WO2021257469A1 - Immunoassay for the detection of anti-sars-cov-2 spike protein antibody in milk samples - Google Patents

Immunoassay for the detection of anti-sars-cov-2 spike protein antibody in milk samples 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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French (fr)
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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

Provided herein are methods of detecting antibodies in milk samples using recombinant SARS-CoV-2 spike proteins in an immunoassay. Also provided herein a methods for treating SARS-CoV-2 infections or COVID-19 comprising administering a composition comprising immunoglobulin purified from a milk sample.

Description

IMMUNOASSAY FOR THE DETECTION OF
ANTI-SARS-COV-2 SPIKE PROTEIN ANTIBODY IN MILK SAMPLES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/039,394, filed June 15, 2020, which is incorporated by reference herein in its entirety.
1. INTRODUCTION
[0002] Provided herein are methods of detecting antibodies in milk samples using recombinant SARS-CoV-2 spike proteins in an immunoassay. Also provided herein a methods for treating SARS-CoV-2 infections or COVID-19 comprising administering a composition comprising immunoglobulin purified from a milk sample.
2. BACKGROUND
[0003] In the six months following the first reported case of coronavirus disease 2019 (COVID- 19) in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected over 5.3 million people and caused more than 340,000 deaths worldwide1. Though COVID-19 pathology in children is typically mild compared to adults, approximately 10% of infants under one year of age who contract the virus will experience severe COVID-19 illness requiring advanced care2,3. Given that COVID-19 pathology does not correlate with transmissibility, infants and young children are likely responsible for a significant amount of SARS-CoV-2 dissemination4 ’ 5. As well, recently it has become evident that a minority of children will experience a ‘Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19’ after SARS-CoV-2 infection, which has been fatal in certain cases6,7. For all of these reasons, protecting this population from infection is essential. One potential protective mechanism might be passive immunity via breastfeeding from a previously-infected mother. [0004] To date, almost nothing is known about the antibody response in human milk to SARS- CoV-28. One non-peer-reviewed preprint by Yu and colleagues (2020)9 reported that two milk samples produced by a 32-year-old Chinese mother of a 13 -month-old boy were positive for SARS-CoV-2 IgG and negative for IgM on days 8 and 24 after hospital admission. Additional research is urgently needed to test human milk for SARS-CoV-2 specific antibodies (Abs) and their functions. Knowing the typology and degree of COVID-19 specific Abs in human milk will help inform smart policy and treatment decisions for the many pregnant and breastfeeding mothers who are or will become infected by SARS-CoV-2. Previous study of placental transfer of maternal SARS Abs suggests that transfer of SARS-CoV-2 Abs may not necessarily occur10, indicating that young infants of mothers infected before or during pregnancy may need to rely on milk Abs for protection.
[0005] Certainly, any evidence of SARS-CoV-2 specific Abs in human milk must also be carefully weighed against the currently unknown risks of potential vertical transmission of SARS-CoV-2 through human milk. Limited, weak evidence suggests that some coronaviruses (including SARS-CoV-2) may be present in human milk11 ’ 12, although existing studies are few in number, rely on small samples or case reports, and typically lack validation of reverse transcriptase polymerase chain reaction (RT-PCR) assays for human milk8.
[0006] Despite the dearth of research, there are strong reasons to expect some SARS-CoV-2- specific Abs to be present in the milk of previously infected mothers. Given that milk IgG originates predominantly from serum, it follows that specific IgG in milk should appear contemporaneously with the previously reported serum SARS-CoV-2 Ab response, though IgG comprises only ~2% of milk Ig13. Approximately 90% of human milk Ab is IgA and ~8% IgM, nearly all in secretory (s) form (slgA/sIgM; polymeric Abs (Abs) complexed to j -chain and secretory component (SC) proteins)13-15. The majority of slgA/sIgM derives from the gut- associated lymphoid tissue (GALT), though there is also homing of B cells from other mucosa (i.e. the respiratory system) to the mammary gland.
[0007] There is a need to determine if SARS-CoV-2-specific slgA/sIgM are present in the milk of previously infected mothers.
3. SUMMARY
[0008] In one aspect, provided herein is a method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample using an immunoassay (e.g., ELISA). In one embodiment, provided herein is a method for detecting antibody that specifically binds to SARS- CoV-2 spike protein in a milk sample, comprising: (a) incubating a milk sample diluted in protein buffered solution (e.g., 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS)) in a well coated with a recombinant SARS-CoV-2 spike protein for a first period time;
(b) washing the well; (c) incubating a labeled secondary antibody that binds to an isotype or subtype of immunoglobulin, or secretory component in the well for a second period of time; (d) washing the well; and (e) detecting the binding of the labeled secondary antibody to antibody present in the milk sample bound to the recombinant SARS-CoV-2 spike protein in the well. In a specific embodiment, the recombinant SARS-CoV-2 spike protein is soluble and comprises amino acid residues 319-541 of GenBank Accession No. MN908947.3 and a tag (e.g. , a hexahistidine tag or flag tag). In another specific embodiment, 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 (e.g., a hexahistidine tag or flag tag), wherein the recombinant soluble SARS-CoV-2 spike protein does not contain a polybasic cleavage site (e.g., the polybasic cleavage site (RRAR) is replaced by a single A) 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. In certain embodiments, the recombinant SARS-CoV-2 spike protein is one described in Amanat F, Stadlbauer D, Strohmeier S, Nguyen T, Chromikova V, McMahon M, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv [Internet], 2020 Jan 1;2020.03.17.20037713. The well may be part of a 96 well microtiter plate.
[0009] In another embodiment, provided herein is a method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample, comprising: (a) incubating a milk sample diluted in protein buffered solution (e.g., 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS)) in a well coated with a first recombinant soluble SARS-CoV-2 spike protein for a first period of time, wherein the first recombinant soluble SARS-CoV-2 spike protein comprises amino acid residues corresponding to amino acid residues 319-541 of GenBank Accession No. MN908947.3 and a tag (e.g., a hexahistidine tag or flag tag) and incubating the milk sample diluted in protein buffered solution (e.g., 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS)) in another 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 comprises amino acid residues corresponding to amino acid residues 1-1213 of GenBank Accession No. MN908947.3, a C-terminal cleavage site (e.g., a C-terminal thrombin cleavage site), trimerization domain (e.g., a T4 foldon trimerization domain), and a tag (e.g., a hexahistidine tag or flag tag), and wherein the second recombinant SARS-CoV-2 spike protein does not contain a polybasic cleavage site (e.g., the polybasic cleavage site (RRAR) is replaced by a single A); (b) washing the wells; (c) incubating a labeled secondary antibody that binds to an isotype or subtype of an immunoglobulin, or secretory component in the wells for a second period of time; (d) washing the wells; and (e) detecting the binding of the labeled secondary antibody to antibody present in the milk sample bound to the first and second recombinant SARS-CoV-2 spike proteins. In certain embodiments, the second recombinant SARS-CoV-2 spike protein includes a stabilizing mutation 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, or a stabilizing mutation of 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. In certain embodiments, the first and second recombinant SARS-CoV-2 spike protein are those described in Amanat F, Stadlbauer D, Strohmeier S, Nguyen T, Chromikova V, McMahon M, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv [Internet], 2020 Jan 1;2020.03.17.20037713. The wells may be part of a 96 well microtiter plate.
[0010] In a specific embodiment, provided herein is a method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample, comprising: (a) incubating a milk sample diluted in 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS) in a well coated with a first recombinant soluble SARS-CoV-2 spike protein for a first period of time, wherein the first recombinant soluble SARS-CoV-2 spike protein comprises amino acid residues 319-541 of GenBank Accession No. MN908947.3 and a tag, and incubating the milk sample diluted in 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS) in another 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 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 (e.g., a hexahistidine tag or flag tag), and wherein the second recombinant soluble SARS-CoV-2 spike protein does not contain a polybasic cleavage site (e.g., the polybasic cleavage site (RRAR) is replaced by a single A); (b) washing the wells; (c) incubating a labeled secondary antibody that binds to an isotype or subtype of an immunoglobulin, or secretory component in the wells for a second period of time; (d) washing the wells; and (e) detecting the binding of the labeled secondary antibody to antibody present in the milk sample bound to the first and second recombinant SARS-CoV-2 spike proteins. In certain embodiments, the second recombinant SARS-CoV-2 spike protein includes a stabilizing mutation 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, or a stabilizing mutation of 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. In certain embodiments, the first and second recombinant SARS-CoV-2 spike protein are those described in Amanat F, Stadlbauer D, Strohmeier S, Nguyen T, Chromikova V, McMahon M, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv [Internet], 2020 Jan 1;2020.03.17.20037713. The wells may be part of a 96 well microtiter plate.
[0011] In a specific embodiment, a milk sample is obtained from a lactating human female. In specific embodiments, a milk sample prior to dilution in a protein buffered solution (e.g., 1% BSA/PBS) is centrifuged at 800 g for 15 minutes once, twice or three times to remove fact and cells. In some embodiments, the supernatant is transferred to a new container in between one, two or all of the centrifugations.
[0012] In certain embodiments of the methods for detection of antibody described herein, the labeled secondary antibody is anti-human IgA, anti-human IgM, anti-human IgG or anti-human secretory component. In certain embodiments of the methods for detection of antibody described herein, the labeled secondary antibody is diluted in 1% BSA/PBS prior to use. In some embodiments of the methods for detection of antibody described herein, the secondary antibody is labeled with a chemiluminescent, fluorescent or radioactive moiety. In a specific embodiment of the methods for detection of antibody described herein, secondary antibody is labeled with horseradish peroxidase. In a specific embodiment of the methods for detection of antibody described herein, the secondary antibody is one described in Section 6, infra.
[0013] In some embodiments of the methods for detection of antibody described herein, the well(s) is/are washed with 0.1% Tween 20/PBS (PBS-T). In certain embodiments of the methods for detection of antibody described herein, the first period of time is 1.5 to 2.5 hours. In a specific embodiment of the methods for detection of antibody described herein, the first period of time is 2 hours. In certain embodiments of the methods for detection of antibody described herein, the second period of time is 30 minutes to 1.5 hours. In a specific embodiment of the methods for detection of antibody described herein, the second period of time is 1 hour. In certain embodiments of the methods for detection of antibody described herein, the third period of time is 1.5 to 2.5 hours. In a specific embodiment of the methods for detection of antibody described herein, the third period of time is 2 hours.
[0014] In certain embodiments of the methods for detection of antibody described herein, the secondary antibody is labeled with horseradish peroxidase. In some embodiments of the methods for detection of antibody described herein, the detecting the binding of the labeled secondary antibody comprises adding 3,3',5,5'-Tetramethylbenzidine (TMB) reagent to the well for a fourth period of time followed by adding 2N hydrochloric acid (HC1) to the well and reading the well at 450 nm. In certain embodiments of the methods for detection of antibody described herein, the fourth period of time is 1 to 10 minutes, 2 to 8 minutes or 5 to 10 minutes. [0015] In a specific embodiment, a method for detecting antibody specific for SARS-CoV-2 spike protein comprises the methods described in Section 6, infra.
[0016] In a specific embodiment, provided herein is a method for determining if a subject has protection against the development of moderate to severe COVID-19, the method 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. In another specific embodiment, provided herein is a method for determining if a subject should be vaccinated with a COVID-19 vaccine or a booster of a COVID-19 vaccine, the method 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. In another specific embodiment, provided herein is a method for determining if a milk sample has use in the prevention or treatment of COVID-19, the method 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. The protection against the development of moderate to severe COVID-19 may not be complete but partial (e.g., at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% protective). In another specific embodiment, provided herein is 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, the method 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 protection against COVID-19 may not be complete but may prevent or reduce the development of moderate or severe COVID-19. In one embodiment, the recombinant SARS- CoV-2 spike protein is soluble and comprises the receptor binding domain of SARS-CoV-2 spike protein. In a specific embodiment, the receptor binding domain comprises amino acid residues corresponding to amino acid residues 319-541 of GenBank Accession No.
MN908947.3. In another embodiment, 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, wherein 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. In a specific embodiment, the ectodomain comprises amino acid residues corresponding to amino acid residues 15-1213 of GenBank Accession No.
MN908947.3, and a tag, In another specific embodiment, the polybasic cleavage site (RRAR) is replaced by a single A. In certain embodiments, the recombinant SARS-CoV-2 spike protein further comprises a tag (e.g., a hexahistidine tag).
[0017] In another aspect, provided herein are the use of immunoglobulin purified from a milk sample from an individual(s) (e.g., a human female(s) or other mammalian female(s)) that has anti-SARS-CoV-2 spike protein immunoglobulin to treat a SARS-CoV-2 infection or COVID-19 in a subject (e.g., a human subject). In one embodiment, provided herein is a method for treating a SARS-CoV-2 infection or COVID-19, comprising administering to a subject (e.g., a human) in need thereof a composition comprising immunoglobulin purified from a milk sample of an individual (e.g., human female(s) or other mammalian female(s)) that tested positive for anti- SARS-CoV-2 spike protein antibody. In certain embodiments, the immunoglobulin is IgA, IgM, or IgG. In a specific embodiment, the immunoglobulin is IgA, IgM, or both. In another specific embodiment, the immunoglobulin is secreted IgA, secreted IgM, or both. The composition may be administered by any route to a subject (e.g., a human). For example, the composition may be administered to a subject intranasally, mucosally, or by pulmonary administration.
[0018] In a specific embodiment, provided herein is a method for treating a SARS-CoV-2 infection or 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. In another specific embodiment, provided herein is 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. In one embodiment, the immunoglobulin comprises IgA. In another embodiment, the immunoglobulin comprises secreted IgA. In certain embodiments, the composition is administered to the subject orally, intranasally, mucosally, or by pulmonary administration. In a specific embodiment, the second subject is a human female. In another specific embodiment, the first subject is a human. In another specific embodiment, the first subject is a human infant or human toddler.
[0019] In a specific embodiment, provided herein is 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. In one embodiment, the anti- SARS-CoV-2 spike protein antibody comprises IgA. In another embodiment, the anti-SARS- CoV-2 spike protein antibody comprises secreted IgA. In another embodiment, the anti-SARS- CoV-2 spike protein antibody comprises secreted immunoglobulin (e.g., secreted IgA, secreted IgM or both). In certain embodiments, the milkd is administered to the subject orally. In a specific embodiment, the second subject is a human female. In another specific embodiment, the first subject is a human. In another specific embodiment, the first subject is a human infant or human toddler. 4. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A-1J. Eighty percent of human milk samples collected from COVID- 19- recovered donors exhibits ELISA reactivity against the receptor binding domain of the SARS-CoV-2 spike protein. Plates were coated with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Milk was titrated and added to the plate. Plates were washed and incubated with the appropriate secondary Ab and read on an ELISA plate reader. (FIGS. 1A-1D) Full titrations with positive cutoff values (dotted line) calculated as the mean OD for undiluted negative control milk samples + 2*SD. NEG/ segmented lines: pre-pandemic controls. COV/solid lines: milk from COVID- 19-recovered donors. (FIG. 1A) IgA (FIG. 1B) SC (FIG. 1C) IgM (FIG. 1D) IgG. (FIGS. 1E-1H) Grouped OD values for undiluted milk. (FIG. 1E) IgA (FIG. 1F) SC (FIG. 1G) IgM (FIG. 1H) IgG. Mean with SEM is shown. (FIGS. 1I, 1J) Correlated IgA/secretory Ab (FIG. 1I) and IgG/IgM (FIG. 1 J) reactivity. OD values for undiluted milk were used in duplicate in a 2-tailed Spearman correlation test. SC: secretory component-containing
Abs.
[0021] FIGS. 2A-2B. All human milk samples collected from COVID- 19-recovered donors exhibits ELISA reactivity against the full SARS-CoV-2 spike protein. Plates were coated with spike protein and ELISA was performed as in FIG. 1. (FIG. 2A) Full titration against spike with positive cutoff value (dotted line) calculated as in FIG. 1. (FIG. 2B) Endpoint dilution titers. Dotted lines show positive cutoff, 5x positive cutoff, and lOx positive cutoff values. Mean with SEM is shown.
[0022] FIGS. 3A-3B. All Human Milk Samples Collected from COVID- 19-Recovered Donors Exhibit IgA Reactivity Against the Full SARS-CoV-2 Spike Trimer. (FIG. 3A) Full titration against Spike. NEG (i.e., negative)/ segmented lines: pre-pandemic controls. COV/solid lines: milk from COVID- 19-recovered donors. (FIG. 3B) Endpoint dilution titers. 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). Segmented line indicates 10× positive cutoff value.
[0023] FIGS. 4A-4D. Eighty Percent of Human Milk Samples Collected from COVID-19- Recovered Donors Exhibit IgA and Secretory Antibody Reactivity Against the Receptor- Binding Domain (RBD) of the SARS-CoV-2 Spike. (FIG. 4A) Full titration against RBD, measuring IgA binding. (FIG. 4B) IgA endpoint dilution titers. (FIG. 4C) Full titration against RBD, measuring secretory antibody binding. (FIG. 4D) Secretory antibody endpoint dilution titers. NEG (i.e., negative)/ segmented lines: pre-pandemic controls. 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).
[0024] 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. NEG (i.e., negative)/ segmented lines: pre-pandemic controls. 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).
[0025] FIGS. 6A-6F. Milk from COVID-19-Recovered Donors Exhibits Significantly Greater IgA, Secretory Antibody, and IgG Binding Against RBD Compared with Controls. (FIG. 6A) IgA reactivity of grouped milk samples against the Receptor Binding Domain. (FIG. 6B) Secretory antibody reactivity of grouped milk samples against the Receptor Binding Domain. (FIG. 6C) IgG reactivity of grouped milk samples against the Receptor Binding Domain. (FIG. 6D) IgM reactivity of grouped milk samples against the Receptor Binding Domain. (FIG. 6E) Correlation test of IgA vs. secretory antibody binding against the Receptor Binding Domain. (FIG. 6F) Correlation test of IgG vs. IgM binding against the Receptor Binding Domain.
[0026] FIGS. 7A-7F. Spike-specific milk Ab profile 14 days post-dose 2 of the Pfizer/BioNT ech or Moderna mRNA-based COVID- 19 vaccine. Milk was obtained 1 day before dose 1 (open circles/ segmented lines) and 14 days post-dose 2 (filled circles/solid lines) from 10 participants. Milk was processed, titrated, and tested by ELISA against the full trimeric Spike for specific IgA (FIG. 7A), secretory Ab (SC: secretory chain) (FIG. 7B), and IgG (FIG. 7C). Dotted lines: positive cutoff values previously determined for each assay as the mean OD of negative control milk samples + 2*SD. (FIGS. 7D-7F) Endpoint binding titers were calculated for each experiment. Segmented lines: positive cutoff values; dotted lines: 5× positive endpoint cutoff values, designating samples as ‘high-titer’ . [0027] FIGS. 8A-8C. A robust, Spike-specific IgA response in milk commonly occurs after SARS-CoV-2 infection. (FIG. 8A) Screening of undiluted milk samples for specific IgA by ELISA against the full-length Spike trimer. Mean values with SEM are shown. Dotted line: positive cutoff value (mean OD of negative control milk samples + 2*SD). (FIG. 8B) Full titration against Spike of 40 milk samples found to be positive by the initial screening. (FIG. 8C) Endpoint dilution titers of the 40 titrated milk samples. Segmented line: positive cutoff value; dotted line: 5x positive endpoint cutoff value, designating samples as ‘high-titer’.
[0028] FIGS. 9A-9E. The dominant Spike-specific IgA response in milk after SARS-CoV-2 infection is strongly correlated with a robust secretory Ab response, while specific IgG activity is relatively modest. Twenty samples assayed for Spike-specific IgA were also assessed for Spike-specific secretory Ab (by detecting for SC), and IgG. (FIGS. 5 A, 5B) Full titration against Spike, detecting (FIG. 9A) secretory Ab, and (FIG. 9B) IgG. NEG (i.e. negative)/ segmented lines: pre-pandemic controls. COV/solid lines: milk from COVID-19- recovered donors. Dotted lines: positive cutoff values. (FIGS. 9C, 9D) Endpoint titer values calculated for (FIG. 9A) secretory Ab, and (FIG. 9B) IgG. Segmented lines: positive cutoff values; dotted lines: 5x positive cutoff (high-titer cutoff). (FIG. 9E) IgA and secretory Ab binding OD values or endpoint titers were used in 2-tailed Spearman correlation tests. SC: secretory component.
