JP2016007145A - Determination method for immune status, increase prediction method for cd4+t cell count, and reduction prediction method for cd4+t cell count, and kit for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a determination method for an immune status which is capable of determining simply and highly accurately an immune status in a patient after the start of treatment with an anti-human immunodeficiency virus drug, an increase prediction method for CD4T cell count which is capable of simply and highly accurately predicting an increase of CD4T cell count in a patient after the start of treatment with an anti-human immunodeficiency virus drug, a reduction prediction method for CD4T cell count which is capable of simply and highly accurately predicting a reduction of CD4T cell count in a patient to whom treatment with an anti-human immunodeficiency virus drug has not been started, and a kit thereof.SOLUTION: A determination method for an immune status in a patient after the start of treatment with an anti-human immunodeficiency virus drug includes: a detection process of detecting a transcription product from a transcription start point of a human immunodeficiency virus RNA to a 200 base in a peripheral blood mononuclear cell; a process of evaluating whether or not immunity in a patient is in an activated state by using whether or not the transcription product has been detected as an indicator.

Description

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.

  Against human immunodeficiency virus (hereinafter sometimes referred to as “HIV”) infection that is the cause of acquired immunodeficiency syndrome (hereinafter sometimes referred to as “AIDS”) 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.

  Currently, diagnosis of HIV infection is performed by measuring HIV RNA and HIV antigen in the plasma of infected persons.

As treatment for HIV, treatment with an anti-human immunodeficiency virus drug is performed. As the treatment, for example, a multi-drug combination therapy (High Active Anti-Retroviral Therapy; hereinafter sometimes referred to as “HAART therapy”) is common. According to the above treatment, the amount of HIV RNA or HIV antigen in the plasma of an infected person can be suppressed to below the detection limit.
However, in the course of treatment with HAART therapy, 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. Since 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.

  Thus, as a method for detecting HIV even when the amount of HIV RNA or HIV antigen is below the detection limit, a method for detecting a transcript of human immunodeficiency virus produced by residual infected cells has been proposed (for example, patents). Reference 1). According to the proposal, HIV can be detected with high accuracy and high sensitivity.

  On the other hand, with the HAART therapy, 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. Examples of 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.

  Therefore, even when the amount of HIV RNA or HIV antigen in plasma is below the detection limit, the method of predicting HIV activity or determining the immune power of an HIV-infected person is quick. Is strongly demanded.

International Publication No. 2013/111800

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.
<3> 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 who has not started treatment with an anti-human immunodeficiency virus drug, comprising the step of evaluating whether or not CD4 ⁺ T cells are decreased using the presence or absence of detection of the transcript as an index This is a method for predicting a decrease in the number of T cells.
<4> 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.

According to the present invention, various conventional problems can be solved, and 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, capable of easily and accurately predicting an increase in the number of CD4 ⁺ T cells, an anti-human immunodeficiency virus 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. 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 of 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.

(Determination method of immune status)
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.

<Detection process>
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.

-HIV short RNA-
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 having about 30 bases, about 50 bases to about 70 bases, and about 90 bases to about 110 bases from the transcription start point.

-Method for detecting HIV short-chain RNA-
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.

-PolyA addition process-
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).
There is no restriction | limiting in particular as the length of said polyA, Although it can select suitably according to the objective, 10 bases-40 bases are preferable, and 20 bases-30 bases are more preferable.

--- cDNA synthesis process-
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).
There is no restriction | limiting in particular as the length of said polyT, Although it can select suitably according to the objective, 10 bases-40 bases are preferable, and 20 bases-30 bases are more preferable. The polyT is annealed to the polyA.
The polyT-containing oligonucleotide may have a degenerate sequence at the 3 ′ end.
By a reverse transcription reaction starting from the 3 ′ end of the polyT-containing oligonucleotide, cDNA complementary to the RNA added with polyA is synthesized.
There is no restriction | limiting in particular as 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.

Since the cDNA forms a hybrid with RNA used as a template, it is preferable to decompose RNA using RNase.
There is no restriction | limiting in particular as said RNase, According to the objective, it can select suitably, For example, RNaseH etc. are mentioned.

