New use of sCD73 as a marker for predicting immune reconstitution in HIV / AIDS patients after antiviral therapy

Abstract

The application provides a new use of soluble CD73 (sCD73) as a marker for predicting the immune reconstruction of HIV / AIDS patients after antiviral treatment, and belongs to the technical field of biological medicine. The sCD73 can be used for predicting the reconstruction of the immune system of HIV / AIDS patients after antiviral treatment, and has the advantages of rapid detection, accuracy, low cost and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the novel use of sCD73 as a biomarker for predicting immune reconstitution after antiretroviral therapy in HIV / AIDS patients. Background Technology

[0002] The morbidity and mortality rates of HIV / AIDS patients have significantly decreased with the widespread use of antiretroviral therapy, which can sustainably suppress HIV replication and gradually restore CD4 T cell counts. However, although viral replication can be completely suppressed after antiretroviral therapy, 10%–40% of patients still do not have their CD4 T cell counts return to normal. These patients are called those with poor immune reconstitution, and they often exhibit immune system deficiencies. Compared to patients with good immune reconstitution, those with poor reconstitution have a higher risk of AIDS-related and non-AIDS-related events and death. To date, many therapies for poor immune reconstitution are under investigation, but no effective treatment has yet emerged due to the complex mechanisms underlying poor immune reconstitution. Early identification / prediction of patients who may have poor immune reconstitution, regular monitoring, and timely intervention with supportive therapies are crucial for improving clinical outcomes. Therefore, it is necessary to further explore scientific and feasible methods to guide clinicians in the early identification / prediction of which patients' immune systems are not reconstituted.

[0003] Previous studies have explored parameters for predicting immune reconstitution in patients after antiviral therapy. Commonly used predictive indicators include baseline CD4 count and the CD4 / CD8 ratio. However, in resource-constrained hospitals, expensive flow cytometry is not widely used, making it challenging to predict immune reconstitution based on the number or ratio of immune cells. Furthermore, routinely measuring cell counts ignores cellular function and other information about the overall immune environment. Studies have shown that after antiviral therapy, some patients may experience a return to normal viral load and CD4 count, but their immune systems remain overactivated, and these individuals are more likely to develop poor immune reconstitution. Cell counts cannot accurately predict the reconstitution of a patient's immune system; therefore, it is necessary to explore and develop biomarkers for predicting immune reconstitution in HIV / AIDS patients, enabling rapid, convenient, and accurate prediction of immune reconstitution status. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a biomarker for early prediction or assessment of immune reconstitution after antiretroviral therapy in HIV / AIDS patients, and a product for predicting or assessing immune reconstitution after antiretroviral therapy in HIV / AIDS patients using the biomarker, so as to monitor and intervene in patients who may have poor immune reconstitution.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention provides a novel use of sCD73 as a biomarker for predicting immune reconstitution in HIV / AIDS patients after antiretroviral therapy.

[0009] Secondly, the present invention provides the use of antibodies that specifically recognize CD73 in the preparation of products for predicting or assisting in predicting whether immune reconstitution is poor in HIV / AIDS patients after antiviral therapy.

[0010] Preferably, the product is a detection reagent, a kit, an antibody chip, an antibody probe, or a detection instrument.

[0011] Preferably, the kit comprises: an antibody that specifically recognizes CD73, a biotin-labeled CD73 antibody, and an avidin-biotin-peroxidase complex.

[0012] Preferably, the kit further includes reagents for obtaining peripheral blood from the patient and / or reagents for separating plasma. Preferably, the plasma is plasma separated from peripheral blood.

[0013] Thirdly, the present invention provides a product for predicting whether immune reconstitution is poor in HIV / AIDS patients after antiviral therapy, the product being able to detect the concentration of sCD73 in plasma.

[0014] Preferably, the product is a detection reagent, kit, antibody chip, antibody probe, or detection instrument. Preferably, the plasma is peripheral blood.

[0015] Preferably, the detection reagent is an ELISA kit that specifically binds to sCD73; this kit can detect the concentration of sCD73 protein in plasma by specifically binding to sCD73 in plasma. For example, the sCD73 ELISA kit used in the examples was purchased from Abcam (ab213761).

[0016] Fourthly, the present invention provides a system for predicting good / poor immune reconstitution in HIV / AIDS patients after antiviral therapy, the system comprising: a detection module and a prediction judgment module;

[0017] The detection module is used to detect the concentration of sCD73 in the plasma of the patient to be tested;

[0018] The prediction and judgment module includes a readable carrier containing judgment rules, which are as follows: the concentration detection value of sCD73 is compared with the reference value, and when the concentration detection value of sCD73 is higher than the reference value, it is determined that the patient under test has poor immune reconstitution after antiviral treatment.

