Evaluating varicella-zoster virus vaccine immunogenicity through Fc-mediated antibody functions: the roles of ADCP and ADCC

Evaluating varicella-zoster virus vaccine immunogenicity through Fc-mediated antibody functions: the roles of ADCP and ADCC | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Evaluating varicella-zoster virus vaccine immunogenicity through Fc-mediated antibody functions: the roles of ADCP and ADCC Sivilay Xayaheuang, Ji-Young Hwang, Yunhwa Kim, Kyung-Min Lee, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8121887/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Mar, 2026 Read the published version in npj Vaccines → Version 1 posted 9 You are reading this latest preprint version Abstract Assessing the functional activity of vaccine-induced antibodies is critical for evaluating immunogenicity. We developed and validated antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) assays to quantify Fc-mediated antibody responses elicited by varicella and zoster vaccines. Both assays demonstrated robust performance and broad linearity. Antibody titers were measured using fluorescent antibody to membrane antigen (FAMA) and ELISA. ADCP and ADCC activities, along with FAMA and ELISA geometric mean titers (GMTs), were significantly increased in post- vs. pre-vaccination sera ( p < 0.0001). Strong correlations were observed between ADCP and ADCC activities and both FAMA and ELISA GMTs. Although children exhibited lower total varicella-zoster virus-specific IgG levels than adults, higher IgG3 subclass levels in children were associated with comparable Fc-mediated activities. These results highlight the utility of ADCP and ADCC as valuable assays for evaluating Fc-mediated antibody function and potential surrogates of protective immunity to varicella and zoster vaccination. Biological sciences/Immunology Biological sciences/Microbiology Antibody-dependent cellular phagocytosis Antibody-dependent cellular cytotoxicity Varicella-zoster virus Immunogenicity Vaccine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Varicella (chickenpox) and herpes zoster (HZ) are distinct clinical manifestations caused by the varicella-zoster virus (VZV), progressing from primary infection and viral reactivation, respectively. During primary infection, VZV spreads systemically through viremia, whereas HZ results from viral reactivation in sensory ganglia followed by spread along peripheral nerves. These pathogenic differences imply distinct immune mechanisms for protection and disease control. Antibodies targeting VZV glycoproteins, which reflect neutralizing antibody activity, correlate strongly with protection against varicella 1 . By contrast, such antibodies do not prevent HZ, for which cellular immunity is critical 2 , 3 . Notably, children with isolated agammaglobulinemia are not prone to severe varicella 4 . However, VZV-specific memory T cells responses after vaccination remain weak or undetectable in children 5 . Severe or disseminated varicella has been reported in individuals with natural killer (NK) or invariant NKT-cell deficiencies, underscoring the importance of innate cellular immunity in controlling infection 6 – 9 . Together, these findings suggest that clearance of VZV-infected cells relies not only on T cells but also on cooperation between adaptive and innate immune responses. Fc-effector functions, such as antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), enhance the clearance of virus-infected cells 10 – 17 . These functions may be particularly relevant for HZ, where the virus is presumed to evade neutralization 18 . Beyond Fab-mediated neutralization, antibodies exert their functions through the Fc-domain, which recruits and activates innate immune cells including macrophages and NK cells 19 , 20 . Fc-mediated effector functions have been implicated in protection against diverse viral infections, including herpes simplex virus-2 (HSV–2), Dengue virus, respiratory syncytial virus (RSV), SARS-CoV-2, Ebola, and influenza 21 – 29 . Immunoglobulin G (IgG) subclasses also differ in their ability to mediate Fc effector activity, with IgG3 generally exhibiting greater potency than IgG1 30 . Differential subclass responses have been reported in varicella and zoster infection 31 , suggesting that host age and disease context may shape Fc-effector profiles. Despite these insights, Fc-mediated antibody functions against VZV remain poorly characterized, and no standardized assays are currently available for their evaluation. This gap limits our understanding of protective immunity and hampers the development of reliable correlates of protection for VZV vaccines. To address this, we established physiologically relevant macrophages-based ADCP and NK cell-based ADCC assays and validated their reproducibility and robustness. Then, we applied these assays to sera from children and adults immunized with varicella and zoster vaccines, respectively. In parallel, antibody levels were measured by fluorescent antibody to membrane antigen (FAMA), ELISA, and IgG subclass analyses (Fig. 1 ). Together, these data provide the most comprehensive characterization to date of VZV-specific antibody functions across age groups and highlight their potential as functional biomarkers for evaluating vaccine-induced immunity. Results Establishment of ADCP and ADCC assay to assess varicella and zoster vaccine immunogenicity We established an ADCP assay using carboxyfluorescein succinimidyl ester (CFSE)-labeled VZV-infected MRC-5 cells as target cells, employing M1-U937 cells as effector cells for evaluating human sera. To optimize the experimental conditions, U937 cells were differentiated into M1 macrophages using 20 ng/ml of each PMA, LPS and IFN-γ (Supplementary Fig. 1). The FcγRII (CD32) and FcγRI (CD64) were particularly upregulated in differentiated M1-U937 cells compared to U937 cells (Supplementary Fig. 2). These findings confirm that M1-U937 exhibit an activated macrophage phenotype. The ADCP activity was quantified as the percentage of CFSE + CD89 + double-positive cells by flow cytometry (Fig. 2 a) and further confirmed by confocal microscopy (Fig. 2 b). Pre-vaccination sera did not induce detectable phagocytosis, as indicated by the absence of double-positive cells (Fig. 2 b, left). In contrast, post-vaccination sera promoted robust phagocytosis of VZV-infected target cells by effector macrophages (Fig. 2 b, right). For ADCC assay, CMTMR-labeled VZV-infected MRC5 cells served as a target cells, employing the no-GFP-CD16.NK-92 cell line as an effector. Granzyme B (GrB)-uptaking target cells (CMTMR + GrB + double-positive cells) were defined as the readout for ADCC detection (Fig. 2 c). ADCC responses were also confirmed by confocal microscopy (Fig. 2 d). Pre-vaccination sera induce negligible GrB uptake (Fig. 2 d, left), but post-vaccination sera facilitated efficient transfer of GrB into target cells (Fig. 2 d, right), confirming vaccine-induced functional antibody responses. Optimization of both assays involved assessing effector to target cell number (E:T) ratio, serum dilution, and the reaction time, using pre- and post-vaccination sera. E:T ratios of 1:2 and 2:1 (for ADCP) or 1:1 and 1:2 (for ADCC) were tested, followed by serial dilutions of sera at the optimal E:T ratios (1:2 for ADCP and 1:1 for ADCC). Incubation times of 2, 3, 4 or 5 hours were also evaluated. The final protocol of ADCP assay was established at an E:T ratio of 1:2, a 1:100 serum dilution, and a 4-hour incubation. The ADCC assay was conducted using an E:T ratio of 1:1, a 1:100 serum dilution, and a 4-hour incubation. (Supplementary Fig. 3, Supplementary Table 1). Validation of ADCP and ADCC assays Comprehensive validation of the ADCP and ADCC assays were performed using VZV plasma panel together with pre- and post-VZV vaccination sera (Supplementary Table 2). Lot-to-lot variability of VZV-infected MRC-5 target cell stocks was assessed across three antibody concentrations. Given the cell-based nature of these assays, coefficient of variation (CV) below 30% was predefined as an acceptance criterion. The range of target-cell lot-to-lot CVs were 0.6%–4.3% for ADCP and 2.8%–7.1% for ADCC, meeting this criterion (Fig. 3 a, b; Supplementary Tables 3, 4). Linearity was evaluated using five serial dilutions of three samples. Simple linear regression analysis showed very strong linearity across a broad range (ADCP r 2 = 0.87–0.96; ADCC r 2 = 0.87–0.97) (Fig. 3 c, d; Supplementary Tables 5, 6), supporting accurate quantification across varying antibody concentrations. Intra-assay precision was assessed by running five replicates of six panel samples on a single plate. For ADCP, intra-assay CVs were 5.0%–28.5% (Fig. 3 e, Supplementary Table 7) and ADCC intra-assay CVs were 2.7%–28.0% across all six panels, meeting the acceptance criterion at low, medium, and high levels (Fig. 3 f, Supplementary Table 8). Inter-assay precision was determined across three independent runs on different days by two operators. Between-operator CVs were 1.9%–28.4% for ADCP (Fig. 3 g, Supplementary Table 9) and 2.3%–18.0% for ADCC (Fig. 3 h, Supplementary Table 10). Inter-day CVs were ranged from 3.7%–16.6% for ADCP (Fig. 3 i, Supplementary Table 11) and 0.2%–25.1% for ADCC (Fig. 3 j, Supplementary Table 12). Finally, inter-laboratory testing across three laboratories using three paired pre-/post-vaccination sera showed comparable readouts, with inter-lab CVs of 5.7%–21.8% for ADCP and 6.3%–21.8% for ADCC (Fig. 3 k, l; Supplementary Tables 13, 14). Together, these data indicate that the ADCP and ADCC assays using VZV-infected MRC-5 cells are robust and reproducible across operators, days, and laboratories, and perform consistently across a broad range of antibody levels, supporting their utility for Fc-mediated antibody assessment in recipients of both varicella and zoster vaccines. Varicella or zoster vaccination induced ADCP and ADCC activities Fc effector functions were evaluated using the ADCP and ADCC in pre- and post-vaccination sera from recipients of varicella or zoster vaccines. In the pre-vaccination sera of 1-year-old children receiving live attenuated varicella vaccines (LAVs): BARYCELA or VARIVAX, ADCP and ADCC activities were at baseline levels: BARYCELA (ADCP: 8.3%, ADCC: 11.3%) and VARIVAX (ADCP: 7.8%, ADCC: 12.8%). However, these activities significantly increased after a single dose of vaccination: BARYCELA (ADCP: 34.3%, ADCC: 24.3%) and VARIVAX (ADCP: 34.1%, ADCC: 21.7%) ( p < 0.0001; Fig. 4 a, b; Supplementary Table 15). Since most Korean adults already possess immunity to VZV, pre-vaccination sera showed similar levels of ADCP and ADCC activities in two zoster vaccine (ZV) groups, ZOSTAVAX (ADCP: 27.0%, ADCC: 20.3%) and SHINGRIX (ADCP: 28.9%, ADCC: 24.8%). Post-vaccination sera showed significant increases in ADCP and ADCC activities in ZOSTAVAX (ADCP: 40.8%, ADCC: 25.9%) after a single dose and SHINGRIX (ADCP: 51.5%, ADCC: 31.9%) after two doses, compared to pre-vaccination sera ( p < 0.0001; Fig. 4 a, b; Supplementary Table 15). A single dose of LAVs in children showed comparable ADCP and ADCC activities to a single dose of ZOSTAVAX in adults. The fold changes between pre- and post-vaccination were as follows BARYCELA: ADCP (4.1-fold), ADCC (2.1-fold), VARIVAX: ADCP (4.4-fold), ADCC (1.7-fold), ZOSTAVAX: ADCP (1.5-fold), ADCC (1.3-fold) and SHINGRIX: ADCP (1.80-fold), ADCC (1.3-fold). Therefore, there were no significant differences in fold changes between BARYCELA and VARIVAX or ZOSTAVAX and SHINGRIX recipients (Fig. 4 c, d; Supplementary Table 15). Antibody titers measured by FAMA test and ELISA Antibody titers were measured by FAMA test, the gold standard method for the varicella vaccine-induced antibody titer, and ELISA. The geometric mean titers (GMTs) of FAMA in post-vaccination sera for all four vaccine groups were significantly higher than those in the pre-vaccination sera ( p < 0.0001) (Fig. 5 a). GMTs of pre-vaccinated children’s sera (BARYCELA and VARIVAX) were negative levels (≤ 2.2). Post-BARYCELA GMT (153.4: ranging from 32 to 512) and post-VARIVAX GMT (164.3: range, 32–1,024) were significantly increased but there were no differences between two vaccines. The FAMA GMTs of pre-vaccinated adults’ sera showed similar levels; pre-ZOSTAVAX (78.8: range, 32–256) and pre-SHINGRIX (87.4: range, 32–256). However, those of post-SHINGRIX (776.0: range, 256–2,048) were significantly higher than post-ZOSTAVAX (215.3: range, 64–512) (Fig. 5 a, Supplementary Table 16). Anti-VZV IgG ELISA antibody titers were measured using VZV IgG Serion ELISA classic kit (detection range, 15–2,000 mIU/mL). The GMTs of anti-VZV IgG ELISA antibodies in the post-vaccinated sera were significantly elevated relative to