WO2016127785A1 - 用于评估机体发生人巨细胞病毒活动性感染风险的方法及相关的试剂盒 - Google Patents

用于评估机体发生人巨细胞病毒活动性感染风险的方法及相关的试剂盒 Download PDF

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WO2016127785A1
WO2016127785A1 PCT/CN2016/072094 CN2016072094W WO2016127785A1 WO 2016127785 A1 WO2016127785 A1 WO 2016127785A1 CN 2016072094 W CN2016072094 W CN 2016072094W WO 2016127785 A1 WO2016127785 A1 WO 2016127785A1
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hcmv
antibody
subject
reference value
active infection
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French (fr)
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葛胜祥
李金洁
黄茜
李廷栋
张军
夏宁邵
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厦门大学
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Priority to EP16748586.1A priority patent/EP3258265B1/en
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • 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
    • G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/03—Herpetoviridae, e.g. pseudorabies virus
    • G01N2333/04—Varicella-zoster virus
    • G01N2333/045—Cytomegalovirus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to the field of medicine and immunology, in particular to the field of immunology diagnosis.
  • the present invention discloses a method for assessing whether a subject is at risk of developing a human cytomegalovirus (HCMV) active infection, comprising the steps of: (1) determining from the subject Level of the antibody against the HCMV protein in the body fluid sample; (2) comparing the level to a predetermined reference value; wherein, if the level is lower than the reference value, determining that the subject is in the presence of HCMV In the risk of active infection; and kits for use in the methods.
  • the present invention also discloses methods for screening for a drug candidate capable of increasing a subject's ability to resist human cytomegalovirus (HCMV) active infection, and kits for use in the method.
  • HCMV Human Cytomegalovirus
  • HCMV congenital infection occurs in a fetus (ie, CMV breaks through the blood-fetal barrier and infects the intrauterine fetus), it can lead to serious clinical hazards, including stillbirth, miscarriage, and birth defects [1, 2] (Dollard SC, Grosse SD, Ross DS. New estimates of the prevalence of neurological and sensory sequelae and mortality associated with congenital cytomegalovirus infection. Rev Med Virol. 2007, 17: 355-363; Jiang Yi.
  • CMV congenital cytomegalovirus
  • CMV-IgM and IgG antibody tests are commonly used in various countries to check prenatal CMV active infection in pregnant women.
  • IgM and IgG antibody assays are of value in the diagnosis of primary infections in pre-pregnancy antibody-negative pregnant women.
  • IgM and IgG antibodies are detected for the re-infection of pregnant women with positive pre-pregnancy antibodies, their sensitivity and specificity are more questioned [10,11] (He Xiaozhou, Wang Xiaofang, Wang Shiwen. Congenital giant cells) Advances in research on viral infection status and detection methods. Acta. 2012, 28: 73-77; Ross SA, Novak Z, Pati S, Boppana SB. Overview of the diagnosis of cytomegalovirus infection. Infect Disord Drug Targets. 2011, 11: 466-474).
  • the term "active infection of cytomegalovirus” means that a subject who has never been infected with CMV is initially infected with CMV; and a subject who has previously been infected with CMV (which typically carries a latent virus, And the serological test results are usually positive) CMV re-infection occurs or the latent CMV virus in the body is activated (also known as "overlapping new strain” or “latent virus reactivation”).
  • the typical manifestation of active infection of cytomegalovirus is The tester produces a significant immune response against the infected cytomegalovirus (first or again), which can result in a significant increase in anti-CMV protein antibody levels in the subject.
  • active infection of cytomegalovirus is also known as a viral event.
  • level of anti-CMV protein antibody in a subject is increased by a factor of 4 or more, it can be determined that the subject has an active infection with cytomegalovirus (or a viral event has occurred).
  • pp150 refers to a phosphorylated protein in the CMV viral envelope layer, which is also referred to as a UL32 protein.
  • the pp150 protein is one of the more abundant CMV proteins, closely related to the assembly and secretion of the virus, and is conserved among the CMV strains and has good immunoreactivity with CMV-infected serum [12] (Jahn G, Scholl BC) , Trauche B, Fleckenstein B. The two major structural phosphoproteins (pp65 and pp150) of human cytomegalovirus and their antigenic properties. J Gen Virol. 1987, 68: 1327-1337).
  • pp150 Since pp150 has good conservation and immunoreactivity, it has been used for the detection of CMV-IgG antibodies [13] (Plachter B, Wieczorek L, Scholl BC, Ziegelmaier R, Jahn G. Detection of cytomegalovirus antibodies by an enzyme -linked immunosorbent assay using recombinant polypeptides of the large phosphorylated tegument protein pp150. J Clin Microbiol. 1992, 30: 201-206).
  • a positive serological test result i.e., the presence of an anti-pp150 antibody in the serum indicates that the individual has been infected with CMV, resulting in an immune response against pp150.
  • the level of anti-pp150 antibody correlates with the probability of a subject's cytomegalovirus active infection.
  • amino acid sequence of the pp150 protein is well known to those skilled in the art, and a typical example thereof can be found, for example, in GenBank Accession No. ACL51112. As used herein, when referring to the amino acid sequence of the pp150 protein, it is described using the sequence shown in SEQ ID NO: 1. For example, the expression "aa 861-1048 of pp150" or “amino acid residue of positions 861-1048 of pp150" means the amino acid residue of positions 861-1048 of the polypeptide of SEQ ID NO: 1.
  • pp150 in the amino acid sequence of pp150, mutations or mutations (including but not limited to, substitutions, deletions and/or additions, such as pp150 of different CMV virus isolates) may be naturally occurring or artificially introduced without Affecting its biological work can.
  • the term "pp150” shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 1 as well as natural or artificial variants thereof. Also, when describing a sequence fragment of pp150, it includes not only the sequence fragment of SEQ ID NO: 1, but also the corresponding sequence fragment in its natural or artificial variant.
  • amino acid residues 861-1048 of pp150 includes amino acid residues 861-1048 of SEQ ID NO: 1, and variants thereof (natural or artificial) The corresponding fragment.
  • pp28 or "pp28 protein” refers to a phosphorylated protein in the CMV viral envelope, also known as the UL99 protein (Gambarino S, Callea S, Rizzo G, Montanari P). , Loiacono E, Bergallo M. Evaluation of UL99 transcript as a target for antiviral treatment efficacy. J Virol Methods. 2014, 207:104-9; John Paul Tomtishen III. Human cytomegalovirus tegument proteins (pp65, pp71, pp150, pp28). Virol J. 2012, 9:22).
  • amino acid sequence of the pp28 protein is well known to those skilled in the art, and a typical example thereof can be found, for example, in GenBank Accession No. ACL 51167.1. As used herein, when referring to the amino acid sequence of the pp28 protein, it is described using the sequence shown in SEQ ID NO: 3. For example, the expression "aa 1-190 of pp28" or "amino acid residue 1-190 of pp28” means the amino acid residues 1-190 of the polypeptide represented by SEQ ID NO: 3.
  • pp28 in the amino acid sequence of pp28, mutations or mutations (including but not limited to, substitutions, deletions and/or additions, such as pp28 of different CMV virus isolates) may be naturally occurring or artificially introduced without Affect its biological function.
  • the term "pp28" shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 3, as well as natural or artificial variants thereof.
  • a sequence fragment of pp28 it includes not only the sequence fragment of SEQ ID NO: 3 but also the corresponding sequence fragment in its natural or artificial variant.
  • the expression "aa 1-190 of pp28" or "amino acid residues 1-190 of pp28” includes amino acid residues 1-190 of SEQ ID NO: 3, and variants thereof (natural or artificial) The corresponding fragment in .
  • pp65 or "pp65 protein” refers to a CMV disease.
  • a phosphorylated protein in the venom layer also known as the UL83 protein (Jahn G, Scholl BC, Traupe B, Fleckenstein B. The two major structural phosphoproteins (pp65 and pp150) of human cytomegalovirus and their antigenic properties. Gen Virol. 1987, 68: 1327-1337; John Paul Tomtishen III. Human cytomegalovirus tegument proteins (pp 65, pp 71, pp 150, pp 28). Virol J. 2012, 9: 22).
  • amino acid sequence of the pp65 protein is well known to those skilled in the art, and a typical example thereof can be found, for example, in GenBank Accession No. ACL 51152.1. As used herein, when referring to the amino acid sequence of the pp65 protein, it is described using the sequence shown in SEQ ID NO: 4. For example, the expression "aa 1-561 of pp65” or “amino acid residues 1-561 of pp65” means the amino acid residues 1-536 of the polypeptide of SEQ ID NO: 4.
  • pp65 in the amino acid sequence of pp65, mutations or mutations (including but not limited to, substitutions, deletions and/or additions, such as pp65 of different CMV virus isolates) may be naturally occurring or artificially introduced without Affect its biological function.
  • the term "pp65” shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 4, as well as natural or artificial variants thereof.
  • a sequence fragment of pp65 it includes not only the sequence fragment of SEQ ID NO: 4 but also the corresponding sequence fragment in its natural or artificial variant.
  • the expression "aa 1-561 of pp65” or “amino acid residues 1-561 of pp65” includes amino acid residues 1-561 of SEQ ID NO: 4, and variants thereof (natural or artificial) The corresponding fragment in .
  • gp52 or "gp52 protein” refers to a phosphorylated protein in the CMV viral envelope, also known as the UL44 protein (Strang B L, Boulant S, Chang L, et al.Human cytomegalovirus UL44concentrates at the periphery of replication compartments, the site of viral DNA synthesis[J].Journal of virology,2012,86:2089-2095;Sinigalia E,Alvisi G,Segr é C V,et al.The human Cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNA-dependent manner [J]. PLoS One, 2012, 7(11): e49630).
  • amino acid sequence of the gp52 protein is well known to those skilled in the art, and a typical example thereof can be found, for example, in GenBank Accession No. ACL 51123.1. As used herein, when referring to the amino acid sequence of the gp52 protein, it is described using the sequence shown in SEQ ID NO: 5. For example, the expression "aa 1-433 of gp52" or “amino acid residues 1-433 of gp52” means the amino acid residues 1-433 of the polypeptide represented by SEQ ID NO: 5.
  • gp52 in the amino acid sequence of gp52, mutations or mutations (including but not limited to, substitutions, deletions and/or additions, such as gp52 of different CMV virus isolates) may be naturally occurring or artificially introduced without Affect its biological function.
  • the term "gp52" shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 5, as well as natural or artificial variants thereof. Also, when describing a sequence fragment of gp52, it includes not only the sequence fragment of SEQ ID NO: 5 but also the corresponding sequence fragment in its natural or artificial variant.
  • amino acid residues 1-433 of gp52 includes amino acid residues 1-433 of SEQ ID NO: 5, and variants thereof (natural or artificial) The corresponding fragment in .
  • pp38 refers to a phosphorylated protein in the CMV viral envelope, also known as UL80.5 protein (Loveland A N, Nguyen N L, Brignole) E J, et al.
  • the amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production. Journal of virology, 2007, 81: 620-628; Wood L J, Baxter M K , Plafker S M, et al.
  • Human cytomegalovirus capsid assembly protein precursor (pUL 80.5) interacts with itself and with the major capsid protein (pUL86) through two different domains. Journal of virology, 1997, 71: 179-190).
  • amino acid sequence of the pp38 protein is well known to those skilled in the art, and a typical example thereof can be found, for example, in GenBank Accession No. ACL51150.1. As used herein, when referring to the amino acid sequence of the pp38 protein, it is described using the sequence set forth in SEQ ID NO: 6. For example, the expression "aa 1-373 of pp38" or “amino acid residues 1-373 of pp38” means the amino acid residues 1-373 of the polypeptide of SEQ ID NO: 6.
  • pp38 in the amino acid sequence of pp38, mutations or mutations (including but not limited to, substitutions, deletions and/or additions, such as pp38 of different CMV virus isolates) may be naturally occurring or artificially introduced without Affect its biological function.
  • the term "pp38” shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 6 as well as natural or artificial variants thereof.
  • a sequence fragment of pp38 it includes not only the sequence fragment of SEQ ID NO: 6, but also the corresponding sequence fragment in its native or artificial variant.
  • the expression "aa 1-373 of pp38” or “amino acid residues 1-373 of pp38” includes amino acid residues 1-373 of SEQ ID NO: 6, and variants thereof (natural or artificial) The corresponding fragment in .
  • UL48a or "UL48a protein” refers to a capsid protein of the CMV virus (Baldick C J, Shenk T. Proteins associated with purified human cytomegalovirus particles. Journal of virology, 1996, 70). :6097-6105).
  • amino acid sequence of the UL48a protein is well known to those skilled in the art, and a typical example thereof can be found, for example, in GenBank Accession No. ACL 51128.1. As used herein, when referring to the amino acid sequence of the UL48a protein, it is described using the sequence set forth in SEQ ID NO: 7. For example, the expression "aa 1-75 of UL48a” or "amino acid residue 1-75 of UL48a” means the amino acid residues 1-75 of the polypeptide represented by SEQ ID NO: 7.
  • UL48a shall include all such sequences, including, for example, the sequences set forth in SEQ ID NO: 7 as well as natural or artificial variants thereof.
  • a sequence fragment of UL48a it includes not only the sequence fragment of SEQ ID NO: 7, but also the corresponding sequence fragment in its natural or artificial variant.
  • the expression "aa 1-75 of UL48a” or "amino acid residues 1-75 of UL48a” includes amino acid residues 1-75 of SEQ ID NO: 7, and variants thereof (natural or artificial) The corresponding fragment in .
  • IE1 protein refers to an immediate early protein of the CMV virus (Torres L, Tang Q.Immediate-Early (IE) gene regulation Of cytomegalovirus: IE1-and pp71-mediated viral strategies against cellular defenses [J].
  • CMV virus Torres L, Tang Q.Immediate-Early (IE) gene regulation Of cytomegalovirus: IE1-and pp71-mediated viral strategies against cellular defenses [J].
  • amino acid sequence of the IE1 protein is well known to those skilled in the art, and a typical example thereof can be found, for example, in GenBank Accession No. ACL51183.1.
  • corresponding sequence fragment or “corresponding fragment” means that when the sequences are optimally aligned, ie when the sequences are aligned to obtain the highest percentage identity, the sequences to be compared are in the equivalent position. Fragment of.
  • identity is used to mean the matching of sequences between two polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two
  • Each position in each of the polypeptides is occupied by lysine, and then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6, or 4 and the length weights of 1, 2, 3, 4, 5 or 6 are determined. Percent identity between two amino acid sequences.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the essential properties of a protein/polypeptide comprising an amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions of amino acid residues with similar side chains in place of amino acid residues, for example, physically or functionally similar to corresponding amino acid residues (eg, having similar size, shape, charge, chemical properties, including Substitution of residues by formation of a covalent bond or a hydrogen bond, etc.).
  • a family of amino acid residues having similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine) , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg alanine, valine, leucine, isoluminescence) Acid, valine, phenylalanine, methionine), beta branch side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Amino acids of phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • antibody refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids.
  • Each heavy chain is comprised of a heavy chain variable region (V H) and a heavy chain constant region (C H) composition.
  • the heavy chain constant region is comprised of three domains (C H 1, C H 2 and C H 3) components.
  • Each light chain is comprised of a light chain variable region (V L) and a light chain constant region (C L) components.
  • the light chain constant region is comprised of one domain, C L composition.
  • the constant region of the antibody mediates binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
  • V H regions may be subdivided into hypervariability regions (termed complementarity determining regions (CDR)), interspersed with regions are more conserved, termed framework regions (FR) of.
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L the following order: FR1, CDR1, FR2, CDR2 , FR3, CDR3, FR4 from the amino terminus to the carboxy terminus arranged three four FR and CDR components.
  • the assignment of amino acids to regions or domains follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.
  • antibody is not limited by any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be antibodies of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "level of antibody” refers to the amount of an antibody that is reactive with a human cytomegalovirus protein (eg, pp150/pp28), which may be, for example, by any relative or absolute quantitative means (including but not It is limited to any detection means capable of obtaining an antibody level by an ELISA, a colloidal gold method, or a chemiluminescence method, and can be described, for example, as an antibody-antigen reaction intensity, an antibody titer, or a quantitative detection value of an antibody (for example, an antibody). The absolute amount) and so on.
  • a human cytomegalovirus protein eg, pp150/pp28
  • any relative or absolute quantitative means including but not It is limited to any detection means capable of obtaining an antibody level by an ELISA, a colloidal gold method, or a chemiluminescence method, and can be described, for example, as an antibody-antigen reaction intensity, an antibody titer, or a quantitative detection value of an antibody (for example, an antibody
  • antibody titer refers to a sample (eg, serum, antibody solution) containing an antibody that is required for recognition of an antigen (or antigenic fragment thereof, eg, an epitope) to which the antibody specifically binds.
  • the lowest concentration i.e., maximum dilution
  • it is generally expressed as the maximum dilution that still produces a positive result.
  • Methods for determining the antibody titer of a sample are well known to those skilled in the art. For example, antibody titers of a sample can generally be detected by an ELISA method.
  • Such an ELISA method may, for example, comprise the steps of: (1) using an antigen-specific protein (or an antigenic fragment thereof) to which the antibody specifically binds as a coating antigen to coat a microplate; (2) a sample containing the antibody (for example, serum) for serial dilution; (3) adding each diluted sample to the well of the coated microplate of step (1); (4) detecting and coating the antigen in the microplate The presence of the bound antibody (eg, using an antibody against the antibody, or a labeled antigenic protein, etc.), and thereby determining the maximum dilution of the sample (ie, the antibody titer of the sample) that still produces a positive result.
  • an antigen-specific protein or an antigenic fragment thereof
  • the term "absolute amount of antibody” refers to the amount of antibody expressed in terms of the biological activity of the antibody, which is typically in IU/ml or variants thereof (eg, IU/L, mIU/ml, etc.) )To represent.
  • Methods for determining the absolute amount of antibody in a sample are well known to those skilled in the art.
  • the absolute amount of antibody in the sample can be determined by comparing the antibody reactivity of a sample containing the antibody (e.g., serum) to the antibody reactivity of a standard having a known antibody content.
  • such methods can include the steps of: (1) determining antibody reactivity of serial dilutions of standards by ELISA, and plotting a standard profile of antibody content-antibody reactivity; (2) determining samples containing antibodies ( Antibody reactivity of or a dilution thereof; and (3) converting the antibody reactivity of the sample (or its dilution) to the absolute amount of antibody contained in the sample using a standard curve.
  • antibody reactivity refers to the ability of an antibody to specifically recognize an antigen, which can be readily determined by immunological assays (eg, ELISA). For example, antibody reactivity can be expressed as an OD value obtained from an ELISA assay that detects antigen-antibody binding.
  • antigenic fragment refers to a fragment of an antigenic protein that retains its ability to specifically bind to an antibody that specifically recognizes the antigenic protein.
  • antigenic fragments can be obtained by those skilled in the art by conventional techniques. For example, such methods can include the steps of: (1) preparing various fragments of an antigenic protein (eg, by genetic engineering techniques, chemical synthesis techniques, enzymatic hydrolysis of antigenic proteins, etc.); and (2) by immunological assays (eg, ELISA) to determine whether the prepared fragment retains the specific binding ability to an antibody that specifically recognizes the antigenic protein, thereby screening for an antigenic fragment of the antigenic protein.
  • immunological assays eg, ELISA
  • immunological assay refers to an assay that utilizes specific interaction/binding affinity between antigen-antibodies, which is generally useful for detecting the presence of a particular antigen or antibody in a sample or Level.
  • immunological assays are well known to those skilled in the art and include, but are not limited to, ELISA assays, Western blots, surface plasmon resonance assays, Elispot assays, and the like.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (K D ) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • K D refers to a particular antibody - antigen interaction dissociation equilibrium constant, which is used to describe the binding affinity between antibody and antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.
  • the antibody has a dissociation equilibrium constant (K D ) of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • the antigen is bound, for example, as measured in a BIACORE instrument using surface plasmon resonance (SPR).
  • subject includes, but is not limited to, various animals, particularly mammals, such as humans.
  • body fluid sample refers to a sample containing bodily fluids obtained from a subject or fractions thereof.
  • the body fluid sample may comprise or be obtained directly from the subject's body fluid (or a fraction thereof), may also comprise or be treated body fluid (eg, an isolated body fluid fraction), and may optionally comprise other components For example, buffers, anticoagulants, diluents, and the like.
  • the various body fluids or fractions thereof obtained from the subject are well known to those skilled in the art and include, but are not limited to, blood, serum, plasma, urine, saliva, and the like.
  • the term "agent capable of determining the level of an antibody against an HCMV protein” refers to an agent capable of determining the level of an anti-HCMV protein antibody in a sample in a quantitative or semi-quantitative manner. In general, it is particularly preferred that such agents are capable of determining the level of anti-HCMV protein antibodies in a sample by immunological assays. The use of immunological assays is particularly advantageous because it exploits the specific interaction/binding affinity between antigen-antibodies.
  • the reagent can be used to determine the level of the anti-HCMV protein antibody in the sample by immunological assay (ie, the reagent can be used)
  • a reagent capable of measuring the level of an antibody against an HCMV protein can be used.
  • agents that retain antigenicity in response to antibodies are readily apparent to those skilled in the art and are readily available, including, but not limited to, the antigenic proteins themselves (in the present application, HCMV proteins) Or an antigenic fragment thereof, comprising the anti- A fusion protein of a proprotein (in the present application, an HCMV protein) or an antigenic fragment thereof, and any combination thereof.
  • test animal refers to an animal for use in an experiment, which is typically a non-human mammal, such as a model animal (eg, mouse, rat, rabbit, primate). Those skilled in the art can select various suitable test animals according to the specific needs of the experiment.
  • a model animal eg, mouse, rat, rabbit, primate.
  • RR relative risk
  • a risk ratio refers to a parameter or indicator (in the present invention, antibody levels, such as antibodies against pp150 or pp28) Level)
  • Relative risk is an indicator that reflects the strength of the association between parameters/indicators and disease.
  • the parameters/indicators are not related to the disease; when the RR is 1.2-1.4, the parameters/indicators are weakly related to the disease; when the RR is 1.5-2.9, the parameters/indicators are moderately related to the disease.
  • the RR 3.0-9.9, the parameters/indicators have a strong correlation with the disease; when the RR is greater than 10, the parameters/indicators have a strong correlation with the disease.
  • the term "Youden index” refers to an indicator used to evaluate the authenticity of a screening test/predictive test.
  • the Yoden index is equal to the sum of the sensitivity and specificity of the test minus one.
  • the Yoden Index indicates that the screening test/predictive test finds the true capacity of both true and non-patients. The greater the value of the Yoden index, the better the effectiveness of the screening test/predictive test and the greater the authenticity.
  • One technical problem to be solved by the present invention is to provide a method for accurately and effectively estimating the probability of occurrence of a cytomegalovirus active infection in a subject (for example, a pregnant woman, an organ transplant patient, an HIV infected person, etc.), which can Interventions are provided to reduce the incidence of congenital cytomegalovirus infection, and can guide clinical medication to prevent the occurrence of complications in patients.
  • a subject for example, a pregnant woman, an organ transplant patient, an HIV infected person, etc.
  • the inventors expressed and purified the detection proteins commonly used in cytomegalovirus diagnostic studies [14] (Plachter B, Wieczorek L, Scholl BC, Ziegelmaier R, Jahn G. Detection of cytomegalovirus antibodies by an enzyme-linked immunosorbent assay Using recombinant polypeptides of the large phosphorylated tegument protein pp150.J Clin Microbiol.
  • the inventors found antibody levels of anti-pp150 (UL32) and/or pp28 (UL99) in the serum of the subject (which may, for example, use pp150 (UL32) and/or pp28 ( UL99) is detected by ELISA as a coating antigen) and can be used to effectively and reliably reflect the probability of a subject's cytomegalovirus active infection.
  • ELISA a coating antigen
  • the invention provides a method of assessing whether a subject is at risk of developing a human cytomegalovirus (HCMV) active infection, comprising the steps of:
  • the subject is judged to be at risk of developing an active HCMV infection.
  • the subject is a mammal, such as a human.
  • the body fluid sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the active infection is that the subject not infected with HCMV is initially infected with HCMV, or the subject who has been infected with HCMV is again activated by HCMV infection or a latent HCMV virus in the body.
  • the HCMV protein is selected from the group consisting of pp150 and/or pp28.
  • the level of antibody against the HCMV protein in the body fluid sample is determined by immunological assay.
  • the Immunological assays were selected from the group consisting of ELISA assays, Western blots, surface plasmon resonance assays, and Elispot assays.
  • the level is an antibody titer (eg, an antibody titer as determined by ELISA) and the reference value is a predetermined antibody titer; alternatively, the level is the absolute amount of antibody (eg, The absolute amount expressed by IU/ml) and the reference value is the absolute amount of the predetermined antibody (for example, the absolute amount expressed in IU/ml).
  • the level is an antibody titer (eg, an antibody titer as determined by ELISA) and the reference value is a predetermined antibody titer; alternatively, the level is the absolute amount of antibody (eg, The absolute amount expressed by IU/ml) and the reference value is the absolute amount of the predetermined antibody (for example, the absolute amount expressed in IU/ml).
  • antibody titers of antibodies against pp150 and/or pp28 in the body fluid sample are determined by ELISA.
  • the reference value is an antibody titer ranging between 40-320.
  • the reference value is an antibody titer ranging between 40-160.
  • the reference value is an antibody titer of 40, 80, or 160.
  • the reference value is an antibody titer of 80.
  • the relative risk of HCMV active infection in the subject whose antibody titer is lower than the reference value exceeds 10
  • the lower limit of 95% CI exceeds 7. This suggests that there is a strong correlation or a strong correlation between this parameter (antibody titer) and HCMV active infection.
