WO2017060988A1 - Procédé de détermination du statut immunitaire, procédé de prédiction de l'augmentation du nombre de cellules cd4+ t, procédé de prédiction de la baisse du nombre des cellules cd4+ t, et trousse à cet effet - Google Patents

Procédé de détermination du statut immunitaire, procédé de prédiction de l'augmentation du nombre de cellules cd4+ t, procédé de prédiction de la baisse du nombre des cellules cd4+ t, et trousse à cet effet Download PDF

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WO2017060988A1
WO2017060988A1 PCT/JP2015/078488 JP2015078488W WO2017060988A1 WO 2017060988 A1 WO2017060988 A1 WO 2017060988A1 JP 2015078488 W JP2015078488 W JP 2015078488W WO 2017060988 A1 WO2017060988 A1 WO 2017060988A1
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cells
human immunodeficiency
patient
immunodeficiency virus
rna
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PCT/JP2015/078488
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Japanese (ja)
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壮利 水谷
彩 石坂
愛 立川
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公益財団法人微生物化学研究会
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the present invention relates to a method for determining an immune state in a patient after starting treatment with an anti-human immunodeficiency virus drug, a method for predicting an increase in the number of CD4 + T cells in a patient after starting treatment with an anti-human immunodeficiency virus drug, A method for predicting a decrease in the number of CD4 + T cells in a patient who has not started treatment with an anti-human immunodeficiency virus drug, and a kit for determining an immune status in a patient after starting treatment with an anti-human immunodeficiency virus drug; A kit for predicting an increase in the number of CD4 + T cells in a patient after starting treatment with an anti-human immunodeficiency virus agent, or the number of CD4 + T cells in a patient who has not started treatment with an anti-human immunodeficiency virus agent
  • the present invention relates to a kit for predicting a decrease in the amount.
  • HIV infection against human immunodeficiency virus (hereinafter sometimes referred to as “HIV”) infection, which is the cause of acquired immunodeficiency syndrome (Acquired Immunity Definition; In addition to fundamental prevention of HIV infection, it is important to establish a technique capable of early diagnosis of the presence or absence of infection in the unlikely event that infection is suspected.
  • HIV human immunodeficiency virus
  • diagnosis of HIV infection is performed by measuring HIV RNA and HIV antigen in the plasma of infected persons.
  • HAART therapy High Active Anti-Retroviral Therapy
  • the amount of HIV RNA or HIV antigen in the plasma of an infected person can be suppressed to below the detection limit.
  • some infected cells escape from the HAART therapy because the anti-human immunodeficiency virus drug is difficult to reach deep in the tissue (hereinafter sometimes referred to as “residual infected cells”). It is known.
  • HAART therapy inhibits each step of virus growth, it is difficult to remove virus from residual infected cells by HAART therapy. Therefore, even if the amount of HIV RNA or HIV antigen in the plasma of an infected person is kept below the detection limit, if the HAART therapy is interrupted, the viral life cycle in the remaining infected cells will restart. There's a problem.
  • HIV infection is not a lethal disease, but the presence of the remaining infected cells can cause low-level virus replication, chronic inflammation, and decreased immunity.
  • the risk of occurring and causing various long-term complications has become a problem.
  • the complication include non-AIDS-related malignant tumors, cardiovascular diseases, chronic kidney diseases, liver diseases, bone-related diseases, HIV-related neurocognitive diseases, and the like.
  • An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention provides a method for determining an immune state that can easily and accurately determine an immune state in a patient after starting treatment with an anti-human immunodeficiency virus drug, and a treatment with an anti-human immunodeficiency virus drug.
  • a method for predicting an increase in the number of CD4 + T cells that can easily and accurately predict an increase in the number of CD4 + T cells in a patient after initiation, in a patient who has not started treatment with an anti-human immunodeficiency virus drug It is an object of the present invention to provide a method for predicting a decrease in the number of CD4 + T cells capable of predicting a decrease in the number of CD4 + T cells easily and with high accuracy, and a kit for them.
  • Means for solving the problems are as follows. That is, ⁇ 1> a detection step of detecting a transcript from the transcription start point of human immunodeficiency virus RNA to 200 bases in peripheral blood mononuclear cells; After the initiation of treatment with an anti-human immunodeficiency virus drug, comprising the step of evaluating whether or not immunity in a patient is in an activated state using the presence or absence of detection of the transcript as an index. This is a method for determining an immune state in a patient.
  • ⁇ 2> a detection step of detecting a transcript from the transcription start point of human immunodeficiency virus RNA to 200 bases in peripheral blood mononuclear cells; CD4 in a patient after initiating treatment with an anti-human immunodeficiency virus drug, comprising evaluating whether or not the number of CD4 + T cells increases using the presence or absence of detection of the transcript as an index + A method for predicting an increase in the number of T cells.
  • An anti-human immunodeficiency virus comprising an oligonucleotide containing a base sequence having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1.
  • kits for determining immune status in a patient after initiating treatment with a drug a kit for predicting an increase in the number of CD4 + T cells in a patient after initiating treatment with an anti-human immunodeficiency virus agent, or anti A kit for predicting a decrease in the number of CD4 + T cells in a patient who has not started treatment with a human immunodeficiency virus drug.
