WO2021257469A1 - Dosage immunologique pour la détection d'anticorps de protéine de spicule anti-sras-cov-2 dans des échantillons de lait - Google Patents

Dosage immunologique pour la détection d'anticorps de protéine de spicule anti-sras-cov-2 dans des échantillons de lait Download PDF

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WO2021257469A1
WO2021257469A1 PCT/US2021/037256 US2021037256W WO2021257469A1 WO 2021257469 A1 WO2021257469 A1 WO 2021257469A1 US 2021037256 W US2021037256 W US 2021037256W WO 2021257469 A1 WO2021257469 A1 WO 2021257469A1
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cov
sars
spike protein
milk
covid
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PCT/US2021/037256
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Rebecca POWELL
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Icahn School Of Medicine At Mount Sinai
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    • 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
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/12—Immunoglobulins specific features characterized by their source of isolation or production isolated from milk
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • 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

  • kits for detecting antibodies in milk samples using recombinant SARS-CoV-2 spike proteins in an immunoassay comprising administering a composition comprising immunoglobulin purified from a milk sample.
  • BSA bovine serum albumin
  • PBS phosphate buffered solution
  • FIG. 1A IgA
  • FIG. 1B SC
  • FIG. 1C IgM
  • FIG. 1D IgG.
  • FIG. 1E-1H Grouped OD values for undiluted milk.
  • FIG. 1E IgA
  • SC SC
  • FIG. 1G IgM
  • FIG. 1H IgG. Mean with SEM is shown.
  • FIGS. 1I, 1J Correlated IgA/secretory Ab
  • IgG/IgM FIG. 1 J
  • FIGS. 5A-5B The RBD-Speciflc IgA Response in Milk is Dominant and Not Necessarily Concurrent with a Measurable IgG or IgM Response.
  • FIGS. 5A and 5B Full titrations against RBD, measuring IgG (A) and IgM (B) binding are shown.
  • COV/solid lines milk from COVID-19- recovered donors. Experiments were performed in duplicate and repeated twice. Mean with SEM is shown. Dotted lines indicate positive cutoff value (mean OD or endpoint titer of negative control milk samples +2 ⁇ SD).
  • a recombinant SARS-CoV-2 spike protein comprises (or consists of) the ectodomain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) known to one of skill in the art, such as the SARS-CoV-2 spike protein found at GenBank Accession No.
  • a recombinant SARS-CoV-2 spike protein described herein further comprises one, two or all of the following: a C-terminal cleavage site (e.g., a C-terminal thrombin cleavage site or other known cleavage site), trimerization domain (e.g., a T4 foldon trimerization domain) and a tag (e.g., a flag tag or histidine tag (such as, e.g., hexahistidine tag)).
  • a C-terminal cleavage site e.g., a C-terminal thrombin cleavage site or other known cleavage site
  • trimerization domain e.g., a T4 foldon trimerization domain
  • a tag e.g., a flag tag or histidine tag (such as, e.g., hexahistidine tag)
  • the two sequences are the same length.
  • the percent identity is determined over the entire length of an amino acid sequence or nucleotide sequence.
  • the length of sequence identity comparison may be over the full-length of the two sequences being compared (e.g., the full-length of a gene coding sequence, or a fragment thereof).
  • a fragment of a nucleotide sequence is at least 25, at least 50, at least 75, or at least 100 nucleotides.
  • Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25:33893402.
  • PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id).
  • a recombinant SARS-CoV-2 spike protein comprises (or consists of) the ectodomain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) known to one of skill in the art, such as the SARS-CoV-2 spike protein found at GenBank Accession No.
  • a stabilizing mutation e.g., lysine to proline at the amino acid residue corresponding to amino acids residue 986 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No.
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) the ectodomain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the ectodomain of the SARS-CoV-2 spike protein disclosed at GenBank Accession No.
