate, without detoxified endotoxins are also contemplated.
- adjuvants that can be conjugated to or admixed with vaccines as described herein, and these include alkyl lysophosphilipids (ALP); BCG; and biotin (including biotinylated derivatives) among others.
- ALP alkyl lysophosphilipids
- BCG BCG
- biotin including biotinylated derivatives
- Certain adjuvants particularly contemplated for use are the teichoic acids from Gram negative bacterial cells. These include the lipoteichoic acids (LTA), ribitol teichoic acids (RTA) and glycerol teichoic acid (GTA). Active forms of their synthetic counterparts can also be used.
- LTA lipoteichoic acids
- RTA ribitol teichoic acids
- GTA glycerol teichoic acid
- Vaccine formulations can comprise a coronavirus polypeptide as described herein or a fusion protein thereof that is microencapsulated or macroencapsulated, using methods well known in the art (e.g., liposomes (see, for example, Garcon and Six, J. Immunol. 146:3697 (1991)), into, for example, the inner capsid protein of bovine rotavirus (Redmond et al., Mol. Immunol. 28:269 (1991)) into immune stimulating molecules (ISCOMS) composed of saponins such as Quil A (Morein et al., Nature 308:457 (1984)); Morein et al., in Immunological Adjuvants and Vaccines (G.
- liposomes see, for example, Garcon and Six, J. Immunol. 146:3697 (1991)
- ICOMS immune stimulating molecules
- the stabilized coronavirus polypeptides described herein or fusion proteins thereof can also be adsorbed to the surface of lipid microspheres containing squalene or squalane emulsions prepared with a PLURONIC block-copolymer such as L-121 and stabilized with a detergent such as TWEEN 80 (see Allison and Byers, Vaccines: New Approaches to Immunological Problems (R. Ellis ed.) pp. 431-449, Butterworth-Hinemann, Stoneman N.Y. (1992)).
- Vaccine formulations described herein comprise a stabilized coronavirus polypeptide as described herein or fusion proteins thereof in an “effective amount” or a “therapeutically effective amount.”
- the phrase “effective amount” or “therapeutically effective amount” refers to a dose sufficient to provide concentrations high enough to generate (or contribute to the generation of) an immune response in the recipient thereof.
- the specific effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated, the severity of the disorder, the activity of the specific compound, the route of administration, the rate of clearance of the administered agent(s), the duration of treatment, the drugs used in combination or coincident with the administered agent(s), the age, body weight, sex, diet, and general health of the subject, and like factors well known in the medical arts and sciences.
- the pH of the formulation can also vary. In general, it is between about pH 6.2 to 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 a range of 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.
- 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.
- 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.
- 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.
- 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, about 7.4 and about 7.6, or between about 7.6 and about 8.0.
- the formulation can include one or more salts, such as sodium chloride, sodium phosphate, or a combination thereof.
- each salt is present in the formulation at about 10 mM to about 200 mM.
- any salt that is present is present at about 10 mM to about 200 mM, about 20 mM to about 200 mM, about 25 mM to about 200 mM, at about 30 mM to about 200 mM, at about 40 mM to about 200 mM, at about 50 mM to about 200 mM, at about 75 mM to about 200 mM, at about 100 mM to about 200 mM, at about 125 mM to about 200 mM, at about 150 mM to about 200 mM, or at about 175 mM to about 200 mM.
- any salt that is present is present at about 10 mM to about 175 mM, about 20 mM to about 175 mM, about 25 mM to about 175 mM, at about 30 mM to about 175 mM, at about 40 mM to about 175 mM, at about 50 mM to about 175 mM, at about 75 mM to about 175 mM, at about 100 mM to about 175 mM, at about 125 mM to about 175 mM, or at about 150 mM to about 175 mM.
- any salt that is present is present at about 10 mM to about 150 mM, about 20 mM to about 150 mM, about 25 mM to about 150 mM, at about 30 mM to about 150 mM, at about 40 mM to about 150 mM, at about 50 mM to about 150 mM, at about 75 mM to about 150 mM, at about 100 mM to about 150 mM, or at about 125 mM to about 150 mM.
- any salt that is present is present at about 10 mM to about 125 mM, about 20 mM to about 125 mM, about 25 mM to about 125 mM, at about 30 mM to about 125 mM, at about 40 mM to about 125 mM, at about 50 mM to about 125 mM, at about 75 mM to about 125 mM, or at about 100 mM to about 125 mM.
- any salt that is present is present at about 10 mM to about 100 mM, about 20 mM to about 100 mM, about 25 mM to about 100 mM, at about 30 mM to about 100 mM, at about 40 mM to about 100 mM, at about 50 mM to about 100 mM, or at about 75 mM to about 100 mM.
- any salt that is present is present at about 10 mM to about 75 mM, about 20 mM to about 75 mM, about 25 mM to about 75 mM, at about 30 mM to about 75 mM, at about 40 mM to about 75 mM, or at about 50 mM to about 75 mM.
- any salt that is present is present at about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, at about 30 mM to about 50 mM, or at about 40 mM to about 50 mM.
- any salt that is present is present at about 10 mM to about 40 mM, about 20 mM to about 40 mM, about 25 mM to about 40 mM, at about 30 mM to about 40 mM, at about 10 mM to about 30 mM, at about 20 mM to about 30, at about 25 mM to about 30 mM, at about 10 mM to about 25 mM, at about 20 mM to about 25 mM, or at about 10 mM to about 20 mM.
- the sodium chloride is present in the formulation at about 100 mM.
- the sodium phosphate is present in the formulation at about 25 mM.
- Formulations comprising the mutated coronavirus proteins described herein may further comprise a solubilizing agent such as a nonionic detergent.
- a solubilizing agent such as a nonionic detergent.
- Such detergents include, but are not limited to polysorbate 80 (Tween® 80), TritonXIOO and polysorbate 20.
- Vaccine formulations as described herein can further comprise a pharmaceutically acceptable carrier, including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to the vertebrate receiving the composition, and which may be administered without undue toxicity.
- a pharmaceutically acceptable carrier including any suitable diluent or excipient, which includes any pharmaceutical agent that does not itself induce the production of an immune response harmful to the vertebrate receiving the composition, and which may be administered without undue toxicity.
- pharmaceutically acceptable means being approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopia, European Pharmacopia or other generally recognized pharmacopia for use in vertebrates, and more particularly in humans. These compositions can be useful as a vaccine and/or antigenic compositions for inducing a protective immune response in a vertebrate.
- compositions include but are not limited to saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
- saline a thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is presented in Remington's Pharmaceutical Sciences (Mack Pub. Co. N.J. current edition).
