Advantages peptide fragment library and kit for differential diagnosis of active tuberculosis and latent tuberculosis infection

Abstract

The invention discloses a dominant peptide fragment library and a kit for differential diagnosis of active tuberculosis and latent tuberculosis infection, which are characterized in that full peptide fragments of tuberculosis virulence antigens ESAT-6 and CFP-10 are screened, dominant peptide fragments are retained, interference peptide fragments are removed, and a new dominant peptide fragment library simultaneously containing two virulence antigens is obtained; according to the present invention, the total peptide fragments of the tuberculosis latent related antigen Rv1733c long peptide and the tuberculosis latent related antigen Rv2028c long peptide are screened, the dominant peptide fragments are retained, the interference peptide fragments are removed, the new dominant peptide fragment library simultaneously containing the two latent related antigens is obtained, the accuracy of differential diagnosis of ATB and LTBI can be improved, the cost can be reduced, and the corresponding kit is developed on the basis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a superior peptide segment library and a kit for differential diagnosis of active tuberculosis and latent tuberculosis infection. Background Art

[0002] Tuberculosis (TB) is a chronic disease caused by infection with Mycobacterium tuberculosis (Mtb), which can invade multiple tissues and 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 has LTBI, and about 5%-10% of them may develop ATB in their lifetime. [1] .

[0003] Early diagnosis of tuberculosis is of great significance for improving cure rates, improving patient prognosis, reducing mortality and transmission risks. However, tuberculosis, especially extrapulmonary tuberculosis, has diverse clinical manifestations and is difficult to diagnose. Traditional diagnostic methods have many shortcomings. Smear acid-fast staining has low sensitivity and mycobacterial culture takes a long time. Nucleic acid testing is not applicable for 30%-50% of patients because they cannot obtain suitable samples. In my country, only about 50% of pulmonary tuberculosis patients are diagnosed with pathogens, and less than 13% of extrapulmonary tuberculosis patients. When pathogenic evidence cannot be obtained, rapid and accurate diagnosis of ATB remains a severe challenge facing the clinic. The interferon-γ release assay (IGRA) is one of the immunological detection methods currently used in clinical diagnosis of tuberculosis infection. It diagnoses tuberculosis infection by detecting IFN-γ released after stimulation with MTB-specific antigens, which is more sensitive and specific than the traditional tuberculin skin test (TST). The specificity of IGRA in diagnosing tuberculosis infection has been 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 proteins are unique to MTB and are absent in BCG and non-tuberculous mycobacteria, which makes IGRA unaffected by BCG vaccination and the vast majority of non-tuberculous mycobacterial infections.

[0004] However, a positive IGRA test only indicates the presence of tuberculosis infection in the body, but cannot differentiate between 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. In the context of the LTBI infection rate of about 20% in my country, 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 body's cellular immune response to tuberculosis-specific antigens ESAT-6 and CFP-10. In fact, the immune response induced by different epitopes of tuberculosis-specific antigens may be different.

[10] Different antigen epitopes may be recognized by different types of specific T cells, activating and initiating specific immune responses, inducing the secretion of different cytokines, and forming specific secretion characteristics and patterns. Therefore, it is an effective way to improve diagnostic sensitivity and specificity to accurately identify and eliminate those interfering peptides through peptide screening, ensure that the selected peptides only contain specific epitopes related to the target antigen, and avoid immune response interference caused by other irrelevant epitopes.

[0006] References:

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

[0008] [2] Zhou Guozhong, Luo Qingyi, Luo Shiqi, et al. Interferonγrelease assays or tuberculin skin test for detection and management of latent tuberculosis infection: A systematic review and Meta-analysis[J]. The LancetInfectious 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 alter-natives[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 Dan, Ma Yanlin, Li Fei, et al. Intervention effect of berberine on T cell exhaustion induced by continuous stimulation of tuberculosis antigens[J]. Journal of Lanzhou University: Medical Edition, 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 of Mycobacterium 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 pluscombined with HBHA-induced IFN-γrelease assay improves the accuracy ofidentifying 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-1273vaccination and boosters. Front Immunol. 2024Mar18;15:1285278. Summary of the invention

[0018] In view of the current difficulties in differential diagnosis of active tuberculosis and latent tuberculosis infection, the present application screens the full peptide segments of tuberculosis virulence antigens ESAT-6 and CFP-10, retains the dominant peptide segments, removes the interfering peptide segments, and obtains a new dominant peptide segment library containing both virulence antigens. The full peptide segments of tuberculosis latent-related antigens Rv1733c long peptide and Rv2028c long peptide are screened, the dominant peptide segments are retained, and the interfering peptide segments are removed to obtain a new dominant peptide segment library containing both latent-related antigens. The accuracy of differential diagnosis of ATB and LTBI can be improved, while reducing costs. On this basis, a corresponding kit is developed. To achieve the above purpose, the specific technical scheme of the present invention is as follows:

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

[0020] (I) Synthesis of toxic antigen peptides

[0021] A complete peptide library of tuberculosis virulence antigen ESAT-6 containing 17 peptides was constructed; a complete peptide library of tuberculosis virulence antigen CFP-10 containing 18 peptides was constructed.

