WO2022108460A2 - Peptide-based antigenic constructs recognized by immunoglobulins that bind to protein epitopes of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) - Google Patents
Peptide-based antigenic constructs recognized by immunoglobulins that bind to protein epitopes of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) Download PDFInfo
- Publication number
- WO2022108460A2 WO2022108460A2 PCT/PH2021/050039 PH2021050039W WO2022108460A2 WO 2022108460 A2 WO2022108460 A2 WO 2022108460A2 PH 2021050039 W PH2021050039 W PH 2021050039W WO 2022108460 A2 WO2022108460 A2 WO 2022108460A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- monomeric units
- artificial sequence
- acid residue
- amino
- target sequence
- Prior art date
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 48
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 45
- 108060003951 Immunoglobulin Proteins 0.000 title claims abstract description 40
- 102000018358 immunoglobulin Human genes 0.000 title claims abstract description 40
- 241001678559 COVID-19 virus Species 0.000 title claims abstract description 34
- 229940072221 immunoglobulins Drugs 0.000 title claims abstract description 32
- 230000000890 antigenic effect Effects 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 29
- 239000002243 precursor Substances 0.000 claims abstract description 21
- 125000000539 amino acid group Chemical group 0.000 claims description 51
- 235000018102 proteins Nutrition 0.000 claims description 42
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 31
- 108010032595 Antibody Binding Sites Proteins 0.000 claims description 17
- 238000006467 substitution reaction Methods 0.000 claims description 16
- 239000000427 antigen Substances 0.000 claims description 12
- 102000036639 antigens Human genes 0.000 claims description 12
- 108091007433 antigens Proteins 0.000 claims description 12
- 239000011347 resin Substances 0.000 claims description 10
- 229920005989 resin Polymers 0.000 claims description 10
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 229930182817 methionine Natural products 0.000 claims description 9
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 claims description 8
- 230000001590 oxidative effect Effects 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- 230000007017 scission Effects 0.000 claims description 3
- 239000007787 solid Substances 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims 2
- 241000494545 Cordyline virus 2 Species 0.000 claims 1
- 244000052769 pathogen Species 0.000 description 16
- 230000001717 pathogenic effect Effects 0.000 description 14
- 238000002649 immunization Methods 0.000 description 12
- 230000003053 immunization Effects 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 210000002966 serum Anatomy 0.000 description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 210000000987 immune system Anatomy 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 239000004202 carbamide Substances 0.000 description 5
- 210000003719 b-lymphocyte Anatomy 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 208000025721 COVID-19 Diseases 0.000 description 3
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 3
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 230000003278 mimic effect Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 239000012723 sample buffer Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 102000019260 B-Cell Antigen Receptors Human genes 0.000 description 2
- 108010012919 B-Cell Antigen Receptors Proteins 0.000 description 2
- 238000011537 Coomassie blue staining Methods 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- 101710172711 Structural protein Proteins 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000006664 bond formation reaction Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 125000005392 carboxamide group Chemical class NC(=O)* 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 239000013024 dilution buffer Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- 210000004180 plasmocyte Anatomy 0.000 description 2
- 238000011533 pre-incubation Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 125000004434 sulfur atom Chemical group 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 102000005367 Carboxypeptidases Human genes 0.000 description 1
- 108010006303 Carboxypeptidases Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241001669573 Galeorhinus galeus Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108091005774 SARS-CoV-2 proteins Proteins 0.000 description 1
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 1
- 101000953880 Severe acute respiratory syndrome coronavirus 2 Membrane protein Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000011545 carbonate/bicarbonate buffer Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 125000003636 chemical group Chemical group 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011359 convalescent plasma therapy Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1002—Coronaviridae
- C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Definitions
- the present invention relates to the field of immunoglobulin (e.g. , antibody) detection and production.
- this invention provides for the use of peptide-based antigenic constructs recognized by immunoglobulins (e.g. , antibodies) that bind to protein epitopes of severe acute respiratory syndrome coronavirus 2 (hereafter referred to as SARS-CoV-2) , said constructs thus serving as replacements for whole SARS-CoV-2 and proteins thereof among biomedical applications such as immunodiagnostics and immunization.
- immunoglobulins e.g. , antibodies
- SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
- Pathogen e.g. , SARS-CoV-2 genomes code for pathogen-associated proteins (e.g. , forming structural components of pathogens themselves) that are produced in the course of pathogen propagation (e.g., viral replication) .
- Said proteins may be recognized by host immune system components such as immunoglobulins (e.g. , antibodies) .
- immunoglobulins e.g. , antibodies
- Said immunoglobulins are thus useful for various biomedical applications, most notably immunodiagnostics and immunization for the control and prevention of infectious diseases.
- the immune system of a vertebrate host typically mounts an immune response that comprises an antibody response, whereby antibodies are produced that recognize pathogen-associated proteins.
- Said antibody response is typically initiated by the binding of surface immunoglobulins to said proteins, said immunoglobulins being physically associated with the plasma membranes of B cells (i.e. , B lymphocytes) of the host immune system.
- B cells i.e. , B lymphocytes
- Said B cells may thus be activated to proliferate and subsequently differentiate into plasma cells, which secrete said antibodies (i.e. , soluble immunoglobulins) .
- Said antibodies are thus analytes of interest from the standpoint of immunodiagnostics, as their presence may indicate current or past infection by said pathogen.
- said antibodies may also be of therapeutic and/or prophylactic value, as they may be useful for immunization that enables the treatment and/or prevention of infection by said pathogen .
- Said immunization may be either active where said antibodies are endogenously produced or passive where they are exogenously supplied. Active immunization may thus occur as the result of infection with a pathogen, such that the infected host mounts an immune response against the pathogen and/or pathogen biomolecules.
- active immunization may be attained via vaccination, with administration of a vaccine that stimulates the host immune system to mount an immune response, possibly even before the host ever encounters the pathogen.
- passive immunization may be achieved by administering preformed antibodies (e.g. , produced via active immunization) to a recipient host, for the treatment and/or prevention of infection by the pathogen, as exemplified by convalescent plasma therapy.
