Sex differences in anti-EBV antibody responses

Abstract

We investigated anti-EBV IgA and IgG responses by sex among 387 cancer-free individuals in Asia. Antibody responses were measured using an EBV proteome array to assess age-adjusted sex-specific associations with 404 EBV-antigens in 86 protein sequences via meta-analysis and pathway analysis by EBV stage. Males were more likely to have elevated IgA responses (P=0.001) and females had higher IgG responses (P=0.003). Significant sex associations were observed across stages of lytic replication. The largest sex differences were seen in latent IgA, but no differences were observed in latent IgG antibodies. Higher IgA responses suggest higher rates of EBV reactivation in males.

Article summary:

Significant sex associations were observed across stages of EBV lytic replication. The largest sex differences were for latent IgA antibodies, but no differences were observed in latent IgG antibodies. Higher IgA responses suggest higher rates of EBV reactivation in males.

Background

Approximately 90% of adults globally are infected with Epstein-Barr virus (EBV).[1] EBV is associated with several malignancies including nasopharyngeal carcinoma (NPC), Hodgkin lymphoma (HL), extranodal natural killer(NK)/T-cell lymphoma, and gastric carcinoma.[1] However, only a small proportion of those infected with EBV develop EBV-related cancers for reasons that are not yet understood.

Despite EBV prevalence being similar between the sexes,[1] EBV-associated cancers occur significantly more frequently in men than women, with incidence rates 2 to 3 times higher in men, even after adjusting for various risk factors that differ in prevalence by sex such as obesity, smoking, alcohol consumption, physical activity, diabetes, and hypertension.[2] The lower incidence of EBV-related cancers in women may be attributed to more effective immunological control of the virus. For example, women typically mount stronger innate and adaptive immune responses to many viral infections compared to men, which could account for the enhanced containment of EBV.[3] Notably, humoral immune responses, such as the production of antibodies by B cells, tend to be more robust in women.[4] In addition, females generally experience stronger cell-mediated immune responses, with higher Th2 activity than their male counterparts.[3] These sex-based differences in adaptive immunity may contribute to the variation in immune response to EBV and the resulting malignancies.

Research has shown that elevated antibody levels against EBV are associated with a higher risk of EBV-related cancers. [5–7] Several studies have reported increased titers of IgA antibodies targeting EBV viral capsid antigen (VCA), early antigen, and EBV nuclear antigen 1 (EBNA-1) in patients with nasopharyngeal carcinoma (NPC).[8] IgG VCA and EBNA-1 antibody levels have consistently been found to be higher in females.[9] However, these earlier studies focused only on two IgG antigens, and did not consider the full range of both IgG and IgA EBV antibodies across the viral lifecycle. To address this gap, leveraging antibody data generated by an EBV antibody array, we aimed to evaluate sex-based differences in both IgG and IgA antibody levels. A deeper understanding of these differences in the immune response to EBV could help explain the immunological mechanisms that make females less susceptible to EBV-associated cancers.

Methods

We used data on 387 EBV-positive non-cancer participants (controls) from three case-control studies conducted in Hong Kong and Taiwan, which have been detailed elsewhere.[6, 10, 11] Briefly, we included 174 community controls (frequency matched to cases on sex, age, and region) from a case-control study of NPC conducted in Taiwan between 1991–1994;[6] and 213 controls recruited from two case-control studies (153 controls examining NK/T-cell lymphoma[10] and 60 controls examining Hodgkin lymphoma[11]) matched to cases on age, enrolment date, and region in the AsiaLymph study, a multicenter, hospital-based study conducted in Hong Kong and Taiwan from 2012–2017. All included individuals were 18 years or older, provided informed consent before any study procedures, and had their blood drawn at study enrollment. All contributing studies were approved by regional scientific ethics committees and data protection agencies.

EBV custom proteome microarray

We used a custom EBV proteome microarray, which has been previously described.[6] Briefly, the array encompassed 404 protein sequences representing nonredundant open reading frames and predicted splice variants from 86 EBV proteins derived from five EBV strains (AG876, Akata, B95–8, Mutu, and Raji). Included in the array were three synthetic EBV peptides (VCAp18, EBNA-1, and EA p47), which are considered the gold standard for detecting EBV-specific antibody responses. Four “no DNA” (no translated protein) spots were included in the array as a measure of non-EBV background reactivity (e.g., E. coli reactivity). Each microarray slide was printed using 16 arrays.