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

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

[0024] (III) Evaluation and screening of immunogenicity of virulence antigen peptide pool

[0025] FluoroSpot was used to detect the secretion of IFN-γ and IL-2 at the single-cell level in peripheral blood mononuclear cells of ATB patients and healthy LTBI subjects, using 5 peptide pools of ESAT-6 and 6 peptide pools of CFP-10 as stimuli respectively. The peptides were screened and eliminated based on the number and proportion of tuberculosis-specific T cells with different cytokine profiles secreting IFN-γ and IL-2 in the two groups.

[0026] (IV) Construction of a library of dominant peptide fragments of virulence antigens

[0027] The dominant peptide pools 1, 2, and 5 of ESAT-6 and the dominant peptide pools 1, 3, and 5 of CFP-10 were selected and combined into a new virulence antigen dominant peptide library.

[0028] Second aspect: Screening and construction of the dominant peptide library of latency-related antigens

[0029] (I) Synthesis of latency-related antigen peptides

[0030] A complete peptide library of Rv1733c long peptide containing 14 peptide segments was constructed; a complete peptide library of Rv2028c long peptide containing 19 peptide segments was constructed.

[0031] (II) Construction of latency-related antigen peptide pool

[0032] The full peptide library of the Rv1733c long peptide (containing 14 peptides) was combined into 5 peptide pools; the full peptide library of the Rv2028c long peptide (containing 19 peptides) was combined into 6 peptide pools.

[0033] (III) Evaluation and screening of immunogenicity of latency-related antigen peptide pools

[0034] Five peptide pools of Rv1733c long peptide and six peptide pools of Rv2028c long peptide were used as stimuli, and FluoroSpot was used to detect the secretion of IFN-γ and IL-2 at the single-cell level in peripheral blood mononuclear cells of ATB patients and healthy LTBI subjects. The peptides were screened and eliminated based on the number and proportion of tuberculosis-specific T cells with different cytokine spectra secreting IFN-γ and IL-2 in the two groups of people.

[0035] (IV) Construction of a library of dominant peptides of latency-related antigens

[0036] 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 latent-associated antigen dominant peptide library.

[0037] Third, the immunogenicity validation of the virulence antigen dominant peptide library and the latency-related antigen dominant peptide library

[0038] The FluoroSpot-IFN-γ / IL-2 method was used to detect the immunogenicity of the dominant peptide library of virulence antigens and the dominant peptide library of latency-related antigens.

[0039] Conclusion: Compared with the Mtb virulence antigen ESAT-6 combined with CFP-10 and the latency-associated antigen Rv1733c long peptide combined with Rv2028c long peptide, the Mtb virulence antigen dominant peptide library and latency-associated antigen dominant peptide library constructed after current screening and integration in the present invention retain good immunogenicity and can be further used in clinical practice to evaluate the accuracy of differential diagnosis of ATB and LTBI.

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

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

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

[0043] Fifth aspect: Comparison of the latency-associated antigen dominant peptide library and latency-associated antigen (Rv1733c long peptide combined with Rv2028c long peptide) in differential diagnosis of active tuberculosis and latent tuberculosis infection

[0044] Results: Under the stimulation of the latency-associated antigen dominant peptide library, the AUC of the tuberculosis activity index for differential diagnosis of ATB and LTBI was 0.906. Under the stimulation of the latency-associated antigens (Rv1733c long peptide and Rv2028c long peptide), the AUC of the tuberculosis activity index for differential diagnosis of ATB and LTBI was 0.795, which was significantly lower than the differential diagnosis accuracy of the latency-associated antigen dominant peptide library (P=0.048).

