POLYPEPTIDE ENCODED BY EB VIRUS BNLF2B GENE AND USE THEREOF IN DETECTION
20230324396 · 2023-10-12
Assignee
- Xiamen University (Xiamen, CN)
- XIAMEN INNODX BIOTECH CO., LTD (Xiamen, CN)
- Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. (Beijing, CN)
Inventors
- Tingdong LI (Xiamen, CN)
- Shengxiang Ge (Xiamen, CN)
- Xiaoyi GUO (Xiamen, CN)
- Congming HONG (Xiamen, CN)
- Liuwei Song (Xiamen, CN)
- Shunhua Wen (Xiamen, CN)
- Jiabao TANG (Xiamen, CN)
- Jun Zhang (Xiamen, CN)
- Ningshao Xia (Xiamen, CN)
Cpc classification
C12N2710/16222
CHEMISTRY; METALLURGY
G01N2469/20
PHYSICS
International classification
Abstract
Provided are a method for diagnosing nasopharyngeal carcinoma on the basis of the anti-EB virus (EBV) antibody level and a kit used for the method. Also provided are a polypeptide encoded by a BNLF2b gene in EB virus used for the above-mentioned diagnosis and the use thereof for diagnosing nasopharyngeal carcinoma.
Claims
1-39.(canceled)
40. An isolated polypeptide or variant thereof, wherein the polypeptide is composed of at least 7 and not more than 74 contiguous amino acid residues of a wild-type protein encoded by BNLF2b gene, and comprises at least 1, at least 2, at least 3, or all 4 sequences selected from the following: amino acid residues 5-11, amino acid residues 16-23, amino acid residues 31-39, or amino acid residues 53-60 of the wild-type protein encoded by BNLF2b gene; wherein the variant differs from the polypeptide from which it is derived only by a substitution of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues, and retains an activity of being recognized and bound by an anti-EBV antibody; and wherein the variant does not comprise an amino acid substitution in amino acid positions corresponding to the following positions: 6, 9, 10, 11, 16, 31, 33, 38, 39, 53, 54, 56, 57, 58, 59, 95, 96, 97 with reference to amino acid positions set forth in the wild-type protein encoded by BNLF2b gene.
41. The isolated polypeptide or variant thereof according to claim 40, wherein the isolated polypeptide comprises: amino acid residues 53-60, amino acid residues 5-23, amino acid residues 16-39, amino acid residues 31-60, amino acid residues 5-39, amino acid residues 16-60, or amino acid residues 5-60 of the wild-type protein encoded by BNLF2b gene.
42. The isolated polypeptide or variant thereof according to claim 40, wherein the isolated polypeptide consists of a sequence selected from the group consisting of amino acids 51-56, amino acid residues 1-25, amino acid residues 14-52, amino acid residues 1-52, amino acid residues 14-74, amino acid residues 11-65, amino acid residue 11-74, or amino acid residues 1-74 of the wild-type protein encoded by BNLF2b gene.
43. The isolated polypeptide or variant thereof according to claim 40, wherein the variant comprise an amino acid substitution at 1, 2, 3 or 4 amino acid positions corresponding to the following positions: 5, 7, 8, 12, 13, 14, 15, 19, 22, 24, 25, 32, 34, 35, 36, 37, 40, 41, 42, 52, 55, 60, 61, 89, 91, 93 or 98 with reference to amino acid positions set forth in the wild-type protein encoded by BNLF2b gene.
44. The isolated polypeptide or variant thereof according to claim 40, wherein the variant comprises 1, 2, 3, or 4 amino acid substitutions selected from the group consisting of: substitution of an amino acid at a position corresponding to position 5 with A, substitution of an amino acid at a position corresponding to position 7 with A, substitution of an amino acid at a position corresponding to position 8 with G, substitution of an amino acid at a position corresponding to position 12 with G, T, D or S, substitution of an amino acid at a position corresponding to position 13 with A, substitution of an amino acid at a position corresponding to position 14 with G, substitution of an amino acid at a position corresponding to position 15 with A, substitution of an amino acid at a position corresponding to position 22 with A, substitution of an amino acid at a position corresponding to position 24 with A, substitution of an amino acid at a position corresponding to position 25 with A, substitution of an amino acid at a position corresponding to position 32 with A, substitution of an amino acid at a position corresponding to position 34 with A, substitution of an amino acid at a position corresponding to position 35 with A, substitution of an amino acid at a position corresponding to position 36 with A, substitution of an amino acid at a position corresponding to position 37 with A, N, Q, S or R, substitution of an amino acid at a position corresponding to position 40 with A, substitution of an amino acid at a position corresponding to position 41 with A, substitution of an amino acid at a position corresponding to position 42 with A, substitution of an amino acid at a position corresponding to position 52 with K, H, A, S or D, substitution of an amino acid at a position corresponding to position 55 with S, substitution of an amino acid at a position corresponding to position 60 with A, substitution of an amino acid at a position corresponding to position 61 with K, H, S or A, substitution of an amino acid at a position corresponding to position 89 with A or T, substitution of an amino acid at a position corresponding to position 91 with A, substitution of an amino acid at a position corresponding to position 93 with Q, substitution of an amino acid at a position corresponding to position 98 with A.
45. The isolated polypeptide or variant thereof according to claim 40, wherein the isolated polypeptide or variant thereof is attached to a surface of a solid support, or has a modifying group capable of being attached to a solid support; or the isolated polypeptide or variant thereof bears a detectable label.
46. A kit, which comprises a capture reagent, in which the capture reagent is selected from the isolated polypeptide or variant thereof according to claim 40; and the kit further comprises an instruction for using the isolated polypeptide or variant thereof as the capture reagent to detect a level of an antibody specific to BNLF2b gene-encoded protein in a sample, optionally determining whether the subject has nasopharyngeal cancer or is at risk for nasopharyngeal cancer.
47. The kit according to claim 46, which further comprises a detection reagent, wherein the detection reagent is selected from the isolated polypeptide or variant thereof bearing a detectable label; or the detection reagent is selected from a secondary antibody bearing a detectable label.
48. The kit according to claim 47, wherein the secondary antibody is selected from the group consisting of anti-IgG antibody, anti-IgM antibody, and anti-IgA antibody.