  • RRAR polybasic cleavage site
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) the ectodomain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the ectodomain of the SARS-CoV-2 spike protein disclosed at GenBank Accession No.
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) the ectodomain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the ectodomain of the SARS-CoV-2 spike protein disclosed at GenBank Accession No.
  • a recombinant SARS-CoV-2 spike protein comprises amino acids 1-1213 (MFIF....IKWP) of the spike protein found at GenBank Accession No. MN908947.3, a C-terminal thrombin cleavage site, T4 foldon trimerization domain and hexahistidine tag, wherein the protein does not contain the polybasic cleavage site ((RRAR ) e.g., RRAR is changed to A).
  • a recombinant SARS-CoV-2 spike protein comprises amino acids 1-1213 (MFIF....IKWP) of the spike protein found at GenBank Accession No.
  • the polybasic cleavage site (RRAR) at amino acid residues 682 to 685 of the amino acid sequence of SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3 is replaced with a single alanine.
  • the polybasic cleavage site (RRAR) at amino acid residues corresponding to amino acid residues 682 to 685 of the amino acid sequence of SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3 is replaced with a single alanine.
  • a recombinant SARS-CoV-2 spike protein comprises amino acids 15-1213 of the spike protein found at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, a C- terminal thrombin cleavage site, T4 foldon trimerization domain and hexahistidine tag, wherein the protein does not contain the polybasic cleavage site ((RRAR ); e.g., RRAR is changed to A).
  • RRAR polybasic cleavage site
  • a recombinant SARS-CoV-2 spike protein comprises amino acids 15-1213 (MFIF....IKWP) of the spike protein found at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, a C-terminal thrombin cleavage site, T4 foldon trimerization domain and hexahistidine tag, wherein the protein does not contain the polybasic cleavage site ((RRAR); e.g., RRAR is changed to A) and the protein contains two stabilizing mutations (K986P and V987P, wild type numbering).
  • RRAR polybasic cleavage site
  • polybasic cleavage site (RRAR) at amino acid residues corresponding to amino acid residues 682 to 685 of the amino acid sequence of SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3 is replaced with a single alanine.
  • a recombinant soluble SARS-CoV-2 spike protein comprises amino acids 1-1213 (MFIF....IKWP) of the spike protein (otherwise known as the S or structural protein) found at GenBank Accession No. MN908947.3.
  • a recombinant soluble SARS-CoV-2 spike protein comprises amino acids 1-1213 (MFIF....IKWP) of the spike protein found at GenBank Accession No. MN908947.3, wherein the protein does not contain the polybasic cleavage site.
  • the polybasic/furin cleavage site (RRAR) 355 is replaced by a single A (RRAR to A).
  • a recombinant soluble SARS- CoV-2 spike protein comprises amino acids 1-1213 (MFIF....IKWP) of the spike protein found at GenBank Accession No. MN908947.3, wherein the protein does not contain the polybasic cleavage site (RRAR to A) and the protein contains two stabilizing mutations (K986P and V987P, wild-type numbering).
  • a recombinant soluble SARS-CoV-2 spike protein comprises amino acids 1-1213 (MFIF....IKWP) of the spike protein found at GenBank Accession No. MN908947.3, a C-terminal thrombin cleavage site, T4 foldon trimerization domain and hexahistidine tag, wherein the protein does not contain the polybasic cleavage site (RRAR to A).
  • a recombinant soluble SARS-CoV-2 spike protein comprises amino acids 1-1213 (MFIF....IKWP) of the spike protein found at GenBank Accession No.
  • such a recombinant soluble spike protein comprises a signal peptide, such as the signal peptide of the spike protein disclosed at GenBank Accession No. MN908947.3.
  • a recombinant soluble SARS-CoV-2 spike protein comprises amino acids 15-1213 of the spike protein (otherwise known as the S or structural protein) found at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558.
  • a recombinant soluble SARS- CoV-2 spike protein comprises amino acids 15-1213 of the spike protein found at GenBank Accession No.