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

[0046] Sixth aspect: The combination of the virulence antigen dominant peptide library and the latency-related antigen dominant peptide library further improves the accuracy of differential diagnosis between active tuberculosis and latent tuberculosis infection

[0047] Results: Under the stimulation of the virulence antigen dominant peptide library combined with the latency-related antigen dominant peptide library, the AUC of the tuberculosis activity index for the differential diagnosis of ATB and LTBI was 0.972. When the tuberculosis activity index was 0.184 as the cutoff value, 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] Aspect 7: Kits 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 beneficial effects of this application are:

[0054] 1. The tuberculosis virulence antigen advantage peptide library of the present application retains good immunogenicity, and has higher accuracy in differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) than virulence antigens (ESAT-6 combined with CFP-10), and reduces costs;

[0055] 2. The tuberculosis latent-related antigen advantage peptide library of the present application retains good immunogenicity, and has higher accuracy in differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI) than the latent-related antigen (Rv1733c long peptide combined with Rv2028c long peptide), and reduces costs;

[0056] 3. The tuberculosis virulence antigen dominant peptide library combined with the tuberculosis latency-related antigen dominant peptide library of the present application 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 1, the number and proportion of specific T cells secreting IFN-γ and IL-2 in ATB and LTBI populations for the whole ESAT-6 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 using the full CFP-10 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 using the CFP-10 full 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, including the full peptide of Rv1733c 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, including the full peptide of Rv2028c and the six peptide pools;

[0062] Figure 6 , validation of immunogenicity of virulence antigen dominant peptide library and tuberculosis virulence antigen ESAT-6 combined with CFP-10;

[0063] Figure 7 , validation of immunogenicity of the latency-related antigen dominant peptide library and the latency-related antigen Rv1733c long peptide combined with the Rv2028c long peptide;

[0064] Figure 8 , comparison of the virulence antigen dominant peptide library and the virulence antigen ESAT-6 combined with CFP-10 for differential diagnosis of ATB and LTBI;

[0065] Fig. 9 , comparison of the latency-associated antigen dominant peptide library and the latency-associated antigen Rv1733c long peptide combined with Rv2028c long peptide in differential diagnosis of ATB and LTBI;

[0066] Fig.10 , the accuracy of the virulence antigen dominant peptide library combined with the latency-related antigen dominant peptide library in differential diagnosis of ATB and LTBI.

[0067] in Figure 1-Figure 10 middle,

[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 was statistically significant. DETAILED DESCRIPTION

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

[0070] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

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

[0072] Main experimental materials

[0073]

[0074] Main experimental equipment

[0075]

[0076] Example 1. Screening and construction of a virulence antigen dominant peptide library

[0077] 1. Synthesis of toxic antigen peptides

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

[0079] 2. Construction of virulence antigen peptide pool

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

[0081] Table 1. Peptide sequences of tuberculosis virulence antigens and their peptide pool grouping information

[0082]

[0083]

[0084] 3. Evaluation and screening of immunogenicity of virulence antigen peptide pool

[0085] 1. Methods

[0086] 1. Patients with etiologically confirmed ATB and healthy LTBI patients (T-SPOT.TB positive) who had not received anti-tuberculosis treatment and had no underlying disease were enrolled, peripheral blood was collected, PBMCs were separated, and cell suspension was prepared;

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

[0088] 3. Peptides were screened and eliminated based on the number and proportion of specific T cells that secreted different cytokines in the two groups. Advantageous peptides were screened based on the number of specific T cells that secreted IFN-γ and IL-2 in total; interfering peptides were eliminated based on the trend that the proportion of specific T cells that secreted IFN-γ alone in the ATB group was higher than that in the LTBI group, and the proportion of specific T cells that secreted IL-2 alone in the LTBI group was 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, such as Figure 1 As shown, in ESAT-6, the number of specific T cells secreting IFN-γ and IL-2 after stimulation with different peptide pools was different. After stimulation with peptide pools 1, 2, 4, and 5, the proportion of specific T cells secreting IFN-γ alone in ATB was higher than that in LTBI, and after stimulation with peptide pool 3, the proportions in ATB and LTBI showed opposite secretion characteristics; after stimulation with peptide pools 1, 2, and 5, the proportion of specific T cells secreting IL-2 alone in LTBI was higher than that in ATB, and after stimulation with peptide pool 3, the proportions showed opposite secretion characteristics, and after stimulation with peptide pool 4, the secretion proportions of the two groups of people were equivalent. Peptide pools 3 and 4 with opposite or equivalent secretion characteristics were eliminated, and the dominant peptide pools 1, 2, and 5 were retained.

