Evaluation of commercial EBV RecombLine assay for diagnosis of nasopharyngeal carcinoma

BACKGROUND: In recent years a number of Epstein-Barr virus (EBV) proteins were defined as being immunodominant for either IgM, IgG or IgA immune responses, yielding promising markers for diagnostic serology. Specific reactivity patterns to these proteins have been described for infectious mononucleosis (IM), nasopharyngeal carcinoma (NPC), various types of lymphoma, and healthy EBV carriers. OBJECTIVES: To compare the NPC-related diagnostic value of EBV RecombLine test (Mikrogen, Germany) with a standardized immunoblot assay [Fachiroh J, Schouten T, Hariwiyanto B, Paramita DK, Harijadi A, Haryana SM, et al. Molecular diversity of Epstein-Barr virus IgG and IgA antibody responses in nasopharyngeal carcinoma: a comparison of Indonesian, Chinese, and European subjects. J Infect Dis 2004;190:53-62] and to define the diagnostic value of individual EBV marker proteins in a population with high incidence of NPC. RESULT: Sera from Indonesian NPC patients taken at primary diagnosis (n=108) were analyzed for IgG and IgA reactivity and compared with regional healthy blood donors (n=62), non-NPC patient controls (n=10) and IM patients (n=10). Most NPC patients and controls showed strong IgG reactivity to VCA-p18, -p23, and EBNA1, limiting their diagnostic use. Few (<20%) healthy donors and patient controls showed IgG reactivity to EA proteins p47/54 and p138, yielding combined sensitivity/specificity and PPV/NPV values of 92.6%/98.3% and 99.0%/88.1%, for diagnosing NPC. NPC sera showed significantly more EBV reactive IgA antibody (>80% positive) than controls (<10% positive), although being less broadly reactive and significantly less strong compared to IgG. For IgA best results were observed for RecombLine EBNA1 with sensitivity/specificity and PPV/NPV values of 92%/89% and 93.4%/85.9%, respectively. CONCLUSION: In high incidence NPC regions with low incidence IM yet high prevalence of EBV infection, both RecombLine IgG and IgA tests provide a useful alternative to the more complex cell-extract based immunoblot assay as confirmation test for NPC diagnosis in particular when using EA and EBNA1 as discriminators in IgG and IgA testing, respectively.     

Native early antigen of Epstein-Barr virus, a promising antigen for diagnosis of nasopharyngeal carcinoma

The Epstein-Barr virus (EBV) early antigen (EA) complex consists of multiple proteins with relevance for diagnosis of acute, chronic and malignant EBV related diseases, including nasopharyngeal carcinoma (NPC). In a recent study, it was found that the molecular diversity of EBV-specific IgG and IgA antibody responses in NPC patients and demonstrated that these reflect independent B-cell triggering leading to distinct EBV antigen-recognition profiles. The fine-specificity of NPC-related IgG and IgA responses was explored further against defined recombinant and synthetic EBV-EA antigens using immunofluorescence, immunoblot and ELISA techniques and determined their diagnostic value in a large panel of sera from NPC (n = 154), non-NPC tumor patients (n = 133), acute mononucleosis patients (n = 70) and healthy EBV carriers (n = 259). Individual recombinant EBV-EA markers yielded sensitivity/specificity values not exceeding 86%, whereas selected EA-specific peptide epitopes were rather poorly recognized by IgG and IgA antibodies in NPC sera. Surprisingly, we found that a "low salt" native EA-protein extract reproducibly prepared from purified nuclei of EA-induced HH514 cells, and containing characteristic EA(D)-polypeptides, such as p47-54 (BMRF1), p138 (BALF2), p55-DNAse (BGLF5), and p65-TK (BXLF1), but without viral capsid (VCA) or nuclear antigen (EBNA) reactivity, gave highest sensitivity (90.4%) and specificity (95.5%) values for NPC diagnosis in both IgG and IgA ELISA. The data support further the notion that EBV-EA reactive IgG and IgA antibodies in NPC patients are directed against distinct conformational and-in part-linear epitopes on EBV-specific proteins, barely recognized in other EBV-related syndromes. The use of a defined native EBV EA-specific antigen opens the way to further improve serological diagnosis of NPC.     

