hr-HPV outcome prediction markers, kits for their preparation and use

Abstract

The application discloses hr-HPV prognosis prediction markers, a kit prepared from the same and application thereof, and belongs to the technical field of biological detection and molecular immunology, and particularly relates to hr-HPV prognosis prediction markers, which include interferon-gamma, interleukin-2, chemokine gamma interferon-induced protein-10, gamma interferon-induced monocyte factor, interferon-induced protein guanosine acid binding protein 1 and ankyrin repeat domain protein 22. The application also discloses application of the hr-HPV prognosis prediction markers in preparation of hr-HPV infection prognosis diagnosis kits. The hr-HPV prognosis prediction markers are screened by the application, and through a "autologous whole blood-signal amplification-background deduction" system, local immune functions of hr-HPV infected persons can be accurately evaluated, so that the prognosis of hr-HPV infection can be predicted.

Description

Technical Field

[0001] This invention belongs to the field of biological detection and molecular immunology technology, and particularly relates to hr-HPV outcome prediction biomarkers, their preparation kits, and applications. Background Technology

[0002] Persistent infection with high-risk human papillomavirus (hr-HPV) is a necessary factor for the development of cervical cancer. Although hr-HPV testing has high sensitivity in secondary prevention of cervical cancer, the clinical outcomes after hr-HPV infection are significantly heterogeneous: most infected individuals achieve spontaneous remission within 12 to 24 months, while only a small number of individuals develop persistent infection and progress to high-grade squamous intraepithelial lesion (HSIL) and even cervical cancer.

[0003] Currently, clinical cervical cancer screening and triage strategies mainly rely on HPV DNA testing, liquid-based cytology (TCT), and colposcopy. However, these methods are largely static assessments, reflecting only the presence of the virus or morphological changes in host epithelial cells, and cannot accurately reflect the "dynamic" immune function of the host in clearing the virus. This means that a positive hr-HPV result only indicates that "infection has occurred," but cannot accurately predict whether an individual has the ability to effectively clear the virus, leading to a large number of low-risk patients being over-referred to colposcopy.

[0004] Mounting evidence suggests that the cervical local immune microenvironment, particularly the antigen-induced response function of local lymphocytes, is a key site determining persistent hr-HPV infection and lesion progression. However, existing methods for studying local immunity have the following drawbacks: (1) Invasiveness of sampling methods: In-depth research on local immunity usually relies on tissue biopsy. This invasive procedure is not only difficult to be repeated in the screening population, but the local trauma and inflammatory response caused by biopsy will interfere with the judgment of natural immune status and is not suitable for continuous and dynamic monitoring.

[0005] (2) Non-specificity of peripheral blood detection: Traditional peripheral blood mononuclear cell (PBMC) immunological studies mainly reflect systemic immune reserves and cannot be accurately equated with the actual immune effect of cervical lesions.

[0006] (3) Technical limitations of non-invasive samples: Although cervical swabs are a routine and non-invasive clinical sample source, the abundance of immune cells (such as T cells) derived from the local mucosa is extremely low (only about 10 per sample). 3 -10 4 The presence of numerous antigens results in extremely weak induced response signals after antigen stimulation, making it difficult for traditional detection methods to achieve stable and sensitive functional analysis amidst complex background noise.

[0007] In conclusion, there is an urgent clinical need for a testing system that can obtain samples non-invasively and sensitively assess the local antiviral immune function of the cervix, in order to achieve accurate risk stratification and individualized triage management of hr-HPV infected individuals. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention proposes hr-HPV outcome prediction biomarkers, their preparation kits, and applications. This invention has screened and obtained hr-HPV outcome prediction biomarkers. Through the "autologous whole blood-signal amplification-background subtraction" system, the local immune function of hr-HPV infected individuals can be accurately assessed, thereby predicting the outcome of hr-HPV infection.

[0009] To achieve the above objectives, the present invention provides a biomarker for predicting the outcome of hr-HPV, wherein the biomarker is interferon-γ, interleukin-2, chemokine-γ interferon-induced protein-10, interferon-γ induced mononuclear factor, interferon-induced protein guanylate-binding protein 1, and ankyrin repeat domain protein 22.

[0010] This invention also provides the application of the hr-HPV outcome prediction biomarker in the preparation of an hr-HPV infection outcome diagnostic kit.