[0091] In CFP-10 ( Figure 2 ), the number of specific T cells secreting IFN-γ and IL-2 after stimulation with different peptide pools was different. After stimulation with peptide pools 1, 3, 5, and 6, the proportion of specific T cells secreting IFN-γ alone in ATB was higher than that in LTBI, and after stimulation with peptide pools 2 and 4, the proportions in ATB and LTBI showed opposite secretion characteristics; after stimulation with peptide pools 1, 3, and 5, the proportion of specific T cells secreting IL-2 alone in LTBI was higher than that in ATB, and after stimulation with peptide pools 2 and 4, the proportions showed opposite secretion characteristics, and the secretion proportions of the two groups were equivalent in peptide pool 6. Peptide pools 2, 4, and 6 with opposite or equivalent secretion characteristics were eliminated, and the dominant peptide pools 1, 3, and 5 were retained.

[0092] Taking a patient with ATB as an example, the specific screening results are as follows Figure 3As shown, taking the characteristics of specific T cells secreting the CFP-10 full peptide library as a reference, the dominant peptide pools 1, 3, 4, 5, and 6 can be screened out through the number of specific T cells that secrete total IFN-γ and total IL-2; peptide pools 4 and 6 are eliminated based on the fact that the proportion of specific T cells that only secrete IFN-γ in ATB is higher than the proportion of specific T cells that only secrete IL-2.

[0093] 4. Construction of the dominant peptide library of virulence antigens

[0094] The dominant peptide pools 1, 2, 5 of ESAT-6 and the dominant peptide pools 1, 3, 5 of CFP-10 were selected and combined into a new virulence antigen dominant peptide library. The peptide sequences of the virulence antigen dominant peptide library are shown in Table 2 and are used as antigen 1.

[0095] Table 2. Peptide sequences of the virulence antigen dominant peptide library

[0096]

[0097]

[0098] Example 2: Screening and construction of a library of dominant peptide segments of latency-related antigens

[0099] 1. Synthesis of Latency-Related Antigenic Peptides

[0100] A Rv1733c long peptide library containing 14 peptides was constructed, each peptide was 28 amino acids long, overlapped 14 amino acids at both ends, and had a purity of ≥80%; a Rv2028c full peptide library containing 19 peptides was constructed, each peptide was 28 amino acids long, overlapped 14 amino acids at both ends, and had a purity of ≥80%.

[0101] 2. Construction of latency-related antigen peptide pool

[0102] The Rv1733c long peptide library (containing 14 peptides) was combined into 5 peptide pools, each containing 2 to 3 peptides, and the Rv2028c long peptide library (containing 19 peptides) was combined into 6 peptide pools, each containing 3 to 4 peptides. The peptide sequences of the tuberculosis latency-related antigens Rv1733c long peptide and Rv2028c long peptide and their peptide pool grouping information are shown in Table 3.

[0103] Table 3. Peptide sequences of tuberculosis latency-related antigens and their peptide pool grouping information

[0104]

[0105]

[0106]

[0107] 3. Evaluation and screening of immunogenicity of latency-related antigen peptide pools

[0108] 1. Methods

[0109] 1. Patients with etiologically confirmed ATB and healthy LTBI patients (T-SPOT.TB positive) who had not received anti-tuberculosis treatment and had no underlying disease were enrolled, peripheral blood was collected, PBMCs were separated, and cell suspension was prepared;

[0110] 2. Use different peptide pools in Table 3 as stimuli, and use FluoroSpot to detect the secretion of IFN-γ and IL-2 at the single cell level;

[0111] 3. Peptides were screened and eliminated based on the number and proportion of specific T cells that secreted different cytokines in the two groups. Advantageous peptides were screened based on the number of specific T cells that secreted IFN-γ and IL-2 in total; interfering peptides were eliminated based on the trend that the proportion of specific T cells that secreted IFN-γ in the ATB group was lower than that in the LTBI group, and the proportion of specific T cells that secreted IL-2 in the LTBI group was higher than that in the ATB group.

[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 the figure, in the Rv1733 long peptide, different peptide pools secreted different numbers of specific T cells that secreted IFN-γ and IL-2 after stimulation. After stimulation with all peptide pools, the proportion of specific T cells that secreted IFN-γ alone in ATB was lower than that in LTBI, and the proportion of specific T cells that secreted IL-2 alone in ATB was higher than that in LTBI, with a consistent trend. Peptide pools 1 and 4 with weak reaction intensity were eliminated, and the 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 with different peptide pools was different. After stimulation with peptide pools 1, 2, 3, and 5, the proportion of specific T cells secreting IFN-γ in ATB was lower than that in LTBI, and after stimulation with peptide pools 4 and 6, the proportions in ATB and LTBI showed opposite secretion characteristics; after stimulation with peptide pools 1, 2, 3, 4, and 5, the proportion of specific T cells secreting IL-2 in ATB was higher than that in LTBI, and after stimulation with peptide pool 6, the proportions 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 the dominant peptide pools 1, 2, and 3 were retained.