  • a recombinant soluble SARS-CoV-2 spike protein comprises amino acids 15-1213 of the spike protein found at GenBank Accession No.
  • such a recombinant soluble spike protein comprises one, two or all of the following: a C-terminal cleavage site (e.g., a C-terminal thrombin cleavage site or other known cleavage site), trimerization domain (e.g., a T4 foldon trimerization domain) and a tag (e.g., a flag tag or histidine tag (such as, e.g., hexahistidine tag)).
  • the C-terminal cleavage site, trimerization domain, and/or tag may be at C-terminus of the ectodomain.
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) a fragment of the ectodomain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the ectodomain of the SARS-CoV-2 spike protein disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558.
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) a fragment of the ectodomain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558.
  • the fragment of the SARS-CoV-2 spike protein ectodomain is at least 1000, 1025, 1075, 1100, 1125, 1150, 1200 or 1215 amino acid residues in length.
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) the SI domain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, orMT334558 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more mutations (e.g., substitutions, deletions, additions or a combination thereof).
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) the SI domain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, or a fragment thereof, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more substitutions (e.g., conservative amino acid substitutions).
  • a recombinant SARS-CoV-2 spike protein described herein further comprises a domain(s) that facilitates purification, folding and/or cleavage of portions of a protein.
  • a recombinant SARS-CoV-2 spike protein described herein further comprises one, two or all of the following: a C-terminal cleavage site (e.g., a C-terminal thrombin cleavage site or other known cleavage site), trimerization domain (e.g., a T4 foldon trimerization domain) and a tag (e.g., a flag tag or histidine tag (such as, e.g., hexahistidine tag)).
  • a C-terminal cleavage site e.g., a C-terminal thrombin cleavage site or other known cleavage site
  • trimerization domain e.g., a T4 foldon trimerization domain
  • a tag e.g.,
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) the S2 domain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the S2 domain of the SARS-CoV-2 spike protein disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, or a fragment thereof.
  • a recombinant SARS-CoV-2 spike protein described herein comprises (or consists of) the S2 domain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, or a fragment thereof, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more substitutions (e.g., conservative amino acid substitutions).
  • the C-terminal cleavage site, trimerization domain, and/or tag may be at C- terminus of the S2 domain or fragment thereof.
  • the fragment of the SARS-CoV-2 spike protein S2 domain is at least 250, 300, 400, 500, or 750 amino acid residues in length.
  • such a recombinant spike protein further comprises one, two or all of the following: a C-terminal cleavage site (e.g., a C-terminal thrombin cleavage site or other known cleavage site), trimerization domain (e.g., a T4 foldon trimerization domain) and a tag (e.g., a flag tag or histidine tag (such as, e.g., hexahistidine tag)).
  • the C-terminal cleavage site, trimerization domain, and/or tag may be at C-terminus of the receptor binding domain.
  • a recombinant SARS-CoV-2 spike protein is one that may be commercially purchased from Genscript. In another specific embodiment, a recombinant SARS-CoV-2 spike protein is one that may be purchased from, e.g., a vendor.
  • an expression vector containing a polynucleotide encoding a SARS-CoV-2 spike protein can be transcribed and translated in vitro using, e.g., T7 promoter regulatory sequences and T7 polymerase.
  • a coupled transcription/translation system such as Promega TNT®, or a cell lysate or cell extract comprising the components necessary for transcription and translation may be used to produce a SARS-CoV-2 spike protein described herein.
  • an antibody response of a subject or a population of subjects that has/have been infected by SARS-CoV-2 or immunized with a vaccine that includes a SARS-CoV-2 spike protein may be assessed in an immunoassay (e.g., an ELISA described herein) to identify the types of antibodies (e.g., IgG, IgA, IgM, etc) in a milk sample from the subject or population of subjects specific for the SARS-CoV-2 spike protein.
