A library of differential diagnosis of active tuberculosis and latent tuberculosis infection and a kit

Abstract

The application discloses a library of superior peptide segments and a kit for differential diagnosis of active tuberculosis and latent tuberculosis infection. Whole peptide segments of tuberculosis virulence antigens ESAT-6 and CFP-10 are screened, the interfering peptide segments are removed, and new libraries of superior peptide segments containing the two virulence antigens are obtained. Whole peptide segments of tuberculosis latency-related antigens Rv1733c long peptide and Rv2028c long peptide are screened, the interfering peptide segments are removed, and new libraries of superior peptide segments containing the two latency-related antigens are obtained. The libraries can improve the accuracy of differential diagnosis of ATB and LTBI and reduce the cost. On this basis, the corresponding kit is developed.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a superior peptide segment library and kit for differential diagnosis of active tuberculosis and latent tuberculosis infection. BACKGROUND

[0002] Tuberculosis (TB) is a chronic disease caused by Mycobacterium Tuberculosis (Mtb) infection, which can invade multiple organs of the human body. It is estimated that about 1 / 4 of the world's population is infected with MTB, including active tuberculosis (ATB) and latent tuberculosis infection (LTBI). About 20% of the general population in China is LTBI, and about 5%-10% of them may progress to ATB in their lifetime [1] .

[0003] Early diagnosis of tuberculosis is of great significance for improving cure rate, improving patient prognosis, reducing mortality and transmission risk. However, tuberculosis, especially extrapulmonary tuberculosis, has various clinical manifestations and is difficult to diagnose. Traditional diagnostic methods have many shortcomings. Acid-fast staining of smear has low sensitivity, and mycobacterium culture takes a long time. Nucleic acid detection is not suitable for 30%-50% of patients who cannot obtain suitable samples. In China, only about 50% of patients with pulmonary tuberculosis are diagnosed by etiology, and less than 13% of patients with extrapulmonary tuberculosis are diagnosed by etiology. When etiological evidence is not available, rapid and accurate diagnosis of ATB remains a serious challenge for clinicians. Interferon-γ release assay (IGRA) is one of the immunological detection methods currently used in clinical diagnosis of tuberculosis infection. It can diagnose tuberculosis infection by detecting IFN-γ released after stimulation by MTB-specific antigens, and is more sensitive and specific than traditional tuberculin skin test (TST). The specificity of IGRA in the diagnosis of tuberculosis infection is improved due to the discovery of tuberculosis-specific antigens, early secreting antigen target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) [7-8] These small molecule proteins are specific to MTB and are absent in BCG and non-tuberculous mycobacteria, which makes IGRA not interfered by BCG vaccination and most non-tuberculous mycobacterial infections.

[0004] However, IGRA positive only indicates the presence of TB infection in the body, but cannot distinguish ATB and LTBI [9]. In the process of clinical diagnosis and treatment, especially in general hospitals, many patients have complex conditions and need to be differentiated from ATB. Under the background of LTBI infection rate of about 20% in China, it is crucial to quickly and accurately distinguish ATB from LTBI for clinical decision-making.

[0005] The detection principle of IGRA is based on the cellular immune response of the body to TB-specific antigens ESAT-6 and CFP-10. In fact, the immune response induced by different epitopes of TB-specific antigens may have differences

[10] . Different antigen epitopes may be recognized by different types of specific T cells, activated and initiated specific immune response, induced to secrete different cytokines, formed specific secretion characteristics and patterns. Therefore, through peptide screening, accurate recognition and elimination of those interfering peptides can ensure that the selected peptides only contain specific epitopes related to the target antigen, avoiding the interference of immune response caused by other irrelevant epitopes, which is an effective way to improve the sensitivity and specificity of diagnosis.

[0006] References:

[0007] [1] Ma Huimin. Antigen-specific cellular immune response of Mycobacterium tuberculosis for differential diagnosis of latent tuberculosis infection and active tuberculosis[D]. Beijing Union Medical College, 2021.

[0008] [2] Zhou Guozhong, Luo Qingyi, Luo Shiqi, et al. Interferon γ release assays or tuberculin skin test for detection and management of latent tuberculosis infection: A systematic review and Meta-analysis[J]. The Lancet Infectious Diseases, 2020, 20(12): 1457-1469

[0009] [3] Carranza C, Pedraza-Sanchez S, de Oyarzabal-Mendez E, et al. Diagnosis for latent tuberculosis infection: New alternatives[J]. Frontiers in Immunology, 2020, 11: 2006-2018.

[0010] [4]Hamada Y,Cirillo DM,Matteelli A,et al.Tests for tuber culosis

[0011] infection:Landscape analysis[J].European Respiratory Journal,2021,58(5):2100167-2100178.

[0012] [5]Zhang D,Ma YL,Li F,et al.Intervention effect of berberine on T cell exhaustion induced by persistent stimulation of tuberculosis antigen[J].Journal of Lanzhou University:Medicine Science,2023,49(5):8-13,21.