Serum/plasma samples from participants were tested blinded for both IgA and IgG antibody responses.[6] Antibody responses were detected using biotin-conjugated goat anti-human IgG (1:1000 dilution) or IgA (1:500 dilution) secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and visualized with streptavidin-conjugated SureLight^® P3 (Columbia Biosciences, Columbia, MD, USA) antibody (1:200 dilution). After testing, air-dried probed slides were scanned using an Axon GenePix 4300 B (Molecular Devices). Raw fluorescence intensities were corrected for spot-specific background using Axon GenePix Pro 7 software, and data were variant-log-transformed using variance stabilizing normalization (VSN) transformation in Gmine.[12] Standardization to the person-specific background was performed by dividing VSN values with the participant’s cutoff (mean ± 1.5 standard deviation of the four “no DNA” spots), with the transformed data referred to as the standardized signal intensity.

Statistical analysis

We fit linear regression models with each antibody as the outcome and sex and age as independent predictors separately for each study and combined the results using meta-analysis. We plotted the coefficients from the meta-analysis representing the female-to-male fold change, against the P-value for each antibody. We used a Bonferroni corrected P-value (0.05/404) of P<0.00012 to adjust for multiple comparisons. In addition, fit a single regression model to all studies combined that included an interaction term for sex by study to determine whether it was appropriate to pool the three studies.

We grouped EBV-targeted antibodies into pathways by immunoglobulin type (IgA and IgG) and life-cycle stage (latent, immediate early lytic, early lytic, late lytic, glycoprotein, and other/unknown) and evaluated their association with sex. For each antibody EBV type and life-cycle stage, we fit linear regression models with sex as the independent variable and levels of antibodies (i.e., standardized signal intensity) as the dependent variable. Using an adaptive rank truncated product statistic, [13] we computed a combined P-value of each EBV life-cycle stage based on the antibody-specific P-values for the estimated coefficients associated with sex. The P-value was determined using a permutation approach, which minimizes the multiple testing burden by treating the EBV life-cycle stage and immunoglobin type as the unit of analysis instead of each individual antibody.

Results

Among the 387 participants from Taiwan and Hong Kong, 64.3% were men and 35.7% were women. Most participants were between 40 and 49 years old, ranging from 18 to 80 years (Supplemental Table 1). Figure 1 illustrates the association between sex and IgA and IgG antibodies at different stages of the EBV lifecycle. Sex was statistically significantly associated with both IgA (P=0.001) and IgG responses (P=0.003). These associations remained statistically significant across all life stages, except for latent IgG antibodies (Supplemental Table 2).

Figure 1.

Associations between sex and anti-EBV antibodies by lifecycle. IgA and IgG antibodies associations depicting P-values on the −log₁₀ scale by viral lifecycle stage (early lytic, glycoprotein, immediate early lytic, late lytic, latent, and other/unknown).

To determine whether males or females were more likely to have higher IgA or IgG responses, we analyzed sex-specific adjusted associations for the 202 IgA and 202 IgG EBV antibodies. Figure 2 shows the results of the meta-analysis, which used linear regression to estimate the associations between sex and EBV antibody levels, adjusting for age. We found that males were more likely to have elevated IgA responses (i.e., female-to-male coefficients below 0), while females had higher overall IgG responses (i.e., female-to-male coefficients above 0). After correcting for multiple comparisons, 13 antibodies (12 IgA and one IgG) were significantly associated with sex (P<0.00012) (Supplemental Table 3). The 12 IgA antibody levels were significantly higher in males – BBLF2/3 (female-to-male coefficient: −0.09; P=0.00011), BDLF3 (−0.08; P=0.00009), BDLF3.5 (−0.09; P=0.000005), BALF3 (−0.07; P=0.00002), BSRF1 (−0.09; P=0.00006), EBNA2 (−0.08; P=0.00003), EBNA3A (−0.07; P=0.00007), EBNA3B (−0.10; P=0.00007), EBNA3C_ver1 (−0.08; P=0.00011), EBNA3C_ver2 (−0.09; P=0.00008), EBNA-LP (−0.07; P=0.00001), and LMP1 (−0.07; P=0.00011). Females had statistically significantly higher IgG antibody levels against BBRF1 (female-to-male coefficient: 0.15; P=0.00002). No heterogeneity was observed between studies for these 13 antibodies (Supplemental Table 3).