49. The kit according to claim 46, wherein the kit further comprises one or more reagents or devices selected from the group consisting of: (i) a device for collecting or storing the sample; (ii) an additional reagent required to perform the detection which is selected from buffer, diluent, blocking solution, and/or standard.
50. A method for determining whether a subject has nasopharyngeal cancer or is at risk for nasopharyngeal cancer, which comprises: (1) deteimining a level of an antibody specific to BNLF2b gene-encoded protein in a sample from the subject; and, (2) comparing the level to a reference value; wherein if the level is higher than the reference value, it is considered that the subject has nasopharyngeal cancer or is at risk for nasopharyngeal cancer.
51. The method according to claim 50, wherein step (1) comprises the steps of: (1a) contacting a sample from the subject with a capture reagent to obtain an antigen-antibody immune complex; (1b) determining an amount of the antigen-antibody immune complex obtained in step (1a); wherein the capture reagent is an isolated polypeptide or variant thereof; wherein the polypeptide is composed of at least 7 contiguous amino acid residues of a wild-type protein encoded by BNLF2b gene, and comprises at least 1, at least 2, at least 3, or all 4 sequences selected from the following: amino acid residues 5-11, amino acid residues 16-23, amino acid residues 31-39, or amino acid residues 53-60 of the wild-type protein encoded by BNLF2b gene; wherein the variant differs from the polypeptide from which it is derived only by a substitution of 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid residues, and retains an activity of being recognized and bound by an anti-EBV antibody; and wherein the variant does not comprise an amino acid substitution in amino acid positions corresponding to the following positions: 6, 9, 10, 11, 16, 31, 33, 38, 39, 53, 54, 56, 57, 58, 59, 95, 96, 97 with reference to amino acid positions set forth in the wild-type protein encoded by BNLF2b gene.
52. The method according to claim 51, wherein, in step (1 b), a detection reagent is used to detect the amount of the immune complex, wherein the detection reagent is selected from the isolated polypeptide or variant thereof bearing a detectable label; or is a secondary antibody bearing a detectable label.
53. The method according to claim 52, wherein the secondary antibody is selected from the group consisting of anti-IgG antibody, anti-IgM antibody or anti-IgA antibody.
54. The method according to claim 50, wherein the sample is a blood sample, such as whole blood, plasma or serum: and/or the subject is a human.
55. The method according to claim 50, which further comprises: prior to step (1), providing a sample from the subject; and/or after step (2), administering to a subject who is considered to have nasopharyngeal cancer or be at risk for nasopharyngeal cancer a therapeutically effective amount of an anti-tumor therapy capable of treating nasopharyngeal cancer.
56. A method for detecting an antibody specific to BNLF2b gene-encoded protein in a sample, comprising the steps of: (1) contacting the sample with a capture reagent to obtain an antigen-antibody immune complex; wherein the capture reagent is selected from the isolated polypeptide or variant thereof according to claim 40; (2) determining an amount of the antigen-antibody immune complex obtained in step (1).
57. The method according to claim 56, wherein, in step (2), a detection reagent is used to determine the amount of the immune complex, wherein the detection reagent is selected from the isolated polypeptide or variant thereof bearing a detectable label; or is a secondary antibody bearing a detectable label.
58. The method according to claim 57, wherein the secondary antibody is selected from the group consisting of anti-IgG antibody, anti-IgM antibody or anti-IgA antibody.
59. The isolated polypeptide or variant thereof according to claim 40, wherein the wild-type protein encoded by BNLF2b gene has a sequence set forth in SEQ ID NO: 101.
60. The kit according to claim 46, wherein the capture reagent is attached to a surface of a solid support or has a modifying group capable of being attached to a solid support.
61. The method according to claim 50, wherein the level of the antibody specific to BNLF2b gene-encoded protein in the sample is determined by enzyme immunoassay, chemiluminescence immunoassay, fluorescent immunoassay or radioimmunoassay.
62. The method according to claim 51, wherein the capture reagent is attached to a surface of a solid support or has a modifying group capable of being attached to a solid support.
63. The method according to claim 56, wherein the capture reagent is attached to a surface of a solid support or has a modifying group capable of being attached to a solid support.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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Sequence Information
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TABLE-US-00001 TABLE 1 Information of sequences referred to in the present application is described in the table below. SEQ ID NO: Description 1 EBV ORF 1, aa467-486 2 EBV ORF 2, aa90-109 3 EBV ORF 3, aa138-157 4 EBV ORF 15, aa12-31 5 EBV ORF 16, aa103-122 6 EBV ORF 16, aa340-359 7 EBV ORF 17, aa26-45 8 EBV ORF 18, aa110-129 9 EBV ORF 19, aa394-413 10 EBV ORF 19, aa372-391 11 EBV ORF 20, aa63-82 12 EBV ORF 20, aa101-120 13 EBV ORF 21, aa670-689 14 EBV ORF 22, aa330-349 15 EBV ORF 23, aa205-224 16 EBV ORF 26, aa110-129 17 EBV ORF 27, aa256-275 18 EBV ORF 28, aa9-28 19 EBV ORF 29, aa1-20 20 EBV ORF 30, aa789-801 21 EBV ORF 31, aa185-204 22 EBV ORF 32, aa246-265 23 EBV ORF 33, aa26-45 24 EBV ORF 34, aa134-153 25 EBV ORF 34, aa61-80 26 EBV ORF 35, aa665-684 27 EBV ORF 35, aa822-841 28 EBV ORF 36, aa60-79 29 EBV ORF 37, aa92-111 30 EBV ORF 38, aa1-20 31 EBV ORF 39, aa105-124 32 EBV ORF 39, aa593-612 33 EBV ORF 40, aa568-587 34 EBV ORF 41, aa63-82 35 EBV ORF 41, aa177-196 36 EBV ORF 42, aa179-198 37 EBV