This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gu, A.-D.
Right arrow Articles by Zeng, Y.-X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gu, A.-D.
Right arrow Articles by Zeng, Y.-X.

 Previous Article  |  Next Article 

Clinical and Vaccine Immunology, April 2009, p. 592-593, Vol. 16, No. 4
1071-412X/09/$08.00+0     doi:10.1128/CVI.00207-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of Antibodies against Different Epstein-Barr Virus Nuclear Antigen 1 Peptides in Diagnosis of Nasopharyngeal Carcinoma{triangledown}

Ai-Di Gu,1,2 Hao-Yuan Mo,3 Jin-Xin Bei,1,2 Yan-Bo Xie,1,2 Li-Zhen Chen,1,2 Qi-Sheng Feng,1,2 Tiebang Kang,1,2 and Yi-Xin Zeng1,2*

State Key Laboratory of Oncology in Southern China,1 Departments of Experimental Research,2 Nasopharyngeal Carcinoma, Sun Yat-sen University Cancer Center, Guangzhou, China3

Received 28 May 2008/ Returned for modification 22 August 2008/ Accepted 9 February 2009


arrow
ABSTRACT
 
Epstein-Barr virus nuclear antigen 1 (EBNA1) is a protein expressed consistently in nasopharyngeal carcinoma (NPC). Although antibody levels against three different EBNA1 peptides were all high in NPC patients, the correlation between any two biomarkers was low. Therefore, the selection of distinct EBNA1 peptides could render different results in serological detection for individuals with NPC.


arrow
TEXT
 
Epstein-Barr virus (EBV) infection is closely associated with several malignant diseases, including nasopharyngeal carcinoma (NPC). NPC is highly prevalent in southern China, and NPC patients usually have higher EBV antibody levels than healthy controls. Examination of the serum immunoglobulin A (IgA) antibodies against EBV capsid antigen, diffuse early antigens (EA-D), and EBV nuclear antigen 1 (EBNA1) are widely used for NPC diagnosis and prognosis (3).

EBNA1 plays a role in the maintenance of latent EBV infection and is expressed in all EBV-associated malignant tissues (7), resulting in the hypothesis that EBNA1 is critical for initiating and developing these tumors (6). Encoded by the BamHI K fragment of the EBV genome, EBNA1 contains a Gly-Ala repeat domain flanked by unique regions (1). The repeat region, C terminus, and N terminus are antigenic (2, 8). Thus, peptides with these motifs may be useful for EBNA1 serology.

To evaluate the commercial EBNA1 proteins for EBV serological examination, two EBNA1 peptides, a recombinant full-length peptide (rEBNA1) (Biodesign, Saco, ME) and a recombinant fusion fragment containing amino acids 1 to 90 and 408 to 498 (fEBNA1) (ProSpec Co., Rehovot, Israel), were chosen to compare antibody responses in NPC patients and healthy controls. Furthermore, to test if a synthesized EBNA1 peptide could substitute for the recombinant EBNA1 proteins in the serological examination, we analyzed the immunodominant epitopes of EBNA1 as described before (4). Briefly, the protein sequences were examined according to the reported EBV proteomes by using DNAStar software, and a sequence with a high possibility of hydrophilicity, surface orientation, and flexibility was chosen. Finally, we selected amino acids 61 to 78 in the BamHI K fragment to be chemically synthesized (sEBNA1, GSGPRHRDGVRRPQKRPS) by adding a biotinylated linker to the N terminus (Hanyu, Shenzhen, China).

Ninety-five patients with newly diagnosed and pathologically confirmed NPC were recruited from Sun Yat-sen University Cancer Center. The stage of disease progression was classified according to the 1996 Union International Cancer Control classification. The NPC case group, including 4 patients with stage I, 10 with stage II, 58 with stage III, and 23 with stage IV cancer, had 72 males and 23 females with an age range of 17 to 68 (mean ± standard deviation, 45.6 ± 10.9) years. Eighty-eight healthy volunteers were also recruited as healthy controls, including 78 males and 10 females with an age range of 25 to 71 (mean, 46.6 ± 13.1) years. Written informed consent was obtained from all participants.

Coupling of rEBNA1 and fEBNA1 to the carboxylated beads (Luminex Corp., Austin, TX) was performed according to our protocols as described previously (5). sEBNA1 was coupled to LumAvidin microspheres (Luminex Corp., Austin, TX) according to the manufacturer's instructions. Serum samples diluted to 1:21 in storage buffer (20 µl/well) were added to the 96-well filtration system (Millipore, Billerica, MA) and incubated with the conjugated beads for 30 min at room temperature in the dark. After three washes, 150 µl of R-phycoerythrin-conjugated goat anti-human IgA or IgG (1:200 in phosphate-buffered saline; SouthernBiotech, Birmingham, AL) was added to each reaction well and incubated for 30 min. The detection analysis was performed by using the Luminex multianalytic 100 system (Bio-Rad, Hercules, CA). All tests were carried out in duplicate.