- the formulation should suit the mode of administration.
- the formulation is suitable for administration to humans, preferably is sterile, non-particulate and/or non-pyrogenic.
- the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
- the composition can be a solid form, such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
- Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
- the coronavirus vaccines described herein are administered in an effective amount or quantity sufficient to stimulate an immune response against one or more strains of coronavirus.
- administration of a vaccine formulation elicits substantial immunity against at least one coronavirus.
- the dose can be adjusted based on, e.g., age, physical condition, body weight, sex, diet, time of administration, and other clinical factors.
- While stimulation of substantial immunity with a single dose is preferred, additional dosages can be administered, by the same or different route, to achieve the desired effect.
- multiple administrations may be required to elicit sufficient levels of immunity. Administration can continue at intervals throughout childhood, as necessary to maintain sufficient levels of protection against a coronavirus infection.
- adults who are particularly susceptible to serious disease or repeat infections such as, for example, health care workers, day care workers, family members of young children, the elderly, and individuals with compromised cardiopulmonary function may require multiple immunizations to establish and/or maintain protective immune responses.
- Levels of induced immunity can be monitored, for example, by measuring amounts of neutralizing secretory and serum antibodies, and dosages adjusted or vaccinations repeated as necessary to elicit and maintain desired levels of protection.
- a prophylactic vaccine formulation can be systemically administered, e.g., by subcutaneous or intramuscular injection using a needle and syringe, or a needle-less injection device.
- the vaccine formulation is administered intranasally, either by drops, large particle aerosol (greater than about 10 microns), or spray into the upper respiratory tract. While any of the above routes of delivery can result in an immune response, intranasal administration may confer the added benefit of eliciting mucosal immunity at one of the sites of entry of the coronavirus.
- Non-limiting methods of administering a vaccine formulation 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).
- parenteral administration e.g., intradermal, intramuscular, intravenous and subcutaneous
- epidural e.g., epidural and mucosal
- mucosal e.g., intranasal and oral or pulmonary routes or by suppositories.
- vaccine compositions as described herein are administered intramuscularly, intravenously, subcutaneously, transdermally or intradermally.
- 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 mucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
- epithelial or mucocutaneous linings e.g., oral mucous, colon, conjunctiva, nasopharynx, oropharynx, vagina, urethra, urinary bladder and intestinal mucosa, etc.
- intranasal or other mucosal routes of administration of a vaccine composition as described herein may induce an antibody or other immune response that is substantially higher than other routes of administration.
- intranasal or other mucosal routes of administration of a vaccine composition as described herein may induce an antibody or other immune response that will induce cross protection against other strains of coronaviruses.
- Administration can be systemic or local.
- the vaccine formulation is administered in such a manner as to target mucosal tissues in order to elicit an immune response at the site of immunization.
- mucosal tissues such as gut associated lymphoid tissue (GALT) can be targeted for immunization by using oral administration of compositions which contain adjuvants with particular mucosal targeting properties. Additional mucosal tissues can also be targeted, such as nasopharyngeal lymphoid tissue (NALT) and bronchial -associated lymphoid tissue (BALT).
- Vaccine formulations as described herein can also be administered on a dosage schedule, for example, an initial administration of the vaccine composition with subsequent booster administrations.
- a second dose of the composition is administered anywhere from two weeks to one year, preferably from about 1, about 2, about 3, about 4, about 5 to about 6 months, after the initial administration.
- a third dose may be administered after the second dose and from about three months to about two years, or even longer, preferably about 4, about 5, or about 6 months, or about 7 months to about one year after the initial administration.
- the third dose may be optionally administered when no or low levels of specific immunoglobulins are detected in the serum and/or urine or mucosal secretions of the subject after the second dose.
- a second dose is administered about one month after the first administration and a third dose is administered about six months after the first administration.
- the second dose is administered about six months after the first administration.
- the dosage of the pharmaceutical formulation can be determined readily by the skilled artisan, for example, by first identifying doses effective to elicit a prophylactic or therapeutic immune response, e.g., by measuring the serum titer of virus specific immunoglobulins or by measuring the inhibitory ratio of antibodies in serum samples, or urine samples, or mucosal secretions. Said dosages can be determined from animal studies.
- a non-limiting list of animals used to study the coronavirus include the guinea pig, Syrian hamster, chinchilla, hedgehog, chicken, rat, mouse, pig, bovine, bat and ferret. Bats in particular are thought to be a natural host to coronaviruses and can be particularly useful in researching or testing vaccines.
- any of the above animals can be dosed with a vaccine formulation as described herein, to partially characterize the immune response induced, and/or to determine if any neutralizing antibodies have been produced.
- Another method of inducing or enhancing an immune response can be accomplished by preparing a vaccine composition as described herein to include one or more immunes stimulators, such as one or more cytokines, lymphokines or chemokines with immunostimulatory, immunopotentiating, and/or pro-inflammatory activities (e.g., interleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growth factors (e.g., granulocyte-macrophage (GM)-colony stimulating factor (CSF)); and other immunostimulatory molecules, such as macrophage inflammatory factor, Flt3 ligand, B7.1; B7.2, etc.)
- immunes stimulators such as one or more cytokines, lymphokines or chemokines with immunostimulatory, immunopotentiating, and/or pro-inflammatory activities (e.g., interleukins (e.g., IL-1, IL-2, IL-3, IL-4,
- the RBD polypeptides or stabilized coronavirus spike proteins described herein can be used in vaccine formulations that can induce substantial immunity in a vertebrate (e.g. a human) when administered to the vertebrate.
- the substantial immunity results from an immune response against the RBD polypeptides or stabilized coronavirus spike proteins and protects against or ameliorates coronavirus infection or at least reduces a symptom of a coronavirus virus infection in said vertebrate.
- a vaccinated subject that subsequently becomes infected will be asymptomatic.
- the response may be not a fully protective response and a partial immune response is also contemplated herein.
- a partially protected subject that is subsequently infected with a coronavirus will experience reduced severity of symptoms or a shorter duration of symptoms compared to a non-immunized vertebrate.
- a subject vaccinated with a formulation comprising a SARS-CoV-2 stabilized spike protein may not require a lengthy hospitalization stay or the use of a ventilator to treat COVID-19.
- kits for inducing substantial immunity to infection by one or more coronaviruses or at least one symptom thereof in a subject comprising administering at least one effective dose of a vaccine formulation comprising a RBD polypeptide or stabilized coronavirus S protein as described herein.
- said induction of substantial immunity reduces the duration of coronavirus-associated disease symptoms (e.g., SARS, COVID-19).
- a vaccine formulation as described herein can elicit an immune response that will provide protection against more than one strain of coronavirus.