[0029] FIGS. 10A-10B. The Spike-specific IgA response in milk after SARS-CoV-2 infection is highly durable over time. (FIG. 10A) IgA endpoint titers determined from Spike ELISA for 28 pairs of milk samples obtained from COVID-19-recovered donors 4-6 weeks and 4-10 months after infection are shown. Blue lines indicate mean endpoint values for each group. (FIG. 10B) IgA endpoint titers for a subset of 14 paired samples obtained 4-6 weeks and 7-10 months after infection. Mean with SEM is shown.
[0030] FIGS. 11A-11D. Extracted milk IgA from COVID-19-recovered donors exhibits SARS-CoV-2 Spike-targeted neutralization potency that is highly correlated with IgA binding activity. (FIG. 11 A) Total IgA was purified from milk by conventional means using peptide M agarose. IgA was titrated and tested in a VSV-based SARS-CoV-2 pseudovirus neutralization assay. NEG/segmented lines: pre-pandemic controls. COV/solid lines: COVID-19- recovered milk samples. Segmented line: 50% neutralization cutoff value. (FIG. 11B) Percent neutralization achieved using 50ug/mL of total extracted milk IgA. Mean values with SEM are shown. (FIG. 11C) Neutralization IC50 values determined from IgA titration curves. (FIG. 11D) Endpoint titer values determined in FIG. 4 and IC50 values were used in a 2-tailed Spearman correlation test.
5. DETAILED DESCRIPTION
5.1 Recombinant SARS-CoV-2 Spike Protein
[0031] In one embodiment, 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) known to one of skill in the art, such as the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3, MT049951, MT093631, or MT121215. In another embodiment, 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. MN908947.3, MT049951, MT093631, or MT121215, wherein the protein does not contain the polybasic cleavage site (RRAR). In a specific embodiment, the polybasic/furin cleavage site (RRAR) is replaced by a single A (RRAR to A) at the amino acid residue corresponding to amino acid residue 355 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3. In another specific embodiment, 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. In another embodiment, 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. MN908947.3, MT049951, MT093631, or MT121215, wherein the protein does not contain the polybasic cleavage site ((RRAR) e.g., RRAR is changed to A) and the protein contains 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. MN908947.3, or valine to proline at the amino acid residue corresponding to amino acids residue 987 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3). In another embodiment, 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. MN908947.3, MT049951, MT093631, or MT121215, 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 (lysine to proline and valine to proline at the amino acid residues corresponding to amino acids residues 986 and 987 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3). In some embodiments, a recombinant SARS-CoV-2 spike protein described herein further comprises a transmembrane domain of a SARS-CoV-2 spike protein known to one of skill in the art, such as disclosed at GenBank Accession No. MN908947.3, MT049951, MT093631, or MT121215. In other embodiments, a recombinant SARS-CoV-2 spike protein described herein does not comprise a transmembrane domain of a SARS-CoV-2 spike protein known to one of skill in the art, such as disclosed at GenBank Accession No. MN908947.3, MT049951, MT093631, or MT121215. In certain embodiments, 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)). In some embodiments, such a recombinant spike protein comprises a signal peptide, such as the signal peptide of a SARS-CoV-2 spike protein known to one of skill in the art, such as disclosed at GenBank Accession No. MN908947.3, MT049951, MT093631, or MT121215.
[0032] In another embodiment, 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) of the spike protein disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558. In certain embodments, 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. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558. In some embodiments, 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) disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558 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). In certain embodiments, 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) disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more substitutions (e.g., conservative amino acid substitutions). In specific embodiments, a recombinant SARS-CoV-2 protein retains one, two or more of the functions of the SARS-CoV-2 spike protein known in the art. In some embodiments, a recombinant SARS-CoV-2 spike protein described herein further comprises a transmembrane domain of a SARS-CoV-2 spike protein known to one of skill in the art, such as disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, or a transmembrane that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the transmembrane of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558. In other embodiments, a recombinant SARS-CoV-2 spike protein described herein does not comprise a transmembrane domain. In certain embodiments, a recombinant SARS- CoV-2 spike protein described herein does not comprise a transmembrane domain of a SARS- CoV-2 spike protein known to one of skill in the art, such as disclosed at GenBank Accession No. GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558. In some embodiments, 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. In certain embodiments, 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)). [0033] In another embodiment, 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) of the spike protein disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558. In certain embodments, 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. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558. In some embodiments, 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) disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558 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). In certain embodiments, 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) disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more substitutions (e.g., conservative amino acid substitutions). In specific embodiments, a recombinant SARS-CoV-2 protein retains one, two or more of the functions of the SARS-CoV-2 spike protein known in the art. In specific embodiments, the recombinant SARS-CoV-2 spike protein described herein does not comprise a transmembrane domain. In some embodiments, 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. In certain embodiments, the 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)).
[0034] Techniques known to one of skill in the art can be used to determine the percent identity between two amino acid sequences or between two nucleotide sequences. Generally, to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions/total number of positions X 100%). In one embodiment, the two sequences are the same length. In a certain embodiment, the percent identity is determined over the entire length of an amino acid sequence or nucleotide sequence. In some embodiments, 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). In some embodiments, a fragment of a nucleotide sequence is at least 25, at least 50, at least 75, or at least 100 nucleotides. Similarly, "percent sequence identity" may be readily determined for amino acid sequences, over the full-length of a protein, or a fragment thereof. In some embodiments, a fragment of a protein comprises at least 20, at least 30, at least 40, at least 50 or more contiguous amino acids of the protein. In certain embodiments, a fragment of a protein comprises at least 75, at least 100, at least 125, at least 150 or more contiguous amino acids of the protein.
[0035] The determination of percent identity between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:22642268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25:33893402. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. [0036] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
[0037] A typical SARS-CoV-2 spike protein comprises domains known to those of skill in the art, including an SI domain, a receptor binding domain, an S2 domain, a transmembrane domain and a cytoplasmic domain. See, e.g., Wrapp et al., 2020, Science 367: 1260-1263 for a description of SARS-CoV-2 spike protein (in particular, the structure of such protein). The SARS-CoV-2 spike protein may be characterized has having a signal peptide (e.,g a signal peptide of 1-14 amino acid residues of the amino acid sequence of GenBank Accession No. MN908947.3), a receptor binding domain (e.g., a receptor binding domain of 319-541 amino acid residues of GenBank Accession No. MN908947.3), an ectodomain (e.g., an ectodomain of 15-1213 amino acid residues of GenBank Accession No. MN908947.3), and a transmembrane and endodomain (e.g,. a transmembrane and endodomain of 1214-1273 amino acid residues of GenBank Accession No. MN908947.3).
[0038] In another embodiment, 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. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, wherein the protein does not contain the polybasic cleavage site ((RRAR); e.g., RRAR is changed to A) and the protein contains 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. MN908947.3, or valine to proline at the amino acid residue corresponding to amino acids residue 987 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3). In another embodiment, 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. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, 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 (lysine to proline and valine to proline at the amino acid residues corresponding to amino acids residues 986 and 987 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3).
[0039] In certain embodments, 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. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, wherein the protein does not contain the polybasic cleavage site ((RRAR); e.g., RRAR is changed to A) and the protein contains 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. MN908947.3, or valine to proline at the amino acid residue corresponding to amino acids residue 987 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3). In certain embodments, 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. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, , 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 (lysine to proline and valine to proline at the amino acid residues corresponding to amino acids residues 986 and 987 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3). In specific embodiments, a recombinant SARS-CoV-2 protein retains one, two or more of the functions of the SARS-CoV-2 spike protein known in the art. In some embodiments, a recombinant SARS- CoV-2 spike protein described herein further comprises a transmembrane domain of a SARS- CoV-2 spike protein known to one of skill in the art, such as disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, or a transmembrane that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the transmembrane of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558. In other embodiments, a recombinant SARS-CoV-2 spike protein described herein does not comprise a transmembrane domain. In certain embodiments, a recombinant SARS-CoV-2 spike protein described herein does not comprise a transmembrane domain of a SARS-CoV-2 spike protein known to one of skill in the art, such as disclosed at GenBank Accession No. GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, orMT334558. In certain embodiments, 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)). The C-terminal cleavage site, trimerization domain, and/or tag may be at C-terminus of the ectodomain.
[0040] In certain embodiments, 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. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, wherein the protein does not contain the polybasic cleavage site ((RRAR); e.g., RRAR is changed to A) and the protein contains 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. MN908947.3, or valine to proline at the amino acid residue corresponding to amino acids residue 987 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3). In certain embodiments, 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. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, , 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 (lysine to proline and valine to proline at the amino acid residues corresponding to amino acids residues 986 and 987 of the amino acid sequence of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3). In specific embodiments, a recombinant SARS-CoV-2 protein retains one, two or more of the functions of the SARS-CoV-2 spike protein known in the art. In some embodiments, a recombinant SARS- CoV-2 spike protein described herein does not comprise a transmembrane domain. In certain embodiments, a recombinant SARS-CoV-2 spike protein described herein does not comprise a transmembrane domain of a SARS-CoV-2 spike protein known to one of skill in the art, such as disclosed at GenBank Accession No. GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, orMT334558. In certain embodiments, 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)). The C-terminal cleavage site, trimerization domain, and/or tag may be at C-terminus of the ectodomain.
[0041] In a specific embodiment, 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). In another specific embodiment, 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) and the protein contains two stabilizing mutations (K986P and V987P, wild type numbering). In a specific embodiment, 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.
In some embodiments, 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. [0042] In another specific embodiment, 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 and T4 foldon trimerization domain, wherein the protein does not contain the polybasic cleavage site ((RRAR ); e.g., RRAR is changed to A). In another specific embodiment, 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 and T4 foldon trimerization domain, 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). In a specific embodiment, 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.
[0043] In another specific embodiment, 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). In another specific embodiment, 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). In another specific embodiment, 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.
[0044] In a specific embodiment, 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. In another embodiment, 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. In a specific embodiment, the polybasic/furin cleavage site (RRAR) 355 is replaced by a single A (RRAR to A). In another embodiment, 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). In certain embodiments, 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)). In some embodiments, 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. [0045] In a specific embodiment, 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). In another specific embodiment, 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) and the protein contains two stabilizing mutations (K986P and V987P, wild type numbering). In some embodiments, 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.
[0046] In a specific embodiment, 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. In another embodiment, a recombinant soluble 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, wherein the protein does not contain the polybasic cleavage site. In a specific embodiment, the polybasic/furin cleavage site (RRAR) 355 is replaced by a single A (RRAR to A). In another embodiment, a recombinant soluble 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, 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). In certain embodiments, 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.
[0047] In certain embodments, 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. In some embodments, 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. In certain embodiments, 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. In some embodiments, 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. In certain embodiments, 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)). The C-terminal cleavage site, trimerization domain, and/or tag may be at C-terminus of the fragment of the ectodomain.
[0048] In another embodiment, 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) of the spike protein disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, or a fragment thereof. In certain embodiments, 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) with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the SI 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. In some embodiments, 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). In certain embodiments, 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). In some embodiments, 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. In certain embodiments, 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)). The C-terminal cleavage site, trimerization domain, and/or tag may be at C-terminus of the SI domain or fragment thereof. In certain embodiments, the fragment of the SARS-CoV-2 spike protein SI domain is at least 250, 300, 400, 500, or 750 amino acid residues in length.
[0049] In another embodiment, 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) of the spike protein disclosed at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, or a fragment thereof. In certain embodments, 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. In some embodiments, 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, 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), or a fragment thereof. In certain embodiments, 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). In some embodiments, 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. In certain embodiments, 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)). The C-terminal cleavage site, trimerization domain, and/or tag may be at C- terminus of the S2 domain or fragment thereof. In certain embodiments, 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.
[0050] In a specific embodiment, a recombinant SARS-CoV-2 spike protein described herein comprises the receptor binding domain 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. MN908947.3, MT049951, MT093631, orMT121215. In another embodiment, a recombinant soluble SARS-CoV-2 spike protein comprises (or consists of) the receptor binding domain of a SARS-CoV-2 protein known to one of skill in art (e.g., the SARS-CoV-2 spike protein found at GenBank Accession No. MT049951, MT093631, or MT 121215) corresponding to amino acid residues corresponding to amino acid residues 319-541 of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3. In certain embodiments, 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)). In some embodiments, such a recombinant SARS-CoV-2 spike protein comprises a signal peptide, such as the signal peptide of the spike protein disclosed at GenBank Accession No. MN908947.3, MT049951, MT093631, or MT121215.
[0051] In a specific embodiment, a recombinant SARS-CoV-2 spike protein described herein comprises the receptor binding domain of a SARS-CoV-2 spike protein (otherwise known as the S or structural protein) of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558, or is at least 75%, at least 80%, at least 85%, at least 90%, at least 98% identical to the receptor binding domain of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558. In another embodiment, a recombinant soluble SARS-CoV-2 spike protein comprises (or consists of) the receptor binding domain of a SARS-CoV-2 protein known to one of skill in art (e.g., the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3, MT447160, MT44636, MT446360, MT444593, MT444529, MT370887, or MT334558) corresponding to amino acid residues corresponding to amino acid residues 319-541 of the SARS-CoV-2 spike protein found at GenBank Accession No. MN908947.3. In some embodiments, 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. In certain embodiments, 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.
[0052] In another embodiment, a recombinant soluble SARS-CoV-2 spike protein comprises amino acid residues 319-541 of the spike protein found at GenBank Accession No.
MN908947.3. In certain embodiments, such a recombinant soluble spike protein comprises a tag, such as a histidine tag (e.g., hexahistidine tag) or flag tag. In some embodiments, such a recombinant soluble SARS-CoV-2 spike protein comprises a signal peptide, such as the signal peptide of the spike protein disclosed at GenBank Accession No. MN908947.3. In a specific embodiment, a recombinant soluble SARS-CoV-2 spike protein comprises the signal peptide of the spike protein disclosed at GenBank Accession No. MN908947.3, amino acid residues 319- 541 of the spike protein found at GenBank Accession No. MN908947.3, and a hexahistidine tag. [0053] In a specific embodiment, a recombinant SARS-CoV-2 spike protein is one described in Section 6, infra. In another specific embodiment, a recombinant SARS-CoV-2 spike protein is one described in Amanat F, Stadlbauer D, Strohmeier S, Nguyen T, Chromikova V, McMahon M, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv [Internet], 2020 Jan 1;2020.03.17.20037713. In another specific embodiment, 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.
[0054] In a specific embodiment, provided herein is a recombinant SARS-CoV-2 spike protein described herein maintains the structure of a SARS-CoV-2 spike protein found in nature. In certain embodiments, a SARS-CoV-2 spike protein is not a full length SARS-CoV-2 spike protein found in nature. In specific embodiments, a SARS-CoV-2 spike protein described herein has been altered by man by, e.g., genetic engineering other techniques. In certain embodiments, a SARS-CoV-2 spike protein described herein is monomeric. In certain embodiments, a SARS- CoV-2 spike protein described herein is multimeric. In a specific embodiment, a SARS-CoV-2 spike protein described herein is trimeric. See, e.g., Example 1, infra, for examples of SARS- CoV-2 spike protein described herein. In some embodiments, a recombinant SARS-CoV-2 spike protein described herein retains the ability to bind to the host cell receptor for SARS-CoV-2 (e.g., ACE-2). In a specific embodiment, a recombinant SARS-CoV-2 spike protein described herein is soluble.
[0055] Techniques known to one of skill in the art may be used to recombinantly produced a SARS-CoV-2 spike protein described herein. For example, a recombinant SARS-CoV-2 spike protein described herein may be produced as described in Amanat F, Stadlbauer D, Strohmeier S, Nguyen T, Chromikova V, McMahon M, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv [Internet], 2020 Jan 1;2020.03.17.20037713. The recombinant SARS-CoV-2 spike protein may be expressed in a cell (e.g., a mammalian cell, bacterial cell, insect cell, or plant cell) in cell culture using techniques known in the art. In certain embodiments, a cell (e.g., a mammalian cell, bacterial cell, insect cell, or plant cell) may be transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a SARS-CoV-2 spike protein described herein such that the cell may produce the recombinant SARS-CoV-2 spike protein. In some embodiments, a nucleotide sequence encoding a SARS-CoV-2 spike protein described herein may be present in a vector (e.g., a viral vector or plasmid) and a cell (e.g., a mammalian cell, bacterial cell, insect cell, or plant cell) may be transfected or transformed such that the cell may produce the recombinant SARS-CoV-2 spike protein. The cell may constitutively express the recombinant SARS-CoV-2 spike protein or it may be induced to express the recombinant protein. In specific embodiments, the cell is present in cell culture. In a specific embodiment, a recombinant SARS-CoV-2 spike protein described herein is purified. Any technique known to one of skill in the art may be used to purify the recombinant SARS-CoV-2 spike protein. In a specific embodiment, the terms “purified” and “isolated” when used in the context of a protein that is obtained from cells refers to a protein which is substantially free of contaminating materials, e.g. cell debris, cell wall materials, membranes, organelles, the bulk of the nucleic acids, carbohydrates, proteins, and/or lipids present in cells. Thus, in certain embodiments, a protein that is isolated includes preparations of a protein having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight) of cellular materials and/or contaminating materials. In a specific embodiment, a recombinant a recombinant soluble SARS-CoV-2 spike protein described herein is purified or isolated.
[0056] As an alternative to recombinant expression of a SARS-CoV-2 spike protein described herein using a cell, 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. In a specific embodiment, 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.
5.2 Methods for Detecting Antibody in a Milk Sample
[0057] In one aspect, provided herein is an immunoassay for the detection of anti-SARS-CoV-2 spike protein in a milk sample. In a specific embodiment, the method involves an ELISA described herein. An ELISA may be run in a high-throughput format. The assay may be used to assess the antibody response of a subject or a population of subjects to a SARS-CoV-2 spike protein. In specific embodiments, a recombinant SARS-CoV-2 spike protein described herein can be used to assess the presence of SARS-CoV-2 spike protein receptor binding domain- specific antibodies in a milk sample from subject (e.g., a human) or population of subjects (e.g., humans). In another specific embodiment, 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. For example, the subject(s) may have been vaccinated with Pfizer’s/ BioNTech’s COVID-19 vaccine, Moderna’s COVID-19 vaccine, Johnson & Johnson’s COVID-19 vaccine, AstraZeneca’s COVID-19 vaccine, or another COVID-19 vaccine.
[0058] In a specific embodiment, provided herein is a method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample, comprising: (a) incubating a milk sample diluted in protein buffered solution (e.g., 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS)) in a well coated with a recombinant SARS-CoV-2 spike protein for a first period time; (b) washing the well; (c) incubating a labeled secondary antibody that binds to an isotype or subtype of immunoglobulin, or secretory component (e.g., 50 μl) in the well for a second period of time; (d) washing the well; and (e) detecting the binding of the labeled secondary antibody to antibody present in the milk sample bound to the recombinant SARS-CoV-2 spike protein in the well. In certain embodiments, 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. In some embodiments, the well(s) is/are washed with 0.1% Tween 20/PBS (PBS-T). The washing may occur at room temperature. In certain embodiments, the first period of time is 1.5 to 2.5 hours. In a specific embodiment, the first period of time is 2 hours. In certain embodiments, the second period of time is 30 minutes to 1.5 hours. In a specific embodiment, the second period of time is 1 hour.
In specific embodiments, the method comprises use of a negative control, such as an antibody(ies) that specifically binds to a spike protein of an alphacoronavirus, such as, e.g., an antibody(ies) that specifically binds to a spike protein of NL63, 229E or both, or a betacoronavirus, such as, e.g., an antibody(ies) that specifically binds to a spike protein of OC43, HKU1 or both. In some embodiments, the method comprises use of a positive control, such as an antibody(ies) that specifically binds to the spike protein of SARS-CoV-2 (e.g., antibodies from COVID-19 patients or monoclonal antibodies (mAbs) like CR3022). In certain embodiments, the method comprises use of a negative control and a positive control. In specific embodiments, when a negative and/or positive control is used the method involves the same steps as with the milk sample in different wells. In certain embodiments, the method is run in a high-throughput format so that the detection of antibody(ies) in multiple milk samples may be conducted concurrently. For example, in certain embodiments, 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.