pre-vaccination levels in all groups ( p < 0.0001; Fig. 5 b). Most pre-vaccinated children’s sera were lower than detection limit; pre-BARYCELA GMT (0.8 mIU/mL: range, 0.1–32.0 mIU/mL) and pre-VARIVAX GMT (0.4 mIU/mL: range, 0.01–24.8 mIU/mL). Post-BARYCELA GMT (118.9 mIU/mL: range, 27.8–435.4 mIU/mL) and post-VARIVAX GMT (139.2 mIU/mL: range, 37.5–893.2 mIU/mL) were significantly increased but there were no differences between two vaccines (Fig. 5 b, Supplementary Table 17). The ELISA GMTs of pre-vaccinated adults’ sera showed similar levels, pre-ZOSTAVAX GMT (520.1 mIU/mL: range, 159.1–1,484.4 mIU/mL) and pre-SHINGRIX GMT (609.6 mIU/mL: range, 145.0–2,176.0 mIU/mL). However, all of the post-SHINGRIX samples, the ELISA titers were over the detection limit; post-SHINGRIX GMT (10,428.9 mIU/mL: range, 4,561.4–36,355.9 mIU/mL) and significantly higher than post-ZOSTAVAX GMT (1,697.3 mIU/mL: range, 415.2–4,599.2 mIU/mL) (Fig. 5 b, Supplementary Table 17). Differential expression of anti-VZV IgG1 and IgG3 in varicella-vaccinated children and zoster-vaccinated adults To characterize subclass-specific antibody responses induced by VZV vaccination, IgG1 and IgG3, key mediators of ADCP and ADCC responses, were quantified by ELISA. Distinct IgG subclass profiles were observed between varicella-vaccinated children and zoster-vaccinated adults. Both IgG1 and IgG3 antibody levels significantly increased after vaccination in all groups ( p < 0.0001, Fig. 5 c, d). Analysis of IgG1 subclass fold-changes revealed that children who received LAVs exhibited significantly lower increases: BARYCELA (1.8) and VARIVAX (2.5), compared with adults vaccinated with HZ vaccines, ZOSTAVAX (3.3) and SHINGRIX (4.9) (Fig. 5 e, Supplementary Table 18). Conversely, IgG3 subclass fold-changes were significantly higher in children immunized with LAVs −BARYCELA (4.8) and VARIVAX (5.8) −than adults vaccinated with ZOSTAVAX (2.1) or SHINGRIX (1.8) (Fig. 5 f, Supplementary Table 18). These findings indicate that varicella vaccination in children preferentially induces IgG3, whereas adult zoster vaccination elicits a predominant IgG1 response. In children, both IgG1 and IgG3 levels showed significant correlations with ADCP, ADCC, FAMA titer, and ELISA titer. However, IgG3 exhibited stronger correlations with ADCP, ADCC, and FAMA titer compared to IgG1 (Fig. 6 a−h). Correlation coefficients ( r ) between IgG1 and ADCP, ADCC, and FAMA were 0.563 ( p < 0.0001), 0.340 ( p < 0.0012), and 0.545 ( p < 0.0001), respectively (Fig. 6 a−c). Whereas those between IgG3 and the same parameters were higher, 0.804, 0.647, and 0.802, respectively (all with p < 0.0001, Fig. 6 e−g). However, the correlations between ELISA titers and IgG1 ( r = 0.775) or IgG3 ( r = 0.744) were comparable (Fig. 6 d, h). In contrast, among zoster-vaccinated adults, strong and significant correlations were found between IgG1 and ADCP ( r = 0.856), ADCC ( r = 0.698), FAMA titer ( r = 0.939), and ELISA titer ( r = 0.791), all with p 0.05, Fig. 6 m−p). These findings suggest that IgG3 may play a more prominent role in vaccine-induced Fc-mediated antiviral functions in children, whereas IgG1 appears to be more critical in adults. Correlation between Fc-mediated antibody functions (ADCP, ADCC) and antibody titers (FAMA, ELISA) across vaccine types Pearson correlations analysis was performed to determine the relationships between Fc-mediated antibody functions (ADCP and ADCC) and antibody titers (FAMA and ELISA) across different vaccine types. A robust and statistically significant correlations were observed between ADCP activity and FAMA titers across all vaccine groups: BARYCELA ( r = 0.947), VARIVAX ( r = 0.936), ZOSTAVAX ( r = 0.809), and SHINGRIX ( r = 0.885) (Fig. 7 a–d; all p < 0.0001), indicating a very strong correlation between these two immunological parameters. Similarly, ADCC activities were strongly correlated with FAMA titers in all vaccine groups: BARYCELA ( r = 0.837), VARIVAX ( r = 0.705), ZOSTAVAX ( r = 0.707), and SHINGRIX ( r = 0.885) (Fig. 7 e–h, all p < 0.0001). In contrast, correlations of ADCP and ADCC with ELISA titers were relatively weaker than those with FAMA titers, though still statistically significant. For ADCP, the correlations with ELISA were: BARYCELA ( r = 0.890), VARIVAX ( r = 0.761), ZOSTAVAX ( r = 0.853, p < 0.001), and SHINGRIX ( r = 0.702) (Fig. 7 i–l, all other p < 0.0001). For ADCC, the correlations with ELISA were: BARYCELA ( r = 0.823), VARIVAX ( r = 0.500), ZOSTAVAX ( r = 0.667), and SHINGRIX ( r = 0.567) (Fig. 7 m–p, p < 0.0001). Moderate to very strong correlations were also noted between ADCC and ADCP within each vaccine group; BARYCELA ( r = 0.854), VARIVAX ( r = 0.632), ZOSTAVAX ( r = 0.655), and SHINGRIX ( r = 0.748) (Fig. 7 q–t, p < 0.0001). In the overall VZV-vaccinated population, ADCP and ADCC activities were strongly correlated with each other ( r = 0.8060, p < 0.0001) and with FAMA titers (ADCP: r = 0.917; ADCC: r = 0.806; both p < 0.0001) (Fig. 8 a−c). In comparison, their correlations with ELISA titer were moderate (ADCP: r = 0.529; ADCC: r = 0.505; both p < 0.0001) (Fig. 8 d, e). These findings suggest that because FAMA titer is a well-established CoP against varicella, the strong correlations of ADCP and ADCC with FAMA indicated that these Fc-mediated activities provide a robust and biologically relevant representation of vaccine-induced immunogenicity, serving as reliable indicators of functional antibody response to VZV vaccines. Discussion Varicella is a highly contagious infectious disease, and live attenuated varicella vaccines have been incorporated into national immunization programs in many developed countries 32 . Meanwhile, global demand for varicella vaccines continues to grow, highlighting the importance of accurate immunogenicity assessment. In addition, HZ remains a significant global health concern, with increasing incidence and the limited vaccine availability 33 . The FAMA test is regarded as the gold standard for assessing protective antibody responses to varicella, as it correlates strongly with neutralizing antibody levels and has long been considered a correlate of protection (CoP) 34 . However, despite its high sensitivity and specificity, FAMA is labor-intensive, impractical for large-scale clinical trials, and does not directly reflect clearance of infected cells. ELISA is more suitable for high-throughput analysis but provides ambiguous cut-off values when applied to vaccine-induced immunity 5 . While these assays assess only acquired humoral immunity, the observations that agammaglobulinemia patients do not progress to severe varicella 4 and that children exhibit minimal VZV-specific T-cell responses 5 , 35 suggest that innate cellular immunity plays a critical role in protection. Therefore, it is important to evaluate the contribution of Fc-mediated antibody functions in cooperation with innate immune effector cells when assessing the protective efficacy of varicella vaccines. HZ, caused by the reactivation of dormant VZV in the ganglia and spreading via nerve fibers, cannot be prevented solely by neutralizing antibodies. Instead, T-cell immunity plays a critical role in protection, typically assessed by cytokine secretion (e.g., IFN-γ, IL-2, and TNF-α) in PBMCs, and the frequency of polyfunctional CD4 + and CD8 + T cells 36 – 40 . Nevertheless, memory CD8⁺ T-cell responses against VZV are rarely detected even in adults, underscoring the limitations of cellular immune readouts 39 , 41 , 42 . This limitation highlights that such immunological measures do not reflect the clearance of virus-infected cells, and no definitive CoP for HZ vaccine has been established. Consequently, making large-scale clinical follow-up studies remain essential for accurate assessment of HZ vaccine efficacy. In this context, the identification of reliable immunogenicity biomarkers that can serve as CoP for HZ vaccines is urgently needed. Therefore, evaluating Fc-mediated effector functions, such as ADCP and ADCC, may not only provide mechanistic insights into virus clearance but also hold potential as exploratory biomarkers that could complement these clinical endpoints, pending further clinical validation. Evidence from other viral infections supports this concept. In nonhuman primates HIV vaccine studies, ADCP and ADCC correlated with reduced infection or improved viral control 43 , 44 . Similar findings have been reported in SARS-CoV-2, where Fc-mediated effector functions contributed to viral clearance and protection against lethal challenge 45 , 46 . In human, impaired ADCP and ADCC responses have been linked to severe COVID-19 outcomes 47 , 48 . In a previous study on varicella, ADCC activity was detected earlier than Nab responses in individuals with natural infection and in live attenuated vaccine recipients, suggesting a key role for Fc-mediated functions in the early phase of recovery from VZV infection 49 . While ADCC induction by zoster vaccination has been speculated 18 , the existing study employed artificial systems with limited physiological relevance. To date, few studies have evaluated the capacity of the zoster vaccines to elicit ADCP or ADCC, and none have done so in a comprehensive manner. To address this gap, we developed physiologically relevant ADCP and ADCC assays using VZV-infected MRC-5 cells as target cells, employing macrophages as effector cells for ADCP and NK cells for ADCC. This design, better reflects the complex antigenic profile of live attenuated VZV vaccines (e.g, BARYCELA, VARIVAX, and ZOSTAVAX), which elicit antibodies against multiple VZV antigens, thereby providing a broader assessment of antibody functionality than gE-transfected system. Both ADCP and ADCC assays demonstrated high reproducibility and robustness. Importantly, their activities correlated strongly with FAMA titers, supporting their validity as functional surrogate marker for protective immunity for varicella. While FAMA primarily reflects acquired humoral immunity, ADCP and ADCC capture the combined contribution of innate cellular and antibody-mediated responses. Our key finding is that all tested VZV vaccines across both live attenuated and subunit platforms and in different age groups elicited antibodies capable of mediating ADCP and ADCC against VZV-infected cells. Interestingly, although the ELISA-based antibody concentrations were significantly higher in adults vaccinated with zoster vaccine than in children who received LAV, Fc-mediated functions (ADCP and ADCC) were comparable across live attenuated platform vaccine groups regardless of age. This indicates that children generate antibodies with enhanced Fc-effector functionality despite lower total IgG levels. IgG subclass analysis revealed higher IgG3 and lower IgG1 levels in children than in adults. This is consistent with previous observations that varicella vaccination or primary varicella infection in children elicits an IgG3-dominant response, whereas adults with HZ exhibit an IgG1-dominant profile 50 . Age-related shifts in IgG subclasses align with reports from other viral infections, including RSV and measles, where IgG3 predominates in young children but declines with age 51 . The subclass-specific differences observed here reflect known distinctions in Fc receptor engagement on effector cells and the biological activities of IgG subclasses 52 . In the RV144 vaccine trial, depletion of IgG3 antibodies significantly reduced ADCP and ADCC activities, underscoring its importance in Fc-effector function 53 . Together, these findings highlight age-related variation in IgG subclass responses and their impact on Fc-mediated immunity, with implications for optimizing vaccine formulations and assessment strategies tailored to different age groups. This study has several limitations. First, the sample size was insufficient to assess the impact of demographic characteristics beyond sex-based differences. Second, we employed M1-differentiated U937 or No-GFP-CD16.NK-92 cell line as effector cells rather than autologous innate immune cells from vaccinees. As a result, we were unable to assess ADCP and ADCC activities mediated by the vaccinees’ own effector cells. Nevertheless, the use of standardized effector cell lines helps to minimize inter-individual variability, which is crucial to evaluating vaccine-induced antibody functions in clinical trials. In conclusion, we developed cell-based ADCP and ADCC assays that provide physiologically relevant measures of Fc-mediated antibody function against VZV. These assays correlated more strongly with FAMA titers than with ELISA antibody levels. Children, despite lower overall IgG levels, mounted potent IgG3-driven ADCP and ADCC activities comparable to adults. These findings underscore the