[0013] [6]Huang Tsishu,Lee SS,Liu Yufan,et al.Evaluation of an AI-based TBAFB smear screening system for laboratory siagnosis on routine practice[J].Sensors,2022,22(21):8497-8506.

[0014] [7]Hong JY,Kim A,Park SY,et al.Screening for Mycobacteriumtuberculosis infection using Beijing / K strain specific peptides in a schooloutbreak cohort[J].Frontiers in Cellular and Infection Microbiology,2021,11:599386-599395.

[0015] [8]Davies LRL,Smith MT,Cizmeci D,et al.IFN-γindependent markers ofMycobacterium tuberculosis exposure among male South African gold miners[J].eBioMedicine,2023,93:104678-104691.

[0016] [9]Tang Jinhua, Huang Yuan, Jiang Shen, et al. QuantiFERO N-TB gold plus combined with HBHA-induced IFN-γ release assay improves the accuracy of identifying tuberculosis disease status[J]. Tuberculosis, 2020, 124: 101966-101972.

[0017]

[10] Girard B, Baum-Jones E, Best RL, et al. Profiling antibody epitopes induced by mRNA-1273 vaccination and boosters. Front Immunol. 2024 Mar 18;15:1285278. SUMMARY

[0018] In view of the difficulty in differentiating active tuberculosis (ATB) from latent tuberculosis infection (LTBI), the present application screens the full peptide segments of tuberculosis virulence antigens ESAT-6 and CFP-10, retains the dominant peptide segments, and eliminates the interfering peptide segments, to obtain a new dominant peptide segment library containing both virulence antigens. The full peptide segments of tuberculosis latency-related antigens Rv1733c long peptide and Rv2028c long peptide are screened, the dominant peptide segments are retained, and the interfering peptide segments are eliminated, to obtain a new dominant peptide segment library containing both latency-related antigens. The accuracy of differentiating ATB from LTBI is improved, and the cost is reduced. On this basis, a corresponding kit is developed. In order to achieve the above-mentioned purposes, the specific technical solutions of the present application are as follows:

[0019] First aspect, screening and construction of virulence antigen dominant peptide segment library

[0020] (I) Virulence antigen peptide segment synthesis

[0021] A full peptide library of tuberculosis virulence antigen ESAT-6 containing 17 peptide segments is constructed; a full peptide library of tuberculosis virulence antigen CFP-10 containing 18 peptide segments is constructed.

[0022] (II) Construction of virulence antigen peptide segment pool

[0023] The ESAT-6 full peptide library (containing 17 peptide segments) is combined into 5 peptide segment pools; the CFP-10 full peptide library (containing 18 peptide segments) is combined into 6 peptide segment pools.

[0024] (III) Immunogenicity evaluation and screening of virulence antigen peptide segment pool

[0025] The FluoroSpot was used to detect the secretion of IFN-γ and IL-2 of peripheral blood mononuclear cells of ATB patients and healthy LTBI persons at the single cell level by using the five peptide pool of ESAT-6 and the six peptide pool of CFP-10 as stimulants respectively; the number and proportion of tubercle-specific T cells secreting different cytokine profiles of IFN-γ and IL-2 in the two groups of people were used to screen and eliminate the peptide segments.

[0026] (IV) Construction of the dominant peptide pool of virulence antigens

[0027] The dominant peptide pool 1, 2, 5 of ESAT-6 and the dominant peptide pool 1, 3, 5 of CFP-10 were selected and combined to form a new dominant peptide pool of virulence antigens.

[0028] Second aspect, screening and construction of the dominant peptide pool of latency-related antigens

[0029] (I) Synthesis of the peptide segments of latency-related antigens

[0030] The Rv1733c long peptide full peptide pool containing 14 peptide segments was constructed; the Rv2028c long peptide full peptide pool containing 19 peptide segments was constructed.

[0031] (II) Construction of the peptide segment pool of latency-related antigens

[0032] The Rv1733c long peptide full peptide pool (containing 14 peptide segments) was combined into five peptide segment pools; the Rv2028c long peptide full peptide pool (containing 19 peptide segments) was combined into six peptide segment pools.

[0033] (III) Immunogenicity evaluation and screening of the peptide segment pool of latency-related antigens

[0034] The FluoroSpot was used to detect the secretion of IFN-γ and IL-2 of peripheral blood mononuclear cells of ATB patients and healthy LTBI persons at the single cell level by using the five peptide segment pools of Rv1733c long peptide and the six peptide segment pools of Rv2028c long peptide as stimulants respectively; the number and proportion of tubercle-specific T cells secreting different cytokine profiles of IFN-γ and IL-2 in the two groups of people were used to screen and eliminate the peptide segments.

[0035] (IV) Construction of the dominant peptide pool of latency-related antigens

[0036] The dominant peptide pool 2, 3, 5 of Rv1733c long peptide and the dominant peptide pool 1, 2, 3 of Rv2028c long peptide were selected and combined to form a new dominant peptide pool of latency-related antigens.