  • an immune status determination method that can easily and accurately determine an immune status in a patient after starting treatment with an anti-human immunodeficiency virus drug.
  • a method for predicting an increase in the number of CD4 + T cells in a patient after the start of treatment with an anti-human immunodeficiency virus drug which can easily and accurately predict the increase in the number of CD4 + T cells
  • reduction prediction method of CD4 + T cell count that can predict the decrease in CD4 + T cell count in patients not started treatment with drugs simple and with high precision, as well as to provide kits for them it can.
  • FIG. 1 is a graph showing the results of Test Example 1.
  • FIG. 2 is a graph showing the results of Test Example 2.
  • FIG. 3 is a graph showing the results of Test Example 3.
  • FIG. 4 is a graph showing the results of Test Example 4.
  • FIG. 5A is a graph showing the results of analyzing the relationship between the presence or absence of detection of HIV short-chain RNA in peripheral blood mononuclear cells and the number of CD4 + T cells in patients who have started treatment in Test Example 5.
  • FIG. 5B is a graph showing the results of analyzing the relationship between the presence or absence of detection of HIV short-chain RNA in peripheral blood mononuclear cells and the increase / decrease in the number of CD4 + T cells in the treatment-start patient in Test Example 5.
  • FIG. 5A is a graph showing the results of analyzing the relationship between the presence or absence of detection of HIV short-chain RNA in peripheral blood mononuclear cells and the increase / decrease in the number of CD4 + T cells in the treatment-start patient in Test Example 5.
  • FIG. 6A is a graph showing the relationship between the amount of HIV short RNA in Test Example 6 and the proportion of HLA-DR + CD38 + cells in total CD8 + T cells.
  • FIG. 6B is a graph showing the relationship between the amount of HIV short RNA in Test Example 6 and the amount of HLA-DR MFI in total CD8 + T cells.
  • FIG. 7 is a graph showing the results of Test Example 7.
  • FIG. 8 is a graph showing the results of Test Example 8.
  • the method for determining an immune state in a patient after starting treatment with the anti-human immunodeficiency virus drug of the present invention includes at least a detection step and an evaluation step, and further includes other steps as necessary.
  • the detection step in the method for determining an immune state is a transcription product from peripheral transcription mononuclear cells of human immunodeficiency virus RNA up to 200 bases (hereinafter sometimes referred to as “HIV short RNA”). It is the process of detecting.
  • the length of the HIV short RNA is not particularly limited as long as it is a transcription product from the transcription start point of human immunodeficiency virus RNA to 200 bases, and can be appropriately selected according to the purpose. Examples include those of about 30 bases, about 50 bases to about 70 bases, about 90 bases to about 110 bases from the transcription start point shown in Test Example 8.
  • the method for detecting the HIV short-chain RNA is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a method comprising polyA addition treatment, cDNA synthesis treatment, amplification treatment, and detection treatment Is mentioned.
  • the polyA addition treatment is a treatment of adding polyA to the 3 ′ end of RNA of 200 bases or less separated and purified from a peripheral blood mononuclear cell fraction using polyA polymerase.
  • the polyA addition process can be performed using, for example, miScript II RT Kit (manufactured by QIAGEN).
  • the length of the polyA is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 to 40 bases, more preferably 20 to 30 bases.
  • the cDNA synthesis treatment is performed by reverse transcription using the oligonucleotide having polyT and an adapter sequence on the 5 ′ side of the polyT (hereinafter sometimes referred to as “polyT-containing oligonucleotide”). This is a process of synthesizing cDNA complementary to the added RNA.
  • the cDNA synthesis process can be performed using, for example, miScript II RT Kit (manufactured by QIAGEN).
  • the length of the polyT is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 to 40 bases, more preferably 20 to 30 bases.
  • the polyT is annealed to the polyA.
  • the polyT-containing oligonucleotide may have a degenerate sequence at the 3 ′ end.
  • a reverse transcription reaction starting from the 3 ′ end of the polyT-containing oligonucleotide, cDNA complementary to the RNA added with polyA is synthesized.
  • a reverse transcriptase used for the said reverse transcription reaction, According to the objective, it can select suitably, For example, the reverse transcriptase derived from Moloney Murine Leukemia Virus etc. are mentioned.
  • the adapter sequence is not particularly limited as long as it does not inhibit the reverse transcription reaction, and can be appropriately selected according to the purpose. However, the adapter sequence is preferably not complementary to a known gene in a living body. More preferably, it is not complementary to.
  • the polyA addition process and the cDNA synthesis process may be performed simultaneously or separately.
  • RNA used as a template it is preferable to decompose RNA using RNase.
  • RNase There is no restriction
  • the amplification treatment includes an oligonucleotide containing a base sequence corresponding to a transcript of HIV RNA (hereinafter sometimes referred to as “forward primer”) and an oligonucleotide containing a sequence complementary to the adapter sequence (hereinafter referred to as “ Is used to amplify a target base sequence having the sequence of HIV cDNA from the cDNA.
  • forward primer an oligonucleotide containing a base sequence corresponding to a transcript of HIV RNA
  • Is used to amplify a target base sequence having the sequence of HIV cDNA from the cDNA.