importance of functional antibody quality over quantity and suggest that incorporating ADCP and ADCC assays alongside conventional serological tests may provide a more comprehensive mechanistically relevant assessment of VZV vaccine-induced immunity. Methods Ethics statement and Sera from vaccinees Human specimens were obtained from pediatric LAV recipients enrolled in the phase 3 clinical trial of MG1111 (Thailand and South Korea, protocol No. NCT03375502) and from adult ZOSTAVAX recipients (IRB file No. YUH-12-0462) conducted in Yeungnam University Medical Center. Written consent for secondary research use of banked specimens was obtained from all participants or their legal guardians. Secondary use for this study was approved by the Institutional Review Board of Yeungnam University Medical Center (IRB No. YUMC 2023-04-034). Blood collection from SHINGRIX recipients was approved by the Institutional Review Board of Korea University Guro Hospital (IRB No. 2023GR0083). All samples were de-identified prior to transfer. The study was conducted in accordance with the Declaration of Helsinki, and ICH E6(R2) Good Clinical Practice guidelines, as well as applicable local regulatory and bioethics requirements. Paired pre- and post-vaccination plasma/serum samples were obtained from four cohorts: 1-year-old children who received a single dose of LAV: BARYCELA® (GC Biopharma) or VARIVAX® (Marck & Co.), and adults aged ≥ 50 years old who received ZOSTAVAX® (Merck & Co.) or SHINGRIX® (GlaxoSmithKline). In total, 176 samples were analyzed: 46 from the BARYCELA® group, 50 from the VARIVAX® group, and 40 samples each from the ZOSTAVAX® and SHINGRIX® groups (Fig. 9 ). Cells MRC-5 cells (ECACC, UK) were cultured in Minimal Essential Medium (MEM; Welgene Inc,) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% non-essential amino acid (NEAA; Sigma-Aldrich), 1% sodium pyruvate (Gibco), and 1× antibiotic-antimycotic (Gibco) at 37°C in 5% CO 2 . U937 cells (ATCC®; CRL-1593.2) were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (SERANA) supplemented with 10% heat-inactivated FBS (HI-FBS; Gibco) and antibiotic. No-GFP-CD16.NK-92 cell line (ATCC®; PTA-6967) were cultured in α-Minimum Essential Medium (α-MEM; Gibco) supplemented with 12.5% HI-FBS, 12.5% Horse Serum (Sigma-Aldrich), 0.2 mM myo-inositol (Sigma-Aldrich), 0.02 mM folic Acid (Sigma-Aldrich), 1× antibiotic, 0.1 mM 2-Mercaptoethanol (Gibco), and 100 U/ml IL-2 (R&D systems). Differentiation of U937 cells into M1 macrophages and Fc receptors expression To generate M1-polarized macrophages from U937 monocytes, 2×10 6 U937 cells in 5 mL of complete culture medium were treated with 20 ng/mL of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 24 h at 37°C in 5% CO 2 . After PMA treatment, non-adherent cells were removed, and the culture was washed twice with Dulbecco’s phosphate buffered saline (DPBS; SERANA), incubated for an additional 24 h in fresh medium. To induce M1 polarization, cells were stimulated with 20 ng/mL of lipopolysaccharide (LPS; Sigma-Aldrich) and 20 ng/mL of interferon-gamma (IFN-γ; R&D system) for 24 h before the ADCP assay. Morphological changes were monitored by optical microscope (Carl Zeiss) (Supplementary Fig. 1). Flow cytometric staining of Fcγ receptors M1-polarized U937 cells were stained separately with fluorochrome-conjugated antibodies against CD16, CD32, and CD64 (BioLegend, Table 1 ) in 2% FBS-DPBS for 30 min on ice. Cells were then washed twice with 2% FBS-DPBS before acquisition on CytoFLEX flow cytometer (Beckman Coulter). Table 1 Antibody panel used for Fc receptors Target Full name Common clone Fluorochrome Catalog No. (BD) CD16 FcγRIII 3G8 FITC 560996 CD32 FcγRII FLI8.26 PE 568913 CD64 FcγRI 10.1 APC 561189 VZV-infected target cell preparation VZV-infected cells were prepared as target cells as follows: MRC-5 cells at 70%–80% confluence were infected with VZV YC03 strain (GenBank Accession No. KJ808816) at a ratio of one infected cell to 100 normal MRC-5 cells. The cells were harvested 72 h post-infection and cryopreserved in liquid nitrogen until use. The percentage of cells expressing VZV antigens on cell surface was determined using a flow cytometry-based FAMA test 54 . The WHO international standard for VZV immunoglobulin (NIBSC code W1044, diluted to 50 mIU/mL) was used as a positive reference, and DPBS served as the negative control. Following FAMA staining, VZV antigen expressed cells were analyzed using CytoFLEX flow cytometer (Beckman Coulter). Cells preparations exhibiting ≥ 80% VZV-positive events were used as target cell in ADCP and ADCC assays. Antibody-dependent cellular phagocytosis assay The phagocytic activity was evaluated using M1-U937 as effector cells. Sera were heat inactivated at 56°C for 30 min before the assay. VZV-infected MRC-5 target cells were labeled with 3 µM CFSE (5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester) in DPBS at 37°C for 10 min and then washed twice with DPBS supplemented with 2% HI-FBS. The CFSE-labeled target cells were incubated with control or vaccinated sera for 30 min; followed by co-incubated with M1-U937 effector cells for 4 h. Sera were applied at final dilutions of 1:100. Prior to flow cytometry, M1-U937 cells were labeled with CD89 antibody conjugated with APC (Miltenyi Biotec). ADCP activity, measured using CytoFLEX flow cytometer (Beckman Coulter), was expressed as the percentage of CSFE + CD89 + double-positive macrophages which phagocytosed CFSE + VZV-infected cells. Antibody-dependent cellular cytotoxicity assay: measuring granzyme B (GrB + ) incorporation into target cell by flow cytometry To measure ADCC activities, GrB + incorporation into the VZV-infected MRC-5 target cell assay was adopted from the ADCC-GTL assay 55 . VZV-infected target cells were labeled with 5 µM CellTracker™ Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (Invitrogen) in serum-free AIM-V medium (Gibco) at 37°C for 30 min. Labeled cells were washed once with serum-free AIM-V medium, and 2 × 10 4 cells per well were plated in 96-well U-bottom plate. Heat-inactivated sera were added and incubated with target cells for 30 min at 37°C. No-GFP-CD16.NK-92® cells were then added at a 1:1 E:T ratio, yielding a final serum dilution of 1:100. Co-cultures were incubated for 4 h at 37°C before harvest. Cells were fixed with 100 µL/well of Fixation/Permeabilization solution (BD Biosciences) for 20 min at 4°C, washed twice with 1× BD Perm/Wash™ buffer (BD Biosciences), and pelleted. The fixed/permeabilized cells were resuspended thoroughly in 100 µL of 1× BD Perm/Wash™ buffer containing FITC conjugated anti-Granzyme B (GrB) antibody at a 1:50 dilution (BioLegend). The samples were incubated at 4°C for 30 min in the dark. After incubation, cells were washed cells twice with 1× BD Perm/Wash™ buffer and resuspended in 1× BD Perm/Wash™ buffer for flow cytometric analysis. ADCC activities, measured using CytoFLEX flow cytometer (Beckman Coulter), was expressed as the percentage of CMTMR + GrB + double-positive target cells. Validation of ADCP and ADCC assays Following established bioanalytical assay validation guidelines 56 , 57 , key performance parameters were selected for methodological evaluation of ADCP and ADCC assays. These parameters included VZV-target cell lot-to-lot variation, linearity, intra-assay precision, inter-assay precision (inter-personal and inter-day precision) and inter-laboratories precision. Lot-to-lot variability was evaluated to determinate the consistency of assay results across different target cell stocks used in the same experiment, ensuring that variations between target cells do not significantly affect assay outcomes. Linearity assay was assessed using five serial dilutions of three different sera, with a simple linear regression analysis performed to confirm linearity. Intra-assay precision was assessed by testing the six samples, each five replicated on the same plate by one operator, to determine the assay’s precision within a single run. Inter-assay precision was assessed by testing the same six samples across three days, potentially by two different operators, to assess reproducibility across independent experiments. Inter-laboratory precision was determined by having three laboratories independently conduct the assay using identical sera and the same stock of VZV-target cells, ensuring reproducibility across different lab setting. The coefficient of variation (CV) was calculated to evaluate the precision. Fluorescent antibody to membrane antigen test FAMA test was conducted as previously described 58 . Briefly, FAMA antigens were prepared as follows: MRC-5 cells at 70%–80% confluence were infected with VZV YC03 strain at a ratio of one infected cell to 200 normal MRC-5 cells and harvested 72 h post-infection, and stored in liquid nitrogen until use. Sera were twofold serially diluted with DPBS, and then incubated with 2 × 10 5 FAMA antigen cells for 30 min at RT, followed by two DPBS washes. After the secondary antibody (goat anti-human IgG-Alexa 488, Invitrogen) reaction for 30 min at RT, cells were washed three times with DPBS. FAMA antigen cells were loaded onto a 14-well slide (Cel-Line®, Thermo Scientific) and then dried. Slides were mounted with Vectashield mounting medium with DAPI (Vector Laboratories) and observed using an Axioscope fluorescence microscope equipped with an HBO 100 mercury lamp (Carl Zeiss). VZV-specific IgG ELISA VZV-specific IgG levels were quantified using the SERION ELISA classic kit (ESR104G, Virion\Serion, Germany) according to the manufacturer’s instructions. Briefly, 100 µL of controls and diluted sera were added to VZV-antigen-coated wells and incubated for 1 h. After washing, 100 µL of goat anti-human IgG conjugated with alkaline phosphatase (AP) was added and incubated for 30 min. Following additional washes, substrate was added and incubated for 30 min. All incubation steps were performed at 37°C. The reaction was then terminated with a stopping solution, and the absorbance was measured at 405 nm using a Multiskan FC 357 microplate photometer (Thermo Scientific). VZV-specific IgG subclass ELISA VZV-specific IgG1 and IgG3 antibodies were measured using the SERION ELISA classic kit (ESR104G, Virion\Serion) with modifications. VZV antigen-coated plates were incubated with 100 µL of diluted sera (1:50) for 1 h at 37°C. After washing, plates were incubated with mouse anti-human IgG1 (1:2000) or IgG3 (1:100) antibodies conjugated with AP (SouthernBiotech) at 37°C for 30 min. Following incubation with pNPP substrate for 30 min, the reaction was stopped, and absorbance was measured at 405 nm using a Multiskan FC 357 microplate Photometer (Thermo Scientific). Subclass-specific responses were expressed as optical densities. Statistical analyses Statistical analyses were performed using GraphPad Prism 10.0.2 (GraphPad Software). Pair t-tests or Wilcoxon matched-paired signed rank tests were used for comparisons between two groups. One-way ANOVA or the Kruskal-Wallis tests were used for multiple group comparisons, with post-hoc pairwise correction as appropriate. Pearson correlation coefficients (r) were calculated to assess relationships among ADCP, ADCC, FAMA and ELISA GMTs. Statistical significance was denoted as ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Declarations Competing interests The authors declare no competing financial or personal interests that could have influenced the work reported in this manuscript. Funding This study was supported by grant 23202MFDS136 from the Ministry of Food and Drug Safety, Republic of Korea, in 2023. The funding agency had no role in study design, data collection and analysis, decision to publish, or manuscript preparation. Author Contribution S.X., J.Y.H., H.S.L., O.S.S., J.Y.N., S.H.H., and H.P. conceived and designed the study. 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Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 25 Mar, 2026 Read the published version in npj Vaccines → Version 1 posted Editorial decision: Revision requested 04 Jan, 2026 Reviews received at journal 25 Dec, 2025 Reviews received at journal 25 Dec, 2025 Reviewers agreed at journal 10 Dec, 2025 Reviewers agreed at journal 09 Dec, 2025 Reviewers invited by journal 09 Dec, 2025 Editor assigned by journal 06 Dec, 2025 Submission checks completed at journal 19 Nov, 2025 First submitted to journal 15 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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1","display":"","copyAsset":false,"role":"figure","size":4917282,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy overview and workflows to quantify Fc-mediated effector functions and humoral immunity to VZV after varicella and zoster vaccination. a \u003c/strong\u003eSchematic representation of ADCP and ADCC assays with key parameters for assay optimization and validation.