[0037] Third aspect, immunogenicity verification of the dominant peptide pool of virulence antigens and the dominant peptide pool of latency-related antigens

[0038] The immunogenicity of the virulence antigen dominant peptide segment library and the latency related antigen dominant peptide segment library was detected using the FluoroSpot-IFN-γ / IL-2 method.

[0039] Conclusion: Compared with the virulence antigen ESAT-6 combined with CFP-10 and the latency related antigen Rv1733c long peptide combined with Rv2028c long peptide, the virulence antigen dominant peptide segment library and the latency related antigen dominant peptide segment library screened and integrated in the present application retain good immunogenicity and can be further used in clinical evaluation of the accuracy of ATB and LTBI differential diagnosis.

[0040] Fourth aspect, comparison of the virulence antigen dominant peptide segment library and the virulence antigen (ESAT-6 combined with CFP-10) in differential diagnosis of active tuberculosis and latent tuberculosis infection

[0041] Result: Under the stimulation of the virulence antigen dominant peptide segment library, the AUC of the tuberculosis activity index in the differential diagnosis of ATB and LTBI is 0.907, and under the stimulation of the virulence antigen (ESAT-6 combined with CFP-10), the AUC of the tuberculosis activity index in the differential diagnosis of ATB and LTBI is 0.768, which is significantly lower than the differential diagnosis accuracy of the virulence antigen dominant peptide segment library (p=0.043).

[0042] Conclusion: The virulence antigen dominant peptide segment library has higher accuracy in the differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) than the virulence antigen (ESAT-6 combined with CFP-10).

[0043] Fifth aspect, comparison of the latency related antigen dominant peptide segment library and the latency related antigen (Rv1733c long peptide combined with Rv2028c long peptide) in differential diagnosis of active tuberculosis and latent tuberculosis infection

[0044] Result: Under the stimulation of the latency related antigen dominant peptide segment library, the AUC of the tuberculosis activity index in the differential diagnosis of ATB and LTBI is 0.906, and under the stimulation of the latency related antigen (Rv1733c long peptide and Rv2028c long peptide), the AUC of the tuberculosis activity index in the differential diagnosis of ATB and LTBI is 0.795, which is significantly lower than the differential diagnosis accuracy of the latency related antigen dominant peptide segment library (P=0.048).

[0045] Conclusion: The latency related antigen dominant peptide segment library has higher accuracy in the differential diagnosis of ATB and LTBI than the latency related antigen (Rv1733c long peptide combined with Rv2028c long peptide).

[0046] Sixth aspect, further improving the differential diagnosis accuracy of active tuberculosis and latent tuberculosis infection by combining the virulence antigen dominant peptide segment library and the latency related antigen dominant peptide segment library

[0047] Results: Under the stimulation of the virulence antigen dominant peptide library combined with the latency-associated antigen dominant peptide library, the tuberculosis activity index had a differential diagnosis area under the curve (AUC) of 0.972 for ATB and LTBI. When the tuberculosis activity index was set at a cutoff value of 0.184, the sensitivity and specificity of differentiating ATB and LTBI were 100% and 90.5%.

[0048] Conclusion: The combination of the dominant peptide library of virulence antigens and the dominant peptide library of latency-related antigens can realize the differential diagnosis of ATB and LTBI.

[0049] Seventh aspect: kit for differential diagnosis of ATB and LTBI

[0050] The kit is composed of tuberculosis-specific antigens, i.e., the virulence antigen dominant peptide library or the virulence antigen dominant peptide library & latency-related antigen dominant peptide library of the present invention, and reagents;

[0051] The reagents include: 96-well plates pre-coated with IFN-γ and IL-2 monoclonal antibodies; anti-CD28, fluorescein-labeled monoclonal antibodies IFN-γ-FITC and IL-2-biotin, and secondary antibodies anti-FITC-490 and SA-550.

[0052] The steps for using the kit include: test sample processing, antigen stimulation, and incubation detection.

[0053] Compared with the prior art, the present invention has the following advantages:

[0054] 1. The tuberculosis virulence antigen dominant peptide library of this application retains good immunogenicity and has higher accuracy in differentiating active tuberculosis (ATB) from latent tuberculosis infection (LTBI) than virulence antigens (ESAT-6 combined with CFP-10), while also reducing costs.

[0055] 2. The tuberculosis latency-associated antigen dominant peptide library of the present application retains good immunogenicity and has higher accuracy in differentiating active tuberculosis (ATB) from latent tuberculosis infection (LTBI) than the latency-associated antigens (Rv1733c long peptide combined with Rv2028c long peptide), while also reducing costs.