  • an immunoassay e.g., an ELISA described herein
  • the method comprises use of a negative control and a positive control.
  • a negative and/or positive control when used the method involves the same steps as with the milk sample in different wells.
  • the method is run in a high-throughput format so that the detection of antibody(ies) in multiple milk samples may be conducted concurrently.
  • a 96 well microtiter plate is used with different milk samples or controls in different wells, or different dilutions of a milk sample in different wells, wherein the wells are coated with a recombinant SARS-CoV-2 spike protein.
  • each well is coated with 50 ⁇ l of 1 ⁇ g of a recombinant SARS-CoV-2 spike protein described herein. In some embodiments, each well is coated with 25-50 ⁇ l, 25 to 75 ⁇ l, 50 to 75 ⁇ l, or 50 to 100 ⁇ l of 1 ⁇ g of a recombinant SARS-CoV-2 spike protein described herein. The well may be coated with recombinant SARS-CoV-2 spike protein overnight at 4° C and then washed with tween/PBS (PBS-T; e.g., 100 ⁇ ;1 of 0.1% PBS-T).
  • PBS-T tween/PBS
  • the method further comprises (f) incubating the milk sample or a diluted milk sample in a well coated with a second recombinant soluble SARS-CoV-2 spike protein for a third period of time, wherein the second recombinant soluble SARS-CoV-2 spike protein is different from the first recombinant soluble SARS-CoV-2 spike protein (e.g., the first recombinant soluble SARS-CoV-2 spike protein may comprise the receptor binding domain of a SARS-CoV-2 spike protein but not the entire ectodomain and the second recombinant soluble SARS-CoV-2 spike protein may comprise the ectodomain of a SARS-CoV-2 spike protein); (g) washing the well; (h) incubating a second labeled antibody that binds to an isotype or subtype of an immunoglobulin (e.g., IgG) for a fourth period of time in well; (i) washing the well; and (j) detecting the binding
  • the first time period, second time period, or both are 30 minutes, 45 minutes, 1 hour, 2 hours, 2.5 hours or 3 hours.
  • the third and fourth time periods are 30 minutes, 45 minutes, 1 hour, 2 hours, 2.5 hours or 3 hours.
  • the second labeled antibody is the same as the first labeled antibody.
  • the well is blocked with PBS/3% serum (e.g., goat serum)/0.5% milk powder/3.5% PBS-T for 30 minutes to 1.5 hours (e.g., 1 hour) before adding a milk sample at room temperature.
  • the well(s) is/are washed with 0.1% Tween 20/PBS (PBS-T). The washing may occur at room temperature.
  • the labeled secondary antibody is labeled with horseradish peroxidase (HRP) conjugated to an antibody that binds to the particular immunoglobulin isotype or subtype (e.g., anti-human IgA antibody), or secretory component and detection of the binding of the labeled antibody to the recombinant SARS-CoV-2 spike protein comprises incubating substrate (e.g., o- phenylenediamine dihydrocloride) in the well, stopping the reaction (e.g., with 3 M HC1 stop solution), and reading the optical density of the well at, e.g., 450 nm.
  • substrate e.g., o- phenylenediamine dihydrocloride
  • a recombinant soluble SARS-CoV-2 spike protein described herein is immobilized (e.g., coated) on a bead (e.g., a glass bead, plastic bead, magnetic bead, or polystyrene bead), a test strip, a microtiter plate, a membrane, a glass surface, a slide (e.g., a microscopy slide), a microarray, a column (e.g., a chromatography column), or a biochip.
  • a bead e.g., a glass bead, plastic bead, magnetic bead, or polystyrene bead
  • test strip e.g., a test strip
  • a microtiter plate e.g., a membrane, a glass surface, a slide (e.g., a microscopy slide), a microarray, a column (e.g., a chromatography column),
  • cells are washed twice (e.g., washed twice with PBS) and the plates are developed (e.g., developed using 100 ⁇ L of SigmaFast OPD substrate).