  • the forward primer is not particularly limited as long as it can detect HIV short-chain RNA, and can be appropriately selected according to the purpose. However, the forward primer has 10 or more consecutive sequences in the base sequence represented by SEQ ID NO: 1.
  • An oligonucleotide containing a base sequence having at least 80% sequence identity with the prepared base sequence (hereinafter, sometimes referred to as “forward primer oligonucleotide”) is preferred.
  • the oligonucleotide for forward primer is a sequence other than a base sequence having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1 (hereinafter referred to as “other sequences”). May be included).
  • the base sequence represented by SEQ ID NO: 1 is a DNA representation of the base sequence of the transcription product from the transcription start point of HIV-1 RNA to 200 bases.
  • the sequence identity of the base sequence portion having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1 in the forward primer oligonucleotide is at least 80% If it is, there is no restriction
  • a nucleotide sequence portion having at least 80% sequence identity with 10 or more consecutive nucleotide sequences in the nucleotide sequence represented by SEQ ID NO: 1 of the oligonucleotide for forward primer is represented by SEQ ID NO: 1.
  • One or a plurality of bases may be substituted, deleted, inserted or added to the base sequence.
  • the length of the base sequence portion having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1 of the forward primer oligonucleotide is 10 bases or more. If there is, there is no restriction
  • the length of the other sequence is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 to 20 bases.
  • the other sequence is preferably added to the 5 ′ end of a base sequence having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1. .
  • a specific example of the other sequence is preferably a sequence added to the 5 ′ end of the oligonucleotide for forward primer (hereinafter sometimes referred to as “Flap sequence”).
  • the flap sequence is not particularly limited and may be appropriately selected depending on the intended purpose.
  • an AT-rich flap sequence is preferable. There is no restriction
  • the AT-rich sequence is not particularly limited and may be appropriately selected depending on the intended purpose. However, the flap sequence represented by SEQ ID NO: 10 is preferable. 5′-AATAAAATCATAA-3 ′ (SEQ ID NO: 10)
  • the length of the forward primer is not particularly limited as long as it is 10 bases or more, and can be appropriately selected according to the purpose, but is preferably 10 bases to 40 bases, more preferably 20 bases to 40 bases.
  • the oligonucleotide for forward primer include an oligonucleotide represented by SEQ ID NO: 2, an oligonucleotide represented by SEQ ID NO: 6, an oligonucleotide represented by SEQ ID NO: 8, and SEQ ID NO: 9
  • SEQ ID NO: 2 an oligonucleotide represented by SEQ ID NO: 2
  • SEQ ID NO: 6 an oligonucleotide represented by SEQ ID NO: 8
  • SEQ ID NO: 9 The oligonucleotide represented by these, etc. are mentioned.
  • the oligonucleotide represented by SEQ ID NO: 2 and the oligonucleotide represented by SEQ ID NO: 6 are preferable, and the oligonucleotide represented by SEQ ID NO: 2 is more preferable.
  • 5′-GGTTAGACCAGATCTGAGCCT-3 ′ (SEQ ID NO: 2) 5'- AATAAACATATAA GGTTAGAGACCAGATCTGAGCTCTG-3 '(SEQ ID NO: 6) The underlined portion indicates a flap sequence.
  • 5′-GGGTCTCTCTGGTTAGACCAG-3 ′ (SEQ ID NO: 8) 5′-CTGGTTAGACCAGATCTGAGCC-3 ′ (SEQ ID NO: 9)
  • the reverse primer may include a sequence other than a sequence complementary to the adapter sequence (hereinafter, also referred to as “other sequence”).
  • the length of the other sequence is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 5 to 20 bases.
  • the other sequence is preferably added to the 5 ′ end of a sequence complementary to the adapter sequence.
  • a specific example of the other sequence is preferably a sequence added to the 5 ′ end of a sequence complementary to the adapter sequence (hereinafter sometimes referred to as “Flap sequence”).
  • the flap sequence is not particularly limited and may be appropriately selected depending on the intended purpose.
  • an AT-rich flap sequence is preferable. There is no restriction
  • the AT-rich sequence is not particularly limited and may be appropriately selected depending on the intended purpose. However, the flap sequence represented by SEQ ID NO: 10 is preferable. 5′-AATAAAATCATAA-3 ′ (SEQ ID NO: 10)
  • the length of the reverse primer is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 10 to 40 bases, more preferably 15 to 30 bases.
  • the reverse primer include an oligonucleotide represented by SEQ ID NO: 3, an oligonucleotide represented by SEQ ID NO: 7, and the like.
  • the oligonucleotide represented by SEQ ID NO: 7 is preferable. 5′-CAGTTACCGCATGCCG-3 ′ (SEQ ID NO: 3) 5′- AATAAACATAA CAGTTACCGCATGCCG-3 ′ (SEQ ID NO: 7)
  • the underlined portion indicates a flap sequence.
  • the combination of the forward primer and the reverse primer is not particularly limited and may be appropriately selected depending on the purpose. However, an embodiment in which only the reverse primer includes a flap sequence is preferable, and is represented by SEQ ID NO: 2. A combination of the oligonucleotide and the oligonucleotide represented by SEQ ID NO: 7 is more preferred.
  • the method for amplifying the target base sequence is not particularly limited, and a known method can be selected as appropriate.