\u003cstrong\u003e b \u003c/strong\u003eApplication of ADCP/ADCC to samples from\u003cstrong\u003e \u003c/strong\u003evaccinated children and adults, and workflow from sample collection to flow-cytometric readout.\u003cstrong\u003e \u003c/strong\u003eAssessment of humoral immunity using FAMA, VZV-specific IgG ELISA, and IgG subclass ELISA (IgG1, IgG3).\u003cstrong\u003e \u003c/strong\u003eLAV, live attenuated varicella vaccine; RZV, recombinant zoster vaccine; VZV, varicella zoster virus;ZVL, live attenuated zoster vaccine.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/9f822710f7146b130125856d.png"},{"id":98181215,"identity":"b711accd-37e9-47d9-b2aa-1296abebd628","added_by":"auto","created_at":"2025-12-15 01:09:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10072982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eADCP and ADCC activities measured by flow cytometry with representative confirmation by confocal microscopy. a\u003c/strong\u003e Antibody-dependent cellular phagocytosis (ADCP) quantified by flow cytometry. VZV-infected MRC-5 target cells were labeled with CFSE and incubated with pre- (left panel) and post-varicella vaccinated (right panel) sera, and then incubated with effector M1-U937. M1-U937 were stained with APC-conjugated anti-CD89 antibody. ADCP activity was defined as the percentage of CFSE\u003csup\u003e+\u003c/sup\u003eCD89\u003csup\u003e+\u003c/sup\u003e double-positive cells. \u003cstrong\u003eb\u003c/strong\u003e Confocal image of ADCP showing effector macrophages (red; anti-CD89-APC) containing CFSE-labeled VZV-infected MRC-5 target cells (green). No double-positive cells were detected with pre-varicella vaccination serum (left), whereas post-varicella vaccination serum induced efficient internalization of target cells into effector macrophages (right). Scale bar, 30 μm. \u003cstrong\u003ec\u003c/strong\u003e Antibody-dependent cellular cytotoxicity (ADCC) was quantified by flow cytometry. VZV-infected MRC-5 target cells were labeled with CMTMR and incubated with pre- (left) and post-varicella vaccinated (right) sera, and then incubated with effector NK cells. GrB was stained with FITC-conjugated anti-GrB antibody. ADCC activity was expressed as the percentage of CMTMR\u003csup\u003e+\u003c/sup\u003eGrB\u003csup\u003e+\u003c/sup\u003e target cells. \u003cstrong\u003ed\u003c/strong\u003e Confocal image of CMTMR-labeled target cells (red) with internalized GrB (green) from effector cells. Pre-vaccination serum showed negligible GrB uptake (left), whereas post-vaccination serum promoted marked GrB internalization (right). Scale bar, 30 μm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/b38613d304b9edfbe649370c.png"},{"id":98181223,"identity":"8d2467ae-c121-43cf-997a-e14f9f1b39cd","added_by":"auto","created_at":"2025-12-15 01:09:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2613002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA series of validation experiments for the ADCP and ADCC assays.\u003c/strong\u003e \u003cstrong\u003ea, b \u003c/strong\u003eLot-to-lot consistency: ADCP (\u003cstrong\u003ea\u003c/strong\u003e) and ADCC (\u003cstrong\u003eb\u003c/strong\u003e) activities were evaluated using two independent lots of VZV-infected MRC-5 cell stocks, demonstrating consistent results across different target cell lots. \u003cstrong\u003ec, d \u003c/strong\u003eLinearity: Five-point serial two-fold dilutions of three representative sera were used to assess the linearity of the ADCP (\u003cstrong\u003ec\u003c/strong\u003e) and ADCC (\u003cstrong\u003ed\u003c/strong\u003e) responses, demonstrating strong proportionality between antibody dilution levels and Fc-mediated functional activities. \u003cstrong\u003ee, f \u003c/strong\u003eIntra-assay precision: Repeatability of ADCP (\u003cstrong\u003ee\u003c/strong\u003e) and ADCC (\u003cstrong\u003ef\u003c/strong\u003e) assays was assessed by five replicates of six samples in a single run, confirming assay reliability under identical conditions. \u003cstrong\u003eg, h\u003c/strong\u003e Inter-operator (inter-personal) precision: ADCP (\u003cstrong\u003eg\u003c/strong\u003e) and ADCC (\u003cstrong\u003eh\u003c/strong\u003e) assays were performed independently by two operators across three separate runs to evaluate variability attributable to operator handling. \u003cstrong\u003ei, j\u003c/strong\u003e Inter-day precision: Day-to-day reproducibility of ADCP (\u003cstrong\u003ei\u003c/strong\u003e) and ADCC (\u003cstrong\u003ej\u003c/strong\u003e) assays was assessed by repeated experiments over three separate days using the same set of samples. \u003cstrong\u003ek, l\u003c/strong\u003eInter-laboratory precision: ADCP (\u003cstrong\u003ek\u003c/strong\u003e) and ADCC (\u003cstrong\u003el\u003c/strong\u003e) assays were conducted in three independent laboratories using paired pre- and post-vaccination sera to assess cross-site reproducibility.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/3e7567224453bf70bad9e79a.png"},{"id":98181209,"identity":"26914542-38f6-4573-ac0c-76ca7d88db93","added_by":"auto","created_at":"2025-12-15 01:09:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":72048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVZV-specific Fc-mediated responses following VZV-immunization were measured by ADCP and ADCC assays.\u003c/strong\u003e \u003cstrong\u003ea, b \u003c/strong\u003eADCP (\u003cstrong\u003ea\u003c/strong\u003e) and ADCC (\u003cstrong\u003eb\u003c/strong\u003e) activities measured in paired pre- and post-vaccination sera. \u003cstrong\u003ec, d \u003c/strong\u003eFold changes (post/pre) in ADCP (c) and ADCC (\u003cstrong\u003ed\u003c/strong\u003e) responses. Sera were collected from individuals who received one dose of BARYCELA (children, n = 23), one dose of VARIVAX (children, n = 25), one dose of ZOSTAVAX (adults, n = 20), or two doses SHINGRIX (adults, n = 20). Statistical analyses were performed using paired t-test, Wilcoxon matched-pairs signed-rank tests, or the Kruskal-Wallis test with post hoc pairwise comparisons and appropriate corrections. Significance levels are indicated as p \u0026lt; 0.05 (*), p \u0026lt; 0.01 (**), p \u0026lt; 0.001 (***), p \u0026lt; 0.0001 (****) and ns = not significant. Data are presented as mean ± standard deviation (SD), with error bars indicating SD.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/3ab8d3925ff7683ff0e52652.png"},{"id":98181169,"identity":"24b4a415-7c05-41fd-9544-319a6a17ed00","added_by":"auto","created_at":"2025-12-15 01:09:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":79375,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVZV-specific humoral immune responses before and after vaccination. \u003c/strong\u003ePaired pre- and post-vaccination sera were analyzed from individuals who received one dose of BARYCELA (children, n = 23), one dose of VARIVAX (children, n = 25), one dose of ZOSTAVAX (adults, n = 20), or two doses of SHINGRIX (adults, n = 20). \u003cstrong\u003ea\u003c/strong\u003e FAMA geometric mean titers (GMTs). \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cstrong\u003eb \u003c/strong\u003eVZV-specific IgG antibody titers quantified using the Serion ELISA Classic VZV IgG kit. \u003cstrong\u003ec-d\u003c/strong\u003e VZV-specific IgG1 (c) and IgG3 (d) subclass levels measured by ELISA. \u003cstrong\u003ee-f\u003c/strong\u003eFold-change comparisons of IgG1 (e) and IgG3 (f) responses in the four vaccine groups.\u003cem\u003e \u003c/em\u003eEach dotrepresents an individual participant; lines indicate medians. Statistical analyses were performed using pairedt-tests,Wilcoxon matched-pairs signed-rank tests, or Kruskal-Wallis test with post hoc pairwise comparisons with appropriate corrections. Significance levels are indicated as \u003cem\u003ep \u0026lt; \u003c/em\u003e0.05 (*), \u003cem\u003ep \u0026lt; \u003c/em\u003e0.01 (**)\u003cem\u003e, p \u0026lt; \u003c/em\u003e0.001 (***),\u003cem\u003e p \u0026lt; \u003c/em\u003e0.0001 (****) and ns = not significant\u003cem\u003e. \u003c/em\u003eData are presented as mean ± SD, with error bars indicating SD.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/738880c6540fd1c752481569.png"},{"id":98181255,"identity":"037386f4-1b59-4557-9ae4-3775392a690a","added_by":"auto","created_at":"2025-12-15 01:09:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":740974,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation of ADCP, ADCC, FAMA, and ELISA titers with VZV-specific IgG1 and IgG3 subclass levels in vaccinated individuals. \u003c/strong\u003eCorrelation analyses were performed using pre- and post-vaccinated sera from children who received varicella vaccines (BARYCELA, VARIVAX) and adults who received zoster vaccines (ZOSTAVAX, SHIGRIX). \u003cstrong\u003ea\u003c/strong\u003e–\u003cstrong\u003ed\u003c/strong\u003eCorrelations between IgG1 levels and ADCP, ADCC, FAMA titers, and ELISA titers in children. \u003cstrong\u003ee\u003c/strong\u003e–\u003cstrong\u003eh\u003c/strong\u003e Correlations between IgG3 levels and the same parameters in children. \u003cstrong\u003ei\u003c/strong\u003e–\u003cstrong\u003el\u003c/strong\u003e Correlations between IgG1 levels and ADCP, ADCC, FAMA titers, and ELISA titers in adults. \u003cstrong\u003em\u003c/strong\u003e–\u003cstrong\u003ep\u003c/strong\u003e Correlations between IgG3 levels and the same parameters in adults. Pearson’s correlation coefficients (r) and corresponding p-values are shown in each panel.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/9c7f8b1d8e625052a97eeb4f.png"},{"id":98181162,"identity":"00a1fd95-6ef3-498d-bb4a-08860bc63bfb","added_by":"auto","created_at":"2025-12-15 01:09:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":946404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelation of ADCP and ADCC activities with FAMA titer, ELISA antibody titer, and between each other, stratified by vaccine type.\u003c/strong\u003e Scatter plots show the correlations between FAMA titer and ADCP (\u003cstrong\u003ea-d\u003c/strong\u003e) or ADCC (\u003cstrong\u003ee-h\u003c/strong\u003e), between ELISA antibody titer and ADCP (\u003cstrong\u003ei-l\u003c/strong\u003e) or ADCC (\u003cstrong\u003em-p\u003c/strong\u003e), and between ADCP and ADCC (\u003cstrong\u003eq-t\u003c/strong\u003e) in recipients of BARYCELA (\u003cstrong\u003ea, e, i, m, q\u003c/strong\u003e), VARIVAX (\u003cstrong\u003eb, f, j, n, r\u003c/strong\u003e), ZOSTAVAX (\u003cstrong\u003ec, g, k, o, s\u003c/strong\u003e), and SHINGRIX (\u003cstrong\u003ed, h, l, p, t\u003c/strong\u003e). Each dot represents an individual recipient. Pearson correlation coefficient (r) and corresponding p-values are indicated in each panel. Positive and statistically significant correlations were observed across all vaccine types.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/8446fd849458f5344b318fc5.png"},{"id":98181167,"identity":"88ccb7ca-a4c0-4c90-b63c-f5554f690add","added_by":"auto","created_at":"2025-12-15 01:09:14","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":365809,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCorrelations among ADCP, ADCC, FAMA titers and ELISA antibody titers across all vaccine groups.\u003c/strong\u003e Scatter plots show correlations between ADCP and ADCC (\u003cstrong\u003ea\u003c/strong\u003e), FAMA titers and ADCP (\u003cstrong\u003eb\u003c/strong\u003e) or ADCC (\u003cstrong\u003ec\u003c/strong\u003e), and ELISA antibody titer and ADCP (\u003cstrong\u003ed\u003c/strong\u003e) or ADCC (\u003cstrong\u003ee\u003c/strong\u003e) using pooled data from all vaccine recipients. Each dot represents an individual serum sample. Pearson correlation coefficients (r) and p-values are indicated in each panel.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/2ac98e82676febe106a4d2c4.png"},{"id":98181211,"identity":"2a025bff-9a60-4ef6-a260-62ef809b4085","added_by":"auto","created_at":"2025-12-15 01:09:22","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1259881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy design for vaccination and sample collection schedules. a\u003c/strong\u003eHealthy children were vaccinated with either BARYCELA (n = 23) or VARIVAX (n = 25); sera were collected at baseline and on Day 42 post-vaccination. \u003cstrong\u003eb\u003c/strong\u003e Healthy adults (n = 20) received a single dose of ZOSTAVAX; sera were collected at baseline and between Days 28 and 42 post-vaccination. \u003cstrong\u003ec\u003c/strong\u003eHealthy adults (n = 20) received two doses of SHINGRIX administered ≥ 2 months apart; sera were collected at baseline and 28 days after the second dose.