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WO2020252365A1 - Systèmes et procédés pour prédire un risque accru d'événements cardiovasculaires indésirables chez des sujets infectés par le virus de l'immunodéficience humaine (vih) - Google Patents

Systèmes et procédés pour prédire un risque accru d'événements cardiovasculaires indésirables chez des sujets infectés par le virus de l'immunodéficience humaine (vih) Download PDF

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WO2020252365A1
WO2020252365A1 PCT/US2020/037565 US2020037565W WO2020252365A1 WO 2020252365 A1 WO2020252365 A1 WO 2020252365A1 US 2020037565 W US2020037565 W US 2020037565W WO 2020252365 A1 WO2020252365 A1 WO 2020252365A1
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lysops
level
cys
protein
biomarkers
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Dominic W. Chung
Xiaoyun Fu
Jose Aron Lopez
Junmei Chen
Yi Wang
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Bloodworks LLC
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Bloodworks LLC
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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
    • G01N33/56988—HIV or HTLV
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the current disclosure provides systems and methods to predict increased risk of adverse cardiovascular events in individuals infected with human immunodeficiency virus (HIV).
  • the systems and methods measure levels of biomarkers including glutathione peroxidase-3 (GPX-3), platelet factor 4 (PF4), platelet basic protein (PBP), thrombospondin-1 (TSP-1), plasma kallikrein (KLKB1), serotonin, factor V (coagulation factor V), protein C (PROC), protein C inhibitor (PCI), C-reactive protein (CRP), linoleic acid (LA), arachidonic acid (AA), 12- hydroxyeicosatetraenoic acid (12-HETE), lysophosphatidylserine (LysoPS) 18:0, lysoPS 18:1 , lysoPS 20:4, Cysteine (Cys), and/or protein bound Cysteine (P-ss-Cys). If an increased risk is detected, a therapeutic intervention can be initiated.
  • Cardiovascular disease accounts for at least 30% of deaths globally. People can be vulnerable to cardiovascular events due to a number of risk factors including age, tobacco use, unhealthy diet, physical inactivity, elevated blood pressure, abnormal blood lipids, and diabetes.
  • a population known to be at risk for cardiovascular events are individuals infected with human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • the current disclosure provides systems and methods to predict the occurrence of adverse cardiovascular events (CVE) in individuals infected with human immunodeficiency virus (HIV). Particular embodiments include initiating treatments before such adverse events occur in HIV-infected individuals identified to be at risk.
  • CVE adverse cardiovascular events
  • Particular embodiments identify an HIV-infected subject as at risk for an adverse CVE by detecting the level of at least one primary biomarker and the levels of at least nine secondary biomarkers in a biological sample obtained from the subject where at least one of the secondary biomarkers is lysophosphatidylserine (lysoPS) 18:1 or lysoPS 20:4.
  • lysoPS lysophosphatidylserine
  • primary biomarkers include glutathione peroxidase-3 (GPX-3); platelet factor 4 (PF4); platelet basic protein (PBP); thrombospondin-1 (TSP-1); and plasma kallikrein (KLKB1); and secondary biomarkers include serotonin; factor V (coagulation factor V); protein C (PROC); protein C inhibitor (PCI); C-reactive protein (CRP); linoleic acid (LA); arachidonic acid (AA); 12- hydroxyeicosatetraenoic acid (12-HETE); lysoPS 18:0; lysoPS 18: 1 ; lysoPS 20:4; Cysteine (Cys); and protein bound Cysteine (P-ss-Cys).
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • KLKB1 plasma kallikrein
  • secondary biomarkers include se
  • the levels of all primary biomarkers tested indicate that the HIV-infected subject does not have an increased risk of an adverse CVE
  • the levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4 confirm that the subject is infected with HIV
  • the combination of those results all “negative” primary biomarkers and at least nine “confirmatory” secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4 indicates or predicts that the HIV-infected subject does not have an increased risk of an adverse CVE.
  • the level of at least one primary biomarker indicates that the HIV-infected subject has an increased risk of an adverse CVE (even if one or more of the other primary markers are negative)
  • the levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4 confirm that the subject is infected with HIV, then the combination of those results (at least one“positive” primary biomarker and at least nine“confirmatory” secondary biomarkers where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4) indicates or predicts that the HIV- infected subject has an increased risk of an adverse CVE.
  • the levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, indicate a confirmatory result
  • the levels of 4 or fewer secondary biomarkers indicate a non-confirmatory result
  • the combination of those results indicates that the primary biomarker result is predictive of whether or not the subject has an increased risk of an adverse CVE.
  • an HIV-infected subject can be identified as at risk for an adverse CVE based on one or more of: a decrease in GPX-3 level; an increase in PF4 level; an increase in PBP level; an increase in TSP-1 level; and/or a decrease in KLKB1 level, so long as at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, confirm that the subject is infected with HIV.
  • those secondary biomarkers in certain embodiments will be: an increase in serotonin level; an increase in factor V level; a decrease in PROC level; a decrease in PCI level; an increase in CRP level; an increase in LA level; an increase in AA level; an increase in 12-HETE level; an increase in lysoPS 18:0 level; an increase in lysoPS 18:1 level; an increase in lysoPS 20:4 level; a decrease in Cys level; and/or an increase in P-ss-Cys level, in a biological sample obtained from the subject.
  • changes in levels of one or more primary biomarkers can predict risk of adverse CVE in HIV patients if levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, confirm that a subject is infected with HIV, such that the detection of the biomarkers can lead to therapeutic interventions before an adverse CVE occurs.
  • FIG. 1 Relative levels of glutathione peoxidase-3 (GPX-3) determined by proteomic analysis, quantifying at least two peptides from GPX-3. The study cohort for FIGs. 1-9B is described in Example 1. Levels of GPX-3 drop in HIV-infected subjects; the levels drop further in HIV-infected subjects with subsequent adverse CVE.
  • GPX-3 Relative levels of glutathione peoxidase-3 (GPX-3) determined by proteomic analysis, quantifying at least two peptides from GPX-3.
  • the study cohort for FIGs. 1-9B is described in Example 1.
  • Levels of GPX-3 drop in HIV-infected subjects; the levels drop further in HIV-infected subjects with subsequent adverse CVE.
  • FIGs. 2A-2C Levels of (FIG. 2A) platelet factor 4 (PF4), (FIG. 2B) platelet basic protein (PBP), and (FIG. 2C) thrombospondin-1 (TSP-1) were determined by proteomic analysis of depleted plasma. The biomarkers are elevated in HIV-infected subjects with subsequent adverse CVE.
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • FIG. 3 Relative levels of plasma kallikrein (KLKB1) determined by proteomic analysis of depleted plasma.
  • FIG. 4 Levels of serotonin were determined by targeted metabolomic analysis using liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC- MS/MS-MRM).
  • FIG. 5A-5C Relative levels of (FIG. 5A) factor V (coagulation factor V), (FIG. 5B) protein C (PROC), and (FIG. 5C) protein C inhibitor (PCI) determined by proteomic analysis of depleted plasma.
  • factor V coagulation factor V
  • PROC protein C
  • PCI protein C inhibitor
  • FIG. 6 Levels of C-reactive protein (CRP) determined by proteomic analysis of depleted plasma.
  • FIGs. 7A-7C Concentrations of (FIG. 7A) linoleic acid (LA), (FIG. 7B) arachidonic acid (AA), and (FIG. 7C) 12-hydroxyeicosatetraenoic acid (12-HETE) in normal control and HIV- infected subjects without CVE, determined by targeted metabolomic analysis using mass spectrometry.
  • LA linoleic acid
  • AA arachidonic acid
  • FIG. 7C 12-hydroxyeicosatetraenoic acid
  • FIGs. 8A-8C Levels of (FIG. 8A) lysophosphatidylserine (lysoPS) 18:0, (FIG. 8B) lysoPS 18:1 , and (FIG. 8C) lysoPS 20:4 determined by targeted metabolomic analysis.
  • lysoPS lysophosphatidylserine
  • FIGs. 9A-9C Levels of (FIG. 9A) cysteine and (FIG. 9B) protein bound Cysteine (P-ss- Cys) determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using multiple reaction monitoring (MRM), as described in Fu et al. (2019) Scientific reports, 9(1): 115 and PCT/US2015/042318.
  • Thiols in a biological sample can be blocked with N-ethylmaleimide (NEM), which minimizes both artefactual oxidation and thiol- disulfide exchange.
  • NEM N-ethylmaleimide
  • 9C shows exemplary workflows to quantify individual small molecule thiols and disulfides, total small molecule thiols (reduced and disulfide forms), and total thiols (including protein-bound forms) in biological samples.
  • Isotopically labeled internal standards were used for absolute quantification. Proteins in the samples were precipitated with methanol and the supernatants were analyzed. The disulfides and thiol-NEM adducts were separated by UPLC and detected by LC-MS/MS-MRM. Product ions from the analyte were quantified by comparison to analogous ions derived from isotopically labeled internal standards.
  • FIG. 10 Exemplary biomarker distribution in HIV-infected patients with and without CVE.
  • a primary biomarker gives a positive result when the level of each primary biomarker is significantly increased or decreased (depending upon the biomarker) as compared to the average of patients with CVE.
  • a secondary biomarker gives a confirmatory result when the level of each secondary biomarker is significantly increased or decreased as compared to the average of normal controls (i.e. , subjects who are not infected with HIV).
  • Primary biomarkers GPX-3, glutathione peroxidase-3; PF4, platelet factor 4; PBP, platelet basic protein; TSP-1 thrombospondin-1 ; KLKB1 , plasma kallikrein.
  • Serotonin; Factor V; PROC protein C
  • PCI protein C inhibitor
  • CRP C-reactive protein
  • LA linoleic acid
  • AA arachidonic acid
  • 12-HETE 12-hydroxyeicosatetraenoic acid
  • LysoPS18:0 lysophatidylserine 18:0
  • LysoPS18:1 lysophatidylserine 18:1
  • Cys Cysteine
  • P-ss-Cys protein-bound Cys.
  • Cardiovascular disease is the leading cause of death for women and men in the U.S. and accounts for at least 30% of deaths globally.
  • the general population can be vulnerable to cardiovascular events due to a number of risk factors including age, tobacco use, unhealthy diet, physical inactivity, elevated blood pressure, abnormal blood lipids, and diabetes.
  • a population known to be at risk for cardiovascular events are individuals infected with human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • HIV human immunodeficiency virus
  • Improvements in anti-retroviral therapy have converted HIV infection to a manageable chronic disease.
  • cardiovascular diseases including myocardial infarction, ischemic stroke, arterial and venous thrombosis, hypertension, and atherosclerosis; type 2 diabetes mellitus; metabolic syndrome; and chronic kidney disease.
  • myocardial infarction ischemic stroke
  • arterial and venous thrombosis hypertension, and atherosclerosis
  • type 2 diabetes mellitus type 2 diabetes mellitus
  • metabolic syndrome and chronic kidney disease.
  • chronic HIV infection itself as well as anti-retroviral therapy regimens contribute to the development of a pro-thrombotic state that promotes thrombosis.
  • CVE adverse cardiovascular events
  • HAV human immunodeficiency virus
  • Particular embodiments include initiating (or modifying) treatments before such adverse events occur in individuals identified to be at risk.
  • CVE and “adverse CVE” are used interchangeably herein.
  • Particular embodiments identify an HIV-infected subject as at risk for an adverse CVE by detecting the level of at least one primary biomarker and the levels of at least nine secondary biomarkers in a biological sample obtained from the subject where at least one of the secondary biomarkers is lysophosphatidylserine (lysoPS) 18:1 or lysoPS 20:4.
  • lysoPS lysophosphatidylserine
  • primary biomarkers include glutathione peroxidase-3 (GPX-3); platelet factor 4 (PF4); platelet basic protein (PBP); thrombospondin-1 (TSP-1); and plasma kallikrein (KLKB1); and secondary biomarkers include serotonin; factor V (coagulation factor V); protein C (PROC); protein C inhibitor (PCI); C-reactive protein (CRP); linoleic acid (LA); arachidonic acid (AA); 12- hydroxyeicosatetraenoic acid (12-HETE); lysophosphatidylserine (lysoPS) 18:0; lysoPS 18:1 ; lysoPS 20:4; Cysteine (Cys); and protein bound Cysteine (P-ss-Cys).
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • KLKB1 plasma
  • the levels of all primary biomarkers tested indicate that the HIV-infected subject does not have an increased risk of an adverse CVE (a“negative” result; Table 1), and the levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, confirm that the subject is infected with HIV (a “confirming” or “confirmatory” result; Table 2), then the combination of those results (all“negative” primary biomarkers and at least nine“confirmatory” secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4) indicates (or predicts) that the HIV- infected subject does not have an increased risk of an adverse CVE.
  • the level of at least one primary biomarker indicates that the HIV-infected subject has an increased risk of an adverse CVE (a“positive” result; see Table 1) (even if one or more of the other primary markers are negative), and the levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, confirm that the subject is infected with HIV (a“confirming” or“confirmatory” result; see Table 2), then the combination of those results (at least one “positive” primary biomarker and at least nine “confirmatory” secondary biomarkers) indicates (or predicts) that the HIV-infected subject has an increased risk of an adverse CVE.
  • the levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, indicate a confirmatory result, and the levels of 4 or fewer secondary biomarkers indicate a non-confirmatory result, then the combination of those results (at least nine confirmatory secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, and 4 or fewer non-confirmatory secondary biomarkers) indicates that the primary biomarker result is predictive of whether or not the subject has an increased risk of an adverse CVE.