  • a PCR Polymerase Chain Reaction
  • LAMP Long-Mediated Isolation Amplification
  • NASBA Nucleic) Acid Sequence Based Amplification
  • ICSR Isomeric and Chimerical-initiated Amplified of Nucleic Acids
  • TRC Transcribion Reverse-Trend-Trend-Residence-Trend
  • TMA Translation Mediated Amplification
  • SMAP Smart Amplification Process
  • RPA Recombines polymerase amplification
  • HAD Helicase-dependent
  • the DNA polymerase used for amplification of the target base sequence is not particularly limited, and a known DNA polymerase can be appropriately selected, and examples thereof include Taq DNA polymerase, Tth DNA polymerase, Vent DNA polymerase, and the like. Among these, Taq DNA polymerase is preferable when the real-time PCR method is performed.
  • the target base sequence is amplified as follows.
  • the forward primer anneals to the 3 ′ end side of the cDNA, an extension reaction is performed, and a complementary strand of the cDNA (hereinafter sometimes referred to as “extension product A”) is synthesized.
  • extension product A a complementary strand of the extension product
  • extension product B a complementary strand of the extension product A
  • extension product C a complementary strand of the extension product B (hereinafter sometimes referred to as “extension product C”) is synthesized.
  • the target base sequence is amplified by repeating the process of synthesizing the extension product B and the process of synthesizing the extension product C.
  • the PCR conditions are not particularly limited and may be appropriately selected depending on the purpose. For example, the reaction is performed at 95 ° C. for 30 seconds for 1 cycle, and then 95 ° C. for 5 seconds and 60 ° C. for 10 seconds. The reaction is performed for 50 cycles.
  • the detection process is a process for detecting an amplification product of the target base sequence.
  • the endpoint assay is a method for evaluating whether or not the target base sequence has been amplified after the reaction.
  • a specific example of the endpoint assay is a method of evaluating by electrophoresis.
  • the electrophoresis the amplification product of the target base sequence and the nucleic acid molecular weight marker are electrophoresed, and the degree of migration of both is compared to evaluate whether the target base sequence having a predetermined molecular weight has been amplified.
  • the method There is no restriction
  • the real-time assay is a method for measuring amplification of a target base sequence over time (real time).
  • a specific example of the real-time assay includes a method for evaluation using a real-time PCR apparatus.
  • the real-time PCR uses a known amount of serially diluted DNA as a standard, the amplification of the standard DNA by PCR and the target base sequence is measured over time, and the number of molecules in which the amplification of the standard DNA occurs exponentially Within the range, the number of molecules of the target base sequence is quantified.
  • the method using a fluorescent dye etc. are mentioned.
  • the method using the fluorescent dye is not particularly limited and may be appropriately selected depending on the intended purpose.
  • Examples include a method using a probe in which a fluorescent dye is bound to an oligonucleotide specific for DNA.
  • a method using a probe in which a fluorescent dye is bound to an oligonucleotide specific for the DNA to be amplified is preferable in that the target base sequence can be detected more specifically.
  • the dye that specifically intercalates with the double-stranded DNA and emits fluorescence is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include cyber green.
  • the probe is not particularly limited and may be appropriately selected depending on the intended purpose. However, one end of an oligonucleotide that can hybridize to at least part of the target sequence is modified with a fluorescent substance, Those whose ends are modified with a quenching substance are preferred. In the preferable probe, since the fluorescent substance and the quenching substance are close to each other, the fluorescence signal and the function of emitting the fluorescent signal are hindered by the quenching substance. In the amplification process, the probe hybridizes to the target base sequence under conditions where the forward primer anneals to the target base sequence.
  • the probe is degraded by the 5 ′ ⁇ 3 ′ exonuclease activity of Taq DNA polymerase.
  • the fluorescent substance and the quenching substance modified in the probe are spatially separated from each other, and the fluorescent substance can emit a fluorescent signal.
  • the fluorescence signal is proportional to the number of molecules of the amplified target base sequence.
  • the fluorescent substance is not particularly limited, and a known fluorescent substance can be appropriately selected.
  • FAM carboxyfluorescein
  • JOE 6-carboxy-4 ′, 5′-dichloro 2 ′, 7′- Dimethoxyfluorescein
  • FITC fluorescein isothiocyanate
  • TET tetrachlorofluorescein
  • HEX 5′-hexachloro-fluorescein-CE phosphoramidite
  • Cy3, Cy5, Alexa568 and the like is preferable.
  • the quenching substance is not particularly limited, and a known quenching substance can be appropriately selected.
  • a known quenching substance can be appropriately selected.
  • BHQ, TAMRA (tetramethyl-rhodamine), DABCYL (4- (4-dimethylaminophenylazo) benzoic acid) Etc a known quenching substance can be appropriately selected.
  • BHQ and TAMRA are preferable, and BHQ is more preferable.
  • the oligonucleotide used as the probe is not particularly limited as long as the HIV short RNA can be detected, and can be appropriately selected according to the purpose.
  • the oligonucleotide represented by SEQ ID NO: 4 is preferable. By using the oligonucleotide represented by SEQ ID NO: 4, the amplification of the target base sequence can be detected more specifically. 5′-CTAGCTAGCCAGAGAGCTCCCAGG-3 ′ (SEQ ID NO: 4)
  • a method using an oligonucleotide labeled with a labeling substance a method using high performance liquid chromatography, or a mass spectrum is used. Examples thereof include a method, a method using melting curve analysis, and a method using growth curve analysis.