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/f6d3d582e038f758b072895b.png"},{"id":105755042,"identity":"1734f99e-e8ff-409d-b824-4f6135aaa2d5","added_by":"auto","created_at":"2026-03-30 16:24:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19963204,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/5bebf529-0b6b-496f-9d94-aa2db04e3769.pdf"},{"id":98181257,"identity":"a76d3925-552d-49de-b75e-623cce06d389","added_by":"auto","created_at":"2025-12-15 01:09:27","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17529333,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8121887/v1/019fa0ab3303ac79e36157ea.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating varicella-zoster virus vaccine immunogenicity through Fc-mediated antibody functions: the roles of ADCP and ADCC","fulltext":[{"header":"Introduction","content":"\u003cp\u003eVaricella (chickenpox) and herpes zoster (HZ) are distinct clinical manifestations caused by the varicella-zoster virus (VZV), progressing from primary infection and viral reactivation, respectively. During primary infection, VZV spreads systemically through viremia, whereas HZ results from viral reactivation in sensory ganglia followed by spread along peripheral nerves. These pathogenic differences imply distinct immune mechanisms for protection and disease control. Antibodies targeting VZV glycoproteins, which reflect neutralizing antibody activity, correlate strongly with protection against varicella\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. By contrast, such antibodies do not prevent HZ, for which cellular immunity is critical\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Notably, children with isolated agammaglobulinemia are not prone to severe varicella\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. However, VZV-specific memory T cells responses after vaccination remain weak or undetectable in children\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Severe or disseminated varicella has been reported in individuals with natural killer (NK) or invariant NKT-cell deficiencies, underscoring the importance of innate cellular immunity in controlling infection\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Together, these findings suggest that clearance of VZV-infected cells relies not only on T cells but also on cooperation between adaptive and innate immune responses.\u003c/p\u003e\u003cp\u003eFc-effector functions, such as antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), enhance the clearance of virus-infected cells\u003csup\u003e\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15 CR16\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. These functions may be particularly relevant for HZ, where the virus is presumed to evade neutralization\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Beyond Fab-mediated neutralization, antibodies exert their functions through the Fc-domain, which recruits and activates innate immune cells including macrophages and NK cells\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Fc-mediated effector functions have been implicated in protection against diverse viral infections, including herpes simplex virus-2 (HSV\u0026ndash;2), Dengue virus, respiratory syncytial virus (RSV), SARS-CoV-2, Ebola, and influenza\u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Immunoglobulin G (IgG) subclasses also differ in their ability to mediate Fc effector activity, with IgG3 generally exhibiting greater potency than IgG1\u003csup\u003e30\u003c/sup\u003e. Differential subclass responses have been reported in varicella and zoster infection\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, suggesting that host age and disease context may shape Fc-effector profiles.\u003c/p\u003e\u003cp\u003eDespite these insights, Fc-mediated antibody functions against VZV remain poorly characterized, and no standardized assays are currently available for their evaluation. This gap limits our understanding of protective immunity and hampers the development of reliable correlates of protection for VZV vaccines. To address this, we established physiologically relevant macrophages-based ADCP and NK cell-based ADCC assays and validated their reproducibility and robustness. Then, we applied these assays to sera from children and adults immunized with varicella and zoster vaccines, respectively. In parallel, antibody levels were measured by fluorescent antibody to membrane antigen (FAMA), ELISA, and IgG subclass analyses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Together, these data provide the most comprehensive characterization to date of VZV-specific antibody functions across age groups and highlight their potential as functional biomarkers for evaluating vaccine-induced immunity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEstablishment of ADCP and ADCC assay to assess varicella and zoster vaccine immunogenicity\u003c/h2\u003e\u003cp\u003eWe established an ADCP assay using carboxyfluorescein succinimidyl ester (CFSE)-labeled VZV-infected MRC-5 cells as target cells, employing M1-U937 cells as effector cells for evaluating human sera. To optimize the experimental conditions, U937 cells were differentiated into M1 macrophages using 20 ng/ml of each PMA, LPS and IFN-γ (Supplementary Fig.\u0026nbsp;1). The FcγRII (CD32) and FcγRI (CD64) were particularly upregulated in differentiated M1-U937 cells compared to U937 cells (Supplementary Fig.\u0026nbsp;2). These findings confirm that M1-U937 exhibit an activated macrophage phenotype. The ADCP activity was quantified as the percentage of CFSE\u003csup\u003e+\u003c/sup\u003eCD89\u003csup\u003e+\u003c/sup\u003e double-positive cells by flow cytometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and further confirmed by confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Pre-vaccination sera did not induce detectable phagocytosis, as indicated by the absence of double-positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, left). In contrast, post-vaccination sera promoted robust phagocytosis of VZV-infected target cells by effector macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, right). For ADCC assay, CMTMR-labeled VZV-infected MRC5 cells served as a target cells, employing the no-GFP-CD16.NK-92 cell line as an effector. Granzyme B (GrB)-uptaking target cells (CMTMR\u003csup\u003e+\u003c/sup\u003eGrB\u003csup\u003e+\u003c/sup\u003e double-positive cells) were defined as the readout for ADCC detection (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). ADCC responses were also confirmed by confocal microscopy (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Pre-vaccination sera induce negligible GrB uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, left), but post-vaccination sera facilitated efficient transfer of GrB into target cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, right), confirming vaccine-induced functional antibody responses.\u003c/p\u003e\u003cp\u003eOptimization of both assays involved assessing effector to target cell number (E:T) ratio, serum dilution, and the reaction time, using pre- and post-vaccination sera. E:T ratios of 1:2 and 2:1 (for ADCP) or 1:1 and 1:2 (for ADCC) were tested, followed by serial dilutions of sera at the optimal E:T ratios (1:2 for ADCP and 1:1 for ADCC). Incubation times of 2, 3, 4 or 5 hours were also evaluated. The final protocol of ADCP assay was established at an E:T ratio of 1:2, a 1:100 serum dilution, and a 4-hour incubation. The ADCC assay was conducted using an E:T ratio of 1:1, a 1:100 serum dilution, and a 4-hour incubation. (Supplementary Fig.\u0026nbsp;3, Supplementary Table\u0026nbsp;1).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eValidation of ADCP and ADCC assays\u003c/h3\u003e\n\u003cp\u003eComprehensive validation of the ADCP and ADCC assays were performed using VZV plasma panel together with pre- and post-VZV vaccination sera (Supplementary Table\u0026nbsp;2). Lot-to-lot variability of VZV-infected MRC-5 target cell stocks was assessed across three antibody concentrations. Given the cell-based nature of these assays, coefficient of variation (CV) below 30% was predefined as an acceptance criterion. The range of target-cell lot-to-lot CVs were 0.6%\u0026ndash;4.3% for ADCP and 2.8%\u0026ndash;7.1% for ADCC, meeting this criterion (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b; Supplementary Tables\u0026nbsp;3, 4). Linearity was evaluated using five serial dilutions of three samples. Simple linear regression analysis showed very strong linearity across a broad range (ADCP \u003cem\u003er\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.87\u0026ndash;0.96; ADCC \u003cem\u003er\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.87\u0026ndash;0.97) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec, d; Supplementary Tables\u0026nbsp;5, 6), supporting accurate quantification across varying antibody concentrations. Intra-assay precision was assessed by running five replicates of six panel samples on a single plate. For ADCP, intra-assay CVs were 5.0%\u0026ndash;28.5% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, Supplementary Table\u0026nbsp;7) and ADCC intra-assay CVs were 2.7%\u0026ndash;28.0% across all six panels, meeting the acceptance criterion at low, medium, and high levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef, Supplementary Table\u0026nbsp;8). Inter-assay precision was determined across three independent runs on different days by two operators. Between-operator CVs were 1.9%\u0026ndash;28.4% for ADCP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg, Supplementary Table\u0026nbsp;9) and 2.3%\u0026ndash;18.0% for ADCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh, Supplementary Table\u0026nbsp;10). Inter-day CVs were ranged from 3.7%\u0026ndash;16.6% for ADCP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, Supplementary Table\u0026nbsp;11) and 0.2%\u0026ndash;25.1% for ADCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej, Supplementary Table\u0026nbsp;12). Finally, inter-laboratory testing across three laboratories using three paired pre-/post-vaccination sera showed comparable readouts, with inter-lab CVs of 5.7%\u0026ndash;21.8% for ADCP and 6.3%\u0026ndash;21.8% for ADCC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek, l; Supplementary Tables\u0026nbsp;13, 14). Together, these data indicate that the ADCP and ADCC assays using VZV-infected MRC-5 cells are robust and reproducible across operators, days, and laboratories, and perform consistently across a broad range of antibody levels, supporting their utility for Fc-mediated antibody assessment in recipients of both varicella and zoster vaccines.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eVaricella or zoster vaccination induced ADCP and ADCC activities\u003c/h3\u003e\n\u003cp\u003eFc effector functions were evaluated using the ADCP and ADCC in pre- and post-vaccination sera from recipients of varicella or zoster vaccines. In the pre-vaccination sera of 1-year-old children receiving live attenuated varicella vaccines (LAVs): BARYCELA or VARIVAX, ADCP and ADCC activities were at baseline levels: BARYCELA (ADCP: 8.3%, ADCC: 11.3%) and VARIVAX (ADCP: 7.8%, ADCC: 12.8%). However, these activities significantly increased after a single dose of vaccination: BARYCELA (ADCP: 34.3%, ADCC: 24.3%) and VARIVAX (ADCP: 34.1%, ADCC: 21.7%) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b; Supplementary Table\u0026nbsp;15).\u003c/p\u003e\u003cp\u003eSince most Korean adults already possess immunity to VZV, pre-vaccination sera showed similar levels of ADCP and ADCC activities in two zoster vaccine (ZV) groups, ZOSTAVAX (ADCP: 27.0%, ADCC: 20.3%) and SHINGRIX (ADCP: 28.9%, ADCC: 24.8%). Post-vaccination sera showed significant increases in ADCP and ADCC activities in ZOSTAVAX (ADCP: 40.8%, ADCC: 25.9%) after a single dose and SHINGRIX (ADCP: 51.5%, ADCC: 31.9%) after two doses, compared to pre-vaccination sera (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b; Supplementary Table\u0026nbsp;15). A single dose of LAVs in children showed comparable ADCP and ADCC activities to a single dose of ZOSTAVAX in adults.\u003c/p\u003e\u003cp\u003eThe fold changes between pre- and post-vaccination were as follows BARYCELA: ADCP (4.1-fold), ADCC (2.1-fold), VARIVAX: ADCP (4.4-fold), ADCC (1.7-fold), ZOSTAVAX: ADCP (1.5-fold), ADCC (1.3-fold) and SHINGRIX: ADCP (1.80-fold), ADCC (1.3-fold). Therefore, there were no significant differences in fold changes between BARYCELA and VARIVAX or ZOSTAVAX and SHINGRIX recipients (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d; Supplementary Table\u0026nbsp;15).