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

Assessment of immunoglobulin A (IgA) antibody responses to various Epstein-Barr virus (EBV) antigen complexes, usually involving multiple serological assays, is important for the early diagnosis of nasopharyngeal carcinoma (NPC). Through combination of two synthetic peptides representing immunodominant epitopes of EBNA1 and viral capsid antigen (VCA)-p18 we developed a one-step sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of EBV reactive IgG and IgA antibodies in NPC patients (EBV IgG/IgA ELISA). Sera were obtained from healthy donors (n = 367), non-NPC head and neck cancer patients (n = 43), and biopsy-proven NPC patients (n = 296) of Indonesian and Chinese origin. Higher values of optical density at 450 nm for EBV IgG were observed in NPC patients compared to the healthy EBV carriers, but the large overlap limits its use for NPC diagnosis. Using either EBNA1 or VCA-p18 peptides alone IgA ELISA correctly identified 88.5% and 79.8% of Indonesian NPC patients, with specificities of 80.1% and 70.9%, whereas combined single-well coating with both peptides yielded sensitivity and specificity values of 90.1 and 85.4%, respectively. The positive and negative predictive values (PPV and NPV, respectively) for the combined EBNA1 plus VCA EBV IgA ELISA were 78.7% and 93.9%, respectively. In the Indonesia panel, the level of EBV IgA reactivity was not associated with NPC tumor size, lymph node involvement, and metastasis stage, sex, and age group. In the China panel the sensitivity/specificity values were 86.2/92.0% (EBNA1 IgA) and 84.1/90.3% (VCA-p18 IgA) for single-peptide assays and 95.1/90.6% for the combined VCA plus EBNA1 IgA ELISA, with a PPV and an NPV for the combined EBV IgA ELISA of 95.6 and 89.3%, respectively. Virtually all NPC patients had abnormal anti-EBV IgG diversity patterns as determined by immunoblot analysis. On the other hand, healthy EBV carriers with positive EBV IgA ELISA result showed normal IgG diversity patterns. By using EBV IgG immunoblot diversity as confirmation assay for EBV IgA ELISA-positive samples, the sensitivity and specificity for NPC diagnosis increased to 98% and 99.2%, respectively, in the Indonesian NPC samples. The use of these combined methods for seroepidemiological screening studies is proposed.     

Humoral immune responses to Epstein-Barr virus encoded tumor associated proteins and their putative extracellular domains in nasopharyngeal carcinoma patients and regional controls

Epstein-Barr virus (EBV) latency proteins EBNA1, LMP1, LMP2, and BARF1 are expressed in tumor cells of nasopharyngeal carcinoma (NPC). IgG and IgA antibody responses to these non-self tumor antigens were analyzed in NPC patients (n=125) and regional controls (n=100) by three approaches, focusing on the putative LMP1, LMP2 extracellular domains. Despite abundant IgG and IgA antibody responses to lytic antigens and EBNA1, patients had low titer (1:25-1:100) IgG to LMP1 (81.2%), LMP2 (95.6%), and BARF1 (84.8%), while immunoblot showed such reactivity in 24.2%, 12.5%, and 12.5% at 1:50 dilution, respectively. Few IgA responses were detected, except for EBNA1. Controls only showed IgG to EBNA1. ELISA using peptides from different domains of LMP1, LMP2, and BARF1 also yielded mostly negative results. When existing, low level IgG to intracellular C-terminus of LMP1 (62.9%) prevailed. Rabbit immunization with peptides representing extracellular (loop) domains yielded loop-specific antibodies serving as positive control. Importantly, these rabbit antibodies stained specifically extracellular domains of LMP1 and LMP2 on viable cells and mediated complement-driven cytolysis. Rabbit anti-LMP1 loop-1 and -3 killed 50.4% and 59.4% of X50/7 and 35.0% and 35.9% of RAJI cells, respectively, and 22% of both lines were lysed by anti-LMP2 loop-2 or -5 antibodies. This demonstrates that (extracellular domains of) EBV-encoded tumor antigens are marginally immunogenic for humoral immune responses. However, peptide-specific immunization may generate such antibodies, which can mediate cell killing via complement activation. This opens options for peptide-based tumor vaccination in patients carrying EBV latency type II tumors such as NPC.     