[0056] 3. The tuberculosis virulence antigen dominant peptide library of the present application combined with the tuberculosis latency-related antigen dominant peptide library can realize the differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI). On this basis, a kit containing tuberculosis-specific antigens, namely the virulence antigen dominant peptide library and the latency-related antigen dominant peptide library of the present invention, has been developed to reduce costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1The number and proportion of specific T cells secreting IFN-γ and IL-2 in ATB and LTBI populations stimulated by ESAT-6 whole peptide and five peptide pools;

[0058] Figure 2 The number and proportion of specific T cells secreting IFN-γ and IL-2 in ATB and LTBI populations stimulated by CFP-10 whole peptide and six peptide pools;

[0059] Figure 3 The number and proportion of specific T cells secreting IFN-γ and IL-2 in a certain ATB patient stimulated by CFP-10 whole peptide and six peptide pools;

[0060] Figure 4 The number and proportion of specific T cells secreting IFN-γ and IL-2 in ATB and LTBI populations stimulated by Rv1733c long peptide whole peptide and five peptide pools;

[0061] Figure 5 The number and proportion of specific T cells secreting IFN-γ and IL-2 in ATB and LTBI populations stimulated by Rv2028c long peptide whole peptide and six peptide pools;

[0062] Figure 6 Immunogenicity verification of the dominant peptide pool of virulence antigens combined with ESAT-6 and CFP-10 of tuberculosis virulence antigens;

[0063] Figure 7 Immunogenicity verification of the dominant peptide pool of latency-related antigens combined with Rv1733c long peptide and Rv2028c long peptide of latency-related antigens;

[0064] Figure 8 Comparison of the diagnostic accuracy of ATB and LTBI by the dominant peptide pool of virulence antigens combined with ESAT-6 and CFP-10 of virulence antigens;

[0065] Figure 9 Comparison of the diagnostic accuracy of ATB and LTBI by the dominant peptide pool of latency-related antigens combined with Rv1733c long peptide and Rv2028c long peptide of latency-related antigens;

[0066] Figure 10 The diagnostic accuracy of ATB and LTBI by the dominant peptide pool of virulence antigens combined with the dominant peptide pool of latency-related antigens.

[0067] Wherein Figures 1-10 In the above table,

[0068] *, **, ***, and **** represent P<0.05, P<0.01, P<0.001, and P<0.0001, respectively, indicating that the difference between the two groups has statistical significance. DETAILED DESCRIPTION

[0069] The following examples are intended to illustrate the application but not to limit the scope of the application. If not specified, the technical means used in the examples are the conventional means known to those skilled in the art.

[0070] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0071] All the materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0072] Main experimental materials

[0073]

[0074] Main experimental equipment

[0075]

[0076] Example 1, screening and construction of a library of dominant peptide segments of virulence antigens

[0077] I. Synthesis of virulence antigen peptide segments

[0078] A library of ESAT-6 full peptides containing 17 peptide segments was constructed, each peptide segment being 15 amino acids long with a 10-amino-acid overlap at both ends and having a purity of ≥ 95%; a library of CFP-10 full peptides containing 18 peptide segments was constructed, each peptide segment being 15 amino acids long with a 10-amino-acid overlap at both ends and having a purity of ≥ 95%.

[0079] II. Construction of virulence antigen peptide segment pools

[0080] The library of ESAT-6 full peptides (containing 17 peptide segments) was combined into 5 peptide segment pools, each containing 3-4 peptide segments, and the library of CFP-10 full peptides (containing 18 peptide segments) was combined into 6 peptide segment pools, each containing 3 peptide segments; the sequences of the peptide segments of the tubercle virulence antigens ESAT-6 and CFP-10 and the grouping information of the peptide segment pools are shown in Table 1.

[0081] Table 1. Sequences of peptide segments of tubercle virulence antigens and grouping information of peptide segment pools

[0082]

[0083]

[0084] III. Immunogenicity evaluation and screening of virulence antigen peptide segment pools

[0085] (I) Methods

[0086] 1. Collect peripheral blood from patients with ATB and healthy people with LTBI (T-SPOT.TB positive) who have not received anti-tuberculosis treatment and have no underlying diseases, isolate PBMC, and prepare cell suspension;

[0087] 2. Use different peptide pools in Table 1 as stimulants respectively, and use FluoroSpot to detect the secretion of IFN-γ and IL-2 at the single cell level.

[0088] 3. According to the number and proportion of specific T cells secreting different cytokines in the two groups of people, screen and eliminate peptide segments. The dominant peptide segment is screened by the number of specific T cells secreting total IFN-γ and total IL-2. The interfering peptide segment is eliminated by the trend that the proportion of specific T cells secreting single IFN-γ in the ATB group is higher than that in the LTBI group, and the proportion of specific T cells secreting single IL-2 in the LTBI group is higher than that in the ATB group.