  • a certain period of time later e.g., ten minutes later, the reactions are stopped (e.g., stopped using 50 ⁇ L per well of 3M HCI) and the OD is read (e.g., OD 492 nM is measured on a Biotek SynergyHl Microplate Reader).
  • Non-linear regression curve fit analysis (The top and bottom constraints may be set at 100% and 0%) over the dilution curve may ve performed to calculate 50% of inhibitory dilution (ID50) of the milk sample using GraphPad Prism 7.0.
  • the detection of a particular isotype (e.g., IgA) or subtype in a milk sample from a subject (e.g., human) using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine if the subject may have some degree of protection against the development of COVID-19.
  • a medical professional e.g., a physician
  • the detection a particular isotype (e.g., IgA) or subtype in a milk sample from a subject (e.g., human) using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine if the subject may be fully protected from developing moderate to severe COVID-19.
  • a medical professional e.g., a physician
  • the antibodies from the milk sample or a particular isotype or subtype are assessed for microneutralization.
  • the anti-SARS-CoV-2 spike protein antibody or a particular isotype or subtype of anti-SARS- CoV-2 spike protein antibody exhibits microneutralization activity.
  • the detection of IgA anti-SARS-CoV-2 spike protein antibody in a milk sample from a subject indicates that the subject has some degree of protection against COVID-19.
  • the detection of IgA anti-SARS-CoV-2 spike protein antibody in a milk sample from a subject, which IgA exhibits microneutralization activity indicates that the subject has some degree of protection against COVID-19.
  • a patient treated in accordance with the methods provided herein is a patient infected with a SARS-CoV-2 that does not manifest any symptoms of the infection or COVID-19.
  • a patient treated in accordance with the methods provided herein is a patient infected with a SARS-CoV-2 that manifests mild symptoms of the infection or COVID-19.
  • a patient treated in accordance with the methods provided herein is a patient infected with a SARS-CoV-2 that manifests moderate symptoms of the infection or COVID-19.
  • a patient treated in accordance with the methods provided herein is a patient infected with a SARS-CoV-2 that manifests moderate to severe symptoms of the infection or COVID-19.
  • milk Ab response would be reflective of systemic immunity (i.e., milk Ab should generally mirror serum Ab)
  • milk Ab should generally mirror serum Ab
  • only a small fraction of milk Ab originates from serum - likely less than 10%, and only -2% of milk Ab is IgG 9 .
  • Human milk Ab is -90% IgA and 8% IgM, nearly all slgA/sIgM.
  • the B cells that ultimately produce slgA/sIgM originate mainly from the GALT, known as the entero-mammary link, with some proportion originating from other mucosa such as the respiratory system 14,18 ’ 19 .
  • GALT known as the entero-mammary link
  • the ELISA used in this study could not determine with certainty that the IgA (or IgM) measured was of the secretory type or not (Brandtzaeg, 2010, J. Pediatr. 156: S8-S15).
  • the assay measuring secretory Ab reactivity employs a secondary Ab specific for the SC, which can be free or bound to Ab. Notably, all samples exhibiting positive IgA reactivity also exhibited positive SC reactivity, and a very strong positive correlation was present when comparing the OD values of undiluted milk for the IgA and SC assays.
  • the purified material would need to be extensively safety-tested, including ensuring it as free of SARS-CoV-2 material.
  • monoclonal or polyclonal Spike- specific slgA could be employed as a similar therapeutic.
  • Example 2 the milk Ab response after SARS-CoV-2 infection demonstrated that Spike-specific IgA in milk after infection is dominant and highly correlated with a secretory Ab response [2], Determining if secretory Abs are elicited in milk is critical, as this Ab class is highly stable and resistant to enzymatic degradation in all mucosae - not only in the infant oral/nasal cavity and gut, but in the airways and GI tract as well [3, 4]. [00155] This Example describes the analysis of vaccine-elicited antibodies in 10 pairs of milk samples obtained from individual donors 1 day before dose 1, and 14 days after dose 2, of either the Pfizer/ZBioNT ech or Moderna mRNA-based COVID-19 vaccines.