ORF 42, aa1-20 38 EBV ORF 43, aa444-463 39 EBV ORF 44, aa126-145 40 EBV ORF 44, aa2-21 41 EBV ORF 45, aa495-514 42 EBV ORF 45, aa10-29 43 EBV ORF 47, aa60-79 44 EBV ORF 48, aa212-231 45 EBV ORF 50, aa45-64 46 EBV ORF 52, aa211-230 47 EBV ORF 53, aa166-185 48 EBV ORF 53, aa1-20 49 EBV ORF 54, aa209-228 50 EBV ORF 54, aa104-123 51 EBV ORF 55, aa357-376 52 EBV ORF 56, aa50-69 53 EBV ORF 57, aa326-345 54 EBV ORF 59, aa55-74 55 EBV ORF 59, aa231-250 56 EBV ORF 60, aa195-214 57 EBV ORF 60, aa82-101 58 EBV ORF 61, aa18-37 59 EBV ORF 62, aa41-60 60 EBV ORF 63, aa280-299 61 EBV ORF 64, aa88-107 62 EBV ORF 64, aa154-173 63 EBV ORF 65, aa118-137 64 EBV ORF 66, aa88-107 65 EBV ORF 66, aa359-378 66 EBV ORF 67, aa244-263 67 EBV ORF 68, aa120-139 68 EBV ORF 69, aa17-36 69 EBV ORF 70, aa171-190 70 EBV ORF 71, aa201-220 71 EBV ORF 72, aa143-162 72 EBV ORF 73, aa195-214 73 EBV ORF 73, aa154-173 74 EBV ORF 75, aa514-533 75 EBV ORF 75, aa432-451 76 EBV ORF 76, aa149-168 77 EBV ORF 77, aa180-199 78 EBV ORF 78, aa19-38 79 EBV ORF 79, aa323-342 80 EBV ORF 79, aa579-598 81 EBV ORF 80, aa32-51 82 EBV ORF 81, aa11-30 83 EBV ORF 82, aa155-174 84 EBV ORF 83, aa66-85 85 EBV ORF 84, aa188-207 86 EBV ORF 85, aa31-50 87 EBV ORF 86, aa19-38 88 EBV ORF 85, aa14-52 89 EBV ORF 85, aa1-74 90 EBV ORF 85, aa14-74 91 EBV ORF 85, aa1-52 92 EBV ORF 85, aa1-15 93 EBV ORF 85, aa11-25 94 EBV ORF 85, aa21-35 95 EBV ORF 85, aa31-45 96 EBV ORF 85, aa41-55 97 EBV ORF 85, aa51-65 98 EBV ORF 85, aa61-75 99 EBV ORF 85, aa71-85 100 EBV ORF 85, aa81-98 101 BNLF2b gene-encoded full-length protein 102 EBV ORF 85, aa1-25 103 EBV ORF 85, aa11-65 104 EBV ORF 85, aa11-74
[0187] EXAMPLES
Example 1: Synthesis of Epstein-Barr Virus Gene-Encoded Polypeptides
[0188] Based on the amino acid sequence information of the proteins encoded by the 86 open reading frames (ORFs) of the EBV B95-8 strain in GenBank (GenBank ID: V01555.2), the B cell epitopes of each protein were predicted online by bioinformatics tools; according to the prediction results, 1 to 2 possible B cell epitope peptides were selected for each protein and entrusted to Xiamen Jingju Biotechnology Co., Ltd. for synthesis. During synthesis, biotin was coupled to the N-terminus of polypeptide to facilitate subsequent experiments. Finally, 87 Epstein-Barr virus gene-encoded polypeptides (SEQ ID NOs: 1 to 87) were successfully synthesized, and these polypeptides were derived from 68 ORFs, and the specific information thereof was shown in Table 1.
Example 2: Evaluation of Reactivity of EBV Polypeptides with Serum IgA Antibody
[0189] The biotin-labeled polypeptides (Nos. 1 to 87) obtained in Example 1 were diluted to 500 ng/ml, respectively, and added to a streptavidin-coated 96-well microwell plate at 100 μL per well, for reaction at 37° C. for 2 hours. After the reaction, washing twice with PBST, and 200 μL of blocking solution was added to each well, for blocking for 2 hours at 37° C. After the blocking, the blocking solution was discarded, and 100 μL of 1:20 diluted nasopharyngeal cancer patient serum or negative control serum was added to each well, and the reaction was performed at 37° C. for 30 minutes. After the reaction, washing 5 times with PBST, and 1:20,000 diluted HRP-labeled goat-anti-human IgA (KPL, Gaithersburg, Md.) was added, and the reaction was continued for 30 minutes at 37° C. After washing 5 times with PBST, 100 μL of TMB chromogenic solution was added to each well; after incubation at 37° C. for 15 minutes, 50 μL of termination solution was added to each well; after mixing, the absorbance at 450 and 630 nm was measured by a microplate reader.
[0190] The results were shown in
[0191] We further evaluated the specificity of these 5 polypeptides through 36 negative serum samples, and the results were shown in
TABLE-US-00002 TABLE 2-1 Reactivity of EBV polypeptides with nasopharyngeal cancer patient serum IgA antibody SEQ ID Open reading Corresponding Positive serum No: Gene frame protein OD.sub.450/630 21 BSRF1 31 — 0.403 27 BLLF1b 35 gp220 0.808 36 BZLF1 42 Zta 0.341 38 BRLF1 43 Rta 0.787 54 BGLF3 59 UL14 0.382 65 BDLF2 66 — 0.390 75 BVRF2 75 Protease 0.783 76 BdRF1 76 VCA-p40 0.424
TABLE-US-00003 TABLE 2-2 Reactivity of EBV polypeptides with negative control serum IgA antibody Negative serum OD.sub.450/630 SEQ ID Geometric mean Ratio of greater No: Gene (range) than 0.1 21 BSRF1 0.027 (0.001, 0.126) 1/36 27 BLLF1b 0.194 (0.031, 2.54) 26/36 54 BGLF3 0.025 (0.006, 0.164) 1/36 65 BDLF2 0.128 (0.036, 1.766) 19/36 75 BVRF2 0.036 (0.011, 0.128) 2/36
Example 3: Evaluation of Reactivity of EBV Polypeptides with Serum IgG Antibody
[0192] The reactivity of 87 polypeptides (1 to 87) synthesized in Example 1 with serum IgG was detected according to the method in Example 2, wherein HRP-labeled mouse anti-human IgG (Wanyu Meilan, Beijing) diluted by 1:5000 was used to replace the goat anti-human IgA. The results were shown in
[0193] We further evaluated the specificity of the BVRF2- and BNLF2b-encoded polypeptides through 36 negative serum samples, and the results were shown in
TABLE-US-00004 TABLE 3-1 Reactivity of EBV polypeptides with positive serum IgG antibody SEQ ID Open reading Corresponding Positive serum No: Gene frame protein OD.sub.450/630 36 BZLF1 42 Zta 0.367 38 BRLF1 43 Rta 0.334 75 BVRF2 75 protease 0.883 77 BILF2 77 gp78 0.171 86 BNLF2b 85 — 0.539
TABLE-US-00005 TABLE 2-2 Reactivity of EBV polypeptides with negative control serum IgG antibody Negative serum OD.sub.450/630 SEQ ID Geometric mean Ratio of greater No: Gene (range) than 0.1 75 BVRF2 0.072 (0.020, 3.139) 8/36 86 BNLF2b 0.019 (0.011, 0.034) 0/36
Example 4: Establishment and Preliminary Performance Evaluation of Indirect Method of Anti-BNLF2b Antibody
[0194] We further analyzed the hydrophilicity, hydrophobicity and antigenicity of the protein encoded by BNLF2b (SEQ ID NO: 101) through the Protean software in the DNASTAR software package, and synthesized two polypeptides aa14-52 (SEQ ID NO: 88) and aa1-74 (SEQ ID NO: 89), which were used as coating antigens for indirect detection.