- Pathogen-associated proteins may thus be used for immunodiagnostics to detect antibodies and/or for immunization (e.g. , with vaccination for active immunization and thus production of antibodies for passive immunization) .
- production of said proteins for such applications is problematic in that it entails safety issues related to the handling of potentially biohazardous materials (e.g., pathogens themselves and/or their associated biomolecules) and more generally to the use of biotechnology (e.g. , with genetic manipulation and microbial propagation) , which pose the problem of dual use (i.e. , utility for both peaceful and military aims) .
- synthetic chemistry provides a safer alternative to biotechnology.
- synthetic chemistry enables the production of relatively short synthetic peptides that comprise amino-acid residue sequences of pathogen-associated proteins, said sequences being recognized by immunoglobulins that bind to said proteins.
- This can obviate dual-use biotechnology while still enabling detection and even production of antibodies, using peptide-based constructs instead of said proteins for immunodiagnostics and vaccines.
- peptide-based approach is applicable only in cases where said immunoglobulins still recognize said sequences when the latter are removed from the structural context of said proteins, which may assume conformations that are difficult to mimic using synthetic peptides (e.g. , due to excessive conformational flexibility of such peptides relative to natively folded protein segments of identical amino-acid residue sequence) .
- binding of said immunoglobulins to said sequences may be too weak to even detect using conventional immunoassay platforms, especially where said sequences are in monomeric form (i.e. , when only a single copy of each sequence is present per molecule of peptide-based construct) .
- the present invention provides: peptide-based antigenic constructs, each comprising a plurality of monomeric units and recognized by immunoglobulins that bind to protein epitopes of SARS-CoV-2; methods for producing said constructs using peptidic precursors that comprise artificial sequences, said monomeric units being derived from said precursors; methods and testing systems for detecting said immunoglobulins; and use of said constructs in processes for detecting and producing said immunoglobulins.
- the present invention enables the preparation of antigenic constructs, each comprising a plurality of monomeric units, each of said monomeric units being derived from a peptidic precursor, said precursor comprising an artificial sequence, said artificial sequence comprising at least one epitope, said epitope being recognizable by at least one paratope, said artificial sequence comprising at least two oxidized cysteine residues, each of said monomeric units being covalently linked to at least one other of said monomeric units by a disulfide bond between oxidized cysteine residues, said paratope recognizing an antigen that comprises at least one target sequence, said artificial sequence being homologous to said target sequence, said artificial sequence comprising at least one amino-acid residue substitution, said substitution entailing replacement of a reference amino-acid residue present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another, said method comprising the step of oxidizing said peptidic precursor, said target sequence occurring within a protein of at least one
- FIG. 1A shows SDS-PAGE (4% stacking, 15% resolving gel) results of mMl and pMl in reducing (1 and 2) and non-reducing (3 and 4) sample buffers (Coomassie blue staining) .
- Lanes 1 and 3 contain mMl.
- Lanes 2 and 4 contain pMl polymerized in 50% DMSO/8M urea.
- FIG. IB shows acid-urea PAGE (15% resolving gel) results of pMl in reducing (1) and non-reducing (2) sample buffers, and of mMl in reducing (3) and nonreducing (4) sample buffers (Coomassie blue staining) .
- FIG. 2 is a plot showing antibody binding of polymerized (pMl) and non-polymeri zed (mMl ) peptide when exposed to serum from recovered COVID-19 patient (convalescent) and negative-control serum.
- FIG. 3 is a plot comparing absorbance values of polymerized peptide pMl-antibody binding in convalescent serum pre-incubated with varying concentrations of either pMl or irrelevant peptide pTlC+ in competition assay.
- peptide refers to a plurality of aminoacid residues covalently linked via the main chain (as opposed to side chain) by a peptide amide bond between consecutive amino-acid residues along a linear molecular sequence of such residues, with unblocked or blocked Nterminus and/or C- terminus (such that the N-terminus is either unblocked with a free main-chain amino group or blocked, whereas the C-terminus is either unblocked with a free main-chain carboxyl group or blocked with C-terminal main-chain carbonyl group forming part of a carboxamide or other non-carboxyl chemical group) .
- polymeric peptide refers to a polymeric macromolecular covalent structure comprising peptide-derived structural components as monomeric units, said components being exemplified by oligopeptide-derived monomeric units, such that said structure can exist as a member of a polydisperse population of similar covalent structures differing in their degree of polymerization.
- antigen refers to a substance recognized by a vertebrate immune-system component such as an immunoglobulin.
- immunoglobulin refers to a protein produced by a vertebrate immune system and capable of recognizing an antigen via binding thereto.
- An immunoglobulin may be either surface immunoglobulin, which is physically bound to the plasma membrane (typically on B lymphocytes) , or antibody, which is secreted (typically by plasma cells) .
- immunoglobulin G refers to a class of immunoglobulin which typically constitutes the majority of circulating antibody in mammalian blood plasma .
- epitope refers to a structural feature (e.g., amino-acid residue sequence) forming part of an antigen and recognized by an immunoglobulin.
- immunoglobulin refers to an epi tope -binding portion of an immunoglobulin.
- antigenic construct refers to an artificially produced antigen.
- oxidized cysteine residue is used to refer to a cysteine residue wherein the sidechain sulfhydryl group has been oxidized, for example, such that its sulfur atom is covalently linked by a disulfide bond to the sulfur atom of another oxidized cysteine residue.
- the present invention provides antigenic constructs comprising epitopes that can be recognized by immunoglobulins (e.g. , antibodies) that, for example, have been produced in response to infection by SARS-CoV-2 or otherwise recognize protein epitopes of SARS-CoV-2 (e.g. , consequent to active immunization with a vaccine comprising protein epitopes of SARSCoV-2) .
- Said constructs each comprise a plurality of monomeric units, each of which is derived from a peptidic precursor that comprises an artificial sequence, itself comprising at least one epitope that is recognized by at least one paratope.