[0089] (ii) Results

[0090] Different peptide pools of virulence antigens have different secretion characteristics in ATB and LTBI populations, as shown in Table 2. Figure 1 As shown in Table 2, the number of specific T cells secreting IFN-γ and IL-2 after stimulation by different peptide pools in ESAT-6 is different. After stimulation by peptide pools 1, 2, 4, and 5, the proportion of specific T cells secreting single IFN-γ in ATB is higher than that in LTBI. After stimulation by peptide pool 3, the proportion in ATB and LTBI shows opposite secretion characteristics. After stimulation by peptide pools 1, 2, and 5, the proportion of specific T cells secreting single IL-2 in LTBI is higher than that in ATB. After stimulation by peptide pool 3, the proportion shows opposite secretion characteristics. After stimulation by peptide pool 4, the proportions of the two groups are similar. Eliminate peptide pools 3 and 4 with opposite or similar secretion characteristics, and retain dominant peptide pools 1, 2, and 5.

[0091] In CFP-10, Figure 2 ), the number of specific T cells secreting IFN-γ and IL-2 after stimulation by different peptide pools is different. After stimulation by peptide pools 1, 3, 5, and 6, the proportion of specific T cells secreting single IFN-γ in ATB is higher than that in LTBI. After stimulation by peptide pools 2 and 4, the proportion in ATB and LTBI shows opposite secretion characteristics. After stimulation by peptide pools 1, 3, and 5, the proportion of specific T cells secreting single IL-2 in LTBI is higher than that in ATB. After stimulation by peptide pools 2 and 4, the proportion shows opposite secretion characteristics. In peptide pool 6, the proportions of the two groups are similar. Eliminate peptide pools 2, 4, and 6 with opposite or similar secretion characteristics, and retain dominant peptide pools 1, 3, and 5.

[0092] Take a certain ATB patient as an example, and the specific screening results are shown in Table 3. Figure 3The dominant peptide pool 1, 3, 4, 5, 6 can be screened by the number of total secreted IFN-γ specific T cells and total secreted IL-2 specific T cells, with the characteristics of CFP-10 full peptide library secreting specific T cells as a reference; according to the proportion of single secreted IFN-γ specific T cells being higher than the proportion of single secreted IL-2 specific T cells in the ATB, the peptide pool 4 and the peptide pool 6 are eliminated.

[0093] Four, construction of the dominant peptide pool of virulence antigens

[0094] The dominant peptide pool 1, 2, 5 of ESAT-6 and the dominant peptide pool 1, 3, 5 of CFP-10 are selected and combined into a new dominant peptide pool of virulence antigens, and the peptide sequence of the dominant peptide pool of virulence antigens is shown in Table 2 as antigen 1.

[0095] Table 2, peptide sequence of the dominant peptide pool of virulence antigens

[0096]

[0097]

[0098] Example 2, screening and construction of the dominant peptide pool of latency-related antigens

[0099] One, synthesis of the peptide segment of latency-related antigens

[0100] A Rv1733c long peptide full peptide library containing 14 peptide segments is constructed, each peptide segment is 28 amino acids long, and the two ends are overlapped by 14 amino acids, and the purity is ≥80%; a Rv2028c full peptide library containing 19 peptide segments is constructed, each peptide segment is 28 amino acids long, and the two ends are overlapped by 14 amino acids, and the purity is ≥80%.

[0101] Two, construction of the peptide segment pool of latency-related antigens

[0102] The Rv1733c long peptide full peptide library (containing 14 peptide segments) is combined into 5 peptide segment pools, each containing 2-3 peptide segments, and the Rv2028c long peptide full peptide library (containing 19 peptide segments) is combined into 6 peptide segment pools, each containing 3-4 peptide segments. The peptide sequence of each peptide segment of the long peptide of the latency-related antigens Rv1733c and Rv2028c and the grouping information of the peptide segment pool are shown in Table 3.

[0103] Table 3, peptide sequence of each peptide segment of the latency-related antigens and grouping information of the peptide segment pool

[0104]

[0105]

[0106]

[0107] Three, the immunogenicity evaluation and screening of latency-related antigen peptide pool

[0108] (I) Method

[0109] 1. Patients with ATB and healthy individuals with LTBI (T-SPOT.TB positive) were enrolled without anti-tuberculosis treatment and underlying diseases. Peripheral blood was collected, PBMC was isolated, and cell suspension was prepared;

[0110] 2. Different peptide pools in Table 3 were used as stimulants respectively, and FluoroSpot was used to detect the secretion of IFN-γ and IL-2 at the single cell level;

[0111] 3. The number and proportion of specific T cells secreting different cytokines in the two groups were used to screen and eliminate peptides. The number of specific T cells secreting total IFN-γ and total IL-2 was used to screen dominant peptides; the trend that the proportion of specific T cells secreting single IFN-γ in the ATB group was lower than that in the LTBI group, and the proportion of specific T cells secreting single IL-2 in the LTBI group was higher than that in the ATB group was used to eliminate interfering peptides.