4.1 Indirect Detection Based on Polypeptide aa14-52
[0195] The polypeptide aa14-52 was diluted to 125 ng/ml with carbonate buffer (pH 9.6), and coating was performed with 100 μL per well. According to the method in Example 3, 86 serum samples of nasopharyngeal cancer patients and 195 serum samples of healthy human were used in the detection. The results were shown in
4.2 Indirect Detection Based on Polypeptide aa1-74
[0196] Using the polypeptide aa1-74 as the coating antigen, 63 serum samples of healthy people and 221 serum samples of nasopharyngeal cancer were detected according to the same method as 4.1. As shown in
[0197] In addition, the polypeptide aa1-74 was also used as the coating antigen to detect the levels of IgA and IgM antibodies in 221 serum samples of healthy control by the indirect method, in which IgA antibody detection used 1:20000 diluted HRP-labeled goat anti-human IgA (KPL, Gaithersburg, Md.), and IgM antibody detection used 1:50000 diluted HRP-labeled goat anti-human IgM (Wanyu Medan, Beijing). The results were shown in
[0198] Table 4 showed the performance of the above methods in distinguishing nasopharyngeal cancer and non-nasopharyngeal cancer. The results showed that whether the indirect method of IgG antibody using polypeptide aa14-52 as the coating antigen, or the indirect methods of IgG, IgA and IgM antibodies using polypeptide aa1-74 as the coating antigen, could effectively distinguish nasopharyngeal cancer from healthy control, and had good detection sensitivity and specificity.
TABLE-US-00006 TABLE 4 Performance of anti-BNLF2b antibody indirect method to distinguish nasopharyngeal cancer from non-nasopharyngeal cancer Coating peptide fragment Antibody type AUC Cut-off Sensitivity Specificity aa14-52 IgG 0.942 0.1 66.3% 100% (57/86) (195/195) aa14-52 IgG 0.942 0.025 80.23% 100% (69/86) (195/195) aa1-74 IgG 0.950 0.1 87.30% 95.48% (55/63) (211/221) aa1-74 IgA — 0.1 — 98.19% (217/221) aa1-74 IgM — 0.1 — 96.83% (214/221)
Example 5: Establishment of Double-Antigen Sandwich Method for Anti-BNLF2b Antibody
[0199] Since the results of Example 4 showed that the 3 kinds of antibodies IgA, IgM and IgG all had good specificity in predicting the risk of nasopharyngeal cancer, the risk of nasopharyngeal cancer could be predicted by detecting the total antibody against BNLF2b. Based on this, in this example, a double-antigen sandwich method for detecting anti-BNLF2b antibody was established to evaluate its performance in screening nasopharyngeal cancer.
[0200] The BNLF2b-encoded polypeptide aa1-74 was diluted to 100 ng/mL with carbonate buffer (pH 9.6), added to a standard 96-well microwell plate at 100 μL per well, and reacted at 37° C. for 2 hours. After the reaction, washing twice with PBST, and 200 μL of blocking solution was added to each well, and the blocking was performed at 37° C. for 2 hours. After blocking, the blocking solution was discarded, and 50 μL of dilution containing 67 ng/ml biotinylated polypeptide aa1-74 and 50 μL of nasopharyngeal cancer patient serum or negative control serum were added to each well, and the reaction was performed at 37° C. for 60 minutes. After the reaction, washing 5 times with PBST, and 1:5000 diluted HRP-labeled streptavidin and 1:15000 diluted HRP-labeled aa1-74 were added, and the reaction was continued for 30 minutes at 37° C. After washing 5 times with PBST, 100 μL of TMB chromogenic solution was added to each well; after incubation at 37° C. for 15 minutes, 50 μL of termination solution was added to each well; after mixing, the absorbance at 450 nm and 630 nm was measured by a microplate reader. This method was used to detect 50 serum samples of nasopharyngeal cancer patient and 500 serum samples of healthy people, and the results were shown in
[0201] In addition, in order to compare the performance of different BNLF2b peptides in the diagnosis of nasopharyngeal cancer, in addition to aa1-74, we also synthesized two peptides aa1-52 (SEQ ID NO: 91) and aa14-74 (SEQ ID NO: 90), and both of them were labeled with biotin at C-terminus. Another 175 serum samples of nasopharyngeal cancer patient were taken and detected according to the same method. The results were shown in the following table. When the cut-off was 0.1, the sensitivity of all of the three polypeptides were higher than 85%, indicating that the method had high detection sensitivity and specificity by using aa1-52, aa14-74 or aa1-74 as the coating antigen.
