# US 2022/0034880 A1 · High specificity and sensitivity immunosorbent diagnostic assays with simultaneous resolution of multiple antibody isotypes > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2022/0034880 A1 - Document ID: US20220034880A1 - Status: Published application - Kind: A1 - Application number: 17/279,012 - Filed: 2019-10-01 - Published: 2022-02-03 - Earliest claimed: 2018-10-01 - Applicants: The Board of Trustees of the Leland Stanford Junior University - Inventors: Irving L. Weissman; Michal Caspi Tal - Web page: https://patentgrep.com/patent/US20220034880A1 ## CPC - G01N 33/56911: {Bacteria} - C12N 1/20: Bacteria; Culture media therefor - C07K 2319/42: containing a HA(hemagglutinin)-tag - G01N 2333/20: from Spirochaetales (O), e.g. Treponema, Leptospira - G01N 2469/20: Detection of antibodies in sample from host which are directed against antigens from microorganisms - G01N 2800/26: Infectious diseases, e.g. generalised sepsis - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## Abstract Compositions and methods are provided for diagnosis of infections. The patterns of antibody isotype, subtype and glycosylation provide for a signature pattern that can identify infective agents and patient response to infection. Patients likely to benefit from therapeutic intervention can be discriminated from patients that have a low probability of responsiveness. Therapies are also provided. ## Claims ### Claim 1 A method of characterizing an immune response to a pathogen by an individual, the method comprising: collecting at least one antibody-containing sample from the individual; contacting said at least one antibody-containing sample from the individual with a diagnostic pathogen; contacting the diagnostic pathogen with one or more isotype-specific or glycosylation-specific reagents, which reagents are operably linked to a detectable moiety; and analyzing the diagnostic pathogen for the presence of bound isotype-specific or glycosylation-specific reagents to determine the presence and type of pathogen-specific antibodies, wherein the presence and type is indicative of a pathogen infection and immune response. ### Claim 2 (depends on 1) The method of claim 1, wherein the diagnostic pathogen is an intact pathogen, genetically modified to express a fluorophore. ### Claim 3 (depends on 2) The method of claim 2, wherein the fluorophore is a fluorescent protein. ### Claim 4 (depends on 3) The method of claim 3, wherein the fluorescent protein is selected from the group consisting of green fluorescent protein (GFP), red fluorescent protein (RFP), and analogs thereof. ### Claim 5 (depends on 1, 2, 3, 4) The method of any of claims 1-4, wherein the diagnostic pathogen is a live pathogen or a fixed pathogen. ### Claim 6 (depends on 1, 2, 3, 4, 5) The method of any of claims 1-5, wherein the diagnostic pathogen is a clinical isolate or an environmental isolate. ### Claim 7 (depends on 1, 2, 3, 4, 5, 6) The method of any of claims 1-6, wherein the diagnostic pathogen is from a cell line or cell culture. ### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7) The method of any of claims 1-7, wherein the diagnostic pathogen is further genetically modified to eliminate expression of proteins and other epitopes that are highly conserved among pathogens. ### Claim 9 (depends on 8) The method of claim 8, wherein the epitopes are present on cell surface proteins. ### Claim 10 (depends on 9) The method of claim 9, wherein the cell surface proteins are flagellar proteins. ### Claim 11 (depends on 10) The method of claim 10, wherein the flagellar proteins are one or both of fliH and flil proteins of Borrelia. ### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) The method of any of claims 1-11, wherein the pathogen is a cellular pathogen. ### Claim 13 (depends on 12) The method of claim 12, wherein the pathogen is selected from a bacterium, a fungus, and a protozoan. ### Claim 14 (depends on 13) The method of claim 13, wherein the bacterium is a Spirochaetes. ### Claim 15 (depends on 14) The method of claim 14, wherein the Spirochaetes is a Borrelia sp. ### Claim 16 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) The method of any of claims 1-15 wherein the infectious pathogen is a tick-borne pathogen. ### Claim 17 (depends on 16) The method of claim 16, wherein the tick-borne pathogen is Borrelia burgdorferi. ### Claim 18 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17) The method of any of claims 1-17, wherein the isotype specific reagent is an antibody that recognizes one of IgM, IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgA1, IgA2, IgE, IgD or Ig specific glycosylation sites (such as IgA1-G2S1 or IgG4-G0F). ### Claim 19 (depends on 18) The method of claim 18, wherein a cocktail of 2 or more uniquely-labeled isotype/subtype/glycosylation-specific reagents is contacted with the sample. ### Claim 20 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19) The method of any of claims 1-19, wherein analysis is performed by a method selected from flow cytometry, mass cytometry, sequencing or PCR of sequence barcoded antibodies, and high dimensional/multi-parameter microscopy. ### Claim 21 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) The method of any of claims 1-20 wherein the pathogen is a vaccine strain and the presence and type of pathogen-specific antibodies is indicative of response to vaccine immunization. An additional claim on bead or chip based protein or peptide arrays as the “pathogen” ### Claim 22 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) The method of any of claims 1-21, further