  • Samples were assayed for specific IgA, IgG, and secretory Ab against the full trimeric SARS-CoV-2 Spike protein. Unlike the post-infection milk antibody profile, IgG dominates after COVID-19 vaccination.
  • One hundred percent of post-vaccine milk contained significant levels of Spike-specific IgG, with 8/10 samples exhibiting high IgG endpoint titers.
  • 6/10 (60%) of post-vaccine samples were positive for Spike specific IgA, with only 1 (10%) exhibiting high IgA endpoint titer.
  • 5/10 (50%) post-vaccine milk samples contained Spike-specific secretory Ab, none of which were found to be high-titer.
  • ELISA Levels of SARS-CoV-2 Abs in human milk were measured as previously described [2], Briefly, before Ab testing, milk samples were thawed, centrifuged at 800g for 15 min at room temperature, fat was removed, and supernatant transferred to a new tube. Centrifugation was repeated 2x to ensure removal of all cells and fat. Skimmed acellular milk was aliquoted and frozen at -80°C until testing. Milk was tested in separate assays measuring IgA, IgG, and secretory-type Ab reactivity (the secondary Ab used in this assay is specific for free and bound SC).
  • Spike-specific secretory Ab was measured. It was found that none of the undiluted pre- vaccine samples and 5/10 undiluted post- vaccine samples contained Spike-specific secretory Ab (50%; FIG. 7B). Notably, 4/5 (90%) of these positive samples exhibited binding just at or just above the positive cutoff. Upon titration, 3/5 (60%) of positive samples exhibited significant secretory Ab endpoint binding, with none of these samples exhibiting a high-titer response (FIG. 7E). Overall, 3/10 (30%) of post-vaccine milk samples contained Spike-specific secretory Ab exhibiting a significant endpoint binding titer.
  • ELISA OD values as well as endpoint binding titers for each assay were compared in separate Spearman correlation analyses (IgG v IgA; IgG v SC; IgA v SC). No correlations were found among any of the parameters measured (data not shown). Additionally, ELISA ODs for each Ab class were compared for milk samples obtained from participants who received the Pfizer vs. Moderna vaccine. No significant differences in values were detected (data not shown).
  • IM vaccines have been shown previously to generate mucosal Ab, including Abs in milk, though whether IM vaccination tends to elicit secretory Abs, which would be expected to be the most protective class in a mucosal environment, has generally not been addressed [16-23], Human milk slgA is naturally dominant (-90% of total), and is derived from B cells that transit mainly from the GALT, with some respiratory MALT trafficking as well [4, 32], The data investigating the SARS-CoV-2-specific Ab response in milk following infection has demonstrated clearly that this response is robust in most people, and dominated by specific IgA that is largely of the secretory class, while the IgG response is detected in fewer people and is generally of a much lower potency ([2] and Example 4).
  • antigen may diffuse differentially from the IM immunization site to local lymph nodes, wherein it is taken up by APCs that initiate a local response followed by migration of these cells and activated lymphocytes to various MALT locales, including Peyer's patches (PP) in the GALT, which would be critical to the ultimate activation of the entero-mammary pathway and eventual secretion of slgA in milk [28, 29],
  • Adjuvants as well as immunomodulatory receptors on vectored vaccines may increase and/or modify APC and lymphocyte recruitment, stimulation and trafficking [29]
  • NHP studies have demonstrated that the vaccine platform and regimen/route is highly significant in terms of the ultimate milk Ab response produced [24-26], Additionally, the passive transfer of serum Ab into milk should not be ignored, and is likely to also differ among these vaccines based on their differential immunogenicity profiles over time [33-37],
  • IgA antibodies and antibodies bearing secretory component were shown to be strongly positively correlated.