- This cross-protection of a vertebrate with a stabilized coronavirus S protein or an RBD polypeptide constructed from a particular strain, of a particular subgroup can induce cross-protection against coronaviruses of different strains and/or subgroups.
- kits for vaccination of an individual or animal comprising one or more containers fdled with one or more of the ingredients of the vaccine formulations as described herein.
- the kit comprises two containers, one containing the stabilized coronavirus polypeptides or fusion proteins thereof and the other containing an adjuvant.
- Associated with such container(s) can be a notice in the form prescribed by a governmental 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 be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity of composition.
- the vaccine composition is supplied as a liquid, in another embodiment, as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water or saline to the appropriate concentration for administration to a subject.
- the vaccine composition is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of preferably, about 1 pg, about 5 pg, about 10 pg, about 20 pg, about 25 pg, about 30 pg, about 50 pg, about 100 pg, about 125 pg, about 150 pg, or about 200 pg.
- the unit dosage of the vaccine composition is less than about 1 pg, (for example about 0.08 pg, about 0.04 pg; about 0.2 pg, about 0.4 pg, about 0.8 pg, about 0.5 pg or less, about 0.25 pg or less, or about 0.1 pg or less), or more than about 125 pg, (for example about 150 pg or more, about 250 pg or more, or about 500 pg or more). These doses can be measured as total coronavirus polypeptides or as pg of 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 being reconstituted from the lyophilized powder.
- a vaccine composition as described herein is supplied in liquid form in a hermetically sealed container indicating the quantity and concentration of the composition.
- the liquid form of the vaccine composition is supplied in a hermetically sealed container at least about 50 pg/ml, more preferably at least about 100 pg/ml, at least about 200 pg/ml, at least 500 pg/ml, or at least 1 mg/ml.
- 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 the at least two mutations are selected from the group consisting of: F338L/Y365W; Y365W/L513M; Y365W/F392W; F338M/A363L/Y365F/F377V; Y365F/F392W; Y365F/V395I;
- I358F/Y365W/L513M I358F/Y365W/F392W; F338M/I358F/A363L/Y365F/F377V;
- 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 the receptor binding domain of a second coronavirus as determined by a 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 the 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.
- RBD coronavirus receptor binding domain
- I358F/Y365W/F392W F338M/I358F/A363L/Y365F/F377V; I358F/Y365F/F392W;
- I358F/Y365F/V395I I358F/Y365F/F392W/V395I; I358F/Y365W/L513I/F515L; and
- the second coronavirus comprises a sequence of a coronavirus selected from: Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2), Severe acute respiratory syndrome associated coronavirus (SARS-CoV); Middle East respiratory syndrome (MERS); 229E; NL63; OC43; or HKU1.
- SARS-CoV-2 Severe acute respiratory syndrome associated coronavirus 2
- SARS-CoV Severe acute respiratory syndrome associated coronavirus
- MERS Middle East respiratory syndrome
- 229E 229E
- NL63; OC43 Middle East respiratory syndrome
- composition comprising the polypeptide of any one of paragraphs 1-17 and a pharmaceutically acceptable carrier.
- 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 -fdling mutation and at least a second mutation.
- coronavirus polypeptide of any one of paragraphs 22-24, wherein the at least one cavity-filling mutation comprises mutation of a residue within residues 328-531 of SEQ ID NO: 1 or at corresponding residues of a second coronavirus spike protein, subunit 1 as determined by a 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.
- coronavirus polypeptide of any one of paragraphs 22-26, wherein the at least one cavity-filling mutation comprises mutation of a residue of SEQ ID NO: 1 at amino acid 336, 338, 341, 342, 358, 361, 363, 365, 368, 374, 377, 387, or 392 or of a corresponding residue of a second coronavirus spike protein, subunit 1 as determined by a sequence alignment of SEQ ID NO: 1 with the sequence of the second coronavirus using Blast-p parameters of protocol 1 or protocol 2.
- coronavirus polypeptide of 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 a receptor binding domain sequence of a second coronavirus spike protein, subunit 1 as determined by a 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.
- coronavirus polypeptide of paragraph 35 the cognate coronavirus receptor comprises an angiotensin converting enzyme (ACE) receptor.
- ACE angiotensin converting enzyme
- SARS-CoV-2 Severe acute respiratory syndrome associated coronavirus 2
- SARS-CoV Severe acute respiratory syndrome associated coronavirus
- MERS Middle East respiratory syndrome
- 229E Middle East respiratory syndrome
- NL63 OC43
- HKUl Middle East respiratory syndrome
- composition comprising the coronavirus polypeptide of any one of paragraphs 22-
- a method of vaccinating a subject against a coronavirus comprising administering a composition of paragraph 21 or paragraph 46 to the subject.
- a method of making a vaccine comprising combining a composition of any one of paragraphs 1-15 or 22-42 with an adjuvant and a pharmaceutically acceptable carrier.
- a coronavirus spike protein comprising the polypeptide of any one of paragraphs 1 -25.
- blastp protein-protein BLAST
- Blast-p parameters of protocol 2 comprise: blastp -query query. fasta -subject sbjct.fasta -matrix BLOSUM62 -evalue 0.1 - word size 6 -gapopen 11 -gapextend 1 -out results.txt
- RBD coronavirus receptor binding domain
- polypeptide of paragraph 54 or paragraph 55 wherein the polypeptide comprises a second mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L5131, L513M, and F515L.
- polypeptide of paragraph 58 wherein the polypeptide comprises a third mutation selected from the group consisting of I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L5131, L513M, and F515L.
- polypeptide of paragraph 57 wherein the polypeptide comprises the polypeptide sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
- heterologous protein scaffold comprises a polypeptide of SEQ ID NO: 3.
- polypeptide of paragraph 61 wherein the polypeptide comprises the polypeptide sequence of SEQ ID NO: 6 or SEQ ID NO: 7.
- 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 that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 13-18.
- a vaccine composition comprising a composition of any one of paragraphs 54-62 or the polypeptide complex of paragraph 63.
- a method of vaccinating a subject against a coronavirus comprising administering a polypeptide of any one of paragraphs 54-62, a protein complex of paragraph 63, or a vaccine composition of any one of paragraphs 64-68 to the subject.
- a method of making a vaccine comprising combining a polypeptide of any one of paragraphs 54-62 with an adjuvant and a pharmaceutically acceptable carrier.
- a method of making a vaccine comprising combining a first component consisting of the polypeptide of any one of paragraphs 59-62; a second component that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NO: 13- 18; a pharmaceutically acceptable carrier; and optionally an adjuvant.