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.

The entire amino acid sequence of the unique region of the EBNA 1 protein was synthesized as a set of 41 20-residue peptides with an overlap of 10 amino acids. The peptides were tested in the enzyme-linked immunosorbent assay for reactivity with immunoglobulin A (IgA) and IgG in sera from 50 patients with nasopharyngeal carcinoma (NPC) as compared with 36 serum samples from healthy Epstein-Barr virus (EBV)-seropositive donors and 5 serum samples from EBV-negative donors. The most immunoreactive peptide for both IgA and IgG binding was localized to the glycine-alanine repeat domain of the antigen. In the unique regions, 16 immunoreactive peptides were found. Of these, four were reactive with IgG but not IgA and three peptides were reactive with IgA but not IgG in NPC sera. In addition, several IgA and IgG epitopes on the carboxy-terminal region were specifically reactive with NPC sera, but unreactive with sera from healthy EBV-positive donors. The results suggest that EBV serology specific for individual epitopes may provide additional useful information not available by conventional serology with whole antigens or the EBNA complex.     

Diagnostic value of measuring Epstein-Barr virus (EBV) DNA load and carcinoma-specific viral mRNA in relation to anti-EBV immunoglobulin A (IgA) and IgG antibody levels in blood of nasopharyngeal carcinoma patients from Indonesia

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and is strongly associated with Epstein-Barr virus (EBV). We investigated the primary diagnostic value of circulating EBV DNA and anti-EBV immunoglobulin G (IgG) and IgA levels in Indonesian NPC patients (n = 149). By a 213-bp Epstein-Barr virus nuclear antigen 1 (EBNA1)-based real-time LightCycler PCR, 72.5% of patients were positive for EBV DNA in whole blood, with 29.5% having levels above a previously determined clinical cutoff value (COV) of 2,000 EBV DNA copies/ml, the upper level in healthy carriers. In a 99-bp LightCycler PCR, 85.9% of patients were positive and 60.4% had levels above the COV. This assay quantified a significantly higher EBV load than the 213-bp PCR assay (P < 0.0001), suggesting that circulating EBV DNA is fragmented. Using data from 11 different studies, we showed a significant inverse correlation between PCR amplicon size and the percentage of patients positive for circulating EBV DNA (Spearman's rho = -0.91; P < 0.0001). EBV DNA loads were unrelated to anti-EBV IgG or IgA levels, as measured by VCA-p18 and EBNA1-specific synthetic peptide-based enzyme-linked immunosorbent assays. The presence of circulating tumor cells was assessed by amplification of BamHI-A rightward frame 1 (BARF1) mRNA, a viral oncogene abundantly expressed in EBV-carrying carcinomas but virtually absent from EBV-associated lymphomas. Despite high EBV DNA loads and the presence of EBNA1 and human U1A small nuclear ribonucleoprotein mRNA, BARF1 mRNA was never detected in blood. We conclude that amplicon size significantly influences EBV DNA load measurement in NPC patients. The circulating EBV DNA load is independent of serological parameters and does not reflect intact tumor cells. The primary diagnostic value of the EBV DNA load for the detection of NPC is limited.     

Comparison of three different serological techniques for primary diagnosis and monitoring of nasopharyngeal carcinoma in two age groups from Tunisia

Nasopharyngeal carcinoma (NPC) in Tunisia is characterized by its bimodal age distribution involving juvenile patients of 10-24 years and adult patients of 40-60 years. Three serological techniques were compared for primary diagnosis (N = 117) and post-treatment monitoring (N = 21) of NPC patients separated in two age groups. Immunofluorescence assay (IFA) was used as the "gold standard" for detection of IgG and IgA antibodies reactive with Epstein-Barr virus (EBV) early (EA) and viral capsid (VCA) antigens. Results were compared with ELISA measuring IgG and IgA antibody reactivity to defined EBNA1, EA, and VCA antigens. Immunoblot was used to reveal the molecular diversity underlying the anti-EBV IgG and IgA antibody responses. The results indicate that young NPC patients have significantly more restricted anti-EBV IgG and IgA antibody responses with aberrant IgG VCA/EA levels in 78% compared to 91.7% in elder patients. IgA VCA/EA was detected in 50% of young patients versus 89.4% for the elder group (P < 0.001). Immunoblot revealed a reduced overall diversity of EBV antigen recognition for both IgG and IgA in young patients. A good concordance was observed between ELISA and IFA for primary NPC diagnosis with 81-91% overall agreement. Even better agreement (95-100%) was found for antibody changes during follow-up monitoring, showing declining reactivity in patients in remission and increasing reactivity in patients with persistent disease or relapse. ELISA for IgA anti-VCA-p18 and immunoblot proved most sensitive for predicting tumor relapse. VCA-p18 IgA ELISA seems suitable for routine diagnosis and early detection of NPC complication.     