  • the secretory IgA response was dominant among the milk samples tested compared to the IgG response, which was present in 75% of samples and found to be of high-titer in only 13% of cases.
  • COVID-19 and pre-pandemic control milk samples were tested for the presence of neutralizing antibodies; 6 of 8 COVID-19 samples exhibited neutralization of Spike-pseudotyped VSV (IC50 range, 2.39 - 89.4ug/mL) compared to 1 of 8 controls.
  • IgA binding and neutralization capacities were found to be strongly positively correlated.
  • Study participants Individuals were eligible to have their milk samples included in this analysis if they were lactating and had a laboratory-confirmed SARS-CoV-2 infection 4-6 weeks prior to the initial milk sample used for analysis. Certain participants were also able to continue participation in the study and provide a follow-up sample 4-10 months after confirmed infection.
  • ELISA Levels of SARS-CoV-2 Abs in human milk were measured as previously described (2). Briefly, before Ab testing, milk samples were thawed, centrifuged at 800g for 15 min at room temperature, fat was removed, and the de-fatted milk transferred to a new tube. Centrifugation was repeated 2x to ensure removal of all cells and fat. Skimmed acellular milk was aliquoted and frozen at -80°C until testing. Both COVID-19 recovered and control milk samples were then tested in separate assays measuring IgA, IgG, and secretoiy-type Abs, in which the secondary Ab used for the latter measurement was specific for free and bound SC.
  • Half-area 96-well plates were coated with the full trimeric recombinant Spike protein produced, as described previously (10). Plates were incubated at 4°C overnight, washed in 0.1% Tween 20/PBS (PBS-T), and blocked in PBS-T/3% goat serum/0.5% milk powder for 1 h at room temperature. Milk was used undiluted or titrated 4-fold in 1% bovine serum albumin (BSA)/PBS and added to the plate.
  • BSA bovine serum albumin
  • IgA extraction from milk Total IgA was extracted from 25 - 100mL of milk using peptide M agarose beads (Pierce) following manufacturer’s protocol, concentrated using Ami con Ultra centrifugal filters (10 kDa cutoff; Millipore Sigma) and quantified by Nanodrop.
  • Pseudovirus neutralization assay Neutralization assays were performed using a standardized SARS-CoV-2 Spike-pseudotyped Vesicular Stomatitis Virus (VSV)-based assay with ACE2- and TMPRSS2-expressing 293T cells as previously described (11).
  • VSV Vesicular Stomatitis Virus
  • a method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample comprising:
  • the recombinant SARS-CoV-2 spike protein comprises amino acid residues 1-1213 of GenBank Accession No. MN908947.3, a C-terminal thrombin cleavage site, T4 foldon trimerization domain, and a tag
  • the recombinant soluble SARS-CoV-2 spike protein does not contain a polybasic cleavage site and includes two stabilizing mutatons of lysine to proline at amino acid residue 986 of the amino acid sequence found at GenBank Accession No. MN908947.3, and valine to proline at amino acid residue 987 of the amino acid sequence found at GenBank Accession No. MN908947.3.
  • a method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample comprising:
  • BSA bovine serum albumin
  • PBS phosphate buffered solution
  • the second recombinant soluble SARS-CoV-2 spike protein comprises amino acid residues 1-1213 of GenBank Accession No. MN908947.3, a C-terminal thrombin cleavage site, T4 foldon trimerization domain, and a tag
  • the second recombinant SARS-CoV-2 spike protein does not contain a polybasic cleavage site
  • a method for treating a SARS-CoV-2 infection or COVID-19 comprising administering to a subject in need thereof a composition comprising immunoglobulin purified from a milk sample of an individual that tested positive for anti-SARS-CoV-2 spike protein antibody.