[0112] (II) Results

[0113] Different peptide pools of latency-related antigens have different secretion characteristics in ATB and LTBI populations, such as Figure 4 As shown in Table 1, in the Rv1733 long peptide, the number of specific T cells secreting IFN-γ and IL-2 after stimulation by different peptide pools was different. After stimulation by all peptide pools, the proportion of specific T cells secreting single IFN-γ in ATB was lower than that in LTBI, and the proportion of specific T cells secreting single IL-2 in ATB was higher than that in LTBI, with consistent trends. Peptide pools 1 and 4 with weak reaction intensity were eliminated, and dominant peptide pools 2, 3, and 5 were retained.

[0114] In the Rv2028c long peptide, Figure 5 ), the number of specific T cells secreting IFN-γ and IL-2 after stimulation by different peptide pools was different. After stimulation by peptide pools 1, 2, 3, and 5, the proportion of specific T cells secreting single IFN-γ in ATB was lower than that in LTBI, and after stimulation by peptide pools 4 and 6, the proportion in ATB and LTBI showed opposite secretion characteristics; after stimulation by peptide pools 1, 2, 3, 4, and 5, the proportion of specific T cells secreting single IL-2 in ATB was higher than that in LTBI, and after stimulation by peptide pool 6, the proportion in ATB and LTBI showed opposite secretion characteristics. Peptide pools 4 and 6 with opposite secretion characteristics and peptide pool 5 with weak reaction intensity were eliminated, and dominant peptide pools 1, 2, and 3 were retained.

[0115] Four, construction of dominant peptide pool of latency-related antigens

[0116] The dominant peptide pools 2, 3, and 5 of the Rv1733c long peptide and the dominant peptide pools 1, 2, and 3 of the Rv2028c long peptide were selected and combined into a new latency-associated antigen dominant peptide library. The dominant peptide sequences of the latency-associated antigen peptide library are shown in Table 4 and are used as antigen 2.

[0117] Table 4. Peptide sequences of the dominant peptide library of latent-associated antigens

[0118]

[0119]

[0120] Example 3: Immunogenicity Verification of Virulence Antigen Dominant Peptide Libraries and Latency-Related Antigen Dominant Peptide Libraries

[0121] 1. Methods

[0122] Seventy clinical patients with positive T-SPOT.TB test were enrolled. Peripheral blood was collected and PBMCs were isolated. The immunogenicity of the dominant peptide library of virulence antigens and the dominant peptide library of latency-associated antigens was detected using the FluoroSpot-IFN-γ / IL-2 method.

[0123] 2. Results

[0124] like Figure 6 As shown, there was no significant difference in the frequencies of specific T cells secreting total IFN-γ, total IL-2, and dual IFN-γ / IL-2 after stimulation with the dominant peptide library of virulence antigens (antigen 1) compared with those secreted by ESAT-6 combined with CFP-10 (p>0.05). The correlation coefficients for the frequencies of specific T cells secreting total IFN-γ, total IL-2, and dual IFN-γ / IL-2 were 0.947 (p<0.001), 0.763 (p<0.001), and 0.993 (p<0.001), respectively.

[0125] like Figure 7 As shown in the figure, compared with the Rv1733c long peptide combined with the Rv2028c long peptide, there was no significant difference in the frequencies of specific T cells that secreted total IFN-γ, total IL-2, and dual IFN-γ / IL-2 after stimulation with the latency-related antigen dominant peptide library (antigen 2) (p>0.05). The correlation coefficients between the two were 0.674 (p<0.001), 0.448 (p=0.025), and 0.692 (p<0.001) in the frequencies of specific T cells that secreted total IFN-γ, total IL-2, and dual IFN-γ / IL-2, respectively.

[0126] 3. Conclusion

[0127] Compared with CFP-10 combined with Mtb virulence antigen ESAT-6, latent related antigen Rv1733c long peptide combined with Rv2028c long peptide, the Mtb virulence antigen dominant peptide library (antigen 1) and the latent related antigen dominant peptide library (antigen 2) screened and integrated by the present application retain good immunogenicity, and can be further used in clinical evaluation of the accuracy of ATB and LTBI differential diagnosis.

[0128] Example 4, Comparison of virulence antigen dominant peptide library (antigen 1) and virulence antigen (ESAT-6 combined with CFP-10, E&C) in differential diagnosis of active tuberculosis and latent tuberculosis infection

[0129] I. Method

[0130] 1. 11 cases of ATB patients diagnosed by etiology and 46 cases of LTBI patients (T-SPOT.TB test positive) needing to be differentiated from ATB were included.

[0131] 2. The virulence antigen dominant peptide library (antigen 1) and the virulence antigen (ESAT-6 and CFP-10) were respectively used as stimulants, and the FluoroSpot method was used to detect the number and proportion of single secretion IFN-γ, single secretion IL-2, and double secretion IFN-γ and IL-2 specific T cells at the single cell level, and the above six indicators were fitted into the tuberculosis activity index by using the binary logistic regression.

