Molecular Diversity of EpsteinBarr Virus IgG and IgA Antibody Responses in Nasopharyngeal Carcinoma.docx

1、Molecular Diversity of EpsteinBarr Virus IgG and IgA Antibody Responses in Nasopharyngeal CarcinomaChapter 2Molecular Diversity of Epstein-Barr Virus IgG and IgA Antibody Responses in Nasopharyngeal Carcinoma: A Comparison of Indonesian, Chinese,and European SubjectsJajah Fachiroh1, Tabitha Schouten

2、3, Bambang Hariwiyanto1, Dewi K Paramita1, Ahmad Harijadi1, Sofia M Haryana1, Mun H Ng2, and Jaap M Middeldorp31 Gadjah Mada University, Yogyakarta, Indonesia,2Department of Microbiology, Queen Marys Hospital, Hongkong, China3Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, The

3、 NetherlandsThe Journal of Infectious Diseases 2004; 190:536271Chapter 2AbstractEpstein-Barr virus (EBV)-specific immunoblot analysis was used to reveal the molecular diversity of immunoglobulin (Ig) G and IgA antibody responses against Epstein-Barr nuclear antigen (EBNA), early antigen (EA), and vi

4、ral capsid antigen (VCA) in serum samples from patients with nasopharyngeal carcinoma (NPC) and control subjects, by use of immunofluorescence assay (IFA). Control donors (np150) showed IgG responses to few EBV proteins VCA-p18, VCA- p40, EBNA1, and Zebra - and sporadically weak IgA reactivity to EB

5、NA1 and VCA- p18. Patients with NPC stage 1 (n p 6) had similar response patterns. Patients with NPC stage 24 (n p 132) showed significantly more diverse IgG and IgA responses to EA and VCA proteins-VCA-p18/-p40, EBNA1, Z-encoded broadly reactive activator, and EAd-p47/54, -DNAse, thymidine kinase,

6、and -p138. No correlation was found between IFA titers and the number of EBV proteins recognized by IgG or IgA. Our results reveal dissimilarity between EBV polypeptides recognized by IgG and IgA antibodies, which suggests independent B cell triggering events.INTRODUCTIONEpstein-Barr virus (EBV), a

7、g-herpesvirus, is well established in the human population and is efficiently transmitted by mucosal secretions. EBV infection usually occurs silently early in life, but it may be symptomatic when infection is delayed until adolescence 13. EBV is also a human carcinogen that has been implicated in t

8、he development of malignancies of lymphoid and epithelial origin, including Burkitt lymphoma, Hodgkin disease (HD), immunodeficiency- related B cell lymphoma, extranodal T/NK cell lymphomas, gastric carcinoma, and nasopharyngeal carcinoma (NPC) 2, 46. Most EBV-associated malignancies show a distinct

9、 gene-expression pattern 7 and are accompanied by aberrant antibody responses to various EBV proteins and antigen complexes 8.Undifferentiated NPC is 100% associated with EBV and forms an unusual tumor with intriguing epidemiological and biological characteristics 610. The highest incidence is found

10、 in persons of Chinese ethnicity living in southern China, Hong Kong, Taiwan, and Singapore. Intermediate incidence occurs in certain African and Mediterranean populations, in Inuits from Greenland and Alaska, and in Malays from Singapore and Malaysia. A low incidence is found in American and Europe

11、an whites, Hispanics, and Japanese. In Indonesia, NPC has an overall intermediate incidence (3.9 cases/100,000 population) similar to that in Malaysia. However, in the Yogyakarta area, NPC constitutes 21.8% of tumors in men and 7.9% of tumors in women, which ranks NPC as the most frequent tumor in m

12、en and the fourth most frequent tumor in women 11. The results of seroepidemiological studies have indicated a close relationship between EBV infection and NPC, as revealed by elevated IgG and, especially, IgA responses to EBV viral capsid antigen (VCA), early antigen (EA), and Epstein-Barr nuclear

13、antigen (EBNA) complexes 8, 12. Elevated total and EBV-specific serum IgA levels are indicative of NPC stage 8, 13, 14 and can precede tumor development by 15 years,72Molecular Diversity of Epstein-Barr Virus IgG and IgA Antibody Responses in NPCwhich suggests that a reactivation of EBV infection pl

14、ays a role in tumor development 14, 15. In addition, a decline in anti-EBV antibody responses after radiotherapy may have prognostic value 16. At present, the indirect immunofluorescence assay (IFA) is still used as the reference standard for the serodiagnosis of EBV in NPC 2, 8, 12, 14. This method

15、, however, is difficult to standardize and is not suitable for large-scale testing in developing countries; it is gradually being replaced by more-defined EIAs 17-20. Of importance, IFA does not provide insight into the molecular basis of anti-EBV responses, because EBV- infected cells each contain

16、a multitude of different EBV proteins that can serve as the target for antibody interaction 21-23.Recent molecular serological testing approaches in the diagnosis of NPC have focused on the use of defined recombinant EBV proteins. Tedeschi et al. 24 showed that antibodies against the Z-encoded broad