As shown in Table 1, the IgA values against the three peptides were significantly higher for samples from the NPC patients than from the healthy controls (P < 0.0001). The areas under the concentration-time curve for IgA xMAP assays were all above 0.8, and the sensitivities and specificities ranged from 80 to 88% for NPC diagnosis according to the optimal cutoff values. The IgG levels against the fusion fragment or synthesized peptide were higher in samples from the NPC patients. However, the IgG levels against full-length EBNA1 were higher in samples from the healthy controls. These results might be due to the nonspecific response to the Gly-Ala repeat region presented in the full-length peptide.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Analysis of antibodies against different EBNA1 peptides in samples from NPC patients and healthy controls

The correlation between any two biomarkers was low, as the correlation coefficient (r) ranged from 0.314 to 0.456. Moreover, the levels of IgG-rEBNA1 had no correlation with the levels of IgG-sEBNA1 (r = –0.066, P = 0.537) or IgG-fEBNA1 (r = 0.072, P = 0.333), indicating that the serum samples recognized the EBNA1 peptides variously. This may be due to various individual immune responses to EBNA1 after EBV infection. Alternatively, the peptides might have different conformations, consequently altering the immunogenic regions and resulting in distinct affinities with the same serum.

Therefore, the selection of distinct EBNA1 peptides could render different results in serological detection for individuals with NPC, and it might be more efficient for NPC screening and diagnosis in regions where the disease is endemic if any combination of these peptides is analyzed. Indeed, when IgA-rEBNA1 and -fEBNA1 were combined, only 7 of 95 NPC patients had IgA levels below both cutoff values, and the sensitivity of NPC diagnosis increased to 92.6% (95% confidence interval [CI], 85.4 to 97.0%), providing a better strategy for NPC screening and diagnosis. As for other combinations, the sensitivities of IgA-rEBNA1 and -sEBNA1, IgA-fEBNA1 and -sEBNA1, and IgG-fEBNA1 and -sEBNA1 reached 93.6% (95% CI, 82.5 to 98.7%), 97.9% (95% CI 88.7 to 99.9%), and 80.9% (66.7 to 90.9%), respectively, with better discriminatory values than the individual peptides. Since the EBV EBNA1 serology examination was performed with members of the Cantonese population, which has the highest NPC incidence, the diagnostic values of these methods await further confirmation in regions where NPC is not endemic.


arrow
ACKNOWLEDGMENTS
 
This study was supported by the Scientific and Technologic Project of Guangzhou City (2007Z-E4021) and the China Postdoctoral Science Foundation (20070410862).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou 510060, China. Phone: 86-20-8734-3333. Fax: 86-20-8734-3295. E-mail: zengyix{at}mail.sysu.edu.cn Back

{triangledown} Published ahead of print on 11 March 2009. Back


arrow
REFERENCES
 
    1
  1. Baer, R., A. T. Bankier, M. D. Biggin, P. L. Deininger, P. J. Farrell, T. J. Gibson, G. Hatfull, G. S. Hudson, S. C. Satchwell, C. Seguin, et al. 1984. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310:207-211.[CrossRef][Medline]
    2. 2
  2. Cheng, H. M., Y. T. Foong, C. K. Sam, U. Prasad, and J. Dillner. 1991. Epstein-Barr virus nuclear antigen 1 linear epitopes that are reactive with immunoglobulin A (IgA) or IgG in sera from nasopharyngeal carcinoma patients or from healthy donors. J. Clin. Microbiol. 29:2180-2186.[Abstract/Free Full Text]
    3. 3
  3. de The, G. 2005. Sero epidemiology of EBV and associated malignancies. Caister Academic Press, Wymondham, United Kingdom.
    4. 4
  4. Fachiroh, J., D. K. Paramita, B. Hariwiyanto, A. Harijadi, H. L. Dahlia, S. R. Indrasari, H. Kusumo, Y. S. Zeng, T. Schouten, S. Mubarika, and J. M. Middeldorp. 2006. 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. J. Clin. Microbiol. 44:1459-1467.[Abstract/Free Full Text]
    5. 5
  5. Gu, A. D., Y. B. Xie, H. Y. Mo, W. H. Jia, M. Y. Li, M. Li, L. Z. Chen, Q. S. Feng, Q. Liu, C. N. Qian, and Y. X. Zeng. 2008. Antibodies against Epstein-Barr virus gp78 antigen: a novel marker for serological diagnosis of nasopharyngeal carcinoma detected by xMAP technology. J. Gen. Virol. 89:1152-1158.[Abstract/Free Full Text]
    6. 6
  6. Horner, D., M. Lewis, and P. J. Farrell. 1995. Novel hypotheses for the roles of EBNA-1 and BHRF1 in EBV-related cancers. Intervirology 38:195-205.[Medline]
    7. 7
  7. Leight, E. R., and B. Sugden. 2000. EBNA-1: a protein pivotal to latent infection by Epstein-Barr virus. Rev. Med. Virol. 10:83-100.[CrossRef][Medline]
    8. 8
  8. Rumpold, H., G. H. Rhodes, P. L. Bloch, D. A. Carson, and J. H. Vaughan. 1987. The glycine-alanine repeating region is the major epitope of the Epstein-Barr nuclear antigen-1 (EBNA-1). J. Immunol. 138:593-599.[Abstract]

Clinical and Vaccine Immunology, April 2009, p. 592-593, Vol. 16, No. 4
1071-412X/09/$08.00+0     doi:10.1128/CVI.00207-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gu, A.-D.
Right arrow Articles by Zeng, Y.-X.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gu, A.-D.
Right arrow Articles by Zeng, Y.-X.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»