  • Table 1 shows each primary biomarker and the change (or lack of change) in level of each primary biomarker that may be indicative (or not indicative) of whether a subject has an increased risk of an adverse CVE.
  • Table 1 Change in level of a primary biomarker in a subject to indicate whether the subject has an increased risk of an adverse CVE
  • the sample is compared to a reference level of that primary biomarker (for instance, the reference level can be an average of levels of the primary biomarker from a group of subjects who have HIV and who have had or who do have CVE).
  • Table 2 shows each secondary biomarker and the change (or lack of change) in level of each secondary biomarker that is confirmatory (or not confirmatory) of an HIV infection in a subject.
  • Table 2 Change in level of a secondary biomarker in a subject to indicate HIV infection status of the subject
  • the sample is compared to a reference level of that secondary biomarker (for instance, the reference level can be an average of levels of the secondary biomarker from a group of subjects who are not infected with HIV).
  • an HIV-infected subject can be identified as at risk for an adverse CVE based on one or more of: a decrease in GPX-3 level; an increase in PF4 level; an increase in PBP level; an increase in TSP-1 level; and/or a decrease in KLKB1 level, in those circumstances where at least nine of the following secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, from the same subject confirm that the subject is infected with HIV: an increase in serotonin level; an increase in factor V level; a decrease in PROC level; a decrease in PCI level; an increase in CRP level; an increase in LA level; an increase in AA level; an increase in 12-HETE level; an increase in lysoPS 18:0 level; an increase in lysoPS 18:1 level; an increase in lysoPS 20:4 level; a decrease in Cys level; and/or an increase in P-s
  • a biomarker of the present disclosure is a molecule expressed and/or released from a cell in (or from) a subject, which is useful for identification or prediction.
  • Such biomarkers are molecules (e.g., proteins, small molecules, nucleic acids, lipids, and carbohydrates) that can be differentially expressed (e.g., overexpressed or underexpressed), differentially functioning, or differentially released (e.g., released from a cell) in response to a health condition of a subject.
  • biomarkers can refer to the biochemical compounds and/or proteins disclosed herein, which are increased or decreased, for instance, 1- fold, 2-fold, 3-fold, 4-fold, 5-fold or more, in HIV-infected subjects who have or will have adverse CVE versus HIV-infected subjects who do not have or will not have adverse CVE.
  • primary biomarkers The following biomarkers are referred to as primary biomarkers within the current disclosure: GPX-3, PF4, PBP, TSP-1 , and KLKB1.
  • primary biomarkers can distinguish between HIV-infected subjects who are at an increased risk of having adverse CVE and HIV-infected subjects who are not at an increased risk of having adverse CVE, given that levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, confirm that each subject is infected with HIV.
  • lysoPS 18:1 or lysoPS 20:4 confirm that each subject is infected with HIV.
  • Glutathione peroxidase-3 (GPX-3).
  • GPX-3 is also known as GPx-P, GSHPx-3, and GSHPx-P.
  • GPX-3 belongs to the glutathione peroxidase family. It protects cells against oxidative damage by catalyzing the reduction of organic hydroperoxides, lipid peroxides, and hydrogen peroxide (H2O2) by glutathione.
  • GPX-3 is secreted from cells and is abundantly found in plasma.
  • GPX-3 is also a selenoprotein, containing the rare amino acid selenocysteine (Sec) at its active site.
  • GPX-3 Downregulation of expression of the gene encoding GPX-3 by promoter hypermethylation has been observed in a wide spectrum of human malignancies, including thyroid cancer, hepatocellular carcinoma and chronic myeloid leukemia. GPX-3 is highly expressed in tissues that include kidney and thyroid.
  • human GPX-3 has an amino acid sequence of UniProt ID P22352, encoded by the nucleotide sequence of NCBI Ref Sequence: NM_002084.4, nucleotides 244-924.
  • PF4 Platelet factor 4
  • PF4 is also known as PF-4, CXCL4, and SCYB4.
  • PF4 is a member of the CXC chemokine family. This chemokine is released from the alpha granules of activated platelets in the form of a homotetramer which has high affinity for heparin and is involved in platelet aggregation.
  • This protein serves as a chemotactic signal for cells such as neutrophils, fibroblasts, and monocytes, and also functions as an inhibitor of hematopoiesis, angiogenesis and T-cell function. The protein also exhibits antimicrobial activity against Plasmodium falciparum.
  • PF4 is highly expressed in tissues that include bone marrow and spleen.
  • human PF-4 has an amino acid sequence of UniProt ID P02776, encoded by the nucleotide sequence of NCBI Ref Sequence: NM_002619.3, nucleotides 172-477.
  • PBP Platelet basic protein
  • b-thromboglobulin b-thromboglobulin
  • PBP pro-platelet basic protein
  • b-thromboglobulin b-TG
  • PBP pro-platelet basic protein
  • TC1 TC2
  • TGB LDGF
  • MDGF TGB1 , B-TG1
  • CTAP3 CXCL7, NAP-2
  • SCYB7 THBGB
  • LA-PF4 THBGB1
  • CTAPIII CTAPIII
  • CTAP-III CTAP-III
  • PBP is a platelet-derived growth factor that belongs to the CXC chemokine family. This growth factor is a potent chemoattractant and activator of neutrophils.
  • PBP is also an antimicrobial protein with bactericidal and antifungal activity. PBP is highly expressed in tissues that include bone marrow and spleen.
  • human b-thromboglobulin has an amino acid sequence of UniProt ID P02775, encoded by the nucleotide sequence of NCBI Ref Sequence: NM_002704.3, nucleotides 88-474.
  • TSP-1 Thrombospondin-1
  • TSP-1 is also known as TSP, THBS, TSP1 , THBS1 , and THBS-1.
  • TSP-1 is a subunit of a disulfide-linked homotrimeric protein. This protein is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions. This protein can bind to fibrinogen, fibronectin, laminin, type V collagen and integrin alpha-V/beta-1.
  • TSP-1 has roles in platelet aggregation, angiogenesis, and tumorigenesis.
  • TSP-1 is broadly expressed in tissues that include appendix, gall bladder, ovary, prostate, lung, placenta, kidney and platelets.
  • human TSP-1 has an amino acid sequence of UniProt ID P07996-1 , encoded by the nucleotide sequence of NCBI Ref Sequence: NM_003246, nucleotides ISO- 3692.
  • KLKB1 Plasma kallikrein
  • KLKB1 is also known as Fletcher factor, kininogenin, and plasma prekallikrein. Blood clots quickly when it comes in contact with foreign surfaces through a process known as contact activation. Contact activation is initiated by the binding of factor XII (Hageman factor) in plasma to negatively charged surfaces, which leads to activation of plasma prekallikrein (Fletcher factor, KLKB1) to the active protease plasma kallikrein. Plasma kallikrein then reciprocally activates factor XII to factor XI la in a reaction that leads to amplification of the surface-dependent reactions.
  • factor XII Heman factor
  • Plasma kallikrein also cleaves high molecular weight kininogen to release the vasoactive peptide bradykinin, which is a pharmacological agent that dilates blood vessels, lowers blood pressure, increases blood vessel permeability, and causes pain and inflammation.
  • Plasma prekallikrein is synthesized in the liver and secreted into the circulation as a single polypeptide chain with an apparent molecular weight of 88,000 daltons. It is present in plasma at a concentration of 35-45 pg/ml and circulates as a non-covalent complex with high molecular weight kininogen.
  • Plasma prekallikrein is 58% identical in amino acid sequence to coagulation factor XI.
  • human KLKB1 has an amino acid sequence of UniProt ID P03952-1 , encoded by the nucleotide sequence of NCBI Ref Sequence: NM_000892.4, nucleotides 119-2035.
  • biomarkers are secondary biomarkers within the current disclosure: serotonin, Factor V, protein C (PROC), protein C inhibitor (PCI), C-reactive protein (CRP), linoleic acid (LA), arachidonic acid (AA), 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cysteine (Cys), and protein bound Cysteine (P-ss-Cys).
  • Results from at least nine secondary biomarkers where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, are used to confirm whether a subject is infected with HIV and thus indicate when at least one positive primary biomarker is predictive of the HIV-infected subject having an increased risk of an adverse CVE or indicate when at least one negative primary biomarker is not predictive of the HIV-infected subject having an increased risk of an adverse CVE.
  • the secondary biomarkers is described below.
  • Serotonin also known as 5-hydroxytryptamine (5-HT) has the following structure:
  • Serotonin is a monoamine neurotransmitter derived from the amino acid tryptophan. Serotonin is primarily found in the enteric nervous system located in the gastrointestinal (Gl) tract, where it is used to regulate intestinal movements. However, it is also produced in the central nervous system (CNS) in the Raphe nuclei located in the brainstem, where it can regulate mood, appetite, sleep memory, and learning. This indoleamine molecule is thought to contribute to feelings of well-being and happiness. Additionally, serotonin is stored in blood platelets and is released during agitation and vasoconstriction, where it then acts as an agonist to other platelets.
  • Factor V Coagulation factor V is also known as blood coagulation factor V, factor V, activated protein C cofactor, and proaccelerin, labile factor.
  • Factor V is an essential cofactor in the coagulation system, which is a series of chemical reactions that forms blood clots in response to injury.
  • Factor V circulates in an inactive form in plasma and is activated upon an injury that damages blood vessels.
  • Activated factor V forms a complex with activated coagulation factor X (designated factor Va and factor Xa, respectively), and the fVa/fXa complex converts an important coagulation protein called prothrombin to its active form, thrombin.
  • Thrombin then converts a protein called fibrinogen into fibrin, which is the material that forms the clot.
  • Factor V also has a role in regulating the coagulation system through its interaction with a protein called activated protein C (APC).
  • APC activated protein C
  • the cutting of factor Va at specific sites by APC inactivates factor V, which slows down the clotting process and prevents clots from growing too large.
  • factor Va cannot be inactivated normally by APC.
  • the clotting process remains active longer than usual, increasing the chance of developing abnormal blood clots that could occlude blood vessels.
  • Factor V protein is made primarily by cells in the liver.
  • human Factor V has an amino acid sequence of UniProt ID P12259, encoded by the nucleotide sequence of NCBI Ref Sequence: NM_000130.4, nucleotides 146-6820.
  • Protein C is also known as anticoagulant protein C, autoprothrombin IIA and blood coagulation factor XIV.
  • Protein C is a serine protease that controls blood clotting by blocking the activity of two proteins that promote the formation of blood clots, activated factor V (factor Va) and activated factor VIII (factor Villa).
  • Inactive protein C is converted to activated protein C (APC) by thrombin in the presence of a cofactor thrombomodulin.
  • APC is responsible for inactivating factor Va.
  • Protein C is a vitamin K-dependent glycoprotein that is also involved in regulating inflammation, cell death, and maintaining the permeability of blood vessel walls.
  • human Protein C has an amino acid sequence of UniProt ID P04070, encoded by the nucleotide sequence of NCBI Ref Sequence: NM_000312.3, nucleotides 95-1480.
  • Protein C Inhibitor is also known as PCI, SERPINA5, and plasminogen activator inhibitor-3 (PAI-3). It is a serine protease inhibitor that limits the activity of activated protein C (APC) to regulate the anticoagulant protein C pathway and fibrinolysis.
  • the inhibitor is a 52 kDa glycoprotein and belongs to the serine protease inhibitor (Serpin) super family of proteins.
  • Protein C Inhibitor was initially identified as an inhibitor of APC; however, this inhibitor can regulate other coagulation factors, such as thrombin and factor Xa. Its inhibitory activity is modulated by binding of glycosaminoglycans and/or phospholipids.
  • Protein C Inhibitor regulates mammalian fertilization by inhibiting prostate-specific antigen and sperm acrosin (a serine protease). It may also function in lung remodeling, tissue regeneration, vascular permeability, proteolysis in the kidney and tumor invasion. Protein C inhibitor is found in most tissues and fluids, including blood plasma, seminal plasma and urine of human. In particular embodiments, human Protein C inhibitor has an amino acid sequence of UniProt ID P05154, encoded by the nucleotide sequence of NCBI Ref Sequence: NM_000624.5, nucleotides 236- 1456.
  • CRP C-Reactive Protein
  • PTX1 C-Reactive Protein
  • the protein belongs to the pentaxin family. It is involved in several host defense related functions based on its ability to recognize foreign pathogens and damaged cells of the host. It can promote agglutination, bacterial capsular swelling, phagocytosis and complement fixation through its calcium- dependent binding to phosphorylcholine. The level of this protein in plasma increases greatly during the acute phase response to tissue injury, infection, or other inflammatory stimuli.
  • CRP is highly expressed in tissues including the liver.
  • human CRP has an amino acid sequence of UniProt ID P02741-1 , encoded by the nucleotide sequence of NCBI Ref Sequence: NM_001329057.1 , nucleotides 105-779.
  • Linoleic Acid is also known as: (9Z,12Z)-octadeca-9,12-dienoic acid; 9 trans,12 trans octadecadienoic acid; 9,12 octadecadienoic acid; acid, 9,12-octadecadienoic; cis,cis-9,12-octadecadienoic Acid; linoelaidic acid; linoelaidic acid, (E,Z)-isomer; linoleate; linoleic acid, (E,E)-isomer; linoleic acid, (Z,E)-isomer; linoleic acid, (Z,Z)-isomer; linoleic acid, (Z,Z)-isomer; linoleic acid, (Z,Z)-isomer; linoleic acid, (Z,Z)-isomer; linoleic acid, (Z
  • Linoleic acid is an essential polyunsaturated fatty acid (PUFA) that must be supplied by the diet since it cannot be synthesized by the body. Rich sources of linoleic acid include nuts, fatty seeds, and their derived vegetable oils, such as safflower, sunflower, corn, and soybean oils. Linoleic acid has essential functions in maintaining the epidermal water barrier of the skin. Linoleic acid is also important as a precursor of the long chain PUFAs (LCPUFAs) that are incorporated into cell membranes in the form of phospholipids and other lipid components which maintain normal membrane fluidity, structure, and function.