BamHI‐A rightward frame 1, an Epstein–Barr virus‐encoded oncogene and immune modulator

Epstein–Barr virus (EBV) causes several benign and malignant disorders of lymphoid and epithelial origin. EBV‐related tumors display distinct patterns of viral latent gene expression, of which the BamHI‐A rightward frame 1 (BARF1) is selectively expressed in carcinomas, regulated by cellular differentiation factors including ΔNp63α. BARF1 functions as a viral oncogene, immortalizing and transforming epithelial cells of different origin by acting as a mitogenic growth factor, inducing cyclin‐D expression, and up‐regulating antiapoptotic Bcl‐2, stimulating host cell growth and survival. In addition, secreted hexameric BARF1 has immune evasive properties, functionally corrupting macrophage colony stimulating factor, as supported by recent functional and structural data. Therefore, BARF1, an intracellular and secreted protein, not only has multiple pathogenic functions but also can function as a target for immune responses. Deciphering the role of BARF1 in EBV biology will contribute to novel diagnostic and treatment options for EBV‐driven carcinomas. Herein, we discuss recent insights on the regulation of BARF1 expression and aspects of structure‐function relating to its oncogenic and immune suppressive properties. © 2013 The Authors. Reviews in Medical Virology published by John Wiley & Sons, Ltd.     

Use of bacterially expressed GST/EBNA-1 fusion proteins for detection of antibodies in sera from patients with nasopharyngeal carcinoma and healthy donors

Epstein-Barr virus nuclear antigen-1 (EBNA-1) is a protein expressed consistently in EBV infected cells and in EBV related malignant tissues. Antibodies against EBNA-1 may therefore possibly be used as a marker for disease screening. Western blot analysis of serum antibodies was performed using GST (glutathione-S-transferase) fusion proteins containing different regions of EBNA-1 as antigens. Serum samples were collected from 38 patients with nasopharyngeal carcinoma (NPC) and 38 healthy individuals in Taiwan. All samples were found IgG positive for EBNA-1 when a truncated protein GST/E1 (70-102, 325-641) was used as the antigen. Thirty-three out of 38 NPC sera (86.8%) were positive for IgA antibody against EBNA-1. The positive rate was higher in comparison with IgA antibody against VCA (65.7%) or antibody against DNase (60.5%). Only 2.6% of sera from normal individuals were positive for an IgA response against EBNA-1. The major antigenic determinants for NPC serum IgA response were between amino acid(aa) 390 to aa 459 when different portions of EBNA-1 were used as antigens. The results suggest that IgA response against EBNA-1 could be used in combination with other EBV serology markers for NPC screening.     

Antibodies to Epstein-Barr virus in nasopharyngeal carcinoma, tonsillar carcinoma and control groups

In the sera of 17 patients with nasopharyngeal carcinoma (NPC) and of 19 patients with tonsillar carcinoma (TC) the titres of IgA, IgG and IgM antibodies to EBV VCA (viral capsid antigen) and of IgG antibodies to EBV EA (early antigen) were determined by the indirect immunofluorescence (IF) method. Significant difference was observed in the frequency of IgA antibodies to EBV VCA and IgG antibodies to EBV EA between NPC patients and controls. There was also a significant difference between the frequency of IgM antibody to EBV VCA and EBV EA antibody titres in TC patients and controls. The geometric mean titre (GMT) of IgG antibodies to EBV VCA was significantly higher in the NPC and TC patients as compared to controls.     