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eAntibody titers measured by FAMA test and ELISA\u003c/h3\u003e\n\u003cp\u003eAntibody titers were measured by FAMA test, the gold standard method for the varicella vaccine-induced antibody titer, and ELISA. The geometric mean titers (GMTs) of FAMA in post-vaccination sera for all four vaccine groups were significantly higher than those in the pre-vaccination sera (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). GMTs of pre-vaccinated children\u0026rsquo;s sera (BARYCELA and VARIVAX) were negative levels (\u0026le;\u0026thinsp;2.2). Post-BARYCELA GMT (153.4: ranging from 32 to 512) and post-VARIVAX GMT (164.3: range, 32\u0026ndash;1,024) were significantly increased but there were no differences between two vaccines. The FAMA GMTs of pre-vaccinated adults\u0026rsquo; sera showed similar levels; pre-ZOSTAVAX (78.8: range, 32\u0026ndash;256) and pre-SHINGRIX (87.4: range, 32\u0026ndash;256). However, those of post-SHINGRIX (776.0: range, 256\u0026ndash;2,048) were significantly higher than post-ZOSTAVAX (215.3: range, 64\u0026ndash;512) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, Supplementary Table\u0026nbsp;16).\u003c/p\u003e\u003cp\u003eAnti-VZV IgG ELISA antibody titers were measured using VZV IgG Serion ELISA classic kit (detection range, 15\u0026ndash;2,000 mIU/mL). The GMTs of anti-VZV IgG ELISA antibodies in the post-vaccinated sera were significantly elevated relative to pre-vaccination levels in all groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Most pre-vaccinated children\u0026rsquo;s sera were lower than detection limit; pre-BARYCELA GMT (0.8 mIU/mL: range, 0.1\u0026ndash;32.0 mIU/mL) and pre-VARIVAX GMT (0.4 mIU/mL: range, 0.01\u0026ndash;24.8 mIU/mL). Post-BARYCELA GMT (118.9 mIU/mL: range, 27.8\u0026ndash;435.4 mIU/mL) and post-VARIVAX GMT (139.2 mIU/mL: range, 37.5\u0026ndash;893.2 mIU/mL) were significantly increased but there were no differences between two vaccines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, Supplementary Table\u0026nbsp;17). The ELISA GMTs of pre-vaccinated adults\u0026rsquo; sera showed similar levels, pre-ZOSTAVAX GMT (520.1 mIU/mL: range, 159.1\u0026ndash;1,484.4 mIU/mL) and pre-SHINGRIX GMT (609.6 mIU/mL: range, 145.0\u0026ndash;2,176.0 mIU/mL). However, all of the post-SHINGRIX samples, the ELISA titers were over the detection limit; post-SHINGRIX GMT (10,428.9 mIU/mL: range, 4,561.4\u0026ndash;36,355.9 mIU/mL) and significantly higher than post-ZOSTAVAX GMT (1,697.3 mIU/mL: range, 415.2\u0026ndash;4,599.2 mIU/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, Supplementary Table\u0026nbsp;17).\u003c/p\u003e\n\u003ch3\u003eDifferential expression of anti-VZV IgG1 and IgG3 in varicella-vaccinated children and zoster-vaccinated adults\u003c/h3\u003e\n\u003cp\u003eTo characterize subclass-specific antibody responses induced by VZV vaccination, IgG1 and IgG3, key mediators of ADCP and ADCC responses, were quantified by ELISA. Distinct IgG subclass profiles were observed between varicella-vaccinated children and zoster-vaccinated adults. Both IgG1 and IgG3 antibody levels significantly increased after vaccination in all groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d). Analysis of IgG1 subclass fold-changes revealed that children who received LAVs exhibited significantly lower increases: BARYCELA (1.8) and VARIVAX (2.5), compared with adults vaccinated with HZ vaccines, ZOSTAVAX (3.3) and SHINGRIX (4.9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, Supplementary Table\u0026nbsp;18). Conversely, IgG3 subclass fold-changes were significantly higher in children immunized with LAVs \u0026minus;BARYCELA (4.8) and VARIVAX (5.8) \u0026minus;than adults vaccinated with ZOSTAVAX (2.1) or SHINGRIX (1.8) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ef, Supplementary Table\u0026nbsp;18). These findings indicate that varicella vaccination in children preferentially induces IgG3, whereas adult zoster vaccination elicits a predominant IgG1 response.\u003c/p\u003e\u003cp\u003eIn children, both IgG1 and IgG3 levels showed significant correlations with ADCP, ADCC, FAMA titer, and ELISA titer. However, IgG3 exhibited stronger correlations with ADCP, ADCC, and FAMA titer compared to IgG1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u0026minus;h). Correlation coefficients (\u003cem\u003er\u003c/em\u003e) between IgG1 and ADCP, ADCC, and FAMA were 0.563 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), 0.340 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0012), and 0.545 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea\u0026minus;c). Whereas those between IgG3 and the same parameters were higher, 0.804, 0.647, and 0.802, respectively (all with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u0026minus;g). However, the correlations between ELISA titers and IgG1 (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.775) or IgG3 (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.744) were comparable (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed, h). In contrast, among zoster-vaccinated adults, strong and significant correlations were found between IgG1 and ADCP (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.856), ADCC (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.698), FAMA titer (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.939), and ELISA titer (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.791), all with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ei\u0026minus;l). However, the correlations between IgG3 and these parameters were weak and not significant (\u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003em\u0026minus;p). These findings suggest that IgG3 may play a more prominent role in vaccine-induced Fc-mediated antiviral functions in children, whereas IgG1 appears to be more critical in adults.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eCorrelation between Fc-mediated antibody functions (ADCP, ADCC) and antibody titers (FAMA, ELISA) across vaccine types\u003c/h2\u003e\u003cp\u003ePearson correlations analysis was performed to determine the relationships between Fc-mediated antibody functions (ADCP and ADCC) and antibody titers (FAMA and ELISA) across different vaccine types. A robust and statistically significant correlations were observed between ADCP activity and FAMA titers across all vaccine groups: BARYCELA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.947), VARIVAX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.936), ZOSTAVAX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.809), and SHINGRIX (\u003cem\u003er\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.885) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea\u0026ndash;d; all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating a very strong correlation between these two immunological parameters. Similarly, ADCC activities were strongly correlated with FAMA titers in all vaccine groups: BARYCELA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.837), VARIVAX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.705), ZOSTAVAX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.707), and SHINGRIX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.885) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ee\u0026ndash;h, all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003eIn contrast, correlations of ADCP and ADCC with ELISA titers were relatively weaker than those with FAMA titers, though still statistically significant. For ADCP, the correlations with ELISA were: BARYCELA (\u003cem\u003er\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.890), VARIVAX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.761), ZOSTAVAX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.853, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and SHINGRIX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.702) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ei\u0026ndash;l, all other \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). For ADCC, the correlations with ELISA were: BARYCELA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.823), VARIVAX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.500), ZOSTAVAX (\u003cem\u003er\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.667), and SHINGRIX (\u003cem\u003er\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.567) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003em\u0026ndash;p, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Moderate to very strong correlations were also noted between ADCC and ADCP within each vaccine group; BARYCELA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.854), VARIVAX (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.632), ZOSTAVAX (\u003cem\u003er\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.655), and SHINGRIX (\u003cem\u003er\u0026thinsp;=\u003c/em\u003e\u0026thinsp;0.748) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eq\u0026ndash;t, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cp\u003eIn the overall VZV-vaccinated population, ADCP and ADCC activities were strongly correlated with each other (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8060, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and with FAMA titers (ADCP: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.917; ADCC: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.806; both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea\u0026minus;c). In comparison, their correlations with ELISA titer were moderate (ADCP: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.529; ADCC: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.505; both \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed, e).\u003c/p\u003e\u003cp\u003eThese findings suggest that because FAMA titer is a well-established CoP against varicella, the strong correlations of ADCP and ADCC with FAMA indicated that these Fc-mediated activities provide a robust and biologically relevant representation of vaccine-induced immunogenicity, serving as reliable indicators of functional antibody response to VZV vaccines.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eVaricella is a highly contagious infectious disease, and live attenuated varicella vaccines have been incorporated into national immunization programs in many developed countries\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Meanwhile, global demand for varicella vaccines continues to grow, highlighting the importance of accurate immunogenicity assessment. In addition, HZ remains a significant global health concern, with increasing incidence and the limited vaccine availability\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe FAMA test is regarded as the gold standard for assessing protective antibody responses to varicella, as it correlates strongly with neutralizing antibody levels and has long been considered a correlate of protection (CoP)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. However, despite its high sensitivity and specificity, FAMA is labor-intensive, impractical for large-scale clinical trials, and does not directly reflect clearance of infected cells. ELISA is more suitable for high-throughput analysis but provides ambiguous cut-off values when applied to vaccine-induced immunity\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. While these assays assess only acquired humoral immunity, the observations that agammaglobulinemia patients do not progress to severe varicella\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e and that children exhibit minimal VZV-specific T-cell responses\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e suggest that innate cellular immunity plays a critical role in protection. Therefore, it is important to evaluate the contribution of Fc-mediated antibody functions in cooperation with innate immune effector cells when assessing the protective efficacy of varicella vaccines.