[0115] 4. Construction of a library of dominant peptides 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 latent-associated antigen dominant peptide library

[0118]

[0119]

[0120] Example 3: Immunogenicity verification of the virulence antigen dominant peptide library and latency-related antigen dominant peptide library

[0121] 1. Methods

[0122] A total of 70 clinical patients with positive T-SPOT.TB test were enrolled, peripheral blood was collected, PBMC was separated, and the immunogenicity of the dominant peptide library of virulence antigens and the dominant peptide library of latency-related antigens was detected using the FluoroSpot-IFN-γ / IL-2 method.

[0123] 2. Results

[0124] like Figure 6 As shown in the figure, compared with ESAT-6 combined with CFP-10, after stimulation with the dominant peptide library of virulence antigens (antigen 1), there was no significant difference in the frequencies of specific T cells that secreted total IFN-γ, total IL-2, and dual secretion of IFN-γ / IL-2 (p>0.05). The correlation coefficients between the two in the frequencies of specific T cells that secreted total IFN-γ, total IL-2, and dual secretion of 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, compared with the Rv1733c long peptide combined with the Rv2028c long peptide, after stimulation with the latency-related antigen dominant peptide library (antigen 2), 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 (p>0.05), and 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] Conclusion

[0127] Compared with the Mtb virulence antigen ESAT-6 combined with CFP-10 and the latency-associated antigen Rv1733c long peptide combined with Rv2028c long peptide, the Mtb virulence antigen dominant peptide library (antigen 1) and the latency-associated antigen dominant peptide library (antigen 2) constructed after the current screening and integration of the present invention retain good immunogenicity and can be further used in clinical practice to evaluate the accuracy of differential diagnosis of ATB and LTBI.

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

[0129] 1. Methods

[0130] 1. Eleven patients with etiologically confirmed ATB and 46 patients with LTBI (T-SPOT.TB positive) who needed to be clinically differentiated from ATB were included.

[0131] 2. The dominant peptide library of virulence antigens (antigen 1) and virulence antigens (ESAT-6 and CFP-10) were used as stimuli, and the FluoroSpot method was used to detect the number and proportion of specific T cells that secreted only IFN-γ, only IL-2, and both IFN-γ and IL-2 at the single-cell level. Binary logistic regression was used to fit the above six indicators into the tuberculosis activity index.

[0132] 2. Results

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

[0134] Conclusion

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

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

[0137] 1. Methods

[0138] 1. A total of 14 patients with etiologically confirmed ATB and 30 patients with LTBI (T-SPOT.TB positive) who needed to be clinically differentiated from ATB were included.

[0139] 2. The latency-associated antigen dominant peptide library and latency-associated antigens (Rv1733c long peptide and Rv2028c long peptide) were used as stimuli, and the FluoroSpot method was used to detect the number and proportion of specific T cells that secreted single IFN-γ, single IL-2, and dual IFN-γ and IL-2 at the single-cell level. Binary logistic regression was used to fit the above six indicators into the tuberculosis activity index.

[0140] 2. Results

[0141] like Fig. 9 As shown, under the stimulation of the latency-associated antigen dominant peptide library (antigen 2), the AUC of the tuberculosis activity index for the differential diagnosis of ATB and LTBI was 0.906. Under the stimulation of the latency-associated antigens (Rv1733c long peptide link and Rv2028c long peptide, 1733&2028), the AUC of the tuberculosis activity index for the differential diagnosis of ATB and LTBI was 0.795, which was significantly lower than the differential diagnosis accuracy of the latency-associated antigen dominant peptide library (antigen 2) (P=0.048).

[0142] Conclusion

[0143] The latency-associated antigen dominant peptide library (antigen 2) has higher accuracy in differential diagnosis of ATB and LTBI than the latency-associated antigen (Rv1733c long peptide combined with Rv2028c long peptide).

[0144] Example 6: The combination of the dominant peptide library of virulence antigens and the dominant peptide library of latency-related antigens (antigen 1 & antigen 2) further improves the accuracy of differential diagnosis between active tuberculosis and latent tuberculosis infection

[0145] 1. Methods

[0146] 1. Eleven patients with etiologically confirmed ATB and 46 patients with LTBI (T-SPOT.TB positive) who needed to be clinically differentiated from ATB were included.