[0132] II. Results

[0133] As shown in Figure 8 , under the stimulation of the virulence antigen dominant peptide library, the tuberculosis activity index had an AUC of 0.907 in the differential diagnosis of ATB and LTBI, and under the stimulation of the virulence antigen (ESAT-6 combined with CFP-10), the tuberculosis activity index had an AUC of 0.768 in the differential diagnosis of ATB and LTBI, which was significantly lower than the differential diagnosis accuracy of the virulence antigen dominant peptide library (P=0.043).

[0134] III. Conclusion

[0135] The virulence antigen dominant peptide library (antigen 1) has higher accuracy in the differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) than the virulence antigen (ESAT-6 combined with CFP-10, E&C).

[0136] Example 5, Comparison of latent related antigen dominant peptide library (antigen 2) and latent related antigen (Rv1733c long peptide combined with Rv2028c long peptide, 1733&2028) in differential diagnosis of active tuberculosis and latent tuberculosis infection

[0137] I. Method

[0138] 1. 14 cases of ATB patients diagnosed by etiology and 30 cases of LTBI patients (T-SPOT.TB test positive) that need to be differentiated from ATB.

[0139] 2. The latent related antigen dominant peptide library and the latent related antigen (Rv1733c long peptide and Rv2028c long peptide) were used as stimulants respectively, and the FluoroSpot method was used to detect the number and proportion of single secretion IFN-γ, single secretion IL-2, double secretion IFN-γ and IL-2 specific T cells at the single cell level, and the above six indicators were fitted into the tuberculosis activity index by using binary logistic regression.

[0140] II. Results

[0141] As shown in Figure 9 , under the stimulation of the latent related antigen dominant peptide library (antigen 2), the tuberculosis activity index had an AUC of 0.906 in the differential diagnosis of ATB and LTBI, and under the stimulation of the latent related antigen (Rv1733c long peptide and Rv2028c long peptide, 1733&2028), the tuberculosis activity index had an AUC of 0.795 in the differential diagnosis of ATB and LTBI, which was significantly lower than the differential diagnosis accuracy of the latent related antigen dominant peptide library (antigen 2) (P=0.048).

[0142] III. Conclusion

[0143] The latent related antigen dominant peptide library (antigen 2) has higher accuracy in the differential diagnosis of ATB and LTBI than the latent related antigen (Rv1733c long peptide and Rv2028c long peptide).

[0144] Example 6, the virulence antigen dominant peptide library combined with the latent related antigen dominant peptide library (antigen 1 & antigen 2) further improves the accuracy of the differential diagnosis of active tuberculosis and latent tuberculosis infection

[0145] I. Methods

[0146] 1. 11 cases of ATB patients diagnosed by etiology and 46 cases of LTBI patients (T-SPOT.TB test positive) that need to be differentiated from ATB.

[0147] 2. The virulence antigen dominant peptide library combined with the latent related antigen dominant peptide library was used as a stimulant, and the FluoroSpot method was used to detect the number and proportion of single secretion IFN-γ, single secretion IL-2, double secretion IFN-γ and IL-2 specific T cells at the single cell level, and the above six indicators were fitted into the tuberculosis activity index by using binary logistic regression.

[0148] II. Results

[0149] AsFigure 10 As shown, under the stimulation of the virulence antigen dominant peptide library combined with the latent related antigen dominant peptide library (antigen 1 & antigen 2), the AUC of the tuberculosis activity index for the differential diagnosis of ATB and LTBI is 0.972, and when the tuberculosis activity index is taken as the cutoff value of 0.184, the sensitivity for the differential diagnosis of ATB and LTBI is 100%, and the specificity is 90.5%.

[0150] III. Conclusion

[0151] The virulence antigen dominant peptide library combined with the latent related antigen dominant peptide library (antigen 1 & antigen 2) can realize the differential diagnosis of ATB and LTBI.

[0152] Example 7, kit for differential diagnosis of ATB and LTBI

[0153] I. Kit composition

[0154] Reagents: pre-coated IFN-γ, IL-2 monoclonal antibody 96-well plate; anti-CD28, fluorescein-labeled monoclonal antibody IFN-γ-FITC and IL-2-biotin, secondary antibody anti-FITC-490 and SA-550;

[0155] Tuberculosis-specific antigens: the virulence antigen dominant peptide library and the latent related antigen dominant peptide library of the present application.

[0156] II. Kit use steps

[0157] 1. Sample processing: collect human peripheral blood, separate and extract peripheral blood mononuclear cells, and prepare a cell suspension with AIM-V medium;

[0158] 2. Antigen stimulation: add the virulence antigen dominant peptide library and the latent related antigen dominant peptide library (antigen 1 or antigen 1 & antigen 2) of the present application, add peripheral blood mononuclear cells and anti-CD28;

[0159] 3. Incubation detection: after antigen stimulation, incubate the sample at 37℃ for 18 hours, add fluorescein-labeled monoclonal antibody IFN-γ-FITC and IL-2-biotin; avoid light and dry at room temperature for 2h; add secondary antibody anti-FITC-490 and SA-550; avoid light and dry at room temperature for 1h; count the specific T cells secreting IFN-γ and IL-2, calculate the number and proportion of single-secreting IFN-γ, single-secreting IL-2, double-secreting IFN-γ and IL-2 specific T cells, and fit the tuberculosis activity index.