- Each of said monomeric units comprises at least two oxidized cysteine residues and is covalently linked to at least one other such monomeric unit by a disulfide bond between oxidized cysteine residues.
- said paratope recognizes an antigen comprising at least one target sequence that occurs within a protein of at least one strain of SARS-CoV-2 and is homologous to said artificial sequence, which thus comprises at least one amino-acid residue substitution relative to said target sequence.
- Said substitution entails replacement of a reference amino-acid residue present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another.
- Said alternative amino-acid residue may be one of said two oxidized cysteine residues.
- said reference amino-acid residue may be a methionine residue and/or located at the N-terminus of said protein.
- said artificial sequence may be CADSNGT I TVEELKKLLEQC , which is homologous to the 20-residue N-terminal sequence of the membrane glycoprotein of SARS-CoV-2 strains exemplified by the SARS-CoV-2 isolate Wuhan-Hu-1, such that the N-terminal methionine residue and the C-terminal tryptophan residue of said N-terminal sequence are each replaced by a cysteine residue in said artificial sequence.
- methionine and/or tryptophan in said antigenic constructs is avoided, which is desirable in that both methionine and tryptophan are sensitive to oxidative damage.
- the present invention also provides resins comprising ma trix- 1 inked peptides covalently attached to a solid support matrices, said resins being suitable for producing free peptides by chemical cleavage from said matrices, said free peptides being suitable for producing said antigenic constructs.
- the present invention likewise provides said free peptides. Accordingly, the present invention provides methods for preparing said antigenic constructs, each of said methods comprising the step of oxidizing said free peptides.
- the present invention provides detection methods for detecting immunoglobulins that bind to at least one protein epitope of SARS-CoV-2. Each of said detection methods comprises the steps o f : (i) contacting said immunoglobulins with one or more of said antigenic constructs, and
- the present invention provides testing systems for detecting immunoglobulins that bind to at least one protein epitope of SARS-CoV-2.
- Each testing system comprises one or more of said antigenic constructs.
- the 20-residue N-terminal sequence of the SARS-CoV-2 membrane glycoprotein (MADSNGTITVEELKKLLEQW) was modified into an artificial homolog, with replacement of the oxidation-sensitive methionine and tryptophan residues by cysteine residues, thereby yielding a 20-residue artificial sequence ( CADSNGT I TVEELKKLLEQC ) consisting of an 18-residue wild-type core sequence between a pair of flanking cysteine residues.
- CADSNGT I TVEELKKLLEQC 20-residue artificial sequence consisting of an 18-residue wild-type core sequence between a pair of flanking cysteine residues.
- a peptide was designed that consisted of said artificial sequence with an unblocked N- terminus (i.e.
- lyophilized crude peptide 40 mg was dissolved in dimethyl sulfoxide (200 pL) to yield a stock solution, which was stored at -20°C until further use.
- a sample of said stock solution (5 pl) was mixed with an equal volume of an 8M aqueous urea solution, and the resulting mixture was incubated (room temperature, 24 hours) to yield an oxidized-peptide solution, which was also stored at -20°C until further use.
- Said stock solution thus contained unoxidized peptide (hereinafter referred to as mMl )
- said oxidized peptide solution contained oxidized peptide (hereinafter referred to as pMl) .
- Samples (40 pg each) of pMl and mMl were analyzed under either non-reducing or reducing conditions using both sodium dodecyl sul fate-polyacrylamide gel electrophoresis (SDSPAGE) and acid-urea PAGE with subsequent Coomassie Blue gel staining.
- SDSPAGE sodium dodecyl sul fate-polyacrylamide gel electrophoresis
- SDS-PAGE analysis revealed stained material forming smears of lower average electrophoretic mobility under nonreducing versus reducing conditions, which is consistent with both the formation of polymeric peptide via oxidant-mediated intermolecular disulfide bond formation between cysteine residues and the degradation of said polymeric peptide.
- Acid- urea PAGE revealed a similar pattern consistent with formation of polymeric peptide.
- Binding of anti-SARS-CoV-2 antibody to peptide targets was evaluated using indirect ELISA with high- binding polystyrene microtiter plates (Costar 3590, Corning Inc., NY, USA) .
- coating 37 ° C , 1 hour
- wells were loaded (100 pL/well) with either polymerized peptide or non-polymeri zed peptide (20 pg/mL) in 0.05 M carbonate-bicarbonate buffer.
- For blocking 37 ° C , 30 minutes
- wells were loaded (100 pL/well) with blocking buffer (5% skim milk in wash buffer) .
- Fresh chromogenic substrate solution was prepared by dissolving 3, 3' , 5, 5' - tetramethylbenzidine in DMSO (200 pL) , diluting with phosphate-ci trate buffer (9.8 mL) and adding 30% hydrogen peroxide (2 pL) .
- Wells were incubated (room temperature, 1 hour) with CSS (50 pL/well) , after which 1 M H2SO4 (50 pL/well) was added to stop the enzymatic reaction.
- Wells were read at 450 nm using a Bio-Rad Model 680 microplate reader.
Abstract
The invention provides: peptide-based antigenic constructs, each comprising a plurality of monomeric units and recognized by immunoglobulins that bind to protein epitopes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); methods for producing said constructs using peptidic precursors that comprise artificial sequences, said monomeric units being derived from said precursors; methods and testing systems for detecting said immunoglobulins; and use of said constructs in processes for detecting and producing said immunoglobulins.
Description
PEPTIDE-BASED ANTIGENIC CONSTRUCTS RECOGNIZED BY IMMUNOGLOBULINS THAT BIND TO PROTEIN EPITOPES OF SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-CoV-2)
Field of the Invention
The present invention relates to the field of immunoglobulin (e.g. , antibody) detection and production. In particular, this invention provides for the use of peptide-based antigenic constructs recognized by immunoglobulins (e.g. , antibodies) that bind to protein epitopes of severe acute respiratory syndrome coronavirus 2 (hereafter referred to as SARS-CoV-2) , said constructs thus serving as replacements for whole SARS-CoV-2 and proteins thereof among biomedical applications such as immunodiagnostics and immunization.