\u003c/p\u003e\u003cp\u003eHZ, caused by the reactivation of dormant VZV in the ganglia and spreading via nerve fibers, cannot be prevented solely by neutralizing antibodies. Instead, T-cell immunity plays a critical role in protection, typically assessed by cytokine secretion (e.g., IFN-γ, IL-2, and TNF-α) in PBMCs, and the frequency of polyfunctional CD4\u003csup\u003e+\u003c/sup\u003e and CD8\u003csup\u003e+\u003c/sup\u003e T cells \u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38 CR39\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Nevertheless, memory CD8⁺ T-cell responses against VZV are rarely detected even in adults, underscoring the limitations of cellular immune readouts\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. This limitation highlights that such immunological measures do not reflect the clearance of virus-infected cells, and no definitive CoP for HZ vaccine has been established. Consequently, making large-scale clinical follow-up studies remain essential for accurate assessment of HZ vaccine efficacy. In this context, the identification of reliable immunogenicity biomarkers that can serve as CoP for HZ vaccines is urgently needed. Therefore, evaluating Fc-mediated effector functions, such as ADCP and ADCC, may not only provide mechanistic insights into virus clearance but also hold potential as exploratory biomarkers that could complement these clinical endpoints, pending further clinical validation. Evidence from other viral infections supports this concept. In nonhuman primates HIV vaccine studies, ADCP and ADCC correlated with reduced infection or improved viral control\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Similar findings have been reported in SARS-CoV-2, where Fc-mediated effector functions contributed to viral clearance and protection against lethal challenge\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In human, impaired ADCP and ADCC responses have been linked to severe COVID-19 outcomes\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In a previous study on varicella, ADCC activity was detected earlier than Nab responses in individuals with natural infection and in live attenuated vaccine recipients, suggesting a key role for Fc-mediated functions in the early phase of recovery from VZV infection\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. While ADCC induction by zoster vaccination has been speculated\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, the existing study employed artificial systems with limited physiological relevance. To date, few studies have evaluated the capacity of the zoster vaccines to elicit ADCP or ADCC, and none have done so in a comprehensive manner.\u003c/p\u003e\u003cp\u003eTo address this gap, we developed physiologically relevant ADCP and ADCC assays using VZV-infected MRC-5 cells as target cells, employing macrophages as effector cells for ADCP and NK cells for ADCC. This design, better reflects the complex antigenic profile of live attenuated VZV vaccines (e.g, BARYCELA, VARIVAX, and ZOSTAVAX), which elicit antibodies against multiple VZV antigens, thereby providing a broader assessment of antibody functionality than gE-transfected system.\u003c/p\u003e\u003cp\u003eBoth ADCP and ADCC assays demonstrated high reproducibility and robustness. Importantly, their activities correlated strongly with FAMA titers, supporting their validity as functional surrogate marker for protective immunity for varicella. While FAMA primarily reflects acquired humoral immunity, ADCP and ADCC capture the combined contribution of innate cellular and antibody-mediated responses. Our key finding is that all tested VZV vaccines across both live attenuated and subunit platforms and in different age groups elicited antibodies capable of mediating ADCP and ADCC against VZV-infected cells.\u003c/p\u003e\u003cp\u003eInterestingly, although the ELISA-based antibody concentrations were significantly higher in adults vaccinated with zoster vaccine than in children who received LAV, Fc-mediated functions (ADCP and ADCC) were comparable across live attenuated platform vaccine groups regardless of age. This indicates that children generate antibodies with enhanced Fc-effector functionality despite lower total IgG levels. IgG subclass analysis revealed higher IgG3 and lower IgG1 levels in children than in adults. This is consistent with previous observations that varicella vaccination or primary varicella infection in children elicits an IgG3-dominant response, whereas adults with HZ exhibit an IgG1-dominant profile\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Age-related shifts in IgG subclasses align with reports from other viral infections, including RSV and measles, where IgG3 predominates in young children but declines with age\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. The subclass-specific differences observed here reflect known distinctions in Fc receptor engagement on effector cells and the biological activities of IgG subclasses\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. In the RV144 vaccine trial, depletion of IgG3 antibodies significantly reduced ADCP and ADCC activities, underscoring its importance in Fc-effector function\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Together, these findings highlight age-related variation in IgG subclass responses and their impact on Fc-mediated immunity, with implications for optimizing vaccine formulations and assessment strategies tailored to different age groups.\u003c/p\u003e\u003cp\u003eThis study has several limitations. First, the sample size was insufficient to assess the impact of demographic characteristics beyond sex-based differences. Second, we employed M1-differentiated U937 or No-GFP-CD16.NK-92 cell line as effector cells rather than autologous innate immune cells from vaccinees. As a result, we were unable to assess ADCP and ADCC activities mediated by the vaccinees\u0026rsquo; own effector cells. Nevertheless, the use of standardized effector cell lines helps to minimize inter-individual variability, which is crucial to evaluating vaccine-induced antibody functions in clinical trials. In conclusion, we developed cell-based ADCP and ADCC assays that provide physiologically relevant measures of Fc-mediated antibody function against VZV. These assays correlated more strongly with FAMA titers than with ELISA antibody levels. Children, despite lower overall IgG levels, mounted potent IgG3-driven ADCP and ADCC activities comparable to adults. These findings underscore the importance of functional antibody quality over quantity and suggest that incorporating ADCP and ADCC assays alongside conventional serological tests may provide a more comprehensive mechanistically relevant assessment of VZV vaccine-induced immunity.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eEthics statement and Sera from vaccinees\u003c/h2\u003e\u003cp\u003eHuman specimens were obtained from pediatric LAV recipients enrolled in the phase 3 clinical trial of MG1111 (Thailand and South Korea, protocol No. NCT03375502) and from adult ZOSTAVAX recipients (IRB file No. YUH-12-0462) conducted in Yeungnam University Medical Center. Written consent for secondary research use of banked specimens was obtained from all participants or their legal guardians. Secondary use for this study was approved by the Institutional Review Board of Yeungnam University Medical Center (IRB No. YUMC 2023-04-034). Blood collection from SHINGRIX recipients was approved by the Institutional Review Board of Korea University Guro Hospital (IRB No. 2023GR0083). All samples were de-identified prior to transfer. The study was conducted in accordance with the Declaration of Helsinki, and ICH E6(R2) Good Clinical Practice guidelines, as well as applicable local regulatory and bioethics requirements.\u003c/p\u003e\u003cp\u003ePaired pre- and post-vaccination plasma/serum samples were obtained from four cohorts: 1-year-old children who received a single dose of LAV: BARYCELA\u0026reg; (GC Biopharma) or VARIVAX\u0026reg; (Marck \u0026amp; Co.), and adults aged\u0026thinsp;\u0026ge;\u0026thinsp;50 years old who received ZOSTAVAX\u0026reg; (Merck \u0026amp; Co.) or SHINGRIX\u0026reg; (GlaxoSmithKline). In total, 176 samples were analyzed: 46 from the BARYCELA\u0026reg; group, 50 from the VARIVAX\u0026reg; group, and 40 samples each from the ZOSTAVAX\u0026reg; and SHINGRIX\u0026reg; groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCells\u003c/h2\u003e\u003cp\u003eMRC-5 cells (ECACC, UK) were cultured in Minimal Essential Medium (MEM; Welgene Inc,) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% non-essential amino acid (NEAA; Sigma-Aldrich), 1% sodium pyruvate (Gibco), and 1\u0026times; antibiotic-antimycotic (Gibco) at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. U937 cells (ATCC\u0026reg;; CRL-1593.2) were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (SERANA) supplemented with 10% heat-inactivated FBS (HI-FBS; Gibco) and antibiotic. No-GFP-CD16.NK-92 cell line (ATCC\u0026reg;; PTA-6967) were cultured in α-Minimum Essential Medium (α-MEM; Gibco) supplemented with 12.5% HI-FBS, 12.5% Horse Serum (Sigma-Aldrich), 0.2 mM myo-inositol (Sigma-Aldrich), 0.02 mM folic Acid (Sigma-Aldrich), 1\u0026times; antibiotic, 0.1 mM 2-Mercaptoethanol (Gibco), and 100 U/ml IL-2 (R\u0026amp;D systems).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDifferentiation of U937 cells into M1 macrophages and Fc receptors expression\u003c/h2\u003e\u003cp\u003eTo generate M1-polarized macrophages from U937 monocytes, 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e U937 cells in 5 mL of complete culture medium were treated with 20 ng/mL of phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 24 h at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. After PMA treatment, non-adherent cells were removed, and the culture was washed twice with Dulbecco\u0026rsquo;s phosphate buffered saline (DPBS; SERANA), incubated for an additional 24 h in fresh medium. To induce M1 polarization, cells were stimulated with 20 ng/mL of lipopolysaccharide (LPS; Sigma-Aldrich) and 20 ng/mL of interferon-gamma (IFN-γ; R\u0026amp;D system) for 24 h before the ADCP assay. Morphological changes were monitored by optical microscope (Carl Zeiss) (Supplementary Fig.\u0026nbsp;1).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eFlow cytometric staining of Fcγ receptors\u003c/h2\u003e\u003cp\u003eM1-polarized U937 cells were stained separately with fluorochrome-conjugated antibodies against CD16, CD32, and CD64 (BioLegend, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) in 2% FBS-DPBS for 30 min on ice. Cells were then washed twice with 2% FBS-DPBS before acquisition on CytoFLEX flow cytometer (Beckman Coulter).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAntibody panel used for Fc receptors\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTarget\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFull name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCommon clone\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFluorochrome\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCatalog No. (BD)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFcγRIII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3G8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFITC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e560996\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD32\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFcγRII\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFLI8.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e568913\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCD64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFcγRI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eAPC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e561189\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eVZV-infected target cell preparation\u003c/h2\u003e\u003cp\u003eVZV-infected cells were prepared as target cells as follows: MRC-5 cells at 70%\u0026ndash;80% confluence were infected with VZV YC03 strain (GenBank Accession No. KJ808816) at a ratio of one infected cell to 100 normal MRC-5 cells. The cells were harvested 72 h post-infection and cryopreserved in liquid nitrogen until use. The percentage of cells expressing VZV antigens on cell surface was determined using a flow cytometry-based FAMA test\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The WHO international standard for VZV immunoglobulin (NIBSC code W1044, diluted to 50 mIU/mL) was used as a positive reference, and DPBS served as the negative control. Following FAMA staining, VZV antigen expressed cells were analyzed using CytoFLEX flow cytometer (Beckman Coulter). Cells preparations exhibiting\u0026thinsp;\u0026ge;\u0026thinsp;80% VZV-positive events were used as target cell in ADCP and ADCC assays.