  • the reference value is 40, and if the antibody titer of the anti-pp150 antibody is less than or equal to 40, the relative risk of determining HCMV active infection in the subject is 11.2. 95% CI is 8.7-14.6. In certain preferred embodiments, the reference value is set to an antibody titer of 40, and the sensitivity of the method for predicting HCMV active infection is 57.3%, specificity is 94.7%, and the Yoden index is 0.52. .
  • the reference value is 80, and if the antibody titer of the anti-pp150 antibody is less than or equal to 80, the relative risk of determining HCMV active infection in the subject is 10.6, 95% CI is 7.7-14.6.
  • the reference value is set to an antibody titer of 80, and the sensitivity of the method for predicting HCMV active infection is 73.1%, specificity is 85.7%, and the Yoden index is 0.59. .
  • the reference value is 160, and if When the antibody titer of the pp150 antibody is less than or equal to 160, the relative risk of the HCMV active infection in the subject is judged to be 14.8, and the 95% CI is 9.0-24.6.
  • the reference value is set to an antibody titer of 160, and the sensitivity of the method for predicting HCMV active infection is 90.6%, specificity is 66.4%, and the Yoden index is 0.57. .
  • the reference value is 320, and if the antibody titer of the anti-pp150 antibody is less than or equal to 320, the relative risk of determining HCMV active infection in the subject is 23.7, 95% CI is 8.8-63.4.
  • the reference value is set to an antibody titer of 320, and the sensitivity of the method for predicting HCMV active infection is 97.7%, specificity is 40.1%, and the Yoden index is 0.37. .
  • the level of antibody titer also directly indicates the risk of HCMV active infection in the subject.
  • the risk of HCMV active infection in the subject may reach 55.37%; if the antibody titer of the anti-pp150 antibody is less than or equal to 80, the risk of HCMV active infection in the subject may reach 36.98%; if the antibody titer of the anti-pp150 antibody is less than or equal to 160, the risk of HCMV active infection in the subject may reach 23.66%; if the antibody titer of the anti-pp150 antibody is less than or equal to 320, the subject may have a risk of HCMV active infection of 15.77%.
  • the reference value is 40, and if the antibody titer of the anti-pp150 antibody is less than or equal to 40, the probability of the subject developing an HCMV active infection is 55.37%.
  • the reference value is 80, and if the antibody titer of the anti-pp150 antibody is less than or equal to 80, the probability of the subject developing HCMV active infection is 36.98%.
  • the reference value is 160, and if the antibody titer of the anti-pp150 antibody is less than or equal to 160, the probability of the subject developing HCMV active infection is 23.66%.
  • the reference value is 320, and if the antibody titer of the anti-pp150 antibody is less than or equal to 320, the probability of the subject developing HCMV active infection is 15.77%.
  • the antibody titer of the anti-pp150 antibody in the body fluid sample is determined by ELISA using pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150); and/or The antibody titer of the anti-pp28 antibody in the body fluid sample was determined by ELISA using pp28 and/or its antigenic fragment.
  • pp150 has the amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has the amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has The amino acid sequence shown as SEQ ID NO: 3.
  • step (1) the absolute amount of antibody against pp150 and/or pp28 (e.g., the absolute amount expressed in IU/ml) in the body fluid sample is determined.
  • the anti-body sample is determined to be resistant by comparing the reactivity of the anti-pp150 and/or pp28 antibody in the body fluid sample to the antibody reactivity of a standard having a known antibody content.
  • the reference value is the absolute amount of antibody ranging between 0.8 and 6.4 IU/ml.
  • the reference value is the absolute amount of antibody ranging between 0.8-3.2 IU/ml.
  • the reference value is an absolute amount of antibody of 0.8, 1.6, or 3.2 IU/ml.
  • the reference value is an absolute amount of antibody of 3.2 IU/ml.
  • the relative risk of HCMV active infection in the subject whose absolute amount of antibody is lower than the reference value exceeds 11 and 95% thereof.
  • the lower limit of CI is more than 7. This suggests that there is a strong correlation or a strong correlation between this parameter (absolute amount of antibody) and HCMV active infection.
  • the reference value is 0.8 IU/ml, and if the absolute amount of the antibody against the pp150 antibody is less than or equal to 0.8 IU/ml, the subject is judged to have an active HCMV infection.
  • the relative risk is 11.6, 95% CI It is 7.7-17.2.
  • the reference value is 0.8 IU/ml and the sensitivity of the method for predicting HCMV active infection is 46.15%, specificity is 96.97%, and the Yoden index is 0.43.
  • the reference value is 1.6 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 1.6 IU/ml, then the subject is judged to have an HCMV active infection.
  • the relative risk is 15.2 and the 95% CI is 9.5-24.3.
  • the reference value is 1.6 IU/ml and the sensitivity of the method for predicting HCMV active infection is 67.69%, specificity is 93.34%, and the Yoden index is 0.61.
  • the reference value is 3.2 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 3.2 IU/ml, then the subject is judged to have an HCMV active infection.
  • the relative risk is 19.0 and the 95% CI is 9.6-37.7.
  • the reference value is 3.2 IU/ml and the sensitivity of the method for predicting HCMV active infection is 86.15%, specificity is 81.39%, and the Yoden index is 0.68.
  • the reference value is 6.4 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 6.4 IU/ml, then the subject is judged to have an HCMV active infection.
  • the relative risk is 25.2 and the 95% CI is 8.0-79.6.
  • the reference value is 6.4 IU/ml and the sensitivity of the method for predicting HCMV active infection is 95.38%, specificity is 59.91%, and the Yoden index is 0.55.
  • the absolute amount of antibody also directly indicates the risk of HCMV active infection in the subject.
  • the risk of HCMV active infection in the subject can reach 60.0%; if the absolute amount of the antibody against the pp150 antibody Less than or equal to 1.6 IU/ml, the subject may have a risk of developing HCMV active infection by 50.0%; if the absolute amount of the anti-pp150 antibody is less than or equal to 3.2 IU/ml, the subject The risk of developing HCMV active infection can reach 31.3%; if the absolute amount of anti-pp150 antibody is less than or equal to 6.4 IU/ml, The subject may have a risk of developing HCMV active infection of 19.0%.
  • the reference value is 0.8 IU/ml, and if the absolute amount of the antibody against the pp150 antibody is less than or equal to 0.8 IU/ml, the subject is judged to have an active HCMV infection.
  • the probability is 60.0%.
  • the reference value is 1.6 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 1.6 IU/ml, the probability of HCMV active infection in the subject is determined. It is 50.0%.
  • the reference value is 3.2 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 3.2 IU/ml, the probability of HCMV active infection in the subject is determined. It is 31.3%. In a preferred embodiment, the reference value is 6.4 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 6.4 IU/ml, the probability of HCMV active infection in the subject is determined. It is 19.0%.
  • the method of the present invention further comprises:
  • a vaccine or antibody that is determined to be at risk of developing an active HCMV infection is administered a vaccine or antibody against HCMV to reduce the risk of HCMV active infection in the subject.
  • the invention provides the use of an agent capable of determining the level of an antibody against an HCMV protein, in a kit for determining the level of an antibody against an HCMV protein in a body fluid sample from a subject The subject is assessed for risk of developing a human cytomegalovirus (HCMV) active infection.
  • HCMV human cytomegalovirus
  • the agent is capable of determining the level of an antibody against an HCMV protein by immunological assay.
  • the immunological assay is selected from the group consisting of an ELISA assay, a Western blot, a surface plasmon resonance method, and Elispot Determination.
  • the agent is selected from the group consisting of: an HCMV protein or antigenic fragment thereof, a fusion protein comprising the HCMV protein or an antigenic fragment thereof, and any combination thereof.
  • the HCMV protein is selected from the group consisting of pp150 and/or pp28.
  • the HCMV protein is pp150
  • the reagent is pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150); alternatively, the HCMV protein is pp28 And the reagent is pp28 and/or an antigenic fragment thereof; or, the HCMV protein is pp150 and pp28, and the reagent comprises: pp150 as a first component and/or an antigenic fragment thereof (for example, comprising Fragment of aa861-1048 of pp150), and pp28 and/or its antigenic fragment as a second component.
  • pp150 has the amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has the amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has An amino acid sequence as shown in SEQ ID NO: 3;
  • the subject is a mammal, such as a human.
  • the body fluid sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the active infection is that the subject not infected with HCMV is initially infected with HCMV, or the subject who has been infected with HCMV is again activated by HCMV infection or a latent HCMV virus in the body.
  • the kit further comprises:
  • the kit assesses whether the subject is at risk of developing a human cytomegalovirus (HCMV) active infection by a method comprising the steps of:
  • the subject is judged to be at risk of developing an active HCMV infection.
  • the level is an antibody titer (eg, an antibody titer as determined by ELISA), and the reference value is a predetermined antibody titer; or, the level is The absolute amount of antibody (e.g., the absolute amount expressed in IU/ml) and the reference value is the absolute amount of the predetermined antibody (e.g., the absolute amount expressed in IU/ml).
  • the antibody titer of the antibody against pp150 and/or pp28 in the body fluid sample is determined by ELISA.
  • the antibody titer of the anti-pp150 antibody in the body fluid sample is determined by ELISA using pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150); and/or The antibody titer of the anti-pp28 antibody in the body fluid sample was determined by ELISA using pp28 and/or its antigenic fragment.
  • the reference value is an antibody titer ranging between 40-320.
  • the reference value is an antibody titer ranging between 40-160.
  • the reference value is an antibody titer of 40, 80, or 160.
  • the reference value is an antibody titer of 80.
  • the reference value is 40, and if the antibody titer of the anti-pp150 antibody is less than or equal to 40, the relative risk of determining HCMV active infection in the subject is 11.2, 95.
  • the %CI is 8.7-14.6.
  • the reference value is 40, and if the antibody titer of the anti-pp150 antibody is less than or equal to 40, the probability of the subject developing an HCMV active infection is 55.37%.
  • the reference value is set to an antibody titer of 40, and the sensitivity of the method for predicting HCMV active infection is 57.3%, specificity is 94.7%, and the Yoden index is 0.52. .
  • the reference value is 80, and if the antibody titer of the anti-pp150 antibody is less than or equal to 80, then the subject is judged to have HCMV activity.
  • the relative risk of active infection was 10.6, 95% CI was 7.7-14.6.
  • the reference value is 80, and if the antibody titer of the anti-pp150 antibody is less than or equal to 80, the probability of the subject developing HCMV active infection is 36.98%.
  • the reference value is set to an antibody titer of 80, and the sensitivity of the method for predicting HCMV active infection is 73.1%, specificity is 85.7%, and the Yoden index is 0.59. .
  • the reference value is 160, and if the antibody titer of the anti-pp150 antibody is less than or equal to 160, the relative risk of determining HCMV active infection in the subject is 14.8, 95% CI is 9.0-24.6. In another preferred embodiment, the reference value is 160, and if the antibody titer of the anti-pp150 antibody is less than or equal to 160, the probability of the subject developing HCMV active infection is 23.66%. In certain preferred embodiments, the reference value is set to an antibody titer of 160, and the sensitivity of the method for predicting HCMV active infection is 90.6%, specificity is 66.4%, and the Yoden index is 0.57. .
  • the reference value is 320, and if the antibody titer of the anti-pp150 antibody is less than or equal to 320, the relative risk of determining HCMV active infection in the subject is 23.7, 95% CI is 8.8-63.4. In another preferred embodiment, the reference value is 320, and if the antibody titer of the anti-pp150 antibody is less than or equal to 320, the probability of the subject developing HCMV active infection is 15.77%. In certain preferred embodiments, the reference value is set to an antibody titer of 320, and the sensitivity of the method for predicting HCMV active infection is 97.7%, specificity is 40.1%, and the Yoden index is 0.37. .
  • the absolute amount of antibody against pp150 and/or pp28 in the body fluid sample (eg, the absolute amount expressed in IU/ml) is determined. .
  • the anti-body sample is determined to be resistant by comparing the reactivity of the anti-pp150 and/or pp28 antibody in the body fluid sample to the antibody reactivity of a standard having a known antibody content.
  • the reference value is the absolute amount of antibody ranging between 0.8 and 6.4 IU/ml.
  • the reference value is The absolute amount of antibody in the range of 0.8-3.2 IU/ml.
  • the reference value is an absolute amount of antibody of 0.8, 1.6, or 3.2 IU/ml.
  • the reference value is an absolute amount of antibody of 3.2 IU/ml.
  • the reference value is 0.8 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 0.8 IU/ml, the relative probability of HCMV active infection in the subject is determined. The risk is 11.6 and the 95% CI is 7.7-17.2.
  • the reference value is 0.8 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 0.8 IU/ml, the probability of HCMV active infection in the subject is determined. It is 60.0%.
  • the reference value is 0.8 IU/ml and the sensitivity of the method for predicting HCMV active infection is 46.15%, specificity is 96.97%, and the Yoden index is 0.43.
  • the reference value is 1.6 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 1.6 IU/ml, then the subject is judged to have an HCMV active infection.
  • the relative risk is 15.2 and the 95% CI is 9.5-24.3.
  • the reference value is 1.6 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 1.6 IU/ml, the probability of HCMV active infection in the subject is determined. It is 50.0%.
  • the reference value is 1.6 IU/ml and the sensitivity of the method for predicting HCMV active infection is 67.69%, specificity is 93.34%, and the Yoden index is 0.61.
  • the reference value is 3.2 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 3.2 IU/ml, then the subject is judged to have an HCMV active infection.
  • the relative risk is 19.0 and the 95% CI is 9.6-37.7.
  • the reference value is 3.2 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 3.2 IU/ml, the probability of HCMV active infection in the subject is determined. It is 31.3%.
  • the reference value is 3.2 IU/ml and the sensitivity of the method for predicting HCMV active infection is 86.15%, specificity is 81.39%, and the Yoden index is 0.68.
  • the reference value is 6.4 IU/ml, and if the absolute amount of the antibody against the pp150 antibody is less than or equal to 6.4 IU/ml, then it is judged The relative risk of developing HCMV active infection in the subject was 25.2, 95% CI was 8.0-79.6.
  • the reference value is 6.4 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 6.4 IU/ml, the probability of HCMV active infection in the subject is determined. It is 19.0%.
  • the reference value is 6.4 IU/ml and the sensitivity of the method for predicting HCMV active infection is 95.38%, specificity is 59.91%, and the Yoden index is 0.55.
  • the invention provides a kit for assessing whether a subject is at risk of developing a human cytomegalovirus (HCMV) active infection, comprising an agent capable of determining the level of an antibody against an HCMV protein, and optionally, the reagent is used to determine the level of an antibody against the HCMV protein in a body fluid sample from the subject, thereby assessing whether the subject is in the risk of developing a human cytomegalovirus (HCMV) active infection .
  • HCMV human cytomegalovirus
  • the agent is capable of determining the level of an antibody against an HCMV protein by immunological assay.
  • the immunological assay is selected from the group consisting of an ELISA assay, a Western blot, a surface plasmon resonance method, and an Elispot assay.
  • the agent is selected from the group consisting of: an HCMV protein or antigenic fragment thereof, a fusion protein comprising the HCMV protein or an antigenic fragment thereof, and any combination thereof.
  • the HCMV protein is selected from the group consisting of pp150 and/or pp28.
  • the HCMV protein is pp150
  • the reagent is pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150); alternatively, the HCMV protein is pp28 And the reagent is pp28 and/or an antigenic fragment thereof; or, the HCMV protein is pp150 and pp28, and the reagent comprises: pp150 as a first component and/or an antigenic fragment thereof (for example, comprising Fragment of aa861-1048 of pp150), and pp28 and/or its antigenic fragment as a second component.
  • pp150 has the amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has the amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has An amino acid sequence as shown in SEQ ID NO: 3;
  • the subject is a mammal, such as a human.
  • the body fluid sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the active infection is that the subject not infected with HCMV is initially infected with HCMV, or the subject who has been infected with HCMV is again activated by HCMV infection or a latent HCMV virus in the body.
  • the kit further comprises:
  • the kit assesses whether the subject is at risk of developing a human cytomegalovirus (HCMV) active infection by a method comprising the steps of:
  • the subject is judged to be at risk of developing an active HCMV infection.
  • the level is an antibody titer (eg, an antibody titer as determined by ELISA), and the reference value is a predetermined antibody titer; or, the level is The absolute amount of antibody (e.g., the absolute amount expressed in IU/ml) and the reference value is the absolute amount of the predetermined antibody (e.g., the absolute amount expressed in IU/ml).
  • the antibody titer of the antibody against pp150 and/or pp28 in the body fluid sample is determined by ELISA; in a preferred embodiment , using pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150) to determine antibody titer of the anti-pp150 antibody in the body fluid sample by ELISA; and/or using pp28 and/or The antigenic fragment thereof was assayed for antibody titer of the anti-pp28 antibody in the body fluid sample by ELISA.
  • the reference value is an antibody titer ranging between 40-320.
  • the reference value is an antibody titer ranging between 40-160.
  • the reference value is an antibody titer of 40, 80, or 160.
  • the reference value is an antibody titer of 80.
  • the reference value is 40, and if the antibody titer of the anti-pp150 antibody is less than or equal to 40, the relative risk of determining HCMV active infection in the subject is 11.2, 95.
  • the %CI is 8.7-14.6.
  • the reference value is 40, and if the antibody titer of the anti-pp150 antibody is less than or equal to 40, the probability of the subject developing an HCMV active infection is 55.37%.
  • the reference value is set to an antibody titer of 40, and the sensitivity of the method for predicting HCMV active infection is 57.3%, specificity is 94.7%, and the Yoden index is 0.52. .
  • the reference value is 80, and if the antibody titer of the anti-pp150 antibody is less than or equal to 80, the relative risk of determining HCMV active infection in the subject is 10.6, 95% CI is 7.7-14.6. In another preferred embodiment, the reference value is 80, and if the antibody titer of the anti-pp150 antibody is less than or equal to 80, the probability of the subject developing HCMV active infection is 36.98%. In certain preferred embodiments, the reference value is set to an antibody titer of 80, and the sensitivity of the method for predicting HCMV active infection is 73.1%, specificity is 85.7%, and the Yoden index is 0.59. .
  • the reference value is 160, and if the antibody titer of the anti-pp150 antibody is less than or equal to 160, the relative risk of determining HCMV active infection in the subject is 14.8, 95% CI is 9.0-24.6. In another preferred embodiment, the reference value is 160, and if the antibody titer of the anti-pp150 antibody is less than or equal to 160, the probability of the subject developing HCMV active infection is 23.66%. In certain preferred embodiments, the reference value is set to an antibody titer of 160, and the sensitivity of the method for predicting HCMV active infection is 90.6%, specificity is 66.4%, and the Yoden index is 0.57. .
  • the reference value is 320, and if When the antibody titer of the pp150 antibody is less than or equal to 320, the relative risk of the HCMV active infection in the subject is judged to be 23.7, and the 95% CI is 8.8-63.4.
  • the reference value is 320, and if the antibody titer of the anti-pp150 antibody is less than or equal to 320, the probability of the subject developing HCMV active infection is 15.77%.
  • the reference value is set to an antibody titer of 320, and the sensitivity of the method for predicting HCMV active infection is 97.7%, specificity is 40.1%, and the Yoden index is 0.37. .
  • the absolute amount of antibody against pp150 and/or pp28 in the body fluid sample is determined. .
  • the anti-body sample is determined to be resistant by comparing the reactivity of the anti-pp150 and/or pp28 antibody in the body fluid sample to the antibody reactivity of a standard having a known antibody content.
  • the reference value is the absolute amount of antibody ranging between 0.8 and 6.4 IU/ml.
  • the reference value is the absolute amount of antibody ranging between 0.8-3.2 IU/ml.
  • the reference value is an absolute amount of antibody of 0.8, 1.6, or 3.2 IU/ml.
  • the reference value is an absolute amount of antibody of 3.2 IU/ml.
  • the reference value is 0.8 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 0.8 IU/ml, the relative probability of HCMV active infection in the subject is determined. The risk is 11.6 and the 95% CI is 7.7-17.2.
  • the reference value is 0.8 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 0.8 IU/ml, the probability of HCMV active infection in the subject is determined. It is 60.0%.
  • the reference value is 0.8 IU/ml and the sensitivity of the method for predicting HCMV active infection is 46.15%, specificity is 96.97%, and the Yoden index is 0.43.
  • the reference value is 1.6 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 1.6 IU/ml, then the subject is judged to have an HCMV active infection.
  • the relative risk is 15.2 and the 95% CI is 9.5-24.3.
  • the reference value is 1.6 IU/ml, and And if the absolute amount of the antibody against the pp150 antibody is less than or equal to 1.6 IU/ml, the probability of the HCMV active infection in the subject is judged to be 50.0%.
  • the reference value is 1.6 IU/ml and the sensitivity of the method for predicting HCMV active infection is 67.69%, specificity is 93.34%, and the Yoden index is 0.61.
  • the reference value is 3.2 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 3.2 IU/ml, then the subject is judged to have an HCMV active infection.
  • the relative risk is 19.0 and the 95% CI is 9.6-37.7.
  • the reference value is 3.2 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 3.2 IU/ml, the probability of HCMV active infection in the subject is determined. It is 31.3%.
  • the reference value is 3.2 IU/ml and the sensitivity of the method for predicting HCMV active infection is 86.15%, specificity is 81.39%, and the Yoden index is 0.68.
  • the reference value is 6.4 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 6.4 IU/ml, then the subject is judged to have an HCMV active infection.
  • the relative risk is 25.2 and the 95% CI is 8.0-79.6.
  • the reference value is 6.4 IU/ml, and if the absolute amount of antibody to the anti-pp150 antibody is less than or equal to 6.4 IU/ml, the probability of HCMV active infection in the subject is determined. It is 19.0%.
  • the reference value is 6.4 IU/ml and the sensitivity of the method for predicting HCMV active infection is 95.38%, specificity is 59.91%, and the Yoden index is 0.55.
  • the method of the present invention is capable of reliably and efficiently assessing the risk of human cytomegalovirus (HCMV) active infection in a subject by measuring the level of an antibody against the HCMV protein in a body fluid sample from the subject. Based on this, if the subject's level of anti-HCMV protein antibody in the body fluid sample is increased after receiving a candidate drug (compared to the level before receiving the drug candidate), then this indicates that the subject The risk of developing human cytomegalovirus (HCMV) active infection is reduced after receiving the candidate drug. It can be reasonably determined that the candidate drug used can enhance the subject's ability to resist active cytomegalovirus (HCMV) active infection and reduce the occurrence of human cytomegalovirus (HCMV) active infection in the subject. risks of.
  • HCMV human cytomegalovirus
  • the invention provides for screening for the ability to increase a subject's ability to resist human cytomegalovirus (HCMV) active infection or to reduce the risk of human cytomegalovirus (HCMV) active infection in a subject
  • a method of drug candidate comprising the steps of:
  • HCMV human cytomegalovirus
  • test animal is a non-human mammal, such as a model animal (eg, mouse, rat, rabbit, primate).
  • body fluid sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the subject is a mammal, such as a human.
  • the active infection is that the subject not infected with HCMV is initially infected with HCMV, or the subject who has been infected with HCMV is again activated by HCMV infection or a latent HCMV virus in the body.
  • the HCMV protein is selected from the group consisting of pp150 and/or pp28.
  • the level of antibody against the HCMV protein in the body fluid sample is determined by immunological assay.
  • the immunological assay is selected from the group consisting of an ELISA assay, a Western blot, a surface plasmon resonance method, and an Elispot assay.
  • the first and second levels are antibody titers (eg, antibody titers determined by ELISA); alternatively, the first and second levels are absolute amounts of antibody (eg, in IU /ml indicates the absolute amount).
  • first and second levels of antibodies against pp150 and/or pp28 in the body fluid sample are determined.
  • first and second levels of the anti-pp150 antibody in the body fluid sample are determined by ELISA using pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150);
  • the first and second levels of the anti-pp28 antibody in the body fluid sample are determined by ELISA using pp28 and/or antigenic fragments thereof.
  • pp150 has the amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has the amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has An amino acid sequence as shown in SEQ ID NO: 3;
  • the anti-body sample is determined to be resistant by comparing the reactivity of the anti-pp150 and/or pp28 antibody in the body fluid sample to the antibody reactivity of a standard having a known antibody content. First and second levels of antibodies to pp150 and/or pp28.
  • the invention provides the use of an agent capable of determining the level of an antibody against an HCMV protein in a kit for screening for enhancing resistance to human cytomegalovirus (HCMV) activity in a subject The ability to infect or reduce the risk of a subject's risk of developing human cytomegalovirus (HCMV) active infection.
  • an agent capable of determining the level of an antibody against an HCMV protein in a kit for screening for enhancing resistance to human cytomegalovirus (HCMV) activity in a subject The ability to infect or reduce the risk of a subject's risk of developing human cytomegalovirus (HCMV) active infection.
  • the agent is capable of determining the level of an antibody against an HCMV protein by immunological assay.
  • the immunological assay is selected from the group consisting of an ELISA assay, a Western blot, a surface plasmon resonance method, and an Elispot assay.
  • the agent is selected from the group consisting of: an HCMV protein or antigenic fragment thereof, a fusion protein comprising the HCMV protein or an antigenic fragment thereof, and any combination thereof.
  • the HCMV protein is selected from the group consisting of pp150 and/or pp28.
  • the HCMV protein is pp150
  • the reagent is pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150); alternatively, the HCMV protein is pp28 And the reagent is pp28 and/or an antigenic fragment thereof; or, the HCMV protein is pp150 and pp28, and the reagent comprises: pp150 as a first component and/or an antigenic fragment thereof (for example, comprising Pp150 aa Fragments of 861-1048), and pp28 and/or antigenic fragments thereof as a second component.
  • pp150 has the amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has the amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has The amino acid sequence shown as SEQ ID NO: 3.
  • the subject is a mammal, such as a human.