Background of the Invention
Pathogen (e.g. , SARS-CoV-2) genomes code for pathogen-associated proteins (e.g. , forming structural components of pathogens themselves) that are produced in the course of pathogen propagation (e.g., viral replication) . Said proteins may be recognized by host immune system components such as immunoglobulins (e.g. , antibodies) . Said immunoglobulins are thus useful for various
biomedical applications, most notably immunodiagnostics and immunization for the control and prevention of infectious diseases.
Consequent to infection with a pathogen, the immune system of a vertebrate host typically mounts an immune response that comprises an antibody response, whereby antibodies are produced that recognize pathogen-associated proteins. Said antibody response is typically initiated by the binding of surface immunoglobulins to said proteins, said immunoglobulins being physically associated with the plasma membranes of B cells (i.e. , B lymphocytes) of the host immune system. Said B cells may thus be activated to proliferate and subsequently differentiate into plasma cells, which secrete said antibodies (i.e. , soluble immunoglobulins) . Said antibodies are thus analytes of interest from the standpoint of immunodiagnostics, as their presence may indicate current or past infection by said pathogen. Furthermore, said antibodies may also be of therapeutic and/or prophylactic value, as they may be useful for immunization that enables the treatment and/or prevention of infection by said pathogen .
Said immunization may be either active where said antibodies are endogenously produced or passive where they are exogenously supplied. Active
immunization may thus occur as the result of infection with a pathogen, such that the infected host mounts an immune response against the pathogen and/or pathogen biomolecules. Alternatively, active immunization may be attained via vaccination, with administration of a vaccine that stimulates the host immune system to mount an immune response, possibly even before the host ever encounters the pathogen. In contrast, passive immunization may be achieved by administering preformed antibodies (e.g. , produced via active immunization) to a recipient host, for the treatment and/or prevention of infection by the pathogen, as exemplified by convalescent plasma therapy.
Pathogen-associated proteins may thus be used for immunodiagnostics to detect antibodies and/or for immunization (e.g. , with vaccination for active immunization and thus production of antibodies for passive immunization) . However, production of said proteins for such applications is problematic in that it entails safety issues related to the handling of potentially biohazardous materials (e.g., pathogens themselves and/or their associated biomolecules) and more generally to the use of biotechnology (e.g. , with genetic manipulation and microbial propagation) , which pose the problem of dual use (i.e. , utility for both peaceful and military aims) .
For detection and production of antibodies, synthetic chemistry provides a safer alternative to biotechnology. In particular, synthetic chemistry enables the production of relatively short synthetic peptides that comprise amino-acid residue sequences of pathogen-associated proteins, said sequences being recognized by immunoglobulins that bind to said proteins. This can obviate dual-use biotechnology while still enabling detection and even production of antibodies, using peptide-based constructs instead of said proteins for immunodiagnostics and vaccines. Yet, such a peptide-based approach is applicable only in cases where said immunoglobulins still recognize said sequences when the latter are removed from the structural context of said proteins, which may assume conformations that are difficult to mimic using synthetic peptides (e.g. , due to excessive conformational flexibility of such peptides relative to natively folded protein segments of identical amino-acid residue sequence) . Hence, binding of said immunoglobulins to said sequences may be too weak to even detect using conventional immunoassay platforms, especially where said sequences are in monomeric form (i.e. , when only a single copy of each sequence is present per molecule of peptide-based construct) .
In principle, adequate strength of binding may
still be realized via an avidity gain with covalent linkage of monomeric units (e.g. , with sequences in polymeric form that facilitates simultaneous binding of covalently linked monomeric units by a single immunoglobulin molecule) . Such covalent linkage may be achieved through intermolecular disulfide-bond formation between artificially introduced cysteine residues (e.g. , flanking the sequences of interest) ; but such covalent linkage may itself introduce steric hindrance and other factors (e.g. , unfavorable electrostatic interactions) that tend to preclude binding of immunoglobulins.
In view of the preceding considerations, a need therefore exists to develop peptide-based constructs recognized by immunoglobulins that bind to proteins of SARS-CoV-2, despite the difficulties of using peptides to mimic immunologically relevant features of protein structure. This need is fulfilled by the present invention as described below.
Summary of the Invention
In order to provide safer alternatives to biotechnological products for the detection and production of antibodies that recognize SARS-CoV-2 and/or SARS-CoV-2 proteins, the present invention provides: peptide-based antigenic constructs, each comprising a plurality of monomeric units and
recognized by immunoglobulins that bind to protein epitopes of SARS-CoV-2; methods for producing said constructs using peptidic precursors that comprise artificial sequences, said monomeric units being derived from said precursors; methods and testing systems for detecting said immunoglobulins; and use of said constructs in processes for detecting and producing said immunoglobulins.
Accordingly, the present invention enables the preparation of antigenic constructs, each comprising a plurality of monomeric units, each of said monomeric units being derived from a peptidic precursor, said precursor comprising an artificial sequence, said artificial sequence comprising at least one epitope, said epitope being recognizable by at least one paratope, said artificial sequence comprising at least two oxidized cysteine residues, each of said monomeric units being covalently linked to at least one other of said monomeric units by a disulfide bond between oxidized cysteine residues, said paratope recognizing an antigen that comprises at least one target sequence, said artificial sequence being homologous to said target sequence, said artificial sequence comprising at least one amino-acid residue substitution, said substitution entailing replacement of a reference amino-acid residue
present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another, said method comprising the step of oxidizing said peptidic precursor, said target sequence occurring within a protein of at least one strain of SARS-CoV-2.
Brief Description of the Drawings
For a more complete understanding of the invention, reference is made to the following description and accompanying drawing, in which:
FIG. 1A shows SDS-PAGE (4% stacking, 15% resolving gel) results of mMl and pMl in reducing (1 and 2) and non-reducing (3 and 4) sample buffers (Coomassie blue staining) . Lanes 1 and 3 contain mMl. Lanes 2 and 4 contain pMl polymerized in 50% DMSO/8M urea.