  • PUFA polyunsaturated fatty acid
  • Omega-6, or n-6, fatty acids are derived from linoleic acid and serve as storage fatty acids in adipose tissue, and in liver, kidney, and muscle cells. Linoleic acid-derived fatty acids found in cell membranes also have interactive roles with regulatory proteins that are important for cell metabolism and signaling.
  • Arachidonic Acid is also known as: (5Z,8Z,11Z,14Z)-icosa-5,8,11 ,14- tetraenoic acid; (all-Z)-5,8,11 ,14-eicosatetraenoic acid; c/s-5,8,11 ,14-eicosatetraenoic acid; arachidonate; and vitamin F.
  • the molecular formula for arachidonic acid is C20H32O2.
  • Arachidonic acid has the following structure:
  • Arachidonic acid is an important LCPUFA produced from linoleic acid, and it is a major cell membrane fatty acid and a precursor of certain types of eicosanoids.
  • the eicosanoids are a diverse group of 20-carbon substances that are produced and released from cell membranes in response to physical or chemical trauma. Eicosanoids have local effects upon immune and inflammatory responses.
  • the four primary types of eicosanoids are the prostaglandins, prostacyclins, thromboxanes, and leukotrienes.
  • a number of enzymes metabolize arachidonic acid to these eicosanoids and eicosanoid metabolites.
  • the enzymes cyclooxygenase- 1 and cyclooxygenase-2 metabolize arachidonic acid to prostaglandin G2 and prostaglandin H2, which in turn may be converted to various prostaglandins, prostacyclin, and thromboxanes.
  • the enzyme arachidonate 5-lipoxygenase metabolizes arachidonic acid to 5- hydroperoxyicosatetraenoic acid (5-HPETE), which in turn is metabolized to various leukotrienes, as well as to 5-hydroxyicosatetraenoic acid (5-HETE).
  • arachidonate 15-lipoxygenase-1 and arachidonate 15-lipoxygenase-2 metabolize arachidonic acid to 15- hydroperoxyicosatetraenoic acid (15-HPETE), which may then be further metabolized to 15- hydroxyicosatetraenoic acid (15-HETE) and lipoxins.
  • the enzyme arachidonate 12- lipoxygenase metabolizes arachidonic acid to 12-hydroperoxyeicosatetraenoic acid (12-HPETE) which may then be metabolized to 12-hydroxyeicosatetraenoic acid (12-HETE) and to hepoxilins.
  • Arachidonic acid is found in animal and human fat as well as in the liver, brain, skeletal muscle, and glandular organs, and is a constituent of animal phosphatides.
  • 12-hydroxyeicosatetraenoic acid (12-HETE).
  • the "S" stereoisomer of 12-HETE is also known as: (5Z,8Z,10E,12S,14Z)-12-hydroxyicosa-5,8,10,14-tetraenoic acid; 12(S)-hydroxy- (5Z,8Z, 10E, 14Z)-eicosatetraenoic acid; (S)-12-HETE; 12(S)-HETE; and 12S-HETE.
  • the "R” stereoisomer of 12-HETE, 12(R)-hydroxy-5Z,8Z,1QE,14Z-eicosatetraenoic acid is also known as 12(R) ⁇ HETE or 12R-HETE 12-HETE, as used in the present disclosure, refers to the“S” stereoisomer of 12-HETE, the“R” stereoisomer of 12-HETE, or both stereoisomers of 12-HETE.
  • the molecular formula for 12-HETE is C20H32O3.
  • 12S-HETE has the following structure:
  • the "S” stereoisomer is made by arachidonate 128-lipoxygenase in platelets.
  • the "R" stereoisomer, 12(R)-hydroxy- 5Z,8Z.10E,14Z-eicosatetraenoic acid also termed 12(R) ⁇ HETE or 12R-HETE
  • 12(R) ⁇ HETE or 12R-HETE made by other tissues through their arachidonate 12R-!ipoxygenase enzyme.
  • the two isomers either directly or after being further metabolized, have been suggested to be involved in a variety of human physiological and pathological conditions including inflammation.
  • 12S-HETE has been implicated in cancer, diabetes, and high blood pressure. These arachidonic acid metabolites may act locally to regulate the behavior of ceils from which they originate or of nearby cells.
  • Lysophosphatidylserine is a lysophospholipid mediator that is derived from hydrolysis of an acyl chain (deacylation) from the phospholipid phosphatidylserine (PS) by phosphatidylserine-specific phospholipase A1 (PS- PLAi) or phospholipase A2 (PS-PLA2) enzymes at the sn- 1 and sn-2 positions of PS, respectively.
  • PS- PLAi phosphatidylserine-specific phospholipase A1
  • PS-PLA2 phospholipase A2
  • LysoPS is upregulated by various inflammatory stimuli. LysoPS has been detected after injury to tissues (tumor growth, graft rejection, burns). When cells are damaged, lysoPS can be generated by a reaction dependent on activation of the NADPH oxidase. It may play a role in resolution of inflammation by enhancing clearance of activated and dying neutrophils. In particular, sn- 2-lysoPS stimulates degranulation of mast cells. LysoPS may have cell signaling functions, for example, in regulating calcium flux and stimulating immune cells through specific receptors that have been identified in mice and humans.
  • lysoPS species tend to organize in non-bilayer structures and are believed to facilitate folding of certain membrane proteins in situ better than bilayer-forming lipids.
  • LysoPS species vary by acyl chain length and saturation, among which the 16:0, 18:0, and 18:1 isoforms are the most abundant in brain, heart, kidney, and lung tissues.
  • PS-PLA1, ABHD6, and ABHD12 can catalyze the degradation of lysoPS, and genetic deficiencies in the latter two enzymes have been linked to metabolic syndrome and inflammatory neurodegenerative disease, respectively. LysoPS also has been linked to certain cancers and to night blindness.
  • LysoPS 18:0 Lysophosphatidylserine 18:0 is also known as: lysoPS; lysophosphatidyl- L-serine; 1 -octadecanoyl-sn-glycero-3-phosphoserine; 1 -stearoyl-sn-glycero-3-phosphoserine; 1-stearoyl-sn-glycero-3-phospho-L-serine; 1-octadecanoyl-sn-glycero-3-phospho-L-serine; O- ⁇ hydroxy[(2R)-2-hydroxy-3-(octadecanoyloxy)propoxy]phosphoryl ⁇ -L-serine; 1-stearoyl-2- hydroxy-sn-glycero-3-phospho-L-serine and (2S)-2-amino-3-[hydroxy-[(2R)-2-hydroxy-3- octadecanoyloxyprop
  • LysoPS 18:1 is also known as: 1-oleoyl-sn-glycero-3- phosphoserine; 1-(9Z)-octadecenoyl-sn-glycero-3-phosphoserine; 1-(9Z-octadecenoyl)-sn- glycero-3-phospho-L-serine; 1-Ci 8:i (w-9)-lysophosphatidylserine; and 1-oleoyl (18:1) lysoPS.
  • the molecular formula for LysoPS 18:1 is C24H46NO9P.
  • LysoPS 18:1 has the following structure. The 18:1 denotes that the remaining acyl group contains 18 carbons and one double bond:
  • Lysophosphatidylserine 20:4 is also known as: 1-(5Z,8Z,11Z,14Z- eicosatetraenoyl)-sn-glycero-3-phosphoserine, (2S)-2-amino-3-[hydroxy-[(2R)-3-
  • LysoPS 20:4 has the following structure. The 20:4 denotes that the remaining acyl group contains 20 carbons and four double bonds:
  • Cysteine is also known as L-cysteine.
  • the molecular formula for cysteine is C 3 H7NO2S. Cysteine has the following structure:
  • Cysteine is a non-essential thiol-containing amino acid in humans.
  • the enzyme cystathionine b-synthase catalyzes condensation of homocysteine with serine to form cystathionine, which is deaminated and hydrolyzed by cystathionine b-lyase to form cysteine and a-ketobutyrate.
  • Cysteine has numerous biological functions due to the nucleophilic nature of its thiol group. The formation of disulfide linkages between the thiol groups of cysteine residues helps to stabilize the tertiary and quaternary structure of proteins. Cysteine is important for protein synthesis, detoxification, and diverse metabolic functions.
  • cysteine is important in collagen production, skin elasticity, and skin texture.
  • Cysteine is important in the manufacture of amino acid taurine and plays a role in the metabolism of essential biochemicals such as coenzyme A, heparin, and biotin. Cysteine is the limiting precursor of the major intracellular antioxidant glutathione. Cysteine, homocysteine and other aminothiols exist in plasma in reduced, oxidized, and protein-bound forms, interacting with each other through redox pathways. Individuals with lower cysteine levels are more prone to damage from reactive oxygen species, which are generally removed either by thiols or by glutathione-linked enzymes. An elevated level of total cysteine can predict adverse outcomes such as cardiovascular diseases and metabolic syndromes. Oxidized cysteine is known as cystine.
  • Protein bound Cysteine (P-ss-Cys). Protein bound Cysteine (P-ss-Cys) refers to cysteine covalently bound to a protein via a disulfide linkage.
  • Table 3 summarizes the primary and secondary biomarkers of the present disclosure.
  • the type of molecule For each primary biomarker, the following are indicated: the type of molecule; whether an increase or a decrease in the level of each primary biomarker predicts occurrence of CVE in HIV-infected subjects, and representative assays to detect each primary biomarker.
  • the type of molecule For each secondary biomarker, the following are indicated: the type of molecule; whether an increase or a decrease in the level of each secondary biomarker confirms that a subject is infected with HIV, and representative assays to detect each secondary biomarker. The representative assays are described below.
  • Biological samples of the present disclosure can include blood, plasma, serum, saliva, and urine.
  • a biological sample includes cells.
  • the biological sample is plasma.
  • the plasma is depleted of high abundant proteins including albumin, IgG, a1 -antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, a2-macroglobulin, a1-acid glycoprotein, IgM, apolipoprotein A1 , apolipoprotein All, complement C3 and transthyretin.
  • the plasma is depleted of high abundant proteins including albumin, IgG, IgA, transferrin, haptoglobin, and a1 -antitrypsin.
  • a control sample includes pooled normal plasma which can be obtained commercially from, e.g., George King Bio-Medical Inc. (#0010-0, Overland Park, KS), Precision BioLogic Inc. (CRYOcheckTM, #CCN-10, Dartmouth, Nova Scotia, CA), and Innovative Research (#IPLA-N, Novi, Michigan).
  • Pooled normal plasma (PNP) can include citrated plasma from at least 5, at least 10, at least 20, or at least 30 or more carefully screened human donors.
  • Individual donor units that form a PNP can be subjected to viral testing and only included in the PNP if found negative for, e.g., HBsAg, HCV, HIV-1 , HIV-2, HIV-1Ag or HIV 1-NAT, ALT, and syphilis by FDA-approved methods.
  • a PNP can be platelet poor with no buffers or preservatives.
  • a PNP can include approximately equal number of male and female donors. The donors for the PNP can be aged 18 to 66.
  • a Certificate of Analysis typically accompanies each shipment of PNP. This lot-specific Certificate of Analysis certifies the normalcy of the PNP satisfying regulatory guidelines.
  • Assay values can be assigned and reported on the Certificate of Analysis for Prothrombin Time (PT) and activated Partial Thromboplastin Time (aPTT). Fibrinogen, prothrombin and Factors V, VII, VIII, IX, X, XI and XII can be assayed and certified to be within the normal range.
  • PT Prothrombin Time
  • aPTT Partial Thromboplastin Time
  • a control sample includes sample(s) derived from subject(s) who do not have HIV infection and who do not have known cardiovascular disease at the time of blood donation or other provision of the sample(s).
  • Particular embodiments provide control samples derived from subjects who: do not have HIV infection; are gender-matched to HIV- infected subjects; are aged 24-33; are nonsmokers; do not have hypertension (systolic ⁇ 140 and diastolic ⁇ 80 mm Hg); are non-anemic; do not have a blood disorder or cancer; do not have a cold or influenza; are not taking any anti-coagulation, anti-platelet, or anti-inflammation medication; and/or have no known cardiovascular disease at the time of blood donation or other provision of the sample(s).
  • a control sample includes sample(s) derived from subject(s) who do not have HIV infection.
  • a control sample includes sample(s) derived from subject(s) who have HIV infection and who have had or who do have an adverse CVE at the time of blood donation or other provision of the sample(s).
  • a biological sample is derived from a subject or source.
  • derived from refers to a biological sample being obtained from a subject or other source and including any modification to the sample, addition to the sample, or removal from the sample, as long as biomarkers of the present disclosure can be measured from the sample using the systems and methods of the present disclosure.
  • biomarkers Up- or down-regulation of the biomarkers, as indicated elsewhere herein for particular biomarkers, can be assessed by comparing a measured value (from a test or subject sample) to a relevant reference level. For example, the quantity of one or more biomarkers can be indicated as a value.
  • the value can be expressed numerically and result from assaying a sample, and can be derived, e.g., by measuring level(s) of the biomarker(s) in the sample by an assay performed in a laboratory, by measuring the ratio or ratios of the levels of two or more of the biomarkers, or from a dataset obtained from a provider such as a laboratory, or from a dataset stored on a server.