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eAntibody-dependent cellular phagocytosis assay\u003c/h2\u003e\u003cp\u003eThe phagocytic activity was evaluated using M1-U937 as effector cells. Sera were heat inactivated at 56\u0026deg;C for 30 min before the assay. VZV-infected MRC-5 target cells were labeled with 3 \u0026micro;M CFSE (5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester) in DPBS at 37\u0026deg;C for 10 min and then washed twice with DPBS supplemented with 2% HI-FBS. The CFSE-labeled target cells were incubated with control or vaccinated sera for 30 min; followed by co-incubated with M1-U937 effector cells for 4 h. Sera were applied at final dilutions of 1:100. Prior to flow cytometry, M1-U937 cells were labeled with CD89 antibody conjugated with APC (Miltenyi Biotec). ADCP activity, measured using CytoFLEX flow cytometer (Beckman Coulter), was expressed as the percentage of CSFE\u003csup\u003e+\u003c/sup\u003eCD89\u003csup\u003e+\u003c/sup\u003e double-positive macrophages which phagocytosed CFSE\u003csup\u003e+\u003c/sup\u003e VZV-infected cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eAntibody-dependent cellular cytotoxicity assay: measuring granzyme B (GrB\u003csup\u003e+\u003c/sup\u003e) incorporation into target cell by flow cytometry\u003c/h2\u003e\u003cp\u003eTo measure ADCC activities, GrB\u003csup\u003e+\u003c/sup\u003e incorporation into the VZV-infected MRC-5 target cell assay was adopted from the ADCC-GTL assay\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. VZV-infected target cells were labeled with 5 \u0026micro;M CellTracker\u0026trade; Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine (Invitrogen) in serum-free AIM-V medium (Gibco) at 37\u0026deg;C for 30 min. Labeled cells were washed once with serum-free AIM-V medium, and 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well were plated in 96-well U-bottom plate. Heat-inactivated sera were added and incubated with target cells for 30 min at 37\u0026deg;C. No-GFP-CD16.NK-92\u0026reg; cells were then added at a 1:1 E:T ratio, yielding a final serum dilution of 1:100. Co-cultures were incubated for 4 h at 37\u0026deg;C before harvest. Cells were fixed with 100 \u0026micro;L/well of Fixation/Permeabilization solution (BD Biosciences) for 20 min at 4\u0026deg;C, washed twice with 1\u0026times; BD Perm/Wash\u0026trade; buffer (BD Biosciences), and pelleted. The fixed/permeabilized cells were resuspended thoroughly in 100 \u0026micro;L of 1\u0026times; BD Perm/Wash\u0026trade; buffer containing FITC conjugated anti-Granzyme B (GrB) antibody at a 1:50 dilution (BioLegend). The samples were incubated at 4\u0026deg;C for 30 min in the dark. After incubation, cells were washed cells twice with 1\u0026times; BD Perm/Wash\u0026trade; buffer and resuspended in 1\u0026times; BD Perm/Wash\u0026trade; buffer for flow cytometric analysis. ADCC activities, measured using CytoFLEX flow cytometer (Beckman Coulter), was expressed as the percentage of CMTMR\u003csup\u003e+\u003c/sup\u003e GrB\u003csup\u003e+\u003c/sup\u003e double-positive target cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eValidation of ADCP and ADCC assays\u003c/h2\u003e\u003cp\u003eFollowing established bioanalytical assay validation guidelines\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, key performance parameters were selected for methodological evaluation of ADCP and ADCC assays. These parameters included VZV-target cell lot-to-lot variation, linearity, intra-assay precision, inter-assay precision (inter-personal and inter-day precision) and inter-laboratories precision.\u003c/p\u003e\u003cp\u003eLot-to-lot variability was evaluated to determinate the consistency of assay results across different target cell stocks used in the same experiment, ensuring that variations between target cells do not significantly affect assay outcomes. Linearity assay was assessed using five serial dilutions of three different sera, with a simple linear regression analysis performed to confirm linearity. Intra-assay precision was assessed by testing the six samples, each five replicated on the same plate by one operator, to determine the assay\u0026rsquo;s precision within a single run. Inter-assay precision was assessed by testing the same six samples across three days, potentially by two different operators, to assess reproducibility across independent experiments. Inter-laboratory precision was determined by having three laboratories independently conduct the assay using identical sera and the same stock of VZV-target cells, ensuring reproducibility across different lab setting. The coefficient of variation (CV) was calculated to evaluate the precision.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eFluorescent antibody to membrane antigen test\u003c/h2\u003e\u003cp\u003eFAMA test was conducted as previously described\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Briefly, FAMA antigens were prepared as follows: MRC-5 cells at 70%\u0026ndash;80% confluence were infected with VZV YC03 strain at a ratio of one infected cell to 200 normal MRC-5 cells and harvested 72 h post-infection, and stored in liquid nitrogen until use. Sera were twofold serially diluted with DPBS, and then incubated with 2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e FAMA antigen cells for 30 min at RT, followed by two DPBS washes. After the secondary antibody (goat anti-human IgG-Alexa 488, Invitrogen) reaction for 30 min at RT, cells were washed three times with DPBS. FAMA antigen cells were loaded onto a 14-well slide (Cel-Line\u0026reg;, Thermo Scientific) and then dried. Slides were mounted with Vectashield mounting medium with DAPI (Vector Laboratories) and observed using an Axioscope fluorescence microscope equipped with an HBO 100 mercury lamp (Carl Zeiss).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eVZV-specific IgG ELISA\u003c/h2\u003e\u003cp\u003eVZV-specific IgG levels were quantified using the SERION ELISA classic kit (ESR104G, Virion\\Serion, Germany) according to the manufacturer\u0026rsquo;s instructions. Briefly, 100 \u0026micro;L of controls and diluted sera were added to VZV-antigen-coated wells and incubated for 1 h. After washing, 100 \u0026micro;L of goat anti-human IgG conjugated with alkaline phosphatase (AP) was added and incubated for 30 min. Following additional washes, substrate was added and incubated for 30 min. All incubation steps were performed at 37\u0026deg;C. The reaction was then terminated with a stopping solution, and the absorbance was measured at 405 nm using a Multiskan FC 357 microplate photometer (Thermo Scientific).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eVZV-specific IgG subclass ELISA\u003c/h2\u003e\u003cp\u003eVZV-specific IgG1 and IgG3 antibodies were measured using the SERION ELISA classic kit (ESR104G, Virion\\Serion) with modifications. VZV antigen-coated plates were incubated with 100 \u0026micro;L of diluted sera (1:50) for 1 h at 37\u0026deg;C. After washing, plates were incubated with mouse anti-human IgG1 (1:2000) or IgG3 (1:100) antibodies conjugated with AP (SouthernBiotech) at 37\u0026deg;C for 30 min. Following incubation with pNPP substrate for 30 min, the reaction was stopped, and absorbance was measured at 405 nm using a Multiskan FC 357 microplate Photometer (Thermo Scientific). Subclass-specific responses were expressed as optical densities.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analyses\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using GraphPad Prism 10.0.2 (GraphPad Software). Pair t-tests or Wilcoxon matched-paired signed rank tests were used for comparisons between two groups. One-way ANOVA or the Kruskal-Wallis tests were used for multiple group comparisons, with post-hoc pairwise correction as appropriate. Pearson correlation coefficients (r) were calculated to assess relationships among ADCP, ADCC, FAMA and ELISA GMTs. Statistical significance was denoted as ns, not significant; * \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; *** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; **** \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing financial or personal interests that could have influenced the work reported in this manuscript.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was supported by grant 23202MFDS136 from the Ministry of Food and Drug Safety, Republic of Korea, in 2023. The funding agency had no role in study design, data collection and analysis, decision to publish, or manuscript preparation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.X., J.Y.H., H.S.L., O.S.S., J.Y.N., S.H.H., and H.P. conceived and designed the study. S.X., J.Y.H., Y.K., K.M.L., E.J.J., S.T.C., J.S.L., J.Y.B., K.K., B.Y., and J.H.L performed the experiments and collected the data. S.X. and Y.K. analyzed the data. S.X., J.Y.H., Y.K., and H.P. drafted the manuscript, which was critically reviewed by all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank the Ministry of Food and Drug Safety, Republic of Korea, for supporting this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its Supplementary Information files. Additional datasets are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHaumont, M. et al. Neutralizing antibody responses induced by varicella-zoster virus gE and gB glycoproteins following infection, reactivation or immunization. \u003cem\u003eJ. Med. Virol.\u003c/em\u003e 53, 63\u0026ndash;68 (1997).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAsada, H. VZV-specific cell-mediated immunity, but not humoral immunity, correlates inversely with the incidence of herpes zoster and the severity of skin symptoms and zoster-associated pain: The SHEZ study. \u003cem\u003eVaccine\u003c/em\u003e 37, 6776\u0026ndash;6781 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLevin, M. J. et al. Varicella-zoster virus-specific immune responses in elderly recipients of a herpes zoster vaccine. \u003cem\u003eJ. Infect. 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Immunother.\u003c/em\u003e 19, 2210961 (2023).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antibody-dependent cellular phagocytosis, Antibody-dependent cellular cytotoxicity, Varicella-zoster virus, Immunogenicity, Vaccine","lastPublishedDoi":"10.21203/rs.3.rs-8121887/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8121887/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAssessing the functional activity of vaccine-induced antibodies is critical for evaluating immunogenicity. We developed and validated antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) assays to quantify Fc-mediated antibody responses elicited by varicella and zoster vaccines. Both assays demonstrated robust performance and broad linearity. Antibody titers were measured using fluorescent antibody to membrane antigen (FAMA) and ELISA. ADCP and ADCC activities, along with FAMA and ELISA geometric mean titers (GMTs), were significantly increased in post- vs. pre-vaccination sera (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.0001). Strong correlations were observed between ADCP and ADCC activities and both FAMA and ELISA GMTs. Although children exhibited lower total varicella-zoster virus-specific IgG levels than adults, higher IgG3 subclass levels in children were associated with comparable Fc-mediated activities. These results highlight the utility of ADCP and ADCC as valuable assays for evaluating Fc-mediated antibody function and potential surrogates of protective immunity to varicella and zoster vaccination.\u003c/p\u003e","manuscriptTitle":"Evaluating varicella-zoster virus vaccine immunogenicity through Fc-mediated antibody functions: the roles of ADCP and ADCC","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-15 01:08:17","doi":"10.21203/rs.3.rs-8121887/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-04T20:08:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-25T09:54:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-25T06:43:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184195728392878703133489550376611831896","date":"2025-12-10T13:49:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"52620587700390067740685470865926192420","date":"2025-12-10T04:59:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-09T16:11:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-06T20:11:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-19T17:30:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2025-11-15T12:04:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b9aa4a60-a329-400d-b44e-17592e717f18","owner":[],"postedDate":"December 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":59426581,"name":"Biological sciences/Immunology"},{"id":59426582,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-30T16:18:29+00:00","versionOfRecord":{"articleIdentity":"rs-8121887","link":"https://doi.org/10.1038/s41541-026-01424-w","journal":{"identity":"npj-vaccines","isVorOnly":false,"title":"npj Vaccines"},"publishedOn":"2026-03-25 16:11:23","publishedOnDateReadable":"March 25th, 2026"},"versionCreatedAt":"2025-12-15 01:08:17","video":"","vorDoi":"10.1038/s41541-026-01424-w","vorDoiUrl":"https://doi.org/10.1038/s41541-026-01424-w","workflowStages":[]},"version":"v1","identity":"rs-8121887","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8121887","identity":"rs-8121887","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}