In certain embodiments, each well is coated with 50 μl of 1 μg of a recombinant SARS-CoV-2 spike protein described herein. In some embodiments, each well is coated with 25-50 μl, 25 to 75 μl, 50 to 75 μl, or 50 to 100 μl of 1 μg 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 μ;1 of 0.1% PBS-T). [0059] In some embodiments, provided herein is a method for the detection of antibody that specifically binds to human SARS-CoV-2 spike protein, comprising: (a) incubating a milk sample or a diluted milk sample in a well coated with a first recombinant soluble SARS-CoV-2 spike protein for a first period of time; (b) washing the well; (c) incubating a labeled antibody that binds to an isotype or subtype of an immunoglobulin (e.g., IgG) for a second period of time in the well; (d) washing the well; and (e) detecting the binding of the labeled antibody to the first recombinant SARS-CoV-2 spike protein. In certain embodiment, 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 of the second labeled antibody to the second recombinant SARS-CoV-2 spike protein. In certain embodiments, the first time period, second time period, or both are 30 minutes, 45 minutes, 1 hour, 2 hours, 2.5 hours or 3 hours. In some embodiments, the third and fourth time periods are 30 minutes, 45 minutes, 1 hour, 2 hours, 2.5 hours or 3 hours. In certain embodiments, the second labeled antibody is the same as the first labeled antibody. In certain embodiments, 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. In some embodiments, the well(s) is/are washed with 0.1% Tween 20/PBS (PBS-T). The washing may occur at room temperature.
[0060] The milk sample may be serially diluted, such as described in Section 6, infra. The milk sample may be from an asymptomic subject, a symptomic subject or those suspected of having a SARS-CoV-2 infection or COVID-19. The milk sample may be from a subject that previously tested positive for SARS-CoV-2 infection or was diagnosed with COVID-19. The milk sample may be from a subject that previously exhibits symptoms of COVID-19. The milk sample may be from a subject that has been vaccinated with a COVID-19 vaccine. [0061] In specific embodiments, a milk sample from a subject or population of subjects may be centrifuged at 800 g for 15 minutes once, twice or three times to remove fat and cells prior to use in an immunoassay described herein. In some embodiments, supernatant from the milk sample is transferred to a new container in between one, two or all of the centrifugations. In certain embodiments, the milk sample is diluted in a protein buffered solution (e.g., 1% bovine serum albumin/phosphate buffered saline). In a specific embodiment, a milk sample from a subject or population of subjects is processed as described in Section 6, infra, prior to use in an immunoassay described herein. A milk sample may be obtained from a human female or other mammalian female. The milk sample may be obtained from a female that is lactating and that has tested positive for anti-SARS-CoV-2 spike protein antibody. The milk sample obtained from a female may be tested using an immunoassay, such as described herein, for anti-SARS-CoV-2 spike protein antibody. The milk sample may be obtained from a female that is lactating and that has tested positive for SARS-CoV-2 (e.g., positive using an RT-PCR assay or other assay, such as an antigen assay). A milk sample may be stored a refrigerator or at -20° C or -80° C before being processed or after processing to remove cells and fat. In a specific embodiment, a milk sample is processed as described in Section 6, infra.
[0062] In certain embodiments, the labeled secondary antibody is anti-human IgA, anti-human IgM, anti-human IgG or anti-human secretory component. In certain embodiments, the labeled secondary antibody is diluted in 1% BS A/PBS prior to use. In some embodiments, the secondary antibody is labeled with a chemiluminescent, fluorescent or radioactive moiety. In a specific embodiment, secondary antibody is labeled with horseradish perioxidase. In a specific embodiment, the secondary antibody is one described in Section 6, infra. In a specific embodiment, 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.
[0063] In certain embodiments, a recombinant soluble SARS-CoV-2 spike protein described herein is immobilized (e.g., coated) on a solid support and the binding of antibody in a sample (e.g., a biological sample) to the recombinant soluble SARS-CoV-2 spike protein is detected. Solid supports include silica gels, resins, derivatized plastic films, glass surfaces, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene, beads (e.g., glass beads, plastic beads, magnetic beads, or polystyrene beads), or alumina gels. In some embodiments, 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.
[0064] In a specific embodiment, a method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample comprises the steps or similar steps as described in Section 6, infra. In another specific embodiment, a method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample comprises the steps or similar steps as described in Example 2, 3, or 4, infra.
[0065] In some embodiments, a microneutralization assay is conducted with a milk sample for which an immunoassay described herein is conducted. Microneutralization assays known in the art or described herein (see, e.g., Example 4). In one example, cells (e.g., Vero E6 cells; e.g., 20,000 cells) are seeded per well overnight in a 96-well cell culture plate. Serial dilutions (e.g.,
3 -fold serial dilutions are prepared in a 96-well cell culture plate and each dilution is mixed with X times (e.g., 600 times) the 50% tissue culture infectious dose (TCID50) of SARS-CoV-2 (e.g., USA-WA1/2020, BEI Resources NR-52281). Milk sample- virus mixture is incubated for lh at room temperature. Milk sample-virus mixture is added to the cells for a certain period of time (e.g., lh) and kept in a 37°C incubator. Next, the milk sample- virus mixture is removed and the corresponding milk sample dilution is added to the cells with addition 1X MEM. The cells are incubated for a certain period of time (e.g. 2 days) and fixed with formaldehyde (e.g., 100 μL 10% formaldehyde) per well for a certain period of time (e.g., 24 h). The formaldehyde is carefully removed from the cells. Cells are washed (e.g., washed with 200 μL PBS) once before being permeabilized (e.g., permabilizing with PBS containing 0.1% Triton X-100 for 15 min at RT). Cells are washed (e.g., washed with PBS) and blocked (e.g., blocked in PBS containing 3% dry milk) for a certain period of time (e.g., lh) at RT. Cells are then stained with a a mouse monoclonal anti-NP antibody (e.g., 100 μL per well of a mouse monoclonal anti-NP antibody (1C7) at lμg/ml) for a certain period of time (e.g., lh) at RT. Cells are washed (e.g., washed with PBS) and incubated with labeled anti-mouse IgG (e.g,. 100 μL per well anti -mouse IgG HRP (Rockland, cat. no. 610-4302) secondary antibody at 1:3,000 dilution in PBS containing 1% dry milk) for a certain period of time (e.g., lh) at RT. Finally, cells are washed twice (e.g., washed twice with PBS) and the plates are developed (e.g., developed using 100 μL of SigmaFast OPD substrate). A certain period of time later (e.g., ten minutes later, the reactions are stopped (e.g., stopped using 50 μL 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.
5.3 Antibody Profile
[0066] In certain embodiments, the detection of anti-SARS-CoV-2 spike protein antibody(ies) (e.g., IgA, IgG, IgM, etc.) 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. In specific embodiments, 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. In some embodiments, 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. In some embodiments, 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 one, two or more symptoms of moderate or severe COVID-19. In some embodiments, the detection a particular isotype (e.g., IgA) or subtype in a milk sample from a subject (e.g., human) indicates that the subject may have some degree of protection against COVID-19, or be fully protected from developing one, two or more symptoms of moderate or severe COVID-19. In certain embodiments, the antibodies from the milk sample or a particular isotype or subtype are assessed for microneutralization. In a specific embodiment, 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. In another specific embodiment, 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. In another specific embodiment, 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. In another specific embodiment, the detection of secretory 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. In another specific embodiment, the detection of secretory 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.
[0067] In some embodiments, anti-SARS-CoV-2 spike protein antibody in a milk sample from a subject pre-infection is compared to anti-SARS-CoV-2 spike protein antibody in a milk sample from the same subject post-infection with SARS-CoV-2. In a specific embodiment, the concentration (e.g., geometric mean titer or geometric mean concentration) anti-SARS-CoV-2 spike protein antibody is higher (e.g., 10%, 20%, 30%, 40%, 50% or higher) in a milk sample from the subject post-infection with SARS-CoV-2 than the concentration (e.g., geometric mean titer) anti-SARS-CoV-2 spike protein antibody pre-infection with SARS-CoV-2. In another specific embodiment, the concentration (e.g., geometric mean titer) a particular isotype (e.g., IgA) or secreted Ig (e.g., secreted IgA) anti-SARS-CoV-2 spike protein antibody is higher (e.g., 10%, 20%, 30%, 40%, 50% or higher) in a milk sample from the subject post-infection with SARS-CoV-2 than the concentration (e.g., geometric mean titer or geometric mean concentration) the isotype or secreted Ig anti-SARS-CoV-2 spike protein antibody pre-infection with SARS-CoV-2.
[0068] In certain embodiments, the detection of anti-SARS-CoV-2 spike protein antibody(ies) (e.g., IgA, IgG, IgM, etc.) 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 should be vaccinated with a COVID-19 vaccine or receive a booster of a COVID-19 vaccine. In specific embodiments, 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 should be vaccinated with a COVID-19 vaccine or receive a booster of a COVID-19 vaccine. In certain embodiments, the antibodies from the milk sample or a particular isotype or subtype are assessed for microneutralization. In another specific embodiment, the detection of IgA anti- SARS-CoV-2 spike protein antibody in a milk sample from a subject indicates that the subject may have some degree of protection against COVID-19 and may not need to be vaccinated with a COVID-19 vaccine or receive a booster of a COVID-19 vaccine. In another specific embodiment, 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 may have some degree of protection against COVID-19 and may not need to be vaccinated with a COVID-19 vaccine or receive a booster of a COVID-19 vaccine. In another specific embodiment, the detection of secretory IgA anti-SARS-CoV-2 spike protein antibody in a milk sample from a subject indicates that the subject may have some degree of protection against COVID-19 and may not need to be vaccinated with a COVID-19 vaccine or receive a booster of a COVID-19 vaccine. In another specific embodiment, the detection of secretory IgA anti- SARS-CoV-2 spike protein antibody in a milk sample from a subject, which IgA exhibits microneutralization activity, indicates that the subject may have some degree of protection against COVID-19 and may not need to be vaccinated with a COVID-19 vaccine or a booster of a COVID-19 vaccine.
[0069] In certain embodiments, the lack of detection of anti-SARS-CoV-2 spike protein antibody(ies) (e.g., IgA, IgG, IgM, etc.) or detection of anti-SARS-CoV-2 spike protein antibody(ies) below a particular threshold level in a milk sample from a subject (e.g., human) using, e.g., an immunoassay, such as described herein, indicates the subject should be vaccinated with a COVID-19 vaccine or receive a booster of a COVID-19 vaccine. In specific embodiments, the lack of detection of a particular isotype (e.g., IgA) or subtype, or 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, indicates the subject should be vaccinated with a COVID-19 vaccine or receive a booster of a COVID-19 vaccine. In certain embodiments, the antibodies from the milk sample or a particular isotype or subtype are assessed for microneutralization. In another specific embodiment, the lack of detection of secretory IgA anti- SARS-CoV-2 spike protein antibody with microneutralization activity, or detection of secretory IgA anti-SARS-CoV-2 spike protein antibody with microneutralization activity below a particular threshold level in a milk sample from a subject, indicates that the subject may need to be vaccinated with a COVID-19 vaccine or receive a booster of a COVID-19 vaccine.
[0070] In some embodiments, anti-SARS-CoV-2 spike protein antibody in a milk sample prevaccination with a COVID-19 vaccine is compared to anti-SARS-CoV-2 spike protein antibody in a milk sample post-vaccination with a COVID-19 vaccine. In a specific embodiment, the concentration (e.g., geometric mean titer or geometric mean concentration) anti-SARS-CoV-2 spike protein antibody is higher (e.g., 10%, 20%, 30%, 40%, 50% or higher) in a milk sample from the subject post-vaccination with a COVID-19 vaccine than the concentration (e.g., geometric mean titer or geometric mean concentration) anti-SARS-CoV-2 spike protein antibody pre-vaccination with a COVID-19 vaccine. In another specific embodiment, the concentration (e.g., geometric mean titer) a particular isotype (e.g., IgA) or secreted Ig (e.g., secreted IgA) anti- SARS-CoV-2 spike protein antibody is higher (e.g., 10%, 20%, 30%, 40%, 50% or higher) in a milk sample from the subject post-vaccination with a COVID-19 vaccine than the concentration (e.g., geometric mean titer or geometric mean concentration) the isotype or secreted Ig anti- SARS-CoV-2 spike protein antibody pre-vaccination with a COVID-19 vaccine.
[0071] In certain embodiments, the detection of anti-SARS-CoV-2 spike protein antibody(ies) (e.g., IgA, IgG, IgM, etc.) in a milk sample using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine if a subject (e.g., human infant) receiving the milk sample may have some degree of protection against the development of COVID-19. In specific embodiments, the detection of a particular isotype (e.g., IgA) or subtype in a milk sample using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine if a subject (e.g., human infant) receiving the milk sample may have some degree of protection against the development of COVID-19. In some embodiments, the detection a particular isotype (e.g., IgA) or subtype in a milk sample using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine if a subject (e.g., human infant) receiving the milk sample may be fully protected from developing moderate to severe COVID-19. In some embodiments, the detection a particular isotype (e.g., IgA) or subtype in a milk sample using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine if a subject (e.g., human infant) receiving the milk sample may be fully protected from developing one, two or more symptoms of moderate or severe COVID-19.
[0072] In certain embodiments, the detection of anti-SARS-CoV-2 spike protein antibody(ies) (e.g., IgA, IgG, IgM, etc.) in a milk sample using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine indicates that a subject (e.g., human infant) receiving the milk sample may have some degree of protection against the development of COVID-19. In specific embodiments, the detection of a particular isotype (e.g., IgA) or subtype in a milk sample using, e.g., an immunoassay, such as described herein, indicates that a subject (e.g., human infant) receiving the milk sample may have some degree of protection against the development of COVID-19. In some embodiments, the detection a particular isotype (e.g., IgA) or subtype in a milk sample using, e.g., an immunoassay, such as described herein, indicates that a subject (e.g., human infant) receiving the milk sample may be fully protected from developing moderate to severe COVID-19. In some embodiments, the detection a particular isotype (e.g., IgA) or subtype in a milk sample using, e.g., an immunoassay, such as described herein, indicates that a subject (e.g., human infant) receiving the milk sample may be fully protected from developing one, two or more symptoms of moderate or severe COVID-19.
[0073] In certain embodiments, the detection of anti-SARS-CoV-2 spike protein antibody(ies) (e.g., IgA, IgG, IgM, etc.) in a milk sample using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine if the milk sample may be used to treat or prevent COVID-19, or antibody isolated from the milk may be used to treat or prevent COVID-19. In specific embodiments, the detection of a particular isotype (e.g., IgA) or subtype in a milk sample using, e.g., an immunoassay, such as described herein, allows a medical professional (e.g., a physician) to determine if a subject (e.g., human infant) receiving the milk sample be used to treat or prevent COVID-19, or antibody isolated from the milk may be used to treat or prevent COVID-19.
[0074] In certain embodiments, the detection of anti-SARS-CoV-2 spike protein antibody(ies) (e.g., IgA, IgG, IgM, etc.) in a milk sample using, e.g., an immunoassay, such as described herein, indicates that the milk sample may be used to treat or prevent COVID-19, or antibody isolated from the milk may be used to treat or prevent COVID-19. In specific embodiments, the detection of a particular isotype (e.g., IgA) or subtype in a milk sample using, e.g., an immunoassay, such as described herein, indicates that a subject (e.g., human infant) receiving the milk sample be used to treat or prevent COVID-19, or antibody isolated from the milk may be used to treat or prevent COVID-19.
[0075] In some embodiments, some degree of protection against the development of COVID-19 means that a subject is partially (e.g., at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%) protected from developing COVID-19. In certain embodiments, some degree of protection against the development of COVID-19 means that a subject is partially (e.g., at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%) protected from developing moderate to severe COVID-19. In some embodiments, some degree of protection against the development of COVID-19 means that a subject is partially (e.g., at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%) protected from developing moderate COVID-19. In some embodiments, some degree of protection against the development of COVID-19 means that a subject is partially (e.g., at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%) protected from developing severe COVID-19. In certain embodiments, some degree of protection against the development of COVID-19 means that a subject is partially (e.g., at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%) protected from developing one, two more symptoms of COVID-19. In some embodiments, some degree of protection against COVID-19 means that a subject is partially (e.g., at least 50%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%) protected from developing one, two, or more symptoms of moderate or severe COVID-19.
[0076] In specific embodiments, the detection of a subject’s (e.g., human subject’s) or a population of subject’s (e.g., a population of human subject’s) of anti-SARS-CoV-2 spike protein antibody response following vaccination with a COVID-19 vaccine, which includes a SARS-CoV-2 spike protein, may be used to identify if the vaccine induces an anti-SARS-CoV-2 spike protein antibody profile that may provide some degree of protection to a subject (e.g., a human infant) receiving a milk sample from a subject vaccinated with the vaccine. The antibody profile may include the relative percentage of certain isotypes of antibodies (e.g., IgG, IgA, IgM, etc), the relative percentage of antibodies that are the secretory type, or a combination thereof.
In certain embodiments, a COVID-19 vaccine may be selected that results in a higher percentage of IgA anti-SARS-CoV-2 spike protein antibody than other anti-SARS-CoV-2 spike protein antibody isotypes. In a specific embodiment, a COVID-19 vaccine may be selected that results in a higher percentage of IgA anti-SARS-CoV-2 spike protein antibody than other anti-SARS- CoV-2 spike protein antibody isotypes. In certain embodiments, the antibodies induced following vaccination with a vaccine are assessed for microneutralization. In a specific embodiment, a COVID-19 vacccine may be selected that results in a higher percentage of IgA anti-SARS-CoV-2 spike protein antibody than other anti-SARS-CoV-2 spike protein antibody isotypes, and the IgA antibody exhibits microneutralization activity. The higher percentage of IgA anti-SARS-CoV-2 spike protein antibody may be 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the amount of other anti-SARS-CoV-2 spike protein antibody isotypes. The higher percentage of IgA anti-SARS-CoV-2 spike protein antibody may be 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the amount of any other anti-SARS-CoV-2 spike protein antibody isotype. In another specific embodiment, a COVID-19 vacccine may be selected that results in a higher percentage of slgA anti-SARS-CoV-2 spike protein antibody than other anti-SARS-CoV- 2 spike protein antibody isotypes, and the slgA antibody exhibits microneutralization activity. T In another specific embodiment, a COVID-19 vaccine may be selected that results in a higher percentage of secretory IgA (slgA) anti-SARS-CoV-2 spike protein antibody than other anti- SARS-CoV-2 spike protein antibody isotypes. The higher percentage of slgA anti-SARS-CoV-2 spike protein antibody may be 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the amount of other anti-SARS-CoV-2 spike protein antibody isotypes. The higher percentage of slgA anti- SARS-CoV-2 spike protein antibody may be 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the amount of any other anti-SARS-CoV-2 spike protein antibody isotype. The COVID-19 vaccine may be Pfizer’s/ BioNTech’s COVID-19 vaccine, Moderna’s COVID-19 vaccine, Johnson & Johnson’s COVID-19 vaccine, AstraZeneca’s COVID-19 vaccine, or another COVID-19 vaccine. The antibody profile may be obtained following 1, 2 or more, or following all administrations of a COVID-19 vaccine.