  • the active infection is that the subject not infected with HCMV is initially infected with HCMV, or the subject who has been infected with HCMV is again activated by HCMV infection or a latent HCMV virus in the body.
  • the kit is screened by a method comprising the steps of increasing the ability of the subject to resist active cytomegalovirus (HCMV) active infection or reducing the occurrence of human cytomegalovirus in the subject ( HCMV) Candidates for the risk of active infections:
  • determining that the candidate drug is capable of increasing a subject's ability to resist human cytomegalovirus (HCMV) active infection or reducing a subject's occurrence of human giant cells The risk of viral (HCMV) active infection.
  • HCMV cytomegalovirus
  • test animal is a non-human mammal, such as a model animal (eg, mouse, rat, rabbit, primate).
  • a model animal eg, mouse, rat, rabbit, primate.
  • the body fluid sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the kit further comprises:
  • the first and second levels are antibody titers (eg, The antibody titer determined by ELISA); alternatively, the first and second levels are the absolute amount of antibody (eg, the absolute amount expressed in IU/ml).
  • the first and second levels of antibodies against pp150 and/or pp28 in the body fluid sample are determined by ELISA.
  • the first and second levels of the anti-pp150 antibody in the body fluid sample are determined by ELISA using pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150);
  • the first and second levels of the anti-pp28 antibody in the body fluid sample are determined by ELISA using pp28 and/or antigenic fragments thereof.
  • pp150 has the amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has the amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has The amino acid sequence shown as SEQ ID NO: 3.
  • the anti-body sample is determined to be resistant by comparing the reactivity of the anti-pp150 and/or pp28 antibody in the body fluid sample to the antibody reactivity of a standard having a known antibody content. First and second levels of antibodies to pp150 and/or pp28.
  • the invention provides a method for screening to increase a subject's ability to resist a human cytomegalovirus (HCMV) active infection or to reduce a subject's risk of developing human cytomegalovirus (HCMV) active infection.
  • a kit of candidate drugs comprising an agent capable of determining the level of an antibody against an HCMV protein, and optionally, the use of the agent for screening to enhance a subject's ability to resist human cytomegalovirus (HCMV) active infection Or instructions for reducing the risk of a candidate drug for a human cytomegalovirus (HCMV) active infection.
  • the agent is capable of determining the level of an antibody against an HCMV protein by immunological assay.
  • the immunological assay is selected from the group consisting of an ELISA assay, a Western blot, a surface plasmon resonance method, and an Elispot assay;
  • the agent is selected from the group consisting of: an HCMV protein or an antigenic fragment thereof, a fusion protein comprising the HCMV protein or an antigenic fragment thereof, and any group thereof Hehe.
  • the HCMV protein is selected from the group consisting of pp150 and/or pp28.
  • the HCMV protein is pp150
  • the reagent is pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150); alternatively, the HCMV protein is pp28 And the reagent is pp28 and/or an antigenic fragment thereof; or, the HCMV protein is pp150 and pp28, and the reagent comprises: pp150 as a first component and/or an antigenic fragment thereof (for example, comprising Fragment of aa861-1048 of pp150), and pp28 and/or its antigenic fragment as a second component.
  • pp150 has the amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has the amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has The amino acid sequence shown as SEQ ID NO: 3.
  • the subject is a mammal, such as a human.
  • the active infection is that the subject not infected with HCMV is initially infected with HCMV, or the subject who has been infected with HCMV is again activated by HCMV infection or a latent HCMV virus in the body.
  • the kit is screened by a method comprising the steps of increasing the ability of the subject to resist active cytomegalovirus (HCMV) active infection or reducing the occurrence of human cytomegalovirus in the subject ( HCMV) Candidates for the risk of active infections:
  • determining that the candidate drug is capable of increasing a subject's ability to resist human cytomegalovirus (HCMV) active infection or reducing a subject's occurrence of human giant cells The risk of viral (HCMV) active infection.
  • HCMV cytomegalovirus
  • test animal is a non-human mammal, such as a model animal (eg, mouse, rat, rabbit, primate).
  • body fluid sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the kit further comprises:
  • the first and second levels are antibody titers (eg, antibody titers determined by ELISA); alternatively, the first and second levels are absolute amounts of antibody (eg, in IU /ml indicates the absolute amount).
  • the first and second levels of antibodies against pp150 and/or pp28 in the body fluid sample are determined by ELISA.
  • the first and second levels of the anti-pp150 antibody in the body fluid sample are determined by ELISA using pp150 and/or an antigenic fragment thereof (eg, a fragment thereof comprising aa 861-1048 of pp150);
  • the first and second levels of the anti-pp28 antibody in the body fluid sample are determined by ELISA using pp28 and/or antigenic fragments thereof.
  • pp150 has the amino acid sequence set forth in SEQ ID NO: 1; and/or, the antigenic fragment of pp150 has the amino acid sequence set forth in SEQ ID NO: 2; and/or, pp28 has The amino acid sequence shown as SEQ ID NO: 3.
  • the anti-body sample is determined to be resistant by comparing the reactivity of the anti-pp150 and/or pp28 antibody in the body fluid sample to the antibody reactivity of a standard having a known antibody content. First and second levels of antibodies to pp150 and/or pp28.
  • the present invention demonstrates for the first time that the antibody level of an anti-HCMV protein (e.g., pp150/pp28) has a significant correlation with the risk of human cytomegalovirus active infection.
  • the present invention first demonstrates that when the antibody titer of an anti-pp150 antibody in a subject's body fluid sample is less than or equal to 40, the probability of HCMV active infection in the subject is 55.37%, and the relative risk is 11.2.
  • 95% CI is 8.7-14.6; when the subject's body fluid sample is resistant to pp150 When the antibody titer of the antibody is less than or equal to 80, the probability of occurrence of HCMV active infection in the subject is 36.98%, the relative risk is 10.6, and the 95% CI is 7.7-14.6; when the subject's body fluid sample is resistant to pp150 When the antibody titer of the antibody is less than or equal to 160, the probability of occurrence of HCMV active infection in the subject is 23.66%, the relative risk is 14.8, 95% CI is 9.0-24.6; when the subject's body fluid sample is resistant to pp150 When the antibody titer of the antibody is less than or equal to 320, the probability of HCMV active infection in the subject is 15.77%, the relative risk is 23.7, and the 95% CI is 8.8-63.4.
  • the present invention also demonstrates for the first time that when the content of the anti-pp150 antibody in the body fluid sample of the subject is less than 0.8 IU/ml, the probability of HCMV active infection in the subject is 60.0%, and the relative risk is 11.6.
  • 95% CI is 7.7-17.2; when the content of anti-pp150 antibody in the body fluid sample is less than 1.6 IU/ml, the probability of HCMV active infection in the subject is 50.0%, and the relative risk is 15.2.
  • 95% CI is 9.5-24.3; when the content of anti-pp150 antibody in the body fluid sample is less than 3.2 IU/ml, the probability of HCMV active infection in the subject is 31.3%, and the relative risk is 19.0.
  • 95% CI is 9.6-37.7; when the content of anti-pp150 antibody in the body fluid sample is less than 6.4 IU/ml, the probability of HCMV active infection in the subject is 19.0%, and the relative risk is 25.2. 95% CI is 8.0-79.6.
  • antibody levels against an anti-HCMV protein can be used as predictors of human cytomegalovirus (HCMV) active infection risk (eg, HCMV reinfection risk in a seronegogenic population); and, the present invention
  • the method can be used to screen individuals (ie, high-risk individuals) prone to human cytomegalovirus active infection in a population of high-risk areas of natural population or human cytomegalovirus active infection.
  • the method of the present invention can provide a basis for taking interventions and can guide clinical medication to prevent the occurrence of complications in patients.
  • the method of the present invention can be used in a pregnant population to screen out high-risk individuals, and the intervention rate and clinical use of these individuals can be used to reduce the risk of neonatal congenital cytomegalovirus infection; or the method of the present invention can be used for organ transplantation.
  • high-risk individuals are screened and reduced by interventions and clinical medications for these individuals. The probability of occurrence of complications.
  • the methods of the invention can be used to screen for drug candidates that increase the subject's ability to resist active cytomegalovirus (HCMV) active infection or reduce the risk of human cytomegalovirus (HCMV) active infection in a subject.
  • HCMV active cytomegalovirus
  • the methods of the present invention provide new ideas and protocols for the development of new drugs for the prevention and/or treatment of HCMV infection.
  • FIG. 1A and 1B show that the ELISA detection platform (based on p150 and pp150-2) established in Example 2 can be used to detect the level of anti-CMV protein antibody (anti-pp150 antibody) in serum samples, thereby determining that the serum sample is positive. Still negative.
  • Figure 1B shows a comparison of the results of ELISA using pp150 and pp150-2 as coating antigens and ELISA using commercially available CMV-IgG reagent (Diasorin-IgG) as coating antigen for 36 serum samples.
  • the results show that the pp150 and pp150-2 based ELISA methods of the present invention are significantly more reactive than the Diasorin based ELISA, which is mostly at a lower level of reactivity to positive sera.
  • the results of Figures 1A and 1B show that the ELISA detection platform established in Example 2 can be used to accurately, reliably and efficiently detect the level of anti-CMV protein antibody (anti-pp150 antibody) in serum samples.
  • Figure 2 shows the correlation between the amount of HCMV virus infection and the Elispot detection value in the IE1 protein-based Elispot assay platform established in Example 3.
  • Figure 3 shows the comparison of the detection results of 61 serum samples (Elispot NT50) with the commercial HCMV IgG antibody detection kit (Diasorin IgG reagent) by the IE1 protein-based Elispot detection platform established in Example 3. Among them, for the 31 sera tested positive in the kit, the Elispot test platform was positive; for the 30 sera negative for the kit, the Elispot test platform was negative; the results were identical. . This indicates the accuracy and reliability of the Elispot detection platform established in Example 3.
  • Figure 4 shows the correlation between the detection results of 61 serum samples (Elispot NT50) and the commercial HCMV IRD neutralization detection platform (IRD NT50) of the IE1 protein-based Elispot detection platform established in Example 3.
  • Figure 5 shows the six ELISA detection platforms established in Example 2 (based on pp150 and pp150-2, HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99), and UL48a, respectively.
  • pp150 refers to an ELISA detection platform based on a mixture of pp150 and pp150-2.
  • the results show that the ELISA detection platform established in Example 2 (especially, ELISA detection platform based on pp150 and pp150-2 and ELISA detection platform based on pp28) can accurately, reliably and effectively detect anti-CMV protein in serum samples.
  • the level of antibody is judged to be negative and positive for serum samples.
  • FIGS. 6A-C show the ROC curve analysis of the pp150 and pp150-2 based ELISA detection platform (abbreviated as pp150 detection platform) in Example 6 under different evaluation criteria for viral events.
  • the abscissa indicates (100% - the detection specificity % of the detection platform), and the ordinate indicates the detection sensitivity % of the detection platform; and,
  • Figure 6A shows the sensitivity of the pp150 detection platform to assess the occurrence of viral events (HCMV active infection) when a test result of multiple other detection platforms is positive for double positive and above as a viral event (HCMV active infection). And specific analysis;
  • Figure 6B shows that when a test result of multiple other detection platforms is positive for Sanyang and above as a viral event (HCMV active infection), the pp150 detection platform evaluates the occurrence of a viral event (HCMV active infection). Analysis of sensitivity and specificity;
  • Figure 6C shows that when a test result of multiple other detection platforms is positive for four positives and above as a viral event (HCMV active infection), the pp150 detection platform evaluates the occurrence of a viral event (HCMV active infection). Analysis of sensitivity and specificity.
  • Figures 7A-C show ROC curve analysis of the pp28-based ELISA detection platform (abbreviated as pp28 detection platform) in Example 6 under different evaluation criteria for viral events.
  • the abscissa indicates (100% - the detection specificity % of the detection platform), and the ordinate indicates the detection sensitivity % of the detection platform; and,
  • Figure 7A shows the sensitivity of the pp28 detection platform to assess the occurrence of viral events (HCMV active infection) when a test result of multiple other detection platforms is positive for double positive and above as a viral event (HCMV active infection). And specific analysis;
  • Figure 7B shows that when a test result of a plurality of other detection platforms is positive for Sanyang and above is defined as a viral event (HCMV active infection), the pp28 detection platform evaluates the occurrence of a viral event (HCMV active infection). Analysis of sensitivity and specificity;
  • Figure 7C shows that when a test result of multiple other detection platforms is positive for four positives and above as a viral event (HCMV active infection), the pp28 detection platform evaluates the occurrence of a viral event (HCMV active infection). Analysis of sensitivity and specificity.
  • Figure 8 is a graph showing changes in antibody titer of anti-pp150 antibody in vivo in a natural population of Guangxi, described in Example 7, at intervals of 12 months (one year), wherein the abscissa indicates baseline antibody level (antibody titer), vertical
  • the coordinates represent the probability that the antibody level will increase by a factor of 4 or a factor of four after one year.
  • the results showed that individuals with higher baseline antibody levels had a 4-fold higher increase in anti-pp150 antibody levels (ie, HCMV active infection) after one year; the lower the baseline antibody level, the more individuals appeared one year later.
  • the risk of HCMV active infection in individuals is inversely related to baseline levels of antibodies.
  • Figure 9 shows the correlation between baseline anti-pp150 antibody levels and HCMV active infection risk in a natural population in Guangxi as described in Example 7.
  • the abscissa indicates the baseline anti-pp150 antibody level (antibody titer), and the ordinate indicates the risk (proportion) of HCMV active infection.
  • the results show that individuals with lower baseline antibody levels have a higher risk of subsequent HCMV active infection (ie, an increase in anti-pp150 antibody levels of 4 fold or more).
  • the risk of HCMV active infection was inversely correlated with baseline antibody levels.
  • the risk of HCMV active infection is as high as 82.6%; and when the antibody baseline level (antibody titer) does not exceed 160, the risk of HCMV active infection is as high as possible. 23.66%.
  • Figure 10 shows a standard curve of antibody content-antibody reactivity plotted with a standard of known antibody content (Paul-Ehrlich-Instltut, Referenz-CMV-IgG, Juli 1996, 110 IU/ml).
  • the abscissa indicates the antibody content (expressed in IU/ml);
  • the ordinate indicates the antibody reactivity (expressed as the OD value obtained from the ELISA).
  • Figure 11 is a graph showing changes in the in vivo anti-pp150 antibody content of a natural population in Guangxi at 12 months (one year) as described in Example 8, wherein the abscissa indicates the baseline antibody level (antibody content) and the ordinate indicates one year. The probability of a post-antibody level increase of 4 fold or a decrease of 4 fold. The results showed that individuals with lower baseline antibody levels developed anti-pp150 antibody water one year later.
  • Figure 12 shows the results of ROC curve analysis of the method described in Example 8, wherein the method predicts HCMV active infection based on the amount/absolute amount of anti-pp150 antibody in serum, and the abscissa indicates (100%- The specificity % of the method, the ordinate indicates the % sensitivity of the method.
  • the results of Figure 12 demonstrate that the methods of the invention can be used to accurately, reliably, and efficiently assess the risk of human cytomegalovirus (HCMV) active infection in a subject.
  • HCMV human cytomegalovirus
  • the present inventors recombinantly expressed HCMV pp150 (UL32), HCMV pp150-2 ( truncated protein of pp150), HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99) and UL48a a total of 7 proteins.
  • Relevant information for recombinantly expressed proteins is listed in Table 2.
  • Primers for PCR amplification of genes encoding the protein of interest are listed in Table 3.
  • Table 3 Information on primers for PCR amplification of genes encoding the protein of interest
  • Example 2 Establishment of an ELISA detection platform for detecting antibodies based on antigenic proteins
  • the inventors are based on HCMV pp150 and pp150-2 (as a combined coating antigen), HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99) or UL48a, respectively.
  • ELISA detection platform for detecting anti-pp150 antibody, anti-pp65 antibody, anti-gp52 antibody, anti-pp38 antibody, anti-pp28 antibody and anti-UL48a antibody (referred to as pp150 detection platform, pp65 detection platform, gp52 detection platform) , pp38 detection platform, pp28 detection platform, UL48a detection platform), each of which includes: microplate coated with antigen protein (ie, coated antigen), coating buffer, blocking solution, washing solution, enzyme-labeled anti-drug Human IgG antibody, enzyme-labeled antibody dilution, negative positive control, chromogenic solution, and stop solution.
  • antigen protein ie, coated antigen
  • Coating antigen HCMV pp150 and pp150-2, HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99) or UL48a;
  • Concentrated washing solution it comprises a phosphate buffer pH 7-7.6 and a surfactant Tween20;
  • Enzyme-labeled anti-human IgG antibody horseradish peroxidase (HRP)-labeled murine anti-human IgG Monoclonal antibodies.
  • Enzyme-labeled antibody dilution phosphate buffer with a pH of 6.8-7.3, containing 0.1-1 M NaCl per 0.3 ml of phosphate buffer and 0.3-1% (mass to volume) of casein, 0.1-0.4% (mass volume) Ratio of TritonX-100, 7-12% (by volume) of fetal bovine serum, 0.2-0.5% (mass to volume ratio) of Geltin;
  • Color developing solution liquid A containing trisodium citrate, citric acid, sodium acetate, glacial acetic acid and hydrogen peroxide; and, including anhydrous ethanol, ethylene glycol, dimethylformamide, 3, 3, 5, 5 - tetramethylbenzidine B solution
  • Stop solution 0.1-1 M sulfuric acid.
  • the pp150 antibody detection platform is taken as an example to illustrate the effect of the ELISA detection platform established in the present embodiment.
  • 288 randomly selected sera were tested in parallel using a pp150 and pp150-2 based ELISA detection platform with a purified virus based ELISA detection platform.
  • the results are shown in Figure 1A.
  • the inventors established an Elispot detection platform based on IE1 protein for detecting anti-HCMV-IE1 antibodies.
  • the Elispot detection method established in this embodiment is a rapid cytomegalovirus drop established by combining the traditional enzyme-linked immunospot assay with the Elispot automated spot counter. Degree detection method. Compared with the traditional method for detecting TCID50, the Elispot detection method established in this embodiment does not rely on cytopathic count or plaque formation to obtain experimental results, but through the specific binding of monoclonal antibodies to early viral proteins. Get the experimental results. Therefore, the Elispot detection method does not require the waiting for the lesion of the virus-infected cells, which greatly shortens the detection time (20h). In addition, the Elispot detection method uses the Elispot detector's automated picture capture and spot count program to read the results, greatly improving the stability and accuracy of the test.
  • Figure 2 shows the correlation between the amount of HCMV virus infection and the Elispot detection value in the IE1 protein-based Elispot detection platform established in this example.
  • the detection results of the Elispot detection platform established in the present example were compared with the detection results of a commercial HCMV IgG antibody detection kit (Diasorin-IgG reagent, DIASORIN, P002033).
  • the results are shown in Figure 3.
  • the results showed that in the 61 serum samples tested in parallel, the Elispot test platform was positive for the 31 sera positive for the kit; the Elispot test platform was tested for 30 sera negative for the kit test. The results were all negative; the results were completely consistent. This indicates the accuracy and reliability of the Elispot detection platform established in this embodiment.
  • Example 4 Detection of known serum in the background using an ELISA assay platform
  • HCMV IRD detection reagent (Aimin Tang, Fengsheng Li, Daniel C. Freed, Adam C. Finnefrock, Danilo R. Casimiro, Dai Wang, Tong-Ming Fu.
  • HCMV IgG antibody detection kit (Diasorin-IgG reagent, DIASORIN, P002033) determined the background of these serum samples.
  • the detection method includes the following steps:
  • the serum to be tested is centrifuged for 5-10 mins (10000 rpm/min) for detection;
  • the components in the kit were equilibrated to room temperature; 90 ul of sample dilution and 10 ul of test serum were added to the sample well of the coated microplate of the ELISA detection platform established in Example 2; A negative control and a positive control were set in the well plate, wherein 10 ul of background-negative serum and 90 ul of sample dilution were added to each well of the negative control, and 10 ul of background-positive serum and 90 ul of sample dilution were added to each well of the positive control; Shake and mix on the shaker; then, attach the sealing film and react in a 37 ° C incubator / constant temperature water bath for 1 hour;
  • Step 3 After the reaction, the sealing film is peeled off, washed 5 times with the washing liquid, and buckled;
  • Step 4 100 ⁇ l of the enzyme-labeled anti-human IgG antibody solution was added to each well, and the sealing film was attached; then, the reaction was carried out for 30 min in a 37 ° C incubator/constant water bath;
  • Step 5 After the reaction, the sealing film is peeled off, washed 5 times with the washing liquid, and buckled;
  • Step 6 Add 50 ul of substrate solution A and 50 ul of substrate solution B to each well, and mix; then react in a 37 ° C incubator / constant temperature water bath for 15 min;
  • Step 7 50 ul of stop solution was added to each well, and then the reading at OD450 was read on a microplate reader, thereby obtaining the antibody reaction OD value of each well.
  • the test results are shown in Fig. 5.
  • Figure 5 shows the six ELISA detection platforms established in Example 2 (based on pp150 and pp150-2, HCMV pp65 (UL83), HCMV gp52 (UL44), HCMV pp38 (UL80a), HCMV pp28 (UL99), and UL48a, respectively.
  • pp150 refers to an ELISA detection platform based on a mixture of pp150 and pp150-2.
  • the results show that the ELISA detection platform established in Example 2 (especially, ELISA detection platform based on pp150 and pp150-2 and ELISA detection platform based on pp28) can accurately, reliably and effectively detect anti-CMV protein in serum samples.
  • the level of antibody is judged to be negative and positive for serum samples.
  • Example 5 Using an ELISA assay platform to determine antibody titers in a sample
  • the inventors used the ELISA detection platform established in Example 2 to determine antibody titers in serum samples to verify the reliability and effectiveness of these ELISA detection platforms.
  • pp150 an ELISA assay platform based on pp150 and pp150-2 was used to perform HCMV IgG antibodies on serial dilutions of two serum (serum 1 and serum 2) samples. Parallel detection of pp150 antibody) (two replicates per sample) to determine antibody titers of anti-pp150 antibodies in both sera. Negative control wells, positive control wells, and blank control wells were set during the assay.
  • the detection method includes the following steps:
  • the serum to be tested is centrifuged for 5-10 mins (10000 rpm/min) for detection;
  • the components in the kit are equilibrated to room temperature
  • a negative control well, a positive control well and a blank control well were set in the microplate, wherein 10 ul of serum with negative background and 90 ul of sample dilution were added to each well of the negative control, and 10 ul of the positive control was added to each well.
  • Positive serum and 90 ul sample dilution, blank control added 100 ul sample dilution per well;
  • the sealing film is pasted and reacted in a 37 ° C incubator / constant temperature water bath for 1 hour;
  • Step 3 After the reaction, the sealing film is peeled off, washed 5 times with the washing liquid, and buckled;
  • Step 4 100 ⁇ l of the enzyme-labeled anti-human IgG antibody solution was added to each well, and the sealing film was attached; then, the reaction was carried out for 30 min in a 37 ° C incubator/constant water bath;
  • Step 5 After the reaction, the sealing film is peeled off, washed 5 times with the washing liquid, and buckled;
  • Step 6 Add 50 ul of substrate solution A and 50 ul of substrate solution B to each well, and mix; then react in a 37 ° C incubator / constant temperature water bath for 15 min;
  • Step 7 50 ul of stop solution was added to each well, and then the reading at OD450 was read on a microplate reader, thereby obtaining the antibody reaction OD value of each well.
  • the test results are shown in Table 4.
  • serum 1 and serum 2 are two separate serum samples, and groups 1 and 2 represent two replicates, and antibody titer is defined as the maximum serum dilution factor with an OD450 of 0.2 or more.
  • the test results showed that the antibody titer of HCMV IgG of serum 1 (i.e., anti-pp150 antibody) was 80, and the antibody titer of serum 2 was 320.
  • Example 4 The results in Table 4 show that the ELISA detection platform established in Example 2 (for example, ELISA detection platform based on pp150 and pp150-2) can accurately, reliably and efficiently detect antibodies against CMV protein in samples (for example, anti-pp150 Antibody titer of antibody).
  • Example 6 Assessment of the risk of HCMV active infection using an ELISA assay platform
  • the protein used in the present example was the protein obtained in Example 1, and the detection method used was the detection method described in Example 5.
  • this example uses the ELISA detection platform established in Example 2 (including ELISA detection platform based on pp150 and pp150-2 (referred to as pp150 detection platform, the same below), pp28 detection platform, pp38 detection platform, UL48a detection platform, gp52 detection platform, pp65 detection platform) and IE1 antibody detection platform (ie, Elispot detection platform for detecting anti-HCMV-IE1 antibody described in Example 3) to detect well-preserved, obtained from Guangxi nature A total of 202 pairs of serum were collected from the population at intervals of 12 months (one year).
  • the number of samples detected by the IE1 antibody detection platform, the pp150 detection platform, the pp28 detection platform, the pp38 detection platform, the UL48a detection platform, the gp52 detection platform, and the pp65 detection platform in the serum samples of 202 (ie, The number of samples in which the antibody titer in the serum collected later was increased by 4 times or more was 8 parts, 77 parts, 21 parts, 29 parts, 30 parts, 24 parts, and 9 parts.
  • the standard for indicating that a viral event has occurred in an individual is defined as that at least 2 of the 6 tests obtained by the other 6 test platforms against the individual's serum pair, at least Three or at least four test results are positive at the same time.
  • Table 5 shows the statistics of the test results of the other six test platforms.
  • Table 6a shows the results of the AUC curve parameter analysis of the pp150 detection platform under different defined criteria for viral events.
  • Table 6b shows the results of the LOGISTIC regression analysis of the pp150 detection platform under different defined criteria for viral events.