  • the biomarkers disclosed herein can be a protein biomarker, or a nucleic acid biomarker (e.g., gene encoding the protein biomarker) and/or a small molecule.
  • the value may be qualitative or quantitative.
  • the systems and methods provide a reading or evaluation, e.g., assessment, of whether or not the biomarker is present in the sample being assayed.
  • the systems and methods provide a quantitative detection of whether the biomarker is present in the sample being assayed, i.e. , an evaluation or assessment of the actual amount or relative abundance of the biomarker in the sample being assayed.
  • the quantitative detection may be absolute or, if the method is a method of detecting two or more different biomarkers in a sample, relative.
  • the term“quantifying” when used in the context of quantifying a biomarker in a sample can refer to absolute or to relative quantification.
  • Absolute quantification can be accomplished by inclusion of known concentration(s) of one or more control biomarkers and referencing, e.g., normalizing, the detected level of the biomarker with the known control biomarkers (e.g., through generation of a standard curve).
  • relative quantification can be accomplished by comparison of detected levels or amounts between two or more different biomarkers to provide a relative quantification of each of the two or more biomarkers, e.g., relative to each other.
  • the actual measurement of values of the biomarkers can be determined using any method known in the art.
  • a biomarker is detected by contacting a sample with reagents (e.g., antibodies or nucleic acid primers), generating complexes of reagent and biomarker(s), and detecting the complexes.
  • reagents e.g., antibodies or nucleic acid primers
  • the reagent can include a probe.
  • a probe is a molecule that binds a target, either directly or indirectly.
  • the target can be a biomarker, a fragment of the biomarker, or any molecule that is to be detected in particular embodiments, the probe includes a nucleic acid or a protein.
  • a protein probe can be an antibody.
  • An antibody can be a whole antibody or a binding fragment of an antibody.
  • a probe can be labeled with a detectable label. Examples of detectable labels include fluorescent chromophores, chemiluminescent emitters, dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and radioactive isotopes.
  • Protein detection includes detection of full-length proteins, mature proteins, pre proteins, polypeptides, isoforms, mutant forms, post-translationally modified proteins and variants thereof, and can be detected in any suitable manner.
  • the following biomarkers discussed herein are proteins: GPX-3, PF4, PBP, TSP-1 , KLKB1 , Factor V, Protein C, Protein C inhibitor, CRP, and P-ss-Cys.
  • Antibodies can be conjugated or immobilized to a solid support suitable for a diagnostic assay (e.g., beads such as protein A or protein G agarose, microspheres, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as passive binding.
  • Antibodies can be conjugated to detectable labels or groups such as radioisotopes (e.g., 35 S, 125 l, 131 1), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein, Alexa, green fluorescent protein, rhodamine) in accordance with known techniques.
  • a diagnostic assay e.g., beads such as protein A or protein G agarose, microspheres, plates, slides or wells formed from materials such as latex or polystyrene
  • detectable labels or groups such as radioisotopes (e.g., 35 S,
  • immunoassays include immunoblotting, immunoprecipitation, immunofluorescence, chemiluminescence, electro-chemiluminescence (ECL), and/or enzyme- linked immunosorbent assays (ELISA).
  • GPX-3, PF4, PBP, TSP-1 , KLKB1 , serotonin, factor V, protein C, protein C inhibitor, CRP, and P-ss-Cys can be measured by ELISAs or by immunoprecipitation.
  • biomarker levels can be assessed by a protein activity assay.
  • protein activity assays include protease assays, kinase assays, phosphatase assays, and reductase assays, among many others.
  • a protein activity assay could include, e.g., assays of oxidative damage (GPX-3); platelet aggregation (PF4); neutrophil activation (PBP); cell-to-matrix interactions (TSP-1); platelet activation (serotonin); etc.
  • Up- or down-regulation of genes also can be detected using, for example, cDNA arrays, cDNA fragment fingerprinting, cDNA sequencing, clone hybridization, differential display, differential screening, fluorescence resonance energy transfer (FRET) detection, liquid microarrays, PCR, RT-PCR, quantitative real-time RT-PCR analysis with TaqMan assays, molecular beacons, microelectric arrays, oligonucleotide arrays, polynucleotide arrays, serial analysis of gene expression (SAGE), and/or subtractive hybridization.
  • FRET fluorescence resonance energy transfer
  • nucleic acid sequences that correspond to nucleic acids encoding biomarkers can be used to construct primers and probes for detecting and/or measuring biomarker nucleic acids.
  • the following biomarkers discussed herein are considered to be measurable by measuring one or more nucleic acids: GPX-3, PF4, PBP, TSP-1 , KLKB1 , Factor V, Protein C, and Protein C inhibitor, and CRP.
  • Northern hybridization analysis using probes which specifically recognize one or more biomarker nucleic acid sequences can be used to determine gene expression.
  • expression can be measured using RT-PCR; e.g., polynucleotide primers specific for the differentially expressed biomarker mRNA sequences are used to reverse-transcribe the mRNA into complementary DNA, which is then amplified in PCR and can be visualized and quantified.
  • Biomarker RNA can also be quantified using, for example, other target amplification methods, such as transcription mediated amplification (TMA), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA), or signal amplification methods (e.g., bDNA), and the like.
  • TMA transcription mediated amplification
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Ribonuclease protection assays can also be used, using probes that specifically recognize one or more biomarker mRNA sequence
  • Proteins and nucleic acids can be conjugated to or immobilized on chips, such as microarray chips. See, for example, U.S. Pat. Nos. 5,143,854; 6,087,112; 5,215,882; 5,707,807; 5,807,522; 5,958,342; 5,994,076; 6,004,755; 6,048,695; 6,060,240; 6,090,556; and 6,040,138.
  • Microarray refers to a solid carrier or support that has a plurality of molecules bound to its surface at defined locations.
  • the solid carrier or support can be made of any material.
  • the material can be hard, such as metal, glass, plastic, silicon, ceramics, and textured and porous materials; or soft materials, such as gels, rubbers, polymers, and other non-rigid materials.
  • the material can also be nylon membranes, epoxy-glass and borofiuorate-glass.
  • the solid carrier or support can be flat, but need not be and can include any type of shape such as spherical shapes (e.g., beads or microspheres).
  • the solid carrier or support can have a fiat surface as in slides and micro-titer plates having one or more wells.
  • an array or microarray can include small molecules.
  • Binding to proteins or nucleic acids on microarrays can be detected by scanning the microarray with a variety of laser or CCD-based scanners, and extracting features with software packages, for example, Imagene (Biodiscovery, Hawthorne, CA), Feature Extraction Software (Agilent), Scanalyze (Eisen, M. 1999. SCANALYZE User Manual; Stanford Univ., Stanford, Calif. Ver 2.32.), or GenePix (Axon Instruments).
  • Imagene Biodiscovery, Hawthorne, CA
  • Feature Extraction Software Agilent
  • Scanalyze Ses, M. 1999. SCANALYZE User Manual; Stanford Univ., Stanford, Calif. Ver 2.32.
  • GenePix GenePix
  • levels, amounts, or ratios of lipids of the present disclosure can be measured by an enzyme-based assay that converts a lipid to an intermediate that can be detected by a probe.
  • levels, amounts, or ratios of biomarkers of the present disclosure can be measured by chromatography, a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.
  • the chromatography is liquid chromatography (LC), a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s).
  • Liquid chromatography includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC).
  • RPLC reverse phase liquid chromatography
  • HPLC high performance liquid chromatography
  • HTLC high turbulence liquid chromatography
  • RPLC reverse phase liquid chromatography
  • HPLC high performance liquid chromatography
  • HTLC high turbulence liquid chromatography
  • the following biomarkers discussed herein are contemplated as being measured using chromatography: serotonin, linoleic acid, arachidonic acid, 12-HETE, LysoPS 18:0, LysoPS 18:1 , and LysoPS 20:4.
  • the chromatography is gas chromatography (GC), a process in which a sample mixture is vaporized and injected into a stream of carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or a particulate solid and is separated into its component compounds according to the affinity of the compounds for the stationary phase.
  • GC gas chromatography
  • the chromatography is thin layer chromatography.
  • Thin layer chromatography separates compounds on a thin layer of adsorbent material typically including a coating of silica gel on a glass plate or plastic sheet.
  • levels, amounts, or ratios of biomarkers of the present disclosure can be measured by UV spectroscopy, which measures the attenuation of a beam of light after it passes through a sample or after the light is reflected from the sample surface.
  • the absorption measurements can be at a single wavelength of light or can be over a spectral range.
  • levels, amounts, or ratios of biomarkers of the present disclosure can be measured by capillary electrophoresis, which separates molecules in submillimeter diameter capillaries, microfluidic, or nanofluidic channels containing electrolyte solutions under the influence of an electric field.
  • Capillary electrophoresis can include gel electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic chromatography. Separated molecules appear as peaks with different retention times in an electropherogram, which reports detector response as a function of time.
  • MS mass spectrometry
  • A“mass spectrometer” generally includes an ionizer and an ion detector. See, e.g., US 6,204,500; US 6,107,623; US 6,268,144; US 6,124,137; Wright et al. , Prostate Cancer and Prostatic Diseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis 21 :1164-67 (2000).
  • Samples may be processed or purified to obtain preparations that are suitable for analysis by mass spectrometry.
  • Such purification will usually include chromatography, such as liquid chromatography, and may also often involve an additional purification procedure that is performed prior to chromatography.
  • chromatography such as liquid chromatography
  • Various procedures may be used for this purpose depending on the type of sample or the type of chromatography. Examples include filtration, extraction, precipitation, centrifugation, delipidization, dilution, combinations thereof and the like.
  • Protein precipitation is one preferred method of preparing a liquid biological sample, such as serum or plasma, for chromatography.
  • Such protein purification methods are well known in the art, e.g., Poison et al., Journal of Chromatography B 785: 263-275 (2003).
  • Protein precipitation may be used to remove most of the protein from the sample leaving compounds of interest soluble in the supernatant.
  • the samples can be centrifuged to separate the liquid supernatant from the precipitated proteins.
  • the resultant supernatant can then be applied to liquid chromatography and subsequent mass spectrometry analysis.
  • the protein precipitation involves adding one volume of the liquid sample (e.g. plasma) to four volumes of methanol.
  • the method involves (1) performing a protein precipitation of the sample of interest; and (2) loading the supernatant directly onto an HPLC- mass spectrometer.
  • Embodiments disclosed herein can be used with high throughput screening (HTS).
  • HTS refers to a format that performs at least 100 assays, at least 500 assays, at least 1000 assays, at least 5000 assays, at least 10,000 assays, or more per day.
  • assays either the number of samples or the number of protein, nucleic acid, or other biomarkers assayed can be considered.
  • HTS methods involve a logical or physical array of either the subject samples, or the protein or nucleic acid biomarkers, or both.
  • an array can include small molecules.
  • Appropriate array formats include both liquid and solid phase arrays.
  • assays employing liquid phase arrays e.g., for hybridization of nucleic acids, binding of antibodies or other receptors to ligand, etc., can be performed in multiwell or microtiter plates.
  • Microtiter plates with 96, 384, or 1536 wells are widely available, and even higher numbers of wells, e.g., 3456 and 9600 can be used.
  • the choice of microtiter plates is determined by the methods and equipment, e.g., robotic handling and loading systems, used for sample preparation and analysis.
  • HTS assays and screening systems are commercially available from, for example, Zymark Corp. (Hopkinton, MA); Air Technical Industries (Mentor, OH); Beckman Instruments, Inc. (Fullerton, CA); Precision Systems, Inc. (Natick, MA), etc. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide HTS as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the various methods of HTS.
  • a "dataset” as used herein is a set of numerical values resulting from evaluation of a sample (or population of samples) under a desired condition. The values of the dataset can be obtained, for example, by experimentally obtaining measures from a sample and constructing a dataset from these measurements.
  • the reference level can be based on e.g., any mathematical or statistical formula useful and known in the art for arriving at a meaningful aggregate reference level from a collection of individual datapoints; e.g., mean, median, median of the mean, etc.
  • a reference level or dataset to create a reference level can be obtained from a service provider such as a laboratory, or from a database or a server on which the dataset has been stored.
  • a dataset of values is determined by measuring biomarkers from a healthy population which can provide a quantitative measure of adverse CVE risk in a subject.
  • Reference levels can be obtained from one or more relevant datasets.
  • a reference level from a dataset can be derived from previous measures derived from a population.
  • a "population" is any grouping of subjects or samples of like specified characteristics. The grouping could be according to, for example, clinical parameters, clinical assessments, therapeutic regimens, disease status, severity of condition, etc.
  • Reference levels can include "normal” or “control” levels or values, defined according to, e.g., discrimination limits or risk defining thresholds, in order to define cut-off points and/or abnormal values for adverse CVE risk.
  • the reference level then is the level of one or more biomarkers or combined biomarker indices typically found in a subject who is not infected with HIV.
  • Other terms for “reference levels” include “index,” “baseline,” “standard,” “healthy,” “uninfected,”“normal,” etc. Such normal levels can vary, based on whether a biomarker is used alone or in a formula combined with other biomarkers to produce a score.
  • the reference level can be a database of biomarker patterns from previously tested subjects who were not infected with HIV over a clinically relevant time period.
  • Reference levels can also be derived from, e.g., a control subject or population whose infection status is known.
  • the reference value can be derived from one or more subjects who have been exposed to HIV, or from subjects who have shown improvements in biomarker levels disclosed herein as a result of exposure to a treatment.