FIG. IB shows acid-urea PAGE (15% resolving gel) results of pMl in reducing (1) and non-reducing (2) sample buffers, and of mMl in reducing (3) and nonreducing (4) sample buffers (Coomassie blue staining) .
FIG. 2 is a plot showing antibody binding of polymerized (pMl) and non-polymeri zed (mMl ) peptide when exposed to serum from recovered COVID-19 patient (convalescent) and negative-control serum.
FIG. 3 is a plot comparing absorbance values of
polymerized peptide pMl-antibody binding in convalescent serum pre-incubated with varying concentrations of either pMl or irrelevant peptide pTlC+ in competition assay.
Definition
The term "peptide" refers to a plurality of aminoacid residues covalently linked via the main chain (as opposed to side chain) by a peptide amide bond between consecutive amino-acid residues along a linear molecular sequence of such residues, with unblocked or blocked Nterminus and/or C- terminus (such that the N-terminus is either unblocked with a free main-chain amino group or blocked, whereas the C-terminus is either unblocked with a free main-chain carboxyl group or blocked with C-terminal main-chain carbonyl group forming part of a carboxamide or other non-carboxyl chemical group) .
The term "polymeric peptide" refers to a polymeric macromolecular covalent structure comprising peptide-derived structural components as monomeric units, said components being exemplified by oligopeptide-derived monomeric units, such that said structure can exist as a member of a polydisperse population of similar covalent structures differing in their degree of polymerization.
The term "antigen" refers to a substance recognized by a vertebrate immune-system component such as an immunoglobulin.
The term "immunoglobulin" refers to a protein produced by a vertebrate immune system and capable of recognizing an antigen via binding thereto. An immunoglobulin may be either surface immunoglobulin, which is physically bound to the plasma membrane (typically on B lymphocytes) , or antibody, which is secreted (typically by plasma cells) .
The term "immunoglobulin G" (hereafter abbreviated as "IgG") refers to a class of immunoglobulin which typically constitutes the majority of circulating antibody in mammalian blood plasma .
The term "epitope" refers to a structural feature (e.g., amino-acid residue sequence) forming part of an antigen and recognized by an immunoglobulin.
The term "paratope" refers to an epi tope -binding portion of an immunoglobulin.
The term "antigenic construct" refers to an artificially produced antigen.
The term "oxidized cysteine residue" is used to refer to a cysteine residue wherein the sidechain sulfhydryl group has been oxidized, for example, such that its sulfur atom is covalently linked by a disulfide bond to the sulfur atom of another oxidized
cysteine residue.
Detailed Description of the Preferred Embodiments
The present invention provides antigenic constructs comprising epitopes that can be recognized by immunoglobulins (e.g. , antibodies) that, for example, have been produced in response to infection by SARS-CoV-2 or otherwise recognize protein epitopes of SARS-CoV-2 (e.g. , consequent to active immunization with a vaccine comprising protein epitopes of SARSCoV-2) . Said constructs each comprise a plurality of monomeric units, each of which is derived from a peptidic precursor that comprises an artificial sequence, itself comprising at least one epitope that is recognized by at least one paratope. Each of said monomeric units comprises at least two oxidized cysteine residues and is covalently linked to at least one other such monomeric unit by a disulfide bond between oxidized cysteine residues. Furthermore, said paratope recognizes an antigen comprising at least one target sequence that occurs within a protein of at least one strain of SARS-CoV-2 and is homologous to said artificial sequence, which thus comprises at least one amino-acid residue substitution relative to said target sequence. Said substitution entails replacement of a reference amino-acid residue
present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another. Said alternative amino-acid residue may be one of said two oxidized cysteine residues. Moreover, said reference amino-acid residue may be a methionine residue and/or located at the N-terminus of said protein. As a case in point, said artificial sequence may be CADSNGT I TVEELKKLLEQC , which is homologous to the 20-residue N-terminal sequence of the membrane glycoprotein of SARS-CoV-2 strains exemplified by the SARS-CoV-2 isolate Wuhan-Hu-1, such that the N-terminal methionine residue and the C-terminal tryptophan residue of said N-terminal sequence are each replaced by a cysteine residue in said artificial sequence. Thus, inclusion of methionine and/or tryptophan in said antigenic constructs is avoided, which is desirable in that both methionine and tryptophan are sensitive to oxidative damage.
The present invention also provides resins comprising ma trix- 1 inked peptides covalently attached to a solid support matrices, said resins being suitable for producing free peptides by chemical cleavage from said matrices, said free peptides being suitable for producing said antigenic constructs. The present invention likewise provides
said free peptides. Accordingly, the present invention provides methods for preparing said antigenic constructs, each of said methods comprising the step of oxidizing said free peptides. Furthermore, the present invention provides detection methods for detecting immunoglobulins that bind to at least one protein epitope of SARS-CoV-2. Each of said detection methods comprises the steps o f : (i) contacting said immunoglobulins with one or more of said antigenic constructs, and
(ii) detecting said immunoglobulins bound to one or more of said antigenic constructs.
Additionally, the present invention provides testing systems for detecting immunoglobulins that bind to at least one protein epitope of SARS-CoV-2. Each testing system comprises one or more of said antigenic constructs.
EXAMPLES
The following examples are provided for illustrative purposes, to set forth the best mode contemplated for reducing the present invention to practice, and therefore shall be interpreted as illustrative and not in a limiting sense.
EXAMPLE 1
Publicly available SARS-CoV-2 genomic sequences of Philippine origin ( "Locat ion"="Phil ippines " ) were retrieved as a single FASTA file from the GISAID EpiFluT Database (https://www.gisaid.org/) on 01 May 2020. Open reading frames (ORFs) were identified and translated using the stranslate (translate) commandline tool, with output thereo captured in a second FASTA file. Additional FASTA files of reference structural-protein sequences translated from the SARS-CoV-2 isolate Wuhan-Hu-1 genomic sequence (Wu et al. , 2020) were downloaded from NCBI (https : / / www .ncbi.nlm.nih.gov/ nuccore/MN908947) and matched with Philippine structural protein sequences using the blast2 command-line tool for protein identification. For the membrane glycoprotein, putative transmembrane sequences were identified using the TMPred online server (https : / / embnet .vitalit.ch/ sof tware/TMPRED_f orm . htm 1) , and the 21-residue Nterminal sequence thus
predicted to be externally located with respect to the viral membrane was considered as a possible target for recognition by immunoglobulins that bind protein epitopes of SARS-CoV-2 (noting that said N- terminal sequence was shared by all the membrane glycoprotein ORFs) .