[0019] Preferably, the reference value is the cut-off value of sCD73 concentration obtained from an HIV / AIDS patient population. Preferably, the cut-off value is specifically 4.111 ng / ml. 4.111 ng / ml is the statistical cutoff value for sCD73 concentration obtained from 171 HIV / AIDS patients.

[0020] Poor immune reconstitution was defined as HIV / AIDS patients who achieved sustained virological suppression (HIV RNA <50 copies / mL) and a CD4 cell count of less than 350 cells / µL after receiving ART; patients with a CD4 cell count of 350 cells / µL or greater were defined as those with good immune reconstitution.

[0021] Preferably, the detection module may include the aforementioned detection reagents, kits, antibody chips, or antibody probes capable of detecting sCD73 concentration. More preferably, the detection module is a fully automated ELISA reader. By coupling the fully automated ELISA reader with a computer, a system for predicting the immune reconstitution status of HIV / AIDS patients after antiviral therapy can be obtained.

[0022] Preferably, the system further includes a sample processing module, which includes a module for obtaining plasma from the patient and a module for preprocessing the plasma so that the preprocessed sample is available for detection by the detection module.

[0023] Preferably, the module for obtaining plasma from a patient obtains peripheral blood from the patient and separates plasma from the peripheral blood.

[0024] (III) Beneficial Effects

[0025] This invention provides an application of sCD73 as a biomarker for predicting the immune reconstitution status (poor / good) after antiretroviral therapy in HIV / AIDS patients. Specifically, this application predicts the immune reconstitution status of HIV / AIDS patients by detecting the concentration of sCD73 in their plasma, enabling real-time monitoring and timely treatment for those with poor immune reconstitution after antiretroviral therapy. Furthermore, the concentration of sCD73 in plasma provides a novel therapeutic target for HIV patients, offering new insights for future patient treatment and drug development.

[0026] This invention uses plasma sCD73 as a biomarker to predict immune reconstitution after antiretroviral therapy in HIV / AIDS patients. It only requires obtaining plasma from the subject and using conventional detection methods such as ELISA kits or enzyme-linked immunosorbent assay (ELISA) readers that specifically bind to sCD73 to predict the immune reconstitution of patients after antiretroviral therapy. Compared with baseline CD4 count and CD4 / CD8 ratio, this invention does not require the use of expensive flow cytometry, thus making prediction simpler, lower in cost, and of widespread applicability.

[0027] Products used to detect the concentration of sCD73 in patient plasma include, but are not limited to, detection reagents, kits, antibody chips, antibody probes, or detection instruments (such as fully automated ELISA readers). This invention also relates to the application of these products in the preparation of a system for predicting good / poor immune reconstitution in HIV / AIDS patients after antiretroviral therapy. Based on this, this invention has developed a system for predicting good / poor immune reconstitution in HIV / AIDS patients after antiretroviral therapy. In addition to a module for detecting the concentration of sCD73 in plasma, this system includes a prediction judgment module with a readable carrier containing judgment rules. This system can automatically predict the immune reconstitution status of the subject. Attached Figure Description

[0028] Figure 1 shows the changes in plasma sCD73 concentration before and after treatment in different types of subjects; among them... Figure 1a The concentration of sCD73 in the plasma of normal individuals (HC), patients who have not received antiviral treatment (TN), and patients who have received antiviral treatment (ART); Figure 1b This represents the plasma sCD73 levels in patients before and after treatment in a cohort study. ***P<0.001.

[0029] Figure 2 middle, Figure 2 a is a graph comparing the probability curves of immune system recovery in patients with different levels of sCD73 plasma concentration when CD4 count is the clinical outcome; Figure 2 b represents the area under the receiver operating characteristic (ROC) curve analysis of the likelihood that plasma sCD73 concentration predicts immune reconstitution in HIV / AIDS patients when CD4 count is the clinical outcome, and is calculated as the area under the curve (AUC).

[0030] Figure 3a This is a graph comparing the probability curves of immune system recovery in patients with different levels of sCD73 plasma concentration when the CD4 / CD8 ratio is the clinical outcome. Figure 3b To assess the likelihood that plasma sCD73 concentration could predict immune reconstitution in HIV / AIDS patients when the CD4 / CD8 ratio was the clinical outcome, the receiver operating characteristic (ROC) curve analysis was used, and the area under the curve (AUC) was calculated. Detailed Implementation

[0031] To better explain the present invention, it will now be described in detail.