  • the reference level can be derived from one or more subjects who have not been exposed to treatment.
  • a reference level is an average of levels of a primary biomarker from a group of subjects who have HIV and who have had or who do have an adverse CVE.
  • a reference level is an average of levels of a secondary biomarker from a group of subjects who are not infected with HIV.
  • a reference level can also be derived from disease activity algorithms or computed indices from population studies.
  • “reference level” can refer to a standardized value for a biomarker disclosed herein which represents a level not associated with any infection; a level associated with recent infection (e.g., less than 2 years); or a level associated with a chronic infection (e.g., more than 2 years).
  • the reference level can be a universal reference level which is useful across a variety of testing locations.
  • the reference level and/or reference weighted score is derived from (i) an individual who is characterized as not having cardiovascular disease; (ii) a group of individuals who are characterized as not having cardiovascular disease; (iii) plasma from an individual or pooled plasma from a group of individuals characterized as not having cardiovascular disease.
  • Characterization of subjects as having or not having cardiovascular disease can include consideration of: any symptoms of cardiovascular disease (e.g., fainting, slow or fast heartbeat, chest tightness, chest pain, shortness of breath, and/or sudden swelling in legs, feet, ankles or abdomen); physical exam and blood tests to measure, e.g., total cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides and/or CRP; results from non-invasive tests including blood pressure test, an electrocardiogram, an echocardiogram, stress test, carotid ultrasound, Holter monitor to continuously monitor heart abnormalities, a chest X-ray, a tilt table test, a CT scan, and/or heart magnetic resonance imaging (MRI); results from invasive procedures including coronary angiography and cardiac catheterization and/or an electrophysiology study; family history of heart disease; history of smoking; presence or absence of obesity; diet; age; and/or lifestyle.
  • any symptoms of cardiovascular disease e.g., fainting, slow or fast heartbeat
  • the reference level and/or reference weighted score is derived from (i) an individual who is not infected with HIV; (ii) a group of individuals who are not infected with HIV; (iii) a subject before diagnosis with HIV; (iv) a subject at the time of diagnosis, at the beginning of a treatment regimen for HIV infection or at particular time points during a treatment; (v) pooled normal plasma (PNP) available commercially from, e.g., George King Bio-Medical Inc. (#0010-0, Overland Park, KS), Precision BioLogic Inc.
  • PNP pooled normal plasma
  • obtained biomarker values can be compared to a reference level, and conclusions can be drawn based on whether a sample value is statistically significantly different or not statistically significantly different from a reference level.
  • a measure is not statistically significantly different if the difference is within a level that would be expected to occur based on chance alone. In contrast, a statistically significant difference or increase is one that is greater than what would be expected to occur by chance alone.
  • Statistical significance or lack thereof can be determined by any of various methods well-known in the art. Examples of commonly used measures of statistical significance include the t-test, the p-value, and other tests described herein.
  • the p-value represents the probability of obtaining a given result equivalent to a particular datapoint, where the datapoint is the result of random chance alone. A result is often considered significant (not random chance) at a p-value less than or equal to 0.05.
  • a patient can be considered to be at risk if at least one of PF4, PBP, and TSP-1 are significantly elevated, provided that at least nine of the disclosed secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, and the remaining secondary biomarkers are selected from serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and/or P-ss- Cys, give confirmatory results for HIV infection.
  • a patient is considered to be at risk for CVE based on the statistically significant elevation of at least two primary biomarkers, provided that at least nine of the disclosed secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, and the remaining secondary biomarkers are selected from serotonin, factor V, PROC, PCI, CRP, LA, AA, 12- HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and/or P-ss-Cys, give confirmatory results for HIV infection; combinations of such primary biomarkers include: PF4 and PBP; PF4 and TSP-1 ; and PBP and TSP-1.
  • a patient is considered to be at risk for CVE based on the significant elevation of PF4, PBP, and TSP-1 , provided that at least nine of the disclosed secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, and the remaining secondary biomarkers are selected from serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and/or P-ss- Cys, give confirmatory results for HIV infection.
  • “Significantly elevated” in various embodiments refers to an increase of more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, or more than 200% compared to a reference level.
  • a patient is considered to be at risk for CVE if GPX-3 or KLKB1 is significantly down-regulated (or significantly reduced or significantly decreased), provided that at least nine of the disclosed secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, and the remaining secondary biomarkers are selected from serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and/or P-ss-Cys, give confirmatory results for HIV infection.
  • a significant down-regulation of GPX-3 and/or KLKB1 is incorporated into any of the biomarker groupings noted above in order to identify a patient as at risk for an adverse CVE.
  • “Significantly down-regulated” can refer to a decrease of more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 150%, or more than 200% compared to a reference level.
  • Table 1 above shows the change in level of a primary biomarker in a subject that is indicative (or not indicative) of whether the subject has an increased risk of an adverse CVE.
  • Particular embodiments may also include measuring the levels of at least nine secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4.
  • levels of 9, 10, 11 , 12, or 13 secondary biomarkers, where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, are measured in conjunction with particular sets of primary biomarkers described above.
  • levels of 9 secondary biomarkers are measured in conjunction with particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, P-ss-Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, P-ss-Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12- HETE, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, P-ss-Cys, and lysoPS 20:4 serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, 12- HETE, P-ss-Cys, and lysoPS 18:1 serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, P-ss- Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, P-ss-Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, P-ss-Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, P-ss-Cys, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, P-ss-Cys, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, P-ss-Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, P-ss-Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, P-ss-Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, P-ss-Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, P-ss-Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, P-ss-Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, 12- HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, P-ss-Cys, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • levels of 10 secondary biomarkers are measured in conjunction with particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 18:1 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4 are added to particular sets of primary biomarkers described above.
  • PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • PROC, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, CRP, LA, AA, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 20:4, Cys, and P-ss- Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , Cys, and P-ss- Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys are added to particular sets of primary biomarkers described above.
  • serotonin, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, 12- HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys are added to particular sets of primary biomarkers described above.
  • levels of serotonin, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys are measured in conjunction with levels of GPX-3 and PF4.
  • levels of PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are measured in conjunction with levels of GPX-3 and KLKB1.
  • serotonin, factor V, PROC, PCI, CRP, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, LA, AA, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • levels of 11 secondary biomarkers are measured in conjunction with particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • levels of serotonin, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are measured in conjunction with levels of TSP-1.
  • serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys are added to particular sets of primary biomarkers described above.
  • levels of serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys are measured in conjunction with levels of GPX-3, PF4, PBP, TSP-1 , and KLKB1.
  • serotonin, PROC, PCI, CRP, AA, 12- HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • the levels of serotonin, PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are measured in conjunction with the levels of GPX-3, PBP, and KLKB1.
  • Other combinations of 11 of the 13 secondary biomarkers can be discerned by one of ordinary skill in the art.
  • levels of 12 secondary biomarkers are measured in conjunction with particular sets of primary biomarkers described above.
  • factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss- Cys are added to particular sets of primary biomarkers described above.
  • serotonin, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • levels of serotonin, factor V, PROC, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are measured in conjunction with levels of PF4 and TSP-1.
  • serotonin, factor V, PROC, PCI, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:1 , lysoPS 20:4, Cys, and P- ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 20:4, Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , Cys, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and P-ss-Cys are added to particular sets of primary biomarkers described above.
  • serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys are added to particular sets of primary biomarkers described above.
  • levels of 13 secondary biomarkers where at least one of the secondary biomarkers is lysoPS 18:1 or lysoPS 20:4, are measured in conjunction with particular sets of primary biomarkers described above.
  • levels of serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are measured in conjunction with levels of GPX-3 and PBP.
  • levels of serotonin, factor V, PROC, PCI, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, Cys, and P-ss-Cys are measured in conjunction with levels of PF4, PBP, TSP-1 , and KLKB1.
  • Table 2 shows the change in level of each secondary biomarker that is indicative (or not indicative) of an HIV infection in a subject.
  • values obtained about the biomarkers and/or other dataset components can be subjected to an analytic process with chosen parameters.
  • the parameters of the analytic process may be those disclosed herein or those derived using guidelines described herein.
  • the analytic process used to generate a result may be any type of process capable of providing a result useful for classifying a sample, for example, comparison of the obtained value with a reference level, a linear algorithm, a quadratic algorithm, a decision tree algorithm, or a voting algorithm.
  • the analytic process may set a threshold for determining the probability that a sample belongs to a given class. The probability preferably is at least at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or higher.
  • Interpretation functions means the transformation of a set of observed data into a meaningful determination of particular interest; e.g., an interpretation function may be a predictive model that is created by utilizing one or more statistical algorithms to transform a dataset of observed biomarker data into a meaningful determination of adverse CVE risk in an HIV-infected subject.
  • values of the detected biomarkers can be calculated into a score.
  • Each value can be weighted evenly within an algorithm generating a score, or the values for particular biomarkers can be weighted more heavily in reaching the score.
  • biomarkers with higher sensitivity and/or specificity can be weighted more heavily than biomarkers with lower sensitivity and/or specificity to determine scores.
  • Biomarkers may also be grouped into classes, and each class given a weighted score.
  • primary biomarker values for diagnosing a patient with increased risk of an adverse CVE may be grouped into classes and weighted as follows (from highest weight to lowest weight): Class 1 : PF4, PBP, and TSP-1 ; and Class 2: GPX-3 and KLKB1.
  • Secondary biomarker values for confirming whether a patient is infected with HIV may be grouped into classes and weighted as follows (from highest weight to lowest weight): Class 1 : serotonin and CRP; Class 2: factor V, PROC, and PCI; Class 3: linoleic acid, arachidonic acid, and 12-HETE; Class 4: lysoPS 18:0, lysoPS 18:1 , and lysoPS 20:4; and Class 5: Cys and P-ss- Cys.
  • weighting scores involves converting the measurement of one biomarker that is identified and quantified in a test sample into one of many potential scores.
  • a receiver operating characteristic (ROC) curve can be used to standardize the scoring between different biomarkers by enabling the use of a weighted score based on the inverse of a false positive % or false negative % defined from the ROC curve.
  • the weighted score can be calculated by multiplying the AUC by a factor for a biomarker and then dividing by the false positive % or false negative % based on a ROC curve.
  • the weighted score can be calculated using the formula:
  • Weighted Score (AUC x X factor)/(1-% specificity x )
  • x is the biomarker
  • the, "factor” is a real number (such as 0, 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 and so on) throughout a particular set of biomarkers; and the specificity is a chosen value that does not exceed 95%. Multiplication of a factor for the particular set of primary biomarkers allows the user to scale the weighted score.
  • a sample e.g., blood, plasma, serum, saliva, urine, a sample including cells
  • testing the sample for up- or down-regulation of one or more biomarkers disclosed herein.
  • the testing includes measuring the level(s) of the one or more biomarkers disclosed herein using one or more of mass spectrometry, immunoassay, chromatography, spectroscopy, electrophoresis, or a combination thereof.
  • methods disclosed herein include obtaining a biological sample derived from an HIV-infected subject; assaying the sample for up- or down-regulation of one or more biomarkers disclosed herein; determining one or more biomarker values based on the assaying; comparing the one or more biomarker values to a reference level; diagnosing or identifying the HIV-infected subject as having an increased risk for an adverse CVE or not at increased risk for an adverse CVE according to the up- or down regulation of biomarkers, as described elsewhere herein.
  • identifying an HIV-infected subject as having an increased risk for an adverse CVE or not having an increased risk for an adverse CVE includes marking a biological sample appropriately so that the HIV-infected subject (from whom the marked biological sample was derived) receives or does not receive a selected, appropriate treatment or disease management procedure or protocol.
  • the marking can include physically marking a container that the biological sample resides in with appropriate information, or entering such appropriate information about the biological sample into a computer.
  • a biological sample can be assessed 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times and every remaining integer up to 100 times or more.
  • Particular embodiments include monitoring HIV-infected patients for levels of one or more biomarkers described herein over a period of time.
  • an HIV patient is selected for adverse CVE risk screening according to the systems and methods disclosed herein because they have been identified as a patient that would benefit from monitoring for risk of occurrence of CVE.
  • Patients who could benefit from monitoring for risk of occurrence of CVE can include those who have been infected with HIV for more than 2 years and/or have been on retroviral therapies for more than 2 years.
  • patients who could benefit from monitoring for risk of occurrence of CVE can include those who have been infected with HIV for more than 6 months and/or have been on retroviral therapies for more than 6 months.
  • patients who could benefit from monitoring for risk of occurrence of CVE can include those who have been infected with HIV for more than 1 year and/or have been on retroviral therapies for more than 1 year.
  • patients who could benefit from monitoring for risk of occurrence of CVE can include those who have been infected with HIV for more than 5 years and/or have been on retroviral therapies for more than 5 years.
  • biological samples can be obtained and assessed from an HIV-infected individual subject weekly, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, or every 11 months, yearly, or at longer spaced periods, to determine if the subject has or has developed an increased risk of an adverse CVE.
  • a diagnosis or identification of an HIV-infected subject at increased risk for adverse CVE can direct a treatment regimen and inform disease management.
  • the results of the methods can be used for clinical decision and support, such as determining whether to defer intervention or treatment, to recommend preventive check-ups (or the frequency of such check-ups) for at-risk patients, to recommend increased (or decreased) visit frequency, to recommend increased (or decreased) testing, and/or to recommend intervention (including in some embodiments the type, frequency, duration, etc. of such intervention).
  • the results of the methods can also be useful for selection of therapy, determining response to treatment, adjustment and dosing of treatment, monitoring ongoing therapeutic efficiency, and indication for change in therapeutic regimens.