Humoral immune response in Epstein-Barr virus infections. I. Elevated serum concentration of the IgG1 subclass in infectious mononucleosis and nasopharyngeal carcinoma

Using radial immunodiffusion we measured IgG subclass concentrations and studied their distribution in serum samples from patients with infectious mononucleosis (IM) and nasopharyngeal carcinoma (NPC), two Epstein-Barr virus (EBV)-associated diseases, in comparison with two control groups [completely anti-EBV negative persons and subjects carrying antibodies to the viral capsid antigen (VCA)]. Antibody titres to VCA and to the early antigen (EA) were determined by indirect immunofluorescence and revealed characteristic patterns for the respective diagnostic groups. Nephelometric assays served for quantitating total protein, albumin, total IgG, IgA and IgM in all the sera. In the IM and NPC groups the concentration of IgG1 was significantly elevated by more than 50% whereas the other three subclasses remained unchanged as compared with the controls. Correspondingly, we found a significant increase of total IgG in IM and NPC. In IM, the only disease where VCA-specific IgM antibodies have been reported to occur, IgM levels were markedly elevated. Our data suggest that the IgG1 subclass plays an important role in the humoral immune response to EBV-determined antigens and that it is possibly involved in the control of virus infection.     

Antibodies to lytic infection proteins in lymphocryptovirus-infected rhesus macaques: a model for humoral immune responses to epstein-barr virus infection

Humoral immune responses to rhesus lymphocryptovirus (rhLCV) lytic infection proteins were evaluated in the rhesus macaque animal model for Epstein-Barr virus (EBV) infection. We found a hierarchy of humoral responses to 14 rhLCV lytic infection proteins in naturally infected rhesus macaques, with (i) widespread and robust responses to four glycoproteins expressed as late proteins, (ii) frequent but less robust responses to a subset of early proteins, and (iii) low-level responses to immediate-early proteins. This hierarchy of humoral responses was similar to that reported for EBV-infected humans, with the notable exception of the response to rhBARF1. Serum antibodies to rhBARF1 were frequently detected in healthy rhLCV-infected macaques, but in humans, anti-BARF1 antibodies have been reported primarily in patients with EBV-positive nasopharyngeal carcinoma (NPC). The macaque data accurately predicted that serum antibodies against BARF1 are a normal response to EBV infection when human serum samples are analyzed. The rhesus macaque animal provides a unique perspective on humoral responses to EBV infection in humans and can be a valuable model for EBV vaccine development.     

Improved sensitivity of detection by avidin-biotin complex (ABC) immunocytochemistry in Epstein-Barr virus serology

Serum antibodies against Epstein-Barr virus (EBV)-determined antigens have traditionally been titrated by the indirect immunofluorescence (IIF) technique. The avidin-biotin complex (ABC) immunocytochemical technique was used to determine the serum levels of IgA against EBV viral capsid antigen (IgA/VCA) and IgA against EBV early antigen (IgA/EA) in sera of 106 nasopharyngeal carcinoma (NPC) patients prior to treatment and 100 normal individuals. The sensitivity of the ABC technique is enhanced by an amplification of the antigen-antibody reaction, which involves the binding of the enzyme-linked ABC to the second biotinylated antibody. There was a good correlation (r = 0.9988) between ABC and IIF-determined IgA/VCA-positive titres, with the ABC technique being more sensitive than IIF in the detection of IgA/VCA in NPC sera: 94% (99/106) and 76% (80/106), respectively. The frequency of IgA/EA reactivity in NPC sera was also markedly increased by immunodetection with the ABC technique as compared with IIF technique: 63% (69/106) and 28% (30/106) respectively. Both the immunocytochemical techniques were equally specific in discriminating between elevated serum titres of IgA/VCA and IgA/EA in NPC sera from normal human sera.     

