CN115877017B - Products and systems for predicting immune reconstitution in HIV/AIDS patients - Google Patents

Abstract

The present invention relates to biologyThe technical field of medicine, in particular to CD70 ⁺ CD4 ⁺ T cell number of total CD4 ⁺ The ratio of the number of T cells to predict the immune reconstitution of HIV/AIDS patients after antiviral treatment. The method has the characteristics of rapidness, low prediction cost, accuracy and early prediction (before antiviral treatment), so that patients with poor immune reconstruction are monitored and clinically intervened in time according to the prediction result, and the long-term survival rate of the patients is improved.

Description

Products and systems for predicting immune reconstitution in HIV/AIDS patients

Technical Field

The invention relates to the technical field of biological medicine, in particular to CD70 ⁺ CD4 ⁺ T cell number of total CD4 ⁺ The ratio of the number of T cells to predict the immune reconstitution of HIV/AIDS patients after antiviral treatment.

Background

AIDS (Acquired immunodeficiency syndrome, AIDS) is CD4 caused by human immunodeficiency virus (human immunodeficiency virus, HIV) ⁺ Reduced T cell numbers, progressive destruction of immune function, and secondary human immunodeficiency syndrome with various opportunistic infections and malignancies. With the wide application of antiviral drugs and the popularization of prevention and treatment concepts, AIDS has become a controllable chronic disease. However, the occurrence of poor immune reconstitution in HIV/AIDS patients has been a major and difficult problem in the field of AIDS research. The immune reconstruction failure is mainly represented by persistent inhibition of HIV virus replication in patients after antiviral treatment, but CD4 ⁺ T cell numbers have not been restored to normal levels; replication is inhibited and CD4 compared to HIV virus ⁺ T cells have a good immune reconstitution for those with good recovery, and the immune system of those with poor reconstitution is deficient, with a higher risk of developing AIDS-related and non-AIDS-related events. Therefore, early prediction of the occurrence of poor immune reconstruction and timely clinical intervention are the clinical problems to be solved urgently for improving the long-term survival rate of patients in the current antiviral treatment process, and are the necessary requirements for comprehensively reducing the complications and the mortality of AIDS.

Disclosure of Invention

First, the technical problem to be solved

In view of the need for early prediction of poor immune reconstitution in HIV/AIDS patients, the present invention provides a product and system for predicting the immune reconstitution of HIV/AIDS patients after antiviral treatment, which employs CD70 ⁺ CD4 ⁺ T cell number of total CD4 ⁺ The ratio of the number of the T cells is used for predicting the immune reconstruction condition (good or bad) of an HIV/AIDS patient after antiviral treatment, and the method has the characteristics of rapidness, low prediction cost, accuracy and early prediction (before antiviral treatment), so that the patient with the immune reconstruction bad is monitored and clinically intervened in time according to the prediction result, and the long-term survival rate of the patient is improved.

(II) technical scheme

In order to achieve the above purpose, the main technical scheme adopted by the invention comprises the following steps:

in a first aspect, the present invention provides a method for detecting CD4 ⁺ Use of an agent that expresses CD70 on T cells for the preparation of a product for predicting immune reconstitution of an HIV/AIDS patient after antiviral treatment.

Preferably, the agent comprises an antibody or antibody fragment (containing a specific binding region) that specifically binds CD 70. For example, a monoclonal antibody or fragment thereof that retains the specific binding capacity of the antibody to CD 70.

Preferably, the agent further comprises an antibody or antibody fragment (containing a specific binding region) that specifically binds CD 4.

Preferably, the product is a detection reagent composition, a kit, a detection chip, an antibody probe or a detection instrument.

Preferably, the kit further comprises reagents or materials for obtaining peripheral blood from the patient, and/or reagents or materials for isolating mononuclear cells from peripheral blood of the patient (e.g., biological magnetic beads), etc.

Preferably, the detection instrument is preferably a flow cytometer.

Wherein the antiviral therapy is ART therapy.