- 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 the at least one mutation is selected from the group consisting of: I358F, Y365F, Y365W, V367F, F392W, G502D, N501F, N501T, Q498Y, F338L, F338M, A363L, Y365M, F377V, V395I, L513I, L513M, and F515L of SEQ ID NO: 1 or a second coronavirus reference sequence wherein corresponding sites in the second coronavirus reference sequence are determined by a sequence alignment of SEQ ID NO: 1 with spike protein sequence of the second coronavirus receptor binding domain using Blast-p parameters of protocol 1 or protocol 2.
- RBD coronavirus receptor binding domain
- polypeptide of paragraph 72 wherein the polypeptide comprises two or more mutations selected from: F338L/Y365W; Y365W/L513M; Y365W/F392W;
- I358F/Y365F/F392W/V395I I358F/Y365W/L513I/F515L
- F338L/I358F/A363L/Y365M I358F/Y365F/F392W/V395I
- I358F/Y365W/L513I/F515L I358F/Y365W/L513I/F515L
- F338L/I358F/A363L/Y365M F358F/Y365F392W/V395I
- I358F/Y365W/L513I/F515L I358F/Y365W/L513I/F515L
- F338L/I358F/A363L/Y365M I358F/Y365F/F392W/V395I
- I358F/Y365W/L513I/F515L I358F/Y365W/L
- An ideal protein-based coronavirus vaccine must be both highly effective and scalably manufacturable, with the latter property largely being affected by vaccine yield and stability. Genetic vaccines using RNA or DNA immunization are also being more frequently explored against SARS- CoV-2, with their efficacy being influenced by expression levels of the target antigen.
- the receptor binding domain (RBD) of many coronaviruses, most particularly SARS-CoV-2 and related sarbecoviruses, is considered to be a highly valuable domain-based vaccine target due to the isolation of many RBD-directed antibodies that are potently neutralizing. While the RBD is amenable to production in many forms, limitations to its yield and stability could hinder the scalable manufacturing and distribution of RBD-based protein vaccines.
- RBD coronavirus polypeptide designed to enhance yield and stability of immunogens containing such coronavirus RBDs.
- Immunogens containing the receptor binding domain were generated with sets of stabilizing mutations that demonstrate highly improved expression and/or yield, in addition to improved stability in solution compared to equivalent immunogens with native (i.e., wild-type) RBD sequences.
- native (i.e., wild-type) RBD sequences As can be understood by those of skill in the art, increased expression of a given protein can be due, in part, to the improved stability of the protein.
- the designed immunogens are antigenically intact as validated by SARS-CoV-2-directed antibodies and the ACE2 receptor. Collectively, these sets of mutations allow for improved ability to scalably manufacture vaccines against diverse coronaviruses, which could also assist the performance of genetic vaccines directed to the RBD.
- SARS-CoV-2 sequence (SEQ ID NO: 1) is used throughout the specification as the basis for mutation numbering in other coronaviruses.
- the receptor binding domain of the SARS-CoV-2 sequence is shown below with bold, underlined text (SEQ ID NO: 2): The following sequence (or receptor binding domain thereof; SEQ ID NO: 2) can be aligned with at least a second coronavirus sequence.
- SEQ ID NO: 2 The following sequence (or receptor binding domain thereof; SEQ ID NO: 2) can be aligned with at least a second coronavirus sequence.
- An exemplary list of mutations that can enhance both yield and stability of a coronavirus protein is provided in the following list (all amino acid residue numbers are based on the “base sequence” above (SEQ ID NO: 1):
- variants 328-531 of SEQ ID NO: 1, or equivalent sequence from variants, e.g., the B.1.351 variant genetically fused to the 153-50 trimeric “A” component with a 16 residue linker were cloned in the pCMV/R vector using the Xbal and Avril restriction sites.
- MGILPSPGMPALLSLVSLLSVLLMGCVAETGT secretion signal and C-terminally tagged with a “GGSHHHHHHHH” sequence to allow for purification.
- the N-terminal methionine of the 153-50 trimeric component is replaced with the helical sequence “EKAAKAEEAAR” to improve accessibility of the antigen. All cysteines in the 153-50 trimeric component were mutated to alanine.
- the human ACE2 ectodomain was genetically fused to a sequence encoding a thrombin cleavage site and a human Fc fragment at the C-terminal end.
- hACE2-Fc was synthesized and cloned by GenScript with a BM40 signal peptide. Genes encoding CR3022 heavy and light chains were ordered from GenScript and cloned into pCMV/R.
- Proteins containing His tags were purified from clarified supernatants via a batch bind method where each clarified supernatant was supplemented with 1 M Tris-HCl pH 8.0 to a final concentration of 45 mM and 5 M NaCl to a final concentration of ⁇ 310 mM.
- Talon cobalt affinity resin Talon cobalt affinity resin (Takara) was added to the treated supernatants and allowed to incubate for 15 minutes with gentle shaking. Resin was collected using vacuum filtration with a 0.2 pm filter and transferred to a gravity column.
- the resin was washed with 20 mM Tris pH 8.0, 300 mM NaCl, and the protein was eluted with 3 column volumes of 20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole. Clarified supernatants of cells expressing monoclonal antibodies and human ACE2-Fc were purified using a MabSelect PrismA 2.6x5 cm column (Cytiva) on an AKTA Avantl50 FPLC (Cytiva).
- Bound antibodies were washed with five column volumes of 20 mM NaPO4, 150 mM NaCl pH 7.2, then five column volumes of 20 mM NaPO4, 1 M NaCl pH 7.4 and eluted with three column volumes of 100 mM glycine at pH 3.0. The eluate was neutralized with 2 M Trizma base to 50 mM final concentration.
- Bio-layer interferometry For measuring secretion levels of proteins in supernatants, all supernatants from protein expressions were diluted 1: 10 into KB1 (25mM Tris pH 8.0, 150mM NaCl, 0.5% bovine serum albumin and 0.01% TWEEN-20). Purified ACE2-Fc or CR3022 antibody were diluted into KB1 at 0.02mg/mL and immobilized on AHC tips (Pall ForteBio/Sartorius) for 300 seconds using an 8-channel Octet system (Pall ForteBio/Sartorius).
- Biosensors were then transferred into an association step with one of five serial two-fold dilutions of wild-type or stabilized monomeric RBDs for 120 s, with RBD concentrations of 125 pM, 62.5 pM, 31.3 pM, 15.6 pM and 7.8 pM used for CV30 and 31.3 pM, 15.6 pM, 7.8 pM, 3.9 pM and 2.0 pM used for CR3022.
- biosensors were transferred into buffer for 300 s of dissociation. Data from the association and dissociation steps were baseline subtracted and kinetics measurements were calculated globally across all five serial dilutions of RBD using a 1 : 1 binding model (ForteBio analysis software, version 12.0).