[0077] In specific embodiments, different groups of subjects are vaccinated with a COVID-19 vaccine, which includes a SARS-CoV-2 spike protein, wherein each group of subjects is vaccinated with a different COVID-19 vaccine, and the detection of the anti-SARS-CoV-2 spike protein antibody response following vaccination in a milk sample from the subjects may be used to identify if a particular vaccine(s) induces an anti-SARS-CoV-2 spike protein antibody profile that may provide some degree of protection to a subject (e.g., human infant) receiving a milk sample from a subject vaccinated with the vaccine(s). The antibody profile may include the relative percentage of a certain isotype(s) of anti-SARS-CoV-2 spike protein antibodies (e.g., IgG, IgA, IgM, etc), the relative percentage of anti-SARS-CoV-2 spike protein antibodies that are the secretory type, or a combination thereof In certain embodiments, a COVID-19 vaccine may be selected that results in a higher percentage of IgA anti-SARS-CoV-2 spike protein antibody than other anti-SARS-CoV-2 spike protein antibody isotypes. In a specific embodiment, a COVID-19 vaccine may be selected that results in a higher percentage of IgA anti-SARS-CoV-2 spike protein antibody than other anti-SARS-CoV-2 spike protein antibody isotypes. In certain embodiments, the antibodies induced following vaccination with a vaccine are assessed for microneutralization. In a specific embodiment, a COVID-19 vacccine may be selected that results in a higher percentage of IgA anti-SARS-CoV-2 spike protein antibody than other anti-SARS-CoV-2 spike protein antibody isotypes, and the IgA antibody exhibits microneutralization activity. The higher percentage of IgA anti-SARS-CoV-2 spike protein antibody may be 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the amount of other anti- SARS-CoV-2 spike protein antibody isotypes. The higher percentage of IgA anti-SARS-CoV-2 spike protein antibody may be 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the amount of any other anti-SARS-CoV-2 spike protein antibody isotype. In another specific embodiment, a COVID-19 vacccine may be selected that results in a higher percentage of slgA anti-SARS- CoV-2 spike protein antibody than other anti-SARS-CoV-2 spike protein antibody isotypes, and the slgA antibody exhibits microneutralization activity. In another specific embodiment, a COVID-19 vaccine may be selected that results in a higher percentage of secretory IgA (slgA) anti-SARS-CoV-2 spike protein antibody than other anti-SARS-CoV-2 spike protein antibody isotypes. The higher percentage of slgA anti-SARS-CoV-2 spike protein antibody may be 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the amount of other anti-SARS-CoV-2 spike protein antibody isotypes. The higher percentage of slgA anti-SARS-CoV-2 spike protein antibody may be 10%, 15%, 20%, 25%, 30%, 35% or 40% higher than the amount of any other anti-SARS-CoV-2 spike protein antibody isotype. The COVID-19 vaccines may include Pfizer’s/ BioNTech’s COVID-19 vaccine, Moderna’s COVID-19 vaccine, Johnson & Johnson’s COVID-19 vaccine, AstraZeneca’s COVID-19 vaccine, and other COVID-19 vaccines. The antibody response may be determined following 1, 2 or more, or following all administrations of a COVID-19 vaccine. [0078] In certain embodiments, anti-SARS-CoV-2 spike protein antibody in milk samples from a subject (e.g., human) is monitored over time to assess the degree of protection the subject may have against COVID-19. In some embodiments, the subject may receive a COVID-19 vaccine or a booster of COVID-19 vaccine if anti-SARS-CoV-2 spike protein in milk samples from the subject decrease below a certain threshold. In certain embodiments, anti-SARS-CoV-2 spike protein antibody in milk samples from a subject (e.g., human) is monitored over time to assess the degree of protection a subject (e.g., human infant) receiving the milk may have against COVID-19. In specific embodiments, anti-SARS-CoV-2 spike protein antibody profile in milk samples from a subject (e.g., human) is monitored over time to assess the degree of protection the subject may have against COVID-19. In some embodiments, the subject may receive a COVID-19 vaccine or a booster of COVID-19 vaccine if isotypes or subtypes of anti-SARS- CoV-2 spike protein in milk samples from the subject decrease below a certain threshold.
[0079] In specific embodiments, anti-SARS-CoV-2 spike protein antibody profile in milk samples from a subject (e.g., human) is monitored over time to assess the degree of protection a subject (e.g., human infant) receiving the milk may have against COVID-19. In some embodiments, the subject from which the milks samples are obtained may receive a COVID-19 vaccine or a booster of COVID-19 vaccine if anti-SARS-CoV-2 spike protein in milk samples from the subject decrease below a certain threshold. In specific embodiments, anti-SARS-CoV-2 spike protein antibody profile in milk samples from a subject (e.g., human) is monitored over time to assess the degree of protection a subject (e.g., human infant) receiving the milk may have against COVID-19. In some embodiments, the subject from which the milk samples are obtained may receive a COVID-19 vaccine or a booster of COVID-19 vaccine if an isotype(s) or subtype(s) of anti-SARS-CoV-2 spike protein in milk samples from the subject decrease below a certain threshold.
[0080] In certain embodiments, an anti-SARS-CoV-2 spike protein antibody profile, such as described in Example 1, 2, 3 or 4, infra, in a milk sample from a subject (e.g., human) indicates that the subject has some degree of protection against COVID-19. In some embodiments, an anti-SARS-CoV-2 spike protein antibody profile, such as described in Example 1, 2, 3 or 4, infra, in a milk sample from a first subject (e.g., human) indicates that a second subject (e.g., human infant) receiving milk from the first subject has some degree of protection against COVID-19. In certain embodiments, an anti-SARS-CoV-2 spike protein antibody profile, such as described in Example 1, 2, 3 or , infra, in a milk sample from a subject (e.g., human) indicates that milk from the subject may be used as to treat or prevent COVID-19 or one or more symptoms thereof In specific embodiments, an anti-SARS-CoV-2 spike protein antibody profile, such as described in Example 1, 2, 3 or 4, infra, in a milk sample from a subject (e.g., human) indicates that antibody (e.g., IgA, IgG, IgM, slgA, etc.) present in the milk may be used as to treat or prevent COVID-19 or one or more symptoms thereof. The antibody or a particular isotype or subclass may be isolated from the milk using techniques known to one of skill in the art or described herein (see, e.g., Example 4).
[0081] In specific embodiments, a subject from which a milk sample is obtained is a human female. In other embodiments, a subject from which a milk sample is obtained is a non-human female, e.g., a non-human mammalian female. In certain embodiments, a subject from which a milk sample is obtained is a non-human animal, e.g., a non-human mammalian female, that is capable of expressing human immunoglobulin. In certain embodiments, a subject from which a milk sample is obtained is a non-human animal, e.g., a non-human mammalian female, that is capable of expressing immunoglobulin of another species. In specific embodiments, a subject from which a milk sample is obtained is a lactacting subject (e.g., human or non-human animal). [0082] The subject from which a milk sample is obtained may be an asymptomic subject, a symptomic subject or those suspected of having a SARS-CoV-2 infection or COVID-19. The subject from which a milk sample is obtained may be a subject that previously tested positive for SARS-CoV-2 infection or was diagnosed with COVID-19. The subject from which a milk sample is obtained may be a subject that previously exhibits symptoms of COVID-19. The subject from which a milk sample is obtained may be a subject that has been vaccinated with a COVID-19 vaccine. In certain embodiments, the subject from which a milk sample is obtained was vaccinated during pregnancy. In some embodiments, the subject from which a milk sample is obtained was vaccinated while lactating. In certain embodiments, the subject from which a milk sample is obtained was vaccinated during pregnancy and after giving birth to an infant (e.g., a human infant).
[0083] In certain embodiments, anti-SARS-CoV-2 spike protein antibody may bind to the ectodomain of the spike protein, the receptor binding domain of the spike protein, or both. In certain embodiments, anti-SARS-CoV-2 spike protein antibody comprises IgA, IgG, or IgM. In some embodiments, anti-SARS-CoV-2 spike protein antibody comprises IgA, IgG, and IgM. In certain embodiments, anti-SARS-CoV-2 spike protein antibody comprises secreted immunoglobulin (e.g., secreted IgA or secreted IgM). In specific embodiments, anti-SARS- CoV-2 spike protein antibody comprises IgA (e.g., secreted IgA). In specific embodiments, anti- SARS-CoV-2 spike protein antibody exhibits microneutralization activity.
5.4 Kits
[0084] In another aspect, provided herein is a kit comprising one or more containers filled with one or more of the ingredients of an ELISA described herein, such as one or more recombinant SARS-CoV-2 spike proteins described herein. In a specific embodiment, the spike protein is a soluble protein described herein. Optionally associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of such kits, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[0085] The kits encompassed herein can be used in accordance with the methods described herein. In one embodiment, a kit comprises a recombinant SARS-CoV-2 spike protein described herein in one or more containers. In a specific embodiment, provided herein are kits comprising a recombinant SARS-CoV-2 spike protein described herein and instructions for using the recombinant SARS-CoV-2 spike protein to assess the antibodies present in a milk sample. In another specific embodiment, provided herein are kits comprising a microtiter plate with wells coated with a recombinant SARS-CoV-2 spike protein described herein for use in detecting SARS-CoV-2 spike protein-specific antibodies in a milk sample. In certain embodiments, a kit may further comprise a negative control, such as an antibody(ies) that specifically binds to a spike protein of an alphacoronavirus, such as, e.g., an antibody(ies) that specifically binds to a spike protein of NL63, 229E or both, or a betacoronavirus, such as, e.g., an antibody(ies) that specifically binds to a spike protein of OC43, HKU1 or both. In some embodiments, a kit may comprise a positive control, such as an antibody(ies) that specifically binds to the spike protein of SARS-CoV-2 (e.g., antibodies from COVID-19 patients or monoclonal antibodies (mAbs) like CR3022). In certain embodiments, a kit may comprise a negative control (such as an antibody(ies) that specifically binds to a spike protein of an alphacoronavirus, such as, e.g., an antibody(ies) that specifically binds to a spike protein of NL63, 229E or both, or a betacoronavirus, such as, e.g., an antibody(ies) that specifically binds to a spike protein of OC43, HKU1 or both) and a positive control (such as an antibody(ies) that specifically binds to the spike protein of SARS-CoV-2, e.g., antibodies from COVID-19 patients or monoclonal antibodies (mAbs) like CR3022). In some embodiments, a kit may comprise one, two or more, or all of the components of the ELISA described in Example 1, infra. In certain embodiments, a kit may comprise one, two or more, or all of the components of the ELISA described in Example 2, 3 or 4, infra.
[0086] In certain embodiments, a kit may comprise a milk sample(s), in one or more containers, from a subject (e.g., human) in which anti-SARS-CoV-2 spike protein antibody has been detected. In some embodiments, a kit may comprise a milk sample(s), in one or more containers, from a subject (e.g., human) in which IgA anti-SARS-CoV-2 spike protein antibody has been detected. In certain embodiments, a kit may comprise a milk sample(s), in one or more containers, from a subject (e.g., human) in which slgA anti-SARS-CoV-2 spike protein antibody has been detected. In certain embodiments, a kit may comprise a milk sample(s), in one or more containers, from a subject (e.g., human) in which secretory anti-SARS-CoV-2 spike protein antibody has been detected.
[0087] In certain embodiments, a kit may comprise anti-SARS-CoV-2 spike protein antibody isolated from a milk sample(s), in one or more containers, wherein the anti-SARS-CoV-2 spike protein antibody has been detected. In some embodiments, a kit may comprise IgA anti-SARS- CoV-2 spike protein antibody isolated from a milk sample(s), in one or more containers, wherein the anti-SARS-CoV-2 spike protein antibody has been detected. In certain embodiments, a kit may comprise slgA anti-SARS-CoV-2 spike protein antibody isolated from a milk sample(s), in one or more containers, wherein the anti-SARS-CoV-2 spike protein antibody has been detected. In some embodiments, a kit may comprise secretory anti-SARS-CoV-2 spike protein antibody isolated from a milk sample(s), in one or more containers, wherein the anti-SARS-CoV-2 spike protein antibody has been detected.
5.5 Methods for Treating COVID-19
[0088] In another aspect, provided herein are the use of immunoglobulin purified from a milk sample from an individual(s) (e.g., a human female(s) or other mammalian female(s)) that has anti-SARS-CoV-2 spike protein immunoglobulin to treat a SARS-CoV-2 infection or COVID-19 in a subject (e.g., a human subject). Immunoglobulin from a milk sample may be administered to a subject in a composition. The composition may contain a pharmaceutically acceptable carrier. In one embodiment, provided herein is a method for treating a SARS-CoV-2 infection or COVID-19, comprising administering to a subject (e.g., a human) in need thereof a composition comprising immunoglobulin purified from a milk sample of an individual (e.g., human female(s) or other mammalian female(s)) that tested positive for anti-SARS-CoV-2 spike protein antibody. In another embodiment, provided herein is a method for treating a SARS-CoV-2 infection or COVID-19, comprising administering to a subject (e.g., a human) in need thereof a composition comprising an effective amount of immunoglobulin purified from a milk sample of an individual (e.g., human female(s) or other mammalian female(s)) that tested positive for anti-SARS-CoV-2 spike protein antibody. Techniques known in the art may be used to purify anti-SARS-CoV-2 spike protein antibody from a milk sample. In certain embodiments, the immunoglobulin is IgA, IgM, or IgG. In a specific embodiment, the immunoglobulin is IgA. In another specific embodiment, the immunoglobulin is secreted IgA.
[0089] In another embodiment, provided herein is a method for treating a SARS-CoV-2 infection or COVID-19, comprising administering to a first subject (e.g., a human) in need thereof a composition comprising an effective amount of immunoglobulin purified from a milk sample of a second subject (e.g., human female(s) or other mammalian female(s)) that tested positive for anti-SARS-CoV-2 spike protein antibody. In another embodiment, provided herein is a method for preventing COVID-19, comprising administering to a first subject (e.g., a human) in need thereof a composition comprising an effective amount of immunoglobulin purified from a milk sample of a second subject (e.g., human female(s) or other mammalian female(s)) that tested positive for anti-SARS-CoV-2 spike protein antibody. See, e.g., Section 5.3 and the Examples for subject from which a milk sample may be obtained. Techniques known in the art may be used to purify anti-SARS-CoV-2 spike protein antibody from a milk sample. In certain embodiments, the immunoglobulin comprises IgA, IgM, or IgG. In certain embodiments, the immunoglobulin comprises a combination of IgA, IgM, and IgG. In a specific embodiment, the immunoglobulin comprises IgA. In another specific embodiment, the immunoglobulin comprises secreted IgA. In another embodiment, the immunoglobulin comprises secreted immunoglobulin (e.g., secreted IgA, secreted IgM or both).
[0090] A “pharmaceutically acceptable” generally means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. In as specific embodiment, the “carried”may be a diluent, adjuvant, excipient, or vehicle with which a composition (e.g., a pharmaceutical composition) is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E.W. Martin. The formulation of a composition (e.g., pharmaceutical composition) should suit the mode of administration.
[0091] In a particular embodiment, the administration of an effective amount of immunoglobulin to the subject inhibits or reduces in the progression of COVID-19. In another embodiment, the administration of an effective amount of immunoglobulin to the subject inhibits or reduces onset, development and/or severity of a symptom (e.g., fever, myalgia, cough, difficulty breathing, tiredness) of COVID-19. In another embodiment, the administration of an effective amount of immunoglobulin to the subject inhibits or reduces duration of COVID-19 or a symptom associated therewith. In another embodiment, the administration of an effective amount of immunoglobulin to the subject reduces organ failure associated with COVID-19. In another embodiment, the administration of an effective amount of immunoglobulin to the subject reduces the hospitalization of the subject. In another embodiment, the administration of an effective amount of immunoglobulin to the subject reduces the length of hospitalization of the subject. In another embodiment, the administration of an effective amount of a composition described herein to the subject increases the overall survival of subjects with COVID-19. In another embodiment, the administration of an effective amount of immunoglobulin to the subject prevents the onset or progression of a secondary infection associated with SARS-CoV-2 infection.
[0092] In a specific embodiment, administration of immunoglobulin to a subject reduces the length of hospitalization by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the length of hospitalization in the absence of administration of the immunoglobulin.
[0093] In a specific embodiment, administration of immunoglobulin to a subject reduces mortality by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the mortality in the absence of administration of a composition described herein .
[0094] In certain embodiments, the administration of an effective amount of immunoglobulin to a subject results in one, two, three, four, five, or more of the following effects: (i) reduction or amelioration in the severity of a SARS-CoV-2 infection, COVID-19 or a symptom associated therewith; (ii) reduction in the duration of a SARS-CoV-2 infection, COVID-19 or a symptom associated therewith; (iii) prevention of the progression of a SARS-CoV-2 infection, COVID-19 or a symptom associated therewith; (iv) regression of a SARS-CoV-2 infection, COVID-19 or a symptom associated therewith; (v) prevention of the development or onset of a symptom of a SARS-CoV-2 infection or COVID-19; (vi) reduction in organ failure associated with a SARS- CoV-2 infection or COVID-19; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a SARS-CoV-2 infection or COVID-19; (x) reduction in SARS-CoV-2 titer; (xi) the reduction in the number of symptoms associated with a SARS-CoV-2 infection or COVID-19; (xxiii) enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy; (xii) prevention of the onset or progression of a secondary infection associated with a SARS-CoV-2 infection or COVID-19; and/or (xiii) prevention of the onset or diminution of disease severity of bacterial pneumonias occurring secondary to a SARS- CoV-2 infection or COVID-19.
[0095] In a specific embodiment, administration of immunoglobulin to a subject reduces the number of and/or the frequency of symptoms of in the subject (exemplary symptoms of a SARS- CoV-2 include, but are not limited to, body aches (especially joints and throat), fever, nausea, headaches, fatigue, sore throat, and difficulty breathing). In another specific embodiment, administration of immunoglobulin to a subject reduces the progression of a SARS-CoV-2 infection or COVID-19 using the WHO ordinal scale. In another specific embodiment, administration of immunoglobulin to a subject reduces the need for invasive mechanical ventilation. In another specific embodiment, administration of immunoglobulin to a subject reduces the need to provide oxygen supplementation to the subject. In another specific embodiment, administration of immunoglobulin to a subject reduces the mortality caused by a SARS-CoV-2 infection or COVID-
19. [0096] Immunoglobulin purified from a milk sample(s) may be administered alone or in combination with another/other type of therapy known in the art. See, e.g., Section 5.5.2 for other therapies.
[0097] In specific embodiment, immunoglobulin from a milk sample(s) may be used as any line of therapy, including, but not limited to, a first, second, third, fourth and/or fifth line of therapy. [0098] In another embodiment, provided herein is a method for treating a SARS-CoV-2 infection or COVID-19, comprising administering to a first subject (e.g., a human) in need thereof a milk sample of a second subject (e.g., human female(s) or other mammalian female(s)) that tested positive for anti-SARS-CoV-2 spike protein antibody. In another embodiment, provided herein is a method for preventing COVID-19, comprising administering to a first subject (e.g., a human) in need thereof a milk sample of a second subject (e.g., human female(s) or other mammalian female(s)) that tested positive for anti-SARS-CoV-2 spike protein antibody. See, e.g., Section 5.3 and the Examples for subject from which a milk sample may be obtained.
In certain embodiments, the milk sample comprises immunoglobulin comprises IgA, IgM, or IgG, or a combination thereof. In a specific embodiment, the milk sample comprises immunoglobulin is IgA. In another specific embodiment, the milk sample comprises immunoglobulin comprises secreted IgA. In another embodiment, the milk sample comprises secreted immunoglobulin (e.g., secreted IgA, secreted IgM or both).
[0099] In a particular embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject inhibits or reduces in the progression of COVID-19. In another embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject inhibits or reduces onset, development and/or severity of a symptom (e.g, fever, myalgia, cough, difficulty breathing, tiredness) of COVID-19. In another embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject inhibits or reduces duration of COVID- 19 or a symptom associated therewith. In another embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces organ failure associated with COVID-19. In another embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces the hospitalization of the subject. In another embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces the length of hospitalization of the subject. In another embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject increases the overall survival of subjects with COVID-19. In another embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject prevents the onset or progression of a secondary infection associated with SARS-CoV-2 infection.
[00100] In a specific embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces the length of hospitalization by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the length of hospitalization in the absence of administration of the immunoglobulin.
[00101] In a specific embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces mortality by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the mortality in the absence of administration of a composition described herein .
