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Annals of Medicine and Surgery logoLink to Annals of Medicine and Surgery
. 2023 Jul 19;85(9):4394–4403. doi: 10.1097/MS9.0000000000000963

EBV-IgA antibody responses in endemic and nonendemic populations with nasopharyngeal carcinoma: tumour marker prognostication study and a cross-sectional study

Insani Budiningsih a,d, Sandra AWM Verkuijlen d, Yoes P Dachlan c, Muhammad V Arfijanto b, Usman Hadi b,*, Jaap M Middeldorp d,*
PMCID: PMC10473314  PMID: 37663720

Abstract

Nasopharyngeal carcinoma (NPC) is the most prevalent head and neck cancer in Indonesia, with 100% Epstein–Barr virus (EBV) infection in tumor cells. NPC is rare in the Netherlands. The involvement of EBV in NPC pathogenesis is reflected by early onset aberrant IgA antibody responses to various EBV proteins. Screening for elevated EBV-IgA levels is proposed for NPC risk assessment in endemic countries but is poorly studied in nonendemic regions. This study analyzed the overall diversity (immunoblot) as well as the prevalence and normalized levels of IgA responses to immunodominant peptide epitopes of EBV proteins VCA P18, EBNA 1, and Zebra (Zta) (N-terminus, P 125, P 130, full-length recombinant Zebra) in Indonesian (n=50) and Dutch (n=50) patients with NPC. The results confirmed that elevated levels of IgA-VCA P18 and IgA-EBNA 1 were found in both NPC populations, but that IgA-Zta was more variable. IgA-Zta responses were more pronounced in Indonesian NPC cases, reflecting more frequent EBV reactivation overall. IgA-VCA P18 and IgA-EBNA are independent tumor markers and are both necessary for NPC risk assessment. Overall, these results confirmed the diagnostic benefit of combined IgA-VCA P18/-EBNA 1 testing for NPC risk assessment in endemic and nonendemic populations.

Keywords: EBNA 1, Epstein–Barr virus, IgA, NPC, VCA P18, Zebra proteins

Introduction

Highlights

  • Nasopharyngeal carcinoma (NPC) is the most prevalent head and neck malignancy in Indonesia, while in the Netherlands, NPC is rare.

  • NPC corresponds to the highest tumor burden that is associated with the Epstein–Barr virus infection.

  • Patients usually arrive at the hospital at a late stage of NPC due to unspecific symptoms in the early stage, and due to the lack of awareness of the disease’s symptoms by both patients and health experts.

  • The treatment of NPC is stage-dependent, and early detection is imperative to achieve higher treatment success.

  • Knowing the markers that can be used to detect NPC at an early stage is a fundamental phase.