In particular, the 20-residue N-terminal sequence of the SARS-CoV-2 membrane glycoprotein (MADSNGTITVEELKKLLEQW) was modified into an artificial homolog, with replacement of the oxidation-sensitive methionine and tryptophan residues by cysteine residues, thereby yielding a 20-residue artificial sequence ( CADSNGT I TVEELKKLLEQC ) consisting of an 18-residue wild-type core sequence between a pair of flanking cysteine residues. Subsequently, a peptide was designed that consisted of said artificial sequence with an unblocked N- terminus (i.e. , with a free N-terminal main-chain amino group) and an amidated C-terminus (i.e. , with a Cterminal main-chain carboxamide group) . The N- terminus was thus left unblocked to mimic the unblocked N-terminus of a typical naturally occurring protein chain and also to facilitate covalent conjugation by means of amine-reactive cross-linking agents such as glutaraldehyde, whereas the C-terminus was blocked by amidation to mitigate hydrolytic degradation by carboxypeptidases.
Said peptide was synthesized by a commercial service provider (GenScript) using PepPower™ peptide synthesis technology (combined liquid- and solidphase synthesis technology [i.e. , with synthesis on a resin and subsequent chemical cleavage from the resin and obtained in the form of lyophilized crude peptide for downstream applications] ) . Said lyophilized crude peptide (40 mg) was dissolved in dimethyl sulfoxide (200 pL) to yield a stock solution, which was stored at -20°C until further use. A sample of said stock solution (5 pl) was mixed with an equal volume of an 8M aqueous urea solution, and the resulting mixture was incubated (room temperature, 24 hours) to yield an oxidized-peptide solution, which was also stored at -20°C until further use. Said stock solution thus contained unoxidized peptide (hereinafter referred to as mMl ) , whereas said oxidized peptide solution contained oxidized peptide (hereinafter referred to as pMl) .
Samples (40 pg each) of pMl and mMl were analyzed under either non-reducing or reducing conditions using both sodium dodecyl sul fate-polyacrylamide gel electrophoresis (SDSPAGE) and acid-urea PAGE with subsequent Coomassie Blue gel staining. SDS-PAGE analysis revealed stained material forming smears of lower average electrophoretic mobility under nonreducing versus reducing conditions, which is
consistent with both the formation of polymeric peptide via oxidant-mediated intermolecular disulfide bond formation between cysteine residues and the degradation of said polymeric peptide. Acid- urea PAGE revealed a similar pattern consistent with formation of polymeric peptide.
Binding of anti-SARS-CoV-2 antibody to peptide targets was evaluated using indirect ELISA with high- binding polystyrene microtiter plates (Costar 3590, Corning Inc., NY, USA) . For coating (37 ° C , 1 hour) , wells were loaded (100 pL/well) with either polymerized peptide or non-polymeri zed peptide (20 pg/mL) in 0.05 M carbonate-bicarbonate buffer. For blocking (37 ° C , 30 minutes) , wells were loaded (100 pL/well) with blocking buffer (5% skim milk in wash buffer) . For antibody binding (room temperature, 1 hour) , wells were loaded (100 pL/well) with either convalescent serum extracted from a recovered COVID- 19 patient and negative-control serum obtained prior to the COVID-19 pandemic (September 2018) in serial two-fold dilution with dilution buffer (0.5% skim milk in wash buffer) starting at 1: 100. For conjugate binding (room temperature, 1 hour) , wells were loaded (50 pL/well) with protein A-peroxidase (0.5 pg/mL in dilution buffer) . Fresh chromogenic substrate solution (CSS) was prepared by dissolving 3, 3' , 5, 5' - tetramethylbenzidine in DMSO (200 pL) , diluting with
phosphate-ci trate buffer (9.8 mL) and adding 30% hydrogen peroxide (2 pL) . Wells were incubated (room temperature, 1 hour) with CSS (50 pL/well) , after which 1 M H2SO4 (50 pL/well) was added to stop the enzymatic reaction. Wells were read at 450 nm using a Bio-Rad Model 680 microplate reader.
Signals of significantly higher intensity were observed in convalescent serum compared to negativecontrol serum for both pMl and mMl, demonstrating binding of convalescent-serum antibody to the peptide. For convalescent serum, higher-intensity signals were observed with pMl compared to mMl.
Competition assays were performed to evaluate the specificity of antibody against the peptide. Using essentially the same ELISA procedure as described above, convalescent serum was tested after preincubation (room temperature, 1 hour) with either pMl or an irrelevant oxidized peptide pTlC+ ( CFI GI TELKKLESKINKVFC ) . Signal intensity was significantly decreased by pre-incubat ion with pMl but not with pTlC+.
Hence, the objects set forth above, among those made apparent from the preceding description, are efficiently attained and, because certain changes may be made in carrying out the above methods and in the constructions set forth without departing from the spirit and scope of the invention, all matter
contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. Furthermore, the following claims are intended to cover all of the generic and specific features of the invention herein described and all statements of the scope of the invention which, as a matter of language, might be said to fall there between.
Claims
1. An antigenic construct comprising a plurality of monomeric units, each of said monomeric units being derived from a peptidic precursor, said precursor comprising an artificial sequence, said artificial sequence comprising at least one epitope, said epitope being recognizable by at least one paratope, each of said monomeric units comprising at least two oxidized cysteine residues, each of said monomeric units being covalently linked to at least one other of said monomeric units by a disulfide bond between oxidized cysteine residues, said paratope recognizing an antigen that comprises at least one target sequence, said artificial sequence being homologous to said target sequence, said artificial sequence comprising at least one amino-acid residue substitution, said substitution entailing replacement of a reference amino-acid residue present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another, characterized by said target sequence occurring within a protein of at least one strain of SARS-CoV-2 .