[00102] In certain embodiments, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject results in one, two, three, four, five, or more of the following effects: (i) reduction or amelioration in the severity of a SARS-CoV-2 infection, COVID-19 or a symptom associated therewith; (ii) reduction in the duration of a SARS-CoV-2 infection, COVID-19 or a symptom associated therewith; (iii) prevention of the progression of a SARS-CoV-2 infection, COVID-19 or a symptom associated therewith; (iv) regression of a SARS- CoV-2 infection, COVID-19 or a symptom associated therewith; (v) prevention of the development or onset of a symptom of a SARS-CoV-2 infection or COVID-19; (vi) reduction in organ failure associated with a SARS-CoV-2 infection or COVID-19; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a SARS-CoV-2 infection or COVID-19; (x) reduction in SARS-CoV-2 titer; (xi) the reduction in the number of symptoms associated with a SARS-CoV-2 infection or COVID-19; (xxiii) enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy; (xii) prevention of the onset or progression of a secondary infection associated with a SARS-CoV-2 infection or COVID- 19; and/or (xiii) prevention of the onset or diminution of disease severity of bacterial pneumonias occurring secondary to a SARS-CoV-2 infection or COVID-19.
[00103] In a specific embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces the number of and/or the frequency of symptoms of in the subject (exemplary symptoms of a SARS-CoV-2 include, but are not limited to, body aches (especially joints and throat), fever, nausea, headaches, fatigue, sore throat, and difficulty breathing). In another specific embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces the progression of a SARS-CoV-2 infection or COVID-19 using the WHO ordinal scale. In another specific embodiment, the administration of a milk sample, which tested positive for anti- SARS-CoV-2 spike protein antibody, to a subject reduces the need for invasive mechanical ventilation. In another specific embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces the need to provide oxygen supplementation to the subject. In another specific embodiment, the administration of a milk sample, which tested positive for anti-SARS-CoV-2 spike protein antibody, to a subject reduces the mortality caused by a SARS-CoV-2 infection or COVID-19.
5.5.1 Routes of Administration and Dosage
[00104] A composition described herein may be delivered to a subject by a variety of routes. These include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, transdermal, intravenous, intranasal and subcutaneous routes. In a specific embodiment, a composition may be administered to a subject intranasally, mucosally, or by pulmonary administration.
[00105] In certain embodiments, a subject is administered a dose of 0.1-100 mg/kg (e.g., 1-15 mg/kg or 10-15 mg/kg) of immunoglobulin purified from a milk sample. An exemplary treatment regime entails administration once per day for a period of 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 2 months, 3 months, or more. In another specific embodiment, administration of a composition described herein to a subject is discontinued if the subject experiences an adverse event.
[00106] In some embodiments, a milk sample may be administered to a subject orally. In certain embodiments, a milk sample is administered by breastfeeding. [00107] In specific embodiments, a milk sample that tested positive for anti-SARS-CoV-2 antibody is administered to a subject once, twice, three times or more per day. The milk sample may be administered to the subject for a period of 2 days, 3 days, 5 days, 7 days, 14 days, 28 days, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months or more.
5.5.2 Combination Therapy
[00108] In various embodiments, a composition described herein may be administered to a subject in combination with one or more other therapies (e.g., antiviral or immunomodulatory therapies). The one or more other therapies may be in the same composition as the immunoglobulin or a different composition. The one or more other therapies may be administered via the same or different routes of administration.
[00109] In some embodiments, the one or more other therapies that are supportive measures, such as pain relievers, anti-fever medications, or therapies that alleviate or assist with breathing. Specific examples of supportive measures include humidification of the air by an ultrasonic nebulizer, aerolized racemic epinephrine, oral dexamethasone, intravenous fluids, intubation, fever reducers (e.g., ibuprofen or acetometaphin), and antibiotic and/or antifungal therapy (i.e., to prevent or treat secondary bacterial and/or fungal infections).
[00110] In certain embodiments, the therapies are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part. In some embodiments, two or more therapies are administered concurrently.
The two or more therapies can be administered in the same composition or a different composition. Further, the two or more therapies can be administered by the same route of administration of a different route of administration. 5.5.3 Subjects
[00111] As used herein, the terms “subject” and “patient” are used interchangeably to refer to an animal (e.g., birds, reptiles, and mammals, such as humans). In one embodiment, a patient treated in accordance with the methods provided herein is a patient suffering from or expected to suffer from a SARS-CoV-2 infection or COVID-19. In another embodiment, a patient treated in accordance with the methods provided herein is a patient exposed to a SARS-CoV-2 infection but not manifesting any symptoms of the infection or COVID-19. In another embodiment, a patient treated in accordance with the methods provided herein is a patient diagnosed with a SARS-CoV-2 infection or COVID-19. In some embodiments, 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. In certain embodiments, 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. In some embodiments, 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. In certain embodiments, 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.
[00112] In another embodiment, a patient treated in accordance with the methods provided herein is a patient experiencing one or more symptoms of COVID-19. Symptoms of COVID-19 include, but are not limited to, body aches (especially joints and throat), fever, nausea, headaches, cough, fatigue, sore throat, lack of smell, lack of taste, congestion, diarrhea, and difficulty breathing. In another embodiment, a patient treated in accordance with the methods provided herein is a patient with COVID-19 who does not manifest symptoms of the disease that are severe enough to require hospitalization. In another embodiment, a patient treated in accordance with the methods provided herein is a patient with COVID-19 manifesting symptoms of the disease that are severe enough to require hospitalization.
[00113] In a specific embodiment, a patient treated in accordance with the methods provided herein is a human. In certain embodiments, a patient treated in accordance with the methods provided herein is a human infant. In some embodiments, a patient treated in accordance with the methods provided herein is a human toddler. In certain embodiments, a patient treated in accordance with the methods provided herein is a human child. In other embodiments, a patient treated in accordance with the methods provided herein is a human adult. In some embodiments, a patient treated in accordance with the methods provided herein is an elderly human. In certain embodiments, a patient treated in accordance with the methods provided herein is patient that is pregnant. As used herein, the term “human adult” refers to a human that is 18 years or older. As used herein, the term “human child” refers to a human that is 1 year to 18 years old. As used herein, the term “human infant” refers to a newborn to 1 year old human. As used herein, the term “human toddleri’ refers to a human that is 1 years to 3 years old. As used herein, the term “elderly human” refers to a human that is 65 years old and older.
[00114] In some embodiments, a patient treated in accordance with the methods provided herein is a patient infected by SARS-CoV-2 with a condition that increases susceptibility to SARS-CoV- 2 complications or for which the virus increases complications associated with the condition such as, e.g., conditions that affect the lung, such as cystic fibrosis, asthma, chronic obstructive pulmonary disease, emphysema, or bacterial infections; cardiovascular disease; or diabetes. Other conditions that may increase SARS-CoV-2 complications include kidney disorders; blood disorders (including anemia or sickle cell disease); or weakened immune systems (including immunosuppression caused by medications, malignancies such as cancer, organ transplant, or HIV infection). In some embodiments, a patient treated in accordance with the methods provided herein is any subject with SARS-CoV-2 infection who is immunocompromised or immunodeficient. [00115] In certain embodiments, patients treated in accordance with the methods provided herein are patients already being treated with antibiotics, antivirals, antifungal s, or other biological therapy/immunotherapy.
6. EXAMPLE 1: EVIDENCE OF A SIGNIFICANT SECRETORY-IGA- DOMINANT SARS-COV-2 IMMUNE RESPONSE IN HUMAN MILK FOLLOWING RECOVERY FROM COVID- 19
[00116] In this Example, the types and magnitude of targeted Abs in human milk against
SARS-CoV-2 were characterized. Specifically, this Example details the findings regarding SARS-CoV-2-reactive IgA, IgG, IgM, and total secretory-type Ab in 15 milk samples obtained from donors previously-infected with COVID-19 between 3-4 weeks after symptoms had abated. To test the antibody components for SARS-CoV-2 in human milk, 15 milk samples were obtained from donors previously-infected with SARS-CoV-2. All milk samples were tested for reactivity to the receptor binding domain (RBD) of the SARS-CoV-2 spike by enzyme linked immunosorbent assays (ELISAs) measuring IgA, IgG, IgM, and secretory Ab, and for IgA reactivity to the full spike. Ten control milk samples obtained prior to December 2019 were used to determine positive cutoff values for each assay, defined as two standard deviations above the mean optical density (OD). Significant binding reactivity to the RBD and the full Spike was exhibited by eighty percent and one-hundred percent of samples, respectively, obtained from COVID- 19-recovered donors. IgA and secretory Ab titers were highly correlated, suggesting most IgA is slgA (>=0.81, p<0.0001). COVID-19 group mean OD values of undiluted milk were significantly greater for IgA (p<0.0001), secretory-type Abs (p<0.0001), and IgG (p=0.004), but not for IgM, compared to pre-pandemic controls. Overall, these data indicate that there is a strong slgA-dominant SARS-CoV-2 immune response in human milk after infection in a vast majority of individuals. Further research is warranted regarding the protectiveness of this response against SARS-CoV-2 infection.
6.1 Background
[00117] Approximately 10% of infants under 1 experience severe coronavirus disease 2019 (COVID-19) requiring advanced care. Passive immunity may be provided via breastfeeding by a previously-infected mother or milk donor. Yet, antibody levels for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have yet to be systematically examined in human milk.
6.2 Materials and Methods
[00118] Procedure and participants
[00119] This study reports on first wave of data collected as part of an ongoing longitudinal study that will examine the typology, durability, and functionality of SARS-CoV-2 specific Abs in the milk of those recovered from COVID-19. COVID- 19-recovered participants were recruited via social media in early April 2020. Interested individuals emailed the study and were screened for eligibility via email and, if eligible, sent to an online consent form. Individuals were eligible to have their milk samples included in this first wave of antibody (Ab) analysis if they resided in New York City (to ease milk sample collection during state- wide restrictions) and had a laboratory-confirmed SARS-CoV-2 infection (n = 8), or highly likely infection based on close contact with a confirmed SARS-CoV-2 case and/or symptoms of infection such as cough, anosmia, malaise, diarrhea, and fever (n = 7) (Table 1). Two participants were infected antenatally during the last 6 weeks of pregnancy. This study was approved by the Institutional Review Board (IRB) at Mount Sinai Hospital.
[00120] Once consented into the study, mothers were asked to collect approximately 30mL of milk into a clean container using electronic or manual pumps at home between 3-4 weeks days after symptoms had abated, in order to allow time for an Ab response to reach its peak and to reduce likelihood of transmission to the investigators. Milk was frozen in participants’ home freezer until samples were picked-up by researchers and transferred on ice to the Mount Sinai Hospital where they were stored at -80°C until Ab testing. As per theIRB-approved protocol, all COVID- 19-recovered participants were informed of the positive or negative Ab status of their milk after testing. Pre-pandemic negative control milk samples were obtained in accordance with IRB-approved protocols prior to December 2019 for other studies, and had been stored in laboratory freezers at -80°C before processing following the same protocol described for COVID- 19 milk samples.
[00121] SARS-CoV-2 Milk Ab Testing
[00122] Before Ab testing, milk samples were thawed, centrifuged at 800g for 15 min, 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. Both COVID-19 recovered and control milk samples were then tested in duplicate in 3 unique experiments for separate assays measuring IgA, IgG, IgM, and secretory-type Ab reactivity (the secondary Ab used in this assay is specific for free and bound SC).
[00123] A SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA) using blood serum/plasma was recently developed and validated and this assay was adapted for use with human milk16,17. Briefly, half-area 96-well plates were coated with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the full trimeric spike protein produced recombinantly as described16. Plates were incubated at 4°C overnight, washed in 0.1% Tween 20/PBS (PBS-T), and blocked in PBS/3% goat serum/0.5% milk powder/3.5% PBS-T for lh at room temperature. Milk was diluted in 1% bovine serum albumin (BSA)/PBS and added to the plate. After 2h incubation at room temperature, plates were washed and incubated for lh at room temperature with horseradish peroxidase-conjugated goat anti-human-IgA, goat anti-human- IgM, goat anti human-IgG (Rockland), or goat anti-human-secretory component (MuBio) diluted in 1% BSA/PBS. Plates were developed with 3,3',5,5'-Tetramethylbenzidine (TMB) reagent followed by 2N hydrochloric acid (HC1) and read at 450nm on a BioTek Powerwave HT plate reader.
[00124] Statistical analysis
[00125] First, control milk samples obtained prior to December 2019 were used to establish positive cutoff values for each assay. Milk was defined as positive for the SARS-CoV-2 Ab type if titer values in COVID-19 recovered milk were two standard deviations above the mean optical density (OD) obtained from pooled control samples. Next, Mann- Whitney U tests were used to determine if the COVID-19 recovered and control milk samples differed in terms of RBD- reactive IgA, secretory Abs, IgG, & IgM. Finally, Spearman correlations were performed to test how Ab factors correlated with one another. For the spike ELISA, endpoint dilution titers were determined from log-transformed titration curves using 4-parameter non-linear regression and an OD value of 1.2. Endpoint dilution positive cutoff values were determined as above. All statistical tests were performed in GraphPad Prism, were 2-tailed, and significance level was set at p-values < 0.05.
6.3 Results
[00126] Control samples exhibited low-level reactivity in all 4 assays; however, this nonspecific or cross-reactive binding was notably greater for the IgA assay. Despite the higher positive cutoff value for this assay, 12/15 milk samples obtained from previously-COVID- 19-infected donors exhibited IgA reactivity to RBD significantly above this cutoff value when undiluted, with wide variation in the ultimate binding endpoint (FIG. 1A). All 12 of these samples were also positive for secretory-type Ab reactivity to RBD (FIG. 1B).
[00127] Individual profiles of Ab subclasses detected in the milk samples from COVID-19 recovered mothers are reported in Table 1. Notably, the RBD-reactive IgA response appears dominant and is not necessarily concurrent with a measurable IgG or IgM response. Of the 12 milk samples shown to be positive for IgA/SC reactivity, 4 samples also exhibited positive IgG and IgM reactivity to RBD (FIG. 1A). An additional 2 samples exhibited positive IgG reactivity but not IgM, and 1 sample also exhibited IgM reactivity but not IgG. One sample exhibited only positive IgG reactivity, but did not exhibited any IgA, IgM, or SC reactivity above the positive cutoff (Table
1). [00128] Overall, OD values of undiluted milk obtained from COVID- 19-recovered donors and pre-pandemic controls for each assay were grouped and compared, and it was found that the COVID- 19-recovered group mean values were significantly greater for IgA (p<0.0001), secretory-type Abs (p<0.0001), and IgG (p=0.017), but not for IgM (FIGS. 1E-1H). OD values for undiluted milk were compared between each Ab subclass. The IgA and secretory Ab (SC)
OD values for undiluted milk were found to be highly correlated (r=0· ., p<0.0001; FIG. 1I).
IgM and IgG OD values were found to be modestly positively correlated (r=0.49, p=0.009; FIG. 1J). No other correlations were found (data not shown).
[00129] Additionally, samples were similarly evaluated for IgA reactivity to the whole SARS- CoV-2 spike protein. It was found that all samples exhibiting reactivity to the RBD were also positive for spike binding; as well, the 3 samples which were negative for RBD-reactive IgA exhibited reactivity to the full spike (FIG. 2A). Endpoint dilution values were also determined for the full spike ELISA. It was found that all COVID- 19-recovered samples exhibited endpoint titers above the positive cutoff and 6/15 were >10× above the cutoff (FIG. 2B).
Table 1. Milk Ab reactivity to SARS-CoV-2 by subclass
Figure imgf000060_0001
* sample was positive against Spike but negative against RBD
Λ SC reactivity against Spike is presumed based on RBD data, but was not tested 6.4 Discussion
[00130] All milk samples obtained from COVID- 19-recovered participants were positive for spike-reactive Ab of at least one subclass. The samples analyzed represent only a snapshot of what is likely a dynamic immune response. A much larger sample size and long-term follow-up study is needed to better understand the time-course of SARS-CoV-2 immunity in milk, as well as whether a typical response is truly protective for breastfed babies. Furthermore, additional testing will be conducted regarding the sufficiency of the amount of Abs (particularly slgA, the highly dominant class) purified from milk and the therapeutic efficacy of those Abs to treat COVID-19. [00131] Though it might be expected that the milk Ab response would be reflective of systemic immunity (i.e., 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 IgG9. 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 system14,18 ’ 19. As such, there is much precedent for milk Ab composition and specificity being unique from that found in blood. Though the milk donors’ blood Ab titers to the milk data was not compared, it was evident that most of the samples contained SARS-CoV-2-reactive IgA without necessarily containing measurable IgG and/or IgM, which particularly in the case of IgG, would likely be derived in large part from the serum. Notably, IgG and IgM reactivities in undiluted milk exhibited a moderate correlation. It may also be that as total IgG and IgM are so much lower in milk than IgA, that this ELISA lacked the sensitivity to pick up very low-titer responses.
[00132] Though it has been determined by previous studies that most IgA in human milk is slgA, the ELISA could not determine with certainty that the IgA (or IgM) measured was of the secretory type or not14. 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. This suggests that a very high proportion of the S ARS-Co V -2-reactive IgA measured herein was slgA. This is extremely relevant to the possibility of using extracted milk Ab as a COVID-19 therapy - for anyone with severe COVID-19 disease, as slgA is highly unique from the IgG- dominant convalescent plasma or purified plasma immunoglobulin being tested currently20. Extracted milk slgA used therapeutically would likely survive well upon targeted respiratory administration, with a much lower dose of Ab likely needed for efficacy compared to systemically-administered convalescent plasma or purified plasma immunoglobulin. With a strong, global recruitment campaign, it would not be difficult to source milk from COVID-19- recovered donors given the ever-increasing number of infections worldwide. Unlike blood, milk can be given every day, and milk supply can be easily increased with increased demand (in this case, using a breast pump) once breastfeeding is successfully established21,22. It is not unrealistic to expect that donors could provide a minimum of 120 mL of milk each, every day, were they be inspired to do so. One hundred such donors would yield ~6000mg of slgA per day21,23. [00133] As comprehensive studies on the human milk immune response to SARS-CoV-2 continues, the aim is to ultimately determine the efficacy of ‘convalescent milk Ab’ as a treatment for COVID-19, and the utility of these Abs to prevent or mitigate infant SARS-CoV-2 infection. These data will have implications beyond the pandemic, as they will serve to fill relatively large knowledge gaps regarding human milk immunology.
6.5 References Cited in Background and Section 6
1. World Health Organization. Coronavirus disease (COVID-2019) Situation Report - 126. SafRiskPharmacother. 2020;8(l):3-8.
2. Dong Y, Mo X, Hu Y, Qi X, Jiang F, Jiang Z, et al. Epidemiological Characteristics of 2143 Pediatric Patients With 2019 Coronavirus Disease in China. Pediatrics [Internet], 2020 Mar 1;e20200702. Available from: http://pediatrics.aappublications.org/content/early/2020/03/16/peds.2020-0702.abstract
3. Coronavirus Disease 2019 in Children - United States, February 12-April 2, 2020.
MMWR Morb Mortal Wkly Rep. 2020 Apr; 69(14): 422-6.
4. Wei WE, Li Z, Chiew CJ, Yong SE, Toh MP, Lee VJ. Presymptomatic Transmission of SARS-CoV-2-Singapore, January 23-March 16, 2020. Morb Mortal Wkly Rep. 2020;69(14):411-5.
5. Li R, Pei S, Chen B, Song Y, Zhang T, Yang W, et al. Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV-2). Science (80- ) [Internet]. 2020 May 1;368(6490):489 LP - 493. Available from: http://science.sciencemag.org/content/368/6490/489.abstract 6. Verdoni L, Mazza A, Gervasoni A, Martelli L, Ruggeri M, Ciuffreda M, et al. An outbreak of severe Kawasaki-like disease at the Italian epicentre of the SARS-CoV-2 epidemic: an observational cohort study. Lancet [Internet], 2020 Jun 3; Available from: https://doi.org/10.1016/S0140-6736(20)31103-X
7. Riphagen S, Gomez X, Gonzalez-Martinez C, Wilkinson N, Theocharis P. Hyperinflammatory shock in children during COVID-19 pandemic. Vol. 395, Lancet (London, England). 2020. p. 1607-8.
8. Lackey KA, Pace RM, Williams JE, Bode L, Donovan SM, Jarvinen KM, et al. SARS-
CoV-2 and human milk: what is the evidence? medRxiv [Internet], 2020; Available from: https://www.medrxiv.org/content/early/2020/04/20/2020.04.07.20056812
9. Yu Y, Xu J, Li Y, Hu Y, Li B. Breast Milk-fed Infant of COVID-19 Pneumonia Mother: a
Case Report [Internet], Research Square; 2020. Available from: https:// doi . org / 10.21203/rs.3.rs-20792/v 1
10. Stockman LJ, Lowther SA, Coy K, Saw J, Parashar UD. SARS during pregnancy, United States. Vol. 10, Emerging infectious diseases. 2004. p. 1689-90.