  • Table 6a AUC curve parameters of the pp150 detection platform under different defined standards for virus events
  • Figure 6A shows the sensitivity of the pp150 detection platform to assess the occurrence of viral events (HCMV active infection) when a test result of multiple other detection platforms is positive for double positive and above as a viral event (HCMV active infection). And specific analysis;
  • Figure 6B shows that when a test result of multiple other detection platforms is positive for Sanyang and above as a viral event (HCMV active infection), the pp150 detection platform evaluates the occurrence of a viral event (HCMV active infection). Analysis of sensitivity and specificity;
  • Figure 6C shows that when a test result of multiple other detection platforms is positive for four positives and above as a viral event (HCMV active infection), the pp150 detection platform evaluates the occurrence of a viral event (HCMV active infection). Analysis of sensitivity and specificity.
  • Tables 6a-6b and 6A-C show that in all three cases, the pp150 detection platform predicts that the incidence of viral events (HCMV active infections) is greater than 80%, which can be used to be accurate, reliable, and effective.
  • the subject is assessed for the risk of developing human cytomegalovirus (HCMV) active infection.
  • Table 7a shows the results of the AUC curve parameter analysis of the pp28 detection platform under different defined criteria for viral events.
  • Table 7b shows the results of the LOGISTIC regression analysis of the pp28 detection platform under different defined criteria for viral events.
  • Table 7a AUC Curve Parameters for the pp28 Detection Platform under Different Definitions of Virus Events
  • Figure 7A shows that when the detection results of various other detection platforms are positive for Shuangyang and above The case is defined as the sensitivity and specificity of the pp28 detection platform to assess the occurrence of viral events (HCMV active infections) when a viral event (HCMV active infection) occurs;
  • Figure 7B shows that when a test result of a plurality of other detection platforms is positive for Sanyang and above is defined as a viral event (HCMV active infection), the pp28 detection platform evaluates the occurrence of a viral event (HCMV active infection). Analysis of sensitivity and specificity;
  • Figure 7C shows that when a test result of multiple other detection platforms is positive for four positives and above as a viral event (HCMV active infection), the pp28 detection platform evaluates the occurrence of a viral event (HCMV active infection). Analysis of sensitivity and specificity.
  • Tables 7a-7b and 7A-C show that in all three cases, the pp28 detection platform predicts that the incidence of viral events (HCMV active infections) is greater than 78%, which can be used to be accurate, reliable, and effective.
  • the subject is assessed for the risk of developing human cytomegalovirus (HCMV) active infection.
  • Example 7 pp150 detection platform for detection results of a natural population in Guangxi
  • the inventors used the ELISA detection platform (pp150 detection platform) established in Example 2 to assess the risk of HCMV active infection in a natural population in Guangxi.
  • the detection method used and the antibody titer calculation method are as described in Example 5.
  • the inventors used the pp150 detection platform to detect duplicate sera of each individual in a natural population (1659 people) in Guangxi at intervals of 12 months (one year).
  • the test results showed that in this population, the positive rate of antibody detection was 98.7% and 98.9%, respectively, and the average antibody titers were 1:269 and 1:260, respectively. This suggests that the vast majority of individuals in this population have previously been infected with HCMV.
  • the inventors further compared the test results obtained by the two tests before and after the population. The results of the comparison are shown in Figures 8-9 and 8a-8b.
  • Figure 8 shows the change in antibody titer of the anti-pp150 antibody in vivo in the natural population at intervals of 12 months (one year).
  • the results showed that the higher the baseline antibody level (ie, the antibody titer obtained in the first first test), the one-fold increase in anti-pp150 antibody levels after one year (ie, the second subsequent test)
  • the antibody titer obtained is at least 4 times higher than the antibody titer obtained by the prior detection, indicating that the probability that the individual has developed HCMV active infection during the 12 month interval is lower; and the lower the baseline antibody level is.
  • Individual The higher the probability of an increase in anti-pp150 antibody levels of 4 times or more after one year.
  • Figure 9 shows the correlation between baseline anti-pp150 antibody levels and the risk of HCMV active infection in the natural population.
  • the abscissa indicates the baseline anti-pp150 antibody level (antibody titer), and the ordinate indicates the proportion (risk) of HCMV active infection.
  • the results show that individuals with lower baseline antibody levels have a higher risk of subsequent HCMV active infection (ie, an increase in anti-pp150 antibody levels of 4 fold or more).
  • the risk of HCMV active infection was inversely correlated with baseline antibody levels.
  • Tables 8a8b and 9 show that: (1) The rate of HCMV active infection (primary infection) in individuals with negative baseline antibody levels (antibody titers ⁇ 10) is as high as 82.6% (19/23); The rate of HCMV active infection (re-infection) in individuals with antibody levels (antibody titers) ⁇ 1:2560 was 0% (0/71) (p ⁇ 0.0001); (2) HCMV active infection rate and baseline antibody There was a significant negative correlation between levels: the higher the baseline antibody level, the lower the HCMV active infection rate.
  • individuals with antibody titers >1:80 (about 80%) had a viral active infection rate of 3.48% within one year, and individuals with antibody titers ⁇ 1:80 (about 20%) had active viral infections. The rate is 36.98%.
  • individuals with antibody titers >1:40 had a viral active infection rate of 4.93% within one year, while individuals with antibody titers ⁇ 1:40 had a viral active infection rate of 55.37%.
  • Individuals with antibody titers >1:160 had a viral active infection rate of 1.59% within one year, while individuals with antibody titers ⁇ 1:160 had a viral active infection rate of 23.66%.
  • the results of Table 8b also show that the method of the present invention can be used to determine the relative risk of infection in a subject, wherein the reference value for determining the level of relative antibody (ie, antibody titer) can be set to Antibody titers ranging between 40-320, such as antibody titers of 40, 80, 160 or 320. If the antibody titer detected in the subject sample is less than or equal to the reference value, then the relative risk of HCMV active infection in the subject is considered to be high. As can be seen from Table 8b, when the reference value is between 40-320, the relative risk of HCMV active infection in subjects with antibody titers below the reference value exceeds 10, and its 95% CI The lower limit exceeds 7. This suggests that there is a strong correlation or a strong correlation between this parameter (antibody titer) and HCMV active infection.
  • the reference value for determining the level of relative antibody ie, antibody titer
  • Antibody titers can be set to Antibody tit
  • the relative risk of developing HCMV active infection is 11.2, 95% compared to subjects with antibody titers above 40.
  • the CI is 8.7-14.6 (ie, the risk of developing HCMV active infections is significantly increased).
  • the relative risk of developing HCMV active infection is 10.6 compared to subjects with antibody titers above 80, 95% CI is 7.7-14.6.
  • the relative risk of developing HCMV active infection is 14.8 compared to subjects with antibody titers above 160, 95% CI is 9.0-24.6.
  • the relative risk of developing HCMV active infection was 23.7 compared to subjects with antibody titers above 320, with a 95% CI of 8.8-63.4.
  • Table 8b also shows the sensitivity, specificity, and Yoden index of the methods of the invention for predicting HCMV active infection.
  • Table 8b when the reference value is set to 40 antibody titers, the sensitivity of the method of the present invention for predicting HCMV active infection is 57.3%, specificity is 94.7%, and the Yoden index is 0.52;
  • the value is set to an antibody titer of 80, the sensitivity of the method of the present invention for predicting HCMV active infection is 73.1%, specificity is 85.7%, and the Yoden index is 0.59;
  • the reference value is set to 160 antibody titer
  • the method of the present invention is used for predicting HCMV active infection, the sensitivity is 90.6%, the specificity is 66.4%, and the Yoden index is 0.57; when the reference value is set to 320 antibody titer, the method of the present invention is used for predicting HCMV.
  • the sensitivity of active infection was 97.7%, specificity was 40.1%, and the Yoden index was 0.37.
  • the method of the present invention has the best predictive effect when the reference value is set to an antibody titer of 80 (i.e., the Yoden index is the highest, 0.59); and, when the reference value is set to 160 and 40, In the case of antibody titers, the methods of the invention also have good predictive effects (i.e., the Yoden index is above 0.5).
  • HCMV active infection rate was not related to gender, age, occupation, education level, and health habits.
  • the inventors used standards to calibrate the amount of antibody in serum samples.
  • the standard used was a CMV IgG standard (Paul-Ehrlich-Instltut, Referenz-CMV-IgG, Juli 1996, 110 IU/ml), and the antibody of interest was an anti-pp150 antibody.
  • the inventors first performed an ELISA assay on serial dilutions of the standard (the ELISA assay used was as described in Example 5) and plotted the antibody content-antibody reactivity standard based on the ELISA assay results. curve.
  • the standard curve is shown in Figure 10; where the abscissa indicates antibody content (expressed in IU/ml) and the ordinate indicates antibody reactivity (expressed as OD values obtained from ELISA).
  • the antibody content in the serum sample can be absolutely quantified by: (1) performing a 10-fold gradient dilution of the serum sample; (2) performing an ELISA test on each diluted serum sample; (3) The detection results falling within the linear range are selected, and the antibody content in the initial serum sample is calculated based on the linear curve equation and the dilution factor described above.
  • Figure 11 shows the changes in the in vivo anti-pp150 antibody content of the natural population at intervals of 12 months (one year).
  • This result is basically consistent with the results shown in Figure 8: individuals with lower baseline antibody levels have a 4-fold increase in anti-pp150 antibody levels after one year (ie, antibody levels obtained in the second post-test are prior) Detection of antibody levels at least 4-fold higher, indicating a higher probability of an individual developing HCMV active infection during a 12-month interval; whereas individuals with higher baseline antibody levels develop anti-pp150 antibody levels one year later The lower the probability of an increase of 4 times or more (ie, HCMV active infection).
  • This result again indicates that the risk of HCMV active infection in individuals is inversely correlated with baseline levels of antibodies in serum. The lower the level of antibody to the anti-HCMV protein in the serum (eg, the amount of anti-pp150 antibody), the higher the risk of an individual being infected with HCMV.
  • Tables 9a-9b show that in the natural population, the lower the baseline antibody level, the higher the risk of subsequent HCMV active infection (ie, an increase in anti-pp150 antibody levels of 4-fold and above); anti-pp150 There was a negative correlation between baseline levels of antibodies and the risk of HCMV active infection.
  • individuals with a baseline antibody level of no more than 0.2 IU/ml have a risk/infection rate of up to 80% of HCMV active infection within one year; individuals with a baseline antibody level of no more than 0.8 IU/ml are at risk of developing HCMV active infection within one year/ The infection rate was 60.0%; the risk of HCMV active infection/infection rate was 50.0% within one year for individuals with baseline antibody levels not exceeding 1.6 IU/ml; HCMV activity occurred within one year for individuals with baseline antibody levels not exceeding 3.2 IU/ml.
  • the risk of infection/infection rate was 31.3%; baseline antibody water Individuals who did not exceed 6.4 IU/ml had a risk/infection rate of 19.0% of HCMV active infection within one year. In contrast, individuals with baseline antibody levels above 0.8 IU/ml had a risk/infection rate of 5.2% of HCMV active infection within one year; individuals with baseline antibody levels above 1.6 IU/ml developed HCMV active infection within one year. The risk/infection rate was 3.3%; individuals with baseline antibody levels above 3.2 IU/ml had a risk/infection rate of 1.6% of HCMV active infection within one year; individuals with baseline antibody levels above 6.4 IU/ml occurred within one year. The risk/infection rate of HCMV active infection was 0.8%; and individuals with baseline antibody levels above 25.6 IU/ml had a risk/infection rate of HCMV active infection within one year.
  • the results of Table 9b also show that the method of the present invention can be used to determine the relative risk of infection in a subject, wherein the reference value for determining the relative risk level of the baseline antibody level (ie, antibody content / antibody absolute amount)
  • the absolute amount of antibody can be set to range between 0.8-6.4 IU/ml, such as an absolute amount of antibody of 0.8, 1.6, 3.2 or 6.4 IU/ml. If the amount of antibody detected in the subject sample is less than or equal to the reference value, then the relative risk of HCMV active infection in the subject is considered to be high.
  • the relative risk of developing HCMV active infection is greater than that of a subject with an antibody content greater than 0.8 IU/ml. 11.6, 95% CI is 7.7-17.2 (ie, the risk of developing HCMV active infection is significantly increased). If the amount of antibody detected in the subject sample is less than or equal to 1.6 IU/ml, the relative risk of developing HCMV active infection is 15.2 compared to subjects with antibody levels above 1.6 IU/ml. 95% CI is 9.5-24.3.
  • the relative risk of developing HCMV active infection is 19.0 compared to subjects with antibody levels above 3.2 IU/ml. 95% CI is 9.6-37.7. If the amount of antibody detected in the subject sample is less than or equal to 6.4 IU/ml, the relative risk of developing HCMV active infection is 25.2 compared to subjects with antibody levels above 6.4 IU/ml. 95% CI is 8.0-79.6.
  • Table 9b also shows that the method of the present invention is used to predict the sensitivity, specificity, and Yoden index of HCMV active infection when the reference value is set to an antibody content of 0.8-6.4 IU/ml.
  • the sensitivity of the method of the present invention for predicting HCMV active infection is 46.15%
  • specificity is 96.97%
  • the Yoden index is 0.43.
  • the sensitivity of the method of the present invention for predicting HCMV active infection is 67.69%, specificity is 93.34%, and the Yoden index is 0.61; when the reference value is set For 3.2 IU/ml resistance
  • the sensitivity of the method of the present invention for predicting HCMV active infection is 86.15%, the specificity is 81.39%, and the Yoden index is 0.68; when the reference value is set to 6.4 IU/ml of the absolute amount of the antibody,
  • the sensitivity of the method of the invention for predicting HCMV active infection was 95.38%, the specificity was 59.91%, and the Yoden index was 0.55.