- hACE2-Fc dimesized receptor
- CR3022 IgGto monomeric RBD and RBD-I53-50 nanoparticles was analyzed at ambient temperature with shaking at 1000 rpm. Protein samples were diluted to 100 nM in Kinetics buffer (Pall ForteBio/Sartorius). Buffer, antibody, receptor, and immunogen were then applied to a black 96-well Greiner Bio-one microplate at 200 ⁇ L per well. Protein A biosensors were first hydrated for 10 min in Kinetics buffer, then dipped into either hACE2-Fc or CR3022 diluted to 10 pg/mL in Kinetics buffer in the immobilization step.
- Kinetics buffer Pall ForteBio/Sartorius
- the tips were transferred to Kinetics buffer for 90 s to reach a baseline.
- the association step was performed by dipping the loaded biosensors into the immunogens for 300 s, and the subsequent dissociation step was performed by dipping the biosensors back into Kinetics buffer for an additional 300 s.
- the data were baseline subtracted prior to plotting using the ForteBio analysis software (version 12.0).
- Thermal melts RBD-based samples were prepared in a buffer containing 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-arginine, 5% glycerol, while HexaPro-foldon-based samples were prepared in a buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 0.25% w/v L-histidine, 5% glycerol.
- Non-equilibrium melting temperatures were determined using an UNcleTM (UNchained Labs) based on the barycentric mean of intrinsic tryptophan fluorescence emission spectra collected from 20-95 °C using a thermal ramp of 1°C per minute. Melting temperatures were defined as the maximum point of the first derivative of the melting curve, with first derivatives calculated using GraphPad Prism software after smoothing with four neighboring points using 2nd order polynomial settings.
- 153-50 nanoparticles were first diluted to 75 pg/mL in 50 mM Tris pH 8, 150 mM NaCl, 100 mM L- arginine, 5% v/v glycerol prior to application of 3 ⁇ L of sample onto freshly glow -discharged 300-mesh copper grids. Sample was incubated on the grid for 1 minute before the grid was dipped in a 50 ⁇ L droplet of water and excess liquid blotted away with filter paper (Whatman). The grids were then dipped into 3 ⁇ L of 0.75% w/v uranyl formate stain.
- Stain was blotted off with filter paper, then the grids were dipped into another 6 ⁇ L of stain and incubated for ⁇ 90 seconds. Finally, the stain was blotted away and the grids were allowed to dry for 1 minute prior to storage or imaging. Prepared grids were imaged in a Tales model L120C transmission electron microscope using a Gatan camera at 57, 000 x.
- Dynamic light scattering was used to measure hydrodynamic diameter (Dh) and polydispersity (%Pd) of RBD-I53-50 nanoparticle samples on an UNcle (UNchained Laboratories). Sample was applied to an 8.8 ⁇ L quartz capillary cassette (UNi, UNchained Laboratories) and measured with 10 acquisitions of 5 s each, using auto-attenuation of the laser. Increased viscosity due to 5% v/v glycerol in the RBD nanoparticle buffer was accounted for by the UNcle Client software in Dh measurements.
- Rpk9-I53-50A trimers were H/D exchanged (HDX) in deuteration buffer (pH* 7.5, 85% D O. Cambridge Isotope Laboratories, Inc.) for 3, 15, 60, 1800, and 72000 seconds at 22°C respectively.
- Exchanged samples were subsequently mixed 1: 1 with ice-chilled quench buffer (200 mM tris(2- chlorethyl) phosphate (TCEP), 8 M Urea, 0.2% formic acid (FA)) for 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°C.
- Deuterium uptake analysis was performed with HX-Express v2. Peaks were identified from the peptide spectra based on the peptide m/z values and binomial fitting was applied. The deuterium uptake level was normalized relative to fully deuterated controls.
- mice Female BALB/c (Stock: 000651) mice were purchased at the age of four weeks from The Jackson Laboratory, Bar Harbor, Maine, and maintained at the Comparative Medicine Facility at the University of Washington, Seattle, WA, accredited by the American Association for the Accreditation of Laboratory Animal Care International (AAALAC). At six weeks of age, 6 mice per dosing group were vaccinated with a prime immunization, and three weeks later mice were boosted with a second vaccination. Prior to inoculation, immunogen suspensions were gently mixed 1: 1 vol/vol with AddaVax adjuvant (Invivogen, San Diego, CA) to reach a final concentration of 0.009 or 0.05 mg/mL antigen.
- AddaVax adjuvant Invivogen, San Diego, CA
- mice were injected intramuscularly into the gastrocnemius muscle of each hind leg using a 27-gauge needle (BD, San Diego, CA) with 50 ⁇ L per injection site (100 ⁇ L total) of immunogen under isoflurane anesthesia.
- BD San Diego, CA
- mice were bled two weeks after prime and boost immunizations.
- Blood was collected via submental venous puncture and rested in 1.5 mL plastic Eppendorf tubes at room temperature for 30 min to allow for coagulation. Serum was separated from hematocrit via centrifugation at 2,000 g for 10 min.
- Complement factors and pathogens in isolated serum were heat-inactivated via incubation at 56°C for 60 min. Serum was stored at 4°C or -80°C until use. All experiments were conducted at the University of Washington, Seattle, WA according to approved Institutional Animal Care and Use Committee protocols.
- ELISA Enzyme-linked immunosorbent assays (ELISA) were used to determine the binding of mouse sera to the delivered antigens.
- Maxisorp (Nunc) ELISA plates were coated overnight at 4°C with 0.08 pg/mL of protein of interest per well in 0.1 M sodium bicarbonate buffer, pH 9.4. Plates were then blocked with a 4% (w/v) solution of dried milk powder (BioRad) in TBS with 0.05% (v/v) Tween 20 (TBST) for 1 hour at room temperature. Serial dilutions of sera were added to the plates and, after washing, antibody binding was revealed using a hydrogen peroxidase coupled horse anti-mouse IgG antibody.
- Pseudovirus neutralization assays Spike-pseudotyped lentivirus neutralization assays used the spike HDM_Spikedelta21_D614G, which is available from Addgene (#158762) or BEI (NR- 53765), with the full sequence available on the world wide web at www.addgene.org/158762Error!
- 293T-ACE2 cells (BEI NR-52511) were seeded at 1.25x 10 4 cells per well in 50 uL DIO growth media (DMEM with 10% heat-inactivated FBS, 2 mM L- glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin) in poly-L-lysine coated black-walled clear-bottom 96-well plates (Greiner 655930). The next day, mouse serum samples were heat inactivated for 30 min at 56°C and then serially diluted in D10 growth media.