Antibody against the Epstein-Barr virus BHRF1 protein,a homologue of Bcl-2, in patients with nasopharyngeal carcinoma

The Epstein-Barr virus (EBV) open reading frame BHRF1, a homologue of the oncogene bcl-2, was cloned from a patient with nasopharyngeal carcinoma (NPC) and overexpressed in Escherichia coli. The resulting recombinant BHRF1 fusion protein, with an apparent molecular weight of 35 KD, was used as antigen in an immunoblotting assay for IgG antibody in human sera. Anti-BHRF1 antibody was detected in 57 (61.3%) of 93 patients with NPC, 5 (5.7%) of 87 patients with nonmalignant diseases of the nasopharynx, and in 1 (1.3%) of 78 healthy blood donors. The positivity rate in these nonmalignant patients was 4.4 times that of the normal controls. Negative results were observed in four patients with infectious mononucleosis and patients with other cancers, including 4 with esophageal cancer, 11 with lung cancer, 10 with lymphoma, 13 with gastric carcinoma, 10 with cervical carcinoma, and 10 with other head and neck cancers. Antibody neutralizing EBV DNase and IgA antibody to viral capsid antigen (VCA) were assayed in parallel. The results showed that 7.5% of the NPC patients were negative for anti-DNase and anti-VCA antibodies and EBV infection could be detected by the anti-BHRF1 antibody alone. The demonstration of anti-BHRF1 antibody in most NPC sera strongly supports the hypothesis that the EBV BHRF1 protein is expressed in most NPC patients and its specific antibody can be a useful marker for the diagnosis of NPC. J. Med. Virol. 56:179–185, 1998. © 1998 Wiley-Liss, Inc.     

基于2008分期标准的壮族鼻咽癌各期患者中EB病毒Zta/IgG等抗体阳性率的比较

探讨桂西地区壮族鼻咽癌患者EB病毒EBNA1/IgA、Zta/IgG及Rta/IgG抗体阳性率与鼻咽癌2008分期的关系。收集140例未经治疗的鼻咽癌患者的血清,用酶联免疫吸附法(ELISA)检测EBNA1/IgA、Zta/IgG及Rta/IgG抗体,按"2008分期法"进行分期,分别计算临床分期的各抗体阳性率并进行两两对比,进行统计学分析。鼻咽癌各临床分期组EB-NA1/IgA、Zta/IgG及Rta/IgG抗体阳性率均有统计学差异(P<0.05)。III期的各抗体阳性率明显低于IV期临床分期(P<0.05),Rta/IgG抗体有显著差异(P=0.001)。壮族鼻咽癌患者EB病毒EBNA1/IgA、Zta/IgG及Rta/IgG抗体阳性率与鼻咽癌临床分期有关。或许EBNA1/IgA、Zta/IgG及Rta/IgG抗体对于评估鼻咽癌临床分期有一定的参考价值。     

Detection of IgA antibodies to Epstein-Barr virus-associated antigens by ELISA

The detection of IgA antibodies to the Epstein-Barr virus (EBV)-associated viral capsid antigen (VCA) and early antigens (EA) is of diagnostic and prognostic importance for patients with nasopharyngeal carcinoma (NPC). An ELISA for the determination of serum IgG antibodies to these antigens has been developed which uses the double antibody method. 136 sera obtained from healthy donors and patients with non-EBV related tumors and lymphomas were tested by ELISA; only 3 sera, from patients with chronic lymphatic leukemia, hairy cell leukemia and Burkitt-like lymphoma, contained antibodies of IgA class to VCA and EA. Ninety-five sera from patients suspected of having NPC were tested. IgA anti-VCA was found in 28 sera (29.5%), 12 of which also contained IgA anti-EA. The assays described are suitable for diagnosis and follow-up of patients with EBV-associated nasopharyngeal carcinoma. Furthermore, isolated EA components may be tested for their reactivity with IgA antibodies, as was shown for the 60 kDa polypeptide associated with the EA complex.     

IgA directed against early antigen of Epstein-Barr virus is no specific marker for the diagnosis of nasopharyngeal carcinoma