[0160] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.

Claims

1. An antigenic peptide segment library for differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI), characterized in that, the amino acid sequence of the antigenic peptide segment library is composed of the peptide segments shown as SEQ ID NO:1-SEQ ID NO:6, SEQ ID NO:14-SEQ ID NO:20, SEQ ID NO:24-SEQ ID NO:26, SEQ ID NO:30-SEQ ID NO:32, SEQ ID NO:39-SEQ ID NO:44 and SEQ ID NO:48-SEQ ID NO:58, the antigenic peptide segment library is composed of the dominant peptide segment pool of virulence antigens and latency-related antigens, the virulence antigens are ESAT-6 and CFP-10, and the latency-related antigens are Rv1733c long peptide and Rv2028c long peptide, the dominant peptide segment pool of the ESAT-6 is: SEQ ID NO:1-SEQ ID NO:3, SEQ ID NO:4-SEQ ID NO:6, SEQ ID NO:14-SEQ ID NO:17, the dominant peptide segment pool of the CFP-10 is: SEQ ID NO:18-SEQ ID NO:20, SEQ ID NO:24-SEQ ID NO:26, SEQ ID NO:30-SEQ ID NO:32, the dominant peptide segment pool of the Rv1733c long peptide is: SEQ ID NO:39-SEQ ID NO:41, SEQ ID NO:42-SEQ ID NO:44, SEQ ID NO:48-SEQ ID NO:49, the dominant peptide segment pool of the Rv2028c long peptide is: SEQ ID NO:50-SEQ ID NO:52, SEQ ID NO:53-SEQ ID NO:55, SEQ ID NO:56-SEQ ID NO:

58.

2. The antigenic peptide pool of claim 1, wherein Under the stimulation of the antigenic peptide segment library, the AUC of the tuberculosis activity index for differentiating ATB from LTBI is 0.972, and when the tuberculosis activity index is taken as the cutoff value of 0.184, the sensitivity for differentiating ATB from LTBI is 100%, and the specificity is 90.5%.

3. An antigenic peptide segment library for differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI), characterized in that, the amino acid sequence of the antigenic peptide segment library is composed of the peptide segments shown as SEQ ID NO:1-SEQ ID NO:6, SEQ ID NO:14-SEQ ID NO:20, SEQ ID NO:24-SEQ ID NO:26 and SEQ ID NO:30-SEQ ID NO:32, the antigenic peptide segment library is composed of the dominant peptide segment pool of virulence antigens ESAT-6 and CFP-10, the dominant peptide segment pool of the ESAT-6 is: SEQ ID NO:1-SEQ ID NO:3, SEQ ID NO:4-SEQ ID NO:6, SEQ ID NO:14-SEQ ID NO:17, The advantage peptide segment pool of the CFP-10 is: SEQ ID NO: 18-SEQ ID NO: 20, SEQ ID NO: 24-SEQ ID NO: 26, SEQ ID NO: 30-SEQ ID NO:

32.

4. The antigenic peptide pool of claim 3, wherein Under the stimulation of the antigen peptide segment library, the AUC of the tuberculosis activity index in the differential diagnosis of ATB and LTBI is 0.

907.

5. A kit for differentiating active tuberculosis, ATB, from latent tuberculosis infection, LTBI, characterized in that, The kit comprises the antigen peptide segment library as claimed in any of claims 1 or 3.

6. The kit of claim 5, wherein The kit further comprises reagents: anti-CD28, pre-coated IFN-γ, IL-2 monoclonal antibody, fluorescein-labeled monoclonal antibody IFN-γ-FITC, IL-2-biotin, secondary antibody anti-FITC-490 and SA-550.

7. The kit of claim 5, wherein The use steps of the kit are: (1) sample detection processing: collecting human peripheral blood, separating and extracting peripheral blood mononuclear cells, and preparing cell suspension with AIM-V medium; (2) antigen stimulation: adding the antigen peptide segment library as claimed in any of claims 1 or 3, adding peripheral blood mononuclear cells and anti-CD28; (3) incubation detection: after antigen stimulation, incubating the sample at 37 DEG C for 18 hours, adding fluorescein-labeled monoclonal antibody IFN-γ-FITC and IL-2-biotin; incubating in a dark dry room at room temperature for 2 hours; adding secondary antibody anti-FITC-490 and SA-550; incubating in a dark dry room at room temperature for 1 hour; counting the specific T cells secreting IFN-γ and IL-2, calculating the number and proportion of single-secretion IFN-γ, single-secretion IL-2, double-secretion IFN-γ and IL-2 specific T cells, and fitting the tuberculosis activity index.

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