  • Examples of the method using the oligonucleotide labeled with the labeling substance include a method of labeling the forward primer or the reverse primer with a labeling substance. According to the method, amplification of a target base sequence can be detected using the labeling substance as an index.
  • marker substance A well-known label
  • the evaluation step in the method for determining an immune state is a step of evaluating whether or not immunity in a patient is activated using the presence or absence of detection of the transcript as an index.
  • the patient in the method for determining an immune state is not particularly limited as long as it is a patient after starting treatment with an anti-human immunodeficiency virus drug, and can be appropriately selected according to the purpose. It is preferred that the immunodeficiency virus RNA and human immunodeficiency virus antigen be a patient below the detection limit.
  • the method for determining immune status of the present invention can also be used to provide information on patients who require prognostic observation among patients in whom the effects of treatment with anti-human immunodeficiency virus drugs are recognized. is there.
  • RNA of 200 bases or less HIV short-chain RNA
  • the peripheral blood mononuclear cell fraction preparation step is a step of separating a peripheral blood mononuclear cell (hereinafter, also referred to as “lymphocyte”) fraction from blood.
  • lymphocyte peripheral blood mononuclear cell
  • the HIV short RNA preparation step is a step of separating and purifying RNA of 200 bases or less from the peripheral blood mononuclear cell fraction.
  • a method for separating and purifying RNA of 200 bases or less from the peripheral blood mononuclear cell fraction is not particularly limited, and a known method can be appropriately selected.
  • an RNA extraction reagent Isogen II (manufactured by Nippon Gene)
  • a method using mirVana miRNA isolation Kit manufactured by Ambion
  • the method for predicting an increase in the number of CD4 + T cells in a patient after starting treatment with the anti-human immunodeficiency virus drug of the present invention includes at least a detection step and an evaluation step, and further includes other steps as necessary. .
  • the detection step in the method for predicting the increase in the number of CD4 + T cells is a step of detecting a transcript from the transcription start point of human immunodeficiency virus RNA to 200 bases in peripheral blood mononuclear cells.
  • the detection step in the method for predicting the increase in the number of CD4 + T cells can be performed in the same manner as the detection step in the method for determining an immune state.
  • Evaluation step in increasing the prediction method of the CD4 + T cell count, as an index the presence or absence of detection of the transcripts, is a step of CD4 + T cell count to assess whether increased.
  • the patient in the method for predicting the increase in the number of CD4 + T cells is not particularly limited as long as it is a patient after starting treatment with an anti-human immunodeficiency virus drug, and can be appropriately selected according to the purpose. It is preferable that the human immunodeficiency virus RNA and the human immunodeficiency virus antigen in plasma are patients whose detection limit is not exceeded.
  • the method for predicting the increase in the number of CD4 + T cells of the present invention provides information on patients who may not recover their immunity among patients who have been treated with anti-human immunodeficiency virus drugs. Can also be used.
  • the method for predicting the decrease in the number of CD4 + T cells in a patient who has not started treatment with the anti-human immunodeficiency virus drug of the present invention includes at least a detection step and an evaluation step, and further includes other steps as necessary. .
  • the detection step in the method for predicting the decrease in the number of CD4 + T cells is a step of detecting a transcript from the transcription start point of human immunodeficiency virus RNA to 200 bases in peripheral blood mononuclear cells.
  • the detection step in the method for predicting the decrease in the number of CD4 + T cells can be performed in the same manner as the detection step in the method for determining an immune state.
  • Evaluation step in reducing the prediction method of the CD4 + T cell count, as an index the presence or absence of detection of the transcripts, is a step of CD4 + T cell count to assess whether reduced.
  • the patient in the method for predicting the decrease in the number of CD4 + T cells is not particularly limited as long as it has not started treatment with an anti-human immunodeficiency virus drug, and can be appropriately selected according to the purpose.
  • test Examples 1 and 2 described later it was also shown that the presence or absence of detection of HIV short-chain RNA and the number of HIV particles or the number of CD4 + T cells are correlated.
  • Test Example 3 to be described later it was also shown that HIV short-chain RNA in peripheral blood mononuclear cells becomes easier to detect as the pathological condition of HIV-infected persons progresses.
  • the number of CD4 + T cells is an index of immunity, and when infected with HIV, the number of CD4 + T cells decreases, and acquired immune deficiency syndrome develops.
  • the method for predicting the decrease in the number of CD4 + T cells of the present invention provides information for determining the timing of starting treatment with an anti-human immunodeficiency virus drug, or grasps the progression of the pathological condition of an HIV-infected person. It is also possible to use it for providing the information.
  • the kit of the present invention comprises at least an oligonucleotide comprising a base sequence having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1, and further if necessary Includes other configurations.
  • the kit of the present invention increases the number of CD4 + T cells in a patient after initiating treatment with an anti-human immunodeficiency virus drug to determine the immune status in the patient after initiating treatment with the anti-human immunodeficiency virus drug. Or to predict a decrease in the number of CD4 + T cells in patients who have not started treatment with anti-human immunodeficiency virus drugs.