  • Adverse CVEs can include nonfatal stroke, nonfatal myocardial infarction, heart failure, malignant dysrhythmia, cardiac shock, and cardiovascular death.
  • a stroke is a medical condition in which poor blood flow to the brain results in cell death.
  • Two main types of stroke include: ischemic, referring to lack of blood flow; and hemorrhagic, referring to bleeding.
  • Myocardial infarction (Ml) is commonly known as a heart attack and occurs when blood flow decreases or stops to a part of the heart, causing damage to the heart muscle.
  • Heart failure also referred to as congestive heart failure, occurs when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs.
  • Cardiac (or cardiogenic) shock is a condition in which the heart suddenly can't pump enough blood to meet the body's needs. The condition is most often caused by a severe heart attack, but not everyone who has a heart attack has cardiogenic shock.
  • Treatments to prevent cardiovascular events include the use of aspirin; anti-hypertensive drugs such as beta-blockers (e.g., propranolol, timolol and metoprolol), angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers, calcium-channel blockers, thiazide-like diuretics, alpha-blockers, central alpha-agonists, and peripheral alpha-agonists; lipid lowering drugs such as statins, fibrates, and nicotinic acid; and modification of lifestyle related risk behaviors. Lifestyle changes include smoking cessation, reduction in alcohol consumption, weight control, improvements in diet, and increase in physical activity.
  • beta-blockers e.g., propranolol, timolol and metoprolol
  • ACEI angiotensin converting enzyme inhibitors
  • angiotensin receptor blockers calcium-channel blockers
  • thiazide-like diuretics alpha-blockers
  • biological samples can be obtained and assessed from an individual patient weekly, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, or every 11 months, or yearly to determine if the increased risk of an adverse CVE has progressed, regressed, or has been successfully or unsuccessfully treated.
  • “stable” measures are measures evaluated in relation to a previous comparison in the same patient and denote a stable biomarker level that has not changed significantly (as determined by a statistical measure known in the art such as a t-test or p-value, e.g., p value >0.05) since the last measurement.
  • “stable” measures are measures evaluated in relation to a previous comparison in the same patient and denote a biomarker level that has not changed significantly (as determined by a statistical measure known in the art such as a t-test or p-value, e.g., p value >0.05) since an aggregated or averaged group of previous measurements (e.g., the last 3, 4, or 5 measurements).
  • “Unchanged” measures are measures evaluated in relation to a previous comparison in the same patient and denote a failure to achieve a statistically significant change (e.g., as determined by a statistical measure known in the art such as a t-test or p-value, e.g., p value >0.05) in a score towards or away from a reference level in the particular subject.
  • “unchanged” measures are measures that have not changed in relation to a previous measurement in the same patient or since an aggregated or averaged group of previous measurements in the patient (e.g., the last 3, 4, or 5 measurements).
  • kits include material(s) and reagent(s) necessary to assay a sample obtained from a subject for one or more biomarkers disclosed herein.
  • the materials and reagents can include those necessary to assay the biomarkers disclosed herein according to any method described herein and/or known to one of ordinary skill in the art.
  • kits include antibodies to biomarker proteins and/or aptamers, epitopes or mimotopes or antigens to bind antibodies.
  • kits additionally or alternatively include oligonucleotides that specifically assay for one or more biomarker nucleic acids based on homology and/or complementarity with biomarker nucleic acids. The oligonucleotide sequences may correspond to fragments of the biomarker nucleic acids.
  • the oligonucleotides can be more than 200, 175, 150, 100, 50, 25, 10, or fewer than 10 nucleotides in length.
  • a biomarker binding agent any molecule (e.g., antibody, aptamer, epitope, mimotope, oligonucleotide) that forms a complex with a biomarker is referred to as a biomarker binding agent herein.
  • the kits include reagents to measure up- or down-regulation of a small molecule biomarker in assays including liquid chromatography, gas chromatography, thin layer chromatography, UV spectroscopy, capillary electrophoresis, and/or mass spectrometry.
  • kits include reagents to measure up- or down-regulation of a biomarker in activity assays such as oxidation/reduction assays, platelet activation assays, platelet aggregation assays, neutrophil activation assays, endothelial cell activation assays, and/or cell-matrix interaction assays.
  • activity assays such as oxidation/reduction assays, platelet activation assays, platelet aggregation assays, neutrophil activation assays, endothelial cell activation assays, and/or cell-matrix interaction assays.
  • kits can contain in separate containers biomarker binding agents either bound to a matrix, or packaged separately with reagents for binding to a matrix.
  • the matrix is, for example, a porous strip.
  • measurement or detection regions of the porous strip can include a plurality of sites containing biomarker binding agents.
  • the porous strip can also contain sites for negative and/or positive controls. Alternatively, control sites can be located on a separate strip from the porous strip.
  • the different detection sites can contain different amounts of biomarker binding agents, e.g., a higher amount in the first detection site and lesser amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of biomarker present in the sample.
  • the detection sites can be configured in any suitably detectable size and shape and can be, e.g., in the shape of a bar or dot spanning the width (or a portion thereof) of a porous strip.
  • the matrix can be a solid substrate, such as a "chip.” See, e.g., U.S. Pat. No. 5,744,305.
  • the matrix can be a solution array; e.g., xMAP (Luminex, Austin, Tex.), Cyvera (lllumina, San Diego, Calif.), RayBio Antibody Arrays (RayBiotech, Inc., Norcross, Ga.), CellCard (Vitra Bioscience, Mountain View, Calif.) and Quantum Dots' Mosaic (Invitrogen, Carlsbad, Calif.).
  • Additional embodiments can include control formulations (positive and/or negative), and/or one or more detectable labels, such as fluorescein, green fluorescent protein, rhodamine, cyanine dyes, dialkylcarbocyanine dyes, Alexa dyes, luciferase, horseradish peroxidase, radiolabels, enzyme reporter, colorimetric label, chemiluminescent label, colored particles, gold nanoparticles, colloids, magnetic bead, or biotin among others.
  • Instructions for carrying out the assay including, optionally, instructions for generating a score, can be included in the kit; e.g., written, tape, VCR, or CD-ROM.
  • kits include materials and reagents necessary to conduct an immunoassay (e.g., ELISA).
  • the kits include materials and reagents necessary to conduct hybridization assays (e.g., PCR).
  • materials and reagents expressly exclude equipment (e.g., plate readers).
  • kits can exclude materials and reagents commonly found in laboratory settings (pipettes; test tubes; buffer solutions, distilled H2O).
  • biomarkers include the protein forms of the biomarkers as well as associated nucleic acids, oligonucleotides, and metabolites, together with their related metabolites, mutations, isoforms, variants, polymorphisms, modifications, fragments, subunits, degradation products, elements, and other analytes or sample-derived measures.
  • Biomarkers can also include mutated proteins, mutated nucleic acids, variations in copy numbers, and/or transcript variants.
  • amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e. , substitutions of similarly charged or uncharged amino acids.
  • a conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains.
  • Naturally occurring amino acids are generally divided into conservative substitution families as follows: Group 1 : Alanine (Ala), Glycine (Gly), Serine (Ser), and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3: (acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), Glutamine (Gin), Asp, and Glu; Group 4: Gin and Asn; Group 5: (basic; also classified as polar, positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (large aliphatic, nonpolar residues): Isoleucine (lie), Leucine (Leu), Methionine (Met), Valine (Val) and Cysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gin, Cys, Ser, and Thr; Group
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Variants of gene sequences can include codon optimized variants, sequence polymorphisms, splice variants, and/or mutations that do not affect the function of an encoded product to a statisti cally-significant degree.
  • Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.
  • % sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences as determined by the match between strings of such sequences.
  • Identity (often referred to as “similarity") can be readily calculated by known methods, including (but not limited to) those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
  • Variants also include nucleic acid molecules that hybridizes under stringent hybridization conditions to a sequence disclosed herein and provide the same function as the reference sequence.
  • Exemplary stringent hybridization conditions include an overnight incubation at 42 °C in a solution including 50% formamide, 5XSSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 pg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C.
  • 5XSSC 750 mM NaCI, 75 mM trisodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5XDenhardt's solution 10% dextran sulfate
  • 20 pg/ml denatured, sheared salmon sperm DNA followed by washing the filters in 0.1XSSC at 50 °C
  • Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5XSSC).
  • Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments.
  • Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • a method of identifying a subject infected with human immunodeficiency virus (HIV) as having an increased risk of an adverse cardiovascular event (CVE) or not having an increased risk of an adverse CVE including:
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • KLKB1 plasma kallikrein
  • the secondary biomarkers is lysophosphatidylserine 18:1 (lysoPS 18:1) or lysophosphatidylserine 20:4 (lysoPS 20:4) and the remaining secondary biomarkers are selected from the group consisting of serotonin; factor V; protein C (PROC); protein C inhibitor (PCI); C-reactive protein (CRP); linoleic acid (LA); arachidonic acid (AA); 12-hydroxyeicosatetraenoic acid (12-HETE); lysophosphatidylserine 18:0 (lysoPS 18:0); lysoPS 18:1 ; lysoPS 20:4; Cysteine (Cys); and protein bound Cysteine (P-ss-Cys);
  • the level of each of one or more of GPX-3 and KLKB1 is decreased compared to the corresponding first reference level
  • the level of each of one or more of PF4, PBP, and TSP-1 is increased compared to the corresponding first reference level
  • the level of each of at least one of lysoPS 18:1 or lysoPS 20:4 is increased compared to the corresponding second reference level
  • the level of each of at least one of serotonin, factor V, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and P-ss-Cys is increased compared to the corresponding reference level; and/or
  • the level of each of one or more of GPX-3 and KLKB1 is increased or unchanged as compared to the corresponding first reference level;
  • the level of each of one or more of PF4, PBP, and TSP-1 is decreased or unchanged as compared to the corresponding first reference level
  • the level of each of at least one of lysoPS 18:1 or lysoPS 20:4 is increased compared to the corresponding second reference level
  • the level of each of at least one of serotonin, factor V, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and P-ss-Cys is increased compared to the corresponding second reference level; and/or
  • the level of each of at least one of PROC, PCI, and Cys is decreased compared to the corresponding second reference level.
  • the first reference level is derived from a control sample including one or more individuals who have HIV and who have had or who have an adverse CVE.
  • control sample includes two or more individuals.
  • the first and/or second reference level includes an average of levels of biomarkers measured in the individuals. 6. The method of any one of embodiments 1-5, wherein the biological sample is plasma.
  • measuring includes measuring the levels of serotonin, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys.
  • a kit for diagnosing an HIV-infected subject with an increased risk of an adverse cardiovascular event including one or more binding agents, each binding agent able to bind a biomarker selected from glutathione peroxidase-3 (GPX-3); platelet factor 4 (PF4); platelet basic protein (PBP); thrombospondin-1 (TSP-1); plasma kallikrein (KLKB1); serotonin; factor V; protein C; protein C inhibitor; C-reactive protein (CRP); Cysteine (Cys); and protein bound Cysteine (P-ss-Cys).
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • KLKB1 plasma kallikrein
  • serotonin factor V
  • protein C protein C inhibitor
  • C-reactive protein C-reactive protein
  • Cysteine Cysteine
  • P-ss-Cys protein bound Cysteine
  • kits of embodiment 11 wherein the binding agents are proteins, antibodies, aptamers, mimotopes, or oligonucleotides.
  • kit of embodiment 11 or 12, further including a detectable label is provided.
  • the detectable label is a radioactive isotope, enzyme reporter, colorimetric label, fluorescent label, chemiluminescent label, colored particles, gold nanoparticles, colloids, magnetic bead, or biotin.
  • kit of any one of embodiments 11-14, wherein the kit includes reagents to perform amplification of nucleic acids, an immunoassay, an immunohistochemical staining, flow cytometry, an enzyme-based colorimetric assay, and/or a protein activity assay.
  • the protein activity assay includes an assay selected from an oxidation/reduction assay, a platelet aggregation assay, a platelet activation assay, an endothelial cell activation assay, and/or a cell-matrix interaction assay.
  • kit of any one of embodiments 11-16 further including reagents to detect a biomarker selected from linoleic acid (LA); arachidonic acid (AA); 12-hydroxyeicosatetraenoic acid (12- HETE); lysophosphatidylserine 18:0 (lysoPS 18:0); lysophosphatidylserine 18:1 (lysoPS 18:1); lysophosphatidylserine 20:4 (lysoPS 20:4); Cys; and P-ss-Cys.
  • a biomarker selected from linoleic acid (LA); arachidonic acid (AA); 12-hydroxyeicosatetraenoic acid (12- HETE); lysophosphatidylserine 18:0 (lysoPS 18:0); lysophosphatidylserine 18:1 (lysoPS 18:1); lysophosphatidylserine 20:4 (lys
  • kits of embodiment 17, wherein the reagents are used in liquid chromatography, gas chromatography, thin layer chromatography, UV spectroscopy, capillary electrophoresis, and/or mass spectrometry.