--Amplification process--
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 ---
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 | limiting in particular, Although it can select suitably according to the objective, 85% or more is preferable, 90% or more is more preferable, and 95% or more is especially preferable.
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 | limiting in particular, According to the objective, it can select suitably.

There is no restriction | limiting in particular as said other arrangement | sequence in the said oligonucleotide for forward primers, According to the objective, it can select suitably.
There is no restriction | limiting in particular as length of the said other sequence | arrangement, Although it can select suitably according to the objective, 5 bases-20 bases are preferable.
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. However, an AT-rich flap sequence is preferable. There is no restriction | limiting in particular as AT content in the said flap arrangement | sequence, Although it can select suitably according to the objective, 80% or more is preferable, 90% or more is more preferable, and 95% or more is especially preferable.
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′-AATAAATCATAA-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.

Preferable specific examples of 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 The oligonucleotide represented by these, etc. are mentioned. Among these, 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′- AATAAATCATAA GGTTAGACCAGATCTGAGCCTG-3 ′ (SEQ ID NO: 6)
The underlined portion indicates a flap sequence.
5′-GGGTCTCTCTGGTTAGACCAG-3 ′ (SEQ ID NO: 8)
5′-CTGGTTAGACCAGATCTGAGC-3 ′ (SEQ ID NO: 9)

--- Reverse primer ---
There is no restriction | limiting in particular as said reverse primer, Although it can select suitably according to the objective, The oligonucleotide containing a sequence complementary to an adapter sequence is preferable.
The reverse primer may include a sequence other than a sequence complementary to the adapter sequence (hereinafter, also referred to as “other sequence”).

There is no restriction | limiting in particular as said other arrangement | sequence in the said reverse primer, According to the objective, it can select suitably.
There is no restriction | limiting in particular as length of the said other sequence | arrangement, Although it can select suitably according to the objective, 5 bases-20 bases are preferable.
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. However, an AT-rich flap sequence is preferable. There is no restriction | limiting in particular as AT content in the said flap arrangement | sequence, Although it can select suitably according to the objective, 80% or more is preferable, 90% or more is more preferable, and 95% or more is especially preferable.
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′-AATAAATCATAA-3 ′ (SEQ ID NO: 10)

  There is no restriction | limiting in particular as the length of the said reverse primer, Although it can select suitably according to the objective, 10 bases-40 bases are preferable, and 15 bases-30 bases are more preferable.

Preferable specific examples of the reverse primer include an oligonucleotide represented by SEQ ID NO: 3, an oligonucleotide represented by SEQ ID NO: 7, and the like. Among these, the oligonucleotide represented by SEQ ID NO: 7 is preferable.
5′-CAGTTACCGCATGCCG-3 ′ (SEQ ID NO: 3)
5′- AATAAATCATAA 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.

---- Amplification ---
The method for amplifying the target base sequence is not particularly limited, and a known method can be appropriately selected. For example, a PCR (Polymerase Chain Reaction) method, a LAMP (Loop-Mediated Isolation Amplification) method, a NASBA (Nucleic) Acid Sequence Based Amplification (ICAN) method, ICI (Isomeric and Chimera-primed-imprinted Amplification of Nucleic Acids) on) method, TMA (Translation Mediated Amplification) method, SMAP (Smart Amplification Process) method, RPA (Recombines polymerase amplification) method, HAD (Helicase-dependent) method and the like.

  There is no restriction | limiting in particular as a DNA polymerase used for amplification of the said target base sequence, A well-known DNA polymerase can be selected suitably, For example, Taq DNA polymerase, Tth DNA polymerase, Vent DNA polymerase etc. are mentioned. Among these, Taq DNA polymerase is preferable when the real-time PCR method is performed.

When the PCR method is used as the amplification method, 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. Next, the reverse primer anneals to the 3 ′ end side of the extension product A, an extension reaction is performed, and a complementary strand of the extension product A (hereinafter sometimes referred to as “extension product B”) is synthesized. . Next, the forward primer anneals to the 3 ′ end side of the extension product B, an extension reaction is performed, and a complementary strand of the extension product B (hereinafter sometimes referred to as “extension product C”) is synthesized. . Thereafter, 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.