  • oligonucleotide containing a base sequence having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1 is the forward according to the method for judging an immune state of the present invention. Oligonucleotide for primer.
  • the other configuration is not particularly limited as long as the effect of the present invention is not impaired, and can be appropriately selected according to the purpose.
  • the reverse primer, probe, and the like described in the method for determining an immune state of the present invention examples include polyA addition treatment reagents, cDNA synthesis treatment reagents, PCR reagents, and the like.
  • Test Example 1 Presence or absence of detection of HIV short RNA in peripheral blood mononuclear cells in patients who have not started treatment with anti-human immunodeficiency virus drugs (hereinafter sometimes referred to as “untreated patients”) (Relationship with plasma HIV RNA levels) ⁇ Detection of HIV short RNA> Approximately 3.5 mL of blood collected from untreated patients (144 hospitals attached to the Institute of Medical Science, The University of Tokyo) was used. A peripheral blood mononuclear cell fraction was separated from the blood by a centrifugal specific gravity method.
  • RNA extraction reagent (Isogen II (manufactured by Nippon Gene) was used to separate a small RNA of 200 bases or less and further mixed into the small RNA. Chromosomal DNA that may come is digested with TURBO DNase (Ambion) and purified.
  • CDNA complementary to RNA obtained by adding polyA to the small RNA was synthesized using miScript II RT Kit (manufactured by QIAGEN).
  • RT-PCR quantitative RT-PCR was performed under the following PCR conditions.
  • a quantitative RT-PCR reagent Premix Ex Taq Kit (manufactured by Takara Bio Inc.) was used, and as a quantitative RT-PCR apparatus, CFX96 (manufactured by Bio Rad) was used.
  • [Primers and probes] -Forward primer 5'-GGTTAGACCAGATCTGAGCCTG-3 '(SEQ ID NO: 2) The forward primer corresponds to the transcript of HIV RNA in the cDNA.
  • ⁇ Probe (Taqman Probe) 5′-CTAGCTAGCCAGAGAGCTCCCAGG-3 ′ (SEQ ID NO: 4) The probe is modified with FAM at the 5 ′ end of the oligonucleotide and BHQ at the 3 ′ end.
  • FAM FAM
  • HIV RNA in plasma A part of blood collected from the patient who detected the HIV short-chain RNA was subjected to a blood biochemical test at a contract laboratory from the University of Tokyo Medical Science Hospital, and HIV RNA in the plasma fraction was requested. The amount was measured.
  • FIG. 1 shows the results of analyzing the relationship between the presence or absence of detection of HIV short-chain RNA and HIV RNA in plasma.
  • the vertical axis indicates the amount of HIV RNA in plasma
  • the horizontal axis indicates the patient in which HIV short RNA was detected (ST +) and the patient in which HIV short RNA was not detected (ST-).
  • the dotted line in FIG. 1 indicates 50 copies / mL
  • the error bar indicates the 95% confidence interval of the average value.
  • FIG. 1 shows that in the patient in which HIV short RNA was detected, compared with the patient in which HIV short RNA was not detected (***: 0.0007 (Mann Whitney test (Mann Whitney test )) Tended to have a high amount of HIV RNA in plasma.
  • Test Example 2 Relationship between presence or absence of detection of HIV short RNA in peripheral blood mononuclear cells and CD4 + T cell count in untreated patients
  • Detection of HIV short RNA> The detection result of HIV short RNA in Test Example 1 was used for the analysis of this test example.
  • ⁇ CD4 + T cell count> In Test Example 1, a portion of blood collected from a patient who detected HIV short-chain RNA was subjected to a blood biochemical test at a contract laboratory from the University of Tokyo Medical Science Hospital, and the plasma fraction The number of CD4 + T cells in the minute was measured.
  • FIG. 2 shows the results of analyzing the relationship between the presence or absence of detection of HIV short RNA and the number of CD4 + T cells.
  • the vertical axis represents the number of CD4 + T cells
  • the horizontal axis represents the patient in which HIV short RNA was detected (ST +) and the patient in which HIV short RNA was not detected (ST ⁇ ).
  • the error bar in FIG. 2 shows the 95% confidence interval of the average value.
  • patients with HIV short RNA detected were significantly (**: 0.0093 (Mann-Whitney test)) immune compared to patients with no HIV short RNA detected.
  • a tendency for the number of CD4 + T cells, which is an index of force, to be small was observed.
  • HIV short RNA is a biomarker reflecting the pathology of HIV-infected persons.
  • Test Example 3 Relationship between presence / absence of detection of HIV short-chain RNA in peripheral blood mononuclear cells and time to start of treatment in untreated patients
  • Detection of HIV short RNA> A test example except that the blood of a patient (104 people) who did not detect HIV short RNA in peripheral blood mononuclear cells and subsequently started treatment with an anti-human immunodeficiency virus drug was used In the same manner as in Example 1, HIV short-chain RNA in peripheral blood mononuclear cells was detected.
  • FIG. 3 shows the results of analyzing the relationship between the presence or absence of detection of HIV short-chain RNA in peripheral blood mononuclear cells and the time until the start of treatment in untreated patients.