[0032] Studies have shown that immune activation is one of the mechanisms underlying poor immune system reconstruction in HIV / AIDS patients. Immune activation may be caused by a combination of factors, including residual HIV viral replication, microbial translocation, immunosenescence, or a decrease in the number of regulatory T lymphocytes. When the immune system is overactivated, the body needs to promptly activate pathways to inhibit excessive immune activation and protect the body from tissue damage caused by persistent immune cell activation. The metabolism of purine nucleotides involving CD73 is one of the important pathways for inhibiting activation. CD73 is a 5'-nucleotidase distributed on the cell membrane, anchored to the cell membrane by glycosylphosphatidylinositol, and widely expressed in various tissues and cells of the human body. CD73 can detach from the cell membrane and enter the plasma as sCD73 (soluble CD73). Studies have shown that sCD73 and CD73 on extracellular vesicles are identical to CD73 on the cell membrane, both possessing enzymatic activity. They can break down extracellular AMP into adenosine, which inhibits cell activity and function by binding to receptors on the cell. Many previous studies have reported that CD73 can participate in HIV disease progression and play an important immunosuppressive role, but the role of sCD73 in HIV disease progression is still unclear.

[0033] The inventors discovered in a cross-sectional study that the plasma sCD73 concentration in HIV / AIDS patients was higher than in healthy individuals, and that the plasma sCD73 concentration decreased after treatment. In a cohort study, the plasma sCD73 concentration in patients decreased significantly after 5.7 years of antiviral treatment. Furthermore, high sCD73 concentration was correlated with clinical outcomes in HIV / AIDS patients; tests revealed that patients with high sCD73 concentrations tended to have poor immune system reconstitution, meaning that poor immune system reconstitution was positively correlated with sCD73 concentration. Therefore, this invention provides the application of sCD73 as a biomarker for predicting the status (poor / good) of immune reconstitution after antiviral treatment in HIV / AIDS patients.

[0034] The relevant technical content includes:

[0035] (1) Research subjects

[0036] With informed consent and meeting inclusion criteria, cases were selected for enrollment, and their basic personal information was recorded. The HIV / AIDS patient cohort was drawn from Beijing Ditan Hospital, Capital Medical University. The study recruited 110 treatment-naïve HIV / AIDS patients (TN), 62 HIV / AIDS patients undergoing antiretroviral therapy (ART), and 25 age- and sex-matched healthy donors (HC). Among the patients undergoing antiretroviral therapy, 171 patients with a baseline CD4 count of less than 350 cells / μL were ultimately selected for the cohort study, and their viral load remained below 50 copies / ml during routine follow-up after ART.

[0037] (2) Sample collection

[0038] In this study, 10 ml of peripheral blood was collected from all participants and placed in an anticoagulated blood collection tube (EDTA). Plasma was separated according to existing standard methods.

[0039] (3) The concentration of sCD73 in plasma was detected by ELISA.

[0040] The sCD73 ELISA kit was purchased from Abcam (ab213761). The concentration of sCD73 in plasma samples from healthy control groups and patients was analyzed according to the kit instructions.

[0041] (4) Statistical analysis

[0042] All data were analyzed using GraphPad 8 or SPSS 24.0 statistical software. The normality of each variable was assessed using the Kolmogorov-Smirnov test. For normally distributed data, unpaired or paired two-tailed Student's t-tests were used to compare two variables. When data were not normally distributed, the Mann-Whitney U test or Wilcoxon paired signed-rank test was used to compare variables for unpaired and paired data, respectively. In comparing more than two independent samples, the Kruskal-Wallis test and Dunn's multiple comparison test were used. The predictive value of sCD73 was assessed using the area under the receiver operating characteristic (ROC) curve (AUC). Comparisons of the probability of immune system recovery in patients were performed using the Kaplan-Meier method. For predictive studies, categorical data were analyzed using the chi-square test or Fisher's exact test. P < 0.05 was considered statistically significant.

[0043] The following embodiments are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise specified, the quantitative experiments in the following embodiments were all performed in triplicate, and the results were averaged.

[0044] Example 1

[0045] 1. The purpose of this experiment is to demonstrate that the concentration of sCD73 in untreated HIV / AIDS patients is higher than that in normal individuals, and that the level of sCD73 is downregulated after antiviral treatment.