--Detection process--
The detection process is a process for detecting an amplification product of the target base sequence.
There is no restriction | limiting in particular as said detection method, According to the objective, it can select suitably, For example, an endpoint assay, a real-time assay, etc. are mentioned. Among these, a real-time assay is preferable.

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.
In 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. Is the method.
There is no restriction | limiting in particular as a reagent used for the said electrophoresis, A well-known reagent can be selected suitably, For example, an ethidium bromide, cyber green, etc. are mentioned.

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.

There is no restriction | limiting in particular as a quantification method in the said real-time PCR, According to the objective, it can select suitably, For example, 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. For example, a method using a dye that specifically intercalates with double-stranded DNA and emits fluorescence, or amplifies. Examples include a method using a probe in which a fluorescent dye is bound to an oligonucleotide specific for DNA. Among these, 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.

  There is no restriction | limiting in particular as a pigment | dye which specifically intercalates with the said double stranded DNA, and it can select suitably according to the objective, For example, cyber green etc. are mentioned.

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. Subsequently, in the course of the extension reaction, for example, the probe is degraded by the 5 ′ → 3 ′ exonuclease activity of Taq DNA polymerase. As a result, 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.

  There is no restriction | limiting in particular as said fluorescent substance, A well-known fluorescent substance can be selected suitably, For example, FAM (carboxy fluorescein), 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. Among these, FAM is preferable.

  The quenching substance is not particularly limited, and a known quenching substance can be appropriately selected. For example, BHQ, TAMRA (tetramethyl-rhodamine), DABCYL (4- (4-dimethylaminophenylazo) benzoic acid) Etc. Among these, 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)

  Other methods for the detection are not particularly limited and may be appropriately selected depending on the purpose. For example, 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.
There is no restriction | limiting in particular as said label | marker substance, A well-known label | marker substance can be selected suitably, For example, fluorescent dye, an energy absorptive substance, a radioisotope, a chemiluminescent body, an enzyme, an antibody etc. are mentioned. There is no restriction | limiting in particular as a labeling site | part in the said oligonucleotide, According to the objective, it can select suitably.

<Evaluation process>
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.

-Patient-
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.

As described in Test Example 6 described later, it was shown that the amount of HIV short-chain RNA was positively correlated with the expression of activation markers for CD8 ⁺ T cells.
Therefore, when the HIV short RNA is detected in the detection step, it can be evaluated that the patient is in an activated state of immunity.

Further, when the HIV short RNA is detected in the detection step, the patient is in a chronic immune activated state, and therefore the CD4 ⁺ T cell count does not recover due to aging or exhaustion of immunity. It can be evaluated that the patient is at high risk, and is at risk of causing opportunistic infections, the appearance of malignant tumors, and cognitive decline.
Therefore, 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.

<Other processes>
The other steps in the method for determining the immune state are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose, for example, a peripheral blood mononuclear cell fraction preparation step, HIV short-chain RNA (hereinafter, sometimes referred to as “small RNA of 200 bases or less”) preparation process and the like.

-Peripheral blood mononuclear cell preparation process-
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.
There is no restriction | limiting in particular as a method of isolate | separating a peripheral blood mononuclear cell fraction from blood, A well-known method can be selected suitably, For example, the method of isolate | separating by the centrifugal specific gravity method etc. are mentioned.

-HIV short RNA preparation process-
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. For example, an RNA extraction reagent (Isogen II (manufactured by Nippon Gene) ), A method using mirVana miRNA isolation Kit (manufactured by Ambion), and the like.

(CD4 ⁺ T cell number increase prediction method)
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. .

<Detection process>
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 process>
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.

-Patient-
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.

As described in Test Example 5 to be described later, the number of CD4 ⁺ T cells significantly decreased in patients who did not detect HIV short RNA compared to patients in which HIV short RNA was detected one year later. Was shown to increase.
Therefore, when the HIV short RNA is not detected in the detection step, it can be evaluated that the CD4 ⁺ T cell number increases (recovers) in the patient. On the other hand, when the HIV short RNA is detected in the detection step, it can be evaluated that the number of CD4 ⁺ T cells does not increase (recover) in the patient.