2. The construct of claim 1, further characterized by said alternative amino-acid residue being one of
said two oxidized cysteine residues.
3. The construct of claim 2, further characterized by said reference amino-acid residue being a methionine or tryptophan residue.
4. The construct of claim 2, further characterized by said reference residue being located at the N- or C-terminus of said target sequence.
5. The construct of claim 3 or 4, further characterized by said artificial sequence being CADSNGTITVEELKKLLEQC .
6. A resin comprising a mat rix- 1 inked peptide covalently attached to a solid support matrix, said resin being suitable for producing a free peptide by chemical cleavage from said matrix, said free peptide being suitable for producing an antigenic construct comprising a plurality of monomeric units, each of said monomeric units being derived from a peptidic precursor, said precursor comprising an artificial sequence, said artificial sequence comprising at least one epitope, said epitope being recognizable by at least one paratope, each of said monomeric units comprising at least two oxidized cysteine residues, each of said monomeric units being
covalently linked to at least one other of said monomeric units by a disulfide bond between oxidized cysteine residues, said paratope recognizing an antigen that comprises at least one target sequence, said artificial sequence being homologous to said target sequence, said artificial sequence comprising at least one amino-acid residue substitution, said substitution entailing replacement of a reference amino-acid residue present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another, characterized by said target sequence occurring within a protein of at least one strain of SARS-CoV- 2.
7. The resin of claim 6, further characterized by said alternative amino-acid residue being one of said two oxidized cysteine residues.
8. The resin of claim 7, further characterized by said reference amino-acid residue being a methionine or tryptophan residue.
9. The resin of claim 7, further characterized by said reference residue being located at the Nor C- terminus of said target sequence.
10. The resin of claim 8 or 9, further characterized by said artificial sequence being CADSNGTITVEELKKLLEQC .
11. A free peptide suitable for producing an antigenic construct comprising a plurality of monomeric units, each of said monomeric units being derived from a peptidic precursor, said precursor comprising an artificial sequence, said artificial sequence comprising at least one epitope, said epitope being recognizable by at least one paratope, each of said monomeric units comprising at least two oxidized cysteine residues, each of said monomeric units being covalently linked to at least one other of said monomeric units by a disulfide bond between oxidized cysteine residues, said paratope recognizing an antigen that comprises at least one target sequence, said artificial sequence being homologous to said target sequence, said artificial sequence comprising at least one amino-acid residue substitution, said substitution entailing replacement of a reference amino-acid residue present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another, characterized by said target sequence occurring within a protein of at least one strain of
SARS-CoV-2 .
12. The peptide of claim 11, further characterized by said alternative amino-acid residue being one of said two oxidized cysteine residues.
13. The peptide of claim 12, further characterized by said reference amino-acid residue being a methionine or tryptophan residue.
14. The peptide of claim 12, further characterized by said reference residue being located at the N- or C-terminus of said target sequence.
15. The peptide of claim 13 or 14, further characterized by said artificial sequence being CADSNGTITVEELKKLLEQC .
16. A method for preparing an antigenic construct, said construct comprising a plurality of monomeric units, each of said monomeric units being derived from a peptidic precursor, said precursor comprising an artificial sequence, said artificial sequence comprising at least one epitope, said epitope being recognizable by at least one paratope, said artificial sequence comprising at least two oxidized cysteine residues, each of said monomeric units being
covalently linked to at least one other of said monomeric units by a disulfide bond between oxidized cysteine residues, said paratope recognizing an antigen that comprises at least one target sequence, said artificial sequence being homologous to said target sequence, said artificial sequence comprising at least one amino-acid residue substitution, said substitution entailing replacement of a reference amino-acid residue present in said target sequence by an alternative amino-acid residue, said reference and alternative aminoacid residues being structurally distinct from one another, said method comprising the step of oxidizing said peptidic precursor, characterized by said target sequence occurring within a protein of at least one strain of
SARS-CoV-2 .
17. The method of claim 16, further characterized by said alternative amino-acid residue being one of said two oxidized cysteine residues.
18. The method of claim 17, further characterized by said reference amino-acid residue being a methionine or tryptophan residue.
19. The method of claim 17, further characterized by said reference residue being located at the N- or C-
terminus of said target sequence.
20. The method of claim 18 or 19, further characterized by said artificial sequence being CADSNGTITVEELKKLLEQC .
21. A method for detecting immunoglobulins that bind to at least one protein epitope of SARSCoV-2, said method comprising the steps of (i) contacting said immunoglobulins with an antigenic construct, and (ii) detecting said immunoglobulins bound to said construct, said construct comprising a plurality of monomeric units, each of said monomeric units being derived from an peptidic precursor, said precursor comprising an artificial sequence, said artificial sequence comprising at least one epitope, said epitope being recognizable by at least one paratope, each of said monomeric units comprising at least two oxidized cysteine residues, each of said monomeric units being covalently linked to at least one other of said monomeric units by a disulfide bond between oxidized cysteine residues, said paratope recognizing an antigen that comprises at least one target sequence, said artificial sequence being homologous to said target sequence, said artificial sequence comprising at least one amino-acid residue substitution, said substitution entailing
replacement of a reference amino-acid residue present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another, characterized by said target sequence occurring within a protein of at least one strain of SARS-CoV-2 .
22. The method of claim 21, further characterized by said alternative amino-acid residue being one of said two oxidized cysteine residues.
23. The method of claim 22, further characterized by said reference amino-acid residue being a methionine or tryptophan residue.
24. The method of claim 22, further characterized by said reference residue being located at the N- or C- terminus of said protein.