[0147] 2. Using the virulence antigen dominant peptide library combined with the latency-related antigen dominant peptide library as stimuli, the FluoroSpot method was used to detect the number and proportion of specific T cells that secreted only IFN-γ, only IL-2, and both IFN-γ and IL-2 at the single-cell level, and binary logistic regression was used to fit the above six indicators into the tuberculosis activity index.

[0148] 2. Results

[0149] like Fig.10 As shown, under the stimulation of the virulence antigen dominant peptide library combined with the latency-related antigen dominant peptide library (antigen 1 & antigen 2), the tuberculosis activity index had a differential diagnosis AUC of 0.972 for ATB and LTBI. When the tuberculosis activity index was cutoff at 0.184, the sensitivity and specificity of differentiating ATB and LTBI were 100% and 90.5%.

[0150] Conclusion

[0151] The combination of the dominant peptide library of virulence antigens and the dominant peptide library of latency-related antigens (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] 1. Kit Composition

[0154] Reagents: 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, secondary antibodies anti-FITC-490 and SA-550;

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

[0156] 2. Steps for using the kit

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

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

[0159] 3. Incubation detection: After antigen stimulation, the sample was incubated at 37°C for 18 hours, and fluorescein-labeled monoclonal antibodies IFN-γ-FITC and IL-2-biotin were added; the sample was incubated for 2 hours at room temperature in a dark place; secondary antibodies anti-FITC-490 and SA-550 were added; the sample was incubated for 1 hour at room temperature in a dark place; the specific T cells secreting IFN-γ and IL-2 were counted, and the number and proportion of specific T cells secreting only IFN-γ, only IL-2, and both IFN-γ and IL-2 were calculated, and the tuberculosis activity index was fitted.

[0160] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An antigen peptide library for differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI), characterized in that: The antigen peptide library includes peptides with amino acid sequences such as SEQ ID NO:1 to SEQ ID NO:6, SEQ ID NO:14 to SEQ ID NO:20, SEQ ID NO:24 to SEQ ID NO:26 and SEQ ID NO:30 to SEQ ID NO:

32.

2. The antigen peptide library according to claim 1, characterized in that: Under the stimulation of the antigen peptide library, the AUC of the tuberculosis activity index for differential diagnosis of ATB and LTBI was 0.

907.

3. An antigen peptide library for differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI), characterized in that: The antigen peptide library includes peptides with amino acid sequences such as SEQ ID NO:1 to SEQ ID NO:6, SEQ ID NO:14 to SEQ ID NO:20, SEQ ID NO:24 to SEQ ID NO:26, SEQ ID NO:30 to SEQ ID NO:32, SEQ ID NO:39 to SEQ ID NO:44 and SEQ ID NO:48 to SEQ ID NO:

58.

4. The antigen peptide library according to claim 3, characterized in that: Under the stimulation of the antigen peptide library, the AUC of the tuberculosis activity index for differential diagnosis of ATB and LTBI was 0.

972. When the tuberculosis activity index was 0.184 as the cutoff value, the sensitivity of differentiating ATB and LTBI was 100% and the specificity was 90.5%.

5. A kit for differential diagnosis of active tuberculosis (ATB) and latent tuberculosis infection (LTBI), characterized in that: The kit comprises the antigen peptide library as described in any of claims 1 or 3.

6. The kit according to claim 5, characterized in that The kit also includes 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 according to claim 5, characterized in that The steps for using the kit are: (1) Sample processing: collect human peripheral blood, separate and extract peripheral blood mononuclear cells, and prepare cell suspension in AIM-V medium; (2) Antigen stimulation: adding the antigen peptide library as described in any one of claims 1 or 3, adding peripheral blood mononuclear cells and anti-CD28; (3) Incubation detection: After antigen stimulation, the sample was incubated at 37°C for 18 hours, and fluorescein-labeled monoclonal antibodies IFN-γ-FITC and IL-2-biotin were added; the sample was incubated for 2 hours at room temperature in a dark place and dried. Secondary antibodies anti-FITC-490 and SA-550 were added; the sample was incubated for 1 hour at room temperature in a dark place and dried. The specific T cells secreting IFN-γ and IL-2 were counted, and the number and proportion of specific T cells secreting only IFN-γ, only IL-2, and both IFN-γ and IL-2 were calculated, and the tuberculosis activity index was fitted.

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