  • the method of the present invention has the best predictive effect when the reference value is set to an absolute amount of antibody of 3.2 IU/ml (i.e., the Yoden index is the highest at 0.68); and, when the reference value is set to 0.8.
  • the method of the present invention also has a good predictive effect when the absolute amount of antibody is 1.6 and 6.4 IU/ml (i.e., the Yoden index is close to or higher than 0.5).
  • results of Table 9b also show that with the increase of the set reference value, the sensitivity of the method of the present invention for predicting HCMV active infection is gradually increased (from 46.15% to 95.38%), and the specificity is gradually decreased (by 99.97% fell to 59.91%).
  • the results of the ROC curve analysis of the above method are shown in Fig. 12, in which the area under the curve was 0.913.
  • the method of the invention is used to predict a gradual decrease in the specificity of HCMV active infection.
  • results of Figure 12 demonstrate that the methods of the present invention can be used to accurately, reliably, and efficiently assess the risk of human cytomegalovirus (HCMV) active infection in a subject.
  • HCMV human cytomegalovirus
  • Yamamoto AY Mussi-Pinhata MM, Isaac Mde L, Amaral FR, Carvalheiro CG, Aragon DC, Manfredi AK, BoppanaSB, Britt WJ. Congenital cytomegalovirus infection as a cause of sensorineural hearing loss in a highly immune population. Pediatr Infect Dis J. 2011, 30: 1043-1046.
  • Boppana SB Rivera LB
  • Fowler KB Mach M
  • Britt WJ Intrauterine transmission of cytomegalovirus to infants of women with preconceptional immunity. N Engl J Med. 2001, 344: 1366-1371.

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Abstract

一种用于评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中的方法及其试剂盒，该方法包括下述步骤：(1)测定来自所述受试者的体液样品中抗HCMV蛋白的抗体的水平，HCMV蛋白选自pp150和／或pp28；(2)将该水平与预先确定的参考值相比较；其中，如果该水平低于参考值的话，则判断受试者处于发生HCMV活动性感染的风险中。以及用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力的候选药物的方法及其试剂盒。

Description

用于评估机体发生人巨细胞病毒活动性感染风险的方法及相关的试剂盒 技术领域
本发明涉及医药领域和免疫学领域，特别是免疫学诊断领域。具体而言，本发明公开了，用于评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中的方法，其包括下述步骤：(1)测定来自所述受试者的体液样品中抗HCMV蛋白的抗体的水平；(2)将所述水平与预先确定的参考值相比较；其中，如果所述水平低于参考值的话，则判断所述受试者处于发生HCMV活动性感染的风险中；以及用于所述方法的试剂盒。此外，本发明还公开了用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力的候选药物的方法，以及用于所述方法的试剂盒。
背景技术
人巨细胞病毒(Human Cytomegalovirus，HCMV)是一种DNA病毒，属于人病原体疱疹病毒科。巨细胞病毒的感染全球普遍存在。人体感染HCMV后将终身携带潜伏病毒，并且这些潜伏病毒可能发生机会性活跃。
HCMV感染绝大多数为隐性感染，但在胎儿及免疫低下的人群中，其将导致严重的、甚至是致死性的疾病。例如，如果胎儿发生CMV先天感染(即，CMV突破血胎屏障，感染宫内胎儿)的话，则将会导致严重的临床危害，包括死胎、流产以及出生缺陷等[1，2](Dollard SC，Grosse SD，Ross DS.New estimates of the prevalence of neurological and sensory sequelae and mortality associated with congenital cytomegalovirus infection.Rev Med Virol.2007，17：355-363；姜毅.先天性巨细胞病毒感染母婴传播及诊断.中国新生儿杂志.2009，24：261-265)。大量研究显示，CMV是全球最常见、危害性最大的新生儿先天性感染病原体，并且是儿童中枢神经性耳聋和 神经发育迟缓的最主要病因。在免疫水平低下的人群中，如器官移植病人、艾滋病患者中，HCMV则会激活感染导致全身性并发症。
因此，有必要筛选出易发生CMV先天性感染的孕妇群体或易发生HCMV激活感染的免疫抑制患者，以便在危害发生前采取干预措施。
过去的研究者认为，抗体阴性母亲易在妊娠期发生巨细胞病毒原发感染，从而有很大可能性引起胎儿先天性感染[3，4](Fowler KB，Stagno S，PassRF，et al.The outcome of congenital cytomegalovirus infection in relation to maternal antibody status.N Engl JMed，1992，326：663-667.2；Kenneson A，CannonMJ.Review and meta-analysis of the epidemi2ology of congenital cytomegalovirus infection.Rev Med Virol，2007，17：253-276)；抗体阳性母亲分娩的新生儿很少出现严重临床后果。然而，近年来，在一些CMV高流行的发展中国家进行的系统研究的结果已逐步扭转了这一错误认知。这些系统研究的结果证实，发展中国家儿童听力障碍大量来自于CMV抗体阳性母亲的母婴传播[5-7](Manicklal S，Emery VC，Lazzarotto T，Boppana SB，Gupta RK.The″silent″global burden of congenital cytomegalovirus.Clin Microbiol Rev.2013，26：86-102；Mussi-Pinhata MM，Yamamoto AY，Moura Brito RM，de Lima Isaac M，de Carvalho e Oliveira PF，Boppana S，Britt WJ.Birth prevalence and natural history of congenital cytomegalovirus infection in a highly seroimmune population.Clin Infect Dis.2009，49：522-528；Yamamoto AY，Mussi-Pinhata MM，Isaac Mde L，Amaral FR，Carvalheiro CG，Aragon DC，Manfredi AK，Boppana SB，Britt WJ.Congenital cytomegalovirus infection as a cause of sensorineural hearing loss in a highly immune population.Pediatr Infect Dis J.2011，30：1043-1046)。研究表明，孕前血清学阳性孕妇的胎儿发生宫内感染与母亲孕期内出现CMV再发感染(recurrent infection，也称为“潜伏病毒再激活(reactivation)”或“重叠感染新毒株(reinfection)”)有关[8，9](Boppana SB，Rivera  LB，Fowler KB，Mach M，Britt WJ.Intrauterine transmission of cytomegalovirus to infants of women with preconceptional immunity.N Engl J Med.2001，344：1366-1371；Ross SA，Arora N，Novak Z，Fowler KB，Britt WJ，Boppana SB.Cytomegalovirus reinfections in healthy seroimmune women.J Infect Dis.2010，201：386-389)。目前各国普遍采用CMV-IgM和IgG抗体检测来检查孕妇产前CMV活动性感染。IgM和IgG抗体检测对于孕前抗体阴性孕妇的原发感染(primary infection)的诊断有一定价值。然而，当将IgM和IgG抗体检测用于诊断孕前抗体阳性的孕妇的再发感染时，其灵敏度和特异性均受到了较多质疑[10，11](何小周，王晓芳，王世文.先天性巨细胞病毒感染状况及检测方法的研究进展.病毒学报.2012，28：73-77；Ross SA，Novak Z，Pati S，Boppana SB.Overview of the diagnosis of cytomegalovirus infection.Infect Disord Drug Targets.2011，11：466-474)。
因此，本领域需要开发新的具有高灵敏度和特异性的方法，以准确、有效地评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“巨细胞病毒的活动性感染”是指，从未感染过CMV的受试者初次感染CMV；以及，之前感染过CMV的受试者(其通常携带潜伏病毒，并且血清学检测结果通常为阳性)发生CMV再发感染或其体内的潜伏CMV病毒被激活(也称为“重叠感染新毒株”或“潜伏病毒再激活”)。巨细胞病毒的活动性感染的典型表现是，受 试者针对感染的巨细胞病毒(首次或再次)产生显著的免疫应答，这可导致受试者体内的抗CMV蛋白抗体水平的显著升高。因此，巨细胞病毒的活动性感染也被称为病毒事件。通常，如果受试者体内的抗CMV蛋白抗体水平升高4倍或更高，则可判定受试者发生了巨细胞病毒的活动性感染(或，发生了病毒事件)。
如本文中所使用的，术语“pp150”或“pp150蛋白”是指，CMV病毒被膜层中的一种磷酸化蛋白，其也被称为UL32蛋白。pp150蛋白是丰度较高的CMV蛋白之一，与病毒的组装和分泌密切相关，并且在各CMV毒株间保守，与CMV感染血清具有良好的免疫反应性[12](Jahn G，Scholl BC，Traupe B，Fleckenstein B.The two major structural phosphoproteins(pp65 and pp150)of human cytomegalovirus and their antigenic properties.J Gen Virol.1987，68：1327-1337)。由于pp150具有良好的保守性以及免疫反应性，目前已将其用于CMV-IgG抗体的检测[13](Plachter B，Wieczorek L，Scholl BC，Ziegelmaier R，Jahn G.Detection of cytomegalovirus antibodies by an enzyme-linked immunosorbent assay using recombinant polypeptides of the large phosphorylated tegument protein pp150.J Clin Microbiol.1992，30：201-206)。阳性的血清学检测结果(即，血清中存在抗pp150抗体)表示，个体已被CMV感染，产生了针对pp150的免疫应答。然而，之前从未教导或暗示，抗pp150抗体的水平与受试者发生巨细胞病毒活动性感染的概率之间的关联性。
pp150蛋白的氨基酸序列是本领域技术人员公知的，并且其典型示例可参见例如GenBank登录号ACL51112。如本文中所使用的，当提及pp150蛋白的氨基酸序列时，其使用SEQ ID NO：1所示的序列来进行描述。例如，表述“pp150的aa 861-1048”或“pp150的第861-1048位氨基酸残基”是指，SEQ ID NO：1所示的多肽的第861-1048位氨基酸残基。然而，本领域技术人员理解，在pp150的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同CMV病毒分离株的pp150)，而不影响其生物学功 能。因此，在本发明中，术语“pp150”应包括所有此类序列，包括例如SEQ ID NO：1所示的序列以及其天然或人工的变体。并且，当描述pp150的序列片段时，其不仅包括SEQ ID NO：1的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“pp150的aa861-1048”或“pp150的第861-1048位氨基酸残基”包括，SEQ ID NO：1的第861-1048位氨基酸残基，以及其变体(天然或人工)中的相应片段。
如本文中所使用的，术语“pp28”或“pp28蛋白”是指，CMV病毒被膜层中的一种磷酸化蛋白，其也被称为UL99蛋白(Gambarino S，Callea S，Rizzo G，Montanari P，Loiacono E，Bergallo M.Evaluation of UL99 transcript as a target for antiviral treatment efficacy.J Virol Methods.2014，207：104-9；John Paul Tomtishen III.Human cytomegalovirus tegument proteins(pp65，pp71，pp150，pp28).Virol J.2012，9：22)。
pp28蛋白的氨基酸序列是本领域技术人员公知的，并且其典型示例可参见例如GenBank登录号ACL51167.1。如本文中所使用的，当提及pp28蛋白的氨基酸序列时，其使用SEQ ID NO：3所示的序列来进行描述。例如，表述“pp28的aa 1-190”或“pp28的第1-190位氨基酸残基”是指，SEQ ID NO：3所示的多肽的第1-190位氨基酸残基。然而，本领域技术人员理解，在pp28的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同CMV病毒分离株的pp28)，而不影响其生物学功能。因此，在本发明中，术语“pp28”应包括所有此类序列，包括例如SEQ ID NO：3所示的序列以及其天然或人工的变体。并且，当描述pp28的序列片段时，其不仅包括SEQ ID NO：3的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“pp28的aa 1-190”或“pp28的第1-190位氨基酸残基”包括，SEQ ID NO：3的第1-190位氨基酸残基，以及其变体(天然或人工)中的相应片段。
如本文中所使用的，术语“pp65”或“pp65蛋白”是指，CMV病 毒被膜层中的一种磷酸化蛋白，其也被称为UL83蛋白(Jahn G，Scholl BC，Traupe B，Fleckenstein B.The two major structural phosphoproteins(pp65 and pp150)of human cytomegalovirus and their antigenic properties.J Gen Virol.1987，68：1327-1337；John Paul Tomtishen III.Human cytomegalovirus tegument proteins(pp65，pp71，pp150，pp28).Virol J.2012，9：22)。
pp65蛋白的氨基酸序列是本领域技术人员公知的，并且其典型示例可参见例如GenBank登录号ACL51152.1。如本文中所使用的，当提及pp65蛋白的氨基酸序列时，其使用SEQ ID NO：4所示的序列来进行描述。例如，表述“pp65的aa 1-561”或“pp65的第1-561位氨基酸残基”是指，SEQ ID NO：4所示的多肽的第1-561位氨基酸残基。然而，本领域技术人员理解，在pp65的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同CMV病毒分离株的pp65)，而不影响其生物学功能。因此，在本发明中，术语“pp65”应包括所有此类序列，包括例如SEQ ID NO：4所示的序列以及其天然或人工的变体。并且，当描述pp65的序列片段时，其不仅包括SEQ ID NO：4的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“pp65的aa 1-561”或“pp65的第1-561位氨基酸残基”包括，SEQ ID NO：4的第1-561位氨基酸残基，以及其变体(天然或人工)中的相应片段。
如本文中所使用的，术语“gp52”或“gp52蛋白”是指，CMV病毒被膜层中的一种磷酸化蛋白，其也被称为UL44蛋白(Strang B L，Boulant S，Chang L，et al.Human cytomegalovirus UL44concentrates at the periphery of replication compartments，the site of viral DNA synthesis[J].Journal of virology，2012，86：2089-2095；Sinigalia E，Alvisi G，Segr é C V，et al.The human cytomegalovirus DNA polymerase processivity factor UL44 is modified by SUMO in a DNA-dependent manner[J].PLoS One，2012，7(11)：e49630)。
gp52蛋白的氨基酸序列是本领域技术人员公知的，并且其典型示例可参见例如GenBank登录号ACL51123.1。如本文中所使用的，当提及gp52蛋白的氨基酸序列时，其使用SEQ ID NO：5所示的序列来进行描述。例如，表述“gp52的aa 1-433”或“gp52的第1-433位氨基酸残基”是指，SEQ ID NO：5所示的多肽的第1-433位氨基酸残基。然而，本领域技术人员理解，在gp52的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同CMV病毒分离株的gp52)，而不影响其生物学功能。因此，在本发明中，术语“gp52”应包括所有此类序列，包括例如SEQ ID NO：5所示的序列以及其天然或人工的变体。并且，当描述gp52的序列片段时，其不仅包括SEQ ID NO：5的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“gp52的aa 1-433”或“gp52的第1-433位氨基酸残基”包括，SEQ ID NO：5的第1-433位氨基酸残基，以及其变体(天然或人工)中的相应片段。
如本文中所使用的，术语“pp38”或“pp38蛋白”是指，CMV病毒被膜层中的一种磷酸化蛋白，其也被称为UL80.5蛋白(Loveland A N，Nguyen N L，Brignole E J，et al.The amino-conserved domain of human cytomegalovirus UL80a proteins is required for key interactions during early stages of capsid formation and virus production.Journal of virology，2007，81：620-628；Wood L J，Baxter M K，Plafker S M，et al.Human cytomegalovirus capsid assembly protein precursor(pUL80.5)interacts with itself and with the major capsid protein(pUL86)through two different domains.Journal of virology，1997，71：179-190)。
pp38蛋白的氨基酸序列是本领域技术人员公知的，并且其典型示例可参见例如GenBank登录号ACL51150.1。如本文中所使用的，当提及pp38蛋白的氨基酸序列时，其使用SEQ ID NO：6所示的序列来进行描述。例如，表述“pp38的aa 1-373”或“pp38的第1-373位氨基酸残基”是指，SEQ ID NO：6所示的多肽的第1-373位氨基酸残基。 然而，本领域技术人员理解，在pp38的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同CMV病毒分离株的pp38)，而不影响其生物学功能。因此，在本发明中，术语“pp38”应包括所有此类序列，包括例如SEQ ID NO：6所示的序列以及其天然或人工的变体。并且，当描述pp38的序列片段时，其不仅包括SEQ ID NO：6的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“pp38的aa 1-373”或“pp38的第1-373位氨基酸残基”包括，SEQ ID NO：6的第1-373位氨基酸残基，以及其变体(天然或人工)中的相应片段。
如本文中所使用的，术语“UL48a”或“UL48a蛋白”是指，CMV病毒的一种衣壳蛋白(Baldick C J，Shenk T.Proteins associated with purified human cytomegalovirus particles.Journal of virology，1996，70：6097-6105)。
UL48a蛋白的氨基酸序列是本领域技术人员公知的，并且其典型示例可参见例如GenBank登录号ACL51128.1。如本文中所使用的，当提及UL48a蛋白的氨基酸序列时，其使用SEQ ID NO：7所示的序列来进行描述。例如，表述“UL48a的aa 1-75”或“UL48a的第1-75位氨基酸残基”是指，SEQ ID NO：7所示的多肽的第1-75位氨基酸残基。然而，本领域技术人员理解，在UL48a的氨基酸序列中，可天然产生或人工引入突变或变异(包括但不限于，置换，缺失和/或添加，例如不同CMV病毒分离株的UL48a)，而不影响其生物学功能。因此，在本发明中，术语“UL48a”应包括所有此类序列，包括例如SEQ ID NO：7所示的序列以及其天然或人工的变体。并且，当描述UL48a的序列片段时，其不仅包括SEQ ID NO：7的序列片段，还包括其天然或人工变体中的相应序列片段。例如，表述“UL48a的aa 1-75”或“UL48a的第1-75位氨基酸残基”包括，SEQ ID NO：7的第1-75位氨基酸残基，以及其变体(天然或人工)中的相应片段。
如本文中所使用的，术语“IE1蛋白”是指，CMV病毒的一种即刻早期蛋白(Torres L，Tang Q.Immediate-Early(IE)gene regulation  of cytomegalovirus：IE1-and pp71-mediated viral strategies against cellular defenses[J].Virologica Sinica，2014，29：343-352；McVoy M A.Cytomegalovirus vaccines.Clinical infectious diseases，2013，57：S196-S199)。
IE1蛋白的氨基酸序列是本领域技术人员公知的，并且其典型示例可参见例如GenBank登录号ACL51183.1。
根据本发明，表述“相应序列片段”或“相应片段”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的片段。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar，Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4：11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48：444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定 两个氨基酸序列之间的百分数同一性。
如本文中使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32：1180-1187(1993)；Kobayashi等人Protein Eng.12(10)：879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94：412-417(1997)，其通过引用并入本文)。
如本文中所使用的，术语“抗体”是指，通常由两对多肽链(每对具有一条“轻”(L)链和一条“重”(H)链)组成的免疫球蛋白分子。抗体轻链可分类为κ和λ轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。抗体的恒定区可介导免疫球蛋白与宿 主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为构架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗体结合部位。氨基酸至各区域或结构域的分配遵循Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health，Bethesda，Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196：901-917；Chothia等人(1989)Nature 342：878-883的定义。术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，特别地，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语“抗体的水平”是指，能与人巨细胞病毒蛋白(例如pp150/pp28)反应的抗体的量，其例如可通过任何相对或绝对定量的方式(包括但不限于ELISA、胶体金法或化学发光法等的能够得到抗体水平的任何检测手段)来进行测定，并例如可被描述为抗体-抗原的反应强度、抗体滴度、抗体的定量检测值(例如抗体的绝对量)等。
如本文中所使用的，术语“抗体滴度”是指，含有抗体的样品(例如血清，抗体溶液)识别所述抗体特异性结合的抗原(或其抗原性片段，例如抗原表位)所需要的最低浓度(也即最大稀释度)；其一般表示为，仍能产生阳性结果的最大稀释度。测定某一样品的抗体滴度的方法是本领域技术人员公知的，例如，通常可通过ELISA方法来检测样品的抗体滴度。此类ELISA方法例如可包括下述步骤：(1)将抗体特异性结合的抗原蛋白(或其抗原性片段)用作包被抗原来包被微孔板；(2)将含有抗体的样品(例如血清)进行连续稀释；(3)将各个经稀释的样品添加到步骤(1)的经包被的微孔板的孔中；(4)检测与微孔板中的包被抗原 结合的抗体的存在(例如，使用抗所述抗体的抗体，或者经标记的抗原蛋白等)，并由此确定仍能产生阳性结果的样品最大稀释度(即，该样品的抗体滴度)。
如本文中所使用的，术语“抗体的绝对量”是指，用抗体的生物活性来表示的抗体的量，其通常以国际单位IU/ml或其变形(例如IU/L，mIU/ml等)来表示。测定某一样品的抗体的绝对量的方法是本领域技术人员公知的。例如，可通过将含有抗体的样品(例如血清)的抗体反应性与具有已知的抗体含量的标准品的抗体反应性进行比较，从而确定所述样品中抗体的绝对量。例如，此类方法可包括下述步骤：(1)通过ELISA来测定标准品的系列稀释物的抗体反应性，并绘制抗体含量-抗体反应性的标准曲线；(2)测定含有抗体的样品(或其稀释物)的抗体反应性；和(3)利用标准曲线，将样品(或其稀释物)的抗体反应性转换为样品所包含的抗体的绝对量。
如本文中所使用的，术语“抗体反应性”是指，抗体特异性识别抗原的能力，其可通过免疫学测定(例如ELISA)来容易地测定。例如，抗体反应性可表示为获自检测抗原-抗体结合的ELISA测定的OD值。
如本文中所使用的，术语“抗原性片段”是指，抗原蛋白中的保留与特异性识别该抗原蛋白的抗体的特异性结合能力的片段。本领域技术人员通过常规的技术手段即可获得此类抗原性片段。例如，此类方法可包括下述步骤：(1)制备抗原蛋白的各种片段(例如通过基因工程技术，化学合成技术，抗原蛋白的酶解等)；和(2)通过免疫学测定(例如ELISA)来确定所制备的片段是否保留了与特异性识别该抗原蛋白的抗体的特异性结合能力，从而筛选得到该抗原蛋白的抗原性片段。
如本文中所使用的，术语“免疫学测定”是指，利用抗原-抗体之间的特异性相互作用/结合亲和力来进行的测定，其一般可用于检测特定抗原或者抗体在样品中的存在或水平。此类免疫学测定是本领域技术人员公知的，包括但不限于，ELISA测定，Western印迹，表面等离子共振法，Elispot测定等。
如本文中使用的，术语“特异性结合”是指，两分子间的非随机 的结合反应，如抗体和其所针对的抗原之间的反应。在某些实施方式中，特异性结合某抗原的抗体(或对某抗原具有特异性的抗体)是指，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的亲和力(KD)结合该抗原。
如本文中所使用的，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。通常，抗体以小于大约10-5M，例如小于大约10-6M、10-7M、10-8M、10-9M或10-10M或更小的解离平衡常数(KD)结合抗原，例如，如使用表面等离子体共振术(SPR)在BIACORE仪中测定的。
如本文中所使用的，术语“受试者”包括但不限于各种动物，特别是哺乳动物，例如人。
如本文中所使用的，术语“体液样品”是指，含有获自受试者的体液或其级分的样品。体液样品可以包含或者是直接获自受试者的体液(或其级分)，也可以包含或者是经处理的体液(例如，经分离的体液级分)，并且可任选地包含其他组分，例如缓冲液，抗凝剂，稀释液等。获自受试者的各种体液或其级分是本领域技术人员公知的，其包括但不限于，血液、血清、血浆、尿液和唾液等。
如本文中所使用的，术语“能够测定抗HCMV蛋白的抗体的水平的试剂”是指，能够以定量或半定量方式确定样品中抗HCMV蛋白抗体的水平的试剂。通常而言，特别优选的是，此类试剂能够通过免疫学测定来确定样品中抗HCMV蛋白抗体的水平。免疫学测定的使用是特别有利的，因为其利用了抗原-抗体之间的特异性相互作用/结合亲和力。因此，只要试剂保留了与抗体(例如，抗HCMV蛋白的抗体)反应的抗原性，那么该试剂即可以通过免疫学测定来确定样品中抗HCMV蛋白抗体的水平(也即，该试剂即可用作能够测定抗HCMV蛋白的抗体的水平的试剂)。保留与抗体(例如，抗HCMV蛋白的抗体)反应的抗原性的各种试剂是本领域技术人员容易想到并且可容易获得的，其包括但不限于，抗原蛋白本身(在本申请中，HCMV蛋白)或其抗原性片段，包含所述抗 原蛋白(在本申请中，HCMV蛋白)或其抗原性片段的融合蛋白，以及其任意组合。
如本文中所使用的，术语“测试动物”是指用于实验的动物，其通常是非人哺乳动物，例如模型动物(例如，小鼠，大鼠，兔，灵长类动物)。本领域技术人员可根据实验的具体需要，选择各种合适的测试动物。
如本文中所使用的，术语“相对风险度(relative risk，RR)”亦称危险度比，其是指，某一参数或指标(在本发明中，抗体水平，例如抗pp150或pp28的抗体水平)低于参考值的受试者发生疾病(在本发明中，HCMV活动性感染)的危险度与所述参数或指标高于该参考值的受试者发生疾病的危险度之比。相对风险度是反映参数/指标与发病的关联强度的指标。通常认为：RR为1.0-1.1时，参数/指标与疾病无关联；RR为1.2-1.4时，参数/指标与疾病有弱的关联；RR为1.5-2.9时，参数/指标与疾病有中等关联；RR为3.0-9.9时，参数/指标与疾病有强的关联；RR大于10时，参数/指标与疾病有极强的关联。
如本文中所使用的，术语“约登指数(Youden index)”是指，用于评价筛查试验/预测试验的真实性的指标。当筛查试验/预测试验的假阴性(漏诊率)和假阳性(误诊率)的危害性具有同等意义时，约登指数即等于所述试验的灵敏度与特异度之和减去1。约登指数表示筛查试验/预测试验发现真正的患者与非患者的总能力。约登指数的数值越大，则筛查试验/预测试验的效果越好，真实性越大。
本发明所要解决的一个技术问题是：提供一种可以准确、有效地评估受试者(例如孕妇、器官移植患者、HIV感染者等)发生巨细胞病毒活动性感染的概率的方法，其能够为采取干预措施以降低先天性巨细胞病毒感染几率提供依据，且能够指导临床用药以预防患者并发症的发生。
为此目的，发明人表达并纯化了巨细胞病毒诊断研究中常用的检测蛋白[14](Plachter B，Wieczorek L，Scholl BC，Ziegelmaier R，Jahn G.Detection of cytomegalovirus antibodies by an  enzyme-linked immunosorbent assay using recombinant polypeptides of the large phosphorylated tegument protein pp150.J Clin Microbiol.1992，30：201-206.)，即pp150(UL32)、pp28(UL99)、pp38(UL80.5)、UL48a、gp52(UL44)和pp65(UL83)；然后尝试利用这几个抗原来建立可靠的用于评估受试者发生巨细胞病毒活动性感染的概率的方法和平台。在经过一系列的研究和实验后，发明人发现，受试者血清中的抗pp150(UL32)和/或pp28(UL99)的抗体水平(其可以例如，使用pp150(UL32)和/或pp28(UL99)作为包被抗原通过ELISA来进行检测)可用于有效地、可靠地反映受试者发生巨细胞病毒活动性感染的概率。由此，发明人基于抗pp150(UL32)和/或pp28(UL99)的抗体水平的检测，成功地建立了用于评估受试者发生巨细胞病毒活动性感染的概率的方法和平台。
因此，在一个方面，本发明提供了评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中的方法，其包括下述步骤：
(1)测定来自所述受试者的体液样品中抗HCMV蛋白的抗体的水平；和
(2)将所述水平与预先确定的参考值相比较；
其中，如果所述水平低于参考值的话，则判断所述受试者处于发生HCMV活动性感染的风险中。
在一个优选的实施方案中，所述受试者是哺乳动物，例如人。在一个优选的实施方案中，所述体液样品选自血液、血清、血浆、尿液和唾液。
在一个优选的实施方案中，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活。
在一个优选的实施方案中，所述HCMV蛋白选自pp150和/或pp28。
在一个优选的实施方案中，通过免疫学测定来测定所述体液样品中抗HCMV蛋白的抗体的水平。在一个进一步优选的实施方案中，所述 免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot测定。