11. Groß R, Conzelmann C, Muller JA, Stenger S, Steinhart K, Kirchhoff F, et al. Detection of SARS-CoV-2 in human breastmilk. Lancet [Internet], 2020 May 26; Available from: https://doi.org/10.1016/S0140-6736(20)31181-8
12. Wu Y, Liu C, Dong L, Zhang C, Chen Y, Liu J, et al. Viral Shedding of COVID-19 in Pregnant Women. SSRN Electron J. 2020;
13. Hurley WL, Theil PK. Perspectives on immunoglobulins in colostrum and milk. Nutrients. 2011 Apr;3(4):442-74.
14. Brandtzaeg P. The mucosal immune system and its integration with the mammary glands. JPediatr. 2010 Feb; 156(2 Suppl):S8-15.
15. Demers-Mathieu V, Underwood MA, Beverly RL, Nielsen SD, Dallas DC. Comparison of Human Milk Immunoglobulin Survival during Gastric Digestion between Preterm and Term Infants. Nutrients. 2018 May; 10(5).
16. Amanat F, Stadlbauer D, Strohmeier S, Nguyen T, Chromikova V, McMahon M, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. medRxiv [Internet], 2020 Jan 1;2020.03.17.20037713. Available from: http://medrxiv.org/content/early/2020/04/16/2020.03.17.20037713.abstract 17. Stadlbauer D, Amanat F, Chromikova V, Jiang K, Strohmeier S, Arunkumar GA, et al. SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup. Curr Protoc Microbiol. 2020 Jun;57(l):el00.
18. Kleinman RE, Walker WA. The enteromammary immune system: an important new concept in breast milk host defense. Dig Dis Sci. 1979 Nov;24(l l):876-82.
19. Ablstedt S, Carlsson B, Fall strom SP, Hanson LA, Holmgren J, Lidin-Janson G, et al. Antibodies in human serum and milk induced by enterobacteria and food proteins. Ciba Found Symp. 1977 Apr;(46): 115-34.
20. Bloch EM, Shoham S, Casadevall A, Sachais BS, Shaz B, Winters JL, et al. Deployment of convalescent plasma for the prevention and treatment of COVID-19. J Clin Invest [Internet], 2020 Apr 7; Available from: https://doi.org/10.1172/JCI138745
21. Hartmann PE, Prosser CG. Physiological basis of longitudinal changes in human milk yield and composition. Fed Proc. 1984 Jun;43 (9) : 2448-53.
22. Peaker M, Wilde CJ. Feedback control of milk secretion from milk. J Mammary Gland Biol Neoplasia. 1996 Jul;l(3):307— 15.
23. Weaver LT, Arthur HM, Bunn JE, Thomas JE. Human milk IgA concentrations during the first year of lactation. Arch Dis Child. 1998 Mar;78(3):235-9.
7. EXAMPLE 2: ROBUST AND SPECIFIC SECRETORY IGA AGAINST SARS-
COV-2 DETECTED IN HUMAN MILK
[00134] In this Example, milk samples from eight COVID- 19-recovered and seven COVID- 19-suspected donors were tested for antibody (Ab) binding to the SARS-CoV-2 Spike protein. All samples exhibited significant specific IgA reactivity to the full Spike, whereas 80% exhibited significant IgA and secretory (s)Ab binding to the Receptor-Binding Domain (RBD). Additionally, 67% samples exhibited IgG and/or IgM binding to RBD. IgA and sAb titers were highly correlated, indicating most IgA to be slgA. Overall, these data indicate that a robust slgA- dominant SARS-CoV-2 Ab response in human milk after infection should be expected in a significant majority of individuals. This research indicates that extracted milk slgA may have therapeutic use. 7.1 Methods
[00135] Study participants
[00136] Fifteen COVID- 19-recovered participants were recruited via social media in early April 2020. Interested individuals emailed the study and were screened for eligibility via email and, if eligible, sent to an online consent form. Individuals were eligible to have their milk samples included in this analysis if they resided in New York City (to ease milk sample collection during state-wide restrictions) and had a laboratory-confirmed SARS-CoV-2 infection (n = 8), or highly likely infection based on close contact with a confirmed SARS-CoV-2 case and/or symptoms of infection such as cough, anosmia, malaise, diarrhea, and fever (n = 7) (see Table 1). Two participants were infected antenatally during the last 6 weeks of pregnancy. This study was approved by the Institutional Review Board (IRB) at Mount Sinai Hospital (IRB 19- 01243).
[00137] Once consented into the study, participants were asked to collect approximately 30mL of milk into a clean container using electronic or manual pumps at home between 3-4 days after symptoms had abated, in order to allow time for an Ab response to reach its peak and to reduce likelihood of transmission to the investigators. Milk was frozen in participants’ home freezer until samples were picked-up by researchers and transferred on ice to the Mount Sinai Hospital where they were stored at -80°C until Ab testing. Pre-pandemic negative control milk samples were obtained in accordance with IRB-approved protocols prior to December 2019 for other studies, and had been stored in laboratory freezers at -80°C before processing following the same protocol described for COVID-19 milk samples.
[00138] ELISA
[00139] To examine the levels of SARS-CoV-2 Abs in human milk, a modified version of an ELISA that was recently developed and validated for use in blood serum/plasma and has been adapted this assay for use with human milk (Amanat et al., 2020; Stadlbauer et al., 2020).
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. Both COVID-19 recovered and control milk samples were then tested in duplicate in 3 unique experiments for separate assays measuring IgA, IgG, IgM, and secretory-type Ab reactivity (the secondary Ab used in this assay is specific for free and bound SC). Half-area 96-well plates were coated with the receptor binding domain (RBD) of the SARS- CoV-2 spike protein, or the full trimeric spike protein produced recombinantly as described (Amanat et al., 2020, medRxiv [Internet], 2020 Jan 1;2020.03.17.20037713). Plates were incubated at 4°C overnight, washed in 0.1% Tween 20/PBS (PBS-T), and blocked in PBS/3% goat serum/0.5% milk powder/3.5% PBS-T for lh at room temperature. Milk was used undiluted or titrated 4-fold in 1% bovine serum albumin (BSA)/PBS and added to the plate. After 2h incubation at room temperature, plates were washed and incubated for lh at room temperature with horseradish peroxidase-conjugated goat anti-human-IgA, goat anti-human-IgM, goat anti- human-IgG (Rockland), or goat anti -human-secretory component (MuBio) diluted in 1% BSA/PBS. Plates were developed with 3,3',5,5'-Tetramethylbenzidine (TMB) reagent followed by 2N hydrochloric acid (HC1) and read at 450nm on a BioTek Powerwave HT plate reader. Assays were performed in duplicate and repeated 2x.
[00140] Analytical Methods
[00141] Control milk samples obtained prior to December 2019 were used to establish positive cutoff values for each assay. Milk was defined as positive for the SARS-CoV-2 Abs if OD values measured using undiluted milk from COVID- 19-recovered donors were two standard deviations (SD) above the mean ODs obtained from control samples. Endpoint dilution titers were determined from log-transformed titration curves using 4-parameter non-linear regression and an OD cutoff value of 1.2. Endpoint dilution positive cutoff values were determined as above. Mann- Whitney U tests were used to determine if the grouped COVID 19-recovered and control milk samples differed in terms of specific reactivity of undiluted milk for each Ab class. Spearman correlations were performed to test how undiluted milk OD values for each Ab class correlated with one another. All statistical tests were performed in GraphPad Prism, were 2- tailed, and significance level was set at p-values < 0.05.
7.2 Results
[00142] All Human Milk Samples Obtained Form COVID-19-Recovered Donors Contain Significant Levels of SARS-CoV-2-Speciflc IgA
[00143] Milk samples were initially evaluated for IgA binding reactivity by human IgA- specific ELISA to the full trimeric SARS-CoV-2 Spike (FIGS. 3A-3B). It was evident that all samples obtained from COVID- 19-recovered donors (100%), in undiluted form, exhibited binding activity significantly above that of the pre-pandemic control milk samples, which did exhibit some low-level non-specific or cross-reactive activity (FIG.3A). Milk samples were titrated and endpoint titer values were determined. It was found that all COVID- 19-recovered samples exhibited endpoint titers significantly higher than control samples (FIG. 3B).
[00144] Most Milk from COVID-19-Recovered Donors Exhibits IgA and Secretory Ab Reactivity against the Receptor-Binding Domain of the SARS-CoV-2 Spike [00145] Samples were further tested separately for Ab binding reactivity to the Receptor- Binding Domain (RBD) of the Spike protein. Of 15 samples 12 (80%) obtained from previously COVID- 19-infected donors exhibited significant IgA binding activity to RBD, as determined by human IgA-specific ELISA, compared with controls in undiluted form (FIG. 4A). Milk samples were titrated, and endpoint titer values were determined. It was found that 9 of the 12 samples with reactive IgA to RBD when undiluted (75%) exhibited significant endpoint titers (FIG. 4C). Notably, all 12 of the milk samples with significant IgA reactivity in undiluted form to RBD (100%) were also positive for RBD-specific secretory Ab reactivity as determined by human SC- specific ELISA (FIG. 4B). All 12 milk samples also exhibited positive secretory Ab endpoint titers upon dilution compared with controls (FIG. 4D).
[00146] The RBD-Specific IgA Response in Milk Is Dominant and Not Necessarily Concurrent with a Measurable IgG or IgM Response
[00147] Individual profiles of Ab subclasses detected in the milk samples from COVID-19- recovered mothers are reported in Table 2. Notably, specific IgG and IgM were only measurable for a subset of samples, as determined by human IgG- and IgM-specific ELISAs, respectively.
Of the 12 milk samples shown to be positive for IgA reactivity, 8 were also positive for IgG and/or IgM activity (67%). Four samples exhibited significant IgG and IgM reactivity to RBD (COV107, COV112, COV113, COV117; FIGS. 5A-5B) An additional 3 samples exhibited significant IgG reactivity but not IgM (COV108b, CO V111, CO V116), and 1 sample exhibited IgM reactivity but not IgG (COV101; FIGS. 5A-5B). [00148] Table 2: Summary Data
Figure imgf000068_0001
Figure imgf000069_0001
a Sample was positive against Spike but negative against RBD. b Secretory component. c SC reactivity against Spike is presumed based on RBD data, but was not tested, d Participants were infected within the last 6 weeks of pregnancy.
[00149] Milk from COVID-19-Recovered Donors Exhibits Significantly Greater IgA, Secretory Ab, and IgG Binding against RBD Compared with Controls [00150] Overall, optical density (OD) values of undiluted milk obtained from COVID-19- recovered donors and pre-pandemic controls for each assay were grouped and compared, and it was found that the COVID-19-recovered group mean values were significantly greater for IgA (p < 0.0001), secretory Abs (p < 0.0001), and IgG (p = 0.004), but not for IgM (FIGS. 6A-6D). OD values for undiluted milk were compared between each Ab subclass. The IgA and secretory Ab OD values for undiluted milk were found to be highly correlated (r = 0.81, p < 0.0001; FIG. 6E). IgM and IgG OD values were found to be modestly positively correlated (r = 0.49, p = 0.009; FIG. 6F). No other correlations were found (data not shown).
7.3 Discussion
[00151] All milk samples obtained from COVID-19-recovered participants were positive for Spike-reactive Ab of at least one subclass, namely, IgA. Eighty percent of these samples were specifically reactive against the RBD, with most (75%) exhibiting RBD binding activity that was quantitatively and/or qualitatively high such that endpoint titers were significantly above the background activity of the pre-pandemic controls. The samples analyzed represent only a snapshot of what is likely a dynamic immune response.
[00152] Although it has been determined by previous studies that most IgA in human milk is slgA, 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. This suggests that a very high proportion of the SARS-CoV-2-reactive IgA measured herein was slgA. This is extremely relevant to the possibility of using extracted milk Ab as a COVID-19 therapy — for anyone with severe COVID- 19 disease, as slgA is unique from the IgG-dominant convalescent plasma or purified plasma immunoglobulin being tested currently (Bloch et al, 2020, J. Clin. Invest. 130: 2757-2765). Extracted milk slgA used therapeutically would likely survive well upon targeted respiratory administration, with a much lower dose of Ab likely needed for efficacy compared with system! cally administered convalescent plasma or purified plasma immunoglobulin. Notably, the purified material would need to be extensively safety-tested, including ensuring it as free of SARS-CoV-2 material. Alternatively, recombinantly produced, monoclonal or polyclonal Spike- specific slgA could be employed as a similar therapeutic.
[00153] The data indicate that “convalescent milk Ab” may be used as a treatment for COVID-19, and that the Abs present in milk may prevent or mitigate infant SARS-CoV-2 infection.
8. EXAMPLE 3: THE VACCINE-ELICITED IMMUNOGLOBULIN PROFILE IN
MILK AFATER COVID-19 MRNA BASED VACCINATION IS IGG-DOMINANT AND LACKS SECRETORY ANTIBODIES
[00154] The Pfizer/BioNTech and Moderna mRNA-based COVID-19 vaccines are licensed under emergency use authorization, with millions of doses already administered globally [1], No COVID-19 vaccines are yet under investigation for use in infants or young children. As such, the passive immunity of the antibodies (Abs) provided through milk from a vaccinated person may be one of the only ways to protect this population until pediatric COVID-19 vaccines are licensed. As described in Example 1 or 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. Conversely, 6/10 (60%) of post-vaccine samples were positive for Spike specific IgA, with only 1 (10%) exhibiting high IgA endpoint titer. Furthermore, 5/10 (50%) post-vaccine milk samples contained Spike-specific secretory Ab, none of which were found to be high-titer.
[00156] These analyses of the immune response in milk to COVID-19 vaccination provide a critical opportunity to address huge knowledge gaps, inform the field as to which COVID-19 vaccine, if any, is likely to provide the best milk Ab response, and highlight the need to design improved vaccines with protection of the breastfeeding infant in mind.
8.1 Background
[00157] Though pediatric COVID-19 is mild in most cases, -10% of infants experience illness requiring advanced care, and even asymptomatic infection can lead to ‘Multisystem Inflammatory Syndrome in Children’ (MIS-C), a rare but potentially deadly inflammatory condition [5-8], Furthermore, infants and young children can also transmit SARS-CoV-2 to others [9-12], Clearly, protecting this population from infection remains essential. Several COVID-19 vaccine candidates employing a variety of novel platforms have entered clinical trials over the past 10 months, including the Pfizer/BioNT ech and Moderna mRNA-based vaccines, which are now licensed under emergency use authorization, with millions of doses already administered globally [1], Importantly, none of these COVID-19 vaccines are authorized or currently under investigation for use in infants or young children. As such, the passive immunity of the antibodies (Abs) provided through breastfeeding by a COVID- 19-vaccinated mother or milk donor may be one of the only ways to protect this population from SARS-CoV-2 infection and pathology until effective pediatric COVID-19 vaccines are licensed and/or herd immunity is achieved. Mature human milk contains ~0.6mg/mL total immunoglobulin (Ig), though there is great variation among women sampled [13], Milk IgG originates predominantly from serum with some local production in specific cases, though IgG comprises only ~2% of total milk Ab [4], Approximately 90% of total milk Ab is IgA and ~8% IgM, nearly all in secretory (s) form (slgA/sIgM; polymeric Abs complexed to j -chain and secretory component (SC) proteins) [4, 14, 15], Nearly all slgA/sIgM derives from the gut-associated lymphoid tissue (GALT), known as the entero-mammary link, though there is also homing of B cells from other mucosal -associated lymphoid tissue (MALT), i.e. the respiratory system to the mammary gland. The SC protein is a cleaved segment of the polymeric immunoglobulin receptor (plgR) which transports this GALT/MALT -derived Abs into the milk.
[00158] As discussed in Example 1 or 2, milk from 88% of COVID- 19-recovered donors 4-6 weeks after infection was positive for Spike-specific IgA, this IgA response was highly potent, and in 95% of cases, correlated very strongly with a Spike-specific secretory (s) Ab response. This slgA response is dominant compared to the IgG response, which was present in 75% of samples but of significantly lower titer than slgA ([2] and newly-submitted data). Determining if secretory Abs are indeed elicited in milk after infection or vaccination 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, but in the airways and GI tract as well [3, 4], Notably, after 2hrs in the infant stomach, total IgA concentration has been found to decrease by <50%, while IgG concentration decreased by >75%; importantly, though total SC concentration decreased by -60%, there was no decrease in the stomach of infants bom pre-term (within the first 3 months of life) - a population highly vulnerable to infection [15],
[00159] Relatively few comprehensive studies exist examining the Ab response in milk after vaccination. The few studies that have examined the milk Ab response after influenza, pertussis, meningococcal and pneumococcal vaccination have generally found specific IgG and/or IgA that tends to mirror the serum Ab response, though none of these studies measured secretory Ab or determined if slgA was elicited, and data regarding the protective capacity of these milk Abs is conflicting or confounded by the effects of placentally-transferred Ab [16-23], It is evident from studies in non-human primates (NHPs) that an intramuscular (IM) vaccine may not elicit a robust slgA response. In a series of experiments with lactating rhesus macaques, an IM DNA prime + IM poxvims and adenovims vector boost vaccine regimen was found to elicit specific IgG but virtually no specific IgA in milk [24]. However, NHPs primed IM with poxvims vector and boosted IM with adjuvanted protein exhibited measurable specific milk IgG and IgA. When alternatively boosted intranasally (IN), specific milk IgA titers were significantly increased [25], These IN-boosted NHPs were later boosted IM/IN, which produced a markedly high specific milk IgA response [26], However, it was subsequently determined that this specific IgA was not slgA, suggesting it did not traffic to milk via the expected route and would be highly susceptible to degradation in the infant mouth and gut. In fact, follow-up study found the IM+IN+IM/IN regimen insufficient to protect breastfeeding infant macaques from oral SHIV acquisition [27], Notably, there is ~3-5x less IgA in macaque milk compared to human milk, and macaque milk IgA appears to naturally be comprised of significantly less slgA compared to human milk; therefore, it is difficult to extrapolate these data onto human vaccination regimens.
[00160] The mechanism of for mucosal secretory Ab production after IM vaccination is not fully elucidated, particularly in terms of the potential for slgA secretion via plgR rather than passive transfer from serum generating mucosal Ab. It has been suggested that antigen may diffuse from the IM immunization site to local lymph nodes, wherein it is taken up by antigen- presenting cells 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],
8.2 Methods
[00161] Study participants: Individuals were eligible to have their milk samples included in this analysis if they were lactating, had no history of a suspected or confirmed SARS-CoV-2 infection, and were scheduled to be or had recently been vaccinated with either the Pfizer or Moderna mRNA-based COVID-19 vaccine. If milk samples were determined to be positive for SARS-CoV-2 IgA prior to vaccination, participants were excluded from this analysis. Milk was frozen in participants’ home freezer until samples were picked up and stored at -80°C until Ab testing.
[00162] 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). Half-area 96-well plates were coated with the full trimeric spike protein produced recombinantly as described [30], 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 lh at room temperature. Milk was used undiluted or titrated 4-fold in 1% bovine serum albumin (BSA)/PBS and added to the plate. After 2h incubation at room temperature, plates were washed and incubated for lh at room temperature with horseradish peroxidase-conjugated goat anti- human-IgA, goat anti-human-IgG (Fisher), or goat anti-human-secretory component (MuBio) diluted in 1% BSA/PBS. Plates were developed with 3,3',5,5'-Tetramethylbenzidine (TMB) reagent followed by 2N hydrochloric acid (HC1) and read at 450nm on a BioTek Powerwave HT plate reader. Assays were performed in duplicate and repeated 2x.