  • a method of treating or not treating an HIV-infected subject for an adverse cardiovascular event (CVE) including:
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • KLKB1 plasma kallikrein
  • the secondary biomarkers is lysophosphatidylserine 18:1 (lysoPS 18:1) or lysophosphatidylserine 20:4 (lysoPS 20:4) and the remaining secondary biomarkers are selected from the group consisting of serotonin; factor V; protein C (PROC); protein C inhibitor (PCI); C-reactive protein (CRP); linoleic acid (LA); arachidonic acid (AA); 12-hydroxyeicosatetraenoic acid (12-HETE); lysophosphatidylserine 18:0 (lysoPS 18:0); lysoPS 18:1 ; lysoPS 20:4; Cysteine (Cys); and protein bound Cysteine (P-ss-Cys);
  • the level of each of one or more of GPX-3 and KLKB1 is decreased compared to the corresponding first reference level
  • the level of each of one or more of PF4, PBP, and TSP-1 is increased compared to the corresponding first reference level
  • the level of each of at least one of lysoPS 18:1 or lysoPS 20:4 is increased compared to the corresponding second reference level
  • the level of each of at least one of serotonin, factor V, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and P-ss-Cys is increased compared to the corresponding reference level; and/or
  • the level of each of at least one of PROC, PCI, and Cys is decreased compared to the corresponding reference level;
  • the level of each of one or more of GPX-3 and KLKB1 is increased or unchanged as compared to the corresponding first reference level;
  • the level of each of one or more of PF4, PBP, and TSP-1 is decreased or unchanged as compared to the corresponding first reference level
  • the level of each of at least one of lysoPS 18:1 or lysoPS 20:4 is increased compared to the corresponding second reference level
  • the level of each of at least one of serotonin, factor V, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and P-ss-Cys is increased compared to the corresponding second reference level; and/or
  • the level of each of at least one of PROC, PCI, and Cys is decreased compared to the corresponding second reference level
  • the treating includes administering drugs selected from aspirin, anti-hypertensive drugs, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers, calcium-channel blockers, thiazide-like diuretics, alpha-blockers, central alpha-agonists, peripheral alpha-agonists, and lipid lowering drugs.
  • drugs selected from aspirin, anti-hypertensive drugs, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers, calcium-channel blockers, thiazide-like diuretics, alpha-blockers, central alpha-agonists, peripheral alpha-agonists, and lipid lowering drugs.
  • drugs selected from aspirin, anti-hypertensive drugs, angiotensin converting enzyme inhibitors (ACEI), angiotensin receptor blockers, calcium-channel blockers, thiazide-like diuretics, alpha-blockers, central alpha-agonists, peripheral alpha-agonists, and lipid
  • control sample includes two or more individuals.
  • first and/or second reference level includes an average of levels of biomarkers measured in the individuals.
  • a method of monitoring an HIV-infected subject for risk of an adverse cardiovascular event including:
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • KLKB1 plasma kallikrein
  • the secondary biomarkers is lysophosphatidylserine 18:1 (lysoPS 18:1) or lysophosphatidylserine 20:4 (lysoPS 20:4) and the remaining secondary biomarkers are selected from the group consisting of serotonin; factor V; protein C (PROC); protein C inhibitor (PCI); C-reactive protein (CRP); linoleic acid (LA); arachidonic acid (AA); 12-hydroxyeicosatetraenoic acid (12-HETE); lysophosphatidylserine 18:0 (lysoPS 18:0); lysoPS 18:1 ; lysoPS 20:4; Cysteine (Cys); and protein bound Cysteine (P-ss-Cys);
  • the level of each of one or more of GPX-3 and KLKB1 is decreased compared to the corresponding first reference level
  • the level of each of one or more of PF4, PBP, and TSP-1 is increased compared to the corresponding first reference level
  • the level of each of at least one of lysoPS 18:1 or lysoPS 20:4 is increased compared to the corresponding second reference level
  • the level of each of at least one of serotonin, factor V, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and P-ss-Cys is increased compared to the corresponding reference level; and/or the level of each of at least one of PROC, PCI, and Cys is decreased compared to the corresponding reference level;
  • the level of each of one or more of GPX-3 and KLKB1 is increased or unchanged as compared to the corresponding first reference level;
  • the level of each of one or more of PF4, PBP, and TSP-1 is decreased or unchanged as compared to the corresponding first reference level
  • the level of each of at least one of lysoPS 18:1 or lysoPS 20:4 is increased compared to the corresponding second reference level
  • the level of each of at least one of serotonin, factor V, CRP, LA, AA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and P-ss-Cys is increased compared to the corresponding second reference level; and/or
  • the level of each of at least one of PROC, PCI, and Cys is decreased compared to the corresponding second reference level
  • a method including:
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • KLKB1 plasma kallikrein
  • the biological sample for levels of nine or more secondary biomarkers wherein at least one of the secondary biomarkers is lysophosphatidylserine 18:1 (lysoPS 18:1) or lysophosphatidylserine 20:4 (lysoPS 20:4) and the remaining secondary biomarkers are selected from the group consisting of serotonin; factor V; protein C (PROC); protein C inhibitor (PCI); C-reactive protein (CRP); linoleic acid (LA); arachidonic acid (AA); 12- hydroxyeicosatetraenoic acid (12-HETE); lysophosphatidylserine 18:0 (lysoPS
  • testing includes measuring the level of the one or more primary biomarkers and measuring the levels of the nine or more secondary biomarkers.
  • the measuring includes measuring the levels of PF4, PPBP, TSP-1 , and KLKB1.
  • measuring includes measuring the levels of serotonin, PROC, PCI, CRP, LA, 12-HETE, lysoPS 18:0, lysoPS 18:1 , lysoPS 20:4, and Cys.
  • 35 The method of embodiment 34, wherein the plasma is depleted of one or more of albumin, IgG, a1 -antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, a2-macroglobulin, a1-acid glycoprotein, IgM, apolipoprotein A1 , apolipoprotein All, complement C3, and transthyretin.
  • An array including a biomarker selected from glutathione peroxidase-3 (GPX-3); platelet factor 4 (PF4); platelet basic protein (PBP); thrombospondin-1 (TSP-1); plasma kallikrein (KLKB1); serotonin; factor V; protein C; protein C inhibitor; C-reactive protein (CRP); and/or protein bound Cys.
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • KLKB1 plasma kallikrein
  • serotonin factor V
  • protein C protein C inhibitor
  • C-reactive protein C-reactive protein
  • An array including a biomarker selected from serotonin; linoleic acid (LA); arachidonic acid (AA); 12-hydroxyeicosatetraenoic acid (12-HETE); lysophosphatidylserine 18:0 (lysoPS 18:0); lysophosphatidylserine 18:1 (lysoPS 18:1); lysophosphatidylserine 20:4 (lysoPS 20:4); and/or Cys.
  • a biomarker selected from serotonin; linoleic acid (LA); arachidonic acid (AA); 12-hydroxyeicosatetraenoic acid (12-HETE); lysophosphatidylserine 18:0 (lysoPS 18:0); lysophosphatidylserine 18:1 (lysoPS 18:1); lysophosphatidylserine 20:4 (lysoPS 20:4); and/or Cys.
  • Example 1 This Example describes targeted metabolomic and proteomic profiles of two subsets of HIV patients with myocardial infarction (Ml) or ischemic stroke compared to corresponding matched controls.
  • Ml myocardial infarction
  • Study cohort included subjects from a national network of 8 HIV clinical care sites. The population is large and diverse with regard to sex, race/ethnicity, age, and transmission risk factor.
  • CVE HIV patients without cardiovascular events
  • proteomic analyses included 4 normal individuals, two HIV patients without CVE, and 3 HIV patients with CVE; two samples from each HIV patient were also analyzed.
  • lipid and oxylipin targets In targeted metabolomic studies, a panel of 47 lipid and oxylipin targets, as well as a panel of 68 targets representing metabolites from arginine and tryptophan metabolic pathways, phosphatidylcholine-related metabolites, and carnitine-related metabolites were analyzed.
  • targeted proteomic studies a panel of 32 plasma proteins indicative of oxidative stress, platelet activation, endothelial activation, inflammation, and thrombosis was analyzed. Additional protein targets were also quantified by ELISA.
  • Protein abundance was determined by peak area of unique peptides detected or spectral counts. For comparison, peak area was normalized to the mean. Reproducibility was monitored with isotopically labeled peptides ( ⁇ 5%).
  • isotopically labeled peptides ⁇ 5%.
  • polyunsaturated fatty acids, oxylipins, and lysophospholipids methods as described in previous publications were used (Fu et al. Transfusion. 2016;56(10):2560-2570; Fu et al. Transfusion. 2017;57(S3)). Briefly, metabolites in plasma containing isotopically labeled internal standards were extracted with 80% methanol.
  • Cystine (Cys-ss-Cys) is an oxidized form of cysteine, in which two cysteine molecules are linked by a disulfide bond.
  • Protein bound Cys (P-ss-Cys) is another oxidized form of cysteine, in which the cysteine is bound by a disulfide bond to another cysteine molecule that is in a protein. Elevation of both forms of oxidized cysteine is consistent with a state of oxidative stress in HIV-infected patients.
  • Targeted metabolomic analyses by mass spectrometry showed that two omega-6 polyunsaturated fatty acids, linoleic acid (LA) and arachidonic acid (AA), were elevated in HIV patients (FIGs. 7A, 7B), while the levels of two other polyunsaturated fatty acids EPA and DHA remained essentially unchanged (not shown). Elevated levels of omega-6 fatty acids including LA and AA correlate with proinflam matory conditions (Simopoulos, World Rev Nutr Diet. 2003;92:1-22) and elevated AA, an agonist for platelets, is prothrombotic.
  • LA linoleic acid
  • AA arachidonic acid
  • 12-HETE 12-hydroxyeicosatetraenoic acid
  • 12-LOX platelet arachidonic lipoxygenase 12
  • High 12-HETE levels correlate with an inflammatory state and pro-aggregation state, characterized by activation of neutrophils, monocytes, eosinophils and platelets.
  • levels of key oxidized polyunsaturated fatty acids may serve as biomarkers of oxidative stress in HIV patients.
  • Biomarkers of platelet activation Proteomic analysis of plasma depleted of the 14 most abundant proteins showed significantly increased levels of platelet factor 4 (PF4), platelet basic protein (PBP), and thrombospondin-1 (TSP-1) in HIV patients with CVE (FIGs. 2A-2C). These three proteins are normally stored in the a-granules of platelets and their increased levels in plasma indicate platelet activation and a-granule release in HIV patients with CVE. Consistent with platelet activation, metabolomic analysis showed increased levels of serotonin in HIV patients (FIG. 4). Since the plasma samples were collected from HIV patients prior to CVE, the elevated levels of serotonin correlate with HIV infection.
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TSP-1 thrombospondin-1
  • Serotonin is stored in dense granules of platelets and elevated serotonin levels in plasma are consistent with platelet activation and dense granule release.
  • Levels of lysoPS The levels of three lysoPS species were elevated in HIV patients (FIGs. 8A-8C). Increased plasma lysoPS levels are highly unusual as lysoPS is normally surface-bound and localized on activated platelet surfaces or apoptotic neutrophil surfaces. Increased lysoPS in plasma may reflect platelet activation (Frasch et al. Prog Lipid Res. 2012;51 (3):199-207), consistent with elevation of other markers of platelet activation described above.
  • Elevated lysoPS levels in plasma may also correlate with the unusual and persistent accumulation of activated neutrophils in the mucosal lining of the gut in HIV patients. This accumulation is suggestive of a clearance defect of apoptotic neutrophils by macrophages, which use lysoPS on apoptotic neutrophil surfaces as targets for efferocytosis (Frasch et al. infra).
  • Example 2 This Example describes how the biomarkers associated with CVE identified in Example 1 will be confirmed or how additional biomarkers associated with CVE will be identified.
  • Evaluation of a molecular cardiovascular profile in a cohort of HIV-infected patients will be used to confirm biomarkers and/or identify additional biomarkers associated with the occurrence of either myocardial infarction or ischemic stroke.
  • Molecular profiling will include unbiased plasma proteomics after removal of the 14 most abundant plasma proteins (listed above in Example 1), which may confound the molecular profiling studies.
  • Molecular profiling will also include quantitative proteomics of proteins involved in blood coagulation, platelet activation, and endothelial activation.
  • Molecular profiling will also include assessments of redox status through the measurement of reduced and oxidized forms of small molecule thiols in plasma, assessment of protein methionine oxidation, and plasma lipid oxidation. 64 cases and 128 controls will be analyzed at minimum.
  • Diagnoses include AIDS-defining diagnoses, non-AIDS malignancies, cardiovascular disease, kidney disease, diabetes, dyslipidemia, liver disease, hypertension, mental illness, and substance use.
  • Laboratory Data include plasma HIV-1 RNA levels, CD4 counts, and viral hepatitis, hematologic, and metabolic markers.
  • Medication Data include ART, anti-microbial, anti-hypertensive, diabetes, lipid lowering, psychiatric medications and transfusion.
  • Demographic Data include sex, race/ethnicity, age, and HIV transmission risk factors.
  • Utilization Data include primary care and specialty care visits and hospitalizations.
  • Vital Status Data include cause of death, death date, and information source.
  • Antiretroviral Resistance Data include full viral genotype, phenotype, and tropism assay results.
  • Patient- reported outcomes (PROs) are collected at the time of routine clinic appointments using web- based software. Domains obtained include substance use, tobacco, medication adherence, physical activity and others.
  • Controls The study will include participants who initiate potent ART after enrollment, attain viral suppression, and have stored plasma samples available for testing, and who have not experienced a primary Ml or stroke. Two controls will be selected for each case, matched to the case based on time since ART initiation (within 12 months) and on current ART regimen.
  • the study will exclude participants with an adjudicated secondary Ml or stroke from both patient groups. Stored plasma specimens from cases and controls meeting the above criteria will be identified. A 400-microliter aliquot of plasma will be shipped from participating sites to the study site on dry ice and stored at -80°C prior to testing.