在一个优选的实施方案中，所述水平为抗体滴度(例如通过ELISA测定的抗体滴度)，并且参考值为预先确定的抗体滴度；或者，所述水平为抗体的绝对量(例如以IU/ml表示的绝对量)并且参考值为预先确定的抗体的绝对量(例如以IU/ml表示的绝对量)。
在一个优选的实施方案中，在步骤(1)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的抗体滴度。在一个优选的实施方案中，所述参考值为范围在40-320之间的抗体滴度。在一个优选的实施方案中，所述参考值为范围在40-160之间的抗体滴度。例如，所述参考值为40、80、或160的抗体滴度。在一个优选的实施方案中，所述参考值为80的抗体滴度。
如本发明所显示的，当参考值在40-320之间时，抗体滴度低于该参考值的受试者发生HCMV活动性感染的相对风险度超过10，并且其95％CI的下限超过7。这表明，该参数(抗体滴度)与HCMV活动性感染之间存在着强关联或者极强关联。
因此，在一个优选的实施方案中，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.2，95％CI为8.7-14.6。在某些优选的实施方案中，所述参考值设定为40的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为57.3％，特异性为94.7％，约登指数为0.52。
在另一个优选的实施方案中，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或等于80的话，则判断所述受试者发生HCMV活动性感染的相对风险度为10.6，95％CI为7.7-14.6。在某些优选的实施方案中，所述参考值设定为80的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为73.1％，特异性为85.7％，约登指数为0.59。
在另一个优选的实施方案中，所述参考值为160，并且如果抗 pp150抗体的抗体滴度小于或者等于160的话，则判断所述受试者发生HCMV活动性感染的相对风险度为14.8，95％CI为9.0-24.6。在某些优选的实施方案中，所述参考值设定为160的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为90.6％，特异性为66.4％，约登指数为0.57。
在另一个优选的实施方案中，所述参考值为320，并且如果抗pp150抗体的抗体滴度小于或者等于320的话，则判断所述受试者发生HCMV活动性感染的相对风险度为23.7，95％CI为8.8-63.4。在某些优选的实施方案中，所述参考值设定为320的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为97.7％，特异性为40.1％，约登指数为0.37。
从另一个方面来说，抗体滴度水平的高低也直接指示着受试者发生HCMV活动性感染的风险的高低。如本发明所显示的，如果抗pp150抗体的抗体滴度小于或者等于40的话，则所述受试者发生HCMV活动性感染的风险可达到55.37％；如果抗pp150抗体的抗体滴度小于或者等于80的话，则所述受试者发生HCMV活动性感染的风险可达到36.98％；如果抗pp150抗体的抗体滴度小于或者等于160的话，则所述受试者发生HCMV活动性感染的风险可达到23.66％；如果抗pp150抗体的抗体滴度小于或者等于320的话，则所述受试者发生HCMV活动性感染的风险可达到15.77％。这些数据是从包含1659个受试者的人群获得的。虽然这些计算的数据在更大的群体中可能略有变化，但是可以明确确定的是，抗pp150抗体的抗体滴度与HCMV活动性感染之间存在着强负相关，抗体滴度可以用于判断受试者发生HCMV活动性感染的风险。
因此，在一个优选的实施方案中，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的概率为55.37％。在另一个优选的实施方案中，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或等于80的话，则判断所述受试者发生HCMV活动性感染的概率为36.98％。在另一个 优选的实施方案中，所述参考值为160，并且如果抗pp150抗体的抗体滴度小于或者等于160的话，则判断所述受试者发生HCMV活动性感染的概率为23.66％。在另一个优选的实施方案中，所述参考值为320，并且如果抗pp150抗体的抗体滴度小于或者等于320的话，则判断所述受试者发生HCMV活动性感染的概率为15.77％。
在一个优选的实施方案中，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的抗体滴度；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的抗体滴度。
在一个优选的实施方案中，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列。
在另一个优选的实施方案中，在步骤(1)中，测定所述体液样品中抗pp150和/或pp28的抗体的绝对量(例如以IU/ml表示的绝对量)。
在一个优选的实施方案中，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的绝对量。在一个优选的实施方案中，所述参考值为范围在0.8-6.4IU/ml之间的抗体绝对量。在一个优选的实施方案中，所述参考值为范围在0.8-3.2IU/ml之间的抗体绝对量。例如，所述参考值为0.8、1.6、或3.2IU/ml的抗体绝对量。在一个优选的实施方案中，所述参考值为3.2IU/ml的抗体绝对量。
如本发明所显示的，当参考值在0.8-6.4IU/ml之间时，抗体绝对量低于该参考值的受试者发生HCMV活动性感染的相对风险度均超过11，并且其95％CI的下限均超过7。这表明，该参数(抗体绝对量)与HCMV活动性感染之间存在着强关联或者极强关联。
因此，在一个优选的实施方案中，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.6，95％CI 为7.8-17.2。在某些优选的实施方案中，所述参考值为0.8IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为46.15％，特异性为96.97％，约登指数为0.43。
在另一个优选的实施方案中，所述参考值为1.6IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为15.2，95％CI为9.5-24.3。在某些优选的实施方案中，所述参考值为1.6IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为67.69％，特异性为93.34％，约登指数为0.61。
在另一个优选的实施方案中，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为19.0，95％CI为9.6-37.7。在某些优选的实施方案中，所述参考值为3.2IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为86.15％，特异性为81.39％，约登指数为0.68。
在另一个优选的实施方案中，所述参考值为6.4IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于6.4IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为25.2，95％CI为8.0-79.6。在某些优选的实施方案中，所述参考值为6.4IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为95.38％，特异性为59.91％，约登指数为0.55。
从另一个方面来说，抗体绝对量的高低也直接指示着受试者发生HCMV活动性感染的风险的高低。如本发明所显示的，如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则所述受试者发生HCMV活动性感染的风险可达到60.0％；如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则所述受试者发生HCMV活动性感染的风险可达到50.0％；如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则所述受试者发生HCMV活动性感染的风险可达到31.3％；如果抗pp150抗体的抗体绝对量小于或者等于6.4IU/ml的话， 则所述受试者发生HCMV活动性感染的风险可达到19.0％。这些数据是从包含726个受试者的人群获得的。虽然这些计算的数据在更大的群体中可能略有变化，但是可以明确确定的是，抗pp150抗体的抗体绝对量与HCMV活动性感染之间存在着强负相关，抗体滴度可以用于判断受试者发生HCMV活动性感染的风险。
因此，在一个优选的实施方案中，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为60.0％。在一个优选的实施方案中，所述参考值为1.6IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为50.0％。在一个优选的实施方案中，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为31.3％。在一个优选的实施方案中，所述参考值为6.4IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于6.4IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为19.0％。
在一个优选的实施方案中，本发明的方法还包括：
在步骤(1)之前，提供来自所述受试者的体液样品；和/或
在步骤(2)之后，给被判断为处于发生HCMV活动性感染的风险中的受试者施用抗HCMV的疫苗或者抗体，以降低所述受试者发生HCMV活动性感染的风险。
在一个方面，本发明提供了能够测定抗HCMV蛋白的抗体的水平的试剂在制备试剂盒中的用途，所述试剂盒用于通过测定来自受试者的体液样品中抗HCMV蛋白的抗体的水平而评估所述受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中。
在一个优选的实施方案中，所述试剂能够通过免疫学测定来测定抗HCMV蛋白的抗体的水平。在一个进一步优选的实施方案中，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot 测定。
在一个优选的实施方案中，所述试剂选自：HCMV蛋白或其抗原性片段，包含所述HCMV蛋白或其抗原性片段的融合蛋白，以及其任意组合。
在一个优选的实施方案中，所述HCMV蛋白选自pp150和/或pp28。
在一个优选的实施方案中，所述HCMV蛋白为pp150，且所述试剂为pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)；或者，所述HCMV蛋白为pp28，且所述试剂为pp28和/或其抗原性片段；或者，所述HCMV蛋白为pp150和pp28，且所述试剂包括：作为第一组分的pp150和/或其抗原性片段(例如其包含pp150的aa861-1048的片段)，以及作为第二组分的pp28和/或其抗原性片段。
在一个优选的实施方案中，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列；
在一个优选的实施方案中，所述受试者是哺乳动物，例如人。
在一个优选的实施方案中，所述体液样品选自血液、血清、血浆、尿液和唾液。
在一个优选的实施方案中，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活。
在一个优选的实施方案中，所述试剂盒还包括：
(i)用于收集或贮存来自受试者的体液样品的装置；
(ii)用于进行所述测定所需的其他试剂(例如缓冲液，稀释液，封闭液，经标记的抗抗体，和/或标准品)；和/或，
(iii)抗HCMV的疫苗或者抗体。
在一个优选的实施方案中，所述试剂盒通过包括下述步骤的方法来评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中：
(1)使用所述试剂来测定来自所述受试者的体液样品中抗HCMV蛋 白的抗体的水平；和，
(2)将所述水平与预先确定的参考值相比较；
其中，如果所述水平低于参考值的话，则判断所述受试者处于发生HCMV活动性感染的风险中。
在一个进一步优选的实施方案中，在所述方法中，所述水平为抗体滴度(例如通过ELISA测定的抗体滴度)，并且参考值为预先确定的抗体滴度；或者，所述水平为抗体的绝对量(例如以IU/ml表示的绝对量)并且参考值为预先确定的抗体的绝对量(例如以IU/ml表示的绝对量)。
在一个进一步优选的实施方案中，在所述方法中，在步骤(1)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的抗体滴度。在一个优选的实施方案中，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的抗体滴度；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的抗体滴度。在一个优选的实施方案中，所述参考值为范围在40-320之间的抗体滴度。在一个优选的实施方案中，所述参考值为范围在40-160之间的抗体滴度。例如，所述参考值为40、80、或160的抗体滴度。在一个优选的实施方案中，所述参考值为80的抗体滴度。
在一个优选的实施方案中，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.2，95％CI为8.7-14.6。在一个优选的实施方案中，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的概率为55.37％。在某些优选的实施方案中，所述参考值设定为40的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为57.3％，特异性为94.7％，约登指数为0.52。
在另一个优选的实施方案中，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或等于80的话，则判断所述受试者发生HCMV活 动性感染的相对风险度为10.6，95％CI为7.7-14.6。在另一个优选的实施方案中，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或等于80的话，则判断所述受试者发生HCMV活动性感染的概率为36.98％。在某些优选的实施方案中，所述参考值设定为80的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为73.1％，特异性为85.7％，约登指数为0.59。
在另一个优选的实施方案中，所述参考值为160，并且如果抗pp150抗体的抗体滴度小于或者等于160的话，则判断所述受试者发生HCMV活动性感染的相对风险度为14.8，95％CI为9.0-24.6。在另一个优选的实施方案中，所述参考值为160，并且如果抗pp150抗体的抗体滴度小于或者等于160的话，则判断所述受试者发生HCMV活动性感染的概率为23.66％。在某些优选的实施方案中，所述参考值设定为160的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为90.6％，特异性为66.4％，约登指数为0.57。
在另一个优选的实施方案中，所述参考值为320，并且如果抗pp150抗体的抗体滴度小于或者等于320的话，则判断所述受试者发生HCMV活动性感染的相对风险度为23.7，95％CI为8.8-63.4。在另一个优选的实施方案中，所述参考值为320，并且如果抗pp150抗体的抗体滴度小于或者等于320的话，则判断所述受试者发生HCMV活动性感染的概率为15.77％。在某些优选的实施方案中，所述参考值设定为320的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为97.7％，特异性为40.1％，约登指数为0.37。
在另一个优选的实施方案中，在所述方法中，在步骤(1)中，测定所述体液样品中抗pp150和/或pp28的抗体的绝对量(例如以IU/ml表示的绝对量)。在一个优选的实施方案中，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的绝对量。在一个优选的实施方案中，所述参考值为范围在0.8-6.4IU/ml之间的抗体绝对量。在一个优选的实施方案中，所述参考值为 范围在0.8-3.2IU/ml之间的抗体绝对量。例如，所述参考值为0.8、1.6、或3.2IU/ml的抗体绝对量。在一个优选的实施方案中，所述参考值为3.2IU/ml的抗体绝对量。
在一个优选的实施方案中，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.6，95％CI为7.8-17.2。在一个优选的实施方案中，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为60.0％。在某些优选的实施方案中，所述参考值为0.8IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为46.15％，特异性为96.97％，约登指数为0.43。
在另一个优选的实施方案中，所述参考值为1.6IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为15.2，95％CI为9.5-24.3。在一个优选的实施方案中，所述参考值为1.6IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为50.0％。在某些优选的实施方案中，所述参考值为1.6IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为67.69％，特异性为93.34％，约登指数为0.61。
在另一个优选的实施方案中，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为19.0，95％CI为9.6-37.7。在一个优选的实施方案中，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为31.3％。在某些优选的实施方案中，所述参考值为3.2IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为86.15％，特异性为81.39％，约登指数为0.68。
在另一个优选的实施方案中，所述参考值为6.4IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于6.4IU/ml的话，则判断 所述受试者发生HCMV活动性感染的相对风险度为25.2，95％CI为8.0-79.6。在一个优选的实施方案中，所述参考值为6.4IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于6.4IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为19.0％。在某些优选的实施方案中，所述参考值为6.4IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为95.38％，特异性为59.91％，约登指数为0.55。
在一个方面，本发明提供了用于评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中的试剂盒，其包括能够测定抗HCMV蛋白的抗体的水平的试剂，以及任选地，使用所述试剂来测定来自受试者的体液样品中抗HCMV蛋白的抗体的水平，从而评估所述受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中的说明书。
在一个优选的实施方案中，所述试剂能够通过免疫学测定来测定抗HCMV蛋白的抗体的水平。在一个进一步优选的实施方案中，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot测定。
在一个优选的实施方案中，所述试剂选自：HCMV蛋白或其抗原性片段，包含所述HCMV蛋白或其抗原性片段的融合蛋白，以及其任意组合。在一个优选的实施方案中，所述HCMV蛋白选自pp150和/或pp28。
在一个优选的实施方案中，所述HCMV蛋白为pp150，且所述试剂为pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)；或者，所述HCMV蛋白为pp28，且所述试剂为pp28和/或其抗原性片段；或者，所述HCMV蛋白为pp150和pp28，且所述试剂包括：作为第一组分的pp150和/或其抗原性片段(例如其包含pp150的aa861-1048的片段)，以及作为第二组分的pp28和/或其抗原性片段。
在一个优选的实施方案中，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列；
在一个优选的实施方案中，所述受试者是哺乳动物，例如人。在 一个优选的实施方案中，所述体液样品选自血液、血清、血浆、尿液和唾液。
在一个优选的实施方案中，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活。
在一个优选的实施方案中，所述试剂盒还包括：
(i)用于收集或贮存来自受试者的体液样品的装置；
(ii)用于进行所述测定所需的其他试剂(例如缓冲液，稀释液，封闭液，经标记的抗抗体，和/或标准品)；和/或，
(iii)抗HCMV的疫苗或者抗体。
在一个优选的实施方案中，所述试剂盒通过包括下述步骤的方法来评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中：
(1)使用所述试剂来测定来自所述受试者的体液样品中抗HCMV蛋白的抗体的水平；和，
(2)将所述水平与预先确定的参考值相比较；
其中，如果所述水平低于参考值的话，则判断所述受试者处于发生HCMV活动性感染的风险中。
在一个进一步优选的实施方案中，在所述方法中，所述水平为抗体滴度(例如通过ELISA测定的抗体滴度)，并且参考值为预先确定的抗体滴度；或者，所述水平为抗体的绝对量(例如以IU/ml表示的绝对量)并且参考值为预先确定的抗体的绝对量(例如以IU/ml表示的绝对量)。
在一个进一步优选的实施方案中，在所述方法中，在步骤(1)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的抗体滴度；在一个优选的实施方案中，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的抗体滴度；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的抗体滴度。在一个优 选的实施方案中，所述参考值为范围在40-320之间的抗体滴度。在一个优选的实施方案中，所述参考值为范围在40-160之间的抗体滴度。例如，所述参考值为40、80、或160的抗体滴度。在一个优选的实施方案中，所述参考值为80的抗体滴度。
在一个优选的实施方案中，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.2，95％CI为8.7-14.6。在一个优选的实施方案中，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的概率为55.37％。在某些优选的实施方案中，所述参考值设定为40的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为57.3％，特异性为94.7％，约登指数为0.52。
在另一个优选的实施方案中，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或等于80的话，则判断所述受试者发生HCMV活动性感染的相对风险度为10.6，95％CI为7.7-14.6。在另一个优选的实施方案中，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或等于80的话，则判断所述受试者发生HCMV活动性感染的概率为36.98％。在某些优选的实施方案中，所述参考值设定为80的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为73.1％，特异性为85.7％，约登指数为0.59。
在另一个优选的实施方案中，所述参考值为160，并且如果抗pp150抗体的抗体滴度小于或者等于160的话，则判断所述受试者发生HCMV活动性感染的相对风险度为14.8，95％CI为9.0-24.6。在另一个优选的实施方案中，所述参考值为160，并且如果抗pp150抗体的抗体滴度小于或者等于160的话，则判断所述受试者发生HCMV活动性感染的概率为23.66％。在某些优选的实施方案中，所述参考值设定为160的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为90.6％，特异性为66.4％，约登指数为0.57。
在另一个优选的实施方案中，所述参考值为320，并且如果抗 pp150抗体的抗体滴度小于或者等于320的话，则判断所述受试者发生HCMV活动性感染的相对风险度为23.7，95％CI为8.8-63.4。在另一个优选的实施方案中，所述参考值为320，并且如果抗pp150抗体的抗体滴度小于或者等于320的话，则判断所述受试者发生HCMV活动性感染的概率为15.77％。在某些优选的实施方案中，所述参考值设定为320的抗体滴度，并且所述方法用于预测HCMV活动性感染的灵敏度为97.7％，特异性为40.1％，约登指数为0.37。
在另一个优选的实施方案中，在所述方法中，在步骤(1)中，测定所述体液样品中抗pp150和/或pp28的抗体的绝对量(例如以IU/ml表示的绝对量)。在一个优选的实施方案中，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的绝对量。在一个优选的实施方案中，所述参考值为范围在0.8-6.4IU/ml之间的抗体绝对量。在一个优选的实施方案中，所述参考值为范围在0.8-3.2IU/ml之间的抗体绝对量。例如，所述参考值为0.8、1.6、或3.2IU/ml的抗体绝对量。在一个优选的实施方案中，所述参考值为3.2IU/ml的抗体绝对量。
在一个优选的实施方案中，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.6，95％CI为7.8-17.2。在一个优选的实施方案中，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为60.0％。在某些优选的实施方案中，所述参考值为0.8IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为46.15％，特异性为96.97％，约登指数为0.43。
在另一个优选的实施方案中，所述参考值为1.6IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为15.2，95％CI为9.5-24.3。在一个优选的实施方案中，所述参考值为1.6IU/ml，并 且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为50.0％。在某些优选的实施方案中，所述参考值为1.6IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为67.69％，特异性为93.34％，约登指数为0.61。
在另一个优选的实施方案中，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为19.0，95％CI为9.6-37.7。在一个优选的实施方案中，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为31.3％。在某些优选的实施方案中，所述参考值为3.2IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为86.15％，特异性为81.39％，约登指数为0.68。
在另一个优选的实施方案中，所述参考值为6.4IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于6.4IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为25.2，95％CI为8.0-79.6。在一个优选的实施方案中，所述参考值为6.4IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于6.4IU/ml的话，则判断所述受试者发生HCMV活动性感染的概率为19.0％。在某些优选的实施方案中，所述参考值为6.4IU/ml，并且所述方法用于预测HCMV活动性感染的灵敏度为95.38％，特异性为59.91％，约登指数为0.55。
本发明的方法能够通过测定来自受试者的体液样品中抗HCMV蛋白的抗体的水平，从而可靠、有效地评估所述受试者发生人巨细胞病毒(HCMV)活动性感染的风险。基于此，如果受试者在接受某一候选药物之后，其体液样品中抗HCMV蛋白的抗体的水平得以升高(与接受该候选药物前的水平相比)，那么这即说明，该受试者在接受该候选药物后，发生人巨细胞病毒(HCMV)活动性感染的风险得以降低。由此可以合理确定，所使用的候选药物能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力，降低受试者发生人巨细胞病毒(HCMV)活动性感染 的风险。
因此，在另一个方面，本发明提供了用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物的方法，其包括下述步骤：
(1)在给测试动物施用候选药物之前，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第一水平；
(2)给所述测试动物施用候选药物；
(3)在给测试动物施用候选药物之后，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第二水平；
(4)将所述第一水平与第二水平进行比较，其中，如果所述第一水平低于所述第二水平的话，则判断所述候选药物能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险。
在一个优选的实施方案中，所述测试动物是非人哺乳动物，例如模型动物(例如，小鼠，大鼠，兔，灵长类动物)。在一个优选的实施方案中，所述体液样品选自血液、血清、血浆、尿液和唾液。
在一个优选的实施方案中，所述受试者是哺乳动物，例如人。
在一个优选的实施方案中，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活。
在一个优选的实施方案中，所述HCMV蛋白选自pp150和/或pp28。
在一个优选的实施方案中，通过免疫学测定来测定所述体液样品中抗HCMV蛋白的抗体的水平。在一个进一步优选的实施方案中，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot测定。
在一个优选的实施方案中，所述第一和第二水平为抗体滴度(例如通过ELISA测定的抗体滴度)；或者，所述第一和第二水平为抗体的绝对量(例如以IU/ml表示的绝对量)。
在一个优选的实施方案中，在步骤(1)和(3)中，通过ELISA来测 定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。在一个优选的实施方案中，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的第一和第二水平；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的第一和第二水平。
在一个优选的实施方案中，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列；
在一个优选的实施方案中，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。
在另一个方面，本发明提供了能够测定抗HCMV蛋白的抗体的水平的试剂在制备试剂盒中的用途，所述试剂盒用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物。
在一个优选的实施方案中，所述试剂能够通过免疫学测定来测定抗HCMV蛋白的抗体的水平。在一个进一步优选的实施方案中，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot测定。
在一个优选的实施方案中，所述试剂选自：HCMV蛋白或其抗原性片段，包含所述HCMV蛋白或其抗原性片段的融合蛋白，以及其任意组合。在一个优选的实施方案中，所述HCMV蛋白选自pp150和/或pp28。
在一个优选的实施方案中，所述HCMV蛋白为pp150，且所述试剂为pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)；或者，所述HCMV蛋白为pp28，且所述试剂为pp28和/或其抗原性片段；或者，所述HCMV蛋白为pp150和pp28，且所述试剂包括：作为第一组分的pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)，以及作为第二组分的pp28和/或其抗原性片段。
在一个优选的实施方案中，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列。
在一个优选的实施方案中，所述受试者是哺乳动物，例如人。
在一个优选的实施方案中，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活。
在一个优选的实施方案中，所述试剂盒通过包括下述步骤的方法来筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物：
(1)在给测试动物施用候选药物之前，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第一水平；
(2)给所述测试动物施用候选药物；
(3)在给测试动物施用候选药物之后，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第二水平；
(4)将所述第一水平与第二水平进行比较，
其中，如果所述第一水平低于所述第二水平的话，则判断所述候选药物能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险。
在一个优选的实施方案中，所述测试动物是非人哺乳动物，例如模型动物(例如，小鼠，大鼠，兔，灵长类动物)。
在一个优选的实施方案中，所述体液样品选自血液、血清、血浆、尿液和唾液。
在一个优选的实施方案中，所述试剂盒还包括：
(i)用于收集或贮存来自测试动物的体液样品的装置；和/或
(ii)用于测定所述第一水平与第二水平所需的其他试剂(例如缓冲液，稀释液，封闭液，经标记的抗抗体，和/或标准品)。
在一个优选的实施方案中，所述第一和第二水平为抗体滴度(例如 通过ELISA测定的抗体滴度)；或者，所述第一和第二水平为抗体的绝对量(例如以IU/ml表示的绝对量)。
在一个优选的实施方案中，在所述方法中，在步骤(1)和(3)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。在一个优选的实施方案中，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的第一和第二水平；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的第一和第二水平。
在一个优选的实施方案中，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列。
在一个优选的实施方案中，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。
在另一个方面，本发明提供了一种用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物的试剂盒，其包括能够测定抗HCMV蛋白的抗体的水平的试剂，以及任选地，使用所述试剂来筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物的说明书。
在一个优选的实施方案中，所述试剂能够通过免疫学测定来测定抗HCMV蛋白的抗体的水平。在一个进一步优选的实施方案中，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot测定；