In addition, when the HIV short RNA is detected in the detection step, the number of CD4 ⁺ T cells does not increase (recover) in the patient, so the patient is highly likely not to recover immunity. It can be evaluated that the patient is at risk of causing an opportunistic infection, the appearance of a malignant tumor, a decline in cognitive function, and the like.
Therefore, 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.

<Other processes>
Other steps in the method for predicting the increase in the number of CD4 ⁺ T cells are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. For example, the method for determining the immune state The same process as the process described in the item of the other process in is mentioned.

(CD4 ⁺ T cell number decrease prediction method)
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. .

<Detection process>
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 process>
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.

-Patient-
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.

As described in Test Example 4 to be described later, the number of CD4 ⁺ T cells significantly increased in the patient in which HIV short RNA was detected, compared with the patient in which HIV short RNA was not detected after one year. Was shown to be decreasing.
Therefore, when the HIV short RNA is detected in the detection step, it can be evaluated that the number of CD4 ⁺ T cells decreases in the patient.
In addition, the time when the number of CD4 ⁺ T cells decreases can be evaluated to decrease after one year at the latest, for example.

Moreover, as described in 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. Moreover, as described in 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. Therefore, 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.

<Other processes>
Other steps in the method for predicting the decrease in the number of CD4 ⁺ T cells are not particularly limited as long as the effects of the present invention are not impaired, and can be appropriately selected according to the purpose. For example, the method for determining the immune state The same process as the process described in the item of the other process in is mentioned.

(kit)
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>
The 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.

<Other configurations>
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. For example, 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.

  The present invention will be specifically described below with reference to test examples, but the present invention is not limited to these test examples.

(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. From the peripheral blood mononuclear cell fraction (1 × 10 ⁶ cells), an 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).

Using the cDNA and the following primers and probes, quantitative RT-PCR was performed under the following PCR conditions. In addition, Premix Ex Taq Kit (manufactured by Takara Bio Inc.) was used as a quantitative RT-PCR reagent, and CFX96 (Bio Rad) was used as a quantitative RT-PCR apparatus.
[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.
Reverse primer 5′-CAGTTACCGCATGCCG-3 ′ (SEQ ID NO: 3)
The reverse primer recognizes the adapter sequence added during the cDNA synthesis.
・ 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.
[PCR conditions]
One cycle of reaction at 95 ° C. for 30 seconds was performed, and then 50 cycles of 2-step reaction at 95 ° C. for 5 seconds and 60 ° C. for 10 seconds were performed.

  As a result of the measurement, 67 untreated patients detected HIV short-chain RNA, and 77 did not detect HIV short-chain RNA.

<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.

<Analysis>
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. In FIG. 1, the vertical axis indicates the amount of HIV RNA in plasma, and the horizontal axis indicates a patient in which HIV short RNA was detected (ST +) and a patient in which HIV short-chain RNA was not detected (ST−). Further, the dotted line in FIG. 1 indicates 50 copies / mL, and the error bar indicates the 95% confidence interval of the average value.
As shown in FIG. 1, 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.

<Analysis>
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. In FIG. 2, the vertical axis represents the number of CD4 ⁺ T cells, and 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−). Moreover, the error bar in FIG. 2 shows the 95% confidence interval of the average value.
As shown in FIG. 2, 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.

From the results of Test Examples 1 and 2, it was shown that the presence or absence of HIV short-chain RNA was correlated with the number of HIV particles or the number of CD4 ⁺ T cells. Therefore, it was suggested that 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.

<Analysis>
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. In FIG. 3, the vertical axis represents the time until the start of treatment, and the horizontal axis represents the ratio of patients (ST +) in which HIV short-chain RNA was detected and patients (ST−) in which HIV short-chain RNA was not detected.
From the results of FIG. 3, it was found that HIV short RNA was detected in peripheral blood mononuclear cells in about half of the 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>
In Test Example 2, 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 / μL was used. Detection was performed.