  • the vertical axis represents the time until the start of treatment
  • the horizontal axis represents the ratio of patients (ST +) in which HIV short RNA was detected and patients (ST ⁇ ) in which HIV short-chain RNA was not detected.
  • ST + the time until the start of treatment
  • ST ⁇ the horizontal axis represents the ratio of patients in which HIV short RNA was detected
  • ST ⁇ the ratio of patients
  • HIV short RNA was detected in peripheral blood mononuclear cells in about half of patients less than 2 weeks to 1 year before the start of treatment with an anti-human immunodeficiency virus drug. . Therefore, it was shown that HIV short-chain RNA in peripheral blood mononuclear cells becomes easier to detect as the pathological condition of HIV-infected persons progresses.
  • Test Example 4 Relationship between presence or absence of detection of HIV short-chain RNA in peripheral blood mononuclear cells in untreated patients and reduction in the number of CD4 + T cells
  • Detection of HIV short RNA> HIV short-chain RNA in peripheral blood mononuclear cells was analyzed in the same manner as in Test Example 1, except that blood one year after the patient whose CD4 + T cell count was greater than 250 cells / ⁇ L was used. Detection was performed.
  • FIG. 4 shows the results of analyzing the relationship between the presence or absence of HIV short RNA detection in peripheral blood mononuclear cells and the decrease in the number of CD4 + T cells in untreated patients.
  • the vertical axis represents the number of CD4 + T cells
  • the horizontal axis represents the blood of patients (six persons) in which HIV short RNA was detected in peripheral blood mononuclear cells at the time of Test Example 2 (analysis).
  • Treatment initiation patient Presence or absence of detection of HIV short RNA in peripheral blood mononuclear cells in patients after initiation of treatment with anti-human immunodeficiency virus drug (hereinafter sometimes referred to as “treatment initiation patient”), CD4 + Relationship with increase or decrease in T cell count or CD4 + T cell count
  • Detection of HIV short RNA in peripheral blood mononuclear cells was carried out in the same manner as in Test Example 1, except that blood one year after the patient who performed the detection of HIV short RNA-1 was used.
  • ⁇ CD4 + T cell count> The number of CD4 + T cells was measured in the same manner as in Test Example 2 except that the blood subjected to detection of HIV short RNA-1 or HIV short RNA detection-2 was used.
  • FIG. 5A shows the results of analyzing the relationship between the presence or absence of detection of HIV short-chain RNA in peripheral blood mononuclear cells and the number of CD4 + T cells in the patient who started treatment.
  • the vertical axis represents the HIV short CD4 + T cell count in the blood of the detection -1 RNA (CD4 + T cell counts during the ST analysis), the horizontal axis is detected HIV short RNA Patients (ST +) and those not detected (ST-) are shown.
  • ST + HIV short RNA Patients
  • ST- those not detected
  • FIG. 5B shows the result of analyzing the relationship between the presence or absence of HIV short-chain RNA detection in peripheral blood mononuclear cells and the increase / decrease in the number of CD4 + T cells in the treatment start patient.
  • the vertical axis represents the increase / decrease in the number of CD4 + T cells in the HIV short RNA detection-2 from the number of CD4 + T cells in the HIV short RNA detection-1 blood (1 year later). Increase and decrease in CD4 + T cell counts), and the horizontal axis indicates the detection of HIV short RNA in the blood of HIV short RNA-1 (ST +) and HIV short RNA detected Patients who did not (ST-) are shown.
  • ST + HIV short RNA-1
  • ST- HIV short RNA detected Patients who did not
  • the patient (ST ⁇ ) in which HIV short RNA was not detected in the blood of HIV short RNA detection-1 was compared with the patient in which HIV short RNA was detected (ST +).
  • the number of CD4 + T cells was significantly increased (recovered) ((* 0.0130 (Mann-Whitney test)). Therefore, it was shown that the recovery of CD4 + T cell count is slow in patients who have started treatment and in which HIV short RNA in peripheral blood mononuclear cells continues to be detected.
  • Such patients may be immunocompromised due to low CD4 + T cell counts due to immunological failure (immunological non-responder), opportunistic infections, and appearance of malignant tumors. There is a risk of causing cognitive decline.
  • Test Example 6 Relationship between the amount of HIV short-chain RNA in peripheral blood mononuclear cells and the expression level of CD8 + T cell activation markers in patients who started treatment
  • ⁇ Expression of CD8 + T cell activation marker> Lymphocytes prepared from blood collected from the treatment-initiated patient who detected the HIV short RNA were immunostained, and the expression level of HLA-DR of CD8 positive T cells satisfying CD3 + and CD8 + (total CD8 + The ratio of HLA-DR + CD38 + cells in T cells, expression intensity) was measured by FACS Aria (BD).
  • FIG. 6A shows the analysis result of the relationship between the amount of HIV short RNA and the proportion of HLA-DR + CD38 + cells in total CD8 + T cells.
  • the vertical axis shows the percentage (%) of HLA-DR + CD38 + cells in total CD8 + T cells
  • FIG. 6B shows the result of analyzing the relationship between the amount of HIV short RNA and the amount of HLA-DR MFI in total CD8 + T cells.