Epstein–Barr virus (EBV) infection is nearly always associated with nasopharyngeal cancer, viral DNA, RNA, and proteins are detected in all forms of tumor cells1, and aberrant IgG and IgA antibody responses can be detected at the early stages of disease development and have diagnostic importance2,3. Indonesia has a population with 100% positive EBV infections4,5. Nasopharyngeal carcinoma (NPC) occurs due to unusual lymphocytic infiltration, and reports have suggested that EBV particles can be transferred from lymphoid cells to nasopharyngeal epithelial cells through cell-to-cell contact. A very rapid progression from a high-grade dysplastic lesion of the nasopharynx to invasive NPC suggests that EBV may have the potential to drive the neoplastic transformation of nasopharyngeal epithelial cells and facilitate the clonal expansion of malignant cells6. NPC is a curable disease if treated early4. The problem in Southeast Asian countries, including Indonesia, is that patients mostly come to the hospital at a late stage of NPC. This is due to the lack of awareness of the disease’s symptoms by both patients and health experts7,8. Early-stage diagnosis is often difficult because the symptoms are not distinctive and the nasopharynx is a difficult area to examine. NPC is initiated in hidden areas that present with limited nonspecific signs and symptoms at an early age. These conditions have caused improper treatment for NPC patients9. NPC is the most common tumor in the head and neck, constituting 23.8% of all head and neck cancer cases10,11. In Europe, the incidence of NPC is increasing due to the migration of people from endemic countries12. Serology is the major method to determine the status of EBV infection; by measuring antibody(ies) reactivity towards EBV antigen(s), distinct antibody patterns have been determined for individuals with primary infection, latent infected carriers, and individuals with disease13. In routine diagnostic settings, no clear functional aspect is considered for IgG and IgA responses, but it is clear that multiple EBV antigens (proteins and epitopes) are involved in such responses, including the VCA, EA, or EBNA complex. Previous studies have found that Zebra and viral membrane antigens can also be used as markers to detect EBV13. However, analysis of the relationship between antigen specificity of IgG and IgA responses has not received much detailed attention. IgG and IgA antibodies may recognize different viral protein antigens, reflecting different antigen activity and immune-triggering events during persistence or disease development, or may differ in epitope recognition, reflecting independent antigen processing and presentation of IgG versus IgA B-cell responses. This analysis may reveal aberrant viral activity (systemic vs. mucosal) at early NPC stages, triggering such responses and the underlying pathogenesis of NPC disease. This study also describes the development and evaluation of antibody tests against Recombinant Zebra protein, Zebra N, Zebra P 125, and Zebra P 130 peptides, as well as VCA P18 and EBNA 1 immunodominant combi peptides, to further understand the epitope binding preference and the relation between IgG and IgA responses in NPC patients and controls, to contribute to new and improved NPC marker tests. By comparing antibody reactivity through IB and ELISA, we can get an insight into whether IgG and IgA responses are epitope-related and triggered by the same antigen interaction event or by (spatially) different triggering events, and which peptide/antigen will be most useful to diagnose NPC at an early stage.

This study was performed to analyze the EBV-specific antibody diversity between NPC patients from the Netherlands and Indonesian backgrounds and has two specific aims: (1) to define the correlation between IgG or IgA against VCA P18 and EBNA 1 (whether they are related to the strength of the response) and IgG versus IgA for these markers (whether they are clonally related to systemic immunity vs. mucosal B-cell triggers), and (2) subsequently to compare IgG and IgA Zebra epitope reactivities (peptides vs. recombinant) (i.e. What is the dominant peptide for measuring anti-Zebra reactivity?). Knowledge of the various proteins or epitopes triggering human anti-EBV IgG and IgA is expected to be utilized in the development of diagnostic tests to improve the detection of NPC at the early stages of the disease.

Materials and methods

Sample collection and NPC serum analysis

This research was a cross-sectional study, and the samples used in this research originated in the Netherlands and Indonesia from 2016 to 2017. The research subjects in this study were 165 patients, among whom were 100 NPC patients from two population backgrounds, and were extracted based on Hospital-Based Cancer Registry (HBCR) data from Cipto Mangunkusumo Hospital, Indonesia, and the Netherlands Cancer Registry (NCR). All studies and the rules of reuse of human materials were performed under the Helsinki Code of Ethics, and the work has been reported in line with the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) Criteria14. All patients subsequently visited the hospital for a more detailed examination by an ENT specialist, and had clinical symptoms characteristic of NPC, such as epistaxis, nasal obstruction, and discharge because of the tumor’s presence in the nasopharynx, and were continued for the biopsy procedures. Subsequently, formalin-fixed paraffin-embedded biopsy specimens were obtained. The NPC tumors were proven to be positive for EBV infection by EBER-RISH immunohistochemical staining of parallel tissue samples, as assessed by a pathologist. After the examinations were concluded, blood from the patients who were confirmed to be NPC-positive was collected to obtain their serum. Samples from Indonesia included 50 serum samples from patients with NPC and 50 serum samples from healthy individuals. The serum of NPC patients and healthy people from Indonesia were placed in a cool box with dry ice and shipped to the Vrije Universitet Medisch Centrum (VUMC), The Netherlands. The serum was immediately aliquoted and frozen at –80°C after arriving at the laboratory in the VUMC, after which, the serum samples were thawed and stored in a refrigerator at +2°−8°C. The samples were stored at −80°C for 7 months before this research was conducted. Healthy control samples were obtained from Indonesian residents who did not suffer from NPC. The samples from the Netherlands were 50 serum samples from NPC patients with the same sampling procedures as those from Indonesia, and 15 serum samples from IM patients. The methods used in this study were SDS–PAGE, Immunoblotting, and ELISA.