17、ly reactive activator (Zebra) protein are regularly found among patients with NPC. Others have proposed the anti-Zebra IgG antibody titer to be a prognostic marker for NPC 16. However, anti-Zebra IgG antibodies are detectable in 74% of healthy EBV carriers, according to the results of a sensitive im

18、munoblot assay 22-this has been confirmed by exchanging blind serum samples (I. Joab and J.M.M., unpublished data). EBV DNAse neutralizing antibodies have been found in 83%-94% of patients with NPC 25, and they appear to be a good marker for NPC screening and prognosis. However, there is no correlat

19、ion between the level of anti-DNAse antibodies and antibody titer to VCA or EA-D, according to the results of IFA serological testing 26. Stolzenberg et al. 27 detected IgA antibody against recombinant DNAse in patients with NPC but rarely in patients with other EBV-related malignancies. Shimakage e

20、t al. 28 suggested the use of the EBNA1-IgA serum level as a prognostic marker for monitoring patients with NPC after radiation therapy, and Foong et al. 29 suggested the use of serum and salivary IgA levels against an EBNA1 Gly-Ala repeat peptide as a suitable NPC marker. Connolly et al. 20 suggest

21、ed the use of thymidine kinase (TK) as the antigen in IgA-ELISA for the diagnosis of and screening for NPC. Dardari et al. 30 indicated that the combination of IgG-Zebra and IgA-EA (p54)+(p138) should be used. Most recently, Chan et al. 31 proposed the combined use of EBNA1-IgA and Zebra-IgG as the

22、best predictor for NPC. It may be obvious from the above that there is no consensus on the use of defined EBV proteins in serological testing for the diagnosis and prognosis of NPC.Still, very little detail is known about the overall molecular diversity (complexity) of anti-EBV IgG and IgA antibody

23、responses in patients with NPC 23. Moreover, a comparison of antibody profiles among patients with NPC who are of different genetic background has not been described. The study described here provides insight into the molecular basis of EBV-specific IgG and IgA antibody responses in patients with NP

24、C of defined tumor stage from Javanese (Indonesia), Chinese (Hong Kong), and white (Europe) origin, compared with those in regional non-NPC control subjects and healthy EBV carriers. Our parallel analysis of IgG and IgA responses revealed differences in EBV antigen recognition profiles, which sugges

25、ts independent B cell triggering.73Chapter 2SUBJECTS, MATERIAL, AND METHODSSerum samples and antibodies. Serum samples from Indonesian Javanese (non Chinese) subjects consisted of samples from a panel of 135 patients with histologically confirmed NPC, 5 patients with non-NPC head and neck cancer (al

26、l of which were collected at the Department of Ear, Nose, and Throat ENT, Dr. Sardjito General Hospital, Yogyakarta), and 70 healthy donors obtained from the local Red Cross blood bank. The NPC serum samples were obtained on the first visit of patients to ENT during 2001-2003. From all patients with

27、 NPC, nasopharyngeal and/or lymph-node biopsy samples were obtained and confirmed histologically for the presence of undifferentiated carcinoma cells and the presence of EBV, by EBER1,2 in situ hybridization, by use of the Dako PNAkit (Dako) and by immunohistochemistry (Labvision) with EBNA1- and la

28、tent membrane protein (LMP) 1 - specific monoclonal antibodies OT1X 32 and OT21C 23, 33, respectively. NPC staging was done by ENT examination and computed tomography scan and was classified according to the 1997 Union International Cancer Control (UICC) classification.Serum samples from persons of

29、Chinese ethnicity living in Hong Kong were provided as a blind panel and included samples from 40 healthy donors, 35 patients with head and neck-related non-NPC tumors, and 40 patients with histologically confirmed NPC (obtained by M.H.N.). The EBV serological profile of the Chinese panel was analyz

30、ed without knowledge of the clinical diagnosis. VCA and EA IgG and IgA antibody titers were determined by standard IFA techniques, according to the method of Henle and Henle 8, and this information was revealed only after breaking the code.Serum samples from 7 white patients with NPC were obtained f

31、rom hospitals in Germany, the United Kingdom, and The Netherlands. One series (n p 5) of follow-up samples from a white Dutch patient with NPC was obtained from the Vrije Universiteit medical center, Amsterdam, The Netherlands. All serum samples were stored at 20C until use.Monoclonal and polyclonal

32、 monospecific antiserum samples wereproduced by the immunization of animals with synthetic peptides or purified recombinant proteins, as described elsewhere. Antibodies to defined EBV proteins consisted of OT13B (anti-EA-p138; BALF2) 34, rabbit anti-EBNA1 (BKRF1) 35, rabbit anti-DNAse (BGLF5) 27,OT14E (anti-EA-p47;BMRF1) 36, BZ-1 (anti-Zebra; BZLF1) 37, OT41A (anti-VCA-p40; BdRF1) 38, and OT15E (anti- VCA-p18; BFRF3) 39.Cell culture and antigen preparation. The superinducible P3HR1-derived cell line HH514.c16 was