在一个优选的实施方案中，所述试剂选自：HCMV蛋白或其抗原性片段，包含所述HCMV蛋白或其抗原性片段的融合蛋白，以及其任意组 合。在一个优选的实施方案中，所述HCMV蛋白选自pp150和/或pp28。
在一个优选的实施方案中，所述HCMV蛋白为pp150，且所述试剂为pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)；或者，所述HCMV蛋白为pp28，且所述试剂为pp28和/或其抗原性片段；或者，所述HCMV蛋白为pp150和pp28，且所述试剂包括：作为第一组分的pp150和/或其抗原性片段(例如其包含pp150的aa861-1048的片段)，以及作为第二组分的pp28和/或其抗原性片段。
在一个优选的实施方案中，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列。
在一个优选的实施方案中，所述受试者是哺乳动物，例如人。
在一个优选的实施方案中，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活。
在一个优选的实施方案中，所述试剂盒通过包括下述步骤的方法来筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物：
(1)在给测试动物施用候选药物之前，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第一水平；
(2)给所述测试动物施用候选药物；
(3)在给测试动物施用候选药物之后，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第二水平；
(4)将所述第一水平与第二水平进行比较；
其中，如果所述第一水平低于所述第二水平的话，则判断所述候选药物能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险。
在一个优选的实施方案中，所述测试动物是非人哺乳动物，例如模型动物(例如，小鼠，大鼠，兔，灵长类动物)。在一个优选的实施方案中，所述体液样品选自血液、血清、血浆、尿液和唾液。
在一个优选的实施方案中，所述试剂盒还包括：
(i)用于收集或贮存来自测试动物的体液样品的装置；和/或
(ii)用于测定所述第一水平与第二水平所需的其他试剂(例如缓冲液，稀释液，封闭液，经标记的抗抗体，和/或标准品)。
在一个优选的实施方案中，所述第一和第二水平为抗体滴度(例如通过ELISA测定的抗体滴度)；或者，所述第一和第二水平为抗体的绝对量(例如以IU/ml表示的绝对量)。
在一个优选的实施方案中，在所述方法中，在步骤(1)和(3)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。在一个优选的实施方案中，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的第一和第二水平；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的第一和第二水平。
在一个优选的实施方案中，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列。
在一个优选的实施方案中，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。
发明的有益效果
与现有技术相比，本发明的技术方案具有以下有益效果：
(1)本发明首次证实，抗HCMV蛋白(例如pp150/pp28)的抗体水平与人巨细胞病毒活动性感染风险具有显著的相关性。特别地，本发明首先证实，当受试者体液样品中抗pp150抗体的抗体滴度小于或者等于40时，所述受试者发生HCMV活动性感染的概率为55.37％，相对风险度为11.2，95％CI为8.7-14.6；当受试者体液样品中抗pp150 抗体的抗体滴度小于或者等于80时，所述受试者发生HCMV活动性感染的概率为36.98％，相对风险度为10.6，95％CI为7.7-14.6；当受试者体液样品中抗pp150抗体的抗体滴度小于或者等于160时，所述受试者发生HCMV活动性感染的概率为23.66％，相对风险度为14.8，95％CI为9.0-24.6；当受试者体液样品中抗pp150抗体的抗体滴度小于或者等于320时，所述受试者发生HCMV活动性感染的概率为15.77％，相对风险度为23.7，95％CI为8.8-63.4。此外，本发明还首次证实，当受试者体液样品中抗pp150抗体的含量低于0.8IU/ml时，所述受试者发生HCMV活动性感染的概率为60.0％，相对风险度为11.6，95％CI为7.8-17.2；当受试者体液样品中抗pp150抗体的含量低于1.6IU/ml时，所述受试者发生HCMV活动性感染的概率为50.0％，相对风险度为15.2，95％CI为9.5-24.3；当受试者体液样品中抗pp150抗体的含量低于3.2IU/ml时，所述受试者发生HCMV活动性感染的概率为31.3％，相对风险度为19.0，95％CI为9.6-37.7；当受试者体液样品中抗pp150抗体的含量低于6.4IU/ml时，所述受试者发生HCMV活动性感染的概率为19.0％，相对风险度为25.2，95％CI为8.0-79.6。这解决了本领域迫切需要解决的技术问题：即，提供了能够准确、可靠、有效地评估受试者发生人巨细胞病毒(HCMV)活动性感染的风险的高灵敏度和特异性的方法。
(2)抗HCMV蛋白(例如pp150/pp28)的抗体水平可作为人巨细胞病毒(HCMV)活动性感染风险(例如，血清学阳性人群的HCMV再发感染风险)的预测指标；并且，本发明的方法可用于筛选自然人群或人巨细胞病毒活动性感染高危害地区的人群中易于发生人巨细胞病毒活动性感染的个体(即，高风险的个体)。由此，本发明的方法能够为采取干预措施提供依据，且能够指导临床用药以预防患者并发症的发生。例如，本发明方法可用于孕妇人群中，筛选出高风险的个体，并通过对这些个体采取干预措施和临床用药来降低新生儿先天性巨细胞病毒感染几率；或者，本发明方法可用于器官移植等免疫抑制患者中，筛选出高风险的个体，并通过对这些个体采取干预措施和临床用药来降低 并发症的发生概率。
(3)本发明的方法可用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物。由此，本发明的方法为开发新的预防和/或治疗HCMV感染的药物提供了新的思路和方案。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1A和1B显示，实施例2中所建立的ELISA检测平台（基于p150和pp150-2)可用于检测血清样品中的抗CMV蛋白的抗体(抗pp150抗体)的水平，进而判断血清样品是阳性的还是阴性的。图1A显示了，使用pp150和pp150-2作为包被抗原的ELISA法(横坐标)以及使用纯化病毒作为包被抗原的ELISA法(纵坐标)对288份血清样品的检测结果的相关性分析。结果显示，两种ELISA法的检测结果高度一致，二者的符合率为99.3％，且反应强度的相关性好(相关系数r＝0.85)。图1B显示了，使用pp150和pp150-2作为包被抗原的ELISA法以及使用商品化CMV-IgG试剂(Diasorin-IgG)作为包被抗原的ELISA法对36份血清样品的检测结果的比较。结果显示，本发明的基于pp150和pp150-2的ELISA法的反应性显著强于基于Diasorin的ELISA法的反应性，后者对阳性血清的反应性大多处于较低的水平。图1A和1B的结果表明，实施例2中所建立的ELISA检测平台可用于准确、可靠、有效地检测血清样品中的抗CMV蛋白的抗体(抗pp150抗体)的水平。
图2显示了，实施例3中建立的基于IE1蛋白的Elispot检测平台中，HCMV病毒感染用量与Elispot检测值之间的相关性。结果显示， Elispot检测平台中的HCMV病毒感染用量与Elispot检测值之间存在显著的线性关系(R2＝0.9988)。这表明，实施例3中所建立的Elispot检测平台可用于准确、可靠、有效地检测血清样品中的抗CMV蛋白的抗体(抗IE1蛋白抗体)的水平。
图3显示，实施例3中建立的基于IE1蛋白的Elispot检测平台对61份血清样品的检测结果(Elispot NT50)与商品化HCMV IgG抗体检测试剂盒(DiasorinIgG试剂)的检测结果的比较。其中，对于试剂盒检测为阳性的31份血清，Elispot检测平台的检测结果均为阳性；对于试剂盒检测为阴性的30份血清，Elispot检测平台的检测结果均为阴性；二者的结果完全一致。这表明，实施例3中所建立的Elispot检测平台的准确性和可靠性。
图4显示，实施例3中建立的基于IE1蛋白的Elispot检测平台对61份血清样品的检测结果(Elispot NT50)与商品化HCMV IRD中和检测平台的检测结果(IRD NT50)的相关性。结果显示，Elispot检测平台的检测结果(Elispot NT50)与IRD中和检测平台的检测结果(IRD NT50)具有良好的相关性(R2＝0.8960)。这表明，实施例3中所建立的Elispot检测平台的准确性和可靠性。
图5显示，实施例2中建立的6种ELISA检测平台(分别基于pp150和pp150-2、HCMV pp65(UL83)、HCMV gp52(UL44)、HCMV pp38(UL80a)、HCMV pp28(UL99)，以及UL48a)对背景明确的61份血清样品的检测结果，其中pp150指基于pp150和pp150-2的混合物的ELISA检测平台。结果显示，实施例2中建立的ELISA检测平台(尤其是，基于pp150和pp150-2的ELISA检测平台和基于pp28的ELISA检测平台)能够准确、可靠、有效地检测血清样品中的抗CMV蛋白的抗体的水平，判断血清样品的阴性和阳性。
图6A-C显示，实施例6中的基于pp150和pp150-2的ELISA检测平台(简称为pp150检测平台)在病毒事件的不同评价标准下的ROC曲线分析。其中，横坐标表示(100％-所述检测平台的检测特异性％)，纵坐标表示所述检测平台的检测灵敏度％；并且，
图6A显示，当将多种其他检测平台的检测结果为双阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp150检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析；
图6B显示，当将多种其他检测平台的检测结果为三阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp150检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析；
图6C显示，当将多种其他检测平台的检测结果为四阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp150检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析。
图6A-C的结果表明，在所有三种情况下，pp150检测平台预测病毒事件(HCMV活动性感染)发生的准确率都大于80％，从而可以用于准确、可靠、有效地评估受试者发生人巨细胞病毒(HCMV)活动性感染的风险。
图7A-C显示，实施例6中的基于pp28的ELISA检测平台(简称为pp28检测平台)在病毒事件的不同评价标准下的ROC曲线分析。其中，横坐标表示(100％-所述检测平台的检测特异性％)，纵坐标表示所述检测平台的检测灵敏度％；并且，
图7A显示，当将多种其他检测平台的检测结果为双阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp28检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析；
图7B显示，当将多种其他检测平台的检测结果为三阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp28检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析；
图7C显示，当将多种其他检测平台的检测结果为四阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp28检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析。
图7A-C的结果表明，在所有三种情况下，pp28检测平台预测病毒事件(HCMV活动性感染)发生的准确率都大于78％，从而可以用于准确、可靠、有效地评估受试者发生人巨细胞病毒(HCMV)活动性感染的 风险。
图8显示，实施例7中描述的广西某自然人群间隔12个月(一年)的体内抗pp150抗体的抗体滴度的变化情况，其中，横坐标表示基线抗体水平(抗体滴度)，纵坐标表示一年后抗体水平升高4倍或降低4倍的概率。结果显示，基线抗体水平越高的个体一年后出现抗pp150抗体水平4倍及以上升高(即，发生HCMV活动性感染)的概率越低；而基线抗体水平越低的个体一年后出现抗pp150抗体水平4倍及以上升高(即，发生HCMV活动性感染)的概率越高。个体发生HCMV活动性感染的风险与抗体基线水平呈负相关。
图9显示，在实施例7中描述的广西某自然人群中，基线抗pp150抗体水平与HCMV活动性感染风险之间的相关性。其中，横坐标表示基线抗pp150抗体水平(抗体滴度)，纵坐标表示发生HCMV活动性感染的风险(比例)。结果显示，基线抗体水平越低的个体随后发生HCMV活动性感染(即，抗pp150抗体水平4倍及以上升高)的风险越高。HCMV活动性感染风险与抗体基线水平呈负相关。其中，当抗体基线水平(抗体滴度)低于10时，发生HCMV活动性感染的风险高达82.6％；并且当抗体基线水平(抗体滴度)不超过160时，发生HCMV活动性感染的风险高达23.66％。
图10显示，用抗体含量已知的标准品(Paul-Ehrlich-Instltut，Referenz-CMV-IgG，Juli 1996，110IU/ml)绘制的抗体含量-抗体反应性的标准曲线。其中，横坐标表示抗体含量(以IU/ml表示)；纵坐标表示抗体反应性(以获自ELISA的OD值表示)。结果显示，抗体含量与抗体反应性之间存在显著的线性关系(R2＝0.9984)，并且线性范围超过1个数量级。因此，使用标准品绘制的抗体含量-抗体反应性的标准曲线可用于准确定量样品中的抗体含量(以IU/ml表示)。
图11显示，实施例8中描述的广西某自然人群间隔12个月(一年)的体内抗pp150抗体含量的变化情况，其中，横坐标表示基线抗体水平(抗体含量)，纵坐标表示一年后抗体水平升高4倍或降低4倍的概率。结果显示，基线抗体水平越低的个体一年后出现抗pp150抗体水 平4倍及以上升高(即，在后的第二次检测获得的抗体水平为在先检测获得的抗体水平的至少4倍高，其指示着个体在12个月的间隔期间发生了HCMV活动性感染)的概率越高；而基线抗体水平越高的个体一年后出现抗pp150抗体水平4倍及以上升高(即，发生HCMV活动性感染)的概率越低。这些结果表明个体发生HCMV活动性感染的风险与血清中的抗体基线水平呈负相关。
图12显示了实施例8中描述的方法的ROC曲线分析结果，其中，所述方法基于血清中抗pp150抗体的含量/绝对量来预测HCMV活动性感染，并且，横坐标表示(100％-所述方法的特异性％)，纵坐标表示，所述方法的灵敏度％。图12的结果表明，本发明方法可以用于准确、可靠、有效地评估受试者发生人巨细胞病毒(HCMV)活动性感染的风险。
序列信息
本发明涉及的序列的信息提供于下面的表1中。
表1：序列信息
Figure PCTCN2016072094-appb-000001
Figure PCTCN2016072094-appb-000002
Figure PCTCN2016072094-appb-000003
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1.蛋白的克隆及表达
在本实施例中，本发明人重组表达了HCMV pp150(UL32)、HCMV pp150-2(pp150的截短蛋白)、HCMV pp65(UL83)、HCMV gp52(UL44)、HCMV pp38(UL80a)、HCMV pp28(UL99)和UL48a共7种蛋白。用于重组表达蛋白的相关信息列于表2中。用于PCR扩增编码目的蛋白的基因的引物列于表3中。
表2：用于重组表达7种蛋白的相关信息
蛋白名称 序列信息 序列登录号 载体 菌株
HCMV pp150 SEQ ID NO：1 ACL51112 B11 BL21
HCMV pp150-2 SEQ ID NO：2 ACL51112 B11 BL21
HCMV pp28 SEQ ID NO：3 ACL51167.1 Pet-GST er感受态
HCMV pp65 SEQ ID NO：4 ACL51152.1 B6 er感受态
HCMV gp52 SEQ ID NO：5 ACL51123.1 B6 er感受态
HCMV pp38 SEQ ID NO：6 ACL51150.1 Pet-GST er感受态
UL48a SEQ ID NO：7 ACL51128.1 Pet-GST er感受态
表3：用于PCR扩增编码目的蛋白的基因的引物的相关信息
Figure PCTCN2016072094-appb-000004
实施例2.基于抗原蛋白的用于检测抗体的ELISA检测平台的建立
在本实施例中，本发明人分别基于HCMV pp150和pp150-2(作为联合包被抗原)、HCMV pp65(UL83)、HCMV gp52(UL44)、HCMV pp38(UL80a)、HCMV pp28(UL99)或UL48a，建立了分别用于检测抗pp150抗体、抗pp65抗体、抗gp52抗体、抗pp38抗体、抗pp28抗体和抗UL48a抗体的ELISA检测平台(分别简称为，pp150检测平台、pp65检测平台、gp52检测平台、pp38检测平台、pp28检测平台、UL48a检测平台)，其各自包括：由抗原蛋白(即，包被抗原)包被的微孔板、包被缓冲液、封闭液、洗涤液、酶标记的抗人IgG抗体、酶标抗体稀释液、阴阳性对照、显色液和终止液。
包被抗原：HCMV pp150和pp150-2、HCMV pp65(UL83)、HCMV gp52(UL44)、HCMV pp38(UL80a)、HCMV pp28(UL99)或UL48a；
样品稀释液：pH为7.8-8.3的Tris-Base缓冲液，其中含有1-5％(质量体积比)的牛血清白蛋白、5-10％(质量体积比)的蔗糖和2-7％(质量体积比)的酪蛋白及7-12％(体积比)的胎牛血清；
浓缩洗涤液：其包含pH 7-7.6的磷酸盐缓冲液和表面活性剂Tween20；
酶标记的抗人IgG抗体：辣根过氧化物酶(HRP)标记的鼠抗人IgG 单克隆抗体。
酶标抗体稀释液：pH为6.8-7.3的磷酸盐缓冲液、其中每1000ml磷酸盐缓冲液含有0.1-1M Nacl及0.3-1％(质量体积比)的酪蛋白、0.1-0.4％(质量体积比)的TritonX-100、7-12％(体积比)的胎牛血清、0.2-0.5％(质量体积比)的Geltin；
显色液：包含有柠檬酸三钠、柠檬酸、乙酸钠、冰乙酸及双氧水的A液；和，包含有无水乙醇、乙二醇、二甲基甲酰胺、3，3，5，5-四甲基联苯胺的B液
终止液：0.1-1M的硫酸。
下面以pp150抗体检测平台为例，举例说明本实施例所建立的ELISA检测平台的效果。使用基于pp150和pp150-2的ELISA检测平台与基于纯化病毒的ELISA检测平台来平行检测288份随机挑选的血清。结果示于图1A中。结果显示，两种ELISA检测平台的检测结果高度一致，二者的符合率为99.3％，且反应强度的相关性好(相关系数r＝0.85)。另外，还使用基于pp150和pp150-2的ELISA检测平台与基于商品化CMV-IgG试剂(Diasorin-IgG)的ELISA检测平台平行检测36份随机挑选的血清。结果示于图1B中。结果显示，基于pp150和pp150-2的ELISA检测平台的反应性显著强于基于Diasorin的ELISA检测平台的反应性，后者对阳性血清的反应性大多处于较低的水平。图1A和1B的结果表明，本实施例中所建立的ELISA检测平台可用于准确、可靠、有效地检测血清样品中的抗CMV蛋白的抗体(抗pp150抗体)的水平。
实施例3.使用Elispot检测平台对HCMV-IE1抗体的检测
在本实施例中，本发明人建立了基于IE1蛋白的、用于检测抗HCMV-IE1抗体的Elispot检测平台。
IE1是HCMV早期蛋白，在感染1h后即出现在感染细胞的细胞核内。本实施例中所建立的Elispot检测方法是，联合传统的酶联免疫斑点法及Elispot自动化斑点计数仪而建立的一种快速巨细胞病毒滴 度检测方法。与传统的检测TCID50的方法相比较，本实施例中所建立的Elispot检测方法不依赖于细胞病变计数或者空斑形成来获取实验结果，而是通过单克隆抗体与病毒早期蛋白的特异性结合来获取实验结果。因此，该Elispot检测方法不需要等待病毒感染的细胞的病变，大大缩短了检测时间(20h)。此外，该Elispot检测方法通过Elispot检测仪的自动化图片摄取及斑点计数程序来进行结果读取，大大提高了检测的稳定性和准确性。
图2显示了，本实施例中建立的基于IE1蛋白的Elispot检测平台中，HCMV病毒感染用量与Elispot检测值之间的相关性。结果显示，Elispot检测平台中的HCMV病毒感染用量与Elispot检测值之间存在显著的线性关系(R2＝0.9988)。这表明，本实施例中所建立的Elispot检测平台可用于准确、可靠、有效地检测血清样品中的抗CMV蛋白的抗体(抗IE1蛋白抗体)的水平。
进一步，还将本实施例中所建立的Elispot检测平台的检测结果与商品化HCMV IgG抗体检测试剂盒(Diasorin-IgG试剂，DIASORIN，P002033)的检测结果进行比较。结果示于图3中。结果显示，在平行检测的61份血清样品中，对于试剂盒检测为阳性的31份血清，Elispot检测平台的检测结果均为阳性；对于试剂盒检测为阴性的30份血清，Elispot检测平台的检测结果均为阴性；二者的结果完全一致。这表明，本实施例中所建立的Elispot检测平台的准确性和可靠性。
进一步，还将本实施例中所建立的Elispot检测平台的检测结果与Aimin Tang等人建立的HCMV IRD中和检测平台(Aimin Tang，Fengsheng Li，Daniel C.Freed，Adam C.Finnefrock，Danilo R.Casimiro，Dai Wang，Tong-Ming Fu.A novel high-throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines.Vaccine.2011Oct 26；29(46)：8350-6)的检测结果进行比较。结果示于图4中。结果显示，Elispot检测平台的检测结果(Elispot NT50)与IRD中和检测平台的检测结果 (IRD NT50)具有良好的相关性(R2＝0.8960)。这表明，本实施例中所建立的Elispot检测平台的准确性和可靠性。
实施例4.使用ELISA检测平台对背景已知的血清的检测
在本实施例中，本发明人利用实施例2中建立的ELISA检测平台来对上述61份背景明确的血清进行抗体检测，以验证这些ELISA检测平台的可靠性和有效性，其中已使用商品化的HCMV IRD检测试剂(Aimin Tang，Fengsheng Li，Daniel C.Freed，Adam C.Finnefrock，Danilo R.Casimiro，Dai Wang，Tong-Ming Fu.A novel high-throughput neutralization assay for supporting clinical evaluations of human cytomegalovirus vaccines.Vaccine.2011，29：8350-6)及商品化的HCMV IgG抗体检测试剂盒(Diasorin-IgG试剂，DIASORIN，P002033)确定了这些血清样品的背景。所述的检测方法包括下述步骤：
步骤1：样品收集
将待测血清离心处理5-10mins(10000rpm/min)，用于检测；
步骤2：加样检测
将所述试剂盒中的成分平衡至室温；在实施例2建立的ELISA检测平台的经包被的微孔板的样品孔中加入90ul样品稀释液及10ul待检血清；同时，在所述微孔板中设置阴性对照和阳性对照，其中阴性对照每孔加入10ul背景明确为阴性的血清及90ul样品稀释液，阳性对照每孔加入10ul背景明确为阳性的血清及90ul样品稀释液；然后在板式振荡器上振荡混匀；然后，贴上封板膜，并在37℃温箱/恒温水浴锅中反应1小时；
步骤3：反应后，揭下封板膜，用洗涤液洗涤5次，扣干；
步骤4：向各个孔中分别加入100ul酶标记的抗人IgG抗体溶液，贴上封板膜；然后在37℃温箱/恒温水浴锅中反应30min；
步骤5：反应后，揭下封板膜，用洗涤液洗涤5次，扣干；
步骤6：向各个孔中分别加入50ul底物溶液A和50ul底物溶液B，混匀；然后在37℃温箱/恒温水浴锅中反应15min；
步骤7：向各个孔中加入50ul终止液，然后在酶标仪上读取OD450处的读值，由此得到各个孔的抗体反应OD值。检测结果示于图5。
图5显示，实施例2中建立的6种ELISA检测平台(分别基于pp150和pp150-2、HCMV pp65(UL83)、HCMV gp52(UL44)、HCMV pp38(UL80a)、HCMV pp28(UL99)，以及UL48a)对背景明确的61份血清样品的检测结果，其中pp150指基于pp150和pp150-2的混合物的ELISA检测平台。结果显示，实施例2中建立的ELISA检测平台(尤其是，基于pp150和pp150-2的ELISA检测平台和基于pp28的ELISA检测平台)能够准确、可靠、有效地检测血清样品中的抗CMV蛋白的抗体的水平，判断血清样品的阴性和阳性。
实施例5.使用ELISA检测平台来确定样品中的抗体滴度
在本实施例中，本发明人利用实施例2中建立的ELISA检测平台来对确定血清样品中的抗体滴度，以验证这些ELISA检测平台的可靠性和有效性。
简而言之，在本实施例中，以pp150为例，使用基于pp150和pp150-2的ELISA检测平台来对两份血清(血清1和血清2)的连续梯度稀释样品进行HCMV IgG抗体(抗pp150抗体)的平行检测(每个样品进行两次重复实验)，以确定这两份血清中的抗pp150抗体的抗体滴度。在测定过程中，设置阴性对照孔、阳性对照孔和空白对照孔。所述的检测方法包括下述步骤：
步骤1：样品收集
将待测血清离心处理5-10mins(10000rpm/min)，用于检测；
步骤2：加样检测
将所述试剂盒中的成分平衡至室温；
向经包被的微孔板的第1样品孔中添加样品稀释液180ul，向第2-10样品孔中添加样品稀释液100ul/孔；然后向第1样品孔中添加20ul待测血清，并在板式振荡器上震荡混匀30s-60s；然后，从第1样品孔中吸出100ul加入第2孔，并震荡混匀；然后，从第2样品孔 中吸出100ul加入第3孔，并震荡混匀；然后，依次稀释到第10孔；然后，从第10孔中吸出100ul弃除；对两份血清中的每一份重复这一过程；
同时，在所述微孔板中设置阴性对照孔、阳性对照孔和空白对照孔，其中阴性对照每孔加入10ul背景明确为阴性的血清及90ul样品稀释液，阳性对照每孔加入10ul背景明确为阳性的血清及90ul样品稀释液，空白对照每孔加入100ul样品稀释液；
然后，贴上封板膜，并在37℃温箱/恒温水浴锅中反应1小时；
步骤3：反应后，揭下封板膜，用洗涤液洗涤5次，扣干；
步骤4：向各个孔中分别加入100ul酶标记的抗人IgG抗体溶液，贴上封板膜；然后在37℃温箱/恒温水浴锅中反应30min；
步骤5：反应后，揭下封板膜，用洗涤液洗涤5次，扣干；
步骤6：向各个孔中分别加入50ul底物溶液A和50ul底物溶液B，混匀；然后在37℃温箱/恒温水浴锅中反应15min；
步骤7：向各个孔中加入50ul终止液，然后在酶标仪上读取OD450处的读值，由此得到各个孔的抗体反应OD值。检测结果示于表4中。
在表4中，血清1和血清2为两份独立的血清样品，第1组和第2组表示两次重复试验，抗体滴度定义为，OD450达到0.2以上的最大血清稀释倍数。检测结果显示，血清1的HCMV IgG(即，抗pp150抗体)的抗体滴度为80，血清2的抗体滴度为320。
表4：使用ELISA检测平台来确定样品中的抗体滴度
Figure PCTCN2016072094-appb-000005
Figure PCTCN2016072094-appb-000006
表4的结果显示，实施例2中建立的ELISA检测平台(例如，基于pp150和pp150-2的ELISA检测平台)能够准确、可靠、有效地检测样品中的抗CMV蛋白的抗体(例如，抗pp150抗体)的抗体滴度。
实施例6.使用ELISA检测平台评估发生HCMV活动性感染的风险
本实施例中所使用的蛋白是实施例1中获得的蛋白，所使用的检测方法是实施例5中描述的检测方法。
简而言之，本实施例使用实施例2中建立的ELISA检测平台(包括，基于pp150和pp150-2的ELISA检测平台(简称为pp150检测平台，下同)，pp28检测平台、pp38检测平台、UL48a检测平台、gp52检测平台、pp65检测平台)以及IE1抗体检测平台(即，实施例3中描述的用于检测抗HCMV-IE1抗体的Elispot检测平台)来分别检测保存良好的、获自广西自然人群的、间隔12个月(一年)采集获得的血清202对。
对于每一对血清，如果与在先采集的血清相比，在后采集的血清中的抗体滴度升高4倍及以上的话，那么定义：该对血清(其来自于同一个个体，因此被当作一个样本)的检测结果为“阳性”。
根据上述定义，在202对血清样品中，IE1抗体检测平台、pp150检测平台、pp28检测平台、pp38检测平台、UL48a检测平台、gp52检测平台和pp65检测平台检测到阳性检测结果的样本数(即，在后采集的血清中的抗体滴度升高4倍及以上的样本数)依次为8份、77份、21份、29份、30份、24份、9份。
进一步，为了评价pp150检测平台评估发生HCMV活动性感染的风险的效力，需要利用其他6个检测平台的检测结果来定义发生了病毒事件(即，发生了HCMV活性感染)的个体。简言之，将指示个体发生了病毒事件(即，发生了HCMV活性感染)的标准定义为，其他6个检测平台针对该个体的血清对所获得的6个检测结果中有至少2个、至少3个或至少4个检测结果同时为阳性。
表5显示了，其他6个检测平台的检测结果的信息统计。
Figure PCTCN2016072094-appb-000007
表6a显示了，在病毒事件的不同定义标准下，pp150检测平台的AUC曲线参数分析的结果。表6b显示了，在病毒事件的不同定义标准下，pp150检测平台的LOGISTIC回归分析的结果。
表6a：病毒事件的不同定义标准下pp150检测平台的AUC曲线参数
Figure PCTCN2016072094-appb-000008
表6b.病毒事件的不同定义标准下pp150检测平台的LOGISTIC回归分析
Figure PCTCN2016072094-appb-000009
pp150检测平台在病毒事件的不同评价标准下的ROC曲线分析还示于图6A-C，其中，
图6A显示，当将多种其他检测平台的检测结果为双阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp150检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析；
图6B显示，当将多种其他检测平台的检测结果为三阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp150检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析；
图6C显示，当将多种其他检测平台的检测结果为四阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp150检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析。
表6a-6b以及图6A-C的结果表明，在所有三种情况下，pp150检测平台预测病毒事件(HCMV活动性感染)发生的准确率都大于80％，从而可以用于准确、可靠、有效地评估受试者发生人巨细胞病毒(HCMV)活动性感染的风险。
表7a显示了，在病毒事件的不同定义标准下，pp28检测平台的AUC曲线参数分析的结果。表7b显示了，在病毒事件的不同定义标准下，pp28检测平台的LOGISTIC回归分析的结果。
表7a：病毒事件的不同定义标准下pp28检测平台的AUC曲线参数
Figure PCTCN2016072094-appb-000010
表7b.病毒事件的不同定义标准下pp28检测平台的LOGISTIC回归分析
Figure PCTCN2016072094-appb-000011
pp28检测平台在病毒事件的不同评价标准下的ROC曲线分析还示于图7A-C，其中，
图7A显示，当将多种其他检测平台的检测结果为双阳及以上阳性 的情况定义为发生了病毒事件(HCMV活动性感染)时，pp28检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析；
图7B显示，当将多种其他检测平台的检测结果为三阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp28检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析；
图7C显示，当将多种其他检测平台的检测结果为四阳及以上阳性的情况定义为发生了病毒事件(HCMV活动性感染)时，pp28检测平台评估病毒事件(HCMV活动性感染)发生的灵敏度和特异性的分析。
表7a-7b以及图7A-C的结果表明，在所有三种情况下，pp28检测平台预测病毒事件(HCMV活动性感染)发生的准确率都大于78％，从而可以用于准确、可靠、有效地评估受试者发生人巨细胞病毒(HCMV)活动性感染的风险。
实施例7.pp150检测平台用于广西某自然人群的检测结果
在本实施例中，本发明人利用实施例2中建立的ELISA检测平台(pp150检测平台)来评估广西某自然人群发生HCMV活动性感染的风险。所使用的检测方法及抗体滴度计算方法如实施例5所述。
简而言之，本发明人利用pp150检测平台检测了广西某自然人群(1659人)中每一个个体间隔12个月(一年)的双份血清。检测结果显示，在该人群中，前后两次检测的抗体阳性率分别为98.7％和98.9％，平均抗体滴度分别为1：269和1：260。这表明，该人群中绝大多数个体之前都曾感染了HCMV。本发明人进一步比较了该人群前后两次检测的所获得的检测结果。比较结果示于图8-9和表8a-8b中。
特别地，图8显示了，所述自然人群间隔12个月(一年)的体内抗pp150抗体的抗体滴度的变化情况。结果显示，基线抗体水平(即，在先的第一次检测获得的抗体滴度)越高的个体一年后出现抗pp150抗体水平4倍及以上升高(即，在后的第二次检测获得的抗体滴度为在先检测获得的抗体滴度的至少4倍高，其指示着个体在12个月的间隔期间发生了HCMV活动性感染)的概率越低；而基线抗体水平越低的个体 一年后出现抗pp150抗体水平4倍及以上升高的概率越高。这一结果再次表明，个体发生HCMV活动性感染的风险与抗体基线水平呈负相关。抗CMV蛋白的抗体水平(例如抗pp150抗体的滴度)越低，个体感染CMV的风险就越高。
图9显示，在所述的自然人群中，基线抗pp150抗体水平与HCMV活动性感染风险之间的相关性。其中，横坐标表示基线抗pp150抗体水平(抗体滴度)，纵坐标表示发生HCMV活动性感染的比例(风险)。结果显示，基线抗体水平越低的个体随后发生HCMV活动性感染(即，抗pp150抗体水平4倍及以上升高)的风险越高。HCMV活动性感染风险与抗体基线水平呈负相关。其中，当抗体基线水平(抗体滴度)低于10时，发生HCMV活动性感染的风险高达82.6％；并且当抗体基线水平(抗体滴度)不超过160时，发生HCMV活动性感染的风险高达23.66％。图9的结果还具体地描述于表8a-8b中。
表8a.广西某自然人群间隔12个月(一年)的两次检测的检测结果的统计分析(一)
Figure PCTCN2016072094-appb-000012
Figure PCTCN2016072094-appb-000013
表8a8b和图9的结果还显示：(1)基线抗体水平为阴性(抗体滴度＜10)的个体发生HCMV活动性感染(原发感染)的比率高达82.6％(19/23)；而基线抗体水平(抗体滴度)≥1∶2560的个体发生HCMV活动性感染(再发感染)的比率为0％(0/71)(p＜0.0001)；(2)HCMV活动性感染率与基线抗体水平呈明显负相关：基线抗体水平越高，HCMV活动性感染率越低。其中，抗体滴度＞1∶80的个体(约占80％)一年内的病毒活动性感染率为3.48％，而抗体滴度≤1∶80的个体(约占20％)的病毒活动性感染率为36.98％。类似地，抗体滴度＞1∶40的个体一年内的病毒活动性感染率为4.93％，而抗体滴度≤1∶40的个体的病毒活动性感染率为55.37％。抗体滴度＞1∶160的个体一年内的病毒活动性感染率为1.59％，而抗体滴度≤1∶160的个体的病毒活动性感染率为23.66％。