[0011] Preferably, the application involves detecting the expression levels of hr-HPV outcome predictive markers in cervical exfoliated cells. Subjects whose hr-HPV infection can heal spontaneously exhibit high expression levels of hr-HPV outcome predictive markers in their cervical exfoliated cells, while subjects whose hr-HPV infection progresses to high-grade squamous intraepithelial lesions show a lack of expression levels of hr-HPV outcome predictive markers in their cervical exfoliated cells. The hr-HPV outcome predictive markers are interferon-γ, interleukin-2, chemokine-γ interferon-induced protein-10, interferon-γ induced mononuclear factor, interferon-induced protein guanylate-binding protein 1, and ankyrin repeat domain protein 22.

[0012] The present invention also provides a kit for preparing the hr-HPV outcome prediction biomarkers, the kit comprising: an hr-HPV specific antigen peptide library and a detection system for detecting the hr-HPV outcome prediction biomarkers; the hr-HPV outcome prediction biomarkers are interferon-γ, interleukin-2, chemokine-γ interferon-induced protein-10, γ interferon-induced mononuclear factor, interferon-induced protein guanylate-binding protein 1, and ankyrin repeat domain protein 22.

[0013] Preferably, the method of using the reagent kit is as follows: 1) Extract cervical exfoliated cells and peripheral venous blood from the subjects; 2) Prepare a cervical exfoliated cell suspension from the cervical exfoliated cells described in step 1); 3) Set up detection groups. Detection group 1 consists of the cervical exfoliated cell suspension prepared in step 2), the peripheral venous blood described in step 1), and the hr-HPV specific antigen peptide library, which are co-incubated. Detection group 2 consists of the cervical exfoliated cell suspension prepared in step 2), the peripheral venous blood described in step 1), and the empty vector solution, which are co-incubated. Detection group 3 consists of the peripheral venous blood described in step 1) and the hr-HPV specific antigen peptide library, which are co-incubated as a whole blood background control. 4) After the incubation described in step 3) is completed, detect the hr-HPV outcome prediction markers in the cells of detection group 1, detection group 2 and detection group 3; 5) Based on the test results described in step 4), calculate the diagnostic index. The formula for calculating the diagnostic index is as follows: DI = (S1 - S3) / S2, where DI is the diagnostic index, S1 is the test result of test group 1, S2 is the test result of test group 2, and S3 is the test result of test group 3. 6) Based on the diagnostic index described in step 5), determine the outcome of the subject's hr-HPV infection. The cervical exfoliated cell suspension collected from the subject is mixed with peripheral venous blood from the same source at a specific ratio. The peripheral venous blood serves as a biosignal amplification medium, providing an antigen-presenting (APC) and co-stimulatory environment for the trace amounts of lymphocytes in the cervical exfoliated cell suspension. The hr-HPV specific antigen peptide library serves as an inducing factor.

[0014] Preferably, the cell concentration of the cervical exfoliated cell suspension described in step 2) of the kit usage method is 9 × 10⁻⁶. 4 cells / mL.

[0015] Preferably, in step 3) of the kit usage method, the volume of cervical exfoliated cell suspension in detection group 1 is 50 μL, and the volume of peripheral venous blood is 1 mL; the volume of cervical exfoliated cell suspension in detection group 2 is 50 μL, and the volume of peripheral venous blood in detection group 3 is 1 mL; the co-incubation environment is 37°C and 5% CO2, and the co-incubation time is 18 h.