TABLE-US-00007 TABLE 5 Comparison of detection sensitivity when using different BNLF2b polypeptides as labeled antigen Coating Mean 95% Confidence Cut- Number of peptide absorbance interval off positives Sensitivity aa1-74 0.604 0.008-2.422 0.1 160 91.43% aa1-52 0.575 0.009-2.809 0.1 155 88.57% aa14-74 0.422 0.007-1.694 0.1 155 88.57%
Example 6: Comparison of the Double-Antigen Sandwich Method for Anti-BNLF2b Antibody and Existing Nasopharyngeal Cancer Screening Reagents
[0202] Parallel detection of 74 serum samples of nasopharyngeal cancer patient and 250 serum samples of healthy people were performed by using the EBNA1/IgA detection kit of Zhongshan Bio, the VCA/IgA detection kit of EU (Cat. No.: EI 2791-9601A) and the double-antigen sandwich method of Example 5 (aa1-74). The detection results of EBNA1/IgA were shown in
[0203] The detection results of anti-BNLF2b antibody were shown in
[0204] The ROC curve analysis was performed for the results of the above anti-BNLF2b antibody detection and the combination of EBNA1/IgA+VCA/IgA, respectively. The results were shown in
[0205] The data in Example 5 and Example 6 were combined for calculation, which showed the anti-BNLF2b antibody detection had a sensitivity of 93.55% (116/124) and a specificity of 99.73% (748/750). According to the incidence calculation formula in the literature (Liu, Z., et al. 2013, Am J Epidemiol), the positive predictive value of the anti-BNLF2b antibody detection was 33.2%, which showed a significant improvement as compared with the positive predictive value (4.4%, 38/862) of the EBNA1/IgA+VCA/IgA combined detection (P<0.0001). The above results indicated that the anti-BNLF2b antibody detection could significantly improve the specificity and positive predictive value of nasopharyngeal cancer screening as compared with the existing combination of EBNA1/IgA+VCA/IgA screening method.
Example 7: Application of Anti-BNLF2b Antibody Double-Antigen Sandwich Method in Nasopharyngeal Cancer Screening
[0206] Population screening was carried out in two towns in high-incidence areas in Zhongshan City, Guangdong Province (Fusha and Nanlang), a total of 1325 people were enrolled, 496 people in Fusha and 829 in Nanlang. According to the method in Example 6 (herein after referred to as the anti-BNLF2b antibody detection), these samples were subjected to parallel detections for EBNA1/IgA, VCA/IgA and anti-BNLF2b antibodies; according to the formula combination of EBNA1/IgA and VCA/IgA (LogitP=−3.934+2.203×VCA/IgA+4.797×EBNA1/IgA), the probability of developing nasopharyngeal cancer was calculated. The results were shown in the table below, which indicated that among the 1325 people, 163, 218 and 32 people were positive for EBNA1/IgA, VCA/IgA and anti-BNLF2b antibodies, respectively. Among the 126 people with probability index of greater than 0.98, 5 were ultimately diagnosed as nasopharyngeal cancer, while among those with a probability of less than 0.98, none was pre-diagnosed to have nasopharyngeal cancer according to the tumor registry system. Among the 5 samples that were diagnosed as nasopharyngeal cancer, the VCA/IgA could detect only 1 sample, while the other methods could detect 100%. In the 1320 non-nasopharyngeal cancer samples, the specificity and positive predictive value of the anti-BNLF2b antibody detection were 97.95% and 15.63%, respectively, which were higher than those of the EBNA1/IgA, VCA/IgA and their combination.
TABLE-US-00008 TABLE 6 Comparison of performance of anti-BNLF2b antibody and existing markers in population screening Positive Negative Screening sensitivity Specificity predictive predictive protocol (n = 5) (n = 1320) value value EBNA1/IgA 100% 88.03% 3.07% 100.00% (5/5) (1162/1320) (5/163) (1162/1162) VCA/IgA 20% 83.56% 0.46% 99.64% (1/5) (1103/1320) (1/218) (1103/1107) Probability 100% 90.83% 3.97% 100.00% (EBNA1/IgA + (5/5) (1199/1320) (5/126) (1199/1199) VCA/IgA) Anti-BNLF2b 100% 97.95% 15.63% 100.00% antibody (5/5) (1293/1320) (5/32) (1293/1293)
Example 8: Combined Use of Anti-BNLF2b Antibody Double-Antigen Sandwich Method and Existing Nasopharyngeal Cancer Screening Reagent
[0207] The data in Example 6 and Example 7 were combined, and there were 79 cases of nasopharyngeal cancer and 1570 cases of non-nasopharyngeal cancer. The detection results of 79 nasopharyngeal cancer samples were shown in Table 7-1, in which there were 70, 67 and 72 cases that were positive in the anti-BNLF2b antibody detection were also positive in EBNA1/IgA, VCA/IgA and probability, respectively. The detection results of the 1570 non-nasopharyngeal cancer cases were shown in Table 7-2, in which there were only 5, 5 and 4 cases that were positive in the anti-BNLF2b antibody detection were also positive in EBNA/IgA, VCA/IgA and probability, respectively. The results of the further combined detection of the anti-BNLF2b antibody detection with EBNA/IgA, VCA/IgA, EBNA/IgA+VCA/IgA were shown in Table 7-3, which indicated that after combined detection with EBNA/IgA, the positive predictive value increased from 15.15% to 50%, and after combined detection with the probability, the positive predictive value increased from 15.15% to 55.56%.
TABLE-US-00009 TABLE 7-1 Detection results of 79 nasopharyngeal cancer samples Anti- EBNA1/IgA VCA/IgA Probability BNLF2b Positive Negative Positive Negative >0.98 ≤0.98 Positive 70 5 67 8 72 3 Negative 3 1 2 2 2 2
TABLE-US-00010 TABLE 7-2 Detection results of 1570 non-nasopharyngeal cancer samples Anti- EBNA1/IgA VCA/IgA Probability BNLF2b Positive Negative Positive Negative >0.98 ≤0.98 Positive 5 23 5 23 4 24 Negative 153 1389 228 1314 125 1417
TABLE-US-00011 TABLE 7-3 Performance of anti-BNLF2b antibody detection and combined detection with existing markers for nasopharyngeal cancer screening Anti-BNLF2b antibody detection Performance — EBNA1/IgA VCA/IgA Probability Sensitivity 94.94% 88.61% 84.81% 91.14% Specificity 98.22% 99.68% 99.68% 99.75% Positive predictive 15.15% 50% 16.67% 55.56% value (5/32) (5/10) (1/6) (5/9) Negative predictive 100% 100% 99.75% 100% value
[0208] We further collected 227 cases (Sample II) that were identified as high-risk in the preliminary screening cohort by the combination of EBNA1/IgA+VCA/IgA of Zhongshan People's Hospital, among which 8 were diagnosed as nasopharyngeal cancer by nasopharyngeal endoscopy. The results were shown in Table 8, which indicated that there were 24 cases of anti-BNLF2b antibody positive, 7 of which were nasopharyngeal cancer, and thus the sensitivity and positive predictive value of the combined detection were 87.50% and 29.17%, respectively.