EBV antigen quantification

EBV antigens applied to the blot consisted of the nuclear fraction of HH514.c16 cells induced to express the full array of EBV antigens, as described in detail elsewhere2. Polypeptides were separated by SDS–PAGE in 10% acrylamide gels using the Bio-Rad minigel system (Bio-Rad) and transferred onto 0.2 μm nitrocellulose membranes (Schleicher & Schuell) that were subsequently cut into 3 mm strips. Marker proteins (Bio-Rad Low MW Markers) were run on the side of the gel, after the nuclear fractions were sonicated and boiled for 5 min in standard Laemmli lysis buffer and clarified by centrifugation at 13 000 rpm. Blot strips were immersed for 1 h in 5% blocking buffer (vol), horse serum (Gibco BRL) (vol), and 5% nonfat dry milk (wt) in phosphate-buffered saline (PBS) (vol), to prevent nonspecific binding. In all experiments, human serum samples were tested at a 1:100 dilution in blocking buffer and incubated with the strips for 1 h at room temperature. Subsequently, the strips were washed three times with PBS containing 0.05% Tween-20 (PBS-T), and horseradish peroxidase (HRP)-conjugated anti-IgG or anti-IgA antibody (DAKO) was added at appropriate dilutions and incubated for 1 h at room temperature. After washing three times with PBS-T and two times with PBS, the bound HRP was visualized using 0.07% 4 chloro-1-naphthol and 0.01% (vol/vol) H2O2 in PBS. The stained strips were washed overnight with 10 ml of H2O, dried, and stored in the dark until photography. Monoclonal and polyclonal monospecific antiserum samples were produced by immunizing animals with synthetic peptides or purified recombinant proteins. The antibodies used to define EBV proteins were rabbit anti-Zebra N (OTP-513), rabbit anti-Zebra P125 (OTP 413), rabbit anti-Zebra P130 (OTP 414), rabbit antiRecombinant Zebra (K150), rabbit anti-EBNA 1 (BKRF1), and rabbit anti-VCA P18 (OT15E).

The position of characteristic EBV antigens was defined by monoclonal or polyclonal antibodies of known specificity, which were detected with HRP-labeled anti-rabbit antibodies (DAKO). In addition, from every batch of blot strips, three strips were stained with reference human serum samples from a healthy seronegative and seropositive donor and serum from a patient with NPC. The two mAbs, P125 and P130 specific for the Zebra protein were employed in a sandwich ELISA for quantifying captured Zebra. Based on recently published results pertaining to a related patient, significant improvements have been made to increase the sensitivity and robustness of the Zebra assay15. Recombinant Zebra was made in the baculovirus expression system. Our prior studies indicated that for serological studies, the expression of EBV protein in the baculovirus system was preferred to Escherichia coli due to the low levels of antibodies reactive to insect cells and baculovirus proteins in human serum. Individual full-length recombinant Zebra proteins have a predicted diagnostic value. TK (BXLF1), and DNase (BGLF5), are early genes that are also expressed in SF9 insect cells and harvested at 48 h postinfection. Protein expression was analyzed using immunofluorescence and IB techniques to assess antibody binding to native and denatured epitopes, respectively16.