[0016] Preferably, the detection described in step 4) of the method of using the kit includes one of the following detection methods: HRT-LAMP, qPCR, ddPCR, ELISA, or chemiluminescent immunoassay.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: This invention provides predictive biomarkers for hr-HPV prognosis, their preparation kits, and applications. It achieves accurate prediction of hr-HPV infection prognosis. Through the selection of six biomarkers (IFN-γ, IL-2, CXCL10, CXCL9, GBP1, and ANKRD22), it can clearly distinguish between patients who can spontaneously recover from hr-HPV infection and those who will progress to high-grade squamous intraepithelial lesions (HSIL). The expression levels of these biomarkers are high in cervical exfoliated cells from patients who have recovered, while they are absent or extremely low in those who have progressed. This invention overcomes the technical bottleneck of weak and undetectable local immune signals in non-invasive prenatal testing (NIPT) samples by constructing an autologous whole blood-cervical exfoliated cell co-incubation amplification system. This system can amplify cervical local immune response signals that are originally close to or below the detection limit by 10 to 100 times, transforming weak signals into quantifiable and analyzable high-quality data. This breakthrough overcomes the limitations of traditional cervical swab samples, which often contain few immune cells and have low response rates. The limitations of weak signals are overcome; background interference from systemic circulating immunity is effectively eliminated; by setting up three parallel incubation groups and combining with the difference algorithm, the antigen-specific immune response signal unique to the cervix can be accurately extracted, avoiding interference from non-specific reactions in peripheral blood; a diagnostic index (DI) for quantitatively assessing local cervical immune function is provided, which can intuitively reflect the net increase in the response intensity of local cervical immune cells to hr-HPV antigens. Clinical validation shows that the DI value of the self-healing group is significantly higher than that of the progression group, and the DI has extremely high diagnostic accuracy and triage efficiency; it has the clinical practicality of being non-invasive, repeatable, and suitable for dynamic monitoring. The samples used are cervical exfoliated cells (routine cervical brush sampling) and peripheral venous blood, both of which are routine non-invasive or minimally invasive clinical samples, avoiding the invasiveness and local trauma of tissue biopsy, facilitating repeated and dynamic monitoring of immune status changes in the screened population, and providing an accurate risk stratification tool for hr-HPV positive individuals. In summary, this invention, through an innovative signal amplification system, background subtraction algorithm, and specific biomarker combination, has for the first time achieved accurate assessment of local antiviral immune function of the cervix based on non-invasive samples, which can effectively predict the outcome of hr-HPV infection and provide a new technical means for cervical cancer screening and individualized triage. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1The results of the co-incubation system in Example 1 of this invention demonstrate the amplification effect of the local immune response signal in the cervix. In the figure, A represents the IFN-γ content, B represents the IP-10 content, C represents the IL-2 content, G1 represents the control group, G2 represents the experimental group, and G3 represents the background group. "Representative difference significance analysis; Figure 2 The results of screening and validation of hr-HPV infection outcome predictive biomarkers in Example 1 of the invention are shown, where A represents the progression group S. 模式2 Signal and S 模式1 -S 模式3 Signal, B represents the self-healing group S 模式2 Signal and S 模式1 -S 模式3 Signals: C represents IFN-γ expression level, D represents IL2 expression level, E represents CXCL10 expression level, F represents CXCL9 expression level, G represents GBP1 expression level, H represents ANKRD22 expression level. In the figure, HSIL represents the progressive group, and self-healing represents the self-healing group. "ns" and " "Representative difference significance analysis; Figure 3 This is the verification result of the cervical local immune shunt diagnostic index based on the HRT-LAMP platform in Example 1 of the present invention. In this figure, A is a four-fold graph comparing the diagnostic index with the pathological results. In the graph, TP represents true positive, FP represents false positive, FN represents false negative, and TN represents true negative. B is the ROC curve analysis of the diagnostic index (DI). C is the diagnostic index (DI) of the progression group and the self-healing group. In the graph, HSIL represents the progression group and self-healing represents the self-healing group. Detailed Implementation

[0020] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The specification and embodiments of this invention are merely exemplary. The terms "comprising," "including," "having," "containing," etc., as used herein are open-ended terms, meaning that they include, but are not limited to, these concepts.

[0021] Example 1 I. Verification of the amplification effect of the co-incubation system on local cervical immune response signals: 1. Experimental objective: To verify whether the "autologous whole blood-cervical exfoliated cells" co-incubation system can effectively amplify the immune response signal of local cervical immune cells to specific antigens (such as the hr-HPV peptide library) and solve the problem of low abundance sample signals being below the limit of detection (LOD).

[0022] 2. Experimental Design: Ten hr-HPV16 positive subjects were selected, and the levels of cytokines (interferon-γ, IFN-γ) produced in the following three culture groups were compared: Control group (G1): 50 μL cervical exfoliated cell suspension + 1 mL culture medium + hr-HPV specific antigen peptide library (HPV16E6 / E7 15 / 11 overlapping peptide, purchased from Shanghai Jietai Biotechnology Co., Ltd.) (simulating the existing conventional swab direct detection mode).