[0209] Taken Sample II and Sample I together, wherein Sample I included 8 cases with probability >0.98 in the 250 healthy controls in Example 6, and 126 cases (5 cases had nasopharyngeal cancer) with probability >0.98 in the screening cohort of 1325 people in Example 7. After merging the two samples, a total of 13 nasopharyngeal cancer cases and 348 non-nasopharyngeal cancer cases were obtained, and the positive predictive value of the combination of EBNA1/IgA+VCA/IgA was 3.60%. On this basis, after the anti-BNLF2b antibody detection, 12 of the 13 nasopharyngeal cancer cases were positive, while only 21 of the 348 non-nasopharyngeal cancers were positive, and thus the positive predictive value of the combined detection was increased to 36.36% (Table 8). Therefore, the combined detection of anti-BNLF2b antibody and EBNA1/IgA+VCA/IgA could further improve the specificity and positive predictive value of nasopharyngeal cancer screening. In order to reduce the workload, anti-BNLF2b antibodies could be detected first, followed by further detection of EBNA1/IgA and VCA/IgA.
TABLE-US-00012 TABLE 8 Performance of anti-BNLF2b antibody detection in high-risk population Number of Positive Total nasopharyngeal predictive Sample number cancer patients Sensitivity Specificity value I 134 5 100% 96.90% 55.56% (5/5) (125/129) (5/9) II 227 8 87.50% 92.24% 29.17% (7/8) (202/219) (7/24) Total 361 13 92.31% 94.24% 36.36% (12/13) (327/348) (12/33)
Example 9: Application of Anti-BNLF2b Antibody Double-Antigen Sandwich Method in Auxiliary Diagnosis of Nasopharyngeal Cancer
[0210] The clinical symptoms of nasopharyngeal cancer are not specific enough to distinguish it from other head and neck diseases. Clinically, suspected cases are mainly diagnosed by nasopharyngeal endoscopy and pathological examination. We collected 63 suspected nasopharyngeal cancer cases, of which 31 cases were ultimately diagnosed as nasopharyngeal cancer. We detected the samples of these 63 cases by the double-antigen sandwich method according to the method in Example 6, the results showed that with a cut-off value of 0.1, 30 of the 31 nasopharyngeal cancer cases were positive for BNLF2b antibody, while only 2 of the 32 non-nasopharyngeal cancer cases were positive for BNLF2b antibody. The BNLF2b antibody detection showed a sensitivity of 96.78% (30/31), a specificity of 93.75% (30/32), and a positive predictive value of 93.75% (30/32). The above results indicated that in outpatient cases, the BNLF2b antibody detection could significantly reduce the number of unnecessary nasopharyngeal endoscopy examinations, thereby reducing the economic and physical burden of patients.
Example 10: Study of Immunodominant Epitope of BNLF2b-Encoded Protein
[0211] The BNLF2b gene encodes a total of 98 amino acids (SEQ ID NO: 101). In order to analyze the immunodominant epitopes of the protein encoded by BNLF2b, we designed 9 overlapping polypeptides. Each polypeptide had 15 amino acids (the last polypeptide had 18 amino acids), and the two adjacent polypeptides were overlapped by 5 amino acids, and the C-terminal was labeled with biotin for subsequent detection (SEQ ID NOs: 92-100). According to the method in Example 3, by using the above-mentioned polypeptide fragment as the coating antigen, the detection of IgG in 1:300 diluted serum sample of nasopharyngeal cancer patient was performed. The results were shown in
[0212] We further preliminarily evaluated the sensitivity of the four segment polypeptides, aa1-25 (SEQ ID NO: 102), aa31-45 (SEQ ID NO: 95), aa51-65 (SEQ ID NO: 97), aa81-98 (SEQ ID NO: 100) in nasopharyngeal cancer screening through 43 serum samples of nasopharyngeal cancer according to the method in Example 3. The results showed that when the cut-off was 0.1, the detection sensitivity was 76.7% (33/43) for aa51-65, 72.1% (31/43) for aa1-25, and the sensitivity of the other two peptides was 55.8% (24/43). The sensitivity of aa14-52 and aa1-74 were 90.7% (39/43) and 93.0% (40/43); and for the 3 serum samples failed in aa1-74 detection, all of the 4 polypeptides were negative, while the reaction of the serum sample of aa14-52 negative and aa1-74 positive with aa51-65 had an OD value of 0.393.
[0213] In order to further determine the key amino acids constituting these epitopes, we further performed N-terminal and C-terminal truncations on the basis of aa1-25, aa31-45, aa51-65 and aa81-98, and synthesized a series of polypeptides. In this experiment, 40 serum samples of nasopharyngeal cancer were mixed to form two samples of pooled serum, and after 1:300 dilution, IgG detection was performed according to the method in Example 3, and the results were shown in
[0214] (1) aa1-25 segment (
[0215] (2) aa31-45 segment (
[0216] (3) aa51-65 segment (
[0217] (4) aa81-98 segment (
[0218] In order to analyze whether the aa1-25 segment contained an epitope spanning the aa1-15 and aa11-25 segments, we detected IgG in 43 serum samples of nasopharyngeal cancer by using aa1-15, aa11-25 and aa1-25 as coating antigens, respectively. The results were shown in
[0219] In addition, for the above four key segments, we also synthesized a series of variants, including natural and artificial variants. According to the method in Example 3, these variants were used as coating antigens to detect their reactivity with IgG in 1:300 diluted pooled serum sample of nasopharyngeal cancer, and the results were shown in
[0220] (1) aa1-25 (
[0221] (2) aa31-45 (
[0222] (3) aa51-65 (
[0223] (4) aa81-98 (
[0224] The above results showed that the aa5-11, aa16-23, aa31-33, aa37-39, aa53-60 and aa89-98 of the BNLF2b-encoded protein were core segments.