  • Plasma proteomics Unbiased proteomics of the samples will be performed as described in Example 1 to determine whether certain plasma proteins are over- or under represented in the patients with events. This analysis may also help to identify alterations produced by ART. Briefly, a very small volume of plasma (10 pi) is first depleted of the 14 most abundant proteins (as listed above in Example 1) using a well characterized affinity spin column (for example, Human 14 Multiple Affinity Removal Spin Cartridge from Agilent, Santa Clara, CA) so as to avoid interference of the high abundance proteins with the much more numerous but less abundant remaining proteins. The proteins remaining in the small plasma volume are then reduced, alkylated, and digested with trypsin.
  • affinity spin column for example, Human 14 Multiple Affinity Removal Spin Cartridge from Agilent, Santa Clara, CA
  • the tryptic peptides thus produced are then analyzed by liquid chromatography/tandem MS. Proteins are then identified, for instance by searching the MS/MS spectra against the human protein database (UniProt) using Proteome Discoverer software. Relative protein abundance is determined by either peak area or spectral counting.
  • Quantitative plasma proteomics The proteins to be quantified by MS are listed in Table 4. Included in this list are proteins involved in modulating plasma oxidative stress, acute phase proteins, proteins secreted from activated platelets, proteins known to be involved in platelet adhesion and thrombotic microangiopathies, coagulation and anticoagulation proteins, and serpins, which regulate the activity of the coagulation proteases.
  • the Quantification concatamers strategy (QconCATs) (Simpson and Beynon, Anal Bioanal Chem. 404(4):977-989, 2012) will be used to quantify the concentrations of proteins of interest.
  • isotopically labeled proteins will be expressed in Escherichia coli, which will serve as internal standards for quantification.
  • the three co-expressed internal standards each representative of a protein expressed at low, medium, and high levels in plasma, would be used at concentrations representative of their physiological concentrations.
  • Three unique peptides from each protein will be expressed.
  • these three isotopically labeled proteins will be mixed in proportion to their concentrations in plasma to create an isotopically labeled internal standard (IS) mixture.
  • IS isotopically labeled internal standard
  • This IS mixture will be added to plasma prior to depletion of either the 14 highest abundance proteins (Human-14, Agilent, Santa Clara, CA) or the six highest abundance proteins (Human-6, Agilent, Santa Clara, CA), including albumin, IgG, IgA, transferrin, haptoglobin, and a1 -antitrypsin.
  • Flow through fractions will be digested by proteases and analyzed by targeted LC-MS/MS for quantification of protein and oxidant modifications. Three unique peptides from each isotopically labeled protein will be used as internal standards.
  • Table 4 List of plasma proteins to be quantified by mass spectrometry
  • Plasma thiol/disulfide redox status to evaluate oxidative stress A highly sensitive, specific, and quantitative assay has been developed by the inventors to measure all of the small molecule thiols present in the blood, in both their reduced and disulfide forms.
  • These small molecule thiols include cysteine, reduced glutathione (GSH), homocysteine, Cys-Gly, yGlu-Cys, N-acetylcysteine (NAC), disulfides, and protein-bound Cys (Fu et al. Blood. 2015; 126: 1044; Cate et al. Blood.
  • a parallel aliquot of plasma will be evaluated by first reducing all disulfide-bound Cys with dithiothreitol (DTT) and then blocking the thiols with NEM before methanol extraction.
  • DTT dithiothreitol
  • Plasma metabolomics Plasma will be analyzed as described in Example 1 for small metabolites, including lipids, lyso-lipids, oxylipins, oxidized lipids, and metabolites derived from the amino acids tryptophan and arginine.
  • QC Quality control
  • Internal standards Collection of metabolites. Pooled plasma samples from controls, HIV patients with or without CVE will be used as quality control (QC), metabolite validation, and identification of new metabolites. A mixture of isotopically labeled standards will be added before extraction. Metabolites in plasma (100 pl_) will be extracted with 80% methanol (Fu et al. Transfusion. 2016;56(10):2560-2570, supra). Extractions will be analyzed by either targeted metabolomics or untargeted metabolomics (Fu et al. Transfusion. 2016;56(10):2560-2570, supra ; Zimring et al. Transfusion. 2016;56(8): 1974-1983; de Wolski et al. Haematologica. 2016;101(5):578-586).
  • LC-MS-tandem mass spectrometry MS/MS
  • MRM multiple reaction monitoring
  • AB Sciex 6500 QTRAP mass spectrometer coupled with a Waters Acquity I- class UPLC system or a system with comparable capability.
  • LC-MS conditions will be optimized, such as use of appropriate LC columns and MS positive/negative ion mode, MRM transitions and collision energy (CE) based on the characteristics of the metabolites to be analyzed.
  • MRM data will be acquired using Analyst 1.6 and peak area of the metabolites will be integrated using MultiQuant 2.1 software and resulting data will be further analyzed with MetaboAnalyst (see below metabolomics data analysis).
  • Targeted metabolomic approaches have been developed to quantify over 100 metabolites involved in pathways related to oxidative stress, amino acids and their metabolites, carboxylic acids, fatty acids, oxylipins, lipids and microbiome-derived metabolites including trimethylamine N-oxide (TMAO) and indoxyl sulfate (Zhang and Davies, Genome Med. 2016;8(1):46).
  • TMAO trimethylamine N-oxide
  • indoxyl sulfate Zhang and Davies, Genome Med. 2016;8(1):46.
  • the study will focus on biomarkers for lipid oxidation and degradation and biomarkers of endothelial activation and VWF dysregulation. The latter biomarkers will be measured as described in Example 1.
  • Biomarkers for lipid oxidation and degradation have been implicated in many biological processes.
  • Polyunsaturated fatty acids are very sensitive to oxidation by either enzymatic or peroxidation pathway.
  • arachidonic acid FA 20:4
  • LOX lipoxygenases
  • HETEs 5-, 12-, 15- hydroxyeicosatetraenoic acids
  • lipid peroxidation is non-specific and can produce 8-, 9-, 11-HETEs.
  • This assay will cover all common polyunsaturated fatty acids and their potential oxidation products, and lysophosphatidylcholines (LysoPCs), lysophosphatidylserines (LysoPSs), lysophosphatidylethanolamines(LysoPEs), lysophosphatidylinositols (LysoPI), and lyso platelet activating factor (lysoPAF).
  • LysoPCs lysophosphatidylcholines
  • LysoPSs lysophosphatidylserines
  • LiysoPEs lysophosphatidylethanolamines
  • LysoPI lysophosphatidylinositols
  • lysoPAF lyso platelet activating factor
  • rTPF TPFA/TPFB
  • rFPF FPFA/FPFB
  • SI spectral index
  • biomarkers disclosed herein and/or identify additional biomarkers that correlate with risk of cardiovascular disease in patients infected with HIV. These biomarkers will not only serve to identify those at risk but also point to physiologic and metabolic pathways involved.
  • each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component.
  • the terms“include” or“including” should be interpreted to recite: “comprise, consist of, or consist essentially of.”
  • the transition term“comprise” or“comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts.
  • the transitional phrase “consisting of’ excludes any element, step, ingredient or component not specified.
  • the transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.
  • a material effect would cause a significant decrease in the ability to use one or more of glutathione peroxidase-3 (GPX-3); platelet factor 4 (PF4); platelet basic protein (PBP); thrombospondin-1 (TSP-1); plasma kallikrein (KLKB1); serotonin; Factor V; Protein C; Protein C inhibitor; C- Reactive Protein (CRP); linoleic acid (LA); arachidonic acid (AA); 12-hydroxyeicosatetraenoic acid (12-HETE); lysophosphatidylserine (lysoPS) 18:0; lysoPS 18:1 ; lysoPS 20:4; Cys; and/or protein bound Cys (P-ss-Cys) to predict the occurrence of adverse CVE in patients infected with human immunodeficiency virus (HIV).
  • GPX-3 glutathione peroxidase-3
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • the term“about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ⁇ 20% of the stated value; ⁇ 19% of the stated value; ⁇ 18% of the stated value; ⁇ 17% of the stated value; ⁇ 16% of the stated value; ⁇ 15% of the stated value; ⁇ 14% of the stated value; ⁇ 13% of the stated value; ⁇ 12% of the stated value; ⁇ 11% of the stated value; ⁇ 10% of the stated value; ⁇ 9% of the stated value; ⁇ 8% of the stated value; ⁇ 7% of the stated value; ⁇ 6% of the stated value; ⁇ 5% of the stated value; ⁇ 4% of the stated value; ⁇ 3% of the stated value; ⁇ 2% of the stated value; or ⁇ 1% of the stated value.

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Abstract

La présente invention concerne des systèmes et des procédés pour prédire un risque accru d'événements cardiovasculaires indésirables chez des sujets infectés par le virus de l'immunodéficience humaine (VIH) présentant un risque d'événements cardiovasculaires. Les systèmes et les procédés mesurent les taux d'un ou plusieurs éléments parmi la glutathion peroxydase-3 (GPX-3), le facteur plaquettaire 4 (PF4), la protéine basique plaquettaire (PBP), la thrombospondine-1 (TSP-1), la kallicréine plasmatique (KLKB1), la sérotonine, le facteur V (facteur de coagulation V), la protéine C (PROC), l'inhibiteur de protéine C (PCI), la protéine C réactive (CRP), l'acide linoléique (LA), l'acide arachidonique (AA), l'acide 12-hydroxyeicosatétraénoïque (12-HETE), la lysophosphatidylsérine (LysoPS) 18:0, la lysoPS 18:1, la lysoPS 20:4, la cystéine (Cys) et/ou une cystéine liée à une protéine (P-ss-Cys). Si un risque accru est détecté, une intervention thérapeutique peut être déclenchée.
PCT/US2020/037565 2019-06-14 2020-06-12 Systèmes et procédés pour prédire un risque accru d'événements cardiovasculaires indésirables chez des sujets infectés par le virus de l'immunodéficience humaine (vih) Ceased WO2020252365A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113324933A (zh) * 2021-06-01 2021-08-31 浙江大学 一种检测血管紧张素转换酶的方法及其应用
EP4276377A1 (fr) 2022-05-12 2023-11-15 France Air Unité terminale de traitement d air pour un bâtiment
EP4286851A1 (fr) * 2022-06-01 2023-12-06 Universität Wien Procédés et moyens pour la caractérisation moléculaire du syndrome de fatigue post-virale

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003089898A2 (fr) * 2002-04-18 2003-10-30 Esa, Inc. Determination de marqueurs biochimiques de progression et de surveillance et de specification de therapie, molecules therapeutiques principales et systemes biochimiques cibles appliques a un accident vasculaire cerebral
WO2015073710A2 (fr) * 2013-11-14 2015-05-21 THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Détection du risque de maladie cardiovasculaire athéroscléreuse et du risque d'infarctus du myocarde
WO2018148567A1 (fr) * 2017-02-09 2018-08-16 Fred Hutchinson Cancer Research Center Biomarqueurs et leurs utilisations permettant la sélection d'une intervention d'immunothérapie
US20180291456A1 (en) * 2014-05-28 2018-10-11 Scripps Health Predictive analysis for myocardial infarction
US20180372744A1 (en) * 2014-08-15 2018-12-27 Case Western Reserve University Methods for determining the risk of cardiovascular disease in a subject having a chronic viral infection

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003089898A2 (fr) * 2002-04-18 2003-10-30 Esa, Inc. Determination de marqueurs biochimiques de progression et de surveillance et de specification de therapie, molecules therapeutiques principales et systemes biochimiques cibles appliques a un accident vasculaire cerebral
WO2015073710A2 (fr) * 2013-11-14 2015-05-21 THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTEMENT OF HEALTH AND HUMAN SERVICES Détection du risque de maladie cardiovasculaire athéroscléreuse et du risque d'infarctus du myocarde
US20180291456A1 (en) * 2014-05-28 2018-10-11 Scripps Health Predictive analysis for myocardial infarction
US20180372744A1 (en) * 2014-08-15 2018-12-27 Case Western Reserve University Methods for determining the risk of cardiovascular disease in a subject having a chronic viral infection
WO2018148567A1 (fr) * 2017-02-09 2018-08-16 Fred Hutchinson Cancer Research Center Biomarqueurs et leurs utilisations permettant la sélection d'une intervention d'immunothérapie

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DRAIN, PK ET AL.: "C-Reactive Protein Independently Predicts HIV-related Outcomes among Women and Children in a Resource-Poor Setting", AIDS, vol. 21, no. 15, 1 October 2007 (2007-10-01), pages 2067 - 207, XP055771002, DOI: 10.1097/QAD.0b013e32826fb6c7 *
SO-ARMAH, K ET AL.: "HIV and Cardiovascular Disease: Update on Clinical Events, Special Populations, and Novel Biomarkers", CURRENT HIV/AIDS REPORTS, vol. 15, no. 3, 2018, pages 233 - 244, XP036527787, DOI: 10.1007/s11904-018-0400-5 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113324933A (zh) * 2021-06-01 2021-08-31 浙江大学 一种检测血管紧张素转换酶的方法及其应用
CN113324933B (zh) * 2021-06-01 2022-07-26 浙江大学 一种检测血管紧张素转换酶的方法及其应用
EP4276377A1 (fr) 2022-05-12 2023-11-15 France Air Unité terminale de traitement d air pour un bâtiment
EP4286851A1 (fr) * 2022-06-01 2023-12-06 Universität Wien Procédés et moyens pour la caractérisation moléculaire du syndrome de fatigue post-virale
WO2023232941A1 (fr) 2022-06-01 2023-12-07 Universitaet Wien Procédés et moyens de caractérisation moléculaire du syndrome de fatigue post-virale

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