  • the vertical axis represents the amount of HLA-DR MFI (fluorescence intensity) in total CD8 + T cells
  • FIGS. 6A and 6B showed that the amount of HIV short RNA was positively correlated with the expression of CD8 + T cell activation markers. Therefore, it was found that in patients who started treatment whose HIV RNA level in plasma was below the detection limit, immunity tended to be strongly activated as the amount of HIV short RNA increased. Therefore, it is considered that detection of HIV short-chain RNA makes it possible to find a patient whose immunity is chronically activated.
  • Test Example 7 Examination of detection primer
  • CDNA was synthesized in the same manner as in Test Example 1 using the following RNA synthesized in vitro as standard RNA.
  • a 10-fold dilution series of the cDNA from 10 7 (1.E + 07) to 10 2 (1.E + 02) copies was prepared, and quantitative PCR was performed as follows to prepare a calibration curve.
  • the results are shown in FIG. -RNA synthesized in vitro (SEQ ID NO: 5)- 5'-GGUCUCUCUUGGUUACGACCAGAUUCGAGCCUGGGAGCUCUCUGGGCUAGCUAGGAGAACCC-3 '
  • Probe (Taqman Probe) 5′-CTAGCTAGCCAGAGAGCTCCCAGG-3 ′ (SEQ ID NO: 4) The probe is modified with FAM at the 5 ′ end of the oligonucleotide and BHQ at the 3 ′ end.
  • FIG. 8 shows the results of deep sequence analysis using GS FLX + / JUNIOR (manufactured by Rosch) using the PCR product after cDNA was synthesized by the method and PCR was performed.
  • HIV short-chain RNA is about 30 bases, about 50 bases to about 70 bases, about 90 bases to about 110 bases from the transcription start point of HIV-1 RNA. It was found that the transcript was stopped.
  • Examples of the aspect of the present invention include the following.
  • ⁇ 1> a detection step of detecting a transcript from the transcription start point of human immunodeficiency virus RNA to 200 bases in peripheral blood mononuclear cells; After the initiation of treatment with an anti-human immunodeficiency virus drug, comprising the step of evaluating whether or not immunity in a patient is in an activated state using the presence or absence of detection of the transcript as an index
  • This is a method for determining an immune state in a patient.
  • ⁇ 2> The method for determining an immune state according to ⁇ 1>, wherein the patient is a patient whose human immunodeficiency virus RNA and human immunodeficiency virus antigen in plasma are below the detection limit.
  • ⁇ 3> From the above ⁇ 1> to ⁇ 1>, wherein the detection step uses an oligonucleotide containing a base sequence having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1.
  • 2> The method for judging an immune state according to any one of 2).
  • ⁇ 4> a detection step of detecting a transcript from the transcription start point of human immunodeficiency virus RNA to 200 bases in peripheral blood mononuclear cells; CD4 in a patient after initiating treatment with an anti-human immunodeficiency virus drug, comprising evaluating whether or not the number of CD4 + T cells increases using the presence or absence of detection of the transcript as an index + A method for predicting an increase in the number of T cells.
  • ⁇ 5> The method for predicting an increase in the number of CD4 + T cells according to the above ⁇ 4>, wherein the patient is a patient whose human immunodeficiency virus RNA and human immunodeficiency virus antigen in plasma are below the detection limit.
  • ⁇ 6> From the above ⁇ 4> to ⁇ 4>, wherein the detection step uses an oligonucleotide containing a base sequence having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1.
  • 5> The method for predicting an increase in the number of CD4 + T cells according to any one of 5>.
  • An anti-human immunodeficiency virus comprising an oligonucleotide containing a base sequence having at least 80% sequence identity with 10 or more consecutive base sequences in the base sequence represented by SEQ ID NO: 1.
  • a kit for determining immune status in a patient after initiating treatment with a drug a kit for predicting an increase in the number of CD4 + T cells in a patient after initiating treatment with an anti-human immunodeficiency virus agent, or anti A kit for predicting a decrease in the number of CD4 + T cells in a patient who has not started treatment with a human immunodeficiency virus drug.

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Abstract

La présente invention concerne un procédé de détermination du statut immunitaire d'un patient après le démarrage d'un traitement utilisant un médicament contre le virus de l'immunodéficience humaine, ledit procédé incluant une étape de détection pour détecter un produit de transcription jusqu'à 200 bases à partir d'un point de démarrage de la transcription de l'ARN du virus de l'immunodéficience humaine dans des cellules mononucléaires de sang périphérique, et une étape d'évaluation afin de déterminer si l'immunité du patient se trouve sous un état activé en utilisant la détection ou la non-détection du produit de transcription comme indice.
PCT/JP2015/078488 2015-10-07 2015-10-07 Procédé de détermination du statut immunitaire, procédé de prédiction de l'augmentation du nombre de cellules cd4+ t, procédé de prédiction de la baisse du nombre des cellules cd4+ t, et trousse à cet effet WO2017060988A1 (fr)

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CN114708989A (zh) * 2022-03-16 2022-07-05 江苏大学 一种基于艾滋病患者的药学服务效果的检测方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114708989A (zh) * 2022-03-16 2022-07-05 江苏大学 一种基于艾滋病患者的药学服务效果的检测方法
CN114708989B (zh) * 2022-03-16 2024-06-11 江苏大学 一种基于艾滋病患者的药学服务效果的检测方法

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