[00163] Analytical Methods: Control milk samples obtained prior to December 2019 were used previously to establish positive cutoff values for each assay [2], Milk was defined as positive for the SARS-CoV-2 Abs if OD values measured using undiluted milk from COVID-19- recovered donors were two standard deviations (SD) above the mean ODs obtained from control samples. Endpoint dilution titers were determined from log-transformed titration curves using 4- parameter non-linear regression and an OD cutoff value of 1.0. Endpoint dilution positive cutoff values were determined as above. Mann- Whitney U tests were used to assess significant differences. Correlation analyses were performed using Spearman correlations. All statistical tests were performed in GraphPad Prism, were 2-tailed, and significance level was set at p-values < 0.05.
8.3 Results
[00164] Ten pairs of milk samples were obtained from vaccine recipients 1 day before dose 1 and 14 days after dose 2. Six participants had received the Pfizer vaccine, and 4 had received the Moderna vaccine. Milk was used in separate ELISAs measuring IgA, secretory Ab, and IgG binding against recombinant trimeric SARS-CoV-2 Spike [30], Milk was defined as positive for SARS-CoV-2 Ab if the measured OD or endpoint titer was greater than two standard deviations above the mean value obtained from pre-pandemic control samples, as previously determined for these assays [2], As part of the screening of participants for this analysis, those with positive Spike-specific IgA in their pre-vaccine sample were excluded; therefore, all pre-vaccine samples in the present study were found to be negative for Spike-specific IgA (FIG.. 7 A, segmented lines). It was found that 6/10 (60%) of undiluted post-vaccine samples were positive for Spike specific IgA (FIG. 7 A, solid lines).
[00165] As a further measure of antibody affinity/ quantity, titrated milk ODs were used to determine endpoint binding titers, finding that of the 6 IgA-positive samples, 5 exhibited positive IgA endpoint binding titers compared to previously-determined cutoff values (83%; FIG. 7D). One sample exhibited Spike-specific IgA endpoint binding ≥5× the positive cutoff, a benchmark designated as ‘high-titer’ (VAX113; FIG. 7D). Overall, 5/10 (50%) of post-vaccine milk samples contained Spike-specific IgA exhibiting a significant endpoint binding titer.
[00166] Next, 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.
[00167] Finally, Spike-specific IgG was measured. It was found that none of the undiluted pre- vaccine samples, and 10/10 (100%) of post- vaccine samples, contained Spike-specific IgG (FIG. 7C). Upon titration, 100% of post-vaccine samples exhibited positive endpoint binding titers, with 8/10 samples designated as high-titer (80%; FIG. 7F).
[00168] 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).
8.4 Discussion
[00169] None of the COVID-19 vaccines currently in clinical trial or authorized for emergency use have been examined for the milk Ab response they elicit. The Pfizer and Moderna COVID-19 vaccines both consist of lipid-encapsulated mRNA delivered IM, both incorporating virtually identical trinucleotide cap analogs, optimized Spike sequences, and Nl- methylpseudouridine; however, their lipid carriers differ and the Pfizer vaccine consists of 30ug of RNA while Moderna includes lOOug [31], In the present study, no differences were detected in milk Ab titers between the groups of participants receiving each vaccine, though numbers in each group were very low and more participants will need to be studied as part of an in-depth longitudinal analysis of each COVID-19 vaccine. 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). This natural milk Ab response fits the profile of a classic entero-mammary immunological link, wherein the baby’s oral-nasal cavity is bathed in highly protective secretory Abs generated in response to the pathogens encountered by the mother in the GALT/MALT, a link that evolved to protect infants from deadly mucosal infections [14], In contrast, the analysis of COVID-19 mRNA vaccine-induced milk Ab described in this Example demonstrates a very distinct Ab profile. Among the 10 samples analyzed, the Spike- specific milk Ab profile was consistently IgG-dominant, with all samples exhibiting significant endpoint binding titers, 80% being notably high-titer. Unlike the post-infection response, only 50% and 30% of samples exhibited significant IgA and secretory Ab titers, respectively, which were lower-titer. IgA and secretory Ab data did not correlate, in contrast to the findings post-infection described in Example 1, wherein IgA and secretory Ab data was highly positively correlated, demonstrating the IgA was largely secretory class ([2] and Example 4).
[00170] Though secretory Abs are vital to the milk Ab defense system, monomeric, non- secretory Abs, originating from serum as well as locally, likely also contribute to the milk Ab defense system, particularly in the case of vaccination [29], Secretory Ab could ultimately arise in milk following IM vaccination, as will be investigated fully by our follow-up analyses. Importantly, certain vaccine platforms, even if delivered IM, may be more ideal for the elicitation of secretory milk Ab. Depending on the vaccine composition, dose, and adjuvant, 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],
8.5 References Cited in Example 3
1. Rawat, K., P. Kumari, and L. Saha, COVID-19 vaccine: A recent update in pipeline vaccines, their design and development strategies. Eur J Pharmacol, 2021. 892: p.
173751.
2. Fox, A., et al., Robust and specific secretory IgA against SARS-CoV-2 detected in human milk. iScience, 2020: p. 101735.
3. Fouda, G.G., et al., Systemic administration of an HIV-1 broadly neutralizing dimeric IgA yields mucosal secretory IgA and virus neutralization. Mucosal Immunol, 2017. 10(1): p. 228-237.
4. Hurley, W.L. and P.K. Theil, Perspectives on immunoglobulins in colostrum and milk. Nutrients, 2011. 3(4): p. 442-74.
5. Dong, Y., et al., Epidemiological Characteristics of 2143 Pediatric Patients With 2019 Coronavirus Disease in China. Pediatrics, 2020.
6. Jones, V.G., et al., COVID-19 and Kawasaki Disease: Novel Virus and Novel Case. Hosp Pediatr, 2020.
7. Kwak, J.H., et al., Clinical features, diagnosis, and outcomes of multisystem inflammatory syndrome in children associated with coronavirus disease 2019. Clin Exp Pediatr, 2020.
8. Dong, Y., et al., Epidemiology of COVID-19 Among Children in China. Pediatrics, 2020. 145(6). 9. Ludvigsson, J.F., Children are unlikely to be the main drivers of the COVID-19 pandemic -A systematic review. Acta Paediatr, 2020. 109(8): p. 1525-1530.
10. WE, W., et al., Presymptomatic Transmission of SARS-CoV-2 — Singapore, January 23- March 16, 2020. ePub: 1 April 2020. , MMWRMorb Mortal Wkly Rep.
11. Li, R., et al., Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2). Science, 2020.
12. Tang, A., et al., Detection of Novel Coronavirus by RT-PCR in Stool Specimen from Asymptomatic Child, China. Emerg Infect Dis, 2020. 26(6).
13. Weaver, L.T., et al., Human milk IgA concentrations during the first year of lactation. Arch Dis Child, 1998. 78(3): p. 235-9.
14. Brandtzaeg, P., The mucosal immune system and its integration with the mammary glands. J Pediatr, 2010. 156(2 Suppl): p. S8-15.
15. Demers-Mathieu, V., et al., Comparison of Human Milk Immunoglobulin Survival during Gastric Digestion between Preterm and Term Infants. Nutrients, 2018. 10(5).
16. Anderson, P.O., Maternal Vaccination and Breastfeeding. Breastfeed Med, 2019. 14(4): p. 215-217.
17. Schlaudecker, E.P., et al., IgA and neutralizing antibodies to influenza a virus in human milk: a randomized trial of antenatal influenza immunization. PLoS One, 2013. 8(8): p. e70867.
18. Henkle, E., et al., The effect of exclusive breast-feeding on respiratory illness in young infants in a maternal immunization trial in Bangladesh. Pediatr Infect Dis J, 2013. 32(5): p. 431-5.
19. Pandolfi, E., et al., Does Breastfeeding Protect Young Infants From Pertussis? Case- control Study and Immunologic Evaluation. Pediatr Infect Dis J, 2017. 36(3): p. e48-e53.
20. Bellido-Blasco, J., et al., A case-control study to assess the effectiveness of pertussis vaccination during pregnancy on newborns, Valencian community, Spain, 1 March 2015 to 29 February 2016. Euro Surveill, 2017. 22(22).
21. Maertens, K., et al., Breastfeeding after maternal immunisation during pregnancy: providing immunological protection to the newborn: a review. Vaccine, 2014. 32(16): p. 1786-92. 22. Shahid, N.S., et al., Serum, breast milk, and infant antibody after maternal immunisation with pneumococcal vaccine. Lancet, 1995. 346(8985): p. 1252-7.
23. Halperin, B.A., et al., Kinetics of the antibody response to tetanus-diphtheria-acellular pertussis vaccine in women of childbearing age and postpartum women. Clin Infect Dis, 2011. 53(9): p. 885-92.
24. Wilks, A.B., et al., Robust vaccine-elicited cellular immune responses in breast milk following systemic simian immunodeficiency virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys. J Immunol, 2010. 185(11): p. 7097-106.
25. Fouda, G.G., et al., Mucosal immunization of lactating female rhesus monkeys with a transmitted/founder HIV-1 envelope induces strong Env-specific IgA antibody responses in breast milk. J Virol, 2013. 87(12): p. 6986-99.
26. Nelson, C. S., et al., Combined HIV-1 Envelope Systemic and Mucosal Immunization of Lactating Rhesus Monkeys Induces a Robust Immunoglobulin A Isotype B Cell Response in Breast Milk. J Virol, 2016. 90(10): p. 4951-65.
27. Eudailey, J. A., et al., Maternal HIV-1 Env Vaccination for Systemic and Breast Milk Immunity To Prevent Oral SHIV Acquisition in Infant Macaques. mSphere, 2018. 3(1).
28. Joo, H.M., et al., Quantitative analysis of influenza virus-specific B cell memory generated by different routes of inactivated virus vaccination. Vaccine, 2010. 28(10): p. 2186-2194.
29. Su, F., et al., Induction of mucosal immunity through systemic immunization: Phantom or reality? Hum Vaccin Immunother, 2016. 12(4): p. 1070-9.
30. Stadlbauer, D., et al., SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup. Curr Protoc Microbiol, 2020. 57(1): p. elOO.
31. Messengers of hope. Nat Biotechnol, 2021. 39(1): p. 1.
32. Wajnberg A , et al., Humoral Immune response and prolonged PCR positivity in a cohort of 1343 SARS-CoV 2 patients in the New York City region. 2020: medRxiv 2020.04.30.20085613; doi: https://doi.org/10.1101/2020.04.30.20085613.
33. Folegatti, P.M., et al., Safety and immunogenicity of the ChAdOxl nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial. Lancet, 2020. 396(10249): p. 467-478. 34. Jackson, L. A., et al., An mRNA Vaccine against SARS-CoV-2 - Preliminary Report. N Engl J Med, 2020. 383(20): p. 1920-1931.
35. Mulligan, M.J., et al., Phase //// study of COVID-19 RNA vaccine BNT162hl in adults. Nature, 2020. 586(7830): p. 589-593.
36. Sadoff, J., et al., Interim Results of a Phase l-2a Trial of Ad26.COV2.S Covid-19 Vaccine. N Engl J Med, 2021.
37. Keech, C., et al., Phase 1-2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine. NEngl JMed, 2020. 383(24): p. 2320-2332.
9. EXAMPLE 4: THE SPIKE-SPECIFIC IGA IN MILK COMMONLY-
ELICITED AFTER SARS-COV-2 INFECTION IS CONCURRENT WITH A ROBUST SECRETORY ANTIBODY RESPONSE, EXHIBITS NEUTRALIZATION POTENCY STRONGLY CORRELATED WITH IGA BINDING, AND IS HIGHLY DURABLE OVER TIME
[00171] Approximately 10% of infants will experience COVID-19 illness requiring advanced care (1). A potential mechanism to protect this population could be provided by passive immunity through the milk of a previously infected mother. As discussed above, the presence of SARS-CoV-2-specific antibodies in human milk has been reported. This Example reports the prevalence of SARS-CoV-2 IgA in the milk of 75 COVID-19-recovered participants, and finds that 88% of samples are positive for Spike-specific IgA. In a subset of these samples, 95% exhibited robust IgA activity as determined by endpoint binding titer, with 50% considered high- titer. These IgA positive specimens were also positive for Spike-specific antibodies bearing the secretory component. Levels of 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. The IgA durability analysis using 28 paired samples, obtained 4-6 weeks and 4-10 months after infection, found that all samples exhibited persistently significant Spike-specific IgA, with 43% of donors exhibiting increasing IgA titers over time. Finally, 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. These data are highly relevant to public health, not only in terms of the protective capacity of these antibodies for breastfed infants, but also for the potential use of such antibodies as a COVID-19 therapeutic, given that secretory IgA is highly stable not only in milk and the infant mouth and gut, but in all mucosa including the gastrointestinal tract, upper airway, and lungs (6).
9.1 Background
[00172] Though COVID-19 pathology among children is typically more mild compared to adults, approximately 10% of infants under the age of one year experience severe COVID-19 illness requiring advanced care, and an ever-growing number of children appear to exhibit signs of “Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19” weeks or months after exposure (1, 4). Furthermore, infants and young children can also transmit SARS-CoV-2 to others and the efficacy of vaccines available for adults have not yet been evaluated for young children or infants (5). Certainly, protecting this population from infection is essential (6).
[00173] One potential mechanism of protection is passive immunity provided through breastfeeding by a previously-infected mother. Mature human milk contains ~0.6mg/mL of total immunoglobulin (7). Approximately 90% of human milk antibody (Ab) is IgA, nearly all in secretory (s) form (slgA, which consists of polymeric Abs complexed to J-chain and secretory component (SC) proteins) (8). Nearly all slgA derives from the gut-associated lymphoid tissue (GALT), via the entero-mammary link, though there is also homing of B cells from other mucosa (e.g., from the respiratory system), and possibly drainage from local lymphatics of systemic IgA to the mammary gland (8). Unlike the Abs found in serum, slgA found in milk is highly stable and resistant to enzymatic degradation not only in milk and the infant mouth and gut, but in all mucosae including the gastrointestinal tract, upper airway, and lungs (3). Notably, after two hours in the infant stomach, the total IgA concentration decreases by <50%, while IgG concentration decreases by >75%; importantly, though total SC concentration decreases by ~60%, there ia no decrease in the stomach of infants bom pre-term (within the first 3 months of life) - a population highly vulnerable to infection (9). 9.2 Methods
[00174] 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.
[00175] Milk was frozen in participants’ home freezers until samples were picked up and stored at -80°C until Ab testing. Pre-pandemic negative control milk samples were obtained in accordance with IRB-approved protocols prior to December, 2019 for other studies, and had been stored in laboratory freezers at -80°C before processing following the same protocol described for COV1D-19 milk samples
[00176] 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. After 2h incubation at room temperature, plates were washed and incubated for lh at room temperature with horseradish peroxidase-conjugated goat anti-human- IgA, goat anti-human-IgG (Fisher), or goat anti-human-secretory component (MuBio) diluted in 1% BSA/PBS. Plates were developed with 3 , 3 ’ , 5 , 5 ’ -T etramethylbenzidine (TMB) reagent followed by 2N hydrochloric acid (HC1) and read at 450nm on a BioTek Powerwave HT plate reader. Assays were performed in duplicate and repeated 2x.
[00177] 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. [00178] 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). Briefly, pseudovirus was produced by transfection of 293T cells with SARS-CoV-2 Spike plasmid, followed 8 h later by infection with a VSVAG-rLuc reporter virus. Two days post-infection, supernatants were collected and clarified by centrifugation (11). A pre-titrated amount of pseudovirus was incubated with serial dilutions of extracted IgA for 30 min at room temperature prior to infection of cells seeded the previous day. Twenty hours post-infection, cells were processed and assessed for luciferase activity as described (11).
[00179] Analytical Methods: Control milk samples obtained prior to December 2019 were used to establish positive cutoff values for each assay. Milk was defined as positive for the SARS-CoV-2 Abs if OD values measured using undiluted milk from COVID-19-recovered donors were two standard deviations (SD) above the mean ODs obtained from control samples. Endpoint dilution titers were determined from log-transformed titration curves using 4-parameter non-linear regression and an OD cutoff value of 1.0. Endpoint dilution positive cutoff values were determined as above. Percent neutralization was calculated as (1- (average luciferase RLU of triplicate test wells - average luciferase expression RLU of 6 ‘virus only’ control wells) *100. Mann-Whitney U tests were used to assess significant differences in the grouped COVID-19- recovered and control extracted IgA neutralization capacities. The concentrati on of milk IgA required to achieve 50% neutralization (ICso) was determined as described above for endpoint determination. Correlation analyses were performed using Spearman correlations. All statistical tests were performed in GraphPad Prism, were 2-tailed, and significance level was set at p-values < 0.05.
9.3 Results
[00180] Ab profile in milk from COVID-19-recovered donors 4-6 weeks after infection. [00181] Skimmed acellular milk was aliquoted and frozen at -80° C until testing. Undiluted milk samples obtained 4-6 weeks post-infection from 75 COVID-19-recovered donors, and 20 pre-pandemic milk samples obtained prior to December, 2019 were screened in our IgA ELISA against recombinant trimeiic SARS-CoV-2 Spike. Sixty-six of 75 samples (88%) were positive for Spike-specific IgA, with the COVID-19 samples exhibiting significantly higher Spike- specific IgA binding compared to controls (FIG. 8A; p<0.0001). Following this initial screening, 40 of the Spike-positive samples were further titrated to determine binding endpoint titers as an assessment of Ab affinity and/or quantity (FIG. 8B). Thirty-eight of 40 (95%) Spike-reactive samples exhibited positive IgA endpoint titers and 19 of these samples (50%) were ≥5 times higher than the endpoint titer of the positive cutoff value, and were therefore designated as ‘high- titer’ (FIG. 8C).
[00182] Additionally, 20 samples assayed for Spike-specific IgA were also assessed for Spike-specific secretory Ab (by detecting for SC), and IgG. Nineteen of these undiluted milk specimens (95%) from convalescent COVID-19 donors were positive for Spike-specific secretory Abs compared to pre-pandemic control milk (FIG. 9A). One sample (COV125) was negative for specific IgA but positive for specific secretory Ab, while another sample (COY 123) was positive for specific IgA but negative for specific secretory Ab. Eighteen undiluted milk samples (95%) exhibiting Spike-specific secretory Ab activity also exhibited positive endpoint titers (FIG. 9C). Of the samples found to be high-titer for Spike-specific IgA, 7 were also high- titer for specific secretory Ab (70%). Mean OD values for undiluted milk and endpoint titers were used in separate Spearman correlation tests to compare IgA and secretory Ab reactivity (FIG. 9E). It was found that IgA and secretory Ab levels were positively correlated (using ODs: r=0.77, p<0.0001; using endpoint titers: r=0.86, p<0.0001). Additionally, 15/20 undiluted milk samples from COVID- 19-recovered donors were positive for Spike-specific IgG compared to pre-pandemic controls (75%; FIG. 9B), with 13/15 of these samples exhibiting a positive endpoint titer (87%; FIG. 9D), and 2/15 designated as high titer with values ≥5 times cutoff (13%). No correlation was found between IgG and IgA titers or between IgG and SC titers (data not shown).
[00183] Durability of the SARS-CoV-2 Spike-specific milk IgA response.
[00184] To assess the durability of this slgA-dominant response, 28 pairs of milk samples obtained from COVID- 19-recovered donors 4-6 weeks and 4-10 months after infection were assessed for Spike-specific IgA. All donors exhibited persistently significant Spike-specific IgA ti ters of the period of follow-up. Mean endpoint titers from the early to the late milk sampl es grouped were not significantly different (FIG. 10A). Fourteen donors (50%) exhibited >10% decrease in IgA titer, 12 donors (43%) exhibited >10% increase in IgA titer, and 2 donors (7%) exhibited no change in titer (FIG. 10A). Notably, only 2 donors (7%) exhibited >50% decrease in titer over time. Furthermore, examining a subset of these samples with the longest follow-up, obtained 7-10 months after infection, mean endpoint titers measured from the early to the late milk samples were also not significantly different (data not shown). These longest follow-up samples included 5 donors (36%) with >10% decrease in IgA titer, 8 donors (57%) with >10% increase in IgA titer, and 1 donor (7%) with no change in titer ( FIG. 10B). Only 1 donor (7%) exhibited >50% decrease in titer, and as with the larger durability cohort, all donors exhibited persistently significant Spike-specific IgA titers.
[00185] SARS-CoV-2 neutralization capacity of total milk IgA from COVID-19- recovered donors.