In a second aspect, the present invention provides a method for predicting HIV after antiviral treatmentProducts for the immune reconstitution of HIV/AIDS patients, capable of detecting CD70 in mononuclear cells isolated from peripheral blood of HIV/AIDS patients ⁺ CD4 ⁺ The proportion of T cells to total number of CD4 positive T cells; the product is a reagent composition, a kit, a detection chip, an antibody probe or a detection instrument.

In a third aspect, the present invention provides a system for predicting the immune reconstitution status of an HIV/AIDS patient after antiviral treatment prior to antiviral treatment, comprising a detection module and a prediction judgment module,

wherein the detection module is used for detecting CD70 in peripheral blood mononuclear cells ⁺ CD4 ⁺ The ratio of T cells to total number of CD4 positive T cells;

the prediction judgment module comprises a readable carrier recorded with judgment rules, wherein the judgment rules are as follows: comparing the ratio obtained by the detection module with a reference value, and judging that the HIV/AIDS patient is more likely to have poor immune reconstruction after antiviral treatment when the ratio is larger than the reference value;

the reference value is set to be CD70 obtained from the HIV/AIDS patient group which is subjected to 5 years of antiviral treatment ⁺ CD4 ⁺ Cut-off of the ratio of T cells to CD4 positive T cells.

Wherein the cut-off value is calculated from the ratio obtained from the HIV/AIDS patient population treated for 5 years with antiviral treatment and the clinical outcome (immunological reconstitution outcome), and the cut-off value is 4.85% when the number of patients in the patient population is 46.

According to a preferred embodiment of the invention, the detection module is a flow cytometer. The flow cytometer is coupled with a computer, and the computer provides the prediction judging module to obtain the system for predicting the immune reconstruction condition of the HIV/AIDS patient after antiviral treatment.

When the flow cytometry is adopted for detection, the CD4 in peripheral blood mononuclear cells needs to be counted respectively ⁺ T cell number and CD70 ⁺ CD4 ⁺ T cell number, CD70 ⁺ CD4 ⁺ T cell number and CD4 ⁺ The ratio of the number of T cells is the target ratio.

According to a preferred embodiment of the invention, the detection module uses detection reagents comprising antibodies or antibody fragments that specifically recognize CD70, and antibodies or antibody fragments that specifically recognize CD 4.

According to a preferred embodiment of the invention, the system further comprises a sample processing module for obtaining peripheral blood mononuclear cells from HIV/AIDS patients.

According to a preferred embodiment of the invention, the system further comprises a sample acquisition module for obtaining a blood sample from an HIV/AIDS patient, preferably the blood sample is peripheral blood. The sample acquisition module may be, for example, an automatic blood collection meter.

Furthermore, the present invention provides the use of CD70 as a biomarker for predicting/assessing the ability of HIV/AIDS patients to reconstruct immunity after antiviral treatment.

Upon judging clinical outcome, poor immune reconstitution is defined as sustained virologic inhibition (HIVRNA) obtained after HIV/AIDS patients received ART<50 copies/mL)，CD4 ⁺ T cell count<350 A patient with cells/μl; CD4 ⁺ Patients with T cell counts of 500 cells/. Mu.L or more were defined as those with good immune reconstitution.

(III) beneficial effects

The inventor found in experimental study that CD4 ⁺ The expression level of CD70 on the surface of T cells is significantly increased in HIV/AIDS patients and is comparable to CD4 ⁺ T cell count and CD4/CD8 ratio are inversely related to CD4 ⁺ T cell activation is positively correlated; untreated HIV/AIDS patient CD70 ⁺ CD4 ⁺ T cell ratio (CD 4) ⁺ The proportion of CD70 positive cells in T cells) is higher than normal, and CD70 ⁺ CD4 ⁺ Increased T cell proportion is associated with disease progression in HIV/AIDS patients; CD70 in patients with poor immune reconstitution in 8 years of antiviral treatment ⁺ CD4 ⁺ The proportion of T cells is significantly higher than that of the patients with good immune reconstruction; further predictive analysis found that CD70 ⁺ CD4 ⁺ Patients with T cell ratios above the cutoff value (. Gtoreq.4.85%) are at significantly higher risk of immune malreconstruction during antiviral treatment than CD70 ⁺ CD4 ⁺ T cell ratio is lower than that of the truncated cellOff (< 4.85%) and CD4 in peripheral blood mononuclear cells of the patient prior to treatment ⁺ Expression of CD70 on T cells can well predict immune reconstitution after antiviral treatment.