[0023] Experimental group (G2): 50 μL cervical exfoliated cell suspension + 1 mL autologous whole blood + hr-HPV specific antigen peptide library (the core amplification system of this invention, including local signal, whole blood background and amplification effect).

[0024] Background group (G3): 1 mL of autologous whole blood + hr-HPV specific antigen peptide library (used to subtract background interference from systemic circulating immunity).

[0025] Evaluation index: Compare the fold difference between the local specific signal intensity extracted from the experimental group (i.e., the result obtained by subtracting the background group G3 signal intensity from the experimental group G2 signal intensity) and the signal intensity (G1) of the control group.

[0026] 3. Experimental steps: (1) Standardized pretreatment of cervical exfoliated cells (to remove mucus interference and enrich immune cells): Stage 1 (Mechanical Elution): Place the collected cervical brush head in 10 mL of RPMI 1640 medium (containing penicillin and antibiotics) (purchased from Thermo Fisher Scientific (China) Co., Ltd.), and vortex at 2000 rpm for 3-5 seconds to allow cells to detach completely. Discard the brush head to obtain a cell suspension. Stage 2 (Washing and Removal): Centrifuge the cell suspension at 800 g for 10 min, discard the supernatant; resuspend in 10 mL of PBS buffer, centrifuge again at 500 g, discard the supernatant containing secretions, and obtain a precipitate. Stage 3 (Physical Sieving and Cell Release): Add 3-5 mL of PBS solution to the precipitate and mix thoroughly. Add the liquid to a 70 μm nylon filter. Use a dropper to draw PBS solution and repeatedly, at high frequency, physically rinse the mucus clumps on the filter surface until the cells in the mucus are observed to be completely released and pass through the filter into the filtrate. Fourth stage (micro-concentration): Collect the filtrate, centrifuge at 800g for 10 min, discard the supernatant, add 50-200 μL of PBS solution to resuspend the filtrate, and prepare a high-concentration local immune cell suspension (cell concentration approximately 9 × 10⁻⁶ cells / mL). 4 (cells / mL).

[0027] (2) Construction of autologous whole blood environment: Simultaneously collect 4 mL of peripheral venous blood from the subject, store it in lithium heparin anticoagulant tubes, keep it at room temperature, and ensure that it enters the incubation program within 4 hours after collection to maintain the optimal activity of immune cells.

[0028] (3) In vitro co-incubation and antigen induction: According to the experimental design groups (G1, G2, G3), the hr-HPV specific antigen peptide library (HPV16 E6 / E7 15 / 11 overlapping peptides, with a final single peptide concentration of 1 μg / mL) was added and incubated at 37℃ and 5% CO2 for 18 h.

[0029] (4) Quantitative reading of the signal: After incubation, samples from each group were collected and centrifuged, and the supernatant was aspirated. The concentration of IFN-γ in the supernatant was quantitatively determined using a high-sensitivity enzyme-linked immunosorbent assay (ELISA) kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.).

[0030] 4. Experimental Results: Overcoming the bottleneck of low abundance detection: Experimental results show that, due to the extremely low local lymphocyte count and lack of necessary antigen presentation and co-stimulatory signals, the IFN-γ signal induced by the control group (G1) is extremely weak, usually near or below the instrument's limit of detection (LOD), making stable quantification impossible (e.g. Figure 1 (As shown in A).

[0031] Significant signal amplification efficiency: In the core amplification co-incubation system constructed in this invention, the local specific signal intensity extracted after background signal subtraction (i.e., the result obtained by subtracting the background group G3 signal intensity from the experimental group G2 signal intensity) was 10 to 100 times higher than that of the control group (G1). This demonstrates that autologous whole blood, as a "biological amplifier," provides a complete immune response microenvironment, achieving cascade amplification of trace local cellular signals (such as...). Figure 1 (As shown in A).

[0032] Validation of the universality of the indicators: Further validation revealed that, in addition to IFN-γ, the core amplification co-incubation system constructed in this invention also exhibited similar amplification effects on key chemokines on the same immune axis, such as interferon-induced protein-10 (IP-10) and interleukin-2 (IL2). Figure 1 China B and Figure 1 (As shown in C).

[0033] In summary, the core amplification co-incubation system described in this invention can transform previously invisible and extremely weak local immune signals into high-quality data that can be quantified and analyzed, providing a key technical foundation for subsequent risk triage of hr-HPV infected individuals.