[00186] Total IgA was extracted from 8 COVID-19 and 8 control milk samples analyzed for Spike-specific Ab profile. All 8 COVID-19 samples had been shown to exhibit positive Spike- specific IgA and secretory Ab titers (FIGS. 8A-8C and 9A-9E). Neutralization capacity was tested using a Vesicular Stomatitis Virus (VSV)-based pseudovirus assay, wherein the native VSV surface protein G is replaced by the SARS-CoV-2 Spike, as described previously ((11); FIGS. 10A-10B). At the maximum concentration tested (200ug/mL total purified milk IgA), 6/8 (75%) COVID-19 samples exhibited >50% neutralization (mean, 87% neutralization; range, 70% - 100%), while only 1/8 control samples (13%) achieved this benchmark (94% neutralization; FIG. 11 A). Mean percent neutralization values at 50 μg/ml extracted IgA were grouped and compared among COVID-19 and pre-pandemic control samples. COVID-19 samples exhibited significantly greater neutralization compared to controls (p=0.0064; FIG.
11B). As well, when the concentration of IgA required to achieve 50% neutralization (IC50) was determined, 7/8 pre-pandemic controls did not achieve 50% neutralization (IC50>200ug/mL while, for the COVID-19 samples, 2/8 did not achieve 50% neutralization, and the mean IC50 for the 6 COVID19 specimens that displayed neutralizing activity was 33.6 μg/mL of total IgA (range, 2.39 - 89.4ug/mL; FIG. 11C). Finally, the neutralization IC50 titers were compared to the IgA endpoint titers measured for these samples (FIGS. 8A-8C). There was a significant positive correlation between IgA binding and neutralization capacities (r=0.83, p=0.0154; FIG. 11D). Notably, the 2 non-neutralizing COVID-19 IgA samples also exhibited the lowest IgA endpoint titers (COV121, COV130; mean IgA endpoint titers of 19 and 17, respectively), while the 6 neutralizing samples exhibited high Spike-specific IgA binding titers (FIGS. 8C, l!C). 9.4 Discussion
[00187] The data presented in this Example demonstrates a SARS-CoV-2 IgA Ab response in milk after infection is very common. This IgA response dominates compared to the measurable but relatively less frequent IgG response that is markedly less robust. Importantly, a very strong positive correlation was found between Spike-specific milk IgA and secretory Abs, using both ELISA OD values of Ab binding in undiluted milk as well as Ab binding endpoint titers, indicating that a very high proportion of the SARS-CoV-2 Spike-specific IgA measured in milk after SARS-CoV-2 infection is slgA, confirming our early reports. This is relevant for the effective protection of a breastfeeding infant, given the high durability of secretory Abs in the relatively harsh mucosal environments of the infant mouth and gut (3, 9). These data are also relevant to the possibility of using extracted milk IgA as a COVID-19 therapy. Extracted milk slgA used therapeutically would likely survive well upon targeted respiratory administration, with a much lower dose of Ab likely needed for efficacy compared to system! cally-administered convalescent plasma or purified plasma immunoglobulin.
[00188] All COVID-19 IgA samples analyzed that had been designated as ‘high titer’ for Spike-specific IgA exhibited significant Spike-directed neutralization capacity, wherein IgA binding endpoint titers and neutralization IC50 values were found to be significantly correlated. Of the 3 samples examined for neutralization capacity that exhibited positive but not high titer Spike-specific IgA, 2 were non-neutralizing. It should be noted that these were all samples obtained 4-6 weeks after infection, and future samples may exhibit neutralization as the Ab response matures.
[00189] Critically, the IgA durability analysis using 28 paired samples obtained 4-6 weeks and 4-10 months after infection revealed that for all donors, Spike-specific IgA titers persisted for as long as 10 months, a finding that is highly relevant for protection of the breastfeeding infant over the course of lactation, and also pertinent to the size of a potential donor pool for collection of milk from COVID- 19-recovered donors for therapeutic use of extracted milk IgA. Notably, even after 7-10 months, only 5 of 14 samples exhibited >10% decrease in specific IgA endpoint titers, while 8 of 14 samples actually exhibited an increase in specific IgA titer. These highly durable or even increased titers may be reflective of long-lived pl asma cells in the GALT and/or mammary gland, as well as continued antigen stimulation in these compartments, possibly by other human coronaviruses, or repeated exposures to SARS-CoV-2. 9.5 References Cited in Example 4
1. Dong Y, Mo X, Hu Y, Qi X, Jiang F, Jiang Z, Tong S. 2020. Epidemiological Characteristics of 2143 Pediatric Patients With 2019 Coronavirus Disease in China. Pediatrics doi:10.1542/peds.2020-0702. Abstract/FREE Full TextGoogle Scolar
2. Fox A, Marino J, Amanat F, Krammer F, Hahn-Holbrook J, Zolla-Pazner S, Powell RL. 2020. Robust and specific secretory IgA against SARS-CoV-2 detected in human milk. iScience doi: 10.1016/j.isci.2020.101735: 101735.
3. Hurley WL, Theil PK. 2011. Perspectives on immunoglobulins in colostrum and milk. Nutrients 3:442^174.
4. Jones VG, Mills M, Suarez D, Hogan CA, Yeh D, Bradley Segal J, Nguyen EL, Barsh GR, Maskatia S, Mathew R. 2020. COVID-19 and Kawasaki Disease: Novel Virus and Novel Case. Hosp Pediatr doi : 10.1542/hpeds.2020-0123.
5. Ludvigsson JF. 2020. Children are unlikely to be the main drivers of the COVID-19 pandemic - A systematic review. Acta Paediatr 109:1525-1530.
6. Li R, Pei S, Chen B, Song Y, Zhang T, Yang W, Shaman J. 2020. Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2). Science doi : 10.1126/ science. abb3221.
7. Weaver LT, Arthur HM, Bunn JE, Thomas JE. 1998. Human milk IgA concentrations during the first year of lactation. Arch Dis Child 78:235-239.
8. Brandtzaeg P. 2010. The mucosal immune system and its integration with the mammary glands. J Pediatr 156:S8-15.
9. Demers-Mathieu V, Underwood MA, Beverly RL, Nielsen SD, Dallas DC. 2018. Comparison of Human Milk Immunoglobulin Survival during Gastric Digestion between Preterm and Term Infants. Nutrients 10.
10. Stadlbauer D, Amanat F, Chromikova V, Jiang K, Strohmeier S, Arunkumar GA, Tan J, Bhavsar D, Capuano C, Kirkpatrick E, Meade P, Brito RN, Teo C, McMahon M, Simon V, Krammer F. 2020. SARS-CoV-2 Seroconversion in Humans: A Detailed Protocol for a Serological Assay, Antigen Production, and Test Setup. Curr Protoc Microbiol 57:el00.
11. Oguntuyo KY, Stevens CS, Hung CT, Ikegame S, Acklin JA, Kowdle SS, Carmichael JC, Chiu HP, Azarm KD, Haas GD, Amanat F, Klingler J, Baine I, Arinsburg S, Bandres JC, Siddiquey MNA, Schilke RM, Woolard MD, Zhang H, Consortium CA, Duty AJ, Kraus TA, Moran TM, Tortorella D, Lim JK, Gamamik AV, Hioe CE, Zolla-Pazner S, Ivanov SS, Kamil JP, Krammer F, Lee B. 2021. Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID- 19 Sera. mBio 12.
10. EMBODIMENTS
[00190] Provided herein are the following non-limiting embodiments.
1. A method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample, comprising:
(a) incubating a milk sample diluted in 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS) in a well coated with a recombinant SARS-CoV-2 spike protein for a first period time;
(b) washing the well;
(c) incubating a labeled secondary antibody that binds to an isotype or subtype of immunoglobulin, or secretory component in the well for a second period of time;
(d) washing the well; and
(e) detecting the binding of the labeled secondary antibody to antibody present in the milk sample bound to the recombinant SARS-CoV-2 spike protein in the well.
2. The method of embodiment 1, wherein the recombinant SARS-CoV-2 spike protein is soluble and comprises amino acid residues 319-541 of GenBank Accession No. MN908947.3 and a tag.
3. The method of embodiment 1, wherein 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, wherein 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.
4. The method of embodiment 3, wherein the polybasic cleavage site (RRAR) is replaced by a single A.
5. The method of embodiment 2, 3 or 4, wherein the tag is a hexahistidine tag. 6. A method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample, comprising:
(a) incubating a milk sample diluted in 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS) in a well coated with a first recombinant soluble SARS-CoV-2 spike protein for a first period of time, wherein the first recombinant soluble SARS-CoV-2 spike protein comprises amino acid residues 319-541 of GenBank Accession No. MN908947.3 and a tag, and incubating the milk sample diluted in 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS) in another 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 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, and wherein the second recombinant SARS-CoV-2 spike protein does not contain a polybasic cleavage site
(b) washing the wells;
(c) incubating a labeled secondary antibody that binds to an isotype or subtype of an immunoglobulin, or secretory component in the wells for a second period of time;
(d) washing the wells; and
(e) detecting the binding of the labeled secondary antibody to antibody present in the milk sample bound to the first and second recombinant SARS-CoV-2 spike proteins.
7. The method of embodiment 6, wherein the polybasic cleavage site (RRAR) is replaced by a single A.
8. The method of embodiment 6 or 7, wherein the tag is a hexahistidine tag.
9. The method of embodiment 6, 7, or 8, wherein the third period of time is 1.5 to 2.5 hours.
10. The method of embodiment 6, 7, or 8, wherein the third period of time is 2 hours.
11. The method of any one of embodiments 1 to 10, wherein the milk sample prior to dilution in 1% BSA/PBS is centrifuged at 800 g for 15 minutes three times to remove fact and cells.
12. The method of any one of embodiments 1 to 11, wherein the milk sample is from a lactating human female.
13. The method of embodiment 12, wherein the labeled secondary antibody is anti-human IgA, anti-human IgM, anti-human IgG or anti-human secretory component.
14. The method of any one of embodiments 1 to 13, wherein the labeled secondary antibody is diluted in 1% BSA/PBS. 15. The method of any one of embodiments 1 to 14, wherein the wells are washed with 0.1% Tween 20/PBS (PBS-T).
16. The method of any one of embodiments 1 to 15, wherein the first period of time is 1.5 to 2.5 hours.
17. The method of any one of embodiments 1 to 15, wherein the first period of time is 2 hours.
18. The method of any one of embodiments 1 to 17, wherein the second period of time is 30 minutes to 1.5 hours.
19. The method of any one of embodiments 1 to 17, wherein the second period of time is 1 hours
20. The method of any one of embodiments 1 to 19, wherein the secondary antibody is labeled with a chemiluminescent, fluorescent or radioactive moiety.
21. The method of any one of embodiments 1 to 19, wherein the secondary antibody is labeled with horseradish perioxidase.
22. The method of embodiment 21, wherein the detecting the binding of the labeled secondary antibody comprises adding 3,3',5,5'-Tetramethylbenzidine (TMB) reagent to the well for a fourth period of time followed by adding 2N hydrochloric acid (HC1) to the well and reading the well at 450 nm.
23. The method of embodiment 22, wherein the fourth period of time is 5 to 10 minutes.
24. 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.
25. The method of embodiment 24, wherein the immunoglobulin is IgA.
26. The method of embodiment 24, wherein the immunoglobulin is secreted IgA.
27. The method of any one of embodiments 24 to 26, wherein the composition is administered to the subject intranasally, mucosally, or by pulmonary administration.
28. The method of any one of embodiments 24 to 27, wherein the individual is a human female.
29. The method of any one of embodiments 24 to 28, wherein the subject is a human.
30. The method of embodiment 1, wherein 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, wherein 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.
31. The method of embodiment 30, wherein the polybasic cleavage site (RRAR) is replaced by a single A.
32. The method of embodiment 30 or 31, wherein the tag is a hexahistidine tag.
33. A method for determining if a subject has protection against the development of moderate to severe COVID-19, the method 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.
34. A method for determining if a subject should be vaccinated with a COVID-19 vaccine or a booster of a COVID-19 vaccine, the method 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.
35. A method for determining if a milk sample has use in the prevention or treatment of COVID-19, the method 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.
36. 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, the method 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.
37. The method of any one of embodiments 33 to 36, wherein the recombinant SARS-CoV-2 spike protein is soluble and comprises the receptor binding domain of SARS-CoV-2 spike protein.
38. The method of embodiment 37, wherein the receptor binding domain comprises amino acid residues corresponding to amino acid residues 319-541 of GenBank Accession No. MN908947.3.
39. The method of any one of embodiments 33 to 36, wherein 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, wherein 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.
40. The method of embodiment 39, wherein the ectodomain comprises amino acid residues corresponding to amino acid residues 15-1213 of GenBank Accession No. MN908947.3, and a tag,
41. The method of embodiment 39 or 40, wherein the polybasic cleavage site (RRAR) is replaced by a single A.
42. The method of any one of embodiments 37 to 41, wherein the recombinant SARS-CoV-2 spike protein further comprises a tag.
43. The method of embodiment 42, wherein the tag is a hexahistidine tag.
44. A method for treating a SARS-CoV-2 infection or 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.
45. 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. 46. 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.
47. The method of embodiment 44 or 45, wherein the immunoglobulin comprises IgA.
48. The method of embodiment 44 or 45, wherein the immunoglobulin comprises secreted IgA.
49. The method of embodiment 46, wherein the anti-SARS-CoV-2 spike protein antibody comprises IgA.
50. The method of embodiment 46, wherein the anti-SARS-CoV-2 spike protein antibody comprises secreted IgA.
51. The method of embodiment 44, 45, 47 or 48, wherein the composition is administered to the subject orally, intranasally, mucosally, or by pulmonary administration.
52. The method of embodiment 46, 50 or 51, wherein the milk sample is administered to the subject orally.
53. The method of any one of embodiments 44 to 52, wherein the second subject is a human female.
54. The method of any one of embodiments 44 to 53, wherein the first subject is a human.
55. The method of any one of embodiments 44 to 53, wherein the first subject is a human infant or human toddler.
[00191] The foregoing is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the antibodies and methods provided herein and their equivalents, in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.
[00192] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically.

Claims

What is claimed is:
1. A method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample, comprising:
(a) incubating a milk sample diluted in 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS) in a well coated with a recombinant SARS-CoV-2 spike protein for a first period time;
(b) washing the well;
(c) incubating a labeled secondary antibody that binds to an isotype or subtype of immunoglobulin, or secretory component in the well for a second period of time;
(d) washing the well; and
(e) detecting the binding of the labeled secondary antibody to antibody present in the milk sample bound to the recombinant SARS-CoV-2 spike protein in the well.
2. The method of claim 1, wherein the recombinant SARS-CoV-2 spike protein is soluble and comprises amino acid residues 319-541 of GenBank Accession No. MN908947.3 and a tag.
3. The method of claim 1, wherein 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, wherein 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.
4. The method of claim 3, wherein the polybasic cleavage site (RRAR) is replaced by a single A.
5. The method of claim 2, 3 or 4, wherein the tag is a hexahistidine tag.
6. A method for detecting antibody that specifically binds to SARS-CoV-2 spike protein in a milk sample, comprising:
(a) incubating a milk sample diluted in 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS) in a well coated with a first recombinant soluble SARS-CoV-2 spike protein for a first period of time, wherein the first recombinant soluble SARS-CoV-2 spike protein comprises amino acid residues 319-541 of GenBank Accession No. MN908947.3 and a tag, and incubating the milk sample diluted in 1% bovine serum albumin (BSA)/phosphate buffered solution (PBS) in another 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 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, and wherein the second recombinant SARS- CoV-2 spike protein does not contain a polybasic cleavage site;
(b) washing the wells;
(c) incubating a labeled secondary antibody that binds to an isotype or subtype of an immunoglobulin, or secretory component in the wells for a second period of time;
(d) washing the wells; and
(e) detecting the binding of the labeled secondary antibody to antibody present in the milk sample bound to the first and second recombinant SARS-CoV-2 spike proteins.
7. The method of claim 6, wherein the polybasic cleavage site (RRAR) is replaced by a single A.
8. The method of claim 6 or 7, wherein the tag is a hexahistidine tag.
9. The method of claim 6, 7, or 8, wherein the third period of time is 1.5 to 2.5 hours.
10. The method of claim 6, 7, or 8, wherein the third period of time is 2 hours.
11. The method of any one of claims 1 to 10, wherein the milk sample prior to dilution in 1% BSA/PBS is centrifuged at 800 g for 15 minutes three times to remove fact and cells.
12. The method of any one of claims 1 to 11, wherein the milk sample is from a lactating human female.
13. The method of claim 12, wherein the labeled secondary antibody is anti-human IgA, anti-human IgM, anti-human IgG or anti-human secretory component.
14. The method of any one of claims 1 to 13, wherein the labeled secondary antibody is diluted in 1% BSA/PBS.
15. The method of any one of claims 1 to 14, wherein the wells are washed with 0.1%
Tween 20/PBS (PBS-T).
16. The method of any one of claims 1 to 15, wherein the first period of time is 1.5 to
2.5 hours.
17. The method of any one of claims 1 to 15, wherein the first period of time is 2 hours.
18. The method of any one of claims 1 to 17, wherein the second period of time is 30 minutes to 1.5 hours.
19. The method of any one of claims 1 to 17, wherein the second period of time is 1 hours
20. The method of any one of claims 1 to 19, wherein the secondary antibody is labeled with a chemiluminescent, fluorescent or radioactive moiety.
21. The method of any one of claims 1 to 19, wherein the secondary antibody is labeled with horseradish perioxidase.
22. The method of claim 21, wherein the detecting the binding of the labeled secondary antibody comprises adding 3,3',5,5'-Tetramethylbenzidine (TMB) reagent to the well for a fourth period of time followed by adding 2N hydrochloric acid (HC1) to the well and reading the well at 450 nm.
23. The method of claim 22, wherein the fourth period of time is 5 to 10 minutes.
24. A method for determining if a subject has protection against the development of moderate to severe COVID-19, the method 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.
25. A method for determining if a subject should be vaccinated with a COVID-19 vaccine or a booster of a COVID-19 vaccine, the method 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.
26. A method for determining if a milk sample has use in the prevention or treatment of COVID-19, the method 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.
27. 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, the method 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.
28. The method of any one of claims 24 to 27, wherein the recombinant SARS-CoV-2 spike protein is soluble and comprises the receptor binding domain of SARS-CoV-2 spike protein.
29. The method of claim 28, wherein the receptor binding domain comprises amino acid residues corresponding to amino acid residues 319-541 of GenBank Accession No. MN908947.3.
30. The method of any one of claims 24 to 27, wherein 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, wherein 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.
31. The method of claim 30, wherein the ectodomain comprises amino acid residues corresponding to amino acid residues 15-1213 of GenBank Accession No. MN908947.3, and a tag,
32. The method of claim 30 or 31, wherein the polybasic cleavage site (RRAR) is replaced by a single A.
33. The method of any one of claims 28 to 32, wherein the recombinant SARS-CoV-2 spike protein further comprises a tag.
34. The method of claim 33, wherein the tag is a hexahistidine tag.
35. A method for treating a SARS-CoV-2 infection or 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.
36. 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.
37. 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.
38. The method of claim 35 or 36, wherein the immunoglobulin comprises IgA.
39. The method of claim 35 or 36, wherein the immunoglobulin comprises secreted
IgA.
40. The method of claim 37, wherein the anti-SARS-CoV-2 spike protein antibody comprises IgA.
41. The method of claim 37, wherein the anti-SARS-CoV-2 spike protein antibody comprises secreted IgA.
42. The method of claim 35, 36, 38 or 39, wherein the composition is administered to the subject orally, intranasally, mucosally, or by pulmonary administration.
43. The method of claim 37, 40 or 41, wherein the milk sample is administered to the subject orally.
44. The method of any one of claims 35 to 43, wherein the second subject is a human female.
45. The method of any one of claims 35 to 44, wherein the first subject is a human.
46. The method of any one of claims 35 to 44, wherein the first subject is a human infant or human toddler.
PCT/US2021/037256 2020-06-15 2021-06-14 Immunoassay for the detection of anti-sars-cov-2 spike protein antibody in milk samples WO2021257469A1 (en)

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