<CD4 ⁺ T cell count>
In Test Example 2, the number of CD4 ⁺ T cells was measured in the same manner as in Test Example 2, except that blood one year after the patient whose CD4 ⁺ T cell count was greater than 250 cells / μL was used.

<Analysis>
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. In FIG. 4, the vertical axis represents the number of CD4 ⁺ T cells, and 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). Sometimes ST +), blood (1 year) of patients (6 people) in which the HIV short RNA was detected, HIV short RNA in peripheral blood mononuclear cells was detected at the time of Test Example 2 Blood of patients who did not (35 people) (ST- at the time of analysis) and blood (1 year after) of patients (35 people) whose HIV short-chain RNA was not detected are shown. Moreover, the error bar in FIG. 4 shows the 95% confidence interval of the average value.
As shown in FIG. 4, at the time of Test Example 2, a patient in which HIV short RNA in peripheral blood mononuclear cells was detected and a patient in which HIV short RNA in peripheral blood mononuclear cells were not detected were detected. There was no significant difference in CD4 ⁺ T cell numbers (NS p = 0.9354 (Mann-Whitney test)). However, after one year, the number of CD4 ⁺ T cells decreased significantly (* p = 0.0112 (Mann-Whitney test)) in patients in whom HIV short RNA was detected in peripheral blood mononuclear cells. .

(Test Example 5: 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-1>
Detection of HIV short-chain RNA in peripheral blood mononuclear cells was carried out in the same manner as in Test Example 1, except that the blood of the treatment starting patient (57 people) whose HIV RNA amount in the plasma was below the detection limit was used. went.

<Detection of HIV short-chain RNA-2>
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 blood subjected to detection of HIV short RNA-1 or HIV short RNA detection-2 was used.

<Analysis-1>
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. In Figure 5A, 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 Patient (ST +) and patient (ST−) not detected.
As shown in FIG. 5A, in the HIV short RNA detection-1 blood, patients with HIV short RNA detected (16 people) and patients with no HIV short RNA detected (41 people) There was no significant difference in the number of CD4 ⁺ T cells (ns 0.6507 (Mann-Whitney test)).

<Analysis-2>
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. In FIG. 5B, the vertical axis indicates the increase or 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 the number of CD4 ⁺ T cells), and the horizontal axis represents the detection of HIV short RNA in the blood of the above-mentioned HIV short RNA detection-1 (ST +) and HIV short RNA detected The patient who did not exist (ST-) is shown.
As shown in FIG. 5B, the patient (ST−) in which HIV short RNA was not detected in the HIV short RNA detection-1 blood compared to the patient in which HIV short RNA was detected (ST +). Thus, 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)
<Detection of HIV short RNA>
Detection of HIV short-chain RNA in peripheral blood mononuclear cells was carried out in the same manner as in Test Example 1, except that blood of the treatment starting patient (34 people) whose HIV RNA amount in the plasma was below the detection limit was used. went.

<Expression of CD8 ⁺ T cell activation marker>
The HIV short chain performs detection lymphocytes immunostaining prepared from the start of treatment was taken from a patient blood go of RNA, the expression amount 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).

<Analysis-1>
FIG. 6A shows the results of analyzing the relationship between the amount of HIV short RNA and the proportion of HLA-DR ⁺ CD38 ⁺ cells in total CD8 ⁺ T cells. In FIG. 6A, the vertical axis shows the percentage (%) of HLA-DR ⁺ CD38 ⁺ cells in total CD8 ⁺ T cells, and the horizontal axis shows the amount of HIV short RNA (rho = 0.5668, p = 0.0004).

<Analysis-2>
The result of analyzing the relationship between the amount of HIV short-chain RNA and the amount of HLA-DR MFI in total CD8 ⁺ T cells is shown in FIG. 6B. In FIG. 6B, the vertical axis represents the amount of HLA-DR MFI (fluorescence intensity) in total CD8 ⁺ T cells, and the horizontal axis represents the amount of HIV short-chain RNA (rho = 0.6539, p <0. 0001).