EBV serum antibody quantification

Ninety-six-well ELISA plates (Greiner Labortechniek) were coated with Zebra N, Zebra P125, Zebra P130, Recombinant Zebra, EBNA 1, and VCA P18 peptides using 135 ml single peptide (1 mg/ml) in 0.05 M Na2CO3, pH 9.6 (Merck). After overnight incubation at 4oC, the antigen was discarded and 200 ml blocking buffer (3% bovine serum albumin; Roche Diagnostic GmbH) in PBS was added to each well. After an hour of incubation at 37oC, the wells were emptied and washed three times with PBS containing 0.05% Tween-20 (PBS-T). Subsequently, 100 ml 1:100-diluted serum was applied (serum dilutions in PBS-T, 1% bovine serum albumin, and 0.1% Triton X-100) and incubated for 1 h at 37oC. All serums were tested in duplicate. After four washes with PBS-T, rabbit antihuman IgA HRP conjugate (diluted 1:4000 in serum dilution buffer) or antihuman IgG-HRP conjugate (diluted 1:8000 in serum dilution buffer) (DAKO) was added and incubated for 1 h at 37oC. After four washes with PBS-T, 100 ml/well of 5-5-3-3-tetramethylbenzidine substrate solution (bioMerieux, Boxtel) was added and incubated in the dark for 15 min for both for IgA and IgG detection. The reaction was stopped by adding 100 ml of 1 M H2SO4 (Merck, Schuchardt). The optical density was determined at 450 nm (OD450) using an ELISA reader (Synergy HT Multi-detection microplate reader). All OD450 values were normalized by subtracting the value for 1:100-diluted EBV-negative serum used in duplicate in each ELISA run. The receiver operating characteristic curve was drawn to determine the cutoff values for Zebra N, Zebra P125, Zebra P130, Recombinant Zebra EBNA 1, and VCA P18, using a large panel of Indonesian healthy subjects (n=50), and NPC and IM patients (n=115).

Statistical analysis

All statistical analyses were calculated by GraphPad Prism version 8.0 software, Ms. Excel and Scatter blot. The Student’s t-test calculation method was used to define the correlation between IgG and IgA markers in a population. Moreover, the R-squared calculation method was used to define the correlation of epitope recognition.

Results

EBV antigens

Figure 1 consists of random blot strips of 10 NPC patients from the Netherlands, 12 NPC patients from Indonesia, and 7 EBV healthy carriers from Indonesia. To show adequate and clear quality pictures this study limits the amount of blot strip pictures. Nasopharyngeal biopsy samples from all patients with NPC were obtained and confirmed histologically for the presence of EBV using EBER-RISH immunohistochemical staining. NPC staging was examined using a computed tomography scan according to the 1997 Union International Cancer Control (UICC) classification and validated by the ENT. All NPC sample profiles were analyzed without knowledge of sex, age, and family history. Confidential information can only be revealed after breaking the code.

Figure 1.

Figure 1

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Epstein–Barr virus antigens.

It is clear from Figure 1 that IgG and IgA antibodies in serum from different NPC individuals recognize different EBV protein antigens, including different epitopes, spread out over the entire blot strip (MW from 200 to 15 kDa), while healthy people who are EBV carriers have limited IgG antibody diversity, recognizing mainly VCA P18 and EBNA 1, and have virtually no IgA response to EBV antigens. NPC patients clearly have more abundant antibody diversity in both IgG and IgA (polyclonal reactivity), compared to EBV carriers in healthy people, suggesting broader EBV antigen exposure during NPC carcinogenesis. The presence of a clear IgA response indicates mucosal epithelial exposure to EBV antigens in NPC patients. Furthermore, it is clear from side-by-side comparisons that IgG (G) and IgA (A) antibodies in the same NPC patient frequently have different antigen recognition patterns (diversity), suggesting that these IgG and IgA responses are triggered by different antigen encounters in the same individual.

EBV serum in Indonesian healthy people EBV carrier antibody

This data set (Fig. 2) illustrates that healthy EBV carriers (blood donors) have low and limited antibody (IgG and IgA) responses to the Zebra protein or its epitopes, whereas they have clearly positive IgG responses to VCA P18 and EBNA 1 (lower panels), reflecting EBV carriership (seropositivity).