- DIO growth media DMEM with 10% heat-inactivated FBS, 2 mM L- glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin
- Spike-pseudotyped lentivirus was diluted 1:50 to yield -200,000 RLUs per well and incubated with the serum dilutions for 1 hr at 37°C. 100 ⁇ L of virus-serum mixture was then added to the cells and -52 hours later luciferase activity was measured using the Bright-Glo Luciferase Assay System (Promega, E2610). Each batch of neutralization assays included a negative control sample of human serum collected in 2017-2018 and a known neutralizing antibody to ensure consistency between batches. Fraction infectivity for each well was calculated compared to two “no-serum” control wells in the same row of the plate.
- the “neutcurve” package (available on the world wide web at j bloomlab .github.io/neutcurve version 0.5.2) was used to calculate the inhibitory concentration 50% (IC 50 ) and the neutralization titer 50% (NT50), which is simply 1/IC 50 , for each serum sample by fitting a Hill curve with the bottom fixed at 0 and the top fixed at 1.
- Expi293F cells are derived from the HEK293F cell line, a female human embryonic kidney cell line transformed and adapted to grow in suspension (Life Technologies). Expi293F cells were grown in Expi293 Expression Medium (Life Technologies), cultured at 36.5°C with 8% CO2 and shaking at 150 rpm. VeroE6 is a female kidney epithelial cell from African green monkey.
- the HEK- ACE2 adherent cell line was obtained through BEI Resources, NIAID, NIH: Human Embryonic Kidney Cells (HEK293T) Expressing Human Angiotensin-Converting Enzyme 2, HEK293T-hACE2 Cell Line, NR-52511.
- mice Female BALB/c mice (four weeks old) were obtained from Jackson Laboratory, Bar
- a design protocol was written using RosettaScripts (58, 59) that takes the aligned protomer and a custom resfde as inputs, with the resfde dictating the side chain identities and conformations sampled during design.
- the protocol applies two rounds of design to a symmetric model based on the input resfde, with side chain minimization applied after each design step.
- the protocol allows for backbone minimization to be simultaneously performed with side chain minimization, and trajectories were performed either with backbone minimization allowed or disallowed. Both design and minimization steps were allowed to repack or minimize residues within 10 A of all mutable or packable residues listed in the resfile.
- Residue positions were manually picked to include positions 358, 365, 392 and surrounding residues based on spike and RBD structures, and possible residue identities were designated for each position in resfiles using the ‘PIKAA’ option.
- Resfile inputs were diversified to include various combinations of I358F, Y365F, Y365W, and/or F392W, while also either restricting or allowing mutations to surrounding residues. Further resfile inputs were similarly set up but did not restrict positions 358, 365, and 392 to specific identities.
- Design models and scores were manually inspected to identify interactions that appeared structurally favorable. Mutations were discarded if they buried polar groups that were natively solvent-exposed or involved in hydrogen bonds. To prevent undesired alterations to antigenicity, mutations to surface-exposed residues were not frequently considered.
- Favorable sets of mutations were iteratively retested from optimized resfiles and manually refined to finalize a diverse set of designs.
- Wild-type and stabilized sequences for the SARS-CoV-2 RBD were genetically fused to the N terminus of the trimeric I53-50A nanoparticle component using linkers of 16 glycine and serine residues.
- Each I53-50A fusion protein also bore a C-terminal octa-histidine tag, and monomeric sequences contained both Avi and octa-histidine tags. All sequences were cloned into pCMV/R using the Xbal and Avril restriction sites and Gibson assembly. All RBD-bearing components contained an N-terminal mu-phosphatase signal peptide.
- hACE2-Fc was synthesized and cloned by GenScript with a BM40 signal peptide.
- the HexaPro-foldon construct used for immunization studies was produced as described (Hsieh et al. Science 369: 1501-05 (2020)) and placed into pCMV/R with an octa-histidine tag.
- HexaPro-foldon constructs used for expression and stability comparisons with and without Rpk9 mutations contained a BM40 signal peptide and were placed into pCMV/R. Plasmids were transformed into the NEB 5 a strain of E.
- Proteins containing His tags were purified from clarified supernatants via a batch bind method where each clarified supernatant was supplemented with 1 M Tris-HCl pH 8.0 to a final concentration of 45 mM and 5 M NaCl to a final concentration of 313 mM.
- Talon cobalt affinity resin Talon cobalt affinity resin (Takara) was added to the treated supernatants and allowed to incubate for 15 min with gentle shaking. Resin was collected using vacuum filtration with a 0.45 pm filter and transferred to a gravity column.
- the resin was washed with 10 column volumes of 20 mM Tris pH 8.0, 300 mM NaCl, and bound protein was eluted with 3 column volumes of 20 mM Tris pH 8.0, 300 mM NaCl, 300 mM imidazole.
- the batch bind process was then repeated on the same supernatant sample and the first and second elutions were combined. SDS-PAGE was used to assess purity.
- IMAC elutions from comparable cell culture conditions and volumes were supplemented with 100 mM L-arginine and 5% glycerol and concentrated to 1.5 mb.
- the concentrated samples were subsequently loaded into a 1 mL loop and applied to a Superdex 75 Increase 10/300 GL column (for monomeric RBDs) or a Superdex 200 Increase 10/300 GL column (for RBD fusions to the I53-50A trimer) pre-equilibrated with 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-arginine, 5% glycerol.
- IMAC elutions from comparable cell culture conditions and volumes were supplemented with 5% glycerol and concentrated to 1.5 mL, which was subsequently loaded into a 1 mL loop and applied to a Superose 6 Increase 10/300 GL column pre-equilibrated with 50 mM Tris pH 8.0, 150 mM NaCl, 0.25% w/v L-histidine, 5% glycerol.
- HexaPro-foldon for immunization studies was purified by IMAC and dialyzed three times against 50 mM Tris pH 8.0, 150 mM NaCl, 0.25% w/v L-histidine, 5% glycerol for four hours at room temperature.
- RBD-based samples were prepared in a buffer containing 50 mM Tris pH 8, 150 mM
- Non-equilibrium melting temperatures were determined using an UNcleTM (UNchained Labs) based on the barycentric mean of intrinsic tryptophan fluorescence emission spectra collected from 20-95 °C using a thermal ramp of 1 °C per minute . Melting temperatures were defined as the maximum point of the first derivative of the melting curve, with first derivatives calculated using GraphPad Prism software after smoothing with four neighboring points using 2nd order polynomial settings.