Based on the above findings, the present invention proposes a scheme (including reagents, reagent compositions, device products, detection instruments and intelligent systems) for predicting the immune reconstitution of HIV/AIDS patients after antiviral treatment, providing early prediction/assessment for prognosis of antiviral treatment of HIV/AIDS patients. Experiments prove that the prediction scheme of the invention has the characteristics of rapidness, simpleness, high prediction accuracy and the like, so that necessary monitoring and timely clinical intervention are given to patients with poor immune reconstruction according to the prediction result, and the long-term survival rate of the patients is improved. In addition, the parameter (CD 70 ⁺ CD4 ⁺ T cell ratio) also provides a new therapeutic target for HIV patients and provides a new therapeutic concept for future patient treatment.

Drawings

FIG. 1A shows CD4 in normal (HC) and different groupings of untreated HIV/AIDS patients (TN) ⁺ CD70 expression levels on T cells; b, C, D of FIG. 1 shows untreated HIV/AIDS patient CD70 ⁺ CD4 ⁺ T cells occupy CD4 ⁺ Ratio of T cells to CD4 ⁺ Correlation analysis between T counts, CD4/CD8 ratio, immune activation; * P (P)<0.05，***P<0.001，****P<0.0001。

FIG. 2A is CD70 in HIV/AIDS patients after HC, TN and antiviral treatment ⁺ CD4 ⁺ Scatter plot of percent T cells; FIG. 2B is a graph showing CD70 between immune well-established (IRs) and poorly established (PIRs) patients in 8 years of treatment ⁺ CD4 ⁺ Differences in T cell ratios; FIG. 2C subject working characteristics (ROC curve) analyze CD70 for untreated patients ⁺ CD4 ⁺ The predictive value of T cell ratio versus patient recovery from each year of immune reconstitution, area under the curve (AUC); FIG. 2D survival curves analysis with different CD70 ⁺ CD4 ⁺ The possibility of recovery of the patient's immune system in proportion to T cell expression; ***P<0.001，****P<0.0001。

Detailed Description

The invention will be better explained by the following detailed description of the embodiments with reference to the drawings.

Previous studies have shown that immune activation is one of the important mechanisms of poor immune reconstitution in HIV/AIDS patients, and that during HIV infection, sustained immune activation can be driven by a number of factors, including residual viral replication, gut microbial translocation, co-infection, and the like. Although antiviral therapy can significantly reduce the level of immune activation and inflammation in HIV/AIDS patients, excessive immune activation persists in patients with poor immune reconstitution compared to those with good immune reconstitution, resulting in abnormal immune responses. While the CD70 molecule is closely related to the activation of T cells, the CD70 molecule is one of family members of tumor necrosis factors, and is highly expressed on the surface of the activated T cells; the ligand of CD70 is CD27, and the interaction of CD27-CD70 can regulate T cell activation, proliferation and differentiation and plays an important role in regulating immune response. Previous studies have shown that in HIV/AIDS patients, CD70 is found in CD4 ⁺ Up-regulation of T cell expression, our study further found that CD70 and CD4 ⁺ T cell count and activation are closely related and CD4 in immunocompromised patients ⁺ The expression level of CD70 on the T cell surface was significantly higher than in patients with well-established immune responses. This suggests that CD70 may be involved in HIV disease progression and in the recovery process of the patient's immune system.