The aim of this study was to evaluate the significance and specificity of IgA directed against Epstein-Barr virus (EBV)-specific early antigens (EA) for the unequivocal diagnosis of nasopharyngeal carcinoma (NPC). Therefore, sera from patients with diseases other than NPC, selected on the basis of elevated antibody titres against EBV antigens, were compared to sera from NPC patients with regard to the presence of IgA directed against EBV viral capsid antigen (VCA-IgA) and IgA directed against EA (EA-IgA). Four hundred forty-seven out of 7,508 non-NPC sera tested showed high titres (>512) of IgG directed against Epstein Barr viral capsid antigen (VCA-IgG) and positive VCA-lgA (?32). Two hundred twenty-seven of these sera were compared to 51 VCA-IgA-positive sera from NPC patients regarding the titre of EA-lgA. 60.7% of VCA-lgA-positive NPC sera showed positive EA-lgA, however 33% of VCA-IgA-positive non-NPC patients also exhibited EA-lgA. This result demonstrates that EA-lgA is not specific for NPC and does not allow an unequivocal serological diagnosis of NPC in individual cases. It seems therefore to be of questionable use for screening programs in NPC low-risk areas. The data do not contradict the usefulness of this marker for monitoring of patients treated for NPC and for screening programmes in high-risk areas. © 1994 Wiley-Liss, Inc.     

Unique variations of Epstein-Barr virus-encoded BARF1 gene in nasopharyngeal carcinoma biopsies

The Epstein-Barr virus (EBV) BamHI-A rightward frame 1 (BARF1) gene is frequently expressed in EBV-associated epithelial malignancies and involves in oncogenicity and immunomodulation. To characterize the variations of BARF1 gene in different populations, the sequences of BARF1 gene in Northern Chinese nasopharyngeal carcinoma (NPC), EBV-associated gastric carcinoma (EBVaGC) and healthy donors were analyzed. The correlation of BARF1 variation with polymorphisms of BamHI F fragment (type F and f variants) and EBV-coded viral interleukin-10 (vIL-10) gene (B95-8 and SPM patterns) was also explored. Two major subtypes of BARF1 gene, designated as B95-8 and V29A, were identified. B95-8 subtype had identical amino acid sequence to B95-8 and was the dominant subtype among the EBV isolates from Northern China. V29A subtype, with one consistent amino acid change at residue 29 (V→A) and several nucleotide changes, showed higher frequency in NPC cases (25.3%, 20/79) than in EBVaGC cases (0/45) or healthy donors (4.3%, 2/46) (NPC vs. EBVaGC: P=0.0001; NPC vs. healthy donor: P=0.004). A preferential linkage between BamHI F and BARF1/vIL-10 polymorphisms was found. Type f isolates was specially correlated with the V29A/SPM genotype in NPC isolates and type f/V29A/SPM was preferentially found in NPC. BARF1/c-fms homology domain, transforming domain and cytotoxic T lymphocyte (CTL) epitopes of BARF1 were highly conserved in most isolates, suggesting the important role of BARF1 in virus infection and the potential usefulness in EBV-targeting immunotherapy of EBV-associated tumors. The relatively higher prevalence of type f/V29A/SPM strains in NPC may also suggest the association between these variations in multiple viral genes and NPC.     

Immunoglobulin class and subclass deficiencies prior to Epstein-Barr virus infection in males with X-linked lymphoproliferative disease.

Patients with X-linked lymphoproliferative (XLP) disease are characterized by extreme vulnerability to Epstein-Barr virus (EBV). Following infection with EBV, affected males develop fatal infectious mononucleosis (IM), hypogammaglobulinemia (H), or non-Hodgkin's lymphoma (NHL). In addition, hyper IgM, red cell aplasia, necrotizing lymphoid vasculitis (NLV), and aplastic anemia occur rarely. The recent use of DNA restriction fragment length polymorphism (RFLP) probes in linkage with the XLP gene now permit detection of affected males prior to primary EBV infection. We have measured immunoglobulin class and subclass levels in sera from EBV-negative males who were either positive or negative for the XLP genotype by RFLP analysis. Elevated IgA or IgM and/or variable deficiency of IgG, IgG1, and IgG3 occurred in the sera of 13/13 RFLP-positive, EBV-negative males. No consistent abnormalities were noted in 14 RFLP-negative, EBV-negative males. We conclude that the immune defect in XLP is not solely EBV-specific, although EBV is responsible for most of the morbidity and all of the mortality. Further, serial measurement of Ig levels may provide information regarding status of EBV-negative males at risk where RFLP analysis is uninformative or in families where sporadic cases of fatal IM, acquired hypogammaglobulinemia or NHL have occurred, but wherein the genotype of XLP cannot be documented.