25. The method of claim 23 or 24, further characterized by said artificial sequence being CADSNGTITVEELKKLLEQC .
26. A testing system for detecting immunoglobulins that bind to at least one protein epitope of SARS- CoV-2, said testing system comprising an antigenic
construct, said construct comprising a plurality of monomeric units, each of said monomeric units being derived from a peptidic precursor, said precursor comprising an artificial sequence, said artificial sequence comprising at least one epitope, said epitope being recognizable by at least one paratope, each of said monomeric units comprising at least two oxidized cysteine residues, each of said monomeric units being covalently linked to at least one other of said monomeric units by a disulfide bond between oxidized cysteine residues, said paratope recognizing an antigen that comprises at least one target sequence, said artificial sequence being homologous to said target sequence, said artificial sequence comprising at least one amino-acid residue substitution, said substitution entailing replacement of a reference amino-acid residue present in said target sequence by an alternative amino-acid residue, said reference and alternative amino-acid residues being structurally distinct from one another, characterized by said target sequence occurring within a protein of at least one strain of SARS-CoV-2 .
27. The method of claim 26, further characterized by said alternative amino-acid residue being one of said two oxidized cysteine residues.
28. The method of claim 27, further characterized by said reference amino-acid residue being a methionine or tryptophan residue.
29. The method of claim 27, further characterized by said reference residue being located at the N- or C- terminus of said protein.
30. The method of claim 28 or 29, further characterized by said artificial sequence being CADSNGTITVEELKKLLEQC .
31. Use of the construct in claims 1, 2, 3 or 4 in a method for detecting immunoglobulins that bind to at least one protein epitope of SARS-CoV-2.
32. Use of the construct in claim 5 in a method for detecting immunoglobulins that bind to at least one protein epitope of SARS-CoV-2.
33. Use of the construct in claims 1, 2, 3 or 4 in a method for producing immunoglobulins that bind to at least one protein epitope of SARS-CoV-2.
34. Use of the construct in claim 5 in a method for producing immunoglobulins that bind to at least one protein epitope of SARS-CoV-2.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PH12020050482 | 2020-11-19 | ||
PH12020050482 | 2020-11-19 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2022108460A2 true WO2022108460A2 (en) | 2022-05-27 |
WO2022108460A3 WO2022108460A3 (en) | 2022-07-21 |
Family
ID=81709564
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/PH2021/050039 WO2022108460A2 (en) | 2020-11-19 | 2021-11-19 | Peptide-based antigenic constructs recognized by immunoglobulins that bind to protein epitopes of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2022108460A2 (en) |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20200347465A1 (en) * | 2020-03-24 | 2020-11-05 | DRK-Blutspendedienst Baden-Württemberg - Hessen gemeinnützige GmbH | Detection of sars-cov-2 in a plurality of biological samples |
IT202000006754A1 (en) * | 2020-03-31 | 2021-10-01 | Diasorin S P A | Assays for the detection of SARS-CoV-2 |
CN111926112A (en) * | 2020-07-01 | 2020-11-13 | 上海速创诊断产品有限公司 | Primer composition, kit and method for detecting SARS-CoV-2 nucleic acid |
-
2021
- 2021-11-19 WO PCT/PH2021/050039 patent/WO2022108460A2/en active Application Filing
Also Published As
Publication number | Publication date |
---|---|
WO2022108460A3 (en) | 2022-07-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3734286A1 (en) | A method for determining the efficacy of a sars-cov-2 vaccine | |
US10323069B2 (en) | Senecavirus A antigens and methods of use | |
Burns et al. | Protein-protein interactions in lymphocytic choriomeningitis virus | |
Clavijo et al. | Development and use of a biotinylated 3ABC recombinant protein in a solid-phase competitive ELISA for the detection of antibodies against foot-and-mouth disease virus | |
Yandar et al. | Immunological profile of a Plasmodium vivax AMA-1 N-terminus peptide-carbon nanotube conjugate in an infected Plasmodium berghei mouse model | |
WO2021180233A1 (en) | Polypeptide, polypeptide vaccine and application thereof | |
Dechamma et al. | Identification of T-helper and linear B epitope in the hypervariable region of nucleocapsid protein of PPRV and its use in the development of specific antibodies to detect viral antigen | |
WO2023083092A1 (en) | Sars-cov-2 s protein polypeptide antigen and application thereof | |
WO2021233885A1 (en) | Mimotope peptides of the spike protein from the sars-cov-2 virus | |
EP2405268A1 (en) | Method for detecting substance in biological sample | |
CN106596964B (en) | Novel assay for diagnosing helminth infection | |
Mirzadeh et al. | Production of refolded Toxoplasma gondii recombinant SAG1-related sequence 3 (SRS3) and its use for serodiagnosis of human toxoplasmosis | |
Borek et al. | Relation between optical configuration and immunogenicity of synthetic polypeptides | |
US20210349090A1 (en) | Corona nucleocapsid antigen for use in antibody-immunoassays | |
Bächi et al. | Staphylococcal protein A in immunoferritin techniques | |
WO2022108460A2 (en) | Peptide-based antigenic constructs recognized by immunoglobulins that bind to protein epitopes of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) | |
AU2018366480C1 (en) | Novel mammalian expressed human immunodeficiency virus envelope protein antigens | |
JP2813768B2 (en) | Foot-and-mouth disease diagnostic peptide and foot-and-mouth disease diagnostic antigen containing the peptide | |
Brown et al. | Antigenic determinants of influenza virus hemagglutinin. V. Antigenicity of the HA2 chain. | |
WO2021209925A1 (en) | Coronavirus serology assay | |
CN113845577A (en) | SARS-CoV-2 specific polypeptide and its application | |
Jiménez-Clavero et al. | Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus | |
AU624066B2 (en) | New peptides and applications thereof | |
US20210333277A1 (en) | Corona nucleocapsid antigen for use in antibody-immunoassays | |
EP3892298A1 (en) | Epitopes having sequence homology to coronavirus spike protein subunit and uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 21895209 Country of ref document: EP Kind code of ref document: A2 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 21895209 Country of ref document: EP Kind code of ref document: A2 |