- 50B.4PT1 (SEQ ID NO: 17), was produced as described in U.S. Patent. Pub. No 2016/0122392, the content of which is incorporated by reference herein in its entirety, and the same protocol was used for purification of the 2OBX non-assembling control pentamer.
- Total protein concentration of purified individual nanoparticle components was determined by measuring absorbance at 280 nm using a UV/vis spectrophotometer (Agilent Cary 8454) and calculated extinction coefficients. The assembly steps were performed at room temperature with addition in the following order: wild-type or stabilized RBD-I53-50A trimeric fusion protein, followed by q.s. with buffer as needed to achieve desired final concentration, and finally I53-50B.4PT1 pentameric component (SEQ ID NO: 17) (in 50 mM Tris pH 8, 500 mM NaCl, 0.75% w/v CHAPS, with a molar ratio of RBD-I53-50A:I53-50B.4PTl of 1.1: 1. q.s.
- Biosensors were then transferred into an association step with one of five serial two-fold dilutions of wild-type or stabilized monomeric RBDs for 120 s, with RBD concentrations of 125 pM, 62.5 pM, 31.3 pM, 15.6 pM and 7.8 pM used for CV30 and 31.3 pM, 15.6 pM, 7.8 pM, 3.9 pM and 2.0 pM used for CR3022.
- biosensors were transferred into buffer for 300 s of dissociation. Data from the association and dissociation steps were baseline subtracted and kinetics measurements were calculated globally across all five serial dilutions of RBD using a 1: 1 binding model (ForteBio® analysis software, version 12.0).
- RBD-I53-50 nanoparticles was analyzed for real-time stability studies using an OctetTM Red 96 System at ambient temperature with shaking at 1000 rpm. Protein samples were diluted to 100 nM in Kinetics buffer (Pall ForteBio/Sartorius®). Buffer, antibody, receptor, and immunogen were then applied to a black 96-well Greiner Bio-one microplate at 200 ⁇ L per well. Protein A biosensors were first hydrated for 10 min in Kinetics buffer, then dipped into either hACE2-Fc or CR3022 diluted to 10 pg/mL in Kinetics buffer in the immobilization step. After 500 s, the tips were transferred to Kinetics buffer for 90 s to reach a baseline.
- Kinetics buffer Pall ForteBio/Sartorius®
- Buffer, antibody, receptor, and immunogen were then applied to a black 96-well Greiner Bio-one microplate at 200 ⁇ L per well. Protein A biosensors were first hydrated for 10 min in Kin
- the association step was performed by dipping the loaded biosensors into the immunogens for 300 s, and the subsequent dissociation steps was performed by dipping the biosensors back into Kinetics buffer for an additional 300 s.
- the data were baseline subtracted prior to plotting using the ForteBio® analysis software (version 12.0).
- Wild-type RBD-I53-50 nanoparticles and Rpk-I53-50 nanoparticles were first diluted to 75 pg/mL in 50 mM Tris pH 8, 150 mM NaCl, 100 mM L-arginine, 5% v/v glycerol prior to application of 3 ⁇ L of sample onto freshly glow-discharged 300-mesh copper grids. Sample was incubated on the grid for 1 minute before the grid was dipped in a 50 ⁇ L droplet of water and excess liquid blotted away with filter paper. The grids were then dipped into 3 ⁇ L of 0.75% w/v uranyl formate stain.
- Stain was blotted off with filter paper, then the grids were dipped into another 6 ⁇ L of stain and incubated for ⁇ 90 seconds. Finally, the stain was blotted away and the grids were allowed to dry for 1 minute prior to storage or imaging. Prepared grids were imaged in a TalosTM model L120C transmission electron microscope using a GatanTM camera at 57,000x.
- DLS Dynamic light scattering
- UV-Vis Ultraviolet- visible spectrophotometry
- RBD-I53-50A, Rpk4-I53-50A, and Rpk9-I53-50A trimers were H/D exchanged (HDX) in the deuteration buffer (pH* 7.5, 85% D2O, Cambridge Isotope Laboratories, Inc.) for 3, 15, 60, 1800, and 72000 seconds at 22°C respectively.
- Exchanged samples were subsequently mixed 1: 1 with ice-chilled quench buffer (200 mM tris(2 -chlorethyl) phosphate (TCEP), 8 M Urea, 0.2% formic acid (FA)) for a final pH of 2.5 and immediately flash frozen in liquid nitrogen.
- TCEP tris(2 -chlorethyl) phosphate
- FA 0.2% formic acid
- a fully deuterated control for each sample series was made by LC eluate collection from pepsin-digested undeuterated sample, speedvac drying, incubation in deuteration buffer for 1 hour at 85°C, and quenching the same as all other HDX samples.
- Internal exchange standards Pro-Pro-Pro-Ile [PPPI] and Pro-Pro-Pro-Phe [PPPF] were added in each sample to ensure consistent labeling conditions for all samples).
- the peptide reference list was updated from wild-type RBD peptide list with addition of new peptides covering mutations. These peptides were manually validated using DriftScopeTM and identified with orthogonal retention time and drift time coordinates. Deuterium uptake analysis was performed with HX-Express v2. Peaks were identified from the peptide spectra based on the peptide m/z values and binomial fitting was applied. The deuterium uptake level was normalized relative to fully deuterated controls.
- mice Female BALB/c (Stock: 000651) mice were purchased at the age of four weeks. At six weeks of age, 6 mice per dosing group were vaccinated with a prime immunization, and three weeks later mice were boosted with a second vaccination. Prior to inoculation, immunogen suspensions were gently mixed 1: 1 vol/vol with AddaVaxTM adjuvant to reach a final concentration of 0.009 or 0.05 mg/mL antigen. Mice were injected intramuscularly into the gastrocnemius muscle of each hind leg using a 27-gauge needle with 50 ⁇ L per injection site (100 ⁇ L total) of immunogen under isoflurane anesthesia.
- Enzyme-linked immunosorbent assays were used to determine the binding of mouse sera to the delivered antigens.
- MaxisorpTM (Nunc®) ELISA plates were coated overnight at 4°C with 0.08 pg/mL of protein of interest per well in 0.1 M sodium bicarbonate buffer, pH 9.4. Plates were then blocked with a 4% (w/v) solution of dried milk powder in TBS with 0.05% (v/v) Tween 20 (TBST) for 1 hour at room temperature. Serial dilutions of sera were added to the plates and, after washing, antibody binding was revealed using a hydrogen peroxidase coupled horse anti- mouse IgG antibody.
- Spike-pseudotyped lentivirus neutralization assays were carried out essentially as described in (67). The protocol was modified for this study to use a SARS-CoV-2 spike with a 21 amino-acid cytoplasmic tail truncation, which increases spike-pseudotyped lentivirus titers, and the D614G mutation, which is now predominant in human SARS-CoV-2.