Figure 2.

Figure 2

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Epstein–Barr virus serum Indonesian healthy people Epstein–Barr virus carrier antibody.

Note that in these healthy EBV carriers, the IgA responses to the immunodominant VCA P18 and EBNA 1 antigens are virtually absent, indicating low or absent mucosal immune triggering, in contrast to NPC cases. Almost all healthy individuals have IgG responses to either VCA P18, EBNA 1, or both. In healthy individuals, occasional VCA P18 IgA responses can be found, but EBNA 1-IgA is usually negative, suggesting that EBNA 1-IgA is the most specific marker for NPC. Overall, 50 healthy Indonesian people did not respond to IgG and IgA of Zebra N, Zebra P125, Zebra P130, and Recombinant Zebra. Most healthy people responded to IgG only on VCA P18 and EBNA 1, except for two people who intermediately responded to IgA-VCA P18. High IgG levels are indicative of EBV lifelong EBV persistence (not a first-time infection). For the two people who had responded to IgA-VCA P18, it might indicate that these two people had a recent or aberrant mucosal infection but are now healed.

EBV serum Indonesian NPC patient antibody

In contrast (Fig. 3), almost all Indonesian patients with NPC had clearly positive IgG and IgA responses to both VCA P18 and EBNA 1, which are diagnostically relevant immunodominant EBV antigens. Thus, anti-Zebra antibody responses are less strong, more diverse, and frequently negative and therefore less useful for NPC diagnosis compared to the VCA P18 and EBNA 1 antigens.

Figure 3.

Figure 3

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Epstein–Barr virus serum Indonesian nasopharyngeal carcinoma patient antibody.

Fifty Indonesian NPC patients barely gave a response in IgG and IgA to Zebra P130, two patients showed an intermediate response in IgA to Zebra N, few patients showed an intermediate response in IgA to Recombinant Zebra, and almost half of the patients showed an intermediate to very high response in both IgA and IgG to Zebra P125. In terms of EBNA 1 epitopes, almost all NPC patients showed a high response in IgG and most also in IgA. Moreover, the majority of patients showed mild-to-high responses in both IgA and IgG to VCA P18. Figure 3 clearly shows the lack of correlation between the levels of IgG and IgA responses to VCA P18 (left) and EBNA 1 (right), illustrating that they can be used as independent markers, reflecting separate antigen-triggering events related to NPC development. Therefore, it has been suggested that both markers should be used in a combined EBV-IgA ELISA test for optimal sensitivity in diagnosing NPC16. Serological EBV-IgA markers are suitable and affordable for NPC risk screening programs17,18.

EBV serum in the Netherlands NPC patients antibodies

Different results from the Netherlands NPC patients are shown in Figure 4 that 50 the Netherlands NPC patients did not show a response to both IgG and IgA of Zebra P130. A few patients responded to IgA of Zebra N, Zebra P125, and Recombinant Zebra, and only a few among them showed an intermediate response to IgG of Zebra P125.

Figure 4.

Figure 4

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Epstein–Barr virus serum the Netherlands nasopharyngeal carcinoma patients antibody.

In terms of EBNA 1 epitopes, all patients showed a high response to IgG, and most of them also to IgA. Furthermore, all patients showed a moderate to high response to IgG VCA P18, and almost half of them had a low to high response to IgA of VCA P18.

Correlation within epitope recognition among Indonesian and the Netherlands NPC patients

To analyze whether antibodies against a protein (i.e. EBV-Zebra) recognize the same epitopes and whether IgG and IgA have the same epitope recognition pattern, this study analyzed IgG and IgA responses to various Zebra peptides in detail, which can be seen in Figure 5. Figure 5 shows the correlations between IgG and IgA reactivity to Zebra proteins in ELISA in serum from Indonesian healthy people (n=50), Indonesian NPC (n=50), and the Netherlands NPC (n=50).

Figure 5.

Figure 5

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Correlation within epitope recognition among Indonesian and the Netherlands nasopharyngeal carcinoma patients.