Example 11: Effect of Combined Use of Different Epitopes of BNLF2b-Encoded Protein on Nasopharyngeal Cancer Screening Performance
[0225] On the basis of the synthesized polypeptides of aa14-52, aa1-74, aa1-52 and aa1-25 above, another two polypeptides aa11-65 (SEQ ID NO: 103) and aa11-74 (SEQ ID NO: 104) were further synthesized, and labeled with biotin at their C-terminus. According to the method in Example 3, the reaction of these polypeptides with 84 serum samples of nasopharyngeal cancer patient and 168 serum samples of healthy people was detected, respectively. The reactivity of the 6 polypeptides with the serum samples of nasopharyngeal cancer was significantly higher than that with the healthy controls (
TABLE-US-00013 TABLE 9 Comparison of performance of different polypeptides as capture antigens in nasopharyngeal cancer screening Geometric mean (95% CI) Nasopharyngeal Healthy ROC curve analysis Coating cancer control Youden polypeptide (n = 84) (n = 168) AUC Cut-off Sensitivity Specificity index aa1-25 0.646 0.016 0.968 0.071 89.29% 96.43% 0.857 (0.014-3.897) (0.007-0.125) aa14-52 0.758 0.011 0.990 0.054 94.05% 97.62% 0.917 (0.030-4.446) (0.004-0.054) aa1-52 1.112 0.011 0.989 0.052 95.24% 97.62% 0.929 (0.026-4.462) (0.004-0.051) aa11-65 1.236 0.011 0.986 0.058 94.05% 97.62% 0.917 (0.029-4.464) (0.005-0.058) aa11-74 1.484 0.013 0.990 0.061 95.24% 97.62% 0.929 (0.032-4.463) (0.005-0.061) aa1-74 1.724 0.013 0.992 0.072 96.43% 97.62% 0.941 (0.045-3.911) (0.005-0.072)
[0226] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and changes can be made to the details in light of all the teachings that have been published, and that these changes are all within the scope of the present invention. The full division of the present invention is given by the appended claims and any equivalents thereof.
REFERENCES
[0227] [1] Torre L A, Bray F, Siegel R L, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65:87-108.
[0228] [2] Tsao S W, Yip Y L, Tsang C M, Pang P S, Lau V M, Zhang G, et al. Etiological factors of nasopharyngeal carcinoma. Oral Oncol. 2014;50:330-8.
[0229] [3] Lee AW, Foo W, Law S C, Poon Y F, Sze W M, O S K, et al. Nasopharyngeal carcinoma: presenting symptoms and duration before diagnosis. Hong Kong medical journal=Xianggang yi xue za zhi. 1997;3:355-61.
[0230] [4] Henle G, Henle W. Epstein-Barr virus-specific IgA serum antibodies as an outstanding feature of nasopharyngeal carcinoma. International journal of cancer. 1976;17:1-7.
[0231] [5] Zhu X X, Zeng Y, Wolf H. Detection of IgG and IgA antibodies to Epstein-Barr virus membrane antigen in sera from patients with nasopharyngeal carcinoma and from normal individuals. International journal of cancer. 1986;37:689-91.
[0232] [6] Zeng J, Gong C H, Jan M G, Fun Z, Zhang L G, Li H Y. Detection of Epstein-Barr virus IgA/EA antibody for diagnosis of nasopharyngeal carcinoma by immunoautoradiography. International journal of cancer. 1983;31:599-601.
[0233] [7] Chen J Y, Liu M Y, Hsu T Y, Cho S M, Yang C S. Use of bacterially-expressed antigen for detection of antibodies to the EBV-specific deoxyribonuclease in sera from patients with nasopharyngeal carcinoma. Journal of virological methods. 1993;45:49-66.
[0234] [8] Connolly Y, Littler E, Sun N, Chen X, Huang P C, Stacey S N, et al. Antibodies to Epstein-Barr virus thymidine kinase: a characteristic marker for the serological detection of nasopharyngeal carcinoma. International journal of cancer. 2001;91:692-7.
[0235] [9] Zhang X, Zhang Y, Nie Y, Wang S, Chen Y, Sun D. Serum Zta antibody of Epstein-Barr virus exerts potential function in the diagnosis of nasopharyngeal cancer. Tumour biology: the journal of the International Society for Oncodevelopmental Biology and Medicine. 2014;35:6879-86.
[0236] [10] Littler E, Newman W, Arrand J R. Immunological response of nasopharyngeal carcinoma patients to the Epstein-Barr-virus-coded thymidine kinase expressed in Escherichia coli. International journal of cancer. 1990;45:1028-32.
[0237] [11] Gu A D, Lu L X, Xie Y B, Chen L Z, Feng Q S, Kang T, et al. Clinical values of multiple Epstein-Barr virus (EBV) serological biomarkers detected by xMAP technology. Journal of translational medicine. 2009;7:73.
[0238] [12] Wout M. J. van Grunsven W J M S, and Jaap M. Middeldorp. Localization and Diagnostic Application of Immunodominant Domains of the BFRF3-Encoded Epstein-Barr Virus Capsid Protein. The Journal of infectious diseases. 1994;170:13-9.
[0239] [13] Zong Y S, Sham J S, Ng M H, Ou X T, Guo Y Q, Zheng S A, et al. Immunoglobulin A against viral capsid antigen of Epstein-Barr virus and indirect mirror examination of the nasopharynx in the detection of asymptomatic nasopharyngeal carcinoma. Cancer. 1992;69:3-7.
[0240] [14] Zeng Y, Zhong J M, Li L Y, Wang P Z, Tang H, Ma Y R, et al. Follow-up studies on Epstein-Barr virus IgA/VCA antibody-positive persons in Zangwu County, China. Intervirology. 1983;20:190-4.
[0241] [15] Zeng Y, Zhang L G, Li H Y, Jan M G, Zhang Q, Wu Y C, et al. Serological mass survey for early detection of nasopharyngeal carcinoma in Wuzhou City, China. International journal of cancer. 1982;29:139-41.