[0034] II. Screening and validation of hr-HPV infection outcome prediction biomarkers based on transcriptome sequencing (RNA-seq): 1. Experimental objective: Using high-throughput transcriptome sequencing (RNA-seq) technology, under the "autologous whole blood amplification-background subtraction" model, we systematically analyzed the characteristic differences in the response of local cervical immune cells to hr-HPV antigen in subjects with different clinical outcomes, thereby screening out key biomarkers that can accurately predict the spontaneous recovery or progression of HPV infection.

[0035] 2. Subject selection and grouping: Twenty female subjects clinically diagnosed with hr-HPV16 infection were included and divided into two groups based on follow-up results and pathological diagnosis: • Self-healing group (self-healing, n=10): those who were previously hr-HPV16 positive, but achieved spontaneous viral clearance during follow-up and had persistently negative cytological examinations.

[0036] • Progression group (HSIL, n=10): Individuals with persistent hr-HPV16 infection and confirmed by histopathology as high-grade squamous intraepithelial lesion (HSIL).

[0037] 3. Experimental Design and Cultivation Model: Cervical exfoliated cells (treated according to standardized pretreatment of cervical exfoliated cells) and autologous whole blood from each subject were subjected to in vitro induction culture in the following three modes (all with hr-HPV16 E6 / E7 peptide library, working concentration 1 μg / mL, incubated for 18 h): Whole blood response system (Mode 1): 50 μL cervical exfoliated cell suspension + 1 mL autologous whole blood + hr-HPV specific antigen peptide library. Total signal (S) of local immune cells amplified in the autologous whole blood environment. 模式1 ).

[0038] Local cell control group (Mode 2): 50 μL cervical exfoliated cell suspension + 1 mL autologous whole blood + empty carrier solvent (PBS / DMSO). The basal response signal (S) of local immune cells amplified in the autologous whole blood environment. 模式2 ).

[0039] Systemic immune control group (Mode 3): 1 mL autologous whole blood + hr-HPV specific antigen peptide library. Background response signal of the peripheral circulating immune system in subjects (S 模式3 ).

[0040] 4. RNA-seq detection and bioinformatics analysis: Library construction and sequencing: Cell pellets from the above incubation modes were collected, and total RNA was extracted. High-throughput sequencing libraries were constructed, and single-end sequencing was performed using the Illumina platform. Locally specific signal extraction algorithm (S...) 局部 Using bioinformatics techniques, the following difference calculation logic was applied to the gene expression matrix of each subject to extract the cervical local specific response intensity after background subtraction: S 局部 =S 模式1 -S 模式2 -S 模式3 This algorithm eliminates systemic circulating immune interference (Mode 3) and background fluctuations from low-abundance local cells (Mode 2), accurately extracting local-specific transcriptional maps induced by the autologous whole blood environment.

[0041] 5. Results of biomarker screening and feature analysis experiments: (1) Differentially expressed genes (DEG) analysis: In the extracted S 局部 In the signaling pathway, by comparing the self-healing group (self-healing) and the progressive group (HSIL), candidate biomarkers with statistically significant differences were screened for in the IFN-γ signaling pathway (fold change > 2.0, P < 0.05, such as...). Figure 2 China A and Figure 2 (As shown in B).

[0042] (2) Identification of core biomarkers: In the cervical local response signaling of the self-healing group (self-healed), the following genes showed significantly high levels of induced expression, while in the progression group (HSIL), the response signaling of these genes was almost absent or extremely low (e.g. Figure 2 C, Figure 2 D, such as Figure 2 Chinese E, Figure 2 Middle F, such as Figure 2 China G and Figure 2 (H in the middle): effector cytokines IFN-γ and IL2, chemokines interferon-induced protein-10 (CXCL10, IP-10) and interferon-induced mononuclear factor (CXCL9), interferon-induced protein guanylate-binding protein 1 (GBP1) and ankyrin repeat domain protein 22 (ANKRD22).

[0043] 6. Conclusion: Through the core amplification co-incubation system and S described in this invention 局部The interpolation algorithm can specifically identify immune markers highly correlated with the clinical outcome of hr-HPV, including effector cytokines IFN-γ and IL2, chemokines CXCL10 and CXCL9, and interferon-inducible proteins GBP1 and ANKRD22. Patients with spontaneous remission exhibit a strong IFN-γ pathway-induced response in the cervix, while HSIL patients show immune dysfunction in this pathway. This finding provides a clear molecular indicator for assessing cervical local immune function using non-invasive swab samples.