The correlation coefficients for IgG versus IgA reactivity in each Zebra protein are shown in the graphs. Overall, there was very little correlation between IgG and IgA responses to individual Zebra peptides in either Indonesian or the Netherlands NPC patients and controls. In contrast to anti-Zebra responses, the response of IgG and IgA antibodies in patients with NPC is much higher compared to controls, reflecting their diagnostic potential19. As expected, all NPC patients had significantly different (higher) levels of IgG and IgA in both VCA P18 and EBNA 1. No differences were found between the IgA responses to either VCA P18 or EBNA 1 between the NPC patients from either country, classifying these responses as specific for NPC in general. The IgG response to VCA P18 and EBNA 1 was higher in Indonesian patients, probably reflecting their higher tumor burden upon the first clinical presentation.

Discussion

NPC is Indonesia’s most prevalent head and neck malignancy10. A report based on a 5-year analysis indicated that patients usually arrive at the hospital to examine their condition at a late stage of NPC due to unspecific symptoms in the early stage2. The treatment of NPC is stage-dependent and early detection is imperative to achieve higher treatment success19. Knowing the markers that can be used to detect NPC at an early stage is essential at this time20,21. NPC corresponds to the highest tumor burden that is associated with the EBV infection9. Indonesia has developed EBV-based serology using IgA-based ELISA and IgG IB as well as EBV-DNA viral quantification, which allowed for the development of improved NPC diagnostic tools19. Serological EBV-IgA markers are suitable and affordable for NPC risk screening programs17,18. These new EBV related diagnostic tools have excellent diagnostic capabilities in screening NPC patients from the population, and can still be used during the follow-up period. The follow-up period of NPC patients from both populations is often shared and informed among the cancer specialists (radiation oncologists, medical oncologists, and head and neck surgeons) from 1–6-month intervals within 5 years, as per standard recommendation regulations. However, it is common in NPC patients to show new symptoms after 5 years, which could indicate tumor recurrence. Therefore, a more than 5-year follow-up period is also necessary for patients with NPC.

Figure 1 reflects the overall diversity of IgG and IgA responses to all EBV proteins on the strips. Both IgG and IgA immunoblots confirmed that the EBV results deviated in NPC patients compared to healthy individuals. The identified NPC cases showed high diversity scores for IgG and IgA immunoblots. EBV IB diversity provides a significant diagnostic value for distinguishing between NPC and non-NPC tumors19. Other additional biomarkers and test options are also available as confirmation assays to establish NPC presence, that is nasopharyngeal brushing, and EBV-DNA load measurement by RT-PCR22. Unfortunately, these three tests are difficult to produce on a large scale and the results are complex and difficult to quantify, making this parameter less favorable as a confirmation test for NPC case findings. In developing countries, financial constraints must be considered when using multiple independent markers with complementary diagnostic values, which may be the best option for early-stage NPC screening. Therefore, this research was continued with an ELISA assessment of IgG and IgA anti-EBV responses, to define a correlation between IgG and IgA reactivity to various EBV markers and their synthetic peptide epitopes.

In this study, Figures 2–4 display that EBV IgG and IgA ELISA were chosen as the reference tests to compare the diversity of response ‘levels’ between defined antigens (EBNA 1, VCA P18, and Zebra). Thus, the EBV IgG and IgA have been tested in two populations from two ethnic backgrounds, the Indonesian NPC and the Netherlands NPC, both compared with healthy people. Both NPC patients from Indonesia and the Netherlands showed no response to both IgG and IgA of Zebra P130. Only a few responded to the IgA of Zebra N and Recombinant Zebra. For IgA and IgG responses of Zebra P125, there were differences between the two populations. Many Indonesian NPC patients responded to the IgA and IgG of Zebra 125, whereas only a few NPC patients in the Netherlands responded to it. These differences might be caused by several factors such as, firstly, the differences in EBV reactivation occurring in the bodies of Indonesian NPC patients that can be explained by environmental and food influences on EBV latency. Certain components in Indonesian (preserved) food may trigger virus replication, thus enhancing Zebra expression, secondly, the differences in HLA between Indonesian and the Netherlands NPC patients. HLA-A2-positive individuals make a strong response to an epitope in LMP2, an example of the impact of MHC polymorphism on immunodominance. This might also apply to Zebra responses23. Indonesian NPC patients responded more to IgA and IgG to Zebra P125 compared to NPC patients from the Netherlands, and whether IgA and IgG of Zebra P125 can be used as an alternative marker to support the diagnostic for NPC cases in Indonesia needs further research.