[0046] 2. Experimental subjects

[0047] (1) With informed consent and meeting the inclusion criteria, cases were selected for enrollment and their basic personal information was recorded. The HIV / AIDS patient cohort was from Beijing Ditan Hospital, Capital Medical University. In the cross-sectional study, 110 untreated HIV / AIDS patients (TN), 62 HIV / AIDS patients who had received antiretroviral therapy (ART), and 25 age- and sex-matched healthy donors (HC) were recruited. In the cohort study, 171 patients with a baseline CD4 count of less than 350 cells / µL were included, and the viral load remained below 50 copies / mL during routine follow-up after ART.

[0048] (2) Collect plasma from subjects before and after antiviral treatment, and store the samples in a -80°C freezer.

[0049] 3. Reagent and Sample Preparation

[0050] (1) Lyophilized recombinant human CD73 standard: Add 1 ml of sample diluent buffer to one lyophilized CD73 standard to prepare a 10000 pg / ml CD73 standard, labeled S1. Mix thoroughly and let stand at room temperature for 10 minutes. Take another 7 tubes and label them S2-S8. Add 300 μL of sample diluent buffer to each of the 7 tubes. Add the above S1 solution to the second tube and mix well, and so on. Use within two hours after preparation.

[0051] (2) Biotinylated anti-human CD73 antibody: Dilute with antibody diluent buffer at a ratio of 1:100 and mix thoroughly and gently (i.e., add 1 μL of biotinylated anti-human CD73 antibody to 99 μL of antibody dilution buffer). Use within two hours after preparation.

[0052] (3) Avidin-Biotin-Peroxidase Complex (ABC): Dilute with ABC diluent buffer at a ratio of 1:100 (i.e., add 1 μL of ABC to 99 μL of ABC dilution buffer) and mix thoroughly. Use within 1 hour after preparation. The ABC dilution solution needs to be incubated at 37°C for 30 minutes before use.

[0053] (4) Take out the sample stored at -80℃ and thaw it. In order to reduce the influence of platelets and lipids, centrifuge at 4℃, 10000xg for 5 minutes, collect the supernatant, and then mix 50μL of sample with sample diluent buffer at 1:1 (i.e., dilute the sample by 2 times).

[0054] 4. Experimental Procedure:

[0055] ① Add 100µL of standard solutions of 10000pg / mL, 5000pg / mL, 2500pg / mL, 1250pg / mL, 625pg / mL, 312pg / mL, 156pg / mL and 0pg / mL to the 96-well plate. Add 100µL of sample diluent buffer (zero well) to the control well. Add 100µL of the prepared sample solution. Seal the plate with the provided adhesive cap and incubate at 37°C for 90 minutes.

[0056] ② Discard the contents of the 96-well plate, then add 100µL of Biotinylated anti-human CD73 antibody solution and incubate at 37°C for 60 minutes;

[0057] ③ Wash the plate three times with a plate washer;

[0058] ④ Add 100µL of ABC working solution to each well and incubate at 37℃ for 30 minutes;

[0059] ⑤ Wash the plate three times with a plate washer;

[0060] ⑥ Add 90µL of TMB color developing agent to each well and incubate at 37℃ in the dark for 15-20 minutes;

[0061] ⑦ Add 100µL of TMB stop solution to each well;

[0062] ⑧ Within 30 minutes of adding the TMB stop solution, read the absorbance value (OD) at 450 nm using a microplate reader.

[0063] 5. Results and Analysis:

[0064] The baseline concentration of sCD73 was calculated based on the OD value read by the machine and the corresponding standard curve.

[0065] like Figure 1a As shown, the test results revealed that, compared with normal individuals (HC), untreated HIV-infected patients (TN) had significantly increased sCD73 levels in their plasma, while sCD73 levels in the plasma of patients receiving ART treatment were significantly downregulated.

[0066] like Figure 1b As shown, a longitudinal analysis was performed on plasma sCD73 data from 38 HIV / AIDS patients at two time points: baseline and 5.7 years after the start of ART. After 5.7 years of ART treatment, the level of sCD73 in the patients' plasma significantly decreased. These results indicate that the concentration of sCD73 in the plasma of HIV / AIDS patients is correlated with disease status.

[0067] Example 2

[0068] The purpose of this embodiment is to verify the predictive role of baseline sCD73 concentration in patient plasma in clinical outcomes after antiviral therapy.

[0069] To further clarify the relationship between plasma sCD73 and disease, this study retrospectively investigated 171 HIV / AIDS patients. Based on baseline plasma sCD73 levels, and using a cut-off value of 4.111 ng / ml, these 171 patients were divided into two groups: a high sCD73 concentration group (n=70) and a low sCD73 concentration group (n=101).