comprising treating the individual in accordance with the analysis, optionally with one or more of an antihistamine, anti-IgE agent or mast cell stabilizing agent where pathogen-specific IgE antibodies are present. ### Claim 23 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) The method of any of claims 1-22, wherein the antibody-containing sample is a blood sample. ### Claim 24 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) The method of any of claims 1-23, further comprising monitoring the immune response to the pathogen by the individual for a period of time by repeating a)-d) at a plurality of time points. ### Claim 25 (depends on 24) The method of claim 24, wherein a first antibody-containing sample is collected from the individual at a first time point and a second antibody-containing sample is collected from the individual at a later second time point, wherein detection of increased levels of one or more pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more pathogen-specific antibodies in the first antibody-containing sample indicates that the infection by the pathogen is worsening, and decreased levels of the one or more pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more pathogen-specific antibodies in the first antibody-containing sample indicates that the infection by the pathogen is improving. ### Claim 26 (depends on 25) The method of claim 25, further comprising monitoring the efficacy of a therapy for treating the infection by the pathogen, wherein the first antibody-containing sample is collected from the individual before the patient undergoes the therapy and the second antibody-containing sample is collected from the individual after the patient undergoes the therapy, wherein detection of increased levels of the one or more pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more pathogen-specific antibodies in the first antibody-containing sample indicates that the infection by the pathogen is worsening or not responding to the therapy, and decreased levels of the one or more pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more pathogen-specific antibodies in the first antibody-containing sample indicates that the infection by the pathogen is improving. ### Claim 27 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26) A diagnostic pathogen for use in the methods of any of claims 1-26. ### Claim 28 A method of diagnosing and treating an individual with Lyme disease, the method comprising: collecting at least one antibody-containing sample from the individual; contacting said at least one antibody-containing sample from the individual with a diagnostic bait displaying a plurality of Borrelia burgdorferi pathogen antigens; contacting the diagnostic bait with one or more isotype-specific or glycosylation-specific reagents, which reagents are operably linked to a detectable moiety; analyzing the diagnostic bait for the presence of bound isotype-specific or glycosylation-specific reagents to determine the presence and type of Borrelia burgdorferi pathogen-specific antibodies, wherein the presence and type is indicative of a Borrelia burgdorferi infection and an immune response to the Borrelia burgdorferi pathogen; diagnosing the individual with Lyme disease if the presence of one or more Borrelia burgdorferi pathogen-specific antibodies is detected, and treating the individual for Lyme disease if the presence of one or more Borrelia burgdorferi pathogen-specific antibodies is detected, wherein one or more of an antihistamine; anti-IgE agent or mast cell stabilizing agent is administered to the individual if the presence of Borrelia burgdorferi pathogen-specific immunoglobulin E (IgE) antibodies is detected. ### Claim 29 (depends on 28) The method of claim 28, wherein the antihistamine inhibits a histamine receptor selected from the group consisting of H1, H2, H3, and H4. ### Claim 30 (depends on 28) The method of claim 28, wherein the antihistamine is selected from the group consisting of cimetidine, ranitidine, Benadryl, diphenhydramine, loratadine, doxepin, thioperamide, and clobenpropit. ### Claim 31 (depends on 28, 29, 30) The method of any of claims 28-30, further comprising depleting or stabilizing mast cells in the individual if the presence of Borrelia burgdorferi pathogen-specific immunoglobulin E (IgE) antibodies is detected. ### Claim 32 (depends on 31) The method of claim 31, wherein said mast cells are depleted by administering anti-c-kit therapy. I think we should have a list of possible mast cell stabilizing drugs as well and/or refer to the tables in that review ### Claim 33 (depends on 32) The method of claim 32, further comprising administering anti-CD47 therapy. ### Claim 34 (depends on 28, 29, 30, 31, 32, 33) The method of any of claims 28-33, further comprising depleting IgE producing B cells in the individual if the presence of Borrelia burgdorferi pathogen-specific immunoglobulin E (IgE) antibodies is detected. ### Claim 35 (depends on 28, 29, 30, 31, 32, 33, 34) The method of any of claims 28-34, wherein anti-IgE therapy comprises IgE blockade or linkage of IgE specific antibodies to a different isotype with beneficial effector functions. ### Claim 36 (depends on 35) The method of claim 35, wherein the isotype is IgG. ### Claim 37 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36) The method of any of claims 28-36, further comprising dampening an IgE response by cytokine blockade of one or more cytokines selected from the group consisting of IL-4, IL-5, and IL-13. ### Claim 38 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37) The method of any of claims 