[0242] [16] Chan K H, Gu Y L, Ng F, Ng P S, Seto W H, Sham J S, et al. EBV specific antibody-based and DNA-based assays in serologic diagnosis of nasopharyngeal carcinoma. International journal of cancer. 2003;105:706-9.
[0243] [17] Chen H, Chi P, Wang W, Li L, Luo Y, Fu J, et al. Evaluation of a semi-quantitative ELISA for IgA antibody against Epstein-Barr virus capsid antigen in the serological diagnosis of nasopharyngeal carcinoma. International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases. 2014;25:110-5.
[0244] [18] Tang J W, Rohwader E, Chu I M, Tsang R K, Steinhagen K, Yeung A C, et al. Evaluation of Epstein-Barr virus antigen-based immunoassays for serological diagnosis of nasopharyngeal carcinoma. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology. 2007;40:284-8.
[0245] [19] Paramita D K, Fachiroh J, Haryana S M, Middeldorp J M. Two-step Epstein-Barr virus immunoglobulin A enzyme-linked immunosorbent assay system for serological screening and confirmation of nasopharyngeal carcinoma. Clinical and vaccine immunology: CVI. 2009;16:706-11.
[0246] [20] Coghill A E, Hsu W L, Pfeiffer R M, Juwana H, Yu K J, Lou P J, et al. Epstein-Barr virus serology as a potential screening marker for nasopharyngeal carcinoma among high-risk individuals from multiplex families in Taiwan. Cancer epidemiology, biomarkers & prevention: a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2014;23:1213-9.
[0247] [21] Coghill A E, Bu W, Nguyen H, Hsu W L, Yu K J, Lou P J, et al. High Levels of Antibody that Neutralize B-cell Infection of Epstein-Barr Virus and that Bind EBV gp350 Are Associated with a Lower Risk of Nasopharyngeal Carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2016;22:3451-7.
[0248] [22] Chang C, Middeldorp J, Yu K J, Juwana H, Hsu W L, Lou P J, et al. Characterization of ELISA detection of broad-spectrum anti-Epstein-Barr virus antibodies associated with nasopharyngeal carcinoma. Journal of medical virology. 2013;85:524-9.
[0249] [23] Li R C, Du Y, Zeng Q Y, Tang L Q, Zhang H, Li Y, et al. Epstein-Barr virus glycoprotein gH/gL antibodies complement IgA-viral capsid antigen for diagnosis of nasopharyngeal carcinoma. Oncotarget. 2016;7:16372-83.
[0250] [24] Fachiroh J, Paramita D K, Hariwiyanto B, Harijadi A, Dahlia H L, Indrasari S R, et al. Single-assay combination of Epstein-Barr Virus (EBV) EBNA1- and viral capsid antigen-p18-derived synthetic peptides for measuring anti-EBV immunoglobulin G (IgG) and IgA antibody levels in sera from nasopharyngeal carcinoma patients: options for field screening. Journal of clinical microbiology. 2006;44:1459-67.
[0251] [25] Cai Y L, Li J, Lu A Y, Zheng Y M, Zhong W M, Wang W, et al. Diagnostic significance of combined detection of Epstein-Barr virus antibodies, VCA/IgA, EA/IgA, Rta/IgG and EBNA1/IgA for nasopharyngeal carcinoma. Asian Pacific journal of cancer prevention: APJCP. 2014;15:2001-6.
[0252] [26] Liu Y, Huang Q, Liu W, Liu Q, Jia W, Chang E, et al. Establishment of VCA and EBNA1 IgA-based combination by enzyme-linked immunosorbent assay as preferred screening method for nasopharyngeal carcinoma: a two-stage design with a preliminary performance study and a mass screening in southern China. International journal of cancer. 2012;131:406-16.
[0253] [27] Ji M F, Wang D K, Yu Y L, Guo Y Q, Liang J S, Cheng W M, et al. Sustained elevation of Epstein-Barr virus antibody levels preceding clinical onset of nasopharyngeal carcinoma. British journal of cancer. 2007;96:623-30.
[0254] [28] Ji M F, Yu Y L, Cheng W M, Zong Y S, Ng P S, Chua D T, et al. Detection of Stage I nasopharyngeal carcinoma by serologic screening and clinical examination. Chinese journal of cancer. 2011;30:120-3.
[0255] [29] Liu Z, Ji M F, Huang Q H, Fang F, Liu Q, Jia W H, et al. Two Epstein-Barr virus-related serologic antibody tests in nasopharyngeal carcinoma screening: results from the initial phase of a cluster randomized controlled trial in Southern China. American journal of epidemiology. 2013;177:242-50.
[0256] [30] Li T, Guo X, Ji M, Li F, Wang H, Cheng W, et al. Establishment and validation of a two-step screening scheme for improved performance of serological screening of nasopharyngeal carcinoma. Cancer Med. 2018;7:1458-67.
[0257] [31] Ji M F, Huang Q H, Yu X, Liu Z, Li X, Zhang L F, et al. Evaluation of plasma Epstein-Barr virus DNA load to distinguish nasopharyngeal carcinoma patients from healthy high-risk populations in Southern China. Cancer. 2014;120:1353-60.
[0258] [32] Chen Y, Zhao W, Lin L, Xiao X, Zhou X, Ming H, et al. Nasopharyngeal Epstein-Barr Virus Load: An Efficient Supplementary Method for Population-Based Nasopharyngeal Carcinoma Screening. PloS one. 2015;10: e0132669.
[0259] [33] Zhang G, Zong J, Lin S, Verhoeven R J, Tong S, Chen Y, et al. Circulating Epstein-Barr virus microRNAs miR-BART7 and miR-BART13 as biomarkers for nasopharyngeal carcinoma diagnosis and treatment. International journal of cancer. 2015;136: E301-12.
[0260] [34] Chan K C A, Woo J K S, King A, Zee B C Y, Lam W K J, Chan S L, et al. Analysis of Plasma Epstein-Barr Virus DNA to Screen for Nasopharyngeal Cancer. The New England journal of medicine. 2017;377:513-22.
[0261] [35] Rao Dongping, Liu Qing, Cao Sumei. Cost-effectiveness evaluation of nasopharyngeal cancer screening using Markov model. Chinese Journal of Oncology. 2012;34:549-53.