The inventors have also found that untreated HIV/AIDS patients CD70 ⁺ CD4 ⁺ T cell ratio is higher than normal and CD70 ⁺ CD4 ⁺ Increased T cell proportion is associated with disease progression in HIV/AIDS patients; CD70 in patients with poor immune reconstitution in 8 years of antiviral treatment ⁺ CD4 ⁺ The proportion of T cells is significantly higher than that of the patients with good immune reconstruction; further predictive analysis found that CD70 ⁺ CD4 ⁺ Patients with T cell ratios above the cutoff value (. Gtoreq.4.85%) are at significantly higher risk of immune malreconstruction during antiviral treatment than CD70 ⁺ CD4 ⁺ T cell ratio is lowIn patients with cut-off values (< 4.85%) and CD4 when the patient is untreated ⁺ The expression level of CD70 on T cells can well predict the immune reconstitution after antiviral treatment.

In order that the above-described aspects may be better understood, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present invention are shown in the drawings, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The experimental methods in the following experimental examples are conventional methods unless otherwise specified. The raw materials and reagent materials used in the examples described below are commercially available products unless otherwise specified. The related technical contents include:

study object (one)

On the premise of informed consent and meeting inclusion conditions, the group of cases are selected and personal basic information is recorded. The HIV/AIDS patient cohort was from the university of capital medical affiliated beijing forum hospital; in a cross-sectional trial, the team recruited 143 untreated HIV/AIDS patients, 57 HIV/AIDS patients treated with 8 years of antiviral treatment, and 51 healthy donors with age-sex matching; of the antiviral treated patients, 49 patients with baseline CD4 less than 350 cells/. Mu.l were finally screened for cohort studies, including 19 immune-well-established (CD 4 ⁺ T cell count ≡500 cells/. Mu.l), 30 cases of poor immune reconstitution (CD 4) ⁺ T cell count<350 cells/. Mu.l). In a retrospective prospective cohort, the team included 80 cases of HIV/AIDS patients whose baseline characteristics were CD4 ⁺ T cell count<350 cells/. Mu.l. Antiviral treatment is applied for 5 years, during which the virus is continuously inhibited and the viral load is increased<50copies/ml; according to patient CD4 ⁺ T cell count, further classifying antiviral treated patients as immunocompetent (CD 4 ⁺ T cell count ≡500 cells/. Mu.l) and immunore-established failure (CD 4) ⁺ T cellCounting<350cells/μl）。

(II) sample collection

In this study, 10ml of peripheral blood was collected for inclusion in all populations and placed in an anticoagulation collection tube (EDTA). Peripheral blood mononuclear cells are isolated according to known methods.

(III) multiparameter flow cytometry analysis

CD4 ⁺ Antibodies for use of T cell surface markers for multiparameter flow cytometry: anti-human CD3-BV786 (clone SK 7), anti-human CD4-BUV395 (clone RPA-T4), anti-human CD8-BV510 (clone SK 1), anti-human CD70-PE-CF594 (clone Ki-24), anti-human CD38-BUV737 (clone HB 7), anti-human HLA-DR-AF700 (clone L243). Experimental data were obtained with LSR-fortesa flow cytometer and analyzed with FlowJo software.

(IV) statistical analysis

All data were analyzed using GraphPad 9 software. The normalization of each variable was assessed using the Kolmogorov-Smirnov test. For normally distributed data, the two variables were compared using the unpaired or paired two-tailed Student t-test. When the data is not normally distributed, the unpaired and paired data are compared for variables using Mann-Whitney U test or Wilcoxon paired symbol rank test, respectively. In the case of comparing two or more independent samples, the Kruskal-Wallis test and Dunn multiple comparison test are employed. The predicted effect of CD70 was assessed by the area under the subject's operating characteristic curve (ROC) curve. The possibility of recovering the patient's immune system was carried out using the Kaplan-Meier method. P <0.05 is considered statistically significant.

Example 1

1. The purpose of this example is to demonstrate that HIV/AIDS patient CD4 ⁺ CD70 expression levels on T cells are associated with disease progression.