For EBNA 1, Indonesian NPC patients responded more highly in IgG than IgA, while NPC patients from the Netherlands responded equally highly in IgG and IgA (Figs 3, 4). EBNA 1 antibodies are found 2–4 months following the onset of EBV infection and persist for life, they simply indicate that a very recent infection has not occurred. Assessing the EBNA 1 test was complicated. For VCA P18, NPC patients from both Indonesia and the Netherlands showed high responses for both IgG and IgA. Since IgG antibodies to a protein on the viral coat (viral capsid antigen) peak early in infection and usually continue throughout life, they denote nothing more than that a person has been exposed to the pathogen. However, the high IgG levels found in combination with other clinical signs of chronic fatigue syndrome accurately reflect EBV reactivation2.

IgA responses reflect mucosal immune responses to a pathogen, and in the case of NPC, a mucosal epithelial cancer IgA-EBV reflects the role of EBV in the pathogenesis of NPC. Early-stage NPC is characterized by abnormal IgG to VCA P18 and EBNA 1 responses, increasing with time as the tumor grows24. Aberrant IgA-EBV responses reflect abnormal (tumor) processes in the mucosal layer of the nasopharynx, normally not seen in healthy EBV carriers, but temporarily encountered in patients with primary infection via saliva (i.e. infectious mononucleosis) (infectious mononucleosis research (n=15) including a picture was not pointed out in this publication). The IgA antibodies against EBV antigens might be used for the diagnosis in the late and early stages of NPC because the high levels of EBV-associated antibodies in NPC patients are generally IgA antibodies from mucosal sites25,26.

Zebra activation domain swap constructs retain Zebra’s native ability to activate specific EBV promoters, disrupt EBV latency, and stimulate replication at the EBV lytic origin. Additional work, employing sequential and internal deletions of Zebra’s N-terminal activation domain, indicates that its separate activities are not attributable to specific subdomains but are spread throughout its N-terminus and therefore cannot be inactivated by deleting localized regions27.

To explore the epitope diversity of anti-Zebra IgG and IgA responses, this study used several synthetic peptide domains and full-length recombinant Zebra protein for antibody detection in patients with NPC. The data and correlations among these responses are shown in Figure 5. The figure shows that Indonesian NPC patients and the Netherlands NPC patients gave somewhat different responses, both being more reactive to Zebra epitopes compared to healthy people. The IgG and IgA responses to Zebra domains were much weaker than antibody responses to the immunodominant antigens of EBV (EBNA 1 and VCA P18). However, a significantly higher IgG reactivity was found in NPC patients as compared with Indonesian EBV-positive and EBV-negative healthy controls. These results, it is indicated that Zebra is a relatively weak immunogenic protein in humans. Thus, the antibody response against the Zebra antigen has limited value for NPC diagnosis. More effort is needed to define whether secreted Zebra can be considered a potential circulating NPC tumor marker. No correlation was found between VCA P18 and EBNA 1 markers (Fig. 5), indicating that the IgG and IgA responses are each independently triggered by different antigen encounters by the IgG and IgA producing B-cells (systemic vs. mucosal immune responses).

NPC cases are very common among indigenous people in Indonesia and have become a prolonged socio-economic problem in this country, with a population of 270 million people. The limitations of this study are that many NPC cases in Indonesia are not registered due to limited hospital facilities, the lack of medical awareness, and inadequate national cancer diagnostic and registration systems.