Figure 2.

Volcano plot representing the association between anti-EBV antibody responses and sex from a meta-analysis based on estimates from linear regression adjusting for age at blood collection. Horizontal lines represent p-value thresholds, P=0.05 and P=0.00012 (Bonferroni corrected P-value). Vertical line represents the line of unity.

Discussion

Our study conducted in an EBV-positive population provides important insights into sex-specific variations in humoral responses to EBV. The higher IgA responses, which reflect recent mucosal infections observed in males, suggest that men may experience a greater frequency of EBV reinfection or reactivation. In contrast, IgG antibodies, which reflect cumulative systemic exposure to EBV, indicating either that females have a more robust initial response or that IgG levels in females decline more slowly over time compared to males. These differences in anti-EBV antibody levels could help explain the higher incidence of EBV-related cancers in males.

Previous studies have shown that elevated IgA antibodies across all stages of the EBV lifecycle are strongly associated with NPC risk.[5, 6, 14] Argirion et al.,[14] using data from the same NPC Taiwanese case-control study as our analysis, along with samples from Ghana, Denmark, Sweden, and the UK, further demonstrated that anti-EBV IgA antibodies—particularly those targeting proteins in the early lytic phase—were linked to EBV-positive classical HL. However, these antibodies were not associated with Burkitt lymphoma or NK/T-cell lymphoma.[14] In our study, only one early lytic-phase anti-EBV IgA protein (BBLF2/3) was significantly associated with sex. Instead, the largest female-to-male differences were found in IgA antibodies against EBV latent proteins (e.g., EBNA2, EBNA3A, EBNA3B, EBNA3C, and LMP1). This observation suggests that elevated IgA reactivity may indicate a general increase in viral activity, potentially increasing cancer risk. The apparent discrepancy between IgA reactivity and oncogenic EBV proteins points to a complex interplay involving viral immune evasion, tissue-specific IgA functions, and hormonal modulation. Furthermore, anti-EBV IgG antibodies have also been shown to be significantly associated with several EBV-related cancers across multiple studies, [5, 6, 14] whereas higher anti-IgG antibody levels were seen in females, though only one anti-EBV IgG antibody (BBRF1) met statistical significance at the Bonferroni corrected level. This finding does not align with the increased risk of EBV-related cancers observed in males, suggesting that increased nonspecific viral activity between sexes may play a more important role than EBV oncoproteins alone in driving cancer risk.

Previous research has consistently shown that adult females have significantly higher anti-EBV IgG titers than adult males and that this did not change with older age.[9] The sex differences in immune responses to viruses are shaped by a combination of factors, including variations in sex-steroid hormone levels, X-linked gene expression, and gender-related life experiences.[15] For instance, estradiol enhances both Th1 and Th2 immune responses and promotes humoral immunity through estrogen receptor binding (ERα in T cells and ERβ in B cells).[4] Conversely, androgens like testosterone suppress immune cell activity, reduce pro-inflammatory responses, and inhibit antiviral cytokines.[4] Females tend to have greater antibody responses and higher B cell counts across all ages,[4] suggesting that their enhanced immune response is not solely regulated by sex steroid hormones. Indeed, sex-based differences in the function of innate immune cells also play a role. For example, males generally have higher natural killer (NK) cell frequencies, while females have more efficient neutrophil and macrophage phagocytic activity, and their antigen-presenting cells are better at presenting peptides.[4] Furthermore, several immune-related genes on the X-chromosome may escape X-inactivation, leading to higher expression levels in females.[4, 15] One such gene, TLR7, enables females to produce more cytokines than males, which can substantially improve viral clearance and enhance the immune response to infection.[4]

The strengths of our analysis include using a custom microarray to comprehensively assess antibody responses to 404 antigens across the EBV lifecycle. However, the study has some limitations, including a relatively small sample size and the inability to adjust for potential confounding factors such as smoking and occupational exposures, which may influence EBV reactivation. Future studies should be replicated in other populations accounting for environmental exposure differences between men and women. Results of our case-control studies can be disaggregated by sex to further elucidate whether sex differences in EBV-antibody response drive the male bias in cancer risk.

In summary, to the best of our knowledge, this is the first study to demonstrate significant sex-based differences in anti-EBV antibody responses across the EBV lifecycle, which may offer new insights into the sex-specific mechanisms underlying EBV-related disease pathogenesis.