2. Study object (see the foregoing)

3. Experimental method

To study CD70 molecules in CD4 ⁺ Correlation of expression levels on T cells with AIDS progression, experiments using flow cytometry to detect CD4 in HC and TN patients ⁺ T cellUpper CD70 expression. CD4 in peripheral blood mononuclear cells according to TN group ⁺ T cell count can be divided into three groups<200 cells/μl, 200-350 cells/μl, ≥350 cells/μl）。

4. Experimental results

Experimental results have found that CD70 in HIV/AIDS patients at various stages of HIV infection ⁺ CD4 ⁺ T cell ratios were higher than normal (see panel A of FIG. 1); and correlation analysis indicated CD70 ⁺ CD4 ⁺ T cell ratio to patient CD4 ⁺ T count and CD4/CD8 ratio are inversely related to CD4 ⁺ T cell activation was positively correlated (see FIG. B, C, D of FIG. 1), demonstrating CD70 ⁺ CD4 ⁺ An increase in the proportion of T cells is closely related to the progression of the disease in HIV/AIDS patients.

Example 2

1. The purpose of this example is to verify CD70 in mononuclear cells obtained from a patient blood sample ⁺ CD4 ⁺ The proportion of T is predictive of the reconstitution (good or bad) of the patient's immunity after antiviral treatment.

2. Study object (see the foregoing)

3. Experimental method

(1) To further investigate the role of CD70 in immune reconstitution following antiviral therapy, experiments using flow cytometry examined cutoff:4.85% of patients CD4 after 8 years of antiviral treatment ⁺ Expression level of CD70 on T cells. The results show that CD70 in patients with long-term antiviral treatment compared with TN patients ⁺ CD4 ⁺ The T cell fraction was not reduced (fig. 2A).

(2) HIV/AIDS patients after antiviral treatment are divided into two subgroups: CD4 when two groups of patients were untreated ⁺ T cell counts were less than 350 cells/. Mu.L, virus was inhibited continuously during treatment, viral load<50 copies/. Mu.L, based on patient current CD4 ⁺ T cell count, two subfractions were classified as 19 in immunocompetent persons (immunological responders, IR, CD 4) ⁺ T cell count ≡500 cells/. Mu.L) and 30 cases of immune dysplasia (poor immunological responders, PIR, CD 4) ⁺ T cell count<350 cells/μL）。

Experimental results found that CD70 ⁺ CD4 ⁺ T cell ratios were significantly higher in the PIR group than in the IR group (FIG. 2B), implying CD70 ⁺ CD4 ⁺ T cell proportion is related to immune reconstitution, and CD70 ⁺ CD4 ⁺ Patients with higher T cell ratios are more prone to poor immune reconstitution.

(3) To further clarify CD70 ⁺ CD4 ⁺ Correlation between T cell ratio and immune reconstitution (good or poor) after antiviral treatment of HIV/AIDS patients, the experimental team also included 80 baseline (initial) CD4 ⁺ Patients with T cell counts below 350 cells/. Mu.L and who received antiviral treatment for 5 years, respond well to the virus, each year based on CD4 in peripheral blood mononuclear cells ⁺ The T cell count measurements and the aforementioned partitioning criteria divide the patients into PIR and IR groups.

The incidence of poor immune reconstitution in HIV/AIDS patients after various durations of antiviral treatment was also analyzed by experiment. The patients were classified as CD70 according to the optimal cut-off (cutoff: 4.85%) obtained from the ROC curve using the protocol ⁺ CD4 ⁺ High T-ratio group (. Gtoreq.4.85%) and CD70 ⁺ CD4 ⁺ Low T ratio group<4.85%). The results showed that CD70 ⁺ CD4 ⁺ Patients with high T proportion are at significantly higher risk of immune malreconstruction than CD70 during 1-5 years of antiviral treatment ⁺ CD4 ⁺ Patients with low T ratios (table 1).