Ethnicities in the Netherlands samples

The Netherlands population is projected to increase in the coming years, with increased migration from NPC risk areas worldwide. NPC samples obtained from the Netherlands originate from various ethnicities. Considering the relatively low presence of non-Dutch citizens overall, the high prevalence of NPC cases of non-Dutch origin reflects the (increasing) immigration contribution to NPC incidence in the Netherlands.

Conclusions

This study showed that IgA responses to EBV VCA P18 and EBNA 1 are independent serological markers for NPC, and each has diagnostic value in both Indonesian and the Netherlands NPC patient populations. Serological EBV-IgA markers are suitable and affordable for NPC risk screening programs17,18. IgG and IgA responses to these proteins did not correlate, indicating independent antigen triggering of systemic (IgG) and mucosal (IgA) B-cells by EBV antigen encounters. This study did not confirm the NPC diagnostic value of the Zebra protein or its epitopes, as was suggested by others previously. Our epitope analysis indicated that IgG and IgA responses to the (sub)epitopes of the Zebra protein were not related, suggesting independent clonal triggering. This study also showed that due to the low immunogenicity of these proteins, antibody responses to Zebra N, Zebra P125, Zebra P130, and Recombinant Zebra are not very effective markers. In this study, a new finding was also discovered, namely, that Indonesian NPC patients were able to give a higher response to Zebra P125 epitope compared to the Netherlands NPC patients. A comprehensive study regarding the Zebra P125 epitope in Indonesian NPC patients is therefore very appealing to pursue further to determine whether the Zebra P125 epitope could be an alternative marker for early-stage detection in the general population, and whether the Zebra P125 epitope could be used as a vaccine.

Ethical approval

All studies were performed under the Helsinki Code of ethics and according to the rules of reuse of human materials under the research code of the VU University Medical Center (AMC–VUmc Research Code 2013).

Consent

Written informed consent was obtained from the patient for publication of this study and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.

Sources of funding

The author(s) received no specific funding for this study.

Author contribution

I.B.: conceptualization, formal analysis, contributed to sample preparation, carried out the experiment, investigation, methodology, designed the model and the computational framework, resources, visualization, writing – original draft, writing – review, and editing; S.V.: resources, visualization, investigation, contributed to sample preparation and experiment, methodology, and supervision; Y.P.D.: resources, visualization, investigation, contributed to manuscript preparation, and supervision; M.V.A.: funding acquisition, project administration, consultation, and supervision; U.H.: funding acquisition, project administration, project management, consultation, and supervision; J.M.M.: funding acquisition, devised the project, the main conceptualization, formal analysis, investigation, methodology, resources, project administration, project management, supervision, visualization, writing – original draft, writing – review, and editing.

Conflicts of interest disclosure

The authors have declared that no conflicting interests exist in this study.

Research registration unique identifying number (UIN)

EBV-IgA antibody responses in endemic and nonendemic populations with Nasopharyngeal Carcinoma: Tumour marker prognostication study and a cross-sectional study, https://www.researchregistry.com/browse-theregistry#home/registrationdetails/642ea2f5c534f10027b2816a/, researchregistry8821.

Guarantor

Insani Budiningsih and Jaap Michiel Middeldorp.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Provenance and peer review

Not commissioned, externally peer reviewed.

Acknowledgements

None.

Footnotes

Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.

Published online 19 July 2023

Contributor Information

Insani Budiningsih, Email: insani.budiningsih-2019@fk.unair.ac.id.

Sandra A.W.M. Verkuijlen, Email: S.Verkuijlen@vumc.nl.

Yoes P. Dachlan, Email: yoesdachlan1930@gmail.com.

Muhammad V. Arfijanto, Email: muhammad-v-a@fk.unair.ac.id.

Usman Hadi, Email: usman.hadi2@fk.unair.ac.id.

Jaap M. Middeldorp, Email: j.middeldorp@amsterdamumc.nl.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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