  • the recombinant SARS-CoV-2 spike protein comprises amino acid residues 15-1213 of GenBank Accession No. MN908947.3, a C-terminal thrombin cleavage site, T4 foldon trimerization domain, and a tag
  • the recombinant soluble SARS-CoV-2 spike protein does not contain a polybasic cleavage site and includes two stabilizing mutatons of lysine to proline at amino acid residue 986 of the amino acid sequence found at GenBank Accession No. MN908947.3, and valine to proline at amino acid residue 987 of the amino acid sequence found at GenBank Accession No. MN908947.3.
  • a method for determining if a subject has protection against the development of moderate to severe COVID-19 comprising contacting a recombinant SARS-CoV-2 spike protein with a milk sample from a human subject or a diluted milk sample and detecting the binding of the recombinant SARS-CoV-2 spike protein to anti-SARS-CoV-2 spike protein antibody in the milk sample, wherein the detection of the antibody indicates that the subject has protection against the development of moderate to severe COVID-19.
  • a method for determining if a subject should be vaccinated with a COVID-19 vaccine or a booster of a COVID-19 vaccine comprising contacting a recombinant SARS-CoV- 2 spike protein with a milk sample from a human subject and detecting the binding of the recombinant SARS-CoV-2 spike protein to anti-SARS-CoV-2 spike protein antibody in the milk sample, wherein the detection of the antibody indicates that the subject has protection against the development of moderate to severe COVID-19.
  • a method for determining if a milk sample has use in the prevention or treatment of COVID-19 comprising contacting a recombinant SARS-CoV-2 spike protein with a milk sample from a human subject and detecting the binding of the recombinant SARS-CoV-2 spike protein to anti-SARS-CoV-2 spike protein antibody in the milk sample, wherein the detection of the antibody indicates that the subject has protection against the development of moderate to severe COVID-19.
  • a method for identifying if a vaccine induces an anti-SARS-CoV-2 spike protein antibody profile that may provide protection to a human subject against COVID-19 comprising contacting a recombinant SARS-CoV-2 spike protein with a milk sample from the human subject and detecting the binding of the recombinant SARS-CoV-2 spike protein to anti- SARS-CoV-2 spike protein antibody in the milk sample, wherein the antibody profile indicates whether the vaccine may provide protection to a human subject against COVID-19.
  • the recombinant SARS-CoV-2 spike protein comprises the ectodomain of SARS-CoV-2 spike protein, a C-terminal thrombin cleavage site, and T4 foldon trimerization domain
  • the recombinant soluble SARS-CoV- 2 spike protein does not contain a polybasic cleavage site and includes two stabilizing mutatons of lysine to proline at the amino acid residue corresponding to amino acid residue 986 of the amino acid sequence found at GenBank Accession No. MN908947.3, and valine to proline at the amino acid residue corresponding to amino acid residue 987 of the amino acid sequence found at GenBank Accession No. MN908947.3.
  • a method for preventing COVID-19 comprising administering to a first subject in need thereof a composition comprising immunoglobulin purified from a milk sample of a second subject that tested positive for anti-SARS-CoV-2 spike protein antibody.
  • a method for treating or preventing COVID-19 comprising administering to a first subject in need thereof a milk sample of a second subject that tested positive for anti-SARS- CoV-2 spike protein antibody.
  • composition is administered to the subject orally, intranasally, mucosally, or by pulmonary administration.

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Abstract

L'invention concerne des méthodes de détection d'anticorps dans des échantillons de lait à l'aide de protéines de spicule de SARS-CoV-2 de recombinaison dans un dosage immunologique. L'invention concerne également des méthodes de traitement d'infections par le SARS-CoV-2 ou COVID-19 consistant à administrer une composition comprenant de l'immunoglobuline purifiée à partir d'un échantillon de lait.
PCT/US2021/037256 2020-06-15 2021-06-14 Dosage immunologique pour la détection d'anticorps de protéine de spicule anti-sras-cov-2 dans des échantillons de lait WO2021257469A1 (fr)

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