[0044] III. Validation of the cervical local immune shunt diagnostic index based on the HRT-LAMP platform: 1. Experimental objective: Based on the aforementioned experiments, by expanding the clinical sample size (n=160) and using the highly sensitive HRT-LAMP technology (detecting CXCL10 mRNA), we verified the practical application value of the "autologous whole blood-cervical exfoliated cells" co-incubation system constructed in this invention in risk triage of patients with hr-HPV infection, and evaluated its predictive efficacy for high-grade lesions.

[0045] 2. Experimental subjects and grouping: 160 subjects with persistent hr-HPV16 positivity were selected and divided into two groups based on their colposcopy biopsy pathology results and subsequent follow-up outcomes: HSIL group (n=66): pathologically diagnosed as high-grade squamous intraepithelial lesion (CIN3).

[0046] Self-healing group (n=94): Patients with normal cytology who achieved spontaneous negative hr-HPV virus within a 12-month follow-up period.

[0047] 3. Experimental methods: (1) Sample collection and preprocessing: A high-concentration cervical exfoliated cell suspension was obtained by processing the cells according to the aforementioned standardized pretreatment method, and autologous heparin-anticoagulated whole blood was collected from the subjects simultaneously.

[0048] (2) Three parallel co-incubation designs: The samples from each subject were divided into three portions and incubated at 37°C and 5% CO2 for 18 hours: First sample (experimental stimulation group, S1): 50 μL cervical exfoliated cell suspension + 1 mL autologous whole blood + hr-HPV specific antigen peptide library.

[0049] The second part (empty control group, S2): 50 μL cervical exfoliated cell suspension + 1 mL autologous whole blood + empty carrier solvent (PBS / DMSO).

[0050] The third sample (whole blood background group, S3): 1 mL of autologous whole blood + hr-HPV specific antigen peptide library.

[0051] (3) Signal detection: mRNA detection: After incubation, total RNA was extracted from each group of cells, and the copy number of CXCL10 mRNA was quantitatively determined using the HRT-LAMP (High Sensitivity Rapid Reverse Transcription Loop-Mediated Isothermal Amplification) kit (Shenzhen Kuaiyi Molecular Diagnostics Technology Co., Ltd.). ddPCR, qPCR, ELISA, and chemiluminescence detection were performed simultaneously. The detection results showed good consistency with HRT-LAMP, but the detection time for all methods exceeded 1 hour; therefore, HRT-LAMP was preferred.

[0052] 4. Signal processing algorithm (diagnostic index calculation): This invention defines a Diagnostic Index (DI) to quantify the specific activation intensity of local immune cells in the cervix. The calculation formula is as follows: DI = (S1 - S3) / S2. Algorithm explanation and logic: S2 represents the background transcription level of cervical cells and autologous whole blood in the unstimulated state of the subject, serving as a baseline reference for each individual. S1-S3 represent the core subtraction logic of this invention. S1 includes "whole blood amplification effect + systemic immune background + local specific immune signal". By subtracting S3 (signals generated only by stimulation of whole blood), interference caused by systemic inflammation or circulating immune reserves in the subject can be effectively eliminated. The Diagnostic Index reflects the "net growth ratio" generated by local immune cells in the cervix after being stimulated by antigens and amplified by autologous whole blood.

[0053] 5. Experimental Results: (1) Clinical triage efficiency: Self-healing group (high index): The DI value of subjects in this group was significantly higher than that in the HSIL group, indicating that there was a highly efficient specific immune response in the cervix of subjects in this group, which could recognize and produce sufficient cytokines to clear the virus (such as...). Figure 3 (As shown in C).

[0054] HSIL group (low index): The DI values ​​of subjects in this group were generally low, even close to 0, indicating that the local immune function of the cervix was impaired or exhausted, and unable to respond effectively to antigen stimulation, suggesting a very high risk of persistent infection and lesion progression (e.g. Figure 3 As shown in C). Figure 3 As shown in Figure A, this is a four-fold table comparing diagnostic indices with pathological results (the gold standard for clinical diagnosis). In the figure, TP represents true positive, FP represents false positive, FN represents false negative, and TN represents true negative.