28-37, wherein the diagnostic bait s a diagnostic Borrelia burgdorferi pathogen. ### Claim 39 (depends on 38) The method of claim 38, wherein the diagnostic Borrelia burgdorferi pathogen is genetically modified to express a fluorophore. ### Claim 40 (depends on 39) The method of claim 39, wherein the fluorophore is a fluorescent protein. ### Claim 41 (depends on 40) The method of claim 40 wherein the fluorescent protein is selected from green fluorescent protein (GFP), red fluorescent protein (RFP), and analogs thereof. ### Claim 42 (depends on 38, 39, 40, 41) The method of any of claims 38-41, wherein the diagnostic Borrelia burgdorferi pathogen is a Borrelia burgdorferi clinical isolate or an environmental isolate. ### Claim 43 (depends on 38, 39, 40, 41) The method of any of claims 38-41, wherein the diagnostic Borrelia burgdorferi pathogen is from a Borrelia burgdorferi cell line or cell culture. ### Claim 44 (depends on 38, 39, 40, 41, 42, 43) The method of any of claims 38-43, wherein the diagnostic pathogen is further genetically modified to eliminate expression of proteins and other epitopes that are highly conserved among pathogens. ### Claim 45 (depends on 44) The method of claim 44, wherein the epitopes are present on cell surface proteins. ### Claim 46 (depends on 45) The method of claim 45, wherein the cell surface proteins are fliH or flil proteins of Borrelia or a combination thereof. ### Claim 47 (depends on 38, 39, 40, 41, 42, 43, 44, 45, 46) The method of any of claims 38-46, wherein the diagnostic pathogen is a live pathogen or a fixed pathogen. ### Claim 48 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47) The method of any of claims 28-47, wherein the diagnostic bait is an antigen array comprising Borrelia burgdorferi pathogen proteins or peptide epitopes. ### Claim 49 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48) The method of any of claims 28-48, wherein the isotype specific reagent is an antibody that recognizes one of IgM, IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgA, IgA1; IgA2, IgE, IgD or Ig specific glycosylation sites (such as IgA1-G2S1 or IgG4-G0F). ### Claim 50 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49) The method of any of claims 28-49, wherein analysis is performed by a method selected from flow cytometry, mass cytometry, sequencing or PCR of sequence barcoded antibodies, and high dimensional/multi-parameter microscopy. ### Claim 51 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) The method of any of claims 28-50, further comprising monitoring the immune response to the Borrelia burgdorferi pathogen by the individual for a period of time by repeating at a plurality of time points. ### Claim 52 (depends on 51) The method of claim 51, wherein a first antibody-containing sample is collected from the individual at a first time point and a second antibody-containing sample is collected from the individual at a later second time point, wherein detection of increased levels of one or more Borrelia burgdorferi pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more Borrelia burgdorferi pathogen-specific antibodies in the first antibody-containing sample indicates that the Lyme disease is worsening, and decreased levels of the one or more Borrelia burgdorferi pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more Borrelia burgdorferi pathogen-specific antibodies in the first antibody-containing sample indicates that the Lyme disease is improving. ### Claim 53 (depends on 52) The method of claim 52, further comprising monitoring the efficacy of a therapy for treating Lyme disease, wherein the first antibody-containing sample is collected from the individual before the patient undergoes the therapy and the second antibody-containing sample is collected from the individual after the patient undergoes the therapy, wherein detection of increased levels of the one or more Borrelia burgdorferi pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more Borrelia burgdorferi pathogen-specific antibodies in the first antibody-containing sample indicates that the Lyme disease is worsening or not responding to the therapy, and decreased levels of the one or more Borrelia burgdorferi pathogen-specific antibodies in the second antibody-containing sample compared to the levels of the one or more Borrelia burgdorferi pathogen-specific antibodies in the first antibody-containing sample indicates that the Lyme disease is improving. ### Claim 54 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53) The method of any of claims 28-53, wherein said treating the individual for Lyme disease further comprises administering an antibiotic. ### Claim 55 (depends on 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54) A Borrelia burgdorferi diagnostic pathogen for use in the methods of any of claims 28-47 and 49-54. ### Claim 56 (depends on 55) A kit comprising the Borrelia burgdorferi diagnostic pathogen of claim 55 and one or more isotype-specific or glycosylation-specific reagents for detecting Borrelia burgdorferi pathogen-specific antibodies. ### Claim 57 (depends on 56) The kit of claim 56, wherein the isotype-specific reagents comprise IgE-specific reagents for detecting Borrelia burgdorferi pathogen-specific IgE antibodies. ### Claim 58 (depends on 56, 57) The kit of claim 56 or 57, further comprising an antihistamine or an anti-IgE therapeutic agent. ### Claim 59 (depends on 58) The kit of claim 58, wherein the antihistamine is selected from the group consisting of cimetidine, ranitidine, Benadryl, diphenhydramine, loratadine, doxepin, thioperamide, and clobenpropit.