- the plasmid encoding this spike, HDM_Spikedelta21_D614G is available from Addgene (#158762) or BEI (NR-53765), and the full sequence is at available on the world wide web at addgene.org/158762).
- DIO growth media DMEM with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 pg/mL streptomycin
- DIO growth media poly-L-lysine coated black-walled clear-bottom 96-well plates
- mouse serum samples were heat inactivated for 30 min at 56°C and then serially diluted in D10 growth media.
- Spike-pseudotyped lentivirus was diluted 1:50 to yield -200,000 RLUs per well and incubated with the serum dilutions for 1 hr at 37°C.
- luciferase activity was measured using the Bright-GloTM Luciferase Assay System (Promega®, E2610).
- Each batch of neutralization assays included a negative control sample of human serum collected in 2017-2018 and a known neutralizing antibody to ensure consistency between batches. Fraction infectivity for each well was calculated compared to two “no-serum” control wells in the same row of the plate.
- the “neutcurve” package (available on the world wide web at jbloomlab.github.io/neutcurve version 0.5.2) was used to calculate the inhibitory concentration 50% (IC 50 ) and the neutralization titer 50% (NT50), which is simply I/IC 50 , for each serum sample by fitting a Hill curve with the bottom fixed at 0 and the top fixed at 1. All neutralization assay data is available on the world wide web at github . com/j bloomlab/RBD nanoparticle vaccine .
- MLV-based SARS-CoV-2 S pseudotypes were prepared as described (AC Walls et al. Cell 181, 281-292.e6 (2020); AC Walls et al. Cell 183, 1367-1382.e 17 (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)).
- HEK293T cells were co-transfected using LipofectamineTM 2000 (Life Technologies) with an SARS-CoV-2 S-encoding plasmid, an MLV Gag-Pol packaging construct, and the MLV transfer vector encoding a luciferase reporter according to the manufacturer’s instructions.
- Cells were washed 3x with Opti-MEM and incubated for 5 h at 37°C with transfection medium.
- DMEM containing 10% FBS was added for 60 h. The supernatants were harvested by spinning at 2,500 g, filtered through a 0.45 pm filter, concentrated with a 100 kDa membrane for 10 min at 2,500 g and then aliquoted and stored at - 80°C.
- HEK-hACE2 cells were cultured in DMEM with 10% FBS
- Rpk4 which features F392W alone
- Rpk9 which combines F392W with the DMS -identified Y365F to remove the buried side chain hydroxyl group and the Rosetta-identified V395I to refill the resulting cavity with hydrophobic packing (data not shown).
- Scaled-up expression from HEK293F cells and purification by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) confirmed increased yields for Rpk4 and Rpk9 both as monomers and as fusions to the I53-50A trimer, with Rpk9 showing a clear advantage for I53-50A trimers (FIGs. 4A & 10A).
- both sets of stabilizing mutations enhanced expression, thermal stability, and structural order of the antigen while minimally impacting antigenicity, with Rpk9 showing superior improvement in all categories for both monomeric RBDs and their genetic fusions to the I53-50A trimer.
- HexaPro refers to a spike protein with four beneficial proline substitutions (F817P, A892P, A899P, A942P) as well as the two proline substitutions in S-2P (prolines at 986 and 987). See Hsieh et al.
- RBD-stabilizing mutations would improve the stability of the nanoparticles in simpler buffers, lacking excipients such as glycerol, L-arginine, and the detergent 3- [(3 -cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS), which otherwise could be used to stabilize preparations of the nanoparticle immunogens.
- the wild-type and stabilized RBD-I53-50A trimers were assembled into nanoparticles (RBD-I53-50, Rpk4-I53-50, and Rpk9-I53- 50) by addition of the complementary I53-50B.4PT1 pentameric component (SEQ ID NO: 17) (FIG. 5A).
- Rpk4-I53-50 and Rpk9-I53-50 were both more resistant to aggregation than RBD- 153-50 and better maintained binding to immobilized human ACE2 (hACE2-Fc) and CR3022 (FIG. 5E).
- Dialysis into TBS alone showed clear evidence of aggregation of all samples and loss of antigenicity, with Rpk9-I53-50 retaining slightly better antigenicity than RBD-I53-50 and Rpk4-I53- 50.
- the improved solution stability observed for the stabilized RBDs appears consistent with their enhanced thermal stability and structural order, and offers a subtle but important improvement in formulation stability that is highly relevant to vaccine manufacturing.
- Binding titers were measured against HexaPro-foldon using enzyme-linked immunosorbent assays (ELISA) and analyzed by measuring area under the curve (AUC) (FIG. 6B) and midpoint titers (FIG. 12A). Sera from all nanoparticle groups showed levels of antigen-specific antibody after the prime that were slightly higher than HexaPro-foldon and markedly higher than the non-assembling controls. Binding signal increased for all groups after the second immunization, with less separation between them.
- ELISA enzyme-linked immunosorbent assays
- RBD variants This thorough biochemical and biophysical characterization of RBD variants enabled the inventors to select designs that enhanced expression; minimized off-target disulfides; improved local structural order; and increased thermal, solution, and shelf-life stability; all while maintaining the potent immunogenicity of the wild-type RBD displayed on the 153-50 nanoparticle.
- Rpk4 F392W
- Rpk9 Y365F, F392W, V395I
- Rpk4- and Rpk9-I53-50 most likely derive from improvements in local structural order and reduced hydrophobic surface area exposure, as indicated by HDX-MS and SYPRO Orange fluorescence. More generally, these results raise the possibility that other RBD antigens may adopt dynamic conformations not observed in existing structures of S ectodomains or isolated RBDs, such as transitions between the open and closed states of the LA binding pocket.
Abstract
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LI, Q ET AL.: "The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity", CELL, vol. 182, no. 5, 3 September 2020 (2020-09-03) - 17 July 2020 (2020-07-17), pages 1284 - 1294, XP055824625, DOI: 10.1016/j.cell.2020.07.012 * |
LONG, SW ET AL.: "Molecular Architecture of Early Dissemination and Massive Second Wave of the SARS-CoV-2 Virus in a Major Metropolitan Area", MBIO, vol. 11, no. 6, 30 October 2020 (2020-10-30), pages 1 - 30, XP055954117, DOI: 10.1128/mBio.02707-20 * |
STARR, TN ET AL.: "Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding", CELL, vol. 182, no. 5, 3 September 2020 (2020-09-03), pages 1295 - 1310, XP055850377, [retrieved on 20200811], DOI: 10.1016/j. cell . 2020.08.01 2 * |
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