[0055] (2) Robustness of detection indicators: ROC curve analysis of the DI index showed that the area under the curve (AUC) for predicting the HSIL group was greater than 0.9 (e.g., ...). Figure 3 (As shown in B).

[0056] The cutoff value for the Diagnostic Index (DI) was determined using receiver operating characteristic (ROC) curve analysis combined with the Youden index method. Specifically, using clinical pathological diagnosis or follow-up outcome as the gold standard, sensitivity and specificity were calculated at different DI thresholds, and the Youden index was further calculated, which is the sum of sensitivity and specificity minus 1. The DI value with the largest Youden index was selected as the optimal cutoff value. In this embodiment, the DI cutoff value of 1.868 was determined using the above method. This threshold corresponds to the best overall performance in terms of sensitivity and specificity, and is therefore used to determine the risk of hr-HPV infection outcome. This cutoff value can be recalibrated as the sample size increases or the applicable population changes.

[0057] According to the diagnostic index in this embodiment, the outcome of the subject's hr-HPV infection is determined as follows: when the diagnostic index is ≥1.868, the outcome of the subject's hr-HPV infection is self-healing; when the diagnostic index is <1.868, the outcome of the subject's hr-HPV infection is high-grade squamous intraepithelial lesion.

[0058] 6. Conclusion: In summary, the "autologous whole blood-signal amplification-background subtraction" system constructed by this invention, combined with HRT-LAMP or ELISA detection, can accurately assess the local immune function of individuals infected with hr-HPV. The obtained diagnostic index (DI) can serve as an important triage tool for clinical cervical cancer screening: a high index indicates a high risk of spontaneous remission, allowing for an "observation and follow-up" strategy; a low index indicates a high risk of lesions, requiring immediate "colposcopy referral or intervention."

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an hr-HPV outcome predictive biomarker in the preparation of an hr-HPV infection outcome diagnostic kit, characterized in that, The hr-HPV outcome prediction biomarker is CXCL10 mRNA; The kit includes an hr-HPV specific antigen peptide library and a detection system for detecting hr-HPV outcome prediction biomarkers; The kit is used to perform the following in vitro detection process: 1) Extract cervical exfoliated cells and peripheral venous blood from the subjects; 2) Prepare a cervical exfoliated cell suspension from the cervical exfoliated cells described in step 1); 3) Set up detection groups. Detection group 1 consists of the cervical exfoliated cell suspension prepared in step 2), the peripheral venous blood described in step 1), and the hr-HPV specific antigen peptide library, which are co-incubated. Detection group 2 consists of the cervical exfoliated cell suspension prepared in step 2), the peripheral venous blood described in step 1), and the empty vector solution, which are co-incubated. Detection group 3 consists of the peripheral venous blood described in step 1) and the hr-HPV specific antigen peptide library, which are co-incubated as a whole blood background control. 4) After the incubation described in step 3) is completed, detect the hr-HPV outcome prediction markers of cells in detection groups 1, 2 and 3; 5) Based on the test results described in step 4), calculate the diagnostic index. The formula for calculating the diagnostic index is as follows: DI = (S1 - S3) / S2, where DI is the diagnostic index, S1 is the test result of test group 1, S2 is the test result of test group 2, and S3 is the test result of test group 3. 6) Based on the diagnostic index described in step 5), obtain the diagnostic index DI for assessing the risk of hr-HPV infection outcome.

2. The application according to claim 1, characterized in that, The cell concentration of the cervical exfoliated cell suspension described in step 2) of the kit usage instructions is 9 × 10⁻⁶. 4 cells / mL.

3. The application according to claim 1, characterized in that, In step 3) of the kit usage method, the volume of cervical exfoliated cell suspension in test group 1 is 50 μL, and the volume of peripheral venous blood is 1 mL; the volume of cervical exfoliated cell suspension in test group 2 is 50 μL, and the volume of peripheral venous blood is 1 mL; the volume of peripheral venous blood in test group 3 is 1 mL; the co-incubation environment is 37℃ and 5% CO2; and the co-incubation time is 18 h.

4. The application according to claim 1, characterized in that, The detection described in step 4) of the kit usage instructions includes one of the following detection methods: HRT-LAMP, qPCR, or ddPCR.

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