表8b的结果还显示：本发明的方法可用于判断受试者感染的相对风险度高低，其中，用于判断相对风险度高低的基线抗体水平(即，抗体滴度)的参考值可设置为范围在40-320之间的抗体滴度，例如40、80、160或320的抗体滴度。如果受试者样品中检测到的抗体滴度小于或者等于该参考值的话，则可认为该受试者发生HCMV活动性感染的相对风险度高。从表8b中可以看出，当参考值在40-320之间时，抗体滴度低于该参考值的受试者发生HCMV活动性感染的相对风险度均超过10，并且其95％CI的下限均超过7。这表明，该参数(抗体滴度)与HCMV活动性感染之间存在着强关联或者极强关联。
例如，如果受试者样品中检测到的抗体滴度小于或者等于40，那么，与抗体滴度高于40的受试者相比，其发生HCMV活动性感染的相对风险度为11.2，95％CI为8.7-14.6(也即，发生HCMV活动性感染的风险显著提高)。如果受试者样品中检测到的抗体滴度小于或者等于80，那么，与抗体滴度高于80的受试者相比，其发生HCMV活动性感染的相对风险度为10.6，95％CI为7.7-14.6。如果受试者样品中检测到的抗体滴度小于或者等于160，那么，与抗体滴度高于160的受试者相比，其发生HCMV活动性感染的相对风险度为14.8，95％CI为9.0-24.6。如果受试者样品中检测到的抗体滴度小于或者等于320， 那么，与抗体滴度高于320的受试者相比，其发生HCMV活动性感染的相对风险度为23.7，95％CI为8.8-63.4。
此外，表8b还显示了本发明的方法用于预测HCMV活动性感染的灵敏度、特异性和约登指数。从表8b可以看出，当参考值设定为40的抗体滴度时，本发明方法用于预测HCMV活动性感染的灵敏度为57.3％，特异性为94.7％，约登指数为0.52；当参考值设定为80的抗体滴度时，本发明方法用于预测HCMV活动性感染的灵敏度为73.1％，特异性为85.7％，约登指数为0.59；当参考值设定为160的抗体滴度时，本发明方法用于预测HCMV活动性感染的灵敏度为90.6％，特异性为66.4％，约登指数为0.57；当参考值设定为320的抗体滴度时，本发明方法用于预测HCMV活动性感染的灵敏度为97.7％，特异性为40.1％，约登指数为0.37。这些结果表明，当参考值设定为80的抗体滴度时，本发明方法具有最佳的预测效果(即，约登指数最高，为0.59)；并且，当参考值设定为160和40的抗体滴度时，本发明方法也都具有良好的预测效果(即，约登指数均高于0.5)。
对所述自然人群的多因素分析还显示，HCMV活动性感染率与性别、年龄、职业、文化程度、卫生习惯等均无关。
实施例8.血清中的抗体含量的标定
在本实施例中，本发明人利用标准品来对血清样品中的抗体含量进行标定。所用标准品为CMV IgG标准品(Paul-Ehrlich-Instltut，Referenz-CMV-IgG，Juli 1996，110IU/ml)，所标定的目的抗体为抗pp150抗体。
为此目的，发明人首先对该标准品的系列稀释物进行了ELISA检测(所使用的ELISA检测方法如实施例5所述)，并根据ELISA的检测结果绘制了抗体含量-抗体反应性的标准曲线。标准曲线示于图10中；其中横坐标表示抗体含量(以IU/ml表示)，纵坐标表示抗体反应性(以获自ELISA的OD值表示)。结果显示，在0.06-0.7IU/ml的范围内，抗体含量与抗体反应性之间存在显著的线性关系(y＝3.163*x-0.073， R2＝0.9984)，线性范围超过1个数量级。因此，可通过下述方案来对对血清样品中的抗体含量进行绝对定量：(1)将血清样品进行10倍梯度稀释；(2)对每一个经稀释的血清样品进行ELISA检测；(3)选取落在线性范围内的检测结果，并根据上述线性曲线方程和稀释倍数计算出初始血清样品中的抗体含量。
按照上述方案，我们对广西地区某自然人群(726人)中的每一个个体的、间隔12个月(一年)采集的双份血清进行检测。检测结果显示，在该人群中，间隔12个月(一年)的前后两次检测的抗体阳性率均为100％，平均抗体滴度分别为6.19IU/ml和5.08IU/ml。本发明人进一步比较了前后两次检测的检测结果。比较结果示于图11和表9中。
特别地，图11显示了，所述自然人群间隔12个月(一年)的体内抗pp150抗体含量的变化情况。该结果与图8中显示的结果基本一致：基线抗体水平越低的个体一年后出现抗pp150抗体水平4倍及以上升高(即，在后的第二次检测获得的抗体水平为在先检测获得的抗体水平的至少4倍高，其指示着个体在12个月的间隔期间发生了HCMV活动性感染)的概率越高；而基线抗体水平越高的个体一年后出现抗pp150抗体水平4倍及以上升高(即，发生HCMV活动性感染)的概率越低。这一结果再次表明，个体发生HCMV活动性感染的风险与血清中的抗体基线水平呈负相关。血清中抗HCMV蛋白的抗体水平(例如抗pp150抗体的含量)越低，个体感染HCMV的风险就越高。
表9a-9b的结果还显示，在所述自然人群中，基线抗体水平越低的个体随后发生HCMV活动性感染(即，抗pp150抗体水平升高4倍及以上)的风险越高；抗pp150抗体的基线水平与HCMV活动性感染的风险之间呈负相关。其中，基线抗体水平不超过0.2IU/ml的个体一年内发生HCMV活动性感染的风险/感染率高达80％；基线抗体水平不超过0.8IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为60.0％；基线抗体水平不超过1.6IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为50.0％；基线抗体水平不超过3.2IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为31.3％；基线抗体水 平不超过6.4IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为19.0％。相比之下，基线抗体水平高于0.8IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为5.2％；基线抗体水平高于1.6IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为3.3％；基线抗体水平高于3.2IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为1.6％；基线抗体水平高于6.4IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为0.8％；并且，基线抗体水平高于25.6IU/ml的个体一年内发生HCMV活动性感染的风险/感染率为0。
表9a.广西某自然人群间隔12个月(一年)的两次检测的检测结果的统计分析(一)
Figure PCTCN2016072094-appb-000014
Figure PCTCN2016072094-appb-000015
表9b的结果还显示：本发明的方法可用于判断受试者感染的相对风险度高低，其中，用于判断相对风险度高低的基线抗体水平(即，抗体含量/抗体绝对量)的参考值可设置为范围在0.8-6.4IU/ml之间的抗体绝对量，例如0.8、1.6、3.2或6.4IU/ml的抗体绝对量。如果受试者样品中检测到的抗体含量小于或者等于该参考值的话，则可认为该受试者发生HCMV活动性感染的相对风险度高。从表9b中可以看出，当参考值在0.8-6.4IU/ml之间时，抗体含量低于该参考值的受试者发生HCMV活动性感染的相对风险度均超过11，并且其95％CI的下限均超过7。这表明，该参数(抗体含量/抗体绝对量)与HCMV活动性感染之间存在着强关联或者极强关联。
例如，如果受试者样品中检测到的抗体含量小于或者等于0.8IU/ml，那么，与抗体含量高于0.8IU/ml的受试者相比，其发生HCMV活动性感染的相对风险度为11.6，95％CI为7.8-17.2(也即，发生HCMV活动性感染的风险显著提高)。如果受试者样品中检测到的抗体含量小于或者等于1.6IU/ml，那么，与抗体含量高于1.6IU/ml的受试者相比，其发生HCMV活动性感染的相对风险度为15.2，95％CI为9.5-24.3。如果受试者样品中检测到的抗体含量小于或者等于3.2IU/ml，那么，与抗体含量高于3.2IU/ml的受试者相比，其发生HCMV活动性感染的相对风险度为19.0，95％CI为9.6-37.7。如果受试者样品中检测到的抗体含量小于或者等于6.4IU/ml，那么，与抗体含量高于6.4IU/ml的受试者相比，其发生HCMV活动性感染的相对风险度为25.2，95％CI为8.0-79.6。
此外，表9b还显示了，当参考值设定为0.8-6.4IU/ml的抗体含量时，本发明的方法用于预测HCMV活动性感染的灵敏度、特异性和约登指数。从表9b可以看出，当参考值设定为0.8IU/ml的抗体绝对量时，本发明方法用于预测HCMV活动性感染的灵敏度为46.15％，特异性为96.97％，约登指数为0.43；当参考值设定为1.6IU/ml的抗体绝对量时，本发明方法用于预测HCMV活动性感染的灵敏度为67.69％，特异性为93.34％，约登指数为0.61；当参考值设定为3.2IU/ml的抗 体绝对量时，本发明方法用于预测HCMV活动性感染的灵敏度为86.15％，特异性为81.39％，约登指数为0.68；当参考值设定为6.4IU/ml的抗体绝对量时，本发明方法用于预测HCMV活动性感染的灵敏度为95.38％，特异性为59.91％，约登指数为0.55。这些结果表明，当参考值设定为3.2IU/ml的抗体绝对量时，本发明方法具有最佳的预测效果(即，约登指数最高，为0.68)；并且，当参考值设定为0.8、1.6和6.4IU/ml的抗体绝对量时，本发明方法也都具有良好的预测效果(即，约登指数均接近或高于0.5)。
此外，表9b的结果还显示，随着设定的参考值的提高，本发明方法用于预测HCMV活动性感染的灵敏度逐渐提高(由46.15％提高到95.38％)，而特异性逐渐下降(由99.97％下降到59.91％)。这与ROC曲线分析的结果是基本上一致的。上述方法的ROC曲线分析结果示于图12，其中，曲线下面积为0.913。从图12可以看出，随着灵敏度的提高，本发明方法用于预测HCMV活动性感染的特异性逐渐降低。并且，图12的结果表明，本发明方法可以用于准确、可靠、有效地评估受试者发生人巨细胞病毒(HCMV)活动性感染的风险。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
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Claims (9)

  1. 评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中的方法，其包括下述步骤：
    (1)测定来自所述受试者的体液样品中抗HCMV蛋白的抗体的水平；
    (2)将所述水平与预先确定的参考值相比较；
    其中，所述HCMV蛋白选自pp150和/或pp28；并且，如果所述水平低于参考值的话，则判断所述受试者处于发生HCMV活动性感染的风险中；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述体液样品选自血液、血清、血浆、尿液和唾液；
    优选地，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活；
    优选地，通过免疫学测定来测定所述体液样品中抗HCMV蛋白的抗体的水平；进一步优选地，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot测定；
    优选地，所述水平为抗体滴度(例如通过ELISA测定的抗体滴度)，并且参考值为预先确定的抗体滴度；或者，所述水平为抗体的绝对量(例如以IU/ml表示的绝对量)并且参考值为预先确定的抗体的绝对量(例如以IU/ml表示的绝对量)。
  2. 权利要求1的方法，其中，在步骤(1)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的抗体滴度；
    优选地，所述参考值为范围在40-320之间的抗体滴度；更优选地，所述参考值为范围在40-160之间的抗体滴度；例如，所述参考值为40、80、或160的抗体滴度；
    优选地，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的相对风险 度为11.2，95％CI为8.7-14.6，和/或，判断所述受试者发生HCMV活动性感染的概率为55.37％；或者，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或者等于80的话，则判断所述受试者发生HCMV活动性感染的相对风险度为10.6，95％CI为7.7-14.6，和/或，判断所述受试者发生HCMV活动性感染的概率为36.98％；或者，所述参考值为160，并且如果抗pp150抗体的抗体滴度小于或等于160的话，则判断所述受试者发生HCMV活动性感染的相对风险度为14.8，95％CI为9.0-24.6，和/或，判断所述受试者发生HCMV活动性感染的概率为23.66％；优选地，所述参考值为80的抗体滴度；
    优选地，使用pp150和/或其抗原性片段(例如其包含pp150的aa861-1048的片段)通过BLISA来测定所述体液样品中抗pp150抗体的抗体滴度；和/或，使用pp28和/或其抗原性片段通过BLISA来测定所述体液样品中抗pp28抗体的抗体滴度；优选地，pp150具有如SBQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列。
  3. 权利要求1的方法，其中，在步骤(1)中，测定所述体液样品中抗pp150和/或pp28的抗体的绝对量(例如以IU/ml表示的绝对量)；
    优选地，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的绝对量；优选地，所述参考值为范围在0.8-6.4IU/ml之间的抗体绝对量；更优选地，所述参考值为范围在0.8-3.2IU/ml之间的抗体绝对量；例如，所述参考值为0.8、1.6、或3.2IU/ml的抗体绝对量；
    优选地，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.6，95％CI为7.8-17.2，和/或，判断所述受试者发生HCMV活动性感染的概率为60.0％；或者，所述参考值为 1.6IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为15.2，95％CI为9.5-24.3，和/或，判断所述受试者发生HCMV活动性感染的概率为50.0％；或者，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为19.0，95％CI为9.6-37.7，和/或，判断所述受试者发生HCMV活动性感染的概率为31.3％；优选地，所述参考值为3.2IU/ml的抗体绝对量。
  4. 权利要求1-3任一项的方法，其还包括：
    在步骤(1)之前，提供来自所述受试者的体液样品；和/或
    在步骤(2)之后，给被判断为处于发生HCMV活动性感染的风险中的受试者施用抗HCMV的疫苗或者抗体，以降低所述受试者发生HCMV活动性感染的风险。
  5. 能够测定抗HCMV蛋白的抗体的水平的试剂在制备试剂盒中的用途，所述试剂盒用于通过测定来自受试者的体液样品中抗HCMV蛋白的抗体的水平而评估所述受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中；其中，所述HCMV蛋白选自pp150和/或pp28；
    优选地，所述试剂能够通过免疫学测定来测定抗HCMV蛋白的抗体的水平；进一步优选地，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot测定；
    优选地，所述试剂选自：HCMV蛋白或其抗原性片段，包含所述HCMV蛋白或其抗原性片段的融合蛋白，以及其任意组合；
    优选地，所述HCMV蛋白为pp150，且所述试剂为pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)；或者，所述HCMV蛋白为pp28，且所述试剂为pp28和/或其抗原性片段；或者，所述HCMV蛋白为pp150和pp28，且所述试剂包括：作为第一组分的pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)，以及作为第 二组分的pp28和/或其抗原性片段；优选地，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述体液样品选自血液、血清、血浆、尿液和唾液；
    优选地，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活；
    优选地，所述试剂盒还包括：(i)用于收集或贮存来自受试者的体液样品的装置；(ii)用于进行所述测定所需的其他试剂(例如缓冲液，稀释液，封闭液，经标记的抗抗体，和/或标准品)；和/或，(iii)抗HCMV的疫苗或者抗体；
    优选地，所述试剂盒通过包括下述步骤的方法来评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中：(1)使用所述试剂来测定来自所述受试者的体液样品中抗HCMV蛋白的抗体的水平；和，(2)将所述水平与预先确定的参考值相比较；其中，如果所述水平低于参考值的话，则判断所述受试者处于发生HCMV活动性感染的风险中；
    进一步优选地，在所述方法中，所述水平为抗体滴度(例如通过ELISA测定的抗体滴度)，并且参考值为预先确定的抗体滴度；或者，所述水平为抗体的绝对量(例如以IU/ml表示的绝对量)并且参考值为预先确定的抗体的绝对量(例如以IU/ml表示的绝对量)；
    优选地，在所述方法中，在步骤(1)中，通过BLISA来测定所述体液样品中抗pp150和/或pp28的抗体的抗体滴度；优选地，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的抗体滴度；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的抗体滴度；优选地，所述参考值为范围在40-320之间的抗体滴度；更优选地，所述参考值为范围在40-160之间的抗体滴度；例如，所述 参考值为40、80、或160的抗体滴度；
    进一步优选地，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.2，95％CI为8.7-14.6，和/或，判断所述受试者发生HCMV活动性感染的概率为55.37％；或者，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或者等于80的话，则判断所述受试者发生HCMV活动性感染的相对风险度为10.6，95％CI为7.7-14.6，和/或，判断所述受试者发生HCMV活动性感染的概率为36.98％；或者，所述参考值为160，并且如果抗pp150抗体的抗体滴度小于或等于160的话，则判断所述受试者发生HCMV活动性感染的相对风险度为14.8，95％CI为9.0-24.6，和/或，判断所述受试者发生HCMV活动性感染的概率为23.66％；优选地，所述参考值为80的抗体滴度；
    优选地，在所述方法中，在步骤(1)中，测定所述体液样品中抗pp150和/或pp28的抗体的绝对量(例如以IU/ml表示的绝对量)；优选地，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的绝对量；优选地，所述参考值为范围在0.8-6.4IU/ml之间的抗体绝对量；更优选地，所述参考值为范围在0.8-3.2IU/ml之间的抗体绝对量；例如，所述参考值为0.8、1.6、或3.2IU/ml的抗体绝对量；
    进一步优选地，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.6，95％CI为7.8-17.2，和/或，判断所述受试者发生HCMV活动性感染的概率为60.0％；或者，所述参考值为1.6IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为15.2，95％CI为9.5-24.3，和/或，判断所述受试者发生HCMV活动性感染的概率为50.0％；或者，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发 生HCMV活动性感染的相对风险度为19.0，95％CI为9.6-37.7，和/或，判断所述受试者发生HCMV活动性感染的概率为31.3％；优选地，所述参考值为3.2IU/ml的抗体绝对量。
  6. 一种用于评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中的试剂盒，其包括能够测定抗HCMV蛋白的抗体的水平的试剂，以及任选地，使用所述试剂来测定来自受试者的体液样品中抗HCMV蛋白的抗体的水平，从而评估所述受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中的说明书；其中，所述HCMV蛋白选自pp150和/或pp28；
    优选地，所述试剂能够通过免疫学测定来测定抗HCMV蛋白的抗体的水平；进一步优选地，所述免疫学测定选自BLISA测定，Western印迹，表面等离子共振法，Elispot测定；
    优选地，所述试剂选自：HCMV蛋白或其抗原性片段，包含所述HCMV蛋白或其抗原性片段的融合蛋白，以及其任意组合；
    优选地，所述HCMV蛋白为pp150，且所述试剂为pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)；或者，所述HCMV蛋白为pp28，且所述试剂为pp28和/或其抗原性片段；或者，所述HCMV蛋白为pp150和pp28，且所述试剂包括：作为第一组分的pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)，以及作为第二组分的pp28和/或其抗原性片段；优选地，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SBQ ID NO：3所示的氨基酸序列；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述体液样品选自血液、血清、血浆、尿液和唾液；
    优选地，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活；
    优选地，所述试剂盒还包括：(i)用于收集或贮存来自受试者的体液样品的装置；(ii)用于进行所述测定所需的其他试剂(例如缓冲液，稀释液，封闭液，经标记的抗抗体，和/或标准品)；和/或，(iii)抗HCMV的疫苗或者抗体；
    优选地，所述试剂盒通过包括下述步骤的方法来评估受试者是否处于发生人巨细胞病毒(HCMV)活动性感染的风险中：(1)使用所述试剂来测定来自所述受试者的体液样品中抗HCMV蛋白的抗体的水平；和，(2)将所述水平与预先确定的参考值相比较；其中，如果所述水平低于参考值的话，则判断所述受试者处于发生HCMV活动性感染的风险中；
    进一步优选地，在所述方法中，所述水平为抗体滴度(例如通过BLISA测定的抗体滴度)，并且参考值为预先确定的抗体滴度；或者，所述水平为抗体的绝对量(例如以IU/ml表示的绝对量)并且参考值为预先确定的抗体的绝对量(例如以IU/ml表示的绝对量)；
    优选地，在所述方法中，在步骤(1)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的抗体滴度；优选地，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过BLISA来测定所述体液样品中抗pp150抗体的抗体滴度；和/或，使用pp28和/或其抗原性片段通过BLISA来测定所述体液样品中抗pp28抗体的抗体滴度；优选地，所述参考值为范围在40-320之间的抗体滴度；更优选地，所述参考值为范围在40-160之间的抗体滴度；例如，所述参考值为40、80、或160的抗体滴度；
    进一步优选地，所述参考值为40，并且如果抗pp150抗体的抗体滴度小于或者等于40的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.2，95％CI为8.7-14.6，和/或，判断所述受试者发生HCMV活动性感染的概率为55.37％；或者，所述参考值为80，并且如果抗pp150抗体的抗体滴度小于或者等于80的话，则判断所述受试者发生HCMV活动性感染的相对风险度为10.6，95％CI为7.7-14.6，和/或，判断所述受试者发生HCMV活动性感染的概率为36.98％；或者，所述参考值为160，并且如果抗pp150抗体的抗体滴度小于或等于160的话， 则判断所述受试者发生HCMV活动性感染的相对风险度为14.8，95％CI为9.0-24.6，和/或，判断所述受试者发生HCMV活动性感染的概率为23.66％；优选地，所述参考值为80的抗体滴度；
    优选地，在所述方法中，在步骤(1)中，测定所述体液样品中抗pp150和/或pp28的抗体的绝对量(例如以IU/ml表示的绝对量)；优选地，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的绝对量；优选地，所述参考值为范围在0.8-6.4IU/ml之间的抗体绝对量；更优选地，所述参考值为范围在0.8-3.2IU/ml之间的抗体绝对量；例如，所述参考值为0.8、1.6、或3.2IU/ml的抗体绝对量；
    进一步优选地，所述参考值为0.8IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于0.8IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为11.6，95％CI为7.8-17.2，和/或，判断所述受试者发生HCMV活动性感染的概率为60.0％；或者，所述参考值为1.6IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于1.6IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为15.2，95％CI为9.5-24.3，和/或，判断所述受试者发生HCMV活动性感染的概率为50.0％；或者，所述参考值为3.2IU/ml，并且如果抗pp150抗体的抗体绝对量小于或者等于3.2IU/ml的话，则判断所述受试者发生HCMV活动性感染的相对风险度为19.0，95％CI为9.6-37.7，和/或，判断所述受试者发生HCMV活动性感染的概率为31.3％；优选地，所述参考值为3.2IU/ml的抗体绝对量。
  7. 用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物的方法，其包括下述步骤：
    (1)在给测试动物施用候选药物之前，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第一水平；
    (2)给所述测试动物施用候选药物；
    (3)在给测试动物施用候选药物之后，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第二水平；
    (4)将所述第一水平与第二水平进行比较，其中，如果所述第一水平低于所述第二水平的话，则判断所述候选药物能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险；
    其中，所述HCMV蛋白选自pp150和/或pp28；
    优选地，所述测试动物是非人哺乳动物，例如模型动物(例如，小鼠，大鼠，兔，灵长类动物)；
    优选地，所述体液样品选自血液、血清、血浆、尿液和唾液；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活；
    优选地，通过免疫学测定来测定所述体液样品中抗HCMV蛋白的抗体的水平；进一步优选地，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Elispot测定；
    优选地，所述第一和第二水平为抗体滴度(例如通过ELISA测定的抗体滴度)；或者，所述第一和第二水平为抗体的绝对量(例如以IU/ml表示的绝对量)；
    优选地，在步骤(1)和(3)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平；优选地，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的第一和第二水平；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的第一和第二水平；优选地，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SBQ ID NO：3所示的氨基酸序列；
    优选地，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。
  8. 能够测定抗HCMV蛋白的抗体的水平的试剂在制备试剂盒中的用途，所述试剂盒用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物；其中，所述HCMV蛋白选自pp150和/或pp28；
    优选地，所述试剂能够通过免疫学测定来测定抗HCMV蛋白的抗体的水平；进一步优选地，所述免疫学测定选自BLISA测定，Western印迹，表面等离子共振法，Blispot测定；
    优选地，所述试剂选自：HCMV蛋白或其抗原性片段，包含所述HCMV蛋白或其抗原性片段的融合蛋白，以及其任意组合；
    优选地，所述HCMV蛋白为pp150，且所述试剂为pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)；或者，所述HCMV蛋白为pp28，且所述试剂为pp28和/或其抗原性片段；或者，所述HCMV蛋白为pp150和pp28，且所述试剂包括：作为第一组分的pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)，以及作为第二组分的pp28和/或其抗原性片段；优选地，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活；
    优选地，所述试剂盒通过包括下述步骤的方法来筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物：(1)在给测试动物施用 候选药物之前，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第一水平；(2)给所述测试动物施用候选药物；(3)在给测试动物施用候选药物之后，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第二水平；(4)将所述第一水平与第二水平进行比较，其中，如果所述第一水平低于所述第二水平的话，则判断所述候选药物能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险；
    优选地，所述测试动物是非人哺乳动物，例如模型动物(例如，小鼠，大鼠，兔，灵长类动物)；
    优选地，所述体液样品选自血液、血清、血浆、尿液和唾液；
    优选地，所述试剂盒还包括：(i)用于收集或贮存来自测试动物的体液样品的装置；和/或(ii)用于测定所述第一水平与第二水平所需的其他试剂(例如缓冲液，稀释液，封闭液，经标记的抗抗体，和/或标准品)；
    优选地，所述第一和第二水平为抗体滴度(例如通过ELIsA测定的抗体滴度)；或者，所述第一和第二水平为抗体的绝对量(例如以IU/ml表示的绝对量)；
    优选地，在所述方法中，在步骤(1)和(3)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平；优选地，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的第一和第二水平；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的第一和第二水平；优选地，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。
  9. 一种用于筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物的试剂盒，其包括能够测定抗HCMV蛋白的抗体的水平的试剂，以及任选地，使用所述试剂来筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物的说明书；其中，所述HCMV蛋白选自pp150和/或pp28；
    优选地，所述试剂能够通过免疫学测定来测定抗HCMV蛋白的抗体的水平；进一步优选地，所述免疫学测定选自ELISA测定，Western印迹，表面等离子共振法，Blispot测定；
    优选地，所述试剂选自：HCMV蛋白或其抗原性片段，包含所述HCMV蛋白或其抗原性片段的融合蛋白，以及其任意组合；
    优选地，所述HCMV蛋白为pp150，且所述试剂为pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)；或者，所述HCMV蛋白为pp28，且所述试剂为pp28和/或其抗原性片段；或者，所述HCMV蛋白为pp150和pp28，且所述试剂包括：作为第一组分的pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)，以及作为第二组分的pp28和/或其抗原性片段；优选地，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，所述受试者是哺乳动物，例如人；
    优选地，所述活动性感染为，未感染HCMV的受试者被HCMV初次感染，或者，已感染HCMV的受试者再次被HCMV感染或其体内的潜伏HCMV病毒被激活；
    优选地，所述试剂盒通过包括下述步骤的方法来筛选能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险的候选药物：(1)在给测试动物施用候选药物之前，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗 体的第一水平；(2)给所述测试动物施用候选药物；(3)在给测试动物施用候选药物之后，测定来自所述测试动物的体液样品中抗HCMV蛋白的抗体的第二水平；(4)将所述第一水平与第二水平进行比较，其中，如果所述第一水平低于所述第二水平的话，则判断所述候选药物能够提高受试者抵抗人巨细胞病毒(HCMV)活动性感染的能力或降低受试者发生人巨细胞病毒(HCMV)活动性感染的风险；
    优选地，所述测试动物是非人哺乳动物，例如模型动物(例如，小鼠，大鼠，兔，灵长类动物)；
    优选地，所述体液样品选自血液、血清、血浆、尿液和唾液；
    优选地，所述试剂盒还包括：(i)用于收集或贮存来自测试动物的体液样品的装置；和/或(ii)用于测定所述第一水平与第二水平所需的其他试剂(例如缓冲液，稀释液，封闭液，经标记的抗抗体，和/或标准品)；
    优选地，所述第一和第二水平为抗体滴度(例如通过ELISA测定的抗体滴度)；或者，所述第一和第二水平为抗体的绝对量(例如以IU/ml表示的绝对量)；
    优选地，在所述方法中，在步骤(1)和(3)中，通过ELISA来测定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平；优选地，使用pp150和/或其抗原性片段(例如其包含pp150的aa 861-1048的片段)通过ELISA来测定所述体液样品中抗pp150抗体的第一和第二水平；和/或，使用pp28和/或其抗原性片段通过ELISA来测定所述体液样品中抗pp28抗体的第一和第二水平；优选地，pp150具有如SEQ ID NO：1所示的氨基酸序列；和/或，pp150的抗原性片段具有如SEQ ID NO：2所示的氨基酸序列；和/或，pp28具有如SEQ ID NO：3所示的氨基酸序列；
    优选地，通过将所述体液样品中抗pp150和/或pp28的抗体的反应性与具有已知的抗体含量的标准品的抗体反应性相比较来确定所述体液样品中抗pp150和/或pp28的抗体的第一和第二水平。
PCT/CN2016/072094 2015-02-09 2016-01-26 用于评估机体发生人巨细胞病毒活动性感染风险的方法及相关的试剂盒 WO2016127785A1 (zh)

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