Table 1: high and low levels of CD70 at various time points during antiviral treatment ⁺ CD4 ⁺ T cell ratio of HIV/AIDS patient to probability of poor reconstruction

	High-CD70(n=35)	Low-CD70(n=45)	P
				1-year of	21/35 (60.0%)	11/45 (24.4%)	0.001
2-year of	18/35 (51.4%)	6/45(13.3%)	0.001
				3-year of	15/35 (42.9%)	4/45 (0.9%)	0.001
4-year old	14/35 (40.0%)	2/45 (0.4%)	<0.0001
				5-year of	11/35 (31.4%)	2/45 (0.4%)	<0.0001

P values were obtained by chi-square test, bold numbers expressing P <0.05.

Subject operating characteristic curve (ROC) results show, CD70 ⁺ CD4 ⁺ T cell ratio can effectively predict immune reconstitution of HIV/AIDS patients every yearIn the case of (5) (fig. 2C). CD70 was found by survival curve analysis (Kaplan-Meier method) ⁺ CD4 ⁺ Patients with a lower proportion of T cells can enter a state of good immune reconstitution earlier than patients with a higher proportion (fig. 2D). The above study conclusion shows that: higher CD70 without antiviral treatment ⁺ CD4 ⁺ T cell ratio means that patients are prone to poor immune reconstitution and therefore high CD70 ⁺ CD4 ⁺ T cell proportion is a sign of patient susceptibility to poor immune reconstitution.

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and are not limiting. Although the present invention has been described in detail with reference to the embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, which is intended to be covered by the appended claims.

Claims (7)

1. CD4 detection ⁺ Use of an agent for the expression level of CD70 on T cells for the preparation of a product for predicting the immune reconstitution of HIV/AIDS patients after antiviral treatment, characterized in that said agent is used for the detection of CD70 in peripheral blood isolated mononuclear cells of HIV/AIDS patients ⁺ CD4 ⁺ The proportion of T cells to the total number of CD4 positive T cells, including antibodies or fragments thereof that specifically bind to CD70 and antibodies or fragments thereof that specifically bind to CD4, wherein patients with a proportion higher than or equal to the cut-off value are taken as a first group of patients, patients with a proportion lower than the cut-off value are taken as a second group of patients, and the risk of developing immune remodeling failure after antiviral treatment is higher in the first group of patients than in the second group of patients, the cut-off value being 4.85%.

2. The use according to claim 1, wherein the product is a detection reagent composition, a kit, a detection chip, an antibody probe or a detection instrument.

3. The use according to claim 2, wherein the kit comprises reagents or materials for obtaining peripheral blood from a patient and/or reagents or materials for isolating mononuclear cells from peripheral blood of a patient.

4. The use according to claim 2, wherein the detection instrument is a flow cytometer.

5. A system for predicting the immune reconstruction condition of an HIV/AIDS patient after antiviral treatment is characterized by comprising a detection module and a prediction judging module, wherein,

the detection module is used for detecting CD70 in peripheral blood mononuclear cells ⁺ CD4 ⁺ The ratio of T cells to total number of CD4 positive T cells;

the prediction judgment module comprises a readable carrier recorded with judgment rules, wherein the judgment rules are as follows: comparing the ratio obtained by the detection module with a reference value, and judging that the HIV/AIDS patient is more likely to have poor immune reconstruction after antiviral treatment when the ratio is larger than the reference value;

the reference value is set to be CD70 obtained from the HIV/AIDS patient group which is subjected to 5 years of antiviral treatment ⁺ CD4 ⁺ T cells account for a cutoff value of the CD4 positive T cell ratio, which is 4.85%.

6. The system of claim 5, wherein the detection module is a flow cytometer.

7. The system of claim 5, wherein the detection module employs a detection reagent comprising an antibody or antibody fragment that specifically recognizes CD70, and an antibody or antibody fragment that specifically recognizes CD 4;

the system further includes a sample processing module for obtaining peripheral blood mononuclear cells from an HIV/AIDS patient.

Images