The results of 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 was prepared from 10 ⁷ (1.E + 07) to 10 ² (1.E + 02) copies of the cDNA, 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′-GGUCUCUCUGUGUUACGACCAGAUUCGAGCCUGGGAGCUCUCUGGGCUAGCUAGGAGAACCC-3 ′

-Quantitative PCR-
Quantitative PCR was performed in the same manner as in Test Example 1 except that the following primers and probes were used.
[Test group 1: No addition of flap sequence]
-Forward primer 5'-GGTTAGACCAGATCTGAGCCTG-3 '(SEQ ID NO: 2)
Reverse primer 5′-CAGTTACCGCATGCCG-3 ′ (SEQ ID NO: 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.
[Test group 2: Flap sequence added only for forward primer]
-Forward primer 5'- AATAAATCATAA GGTTAGACCAGATCTGAGCCTG-3 '(SEQ ID NO: 6)
The underlined portion indicates a flap sequence.
Reverse primer 5′-CAGTTACCGCATGCCG-3 ′ (SEQ ID NO: 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.
[Test group 3: Flap sequence added only for reverse primer]
-Forward primer 5'-GGTTAGACCAGATCTGAGCCTG-3 '(SEQ ID NO: 2)
Reverse primer 5′- AATAAAATCATAACAGTTACCGCATGCCG -3 ′ (SEQ ID NO: 7)
The underlined portion indicates a flap sequence.
・ 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.
[Test group 4: Addition of flap sequence to both forward primer and reverse primer]
-Forward primer 5'- AATAAATCATAA GGTTAGACCAGATCTGAGCCTG-3 '(SEQ ID NO: 6)
The underlined portion indicates a flap sequence.
Reverse primer 5′- AATAAAATCATAACAGTTACCGCATGCCG -3 ′ (SEQ ID NO: 7)
The underlined portion indicates a flap sequence.
・ 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.

  From the results of FIG. 7, it was found that in the test group 3 (only the reverse primer had a flap sequence added), the calibration curve was applied even at a concentration 10 times lower than the other test groups.

(Test Example 8: transcription stop position of HIV short RNA)
J Virol. 72, pages 1,666 to 1,670, as a model cell of the remaining infected cells published in 1998, from the U1 cell line having wild type HIV-1, the same as in Test Example 1 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.
As shown in FIG. 8, 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>.
<7> 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 who has not started treatment with an anti-human immunodeficiency virus drug, comprising the step of evaluating whether or not CD4 ⁺ T cells are decreased using the presence or absence of detection of the transcript as an index This is a method for predicting a decrease in the number of T cells.
<8> The detection step according to <7>, 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. This is a method for predicting a decrease in the number of CD4 ⁺ T cells.
<9> 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.

Claims (9)

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 Method for determining immune status in a patient.   The method for determining an immune state according to claim 1, wherein the patient is a patient whose human immunodeficiency virus RNA and human immunodeficiency virus antigen in plasma are below the detection limit.   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. The method for determining an immune state as described. 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. The method for predicting an increase in the number of CD4 ⁺ T cells according to claim 4, wherein the patient is a patient whose human immunodeficiency virus RNA and human immunodeficiency virus antigen in plasma are below the detection limit. 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. The method for predicting an increase in the number of CD4 ⁺ T cells described. 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 who has not started treatment with an anti-human immunodeficiency virus drug, comprising the step of evaluating whether or not CD4 ⁺ T cells are decreased using the presence or absence of detection of the transcript as an index A method for predicting a decrease in the number of T cells. The CD4 ⁺ T according to claim 7, wherein the detection step uses 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. A method for predicting a decrease in cell number. Treatment with an anti-human immunodeficiency virus drug comprising 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. Kit for determination of immune status in patients after initiation of treatment, kit for predicting increase in CD4 ⁺ T cell count in patients after initiation of treatment with anti-human immunodeficiency virus drug, or anti-human immunodeficiency A kit for predicting a decrease in the number of CD4 ⁺ T cells in a patient who has not started treatment with a viral drug.