[0070] The incidence of poor immune reconstitution in these two groups after different durations of ART was analyzed. Poor immune reconstitution was defined as HIV / AIDS patients who achieved sustained virological suppression (HIV RNA <50 copies / mL) and a CD4 cell count less than 350 cells / µL after ART. Conversely, patients with a CD4 cell count greater than or equal to 350 cells / µL after ART were defined as those with good immune reconstitution. The study showed that patients with high sCD73 concentrations had a higher risk of poor immune reconstitution after 1 to 5 years of ART compared to the low sCD73 concentration group (Table 1).

[0071] Table 1. Probability of poor immune reconstitution in patients with low sCD73 and high sCD73 at different time points after ART treatment

[0072]

[0073] Note: The percentages in the table above represent the probability of poor immune reconstitution.

[0074] a The CD4 count result of one patient was missing in the high sCD73 group.

[0075] b The low sCD73 group lacked the CD4 count result of one patient.

[0076] c The CD4 count results for two patients in the high sCD73 group were missing.

[0077] d The CD4 count result of one patient was missing in the high sCD73 group.

[0078] e The high sCD73 group had missing CD4 counts from ten patients, while the low sCD73 group had missing CD4 counts from eight patients.

[0079] After dividing patients into two groups—one with high sCD73 levels and the other with low sCD73 levels—the Kaplan-Meier (KM) method was used to analyze the recovery possibility of the immune system in both groups. The outcome of the Kaplan-Meier analysis was the recovery of CD4 cell count or CD4 / CD8 ratio, defined as the time point at which the CD4 cell count first reached ≥350 cells / µL or the CD4 / CD8 ratio reached ≥0.7 during treatment.

[0080] When CD4 cell count during treatment was used as a marker of immune system recovery after antiviral therapy, experimental results showed that patients with high baseline sCD73 levels (high group) were more prone to poor immune reconstitution (see [link to study]). Figure 2 (a, low indicates low sCD73 level group, high indicates high sCD73 level group). Furthermore, the predictive potential was assessed using receiver operating characteristic (ROC) curves. The area under the curve (AUC) for sCD73 (0.7122 [95% CI 0.6291–0.7953]) was considered statistically significant (P < 0.05). Figure 2 b). The above results suggest that baseline plasma sCD73 levels in HIV patients can effectively predict immune reconstitution after ART.

[0081] When the CD4 / CD8 ratio during treatment was used as a marker of immune system recovery after antiviral therapy, experimental results showed that patients with high baseline sCD73 levels (high group) were more prone to poor immune reconstitution (see [link to study]). Figure 3a (low indicates low sCD73 level group, high indicates high sCD73 level group). Furthermore, the predictive potential was assessed using receiver operating characteristic (ROC) curves. The area under the curve (AUC) for sCD73 (0.6857 [95% CI 0.5986–0.7728]) was considered statistically significant (P < 0.05). Figure 3b This is basically consistent with the experimental results mentioned above.

[0082] The experiment further divided patients into two subgroups: a high-recovery group with a CD4 / CD8 ratio of 0.7 or higher after 4.9 years of ART treatment, and a low-recovery group with a CD4 / CD8 ratio that failed to reach 0.7 after 4.9 years of ART treatment. The results showed that grouping patients using baseline sCD73 levels in their plasma to predict CD4 / CD8 ratio recovery yielded largely the same results as the previous grouping. This indicates that patients with high baseline sCD73 levels are more likely to experience poor immune reconstitution (CD4 / CD8 ratio failing to reach 0.7 after 4.9 years of ART treatment).

[0083] All of the above results indicate that plasma sCD73 can serve as a potential biomarker for immune reconstitution after ART.

[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a reagent capable of detecting sCD73 concentration in plasma in the preparation of products for predicting or assisting in predicting whether immune reconstitution is poor in HIV / AIDS patients after antiviral therapy, characterized in that, The concentration of sCD73 in plasma isolated from the peripheral blood of the patient to be tested was detected, and the detected sCD73 concentration value was compared with a reference value. When the detected sCD73 concentration value was higher than the reference value, it was determined that the patient to be tested had poor immune reconstitution after antiviral treatment. The reference value was the cut-off value of sCD73 concentration obtained from the HIV / AIDS patient population, and the cut-off value was 4.111 ng / ml.

2. The application according to claim 1, characterized in that, The products mentioned are reagent kits, antibody chips, antibody probes, or detection instruments.

3. The application according to claim 2, characterized in that, The kit includes reagents for obtaining peripheral blood from the patient, and / or reagents for separating plasma.

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